Antibody-drug conjugates comprising anti-human ROR1 antibodies and their uses

Abstract

This invention application relates to antibody-drug conjugates comprising anti-human ROR1 antibodies and their uses. The present invention relates to novel antibody-drug conjugate ADCs targeting ROR1; active metabolites of such ADCs; methods for preparing such ADCs; the use of such ADCs in the treatment and / or prevention of diseases; and the use of such ADCs in the production of drugs for the treatment and / or prevention of diseases, more specifically diseases associated with the overexpression of ROR1, such as cancer. More specifically, the present invention relates to an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof that binds to ROR1, and a pharmaceutical composition comprising the same.

Description

[0001] This application is a divisional application of the application with the filing date of August 21, 2020, application number 202010852078.1, and invention title "Antibody-drug conjugate comprising anti-human ROR1 antibody and its use".

[0002] Related Applications

[0003] This application claims the priority of Korean Application No. 10-2019-0109807, filed on September 4, 2019, which is incorporated herein by reference in its entirety. Technical Field

[0004] The present invention relates to novel antibody-drug conjugates (ADCs) targeting ROR1; active metabolites of such ADCs; methods for preparing such ADCs; the use of such ADCs in the treatment and / or prevention of diseases; and the use of such ADCs in the manufacture of drugs for the treatment and / or prevention of diseases, more specifically diseases associated with the overexpression of ROR1, such as cancer. More specifically, the present invention relates to an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof that binds to ROR1, and a pharmaceutical composition comprising the same. Background Art

[0005] Cancer is a disease caused by abnormal and uncontrolled cell growth in body tissues and is the result of uncontrolled cell growth in various tissues. Tumors in early-stage cancer can be removed by surgical and radiotherapy measures, and metastatic tumors are generally palliated with chemotherapy.

[0006] Most chemotherapeutic agents administered parenterally may induce unwanted side effects or severe toxicity due to systemic administration. Therefore, the focus of development has been on the development of novel chemotherapeutic agents to achieve increased efficacy and minimal toxicity / side effects through the modified and selective action of chemotherapeutic agents on tumor cells or adjacent tissues.

[0007] Antibody-drug conjugates (ADCs) are a targeting technology in which a toxin or drug is conjugated to an antibody, which then binds to an antigen, and the toxin or drug is released into tumor cells, etc., to cause cell death. This technology has excellent efficacy superior to antibody drugs and can significantly reduce the risk of side effects compared to conventional anticancer agents because it specifically delivers the drug to target cancer cells with minimal impact on healthy cells and releases the drug only under specific conditions.

[0008] The basic structure of an antibody-drug conjugate is "antibody-linker-small molecule drug (toxin)". Here, the linker not only needs to function to connect the antibody and the drug, but also ensure that the drug is properly released by antibody-drug dissociation (e.g., due to enzymatic hydrolysis) after circulating through the body and reaching the target cell, and exhibit efficacy against the targeted cancer cells. That is to say, the stability of the linker plays an extremely crucial role in the efficacy and systemic toxicity of the antibody-drug conjugate (Discovery Medicine 2010, 10(53):329-39)85-8 2018-08-14).

[0009] The use of monoclonal antibodies for cancer treatment has had considerable success. Monoclonal antibodies are suitable for the targeted localization of tumor tissues and tumor cells. Antibody-drug conjugates have become a novel and powerful option for the treatment of lymphoma and solid tumors, and recently, immunomodulatory antibodies have appeared to be significantly successful in clinical trials. The development of therapeutic antibodies is based on a profound understanding of cancer serology, protein engineering techniques, mechanisms of action and drug resistance, and the interaction between the immune system and cancer cells.

[0010] Antigens expressed on the surface of human cancer cells cover a wide range of targets that are overexpressed, mutated, or selectively expressed compared to normal tissues. The key issue is to identify the appropriate antigens for antibody-based therapies. These therapeutic agents mediate changes in antigen or receptor function (i.e., as agonists or antagonists), regulate the immune system through Fc and T cell activation, and exhibit efficacy by delivering specific drugs conjugated to antibodies targeting specific antigens. Molecular techniques that can alter antibody pharmacokinetics, function, size, and immune stimulation have emerged as key elements in the development of novel antibody-based therapies. Evidence from clinical trials of therapeutic antibodies in cancer patients emphasizes the importance of methods for optimizing antibodies, including the affinity and binding of the target antigen and the antibody, the choice of antibody structure, and the route of treatment (blocking signal transduction or immune function).

[0011] ROR1 is expressed during embryonic and fetal development and controls cell polarity, cell migration, neurite outgrowth, etc. Expression gradually decreases as development progresses, and it is barely expressed in adults. It is transiently expressed during the development of B cells and is only reported to be minimally expressed in adipocytes (Hudecek et al., 2010, Blood 116:4532, Matsuda et al., 2001, Mech. Dev. 105:153).

[0012] However, when overexpression of ROR1 is observed in various cancer cells, it is classified as an oncogene. Specifically, after ROR1 was found to be overexpressed in chronic lymphocytic leukemia (CLL), it has received attention as a target for anti-cancer antibodies (Klein et al., 2001, J. Exp. Med 194:1625). It has been reported that it is overexpressed not only in blood cancers such as B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt lymphoma, mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and marginal zone lymphoma (MZL), but also in solid cancers including breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer. The expression of ROR1 in such cancers has been reported to be associated with poor prognosis in cancer patients and to affect cancer metastasis.

[0013] Such cancer-specific expression of ROR1 indicates that ROR1 can be an effective cancer target and that antibodies capable of specifically recognizing it need to be developed.

[0014] U.S. Patent No. 9,316,646 relates to anti-ROR1 antibodies and discloses monoclonal antibodies that specifically recognize the human extracellular domain of ROR1.

[0015] U.S. Patent No. 9,266,952 relates to antibodies against ROR1 and their uses and discloses an antibody that induces the death of CLL cells by specifically binding to CLL cells.

[0016] Since various anti-cancer antibodies against the same ROR1 antigen can be developed according to the characteristics or uses of each antibody, when considering the cancer-specific expression of ROR1 and its expression in various cancers, it is necessary to develop various antibodies that can replace or supplement existing antibodies. SUMMARY OF THE INVENTION

[0017] Regarding this technical background, the inventors of the present application have been dedicated to developing antibodies that specifically bind to ROR1, and as a result, anti-ROR1 antibodies have been developed that exhibit excellent binding to ROR1. By applying a linker comprising an effective self-immolative group, which is stable in plasma and in circulation but allows for easy release of the drug and exhibits efficacy in cancer cells, to the anti-ROR1 antibody to further enhance the antibody's effect, it is possible to provide an applicable antibody-drug conjugate (ADC) that targets ROR1 and effectively treats and / or prevents cancer diseases.

[0018] The present invention provides novel antibody-drug conjugates that target ROR1 or a salt thereof.

[0019] In certain embodiments, the present invention provides a drug-antibody conjugate comprising an antibody that specifically binds to ROR1 and a drug linked thereto, and a pharmaceutical composition comprising the same.

[0020] Furthermore, the present invention provides an antibody-linker-drug (toxin) system that allows the drug and / or toxin to reach the target cells and effectively exhibit efficacy, while substantially reducing toxicity for a linker comprising a self-immolative group that is stable in plasma and in circulation, but allowing for easy release of the drug in cancer cells to exhibit efficacy.

[0021] Technical Solution

[0022] One aspect of the present invention provides an antibody conjugate of Formula I or a pharmaceutically acceptable salt or solvate thereof.

[0023] [Formula I]

[0024] Ab-(X)y

[0025] Wherein:

[0026] A is an antibody that specifically binds to the extracellular domain of ROR1, the antibody comprising a heavy chain variable region and a light chain variable region or an antigen-binding fragment thereof,

[0027] Wherein the antibody or an antigen-binding fragment thereof comprises:

[0028] A heavy chain variable region comprising a CDRH1 comprising an amino acid sequence of any one of SEQ ID NOs. 1 to 5;

[0029] A CDRH2 comprising an amino acid sequence of any one of SEQ ID NOs. 6 to 13 and 96; and

[0030] A CDRH3 comprising an amino acid sequence of any one of SEQ ID NOs. 14 to 21 and 97; and

[0031] A light chain variable region comprising CDRL1 having an amino acid sequence comprising any one of SEQ ID NOs. 22 to 29;

[0032] CDRL2 having an amino acid sequence comprising any one of SEQ ID NOs. 30 to 37; and

[0033] CDRL3 having an amino acid sequence comprising any one of SEQ ID NOs. 38 to 42;

[0034] X is a chemical residue independently comprising at least one active agent and a linker;

[0035] The linker connects the Ab and the at least one active agent; and

[0036] y is an integer from 1 to 20.

[0037] In some embodiments, the antibody specifically recognizes the extracellular domain of, for example, human ROR1 and exhibits cross-reactivity to murine ROR1.

[0038] Included within the scope of the present invention are not only intact antibody forms that specifically bind to ROR1, but also binding-resistant fragments of the antibody molecule.

[0039] In some embodiments, the antibody or antigen-binding fragment according to the present invention comprises (i) the complementarity-determining regions of CDRH1, CDRH2, and CDRH3, and / or (ii) the complementarity-determining regions of CDRL1, CDRL2, and CDRL3, wherein CDRH1 has an amino acid sequence comprising any one of SEQ ID NOs. 1 to 5; CDRH2 has an amino acid sequence comprising any one of SEQ ID NOs. 6 to 13 and 96; CDRH3 has an amino acid sequence comprising any one of SEQ ID NOs. 14 to 21 and 97; CDRL1 has an amino acid sequence comprising any one of SEQ ID NOs. 22 to 29; CDRL2 has an amino acid sequence comprising any one of SEQ ID NOs. 30 to 37; and CDRL3 has an amino acid sequence comprising any one of SEQ ID NOs. 38 to 42. CDRH indicates the CDR included in the heavy chain variable region, and CDRL indicates the CDR included in the light chain variable region.

[0040] From this perspective, in other embodiments, an antibody or antigen-binding fragment according to the present invention comprises (i) the complementarity-determining regions of CDRH1, CDRH2, and CDRH3, and / or (ii) the complementarity-determining regions of CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 5; CDRH2 comprises the amino acid sequence of any one of SEQ ID NOs: 6 to 13 and 96; CDRH3 comprises the amino acid sequence of any one of SEQ ID NOs: 14 to 21 and 97; CDRL1 comprises the amino acid sequence of any one of SEQ ID NOs: 22 to 29; CDRL2 comprises the amino acid sequence of any one of SEQ ID NOs: 30 to 37; and CDRL3 comprises the amino acid sequence of any one of SEQ ID NOs: 38 to 42.

[0041] In other embodiments, CDRH1, CDRH2, and CDRH3 comprise the following amino acid sequences: SEQ ID NOs: 1, 6, and 14, respectively; SEQ ID NOs: 2, 7, and 15, respectively; SEQ ID NOs: 1, 8, and 16, respectively; SEQ ID NOs: 3, 9, and 17, respectively; SEQ ID NOs: 1, 10, and 18, respectively; SEQ ID NOs: 4, 11, and 19, respectively; SEQ ID NOs: 5, 12, and 20, respectively; SEQ ID NOs: 3, 13, and 21, respectively; SEQ ID NOs: 2, 96, and 15, respectively; or SEQ ID NOs: 3, 9, and 97, respectively.

[0042] In other embodiments, CDRL1, CDRL2, and CDRL3 respectively comprise the following amino acid sequences: SEQ ID NOs: 22, 30, and 38, respectively; SEQ ID NOs: 23, 31, and 39, respectively; SEQ ID NOs: 24, 32, and 40, respectively; SEQ ID NOs: 25, 33, and 41, respectively; SEQ ID NOs: 26, 34, and 41, respectively; SEQ ID NOs: 27, 35, and 42, respectively; SEQ ID NOs: 28, 36, and 41, respectively; or SEQ ID NOs: 29, 37, and 41, respectively.

[0043] In other embodiments, the antibody comprises a set of CDRs: CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein:

[0044] (a) CDRH1, CDRH2, and CDRH3 of SEQ ID NOs: 1, 6, and 14, respectively, and CDRL1, CDRL2, and CDRL3 of SEQ ID NOs: 22, 30, and 38, respectively;

[0045] (b) CDRH1, CDRH2, and CDRH3 contain the amino acid sequences of SEQ ID NO: 2, 7, and 15 respectively, and CDRL1, CDRL2, and CDRL3 contain the amino acid sequences of SEQ ID NO: 23, 31, and 39 respectively;

[0046] (c) CDRH1, CDRH2, and CDRH3 contain the amino acid sequences of SEQ ID NO: 1, 8, and 16 respectively, and CDRL1, CDRL2, and CDRL3 contain the amino acid sequences of SEQ ID NO: 24, 32, and 40 respectively;

[0047] (d) CDRH1, CDRH2, and CDRH3 contain the amino acid sequences of SEQ ID NO: 3, 9, and 17 respectively, and CDRL1, CDRL2, and CDRL3 contain the amino acid sequences of SEQ ID NO: 25, 33, and 41 respectively;

[0048] (e) CDRH1, CDRH2, and CDRH3 contain the amino acid sequences of SEQ ID NO: 1, 10, and 18 respectively, and CDRL1, CDRL2, and CDRL3 contain the amino acid sequences of SEQ ID NO: 26, 34, and 41 respectively;

[0049] (f) CDRH1, CDRH2, and CDRH3 contain the amino acid sequences of SEQ ID NO: 4, 11, and 19 respectively, and CDRL1, CDRL2, and CDRL3 contain the amino acid sequences of SEQ ID NO: 27, 35, and 42 respectively;

[0050] (g) CDRH1, CDRH2, and CDRH3 contain the amino acid sequences of SEQ ID NO: 5, 12, and 20 respectively, and CDRL1, CDRL2, and CDRL3 contain the amino acid sequences of SEQ ID NO: 28, 36, and 41 respectively;

[0051] (h) CDRH1, CDRH2, and CDRH3 contain the amino acid sequences of SEQ ID NO: 3, 13, and 21 respectively, and CDRL1, CDRL2, and CDRL3 contain the amino acid sequences of SEQ ID NO: 29, 37, and 41 respectively;

[0052] (i) CDRH1, CDRH2, and CDRH3 contain the amino acid sequences of SEQ ID NO: 2, 96, and 15 respectively, and CDRL1, CDRL2, and CDRL3 contain the amino acid sequences of SEQ ID NO: 23, 31, and 39 respectively; or

[0053] (j) CDRH1, CDRH2 and CDRH3 contain the amino acid sequences of SEQ ID NO 3, 9 and 97 respectively, and CDRL1, CDRL2 and CDRL3 contain the amino acid sequences of SEQ ID NO 25, 33 and 41 respectively.

[0054] In some embodiments, the antibody or antigen-binding fragment according to the present invention comprises a heavy chain variable region i, which comprises: the amino acid sequence of any one of SEQ ID NOs 43 to 50, 98 and 99; at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NOs 43 to 50, 98 and 99; or at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs 43 to 50, 98 and 99.

[0055] In some embodiments, the antibody or antigen-binding fragment according to the present invention comprises a light chain variable region, which comprises: the amino acid sequence of any one of SEQ ID NOs 51 to 58; at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NOs 51 to 58; or at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs 51 to 58.

[0056] In some embodiments, the antibody or antigen-binding fragment according to the present invention comprises a combination of heavy and light chain variable regions, the heavy and light chain variable regions comprising the following amino acid sequences: the sequences indicated by: SEQ ID NOs 43 and 51 respectively; SEQ ID NOs 44 and 52 respectively; SEQ ID NOs 45 and 53 respectively; SEQ ID NOs 46 and 54 respectively; SEQ ID NOs 47 and 55 respectively; SEQ ID NOs 48 and 56 respectively; SEQ ID NOs 49 and 57 respectively; SEQ ID NOs 50 and 58 respectively; SEQ ID NOs 98 and 52 or SEQ ID NOs 99 and 54 respectively; at least 90% complementary to these amino acid sequences; or at least 95% complementary to these amino acid sequences.

[0057] In some embodiments, the antibody or antigen-binding fragment according to the present invention is a human antibody, a fully human antibody or an antigen-binding fragment that cross-reacts with murine ROR1.

[0058] In some embodiments, the antibody according to the present invention is a monoclonal antibody, particularly a human monoclonal antibody, which cross-reacts with murine ROR1.

[0059] In some embodiments, the antibody or antigen-binding fragment according to the present invention specifically recognizes and / or binds to human and murine ROR1.

[0060] In some embodiments, the antibody according to the present invention is of the IgG1, IgG2, IgG3 or IgG4 type.

[0061] In some embodiments, the antibody according to the present invention comprises a combination of a heavy chain and a light chain, which comprises the following amino acid sequences: SEQ ID NO 59 and 67 respectively; SEQ ID NO 60 and 68 respectively; SEQ ID NO 61 and 69 respectively; SEQ ID NO 62 and 70 respectively; SEQ ID NO 63 and 71 respectively; SEQ ID NO 64 and 72 respectively; SEQ ID NO 65 and 73 respectively or SEQ ID NO 66 and 74 respectively.

[0062] In some embodiments, the antibody or antigen-binding fragment according to the present invention may include, but is not limited to: monoclonal antibody, domain antibody (dAb), single-chain antibody (scab), Fab fragment, Fab' fragment, F(ab')2 fragment, scFab fragment, Fv fragment, dsFv fragment, single-chain variable fragment (scFv), ScFv-Fc fragment, single-domain heavy-chain antibody, single-domain light-chain antibody, variant antibody, multimeric antibody, microantibody, bifunctional antibody, bispecific antibody or multispecific antibody.

[0063] In some embodiments, in addition to the ROR1 antigen, the antibody or antigen-binding fragment is also specific for at least one or more antigens. In some embodiments, the at least one or more antigens are different from ROR1 and may include, for example, cancer antigens, in which case the antibody may be bispecific. Depending on the specific purpose of the bispecific antibody, those skilled in the art will be able to select a suitable cancer antigen.

[0064] In another aspect, the present invention provides an isolated polynucleotide encoding the antibody or antigen-binding fragment according to the present invention.

[0065] In some embodiments, the polynucleotide according to the present invention is a polynucleotide encoding the CDR disclosed in the present invention.

[0066] In some embodiments, the polynucleotide according to the present invention is a polynucleotide encoding the variable region of the heavy chain or light chain disclosed in the present invention.

[0067] In still other embodiments, the polynucleotide according to the present invention is a polynucleotide encoding the heavy chain or light chain disclosed in the present invention.

[0068] In some embodiments, the polynucleotides according to the invention comprise a sequence encoding any one of SEQ ID NOs: 75 to 82, 102 and 103 of the full-length heavy chain disclosed in the present invention.

[0069] In some embodiments, the polynucleotides according to the invention comprise a sequence encoding any one of SEQ ID NOs: 83 to 90 of the full-length light chain disclosed in the present invention.

[0070] Based on the CDR and variable region amino acid sequences disclosed in the present invention, the polynucleotides encoding the CDRs and variable regions according to the invention can be readily determined from the above nucleic acid sequences encoding the heavy and light chains.

[0071] In another aspect, there is provided a vector comprising a polynucleotide according to the invention. In some embodiments, the vector according to the invention comprises an expression vector for antibody production or a vector for CAR-T cells (chimeric antigen receptor redirected T cells) or CAR-NK (natural killer) cells.

[0072] In another aspect, there is provided a cell line transformed with a vector according to the invention.

[0073] In yet another aspect, the present invention provides a method for preparing an isolated antibody that specifically binds to ROR1 or an antigen-binding fragment thereof, the method comprising the step of isolating an antigen or an antigen-binding fragment thereof from a cell line according to the invention.

[0074] In certain embodiments, the antibody-drug conjugate has a structure represented by formula IIa:

[0075] [Formula (IIa)]

[0076]

[0077] or a pharmaceutically acceptable salt thereof, wherein

[0078] G is a sugar, sugar acid or sugar derivative;

[0079] W is -C(O)-, -C(O)NR'-, -C(O)O-, SO 2 NR'-, -P(O)R"NR'-, -SONR'- or -PO 2 NR'-; wherein C, S or P is directly bonded to the phenyl ring;

[0080] Each Z is independently C 1 -C 8 alkyl, halogen, cyano or nitro;

[0081] n is an integer from 0 to 3; and

[0082] m is 0 or 1;

[0083] L is absent, is C 1 -C 50 an alkylene or heteroalkylene of 1 to 50 atoms, or L contains at least one branching unit (BR) and at least one linking unit;

[0084] R 1 and R 2 are independently hydrogen, C 1 -C 8 alkyl or C 3 -C 8 cycloalkyl; or R 1 and R 2 combine to complete a (C 3 -C 8 ) cycloalkyl ring;

[0085] * represents the point of attachment to the active agent; and

[0086] represents the point of attachment to the antibody.

[0087] In certain preferred embodiments, the antibody-drug conjugate has a structure represented by Formula II:

[0088] [Formula II]

[0089]

[0090] or a pharmaceutically acceptable salt thereof, wherein

[0091] G is a glucuronic acid moiety or

[0092] R 3 is hydrogen or a carboxyl protecting group;

[0093] Each of R 4 is independently hydrogen or a hydroxyl protecting group;

[0094] B is an active agent;

[0095] R 1 and R 2 are each independently hydrogen, C 1 -C 8 alkyl or C 3 -C 8 cycloalkyl;

[0096] W is -C(O)-, -C(O)NR'-, -C(O)O-, SO 2 NR'-, -P(O)R"NR'-, -SONR'- or -PO 2NR'-; wherein C, S or P is directly bonded to the phenyl ring;

[0097] R' and R" are each independently hydrogen, C 1 -C 8 alkyl, C 3 -C 8 cycloalkyl, C 1 -C 8 alkoxy, C 1 -C 8 alkylthio, mono-C 1 -C 8 alkylamino or di-C 1 -C 8 alkylamino, C 3 -C 20 heteroaryl or C 6 -C 20 aryl;

[0098] Each Z is independently C 1 -C 8 alkyl, halogen, cyano or nitro;

[0099] n is an integer from 0 to 3; and

[0100] L comprises:

[0101] A) A C 1 -C 50 alkylene or 1 to 50 atom heteroalkylene that satisfies at least one of the following:

[0102] (i) L includes at least one unsaturated bond;

[0103] (ii) L is substituted by a divalent substituent, where 2 atoms in L are the same as those in the substituent, thus completing a heteroaryl;

[0104] (iii) L is interspersed with a divalent heteroaryl;

[0105] (iv) L is a 1 to 50 atom heteroalkylene;

[0106] (v) L is substituted by at least one C 1-20 alkyl; or

[0107] B) At least one isoprenyl-derived unit of the following general formula III, which can be recognized by a prenyltransferase:

[0108] [General formula III]

[0109]

[0110] In certain embodiments, G is

[0111] R 3 is a hydrogen or carboxyl protecting group; and

[0112] each R 4 is, independently of one another, a hydrogen or hydroxyl protecting group.

[0113] In certain preferred embodiments, R 3 is hydrogen, and each R 4 is hydrogen.

[0114] In certain embodiments, R 1 and R 2 are hydrogen.

[0115] In certain embodiments, Z is independently C 1 -C 8 alkyl, halogen, cyano or nitro.

[0116] In certain embodiments, n is 0.

[0117] In certain embodiments, W is -C(O)-, -C(O)NR'-, -C(O)O-, SO 2 NR'-, -P(O)R"NR'-, -SONR'- or -PO 2 NR'-; said C, S or P is directly bonded to said phenyl ring; said R' and R" are, independently of one another, compounds which are hydrogen, C 1 -C 8 alkyl, C 3 -C 8 cycloalkyl, C 1 -C 8 alkoxy, C 1 -C 8 alkylthio, mono-C 1 -C 8 alkylamino or di-C 1 -C 8 alkylamino, C 3 -C 20 heteroaryl or C 6 -C 20 aryl. In certain embodiments, W is -C(O)-, -C(O)NR'- or -C(O)O-. In certain preferred embodiments, W is -C(O)NR'-; and C(O) is directly bonded to the phenyl ring and NR' is bonded to L.

[0118] In certain embodiments, G is

[0119] W is -C(O)NR'-, the C(O) being bonded to the phenyl ring and NR' being bonded to L; and

[0120] R3 and R 4 is hydrogen.

[0121] In certain embodiments, L is a C containing at least one of the following 1 -C 50 alkylene or 1-50 atom heteroalkylene:

[0122] (i) an unsaturated bond;

[0123] (ii) a divalent substituent, where 2 atoms in L are the same as those in the substituent, which completes a heteroaryl;

[0124] (iii) a divalent heteroaryl interspersed with L;

[0125] (iv) 1 to 50 heteroalkylenes; or

[0126] (v) at least one C 1-20 alkyl substituent.

[0127] In certain embodiments, L is a C containing at least one of the following 1 -C 50 alkylene or 1-50 atom heteroalkylene:

[0128] (i) an unsaturated bond;

[0129] (ii) a heteroaryl (e.g., interspersed with L);

[0130] (iii) 1 to 50 heteroalkylenes; or

[0131] (iv) at least one C 1-20 alkyl substituent.

[0132] In certain embodiments, L is a heteroalkyl group of 1 to 50 atoms containing nitrogen, the linker comprises at least 2 atoms of a hydrophilic amino acid; and the nitrogen forms a peptide bond with the carbonyl group of the hydrophilic amino acid. In certain embodiments, W is -C(O)NR'-, and the nitrogen of W is the nitrogen atom of a hydrophilic amino acid. In certain preferred embodiments, the hydrophilic amino acid is any one selected from the group consisting of: arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, lysine, ornithine, proline, serine, and threonine. In certain embodiments, the amino acid covalently bonds the oxime of the linker to the polyethylene glycol unit of the linker. In certain embodiments, the amino acid is selected from arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, lysine, ornithine, proline, serine, and threonine. In certain embodiments, the hydrophilic amino acid comprises a side chain having a moiety that has a charge in aqueous solution at neutral pH. In certain embodiments, the hydrophilic amino acid is aspartic acid or glutamic acid. In other embodiments, the hydrophilic amino acid is ornithine or lysine. In still other embodiments, the hydrophilic amino acid is arginine.

[0133] In certain embodiments, L further comprises a peptide, and the peptide comprises at least one hydrophilic amino acid and includes a side chain having a moiety that has a charge in aqueous solution at neutral pH. In certain embodiments, each amino acid of the peptide is independently selected from alanine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, lysine, ornithine, proline, serine, and threonine. In certain preferred embodiments, the peptide comprises at least one aspartic acid or glutamic acid. In certain preferred embodiments, W is -C(O)NR'-, and wherein the nitrogen of W is the nitrogen atom of the N-terminal amino acid of the peptide. In certain embodiments, the peptide covalently bonds the oxime of the linker to the polyethylene glycol unit of the linker. In certain embodiments, the peptide comprises 2 to 20 amino acids.

[0134] In certain embodiments, L is covalently bonded to the antibody via a thioether bond, and the thioether bond includes a sulfur atom of a cysteine of the antibody. In certain embodiments, the amino acid motif is a CYYX sequence; C is cysteine; Y is an aliphatic amino acid; X is any one selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine; and the thioether bond includes the sulfur atom of the cysteine of the antibody. In certain embodiments, the amino acid motif is a CYYX sequence; and Y is any one selected from the following: alanine, isoleucine, leucine, methionine, and valine. In certain embodiments, the amino acid motif is a CVIM or CVLL sequence. In certain embodiments, at least one of the 1 to 20 amino acids preceding the amino acid motif is glycine. In certain embodiments, at least three of the 1 to 20 amino acids preceding the amino acid motif are glycine or proline. In certain embodiments, at least one of the 1 to 20 amino acids preceding the amino acid motif is selected from glycine, aspartic acid, arginine, and serine. In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids preceding the amino acid motif are glycine, respectively.

[0135] In certain embodiments, L further comprises the amino acid sequence GGGGGGGCVIM at the C-terminus.

[0136] In certain embodiments, L further comprises at least one isoprenyl-derived unit of Formula III:

[0137] [Formula III]

[0138]

[0139] In certain embodiments, L further comprises at least one isoprenyl-derived unit of Formula III, which can be recognized by an isoprenyltransferase.

[0140] [Formula III]

[0141]

[0142] In certain embodiments, L is a 3- to 50-membered heteroalkyl group containing an oxime, and the oxygen atom of the oxime is on the side where L is connected to W and the carbon atom of the oxime is on the side where L is connected to Ab; or the carbon atom of the oxime is on the side where L is connected to W and the oxygen atom of the oxime is on the side where L is connected to Ab.

[0143] In certain embodiments, L further comprises an oxime and at least one isoprenyl unit covalently bonds the oxime to Ab.

[0144] In certain embodiments, L further comprises a linking unit represented by General Formula VIII or General Formula IX:

[0145] [General Formula VIII]

[0146] -(CH 2 ) r (V(CH 2 ) p ) q -

[0147] [General Formula IX]

[0148] -(CH 2 CH 2 X) w -

[0149] Wherein

[0150] V is a single bond, -O-, -S-, -NR 21 -, -C(O)NR 22 -, NR 23 C(O)-, NR 24 SO 2 -, or -SO 2 NR 25 -;

[0151] X is -O-, C 1 -C 8 alkylene or -NR 21 -;

[0152] R 21 to R 25 are independently and respectively hydrogen, (C 1 -C 6 )alkyl, (C 1 -C 6 )alkyl(C 6 -C 20 )aryl or (C 1 -C 6 )alkyl(C 3 -C 20 )heteroaryl;

[0153] r is an integer from 0 to 10;

[0154] p is an integer from 0 to 10;

[0155] q is an integer from 1 to 20; and

[0156] w is an integer from 1 to 20.

[0157] In certain embodiments, q is an integer from 4 to 20. In certain embodiments, q is an integer from 2 to 12. In certain embodiments, q is an integer from 6 to 20. In certain preferred embodiments, q is the integer 2, 5, or 11.

[0158] In certain embodiments, r is the integer 2.

[0159] In certain embodiments, p is the integer 2.

[0160] In certain preferred embodiments, V is -O-.

[0161] In certain embodiments, r is the integer 2; p is the integer 2; q is the integer 2, 5, or 11; and V is -O-.

[0162] In certain preferred embodiments, X is -O-.

[0163] In certain embodiments, w is an integer from 6 to 20.

[0164] In certain embodiments, L further comprises at least one polyethylene glycol unit represented by: In certain embodiments, L further comprises 1 to 12 -OCH 2 CH 2 - units. In certain embodiments, L further comprises 3 to 12 -OCH 2 CH 2 - units. In certain embodiments, 5 to 12 -OCH 2 CH 2 - units. In certain embodiments, L comprises 6 to 12 -OCH 2 CH 2 - units. In certain preferred embodiments, L further comprises 3 -OCH 2 CH 2 - units.

[0165] In certain embodiments, L further comprises an oxime and at least one polyethylene glycol unit covalently bonds the oxime to the active agent.

[0166] In certain embodiments, L further comprises units formed by 1,3 - dipolar cycloaddition reaction, hetero - diels reaction, nucleophilic substitution reaction, non - aldol - type carbonyl reaction, addition of carbon - carbon multiple bonds, oxidation reaction, or click reaction. In certain preferred embodiments, the linking unit is formed via the reaction of acetylene with azide, or via the reaction of aldehyde or ketone group with hydrazine or hydroxylamine.

[0167] In certain embodiments, L further comprises a linking unit represented by the following general formula IV, V, VI, or VII:

[0168] [General formula IV]

[0169]

[0170] [General formula V]

[0171]

[0172] [General formula VI]

[0173]

[0174] [General formula VII]

[0175]

[0176] wherein

[0177] L 1 is a single bond or a C 1 -C 30 alkylene; and

[0178] R 11 is hydrogen or a C 1 -C 10 alkyl.

[0179] In certain embodiments, L 1 is a single bond. In other embodiments, L 1 is a C 11 alkylene. In other embodiments, L 1 is a C 12 alkylene.

[0180] In certain embodiments, L further comprises wherein

[0181] V is a single bond, -O-, -S-, -NR 21 -, -C(O)NR 22 -, NR 23 C(O)-, NR 24 SO 2 -, or -SO 2 NR 25 -;

[0182] R 21 to R 25 are independently and respectively hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkyl C 6 -C 20 aryl or C1 -C 6 alkyl C 3 -C 20 heteroaryl;

[0183] r is an integer from 1 to 10;

[0184] p is an integer from 0 to 10;

[0185] q is an integer from 1 to 20; and

[0186] L 1 is a single bond.

[0187] In certain embodiments, r is the integer 2 or 3.

[0188] In certain embodiments, p is the integer 1 or 2.

[0189] In certain embodiments, q is an integer from 1 to 6.

[0190] In certain embodiments, r is the integer 2 or 3; p is the integer 1 or 2; and q is an integer from 1 to 6.

[0191] In certain embodiments, the isoprenoid transferase is farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase).

[0192] In certain embodiments, L further comprises one or more branched linkers covalently coupled to the Ab, wherein

[0193] i) each branched linker comprises a branched unit (BR) covalently coupled to the Ab through a primary linker (PL);

[0194] ii) each branched linker comprises a first branch (B1), wherein a first active agent is covalently coupled to the branched unit through a secondary linker (SL) and a cleavage group (CG); and

[0195] iii) each branched linker further comprises a second branch (B2), wherein a) a second active agent is covalently coupled to the branched unit through a secondary linker (SL) and a cleavage group (CG), or b) a polyethylene glycol moiety is covalently coupled to the branched unit,

[0196] wherein each cleavage group is hydrolysable to release the active agent from the antibody-drug conjugate.

[0197] In certain embodiments, the branched unit is represented by:

[0198]

[0199] L 2 、L 3and L 4 each independently is a bond or -C n H 2n -;

[0200] n is an integer from 1 to 30;

[0201] G 1 、G 2 and G 3 each independently is a bond,

[0202] R 30 is hydrogen or C 1-30 alkyl;

[0203] L 5 is a direct bond or C 1-10 alkylene; and

[0204] R 50 is hydrogen C 1-30 alkyl.

[0205] In certain embodiments, the antibody conjugate comprises at least one branched linker covalently linked to the Ab; and at least two active agents are covalently linked to the branched linker. In certain embodiments, the antibody conjugate comprises two or more branched linkers covalently linked to the Ab; and the branched linkers are linked to at least two active agents. In certain embodiments, the antibody conjugate comprises 3 branched linkers. In other embodiments, the antibody conjugate comprises 4 branched linkers. In still other embodiments, the antibody conjugate comprises 1 branched linker. In certain embodiments, the respective branched linkers are each linked to two active agents. In certain embodiments, the conjugate comprises at least two different active agents. In certain embodiments, the branched linker is linked to at least two active agents. In certain embodiments, the active agent is linked to the branched unit via a second linker; and the branched unit is linked to the anti-ROR1 antibody via a first linker.

[0206] In certain preferred embodiments, the branched unit is a nitrogen atom. In certain even other preferred embodiments, the branched unit is an amide, and the first linker comprises the carbonyl of the amide. In the most preferred embodiments, the branched unit is a lysine unit.

[0207] In certain embodiments, the antibody conjugate comprises a structure represented by:

[0208]

[0209] wherein

[0210] each B is an active agent;

[0211] Each n is independently an integer from 0 to 30; and

[0212] Each n is independently an integer from 0 to 30.

[0213] In certain embodiments, n is an integer from 1 to 10. In other embodiments, n is an integer from 4 to 20.

[0214] In certain embodiments, L comprises an oxime and at least one polyethylene glycol unit covalently bonding the oxime to the active agent.

[0215] In certain embodiments, the cleavable bond can be cleaved within the target cell. In certain embodiments, the cleavable bond can be cleaved by an activator (e.g., radiation, acid, base, or enzyme).

[0216] In certain embodiments, the conjugate is represented by the following structure or a pharmaceutically acceptable salt thereof:

[0217]

[0218] where Ab is an anti-ROR1 antibody; B is an active agent; and n is an integer from 1 to 20.

[0219] In certain embodiments, the conjugate is represented by the following structure or a pharmaceutically acceptable salt thereof:

[0220]

[0221] where Ab is an anti-ROR1 antibody; B is an active agent; and n is an integer from 1 to 20. In certain embodiments, the conjugate is represented by the following structure or a pharmaceutically acceptable salt thereof:

[0222]

[0223] where Ab is an anti-ROR1 antibody; B is an active agent; and n is an integer from 1 to 20. In certain embodiments, the conjugate is represented by the following structure or a pharmaceutically acceptable salt thereof:

[0224]

[0225] where Ab is an anti-ROR1 antibody; B is an active agent; and n is an integer from 1 to 20. In some embodiments, the linker has the structure of the following chemical formula (IIa),

[0226] [Chemical formula (IIa)]

[0227]

[0228] where

[0229] G is a sugar, sugar acid or sugar derivative;

[0230] W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO 2 NR'-, -P(O)R"NR'-, -SONR'- or -PO 2 NR'-; wherein C(O), S or P is directly connected to the phenyl ring; R' and R" are each independently hydrogen, (C 1 -C 8 )alkyl, (C 3 -C 8 )cycloalkyl, (C 1 -C 8 )alkoxy, (C 1 -C 8 )alkylthio, mono-(C 1 -C 8 )alkylamino or di-(C 1 -C 8 )alkylamino, (C 3 -C 20 )heteroaryl or (C 6 -C 20 )aryl;

[0231] Each Z is independently (C 1 -C 8 )alkyl, halogen, cyano or nitro;

[0232] n is an integer from 1 to 3;

[0233] m is 0 or 1;

[0234] L is absent or contains at least one branching unit (BR) and at least one linking unit;

[0235] R 1 and R 2 are each independently hydrogen, (C 1 -C 8 )alkyl or (C 3 -C 8 )cycloalkyl, or the R 1 and R 2 together with the carbon atom to which they are attached form a (C 3 -C 8 )cycloalkyl ring; and

[0236] In the above formula, * indicates the region bound to the antibody or its antigen-binding fragment, and indicates the region bound to the drug or toxin.

[0237] In some embodiments, the sugar or sugar acid is a monosaccharide.

[0238] In some embodiments, G is a compound having the structure of formula (IIIa):

[0239] [Formula (IIIa)]

[0240]

[0241] wherein

[0242] R 3 is hydrogen or a carboxyl protecting group; and

[0243] each R 4 is, independently of one another, hydrogen or a hydroxyl protecting group.

[0244] In some embodiments, R 3 is hydrogen, and each R 4 is hydrogen.

[0245] In some embodiments, W is -C(O)NR'-, where C(O) is attached to the phenyl ring and NR' is attached to L.

[0246] In some embodiments, Z is hydrogen and n is 3.

[0247] In some embodiments, R 1 and R 2 are each hydrogen.

[0248] In some embodiments, G is a compound having the structure of formula (IIIa):

[0249] [Formula (IIIa)]

[0250]

[0251] wherein

[0252] R 3 is hydrogen or a carboxyl protecting group;

[0253] each R 4 is, independently of one another, hydrogen or a hydroxyl protecting group; and

[0254] W is -C(O)NR'-, where C(O) is attached to the phenyl ring and NR' is attached to L, each Z is hydrogen, n is 3, m is 1, and R 1 and R 2 are each hydrogen.

[0255] In some embodiments, at least one branching unit is an alkylene having 1 to 100 carbon atoms, wherein the carbon atoms of the alkylene may be substituted by one or more heteroatoms selected from N, O, and S, and the alkylene may be further substituted by an alkyl group having 1 to 20 carbon atoms.

[0256] In some embodiments, at least one branching unit is a hydrophilic amino acid.

[0257] In some embodiments, the hydrophilic amino acid may be arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, lysine, ornithine, proline, serine, and threonine.

[0258] In some embodiments, the hydrophilic amino acid may be an amino acid including a side chain having a residue that has a charge in an aqueous solution at neutral pH.

[0259] In some embodiments, the hydrophilic amino acid is aspartic acid or glutamic acid.

[0260] In some embodiments, the hydrophilic amino acid is ornithine or lysine.

[0261] In some embodiments, the hydrophilic amino acid is arginine.

[0262] In some embodiments, at least one branching unit is -C(O)-, -C(O)NR'-, -C(O)O-, -S(O) 2 NR'-, -P(O)R"NR'-, -S(O)NR'-, or -PO 2 NR'-, and R' and R" are each independently hydrogen, (C 1 -C 8 ) alkyl, (C 3 -C 8 ) cycloalkyl, (C 1 -C 8 ) alkoxy, (C 1 -C 8 ) alkylthio, mono-(C 1 -C 8 ) alkylamino, or di-(C 1 -C 8 ) alkylamino, (C 3 -C 20 ) heteroaryl, or (C 6 -C 20 ) aryl.

[0263] In some embodiments, at least one branching unit is -C(O)NR'-, and R' is hydrogen.

[0264] In some embodiments, at least one linking unit is -(CH2 ) r (V(CH 2 ) p ) q -, where r is an integer from 0 to 10, p is an integer from 0 to 12, q is an integer from 1 to 20, and V is a single bond -O- or -S-.

[0265] In some embodiments, r is 2.

[0266] In some embodiments, p is 2.

[0267] In some embodiments, q is an integer from 6 to 20.

[0268] In some embodiments, r is 2, p is 2, q is 2, 4, 5 or 11, and V is -O-.

[0269] In some embodiments, at least one linking unit is at least one polyethylene glycol unit and has the following structure:

[0270] In some embodiments, at least one linking unit is 1 to 12 -OCH 2 CH 2 - units or 5 to 12 -OCH 2 CH 2 - units or 6 to 12 -OCH 2 CH 2 - units.

[0271] In some embodiments, at least one linking unit is -(CH 2 CH 2 ) w -,

[0272] where X is a single bond -O-, (C 1 -C 8 ) alkylene or -NR 21 -;

[0273] R 21 is hydrogen, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkyl (C 6 -C 20 ) aryl or (C 1 -C 6 ) alkyl (C 3 -C 20 ) heteroaryl; and

[0274] w is an integer from 1 to 20, specifically 1, 3, 6 or 12.

[0275] In some embodiments, X is -O-, and w is an integer from 6 to 20.

[0276] In some embodiments, the linker further comprises a linking unit formed by a 1,3-dipolar cycloaddition reaction, a hetero-Diels-Alder reaction, a nucleophilic substitution reaction, a non-aldol type carbonyl reaction, an addition reaction of a carbon-carbon multiple bond, an oxidation reaction, or a click reaction.

[0277] In some embodiments, the linking unit is formed via a reaction between acetylene and azide, or a reaction between an aldehyde or ketone group and hydrazine or alkoxyamine.

[0278] In some embodiments, the linking unit is represented as:

[0279] [General formula IV]

[0280]

[0281] [General formula V]

[0282]

[0283] [General formula VI]

[0284]

[0285] [General formula VII]

[0286]

[0287] wherein

[0288] L 1 is a single bond or an alkylene group having 1 to 30 carbon atoms;

[0289] R 11 is hydrogen or an alkyl group having 1 to 10 carbon atoms, specifically methyl; and

[0290] L 2 is an alkylene group having 1 to 30 carbon atoms;

[0291] In some embodiments, the linker may further contain at least one isoprenyl unit having the following structure: where n is at least 2.

[0292] In some embodiments, at least one isoprenyl unit is a substrate of an isoprenyltransferase or a product of an isoprenyltransferase.

[0293] In some embodiments, the isoprenyl unit of the linker is covalently linked to the antibody via a thioether bond, and the thioether bond includes the sulfur atom of cysteine. In some embodiments, the antibody includes an amino acid motif recognized by a prenyltransferase, and the thioether bond includes the sulfur atom of cysteine of the amino acid motif.

[0294] In some embodiments, an antibody that binds to ROR1 or an antigen-binding fragment thereof includes an amino acid motif recognized by a prenyltransferase, and the thioether bond includes the sulfur atom of cysteine of the amino acid motif.

[0295] In some embodiments, the amino acid motif is a sequence selected from the group consisting of CXX, CXC, XCXC, XXCC, and CYYX, wherein C indicates cysteine, Y independently indicates an aliphatic amino acid in different cases, X independently indicates glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine in different cases, and the thioether bond includes the sulfur atom of cysteine of the amino acid motif.

[0296] In some embodiments, the amino acid motif is a CYYX sequence; and Y is independently alanine, isoleucine, leucine, methionine, or valine in different cases.

[0297] In some embodiments, the amino acid motif is a CVIM or CVLL sequence.

[0298] In some embodiments, at least one of the 1 to 20 amino acids before the amino acid motif is glycine.

[0299] In some embodiments, at least one of the 1 to 20 amino acids before the amino acid motif is independently selected from glycine, arginine, aspartic acid, and serine, respectively.

[0300] In some embodiments, the 1 to 20 amino acids before the amino acid motif are glycine. More specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 of the 20 amino acids before the amino acid motif are glycine.

[0301] In some embodiments, the antibody may include the amino acid sequence GGGGGGGCVIM.

[0302] In some embodiments, the linker may include:

[0303] (a) at least one branching unit, (b) at least one linking unit, (c) at least one binding unit (BU), and (d) at least one triggering unit (TU).

[0304] Here, a linking unit links a triggering unit and a conjugating unit, a triggering unit and a branching unit, or a branching unit and a conjugating unit;

[0305] at least one triggering unit is capable of releasing at least one drug or toxin; and

[0306] a branching unit links a linking unit and a triggering unit or a linking unit and another linking unit.

[0307] In some embodiments, the triggering unit has a structure of the following chemical formula (IIb):

[0308] [Chemical formula (IIb)]

[0309]

[0310] wherein

[0311] G is a sugar, sugar acid or sugar derivative;

[0312] W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO 2 NR'-, -P(O)R"NR'-, -SONR'- or -PO 2 NR'-; wherein C(O), S or P is directly linked to the phenyl ring; R' and R" are each independently hydrogen, (C 1 -C 8 )alkyl, (C 3 -C 8 )cycloalkyl, (C 1 -C 8 )alkoxy, (C 1 -C 8 )alkylthio, mono-(C 1 -C 8 )alkylamino or di-(C 1 -C 8 )alkylamino, (C 3 -C 20 )heteroaryl or (C 6 -C 20 )aryl, and W is linked to a linking unit or a branching unit;

[0313] each Z is independently (C 1 -C 8 )alkyl, halogen, cyano or nitro;

[0314] n is an integer from 1 to 3;

[0315] m is 0 or 1; and

[0316] R 1 and R 2Each is independently hydrogen, (C 1 -C 8 )alkyl or (C 3 -C 8 )cycloalkyl, or said R 1 and R 2 together with the carbon atom to which they are attached form a (C 3 -C 8 )cycloalkyl ring.

[0317] In some embodiments, the sugar or sugar acid is a monosaccharide.

[0318] In some embodiments, G is a compound having the structure of formula (IIIa):

[0319] [Formula (IIIa)]

[0320]

[0321] Wherein

[0322] R 3 is hydrogen or a carboxyl protecting group; and

[0323] each R 4 is independently hydrogen or a hydroxyl protecting group.

[0324] In some embodiments, R 3 is hydrogen, and each R 4 is hydrogen.

[0325] In some embodiments, W is -C(O)NR'-, where C(O) is attached to the phenyl ring and NR' is attached to L.

[0326] In some embodiments, Z is hydrogen.

[0327] In some embodiments, R 1 and R 2 are each hydrogen.

[0328] In some embodiments, the linking unit is represented as: -(CH 2 ) r (V(CH 2 ) p ) q -, -((CH 2 ) p V) q -, -(CH 2 ) r (V(CH 2 ) p ) q Y-, -((CH 2 ) p V)q (CH 2 ) r -,-Y((CH 2 ) p V) q -or-(CH 2 ) r (V(CH 2 ) p ) q YCH 2 -,

[0329] where

[0330] r is an integer from 0 to 10;

[0331] p is an integer from 1 to 10;

[0332] q is an integer from 1 to 20;

[0333] V and Y are independent and each represents a single bond, -O-, -S-, -NR 21 -,-C(O)NR 22 -,NR 23 C(O)-,NR 24 SO 2 -,or -SO 2 NR 25 -; and

[0334] R 21 to R 25 are independently and each hydrogen, (C 1 -C 6 )alkyl, (C 1 -C 6 )alkyl(C 6 -C 20 )aryl or (C 1 -C 6 )alkyl(C 3 -C 20 )heteroaryl.

[0335] In some embodiments, r is 2.

[0336] In some embodiments, p is 2.

[0337] In some embodiments, q is an integer from 6 to 20.

[0338] In some embodiments, q is 2, 5 or 11.

[0339] In some embodiments, V and Y are each independently -O-.

[0340] In some embodiments, the branching unit is:

[0341]

[0342] wherein

[0343] L 1 、L 2 and L 3 are each independently a direct bond or -C n H 2n -;

[0344] n is an integer from 1 to 30;

[0345] G 1 、G 2 and G 3 are each independently a direct bond,

[0346]

[0347] R 3 is hydrogen or C 1-30 alkyl;

[0348] R 4 is hydrogen or L 4 -COOR 5 ; L 4 is a direct bond or -C n H 2n -; n is an integer from 1 to 10, and R 5 is hydrogen or C 1 -C 30 alkyl.

[0349] In some embodiments, the branching unit is:

[0350]

[0351] wherein

[0352] L 1 、L 2 and L 3 are each independently a direct bond or -C n H 2n -;

[0353] n is an integer from 1 to 30;

[0354] G 1 、G 2 and G 3 are each independently a direct bond,

[0355]

[0356] R 3 is hydrogen or C 1-C 30 alkyl;

[0357] R 4 is hydrogen or L 4 -COOR 5 ; L 4 is a direct bond or -C n H 2n -; n is an integer from 1 to 10, and R 5 is hydrogen or C 1 -C 30 alkyl.

[0358] In some embodiments, the branching unit is

[0359]

[0360] wherein L 1 is a direct bond or an alkylene having 1 to 30 carbon atoms;

[0361] R 11 is hydrogen or an alkyl having 1 to 10 carbon atoms, specifically methyl;

[0362] L 2 is an alkylene having 1 to 30 carbon atoms; and

[0363] the branching unit connects the linking unit and the antibody.

[0364] In some embodiments, L 1 is an alkylene having 12 carbon atoms.

[0365] In some embodiments, R 11 is methyl.

[0366] In some embodiments, L 2 is an alkylene having 11 carbon atoms.

[0367] In some embodiments, the linking unit is covalently bound to the antibody via a thioether bond, and the thioether bond includes the sulfur atom of cysteine. In some such embodiments, the antibody includes an amino acid motif recognized by a prenyltransferase, and the thioether bond includes the sulfur atom of the cysteine of the amino acid motif.

[0368] In some embodiments, the amino acid motif is a sequence selected from the group consisting of CXX, CXC, XCXC, XXCC, and CYYX, wherein C indicates cysteine, Y independently indicates an aliphatic amino acid in different cases, X independently indicates glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine in different cases, and the thioether bond includes the sulfur atom of the cysteine of the amino acid motif.

[0369] In some embodiments, the amino acid motif is a CYYX sequence; and Y is independently alanine, isoleucine, leucine, methionine, or valine in different cases.

[0370] In some embodiments, the amino acid motif is a CVIM or CVLL sequence.

[0371] In some embodiments, at least one of the 1 to 20 amino acids before the amino acid motif is glycine.

[0372] In some embodiments, at least one of the 1 to 20 amino acids before the amino acid motif is independently selected from glycine, arginine, aspartic acid, and serine.

[0373] In some embodiments, the 1 to 20 amino acids before the amino acid motif are glycine. More specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 of the 20 amino acids before the amino acid motif are glycine.

[0374] In some embodiments, the antibody may comprise the amino acid sequence GGGGGGGGCVIM.

[0375] In certain embodiments, the active agent is an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or a combination thereof.

[0376] In certain embodiments, the active agent is selected from:

[0377] (a) Erlotinib, bortezomib, fulvestrant, sunitinib, letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, ethyleneimine, hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine, bullatacin, bullataciionone, camptothecin, topotecan, bryostatin, callystatin, CC-1065, adozelesin, carzelesin, bizelesin, nostocyclopeptide 1, nostocyclopeptide 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, phosphamide mustard, estramustine, ifosfamide, nitrogen mustard, melphalan, neo-nitrogen mustard, phenyl mustard sterol, prednimustine, trofosfamide, uracil mustard, carmustine, clofarabine, fotemustine, lomustine, nimustine, ranimustine, calicheamicin, calicheamicin γ1, calicheamicin ω1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore, aclacinomycin, actinomycin, antrymycin, azaserine, bleomycin, catcinomycin, carabicin, erythromycin rubidomycin, carcidin, chromomycin, actinomycin D, daunorubicin, detorubucin, 6-diazo-5-oxo-L-norleucine, doxorubicin, N-morpholino-doxorubicin, cyano-N-morpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin,Deoxydoxorubicin, epirubicin, esorubicin, maridomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quinamycin, rodorubicin, streptothricin, streptozocin, tudercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, tiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didanosine, doxifluridine, enocitabine, floxuridine, dimethandrolone, methyltestosterone propionate, cythioate, mesterolone, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, acetylcysteine, aldophosphamide glycoside, aminolevulinic acid, eniluracil, aclarubicin, bestrabucil, bisantrene, edatrexate, defofamine, democolcine, diaziquone, eflornithine, elisuride, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocin, mitoguazone, mitoxantrone, mopidanmol, nitralverine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-acetylhydrazine, procarbazine, polysaccharide-k, razoxane, rhizoxin, sizofiran, germanium sesquioxide, tenuazonic acid, triaziquone, 2,2',2"-trichloroethylamine, T-2 toxin, myxothiazol A, bacillisporin A, anguidine, carbamate, vindesine, dacarbazine, mannomustine, dibromomannitol, dibromodulcitol, pipobroman, metoprine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposideIfosfamide, vincristine, vinorelbine, nimustine, teniposide, edatrexate, daunorubicin, aminopterin, capecitabine, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine, retinoic acid, capecitabine or a pharmaceutically acceptable salt, solvate or acid thereof;

[0378] (b) Monokines, lymphokines, traditional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatocyte growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor, tumor necrosis factor-α, tumor necrosis factor-β, Müllerian inhibitory substance, murine gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, osteogenic factor, interferon, interferon-α, interferon-β, interferon-γ, colony-stimulating factor (CSF), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukin (IL), IL-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, tumor necrosis factor, polypeptide factor, LIF, kit ligand or a mixture thereof;

[0379] (c) Diphtheria toxin, botulinum toxin, tetanus toxin, shiga toxin, cholera toxin, amanitin, α-amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivative, indolodiazepine, pyridodiazepine, saxitoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansine, calicheamicin, daunorubicin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, dolastatin analog, microcystin, camptothecin, rhizoxin, rhizoxin derivative, CC-1065, CC-1065 analog or derivative, duocarmycin, enediyne antibiotic, esperamicin, epothilone, toxoid or a mixture thereof;

[0380] (d) An affinity ligand, wherein the affinity ligand is a substrate, inhibitor, activator, neurotransmitter, radioisotope or a mixture thereof;

[0381] (e) A radioactive label, 32P, 35S, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, streptavidin, digoxin, hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to a target or a mixture thereof;

[0382] (f) An immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or a mixture thereof;

[0383] (g) Tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, raloxifene hydrochloride, LY117018, onapristone, or toremifene;

[0384] (h) 4(5)-Imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole, or anastrozole;

[0385] (i) Flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine;

[0386] (j) An aromatase inhibitor;

[0387] (k) A protein kinase inhibitor;

[0388] (l) A lipid kinase inhibitor;

[0389] (m) An antisense oligonucleotide;

[0390] (n) A ribozyme;

[0391] (o) A vaccine; and

[0392] (p) An anti-angiogenic agent.

[0393] In certain preferred embodiments, Ab is an anti-ROR1 antibody;

[0394] The active agent is a pyrrolobenzodiazepine dimer;

[0395] The linker connects Ab to the N10 or N'10 position of the pyrrolobenzodiazepine dimer; and

[0396] y is an integer from 1 to 20.

[0397] In certain embodiments, the active agent is a pyrrolobenzodiazepine dimer;

[0398] The pyrrolobenzodiazepine dimer is substituted at the N10 position by X or at the N'10 position by X', where X or X' connects the pyrrolobenzodiazepine dimer to the linker;

[0399] X and X' are each independently -C(O)O*, -S(O)O-*, -C(O)-*, -C(O)NR X -*, -S(O) 2 NR X -*, -P(O)R'NR X -*, -S(O)NR X -* or -PO 2 NR X -*;

[0400] R X is C 1-8 alkyl, C 3-8 cycloalkyl, C 3-20 heteroaryl or C 5-20 aryl;

[0401] R X' is OH, N 3 -, CN, SH, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1-8 alkylthio, C 3-20 heteroaryl, C 5-20 aryl or amino; and

[0402] * is the binding site between the pyrrolobenzodiazepine dimer and the linker.

[0403] In certain embodiments, X and X' are each independently -C(O)O*, -C(O)-* or -C(O)NR X -*.

[0404] In certain embodiments, the pyrrolobenzodiazepine dimer is represented by the following general formula X or general formula XI:

[0405] [General formula X]

[0406]

[0407] [General formula XI]

[0408]

[0409] wherein:

[0410] The dashed line represents an optional double bond as permitted by the valence;

[0411] R X1 and R X1' are independently selected from H, OH, =O, =CH 2 CN, R m , OR m , =CH-R m' , =C(R m' ) 2 , OSO 2 -R m , CO 2 R m , COR m , a halogen group and a dihalogen group;

[0412] R m' is selected from R m , CO 2 R m , COR m , CHO, CO 2 H and a halogen group;

[0413] Each R m is independently selected from C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 5-20 aryl, C 5-20 heteroaryl, C 3-6 cycloalkyl, a 3-7 membered heterocyclic group, a 3-7 membered heterocycloalkyl group and a 5 to 7 membered heteroaryl group; or R m is X or X`;

[0414] R X2 , R X2' , R X3 , R X3' , R X5 and R X5' are independently selected from H, R m , OH, OR m , SH, SR m , NH 2 , NHR m , NR m 2 , NO 2 , Me 3 , Sn and a halogen group;

[0415] R X4 and R X4' are independently selected from H, R m , OH, OR m , SH, SR m , NH 2 , NHR m , NR m2 , NO 2 , Me 3 Sn, halogen group, C 1-6 alkyl C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3 - 7 membered heteroalkyl, C 5-12 aryl, 5 - 7 membered heteroaryl, -CN, -NCO, -OR n , -OC(O)R n , -OC(O)NR n R n' , -OS(O)R n , -OS(O) 2 R n , -SR n , -S(O)R n , -S(O) 2 R n , -S(O)NR n R n' , -S(O) 2 NR n R n' , -OS(O)NR n R n' , -OS(O) 2 NR n R n' , -NR n R n' , -NR n C(O)R o , -NR n C(O)OR o , -NR n C(O)NR o R o' , -NR n S(O)R o , -NR n , -S(O) 2 R o , -NR n S(O)NR o R o' , -NR n , -S(O) 2 NR o R o' , -C(O)R n , -C(O)OR n and -C(O)NR n R n' ;

[0416] RX and R X' each independently selected from H, OH, N 3 , CN, NO 2 , SH, NH 2 , ONH 2 , NHNH 2 , halo group, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1-8 alkylthio, C 3-20 heteroaryl, C 5-20 aryl or mono-C 1-8 alkylamino or di-C 1-8 alkylamino;

[0417] Y and Y' each independently selected from O, S and N(H);

[0418] each R x6 independently selected from C 3-12 alkylene, C 3-12 alkenylene or C 3-12 heteroalkylene;

[0419] R X7 and R X7' independently selected from H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-7 membered hetero cycloalkyl, C 6-10 aryl, 5 to 7 membered heteroaryl, -OR r , -OC(O)R r , -OC(O)NR r R r' , -OS(O)R r , -OS(O) 2 R r , -SR r , -S(O)R r , -S(O) 2 R r , -S(O)NR r R r' , -S(O) 2 NR r R r' , -OS(O)NR r R r' , -OS(O) 2 NR r R r' , -NR r R r' , -NR rC(O)R s 、-NR r C(O)OR s 、-NR r C(O)NR s R s' 、-NR r S(O)R s 、-NR r S(O) 2 R s 、-NR r S(O)NR s R s' 、-NR r S(O) 2 NR s R s 、-C(O)R r 、-C(O)OR s or -C(O)NR r R r' ;

[0420] Each R r , R r' , R s and R s' Independently selected from H, C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkynyl, C 3-13 Cycloalkyl, 3-7 membered heterocycloalkyl, C 5-10 Aryl and 5- to 7-membered heteroaryl;

[0421] Each R X8 and R X8' Independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Heteroalkyl, 3-7 membered heterocycloalkyl, C 5-10 Aryl, 5- to 7-membered heteroaryl, -S(O)R m 、-S(O) 2 R m 、-S(O)NR m R m' 、-S(O) 2 NR m R m' 、-NR m R m' 、-NR m C(O)R m NR m C(O)OR n 、-NR m C(O)NRn R n' 、 -NR m S(O)R n 、 -NR m S(O) 2 R n 、 -NR m S(O)NR n R n' 、 -NR m S(O) 2 NR n R n' 、 -C(O)R m 、 -C(O)OR m and -C(O)NR m R m' ;

[0422] Z a is selected from OR X12a 、 NR X12a R X12a or SR X12a ;

[0423] Z b is selected from OR X13a 、 NR X13a R X13a or SR X13a ;

[0424] Z a' is selected from OR X12a 、 NR X12a R X12a or SR X12a ;

[0425] Z b' is selected from OR X13a' 、 NR X13a' R X13a' or SR X13a' ;

[0426] Each R X12a 、 R X12a' 、 R X13a' and R x13a' is independently selected from H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3 - 7 - membered heteroalkyl, C 5-10 aryl, 5 - to 7 - membered heteroaryl, -C(O)R X15a 、 -C(O)OR X15a and -C(O)NR X15a R X15a' ;

[0427] Each R X15a and R x15a' are independently selected from C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 5-20 aryl, C 5-20 heteroaryl, C 3-6 cycloalkyl, 3-7 membered heterocyclic group, 3-7 membered heterocycloalkyl and 5 to 7 membered heteroaryl;

[0428] Each R X13a and R X14a are independently H or alkyl; or R X13a and R X14a together with the atoms to which they are attached form a 3-7 membered heterocyclic group, form a 3-7 membered heterocycloalkyl or form a 3-7 membered heteroaryl, and R X13a' and R X14a' are optionally bonded to the atoms to which they are attached to form a 3-7 membered heterocyclic group, a 3-7 membered heterocycloalkyl or a 3-7 membered heteroaryl; and

[0429] R n , R n' , R o , R o' , R p and R p' each independently is selected from H, C 1-7 alkyl, C 2-7 alkenyl, C 2-7 alkynyl, C 3-13 cycloalkyl, 3-7 membered heterocycloalkyl, C 5-10 aryl and 5 to 7 membered heteroaryl.

[0430] In certain embodiments, R m is independently selected from C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 5-20 aryl, C 5-20 heteroaryl, C 3-6 cycloalkyl, 3-7 membered heterocyclic group, 3-7 membered heterocycloalkyl and 5 to 7 membered heteroaryl; and

[0431] R m is a substituted C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 5-20 aryl, C 5-20 heteroaryl, C 3-6 cycloalkyl, 3-7 membered heterocyclic group, 3-7 membered heterocycloalkyl or 5 to 7 membered heteroaryl.

[0432] In certain embodiments, R X4 and R X4' are independently selected from H, R m OH, OR m SH, SR m NH 2 NHR m NR m R m' NO 2 Me 3 SN, halo, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-7 membered heteroalkyl, C 5-12 5 to 7 membered heteroaryl, -CN, -NCO, -OR n OC(O)R n OC(O)NR n R n' OS(O)R n OS(O) 2 R n SR n S(O)R n S(O) 2 R n S(O)NR n R n' S(O) 2 NR n R n' OS(O)NR n R n' OS(O) 2 NR n R n' NR n R n' NR n C(O)R o NR n C(O)OR o NR n C(O)NR o R o' NR n S(O)R o NR n S(O) 2 R o NR n S(O)NR o R o' NR n S(O)2 NR o R o' 、-C(O)R n 、-C(O)OR n and -C(O)NR n R n' ;and

[0433] R X4 or R X4' C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-7 membered heterocycloalkyl, C 5-12 aryl or 5- to 7-membered heteroaryl, and is further substituted with at least one of: C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-7 membered heterocycloalkyl, C 5-10 Aryl, 5- to 7-membered heteroaryl, -OR p 、-OC(O)R p 、-C(O)NR p R p' 、-OS(O)R p 、-OS(O) 2 R p 、-SR p 、-S(O)R p 、-S(O) 2 R p 、-S(O)NR p R p' 、-S(O) 2 NR p R p' 、-OS(O)NR p R p' 、-OS(O) 2 NR p R p' 、-NR p R p' 、-NR p C(O)R q 、-NR p C(O)OR q 、-NR p C(O)NR q R q' 、-NR p S(O)R q 、-NR pS(O) 2 R q 、-NR p S(O)NR q R q' 、-NR p S(O) 2 NR q R q' 、-C(O)R p 、-C(O)OR p or -C(O)NR p R p .

[0434] In certain embodiments, R X1 and R X1' All are R m ; and R m C 1-6 Alkyl, C 2-6 Alkenyl, C 5-7 Aryl or C 3-6 Heteroaryl.

[0435] In certain embodiments, R X2 , R X2' , R X3 , R X3' , R X5 and R X5' Independently selected from H or OH.

[0436] In certain embodiments, R X4 and R X4' All are R m ; and R m C 1-6 In certain preferred embodiments, R X4 and R X4' Each is independently selected from methoxy, ethoxy and butoxy.

[0437] In certain embodiments, Y and Y' are O.

[0438] In certain embodiments, R x6 C 3-12 Alkylene, C 3-12 Alkenylene or C 3-12 Heteroalkylene, wherein R x6 Replaced by: -NH 2 、-NHR m 、-NHC(O)R m 、-NHC(O)R m 、-NHC(O)CH 2 -[OCH 2 CH 2 ]n -R XX or -[CH 2 CH 2 O] n -R XX ;

[0439] R XX is H, OH, N 3 , CN, NO 2 , SH, NH 2 , ONH 2 , NHNH 2 , a halogen group, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1-8 alkylthio, C 3-20 heteroaryl, C 5-20 aryl or mono-C 1-8 alkylamino or di-C 1-8 alkylamino; and

[0440] n is an integer from 1 to 6.

[0441] In certain embodiments, the active agent has a structure represented by General Formula XII or General Formula XIII;

[0442] [General Formula XII]

[0443]

[0444] [General Formula XIII]

[0445]

[0446] wherein

[0447] X a and X a' are each independently a bond or C 1-6 alkylene;

[0448] Z X' and Z X are each independently selected from hydrogen, C 1-8 alkyl, a halogen group, cyano, nitro, and -(CH 2 ) m -OCH 3 ;

[0449] Each R 80 , R 90 and R 100 is independently selected from hydrogen, C 1-8 alkyl, C 2-6Alkenyl and C 1-6 alkoxy; and

[0450] m is an integer from 0 to 12.

[0451] In certain embodiments, Z X' and Z X are each independently selected from hydrogen, and -(CH 2 ) m -OCH 3 ;

[0452] R 80 、R 90 and R 100 are each independently selected from hydrogen, C 1-3 alkyl and C 1-3 alkoxy; and

[0453] m is an integer from 1 to 6.

[0454] In certain preferred embodiments, the active agent is selected from

[0455]

[0456]

[0457]

[0458]

[0459] or a pharmaceutically acceptable salt thereof; and

[0460] The bond superimposed with a dashed line represents the point of attachment to L.

[0461] In another aspect, the present invention provides a method for treating a disease or disorder associated with overexpression of ROR1 in an individual, which comprises administering an antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt thereof. In certain embodiments, the disease or disorder associated with overexpression of ROR1 is cancer. In certain embodiments, the cancer is selected from chronic lymphocytic leukemia (CLL), B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt lymphoma, mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer.

[0462] In another aspect, the present invention provides a method for treating cancer in an individual, which comprises administering an antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt thereof. In certain embodiments, the cancer is selected from chronic lymphocytic leukemia (CLL), B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt lymphoma, mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer.

[0463] In another aspect, the present invention provides an antibody-drug conjugate having the following general formula Ia:

[0464] [General formula Ia]

[0465] Ab-(Linker-D) n ,

[0466] Wherein:

[0467] Ab is an anti-ROR1 antibody;

[0468] The linker is a linker;

[0469] D is a pyrrolobenzodiazepine dimer as an active agent; and

[0470] The linker and the antibody are linked through the N10 or N10' position of the pyrrolobenzodiazepine dimer.

[0471] Here, the pyrrolobenzodiazepine dimer prodrugs at the N10 and N10' positions of the pyrrolobenzodiazepine dimer are each independently attached to any one selected from the group consisting of: -C(O)O-*, -S(O)O-*, -C(O)-*, -C(O)NR-*, -S(O) 2 NR-*, -(P(O)R')NR-*, -S(O)NR-* and -PO 2 NR-* groups;

[0472] R X and R X' are independently selected from H, OH, N 3 , CN, NO 2 , SH, NH 2 , ONH 2 , NHNH 2 , halogen, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1-8 alkylthio, C 3-20 heteroaryl, C 5-20 aryl or mono-C 1-8 alkylamino or (di)-C 1-8 alkylamino;

[0473] where * is the region to which the linker is attached;

[0474] R and R' are each independently H, OH, N 3 , CN, NO 2 , SH, NH 2 , ONH 2 , NHNH 2 , halogen, substituted or unsubstituted C 1-8 alkyl, substituted or unsubstituted C 3-8 cycloalkyl, substituted or unsubstituted C 1-8 alkoxy, substituted or unsubstituted C 1-8 alkylthio, substituted or unsubstituted C 3-20 heteroaryl, substituted or unsubstituted C 5-20 aryl or mono-C 1-8 alkylamino or di-C 1-8 alkylamino; and

[0475] wherein, in C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C1-8 Alkylthio, C 3-20 heteroaryl or C 5-20 When the aryl is substituted, the substitution is by a substituent selected from the group consisting of: H, OH, N 3 , CN, NO 2 , SH, NH 2 , ONH 2 , NHNH 2 , halogen, C 1-6 alkyl, C 1-6 alkoxy and C 6-12 aryl, or a pharmaceutically acceptable salt or solvate thereof can be used as an active agent.

[0476] More specifically, the pyrrolobenzodiazepine dimer is substituted at the N10 position by X, or at the N'10 position by X', where X or X' connects the pyrrolobenzodiazepine dimer to a linker;

[0477] X and X' are each independently selected from: -C(O)O*, -S(O)O-*, -C(O)-*, -C(O)NR X -*, -S(O) 2 NR X -*, -P(O)R'NR X -*, -S(O)NR X -* or -PO 2 NR X -*; and

[0478] R X and R X' are independently selected from H, OH, N 3 , CN, NO 2 , SH, NH 2 , ONH 2 , NHNH 2 , halogen, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1-8 alkylthio, C 3-20 heteroaryl, C 5-20 aryl or mono-C 1-8 alkylamino or di-C 1-8 alkylamino;

[0479] In some embodiments, pyrrolobenzodiazepine dimer precursors are provided. When administered in the form of the precursors according to the present invention, there are advantages compared to conventional PBD drugs in that: additional reactions are necessary for conversion to the active drug after exposure to blood, thereby preventing the possibility of adverse reactions that may occur when the linker cleaves prematurely; because the toxicity to normal cells is reduced, and because the drug is more stable.

[0480] In addition, when preparing antibody-drug conjugates, although antibody-drug conjugates prepared using conventional methods have a high impurity content, and the exposed imine groups are subject to nucleophile attack, thereby creating a risk of drugs having an unwanted structure. On the other hand, drug-drug conjugates prepared using the method according to the present invention have the advantage of being easily separable due to their high purity, and further improve the physical properties compared to conventional PBDs or PBD dimers.

[0481] In some embodiments, the pyrrolobenzodiazepine dimer precursor is a pyrrolobenzodiazepine dimer precursor having the structure of the following general formula X or general formula XI, or a pharmaceutically acceptable salt or solvate thereof:

[0482] [General formula X]

[0483]

[0484] [General formula XI]

[0485]

[0486] In the above formula:

[0487] The dashed line indicates the optional presence of a double bond between C1 and C2, C2 and C3, C'1 and C'2, or C'2 and C'3;

[0488] R X1 and R X1' independently selected from H, OH, =O, =CH 2 , CN, R m OR m , =CH-R m' ,

[0489] =C(R m' ) 2 , O-SO 2 -R m , CO 2 R m , COR m , halogen and dihalogen;

[0490] R m' selected from R m, CO 2 R m , COR m , CHO, CO 2 H and halogen groups;

[0491] Each R m is independently selected from C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 5-20 aryl, C 5-20 heteroaryl, C 3-6 cycloalkyl, 3-7 membered heterocyclic group, 3-7 membered heterocycloalkyl and 5 to 7 membered heteroaryl;

[0492] R X2 , R X2' , R X3 , R X3' , R X5 and R X5' are independently selected from H, R m , OH, OR m , SH, SR m , NH 2 , NHR m , NR m 2 , NO 2 , Me 3 Sn and halogen groups;

[0493] R X4 and R X4' are independently selected from H, R m , OH, OR m , SH, SR m , NH 2 , NHR m , NR m 2 , NO 2 , Me 3 Sn, halogen groups, C 1-6 alkyl C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, 3-7 membered heterocyclic group, C 5-12 aryl, 5 to 7 membered heteroaryl,

[0494] -CN, -NCO, -OR n , -OC(O)R n , -OC(O)NR n R n' , -OS(O)R n , -OS(O) 2 R n, -SR n , -S(O)R n , -S(O) 2 R n , -S(O)NR n R n' , -S(O) 2 NR n R n' , -OS(O)NR n R n' , -OS(O) 2 NR n R n' , -NR n R n' , -NR n C(O)R o , -NR n C(O)OR o , -NR n C(O)NR o R o' , -NR n S(O)R o , -NR n S(O) 2 R o , -NR n S(O)NR o R o' , -NR n S(O) 2 NR o R o' , -C(O)R n , -C(O)OR n and -C(O)NR n R n' ;

[0495] X and X' are independently selected from -C(O)O*, -S(O)O-*, -C(O)-*, -C(O)NR X -*, -S(O) 2 NR X -*, -P(O)R'NR X -*, -S(O)NR X -* or -PO 2 NR X -*;

[0496] R X and R X' are independently selected from H, OH, N 3 , CN, NO 2 , SH, NH 2 , ONH 2, NHNH 2 , halo group, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1-8 alkylthio, C 3-20 heteroaryl, C 5-20 aryl or mono-C 1-8 alkylamino or di-C 1-8 alkylamino;

[0497] Y and Y' are independently selected from O, S, and N(H);

[0498] R x6 is independently selected from C 3-12 alkylene, C 3-12 alkenylene or C 3-12 heteroalkylene;

[0499] R X7 and R X7' are independently selected from H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3- to 7-membered heteroalkyl, C 6-10 aryl, 5- to 7-membered heteroaryl, -OR r , -OC(O)R r , -OC(O)NR r R r' , -OS(O)R r , -OS(O) 2 R r , -SR r , -S(O)R r , -S(O) 2 R r , -S(O)NR r R r' , -S(O) 2 NR r R r' , -OS(O)NR r R r' , -OS(O) 2 NR r R r' , -NR r R r' , -NR r C(O)R s , -NR r C(O)OR s , -NR r C(O)NR s R s' , -NRrS (O)R s 、-NRr S (O) 2 R s 、-NRr S (O)NR s R s' 、-NRr S (O) 2 NR s R s 、-C(O)R r 、-C(O)OR s or -C(O)NR r R r' ;

[0500] Each R r , R r' , R s and R s' Independently selected from H, C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkynyl, C 3-13 Cycloalkyl, 3-7 membered heterocyclyl, 3-7 membered heterocycloalkyl, C 5-10 Aryl and 5- to 7-membered heteroaryl;

[0501] Each R X8 and R X8' Independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Heteroalkyl, 3-7 membered heterocycloalkyl, C 5-10 Aryl, 5- to 7-membered heteroaryl, -S(O)R m 、-S(O) 2 R m 、-S(O)NR m R m' 、-S(O) 2 NR m R m' 、-NR m R m' 、-NR m C(O)R m 、-NR m C(O)OR n 、-NR m C(O)NR n R n' 、-NR m S(O)R n 、-NR m S(O) 2 Rn 、 -NR m S(O)NR n R n' 、 -NR m S(O) 2 NR n R n' 、 -C(O)R m 、 -C(O)OR m and -C(O)NR m R m' ;

[0502] Z a is selected from OR X12a 、NR X12a R X12a or SR X12a ;

[0503] Z b is selected from OR X13a 、NR X13a R X13a or SR X13a ;

[0504] Z a' is selected from OR X12a 、NR X12a R X12a or SR X12a ;

[0505] Z b' is selected from OR X13a' 、NR X13a' R X13a' or SR X13a' ;

[0506] Each R X12a 、R X12a' 、R X13a' and R x13a' is independently selected from H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3 - 7 - membered heteroalkyl, C 5-10 aryl, 5 - to 7 - membered heteroaryl, -C(O)R X15a 、 -C(O)OR X15a and -C(O)NR X15a R X15a' ; and

[0507] Each R X15a and R x15a' is independently selected from C 1-12 alkyl, C 2-12 alkenyl, C 2-12Alkynyl, C 5-20 Aryl, C 5-20 Heteroaryl, C 3-6 Cycloalkyl, 3-7 membered heterocyclic group, 3-7 membered heterocycloalkyl group, and 5 to 7 membered heteroaryl group, and:

[0508] R X13a and R X14a optionally bond to the atom to which they are attached to form a 3-7 membered heterocyclic group, 3-7 membered heterocycloalkyl group, or 3-7 membered heteroaryl group, and R X13a' and R X14a' optionally bond to the atom to which they are attached to form a 3-7 membered heterocyclic group, 3-7 membered heterocycloalkyl group, or 3-7 membered heteroaryl group; and

[0509] R n 、R n' 、R o 、R o' 、R p and R p' each independently selected from H, C 1-7 alkyl, C 2-7 alkenyl, C 2-7 alkynyl, C 3-13 cycloalkyl, 3-7 membered heterocycloalkyl, C 5-10 aryl, and 5 to 7 membered heteroaryl.

[0510] In addition, R m is independently selected from C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 5-20 aryl, C 5-20 heteroaryl, C 3-6 cycloalkyl, 3-7 membered heterocyclic group, 3-7 membered heterocycloalkyl group, and 5 to 7 membered heteroaryl group; and

[0511] R m is further substituted by C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 5-20 aryl, C 5-20 heteroaryl, C 3-6 cycloalkyl, 3-7 membered heterocyclic group, 3-7 membered heterocycloalkyl group, and 5 to 7 membered heteroaryl group.

[0512] In addition, R X4 and R X4' are independently selected from H, R m 、OH、OR m 、SH、SR m 、NH 2 、NHR m 、NR mR m' 、 NO 2 、 Me 3 Sn, halo group, C 1-6 alkyl C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-7 membered heteroalkyl, C 5-12 aryl, 5 to 7 membered heteroaryl, -CN, -NCO, -OR n 、 -OC(O)R n 、 -OC(O)NR n R n' 、 -OS(O)R n 、 -OS(O) 2 R n 、 -SR n 、 -S(O)R n 、 -S(O) 2 R n 、 -S(O)NR n R n' 、 -S(O) 2 NR n R n' 、 -OS(O)NR n R n' 、 -OS(O) 2 NR n R n' 、 -NR n R n' 、 -NR n C(O)Ro, -NR n C(O)ORo, -NR n C(O)NR o R o' 、 -NR n S(O)R o 、 -NR n S(O) 2 R o 、 -NR n S(O)NR o R o' , -NR n S(O) 2 NR o R o' 、 -C(O)R n 、 -C(O)OR n and -C(O)NR n R n '; and

[0513] R X4 or RX4' is C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-7 membered heteroalkyl, C 5-12 aryl or 5 to 7 membered heteroaryl, and further substituted by at least one of the following: C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-7 membered heteroalkyl, C 5-10 aryl, 5 to 7 membered heteroaryl, -OR p , -OC(O)R p , -OC(O)NR p R p' , -OS(O)R p , -OS(O) 2 R p , -SR p , -S(O)R p , -S(O) 2 R p , -S(O)NR p R p' , -S(O) 2 NR p R p' , -OS(O)NR p R p' , -OS(O) 2 NR p R p' , -NR p R p' , -NR p C(O)R q , -NR p C(O)OR q , -NR p C(O)NR q R q' , -NR p S(O)R q , -NR p , -S(O) 2 R q , -NR p , -S(O)NR q R q' , -NR p , -S(O) 2 NR q R q' , -C(O)R p、 -C(O)OR p or -C(O)NR p R p 。

[0514] In addition, R X7 and R X7' are independently selected from H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3 - 7 - membered heteroalkyl, C 6-10 aryl, 5 - to 7 - membered heteroaryl, -OR r 、 -OC(O)R r 、 -OC(O)NR r R r' 、 -OS(O)R r 、 -OS(O) 2 R r 、 -SR r 、 -S(O)R r 、 -S(O) 2 R r 、 -S(O)NR r R r' 、 -S(O) 2 NR r R r' 、 -OS(O)NR r R r' 、 -OS(O) 2 NR r R r' 、 -NR r R r' 、 -NR r C(O)R s 、 -NR r C(O)OR s 、 -NR r C(O)NR s R s' 、 -NR r S(O)R s 、 -NR r S(O) 2 R s 、 -NR r S(O)NR s R s' 、 -NR r S(O) 2 NR s R s 、 -C(O)R r 、 -C(O)OR s or -C(O)NR rR r' ;

[0515] R X7 and R X7' are independently selected from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-7 membered heterocycloalkyl, C 6-10 aryl, 5 to 7 membered heteroaryl, and C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-7 membered heterocycloalkyl, C 6-10 aryl or 5 to 7 membered heteroaryl, -OR t , -OC(O)R t , -OC(O)NR t R t' , -OS(O)R t , -OS(O) 2 R t , -SR t , -S(O)R t , -S(O) 2 R t , -S(O)NR t R t' , -S(O) 2 NR t R t' , -OS(O)NR t R t' , -OS(O) 2 NR t R t' , -NR t R t' , -NR t C(O)R u , -NR t C(O)OR u , -NR t C(O)NR u R u' , -NR t S(O)R u , -NR t , -NR 2 R u , -NR t , -NR u S(O)NR u' , -NR t , -NR 2 S(O)NR u R u', -C(O)R t , -C(O)OR t or -C(O)NR t R t' ; and

[0516] R r 、R r' 、R s 、R s' 、R t 、R t' 、R u and R u' are independently selected from H, C 1-7 alkyl, C 2-7 alkenyl, C 2-7 alkynyl, C 3-13 cycloalkyl, 3 - 7 - membered heteroalkyl, C 5-10 aryl, and 5 - to 7 - membered heteroaryl.

[0517] In addition, R X1 and R X1' are independently selected from R m ; and

[0518] R m is selected from C 1-6 alkyl, C 2-6 alkenyl, C 5-7 aryl, and C 3-6 heteroaryl.

[0519] In addition, R X2 、R X2' 、R X3 、R X3' 、R X5 and R X5' are independently selected from H or OH.

[0520] In addition, R X4 and R X4' are independently selected from R m ; and

[0521] R m is C 1-6 alkoxy.

[0522] In addition, R X4 and R X4' are independently any one selected from methoxy, ethoxy, and butoxy.

[0523] In addition, Y and Y' are O.

[0524] In addition, R x6 is C 3-12 alkylene, C 3-12 alkenylene, or C3-12 Azalkylene, where R x6 is substituted by: -NH 2 , -NHR m , -NHC(O)R m , -NHC(O)CH 2 -[OCH 2 CH 2 n -R XX or -[CH 2 CH 2 O] n -R XX ;

[0525] R XX is H, OH, N 3 , CN, NO 2 , SH, NH 2 , ONH 2 , NHNH 2 , halogen, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1-8 alkylthio, C 3-20 heteroaryl, C 5-20 aryl or mono-C 1-8 alkylamino or di-C 1-8 alkylamino; and

[0526] n is an integer from 1 to 6.

[0527] In addition, in some embodiments, the active agent is a pyrrolobenzodiazepine dimer represented by the following general formula XII or general formula XIII;

[0528] [General formula XII]

[0529]

[0530] [General formula XIII]

[0531]

[0532] X a and X a' are independently selected from a bond or C 1-6 alkylene;

[0533] Z X' and Z X are independently selected from hydrogen, C 1-8 alkyl, halogen, cyano, nitro, or -(CH 2 ) m -OCH​3 ;

[0534] R 80 , R 90 and R 100 are each independently selected from hydrogen, C 1-8 Alkyl, C 2-6 Alkenyl and C 1-6 alkoxy; and

[0535] m is an integer from 0 to 12.

[0536] Z X' and Z X are independently selected from hydrogen and -(CH 2 ) m -OCH 3 Any of;

[0537]

[0538] R 80 , R 90 and R 100 are each independently selected from hydrogen, C 1-3 Alkyl and C 1-3 Alkoxy;

[0539] m is an integer from 1 to 6; and

[0540] The active agent is any one selected from the following:

[0541]

[0542]

[0543]

[0544]

[0545]

[0546]

[0547]

[0548]

[0549]

[0550] In another aspect, the present invention provides a method for preparing a medicament for preventing or treating a disease associated with overexpression of ROR1, a use of the antibody-drug conjugate according to the present invention, or a pharmaceutically acceptable salt or solvate thereof.

[0551] Here, the diseases associated with overexpression of ROR1 are cancers, examples of which include but are not limited to: chronic lymphocytic leukemia (CLL), B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt lymphoma, mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer.

[0552] In another aspect, the present invention provides a pharmaceutical composition for preventing or treating a disease associated with overexpression of ROR1, the composition comprising an antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvate thereof.

[0553] In addition, a pharmaceutically effective amount of a chemotherapeutic agent, such as at least one type of therapeutic adjuvant and a pharmaceutically acceptable excipient, may be further included.

[0554] In some embodiments, the therapeutic adjuvant may be used in combination with: an agent that exhibits prevention, improvement, or treatment of a disease associated with overexpression of ROR1; an agent that can reduce the adverse effects presented when administering a therapeutic agent for a disease associated with overexpression of ROR1; or an agent that exhibits an immune-enhancing effect; but is not limited to the aforementioned therapeutic adjuvants. This means that the therapeutic adjuvant can be used in combination with any drug that exhibits a therapeutically useful effect, further improving the stability of pyrrolobenzodiazepine, reducing the side effects that may occur when administering pyrrolobenzodiazepine, or inducing immunity to maximize the therapeutic effect when administered in the form of a compound drug having pyrrolobenzodiazepine.

[0555] In another aspect of the present invention, the present invention provides a method for treating a disease associated with overexpression of ROR1 in an individual suffering from a disease associated with overexpression of ROR1, the method comprising the step of administering to the individual an effective dose of an antibody-drug composition according to the present invention or a pharmaceutically acceptable salt or solvate thereof to treat a disease associated with overexpression of ROR1; and a method of administering [the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvate thereof] by mixing with one or more anti-proliferative, cell growth inhibitory, or cytotoxic substances.

[0556] In one aspect of the present invention, there is provided a method for treating cancer, the method comprising the step of administering the above drug composition to a patient.

[0557] The antibody-drug conjugate according to the present invention is suitable for delivering an active agent, specifically pyrrolobenzodiazepine, to a target site on a target object. The antibody-drug conjugate according to the present invention releases active pyrrolobenzodiazepine without any linker and does not contain any modification that can affect the reactivity of the pyrrolobenzodiazepine compound.

[0558] The antibody-drug conjugates described herein are capable of effectively and specifically delivering the drug to cells expressing ROR1, by including an antibody as described above. By stably binding the antibody to the drug to maintain in vivo stability while exhibiting the expected cytotoxicity, and specifically being more stable in serum and stable in circulation, and by employing linker technology including self-immolative groups, which allows the drug to be easily released inside cancer cells to maximize efficacy, these conjugates exhibit the benefits of providing a drug-linker-ligand system that allows the drug and / or toxin to safely reach the target cells and exhibit efficacy efficiently while greatly reducing toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0559] Figure 1 The results of analyzing (ELISA) the binding ability of an anti-ROR1 monoclonal phage antibody prepared according to an example of the present invention to the ROR1 antigen are shown. It shows that individual anti-ROR1 monoclonal antibodies specifically bind to the extracellular domain ROR1 antigen. BCMA-Fc is a negative control group, and specifically, it shows that individual anti-ROR1 monoclonal antibodies only specifically bind to the ROR1 antigen and do not bind to the BCMA protein or Fc used as a label.

[0560] Figure 2 The results of measuring (FACS) the binding ability of an anti-ROR1 monoclonal phage antibody according to an example of the present invention to cells expressing the ROR1 antigen on the cell surface are shown, and the JeKo-1 cell line is used as the cells expressing ROR1 on the cell surface. It shows that each anti-ROR1 monoclonal antibody specifically binds to ROR1 expressed on the cell surface.

[0561] Figure 3a And b show the results of analyzing (ELISA) the binding ability of an anti-ROR1 IgG antibody prepared according to an example of the present invention to the human ROR1 antigen. It shows that individual antibodies bind to the human ROR1 antigen in a concentration-dependent manner. The results show that the binding ability to ROR1 is maintained even after changing the monoclonal phage antibody to the IgG form.

[0562] Figure 4 Shown is the result of an analysis (ELISA) of the binding ability of an anti-ROR1 IgG antibody prepared according to an example of the present invention to a murine ROR1 antigen. It shows that each antibody binds to the murine ROR1 antigen in a concentration-dependent manner. Through this experiment, it was confirmed that the anti-ROR1 antibody of the present invention has cross-reactivity with murine ROR1. For the 2A2 antibody used as a reference group, although it has cross-reactivity with murine ROR1, the degree of binding is relatively weak compared to the anti-ROR1 antibody of the present invention.

[0563] Figure 5 Shown is the result of a measurement (FACS) of the binding ability of an anti-ROR1 antibody prepared according to an example of the present invention to an ROR1 antigen expressed on the cell surface, wherein human ROR1 is artificially overexpressed in the CHO human ROR1 cell line, human ROR2 is overexpressed in CHO human ROR2, and murine ROR1 is overexpressed in CHO murine ROR1. It has been shown that individual antibodies specifically bind to human ROR1 expressed on the cell surface, rather than binding to the family protein human ROR2. Furthermore, by confirming the binding to the cell line in which murine ROR1 is artificially overexpressed, it was confirmed that the anti-ROR1 antibody of the present invention has interspecies cross-reactivity with murine ROR1. For the 2A2 antibody used as a reference group, although it has cross-reactivity with murine ROR1, the degree of binding is relatively weak compared to the anti-ROR1 antibody of the present invention.

[0564] Figure 6 Shown is the result of a measurement (FACS) of the binding ability of an anti-ROR1 antibody according to an example of the present invention to an ROR1 antigen expressed on the cell surface, using the JeKo-1 and Mino cell lines as ROR1-expressing positive cell lines and using the MCF7 cell line as an ROR1-negative cell line. It was found that individual antibodies specifically bind to ROR1 expressed on the cell surface and do not bind to MCF7, a cell line that does not express ROR1.

[0565] Figure 7 Shown is the result of a measurement (FACS) of the binding ability of an anti-ROR1 antibody prepared according to an example of the present invention to an ROR1 antigen expressed on the cell surface. The MC38 human ROR1 cell line was used, in which human ROR1 was artificially overexpressed on the murine colon cancer cell line MC38. It was found that individual antibodies bind to the cell line with overexpression of human ROR1 in a concentration-dependent manner.

[0566] Figure 8 Shown is the result of a measurement (FACS) of the binding ability of an anti-ROR1 antibody prepared according to an example of the present invention to an ROR1 antigen expressed on the cell surface in various cancer cell lines.

[0567] Figure 9a And b show the results of analyzing the cancer inhibitory efficacy of an anti-ROR1 antibody according to an example of the present invention in a mouse tumor xenograft model.

[0568] Figure 10 Show the results of analyzing the mechanism of action of an anti-ROR1 antibody prepared according to an example of the present invention. The inhibition of cancer growth by the antibody can manifest, for example, in the form of inducing autophagic cell death, inhibiting cancer cell division, inhibiting tumor angiogenesis, and / or immune cell activation, and the same or different mechanisms of action can manifest depending on the antibody. In Figure 10 , the occurrence of cell death was analyzed using a single mechanism of action whenever possible. The results of treating a cell line expressing ROR1 with an anti-ROR1 antibody according to the present invention were found, and the antibody forms a multimer and is capable of inducing cell death. For the 2A2 antibody used as a reference group, it was found that even when forming a multimer, it does not induce cell death.

[0569] Figure 11a A schema showing the characteristics of an anti-ROR1 antibody-MMAE conjugate (DAR4) prepared according to an example of the present invention.

[0570] Figure 11b A schema showing the characteristics of an anti-ROR1 antibody-dPBD conjugate (DAR2) prepared according to an example of the present invention.

[0571] Figure 12 Show the results of analyzing the cancer inhibitory efficacy of an anti-ROR1 antibody-dPBD conjugate (DAR2) prepared according to an example of the present invention in a mouse model transplanted with MDA-MB-468 breast cancer cell line. It is shown that the anti-ROR1 antibody-dPBD conjugate (DAR2) according to the present invention is effective in tumor elimination in a mouse model transplanted with a breast cancer cell line expressing ROR1.

[0572] Figure 13 Show the results of analyzing the cancer inhibitory efficacy of an anti-ROR1 antibody-dPBD conjugate (DAR2) prepared according to an example of the present invention in a mouse model transplanted with MDA-MB-231 breast cancer cell line. It is shown that the anti-ROR1 antibody-dPBD conjugate (DAR2) according to the present invention is effective in tumor elimination in a mouse model transplanted with a breast cancer cell line expressing ROR1.

[0573] Figure 14Results showing the cancer inhibitory efficacy of an anti-ROR1 antibody-dPBD conjugate (DAR2) prepared according to an example of the present invention were analyzed in a mouse model transplanted with HCC1187 lung cancer cell line. It is shown that the anti-ROR1 antibody-dPBD conjugate (DAR2) according to the present invention is effective in tumor regression in a mouse model transplanted with a lung cancer cell line expressing ROR1.

[0574] Figure 15 Results showing the cancer inhibitory efficacy of an anti-ROR1 antibody-dPBD conjugate (DAR2) and an anti-ROR1 antibody-MMAE conjugate (DAR4) prepared according to an example of the present invention were analyzed in a mouse model transplanted with Calu-3 breast cancer cell line. It is shown that the anti-ROR1 antibody-dPBD conjugate (DAR2) and the anti-ROR1 antibody-MMAE conjugate (DAR4) according to the present invention are effective in tumor regression in a mouse model transplanted with a breast cancer cell line expressing ROR1.

[0575] Figure 16 Results showing the comparison of the cancer inhibitory efficacy of an anti-ROR1 anti-drug conjugate according to the present invention in a mouse model transplanted with Jeko-1 mantle cell lymphoma cell line were presented.

[0576] Figure 17 Results of the anti-ROR1 antibody-dPBD conjugate (DAR2) and the anti-ROR1 antibody-MMAE conjugate (DAR4) according to the present invention were presented. It is shown that the anti-ROR1 antibody-dPBD conjugate (DAR2) and the anti-ROR1 antibody-MMAE conjugate (DAR4) according to the present invention are effective in tumor regression in a mouse model transplanted with a mantle cell lymphoma cell line expressing ROR1. Detailed Description

[0577] The present invention is based on the development of an antibody capable of specifically binding to ROR1.

[0578] The headings used under this section are for convenience in presenting the specification, and the present invention is not limited thereto.

[0579] Unless otherwise defined in the present invention, scientific and technical terms used in the present invention shall be defined as commonly understood by those skilled in the technical field of the present invention. In addition, unless particularly required in the context, singular expressions include plural expressions, and plural expressions include singular expressions.

[0580] [Definitions]

[0581] The following definitions apply to this specification:

[0582] In this specification, a "conjugate" refers to a cell-binding agent that is covalently linked to one or more molecules of a cytotoxin compound. Here, a "cell-binding agent" is a molecule that has an affinity for a biological target, such as a ligand, protein, antibody, specifically a monoclonal antibody, protein, or antibody fragment, and the binding agent is used to direct a bioactive compound to a biological target. In some embodiments, the conjugate can be designed to target tumor cells through a cell surface antigen. The antigen can be a cell surface antigen that is overexpressed or expressed by abnormal cell types. Specifically, the target antigen can be expressed only on proliferating cells (such as tumor cells). The target antigen can be selected based on differential expression, typically between proliferating tissue and normal tissue. In the present invention, a ligand is bound to a linker.

[0583] In the present invention, a "variant" of a polypeptide (such as an antigen-binding fragment, protein, or antibody) is a polypeptide in which an insertion, deletion, addition, and / or substitution has occurred in at least one amino acid residue (including a fusion polypeptide) compared to another polypeptide sequence. In addition, protein variants include proteins that have been modified by proteolytic cleavage, phosphorylation, or other post-translational modifications but maintain the biological activity of the disclosed antibody, such as the binding and specificity for ROR1. The variant can be about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identical to the sequence of the antibody or its antigen-binding fragment disclosed in the present invention. The following can be referred to for calculating the percentage of identity (%) or homology.

[0584] In one example, the percentage of identity of a peptide sequence can be calculated, for example, as 100×[(number of identical positions) / min(TG A , TG B )], where TG A and TG B are the total number of residues and internal gap positions in peptide sequences A and B in an alignment that minimizes TG A and TG B . (Russell et al., Journal of Molecular Biology (J. Mol Biol.), 244:332-350 (1994)).

[0585] In the present invention, a conservative amino acid substitution refers to a substitution that does not significantly affect the activity or antigenicity of a polypeptide. A polypeptide can include one or more conservative substitutions. Non-limiting examples are disclosed in Table 3 below.

[0586] In the present invention, a "derivative" of a polypeptide is a polypeptide in which at least one residue has been chemically modified by binding to another chemical moiety and is different from an insertion, deletion, addition, or substitution variant.

[0587] In the present invention, the term "found in nature" as used with respect to polypeptides, nucleic acids, host cells, etc. means that the material occurs naturally.

[0588] ROR1 (receptor tyrosine kinase-like orphan receptor) recognized by an antibody according to the present invention refers to a transmembrane protein of the RTK (receptor tyrosine kinase) family. In one instance, particularly the extracellular domain is recognized. ROR1 recognized by an antibody according to the present invention may be the extracellular domain present or not present on the cell membrane. The human protein of ROR1 consists of 937 amino acids, wherein the amino acid sequence is NCBI reference sequence ID: NP_005003.2 and the nucleic acid sequence is NM_005012.3. Unless clear from the context in which it is used in the present invention, ROR1 denotes hROR1, but the antibodies according to the present invention also have the ability to specifically bind mouse ROR1. The mouse ROR1 amino acids are GenBank: BAA75480.1.

[0589] In the present invention, "identity" means the sequence similarity of two or more polypeptides or two or more polynucleotides, as determined by aligning and comparing the sequences of two or more polypeptides or two or more polynucleotides. Such intermediate sequence identity is typically expressed as "percent identity", which means the ratio of identical amino acids or nucleotides between the molecules being compared, and is calculated based on the smallest molecule of the molecules being compared. Methods that can be used to align nucleic acids or polypeptides to calculate multi-molecule identity are known in the art.

[0590] In the present invention, "affinity" or ["affinity"] is the strength of the interaction between an antibody or an antigen-binding fragment thereof and an antigen, and is determined by the characteristics of the antigen (such as the size, shape, and / or charge of the antigen) and the CDR sequences of the antibody or antigen-binding fragment. Methods for determining affinity are known in the art, and the following can be used as a reference.

[0591] When the dissociation constant (K D ) < 10 -6 M, an antibody or an antigen-binding fragment thereof is said to "specifically bind" to its target (such as an antigen). When K D < 1x -8 M, the antibody specifically binds to a target with "high affinity".

[0592] As used herein, an "antigen-binding fragment" of an antibody or immunoglobulin chain (heavy or light chain) comprises a portion of the antibody that lacks some amino acids compared to the full-length chain but can specifically bind to an antigen. This fragment can be considered biologically active as it can specifically bind to a target antigen or can compete with other antibodies or antigen-binding fragments for binding to a specific epitope. In some embodiments, this fragment contains at least one CDR present in the full-length light or heavy chain, and in some instances, it comprises a short-chain heavy and / or light chain or a portion thereof. This biologically active fragment can be produced by recombinant DNA techniques or can be generated, for example, by enzymatically or chemically cleaving the intact antibody. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, scFab, dsFv, Fv, scFv, scFv-Fc, bispecific antibodies, minibodies, scAb, and dAb, and can be derived from any mammal, including but not limited to humans, mice, rats, camels, or rabbits. The functional portion of an antibody (such as one or more CDRs disclosed herein) can be covalently linked to a secondary protein or small molecule compound and thereby used as a targeted therapeutic agent for a specific target.

[0593] In the present invention, the "Fc" region comprises two heavy chain fragments containing the CH2 and CH3 domains of the antibody. These two heavy chain fragments associate with each other through disulfide bonds and hydrophobic interactions of two or more in the CH3 domain.

[0594] In the present invention, a "Fab fragment" consists of one light chain and one heavy chain containing only the variable region and CH1. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule. In scFab, two molecules of Fab are linked by a flexible linker.

[0595] In the present invention, a "Fab' fragment" includes the Fab fragment between the CH1 and CH2 domains of the heavy chain and an additional region. Disulfide bonds can form between the two heavy chains of the Fab' fragments of two molecules, forming an F(ab') 2 molecule.

[0596] In the present invention, as described above, "F(ab') 2 fragment" contains two light chains and two heavy chains, the two light chains and two heavy chains containing the variable region CH1 and a portion of the constant region between CH1 and CH2 domains, with an interchain disulfide bond forming between the two heavy chains. Thus, the F(ab') 2 fragment consists of two Fab' fragments, and the two Fab' fragments are joined to each other by a disulfide bond therebetween.

[0597] In the present invention, the "Fv region" is a fragment of an antibody that includes each variable region of the heavy and light chains, but does not include the constant regions. In sdFV, the heavy and light chains are linked by a disulfide bond. In scFc, the Fv is linked by a flexible linker. In scFv-Fc, the Fc is linked to the scFv. In a microantibody, the CH3 is linked to the scFv. A bispecific antibody contains two molecules of scFv.

[0598] In the present invention, a "single-chain" or "scFv" antibody fragment includes the VH and VL domains of an antibody that are present within a single polypeptide chain. The Fv polypeptide may additionally include a polypeptide linker between the Vh and VL domains that allows the scFv to form a target structure for antigen binding.

[0599] In the present invention, a "single-chain antibody (scAb)" is a single polypeptide chain that includes one constant region of the light chain or one constant region of the heavy chain, wherein the heavy and light chain variable regions are linked by a flexible linker. References to single-chain antibodies can be found in U.S. Patent No. 5,260,203, which is hereby incorporated by reference in the present invention.

[0600] In the present invention, a "domain antibody (dAb)" is an immunologically functional immunoglobulin fragment that includes only the variable region of the heavy chain or the variable region of the light chain. In one embodiment, two or more VH regions are linked by a peptide bond through a peptide linker to form a bivalent domain antibody. The two VH regions of this bivalent domain antibody may target the same or different antigens.

[0601] In the present invention, "complementary determining regions" (CDRs; that is, CDR1, CDR2, and CDR3) represent the amino acid residues of the variable domain of an antibody that are necessary for binding to an antigen. Each variable domain typically has three CDR domains, identified as CDR1, CDR2, and CDR3.

[0602] In the present invention, the "framework region" (FR) refers to the variable domain residues that are different from the CDR residues. Each variable domain typically has four FRs, identified as FR1, FR2, FR3, and FR4.

[0603] In the present invention, a "bivalent antigen-binding protein" or "bivalent antibody" contains 2 antigen-binding sites. The two antigen-binding sites included in the bivalent antibody may have the same antigen specificity, or the antibody may be a bispecific antibody in which the antigen-binding sites bind to different antigens.

[0604] In the present invention, a "multispecific antigen-binding protein" or "multispecific antibody" targets two or more antigens or antigen determinants.

[0605] In the present invention, a "linker" refers to a compound that covalently bonds a cytotoxin compound to a ligand or an antibody. In some embodiments, the linkers disclosed in PCT / US2016 / 063564 and PCT / US2016 / 063595 can be used, both of which are incorporated herein by reference in their entireties, and particularly for the linkers disclosed therein.

[0606] In the present invention, "unsubstituted or substituted" is used to refer to a group that can be unsubstituted or substituted; "substituted" refers to a group having at least one substituent; and "substituent" refers to a chemical moiety that is covalently bonded to or joined with a parent group. It should be understood that the substituents and substitution patterns on the compounds of the present invention can be selected by those skilled in the art to produce chemically stable compounds, which can be readily synthesized from readily available starting materials by techniques known in the art and those methods described below. If the substituent itself is substituted by more than one group, it should be understood that these multiple groups can be on the same carbon or on different carbons, as long as a stable structure is produced. For example, the substituent can be, but is not limited to, a hydroxyl group, a hydroxyalkyl group, an alkoxy group, a halogen, an alkyl group, a nitro group, a silyl group, an acyl group, an acyloxy group, an aryl group, a cycloalkyl group, a heterocyclic group, an amino group, an aminoalkyl group, a cyano group, a haloalkyl group, a haloalkoxy group, -OCO-CH 2 -O-alkyl, -OP(O)(O-alkyl) 2 or -CH 2 -OP(O)(O-alkyl) 2 . In certain embodiments, the groups mentioned in the structural formulas herein (such as alkyl, cycloalkyl, heteroaryl, heterocyclic or aryl) are optionally substituted, i.e., unsubstituted or substituted. In certain embodiments, the groups mentioned in the structural formulas herein (such as alkyl, cycloalkyl, heteroaryl, heterocyclic or aryl) are unsubstituted.

[0607] As used herein, the term "optionally substituted" or "unsubstituted or substituted" means that one to six hydrogen groups in a given structure are replaced by a group of designated substituents, the designated substituents including, but not limited to: a hydroxyl group, a hydroxyalkyl group, an alkoxy group, a halogen, an alkyl group, a nitro group, a silyl group, an acyl group, an acyloxy group, an aryl group, a cycloalkyl group, a heterocyclic group, an amino group, an aminoalkyl group, a cyano group, a haloalkyl group, a haloalkoxy group, -OCO-CH 2 -O-alkyl, -OP(O)(O-alkyl) 2 or -CH 2 -OP(O)(O-alkyl) 2. Preferably, "optionally substituted" or "unsubstituted or substituted" means replacing one to four hydrogen groups with the substituents mentioned above in a given structure. More preferably, one to three hydrogen groups are replaced with the substituents mentioned above. It should be understood that the substituents can be further substituted.

[0608] In the present invention, "halogen group" refers to fluorine, chlorine, bromine, iodine, etc.

[0609] In certain embodiments of the present invention, "alkyl group" is a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of an aliphatic or cycloaliphatic saturated or unsaturated (unsaturated, completely unsaturated) hydrocarbon compound. Examples of saturated alkyl groups include methyl, ethyl, butyl, n-pentyl (pentyl), n-hexyl, n-heptyl, etc.; saturated cyclic alkyl groups, such as methyl, ethyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, and neopentyl, etc.

[0610] In other embodiments of the present invention, the term "alkyl group" refers to a saturated aliphatic group, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (cycloaliphatic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In a preferred embodiment, the straight-chain or branched-chain alkyl group has 30 or fewer carbon atoms in its main chain (e.g., C 1-30 , for the straight-chain, and C 3-30 ) for the branched-chain), and more preferably 20 or fewer.

[0611] In the above embodiments and throughout the specification, examples, and claims, "alkyl group" is intended to include both unsubstituted and substituted alkyl groups, the latter referring to an alkyl moiety having substituents replacing the hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups, such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.

[0612] In the present invention, "alkoxy group" refers to -OR where R is an alkyl group, and examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-propoxy, isobutoxy, and tert-butoxy, etc.

[0613] In the present invention, "alkenyl group" is an alkyl group having at least one carbon-carbon double bond. Examples of unsaturated alkenyl groups are vinyl (ethenyl, -CH=CH 2 ), 1-propenyl (-CH=CHCH 3 ), 2-propenyl, isopropenyl, butenyl, pentenyl, and hexenyl, etc.

[0614] In the present invention, "alkynyl group" is an alkyl group having at least one carbon-carbon triple bond, and examples of unsaturated alkynyl groups include ethynyl and 2-propynyl, etc.

[0615] In the present invention, "carboxyl group" refers to -C(=O)OH.

[0616] In the present invention, "formyl" means -C(=O)H.

[0617] In certain embodiments of the present invention, "aryl" means a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound. For example, "C 5-7 aryl" is a moiety having 5 to 7 ring atoms, which is a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, and "C 5-10 aryl" is a moiety having 5 to 10 ring atoms, which is a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound. Here, the prefixes (C 5-7 、C 5-10 etc.) refer to the number of ring atoms or the range of the number of ring atoms, regardless of whether they are carbon atoms or heteroatoms. For example, "C 5-6 aryl" refers to an aryl having 5 or 6 ring atoms. Here, the ring atoms can be all carbon atoms as in "carbocyclic aryl". Examples of carbocyclic aryl include, but are not limited to, carbocyclic aryl derived from benzene, naphthalene, azulene, anthracene, phenanthrene, tetracene, and pyrene. Examples of aryl including fused rings in which at least one is an aromatic ring include, but are not limited to, groups derived from indane, indene, isoindene, tetralin, acenaphthene, benzindene, propylene naphthalene, acetylphenanthrene, and acetyl group. Alternatively, the ring atoms may contain one or more heteroatoms, as in "heteroaryl".

[0618] In other embodiments of the present invention, "aryl" as used herein includes substituted or unsubstituted monocyclic aromatic groups, wherein each atom of the ring is carbon. Preferably, the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic systems having two or more rings, wherein two or more carbons are shared by two adjacent rings, wherein at least one of the rings is an aromatic ring. For example, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclic group. Aryl includes benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0619] In the present invention, "heteroaryl" means an aryl containing one or more heteroatoms, such as pyridine, pyrimidine, benzothiophene, furyl, dioxolanyl, pyrrolyl, oxazolyl, pyridyl, pyridazinyl, more precisely benzofuran, isobenzofuran, indole, isoindole, indolizine, indoline, isoindoline, purine (adenine or guanine), benzimidazole, indazole, benzoxazole, benzisoxazole, benzodioxolene, benzofuran, benzotriazole, benzothiofuran, benzothiazole, C having two fused rings derived from benzothiazole 9, benzopyran, isobenzopyran, chroman, isochroman, benzodioxane, quinoline, isoquinoline, quinazoline, benzoxazine, benzodiazine, pyridopyridine, quinoxaline, quinazoline, cinnoline, phthalazine, naphthyridine, C having two fused rings derived from pyridine 10 , C having two fused rings derived from benzodiazepine 11 , azacarbazole, dibenzofuran, dibenzothiophene, carboline, pyrimidine, C having three fused rings derived from pyridoindole 13 , acridine, xanthene, thioxanthene, phenoxathiin, phenazine, phenoxazine, phenothiazine, thiophene, phenanthridine, phenanthroline, and C having three fused rings derived from phenazine 14 .

[0620] In the present invention, "cycloalkyl" refers to an alkyl that is cycloalkyl and refers to a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cycloalkane compound. Examples of cycloalkyl include, but are not limited to, those derived from:

[0621] Saturated monocyclic hydrocarbon compounds, such as: cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, methylcyclopropane, dimethylcyclopropane, methylcyclobutane, dimethylcyclobutane, methylcyclopentane, dimethylcyclopentane, and methylcyclohexane; or

[0622] Unsaturated monocyclic hydrocarbon compounds, such as: cyclopropene, cyclobutene, cyclopentene, cyclohexene, methylcyclopropene, dimethylcyclopropene, methylcyclobutene, dimethylcyclobutene, methylcyclopentene, dimethylcyclopentene, and methylcyclohexene; and saturated heterocyclic hydrocarbon compounds: norcarane, norphenene, and norbornene.

[0623] In the present invention, "heterocyclic group" refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound.

[0624] In the present invention, the prefixes (C 1-12 , C 3-8 , etc.) refer to the number or range of the number of ring atoms, regardless of whether they are carbon atoms or heteroatoms. For example, the term "C 3-6 heterocyclic group" used in this specification refers to a heterocyclic group having 3 to 6 ring atoms.

[0625] Examples of monocyclic heterocyclic groups include, but are not limited to, those groups derived from:

[0626] 1N: aziridine, azetidine, pyrrolidine, pyrroline, 2H-pyrrole or 3H-pyrrole, piperidine, dihydropyridine, tetrahydropyridine, azepane;

[0627] 2N: imidazolidine, pyrazolidine, imidazoline, pyrazoline, piperazine;

[0628] 1O: ethylene oxide, oxetane, oxolane, synthetic alcohol, oxane, dihydropyran, pyran, oxepin;

[0629] 2O: dioxolane, dioxane and dioxepane;

[0630] 3O: trioxane;

[0631] 1N1O: tetrahydrooxazole, dihydrooxazole, tetrahydroisoxazole, dihydroisoxazole, morpholine, tetrahydrooxazine, dihydrooxazine;

[0632] 1S: ethylene sulfide, thietane, thiolane, thiane, oxepane;

[0633] 1N1S: thiazoline, thiazolidine, thiomorpholine;

[0634] 2N1O: oxadiazine;

[0635] 1O1S: oxathiol, oxathiane; and

[0636] 1N1O1S: oxathiazine.

[0637] In the present invention, a "prodrug" refers to a compound that can be directly or indirectly converted into a pyrrolobenzodiazepine drug by the action of enzymes or gastric acid under physiological conditions in vivo (such as enzymatic oxidation, reduction, and / or hydrolysis).

[0638] In the present invention, a "pharmaceutically acceptable salt" can be an acid addition salt formed from a pharmaceutically acceptable free acid, where the free acid is an organic acid or an inorganic acid.

[0639] Organic acids include, but are not limited to, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, glutamic acid, and aspartic acid. In addition, inorganic acids include, but are not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

[0640] For example, if a compound has a functional group that is an anion or can be an anion (e.g., -COOH can be -COO-), then suitable cations can be used to form salts. Examples of suitable inorganic cations include, but are not limited to, Na + and K + , alkaline earth metal cations (such as Ca 2+ and Mg 2+ ), and other cations (such as Al 3+ ). Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH 4 + ) and substituted ammonium ions (e.g., NH 3 R+ , NH 2 R 2 + , NHR 3 + , NR 4 + ).

[0641] Examples of some suitable substituted ammonium ions are derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, and amino acids such as lysine and arginine. An example of a typical quaternary ammonium ion is N(CH 3 ) 4+ .

[0642] If the compound has a functional group that can be cationic (e.g., -NH 2 can be -NH 3 + ), then salts can be formed with suitable anions. Examples of suitable inorganic anions include but are not limited to those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

[0643] Examples of suitable organic anions include but are not limited to those derived from organic acids such as: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, disulfonic acid, ethanesulfonic acid, fumaric acid, glutaric acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthoic acid, hydroxyethylsulfonic acid, lactic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, benzenesulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, p-aminobenzenesulfonic acid, and tartaric acid, etc. Examples of suitable polymeric organic anions include but are not limited to those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose, etc.

[0644] In the present invention, "solvate" refers to a molecular complex between a compound according to the present invention and a solvent molecule (examples of which include but are not limited to water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, ethanolamine) or a compound according to the present invention bound to its mixed solvent.

[0645] The following may be convenient or necessary: preparing, purifying, and / or processing solvates corresponding to the active compounds. In this specification, the term "solvate" is used in its conventional sense to refer to a solute containing a solvent (e.g., an active compound and a salt of an active compound) and a complex. When the solvent is water, the solvate can be conveniently called a hydrate, such as a monohydrate, dihydrate, or trihydrate, etc.

[0646] In the present invention, an "effective dose" or "effective therapeutic dose" refers to the dose (with respect to the dosing amount, dosing period, and means) necessary to achieve a targeted therapeutic effect. The effective dose is the minimum amount necessary to provide a minimal therapeutic benefit to an object and is an activator below the toxic dose. For example, the dosing amount can range from about 100 ng to about 100 mg / kg per patient, more typically in the range of about 1 μg / kg to about 10 mg / kg. In the case where the activating compound is a salt, ester, amide, or prodrug, etc., the dosing amount is calculated based on the parent compound, and thus the actual mass used increases proportionally. The pyrrolobenzodiazepine compounds according to the present invention can be formulated to include 0.1 mg to 3000 mg, 1 mg to 2000 mg, or 10 mg to 1000 mg of active ingredient per unit dosage form, but is not limited thereto.

[0647] The active ingredient can be administered to obtain a peak plasma concentration of the active compound in the range of about 0.05 μM to 100 μM, 1 μM to 50 μM, or 5 μM to 30 μM. For example, a solution of 0.1 w / v% to 5 w / v% active ingredient in a physiological saline solution can be optionally administered by intravenous injection.

[0648] The concentration of the active compound in the pharmaceutical composition can be determined by the absorption, inactivation, and excretion rates of the drug and other factors known to those skilled in the art. The dosing amount can vary depending on the severity of the symptoms or disease. In addition, the dosing amount and method for a given patient can be adjusted according to the professional judgment of the administrator, generally considering the patient's symptom / disease degree, necessity, age, and reactivity to the drug, etc., and the concentration ranges stated in the present invention are only exemplary and are not intended to limit the present invention to the examples presented by the claimed compositions. In addition, the active ingredient can be administered in a single dose, or smaller doses can be administered in multiple doses.

[0649] Antibody or antigen-binding fragment

[0650] The present invention discloses an antibody that specifically binds to the extracellular domain of the ROR1 protein. As disclosed in the present invention, the antibody according to the present invention is a polypeptide comprising six complementary determining regions or domains (CDRs).

[0651] In some instances, the CDRs are included in "framework" regions, and the framework orients the CDRs such that the CDRs can have appropriate antigen-binding properties.

[0652] The antibody according to the present invention specifically binds to the extracellular domains of human and mouse-derived ROR1 and can specifically bind to the extracellular domain in isolated form or to the extracellular domain of ROR1 expressed on the cell surface.

[0653] The antibodies disclosed in the present invention bind to ROR1, specifically, human ROR1 and mouse ROR1. The antibodies disclosed in the present invention that can specifically bind to the extracellular domain of human or mouse-derived ROR1 or ROR1 expressed on the cell surface can be applied to the targeted treatment of cancers targeting ROR1. For example, the antibodies according to the present invention can be conjugated to anticancer drugs and used to treat specific cancers.

[0654] In addition, when the antibody binds to mouse ROR1, the toxicity to the target can be tested by mouse assays, and the in vivo efficacy can be tested by a syngeneic model using a mouse cancer cell line overexpressing ROR1. Therefore, the antibodies can be applied to the development of various drugs related to ROR1.

[0655] The antibodies can include, but are not limited to, monoclonal antibodies, bispecific antibodies, diabodies, multispecific antibodies, multibodies, microantibodies, domain antibodies, antibody mimetics (or synthetic antibodies), chimeric antibodies or antibody fusions (or antibody conjugates) and fragments thereof, and include various forms of antibodies disclosed herein.

[0656] In some embodiments, the antibody fragments of the antibodies disclosed in the present invention can include Fab, Fab', F(ab') 2 , scFab, Fv, dsFv, scFV, scFV-Fc, microantibodies, bifunctional antibodies, scAb or dAb.

[0657] In some embodiments, the antibodies disclosed in the present invention can be composed of polypeptides containing only the light chain or only the heavy chain of the variable regions disclosed in Tables 2a and 2b.

[0658] An antibody disclosed in the present invention can share a specific region or sequence with another antibody disclosed in the present invention. In some embodiments, it can share the constant region of the antibody or antigen-binding fragment. In some embodiments, it can share the Fc region. In some embodiments, it can share the framework of the variable region.

[0659] In some embodiments, the antibody has the typical structure of an antibody found in nature. Camelids produce antibodies consisting of a single heavy chain, but the structural unit of such an antibody typically comprises a tetrameric polypeptide, where the tetramer comprises a pair of two polypeptide chain bodies consisting of different 2 polypeptide chains. In a typical antibody, a pair of polypeptide chain bodies comprises a full-length light chain (about 25 kDa) and a full-length heavy chain (about 50 to 70 kDa). Each chain exhibits a characteristic folding pattern and consists of several immunoglobulin domains, which are composed of about 90 to 110 amino acids. These domains are the basic units that make up the antibody polypeptide. The amino-terminal portion of each chain typically comprises a portion called the variable region or V region that recognizes an antigen. The carboxyl-terminal portion is more evolutionarily conserved than the amino-terminal portion and includes a portion called the constant region or C region. Human light chains are typically classified as kappa (κ) or lambda (λ) light chains, and these contain one variable region and one constant region, respectively.

[0660] Heavy chains are typically classified as mu (μ), delta (δ), gamma (γ), alpha (α), or epsilon (ε) chains, and these are defined as IgM, IgD, IgG, IgA, and IgE isotypes, respectively. IgG has multiple subtypes, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM subtypes include IgM and IgM2. IgA subtypes include IgA1 and IgA2. In humans, IgA and IgD isotypes contain 4 heavy chains and 4 light chains.

[0661] IgG and IgE isotypes contain 2 heavy chains and 2 light chains, and the IgM isotype contains 5 heavy chains and 5 light chains. The heavy chain constant region typically includes at least one domain that exhibits effector functions. The number of heavy chain constant regions varies depending on the isotype. IgG heavy chains, for example, contain 3 C regions called CH1, CH2, and CH3, respectively. The antibodies disclosed in the present invention can be any of these isotypes and subtypes. In some embodiments, the antibody is an IgG1, IgG2a, IgG2b, IgG3, or IgG4 subtype. In some embodiments, the antibody of the present invention is of the IgG1 type or IgG2 type. In another embodiment, the antibody of the present invention is of the IgG1 type.

[0662] The heavy chain variable region and the light chain variable region according to the present invention can be linked to at least a portion of a human constant region. The choice of the constant region can be determined in part by whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis, and / or complement-dependent cytotoxicity are required. For example, human isotypes IgG1 and IgG3 have complement-dependent cytotoxicity and human isotypes IgG2 and IgG4 do not have this cytotoxicity. In addition, human IgG1 and IgG3 induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region can be λ or κ.

[0663] In some embodiments, the antibody can be a human antibody, and the heavy chain constant region can be of the IgG1 type, IgG2 type, IgG3 type, or IgG4 type. In some embodiments, the antibody of the present invention is of the IgG1 type or IgG2 type.

[0664] In some embodiments, the antibody is a human antibody and specifically recognizes murine ROR1.

[0665] In the full-length light and heavy chains, the variable and constant regions are joined by a region "J" of about 12 or more amino acids in length, and the heavy chain contains a "D" region of about 10 or more amino acids. By way of example, see Fundamental Immunology, 2nd Edition, Ch. 7 (Paul, W. ed.) 1989, New York: Raven Press. Generally, the variable regions of the light / heavy chain pair of an antibody form the antigen-binding site.

[0666] The variable regions of immunoglobulin chains generally have the same overall structure and contain relatively conserved framework regions (FRs) joined by three hypervariable regions called "complementary determining sites or regions or domains" or CDRs (complementary determining regions). The CDRs derived from the variable regions of each chain of a heavy / light chain pair are typically arranged by the framework regions to form a structure that specifically binds to a particular antigenic determinant of a target protein (ROR1). These elements of naturally occurring light and heavy chain regions generally include, in order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The positions of the amino acid sequences corresponding to each variable region can be determined by the Kabat assay. When compared to each other, the CDRs defined by each determination can be overlapping subsets or one can include the other as a subset. However, in the present invention, all CDRs to be defined by each of the above methods are included within the scope of the present invention. Those skilled in the art will readily be able to select the CDR sequences, given the variable region sequences of the antibody, in accordance with the above definitions.

[0667] According to some embodiments of the present invention, the CDR sequences that can be included in the variable regions of the heavy and light chains of an antibody or antigen-binding fragment are disclosed in Tables 1a to 1f, respectively.

[0668] [Table 1a]

[0669]

[0670] [Table 1b]

[0671]

[0672]

[0673] [Table 1c]

[0674]

[0675] [Table 1d]

[0676]

[0677] [Table 1e]

[0678]

[0679]

[0680] [Table 1f]

[0681]

[0682] In some embodiments of the present invention, the heavy and light chain variable region sequences of the antibody or antigen-binding fragment comprising the above light and heavy chain CDR sequences are respectively disclosed in Table 2a and Table 2b below.

[0683] [Table 2a]

[0684]

[0685]

[0686] [Table 2b]

[0687]

[0688] In some embodiments, the CDRs of the respective light chain variable regions and the CDRs of the respective heavy chain variable regions disclosed in Tables 1a to 1f above can be freely combined.

[0689] In some embodiments, the heavy and light chain variable regions disclosed in Tables 2a and 2b can be freely combined to prepare various forms of antibodies, and for example, a single antibody such as ScFV or domain antibody or full-length antibody can be formed.

[0690] Each of the heavy and light chain variable regions disclosed in the present invention can bind to various target heavy and light chain constant regions to respectively form the heavy and light chains of a complete antibody. In addition, the respective heavy and light chain sequences bound to the constant regions can be further combined to form a complete antibody structure.

[0691] Any variable region of the heavy or light chain of an antibody according to the present invention can be linked to at least a portion of a constant region. The constant region can be selected according to whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis, and / or complement-dependent cytotoxicity are required, etc.

[0692] Depending on the target, an appropriate region of the above constant regions can be used, such as constant regions of human or murine origin. In some embodiments, the human heavy chain constant region IgG1 represented by SEQ ID No. 91 is used. In some embodiments, the human lambda region represented by SEQ ID No. 93 is used as the light chain constant region.

[0693] Any variable region disclosed in the present invention can be combined with a constant region to form heavy and light chain sequences. In some embodiments, the heavy chain variable regions disclosed in the present invention can be linked to the human IgG1 constant regions represented by SEQ ID NOs. 59 to 66, 100, and 101. In some embodiments, the light chain variable regions disclosed in the present invention can be linked to the human lambda constant regions, which are represented by SEQ ID No. 67 to 74, respectively. The light and heavy chains according to the present invention can be combined in various combinations to form a complete antibody comprising two light chains and two heavy chains.

[0694] However, these constant region sequences that can be combined with the variable regions disclosed in the present invention are intended to be exemplary, and those skilled in the art should be able to know other variable regions, including IgG1 heavy chain constant regions, IgG3 or IgG4 heavy chain constant regions, any kappa or lambda light chain constant regions, or other modified variable regions, to obtain target characteristics (such as stability, expression, manufacturability, or others).

[0695] The present invention also includes one or more nucleic acid sequences having substantial sequence identity with one or more of the nucleic acid sequences disclosed in the present invention. Substantial identity means that the antibody or antigen-binding fragment encoded by the nucleic acid maintains the function disclosed in the present invention even if the sequence varies. In some embodiments, the sequence has about 90%, 95%, or 99% identity with the heavy chain variable regions disclosed in Table 2a. In some embodiments, the sequence has about 90%, 95%, or 99% identity with the light chain variable regions disclosed in Table 2b. For example, in the case of variants showing 90%, 95%, or 99% identity with the antibodies or antigen-binding fragments disclosed in the present invention, the variation occurs in the framework rather than the CDR of the variable region.

[0696] In some embodiments, the nucleic acid encoding the antibody or fragment thereof disclosed in the present invention is the nucleic acid encoding the CDRs disclosed in the present invention, the variable region including the CDRs, and the full-length antibody including the variable region and the constant region. After determining the amino acid sequence, the nucleic acid sequence can be easily determined using known reverse transcription procedures and taking into account codon usage, etc. An example of the nucleic acid sequence encoding the heavy chain constant region of human IgG1 can be represented by SEQ ID No. 92. An example of the nucleic acid sequence encoding the light chain constant region of human lambda can be represented by SEQ ID No. 94 or 95. Examples of the nucleic acid sequences of the full-length heavy chains including the above constant region nucleic acids can be represented by SEQ ID NOs. 75 to 82, 102, and 103 (heavy chains including the human IgG1 constant region), and examples of the nucleic acid sequences for the full-length light chains can be SEQ ID NOs. 83 to 90 (light chains including the human lambda constant region).

[0697] In addition, nucleic acid sequences including those encoding the CDR sequences of Tables 1a to 1f and the variable regions of Tables 2a and 2b. These nucleic acids are included in the nucleic acid sequences encoding the full-length antibodies disclosed above and are not indicated separately. Those skilled in the art will be able to easily identify the nucleic acid sequences encoding them from SEQ ID No. 75 to 90 based on the protein sequences of the CDRs and variable regions disclosed in the present invention.

[0698] The present invention further includes at least one nucleic acid sequence having substantial sequence identity with at least one nucleic acid disclosed in the present invention. Substantial identity means that the antibody or antigen-binding fragment encoded by the nucleic acid maintains the function disclosed in the present invention, even in cases where nucleic acid differences result in conservative substitutions or amino acid differences, where the change in the nucleic acid is not accompanied by an amino acid substitution.

[0699] Specificity and affinity of the antibody for the antigen

[0700] The antibody or antigen-binding fragment according to the present invention, specifically, is specific for the ECD of the ROR1 antigen and has an affinity suitable for use as an antibody therapeutic or diagnostic agent. In one embodiment, according to Table 6, the affinity for the aggregate is KD < 1.0×10 -9 M; and in another embodiment, KD ≤ 1.0×10 -10 M. Compared with antibodies having lower affinity, the antibody or antigen-binding fragment having such affinity according to the present invention has the advantage of being administered at a reduced dose, such as 10 -8 M or 10 -9 M. Although the antibodies are not limited to those described above, [those described above] have greater clinical advantages because sufficient efficacy can be achieved by a more convenient administration method, such as subcutaneous injection.

[0701] Variable region of an antibody

[0702] The present invention includes the variable regions of the heavy and light chains disclosed in Tables 2a and 2b above. In addition, the present invention includes an antibody that includes immunologically functional fragments, derivatives, mutant proteins, and variants of the variable regions of the light and heavy chains (and corresponding nucleic acid sequences). Antibodies that combine the variable regions of the heavy and light chains according to the present invention in different ways can be expressed as "VHx / VLy", where "x" is the SEQ ID NO. of the heavy chain variable region, and "y" corresponds to the light chain. In one example, the variable regions can include the following combinations: VH43 / VL51, VH43 / VL52, VH43 / VL53, VH43 / VL54, VH43 / VL55, VH43 / VL56, VH43 / VL57, VH43 / VL58, VH44 / VL51, VH44 / VL52, VH44 / VL53, VH44 / VL54, VH44 / VL55, VH44 / VL56, VH44 / VL57, VH44 / VL58, VH45 / VL51, VH45 / VL52, VH45 / VL53, VH45 / VL54, VH45 / VL55, VH45 / VL56, VH45 / VL57, VH45 / VL58, VH46 / VL51, VH46 / VL52, VH46 / VL53, VH46 / VL54, VH46 / VL55, VH46 / VL56, VH46 / VL57, VH46 / VL58, VH47 / VL51, VH47 / VL52, VH47 / VL53, VH47 / VL54, VH47 / VL55, VH47 / VL56, VH47 / VL57, VH47 / VL58, VH48 / VL51, VH48 / VL52, VH48 / VL53, VH48 / VL54, VH48 / VL55, VH48 / VL56, VH48 / VL57, VH48 / VL58, VH49 / VL51, VH49 / VL52, VH49 / VL53, VH49 / VL54, VH49 / VL55, VH49 / VL56, VH49 / VL57, VH49 / VL58, VH50 / VL51, VH50 / VL52, VH50 / VL53, VH50 / VL54, VH50 / VL55, VH50 / VL56, VH50 / VL57, VH50 / VL58, VH98 / VL51, VH98 / VL52, VH98 / VL53, VH98 / VL54, VH98 / VL55, VH98 / VL56, VH98 / VL57, VH98 / VL58, VH99 / VL51, VH99 / VL52, VH99 / VL53, VH99 / VL54, VH99 / VL55, VH99 / VL56, VH99 / VL57 or VH99 / VL58.

[0703] Various other forms of antibodies

[0704] The antibodies disclosed in the present invention are also variants of the antibodies disclosed in the present invention. For example, a portion of the antigen contains conservative amino acid substitutions in one or more of the residues of the heavy chain, light chain, variable region, or CDR sequences disclosed above. Conservative amino acid substitutions refer to substitutions that substantially do not affect the activity or antigenicity of the polypeptide. In some embodiments, conservative amino acid substitutions refer to substituting with another residue belonging to the same category of the following amino acid classifications. Naturally occurring amino acids can be classified according to the common characteristics of their side chain properties as follows: 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; 2) neutral hydrophilic: Cys, Ser, Thr, Gln; 3) acidic: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues affecting chain direction: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe. Conservative amino acid substitutions can also include non-naturally occurring amino acid residues, such as peptidomimetics, and such residues are usually introduced by chemical synthesis rather than by cells.

[0705] Non-limiting examples of conservative amino acid substitutions are shown in Table 3 and are not limited to Table 3.

[0706] [Table 3] Conservative amino acid substitutions

[0707] Original Residue Exemplary Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn GIu Asp Gly Pro His Asn, Gln lle Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

[0708] Non-conservative substitutions include substituting with residues belonging to other categories in the above classifications. Such substitutions can be employed in regions of antibodies homologous to human antibodies or their non-homologous regions.

[0709] Preparation of antibodies

[0710] In the present invention, non-human antibodies can be derived from, for example, any antibody-producing animal, such as mice, rats, rabbits, goats, donkeys, or non-human primates (such as monkeys, such as cynomolgus monkeys or rhesus monkeys) or monkeys (such as chimpanzees). Non-human antibodies can be produced by immunizing the animals using methods known in the art. The antibodies can be polyclonal or monoclonal, or can be synthesized in a cell host by expression of recombinant DNA. Fully human antibodies can be produced by administering an antigen to a transgenic animal comprising a human immunoglobulin gene locus, or by treating a phage display library expressing a human antibody repertoire with an antigen and then selecting the target antibody.

[0711] Monoclonal antibodies (mAbs) can be produced using conventional monoclonal antibody methods, such as the standard somatic cell hybridization method in the literature (see: Kohler and Milstein, 1975, Nature 256:495).

[0712] The single-chain antibodies disclosed in the present invention can be produced using an amino acid bridge (a short peptide linker) to link the heavy and light chain variable domain (Fv region) fragments. The single-chain antibodies disclosed in the present invention include scFv, which includes combinations of heavy and light chain variable regions listed in Tables 1a to 1f, or CDRs disclosed in Tables 1a to 1f, but are not limited thereto.

[0713] In addition, the antibodies disclosed in the present invention can be modified into different subtype antibodies by subtype switching. Thus, for example, IgG antibodies can be derived from IgM antibodies, and vice versa.

[0714] Therefore, included among the antibodies disclosed in the present invention are, for example, variable domain combinatorial antibodies according to the present invention, switched to a target isotype (such as IgA, IgG1, IgG2, IgG3, IgG4, IgE, and IgD).

[0715] Methods for expressing antibodies

[0716] The present invention further relates to expression systems and constructs in the form of plasmids, expression vectors, and transcription or expression cassettes, which include at least one polynucleotide as described above; host cells containing such expression systems or constructs; and methods for producing antibodies using the expression systems or host cells.

[0717] The antibodies disclosed in the present invention can be expressed in a hybridoma cell line or a cell line other than a hybridoma. The expression construct encoding the antibody can be used to transform mammalian, insect, or microbial host cells. Constructs such as plasmids can be prepared for any of the various known methods for introducing polynucleotides into host cells as described in the foregoing. The specific method can vary depending on the type of host cell. Methods for introducing heterogeneous polynucleotides into mammalian cells are well known in the art and include (and are not limited to) for example, dextran-mediated transfer, calcium phosphate precipitation, polybrene-mediated transfer, protoplast fusion, electroporation, encapsulation of the transferred polynucleotide using liposomes, mixing of nucleic acids with positively charged lipids, and direct microinjection of DNA into the cell nucleus.

[0718] Use of human ROR1 antibodies for therapeutic and treatment purposes

[0719] In cancer, ROR1 is associated with poor prognosis in cancer patients and is also reported to affect cancer metastasis. ROR1 is overexpressed not only in hematological malignancies such as B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt lymphoma, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and marginal zone lymphoma (MZL), but also in solid cancers including breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer. For anti-cancer antibody therapies, for example, ROR1 antibodies can be used alone or in the form conjugated to various cytotoxic agents as described in the present invention to eliminate cancer cells overexpressing ROR1. Thus, antibodies conjugated to ROR1 can be used alone or as conjugates to anti-cancer chemotherapeutic agents, cytotoxic agents, or radioactive substances, or can be implemented as cell therapeutic agents such as CAR-T cells, for example, to target anti-cancer targets, and thereby used as targeting therapeutic agents to direct therapeutic agents derived from the antibodies disclosed in the present invention to cells expressing ROR1.

[0720] Therapeutic methods: pharmaceutical formulations and routes of administration

[0721] Also provided is a therapeutic method using an antibody, antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof. In certain embodiments, the antibody, antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof is provided to a patient. The antibody, antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof binds to human ROR1 expressed on the surface of cancer cells, thereby inhibiting cancer metastasis of the cancer cells. In some embodiments, the antibody in the form conjugated to a cytotoxic agent binds to human ROR1 expressed on the surface of cancer cells, specifically delivering the cytotoxic agent to the cancer cells to induce cell death of the cancer cells. In some embodiments, the antibody in the form of an antibody specific to the same or different targets binds to human ROR1 expressed on the surface of cancer cells, thereby enhancing the specificity of the multispecific antibody to the cancer cells or inducing the connection of the cancer cells with other types of cells such as immune cells, and inducing cancer cell death. In certain embodiments, the antibody is expressed on the surface of a cell therapeutic agent such as a CAR-T cell, which binds to human ROR1 and further specifically delivers the cell therapeutic agent to the cancer cells to induce their death.

[0722] Also provided is a pharmaceutical composition comprising a therapeutically effective dose of the antibody-drug conjugate and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or supplement. Additionally, for example, a method of treating a cancer patient by administering such a pharmaceutical composition is included. The term "patient" includes human patients.

[0723] The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. Here, a carrier refers to an excipient, diluent, or adjuvant. The carrier may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, saline, PBS, and other buffer solutions, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. The composition may include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, or combinations thereof.

[0724] The pharmaceutical composition may be formulated into any dosage form according to known methods. The composition may be formulated for oral administration (such as powders, tablets, capsules, syrups, pills, or granules) or non-oral administration (such as injections). Additionally, the composition may be prepared as a systemic or topical formulation.

[0725] The pharmaceutical composition may comprise an effective amount of the antibody conjugate, its drug-binding fragment, an anticancer agent, or a combination of these. The term "effective amount" refers to an amount sufficient to exhibit a prophylactic or therapeutic effect when administered to an individual in need of prophylaxis or treatment. Depending on the cell or individual, the effective amount may be appropriately selected by a PHOSITA and may be determined based on factors including the severity of the disease, the age, weight, health, gender, sensitivity to the drug, duration of administration, route of administration, excretion ratio, duration of therapy, and other factors well known in the medical field of drugs mixed with or used in conjunction with the composition. The effective amount may be from about 0.1 μg to about 2 g / [unit missing] of the pharmaceutical composition.

[0726] For adults, the dosage of the pharmaceutical composition may be, for example, 10 μg / kg to about 30 mg / kg, optionally 0.1 mg / kg to about 30 mg / kg, or alternatively 0.3 mg / kg to about 20 mg / kg. The administration may be carried out once a day, multiple times a day, or once a week to once every four weeks, or once a year to twelve times a year.

[0727] Hereinafter, preferred embodiments are presented to assist in understanding the present invention. However, the following embodiments are provided to facilitate understanding of the present invention, and the present invention is not limited to these embodiments.

[0728] Example 1: Preparation of ROR1 Antibody

[0729] Example 1-1: Antigen

[0730] As an antigen, an ROR1 ECD-Fc type protein is used, in which Fc is linked to the C-terminus of the extracellular domain (ECD) of human ROR1.

[0731] Specifically, to prepare the antigen, residues of amino acids 1 to amino acid 406 corresponding to the ROR1 amino acid sequence represented by NCBI reference number NP_0050032 and including the extracellular domain of ROR1 are used. Regarding the gene encoding the extracellular domain of ROR1, cDNA from Origene (Origene, RC214967) is purchased and used. In addition, to purify the ROR1 extracellular domain thereafter, a gene encoding an Fc protein derived from human IgG1 is synthesized and linked to the 3'-end of the gene encoding the ROR1 extracellular domain (hereinafter referred to as 'ROR1-Fc'). By introducing the gene into the pcDNA3.1 vector, a vector encoding the ROR1-Fc nucleic acid in a mammalian cell line is obtained.

[0732] The expression vector is transiently transfected into HEK 293E cells to express ROR1-Fc by culturing in DMEM- / F12 medium at 8% CO2 and 37 °C and collecting the medium every 72 hours. Protein A affinity chromatography is used to purify the Fc-ROR1 ECD protein.

[0733] Example 1-2: Selection of Antibodies by Phage Library Screening

[0734] Prepare library phages

[0735] Escherichia coli with a human-derived single-chain variable fragment (scFv) library (Yang et al., 2009 Mol. Cells 27:225) at 37 °C 2×10 10Cultivate for 2 to 3 hours (OD600 = 0.5 to 0.7) in a medium containing 2×YT (Amresco, J902 - 500G), ampicillin (Duchefa, C01090025) at 100 μg / mL and 2% glucose (Sigma, G7021), then transfect with helper phage and cultivate at 30 °C in 2×YT medium [2X YT, ampicillin, 70 μg / mL kanamycin (Duchefa, K0126), 1 mM IPTG (Duchefa, I1401)] for 16 hours to induce phage encapsulation. Thereafter, centrifuge the cultured cells (6000 rpm, 15 minutes, 4 °C), and then add 4% PEG8000 (Sigma, P2139) and 3% NaCl (Samchun, S2097) to the supernatant, dissolve thoroughly and react on ice for 1 hour. After centrifuging again (8000 rpm, 20 minutes, 4 °C), add PBS (phosphate buffered saline, Gibco 10010 - 023) to the pellet to produce a suspension, which is centrifuged again (12000 rpm, 10 minutes, 4 °C). Place the supernatant containing the library phage in a new tube and keep at 4 °C until use.

[0736] Translation via phage display

[0737] To screen for antibodies that bind to the human ROR1 protein, the ROR1-Fc protein prepared in Example 1-1 was used for translation a total of three times, as explained below.

[0738] Specifically, in an immunotube (maxisorp 444202), add ROR1-Fc at a concentration of 10 μg / mL and negative control-Fc (BCMA-Fc) to PBS. Allow the protein to adsorb to the surface of the immunotube overnight at 4 °C, after which add a 3% BSA (bovine serum albumin) solution to the immunotube to protect the unadsorbed surface of ROR1-Fc. Empty the immunotube and add 10 12The CFU antibody phage library was placed in an immunotube in which a control Fc protein was adsorbed, and then allowed to react at room temperature for 1 hour (negative selection). Thereafter, phages that did not bind to the negative control Fc were recovered and bound to an immunotube adsorbed with ROR1-Fc. Nonspecifically bound phages were removed by washing 5 to 30 times with a PBS-T solution (phosphate-buffered saline - 0.05% Tween 20), and then the remaining antigen-specific phage antibodies were recovered using a 100 mM triethylamine solution. The recovered phages were neutralized with 1 M Tris buffer (pH 7.4). Escherichia coli ER2537 was infected at 37 °C for 1 hour, and the infected Escherichia coli was inoculated onto 2YT agar medium and cultured overnight at 37 °C. The next day, the cultured Escherichia coli was suspended in 4 ml of 2YT ampicillin culture, and 15% glycerol was added. Some were stored at -80 °C, and the rest were used to prepare phages for the next experiment. This process was repeated a total of three times to amplify and concentrate the ROR1 antigen-specific phage library.

[0739] Monoclonal phage antibody screening (single clone screening)

[0740] The following experiment was conducted to select monoclonal antibodies specifically binding to ROR1 from the phage library obtained by translation.

[0741] To isolate single strains from the concentrated library, the phage library was inoculated into LB-tetracycline / ampicillin agar medium and cultured to obtain single colonies. Then the monoclonal bodies were inoculated into each well of a 96-deep well culture plate containing 400 μl of 2×YT-tetracycline / ampicillin medium and grown overnight, and then 10 μl of the culture was added to a new 96-deep well culture plate containing 390 μl of 2×YT-tetracycline / ampicillin and incubated at 37 °C for 4 hours. 1 mM IPTG was added to the culture solution and incubated overnight at 30 °C. The cultured culture solution was centrifuged overnight to obtain the supernatant.

[0742] Thereafter, the ELISA method was used as follows to select clones expressing monoclonal soluble scFv that bind to the ROR1-Fc antigen (Steinberger Rader and Barbas III 2000 Phage display vectors In: Phage display laboratory manual 1st edition Cold Spring Harbor Laboratory Press New York, USA pp. 119 - 1112). Specifically, 100 ng of recombinant human ROR1-Fc prepared in Example 1-1 or FBMA-Fc was placed in a 96-well microtiter plate (Nunc-Immuno Plate, NUNC, USA) and coated overnight at 4°C. BCMA-Fc is a protein used as a negative control, which is a recombinant protein in which the extracellular domain of the human BCMA protein is linked to human Fc. 200 μL of 3% BSA was placed in each well and blocked for 2 hours at 37°C.

[0743] The monoclonal phage supernatant was prepared by mixing 1:1 with 3% BSA, and 100 μL of each of this mixture was loaded into the wells and reacted for 2 hours at 37°C. After washing 5 times with 300 μL of PBST, anti-HA HRP-conjugated antibody was added and reacted for 1 hour at 37°C, followed by washing 5 times with PBST. 100 μL of TMB (tetramethylbenzidine, Sigma, T0440) was added for color development, and then 50 μL of 1N H2SO4 was added to terminate the reaction. The absorbance was measured at 450 nm and 650 nm, and clones with an absorbance of 10 or higher at 450 nm and 650 nm were screened when coated with 1 μg / mL ROR1 ( Figure 1 ).

[0744] Next, flow cytometry was used to screen for clone lines that bind to the cell line expressing ROR1. Specifically, 100 μL of the monoclonal scFv supernatant was reacted with the cell line (JeKo-1) overexpressing ROR1, and then washed twice with PBS. After reacting with anti-HA-FITC antibody (Sigma, H7411) for 30 minutes at 4°C and washing twice with PBS, and then suspended in 200 μL of PBS, the FACSCalibur flow cytometer (BD Biosciences) was used to screen for clone lines that bind to the JeKo-1 cell line ( Figure 2 ).

[0745] This process screened 10 antibody clone lines (AB4, A2F2, A2F3, BA6, CC9, C2E3, DG6, D2B12, A2F2 M1, and BA6 M1) that bind to the recombinant human ROR1 protein and the cell line expressing ROR1. The amino acid sequences and CDR sequences of the heavy-chain variable region and light-chain variable region of each of these antibodies are shown in the following table.

[0746] [Table 4a]

[0747]

[0748]

[0749] [Table 4b]

[0750]

[0751] The nucleic acid sequences encoding the variable regions and the CDR sequences are included in the order of AB4, A2F2, A2F3, BA6, CC9, C2E3, DG6, D2B12, A2F2 M1, and BA6 M1 as part of the nucleic acid sequences encoding the following full-length heavy and light chains: SEQ ID NO.75 (heavy chain) and SEQ ID NO.83 (light chain), SEQ ID NO.76 (heavy chain) and SEQ ID NO.84 (light chain), SEQ ID NO.77 (heavy chain) and SEQ ID NO.85 (light chain), SEQ ID NO.78 (heavy chain) and SEQ ID NO.86 (light chain), SEQ ID NO.79 (heavy chain) and SEQ ID NO.87 (light chain), SEQ ID NO.80 (heavy chain) and SEQ ID NO.88 (light chain), SEQ ID NO.81 (heavy chain) and SEQ ID NO.89 (light chain), SEQ ID NO.82 (heavy chain) and SEQ ID NO.90 (light chain), SEQ ID NO:102 (heavy chain) and SEQ ID NO.84 (light chain), SEQ ID NO:103 (heavy chain) and SEQ ID NO.86 (light chain). The nucleic acid sequences encoding the constant regions in the encoding nucleic acid sequences are SEQ ID NO.92 (heavy chain) and SEQ ID NO.94 (light chain) or SEQ ID NO.95 (light chain).

[0752] Example 2: Conversion of anti-ROR1 scFv to the full IgG form and its production

[0753] Example 2-1: Cloning of anti-ROR1 scFV into the full IgG form

[0754] To convert the sequences of each of the ROR1-specific monoclonal phage antibodies obtained in Example 1 into the full IgG form, nucleic acids encoding the heavy and light chain variable regions of each of the individual clone lines obtained in Example 1 were synthesized (Genotech, Korea). After synthesizing the genes encoding the heavy and light chain constant regions of the human IgG1 subtype (SEQ ID NOs. 91 and 93, respectively), the genes were ligated to the nucleic acids encoding the individual heavy and light chain variable regions. The nucleic acids encoding the light and heavy chains of each antibody were separately cloned into pcDNA3.1-based expression vectors to obtain vectors encoding the antibody nucleic acids in the CHO-S mammalian cell line.

[0755] For the reference group, a chimeric antibody in which human IgG1 is bound to the variable region of the conventional anti-ROR1 antibody 2A2 (US9,316,646) was used.

[0756] The antibodies according to the present invention in IgG form were disclosed in the order of AB4, A2F2, A2F3, BA6, CC9, C2E3, DG6, D2B12A2F2 M1, and BA6 M1 with the following heavy and light chain full-length sequences: SEQ ID NO. 59 (heavy chain) and SEQ ID NO. 67 (light chain), SEQ ID NO. 60 (heavy chain) and SEQ ID NO. 68 (light chain), SEQ ID NO. 61 (heavy chain) and SEQ ID NO. 69 (light chain), SEQ ID NO. 62 (heavy chain) and SEQ ID NO. 70 (light chain), SEQ ID NO. 63 (heavy chain) and SEQ ID NO. 71 (light chain), SEQ ID NO. 64 (heavy chain) and SEQ ID NO. 72 (light chain), SEQ ID NO. 65 (heavy chain) and SEQ ID NO. 73 (light chain), SEQ ID NO. 66 (heavy chain) and SEQ ID NO. 74 (light chain), SEQ ID NO: 100 (heavy chain) and SEQ ID NO. 68 (light chain), SEQ ID NO: 101 (heavy chain) and SEQ ID NO. 70 (light chain).

[0757] Example 2-2: Expression of anti-ROR1 IgG antibody

[0758] Adjust CHO-S cells to a concentration of 1.5×106 cells / ml in CD-CHO (Gibco, 10743) medium, and then culture them at 37°C and 8% CO2 for 1 day. On the day of DNA transfection, cells grown to a concentration of 2.5 to 3×106 cells / ml were prepared to a concentration of 2.1×106 cells / ml using CD-CHO medium containing 1% DMSO, and then cultured at 37°C and 8% CO2 for 3 hours. After centrifugation at 3000 rpm for 15 minutes, the supernatant was removed and resuspended in RPMI 1640 medium containing 25% FBS. Thereafter, the vectors expressing the heavy and light chains of Example 2-1 were diluted in Opti-MEM medium at a rate of 1 μg per ml of medium, and PEI (Polysciences, 23966, stock concentration: 1 mg / ml) was diluted at a rate of 8 μg per ml of medium.

[0759] Fix the vector and PEI mixture at room temperature for 10 minutes, and then place it in a flask containing the cells prepared as described above. After culturing at 5% CO2, 37°C and 100 rpm for 4 hours, add CD-CHO in the same volume as the culture, and then culture at 8% CO2, 37°C and 110 rpm for 4 days.

[0760] Example 2-3: Isolation and purification of anti-ROR1 IgG antibody

[0761] Equilibrate a MabSelect column (GE Healthcare, 5 mL) with an equilibration buffer solution (50 mM Tris-HCl, pH 7.5, 100 mM NaCl), and pass the culture broth of Example 3-2 through the column (MabSelect (GE Healthcare, 5 mL) so that the expressed antibody will bind to the column. Thereafter, after elution with 50 mM Na-citrate (pH 3.4) and 100 mM NaCl solution, neutralize with 1 M Tris-HCl (pH 9.0) and obtain a final pH of 7.2. Exchange the buffer solution with PBS (phosphate buffered saline, pH 7.4).

[0762] Example 3: Analysis of the binding specificity of anti-ROR1 IgG antibody to ROR1

[0763] Example 3-1: Analysis of the binding ability of anti-ROR1 IgG antibody to ROR1 antigen (extracellular domain) (ELISA)

[0764] Analyze the specific binding ability of the IgG antibodies of each individual clone line prepared and screened in Example 2 to the antigen as follows.

[0765] The anti-ROR1 antibody-antigen binding affinity was evaluated using an ELISA-based solution binding assay. Specifically, a 96-well microtiter plate (Nunc-Immuno Plate, NUNC) was coated with the ROR1 protein described below at a concentration of 1 μg / ml in PBS solution at 4°C for 16 hours, and non-specific binding sites were blocked with 3% BSA (bovine serum albumin) for 2 hours. Here, in the case of human ROR1, the ROR1 protein used was ROR1-Fc of Example 1 or recombinant human ROR1-His (Beijing Sino Biological Inc., 13968-H08H). As stated in the above statement, the ROR1-His used in ELISA is a protein from Beijing Sino Biological Inc. (13968-H08H), and ROR1-His of Example 1 or recombinant mouse ROR1 protein (Acrobiosystems, RO1-M5221-100 μg) was used.

[0766] Subsequently, the anti-ROR1 antibody prepared in Example 3 was added to the 96-well microtiter plate at the concentration stated in Figure 2 and the binding ability was analyzed using ELISA as follows. Specifically, after incubation for 2 hours, the plate was washed 5 times with PBS containing 0.05% Tween 20, and then the HRP-conjugated Fab polyclonal antibody reagent (Perce, 31414) was diluted to 1:10,000 and placed in the washed microtiter plate. After reacting at 37°C for 1 hour, the ROR1 antibody bound to the plate was detected. After the reaction, color development was performed using TMB (tetramethylbenzidine, Sigma, T0440). The enzyme reaction was stopped using 0.5 mol / L sulfuric acid, and the absorbance (450 nm - 650 nm) was measured using a microplate reader (Molecular Devices) at 450 nm and 650 nm.

[0767] The results are shown in Figure 3a , Figure 3b and Figure 4 and it was confirmed that the anti-ROR1 antibody of the present invention binds to human ROR1 and mouse ROR1 in a concentration-dependent manner. In addition, when comparing the cross-reactivity with mouse ROR1 protein, it was found that the ROR1 antibody according to the present invention has excellent binding ability compared to the 2A2 antibody used as a reference group.

[0768] Example 3-2: Measurement of the specific binding ability (FACS) of anti-ROR1 IgG antibody to ROR1 antigen expressed on the cell surface

[0769] For antibodies, it is crucial for a specific antigen used as a therapeutic antibody in the body (e.g., which binds to an antigen expressed on the cell surface). Some antigens will bind to the purified antigen but will not bind to the antigen expressed on the cell surface. In such cases, even when the antibody is administered in vivo, it cannot bind to the antigen, meaning that the antibody cannot bind to the cells expressing the antigen and therapeutic antibodies etc. cannot exhibit in vivo activity.

[0770] Therefore, FACS analysis was performed to confirm that the anti-ROR1 antibody of the present invention binds to ROR1 expressed on the cell surface.

[0771] For this experiment, cell lines ( Figure 5 and Figure 7 , respectively) were transiently (CHO-human ROR1, CHO-human ROR2, CHO-mouse ROR1) or stably (MC38-human ROR1) transfected with the ROR1 gene to achieve artificial overexpression of ROR1 protein, ROR1-expressing cell lines (JeKo-1, Mino) ( Figure 6 ) non-ROR1-expressing cell line (MCF7) ( Figure 6 ), and the FACSCalibur (BD Biosciences) device was used to measure the degree of binding of the anti-ROR1 antibody and ROR1 as follows. MCF7 is a negative control that does not express ROR1, and CHO-human ROR2 is a negative control that expresses human ROR2. JeKo-1, Mino, CHO-human ROR1, CHO-mouse ROR1, and MC38-human ROR1 are all cell lines that express human ROR1 or mouse ROR1.

[0772] Specifically, after dissociating each cell line and washing in PBS, the number of cells was counted and adjusted to 2×10 5 cells / 200 μl PBS. Then, each individual ROR1 monoclonal antibody prepared in Example 3 was diluted 5 times from 10 μg / mL or 10 μg / mL and then reacted at 4 °C for 1 hour. After the reaction, the cells were washed in PBS, and then an FITC-labeled constant region (Fc)-specific antibody (goat anti-human IgG FITC conjugate, Fc-specific, Sigma, F9512, concentration 20 mg / ml) was suspended at 2 μl / 1×10 5cells / 200 μl PBS and reacted at 4°C for 1 hour. Regarding the identification of the expression levels of transiently overexpressed human ROR1, human ROR2, and mouse ROR1, commercially available FACS analysis antibodies were used (anti-ROR1: Andi Biotech, FAB 2000G, anti-ROR2: R & D Department, FAB20641P). After reacting the cells, the cells were washed in PBS and analyzed using a FACSCalibur device. The negative control (2nd Ab) was treated only with a FITC-labeled constant region (Fc)-specific antibody. For the offset readings of the control group (MFI ratio: MFI of anti-ROR1 / MFI of 2nd Ab), the offset readings of each experimental group treated with the ROR1 monoclonal antibody were compared.

[0773] The results are shown in Figure 5 , Figure 6 and Figure 7 . In the results, it was found that the anti-ROR1 antibody of the present invention specifically and in a concentration-dependent manner binds to human ROR1 ( Figure 6 ), which is initially expressed in cells, and the extracellular domain of human ROR1 ( Figure 5 and Figure 7 ) is artificially overexpressed in cells.

[0774] Furthermore, it was confirmed that it does not bind to the family protein human ROR2, and it has interspecies cross-reactivity with mouse ROR1 ( Figure 5 ). When comparing the cross-reactivity of mouse ROR1 expressed on the cell surface, it was confirmed that the ROR1 antibody of the present invention has an excellent binding degree compared to the 2A2 antibody used as a reference group ( Figure 5 ).

[0775] Example 3-3: Measurement of the binding ability of anti-ROR1 IgG antibody to ROR1 antigen expressed on the cell surface in various cancers (FACS)

[0776] Accordingly, FACS analysis was performed to verify that the anti-ROR1 antibody of the present invention binds to cell surface-expressed ROR1 in various types of cancer cell lines. ROR1 is expressed in a variety of cancer cells, including not only blood cancers such as B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt lymphoma, mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and marginal zone lymphoma (MZL), but also solid cancers including breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer, etc.

[0777] For this experiment, the following various cancer cell lines were used: AGS (ATCC CRL-1739 TM , human gastric adenocarcinoma), NCI-N87 (ATCC CRL-5822 TM , human gastric cancer), MKN-28 (KCLB 80102, human gastric adenocarcinoma), SNU-1750 (KCLB01750, human gastric adenocarcinoma), SNU-16 (ATCC CRL5974 TM , human gastric cancer), HCC1187 (ATCC CRL-2322 TM , human breast cancer TNM stage IIA grade 3), MDA-MB-231ATCC HTB-26 TM , human breast cancer), MDA-MB-468 (ATCCHTB-132 TM , human breast cancer), HCC70 (ATCC CRL2315 TM , human breast cancer TNM stage IIIA, grade 3), HCC1143 (ATCC CRL-2321 TM , TNM stage IIA, grade 3, primary ductal carcinoma), BT20 (ATCC HTB-19 TM human breast cancer), HCC1806 (ATCC CRL-2335 TM , human breast cancer TNM stage IIB grade 2), HCC1937 (ATCCCRL2336 TM , TNM stage IIB, grade 3, primary ductal carcinoma), BT474 (ATCC HTB-20 TM , ductal carcinoma), MCF7 (ATCCHTB-22 TM, metastatic sites of breast cancer), H460 (ATCC HTB-177 TM , large cell lung cancer), A549 (ATCC CCL-185 TM , lung cancer), NCI-H1975 (ATCC CRL-5908 TM , non-small cell lung cancer), H1437 (ATCC CRL5872 TM Stage 1, adenocarcinoma small cell lung cancer), Calu-6 (ATCC HTB-56 TM , anaplastic lung cancer), HCT116 (ATCC CCL-247 TM , colorectal cancer), DLD-1 (ATCC CCL-221 TM , Duke type C, colorectal adenocarcinoma), HT29 (ATCC HTB-38 TM , colorectal adenocarcinoma), 697 (DSMZ ACC 42, acute myeloblastic leukemia), Kasumi-2 (ATCC CRL-2724 TM , acute myeloblastic leukemia), Mino (ATCC CRL3000 TM , mantle cell lymphoma), JeKo-1 (ATCC CRL3006 TM , mantle cell lymphoma), Jurkat (ATCC TIB-152 TM , acute T cell leukemia). FACS analysis (FACSCalibur, BD Biosciences) was used to analyze the binding of the anti-ROR1 antibody of the present invention to ROR1 in the above cell lines.

[0778] Specifically, individual cell lines were isolated and washed in PBS, then the number of cells was counted and adjusted to 2×10 5 cells / 200 μl PBS. Then, it was treated with an antibody of clone name C2E3 of the ROR1 monoclonal antibody prepared in Example 3 at 10 μg / mL, and allowed to react at 4°C for 1 hour. After the reaction, the cells were washed in PBS, and then the FITC-labeled constant region (Fc)-specific antibody (goat anti-human IgG FITC conjugate, Fc-specific, Sigma, F9512, concentration 20 mg / ml) was suspended at 2 μl / 1×10 5Cells were resuspended in 200 μl of PBS at a density of [number of cells] cells / 200 μl PBS and reacted at 4°C for 1 hour. After the reaction, the cells were washed with PBS and analyzed using a FACSCalibur device. The negative control was treated only with an FITC-labeled constant region (Fc)-specific antibody. To compare the degree of ROR1 expression between individual cancer cell lines, the quotient of the shifted readings of the experimental group treated with the ROR1 monoclonal antibody (C2E3) of the present invention divided by the shifted readings of the control group (MFI ratio: MFI of anti-ROR1 / MFI of 2nd Ab) was indicated.

[0779] The results are shown in Figure 8 It was confirmed that the anti-ROR1 antibody of the present invention binds to ROR1 expressed in various cancer cell lines derived from gastric cancer, breast cancer, lung cancer, colon cancer, acute lymphoblastic leukemia (ALL), and mantle cell lymphoma (MCL).

[0780] Example 4: Measurement of the affinity of anti-ROR1 IgG antibody for ROR1

[0781] A 96-well black microculture plate (greiner bio one) was placed on a biosensor tray box, and 200 μl of 10X KB or DW was added to each of 8 wells. Then, an anti-Penta His biosensor or an 8AR2G biosensor (ForteBio, USA) was inserted into the hydrate for 10 minutes. The 600 μl of the analyte sample was diluted to 2-fold or 3-fold of the appropriate analytical concentration from 30 to 0.021 nM using 1×KB or 10×KB. To immobilize the antigen, recombinant human ROR1-His (Sino Biological, 13968-H08H) was diluted to 1 μg / ML using 10×KB or sodium acetate pH 5 buffer. In the loading step, the immersion was fixed below a threshold of 0.3 nm. The binding analysis was performed for 3 to 10 minutes, and the dissociation analysis was performed for 20 minutes. According to the octet program template, the buffers were placed in a new 96-well black microculture plate in sequence. As baseline 1, 200 μl of 10X KB or DW was placed. Then, 200 μl of 1 μl / mL of the antigen to be loaded, each of the ROR1-HIS proteins, was added. As baseline 2, 200 μl of 10×KB or 1×KB was placed. Then, 30 to 0.021 nM of the diluted antibody and 200 μl of 10×KB buffer or 1×KB corresponding to each in the reference sheet were added to each well. The temperature of the test plate was fixed at 30°C. After introducing all the samples, the device was started, and after the analysis was completed, the results were uploaded to the octet analysis 90 software, where the KD value was obtained via 1:1 fitting analysis. The results are shown in Table 6 below. By obtaining the KD value via the octet analysis method, it was confirmed that the anti-ROR1 antibody has a strong binding potential to the ROR1 antigen.

[0782] [Table 6] Measurement of the affinity of anti-ROR1 IgG antibody for ROR1

[0783] Number Clone KD(M) kon(1 / Msec) koff(l / sec) Chi R^2 1 AB4 6.39E-11 1.81E+06 1.16E-04 0.016 0.996 2 A2F2 7.73E-11 1.53E+06 1.19E-04 0.106 0.997 3 A2F3 2.40E-11 1.38E+06 3.31E-05 0.131 0.992 4 BA6 9.37E-11 2.47E+06 2.31E-04 0.138 0.993 5 C2E3 4.24E-10 5.46E+06 2.31E-03 0.176 0.966 6 CC9 7.54E-10 7.31E+05 5.51E-04 0.200 0.992 7 DG6 1.52E-10 2.23E+06 3.38E-04 0.506 0.964 8 D2B12 8.03E-10 4.50E+05 3.61E-04 0.341 0.952

[0784] Example 5: Analysis of the cancer growth inhibitory efficacy of anti-ROR1 IgG antibody in a mouse tumor xenograft model

[0785] By transplanting 1×10 7 cells / head of the human mantle cell lymphoma cell line JeKo-1 expressing ROR1 into severe combined immunodeficient mice (SCID), human cancer xenograft mice were prepared. After xenotransplantation, when the tumor size reached an average of 170 mm 3 (Day 1), the mice were grouped, and five types of anti-ROR-1 antibodies were intraperitoneally injected into the mice at 10 mg / kg twice a week for a total of 5 administrations (Days 1, 4, 7, 10, and 14) using a 1 mL syringe. The negative control group was intraperitoneally administered each of 10 mg / kg of human IgG1 (InVivoPlus human IgG1 isotype control, BioXCell, BP0297) twice a week for a total of 5 administrations, and the human IgG1 has a structure similar to that of the ROR-1 antibody. Immediately before the initial administration (Day 1), immediately before each administration date thereafter, and two days after the last administration (Day 16), the tumor size transplanted into the mice and the body weight of the mice were measured.

[0786] The results are presented in Figure 9a and Figure 9b . The anti-ROR-1 antibody according to the present invention inhibits cancer growth, and when the experiment was ended, compared with the human IgG1 antibody (HuIgG1) negative control, the tumor growth inhibition % (TGI%) of [the antibody according to the present invention] was 360% for C2E3, 289% for A2F2, 361% for AB4, 317% for BA6, and 294% for CC9 on Day 16. It was found that the anti-ROR1 antibody according to the present invention had a statistically significant difference compared with the HuIgG administration group (one-way ANOVA, P value < 0.05)( Figure 9a ). The results of body weight measurement ( Figure 9b)No significant differences were shown between the dosing groups. As stated in Examples 3-1 and 3-2, the anti-ROR1 antibodies of the present invention have cross-reactivity with mouse ROR1 antigen. Thus, the administered anti-ROR1 antibodies of the present invention can bind to human ROR1 expressed by xenografted human mantle cell lymphoma cell line JeKo-1, as well as bind to mouse ROR1 expressed by the mouse itself. Measuring body weight indicated a similar increasing trend of body weight between the negative control group (HuIgG1) of the present invention and the anti-ROR1 antibody, and this can be called indicating that the administration of the anti-ROR1 antibody of the present invention does not induce toxicity. These results suggest that the antibody according to the present invention can be applicable as a cancer therapeutic agent.

[0787] In a mouse tumor xenograft model, it was found that all 5 ROR1 antibodies of the present invention inhibited cancer growth. Among them, as shown in Example 6 and Figure 10 as shown, it was found that some antibodies (C2E3 and AB4) were able to induce autologous activation cell death of ROR1 overexpressing cancer cell lines when polymerized by anti-human Fc antibody. However, various cancer inhibitory mechanisms are possible, such as inducing autologous activation cell death, inhibiting cancer cell division and growth, inhibiting tumor angiogenesis and activating immune cells. Thus, the following Figure 10 results indicate that each of the anti-ROR1 antibodies according to the present invention can inhibit cancer growth in vivo via different mechanisms of action.

[0788] Example 6: Analysis of the ability of anti-ROR1 IgG antibodies to induce autologous activation cell death

[0789] To analyze the possible mechanism of the antibody according to the present invention that exhibits tumor inhibitory ability as shown in Example 5, its ability to induce cell death was analyzed.

[0790] For this purpose, the ROR1 overexpressing cell line JeKo-1 was centrifuged to remove the medium containing serum. After washing once with PBS, 5×10 6 cells were seeded in each well of a 6-well plate using serum-free RPMI1640 medium. 100 μg / mL of the anti-ROR1 antibody according to the present invention and 300 μg / ml of anti-human Fc antibody (Thermo Fisher, 31125) were placed in an equivalent test tube at a 1:1 ratio and reacted at room temperature for 10 minutes so that the anti-ROR1 antibody was crosslinked by the anti-human Fc antibody. 150 μl of each mixture was placed in a well containing 15 ml of medium so that the final amount of the treated antibody was 10 μl / mL for the ROR1 antibody and 30 μl / mL for the anti-human Fc antibody, and then reacted by culturing at 5% CO 2 and 37 °C for 24 hours.

[0791] Next, to confirm whether the individual ROR1 antibody and the cross-linked anti-ROR1 antibody have the ability to induce autologous activated cell death, the cells in each well were collected and washed once with PBS. Thereafter, each group was reacted with the autologous activated cell death markers Annexin V and the cell death marker PI, and the degree of staining of the group was observed by FACS analysis.

[0792] The results are shown in Figure 10 . In the results of the experiment, for the group treated with the individual anti-ROR1 antibody, autologous activated cell death was not observed, but in the case of cross-linking of some anti-ROR1 antibody clone lines (C2E3 and AB4) by anti-human Fc, the degree of staining by Annexin V and PI increased over the control group. Specifically, with respect to staining by the autologous activated cell death marker Annexin V, the C2E3 clone line increased by 23% and the AB4 clone line increased by 10% over the control. To see that such an ability to induce autologous activated cell death is a response specific to ROR1, the same method was used with the C2E3 clone line on the cell line U266 that does not express ROR1 to examine the degree of staining by Annexin V and PI. It has been confirmed that the cell line U266 that does not express ROR1 is not able to induce autologous activated cell death, demonstrating that the ability of a cross-linked anti-ROR1 antibody to induce autologous activated cell death is a ROR1-specific response. Antibodies are able to form multimers in vivo by binding to Fcλ receptors via their Fc region, and thus the formation of anti-ROR1 antibody multimers using anti-human Fc antibodies can be said to represent a condition similar to the in vivo phenomenon. The above analysis results relate to a single mechanism, which indicates that the anti-ROR1 antibody according to the present invention can induce autologous activated cell death in ROR1-overexpressing cancer cell lines.

[0793] It should be noted that the induction of autologous activated cell death of cancer cell lines by the multimerization of ROR1 antibodies is not a phenomenon observed in all types of ROR1 antibodies. For example, in the case of the BA6 clone line between the ROR1 antibody of the present invention and the 2A2 antibody used as a reference group, these cells do not induce autologous activated cell death of ROR1-overexpressing cancer cell lines even when multimerized by anti-human Fc antibodies. This indicates that the ROR1 antibody according to the present invention has the ability to inhibit cancer cells through different mechanisms of action. This difference may be attributed to differences in the antigenic determinants bound by the individual ROR1 antibodies, but this theory is not the only explanation.

[0794] Example 7: Preparation of Compounds 1, 2, 3 and 4

[0795]

[0796]

[0797] The above compounds 1, 2, 3, and 4 were prepared using the method described in Patent WO 2017-089895. In the above compounds 1, 2, 3, and 4, the structures of MMAE or MMAF are as follows:

[0798]

[0799] Example 8: Preparation of Compounds 5, 6, and 7

[0800]

[0801]

[0802] The above compounds 5, 6, and 7 were prepared using the method described in Korean Patent Application No. 10-2018-0036895.

[0803] Example 9: Preparation of ADC

[0804] The ADC was prepared through the following two steps, and the commonly used LCB14-0511 and LCB14-0606 were prepared using the method described in Korean Published Patent No. 10-2014-0035393.

[0805] The structural formulas of LCB14-0511 and LCB14-0606 are as follows:

[0806]

[0807] Step 1: Preparation of Isoprenylated Antibody

[0808] The prenylation reaction mixture of the ROR1 monoclonal antibody (C2E3) of the present invention was prepared and allowed to react at 30 °C for 16 hours. The reaction mixture consisted of 24 μM antibody, 200 nM FTase (Calbiochem #344145), and a buffer solution (50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 10 μM ZnCl2, 0.144 mM DTT) containing 0.144 mM LCB14-0511 or LCB14-0606. After the reaction was completed, the isoprenylated antibody was desalted using a G25 Sepharose gel column (AKTA purifier, GE Healthcare) equilibrated with PBS buffer solution.

[0809] As a reference group antibody, a chimeric antibody (2A2) in which human IgG1 was bound to the variable region of the existing anti-ROR1 antibody 2A2 (US 9,316,646) was used.

[0810] Step 2: Method of Drug Conjugation

[0811] <Binding formed through an oxime bond>

[0812] A mixture for the formation reaction of an oxime bond between a prenylated antibody and a linker-drug was prepared by mixing 100 mM sodium acetate buffer solution at pH 5.2, 10 wt% DMSO, 20 μM antibody, and 200 μM linker-drug (compounds 1, 2, 3, 4, 5, and 7 from Examples 7 and 8 in-house), and gently stirring at 30 °C. After reacting for 6 or 24 hours, an FPLC (AKTA purifier, GE Healthcare) process was carried out to remove the excess smaller compounds used. The protein fraction was collected and concentrated.

[0813] <Binding through a click reaction>

[0814] A mixture for the formation reaction of an oxime bond between a prenylated antibody and a linker-drug was prepared by mixing 10% DMSO, 20 μM antibody, and 200 μM linker-drug (compound 6 from Example 8 in-house), 1 mM copper(II) sulfate pentahydrate, 2 mM (BimC4A)3 (Sigma-Aldrich 696854), 10 mM sodium ascorbate, and 10 mM guanidine hydrochloride, reacting at 25 °C for 3 hours, then treating with 20 mM EDTA and reacting for 30 minutes. After the reaction, an FPLC (AKTA purifier, GE Healthcare) process was carried out to remove the excess smaller compounds used. The protein fraction was collected and concentrated.

[0815] [Table 7]

[0816] List of the prepared ADCs

[0817]

[0818] Example 10: Evaluation of ADC properties

[0819] Using the ADCs prepared in Example 9, the properties of the ADCs according to the present invention were analyzed.

[0820] For this purpose, hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) analysis was performed. The ADC was subjected to HIC-HPLC using a phenyl-5PW column (7.5×75 mm, 10 μm, Tosoh Bioscience, USA). A 50 mM potassium phosphate buffer solution (pH 7.0) containing 1.5 M ammonium sulfate was used as buffer solution A, and a 50 mM potassium phosphate buffer solution (pH 7.0) containing 30% acetonitrile was used as buffer solution B, and 70% A and 30% B were stabilized as the initial conditions. Elution was performed using a linear gradient of 70% A / 30% B compared to 10% A / 90% B for the next 25 minutes, with an additional 5-minute elution at 10% A / 90% B. The flow rate and temperature were set at 1.0 ml / min and 25 °C, respectively. Detection was then carried out at 254 and 280 nm. The ROR1 antibody was used, and the prenylated ROR1 antibody was used as the reference group.

[0821] In addition, size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis was performed. SEC-HPLC was carried out on the ADC using a SWXL guard column (6.0×40 mm, Tosoh Bioscience, USA) and a G3000SWxl column (7.8×300 mm, 5 μm, Tosoh Bioscience, USA). Analysis was performed for 30 minutes at a flow rate of 0.5 ml / min and 25 °C using a 200 mM potassium phosphate buffer solution (pH 7.0) containing 250 mM potassium chloride phosphate and 15% isopropanol as the mobile phase. Detection was then carried out at 254 and 280 nm. The ROR1 antibody was used, and the prenylated ROR1 antibody was used as the reference group.

[0822] Results of analyzing the characteristics of ADC2 and ADC5 among the ADCs prepared in Example 9 are shown in Figure 11a and Figure 11b .

[0823] Example 11: In vitro cytotoxicity assessment

[0824] The cancer cell line cell proliferation inhibitory activity of the ADC prepared in Example 9 was measured.

[0825] For this purpose, commercially available cancer cell lines (Mino, Jeko-1, REC-1, H2228, NCIN87, HCC1806, MDA-MB-231, MCF-7, and Daudi cell lines) were used. In a 96-well plate, each well was seeded with 4,000 to 5,000 individual cancer cell lines. After culturing for 24 hours, they were treated with the ADCs in Table 8 at a concentration of 0.0015 to 10.0 nM (serial diluted three times). After 72 hours, the number of live cells was measured using the WST-8 (Dojindo Molecular Technologies) dye.

[0826] The results are presented in Table 8 below. In cancer cell lines overexpressing ROR1, it was confirmed that the anti-ROR1 monoclonal antibody of the present invention has superior cytotoxicity compared to ADCs (ADC 8, ADC 9, and ADC 10) conjugated with conventional anti-ROR1 antibodies. In addition, it was demonstrated that the pyrrolobenzodiazepine-based ADC exhibits strong cytotoxicity compared to auristatin-based ADCs.

[0827] [Table 8]

[0828] Cytotoxicity comparison of ADC samples

[0829]

[0830]

[0831] *n.d: Cytotoxicity was observed with increasing concentration, but the cells did not completely die at the maximum concentration, and an IC50 value may not be found.

[0832] *-: Weak or no cytotoxicity was exhibited at the maximum concentration.

[0833] *N / A: No data

[0834] Example 12: Analysis of in vivo cancer growth inhibitory efficacy

[0835] Example 12-1: Analysis of the cancer growth inhibitory efficacy of ADCs in a mouse model transplanted with a breast cancer cell line expressing ROR1

[0836] 1×10 7 cells / head of the human ROR-1-expressing breast cancer cell line MDA-MB-468 were transplanted into female Balb / C nude mice to prepare human cancer-transplanted mice. After transplantation, when the tumor size reached an average of 166 mm 3 (day 1), the mice were grouped, and 1 mg / kg of ADC5 prepared in Example 9 was intravenously injected into the mice. In the control group, 10 ml / kg of PBS was intravenously injected into the mice.

[0837] Immediately before the first administration (day 1), and at regular intervals thereafter for 56 days, the tumor size in the transplanted mice and the body weight of the mice were measured.

[0838] And 1×10 7 cells / head of the human ROR-1 breast cancer cell line MDA-MB-231 were transplanted into severe combined immunodeficiency (SCID) mice to prepare mice with human cancer transplantation. After transplantation, when the average tumor size reached 110 mm 3 (day 1), the mice were grouped, and 0.5, 1.0, or 2.0 mg / kg of ADC5 prepared in Example 9 was intravenously injected into the mice once, or 0.33 mg / kg of ADC5 was intravenously injected twice a week for a total of three times (day 1, day 7, and day 14). In the control group, 10 ml / kg of PBS was intravenously injected into the mice. Immediately before the first administration (day 1), and at regular intervals thereafter for 22 days, the tumor size in the transplanted mice and the body weight of the mice were measured.

[0839] And 1×10 7 cells / head of the human ROR-1 breast cancer cell line HCC1187 were transplanted into severe combined immunodeficiency (SCID) mice to prepare mice with human cancer transplantation. After transplantation, when the average tumor size reached 110 mm 3 (day 1), the mice were grouped, and 1.25 or 0.31 mg / kg of ADC5 prepared in Example 9 was intravenously injected into the mice once, or 3.75 mg / kg of ADC2 was intravenously injected once. In the control group, 10 ml / kg of PBS was intravenously injected into the mice. Immediately before the first administration (day 1), and at regular intervals thereafter for 41 days, the tumor size in the transplanted mice and the body weight of the mice were measured.

[0840] The results are stated in Figure 12 , Figure 13 and Figure 14 . It was confirmed that the ADC of the present invention inhibits the growth of cancer in a transplanted mouse model of a breast cancer cell line expressing ROR1.

[0841] Example 12-2: Analysis of the cancer growth inhibitory effect of ADC in a mouse model transplanted with a breast cancer cell line expressing ROR1

[0842] 1×10 7 cells / head of the human ROR-1 lung cancer cell line Calu-3 were transplanted into female Balb / C nude mice to prepare mice with human cancer transplantation. After transplantation, when the average tumor size reached 184 mm 3At time (day 1), the mice were grouped, and 3 mg / kg of ADC2 prepared in Example 9 was intravenously injected into the mice once, or 0.25 or 1.0 mg / kg of ADC5 prepared in Example 9 was intravenously injected once. In the control group, 10 ml / kg of PBS was intravenously injected into the mice. Immediately before the first administration (day 1), and at regular intervals thereafter for 35 days, the tumor size in the transplanted mice and the body weight of the mice were measured.

[0843] The results are presented in Figure 15 It was confirmed that the ADC of the present invention inhibits cancer growth in a transplanted mouse model of a lung cancer cell line expressing ROR1.

[0844] Example 12-3: Analysis of the cancer growth inhibitory efficacy of ADCs in a mouse model transplanted with a mantle cell lymphoma cell line expressing ROR1

[0845] 1×10 7 cells / head of the human ROR-1 mantle cell lymphoma cell line Jeko-1 were transplanted into severe combined immunodeficiency (SCID) mice to prepare mice transplanted with human cancer. After transplantation, when the tumor size reached an average of 110 mm 3 At time (day 1), the mice were grouped, and 3 mg / kg of ADC2 prepared in Example 9 was intravenously injected into the mice once, or 0.25 or 1.0 mg / kg of ADC5 prepared in Example 9 was intravenously injected once. In the control group, 10 ml / kg of PBS was intravenously injected into the mice. Immediately before the first administration (day 1), and at regular intervals thereafter for 33 days, the tumor size in the transplanted mice and the body weight of the mice were measured.

[0846] The results are presented in Figure 16 It was confirmed that the ADC of the present invention inhibits cancer growth in a transplanted mouse model of a mantle cell lymphoma cell line expressing ROR1.

[0847] Example 12-4: Analysis of comparing the cancer growth inhibitory efficacy of ADCs in a mouse model transplanted with a mantle cell lymphoma cell line expressing ROR1

[0848] The cancer growth inhibitory efficacy depending on the antibody and drug containing the ADC was compared.

[0849] For this purpose, 1×10 7 cells / head of the human ROR-1 mantle cell lymphoma cell line Calu-3 were transplanted into severe combined immunodeficiency (SCID) mice to prepare mice transplanted with human cancer. After transplantation, when the tumor size reached an average of 110 mm 3At time (day 1), the mice were grouped, and 1 mg / kg or 4 mg / kg of ADC2 (C2MMAE) or ADC9 (2A2 MMAE) prepared in Example 9 was intravenously injected into the mice once, or 0.25 or 1.0 mg / kg of ADC5 (dPBD-ADC) or ADC10 (2A2 dPBD) prepared in Example 9 was intravenously injected once. In the control group, 10 ml / kg of PBS was intravenously injected into the mice. Immediately before the first administration (day 1), and at regular intervals thereafter for 37 days, the tumor size transplanted into the mice and the body weight of the mice were measured.

[0850] The results are stated in Figure 17 . The ADCs conjugated with the anti-ROR1 monoclonal antibody of the present invention were confirmed to have superior cancer growth inhibitory efficacy compared to the ADCs conjugated with conventional anti-ROR1 antibodies (ADC 9 and ADC 10). In addition, it was confirmed that the pyrrolobenzodiazepine-based ADCs exhibited stronger cancer growth inhibition compared to auristatin-based ADCs. <110> ABL Biopharma Co., Ltd. Lego Chemical Bioscience Co., Ltd. <120> Antibody-drug conjugates comprising anti-human ROR1 antibodies and their uses <130> LCH-01225 <150> KR 10-2019-0109807 <151> 2019-09-04 <160> 103 <170> KopatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic: Heavy chain CDR1 <400> 1 Ser Tyr Asp Met Ser 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic: Heavy chain CDR1 <400> 2 Asp Tyr Tyr Met Ser 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthesis: heavy chain CDR1 <400> 3 Asn Tyr Asp Met Ser 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthesis: heavy chain CDR1 <400> 4 Asn Tyr Ala Met Ser 1 5 <210> 5 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthesis: heavy chain CDR1 <400> 5 Asp Tyr Asp Met Ser 1 5 <210> 6 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthesis: heavy chain CDR2 <400> 6 Trp Ile Ser Pro Asp Ser Gly Ser Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 7 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain CDR2 <400> 7 Ser Ile Ser Pro Asp Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 8 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain CDR2 <400> 8 Trp Ile Ser Pro Gly Gly Gly Ser Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 9 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain CDR2 <400> 9 Ala Ile Tyr His Ser Gly Ser Ser Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 10 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain CDR2 <400> 10 Gly Ile Ser His Gly Ser Gly Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 11 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain CDR2 <400> 11 Ser Ile Ser His Asn Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 12 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain CDR2 <400> 12 Val Ile Ser Pro Asp Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 13 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain CDR2 <400> 13 Ser Ile Ser Pro Ser Ser Gly Ser Ser Ile Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly <210> 14 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain CDR3 <400> 14 Pro Thr Gly Arg Phe Asp Tyr 1 5 <210> 15 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthesis: heavy chain CDR3 <400> 15 Asn Leu Arg Ala Phe Asp Tyr 1 5 <210> 16 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthesis: heavy chain CDR3 <400> 16 Val Asn Gly Arg Phe Asp Tyr 1 5 <210> 17 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthesis: heavy chain CDR3 <400> 17 Gly Gly Asn Gly Ala Trp Asp Thr Gly Phe Asp Tyr 1 5 10 <210> 18 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthesis: heavy chain CDR3 <400> 18 Arg Leu Ser Leu Arg Arg Arg Pro Ser Tyr Tyr Ser Asp Asn Ala Met 1 5 10 15 Asp Val <210> 19 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain CDR3 <400> 19 Phe Ile Ser Ala Arg Lys Ser Leu Gly Arg Ser Tyr Ser Asn Gly Met 1 5 10 15 Asp Val <210> 20 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain CDR3 <400> 20 Asp Val Val Glu Cys Asn Met Asn Pro Cys Ser Tyr Asp Asn Ala Met 1 5 10 15 Asp Val <210> 21 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain CDR3 <400> 21 Ala Pro Gly Trp Cys Gln Ala Pro Ser Cys Tyr Tyr Asp Asn Ala Met 1 5 10 15 Asp Val <210> 22 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain CDR1 <400> 22 Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Asn Val Asn 1 5 10 <210> 23 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain CDR1 <400> 23 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Tyr 1 5 10 <210> 24 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain CDR1 <400> 24 Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Asn Val Ser 1 5 10 <210> 25 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain CDR1 <400> 25 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Asp Val Ser 1 5 10 <210> 26 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain CDR1 <400> 26 Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Asn 1 5 10 <210> 27 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain CDR1 <400> 27 Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn Asp Val Thr 1 5 10 <210> 28 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain CDR1 <400> 28 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Ser 1 5 10 <210> 29 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain CDR1 <400> 29 Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Asp Val Ser 1 5 10 <210> 30 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain CDR2 <400> 30 Tyr Asp Asn Lys Arg Pro Ser 1 5 <210> 31 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain CDR2 <400> 31 Ala Asn Ser Gln Arg Pro Ser 1 5 <210> 32 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Light Chain CDR2 <400> 32 Ala Asp Ser His Arg Pro Ser 1 5 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Light Chain CDR2 <400> 33 Tyr Asp Asn Asn Arg Pro Ser 1 5 <210> 34 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Light Chain CDR2 <400> 34 Tyr Asp Ser Asn Arg Pro Ser 1 5 <210> 35 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Light Chain CDR2 <400> 35 Ala Asp Ser Lys Arg Pro Ser 1 5 <210> 36 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: Light chain CDR2 <400> 36 Asp Asp Ser His Arg Pro Ser 1 5 <210> 37 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain CDR2 <400> 37 Asp Asp Ser Gln Arg Pro Ser 1 5 <210> 38 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain CDR3 <400> 38 Gly Thr Trp Asp Ala Ser Leu Ser Gly Tyr Val 1 5 10 <210> 39 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain CDR3 <400> 39 Gly Ser Trp Asp Tyr Ser Leu Ser Gly Tyr Val 1 5 10 <210> 40 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain CDR3 <400> 40 Ala Thr Trp Asp Tyr Ser Leu Ser Gly Tyr Val 1 5 10 <210> 41 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain CDR3 <400> 41 Gly Ala Trp Asp Asp Ser Leu Ser Gly Tyr Val 1 5 10 <210> 42 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain CDR3 <400> 42 Gly Thr Trp Asp Tyr Ser Leu Ser Gly Tyr Val 1 5 10 <210> 43 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain variable region <400> 43 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Ser Pro Asp Ser Gly Ser Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Pro Thr Gly Arg Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 44 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Synthetic: heavy chain variable region <400> 44 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Pro Asp Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Leu Arg Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 45 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Heavy Chain Variable Region <400> 45 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Ser Pro Gly Gly Gly Ser Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Asn Gly Arg Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 46 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: Heavy Chain Variable Region <400> 46 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Tyr His Ser Gly Ser Ser Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Asn Gly Ala Trp Asp Thr Gly Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 47 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: Heavy Chain Variable Region <400> 47 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser His Gly Ser Gly Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Leu Ser Leu Arg Arg Arg Pro Ser Tyr Tyr Ser Asp Asn 100 105 110 Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 48 <211> 127 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain variable region <400> 48 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser His Asn Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Phe Ile Ser Ala Arg Lys Ser Leu Gly Arg Ser Tyr Ser Asn 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 49 <211> 127 <212> PRT <213> Artificial sequence <220> <223> Synthesis: heavy chain variable region <400> 49 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Pro Asp Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Val Val Glu Cys Asn Met Asn Pro Cys Ser Tyr Asp Asn 100 105 110 Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 50 <211> 128 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain variable region <400> 50 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Pro Ser Ser Gly Ser Ser Ile Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Lys Ala Pro Gly Trp Cys Gln Ala Pro Ser Cys Tyr Tyr Asp 100 105 110 Asn Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 51 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Light chain variable region <400> 51 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Asn Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asp Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ala Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 52 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Light Chain Variable Region <400> 52 Gln Ser Val Leu Thr Gln Pro Pro Pro Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asn Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Tyr Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 53 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Light Chain Variable Region <400> 53 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Asn Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asp Ser His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Tyr Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 54 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Light Chain Variable Region <400> 54 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asp Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 55 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: Light Chain Variable Region <400> 55 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asp Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 56 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: Light Chain Variable Region <400> 56 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Asp Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asp Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Tyr Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 57 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Light Chain Variable Region <400> 57 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asp Ser His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 58 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Light Chain Variable Region <400> 58 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asp Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 59 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Heavy Chain <400> 59 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Ser Pro Asp Ser Gly Ser Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Pro Thr Gly Arg Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 60 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Heavy Chain <400> 60 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Pro Asp Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Leu Arg Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 61 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Heavy Chain <400> 61 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Ser Pro Gly Gly Gly Ser Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Asn Gly Arg Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 62 <211> 451 <212> PRT <213> Artificial sequence <220> <223> Synthetic: heavy chain <400> 62 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Tyr His Ser Gly Ser Ser Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Asn Gly Ala Trp Asp Thr Gly Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 63 <211> 457 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Heavy Chain <400> 63 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser His Gly Ser Gly Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Leu Ser Leu Arg Arg Arg Pro Ser Tyr Tyr Ser Asp Asn 100 105 110 Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 130 135 140 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 195 200 205 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 210 215 220 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 225 230 235 240 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 245 250 255 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 260 265 270 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 275 280 285 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 290 295 300 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 305 310 315 320 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 325 330 335 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 340 345 350 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 355 360 365 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 370 375 380 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 385 390 395 400 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 405 410 415 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 420 425 430 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 435 440 445 Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 64 <211> 457 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Heavy Chain <400> 64 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser His Asn Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Phe Ile Ser Ala Arg Lys Ser Leu Gly Arg Ser Tyr Ser Asn 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 130 135 140 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 195 200 205 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 210 215 220 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 225 230 235 240 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 245 250 255 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 260 265 270 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 275 280 285 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 290 295 300 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 305 310 315 320 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 325 330 335 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 340 345 350 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 355 360 365 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 370 375 380 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 385 390 395 400 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 405 410 415 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 420 425 430 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 435 440 445 Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 65 <211> 457 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Heavy Chain <400> 65 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Pro Asp Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Val Val Glu Cys Asn Met Asn Pro Cys Ser Tyr Asp Asn 100 105 110 Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 130 135 140 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 195 200 205 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 210 215 220 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 225 230 235 240 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 245 250 255 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 260 265 270 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 275 280 285 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 290 295 300 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 305 310 315 320 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 325 330 335 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 340 345 350 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 355 360 365 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 370 375 380 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 385 390 395 400 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 405 410 415 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 420 425 430 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 435 440 445 Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 66 <211> 458 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Heavy Chain <400> 66 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Pro Ser Ser Gly Ser Ser Ile Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Lys Ala Pro Gly Trp Cys Gln Ala Pro Ser Cys Tyr Tyr Asp 100 105 110 Asn Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 130 135 140 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 145 150 155 160 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 165 170 175 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 180 185 190 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 195 200 205 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 210 215 220 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 225 230 235 240 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 245 250 255 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 260 265 270 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 275 280 285 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 290 295 300 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 305 310 315 320 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 325 330 335 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 340 345 350 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 355 360 365 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 370 375 380 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 385 390 395 400 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 405 410 415 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 420 425 430 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 435 440 445 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 67 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Light Chain <400> 67 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Asn Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asp Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ala Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Ala Glu Cys Ser 210 215 <210> 68 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Light Chain <400> 68 Gln Ser Val Leu Thr Gln Pro Pro Pro Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asn Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Tyr Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Ala Glu Cys Ser 210 215 <210> 69 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: Light Chain <400> 69 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Asn Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asp Ser His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Tyr Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Ala Glu Cys Ser 210 215 <210> 70 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: Light Chain <400> 70 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asp Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Ala Glu Cys Ser 210 215 <210> 71 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: Light chain <400> 71 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asp Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Ala Glu Cys Ser 210 215 <210> 72 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Light Chain <400> 72 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Asp Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asp Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Tyr Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Ala Glu Cys Ser 210 215 <210> 73 <211> 216 <212> PRT <213> Artificial sequence <220> <223> Synthesis: light chain <400> 73 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asp Ser His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Ala Glu Cys Ser 210 215 <210> 74 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Light Chain <400> 74 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asp Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Ala Glu Cys Ser 210 215 <210> 75 <211> 1338 <212> DNA <213> Artificial sequence <220> <223> Synthesis: Heavy chain coding gene <400> 75 gaagtacaac ttctggagtc aggtggagga cttgttcagc ccggcgggtc cctgaggctg 60 agttgcgcag caagcgggtt cacattctcc tcttatgata tgtcttgggt aagacaggct 120 cctggtaagg gtctggaatg ggtatcctgg ataagtcctg actccggttc aatatactac 180 gccgatagtg tgaagggacg tttcaccatc agccgggaca acagcaaaaa taccttgtat 240 ctccaaatga atagcctccg ggctgaagac actgccgtat attactgcgc cagacctact 300 ggtcgttttg actattgggg gcaaggaaca ctggtaaccg tttcaagcgc ctccaccaag 360 ggcccctccg tgttccccct ggccccctcc tccaagtcca cctccggcgg caccgccgcc 420 ctgggctgcc tggtgaagga ctacttcccc gagcccgtga ccgtgtcctg gaactccggc 480 gccctgacct ccggcgtgca caccttcccc gccgtgctgc agtcctccgg cctgtactcc 540 ctgtcctccg tcgtgaccgt gccctcctcc tccctgggca cccagaccta catctgcaac 600 gtgaaccaca agccctccaa caccaaggtg gacaagaagg tggagcccaa gtcctgcgac 660 aagacccaca cctgccctcc ctgccccgcc cccgagctgc tgggcggccc ctccgtgttc 720 ctgttccctc ctaagcccaa ggacaccctg atgatctccc ggacccccga ggtgacttgc 780 gtggtggtgg acgtgtccca cgaggacccc gaggtgaagt tcaactggta cgtggacggc 840 gtggaggtgc acaacgccaa gaccaagccc cgggaggagc agtacaactc cacctaccgg 900 gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaagga gtacaagtgc 960 aaggtgtcca acaaggccct gcccgccccc atcgagaaga ccatctccaa ggccaagggc 1020 cagccccggg agccccaggt gtacaccctg cccccctccc gggaggagat gaccaagaac 1080 caggtgtccc tgacctgcct ggtgaagggc ttctacccct ccgacatcgc cgtggagtgg 1140 gagtccaacg gccagcccga gaacaactac aagaccaccc cccccgtgct ggactccgac 1200 ggctccttct tcctgtactc caagctgacc gtggacaagt cccggtggca gcagggcaac 1260 gtgttctcct gctccgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1320 tccctgtccc ccggcaag 1338 <210> 76 <211> 1338 <212> DNA <213> Artificial Sequence <220> <223> Synthesis: Heavy Chain Encoding Gene <400> 76 gaagtgcagc tgctggaatc cggcggaggc ctggtgcagc ctggcggctc tctgagactg 60 tcttgcgccg cctccggctt caccttctcc gactactaca tgtcctgggt gcgacaggcc 120 cctggcaagg gcctggaatg ggtgtcctcc atctcccccg acggctccaa cacctactac 180 gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240 ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc caagaacctg 300 cgggccttcg actactgggg ccagggcaca ctggtgaccg tgtcctccgc ctccaccaag 360 ggcccctccg tgttccccct ggccccctcc tccaagtcca cctccggcgg caccgccgcc 420 ctgggctgcc tggtgaagga ctacttcccc gagcccgtga ccgtgtcctg gaactccggc 480 gccctgacct ccggcgtgca caccttcccc gccgtgctgc agtcctccgg cctgtactcc 540 ctgtcctccg tcgtgaccgt gccctcctcc tccctgggca cccagaccta catctgcaac 600 gtgaaccaca agccctccaa caccaaggtg gacaagaagg tggagcccaa gtcctgcgac 660 gtgaaccaca agccctccaa caccaaggtg gacaagaagg tggagcccaa gtcctgcgac 660 aagacccaca cctgccctcc ctgccccgcc cccgagctgc tgggcggccc ctccgtgttc 720 aagacccaca cctgccctcc ctgccccgcc cccgagctgc tgggcggccc ctccgtgttc 720 ctgttccctc ctaagcccaa ggacaccctg atgatctccc ggacccccga ggtgacttgc 780 ctgttccctc ctaagcccaa ggacaccctg atgatctccc ggacccccga ggtgacttgc 780 gtggtggtgg acgtgtccca cgaggacccc gaggtgaagt tcaactggta cgtggacggc 840 gtggtggtgg acgtgtccca cgaggacccc gaggtgaagt tcaactggta cgtggacggc 840 gtggaggtgc acaacgccaa gaccaagccc cgggaggagc agtacaactc cacctaccgg 900 gtggaggtgc acaacgccaa gaccaagccc cgggaggagc agtacaactc cacctaccgg 900 gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaagga gtacaagtgc 960 gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaagga gtacaagtgc 960 aaggtgtcca acaaggccct gcccgccccc atcgagaaga ccatctccaa ggccaagggc 1020 aaggtgtcca acaaggccct gcccgccccc atcgagaaga ccatctccaa ggccaagggc 1020 cagccccggg agccccaggt gtacaccctg cccccctccc gggaggagat gaccaagaac 1080 cagccccggg agccccaggt gtacaccctg cccccctccc gggaggagat gaccaagaac 1080 caggtgtccc tgacctgcct ggtgaagggc ttctacccct ccgacatcgc cgtggagtgg 1140 caggtgtccc tgacctgcct ggtgaagggc ttctacccct ccgacatcgc cgtggagtgg 1140 gagtccaacg gccagcccga gaacaactac aagaccaccc cccccgtgct ggactccgac 1200 gagtccaacg gccagcccga gaacaactac aagaccaccc cccccgtgct ggactccgac 1200 ggctccttct tcctgtactc caagctgacc gtggacaagt cccggtggca gcagggcaac 1260 ggctccttct tcctgtactc caagctgacc gtggacaagt cccggtggca gcagggcaac 1260 gtgttctcct gctccgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1320 gtgttctcct gctccgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1320 tccctgtccc ccggcaag 1338 <210> 77 <211> 1341 <212> DNA <213> Artificial Sequence <220> <223> Synthesis: Heavy Chain Coding Gene <400> 77 gaagtgcaac ttcttgagag tggtggagga ttggtacaac ctgggggtag tttgcgtctc 60 tcctgtgctg cttctggttt cacattttcc tcctatgaca tgagctgggt acggcaagct 120 ccaggaaaag ggcttgagtg ggtctcctgg atctctcccg gtggaggcag caagtattat 180 gcagactctg taaagggtag gtttactata tcacgcgata atagtaagaa tactttgtat 240 ttgcaaatga actccctccg agctgaggac acagcagtct attattgcgc ccgagttaac 300 ggtcgcttcg attactgggg ccaaggcaca ctggttacag tgtcctcagc ctccaccaag 360 ggcccctccg tgttccccct ggccccctcc tccaagtcca cctccggcgg caccgccgcc 420 ctgggctgcc tggtgaagga ctacttcccc gagcccgtga ccgtgtcctg gaactccggc 480 gccctgacct ccggcgtgca caccttcccc gccgtgctgc agtcctccgg cctgtactcc 540 ctgtcctccg tcgtgaccgt gccctcctcc tccctgggca cccagaccta catctgcaac 600 gtgaaccaca agccctccaa caccaaggtg gacaagaagg tggagcccaa gtcctgcgac 660 gtgaaccaca agccctccaa caccaaggtg gacaagaagg tggagcccaa gtcctgcgac 660 aagacccaca cctgccctcc ctgccccgcc cccgagctgc tgggcggccc ctccgtgttc 720 aagacccaca cctgccctcc ctgccccgcc cccgagctgc tgggcggccc ctccgtgttc 720 ctgttccctc ctaagcccaa ggacaccctg atgatctccc ggacccccga ggtgacttgc 780 ctgttccctc ctaagcccaa ggacaccctg atgatctccc ggacccccga ggtgacttgc 780 gtggtggtgg acgtgtccca cgaggacccc gaggtgaagt tcaactggta cgtggacggc 840 gtggtggtgg acgtgtccca cgaggacccc gaggtgaagt tcaactggta cgtggacggc 840 gtggaggtgc acaacgccaa gaccaagccc cgggaggagc agtacaactc cacctaccgg 900 gtggaggtgc acaacgccaa gaccaagccc cgggaggagc agtacaactc cacctaccgg 900 gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaagga gtacaagtgc 960 gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaagga gtacaagtgc 960 aaggtgtcca acaaggccct gcccgccccc atcgagaaga ccatctccaa ggccaagggc 1020 aaggtgtcca acaaggccct gcccgccccc atcgagaaga ccatctccaa ggccaagggc 1020 cagccccggg agccccaggt gtacaccctg cccccctccc gggaggagat gaccaagaac 1080 cagccccggg agccccaggt gtacaccctg cccccctccc gggaggagat gaccaagaac 1080 caggtgtccc tgacctgcct ggtgaagggc ttctacccct ccgacatcgc cgtggagtgg 1140 caggtgtccc tgacctgcct ggtgaagggc ttctacccct ccgacatcgc cgtggagtgg 1140 gagtccaacg gccagcccga gaacaactac aagaccaccc cccccgtgct ggactccgac 1200 gagtccaacg gccagcccga gaacaactac aagaccaccc cccccgtgct ggactccgac 1200 ggctccttct tcctgtactc caagctgacc gtggacaagt cccggtggca gcagggcaac 1260 ggctccttct tcctgtactc caagctgacc gtggacaagt cccggtggca gcagggcaac 1260 gtgttctcct gctccgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1320 gtgttctcct gctccgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1320 tccctgtccc ccggcaagtg a 1341 <210> 78 <211> 1353 <212> DNA <213> Artificial Sequence <220> <223> Synthesis: Heavy Chain Encoding Gene <400> 78 gaggtgcagc tgctggagtc cggcggcggc ctggtgcagc ccggcggctc cctgcggctg 60 tcctgcgccg cctccggctt caccttctcc aactacgaca tgtcctgggt gcggcaggcc 120 cccggcaagg gcctggagtg ggtgtccgcc atctaccact ccggctcctc caagtactac 180 gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240 ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc ccggggcggc 300 aacggcgcct gggacaccgg cttcgactac tggggccagg gcaccctggt gaccgtgtcc 360 tccgcctcca ccaagggccc ctccgtgttc cccctggccc cctcctccaa gtccacctcc 420 ggcggcaccg ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg 480 tcctggaact ccggcgccct gacctccggc gtgcacacct tccccgccgt gctgcagtcc 540 tccggcctgt actccctgtc ctccgtcgtg accgtgccct cctcctccct gggcacccag 600 acctacatct gcaacgtgaa ccacaagccc tccaacacca aggtggacaa gaaggtggag 660 cccaagtcct gcgacaagac ccacacctgc cctccctgcc ccgcccccga gctgctgggc 720 ggcccctccg tgttcctgtt ccctcctaag cccaaggaca ccctgatgat ctcccggacc 780 cccgaggtga cttgcgtggt ggtggacgtg tcccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac 900 aactccacct accgggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 960 aaggagtaca agtgcaaggt gtccaacaag gccctgcccg cccccatcga gaagaccatc 1020 tccaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc ctcccgggag 1080 gagatgacca agaaccaggt gtccctgacc tgcctggtga agggcttcta cccctccgac 1140 atcgccgtgg agtgggagtc caacggccag cccgagaaca actacaagac cacccccccc 1200 gtgctggact ccgacggctc cttcttcctg tactccaagc tgaccgtgga caagtcccgg 1260 tggcagcagg gcaacgtgtt ctcctgctcc gtgatgcacg aggccctgca caaccactac 1320 acccagaagt ccctgtccct gtcccccggc aag 1353 <210> 79 <211> 1374 <212> DNA <213> Artificial Sequence <220> <223> Synthesis: Heavy Chain Encoding Gene <400> 79 gaagttcaac tgttggaatc cgggggtggt ctggtccaac ctggagggtc tcttagactg 60 agttgtgctg cttcaggctt cacatttagc tcatatgata tgtcctgggt cagacaggcc 120 cccggcaaag gtcttgaatg ggtatctggt attagtcatg gatctggcaa caagtactac 180 gctgatagtg tcaaaggacg attcaccata tctcgtgaca actctaaaaa cactttgtac 240 ttgcagatga actcactgcg tgccgaagac acagccgtgt attattgcgc taagcgtctc 300 tcactccgca ggcgaccttc ctattacagc gacaacgcta tggatgtctg ggggcagggt 360 acactcgtca ccgtgtcatc agcctccacc aagggcccct ccgtgttccc cctggccccc 420 tcctccaagt ccacctccgg cggcaccgcc gccctgggct gcctggtgaa ggactacttc 480 cccgagcccg tgaccgtgtc ctggaactcc ggcgccctga cctccggcgt gcacaccttc 540 cccgccgtgc tgcagtcctc cggcctgtac tccctgtcct ccgtcgtgac cgtgccctcc 600 tcctccctgg gcacccagac ctacatctgc aacgtgaacc acaagccctc caacaccaag 660 gtggacaaga aggtggagcc caagtcctgc gacaagaccc acacctgccc tccctgcccc 720 gcccccgagc tgctgggcgg cccctccgtg ttcctgttcc ctcctaagcc caaggacacc 780 ctgatgatct cccggacccc cgaggtgact tgcgtggtgg tggacgtgtc ccacgaggac 840 cccgaggtga agttcaactg gtacgtggac ggcgtggagg tgcacaacgc caagaccaag 900 ccccgggagg agcagtacaa ctccacctac cgggtggtgt ccgtgctgac cgtgctgcac 960 caggactggc tgaacggcaa ggagtacaag tgcaaggtgt ccaacaaggc cctgcccgcc 1020 cccatcgaga agaccatctc caaggccaag ggccagcccc gggagcccca ggtgtacacc 1080 ctgcccccct cccgggagga gatgaccaag aaccaggtgt ccctgacctg cctggtgaag 1140 ggcttctacc cctccgacat cgccgtggag tgggagtcca acggccagcc cgagaacaac 1200 tacaagacca ccccccccgt gctggactcc gacggctcct tcttcctgta ctccaagctg 1260 accgtggaca agtcccggtg gcagcagggc aacgtgttct cctgctccgt gatgcacgag 1320 gccctgcaca accactacac ccagaagtcc ctgtccctgt cccccggcaa gtga 1374 <210> 80 <211> 1371 <212> DNA <213> Artificial Sequence <220> <223> Synthesis: Heavy Chain Encoding Gene <400> 80 gaggtgcagc tgctggagtc cggcggcggc ctggtgcagc ccggcggctc cctgcggctg 60 tcctgcgccg cctccggctt caccttctcc aactacgcca tgtcctgggt gcggcaggcc 120 cccggcaagg gcctggagtg ggtgtcctcc atctcccaca actccggctc cacctactac 180 gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240 ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc caagttcatc 300 tccgcccgga agtccctggg ccggtcctac tccaacggca tggacgtgtg gggccagggc 360 accctggtga ccgtgtcctc cgcctccacc aagggcccct ccgtgttccc cctggccccc 420 tcctccaagt ccacctccgg cggcaccgcc gccctgggct gcctggtgaa ggactacttc 480 cccgagcccg tgaccgtgtc ctggaactcc ggcgccctga cctccggcgt gcacaccttc 540 cccgccgtgc tgcagtcctc cggcctgtac tccctgtcct ccgtcgtgac cgtgccctcc 600 tcctccctgg gcacccagac ctacatctgc aacgtgaacc acaagccctc caacaccaag 660 gtggacaaga aggtggagcc caagtcctgc gacaagaccc acacctgccc tccctgcccc 720 gcccccgagc tgctgggcgg cccctccgtg ttcctgttcc ctcctaagcc caaggacacc 780 ctgatgatct cccggacccc cgaggtgact tgcgtggtgg tggacgtgtc ccacgaggac 840 cccgaggtga agttcaactg gtacgtggac ggcgtggagg tgcacaacgc caagaccaag 900 ccccgggagg agcagtacaa ctccacctac cgggtggtgt ccgtgctgac cgtgctgcac 960 caggactggc tgaacggcaa ggagtacaag tgcaaggtgt ccaacaaggc cctgcccgcc 1020 cccatcgaga agaccatctc caaggccaag ggccagcccc gggagcccca ggtgtacacc 1080 ctgcccccct cccgggagga gatgaccaag aaccaggtgt ccctgacctg cctggtgaag 1140 ggcttctacc cctccgacat cgccgtggag tgggagtcca acggccagcc cgagaacaac 1200 tacaagacca ccccccccgt gctggactcc gacggctcct tcttcctgta ctccaagctg 1260 accgtggaca agtcccggtg gcagcagggc aacgtgttct cctgctccgt gatgcacgag 1320 gccctgcaca accactacac ccagaagtcc ctgtccctgt cccccggcaa g 1371 <210> 81 <211> 1371 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: Heavy Chain Coding Gene <400> 81 gaagtacagt tgcttgaaag tggcggtggt cttgtccagc caggcggttc ccttcggctg 60 tcttgcgccg caagtggctt cactttcagc gactatgata tgtcttgggt ccgccaagca 120 ccaggaaagg gacttgaatg ggtgagtgta atcagtcctg acggagggtc aatttattat 180 gcagattcag tcaagggtcg attcactata tcccgagaca actccaaaaa tactctttat 240 cttcagatga actctttgag agctgaagac accgcagttt attactgtgc tcgggatgta 300 gtggagtgca atatgaatcc ctgctcatac gacaacgcaa tggatgtttg ggggcagggg 360 actctggtga cagtcagctc tgcctccacc aagggcccct ccgtgttccc cctggccccc 420 tcctccaagt ccacctccgg cggcaccgcc gccctgggct gcctggtgaa ggactacttc 480 cccgagcccg tgaccgtgtc ctggaactcc ggcgccctga cctccggcgt gcacaccttc 540 cccgccgtgc tgcagtcctc cggcctgtac tccctgtcct ccgtcgtgac cgtgccctcc 600 tcctccctgg gcacccagac ctacatctgc aacgtgaacc acaagccctc caacaccaag 660 gtggacaaga aggtggagcc caagtcctgc gacaagaccc acacctgccc tccctgcccc 720 gcccccgagc tgctgggcgg cccctccgtg ttcctgttcc ctcctaagcc caaggacacc 780 ctgatgatct cccggacccc cgaggtgact tgcgtggtgg tggacgtgtc ccacgaggac 840 cccgaggtga agttcaactg gtacgtggac ggcgtggagg tgcacaacgc caagaccaag 900 ccccgggagg agcagtacaa ctccacctac cgggtggtgt ccgtgctgac cgtgctgcac 960 caggactggc tgaacggcaa ggagtacaag tgcaaggtgt ccaacaaggc cctgcccgcc 1020 cccatcgaga agaccatctc caaggccaag ggccagcccc gggagcccca ggtgtacacc 1080 ctgcccccct cccgggagga gatgaccaag aaccaggtgt ccctgacctg cctggtgaag 1140 ggcttctacc cctccgacat cgccgtggag tgggagtcca acggccagcc cgagaacaac 1200 tacaagacca ccccccccgt gctggactcc gacggctcct tcttcctgta ctccaagctg 1260 accgtggaca agtcccggtg gcagcagggc aacgtgttct cctgctccgt gatgcacgag 1320 gccctgcaca accactacac ccagaagtcc ctgtccctgt cccccggcaa g 1371 <210> 82 <211> 1374 <212> DNA <213> Artificial Sequence <220> <223> Synthesis: Heavy Chain Coding Gene <400> 82 gaagtgcagc tgcttgaatc aggaggcggc ctcgtacaac cagggggatc tctcagactg 60 tcctgcgctg ccagtggctt cactttcagc aactacgata tgtcatgggt gaggcaggca 120 cctggcaagg gtctggagtg ggtctcaagc ataagtccca gtagtggaag ctcaatttat 180 tacgccgaca gtgtaaaggg ccggttcacc attagtagag acaattctaa gaataccttg 240 taccttcaaa tgaatagtct gagagccgaa gataccgcag tttattattg cgctaaggcc 300 ccagggtggt gtcaggcccc ttcatgctat tatgataatg caatggacgt gtggggtcag 360 ggtactctgg tcacagtcag tagtgcctcc accaagggcc cctccgtgtt ccccctggcc 420 ccctcctcca agtccacctc cggcggcacc gccgccctgg gctgcctggt gaaggactac 480 ttccccgagc ccgtgaccgt gtcctggaac tccggcgccc tgacctccgg cgtgcacacc 540 ttccccgccg tgctgcagtc ctccggcctg tactccctgt cctccgtcgt gaccgtgccc 600 tcctcctccc tgggcaccca gacctacatc tgcaacgtga accacaagcc ctccaacacc 660 aaggtggaca agaaggtgga gcccaagtcc tgcgacaaga cccacacctg ccctccctgc 720 cccgcccccg agctgctggg cggcccctcc gtgttcctgt tccctcctaa gcccaaggac 780 accctgatga tctcccggac ccccgaggtg acttgcgtgg tggtggacgt gtcccacgag 840 gaccccgagg tgaagttcaa ctggtacgtg gacggcgtgg aggtgcacaa cgccaagacc 900 aagccccggg aggagcagta caactccacc taccgggtgg tgtccgtgct gaccgtgctg 960 caccaggact ggctgaacgg caaggagtac aagtgcaagg tgtccaacaa ggccctgccc 1020 gcccccatcg agaagaccat ctccaaggcc aagggccagc cccgggagcc ccaggtgtac 1080 accctgcccc cctcccggga ggagatgacc aagaaccagg tgtccctgac ctgcctggtg 1140 aagggcttct acccctccga catcgccgtg gagtgggagt ccaacggcca gcccgagaac 1200 aactacaaga ccaccccccc cgtgctggac tccgacggct ccttcttcct gtactccaag 1260 ctgaccgtgg acaagtcccg gtggcagcag ggcaacgtgt tctcctgctc cgtgatgcac 1320 gaggccctgc acaaccacta cacccagaag tccctgtccc tgtcccccgg caag 1374 <210> 83 <211> 648 <212> DNA <213> Artificial Sequence <220> <223> Synthesis: Light Chain Coding Gene <400> 83 cagtctgtgc tgacacaacc accttctgcc tctgggactc caggccagcg ggttaccatt 60 agctgttctg gtagttctag taatatcggt aacaacaatg tgaattggta tcaacaactg 120 ccaggaaccg cccctaagtt gctcatatat tatgataaca agcggccttc aggcgttcct 180 gatcgtttct ccggctctaa aagtggcaca tccgccagtc ttgctatcag cggtctcaga 240 tccgaggacg aggccgacta ttattgtggt acatgggacg cttccctgtc aggttacgtc 300 tttggcggcg gcacaaaact gacagttctt ggccagccca aggccgcccc ctccgtgacc 360 ctgttccccc cctcctccga ggagctgcag gccaacaagg ccaccctggt gtgcctgatc 420 tccgacttct accccggcgc cgtgaccgtg gcctggaagg ccgactcctc ccccgtgaag 480 gccggcgtgg agaccaccac cccctccaag cagtccaaca acaagtacgc cgcctcctcc 540 tacctgtccc tgacccccga gcagtggaag tcccaccggt cctactcctg ccaggtgacc 600 cacgagggct ccaccgtgga gaagaccgtg gcccccgccg agtgctcc 648 <210> 84 <211> 648 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: Light Chain Coding Gene <400> 84 cagtctgtgc tgacccagcc tccccctgct tctggcaccc ctggccagag agtgaccatc 60 tcctgctccg gctcctcctc caacatcggc tccaacaccg tgtactggta tcagcagctg 120 cccggcaccg cccccaagct gctgatctac gccaactccc agcggccctc cggcgtgccc 180 gacagattct ccggctccaa gtccggcacc tccgcctccc tggccatctc cggcctgaga 240 tctgaggacg aggccgacta ctactgcggc tcctgggact actccctgtc cggctacgtg 300 ttcggcggag gcaccaagct gaccgtgctg ggccagccta aggccgctcc ctccgtgacc 360 ctgttccccc catcctccga ggaactgcag gccaacaagg ccaccctggt ctgcctgatc 420 tccgacttct accctggcgc cgtgaccgtg gcctggaagg ccgacagctc tcctgtgaag 480 gccggcgtgg aaaccaccac cccctccaag cagtccaaca acaaatacgc cgcctcctcc 540 tacctgtccc tgacccccga gcagtggaag tcccaccggt cctacagctg ccaggtcaca 600 cacgagggct ccaccgtgga aaagaccgtg gcccctgccg agtgctcc 648 <210> 85 <211> 648 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: Light Chain Encoding Gene <400> 85 cagagtgttt tgacccagcc tccttccgcc agcggcaccc ctgggcaacg ggttacaatc 60 agctgttccg ggagcagcag taacattggt aataataacg tctcttggta tcagcagttg 120 cctggcacag cacctaagct cctgatttac gctgactccc accggccttc cggcgtccct 180 gatcgtttct ccgggtcaaa aagtggaacc tcagcaagcc ttgcaatcag cggactgcgg 240 tccgaagatg aagctgacta ctactgcgct acctgggatt actcattgtc cggctacgtc 300 tttggggggg gaaccaaatt gacagtcttg ggtcagccca aggccgcccc ctccgtgacc 360 ctgttccccc cctcctccga ggagctgcag gccaacaagg ccaccctggt gtgcctgatc 420 tccgacttct accccggcgc cgtgaccgtg gcctggaagg ccgactcctc ccccgtgaag 480 gccggcgtgg agaccaccac cccctccaag cagtccaaca acaagtacgc cgcctcctcc 540 tacctgtccc tgacccccga gcagtggaag tcccaccggt cctactcctg ccaggtgacc 600 cacgagggct ccaccgtgga gaagaccgtg gcccccgccg agtgctcc 648 <210> 86 <211> 648 <212> DNA <213> Artificial Sequence <220> <223> Synthesis: Light Chain Coding Gene <400> 86 cagtccgtgc tgacccagcc cccctccgcc tccggcaccc ccggccagcg ggtgaccatc 60 tcctgctccg gctcctcctc caacatcggc tccaacgacg tgtcctggta ccagcagctg 120 cccggcaccg cccccaagct gctgatctac tacgacaaca accggccctc cggcgtgccc 180 gaccggttct ccggctccaa gtccggcacc tccgcctccc tggccatctc cggcctgcgg 240 tccgaggacg aggccgacta ctactgcggc gcctgggacg actccctgtc cggctacgtg 300 ttcggcggcg gcaccaagct gaccgtgctg ggccagccca aggccgcccc ctccgtgacc 360 ctgttccccc cctcctccga ggagctgcag gccaacaagg ccaccctggt gtgcctgatc 420 tccgacttct accccggcgc cgtgaccgtg gcctggaagg ccgactcctc ccccgtgaag 480 gccggcgtgg agaccaccac cccctccaag cagtccaaca acaagtacgc cgcctcctcc 540 tacctgtccc tgacccccga gcagtggaag tcccaccggt cctactcctg ccaggtgacc 600 cacgagggct ccaccgtgga gaagaccgtg gcccccgccg agtgctcc 648 <210> 87 <211> 651 <212> DNA <213> Artificial sequence <220> <223> Synthetic: Light chain coding gene <400> 87 caaagcgtac tcacccagcc cccatccgca tctggcactc ctggtcaacg ggttacaatc 60 tcttgtactg ggtcaagttc caatattgga aataacgcag tgaactggta tcagcagctc 120 cctggcaccg cccctaaact cttgatatac tatgactcta atcggccaag tggagtcccc 180 gataggttct caggttctaa gagtggcaca agtgccagcc tggcaatctc agggctcagg 240 tccgaagatg aggctgatta ttactgcgga gcttgggatg atagcctgag tggctacgtc 300 ttcgggggag gaacaaaatt gaccgtactt ggccagccca aggccgcccc ctccgtgacc 360 ctgttccccc cctcctccga ggagctgcag gccaacaagg ccaccctggt gtgcctgatc 420 tccgacttct accccggcgc cgtgaccgtg gcctggaagg ccgactcctc ccccgtgaag 480 gccggcgtgg agaccaccac cccctccaag cagtccaaca acaagtacgc cgcctcctcc 540 tacctgtccc tgacccccga gcagtggaag tcccaccggt cctactcctg ccaggtgacc 600 cacgagggct ccaccgtgga gaagaccgtg gcccccgccg agtgctcctg a 651 <210> 88 <211> 648 <212> DNA <213> Artificial Sequence <220> <223> Synthesis: Light chain encoding gene <400> 88 cagtccgtgc tgacccagcc cccctccgcc tccggcaccc ccggccagcg ggtgaccatc 60 tcctgcaccg gctcctcctc caacatcggc tccaacgacg tgacctggta ccagcagctg 120 cccggcaccg cccccaagct gctgatctac gccgactcca agcggccctc cggcgtgccc 180 gaccggttct ccggctccaa gtccggcacc tccgcctccc tggccatctc cggcctgcgg 240 tccgaggacg aggccgacta ctactgcggc acctgggact actccctgtc cggctacgtg 300 ttcggcggcg gcaccaagct gaccgtgctg ggccagccca aggccgcccc ctccgtgacc 360 ctgttccccc cctcctccga ggagctgcag gccaacaagg ccaccctggt gtgcctgatc 420 tccgacttct accccggcgc cgtgaccgtg gcctggaagg ccgactcctc ccccgtgaag 480 gccggcgtgg agaccaccac cccctccaag cagtccaaca acaagtacgc cgcctcctcc 540 tacctgtccc tgacccccga gcagtggaag tcccaccggt cctactcctg ccaggtgacc 600 cacgagggct ccaccgtgga gaagaccgtg gcccccgccg agtgctcc 648 <210> 89 <211> 648 <212> DNA <213> Artificial Sequence <220> <223> Synthesis: Light Chain Encoding Gene <400> 89 caaagtgtat tgactcaacc tccctccgct tccggtacac cagggcagcg agtaaccatc 60 agttgcagtg gcagcagctc caatatcgga agcaattatg taagttggta tcaacagttg 120 ccagggaccg ctccaaaact gttgatctat gacgacagtc accgtccttc aggtgtgccc 180 gaccgatttt caggcagcaa gagcggcaca tccgcctccc tcgctatctc cggcctccga 240 tccgaagatg aggccgacta ctattgtgga gcctgggacg actcccttag tggctatgtg 300 tttgggggag ggacaaagtt gaccgtactt ggccagccca aggccgcccc ctccgtgacc 360 ctgttccccc cctcctccga ggagctgcag gccaacaagg ccaccctggt gtgcctgatc 420 tccgacttct accccggcgc cgtgaccgtg gcctggaagg ccgactcctc ccccgtgaag 480 gccggcgtgg agaccaccac cccctccaag cagtccaaca acaagtacgc cgcctcctcc 540 tacctgtccc tgacccccga gcagtggaag tcccaccggt cctactcctg ccaggtgacc 600 cacgagggct ccaccgtgga gaagaccgtg gcccccgccg agtgctcc 648 <210> 90 <211> 648 <212> DNA <213> Artificial Sequence <220> <223> Synthesis: Light Chain Encoding Gene <400> 90 cagtcagttc ttacacaacc cccatccgct tctggcactc ccggccagcg cgtaactata 60 tcttgctctg ggagtagtag caatatcggt aataatgatg tctcatggta ccaacagctg 120 cctggaacag cccccaaact cctcatttat gatgactctc aaaggccaag tggtgtgcca 180 gacagatttt ccggtagcaa gagtggaaca tcagcaagtc ttgctataag tggcttgcgt 240 tccgaggacg aggccgacta ttattgtggc gcatgggatg actcactgag cggctacgtt 300 ttcgggggcg gtactaagtt gaccgttttg ggacagccca aggccgcccc ctccgtgacc 360 ctgttccccc cctcctccga ggagctgcag gccaacaagg ccaccctggt gtgcctgatc 420 tccgacttct accccggcgc cgtgaccgtg gcctggaagg ccgactcctc ccccgtgaag 480 gccggcgtgg agaccaccac cccctccaag cagtccaaca acaagtacgc cgcctcctcc 540 tacctgtccc tgacccccga gcagtggaag tcccaccggt cctactcctg ccaggtgacc 600 cacgagggct ccaccgtgga gaagaccgtg gcccccgccg agtgctcc 648 <210> 91 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Heavy Chain Constant Region <400> 91 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 92 <211> 993 <212> DNA <213> Artificial sequence <220> <223> Synthetic: Heavy chain constant region encoding gene <400> 92 gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc cacctccggc 60 ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgtcc 120 tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagtcctcc 180 ggcctgtact ccctgtcctc cgtcgtgacc gtgccctcct cctccctggg cacccagacc 240 tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa ggtggagccc 300 aagtcctgcg acaagaccca cacctgccct ccctgccccg cccccgagct gctgggcggc 360 ccctccgtgt tcctgttccc tcctaagccc aaggacaccc tgatgatctc ccggaccccc 420 gaggtgactt gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa gttcaactgg 480 tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac 540 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaag 600 gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccatcgagaa gaccatctcc 660 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgcccccctc ccgggaggag 720 atgaccaaga accaggtgtc cctgacctgc ctggtgaagg gcttctaccc ctccgacatc 780 gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac cccccccgtg 840 ctggactccg acggctcctt cttcctgtac tccaagctga ccgtggacaa gtcccggtgg 900 cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 960 cagaagtccc tgtccctgtc ccccggcaag tga 993 <210> 93 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Light Chain Constant Region <400> 93 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 1 5 10 15 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 20 25 30 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40 45 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 65 70 75 80 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90 95 Lys Thr Val Ala Pro Ala Glu Cys Ser 100 105 <210> 94 <211> 318 <212> DNA <213> Artificial Sequence <220> <223> Synthesis: Light chain constant region encoding gene <400> 94 cagcccaagg ccgccccctc cgtgaccctg ttccccccct cctccgagga gctgcaggcc 60 aacaaggcca ccctggtgtg cctgatctcc gacttctacc ccggcgccgt gaccgtggcc 120 tggaaggccg actcctcccc cgtgaaggcc ggcgtggaga ccaccacccc ctccaagcag 180 tccaacaaca agtacgccgc ctcctcctac ctgtccctga cccccgagca gtggaagtcc 240 caccggtcct actcctgcca ggtgacccac gagggctcca ccgtggagaa gaccgtggcc 300 cccgccgagt gctcctga 318 <210> 95 <211> 318 <212> DNA <213> Artificial sequence <220> <223> Synthetic: Light chain constant region encoding gene <400> 95 cagcctaagg ccgctccctc cgtgaccctg ttccccccat cctccgagga actgcaggcc 60 aacaaggcca ccctggtctg cctgatctcc gacttctacc ctggcgccgt gaccgtggcc 120 tggaaggccg acagctctcc tgtgaaggcc ggcgtggaaa ccaccacccc ctccaagcag 180 tccaacaaca aatacgccgc ctcctcctac ctgtccctga cccccgagca gtggaagtcc 240 caccggtcct acagctgcca ggtcacacac gagggctcca ccgtggaaaa gaccgtggcc 300 cctgccgagt gctcctga 318 <210> 96 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain CDR2 <400> 96 Ser Ile Ser Pro Asp Ala Ser Asn Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 97 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain CDR3 <400> 97 Gly Gly Asn Ala Ala Trp Asp Thr Gly Phe Asp Tyr 1 5 10 <210> 98 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Synthesis: Heavy chain variable region <400> 98 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Pro Asp Ala Ser Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Leu Arg Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 99 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Heavy Chain Variable Region <400> 99 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Tyr His Ser Gly Ser Ser Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Asn Ala Ala Trp Asp Thr Gly Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 100 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: Heavy Chain <400> 100 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Pro Asp Ala Ser Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Leu Arg Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 101 <211> 451 <212> PRT <213> Artificial sequence <220> <223> Synthesis: heavy chain <400> 101 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Tyr His Ser Gly Ser Ser Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Asn Ala Ala Trp Asp Thr Gly Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 102 <211> 1338 <212> DNA <213> Artificial Sequence <220> <223> Synthesis: Heavy Chain Encoding Gene <400> 102 gaagtgcagc tgctggaatc cggcggaggc ctggtgcagc ctggcggctc tctgagactg 60 tcttgcgccg cctccggctt caccttctcc gactactaca tgtcctgggt gcgacaggcc 120 cctggcaagg gcctggaatg ggtgtcctcc atctcccccg acgcctccaa cacctactac 180 gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240 ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc caagaacctg 300 cgggccttcg actactgggg ccagggcaca ctggtgaccg tgtcctccgc ctccaccaag 360 ggcccctccg tgttccccct ggccccctcc tccaagtcca cctccggcgg caccgccgcc 420 ctgggctgcc tggtgaagga ctacttcccc gagcccgtga ccgtgtcctg gaactccggc 480 gccctgacct ccggcgtgca caccttcccc gccgtgctgc agtcctccgg cctgtactcc 540 ctgtcctccg tcgtgaccgt gccctcctcc tccctgggca cccagaccta catctgcaac 600 gtgaaccaca agccctccaa caccaaggtg gacaagaagg tggagcccaa gtcctgcgac 660 aagacccaca cctgccctcc ctgccccgcc cccgagctgc tgggcggccc ctccgtgttc 720 ctgttccctc ctaagcccaa ggacaccctg atgatctccc ggacccccga ggtgacttgc 780 gtggtggtgg acgtgtccca cgaggacccc gaggtgaagt tcaactggta cgtggacggc 840 gtggaggtgc acaacgccaa gaccaagccc cgggaggagc agtacaactc cacctaccgg 900 gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaagga gtacaagtgc 960 aaggtgtcca acaaggccct gcccgccccc atcgagaaga ccatctccaa ggccaagggc 1020 cagccccggg agccccaggt gtacaccctg cccccctccc gggaggagat gaccaagaac 1080 caggtgtccc tgacctgcct ggtgaagggc ttctacccct ccgacatcgc cgtggagtgg 1140 gagtccaacg gccagcccga gaacaactac aagaccaccc cccccgtgct ggactccgac 1200 ggctccttct tcctgtactc caagctgacc gtggacaagt cccggtggca gcagggcaac 1260 gtgttctcct gctccgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1320 tccctgtccc ccggcaag 1338 <210> 103 <211> 1353 <212> DNA <213> Artificial sequence <220> <223> Synthesis: Heavy chain coding gene <400> 103 gaggtgcagc tgctggagtc cggcggcggc ctggtgcagc ccggcggctc cctgcggctg 60 tcctgcgccg cctccggctt caccttctcc aactacgaca tgtcctgggt gcggcaggcc 120 cccggcaagg gcctggagtg ggtgtccgcc atctaccact ccggctcctc caagtactac 180 gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240 ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc ccggggcggc 300 aacgccgcct gggacaccgg cttcgactac tggggccagg gcaccctggt gaccgtgtcc 360 tccgcctcca ccaagggccc ctccgtgttc cccctggccc cctcctccaa gtccacctcc 420 ggcggcaccg ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg 480 tcctggaact ccggcgccct gacctccggc gtgcacacct tccccgccgt gctgcagtcc 540 tccggcctgt actccctgtc ctccgtcgtg accgtgccct cctcctccct gggcacccag 600 acctacatct gcaacgtgaa ccacaagccc tccaacacca aggtggacaa gaaggtggag 660 cccaagtcct gcgacaagac ccacacctgc cctccctgcc ccgcccccga gctgctgggc 720 ggcccctccg tgttcctgtt ccctcctaag cccaaggaca ccctgatgat ctcccggacc 780 cccgaggtga cttgcgtggt ggtggacgtg tcccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac 900 aactccacct accgggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 960 aaggagtaca agtgcaaggt gtccaacaag gccctgcccg cccccatcga gaagaccatc 1020 tccaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc ctcccgggag 1080 gagatgacca agaaccaggt gtccctgacc tgcctggtga agggcttcta cccctccgac 1140 atcgccgtgg agtgggagtc caacggccag cccgagaaca actacaagac cacccccccc 1200 gtgctggact ccgacggctc cttcttcctg tactccaagc tgaccgtgga caagtcccgg 1260 tggcagcagg gcaacgtgtt ctcctgctcc gtgatgcacg aggccctgca caaccactac 1320 acccagaagt ccctgtccct gtcccccggc aag 1353

Claims

1. An antibody-drug conjugate represented by Formula I, or a pharmaceutically acceptable salt thereof: [Formula I] Ab-(X) y Wherein: Ab is an anti-ROR1 antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, and the light chain variable region comprising CDRL1, CDRL2, and CDRL3; wherein: CDRH1 consists of the amino acid sequence of SEQ ID NOs: 4; CDRH2 consists of the amino acid sequence of SEQ ID NOs: 11; CDRH3 consists of the amino acid sequence of SEQ ID NOs: 19; CDRL1 consists of the amino acid sequence of SEQ ID NOs: 27; CDRL2 consists of the amino acid sequence of SEQ ID NOs: 35; CDRL3 consists of the amino acid sequence of SEQ ID NOs: 42; X is Bound to Ab; and y is 2.

2. The antibody-drug conjugate according to claim 1, wherein the antibody comprises a heavy chain variable region, and the heavy chain variable region comprises one of the following: i) The sequence according to SEQ ID NO: 48; or ii) A sequence having at least 90% or at least 95% or higher identity with the amino acid sequence of SEQ ID NO:

48.

3. The antibody-drug conjugate according to claim 1, wherein the antibody comprises a light chain variable region, and the light chain variable region comprises one of the following: i) The sequence according to SEQ ID NO: 56; or ii) A sequence having at least 90% or at least 95% or higher identity with the amino acid sequence of SEQ ID NO:

56.

4. The antibody-drug conjugate according to claim 1, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 48, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

56.

5. The antibody-drug conjugate according to claim 1, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 64, and the light chain comprises the amino acid sequence of SEQ ID NO:

72.

6. The antibody-drug conjugate according to claim 1, wherein the antibody is an IgG1 fully human monoclonal antibody and the ROR1 is human ROR1 or mouse ROR1.

7. The antibody-drug conjugate according to claim 1, wherein the antibody is bonded to X via a thioether bond; the thioether bond includes the sulfur atom of a cysteine of the antibody; and the antibody comprises an amino acid motif at the C-terminus of the antibody that can be recognized by a prenyltransferase.

8. The antibody-drug conjugate according to claim 7, wherein the amino acid motif has the sequence CVIM.

9. Use of the antibody-drug conjugate according to any one of claims 1 to 8 in the preparation of a drug for the treatment of cancer, wherein the cancer is lymphoma, breast cancer, or non-small cell lung cancer (NSCLC).

10. The use according to claim 9, wherein the cancer is lymphoma, and wherein the lymphoma is Burkitt lymphoma, mantle cell lymphoma MCL, diffuse large B-cell lymphoma DLBCL, or follicular lymphoma FL and marginal zone lymphoma MZL.

11. The use according to claim 9, wherein the drug comprises an additional chemotherapeutic agent.

Images