Anthracycline derivatives

Abstract

The present application relates to anthracycline derivatives and their use in forming conjugates with target-binding molecules, including, but not limited to, antibodies. Conjugates of target-binding molecules and anthracycline derivatives are also provided. Medical uses of the conjugates and pharmaceutical compositions containing them are also provided.

Description

[Technical Field]

[0001] This invention relates to the use of anthracycline derivatives and target-binding molecules, including but not limited to antibodies, in conjugate formation. Conjugates of target-binding molecules and anthracycline derivatives are also provided. Introduction

[0002] Conjugating low molecular weight toxins to specific binding proteins such as antibodies is a powerful tool for directing toxic payloads to targets within the body. For example, such conjugates can be used to target toxic payloads to cancer cells, demonstrating their significant potential in cancer treatment.

[0003] Developing effective and safe conjugates for cancer treatment requires addressing several aspects. First, the binding protein or antibody must be specific to a given tumor-specific antigen (TSA), which should be expressed little to no by normal or healthy tissue cells.

[0004] Secondly, the covalent bond or linkage between the drug and the binding protein must be stable in circulation to prevent undesirable release of the toxin payload into the bloodstream, but must effectively release the drug upon binding to and / or internalization of cancer cells. Thirdly, even if a potentially limited amount of TSA is expressed on cancer cells, and therefore only a limited amount of ADC is internalized, or if the release of the toxin payload is not achieved with sufficiently high efficacy upon binding to or internalization of cancer cells, the toxin payload must have sufficiently high toxicity or potency to achieve destruction of cancer cells.

[0005] The anthracycline derivative PNU-159682, described as a metabolite of nemorubicin (Quintieri et al. (2005) Clin) Cancer Res. 11, 1608-1617), has been reported to exhibit extremely high efficacy in in vitro cell cytotoxicity in one ovarian (A2780) cell line and one breast cancer (MCF7) cell line (WO2012 / 073217 A1) within the pico-femtomole range. A derivative of PNU-159682 is also described in WO2016 / 102679.

[0006] The binding of PNU-159682 derivatives to antibodies is described in WO2009 / 099741, WO2016 / 127081 and WO2016 / 102679, Yu et al, Clin. Cancer Res 2015, 21, 3298 and Stefan et al, Mol. Cancer. Ther., 2017, 16,879. Summary of the Invention

[0007] The present invention provides anthracycline (PNU) derivatives suitable for use in drug conjugates. Specifically, derivatives of PNU159682 are provided, which lack a C14 carbon and a bonded hydroxyl functional group, and an ethylenediamino (EDA) group forms part of the linker region between the C13 carbonyl and maleimide groups of PNU159682. If the linker contains val-cit-PAB, the maleimide group can be replaced with any reactive group suitable for the conjugation reaction. Such a payload can react with a free thiol group on another molecule. If the free thiol is on a protein, a protein-drug conjugate (PDC) can be formed.

[0008] Accordingly, in the first embodiment, an anthracycline (PNU) derivative of formula (I) is provided. JPEG0007923099000001.jpg65170 In the formula, [X] is any spacer selected from the group consisting of a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof, [L1] and [L2] are selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), a peptide, -(CH2) n -, -(CH2CH2O) n -, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, any amino acid except glycine, and any linker selected from the group consisting of combinations thereof.

[0009] The anthracycline (PNU) derivative of formula (I) may comprise [L1], [L2], or [L1] and [L2].

[0010] Preferably, [L1] and / or [L2] is a peptide, and said peptide does not comprise glycine.

[0011] It will be apparent to those skilled in the art that if any spacer and / or any linker is not present, a bond remains in its place instead.

[0012] Preferably, [X] is selected from the group consisting of polyethylene glycol, JPEG0007923099000002.jpg21170 JPEG0007923099000003.jpg6170 represents the point of attachment to the rest of the molecule, and [R] is any spacer selected from the group consisting of a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof.

[0013] More preferably, [X] is polyethylene glycol. The polyethylene glycol may be PEG4.

[0014] Preferably, [L2] is p-aminobenzyloxycarbonyl (PAB) or alanine. Preferably, the PNU derivative has a structure selected from the following. JPEG0007923099000004.jpg181170JPEG0007923099000005.jpg57170

[0015] In a second aspect, there is provided an anthracycline (PNU) derivative of formula (IV). JPEG0007923099000006.jpg83170wherein [X] is any spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof, and wherein [Z] is a reactive group. The reactive group is any reactive group suitable for use in a conjugation reaction, particularly a conjugation reaction to a target binding molecule.

[0016] Accordingly, [Z] may be a moiety comprising a functional group for use in a bioconjugation reaction. Functional groups for use in bioconjugation reactions include, but are not limited to • maleimide or alkyl halide for reaction with a thiol group or a selenol group on a protein via thioether and selenoether reactions; • a sulfhydryl group for reaction with maleimide, alkyl halide or thiol-functionalized molecules comprising the thiol group of a protein cysteine residue; • an activated disulfide such as pyridyl dithiol (Npys thiol) or TNB thiol (5-thiol-2-nitrobenzoic acid) for reacting with a thiol group to form a disulfide bond via thiol-disulfide exchange; ● Amino groups for bonding to carboxyl groups on proteins and biomolecules via amide bond formation reactions; ● Alkyne groups, particularly ring-bound alkynes such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonine (BCN), react with azide-functionalized biomolecules through copper-free strain-promoting alkyne-azide cycloaddition chemistry. Azide functional groups can be introduced, for example, through the incorporation of the unnatural amino acid para-azidomethyl-L-phenylalanine using enzyme-mediated glycoengineering to attach azide-containing sugar analogs, or into protein glycans; ● Azide groups for reaction with alkyne-activating target-binding molecules via copper-free strain-promoting alkyne-azide cycloaddition chemistry; ● Aminoxy groups for reactions via oxime-forming ligation with aldehyde and ketone groups on biomolecules. Ketones can be introduced into proteins through the use of amber-stop codon techniques, such as the incorporation of the non-natural amino acid, paraacetylphenylalanine. Aldehydes can be found on biomolecules in the presence of reducing sugars and can be introduced into proteins by periodic oxidation of the N-terminal serine residue or by periodic oxidation of the cis-glycol group of the carbohydrate. Aldehyde groups can also be incorporated into proteins via the conversion of protein cysteine ​​to formylglycine within specific sequences by formylglycinase. In addition, proteins containing formylglycine are bound to the payload via hydrazino-pictet-spengler (HIPS) ligation; ● Aldehyde or ketone groups for reacting with aminooxy, hydrazide, and hydrazinyl-functionalized biomolecules via oxime or hydrazine bond formation ligation reactions. Protein aminooxy and hydrazide-functionalized proteins can be produced by cleavage of intein fusion proteins.

[0017] Therefore, [Z] is selected from the group consisting of maleimide, alkyl halides, sulfhydryl groups, activated disulfides (such as pyridyldithiol (Npys thiol) or TNB thiol (5-thiol-2-nitronitrobenzoic acid)), amino groups, alkyne groups (such as dibenzocyclooctin (DBCO) or ring-restricted alkynes such as bicyclo[6.1.0]nonine (BCN)), azide groups, aminooxy groups, aldehyde groups, and ketone groups.

[0018] Furthermore, [Z] may be a portion for enzyme-mediated bioconjugation reactions. The portion for use in enzyme-mediated conjugation reactions may be poly(Gly)[(Gly) for use in sortase enzyme-mediated antibody conjugation. n This includes, but is not limited to, a suitable primary amine for bacterial transglutaminase-mediated conjugation to a glutamine γ-carboxyamide group, including sequences such as ], or Lys-Lys-Gln-Gly and Lys-Pro-Glu-Thr-Gly.

[0019] Therefore, [Z] can be selected from the group consisting of polyglycerides and primary amines.

[0020] Therefore, the PNU derivative according to the second aspect of the present invention may correspond to the PNU derivative according to the first aspect of the present invention, wherein L1 is Val-Cit-PAB, L2 is absent, and the maleimide group may be substituted with another reactive group as defined above.

[0021] Preferably, [X] is polyethylene glycol. Selected from the group including JPEG0007923099000007.jpg21170, JPEG0007923099000008.jpg6170 represents a bond site to the rest of the molecule, and [R] is any spacer selected from the group including substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or a combination thereof.

[0022] More preferably, [X] is polyethylene glycol. Polyethylene glycol may be PEG4.

[0023] The PNU derivative of the first embodiment can be conjugated to various parts. In particular, the PNU derivative of the first embodiment can be conjugated to a target-binding molecule, which is referred to herein as a target-binding molecule. The PNU derivative of the second embodiment can be conjugated to various parts. In particular, the PNU derivative of the second embodiment can be conjugated to a target-binding molecule, which is referred to herein as a target-binding molecule. Examples of target-binding molecules include, but are not limited to, biomolecules, peptides, small molecules, proteins, and nucleic acids (including, but not limited to, aptamers). In some cases, the target-binding molecule may be a multimer (e.g., dimers, trimers, and higher-order multimers or multi-subunit proteins).

[0024] Accordingly, in a further embodiment, a target-binding molecule-drug conjugate comprising a PNU and a binding molecule according to a first embodiment is provided. Alternatively, in this embodiment, a target-binding molecule-drug conjugate comprising a PNU and a binding molecule according to a second embodiment is provided. Suitable binding molecules for use in this embodiment include, but are not limited to, biomolecules, peptides, small molecules, proteins, and nucleic acids (including, but not limited to, aptamers).

[0025] According to this embodiment, a target-binding molecule-drug conjugate is provided, comprising a target-binding molecule and an anthracycline (PNU) derivative, wherein the target-binding molecule-drug conjugate has the structure of formula (II). In formula JPEG0007923099000009.jpg59170, [X] is any spacer selected from the group including substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or combinations thereof. [L1] and [L2] are valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, -(CH2) n -,-(CH2CH2O) n -, any linker selected from the group consisting of p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, any amino acid other than glycine, and combinations thereof, Y is the target-binding molecule.

[0026] The target-binding molecule-drug conjugate of formula (II) may include [L1], [L2], or [L1] and [L2].

[0027] Preferably, [L1] and / or [L2] are peptides, wherein the peptides are glycine-free target-binding molecule-drug conjugates.

[0028] It will be obvious to those skilled in the art that if no spacers and / or linkers are present, a coupling will remain instead.

[0029] In one embodiment, the anthracycline (PNU) derivative comprises [L1] and / or [L2], where [X] is optional. Thus, [L1] and / or [L2] are valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, -(CH2) n -,-(CH2CH2O) n -, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, any amino acid other than glycine, and combinations thereof may be selected from the group. An anthracycline (PNU) derivative of chemical formula (I) may include [L1], [L2], or [L1] and [L2]. An anthracycline (PNU) derivative of chemical formula (I) may include [L1] and / or [L2].

[0030] In one embodiment, an anthracycline (PNU) derivative of formula (I) is provided. JPEG0007923099000010.jpg66170wherein [X] is any spacer selected from the group consisting of a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or combinations thereof, [L1] and / or [L2] is valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), a peptide, -(CH2) n -, -(CH2CH2O) n -, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, any amino acid except glycine, and any linker selected from the group consisting of combinations thereof, the anthracycline (PNU) derivative of formula (I) comprises [L1], [L2] or [L1] and [L2].

[0031] Preferably, [X] is selected from the group consisting of polyethylene glycol, JPEG0007923099000011.jpg21170 JPEG0007923099000012.jpg6170represents a point of attachment to the rest of the molecule, and [R] is any spacer selected from the group consisting of a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or combinations thereof.

[0032] More preferably, [X] is polyethylene glycol. The polyethylene glycol may be PEG4.

[0033] Preferably, [L2] is p-aminobenzyloxycarbonyl (PAB) or alanine. Preferably, the PNU derivative has a structure selected from the following: JPEG0007923099000013.jpg217170

[0034] According to this embodiment, a target-binding molecule-drug conjugate is provided, comprising a target-binding molecule and an anthracycline (PNU) derivative, wherein the target-binding molecule-drug conjugate has the structure of formula (V). In formula JPEG0007923099000014.jpg76170, [X] is any spacer selected from the group including substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or combinations thereof. [Z] is a linker derived from a reactive group used to bond an anthracycline (PNU) derivative to the target binding molecule. Y is the target-binding molecule.

[0035] [Z] is a moiety derived from a reactive group typically used to bind anthracycline (PNU) derivatives and target-binding molecules. [Z] may also be a moiety derived from a reactive group selected from the group consisting of maleimide, alkyl halide, sulfhydryl group, activated disulfide, amino group, alkyne group, azide group, aminooxy group, aldehyde group, and ketone group.

[0036] Therefore, [Z] can be selected from the group consisting of disulfide bonds, amide bonds, oxime bonds, hydrazone bonds, thioether bonds, 1, 2, 3-triazoles, and polyglycols.

[0037] Preferably, [X] is polyethylene glycol. Selected from the group including JPEG0007923099000015.jpg19170, JPEG0007923099000016.jpg6170 represents a bond site to the rest of the molecule, and [R] is any spacer selected from the group including substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or a combination thereof.

[0038] More preferably, [X] is polyethylene glycol. Polyethylene glycol may be PEG4.

[0039] Preferably, the target-binding molecule is a protein or nucleic acid. Examples of target-binding proteins (also called specific antigen-binding proteins) include, but are not limited to, immunoglobulins or antibodies, immunoglobulin Fc regions, immunoglobulin Fab regions, Fab', Fv, Fv-Fc, single-chain Fv(scFv), scFv-Fc, (scFv)2, diabodies, triabodies, tetrabodies, bispecific T cell engagers (BiTEs), inteins, VNAR domains, single-domain antibodies (sdAb), VH domains, and scaffold proteins (afibodies, centinrin, dalpin, etc.). Examples of target-binding nucleic acids include, but are not limited to, aptamers.

[0040] Preferably, the target-binding molecule-drug conjugate is a protein, and the anthracycline (PNU) derivative is incorporated, for example, at the N-terminus or C-terminus of the amino acid sequence of a specific antigen-binding protein, or is introduced by chemical modification of the protein, or is bound to a thiol-containing amino acid residue in the amino acid sequence of a protein with a thiol group. The thiol group can also be introduced into other target-binding molecules (e.g., nucleic acids).

[0041] Target-binding proteins (also called specific antigen-binding proteins) may be selected from a group including immunoglobulins or antibodies, immunoglobulin Fc regions, immunoglobulin Fab regions, Fab', Fv, Fv-Fc, single-chain Fv (scFv), scFv-Fc, (scFv)2, diabodies, triabodies, tetrabodies, bispecific T cell engagers (BiTEs), inteins, VNAR domains, single-domain antibodies (sdAbs), VH domains, or scaffold proteins (such as afibodies, centinrin, and dalpin).

[0042] In a preferred embodiment, the target binding molecule may include a specific antigen-binding protein that may contain an amino acid sequence represented by formula (III). In the JPEG0007923099000017.jpg9170 formula, FW1 is the framework area. CDR1 is a CDR sequence, FW2 is the framework domain, HV2 is a highly variable array, FW3a is the framework domain, HV4 is a highly variable array, FW3b is a framework domain, CDR3 is a CDR sequence, FW4 is the framework domain.

[0043] Preferably, the specific antigen-binding protein binds to receptor tyrosine kinase-like orphan receptor 1 (ROR1). Preferably, the ROR1-specific antigen-binding molecule does not bind to receptor tyrosine kinase-like orphan receptor 2 (ROR2). Preferably, the ROR1-specific antigen-binding molecule binds to both human ROR1 and mouse ROR1 (mROR1). Preferably, the ROR1-specific antigen-binding molecule binds to deglycosylated ROR1. Such molecules are described in concurrently pending international patent application no. PCT / EP2018 / 086823 (the contents of which are incorporated herein by reference).

[0044] More preferably, a ROR1-specific antigen-binding protein, YMESLHMQGEIENQI (Sequence ID 34), CQPWNSQYPHTHTFTALRFP (Sequence ID 35), RSTIYGSRLRIRNLDTTDTGYFQ (Sequence ID 36), QCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYE (Sequence No. 37), It does not bind to the linear peptide sequence selected from the available options.

[0045] In this embodiment of the target-binding molecule-drug conjugate, the specific antigen-binding protein is FW1 is a framework region of 20-28 amino acids. CDR1 is a CDR sequence selected from DTSYGLYS (SEQ ID NO: 1), GAKYGLAA (SEQ ID NO: 2), GAKYGLFA (SEQ ID NO: 3), GANYGLAA (SEQ ID NO: 4), or GANYGLAS (SEQ ID NO: 5). FW2 is a framework region of 6-14 amino acids. HV2 is a hypervariable sequence selected from TTDWERMSIG (sequence number 6), SSNQERISIS (sequence number 7), or SSNKEQISIS (sequence number 8). FW3a is a framework region of 6-10 amino acids. HV4 is a hypervariable sequence selected from NKRAK (sequence number 9), NKRTM (sequence number 10), NKGAK (sequence number 11), or NKGTK (sequence number 12). FW3b is a framework region consisting of 17-24 amino acids. CDR3 is a CDR sequence selected from QSGMAISTGSGHGYNWY (SEQ ID NO: 13), QSGMAIDIGSGHGYNWY (SEQ ID NO: 14), YPWAMWGQWY (SEQ ID NO: 15), VFMPQHWHPAAHWY (SEQ ID NO: 16), REARHPWLRQWY (SEQ ID NO: 17), or YPWGAGAPWLVQWY (SEQ ID NO: 18). FW4 is a framework region of 7-14 amino acids, or a functional variant thereof that has at least 45% sequence identity with respect to them. It may include.

[0046] More preferably, FW1 is selected from ASVNQTPRTATKETGESLTINCVLT (SEQ ID NO: 19), AKVDQTPRTATKETGESLTINCVLT (SEQ ID NO: 20), TRVDQTPRTATKETGESLTINCVVT (SEQ ID NO: 21), TRVDQTPRTATKETGESLTINCVLT (SEQ ID NO: 22), ASVNQTPRTATKETGESLTINCVVT (SEQ ID NO: 23), TRVDQSPSSLSASVGDRVTITCVLT (SEQ ID NO: 24), or ASVTQSPRSASKETGESLTITCRVT (SEQ ID NO: 56). FW2 is selected from TSWFRKNPG (sequence number 25) or TYWYRKNPG (sequence number 26), FW3a is selected from GRYVESV (sequence number 27) or GRYSESV (sequence number 28), FW3b is selected from SFSLRIKDLTVADSATYYCKA (SEQ ID NO: 29), SFTLTISSLQPEDSATYYCRA (SEQ ID NO: 30), SFTLTISSLQPEDFATYYCKA (SEQ ID NO: 31), or SFSLRISSLTVEDSATYYCKA (SEQ ID NO: 57), and FW4 is selected from DGAGTVLTVN (SEQ ID NO: 32), DGAGTKVEIK (SEQ ID NO: 33), or DGQGTKLEVK (SEQ ID NO: 58), or is a functional variant thereof having at least 45% sequence identity.

[0047] More preferably, a ROR1-specific antigen-binding molecule ASVNQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKRAKSFSLRIKDLTVADSATYYCKAQSGMAISTGSGHGYNWYDGAGTVLTVN(Sequence ID 39); AKVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKRAKSFSLRIKDLTVADSATYYCKAQSGMAIDIGSGHGYNWYDGAGTVLTVN(Sequence ID 40); TRVDQTPRTATKETGESLTINCVVTGAKYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWAMWGQWYDGAGTVLTVN(Sequence ID 41); TRVDQTPRTATKETGESLTINCVVTGAKYGLFATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAVFMPQHWHPAAHWYDGAGTVLTVN(Sequence ID 42); TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVN(Sequence ID 43); ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWLVQWYDGAGTVLTVN(Sequence ID 44); TRVDQSPSSLSASVGDRVTITCVLTGANYGLASTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK(Sequence ID 45); TRVDQSPSSLSASVGDRVTITCVLTGANYGLASTYWYRKNPGSSNQERISISGRYSESVNKRTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK(Sequence ID 46); TRVDQSPSSLSASVGDRVTITCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFTLTISSLQPEDFATYYCKAREARHPWLRQWYDGAGTKVEIK(Sequence ID 47); TRVDQSPSSLSASVGDRVTITCVLTDTSYGLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK(Sequence ID 48); TRVDQSPSSLSASVGDRVTITCVLTDTSYGLYSTYWYRKNPGTTDWERMSIGGRYSESVNKGAKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK(Sequence ID 49); ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPWLVQWYDGQGTKLEVK(Sequence ID 59); Or, it includes an amino acid sequence selected from functional variants thereof that have at least 45% sequence identity with respect to them.

[0048] ROR1-specific antigen-binding proteins can be humanized. ROR1-specific antigen-binding proteins can be deimmunized.

[0049] The ROR1-specific antigen-binding molecule may also be part of a fusion protein. A preferred fusion protein is a ROR1-specific antigen-binding molecule fused to an immunoglobulin Fc region. Preferably, the immunoglobulin Fc region is a human immunoglobulin Fc region. In some cases, the ROR1-specific antigen-binding molecule may be a dimer, trimer, or higher-order multimer. Such multimers may also be fusion proteins with other molecules, including but not limited to immunoglobulin Fc. The individual domains of the fusion protein may be linked by any linker. The linkers may include, but are not limited to, (G4S)5, PGVQPSPGGGGS (referred to as WbG4S) (SEQ ID NO: 50), and PGVQPAPGGGGS (referred to as WbG4SGM) (SEQ ID NO: 51).

[0050] Target-binding molecule-drug conjugates according to the above embodiments for use in therapeutic applications are also provided herein.

[0051] Furthermore, this specification provides target-binding molecule-drug conjugates according to the above embodiments for use in the treatment of cancer.

[0052] Furthermore, this specification provides the use of the above-described target-binding molecule-drug conjugate in the manufacture of a pharmaceutical product for the treatment of a disease in a patient who requires it.

[0053] Treatments for diseases in patients requiring treatment are also provided herein, and such treatments include administering to the patient a therapeutically effective dose of a target-binding molecule-drug conjugate according to the above embodiment. The disease may be cancer.

[0054] Preferably, the cancer is a ROR1-positive oncological type. More preferably, the cancer is selected from the group of cancers including lymphomas and leukemias, hematological cancers such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphoma (NHL), and acute myeloid leukemia (AML), as well as solid tumors including neuroblastoma, kidney cancer, lung cancer, colorectal cancer, ovarian cancer, pancreatic cancer, breast cancer, skin cancer, uterine cancer, prostate cancer, thyroid cancer, head and neck cancer, bladder cancer, stomach cancer, or liver cancer. The cancer may be mesothelioma or triple-negative breast cancer (TNBC). Mesothelioma may be pleural mesothelioma.

[0055] In the embodiments described above, the target-binding molecule is an antibody. In another embodiment described above, the target-binding molecule binds to HER-2. Preferably, the target-binding molecule is an antibody that binds to HER-2. More preferably, the antibody is trastuzumab or a derivative thereof.

[0056] Furthermore, this specification also provides pharmaceutical compositions comprising a target-binding molecule-drug conjugate as described in any of the above embodiments, and at least one other pharmaceutically acceptable component. [Brief explanation of the drawing]

[0057] [Figure 1] An example of a payload emitted from a Val-Cit(vc)PAB PNU conjugate. [Figure 2] The PNU derivative of the present invention. [Figure 3] Efficacy of B1hFc-na-EDA-PNU conjugate in killing ROR1-positive PA-1 cell lines compared to unbound control (2VhFc-na-EDA-PNU).

[0058] [Figure 4] Efficacy of the B1hFc-va-EDA-PNU conjugate in killing ROR1-positive PA-1 cell lines compared to an unbound control (2VhFc-va-EDA-PNU). [Figure 5] Efficacy of the B1hFc-na-EDA-PNU conjugate in killing PA-1 cell lines with ROR1 knockout compared to an unbound control (2VhFc-va-EDA-PNU). The B1hFc-na-EDA-PNU conjugate in PA-1 cell lines with ROR1 knockout did not show cytotoxicity.

[0059] [Figure 6]Efficacy of B1hFc-va-EDA-PNU conjugate in killing PA-1 cell lines with ROR1 knockout compared to an unbound control (2VhFc-va-EDA-PNU). B1hFc-va-EDA-PNU conjugate in PA-1 cell lines with ROR1 knockout did not show cytotoxicity. [Figure 7] Efficacy of the B1hFc-na-EDA-PNU conjugate in killing Kasumi-2 compared to the unbound control (2VhFc-na-EDA-PNU). [Figure 8] Efficacy of the B1hFc-va-EDA-PNU conjugate in killing Kasumi-2 cell line compared to an unbound control (2VhFc-va-EDA-PNU).

[0060] [Figure 9] Efficacy of the B1hFc-na-EDA-PNU conjugate in killing MHH-ES1 cell lines compared to the unbound control (2VhFc-na-EDA-PNU). [Figure 10] Efficacy of the B1hFc-va-EDA-PNU conjugate in killing MHH-ES1 cell lines compared to an unbound control (2VhFc-va-EDA-PNU). [Figure 11] Efficacy of the B1hFc-vc-PAB-EDA-PNU conjugate in killing ROR1-positive PA-1 cell lines compared to an unbound control (2VhFc-vc-PAB-EDA-PNU).

[0061] [Figure 12] Efficacy of the B1hFc-vc-PAB-EDA-PNU conjugate in PA-1 cell lines with ROR1 knockout compared to an unbound control (2VhFc-va-EDA-PNU). The B1hFc-vc-PAB-EDA-PNU conjugate in PA-1 cell lines with ROR1 knockout did not show cytotoxicity. [Figure 13] Cell death data of tras(S442C)-vc-PAB-EDA-PNU bound to Her2-positive cell line SK-BR-3 and Her2-negative cell line MDA-MB-468. [Figure 14] Cell mortality rate of tras(S442C)-va-EDA-PNU conjugated to Her2-positive cell line SK-BR-3 and Her2-negative cell line MDA-MB-468.

[0062] [Figure 15] Efficacy of the P3A1hFc(S442C)-va-EDA-PNU conjugate in ROR1-positive PA-1 cell lines with ROR1 knockout and in the killing of PA-1 cell lines. [Figure 16] Binding of multimeric VNAR conjugates to cell surface ROR1 (A549 cancer cell line). (A) BA11-B1-D3 vs BA11-B1-D3-PNU conjugate; (B) P3A1-BA11-D3 vs P3A1-BA11-D3-PNU conjugate; (C) P3A1-BA11-P3A1 vs P3A1-BA11-P3A1-PNU conjugate. Binding to ROR1 is maintained even after binding with vc-PAB-EDA-PNU or va-EDA-PNU linker payloads.

[0063] [Figure 17] In vivo efficacy of B1-hFc-vc-PAB-EDA-PNU and B1-hFc-va-EDA-PNU evaluated in a PDX model of pleural mesothelioma in vehicle-treated mice. Absolute mean tumor volume was plotted as mean + / standard error (n=5). Treatment was initiated with a mean tumor volume of 124 mm3. Treatment with PDC molecules was administered by intravenous injection on days 1, 4, 7, 10, and 18 (indicated by arrows); all mice were pre-primed with mIgG 20 hours prior to the first PDC administration. Vehicle data were plotted on the final day all mice were alive and present in the group.

[0064] [Figure 18]In vivo efficacy of B1-hFc-vc-PAB-EDA-PNU and B1-hFc-va-EDA-PNU evaluated in a PDX model of TNBC in vehicle-treated mice. Absolute mean tumor volume was plotted as mean + / standard error (n=5). Treatment was initiated at a mean tumor volume of 180 mm3. Treatment with PDC molecules was administered by intravenous injection on days 2, 5, 8, 12, and 15 (indicated by arrows); all mice were pre-primed with mIgG 20 hours prior to the first PDC administration. Vehicle data were plotted on the final day all mice were alive and present in the group. Detailed description

[0065] Anthracyclines are a very interesting type of DNA intercalate toxin to use as a payload for drug conjugates, due to their proven efficacy as chemotherapeutic agents in cancer treatment. The anthracycline derivative PNU-159682 has been described as a metabolite of nemorubicin (Quintieri et al. (2005) Clin.11, 1608-1617). It has been reported to exhibit extremely high efficacy in in vitro cell killing in the pico-femtomole region using one ovarian (A2780) and one breast cancer (MCF7) cell line (WO2012 / 073217 A1).

[0066] The stability of chemically bound protein-drug conjugates is a critical consideration because unintended release of highly potent anthracycline toxins like PNU-159682 in patient circulation before targeting of tumor cells can lead to out-of-target effects and undesirable side effects. Several exemplary molecules released from PNU conjugates are shown in Figure 1, which illustrates the release of PNU159682 and EDA-PNU159682 derivatives from drug linkers containing various Val-Cit-PABs.

[0067] Therefore, a highly stable potential toxin that can link to the targeted protein is required to avoid or at least reduce undesirable side effects. Alternatively, the linker payload may be designed so that extracellular cleavage releases a derivative of the payload with attenuated potency. However, sufficient potency must be maintained so as not to negate the reduction in side effects, as higher doses are required to achieve the desired effect.

[0068] The ease of conjugation is a crucial factor in producing easily manufacturable products. The payload of the first embodiment uses a maleimide group, which can react directly with any available thiol group on the conjugate using standard conditions. Furthermore, the use of maleimide / thiol chemistry for conjugation allows for site-specific binding of the introduced thiol group, for example, to the side chain of a modified cysteine ​​residue in a protein sequence. In some embodiments described herein, cysteine ​​may be introduced to the C-terminus or N-terminus of a protein via the introduction of a his myc tag containing a modified cysteine ​​(example sequences include, but are not limited to, QACKAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 52) or QACGAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 53)).

[0069] Using non-selective labeling methods, antibody / protein-drug conjugates generated, for example, through reaction with amino functional groups in proteins, deliver products containing numerous different species with varying drug-to-antibody ratios. This affects the properties of the conjugate, including its potency and pharmacokinetic (PK) properties, which influence its in vivo efficacy and toxicity. Therefore, thiol-reactive payloads are crucial because they can react with naturally occurring cysteine ​​residues in proteins, or with cysteine ​​residues manipulated to specific sites at any point in the protein sequence, either using molecular biology / recombinant protein expression or chemical synthesis, or through expression, synthesis, or chemical modification of natural proteins, in high yield and in a simple manner.

[0070] The present invention provides anthracycline (PNU) derivatives suitable for use in drug conjugates, including but not limited to protein-drug conjugates (PDCs). Specifically, derivatives of PNU159682 are provided, which lack a C14 carbon and a bonded hydroxyl functional group, and an ethylenediamino (EDA) group forms part of the linker region between the C13 carbonyl and maleimide group of PNU159682. The maleimide group is present in the anthracycline (PNU) derivative of the first embodiment of the present invention and may be present in the anthracycline (PNU) derivative of the second embodiment of the present invention. Such payloads can react with a free thiol group on another molecule. If the free thiol is on a protein, a protein-drug conjugate (PDC) can be formed.

[0071] Surprisingly, derivatives of PNU159682 functionalized with ethylenediamino (EDA) groups and linked to thiol groups via maleimide groups exhibit higher stability with slightly lower potency compared to non-EDA payload or free payload derivatives. A more stable payload may be advantageous for reduced off-target effects, which in turn may result in reduced side effects and increased patient adherence.

[0072] The present invention further provides a target-binding molecule-drug conjugate comprising the anthracycline derivative conjugate and target-binding molecule described above.

[0073] According to another embodiment of this target-binding molecule-drug conjugate, the target-binding molecule is a protein, and an anthracycline (PNU) derivative is conjugated by one or more linkers to a thiol group introduced into the amino acid sequence of the protein. The introduced thiol may be introduced to the amino or carboxy terminus of the protein, or to the amino or carboxy terminus of a domain or its subunits. In another embodiment, the conjugation is to a thiol group in the sequence introduced to the amino or carboxy terminus of the protein, or to the amino or carboxy terminus of a domain or its subunits.

[0074] The target binding molecule may be a protein such as a VNAR domain derived from a novel or new antigen receptor (IgNAR) found in the serum of cartilaginous fish (Greenberg AS, et al., Nature, 1995. 374(6518): p. 168-173, Dooley, H., et al, Mol.Immunol, 2003.40(1): p. 25-33; Muller, MR, et al., mAbs, 2012.4(6): p. 673-685).

[0075] Therefore, the target binding molecule may include a specific antigen-binding protein containing the amino acid sequence represented by formula (III). In the JPEG0007923099000018.jpg8170 formula, FW1 is the framework area. CDR1 is a CDR sequence, FW2 is the framework domain, HV2 is a highly variable array, FW3a is the framework domain, HV4 is a highly variable array, FW3b is a framework domain, CDR3 is a CDR sequence, FW4 is the framework domain.

[0076] The framework region FW1 is preferably 20 to 28 amino acid lengths, more preferably 22 to 26 amino acid lengths, and even more preferably 23 to 25 amino acid lengths. In a particular preferred embodiment, FW1 is 26 amino acid lengths. In another preferred embodiment, FW1 is 25 amino acid lengths. In yet another preferred embodiment, FW1 is 24 amino acid lengths.

[0077] The CDR region CDR1 is preferably 7 to 11 amino acids long, and more preferably 8 to 10 amino acids long. In a particular preferred embodiment, CDR1 is 9 amino acids long. In another preferred embodiment, CDR1 is 8 amino acids long.

[0078] The framework region FW2 is preferably 6 to 14 amino acid long, and more preferably 8 to 12 amino acid long. In a particular preferred embodiment, FW2 is 12 amino acid long. In another preferred embodiment, FW2 is 10 amino acid long. In another preferred embodiment, FW2 is 9 amino acid long. In another preferred embodiment, FW2 is 8 amino acid long.

[0079] The hypervariable sequence HV2 is preferably 4 to 11 amino acids long, more preferably 5 to 10 amino acids long. In a particular preferred embodiment, HV2 is 10 amino acids long. In a particular preferred embodiment, HV2 is 9 amino acids long. In another preferred embodiment, HV2 is 6 amino acids long.

[0080] The framework region FW3a is preferably 6 to 10 amino acid lengths, more preferably 7 to 9 amino acid lengths. In a particular preferred embodiment, FW3a is 8 amino acid lengths. In a particular preferred embodiment, FW3a is 7 amino acid lengths.

[0081] The hypervariable sequence HV4 is preferably 3 to 7 amino acid long, more preferably 4 to 6 amino acid long. In a particular preferred embodiment, HV4 is 5 amino acid long. In another preferred embodiment, HV4 is 4 amino acid long.

[0082] The framework region FW3b is preferably 17 to 24 amino acids long, more preferably 18 to 23 amino acids long, and even more preferably 19 to 22 amino acids long. In a particular preferred embodiment, FW3b is 21 amino acids long. In another preferred embodiment, FW3b is 20 amino acids long.

[0083] The CDR region CDR3 is preferably 8 to 21 amino acids long, more preferably 9 to 20 amino acids long, and even more preferably 10 to 19 amino acids long. In a particular preferred embodiment, CDR3 is 17 amino acids long. In another preferred embodiment, CDR3 is 14 amino acids long. In yet another preferred embodiment, CDR3 is 12 amino acids long. In yet another preferred embodiment, CDR3 is 10 amino acids long.

[0084] The framework region FW4 is preferably 7 to 14 amino acid long, more preferably 8 to 13 amino acid long, and even more preferably 9 to 12 amino acid long. In a particular preferred embodiment, FW4 is 12 amino acid long. In another preferred embodiment, FW4 is 11 amino acid long. In yet another preferred embodiment, FW4 is 10 amino acid long. In yet another preferred embodiment, FW4 is 9 amino acid long.

[0085] All possible combinations and substitutions of the above-mentioned framework region, complementarity determination region, and hypervariable region are expressly intended herein.

[0086] Preferred VNAR domains for use in the present invention include B1, P3A1, D3, BA11, and E9, the sequences of which are described below. B1, P3A1, D3, and E9 bind to ROR1 (data is shown in concurrently pending international patent application number PCT / EP2018 / 086823 (published as WO 2019 / 122447), the contents of which are incorporated herein by reference). BA11 is a humanized VNAR that binds with high affinity to human serum albumin (Kovalenko et al., J. Biol. Chem., 2013 JBC). Furthermore, the non-binding VNAR domain 2V is also described below.

[0087] ● B1 is as follows: ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWLVQWYDGAGTVLTVN (Sequence ID 44) ● 2V is as follows: TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAQSLAISTRSYWYDGAGTVLTVN (Sequence ID 54) ● P3A1 is as follows: TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVN (Sequence ID 43) ● D3 is as follows: ASVNQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKRAKSFSLRIKDLTVADSATYYCKAQSGMAISTGSGHGYNWYDGAGTVLTVN (Sequence ID 39) ● BA11 is as follows: TRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIK (Sequence ID 55) ● E9 is as follows: AKVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKRAKSFSLRIKDLTVADSATYYCKAQSGMAIDIGSGHGYNWYDGAGTVLTVN (Sequence ID 40)

[0088] The VNAR domains for use in the present invention include the VNARs identified in SEQ ID NOs. 40-49 and 59. More preferred VNARs include the humanized VNARs identified in SEQ ID NOs. 45-49 and 59. A particularly preferred humanized VNAR is B1V15, which has the following amino acid sequence: ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPWLVQWYDGQGTKLEVK (SEQ ID NOs. 59).

[0089] The sequence identity referred to with respect to the molecules of the present invention may be determined at the level of individual CDRs, HVs, or FWs, or it may be determined over the length of the entire molecule. The described CDR, HV, and FW sequences may also be longer or shorter, whether due to the addition or deletion of amino acids at the N-terminus or C-terminus of the sequence, or due to the insertion or deletion of amino acids from the sequence.

[0090] Any portion of the specific binding protein may be modified to enable conjugation in the PDC of the present invention. In a preferred example, when an immunoglobulin Fc region is used, it may be modified to include a cysteine ​​residue as a binding site. Preferred introduced cysteine ​​residues include, but are not limited to, S252C and S473C (Kabat numbering), which correspond to S239C and S442C in EU numbering, respectively.

[0091] The target-binding molecule-drug conjugate may be any target-binding molecule-drug conjugate disclosed herein. For example, the target-binding molecule-drug conjugate may be selected from the group consisting of: ● B1-hFc-vc-PAB-EDA-PNU ● B1-hFc-va-EDA-PNU ● B1-hFc-na-EDA-PNU

[0092] B1-hFc-vc-PAB-EDA-PNU and B1-hFc-va-EDA-PNU have been shown herein to be particularly effective for use in the treatment of mesothelioma and TNBC. Preferably, the target-binding molecule-drug conjugate includes a PEG4 spacer. For example, the target-binding molecule-drug conjugate may be selected from the group consisting of: ● B1-hFc-PEG4-vc-PAB-EDA-PNU ● B1-hFc-PEG4-va-EDA-PNU ● B1-hFc-PEG4-na-EDA-PNU

[0093] Preferably, hFc contains a cysteine ​​residue introduced at S239C (EU numbering). Therefore, for example, the target-binding molecule-drug conjugate may be selected from the group consisting of: ● B1-hFc(S239C)-vc-PAB-EDA-PNU ● B1-hFc(S239C)-va-EDA-PNU ● B1-hFc(S239C)-na-EDA-PNU Target-binding molecule-drug conjugates can be selected from the following group: ● B1-hFc(S239C)-PEG4-vc-PAB-EDA-PNU ● B1-hFc(S239C)-PEG4-va-EDA-PNU ● B1-hFc(S239C)-PEG4-na-EDA-PNU

[0094] In one embodiment of the target-binding molecule-drug conjugate, the target-binding molecule is an antibody. In another embodiment of the target-binding molecule-drug conjugate, the target-binding molecule binds to HER-2. Preferably, the target-binding molecule is an antibody specific to HER-2.

[0095] Preferably, the target-binding molecule-drug conjugate includes a PEG4 spacer. For example, the target-binding molecule-drug conjugate may be selected from the group consisting of: ● Tras-PEG4-vc-PAB-EDA-PNU ● Tras-PEG4-va-EDA-PNU Target-binding molecule-drug conjugates can be selected from the following group: ● Tras(S442C)-PEG4-vc-PAB-EDA-PNU ● Tras(S442C)-PEG4-va-EDA-PNU

[0096] Any of the features described in relation to any of the above-described aspects of the present invention can be combined with other aspects of the present invention with necessary modifications.

[0097] definition As used herein, alkyl groups are linear or branched, substituted or unsubstituted (preferably unsubstituted) groups containing 1 to 40 carbon atoms. Alkyl groups may be substituted at any position. As used herein, the term "alkenyl" refers to a group derived from the elimination of a single hydrogen atom from a linear or branched aliphatic moiety having at least one carbon-carbon double bond. As used herein, the term "alkynyl" refers to a group derived from the elimination of a single hydrogen atom from a linear or branched aliphatic moiety having at least one carbon-carbon triple bond.

[0098] The terms "alkyl," "aryl," and "heteroaryl" also include polyvalent species such as alkylene, arylene, and "heteroarylene." Examples of alkylene groups include ethylene (-CH2-CH2-) and propylene (-CH2-CH2-CH2-). An exemplary arylene group is phenylene (-C6H4-), and an exemplary heteroarylene group is pyridinylene (-C5H3N-).

[0099] The aromatic ring is a cyclic aromatic group that may have 0, 1, 2 or more, preferably 0, 1 or 2 ring heteroatoms. The aromatic ring may be optionally substituted and / or condensed with one or more aromatic or non-aromatic rings (preferably aromatic), which may contain 0, 1, 2 or more ring heteroatoms, forming a polycyclic ring system.

[0100] Aromatic rings include both aryl and heteroaryl groups. Aryl and heteroaryl groups can be mononuclear, i.e., just one aromatic ring (e.g., phenyl or phenylene), or polynuclear, i.e., two or more aromatic rings that can be condensed (e.g., naphthyl or naphthylene), individually covalently bonded (e.g., biphenyl), and / or combinations of both condensed and individually linked aromatic rings. Preferably, the aromatic group is one in which the aryl or heteroaryl group is substantially conjugated substantially throughout the entire group. The aryl group may contain 5 to 40 ring carbon atoms, 5 to 25 carbon atoms, 5 to 20 carbon atoms, or 5 to 12 carbon atoms. The heteroaryl group may be a 5 to 40-membered, 5 to 25-membered, 5 to 20-membered, or 5 to 12-membered ring and contain one or more ring heteroatoms selected from N, O, S, and P. The aryl or heteroaryl may condense with one or more aromatic or non-aromatic rings (preferably aromatic rings) to form a polycyclic ring system.

[0101] The aryl and heteroaryl terms preferably represent monocyclic, dicyclic, or tricyclic aromatic or heteroaromatic groups having up to 25 ring atoms, which may include a fused ring and may be optionally substituted.

[0102] Preferred aryl groups include, but are not limited to, benzene, biphenylene, triphenylene, [1,1':3',1"]terphenyl-2'-ylene, naphthalene, anthracene, binaphthylene, phenanthrene, pyrene, dihydropyrene, chrysene, perylene, tetracene, pentacene, benzopyrene, fluorene, indene, indenofluorene, and spirobifluorene.

[0103] The preferred heteroaryl groups are not limited to five-membered rings such as pyrrole, pyrazole, silole, imidazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, furan, thiophene, selenophene, oxazole, isoxazole, 1,2-thiazole, 1,3-thiazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, as well as pyridine. Six-membered rings such as pyridazine, pyrimidine, pyrazine, 1,3,5-triazine, 1,2,4-triazine, 1,2,3-triazine, 1,2,4,5-tetrazine, 1,2,3,4-tetrazine, and 1,2,3,5-tetrazine, as well as carbazole, indole, isoindole, indoridine, indazole, benzimidazole, benzotriazole, purine, naphthimidazole, phenanthrimidazole, pyridimidazole, pyrazinimidazole, quinoxalineimidazole, benzoxazole, naphthoxazole, anthroxazole, phenanthroxazole, isoxazole, benzothiazole, benzofuran, isobenzofuran, dibenzofuran, quinoline, isoquinoline, pteridine, benzo-5 ,6-Quinoline, Benzo-6,7-Quinoline, Benzo-7,8-Quinoline, Benzoisoquinoline, Acridine, Phenothiazine, Phenoxazine, Benzopyridazine, Benzopyrimidine, Quinoxaline, Phenazine, Naphthyridine, Azacarbazole, Benzocarborin, Phenanthidine, Phenanthroline, Thieno[2,3b]thiophene, Thieno[3,2b]thiophene, Dithienohyridine, Isobenzothiophene, Dibenzothiophene, Benzothiadiazothiophene, 2,5-Dihydropyrrolo[3,4-c]pyrrole-1,4-dione (Diketopyrrolopyrrole, DPP), 2-Oxo-1H-Indole-3-Ilidene, [3,3'-Bipyrrolo[2,3b]pyridinylidene]-2,2'(1H,This includes condensation systems or combinations thereof such as 1'H)-dione (pyridine isoindigo) and (3E)-3-(2-oxo-1H-indole-3-ylidene)-1H-indole-2-one (isoindigo). The heteroaryl group may be substituted with alkyl, alkoxy, thioalkyl, fluoro, fluoroalkyl, or further aryl or heteroaryl substituents. Preferably, the heteroaryl group is thiophene.

[0104] Particularly preferred heteroatoms are selected from O, S, N, P, and Si, typically with hydrogen completing the valence of the heteroatoms in the molecule of the present invention, and for example, with N, one or two other groups may be involved, resulting in -NH- or -NH2.

[0105] As used herein, the term “optionally substituted” means that one or more hydrogen atoms in the optionally substituted moiety are substituted by a preferred substituent. Unless otherwise specified, an “optionally substituted” group may have preferred substituents at each of its substituted positions, and the substituents may be the same or different at all positions if two or more positions in any given structure are substituted by two or more substituents selected from the specified group. The substituent combinations envisioned by the present invention preferably result in the formation of stable compounds. As used herein, “stable” means a compound that is chemically viable and exists for a sufficiently long time at room temperature (i.e., 16–25°C) to enable their detection, isolation, and / or use in chemical synthesis.

[0106] Any of the above groups (including, for example, those described herein as "optionally substituted," alkyl, aryl, and heteroaryl groups) may optionally contain one or more substituents, preferably silyl, sulfo, sulfonyl, formyl, amino, imino, nitrilo, mercapto, cyano, nitro, halogen, -NCO, -NCS, -OCN, -SCN, and -C(=O)NR 0 R 00 -C(=O)X0 , C(=O)R 0 , -NR 0 R 00 , C 1-12 Alkyl, C 1-12 Alkenil, C 1-12 Alkinyl, C 6-12 Ariel, C 3-12 Cycloalkyl, heterocycloalkyl with 4-12 ring atoms, heteroaryl with 5-12 ring atoms, C 1-12 Alkoxy, hydroxy, C 1-12 Alkylcarbonyl, C 1-12 Alkoxycarbonyl, C 1-12 Alkylcarbonyloxy or C 1-12 Selected from alkoxycarbonyloxy, where one or more H atoms are optionally substituted with F or Cl and / or a combination thereof, X 0 is a halogen, and R 0 and R 00 This is independently H or optionally substituted C 1-12 It is alkyl. Any optional substituent can include any chemically conceivable combination in the same group and / or multiple of the aforementioned groups (for example, amino and sulfonyl represent a sulfamoyl group when directly bonded to each other). In one embodiment, the substituent is not acyl. As used herein, acyl refers to an acyl group which is a moiety derived by removing one or more hydroxyl groups from an oxo acid such as a carboxylic acid. This contains a double-bonded oxygen atom and an alkyl group.

[0107] In some embodiments, the group may be unsubstituted. For example, in one aspect of the present invention, the anthracycline (PNU) derivative may be of formula (I). In formula JPEG0007923099000019.jpg65170, [X] is any spacer selected from the group including unsubstituted alkyl groups, unsubstituted heteroalkyl groups, unsubstituted aryl groups, unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or combinations thereof. [L1] and [L2] are valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, -(CH2) n -,-(CH2CH2O) n - is any linker selected from the group consisting of any amino acid other than p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, glycine, and combinations thereof.

[0108] In embodiments where the group may be unsubstituted, [X] is preferably polyethylene glycol and Selected from the group including JPEG0007923099000020.jpg15170, JPEG0007923099000021.jpg6170 represents the bond site to the rest of the molecule, and [R] is any spacer selected from the group including unsubstituted alkyl groups, unsubstituted heteroalkyl groups, unsubstituted aryl groups, unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or combinations thereof.

[0109] Generally, the term PAB is intended to mean p-aminobenzyloxycarbonyl. Sometimes, in the literature, the term PAB may be used to refer to p-aminobenzyl. In this specification, PAB is intended to refer to p-aminobenzyloxycarbonyl.

[0110] The term "protein" generally refers to a group of amino acid residues linked together by peptide bonds. It is used interchangeably and means the same thing as peptides, oligopeptides, oligomers, or polypeptides, including glycoproteins and their derivatives. The term "protein" is also intended to include protein fragments, analogs, variants, and derivatives that possess essentially the same biological activity or function as a reference protein. These fragments, analogs, variants, or derivatives are essentially identical. Examples of protein analogs and derivatives include peptide nucleic acids and DARPin (designed ankyrin repeat protein).

[0111] The term “target-binding molecule” refers to any molecule that binds to a given target. In this context, “target” and “antigen” may be used interchangeably. Examples of target-binding molecules include native or recombinant proteins containing immunoglobulins or antibodies, immunoglobulin Fc regions, immunoglobulin Fab regions, Fab, Fab', Fv, Fv-Fc, single-chain Fv(scFv), scFv-Fc, (scFv)2, diabodies, triabodies, tetrabodies, bispecific T cell engagers (BiTEs), inteins, intein fusions, VNAR domains, single-domain antibodies (sdAb), VH domains, scaffold proteins (such as afibodies, centinrin, and dalpin), and nucleic acids containing aptamers or small molecules or natural products that have been developed to bind to a target or that bind naturally to a target.

[0112] Antigen-specific binding proteins can include any protein that binds to a given antigen. Preferred examples include immunoglobulins or antibodies, immunoglobulin Fc regions, immunoglobulin Fab regions, Fab', Fv, Fv-Fc, single-chain Fv(scFv), scFv-Fc, (scFv)2, diabodies, triabodies, tetrabodies, bispecific T cell engagers (BiTEs), inteins, intein fusions, VNAR domains, single-domain antibodies (sdAb), VH domains, or scaffold proteins (such as afibodies, centinrin, and dalpin). Particularly preferred examples include VNAR domains containing amino acid sequences derived from synthetic libraries of VNAR molecules or libraries derived from immunization of cartilaginous fish. The terms VNAR, IgNAR, and NAR can also be used interchangeably.

[0113] In this specification, amino acids are represented by either a single-letter code, a three-letter code, or both.

[0114] Chemical modifications of proteins and biomolecules for introducing thiols are well established. Methods include the reaction of an amine group with 2-iminothiolane (Trout's reagent), modification of the amine group with an NHS ester containing a heterobifunctional agent such as N-succinimidyl S-acetylthiolate (SATA) or N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), followed by treatment with hydroxylamine and a reducing agent, respectively, and cleavage of the modified intein fusion protein with cysteamine to generate C-terminal thiol proteins and peptides.

[0115] The term "affinity purification" refers to the purification of molecules based on the specific attraction or binding of a molecule to a chemical or binding partner, enabling the molecule to be separated from impurities by forming a combination or complex that allows the molecule to remain bound to or attracted to a partner portion.

[0116] The terms “complementarity-determining regions” or CDRs (i.e., CDR1 and CDR3) refer to amino acid residues in the VNAR domain whose presence is typically involved in antigen binding. Each VNAR has two CDR regions, typically identified as CDR1 and CDR3. Furthermore, each VNAR domain contains amino acids derived from a “hypervariable loop” (HV), which may also be involved in antigen binding. In some examples, the complementarity-determining region may contain amino acids from both the CDR region and the hypervariable loop. In other examples, antigen binding may involve only a single CDR or HV-derived residue. According to generally accepted nomenclature for VNAR molecules, there is no CDR2 region.

[0117] A "framework region" (FW) is a VNAR residue other than a CDR residue. Each VNAR typically has five framework regions, identified as FW1, FW2, FW3a, FW3b, and FW4.

[0118] The boundaries between the FW, CDR, and HV regions in the VNAR are not intended to be fixed, and therefore some variation in the length and composition of these regions is expected. This will be understood by those skilled in the art, particularly by referring to the work done in analyzing the technical domain. (Anderson et al., PLoS ONE (2016)11(8); Lui et al., Mol Immun (2014)59, 194-199; Zielonka et al., Mar Biotechnol (2015).17, (4)386-392; Fennell et al., J Mol Biol (2010)400.155-170; Kovalenko et al., J Biol Chem (2013)288.17408-17419; Dooley et al., (2006)PNAS 103(6).1846-1851). The molecules of the present invention are defined herein by reference to the FW, CDR, and HV regions, but are not limited to these strict definitions. Accordingly, variations in line with the understanding in the art of the structure of the VNAR domain are explicitly intended herein.

[0119] A "codon set" refers to a set of various nucleotide triplet sequences used to encode a desired variant amino acid. Sets of oligonucleotides can be synthesized, for example, by solid-phase synthesis, and include sequences representing all possible combinations of nucleotide triplets provided by the codon set, and encoding a desired group of amino acids. The standard form of codon designation is that of the IUB code, which is publicly known in the art and is described herein.

[0120] Codon sets are typically represented by three capital letters in italics, e.g., NNK, NNS, XYZ, DVK, etc. Therefore, a “non-random codon set” refers to a codon set encoding a selected amino acid that partially, preferably completely, satisfies the amino acid selection criteria described herein. The synthesis of oligonucleotides having selected nucleotide “degeneracy” at specific positions is well known in the art (e.g., the TRIM approach (Knappek et al.; J. Mol. Biol. (1999), 296, 57-86); Garrard & Henner, Genetics (1993), 128, 103). Such sets of oligonucleotides having a specific codon set can be synthesized using commercially available nucleic acid synthesizers (e.g., available from Applied Biosystems, Foster City, CA) or are commercially available (e.g., from Life Technologies, Rockville, MD). A synthesized set of oligonucleotides having a specific codon set typically contains multiple oligonucleotides with different sequences, the differences established by the codon set within the overall sequence. The oligonucleotides used in accordance with the present invention have sequences that enable hybridization to a VNAR nucleic acid template, and may also include restriction enzyme sites if convenient.

[0121] The terms "cells," "cell lines," and "cell cultures" are used interchangeably (unless the context indicates otherwise), and such designations include all progeny of cells or cell lines. Therefore, terms such as "transformed organisms" and "transformed cells" include primary target cells and cultures derived therefrom, regardless of the number of passages. It is also understood that, due to intentional or accidental mutations, not all progeny will have exactly the same DNA quantity. This includes mutant progeny that possess the same function or biological activity as those screened in the original transformed cells.

[0122] The "detection limit" for a chemical substance in a particular assay is the lowest concentration of that substance that can be detected above the background level for that assay. For example, in phage ELISA, the "detection limit" for a particular phage showing a specific antigen-binding fragment is the phage concentration at which that particular phage produces a higher ELISA signal than that produced by a control phage that does not show the antigen-binding fragment.

[0123] A "fusion protein" and a "fusion polypeptide" are polypeptides having two covalently linked parts, where each part is a polypeptide with different properties. These properties may be biological properties such as activity in vitro or in vivo. Alternatively, these properties may be simple chemical or physical properties such as binding to a target antigen or catalysis of a reaction. The two parts can be linked directly by a single peptide bond or via a peptide linker containing one or more amino acid residues. Generally, the two parts and the linker are in a leading frame relative to each other. Preferably, the two parts of the polypeptide are obtained from heterogeneous or different polypeptides.

[0124] In this specification, the term "fusion protein" generally refers to one or more proteins linked together by chemical means including hydrogen bonds or salt bridges, by peptide bonds obtained through protein synthesis, or both. Typically, fusion proteins are prepared by DNA recombination techniques and may be referred to herein as recombinant fusion proteins.

[0125] "Identity" refers to the relationship between two or more polypeptide sequences or polynucleotide sequences, determined by comparing their sequences. Identity also sometimes refers to the degree of sequence relevance (homology) between polypeptide or polynucleotide sequences, such as that determined by the fit between strings of such sequences. While many methods exist for measuring identity between two polypeptide or polynucleotide sequences, the methods commonly used to determine identity are systematized into computer programs. Preferred computer programs for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. (1990) 215, 403).

[0126] Preferably, the amino acid sequence of the protein has at least 45% identity at the amino acid level to the amino acid sequence disclosed herein, using the default parameters of the BLAST computer program provided by HGMP (Human Genome Mapping Project) (Atschul et al., J. Mol. Biol. (1990) 215, 403-410).

[0127] More preferably, the protein sequence may have identity at the nucleic acid or amino acid level with respect to the amino acid sequence of at least 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, and more preferably 95% (more preferably at least 96%, 97%, 98%, or 99%) as shown herein.

[0128] Furthermore, the protein may also contain sequences having at least 45%, 46%, 47%, 48%, 49%, 50%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequences disclosed herein, using the default parameters of the BLAST computer program provided by HGMP.

[0129] A "mutation" is a deletion, insertion, or substitution of a nucleotide relative to a reference nucleotide sequence, such as the wild-type sequence.

[0130] “Natural” or “naturally occurring” VNARs refer to VNARs identified from non-synthetic sources, such as tissue sources obtained ex vivo, or from the serum of animals of the Elasmobranchii subclass. These VNARs may include VNARs produced in any type of immunoassay, whether natural or induced. Natural VNARs include the amino acid and nucleotide sequences that constitute or encode these antibodies. As used herein, natural VNARs are distinct from “synthetic VNARs.” Synthetic VNARs refer to VNAR sequences that have been altered from a source or template sequence, for example, by substitution, deletion, or addition of one or more amino acids at specific positions with different amino acids, where different amino acids provide an antibody sequence different from the source antibody sequence.

[0131] The fragments, analogs, variants, or derivatives of this protein may be at least 25, preferably 30 or 40, or up to 50 or 100, or 60 to 120 amino acids long, depending on the length of the original protein sequence from which they are derived. In some cases, lengths of 90 to 120 or 100 to 110 amino acids may be advantageous.

[0132] The fragments, derivatives, variants, or analogs of this protein are (i) in which one or more amino acid residues are substituted with conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) one or more amino acid residues contain substituents; or (iii) additional amino acids are fused to a mature polypeptide, for example, a leader or auxiliary sequence used for polypeptide purification. Such fragments, derivatives, variants, and analogs are considered to be within the scope of the art in relation to the teachings herein.

[0133] Oligonucleotides are short-chain, single-chain, or double-chain polydeoxynucleotides that are chemically synthesized by known methods (such as phosphotriester, phosphite, or phosphoramidite chemistry using solid-phase techniques). Further methods include polymerase chain reaction (PCR), which is used when the entire nucleic acid sequence of a gene is known or when a nucleic acid sequence complementary to the coding strand is available. Alternatively, if the target amino acid sequence is known, the potential nucleic acid sequence can be inferred using known and preferred coding residues for each amino acid residue. Oligonucleotides can be purified on a polyacrylamide gel or molecular sizing column, or by precipitation. DNA is "purified" when it is separated from non-nucleic acid impurities (which may be polar, nonpolar, ionic, etc.).

[0134] A “variant” or “mutant” of a starting or reference polypeptide (e.g., a source VNAR or its CDR), such as a fusion protein (polypeptide) or a heterologous polypeptide (heterologous to a phage), is a polypeptide that (1) has a different amino acid sequence from that of the starting or reference polypeptide, and (2) is a polypeptide derived from the starting or reference polypeptide by either natural or induced mutagenesis. Such variants include, for example, deletions of residues in the amino acid sequence of the target polypeptide from the polypeptide, and / or insertions into the polypeptide, and / or substitutions of the polypeptide. For example, a fusion polypeptide of the present invention produced using an oligonucleotide containing a non-random codon set encoding a sequence having variant amino acids (with respect to the amino acids found at the corresponding positions in the source VNAR or antigen-binding fragment) is a variant polypeptide with respect to the source VNAR or antigen-binding fragment. Thus, a variant CDR means a CDR containing a variant sequence with respect to the starting or reference polypeptide sequence (e.g., the sequence of the source VNAR or antigen-binding fragment). In this context, a variant amino acid refers to an amino acid that differs from the amino acid at the corresponding position in the starting or reference polypeptide sequence (e.g., that of a source VNAR or antigen-binding fragment). Any combination of deletions, insertions, and substitutions may be made to arrive at the final variant or mutant construct, provided that the final construct possesses the desired functional properties. Amino acid changes can also alter the post-translational processes of the polypeptide (e.g., changes in the number or position of glycosylation sites).

[0135] The “wild-type” or “reference” sequence, or the sequence of a “wild-type” or “reference” protein / polypeptide (e.g., a coat protein, or a CDR of a source VNAR), can be the reference sequence from which a variant polypeptide is derived through the introduction of a mutation. Generally, the “wild-type” sequence for a given protein is the most common sequence in nature. Similarly, the “wild-type” gene sequence is the most commonly found sequence of that gene in nature. Mutations can be introduced into a “wild-type” gene (and therefore the protein it encodes) either through natural processes or human-induced means. The product of such processes is a “variant” or “mutant” form of the original “wild-type” protein or gene.

[0136] As used herein, the term “conjugation” can refer to any method of chemically linking two or more chemical moieties. Typically, conjugations are via covalent bonds. In the context of the present invention, at least one of the chemical moieties is a target binding molecule, and another or the molecule is a PNU derivative of the present invention. In some cases, the conjugation comprises two or more target binding molecules in addition to the PNU derivative of the present invention, in which case the conjugation may be direct between target binding molecules having a PNU derivative conjugated to one of the target binding molecules.

[0137] The phrase "selected from a group containing" may be replaced with the phrase "selected from a group consisting of" whenever it occurs in this specification, and vice versa.

[0138] The present invention will be further understood by referring to the following embodiments. [Examples]

[0139] Example 1: Characterization of PNU derivatives The PNU derivatives of the present invention were prepared according to standard synthesis methods. Mass spectrometry was used to confirm that the correct molecules were produced (Table 1). JPEG0007923099000022.jpg187170JPEG0007923099000023.jpg101170

[0140] The efficacy of the EDA-PNU159682 derivative (Figure 1) was tested in various cell lines. The results are summarized in Table 2. JPEG0007923099000024.jpg121170

[0141] Example 2: VNAR-hFc PNU Conjugate

[0142] Numerous VNAR-hFc-PNU conjugates were prepared, and PNU derivatives were investigated. Two VNARs were specific to ROR1 (B1 and P3A1). Furthermore, unbound VNAR (2V) was used as a control molecule.

[0143] VNAR was genetically fused to a modified hIgG1 Fc domain containing a cysteine ​​substitution at the hIgG1 Fc sequence, S239C (EU numbering), via the standard [G4S]3. The VNAR Fc fusion protein was expressed as a secreted protein in CHO K1 cells and purified from the culture medium using MabSelect® SuRe® (Evitria, Switzerland). The purified protein was analyzed by SEC (Advance Bio, Agilent), SDS-PAGE, and mass spectrometry to confirm sequence and protein integrity. Binding rates were determined using a Pioneer Surface Plasmon Resonance (SPR) instrument (SensiQ / Pall ForteBio) or a Biolayer Interferometry (BLI) Octet K2 system (ForteBio). ROR1-hFc or ROR2-hFc fusion proteins (extracellular domains) were immobilized on a COOH2 chip or AR2G sensor in sodium acetate pH 5 buffer using amine linkage. VNAR and VNAR-Fc molecules were tested at various concentrations, and Ka(M -1 s -1 ), Kd(s -1The ) and KD(nM) values ​​were determined for biolayer interference measurements using QDat software (SensiQ / Pall ForteBio) or Octet Data Analysis High Throughput software (ForteBio). ROR1 2A2 mAb (Biolegend) and ROR2 mAb (R&D Systems) were included as controls for positive / negative binding to ROR1 and ROR2. 2V is a control VNAR sequence derived from a naive VNAR library and therefore represents this protein class but has no known targets. Tables 3 and 3b summarize the surface plasmon resonance data for the affinity of these molecules for human ROR1 and human ROR2.

[0144] JPEG0007923099000025.jpg161170

[0145] The same process was repeated for P3A1 hFc(442) to obtain comparable binding data. For this derivative, a VNAR was genetically fused to a modified hIgG1 Fc domain containing a cysteine ​​substitution at the hIgG1 Fc sequence, S442C (EU numbering). Surface plasmon resonance data for the affinity of this molecule to human ROR1 and human ROR2 are shown in Table 3b. JPEG0007923099000026.jpg62170

[0146] These proteins were site-specifically labeled with maleimide PNU derivatives using partial reduction, refolding, and labeling methods adapted from the literature [Junutula et al., 2008 Nat Biotech; Jeffrey et al., 2013 Bioconj Chem] (Figures 1 and 2). Briefly, a 1 mg / ml VNAR hFc solution was prepared in PBS + 100 mM L-arginine pH 7.4 containing 1 mM EDTA. 20 molar equivalents of TCEP were added and incubated at 4°C for a minimum of 48 hours. 30 molar equivalents of DHAA were added, the pH was adjusted to 6.5, and incubated at room temperature for 1 hour. Refolded VNAR Fc S239C was broadly dialyzed or buffer-exchanged with PBS + 50 mM L-arginine, quantified by UV light, and then reacted overnight at room temperature with 4 or 5 molar equivalents of maleimide PNU solution. The conjugates were purified by SEC and analyzed by analytical HIC, analytical SEC, and LC-MS. Table 4 summarizes the prepared conjugates.

[0147] JPEG0007923099000027.jpg191170

[0148] The same process was repeated for the P3A1 hFc(442)-va-EDA-PNU conjugate. Table 4b summarizes the prepared conjugates. JPEG0007923099000028.jpg48170

[0149] In vitro cell viability assay for cancer cells treated with anti-ROR1 VNAR drug conjugates

[0150] Cells were seeded in a white, clear-bottomed 96-well plate (Coster) and incubated at 37°C, 5% CO2 for 24 hours. The following day, a dilution series was established for each test drug using ×10 working stocks. The dose-response X10 stocks were 10000, 5000, 1000, 500, 100, 50, 10, 5, 1, 0.5 nM, etc. 10 μL of the X10 stock solution was added to the cell plate using a multichannel pipette (90 μL per well). This resulted in 1:10 dilutions to wells and dose-responses ranging from 1000 nM (column 1) to 0.05 nM (column 10), or continued down to 0.5 fM if necessary for the most sensitive cell lines. 10 μL of vehicle control (PBS) was added to the control wells (columns 11 and 12). The plates were incubated at 37°C, 5% CO2 for 72–96 hours. The Promega Cell Titre Glo reagent was used to evaluate cell viability according to the manufacturer's instructions. Briefly, the assay plate was removed from the incubator and equilibrated to room temperature, after which 100 μl of room-temperature Cell Titre Glo reagent was added to each 100 μl assay well. The plate was placed on a plate shaker at 600 rpm for 2 minutes. After the plate was left at room temperature for a further 10 minutes, luminescence readings were measured using a Clariostar plate reader (BMG). Data were analyzed by calculating the mean for the untreated (vehicle only) control wells and determining the percentage of control for each treated well. The percentage of control data was then plotted against the Log[treatment] concentration, and IC50 values ​​were derived using nonlinear regression fitting in GraphPad Prism software.

[0151] The following cell lines were used: ● Kasumi-2 human B-cell leukemia progenitor cells; ● PA-1 - Human ovarian cancer cell line; ● PA-1 ROR1 ko-ROR1 knockout human ovarian cancer cell line; ● 697 - Human B-cell leukemia progenitor cells, and ● MHH-ES1 - Human Ewing sarcoma cell line. JPEG0007923099000029.jpg255170

[0152] JPEG0007923099000030.jpg71170

[0153] Tables 5 and 6, and Figures 3-12 and 15, show that ROR1 targeting a protein-drug conjugate using the payload of the present invention is highly potent in killing ROR1-expressing cancer cells in a ROR1-dependent manner, exhibiting a large window compared to the corresponding unbound protein-drug conjugate (2V hFc).

[0154] Table 7: Efficacy of uncoupled VNAR(2V)-hFc-PNU159682 conjugate 2V is a control VNAR sequence derived from a naive VNAR library, and therefore has no known targets and does not bind to cancer cell lines by flow cytometry. A 2V-hFc-PNU conjugate was constructed and evaluated for nonselective cell death using a panel of cancer cell lines, as previously described. JPEG0007923099000031.jpg107170

[0155] The data in Table 7 demonstrate that the PNU conjugate of the present invention consistently exhibits higher IC50 values ​​than the prior art PEG4-vc-PAB-DMAE-PNU159682. Therefore, the linker-payload produces a more stable conjugate and / or less potent byproducts, and these protein-drug conjugates should be less toxic to normal tissues.

[0156] Example 3: Trastuzumab Conjugate A trastuzumab mutant was produced in which a single serine residue in the Fc moiety (position 442) was replaced with a cysteine ​​residue. This produces a trastuzumab molecule with a unique thiol at position 442 of the heavy chain within the Fc moiety for binding. Such mutants are sometimes called trastuzumab S442C or tras(S442C).

[0157] Two of the novel PNU payloads were conjugated to this molecule via modified cysteine ​​using the method described above, yielding the corresponding conjugates in good overall yield. Table 8 below outlines the properties of these conjugates.

[0158] JPEG0007923099000032.jpg61170

[0159] Furthermore, the efficacy of the tras(S442C)-PNU conjugate in killing HER2-positive cell lines was investigated. Figures 13 and 14 show that both tras(S442C)-PNU conjugates selectively killed the HER2-positive cell line SK-BR-3 and had little efficacy against the HER2-negative cell line MDA-MB-468.

[0160] Example 4: VNAR-PNU Conjugate Multimeric and bi-paratopic VNAR constructs were constructed using a modified cysteine-containing C-terminal his myc tag for site-specific labeling. The proteins were treated with 2 mM TCEP and purified by IMAC.

[0161] The binding dynamics for the binding of multimeric VNAR proteins to ROR1-hFc or ROR2-hFc fusion proteins (extracellular domains) were determined, as previously described, using a Pioneer surface plasmon resonance (SPR) instrument (SensiQ / Pall ForteBio) or a biolayer interferometry (BLI) Octet K2 system (ForteBio). JPEG0007923099000033.jpg106170

[0162] The conjugate was bound using 4 equivalents of PNU at room temperature for 1 hour and purified by SEC. The final conjugate was analyzed by analytical HIC, analytical SEC, and LC-MS.

[0163] JPEG0007923099000034.jpg158170

[0164] The linkers between VNAR domains are (G4S)5 ([G4S]5); PGVQPSPGGGGS[(WbG4S)] (Sequence ID 50); and PGVQPAPGGGGS[(WbG4SGM)] (Sequence ID 51).

[0165] The payload was conjugated to a unique free thiol introduced into the C-terminal region of the protein by incorporating a C-terminal his myc tag containing a modified cysteine ​​(QACKAHHHHHHGAEFEQKLISEEDL of SEQ ID NO: 52 or QACGAHHHHHHGAEFEQKLISEEDL of SEQ ID NO: 53).

[0166] ROR1 hiThe binding of multimer conjugates to the surface of A549 lung adenocarcinoma cells was evaluated by flow cytometry. Adhered human cancer cells were isolated from tissue culture flasks by incubation with 0.1% EDTA / PBS solution at 37°C for ~10 minutes, or until cells easily separated. Cells were resuspended in 5 ml of ice-cold PBS / 2% FCS in a 15 ml tube and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was removed, and the cell precipitate was resuspended in 1-2 ml of PBS / 2% FCS. Cell counting was performed using a Z1 Coulter particle counter (Beckman Coulter), and 5 × 10^5 cells were dispensed into 96-well plates for each test sample. Cells were incubated on ice for 1 hour with 100 μl of VNAR (His6Myc labeled) or the corresponding VNAR conjugate at the indicated concentrations, and with the control. Sample plates were centrifuged at 2000 rpm for 5 minutes. The supernatant was removed and the cell pellet was washed by resuspending it in 0.25 mL of ice-cold PBS / 2% FCS using a multichannel pipette. The sample was again centrifuged at 2000 rpm for 5 minutes at 4°C. The supernatant was removed and two further washes were performed as described. After the final wash and centrifugation, excess liquid was removed by blotting the plate onto tissue paper. 100 μl of anti-x6His tagged Ab (Abcam) was added per cell pellet in an appropriate amount to bind to VNAR (His6Myc tagged) or the corresponding VNAR conjugate, and incubated on ice for 30 minutes. The washing procedure was performed as described above. Binding of VNAR (His6Myc tagged) and the corresponding VNAR conjugate was detected by incubating with the appropriate sample on ice in the dark for 30 minutes using PE-anti-mouse antibody (JIR). The washing procedure was performed as described above. The cell pellet was resuspended in 0.3 ml of ice-cold PBS / 2% FCS, left on ice in the dark, and then analyzed using a Merck-Millipore Guava EasyCyte HT flow cytometer.

[0167] Figure 16 shows that VNAR multimers bound to either vc-PAB-EDA-PNU or va-EDA-PNU maintain their binding to ROR1 on the surface of cancer cells.

[0168] Example 5: Cathepsin B treatment of protein-PNU conjugates When the VNAR-hFc-vc-PAB-EDA-PNU conjugate and the conjugate of the corresponding multimeric protein were treated with cathepsin B, the free EDA-PNU159682 derivative was quantitatively released. On the other hand, the VNAR-hFc-va-EDA-PNU conjugate and the conjugate of the corresponding multimeric protein were completely stable to cathepsin B treatment. JPEG0007923099000035.jpg85170

[0169] Only vc-PAB-EDA-PNU releases its payload upon treatment with CatB, as expected for the conditions used in this in vitro assay. The release is quantitative, and since the conjugate has an antibody-to-drug ratio of 2, a concentration of the released payload versus twice the concentration of the conjugate is expected after CatB treatment. This expectation is consistent with the data for the vc-PAB-EDA-PNU conjugate shown in Table 11.

[0170] Example 6: Plasma stability of protein-PNU conjugates Various P3A1-hFc-PNU conjugates were incubated in mouse plasma at 37°C at a final protein concentration of 4 μM. The samples were analyzed over time by LC-MS, and the amounts of released PNU159682 and EDA-PNU159682 derivatives were quantified relative to a calibration standard. JPEG0007923099000036.jpg53170

[0171] As shown in Table 12, the conjugate with the va-EDA-PNU159682 payload exhibited excellent mouse plasma stability, with little to no PNU derivatives released over time. For both the vc-PAB-DMAE-PNU and vc-PAB-EDA-PNU conjugates, some free payload release was detectable over time, with 217 nM of PNU159682 and 114 nM of EDA-PNU159682 derivatives detected at 120 hours, respectively. Note that in a parallel study, the inventors calculated the mouse plasma half-lives of free PNU159682 and EDA-PNU159682 derivatives to be 33 hours and 116 hours, respectively. This indicates that the absolute amount of free PNU payload released for the vc-PAB-DMAE-PNU conjugate is underestimated compared to the vc-PAB-EDA-PNU conjugate.

[0172] Various conjugates were incubated in human plasma at 37°C for 168 hours with a final concentration of 4 μM or 2 μM protein. Samples were analyzed by LC-MS as a function of time up to 168 hours, and the amounts of released PNU159682 and EDA-PNU159682 derivatives were quantified by reference to a correction standard. JPEG0007923099000037.jpg127170

[0173] As shown in Table 13, the conjugate exhibited excellent human plasma stability, and no detectable amounts of PNU derivative were released over the course of the study. The half-life of EDA-PNU in human plasma is 172.25 hours (average of four different experiments).

[0174] Example 7: In vivo effects of protein-drug conjugates in a patient-derived xenograft model of pleural mesothelioma. An efficacy trial in a xenograft model of pleural mesothelioma derived from a ROR1+ PXF-1118 patient was conducted by Charles River Laboratories (Freiburg).

[0175] Tumor fragments obtained from xenografts in successive passages of nude mice were used in female NMRI nu / nu mice (Cr:NMRI-Foxn1). nu The tumor graft was subcutaneously transplanted to the animal. In a sufficient number of animals, the tumor graft was 50-250 mm. 3 Preferably 150-200 mm 3 Mice were monitored until the study volume inclusion criteria were met. Mice were randomly assigned to treatment groups to ensure statistically minimal differences in tumor volume between groups. Randomization took place on day 0 of the experiment. Mice were treated intravenously with 0.3 mg / kg of vehicle or protein-drug conjugate B1-hFc-vc-PAB-EDA-PNU or B1-hFc-va-EDA-PNU on days 1, 4, 7, 10, and 18. All mice received single-dose priming with mouse IgG administered intravenously (iv) at 29 mg / kg 20 hours prior to the first PDC administration.

[0176] Absolute tumor volume (ATV) was determined by two-dimensional measurement using a digital caliper on the day of randomization, followed by measurements three times per week. Tumor volume was calculated according to the following formula: Tumor volume = (L × W) 2 ) × 0.5

[0177] Here, L = maximum diameter, W = tumor width (vertical diameter) (mm).

[0178] The animals' weight was measured three times a week, always on the day of administration. The mice were observed and recorded for changes in physical appearance, behavior, adverse clinical signs, and overall well-being, in accordance with the best local veterinary practice guidelines.

[0179] Figure 17 shows the effect of protein-drug conjugates on tumor growth compared to vehicle controls. Both B1-hFc-vc-PAB-EDA-PNU and B1-hFc-va-EDA-PNU were well-tolerated and significantly inhibited tumor growth in this ROR1+ pleural mesothelioma PDX model. PDCs targeting tumors derived from ROR1+ve pleural mesothelioma patients exhibit favorable antitumor effects.

[0180] Example 8: In vivo effects of protein-drug conjugates in a patient-derived xenograft model of triple-negative breast cancer (TNBC). XenTech (Paris) conducted an efficacy trial in a TNBC xenograft model derived from ROR1+ HBCx-28 patients.

[0181] Outcrossed athymoid (nu / nu) female mouse (HSD: Nude-Foxn1) nu The same in vivo passaged tumor was subcutaneously transplanted into the animals. The tumor graft was 60-200 mm in a sufficient number of animals. 3 Preferably 75-196mm 3 Mice were monitored until the study volume inclusion criteria were met. Mice were randomly assigned to treatment groups to ensure statistically minimal differences in tumor volume between groups. Randomization took place on day 0 of the experiment. Mice were treated intravenously on days 2, 5, 8, 12, and 15 with a vehicle or protein-drug conjugate B1-hFc-vc-PAB-EDA-PNU or B1-hFc-va-EDA-PNU at a dose of 0.3 mg / kg, with all mice primed with mouse IgG 20 hours prior to treatment. Tumor volume was assessed by measuring the vertical tumor diameter using calipers three times per week during the experiment until day 55, and then twice per week until the end of the experiment (i.e., Day 103), and weighed. Absolute tumor volume (ATV) was expressed using the formula TV(mm²). 3 ) = [Length (mm) × Width (mm)] 2 The calculation was performed using ] × 0.5, where length and width are the longest and shortest vertical diameters of the tumor, measured vertically, respectively. All animals were weighed at the same time as tumor size measurement. Mice were observed and recorded daily for changes in physical appearance, behavior, adverse general conditions, and overall welfare status in accordance with local welfare and best veterinary practice guidelines.

[0182] Figure 18 shows the effect of protein-drug conjugates on tumor growth compared to vehicle controls. Both B1-hFc-vc-PAB-EDA-PNU and B1-hFc-va-EDA-PNU were well-tolerated and showed statistically significant in vivo effects in this ROR1+ TNBC PDX model. Furthermore, both drugs resulted in complete and sustained tumor regression, and sustained tumor "cure" was confirmed during the study period (103 days). PDC molecules targeting tumors derived from ROR1+ve TNBC patients exhibit robust antitumor effects, resulting in complete and sustained tumor regression (including cure).

Claims

1. An anthracycline (PNU) derivative of formula (I), In the formula, [X] does not exist. The anthracycline (PNU) derivative comprises [L1], [L2], or [L1] and [L2]. [L1] and [L2] are (1) Valine (Val)-alanine (Ala), (2) Asparagine (Asn)-Ala, (3)-(CH ２ CH ２ O) ｎ -CH₂CH₂CO-, wherein n=4, (4) Val-citrulline (Cit)-p-aminobenzyloxycarbonyl (PAB), (5) Val-Ala-PAB, and (6) Ala-Ala-Asn-PAB A linker selected from the group consisting of the following: An anthracycline (PNU) derivative of formula (I).

2. The anthracycline derivative according to claim 1, wherein the PNU derivative has a structure selected from the following:

3. A target-binding molecule-drug conjugate comprising a specific antigen-binding protein and an anthracycline (PNU) derivative, wherein the target-binding molecule-drug conjugate has formula (II), In the formula, [X] does not exist. The target-binding molecule-drug conjugate of formula (II) comprises [L1], [L2], or [L1] and [L2], [L1] and [L2] are (1) Valine (Val)-alanine (Ala), (2) Asparagine (Asn)-Ala, (3)-(CH ２ CH ２ O) ｎ -CH 2 CH 2 CO-, wherein n=4, (4) Val-citrulline (Cit)-p-aminobenzyloxycarbonyl (PAB), (5) Val-Ala-PAB, and (6) Ala-Ala-Asn-PAB A linker selected from the group consisting of, Y is the target-binding molecule, a target-binding molecule-drug conjugate.

4. The target-binding molecule-drug conjugate according to claim 3, wherein the PNU derivative has a structure selected from the following.

5. The target-binding molecule-drug conjugate according to claim 3 or 4, wherein the target-binding molecule is a protein, and the anthracycline (PNU) derivative is bound to a thiol-containing amino acid residue in the amino acid sequence of the protein, or the PNU derivative is bound via a thiol moiety incorporated by chemical modification at the N-terminus or C-terminus of the amino acid sequence of the protein.

6. The target-binding molecule is an immunoglobulin or antibody, an immunoglobulin Fc region, an immunoglobulin Fab region, Fab', Fv, Fv-Fc, single-chain Fv (scFv), scFv-Fc, (scFv) ２ A target-binding molecule-drug conjugate according to any one of claims 3 to 5, wherein the binding protein is selected from the group comprising a diabody, triabody, tetrabody, bispecific T cell engager (BiTE), intein, VNAR domain, single-domain antibody (sdAb), VH domain, or scaffold protein.

7. A target-binding molecule-drug conjugate according to any one of claims 3 to 6, wherein the target-binding molecule binds to receptor tyrosine kinase-like orphan receptor 1 (ROR1).

8. The target-binding molecule-drug conjugate according to any one of claims 3 to 6, wherein the target-binding molecule is a specific antigen-binding protein comprising an amino acid sequence represented by formula (III). In the formula, FW1 is the framework domain, CDR1 is the CDR sequence, FW2 is the framework domain, HV2 is a highly variable array, FW3a is the framework domain, HV4 is a highly variable array, FW3b is the framework domain, CDR3 is a CDR sequence, FW4 is the framework domain.

9. The target-binding molecule-drug conjugate according to claim 8, wherein a specific antigen-binding protein binds to receptor tyrosine kinase-like orphan receptor 1 (ROR1).

10. The target-binding molecule-drug conjugate according to claim 9, wherein the ROR1-specific antigen-binding protein does not bind to receptor tyrosine kinase-like orphan receptor 2 (ROR2).

11. The target-binding molecule-drug conjugate according to claim 9 or 10, wherein the ROR1-specific antigen-binding protein binds to both human ROR1 and mouse ROR1 (mROR1).

12. A target-binding molecule-drug conjugate according to any one of claims 7 to 11, wherein a ROR1-specific antigen-binding protein binds to deglycosylated ROR1.

13. ROR1-specific antigen-binding protein, YMESLHMQGEIENQI (Sequence ID 34), CQPWNSQYPHTHTFTALRFP (Sequence ID 35), RSTIYGSRLRIRNLDTTDTGYFQ (Sequence ID 36), QCVATNGKEVVSSTGVLFVKFGPPPPTASPGYSDEYE (Sequence No. 37), A target-binding molecule-drug conjugate according to claim 9 or 10, which does not bind to a linear peptide sequence selected from.

14. A target-binding molecule-drug conjugate according to any one of claims 9 to 13, FW1 is a framework region of 20-28 amino acids. CDR1 is a CDR sequence selected from DTSYGLYS (SEQ ID NO: 1), GAKYGLAA (SEQ ID NO: 2), GAKYGLFA (SEQ ID NO: 3), GANYGLAA (SEQ ID NO: 4), or GANYGLAS (SEQ ID NO: 5). FW2 is a framework region consisting of 6 to 14 amino acids. HV2 is a hypervariable sequence selected from TTDWERMSIG (SEQ ID NO: 6), SSNQERISIS (SEQ ID NO: 7), or SSNKEQISIS (SEQ ID NO: 8). FW3a is a framework region of 6 to 10 amino acids. HV4 is a hypervariable sequence selected from NKRAK (sequence number 9), NKRTM (sequence number 10), NKGAK (sequence number 11), or NKGTK (sequence number 12). FW3b is a framework region consisting of 17-24 amino acids. CDR3 is a CDR sequence selected from QSGMAISTGSGHGYNWY (SEQ ID NO: 13), QSGMAIDIGSGGHGYNWY (SEQ ID NO: 14), YPWAMWGQWY (SEQ ID NO: 15), VFMPQHWHPAAHWY (SEQ ID NO: 16), REARHPWLRQWY (SEQ ID NO: 17), or YPWGAGAAPWLVQWY (SEQ ID NO: 18). A target-binding molecule-drug conjugate in which FW4 is a framework region of 7-14 amino acids, or a functional variant thereof having at least 45% sequence identity.

15. A target-binding molecule-drug conjugate according to any one of claims 8 to 14, FW1 is selected from ASVNQTPRTATKETGESLTIINCVLT (SEQ ID NO: 19), AKVDQTPRTATKETGESLTIINCVLT (SEQ ID NO: 20), TRVDQTPRTATKETGESLTIINCVVT (SEQ ID NO: 21), TRVDQTPRTATKETGESLTIINCVLT (SEQ ID NO: 22), ASVNQTPRTATKETGESLTIINCVVT (SEQ ID NO: 23), TRVDQSPSSLSASVGDRVTITTCVLT (SEQ ID NO: 24), or ASVTQSPRSASKETGESLTITCRVLT (SEQ ID NO: 56). FW2 is selected from TSWFRKNPG (Sequence ID 25) or TYWYRKNPG (Sequence ID 26), FW3a is selected from GRYVESV (SEQ ID NO: 27) or GRYSESV (SEQ ID NO: 28), FW3b is selected from SFSLRIKDLTVADSATYYCKA (SEQ ID NO: 29), SFTLTISSLQPEDSATYYCRA (SEQ ID NO: 30), SFTLTISSLQPEDFATYYCKA (SEQ ID NO: 31), or SFSLRISSLTVEDSATYYCKA (SEQ ID NO: 57), and A target-binding molecule-drug conjugate in which FW4 is selected from DGAGTTVLTVN (SEQ ID NO: 32), DGAGTKVEIK (SEQ ID NO: 33), or DGQGTKLEVK (SEQ ID NO: 58), or a functional variant thereof having at least 45% sequence identity.

16. ROR1-specific antigen-binding molecules, ASVNQTPRTATKETGESLTIINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKRAKSFSLRIKDLTVADSATYYCKAQSGMAISTGSGHGYNWYDGAGTVLTVN (Sequence ID 39); AKVDQTPRTATKETGESLTIINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKRAKSFSLRIKDLTVADSATYYCKAQSGMAIDIGSGHGYNWYDGAGTVLTVN (Sequence ID 40); TRVDQTPRTATKETGESLTIINCVVTGAKYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWAMWGQWYDGAGTVLTVN (Sequence ID 41); TRVDQTPRTATKETGESLTIINCVVTGAKYGLFATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAVFMPQHWHPAAHWYDGAGTVLTVN (Sequence ID 42); TRVDQTPRTATKETGESLTIINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKARHPWLRQWYDGAGTVLTVN (Sequence ID 43); ASVNQTPRTATKETGESLTIINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAAPWLVQWYDGAGTVLTVN (Sequence ID 44); TRVDQSPSSLSASVGDRVTITCVLTGANYGLASTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAYPWGAGAAPWLVQWYDGAGTKVEIK (Sequence ID 45); TRVDQSPSSLSASVGDRVTITCVLTGANYGLASTYWYRKNPGSSNQERISISGRYSESVNKRTMSFTLTISSLQPEDSATYYCRAYPWGAGAAPWLVQWYDGAGTKVEIK (Sequence ID 46); TRVDQSPSSLSASVGDRVTITCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFTLTISSLQPEDFATYYCKARARPWLRQWYDGAGTKVEIK (Sequence ID 47); TRVDQSPSSLSASVGDRVTITCVLTDTSYGLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (Sequence ID 48); TRVDQSPSSLSASVGDRVTITCVLTDTSYGLYSTYWYRKNPGTTDWERMSIGGRYSESVNKGAKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (Sequence ID 49); ASVTQSPRSASKETGESLTITCCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAAPWLVQWYDGQGTKLEVK (Sequence ID 59); A target-binding molecule-drug conjugate according to any one of claims 9 to 15, comprising an amino acid sequence selected from functional variants thereof having at least 45% sequence identity thereto.

17. A target-binding molecule-drug conjugate according to any one of claims 9 to 16, wherein the ROR1-specific antigen-binding protein is humanized.

18. A target-binding molecule-drug conjugate according to any one of claims 9 to 16, wherein the ROR1-specific antigen-binding protein is deimmunized.

19. A target-binding molecule-drug conjugate according to any one of claims 7 to 18, for use in therapeutic purposes.

20. A target-binding molecule-drug conjugate according to any one of claims 7 to 18, for use in the treatment of cancer.

21. A target-binding molecule-drug conjugate according to any one of claims 3 to 6, wherein the target-binding molecule is an antibody.

22. The target-binding molecule-drug conjugate according to claim 21, wherein the antibody is an antibody that binds to HER-2.

23. The target-binding molecule-drug conjugate according to claim 22, wherein the antibody is trastuzumab or a derivative thereof.

24. A pharmaceutical composition comprising a target-binding molecule-drug conjugate according to any one of claims 7 to 18 or 21 to 23, and at least one other pharmaceutically acceptable component.

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