CN114569739A

CN114569739A - Antibody drug conjugates

Info

Publication number: CN114569739A
Application number: CN202111446643.5A
Authority: CN
Inventors: 丰巍伟; 魏强强; 张喜全; 陈天玺; 唐小齐; 赵伟; 刘铮; 赵娜; 刘飞; 张兵; 王�华; 盛化策; 陈晓; 刘杰
Original assignee: Chia Tai Tianqing Pharmaceutical Group Co Ltd
Current assignee: Chia Tai Tianqing Pharmaceutical Group Co Ltd
Priority date: 2020-12-01
Filing date: 2021-11-30
Publication date: 2022-06-03

Abstract

The invention provides an antibody drug conjugate which specifically comprises an antibody part, an intermediate linker part and a cytotoxic drug part which are connected, wherein the antibody part is an antibody aiming at a HER2 target, and the cytotoxic drug part is a camptothecin topoisomerase I inhibitor or a derivative thereof. The antibody drug conjugate of the present invention can be used for the prevention or treatment of cancer.

Description

Antibody drug conjugates

Technical Field

The present invention relates to antibody drug conjugates comprising a linked antibody moiety, an intermediate linker moiety and a cytotoxic drug moiety. The invention also relates to application of the antibody drug conjugate in preparing a drug for preventing and treating cancer.

Background

Antibody-Drug conjugates (ADCs) are a class of drugs that combine the high specificity of therapeutic antibodies with the high killing activity of cytotoxic drugs, where the Antibody moiety is linked to the cytotoxic Drug moiety through an intermediate linker moiety. At least eight ADC drugs are currently on the world, wherein antibody parts of brentuximab vedotin, polatuzumab vedotin and enfortuzumab vedotin are respectively directed to CD30, CD79b and Nectin-4, antibody parts of trastuzumab emtansine and trastuzumab derxecan be directed to HER2 target, antibody parts of gemtuzumab ozogamicin and inotuzumab ozogamicin are respectively directed to CD33 and CD22 target, and antibody part of sacituzumab govitetecan is directed to TROP2 target. Cytotoxic drug moiety: brentuximab vedotin, polatuzumab vedotin and enfortuzumab vedotin use olylisine (auristatins) toxin molecules acting on microtubules, trastuzumab emtansine uses maytansinoid (maytansinoid) toxin molecules acting on microtubules, gemtuzumab ozogamicin and inotuzumab ozogamicin use calicheamicin (calicheamicins) toxin molecules acting on DNA, and most recently marketed trastuzumab deuxtecan and sacituzumab govitertec use camptothecin toxin molecules. An intermediate linker moiety: the trastuzumab emtansine employs a non-cleavable linker, with the remaining seven molecules employing cleavable linkers.

Camptothecin (CPT) analogues and derivatives exert antitumor activity by binding to topoisomerase I, which shows significant activity against many tumor types. To overcome the poor water solubility of CPT, researchers have synthesized a variety of CPT derivatives in which irinotecan hydrochloride (CPT-11) is a water-soluble prodrug approved for the treatment of metastatic colorectal cancer. However, CPT-11 must be converted to its active form SN38 (formula I) in vivo by carboxylesterase catalysis, which is extremely inefficient, and SN38 itself is difficult to prepare due to its poor solubility. Irinotecan (formula II) (common name: exatecan) is another water-soluble CPT derivative and has been attempted to be developed as an antitumor drug, but the development has been stopped by 2004, and it does not require activation by an enzyme, and in addition, it has a stronger inhibitory activity against topoisomerase I than SN-38, which is the drug-active substance of irinotecan.

ADC class of drugs combines the dual advantages of high potency of cytotoxic small molecules and high selectivity of antibodies to specific tumor cells, and there is still a need to develop highly potent and low-toxic ADC drugs that can be targeted at more indications.

Disclosure of Invention

In one aspect, the present invention provides an antibody drug conjugate comprising an antibody moiety, an intermediate linker moiety and a cytotoxic drug moiety, said antibody moiety and said cytotoxic drug moiety being linked through said intermediate linker moiety, or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the antibody moiety of the invention is coupled to one or more cytotoxic drug moieties, which may be selected from, for example, alkaloids, antimetabolites, antitumor antibiotics, alkylating agents, platinates, and the like, with preferred cytotoxic drugs being microtubule inhibitor cytotoxic drugs (including maytansinoids, orlistatins) or DNA-acting cytotoxic drugs (including calicheamicins, duocarmycins, pbd (pyrazolobendiazepines), topoisomerase I inhibitors, and the like).

In some embodiments, the cytotoxic drug moiety of the invention is a topoisomerase I inhibitor. In some embodiments, the cytotoxic drug moiety of the present invention is a camptothecin topoisomerase I inhibitor or a derivative thereof.

In one aspect, the present invention provides an antibody drug conjugate comprising an antibody moiety, an intermediate linker moiety and a cytotoxic drug moiety, said antibody moiety being linked to said cytotoxic drug moiety through said intermediate linker moiety, wherein said antibody drug conjugate comprises the structure shown in formula III below:

wherein:

l is selected from-NR^a-(CR¹R²)n¹-C＝O-、-NR^b-(CR¹R²)n²-O-(CR³R⁴)n³-、-NR^b-(CR¹R²)n²-O-CR³R⁴-CR⁵R⁶-、-NR^b-(CR¹R²)n²-O-CR³R⁴-CR⁵R⁶-CR⁷R⁸-or-NR^b-(CR¹R²)n²-O-L^a-C＝O-，

R^aIs selected from C_1-6Alkyl, optionally deuterated C_1-6Alkyl, optionally halogenated C_1-6Alkyl, C optionally substituted by hydroxy_1-6Alkyl, C optionally substituted by amino_1-6Alkyl, C optionally substituted by nitro_1-6An alkyl group, a carboxyl group,

R^bselected from hydrogen atoms, C_1-6Alkyl, optionally deuterated C_1-6Alkyl, optionally halogenated C_1-6Alkyl, C optionally substituted by hydroxy_1-6Alkyl, C optionally substituted by amino_1-6Alkyl, C optionally substituted by nitro_1-6An alkyl group, a carboxyl group,

R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸each independently selected from hydrogen atom, deuterium atom, halogen, C_1-6Alkyl, optionally deuterated C_1-6Alkyl, optionally halogenated C_1-6Alkyl, C optionally substituted by hydroxy_1-6Alkyl, C optionally substituted by amino_1-6Alkyl, C optionally substituted by nitro_1-6Alkyl, optionally substituted C_3-7Cycloalkyl, optionally substituted C_3-7Heterocyclyl group, optionally substituted C_6-10Aryl, optionally substituted C_5-12(ii) a heteroaryl group, wherein,

n¹is an integer of 0 to 6, and,

n²is an integer of 1 to 6, and,

n³is an integer of 1 to 6, and,

L^ais selected from-CR⁹R¹⁰-、-(CR³R⁴)n³-CR⁹R¹⁰-、-CR⁹R¹⁰-(CR³R⁴)n³-or- (CR)³R⁴)n³-CR⁹R¹⁰-(CR³R⁴)n³-,

Wherein:

i)R⁹selected from hydrogen atom, deuterium atom, halogen, cyano, C_1-6Alkyl, optionally deuterated C_1-6Alkyl, optionally halogenated C_1-6Alkyl, C optionally substituted by hydroxy_1-6Alkyl, C optionally substituted by amino_1-6Alkyl, C optionally substituted by nitro_1-6Alkyl, optionally substituted C_3-7Cycloalkyl, optionally substituted C_3-7Heterocyclyl group, optionally substituted C_6-10Aryl, optionally substituted C_5-12(ii) a heteroaryl group, wherein,

R¹⁰selected from cyano, C_1-6Alkyl, optionally deuterated C_1-6Alkyl, optionally halogenated C_1-6Alkyl, C optionally substituted by hydroxy_1-6Alkyl, C optionally substituted by amino_1-6Alkyl, C optionally substituted by nitro_1-6Alkyl, optionally substituted C_3-7Cycloalkyl, optionally substituted C_3-7Heterocyclyl group, optionally substituted C_6-10Aryl, optionally substituted C_5-12A heteroaryl group;

or,

ii)R⁹and R¹⁰Together with the carbon atom to which they are attached form optionally substituted C_3-7A heterocyclic group.

In one aspect, the present invention provides an antibody drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody drug conjugate comprises a structure represented by formula IV below:

wherein:

n¹is an integer of 0 to 6, and,

n²is an integer of 1 to 6, and,

n³is an integer of 1 to 6, and,

Wherein:

or,

In one aspect, the present invention provides an antibody drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody drug conjugate is of the structure shown in formula V below:

wherein:

n¹is an integer of 0 to 6, and,

n²is an integer of 1 to 6, and,

n³is an integer of 1 to 6, and,

Wherein:

or,

ii)R⁹and R¹⁰Together with the carbon atom to which they are attached form optionally substituted C_3-7A heterocyclic group,

ab represents an antibody moiety of the group,

n is an integer or decimal number selected from 1 to 10.

In some embodiments, the antibody drug conjugate comprises a structure of formula III or formula IV above or the antibody drug conjugate is a structure of formula V above, wherein L is-NR^a-(CH₂)n¹-C＝O-。

In some embodiments, the antibody drug conjugate comprises formula III or formula III aboveIV or the antibody drug conjugate is shown as the formula V, wherein L is-NR^a-(CH₂)n¹-C ═ O-, and R^aIs methyl.

In some embodiments, the antibody drug conjugate comprises a structure of formula III or formula IV above or the antibody drug conjugate is a structure of formula V above, wherein L is-NR^a-(CH₂)n¹-C ═ O-, and R^aIs ethyl.

In some embodiments, the antibody drug conjugate comprises a structure of formula III or formula IV above or the antibody drug conjugate is a structure of formula V above, wherein L is-NR^a-(CH₂)n¹-C ═ O-, and R^aIs n-propyl or isopropyl.

In some embodiments, the antibody drug conjugate comprises a structure of formula III or formula IV above or the antibody drug conjugate is a structure of formula V above, wherein L is

Or

In some embodiments, the antibody drug conjugate comprises a structure of formula III or formula IV above or the antibody drug conjugate is a structure of formula V above, wherein L is-NH-CH₂-O-(CH₂)n³-。

In some embodiments, the antibody drug conjugate comprises a structure of formula III or formula IV above or the antibody drug conjugate is a structure of formula V aboveWherein L is-NR^b-(CR¹R²)n²-O-L^a-C ═ O-, and L^aIs selected from-CR⁹R¹⁰-。

In some embodiments, the antibody drug conjugate comprises a structure of formula III or formula IV above or the antibody drug conjugate is a structure of formula V above, wherein L is-NH- (CH)₂)n²-O-CR⁹R¹⁰-C＝O-。

In some embodiments, the antibody drug conjugate comprises a structure of formula III or formula IV above or the antibody drug conjugate is a structure of formula V above, wherein L is-NH- (CH)₂)n²-O-CR⁹R¹⁰-C ═ O-, and R⁹Is C_1-6Alkyl or hydrogen atoms, R¹⁰Is C_1-6An alkyl group.

In some embodiments, the antibody drug conjugate comprises a structure of formula III or formula IV above or the antibody drug conjugate is a structure of formula V above, wherein L is-NH- (CH)₂)n²-O-CR⁹R¹⁰-C ═ O-, and R⁹Is a hydrogen atom, R¹⁰Is phenyl.

In some embodiments, the antibody drug conjugate comprises a structure of formula III or formula IV above or the antibody drug conjugate is a structure of formula V above, wherein L is-NR^b-(CR¹R²)n²-O-L^a-C ═ O-, and L^aIs selected from-CR⁹R¹⁰-(CR³R⁴)n³-。

In some embodiments, the antibody drug conjugate comprises a structure of formula III or formula IV above or the antibody drug conjugate is a structure of formula V above, wherein L is-NH- (CH)₂)n²-O-CR⁹R¹⁰-(CR³R⁴)n³-C＝O-。

In some embodiments, the antibody drug conjugate comprises a structure of formula III or formula IV above or the antibody drug conjugate is a structure of formula V above, wherein L is-NH- (CH)₂)n²-O-CR⁹R¹⁰-(CR³R⁴)n³-C ═ O-, and R⁹Is a hydrogen atom, R¹⁰Is methyl, R³Is a hydrogen atom, R⁴Is a hydrogen atom.

In some embodiments, the right terminus of L is linked to the amino group at position 1 of irinotecan.

In some embodiments, all hydrogen atoms in the antibody drug conjugate structure are not substituted with deuterium.

In one aspect, the invention provides an antibody drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety can specifically bind to a tumor antigen, which can be selected from any tumor prevention or treatment target known in the art, e.g., can be selected from HER2, EGFR, CD20, CD30, CD33, CD47, CD79b, VEGF, VEGFR, MET, RET, PD-1, PD-L1, and the like.

In some embodiments, the antibody portion may be modified, e.g., include one or more changes, additions or subtractions of the amino acid sequence.

In some embodiments, the antibody moiety is an antibody capable of specifically binding HER 2.

In some embodiments, the antibody moiety is trastuzumab having the sequence shown in table S1 below.

TABLE S1 Trastuzumab sequences

In some embodiments, the antibody moiety is pertuzumab having the sequence shown in table S2 below.

TABLE S2 pertuzumab sequences

In some embodiments, the antibody portion comprises a first antigen-binding fragment that monovalently and specifically binds to the ECD4 antigen of HER2 on a HER 2-expressing cell, the first antigen-binding fragment being an scFv, the first antigen-binding fragment comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprising SEQ ID NOs: 14. 15, 16, the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17. 21, 19; wherein SEQ ID NO: the sequence shown in 14 is: GFNIX₂DTYIH，X₂Is K or E; SEQ ID NO: the sequence shown in 21 is: SASX₁LYS，X₁Is F or Y.

In some embodiments, the antibody portion comprises a first antigen-binding fragment that monovalently and specifically binds to the ECD4 antigen of HER2 on a HER 2-expressing cell, the first antigen-binding fragment being an scFv, the first antigen-binding fragment comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprising SEQ ID NOs: 30. 15, 16, the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17. 18 and 19.

In some embodiments, the antibody moiety comprises a first antigen-binding fragment that monovalently and specifically binds to the ECD4 antigen of HER2 on HER 2-expressing cells, said first antigen-binding fragment being an scFv and selected from the group consisting of:

i. the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region comprising SEQ ID NOs: 28. 29; or

The first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region comprising a heavy chain variable region and a light chain variable region, respectively, identical to SEQ ID NO: 28. 29, having at least 80% identity to the amino acid sequence set forth in seq id no;

wherein SEQ ID NO: the sequence shown in 28 is:

EVQLVESGGGLVQPGGSLRLSCAASGFNIX₂DTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFT ISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS，X₂is K or E;

SEQ ID NO: the sequence shown in 29 is:

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASX₁LYSGVPSRFSGSRSGTDF TLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK，X₁is F or Y.

In some embodiments, the antibody portion comprises a first antigen-binding fragment that is a scFv that monovalent and specifically binds the ECD4 antigen of HER2 on a HER 2-expressing cell, the first antigen-binding fragment comprising a heavy chain variable region and a light chain variable region that comprise an amino acid sequence according to SEQ ID NO: 22. 23, or a pharmaceutically acceptable salt thereof.

In some embodiments, the portion comprises a first antigen-binding fragment that monovalently and specifically binds to the ECD4 antigen of HER2 on a HER 2-expressing cell, the first antigen-binding fragment being an scFv, the VH and VL of the first antigen-binding fragment being arranged from N-terminus to C-terminus in the following order: VH-linker-VL.

In some embodiments, the antibody portion further comprises a second antigen binding fragment that monovalently and specifically binds the ECD2 antigen of HER2 on HER 2-expressing cells, said second antigen binding fragment being a Fab.

In some embodiments, the antibody portion further comprises a second antigen-binding fragment that binds monovalent and specifically to the ECD2 antigen of HER2 on HER 2-expressing cells, the second antigen-binding fragment being Fab and comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprising SEQ ID NOs: 32. 33, 34, the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 35. 36 and 37.

In some embodiments, the antibody portion further comprises a second antigen binding fragment that binds ECD2 antigen of HER2 monovalent and specifically on HER2 expressing cells, said second antigen binding fragment being Fab and comprising a heavy chain variable region and a light chain variable region comprising SEQ ID NO: 24. 25, or a pharmaceutically acceptable salt thereof.

In some specific embodiments, the antibody portion of the antibody drug conjugates provided herein, or pharmaceutically acceptable salts or solvates thereof, is shown in table S3.

Table S3 antibody moieties of exemplary antibody drug conjugates or pharmaceutically acceptable salts or solvates thereof

In some embodiments, the antibody portion comprises an immunoglobulin domain operably linked to the first antigen-binding fragment and/or the second antigen-binding fragment, the immunoglobulin domain comprising: one or more of i.cl, CH1, CH2, or CH3, or ii.fc.

In some embodiments, the antibody portion comprises an immunoglobulin domain operably linked to the first antigen-binding fragment and/or the second antigen-binding fragment, the immunoglobulin domain comprising: CL, CH1, CH2, or CH3, or ii.fc, wherein said CL, CH1, CH2, CH3, Fc are derived from human IgG's CL, CH1, CH2, CH3, Fc, respectively.

In some embodiments, the antibody portion comprises an immunoglobulin domain operably linked to the first antigen-binding fragment and/or the second antigen-binding fragment, the immunoglobulin domain comprising: one or more of CL, CH1, CH2, or CH3, or ii.fc, wherein the CL, CH1, CH2, CH3, or Fc has or does not have a modification; preferably, the CH3 or Fc has a modification such as a substitution of amino acid 435 and/or 436 according to the Kabat numbering system.

In some embodiments, the antibody portion comprises an immunoglobulin domain operably linked to the first antigen-binding fragment and/or the second antigen-binding fragment, the immunoglobulin domain comprising: one or more of i.cl, CH1, CH2, or CH3, or ii.fc, wherein the Fc is a dimeric Fc comprising a first Fc polypeptide to which a first antigen-binding fragment is operably linked and a second Fc polypeptide to which a second antigen-binding fragment is operably linked.

In some embodiments, the antibody portion comprises a constant region operably linked to the first antigen-binding fragment and/or the second antigen-binding fragment, which constant region may be an immunoglobulin native sequence constant region or a mutated constant region, such as one or more of native or mutated CL, CH1, CH2, and/or CH3 functional domains, in some instances these domains are operably linked by means conventional in the art, the constant region may be derived from a constant region of a human immunoglobulin, such as from IgG1, IgG2, IgG3, or IgG4, in some instances the constant region may have modifications to improve its ability to mediate effector function.

In some embodiments, the antibody portion comprises an immunoglobulin functional domain or backbone, e.g., Fc, operably linked to the first antigen-binding fragment and/or the second antigen-binding fragment, which term includes native sequence Fc regions and variant Fc regions, Fc can be human Fc such as from IgG1, IgG2, IgG3, or IgG4, Fc can have modifications to improve its ability to mediate effector functions, e.g., in some embodiments, the backbone has modifications such as knob intehole, H435R, Y436F, defucose, etc., in some embodiments, the mutation sites for knob intehole of the backbone described above include, e.g., Y349C, T366S, L368A, Y407V, S354C, and T366W, etc.

In some embodiments, the antibody portion comprises a scaffold operably linked to the first antigen-binding fragment and/or the second antigen-binding fragment, in some embodiments a dimeric Fc comprising the first Fc polypeptide and the second Fc polypeptide, in some embodiments the dimeric Fc has a modification, in some embodiments the dimeric Fc has H435R and/or Y436F, which modification may be on either polypeptide chain of the first Fc polypeptide, the second Fc polypeptide, in some embodiments the dimeric Fc has H435R and/or Y436F, which modification occurs on only one Fc polypeptide and not on the other Fc polypeptide, in some embodiments the dimeric Fc has a knob-into-865e mutation site, e.g., Y349 2, T366, L368A, Y407V, S C, and T366W, etc., in some embodiments one chain of the Fc has T W and/or S366, and the other chain has Y407V, Y349C, T366S or/and L368A.

In some embodiments, the antibody portion is a bivalent, bispecific antibody comprising: comprises the amino acid sequence of SEQ ID NO: 6, comprising the heavy chain of SEQ ID NO: 7 and a light chain comprising SEQ ID NO: 8, light chain.

In some specific embodiments, the antibody moiety of an antibody drug conjugate provided herein, or a pharmaceutically acceptable salt or solvate thereof, is as set forth in table S4.

Table S4 antibody moieties of exemplary antibody drug conjugates or pharmaceutically acceptable salts or solvates thereof

In one aspect, the invention provides an antibody drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein the cytotoxic drug moiety is conjugated to the antibody moiety via a linker moiety. The linker moiety of the invention may be attached to the antibody moiety by any method known in the art, preferably the linker moiety is attached to the antibody moiety via a thiol and/or amino group. In some more preferred embodiments, the linker moiety of the invention is attached to the antibody moiety through a thiol group.

In another aspect, the present invention provides an antibody drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein the cytotoxic drug moiety is conjugated to the antibody moiety via a linker moiety, which may be a cleavable linker or a non-cleavable linker, in some embodiments the linker moiety of the present invention is a cleavable linker, such as may be low pH dependent degradation (including hydrazone bonds, carbonate bonds, etc.), proteolytic degradation (including peptide based bonds), or high glutathione concentration dependent degradation (including disulfide bonds), and the like, in other embodiments the linker of the present invention is a non-cleavable linker, such as may be maleimidocaproyl, and the like.

In some embodiments, the invention provides an antibody drug conjugate of the formula or a pharmaceutically acceptable salt or solvate thereof,

wherein antibody moiety Ab comprises a first antigen-binding fragment that is an scFv and comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 30. 15, 16, the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17. 18, 19 and a second antigen binding fragment which is Fab and comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 32. 33, 34, the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 35. 36 and 37, or a pharmaceutically acceptable salt thereof,

n is an integer or decimal number selected from 1 to 10.

wherein, the antibody part Ab is trastuzumab,

n is an integer or decimal number selected from 1 to 10.

wherein antibody moiety Ab comprises a first antigen-binding fragment that is an scFv and comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 30. 15, 16, the light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 17. 18, 19 and a second antigen binding fragment which is Fab and comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 32. 33, 34, the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 35. 36 and 37, or a pharmaceutically acceptable salt thereof,

n is an integer or decimal number selected from 1 to 10.

wherein, the antibody part Ab is trastuzumab,

n is an integer or decimal number selected from 1 to 10.

n is an integer or decimal number selected from 1 to 10.

wherein, the antibody part Ab is trastuzumab,

n is an integer or decimal number selected from 1 to 10.

n is an integer or decimal number selected from 1 to 10.

wherein, the antibody part Ab is trastuzumab,

n is an integer or decimal number selected from 1 to 10.

n is an integer or decimal number selected from 1 to 10.

wherein, the antibody part Ab is trastuzumab,

n is an integer or decimal number selected from 1 to 10.

n is an integer or decimal number selected from 1 to 10.

wherein, the antibody part Ab is trastuzumab,

n is an integer or decimal number selected from 1 to 10.

n is an integer or decimal number selected from 1 to 10.

wherein, the antibody part Ab is trastuzumab,

n is an integer or decimal number selected from 1 to 10.

wherein antibody moiety Ab comprises a first antigen-binding fragment that is an scFv and comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 30. 15, 16, the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17. 18, 19, and a second antigen-binding fragment that is a Fab and comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 32. 33, 34, the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 35. 36 and 37, or a pharmaceutically acceptable salt thereof,

n is an integer or decimal number selected from 1 to 10.

wherein, the antibody part Ab is trastuzumab,

n is an integer or decimal number selected from 1 to 10.

In one aspect, the invention provides a pharmaceutical composition comprising an antibody drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.

In one aspect, the invention provides the use of an antibody drug conjugate of the invention, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the prevention or treatment of cancer.

In one aspect, the present invention provides the use of a pharmaceutical composition comprising an antibody drug conjugate of the present invention, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier for the manufacture of a medicament for the prevention or treatment of cancer.

In one aspect, the present invention provides an antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof for use in the prevention or treatment of cancer.

In one aspect, the present invention provides a method of treating or preventing cancer, the method comprising administering to a patient in need thereof a therapeutically effective amount of an antibody drug conjugate of the present invention, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising an antibody drug conjugate of the present invention, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.

In some embodiments, the antibody drug conjugates of the invention, or pharmaceutically acceptable salts or solvates thereof, may be used for the prevention or treatment of HER2 positive cancers, HER2 negative cancers (including triple negative breast cancer), and cancers that show HER2 expression as IHC2+ as detected by immunohistochemical detection.

In some aspects, the invention provides linker-drug intermediate compounds having the structure shown in formula IVa below:

wherein:

R^bselected from hydrogen atoms, C_1-6Alkyl, optionally deuterated C_1-6Alkyl, optionally halogenated C_1-6Alkyl, C optionally substituted by hydroxy_1-6Alkyl, C optionally substituted by amino_1-6Alkyl, C optionally substituted by nitro_1-6An alkyl group, which is a radical of an alkyl group,

R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸each independently selected from hydrogen atom, deuterium atom, halogen, C_1-6Alkyl, optionally deuterated C_1-6Alkyl, optionally halogenated C_1-6Alkyl, C optionally substituted by hydroxy_1-6Alkyl, C optionally substituted by amino_1-6Alkyl, optionally nitroSubstituted C_1-6Alkyl, optionally substituted C_3-7Cycloalkyl, optionally substituted C_3-7Heterocyclyl group, optionally substituted C_6-10Aryl, optionally substituted C_5-12(ii) a heteroaryl group, wherein,

n¹is an integer of 0 to 6, and,

n²is an integer of 1 to 6, and,

n³is an integer of 1 to 6, and,

Wherein:

or,

In some embodiments, the present invention provides linker-drug intermediate compounds of the structure shown above in formula IVa, wherein L is selected from the group consisting of:

-NR^a-(CH₂)n¹-C＝O-、-NH-CH₂-O-(CH₂)n³-、-NH-(CH₂)n²-O-CR⁹R¹⁰-C ═ O-or-NH- (CH)₂)n²-O-CR⁹R¹⁰-(CR³R⁴)n³-C＝O-。

In some embodiments, the present invention provides linker-drug intermediate compounds of the structure shown above in formula IVa, wherein L is selected from the following structures:

and wherein the right end of L is linked to the amino group at position 1 of irinotecan.

In some embodiments, the present invention provides a compound having the structure shown in formula IIIa below:

wherein:

l is selected from NHR^a-(CR¹R²)n¹-C＝O-、NHR^b-(CR¹R²)n²-O-(CR³R⁴)n³-、NHR^b-(CR¹R²)n²-O-CR³R⁴-CR⁵R⁶-、NHR^b-(CR¹R²)n²-O-CR³R⁴-CR⁵R⁶-CR⁷R⁸-or NHR^b-(CR¹R²)n²-O-L^a-C＝O-，

R^aIs selected from C_1-6Alkyl, optionally deuterated C_1-6Alkyl, optionally halogenated C_1-6Alkyl, optionally substituted by hydroxySubstituted C_1-6Alkyl, C optionally substituted by amino_1-6Alkyl, C optionally substituted by nitro_1-6An alkyl group, a carboxyl group,

n¹is an integer of 0 to 6, and,

n²is an integer of 1 to 6, and,

n³is an integer of 1 to 6, and,

Wherein:

i)R⁹selected from hydrogen atom, deuterium atom, halogen, cyano, C_1-6Alkyl, optionally deuterated C_1-6Alkyl, optionally halogenated C_1-6Alkyl, C optionally substituted by hydroxy_1-6Alkyl, C optionally substituted by amino_1-6Alkyl, C optionally substituted by nitro_1-6Alkyl radical, NSelected substituted C_3-7Cycloalkyl, optionally substituted C_3-7Heterocyclyl group, optionally substituted C_6-10Aryl, optionally substituted C_5-12(ii) a heteroaryl group, wherein,

or,

In some embodiments, the present invention provides compounds of the structure shown above in formula V, wherein L is selected from: NHR^a-(CH₂)n¹-C＝O-、NH₂-CH₂-O-(CH₂)n³-、NH₂-(CH₂)n²-O-CR⁹R¹⁰-C ═ O-or NH₂-(CH₂)n²-O-CR⁹R¹⁰-(CR³R⁴)n³-C＝O-。

In some embodiments, the present invention provides compounds of the structure shown in formula IIIa above, wherein L is selected from the following structures:

In some embodiments, the present invention provides the following compounds:

drawings

FIG. 1 shows the killing rate of anti-HER2 bispecific antibody against BT474 tumor cells;

FIG. 2 shows the killing rate of anti-HER2 bispecific antibody on NCI-N87 tumor cells;

FIG. 3 shows the killing rate of anti-HER2 bispecific antibody against JIMT-1 tumor cells;

FIG. 4 shows the proliferation inhibition of BT474 tumor cells by anti-HER2 bispecific antibody;

FIG. 5 shows the tumor volume change of anti-HER2 bispecific antibody in mice transplanted with gastric carcinoma N87 mouse xenograft;

FIG. 6 shows the body weight changes of mice in the drug effect of anti-HER2 bispecific antibody on gastric cancer N87 mouse xenogeneic tumor;

figure 7 is the structure of some exemplary anti-HER2 bispecific antibodies; wherein the dimeric Fc is depicted with one chain (first Fc polypeptide) shown in black and another chain (second Fc polypeptide) shown in gray, while one antigen binding domain (first antigen binding fragment) is shown hatched and the other antigen binding domain (second antigen binding fragment) is shown white; the first antigen-binding fragment is an scFv fused to a first Fc polypeptide, and the second antigen-binding fragment is a Fab fused to a second Fc polypeptide.

Interpretation and definition

The following terms used in the present application have the following meanings, unless otherwise specified. A particular term should not be considered as ambiguous or unclear without special definition, but rather construed according to ordinary meaning in the art. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient.

The term "substituted" means that any one or more hydrogen atoms on a particular atom is replaced with a substituent, so long as the valence of the particular atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., ═ O), meaning that two hydrogen atoms are substituted, oxo does not occur on the aryl.

The terms "optionally" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, ethyl is "optionally" substituted with halo, meaning that ethyl may be unsubstituted (CH)₂CH₃) Monosubstituted (e.g. CH)₂CH₂F) Polysubstituted (e.g. CHFCH)₂F，CH₂CHF₂Etc.) or completely substituted (CF)₂CF₃). It will be appreciated by those skilled in the art that any group containing one or more substituents will not incorporate any substitution or substitution pattern which is sterically impossible and/or cannot be synthesized.

Herein C_m-nIs that the moiety has an integer or fractional number of carbon atoms in the given range. E.g. "C_1-6By "is meant that the group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

When any variable (e.g., R) occurs more than one time in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 2R, then each R has separate options.

When the number of one linking group is 0, e.g. - (CH)₂)₀-, indicates that the linking group is a covalent bond.

When one of the variables is selected from a covalent bond, it means that the two groups to which it is attached are directly linked, for example, where L represents a covalent bond in A-L-Z, it means that the structure is actually A-Z.

The term "halo" or "halogen" refers to fluoro, chloro, bromo and iodo.

The term "hydroxy" refers to an-OH group.

The term "cyano" refers to the group — CN.

The term "mercapto" refers to the-SH group.

The term "amino" refers to the group-NH₂A group.

The term "nitro" means-NO₂A group.

The term "alkyl" refers to a group of the formula C_nH_2n+1A hydrocarbon group of (2). The alkyl group may be linear or branched. For example, the term "C_1-6Alkyl "means an alkyl group having 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, and the like). Similarly, the alkyl portion (i.e., alkyl) of alkoxy, alkylamino, dialkylamino, alkylsulfonyl and alkylthio groups have the same definitions as above.

The term "alkoxy" refers to-O-alkyl.

The term "alkylamino" refers to-NH-alkyl.

The term "dialkylamino" refers to-N (alkyl)₂。

The term "alkylsulfonyl" refers to-SO₂-an alkyl group.

The term "alkylthio" refers to-S-alkyl.

The term "alkenyl" refers to a straight or branched chain unsaturated aliphatic hydrocarbon group having at least one double bond, consisting of carbon atoms and hydrogen atoms. Non-limiting examples of alkenyl groups include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1, 3-butadienyl, and the like.

The term "alkynyl" refers to a straight or branched chain unsaturated aliphatic hydrocarbon group having at least one triple bond composed of carbon atoms and hydrogen atoms. Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl (-C ≡ CH), 1-propynyl (-C ≡ C-CH)₃) 2-propynyl (-CH)₂-C.ident.CH), 1, 3-butadiynyl (-C.ident.C-C.ident.CH), and the like.

The term "cycloalkyl" refers to a carbon ring which is fully saturated and may be present as a single ring, a bridged ring or a spiro ring. Unless otherwise indicated, the carbocycle is typically a3 to 10 membered ring. Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo [2.2.1] heptyl), bicyclo [2.2.2] octyl, adamantyl, and the like.

The term "cycloalkenyl" refers to a non-aromatic carbocyclic ring that is not fully saturated and may exist as a single ring, bridged ring or spiro ring. Unless otherwise indicated, the carbocycle is typically a5 to 8 membered ring. Non-limiting examples of cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptadienyl, and the like.

The term "heterocyclyl" refers to a non-aromatic ring that is fully saturated or partially unsaturated (but not fully unsaturated heteroaromatic) and may exist as a single ring, a bridged ring, or a spiro ring. Unless otherwise indicated, the heterocyclic ring is typically a3 to 7 membered ring containing 1 to 3 heteroatoms (preferably 1 or 2 heteroatoms) independently selected from sulfur, oxygen and/or nitrogen. Non-limiting examples of heterocyclyl groups include, but are not limited to, oxiranyl, tetrahydrofuryl, dihydrofuranyl, pyrrolidinyl, N-methylpyrrolidinyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyrazolidinyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothienyl, and the like.

The term "heterocycloalkyl" refers to a cyclic group that is fully saturated and may exist as a single ring, a bridged ring, or a spiro ring. Unless otherwise indicated, the heterocyclic ring is typically a3 to 7 membered ring containing 1 to 3 heteroatoms (preferably 1 or 2 heteroatoms) independently selected from sulfur, oxygen and/or nitrogen. Examples of 3-membered heterocycloalkyl include, but are not limited to, oxiranyl, thietanyl, cycloazenyl, non-limiting examples of 4-membered heterocycloalkyl include, but are not limited to, azetidinyl, oxetanyl, thiabutinyl, examples of 5-membered heterocycloalkyl include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidinyl, imidazolidinyl, tetrahydropyrazolyl, examples of 6-membered heterocycloalkyl include, but are not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1, 4-thiaxanyl, 1, 4-dioxanyl, thiomorpholinyl, 1, 3-dithianyl, 1, 4-dithianyl, examples of 7-membered heterocycloalkyl include, but are not limited to, azepanyl, oxepinyl, thiepanyl. Monocyclic heterocycloalkyl groups having 5 or 6 ring atoms are preferred.

The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated pi-electron system. For example, the aryl group can have 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 12 carbon atoms. Non-limiting examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, and 1,2,3, 4-tetrahydronaphthalene, and the like.

The term "heteroaryl" refers to a monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, S, the remaining ring atoms being C, and having at least one aromatic ring. Preferred heteroaryls have a single 4-to 8-membered ring, especially a 5-to 8-membered ring, or multiple fused rings containing 6 to 14, especially 6 to 10 ring atoms. Non-limiting examples of heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, and the like.

"derivatives": compounds formed by substituting atoms or groups of atoms in the molecule of the parent compound with other atoms or groups of atoms are referred to as derivatives of the parent compound.

The compounds and intermediates of the present application may also exist in different tautomeric forms, and all such forms are included within the scope of the present application. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also referred to as proton transfer tautomers) include interconversion via proton migration, such as keto-enol and imine-enamine isomerizations. A specific example of a proton tautomer is an imidazole moiety, wherein the proton can migrate between two ring nitrogens. Valence tautomers include interconversion by a recombination of some of the bonding electrons.

The compounds of the present application may be asymmetric, e.g., having one or more stereoisomers. Unless otherwise indicated, all stereoisomers include, for example, enantiomers and diastereomers. The compounds of the present application containing asymmetric carbon atoms can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from a racemic mixture or synthesized by using chiral starting materials or chiral reagents.

Any atom of the labeled synthetic compounds of the present invention may represent any stable isotope of that atom, if not specifically designated. Unless otherwise specified, when a position in a structure is defined as H, i.e., hydrogen (H-1), that position contains only the naturally occurring isotope. Also, unless otherwise specified, when a position in a structure is defined as D, deuterium (H-2), the position contains an isotopic amount that is at least 3340 times greater than the naturally occurring isotopic amount (0.015%) (i.e., at least 50.1% deuterium isotopes), and when one or more positions in the structure of a labeled synthetic compound are defined as D, deuterium (H-2), the content of the compound represented by the structure may be at least 52.5%, at least 60%, at least 67.5%, at least 75%, at least 82.5%, at least 90%, at least 95%, at least 97%, at least 98.5%, at least 99%, or at least 99.5%. The deuteration ratio of a labeled synthetic compound in the present invention refers to the ratio of the content of the labeled synthetic isotope to the amount of the naturally occurring isotope. The deuteration rate per given deuterium atom of a labeled synthetic compound of the present invention can be at least 3500 times (52.5%), at least 4000 times (60%), at least 4500 times (67.5%), at least 5000 times (75%), at least 5500 times (82.5%), at least 6000 times (90%), at least 6333.3 times (95%), at least 6466.7 times (97%), at least 6566.7 times (98.5%), at least 6600 times (99%), at least 6633.3 times (99.5%). Isotopologues in the present invention refer to compounds that differ only in isotopic composition in terms of chemical structure. The labeled synthetic compounds of the present invention have the same chemical structure, with only isotopic variations in the atomic composition of the molecule. Thus, a compound labeled and synthesized in the present invention that contains deuterium at a particular position will also contain very little hydrogen isotope at that position, and the amount of hydrogen isotope at a position in the compound labeled and synthesized in the present invention depends on many factors, including the deuterium isotopic purity of the deuterated reagent (D2O, D2, NaBD4, LiAlD4, etc.) and the effectiveness of the method of synthesis to incorporate deuterium isotopes. However, as previously mentioned, the total amount of such site hydrogen isotopologues will be less than 49.9%. The total number of hydrogen isotopologues at a position in the labeled synthetic compounds of the invention will be less than 47.5%, 40%, 32.5%, 25%, 17.5%, 10%, 5%, 3%, 1%, or 0.5%.

In the present invention, any individual atom not designated as deuterium is present in its natural isotopic abundance.

The term "treating" means administering a compound or formulation described herein to prevent, ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:

(i) preventing the occurrence of a disease or condition in a mammal, particularly when such mammal is susceptible to the disease condition, but has not yet been diagnosed as having the disease condition;

(ii) inhibiting the disease or disease state, i.e., arresting its development;

(iii) alleviating the disease or condition, i.e., causing regression of the disease or condition.

The term "therapeutically effective amount" means an amount of a compound of the present application that (i) treats or prevents a particular disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of a compound of the present application that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art with their own knowledge and this disclosure.

The term "pharmaceutically acceptable" is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As the pharmaceutically acceptable salt, for example, a metal salt, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, and the like can be mentioned.

The term "solvate" refers to a substance formed by association of a compound with a solvent molecule.

The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present application or salts thereof and pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate administration of the compounds of the present application to an organism.

The term "pharmaceutically acceptable adjuvants" refers to those adjuvants which do not have a significant irritating effect on the organism and do not impair the biological activity and properties of the active compound. Suitable adjuvants are well known to the person skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

The term "antibody": in its broadest sense, it is intended to cover in particular intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, multifunctional antibodies, and antibody fragments, so long as they possess the desired biological activity.

The term "humanized antibody": refers to an antibody comprising CDR regions derived from a non-human antibody, and the remainder of the antibody molecule is derived from one or more human antibodies.

The term "mutant": used to refer to a peptide comprising an amino acid sequence derived from the amino acid sequence of the peptide as follows: the peptide may be obtained by substituting one or two or more amino acids with amino acids different from the original peptide, deleting one or two or more wild-type amino acids, inserting one or two or more amino acids that do not exist in the wild type, and/or adding amino acids that do not exist in the wild type to the amino terminus (N-terminus) and/or the carboxy terminus (C-terminus) of the wild type (hereinafter, collectively referred to as "mutation"). In the present invention, "insertion" may also be included in "addition".

The term "CDR" (complementarity determining region), also known as "hypervariable region", refers to each region of an antibody variable domain which is highly variable in sequence and/or forms structurally defined loops. Natural four-chain antibodies typically comprise six CDRs, three in the heavy chain variable region and three in the light chain variable region.

The term "variable region": the antibody structural unit is composed of two pairs of polypeptide chains, each pair having one heavy chain and one light chain, the N-terminal domain of each chain defining the region of about 100 to 110 or more amino acids primarily responsible for antigen recognition as the variable region.

The term "Fab" refers to a Fab that contains the constant domain of the light Chain (CL) and the first constant domain of the heavy chain (CH1) along with the variable domains VL (light chain variable region) and VH (heavy chain variable region) on the light and heavy chains, respectively. The variable domain comprises Complementarity Determining Regions (CDRs) that are involved in antigen binding.

The term "scFv" includes the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. In some embodiments, the scFv further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the structure required for antigen binding.

The term "ECD" is an extracellular domain. HER2 is a HER receptor that is a receptor protein tyrosine kinase belonging to the family of human epidermal growth factor receptors (HER) and includes the EGFR, HER2, HER3 and HER4 receptors, HER2 receptors generally comprise an extracellular domain that can bind HER ligands, a lipophilic transmembrane domain, a conserved intracellular tyrosine kinase domain and a carboxy-terminal signaling domain with several tyrosine residues that can be phosphorylated, and the extracellular domain of HER2 includes four domains, ECD1, ECD2, ECD3 and ECD4, respectively.

The term "antibody moiety": refers to an antibody moiety in an antibody drug conjugate, which, in certain embodiments, is linked to an intermediate linker moiety through a specific functional group, which antibody moiety can specifically bind to an antigen.

The term "linker moiety": refers to the portion of the antibody drug conjugate that links the antibody moiety to the cytotoxic drug moiety, and may or may not be cleavable, with a cleavable linker that can be cleaved within the target cell to release the cytotoxic drug.

The term "cytotoxic drug moiety": refers to a cytotoxic drug part in an antibody drug conjugate, and in certain specific schemes, the cytotoxic drug part is connected with an intermediate linker part through a functional group, so that cytotoxic drug molecules can be liberated in tumor cells, and an anti-tumor effect can be achieved.

The term "ECD": the extracellular domain, the HER receptor is a receptor protein tyrosine kinase belonging to the family of human epidermal growth factor receptors (HER) and includes the EGFR, HER2, HER3 and HER4 receptors, the HER2 receptor generally comprises an extracellular domain that can bind to HER ligands, a lipophilic transmembrane domain, a conserved intracellular tyrosine kinase domain and a carboxy-terminal signaling domain with several tyrosine residues that can be phosphorylated, the extracellular domain of HER2 comprises four domains, ECD1, ECD2, ECD3 and ECD4, respectively.

The term "trastuzumab": the generic name Trastuzumab, a recombinant humanized monoclonal antibody that selectively acts on the extracellular domain of human epidermal growth factor receptor-4 (HER4) and is useful for treating HER2 positive cancers, an example of which is known under the trade name Trastuzumab

Therapeutic monoclonal antibody products are marketed.

The term "pertuzumab": pertuzumab, a common name, is a recombinant humanized monoclonal antibody that selectively acts on the extracellular domain of human epidermal growth factor receptor-2 (HER2) and is useful for treating HER2 positive cancers.

The term "HER 2": is a second member of the EGFR family and has tyrosine kinase activity, wherein HER2 expression levels can be measured by immunohistochemical assay, HER2 positive means IHC3+, HER2 negative means IHC1+/0, and in the case of IHC2+, ISH detection should be performed again for further clarification.

The term "cancer": refers to a physiological condition in mammals that is generally characterized by unregulated cell growth.

The term "triple negative breast cancer": refers to breast cancer in which the expression of estrogen receptor, progestogen receptor and human epidermal growth factor receptor 2 is negative.

Detailed Description

For clarity, the invention is further illustrated by examples, which do not limit the scope of the application. Reagents used herein are generally commercially available and can be used without further purification.

Trastuzumab (Trastuzumab) and Pertuzumab (Pertuzumab) used in the examples of the present application were prepared according to the conventional methods for antibodies, vector construction was performed first, eukaryotic cells were transfected and then purified for expression, the sequence of Trastuzumab is referred to as WHO DRUG INFORMATION INN RL78, and the sequence of Pertuzumab is referred to as the example section of WO 0100245. DS-8201 is the active ingredient of Enhertu, a commercially available formulation from the first Co., Ltd, having the same structure as Trastuzumab-DXD prepared herein (see example 16 for structure).

Example 1 construction, expression, purification of anti-Her2 scFv-Fc and variants thereof

When the anti-Her2 scFv-Fc is constructed, the Fc part adopts human IgG1, and the anti-Her2 arm variable region sequence is based on

The sequence of the monoclonal antibody is shown by a designed linker 1(GGGGS)3

The light chain variable regions of the monoclonal antibodies are connected in series to form anti-Her2-scFv-Fc (SEQ ID NO: 1), and the amino acid sequences are as follows:

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

in addition, point mutations were constructed on the anti-Her2 scFv-Fc sequence to construct the following variants:

anti-Her2-scFv-VL-F53Y-Fc (SEQ ID NO: 2): derived from wild-type anti-Her2-scFv-Fc, the VL region has F53Y; the amino acid sequence is as follows:

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASYLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

anti-Her2-scFv-VL-F53A-Fc (SEQ ID NO: 3): derived from wild-type anti-Her2-scFv-Fc, VL region with F53A; the amino acid sequence is as follows:

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASALYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

anti-Her2-scFv-VL-F53R-Fc (SEQ ID NO: 4): derived from wild-type anti-Her2-scFv-Fc, VL region with F53R; the amino acid sequence is as follows:

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASRLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

anti-Her2-scFv-VH-K30E-Fc (SEQ ID NO: 5): derived from wild-type anti-Her2-scFv-Fc, the VH region has K30E; the amino acid sequence is as follows:

EVQLVESGGGLVQPGGSLRLSCAASGFNIEDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

DNA sequences of anti-Her2 scFv-Fc and variants thereof were synthesized and cloned into pcDNA3.1 expression vectors, respectively. By using ExpicCHO^TMExpression kit (Thermo Fisher, Cat. A29133) expression vectors for anti-Her2 scFv-Fc or variants thereof were co-transfected into ExpicHO cells (CHO-S, Thermo Co.). Cells were grown in ExpicHO expression medium at 37 ℃ with 8% CO₂In an incubator with a humid atmosphere and on a rail-mounted shaker platform rotating at 130 rpm. The culture supernatant was collected and protein purification was performed using protein a magnetic beads (Genscript, catalog No. L00273). Protein concentration was measured by UV-Vis Spectrophotometer (NanoDrop lite, Thermo Scientific).

Table 1: sequence information for anti-Her2 scFv-Fc and variants thereof

Example 2 aggregate validation of anti-Her2 scFv-Fc and variants thereof and detection of binding to human Her2 antigen

Expression of purified anti-Her2 scFv-Fc and variants thereof the affinity of the test molecules for human Her2 protein was determined by Biacore T200(GE Co.) and the experimental procedure is described below:

capturing a certain amount of Anti-Her2 scFv-Fc or its variant with Anti-hIgG coupled chipIn this case, an antigen human HER2 protein (Sino Biological, catalog No. 10004-H08H) was passed over the chip surface, and a reaction signal was detected in real time by Biacore T200, thereby obtaining binding and dissociation curves. The buffer used in the experiment was Biacore Universal buffer (137mM NaCl, 2.7mM KCl, 10mM Na)₂HPO₄·12H₂O，1.8mM KH₂PO₄0.05% surfactant P20(GE, cat # BR-1000-54), pH 7.4). Anti-hIgG (original human antibody capture kit, GE, catalog No. 29-2346-00) was coupled to CM5 chip surface to reach around 9000RU, Anti-Her2 scFv-Fc or its variants were captured at about 200RU, and then signal values of interaction between human HER2 protein (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM) and Anti-Her2-scFv-Fc or its variants at different concentrations were measured. Flow cell flow rate of 50. mu.l/min, association time 240s, dissociation time 1400s, using 3M MgCl₂(GE Co.) regenerate for 60s with a smooth baseline. According to affinity and dynamics 1 in biacore evaluation software: 1 combining the mode operation to obtain the result. The affinity of anti-Her2 scFv-Fc or variants with the antigen human Her2 protein is shown in Table 2. The binding affinity of the variant anti-Her2-scFv-VL-F53A-Fc (KD ═ 1.26nM) to the antigen human Her2 protein was more affected than that of anti-Her2 scFv-Fc (KD ═ 0.77 nM); the binding affinity of the variant anti-Her2-scFv-VL-F53Y-Fc (KD ═ 0.8nM) for antigen human Her2 protein was similar to that of anti-Her2-scFv-Fc (KD ═ 0.77nM), and the variant anti-Her2-scFv-VH-K30E-Fc binds to antigen human Her2 protein by KD ═ 0.54 nM. It is known that the introduction of F53Y and K30E mutations does not reduce the binding affinity to the antigen human Her2 protein.

And separating each component of the anti-Her2 scFv-Fc or the variant thereof by using a gel chromatographic column. Eluting with buffer solution with neutral pH as mobile phase, and sequentially eluting the components with different molecular weights from large molecular weight to small molecular weight. The chromatographic Column is ACQUITY UPLC Protein BEH SEC Column

1.7 μm, 4.6 × 300mm specification gel chromatography column, column temperature 25 ℃. The mobile phase is 50mmol/L phosphate buffer solution-200 mmol/L sodium chloride, pH 7.0 (weighing 2.33g sodium dihydrogen phosphate dihydrate, twelve)12.53g of disodium hydrogen phosphate hydrate and 11.69g of sodium chloride, adding about 800mL of ultrapure water, stirring until the mixture is fully dissolved, adding the ultrapure water to 1000mL, uniformly mixing, and filtering by using a 0.22 mu m filter membrane). Diluting the sample to 10mg/mL with mobile phase as test solution, precisely measuring 2 μ l, injecting into liquid chromatograph (if the sample concentration is less than 10mg/mL, adjusting injection volume to inject 20 μ g protein), and detecting at 280nm wavelength. The flow rate was 0.30mL/min, and isocratic elution was 15 min. And (4) data processing, and carrying out quantitative analysis on the result by adopting an area normalization method. And respectively calculating the peak area percentages of the aggregate, the immunoglobulin monomer and the low-molecular-weight impurity, wherein the aggregate is arranged before the main peak, the immunoglobulin monomer is arranged at the main peak, and the low-molecular-weight impurity is arranged after the main peak. The ratio of main peak to aggregate content of anti-Her2 scFv-Fc or its variant is shown in Table 2. The variant anti-Her2-scFv-VL-F53Y-Fc and anti-Her2-scFv-VH-K30E-Fc significantly reduced aggregation; the aggregate was reduced from 6.31% to 4.94% and 3.39% respectively for anti-Her2 scFv-Fc.

Table 2: aggregate content and binding affinity to antigen Her2 of anti-Her2 scFv-Fc and variants thereof

Example 3 construction, expression, purification of anti-Her2 bispecific antibody

anti-Her2 bispecific antibodies were generated as human IgG1 by Fc engineering of the Knobs-int-holes (Ridgway, et al, 1996). The Fc region sequence of one heavy chain is designed with H435R and Y436F mutations (Jendeberg et al, 1997) to reduce the affinity of Fc to protein A, which is beneficial to removing homodimers formed during bispecific antibody assembly in protein A affinity purification (patent US 5945311A). From example 2, it is known that anti-Her2-scFv-VL-F53Y-Fc mutation and anti-Her2-scFv-VH-K30E-Fc mutation can significantly reduce anti-Her2-scFv aggregate, meanwhile, the affinity of the antigen to Her2 is not reduced, the antigen binding structural domain of one anti-Her2 arm of the anti-Her2 bispecific antibody in the embodiment is in the form of scFv (VH-linker-VL structure), the variable region sequence comprises the mutation K30E (b-anti-Her2-scFv-VH-K30E-Fc, SEQ ID NO: 11), the mutation F53Y (b-anti-Her2-scFv-VL-F53Y-Fc, SEQ ID NO: 12) or both the two point mutations anti-Her2-scFv-VH-K30E-VL-F53Y-Fc (SEQ ID NO: 6); the antigen binding domain of the other anti-Her2 arm of the anti-Her2 bispecific antibody of this example is in Fab form, including anti-Her2-domain2-HC-Fc (SEQ ID NO: 7) and anti-Her2-domain2-LC (SEQ ID NO: 8); in addition, an anti-Her2 bispecific antibody was constructed without mutation as a control by scFv (VH-linker-VL structure or VL-linker-VH structure).

Table 3: sequence information for anti-Her2 bispecific antibodies

The DNA sequence of the anti-Her2 bispecific antibody was synthesized and cloned into pcDNA3.1 expression vectors, respectively. By using

High-yield expression System (cat # MIR 6270) expression vectors for anti-Her2-scFv-VH-K30E-VL-F53Y-Fc (SEQ ID NO: 6), anti-Her2-domain2-HC-Fc (SEQ ID NO: 7) and anti-Her2-domain2-LC (SEQ ID NO: 8) were transfected in the ratio 1: 1: 1.5 Co-transfection into ExpCHO cells. The transfection density was 6X 10⁶cells/mL. The culture medium is

Expression medium (cat # MIR 6200, manufacturer Mirus). Cell culture supernatants were harvested by centrifugation from continuous culture to day 10 post transfection. Protein purification was performed using protein a magnetic beads (Genscript, catalog No. L00273). Measurement by UV-Vis Spectrophotometer (NanoDrop lite, Thermo Scientific)The protein concentration. This sample was named Expi Her 2-2. By reference to the method, other bispecific antibodies Expi Her2-1, Expi Her2-3, Expi Her2-4 and Expi Her2-5 were obtained by expression and purification.

Example 4 fucose knockout bispecific antibody preparation and validation

The interaction between IgG1 and FcgRIIa can be improved by knocking out fucose expression-related gene FUT8, thereby enhancing the ADCC effect of the antibody (Shields et al, 2002; Yamane-Ohnuki et al, 2004). In this example, fucose knock-out anti-Her2 bispecific antibodies were prepared using CHO-S cells (designated CHO FUT 8-/-cells) knock-out of FUT 8-. DNA sequences of the anti-Her2 bispecific antibody were synthesized and cloned into pcDNA3.1 expression vectors, respectively. By using

High-yield expression System (cat # MIR 6270) expression vectors for anti-Her2-scFv-VH-K30E-VL-F53Y-Fc, anti-Her2-domain2-HC-Fc and anti-Her2-domain2-LC were transfected at a ratio of 1: 1: 1.5 Co-transfection into CHO-S cells knock-out of FUT 8-. The transfection density was 6X 10⁶cells/mL. The culture medium is

Expression medium (cat # MIR 6200, manufacturer Mirus). Cell culture supernatants were harvested by centrifugation from continuous culture to day 10 post transfection. Protein purification was performed using protein a magnetic beads (Genscript, catalog No. L00273). Protein concentration was measured by UV-Vis Spectrophotometer (NanoDrop lite, Thermo Scientific). This sample was named 23C2 Her 2-2. Referring to the method, the sequences of Expi Her2-1, Expi Her2-3, Expi Her2-4 and Expi Her2-5 are expressed in CHO FUT 8-/-cells, so as to obtain fucose knockout anti-Her2 bispecific antibodies 23C2 Her2-1, 23C2 Her2-3, 23C2 Her2-4 and 23C2 Her 2-5.

Samples of anti-Her2 bispecific antibody expressed by CHO FUT 8-/-cells and CHO-S cells were treated with GlycoWorks Rapifluor-MS N-carbohydrate kit (Waters, Milford, Mass., USA) to release N-sugars from proteins, labeled with N-sugars, separated by chromatography column, and analyzed with FLR detector (Waters, Milford, Mass., USA) to obtain the structure and content of N-sugars, with the normal anti-Her2 bispecific antibody Expi HER2-2 containing no fucose at 21.85% and the knockout FUT 8-CHO-S cell expressing anti-Her2 bispecific antibody 23C2 HER2-2 containing no fucose at 99.40%.

Example 5 aggregate validation of bispecific antibodies

This example relates to aggregate validation of anti-Her2 bispecific antibodies 23C2 Her2-1, 23C2 Her2-2, 23C2 Her2-3, 23C2 Her2-4, 23C2 Her 2-5.

The aggregate content was verified by separation of the anti-her2 bispecific antibody using a gel chromatography column. Eluting with buffer solution with neutral pH as mobile phase, and sequentially eluting the components with different molecular weights from large molecular weight to small molecular weight. The chromatographic Column is ACQUITY UPLC Protein BEH SEC Column

1.7 μm, 4.6 × 300mm size gel chromatography column, 25 ℃ column temperature. The mobile phase was 50mmol/L phosphate buffer solution-200 mmol/L sodium chloride, pH 7.0 (2.33 g sodium dihydrogen phosphate dihydrate, 12.53g disodium hydrogen phosphate dodecahydrate, 11.69g sodium chloride were weighed, about 800mL ultrapure water was added, stirred until fully dissolved, 1000mL ultrapure water was added, mixed well and filtered through a 0.22 μm filter membrane). Diluting the sample to 10mg/mL with mobile phase as test solution, precisely measuring 2 μ l, injecting into liquid chromatograph (if the sample concentration is less than 10mg/mL, adjusting injection volume to inject 20 μ g protein), and detecting at 280nm wavelength. The flow rate was 0.30mL/min, and isocratic elution was 15 min. And (4) data processing, and carrying out quantitative analysis on the result by adopting an area normalization method. And respectively calculating the peak area percentages of the aggregate, the immunoglobulin monomer and the low-molecular-weight impurity, wherein the aggregate is arranged before the main peak, the immunoglobulin monomer is arranged at the main peak, and the low-molecular-weight impurity is arranged after the main peak.

Table 4: aggregate content of anti-Her2 bispecific antibody

Sample name	Aggregate	Monomer	Low molecular fragments
				23C2 Her2-2	8.51％	91.02％	0.47％
23C2 Her2-1	19.55％	80.05％	0.40％

The results show that when scFV was assembled into bispecific antibody, bispecific antibody still produced large amount of aggregates, whereas by introducing mutations, aggregate content of bispecific antibody could be reduced, compared to 23C2 Her2-1 without mutations, aggregate content of 23C2 Her2-2 could be reduced to 8.51% (table 4).

Example 6 antigen binding detection of anti-Her2 bispecific antibodies

Expression of purified anti-Her2 bispecific antibody the affinity of the test molecule for Her2 protein was determined by Biacore T200(GE corporation), the experimental procedure is described below:

an amount of Anti-Her2 bispecific antibody was captured on an Anti-hIgG coupled chip, and then the binding and dissociation curves were obtained by passing human Her2(Sino Biological, catalog No. 10004-H08H) on the chip surface and detecting the reaction signal in real time using Biacore T200. The buffer used in the experiment was Biacore Universal buffer (137mM NaCl, 2.7mM KCl, 10mM Na)₂HPO₄·12H₂O，1.8mM KH₂PO₄0.05% surfactant P20, pH 7.4). Anti-hIgG (source human antibody capture kit, GE, catalog No. 29-2346-00) was coupled to CM5 chip surface to reach around 9000RU, and Anti-Her2 bispecific antibody was captured at about 200RU, and then signal values of different concentrations of Her2 protein (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM) interacting with Anti-Her2 bispecific antibody were measured. Flow cell flow rate of 50. mu.l/min, association time 240s, dissociation time 1400s, using 3M MgCl₂(GE) regeneration was 60s with a plateau baseline.

The results were obtained according to biacore evaluation software algorithms. The anti-Her2 bispecific antibody 23C2 Her2-2 had a binding affinity for antigen Her2 and the control trastuzumab and pertuzumab are shown in Table 5, the anti-Her2 bispecific antibody 23C2 Her2-2 had a binding KD for antigen Her2 of 6.11E-10M, the trastuzumab control had a binding KD for antigen Her2 of 1.22E-09M, the pertuzumab had a binding KD for antigen Her2 of 2.35E-09M, and the anti-Her2 bispecific antibody 23C2 Her2-2 had a higher affinity for antigen Her2 than the trastuzumab and pertuzumab.

Table 5: affinity of anti-Her2 bispecific antibodies to human Her2 antigen

	ka(1/Ms)	kd(1/s)	KD(M)
				23C2 Her2-2	1.79E+05	1.09E-04	6.11E-10
Trastuzumab control	1.73E+05	2.12E-04	1.22E-09
				Pertuzumab control	1.15E+05	2.69E-04	2.35E-09

Example 7 antigen binding detection of anti-Her2 bispecific antibodies

The affinity of the bispecific antibody 23C2 Her2-1, 23C2 Her2-2, 23C2 Her2-3, 23C2 Her2-4, 23C2 Her2-5 expressing purified anti-Her2 was determined for the HER2 protein by Biacore T200(GE) with reference to the experimental procedure of example 6.

The results of the affinity assay of the anti-Her2 bispecific antibody 23C2 Her2-1 with the antigen human Her2 protein are shown in Table 6. The results in tables 6 and 7 show that the introduction of F53Y and K30E mutations can improve the binding affinity of the anti-Her2 bispecific antibody to the antigen human Her2 protein.

Table 6: affinity of anti-Her2 bispecific antibodies to human Her2 antigen

Sample name	23C2 Her2-1
		Antigen Her2 KD	4.02nM

Example 8 killing of Her 2-positive target cell BT474 by anti-Her2 bispecific antibody

The killing effect of the anti-Her2 bispecific antibody on target cells (BT474 Her2+ + +, source: China academy of sciences type culture Collection cell Bank) was studied by providing NK cells by human PBMC (peripheral blood mononuclear cells) and the in vitro activity of the anti-Her2 bispecific antibody was evaluated according to the EC50 size.

The specific experimental process is as follows:

BT474 cells cell density was adjusted to 3x10 using 1640 experimental medium containing 2% FBS (fetal bovine serum)⁵cells/mL, 50. mu.l per well, were seeded in 96-well cell culture plates (eppendorf, cat # 0030730199). Different concentrations of anti-Her2 bispecific antibody were formulated using experimental media and different concentrations of antibody were added to the above 96 well cell culture plates at 50. mu.l per well. Human PBMC cell density was adjusted to 1.5x10 using experimental media⁶cells/mL, 100. mu.l per well. Setting an administration group (target cells + effector cells + antibody), a target cell group (BT474 cells), an effector cell group (human PBMC), a target cell + effector cell group, a blank control group (culture medium) and a lysate control group, a target cell maximum release group (target cells + lysate), an effective-to-target ratio of 10: 1. 45min before the assay, 20. mu.l/well of lysis solution (Promega, cat # G182A) was added to the target cell maximum release group and the lysate control group. Used after 45min

The cell lysis rate was measured with a nonradioactive cytotoxicity assay kit (cytox 96nonradioactive cytotoxicity assay, Promega, G1780).

Cracking rate (%) - (OD)_{Administration set}-OD_{Target cell + group of effector cells})/(OD_{Maximum release group of target cells}-OD_{Target cell group})×100％

FIG. 1 shows the killing rate of anti-Her2 bispecific antibodies against BT474 Her2+ + + tumor cells. ADCC enhances the killing of the anti-Her2 bispecific antibody on BT474 tumor cells better than the combination of trastuzumab and pertuzumab, and better than the CHO-S expression anti-Her2 bispecific antibody; wherein the combined EC50 of trastuzumab and pertuzumab (1:1) is 8.627ng/mL, the EC50 of Expi HER2-1 is 38.05ng/mL, Expi HER2-2 is superior to Expi HER2-1, and the EC50 is 35.17 ng/mL; the EC50 of 23C2 HER2-1 with enhanced ADCC is 4.728ng/mL, the EC 2 HER2-2 with enhanced ADCC is better than that of 23C2 HER2-1, and the EC50 of the antibody is 3.658 ng/mL.

Example 9 killing of Her2 Positive target cells NCI-N87 by anti-Her2 bispecific antibody

The killing effect of the anti-Her2 bispecific antibody on target cells (NCI-N87 Her2+ +, source: cell bank of the national academy of sciences' typical culture Collection) was studied by providing NK cells by human PBMC (peripheral blood mononuclear cells), and the in vitro activity of the anti-Her2 bispecific antibody was evaluated according to the EC50 size.

The specific experimental process is as follows:

NCI-N87 cells cell density was adjusted to 3X10 using 1640 experimental medium containing 2% FBS (fetal bovine serum)⁵cells/mL, 50. mu.l per well, were seeded in 96-well cell culture plates (eppendorf, cat # 0030730199). Different concentrations of anti-Her2 bispecific antibody or control were formulated using experimental media and added to the above 96 well cell culture plates at 50 μ l per well. Human PBMC cell density was adjusted to 1.5x10 using experimental media⁶cells/mL, 100. mu.l per well. Setting an administration group (target cells + effector cells + antibody), a target cell group (NCI-N87 cells), an effector cell group (human PBMC), a target cell + effector cell group, a blank control group (culture medium) and a lysate control group, a target cell maximum release group (target cells + lysate), an effective-to-target ratio of 10: 1. 45min before the assay, 20. mu.l/well of lysis solution (Promega, cat # G182A) was added to the target cell maximum release group and the lysate control group. Used after 45min

The cell lysis rate was measured with a nonradioactive cytotoxicity assay kit (cytox 96nonradioactive cytotoxicity assay, Promega, G1780). Using trastuzumab and T-DM1 (trastuzumab-maytansine conjugate, trade name)

) Trituzumab + Partuzumab (1:1), Expi HER2-1 as control drugs.

FIG. 2 shows the killing rate of anti-Her2 bispecific antibodies against NCI-N87 tumor cells. ADCC enhances the killing of the anti-Her2 bispecific antibody 23C2 HER2-2 on NCI-N87 tumor cells better than trastuzumab + pertuzumab combination, trastuzumab, T-DM1 and Expi HER 2-1; wherein the EC50 of 23C2 HER2-2 with enhanced ADCC is 0.02447nM, the EC50 of trastuzumab + pertuzumab combination is 0.08267nM, the EC50 of Expi HER2-1 is 0.1048nM, the EC50 of T-DM1 is 0.07392nM, and the EC50 of trastuzumab is 0.07468 nM.

Example 10 killing of Trastuzole resistant cells JIMT-1 by an anti-Her2 bispecific antibody

The killing effect of the anti-Her2 bispecific antibody on target cells (JIMT-1, source: AddexBio, Catalog #: C0006005) was studied by providing NK cells by human PBMC (peripheral blood mononuclear cells), and the in vitro activity of the anti-Her2 bispecific antibody was evaluated according to the EC50 size.

The specific experimental process is as follows:

JIMT-1 cells cell density was adjusted to 3X10 using 1640 experimental medium containing 2% FBS (fetal bovine serum)⁵cells/mL, 50. mu.l per well, were seeded in 96-well cell culture plates (eppendorf, cat # 0030730199). Different concentrations of anti-Her2 bispecific antibody or control were formulated using experimental media and added to the above 96 well cell culture plates at 50 μ l per well. Human PBMC cell density was adjusted to 1.5x10 using experimental media⁶cells/mL, 100. mu.l per well. Setting an administration group (target cells + effector cells + antibody or control drug), a target cell group (BT474 cells), an effector cell group (human PBMC), a target cell + effector cell group, a blank control group (culture medium) and a lysate control group, a target cell maximum release group (target cells + lysate), an effective-to-target ratio of 20: 1. 45min before the detection, the detection time is 45min,the maximum release group of target cells and the lysate control group were added with 20. mu.l/well of lysate (Promega, cat # G182A). Used after 45min

The cell lysis rate was measured with a nonradioactive cytotoxicity assay kit (cytox 96nonradioactive cytotoxicity assay, Promega, G1780). Trastuzumab, T-DM1, trastuzumab + pertuzumab (1:1), Expi HER2-1 were used as control drugs.

FIG. 3 shows the killing rate of anti-Her2 bispecific antibody against JIMT-1 tumor cells. ADCC enhances the killing of JIMT-1 tumor cells by the anti-Her2 bispecific antibody 23C2 HER2-2 better than the combination of trastuzumab and pertuzumab, trastuzumab, T-DM1 and Expi HER 2-1; wherein the EC50 of 23C2 HER2-2 with enhanced ADCC is 0.01006nM, the EC50 of trastuzumab + pertuzumab combination is 0.06727nM, the EC50 of Expi HER2-1 is 0.08066nM, the EC50 of T-DM1 is 0.08357nM, and the EC50 of trastuzumab is 0.07443 nM.

Example 11 inhibition of proliferation of BT474 Her2+ + + tumor cells by anti-Her2 bispecific antibodies

23C2 Her2-2, trastuzumab, pertuzumab were diluted to a final concentration of 3.2. mu.g/mL using an assay medium, DMEM/F12 medium (GIBCO, cat # 11330-032) containing 2% FBS (fetal bovine serum, manufacturer GIBCO, cat # 10099-141), followed by 1: a ratio gradient of 1 diluted 9 concentrations. Taking BT474 Her2+ + + cells in logarithmic growth phase, adjusting the density to 1 × 10⁵cells/mL were plated, 100. mu.l was added to each well, and a blank well without cells was set as a control. A gradient of diluted sample was added, 50. mu.l per well. At 37 ℃ with 5% CO₂Culturing in carbon dioxide incubator for 5 days. Discarding the culture solution, adding CCK-8 (Japan Dojindo chemical, cat # CK04) working solution 100 μ l per well, incubating and developing for 4-5 hr, placing in enzyme labeling instrument (manufacturer Thermo, model: Varioska nWalsh), reading and recording the absorption of the well plate with wavelength of 450nm using 630nm as reference wavelengthAnd (4) light value. And (4) calculating the proliferation inhibition rate of the tumor cells.

The results are shown in figure 4, the ADCC enhanced anti-Her2 bispecific antibody 23C2 Her2-2 has 78.38% of inhibition rate on BT474 tumor cell proliferation, which is better than 54.12% of inhibition rate on trastuzumab proliferation or 53.7% of inhibition rate on trastuzumab + patubarb combined proliferation.

Example 12 anti-Her2 bispecific antibody inhibition of NCI-N87 Her2+ + gastric carcinoma nude mouse xenograft tumor

The in vivo efficacy of the anti-Her2 bispecific antibody was evaluated in a mouse xenograft model by NCI-N87 Her2+ + gastric cancer cells (cell bank of the culture Collection type, national academy of sciences). NCI-N87 Her2+ + gastric cancer cells were prepared at a concentration of 5X10⁷0.1 mL/mouse, under sterile conditions, was inoculated under the right axilla of nude mice (from Calvens laboratory animals Co., Ltd., Hezhou, nude mice, 14-17g, male, rearing environment: SPF grade). Measuring the diameter of the transplanted tumor by using a vernier caliper for the transplanted tumor of the nude mouse until the tumor grows to 100-250mm³The animals were then divided into 5 groups:

group 1: control

Group 2: expi Her2-1, 10mg/kg

Group 3: 23C2 Her2-2, 5mg/kg

Group 4: 23C2 Her2-2, 10mg/kg

Group 5: per + Tra (control trastuzumab + control pertuzumab), 5+5mg/kg

Each administration group was administered by intravenous injection at the corresponding dose 2 times per week for about 3 weeks (6 times). The two drugs except the group 5 combined group are respectively administered with the volume of 5mL/kg, and the administration volume of the other groups is 10 mL/kg. Group 1 was also i.v.10mL/kg PBS (Hyclone; cat # sh 30256.01). When the combination is administered, trastuzumab is administered at least 30min after pertuzumab is administered.

Dynamically observing the anti-tumor effect of the tested object by using a method for measuring tumor diameter, measuring the tumor volume 2-3 times per week, weighing the mouse, and recording data; mice were observed daily for general performance.

Detection indexes are as follows:

tumor Volume (TV), calculated by the formula: TV ═1/2×a×b²Wherein a and b represent length and width, respectively.

The experiment was started on day d0 and was administered 6 times in total (days d0, d3, d7, d10, d14, d 17). By day d21 of the experiment, no animals died. The average body weight of each group of mice showed a tendency to increase (fig. 6). The medicine has no obvious toxic effect.

By day d21, the effect on xenograft volume in NCI-N87 gastric cancer nude mice of group 2(10mg/kg), group 3(5mg/kg), group 4(10mg/kg), and Per + Tra combination group (5+5mg/kg) is shown in Table 7 and FIG. 5, and the inhibition of xenograft tumor in NCI-N87 gastric cancer nude mice by 23C2 Her2-2 (10mg/kg) is better than that in Expi Her2-1(10mg/kg) and Per + Tra combination group (5+5 mg/kg).

Table 7: effect of anti-HER2 double antibody on the volume of xenograft tumor in NCI-N87 gastric carcinoma nude mice (mean + -SD)

Remarking: the administration is carried out for 6 times on d0, d3, d7, d10, d14 and d17 days.

Synthesis of example 13 Dxd

The title compound was synthesized with reference to example 76 in patent "CN 104755494A".

Example 14 Synthesis of MC-GGFG-Dxd

The title compound was synthesized with reference to example 73 in patent "CN 104755494A".

Example 15 preparation of toxins and linker-toxin intermediates of the invention

1) Preparation of Compound I-1

Dissolving 5mg of irinotecan mesylate dihydrate, 1mg of 2-methyl-2-hydroxypropionic acid and 5.1mg of HBTU (benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate) in 3mL of DMF (N, N-dimethylformamide) at room temperature, adding 3.5mg of DIPEA (N, N-diisopropylethylamine) dropwise, stirring at room temperature for 1 hour, slowly adding 10mL of saturated NaCl solution and 15mL of ethyl acetate, repeatedly extracting for 3 times, combining the organic phases, concentrating, preparing a liquid phase to obtain 3.2mg of the title compound, LC-MS: M/z 522.11(M + H)⁺。

2) Preparation of Compound I-2

Dissolving 10mg of irinotecan mesylate dihydrate, 2.1mg of L-lactic acid and 10.1mg of HBTU in 6mL of DMF at room temperature, dropwise adding 3.5mg of DIPEA, stirring at room temperature for 1H, slowly adding 10mL of saturated NaCl solution and 20mL of ethyl acetate, repeatedly extracting for 3 times, combining organic phases, concentrating, and preparing a liquid phase to obtain 5.2mg of the title compound, LC-MS M/z 508.08(M + H)⁺。

3) Preparation of Compound I-3

Dissolving 10mg of irinotecan mesylate dihydrate, 2.1mg of D-lactic acid and 10.1mg of HBTU in 6mL of DMF at room temperature, dropwise adding 3.5mg of DIPEA, stirring at room temperature for 1H, slowly adding 10mL of saturated NaCl solution and 20mL of ethyl acetate, repeatedly extracting for 3 times, combining organic phases, concentrating, and preparing a liquid phase to obtain 5.2mg of the title compound, LC-MS: M/z 508.08(M + H)⁺。

4) Preparation of Compound I-4

100mg of SM1 (irinotecan mesylate dihydrate) and 43mg of (S) - (+) -mandelic acid were weighed into a 100mL single-neck flask, 3mL of DMF was added, the temperature was reduced to 0 ℃ and 107mg of HBTU and 190mg of DIPEA were added. Return to room temperature and react for 3 hours. 100mL of ethyl acetate and 100mL of water were added, the mixture was stirred for 20 minutes, and the organic phase was separated. 50mL of saturated brine was added thereto, and the mixture was stirred for 20 minutes to separate an organic phase. Dried for 30 minutes by adding 10g of anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness at 38 ℃. The title compound was prepared via liquid phase. ESI-MS, M/z 570.13[ M + H ]]⁺。

5) Preparation of Compound I-5

The method comprises the following steps: synthesis of Boc-E-Gly

1.16mg of E-Gly (N-ethylglycine) was weighed into a 250mL eggplant-shaped bottle, 50mL of purified water was added, and then 2.6mL of di-tert-butyl dicarbonate and 4.7mL of trifluoroacetic acid were sequentially added, and the mixture was stirred at room temperature for 23 hours. 100mL of 10% aqueous hydrochloric acid was added and stirred for 30 minutes, extracted twice with 100mL of ethyl acetate, the organic phases were combined, washed 2 times with 100mL of saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, filtered to remove solids, and the filtrate was concentrated to dryness at 40 ℃ to give 1.737g of Boc-E-Gly.

Step two: synthesis of Boc-E-Gly-Exa

70mg of Boc-E-Gly and 100mg of SM1 were weighed out and put in a 50mL eggplant-shaped bottle, 5mL of DMF was added and dissolved, followed by stirring at 0 ℃ and 89mg of DIPEA, followed by stirring for 10 minutes, 131mg of HATU (2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate) was added, stirring was continued for 10 minutes, and then the mixture was transferred to room temperature and stirred overnight. The reaction was diluted with 200mL ethyl acetate, washed 4 times with 100mL saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, filtered to remove solids, and the filtrate was concentrated to dryness at 40 ℃ to give 135mg Boc-E-Gly-Exa.

Step three: synthesis of the title Compound

135mg of Boc-E-Gly-Exa was weighed out and added to a 50mL round bottom flask, and dissolved in 4mL of methylene chloride, followed by addition of 0.7mL of trifluoroacetic acid and stirring at room temperature overnight. The reaction was concentrated to dryness at 40 ℃ to prepare the title compound from the liquid phase. ESI-MS, M/z 521.15[ M + H ]]⁺。

6) Preparation of Compound I-6

The method comprises the following steps: synthesis of N-isopropylglycine

6g of glyoxylic acid is weighed and added into a 250mL eggplant-shaped bottle, 50mL of dichloromethane is added for dissolution, 2.9mL of isopropylamine is added, the vacuum pumping and the nitrogen filling are carried out, the circulation is carried out for three times, and the stirring is carried out for 22 hours at room temperature. The reaction solution was concentrated to dryness at 40 ℃ and 130mL of 1mol/L hydrochloric acid solution was added, the temperature was raised to 105 ℃ and the mixture was stirred for 16 hours under condensation in a water bath. Cooling to room temperature, concentrating the reaction solution at 60 deg.C, adding 5mL methanol to dissolve, adding 200mL ethyl acetate, shaking, precipitating solid, filtering to obtain white crystal, and discarding the filtrate to obtain 2.236g N-isopropylglycine.

Step two: synthesis of Boc-IP-Gly

2.236g N-isopropylglycine was weighed out and put into a 250mL eggplant type bottle, and 30mL of purified water was added, followed by sequentially adding 4.1mL of di-tert-butyl dicarbonate and 7.9mL of trifluoroacetic acid and stirring at room temperature for 20 hours. 100mL of 10% aqueous hydrochloric acid was added and stirred for 20 minutes, extracted twice with 100mL of ethyl acetate, the organic phases were combined, washed 2 times with 100mL of saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, filtered to remove solids, and the filtrate was concentrated to dryness at 40 ℃ to give 4.1g of Boc-IP-Gly.

Step three: synthesis of Boc-IP-Gly-Exa

75mg of Boc-IP-Gly and 100mg of SM1 were weighed out and put in a 50mL eggplant-shaped bottle, and 4.5mL of DMF was added to dissolve the mixture, and the mixture was stirred at 0 ℃ followed by 89mg of DIPEA, and after stirring for 10 minutes, 131mg of HATU was added, and after stirring for 10 minutes again, the mixture was transferred to room temperature and stirred overnight. The reaction solution was diluted with 200mL of ethyl acetate, washed 4 times with 100mL of saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, the solid was removed by filtration, the filtrate was concentrated to dryness at 40 ℃, and column chromatography (dichloromethane: methanol ═ 20:1) gave 122mg of Boc-IP-Gly-Exa.

Step four: synthesis of the title Compound

122mg of Boc-IP-Gly-Exa was weighed out and put in a 50mL eggplant-shaped bottle, and dissolved in 5mL of methylene chloride, followed by addition of 0.4mL of trifluoroacetic acid and stirring at room temperature overnight. The reaction solution was concentrated to dryness at 40 ℃ to prepare 22mg of the title compound from the liquid phase. ESI-MS: M/z 535.16[ M + H ]]⁺。

7) Preparation of Compound I-7

200mg of SM1 and 103mg of iodopropanol were weighed into a 100mL single-neck flask, 179mg of DIPEA and 20mL of DMF were added, and the temperature was raised to 55 ℃ to react for 18 hours. 200mL of ethyl acetate and 200mL of water were added, stirred for 20 minutes, and the organic phase was separated. The organic phase was added with 200mL of saturated brine, stirred for 20 minutes, and the organic phase was separated. The organic phase was concentrated to dryness. Liquid phase preparation gave 24mg of the title compound. ESI-MS: M/z 494.2[ M + H ]]⁺。

8) Preparation of Compound I-8

At room temperature, 20mg of irinotecan ADissolving sulfonate dihydrate, 3.7mg of (S) -3-hydroxybutyric acid and 20mg of HBTU in 8mL of DMF, dropwise adding 13.6mg of DIPEA, stirring at normal temperature for 1H, slowly adding 15mL of saturated NaCl solution and 30mL of ethyl acetate, repeatedly extracting for 3 times, combining organic phases, concentrating, and preparing a liquid phase to obtain 14mg of the title compound, LC-MS: M/z ═ 522.13(M + H)⁺。

9) Preparation of intermediate II-1

The method comprises the following steps: synthesis of II-1-A2

1.0g A5 and 1.1g of II-1-A1 were weighed into a 50mL eggplant-shaped bottle, 20mL of ethylene glycol dimethyl ether was added thereto, and the mixture was stirred at 0 ℃ for 10 minutes. 108mg of NaOH was weighed, 0.27mL of water was added to prepare an aqueous sodium hydroxide solution, and the aqueous sodium hydroxide solution was added to the reaction mixture and stirred at 0 ℃ for 3 hours. 160mg of glacial acetic acid are added and stirred for 1 hour. The reaction was diluted with 200mL of ethyl acetate. The reaction mixture was washed with 100mL of saturated brine 3 times to obtain an organic phase. The organic phase was dried over anhydrous sodium sulfate. The solid was removed by filtration. The filtrate was concentrated to dryness at 40 ℃. Column chromatography (petroleum ether: ethyl acetate 1:1) gave 650mg of ii-1-a 2. ESI-MS: M/z 525.13[ M + Na ]]⁺。

Step two: synthesis of II-1-A3

650mg of II-1-A2 was weighed into a 50mL round bottom flask, 10mL of THF and 10mL of ethyl acetate were added, and 65mg of 10% wet Pd/C was added. Replace three times with hydrogen and stir at room temperature for 2 h. Pd/C was removed by filtration, and the filtrate was concentrated to dryness at 40 ℃ to give 350mg of II-1-A3. ESI-MS: M/z 435.21[ M + Na ]]⁺。

Step three: synthesis of II-1-A4

137mg of II-1-A3 and 122mg of irinotecan mesylate dihydrate were weighed into a 50mL eggplant-shaped bottle, 8mL of DMF was added thereto, the mixture was stirred at 0 ℃ and then 110mg of N, N-diiso-acetate was added theretoPropylethylamine and 131mg of 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate were transferred to room temperature and stirred for 3 hours. The reaction mixture was diluted with 200mL of ethyl acetate, washed 3 times with 100mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, the solid was removed by filtration, the filtrate was concentrated to dryness at 40 ℃, and column chromatography (dichloromethane: methanol 15:1) was performed to give 170mg of ii-1-a 4. ESI-MS: M/z 830.38[ M + H ]]⁺。

Step four: II-1-A5

200mg of II-1-A4 was weighed into a 50mL eggplant-shaped bottle, and 5mL of THF was added to dissolve it, followed by addition of 60mg of 1, 8-diazabicycloundecen-7-ene and stirring at room temperature for 1 hour. 5mL of n-hexane was added, stirred for 5 minutes, filtered and concentrated to dryness at 40 ℃ to give 130mg of II-1-A5. ESI-MS: M/z 608.25[ M + H ]]⁺。

Step five: synthesis of II-1

100mg of MC-GGF-OH and 125mg of II-1-A5 were weighed into a 50mL eggplant-shaped bottle, dissolved in 5mL of DMF, and stirred in an ice-water bath, followed by sequentially adding 35mg of 1-hydroxybenzotriazole, 80. mu. L N, N-diisopropylethylamine and 49mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, removing the ice-water bath, and stirring at room temperature for 2 hours. The reaction was diluted with 250mL of ethyl acetate, washed 1 time with 100mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered to remove solids, and the filtrate was concentrated to dryness at 40 ℃ to afford 14mg of II-1 by preparative liquid phase. ESI-MS: M/z 1062.45[ M + H ]]⁺。

10) Preparation of intermediate II-2

The method comprises the following steps: synthesis of Compound II-2-A2

1.0g of Compound A5 was weighed into a bottle of eggplant shape, 25ml of ethylene glycol dimethyl ether was added to dissolve it, 0.98g of II-2-A1 (benzyl L-lactate) was added thereto, and the mixture was stirred at 0 ℃. 108mg of NaOH was added to 0.27mL of water to prepare an aqueous NaOH solution. The prepared aqueous solution of NaOH was added dropwise to the reaction solution. Reacting at 0 deg.C for 1 hr, adding 0.075ml glacial acetic acid, and stirring at 0 deg.C for 20 min. The reaction solution was concentrated to dryness. Column chromatography (petroleum ether: ethyl acetate 1:1) gave 1.08g of compound II-2-a 2. ESI-MS: M/z 511.09[ M + Na ]]⁺。

Step two: synthesis of Compound II-2-A3

1g of the compound II-2-A2 was weighed out in a bottle shaped like a eggplant, dissolved in 20ml of methanol, and reacted under hydrogen for 1 hour with 0.5g of wet palladium on carbon (palladium content: 10%) to complete the reaction. The reaction solution was filtered through celite, and the filtrate was concentrated to obtain 0.5g of compound II-2-A3. ESI-MS: M/z 399.16[ M + H ]]⁺。

Step three: synthesis of Compound II-2-A4

75mg of irinotecan mesylate dihydrate and 50mg of the intermediate II-2-A3 are weighed and placed in an eggplant-shaped bottle, 5ml of DMF is added for dissolution, 48mg of N, N-diisopropylethylamine and 71mg of 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate are added, and the mixture is stirred at room temperature for reaction for 3 hours and completely reacted. The reaction was diluted with 50mL of ethyl acetate, washed 2 times with 50mL of saturated sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give 113mg of intermediate II-2-A4. ESI-MS: M/z 816.47[ M + H ]]⁺。

Step four: synthesis of Compound II-2-A5

58mg of intermediate II-2-A4 was weighed into a bottle shaped like a eggplant, and 5ml of tetrahydrofuran was added to dissolve it, and 13mg of 1, 8-diazabicycloundecen-7-ene was added thereto and stirred at room temperature for 1 hour. 5ml of n-hexane was added to the reaction solution to precipitate a solid, which was filtered to obtain 49mg of Compound II-2-A5. ESI-MS: M/z 549.17[ M + H ]]⁺。

Step five: synthesis of Compound II-2

105mg of the compound II-2-A5 was weighed out in a eggplant-shaped bottle, 10ml of DMF was added and dissolved, 63mg of MC-GGF-OH, 27mg of 1-hydroxybenzotriazole and 52mg of N, N-diisopropylethylamine were added, and after stirring uniformly, 51mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was added and stirred at room temperature for 3 hours. The reaction solution was diluted with 50ml of ethyl acetate, washed 2 times with 50ml of saturated sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain 39mg of compound II-2 by preparative liquid phase. ESI-MS: M/z 1048.45[ M + H ]]⁺。

11) Preparation of intermediate II-3

The method comprises the following steps: synthesis of II-3-A1 (benzyl D-lactate)

Weighing 1g D-lactic acid, adding into 50mL eggplant-shaped bottle, adding 15mL N, N-dimethylformamide, stirring at 0 deg.C, and adding 4.34g Cs₂CO₃After stirring for 30 minutes, 1.5mL of benzyl bromide was added and the mixture was allowed to warm to room temperature and stirred for 18 h. To the reaction solution was added 35mL of 5% citric acid solution and stirred for 5 minutes, followed by extraction with 150mL of ethyl acetate, washing with 50mL of 5% citric acid solution 3 times, washing with 50mL of saturated brine 1 time, drying the organic phase with anhydrous sodium sulfate, filtering to remove solids, concentrating the filtrate at 40 ℃ to dryness, and column chromatography (petroleum ether: ethyl acetate 5:1) to give 1.2g of II-3-a 1. ESI-MS: 90.99[ M +2H ] M/z]⁺。

Step two: synthesis of II-3-A2

1.2g of II-3-A1 and 1g A5 were weighed and added to a 50mL eggplant-shaped bottle, 2mL of ethylene glycol dimethyl ether was added, the mixture was stirred at 0 ℃ for 10 minutes, 109mg of NaOH was weighed and 0.27mL of water was added to prepare an aqueous NaOH solution, and the aqueous NaOH solution was added to the reaction mixture and stirred at 0 ℃ for 3.5 hours. To the reaction solution was added 78. mu.L of glacial acetic acid, followed by stirring for 1 hour, diluting the reaction solution with 200mL of ethyl acetate, washing with 100mL of saturated brine 3 times, drying the organic phase over anhydrous sodium sulfate, filtering to remove the solid, concentrating the filtrate at 40 ℃ to dryness, and performing column chromatography (petroleum ether: ethyl acetate 1:1) to give 659mg of II-3-A2. ESI-MS: M/z 511.13[ M + Na ]]⁺。

Step three: synthesis of II-3-A3

659mg of II-3-A2 was weighed and put into a 50mL round bottom flask, 264mL of 10% Pd/C was added, 10mL of THF and 5mL of ethyl acetate were added, the air was replaced with hydrogen gas three times, and the mixture was stirred at room temperature for 4 hours. Filtering to remove solid, filteringThe solution was concentrated to dryness at 40 ℃ to give 400mg of II-3-A3. ESI-MS: M/z 421.14[ M + Na ]]⁺。

Step four: synthesis of II-3-A4

137mg of II-3-A3 and 122mg of irinotecan mesylate dihydrate were weighed into a 50mL eggplant-shaped bottle, 8mL of DMF was added, and the mixture was stirred at 0 ℃ followed by sequentially adding 114. mu. L N, N-diisopropylethylamine and 131mg of 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, and the mixture was allowed to warm to room temperature and stirred for 3 hours. The reaction mixture was diluted with 200mL of ethyl acetate, washed 5 times with 100mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, the solid was removed by filtration, the filtrate was concentrated to dryness at 40 ℃, and column chromatography (dichloromethane: methanol 10: 1) was performed to give 216mg of II-3-a 4. ESI-MS: M/z 816.38[ M + H ]]⁺。

Step five: synthesis of II-3-A5

216mg of II-3-A4 was weighed into a 50mL eggplant-shaped bottle, and 5mL of THF was added to dissolve it, followed by addition of 59. mu.L of 1, 8-diazabicycloundecen-7-ene and stirring at room temperature for 1 hour. 5mL of n-hexane was added, stirred for 5 minutes, filtered, and the mixture was purified by filtration using n-hexane: the solid was rinsed in THF 1:1 and dissolved in 50mL dichloromethane: methanol 2:1, and concentrated to dryness at 40 ℃ to give 188mg of II-3-A5. ESI-MS: M/z 594.15[ M + H ]]⁺。

Step six: synthesis of II-3

101mg of MC-GGF-OH and 127mg of II-3-A5 were weighed into a 50mL eggplant-shaped bottle, dissolved in 5mL of DMF, placed in an ice-water bath and stirred, followed by the sequential addition of 35mg of 1-hydroxybenzotriazole, 88. mu. L N, N-diisopropylethylamine and 49mg of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride, the ice-water bath was then removed and stirred at room temperature for 2 hours. The reaction was diluted with 250mL of ethyl acetate, washed 5 times with 100mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, the solid was removed by filtration, and the filtrate was concentrated to dryness at 40 ℃ to give 14mg of II-3 by preparative liquid phase. ESI-MS: M/z 1048.45[ M + H ]]⁺。

12) Preparation of intermediate II-4

The method comprises the following steps: synthesis of II-4-A2

1.0g A5 and 1.3g of II-4-A1 were weighed into a 50mL round bottom flask, 20mL of ethylene glycol dimethyl ether was added, and the mixture was stirred at 0 ℃ for 10 minutes. 108mg of NaOH was weighed, 0.27mL of water was added to prepare an aqueous sodium hydroxide solution, and the aqueous sodium hydroxide solution was added to the reaction mixture and stirred at 0 ℃ for 3 hours. 160mg of glacial acetic acid are added and stirred for 1 hour. The reaction was diluted with 200mL of ethyl acetate. The reaction mixture was washed with 100mL of saturated brine 3 times to obtain an organic phase. The organic phase was dried over anhydrous sodium sulfate. The solid was removed by filtration. The filtrate was concentrated to dryness at 40 ℃. Column chromatography (petroleum ether: ethyl acetate 1:1) gave 350mg of II-4-a 2. ESI-MS: M/z 573.21[ M + Na ]]⁺。

Step two: synthesis of II-4-A3

325mg of II-4-A2 was weighed and put into a 50mL eggplant type bottle, 10mL of THF and 10mL of ethyl acetate were added, and 32mg of 10% wet Pd/C was added. The air was replaced three times with hydrogen and stirred at room temperature for 2 h. Pd/C was removed by filtration, and the filtrate was concentrated to dryness at 40 ℃ to give 230mg of II-4-A3. ESI-MS: M/z 483.35[ M + Na ]]⁺。

Step three: synthesis of II-4-A4

150mg of II-4-A3 and 154mg of irinotecan mesylate dihydrate were weighed into a 50mL eggplant-shaped bottle, 8mL of DMF was added, the mixture was stirred at 0 ℃ and 140mg of N, N-diisopropylethylamine and 153mg of 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate were sequentially added, the temperature was raised to room temperature and the mixture was stirred for 3 hours. The reaction mixture was diluted with 200mL of ethyl acetate, washed 3 times with 100mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, the solid was removed by filtration, the filtrate was concentrated to dryness at 40 ℃, and column chromatography was performed (dichloromethane: methanol 15:1) to give 160mg of II-4-a 4. ESI-MS: M/z 878.38[ M + H ]]⁺。

Step four: II-4-A5

200mg of II-4-A4 was weighed into a 50mL round bottom bottle, and 5mL of T was addedHF was dissolved, and 35mg of 1, 8-diazabicycloundecen-7-ene was added, followed by stirring at room temperature for 1 hour. 5mL of n-hexane was added thereto, and the mixture was stirred for 5 minutes and filtered to obtain 150mg of II-4-A5. ESI-MS: M/z 656.25[ M + H ]]⁺。

Step five: synthesis of II-4

86mg of MC-GGF-OH and 100mg of II-4-A5 were weighed into a 50mL eggplant-shaped bottle, dissolved in 5mL of DMF, placed in an ice-water bath and stirred, and then 30mg of 1-hydroxybenzotriazole and 42mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride were added thereto in this order, and the ice-water bath was removed and stirred at room temperature for 2 hours. The reaction was diluted with 250mL of ethyl acetate, washed 1 time with 100mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered to remove solids, and the filtrate was concentrated to dryness at 40 ℃ to give 20mg of II-4 by preparative liquid phase. ESI-MS: M/z 1110.62[ M + H ]]⁺。

13) Preparation of intermediate II-5

14) Preparation of intermediate II-6

15) Preparation of intermediate II-7

The method comprises the following steps: synthesis of Compound II-7-A2

Weighing 10g of compound II-7-A1 in a eggplant-shaped bottle, adding 150mL of ethylene glycol dimethyl ether for dissolving, adding 54.3g of 1, 3-propylene glycol, stirring at 0 ℃, weighing 1.43g of NaOH, adding 3mL of water to prepare NaOH aqueous solution, and dropwise adding the NaOH aqueous solution into the reaction solutionThe reaction mixture was stirred at 0 ℃ for 2 hours, 1.07g of glacial acetic acid was added to the reaction mixture, and the mixture was stirred at room temperature for 1 hour to complete the reaction. The reaction mixture was concentrated, and 500mL of ethyl acetate was added, and the mixture was washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, and filtered to obtain 5.8g of compound II-7-A2. ESI-MS: M/z 319.16[ M + Na ]]⁺。

Step two: synthesis of Compound II-7-A3

Weighing 5g of compound II-7-A2 in a solanaceous bottle, adding 150mL of dichloromethane to dissolve, adding 5.12g of triethylamine, adding 3.93g of p-nitrobenzenesulfonyl chloride and 0.1g of 4-dimethylaminopyridine under the ice bath condition, stirring at room temperature to react for 3h, and reacting completely. 50mL of dichloromethane was added to the reaction solution, and the reaction solution was washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate: 2:1) to obtain 6.1g of compound II-7-A3. ESI-MS: M/z 482.00[ M + H ]]⁺。

Step three: synthesis of Compound II-7-A4

680mg of compound II-7-A3 and 250mg of irinotecan mesylate dihydrate are weighed and placed in a solanaceous bottle, 1mL of N-methylpyrrolidone is added for dissolution, 240mg of N, N-diisopropylethylamine is added, the mixture is heated and stirred at 60 ℃ for reaction for 4 hours, and the reaction liquid is directly purified by column chromatography (ethyl acetate: methanol-20: 1) to obtain 335mg of compound II-7-A4. ESI-MS: M/z 714.24[ M + H ]]⁺。

Step four: synthesis of Compound II-7-A5

335mg of intermediate II-7-a4 was weighed into a flask shaped like a eggplant, and 16mL of a dichloromethane methanol solution (dichloromethane: methanol 15:1) was added to dissolve the intermediate, and 300mg of wet palladium on carbon (palladium content 10%) was added thereto, and the mixture was stirred under hydrogen conditions for reaction for 3 hours to complete the reaction. The reaction solution was filtered through celite, and the filtrate was concentrated to obtain 239mg of compound II-7-A5. ESI-MS, M/z 580.12[ M + H ]]⁺。

Step five: synthesis of Compound II-7

Weighing 240mg of intermediate II-7-A5 in a solanaceous bottle, adding 5mL of DMF for dissolving, sequentially adding 235mg of compound MC-GGF-OH, 119mg of 1-hydroxybenzotriazole and 160mg of N, N-diisopropylethylamine, adding 84mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride under ice bath conditions, and carrying out strip at room temperatureStirring the mixture for reaction for 2 hours under the condition of complete reaction, and obtaining 21mg of final product II-7 by directly preparing a liquid phase from the reaction liquid. ESI-MS: M/z 1034.37[ M + H ]]⁺。

16) Preparation of intermediate II-8

EXAMPLE 16 preparation of antibody drug conjugates

Reagent:

solution A: ph7.4 PBS buffer, solution B: 10mM aqueous TCEP (tris (2-carboxyethyl) phosphine hydrochloride), solution C: DMSO (dimethyl sulfoxide), solution D: histidine buffer (containing 0.89mg/ml L-histidine and 4.04mg/ml L-histidine hydrochloride monohydrate), solution E: 700mg/ml sucrose solution (prepared with solution D), solution F: 20mg/ml Tween 80 (prepared from solution D)

Antibody: trastuzumab, 23C2 Her2-2

linker-payload (linker-cytotoxic drug moiety): II-1 to II-8

The experimental process comprises the following steps:

1. replacing the antibody, and fully wetting an ultrafiltration centrifugal tube with 30KD with the solution A; b. displacing the antibody into solution a; c. adding a proper amount of solution A to adjust the antibody concentration

2. Reducing the antibody a, calculating the molar weight of the antibody, and marking as N1; b. adding a proper amount of solution B into the antibody solution to ensure that the molar weight of TCEP in the reaction system is N2; c. wrapped with aluminum foil, placed on a rotary culture instrument and shaken at low speed (20rpm), and reacted for 1h at 37 ℃ in the dark.

3. Coupling a, taking a proper amount of linker-payload, dissolving the linker-payload by DMSO to obtain a final concentration of 10 mg/ml; b. adding DMSO into the antibody solution to make the antibody concentration be 5 wt%, and adding a proper amount of linker-payload solution to make the molar concentration be N3; c. wrapped with aluminum foil, placed on a rotary culture instrument and shaken at low speed (20rpm), and reacted for 2 hours at 20 ℃ in the dark.

4. Stopping coupling, namely a, wetting an ultrafiltration centrifugal tube by using a solution D; b. the antibody was replaced into solution D, and appropriate amount of solution E, F was added and the concentrations of sucrose and Tween 80 were adjusted to 90mg/ml and 0.3mg/ml, respectively, and frozen at-80 ℃.

Determination of DAR value (average number of drug-links per molecule of antibody) of antibody drug conjugates

DAR values were determined by LC-MS method. A50. mu.g sample of the prepared ADC was taken, and 1. mu.l of glycosidase PNGase F (Ruian biosciences, China) was added thereto and incubated at 37 ℃ for 20 hours. The mass spectrometer used in the experiment was a high resolution Xevo G2-XS (Waters, USA). Adjusting the concentration of the sample to 5 mu M, and collecting mass spectrum data in a positive ion mode by adopting a direct injection method. The collected non-denaturing mass spectral data were processed analytically using the software UNIFI 1.8.2.169(Waters, usa).

The following antibody drug conjugates were prepared by the above method:

example 17 in vitro enzyme Activity

1. Reagent material preparation

a. Preparing 1% agarose electrophoresis gel; b. preparing gelred foam dyeing solution, and storing in dark; c. preparing a working solution of topoisomerase I: prepared by ultrapure water and buffer solution.

2. Sample preparation

a. Compounds were reconstituted and diluted with DMSO, starting at 200 μ M, 10-fold diluted, and 5 gradients were set.

3. Reaction system

a. Positive control: 15 μ l of ultrapure water +2 μ l of 10xDNA Toposisomerase Buffer +2 μ l of 0.1% BSA +1 μ l of pBR322 DNA;

b. negative control: 14. mu.l of ultrapure water + 2. mu.l of 10xDNA Toposisomerasel Buffer + 2. mu.l of 0.1% BSA + 1. mu.l of pBR322DNA + 1. mu.l of topoisomerase I working solution;

c. sample group: mu.l of ultrapure water + 2. mu.l of 10xDNA Topoismerasel Buffer + 2. mu.l of 0.1% BSA + 1. mu.l of pBR322DNA + 1. mu.l of topoisomerase I working solution + 2. mu.l of compound.

4. Experimental procedure

a. Water bath at 37 deg.C for 30 min; b. adding 2 mul of loading buffer into each tube system to terminate the reaction; c. voltage is 2-2.5V/cm, electrophoresis is carried out for 1.5 h; d. the gel was stained with gelred photophobic stains for 1.5h and photographed with a gel imager.

EXAMPLE 18 cellular Activity of cytotoxic molecules and antibody drug conjugates

The reference samples S1-S9 were obtained by prediluting I-1 to I-8 to 140000ng/ml, labeled S1, using medium, respectively, followed by a quintupling dilution, with a final drug concentration ranging from 35000ng/ml to 0.0896ng/ml, for a total of 9 concentrations. Collecting HER2 positive tumor cells in logarithmic growth phase, and adjusting density to 1 × 10⁵cells/ml were plated with 100. mu.l per well and a blank well without cells was set as a control. A gradient of 50. mu.l of each of the two samples was added. At 37 ℃ with 5% CO₂Culturing in carbon dioxide incubator for 5 days. The culture solution is discarded, CCK-8 (Japan Dojindo chemical, cat # CK04) working solution is added into each well in an amount of 100. mu.l, the mixture is incubated and developed for 4 to 5 hours, then the obtained product is placed into an enzyme-linked immunosorbent assay (manufacturer Thermo, model: VarioskanWalsh), and the absorbance value of the well plate under the wavelength of 450nm is read and recorded by taking 630nm as a reference wavelength. Computing IC₅₀The value is obtained. See in particular table 8 below.

IC of tables 8-1.I-5, I-6, I-7₅₀Value of

IC of tables 8-2.I-1, I-2, I-3₅₀Value of

IC of tables 8-3.I-8₅₀Value of

IC of tables 8-4.I-4₅₀Value of

The antibody drug conjugates prepared in example 16 were pre-diluted to 20. mu.g/ml, labeled S1, using culture media, respectively, and then five-fold gradient-diluted to obtain respective corresponding samples S1-S9. The final concentration of the medicine is 5000ng/ml-0.0128ng/ml, and the total concentration is 9. Collecting HER2 positive tumor cells in logarithmic growth phase, and adjusting density to 2 × 10⁴cells/ml were plated with 100. mu.l per well and a blank well without cells was set as a control. A gradient of diluted sample was added, 50. mu.l per well. At 37 ℃ with 5% CO₂Culturing in a carbon dioxide incubator. The culture medium was discarded, CTG detection solution (Promega, cat # G7572) was added to each well in an amount of 100. mu.l, incubated and developed for 10min, and then placed on a multifunctional plate reader (manufacturer Thermo, model: Varioska nWalsh) to read the chemiluminescence value. Computing IC₅₀The value is obtained. See in particular table 9 below.

TABLE 9 IC of each ADC molecule₅₀Value of

ND:not determined

Example 19 in vivo pharmacokinetic experiments with antibody drug conjugates

The in vivo metabolic pathways as well as pharmacokinetic parameters of the antibody drug conjugates of the invention were determined.

EXAMPLE 20 therapeutic Effect of antibody drug conjugates on nude mouse transplantable tumors

The in vivo efficacy of the antibody drug conjugate was evaluated by the method of reference example 12.

While the methods of the present invention have been described in terms of preferred embodiments in light of the present disclosure, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention.

The disclosures of all documents cited herein are incorporated by reference herein, to the extent that they provide exemplary, procedural and other details supplementary to those set forth herein.

Sequence listing

<110> Ningda Ningqing pharmaceutical industry group, Inc

<120> antibody drug conjugates

<150> 202011389955.2

<151> 2020-12-01

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<213> Artificial Sequence (Artificial Sequence)

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser

130 135 140

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala

145 150 155 160

Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly

180 185 190

Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

210 215 220

Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu

225 230 235 240

Ile Lys Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro

245 250 255

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

260 265 270

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

275 280 285

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

290 295 300

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

305 310 315 320

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

325 330 335

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

340 345 350

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

355 360 365

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

370 375 380

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

385 390 395 400

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

405 410 415

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

420 425 430

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

435 440 445

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

450 455 460

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470 475

<210> 2

<211> 475

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser

130 135 140

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala

145 150 155 160

Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Leu Tyr Ser Gly

180 185 190

Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

210 215 220

Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu

225 230 235 240

Ile Lys Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro

245 250 255

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

260 265 270

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

275 280 285

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

290 295 300

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

305 310 315 320

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

325 330 335

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

340 345 350

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

355 360 365

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

370 375 380

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

385 390 395 400

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

405 410 415

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

420 425 430

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

435 440 445

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

450 455 460

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470 475

<210> 3

<211> 475

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser

130 135 140

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala

145 150 155 160

Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ala Leu Tyr Ser Gly

180 185 190

Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

210 215 220

Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu

225 230 235 240

Ile Lys Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro

245 250 255

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

260 265 270

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

275 280 285

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

290 295 300

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

305 310 315 320

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

325 330 335

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

340 345 350

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

355 360 365

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

370 375 380

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

385 390 395 400

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

405 410 415

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

420 425 430

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

435 440 445

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

450 455 460

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470 475

<210> 4

<211> 475

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser

130 135 140

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala

145 150 155 160

Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Arg Leu Tyr Ser Gly

180 185 190

Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

210 215 220

Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu

225 230 235 240

Ile Lys Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro

245 250 255

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

260 265 270

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

275 280 285

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

290 295 300

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

305 310 315 320

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

325 330 335

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

340 345 350

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

355 360 365

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

370 375 380

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

385 390 395 400

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

405 410 415

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

420 425 430

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

435 440 445

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

450 455 460

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470 475

<210> 5

<211> 475

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Glu Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser

130 135 140

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala

145 150 155 160

Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly

180 185 190

Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

210 215 220

Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu

225 230 235 240

Ile Lys Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro

245 250 255

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

260 265 270

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

275 280 285

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

290 295 300

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

305 310 315 320

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

325 330 335

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

340 345 350

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

355 360 365

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

370 375 380

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

385 390 395 400

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

405 410 415

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

420 425 430

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

435 440 445

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

450 455 460

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470 475

<210> 6

<211> 480

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Glu Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

130 135 140

Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

145 150 155 160

Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser

180 185 190

Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly

195 200 205

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Val Glu Ile Lys Gly Glu Pro Lys Ser Ser Asp Lys Thr

245 250 255

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

260 265 270

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

275 280 285

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

290 295 300

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

305 310 315 320

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

325 330 335

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

340 345 350

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

355 360 365

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

370 375 380

Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys

385 390 395 400

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

405 410 415

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

420 425 430

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

435 440 445

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

450 455 460

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470 475 480

<210> 7

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe

50 55 60

Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 8

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 9

<211> 481

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Ser Gly Gly

100 105 110

Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Glu

115 120 125

Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

130 135 140

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr

145 150 155 160

Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

165 170 175

Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys

180 185 190

Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu

195 200 205

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser

210 215 220

Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ser Ala Ala Glu Pro Lys Ser Ser Asp Lys

245 250 255

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

260 265 270

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

275 280 285

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

290 295 300

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

305 310 315 320

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

325 330 335

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

340 345 350

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

355 360 365

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

370 375 380

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ile

385 390 395 400

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

405 410 415

Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu

420 425 430

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

435 440 445

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

450 455 460

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470 475 480

Lys

<210> 10

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe

50 55 60

Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val

340 345 350

Tyr Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 11

<211> 480

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Glu Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

130 135 140

Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

145 150 155 160

Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser

180 185 190

Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly

195 200 205

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Val Glu Ile Lys Gly Glu Pro Lys Ser Ser Asp Lys Thr

245 250 255

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

260 265 270

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

275 280 285

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

290 295 300

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

305 310 315 320

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

325 330 335

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

340 345 350

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

355 360 365

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

370 375 380

Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys

385 390 395 400

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

405 410 415

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

420 425 430

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

435 440 445

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

450 455 460

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470 475 480

<210> 12

<211> 480

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

130 135 140

Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

145 150 155 160

Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser

180 185 190

Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly

195 200 205

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Val Glu Ile Lys Gly Glu Pro Lys Ser Ser Asp Lys Thr

245 250 255

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

260 265 270

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

275 280 285

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

290 295 300

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

305 310 315 320

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

325 330 335

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

340 345 350

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

355 360 365

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

370 375 380

Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys

385 390 395 400

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

405 410 415

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

420 425 430

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

435 440 445

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

450 455 460

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470 475 480

<210> 13

<211> 480

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

130 135 140

Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

145 150 155 160

Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser

180 185 190

Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly

195 200 205

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Val Glu Ile Lys Gly Glu Pro Lys Ser Ser Asp Lys Thr

245 250 255

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

260 265 270

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

275 280 285

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

290 295 300

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

305 310 315 320

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

325 330 335

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

340 345 350

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

355 360 365

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

370 375 380

Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys

385 390 395 400

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

405 410 415

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

420 425 430

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

435 440 445

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

450 455 460

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470 475 480

<210> 14

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> UNSURE

<222> (5)

<223> X is K or E

<400> 14

Gly Phe Asn Ile Xaa Asp Thr Tyr Ile His

1 5 10

<210> 15

<211> 17

<212> PRT

<213> Mus musculus

<400> 15

Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 16

<211> 12

<212> PRT

<213> Mus musculus

<400> 16

Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp

1 5 10

<210> 17

<211> 13

<212> PRT

<213> Mus musculus

<400> 17

Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp

1 5 10

<210> 18

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Ser Ala Ser Tyr Leu Tyr Ser

1 5

<210> 19

<211> 9

<212> PRT

<213> Mus musculus

<400> 19

Gln Gln His Tyr Thr Thr Pro Pro Thr

1 5

<210> 20

<211> 7

<212> PRT

<213> Mus musculus

<400> 20

Ser Ala Ser Phe Leu Tyr Ser

1 5

<210> 21

<211> 7

<212> PRT

<213> Mus musculus

<220>

<221> UNSURE

<222> (4)

<223> X is F or Y

<400> 21

Ser Ala Ser Xaa Leu Tyr Ser

1 5

<210> 22

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Glu Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 23

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 24

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe

50 55 60

Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 25

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 26

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 27

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 28

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> UNSURE

<222> (30)

<223> X is K or E

<400> 28

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Xaa Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 29

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> UNSURE

<222> (53)

<223> X is F or Y

<400> 29

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Xaa Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 30

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Gly Phe Asn Ile Glu Asp Thr Tyr Ile His

1 5 10

<210> 31

<211> 10

<212> PRT

<213> Mus musculus

<400> 31

Gly Phe Asn Ile Lys Asp Thr Tyr Ile His

1 5 10

<210> 32

<211> 10

<212> PRT

<213> Mus musculus

<400> 32

Gly Phe Thr Phe Thr Asp Tyr Thr Met Asp

1 5 10

<210> 33

<211> 17

<212> PRT

<213> Mus musculus

<400> 33

Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe Lys

1 5 10 15

Gly

<210> 34

<211> 10

<212> PRT

<213> Mus musculus

<400> 34

Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr

1 5 10

<210> 35

<211> 11

<212> PRT

<213> Mus musculus

<400> 35

Lys Ala Ser Gln Asp Val Ser Ile Gly Val Ala

1 5 10

<210> 36

<211> 7

<212> PRT

<213> Mus musculus

<400> 36

Ser Ala Ser Tyr Arg Tyr Thr

1 5

<210> 37

<211> 9

<212> PRT

<213> Mus musculus

<400> 37

Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr

1 5

Claims

1. An antibody drug conjugate comprising an antibody moiety, an intermediate linker moiety and a cytotoxic drug moiety, said antibody moiety being linked to said cytotoxic drug moiety through said intermediate linker moiety, or a pharmaceutically acceptable salt or solvate thereof, wherein said antibody drug conjugate is of the structure shown below formula V:

wherein,

n¹is an integer of 0 to 6, and,

n²is an integer of 1 to 6, and,

n³is an integer of 1 to 6, and,

Wherein:

or,

ab represents an antibody moiety of the group,

n is an integer or decimal number selected from 1 to 10.

2. The antibody drug conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein L is selected from:

3. the antibody drug conjugate of any one of claims 1-2, or a pharmaceutically acceptable salt or solvate thereof, wherein the right terminus of L is linked to the amino group at position 1 of irinotecan.

4. The antibody drug conjugate of any one of claims 1-3, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody drug conjugate is of the structure:

wherein,

ab represents an antibody moiety of the group,

n is an integer or decimal number selected from 1 to 10.

5. The antibody drug conjugate of any one of claims 1 to 4, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab of the antibody drug conjugate is trastuzumab.

6. Use of an antibody drug conjugate according to any one of claims 1 to 5, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the prevention or treatment of cancer.

7. A compound having the structure shown in formula IIIa below:

wherein:

n¹is an integer of 0 to 6, and,

n²is an integer of 1 to 6, and,

n³is an integer of 1 to 6, and,

Wherein:

or,

8. The compound of claim 7, wherein L is selected from the structures:

9. The following compounds: