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CN112138171B - Antibody coupling drug, intermediate thereof, preparation method and application - Google Patents

Antibody coupling drug, intermediate thereof, preparation method and application Download PDF

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Publication number
CN112138171B
CN112138171B CN201910576227.3A CN201910576227A CN112138171B CN 112138171 B CN112138171 B CN 112138171B CN 201910576227 A CN201910576227 A CN 201910576227A CN 112138171 B CN112138171 B CN 112138171B
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CN112138171A (en
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鲍彬
郭青松
高贝
张一帆
邱雪飞
杨彤
沈毅珺
张文伯
吕伟
王磊
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SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD
Taizhou Fudan Zhangjiang Pharmaceutical Co Ltd
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SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD
Taizhou Fudan Zhangjiang Pharmaceutical Co Ltd
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Priority to CN201910576227.3A priority Critical patent/CN112138171B/en
Priority to CN202311489217.9A priority patent/CN117731798A/en
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6873—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an immunoglobulin; the antibody being an anti-idiotypic antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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Abstract

The invention discloses an antibody coupling drug, an intermediate thereof, a preparation method and application. The invention discloses an antibody coupling drug, which has a structural general formula of Ab- (L 3-L2-L1-D)m. The antibody coupling drug can realize the wide application of cytotoxic drugs, especially camptothecine in the ADC field, and treat tumor patients with microtubule ADC drug resistance.

Description

Antibody coupling drug, intermediate thereof, preparation method and application
Technical Field
The invention belongs to the field of biotechnology and medicine, and particularly relates to an antibody coupling drug, an intermediate thereof, a preparation method and application.
Background
Antibody-conjugated drugs (ADCs) are one of the hot spots of recent interest in the pharmaceutical industry. Because of the inadequate clinical efficacy of many antibody drugs, many industries are increasingly turning their eyes to ADC drugs. Currently, four ADC drugs are commercially available in the world. The FDA approved for use in treating Acute Myelogenous Leukemia (AML) patients with first relapse, over 60 years old, cd33+, unsuitable for cytotoxic chemotherapy at Gemtuzumab Ozogamicin/17 th 2000, while the drug was withdrawn in 2010 but re-marketed in 2017, inotuzumab ozogamicin (trade name Besponsa) for the same year of the fei was also FDA approved for use in treating adult relapsed refractory B-cell ALL. Brentuximab Vedotin (trade name Adcetris) developed by the FDA approved SEATTLE GENETICS company was marketed for the treatment of CD30 positive Hodgkin's Lymphoma (HL) and rare disease Systemic Anaplastic Large Cell Lymphoma (SALCL) at month 8 and 19 2011. 22.2013, ado-trastuzumab emtansine (T-DM 1, trade name Kadcyla) developed by Genntech corporation was approved by the FDA for marketing and was used mainly for the treatment of Her2 positive advanced (metastatic) breast cancer. In addition, more than 100 ADC drugs are internationally in clinical and preclinical development stages.
The linker of ADC is a vital component, and its stability not only affects the release of small molecules in tumor cells, but also is related to toxic side effects of ADC. An over-stable linker is generally not readily releasable, such as T-DM1; too labile linkers tend to release the drug in the plasma, thereby bringing about toxicity, such as IMMU-132. The first co-company published patent (patent number ZL 201380053256) discloses a connecting group which is quite stable in blood plasma, namely, a camptothecine compound is connected with an antibody through a section of tetrapeptide which can be digested and cleaved by self-cleaving aminomethylether structure, so that the camptothecine compound has a better anti-tumor effect. Although this structure maintains good stability in plasma, the aminomethyl ether structure is acid-labile, and there are many acidic environments in the body, including both part of the tumor tissue and some normal tissue. Thus ADC releases part of the drug molecule without endocytosis by tumor cells, resulting in toxicity.
The present invention is directed to solving the above-mentioned problems of the prior art. The invention provides two technical schemes, one is to replace an aminomethyl ether structure with a carbon amide structure, so that the stability is enhanced. The other is to directly connect the self-cleavage fragments to the drug molecules through alkyl groups, and the structure has good stability and can generate rapid 1, 6-elimination after enzyme digestion so as to release the drug. Therefore, the invention can solve the defects of the ADC, can keep better biological activity, and can obtain better anti-tumor activity and lower toxicity in vivo.
Disclosure of Invention
The invention aims to overcome the defect of single type of the existing antibody coupling drug, and provides an antibody coupling drug, an intermediate, a preparation method and application. The antibody coupling medicine can realize the wide application of the cytotoxicity medicine in the ADC field and treat tumor patients with microtubule ADC drug resistance.
The invention solves the technical problems through the following technical proposal.
The invention provides an antibody coupling drug, which has a structural general formula of Ab- (L 3-L2-L1-D)m);
Wherein Ab is an antibody;
D is a cytotoxic drug;
m is 2-8;
The structure of L 1 is shown as a formula I or II; wherein the a end is connected with the cytotoxic drug, the e end is connected with the c end of the L 2,
L is independently a phenylalanine residue, glycine residue, glutamic acid residue, aspartic acid residue, cysteine residue, glutamic acid residue, histidine residue, isoleucine residue, leucine residue, lysine residue, methionine residue, proline residue, serine residue, threonine residue, tryptophan residue, tyrosine residue or valine residue; p is 0 to 4;
R 1 is hydrogen, substituted or unsubstituted C 1～C10 alkyl, substituted or unsubstituted C 6～C14 aryl, substituted or unsubstituted 5-to 10-membered heterocyclyl, or substituted or unsubstituted phenoxy; the substituents in the substituted C 1～C10 alkyl, substituted C 6～C14 aryl, substituted 5-to 10-membered heterocyclyl, and substituted phenoxy are one or more groups selected from the group consisting of, when multiple substituents are present, the substituents are the same or different: halogen, hydroxy, -NR 1-1R1-2、-S(O)2R1-3、C1～C4 alkyl, C 1～C4 alkoxy, hydroxy-substituted C 1～C4 alkoxy, C 3～C8 cycloalkyl, 5-to 8-membered heterocyclyl, C 6～C10 aryl, and 5-to 10-membered heteroaryl; the heteroatom in the substituted or unsubstituted 5-10 membered heterocyclic group, 5-8 membered heterocyclic group or 5-10 membered heteroaryl group is selected from one or more of N, O and S, and the number of the heteroatom is 1,2,3 or 4; r 1-1、R1-2 and R 1-3 are independently C 1～C4 alkyl;
R 2 is independently hydrogen, substituted or unsubstituted C 1～C10 alkyl, substituted or unsubstituted C 3～C10 cycloalkyl, substituted or unsubstituted C 6～C14 aryl, substituted or unsubstituted 5-to 10-membered heteroaryl; the substituents in the substituted C 1～C10 alkyl, substituted C 3～C10 cycloalkyl, substituted C 6～C14 aryl, and substituted 5-to 10-membered heteroaryl are one or more groups selected from the group consisting of, when multiple substituents are present, the substituents are the same or different: halogen, hydroxy, C 1～C4 alkyl, C 1～C4 alkoxy, C 3～C8 cycloalkyl, 5-to 8-membered heterocyclyl, C 6～C10 aryl, and 5-to 10-membered heteroaryl; the hetero atoms in the 5-8 membered heterocyclic group, the 5-10 membered heteroaryl group and the substituted or unsubstituted 5-10 membered heterocyclic group are selected from one or more of N, O and S, and the number of the hetero atoms is 1, 2, 3 or 4;
n 1 is 2, 3 or 4;
L' is independently a phenylalanine residue, glycine residue, glutamic acid residue, aspartic acid residue, cysteine residue, glutamic acid residue, histidine residue, isoleucine residue, leucine residue, lysine residue, methionine residue, proline residue, serine residue, threonine residue, tryptophan residue, tyrosine residue or valine residue; p' is 2-4;
l 2 is Wherein, the c end is connected with the e end of L 1, the f end is connected with the d end of L 3, and n 2 is independently 1-8;
L 3 is Wherein the b end is connected with the Ab, and the d end is connected with the f end of the L 2.
In a preferred embodiment of the invention, the antibody may be an antibody conventional in the field of anti-tumor ADCs, preferably Trastuzumab or a variant thereof, anti-B7-H3 antibody P2E5 or a variant thereof, anti-claudin 18.2 antibody IMAB362 or a variant thereof, or anti-Trop 2 antibody RS7 or a variant thereof, more preferably Trastuzumab or a variant thereof, most preferably Trastuzumab. The amino acid sequence of the light chain in the anti-HER 2 antibody Trastuzumab is preferably shown as SEQ ID No.5 in a sequence table, and the amino acid sequence of the heavy chain is preferably shown as SEQ ID No.6 in the sequence table. The amino acid sequence of the light chain in the anti-B7-H3 antibody P2E5 is preferably shown as SEQ ID No.7 in a sequence table, and the amino acid sequence of the heavy chain is preferably shown as SEQ ID No.8 in the sequence table. The amino acid sequence of the light chain in the Claudin18.2 antibody IMAB362 is preferably shown as SEQ ID No.1 in a sequence table, and the amino acid sequence of the heavy chain is preferably shown as SEQ ID No.2 in the sequence table. The amino acid sequence of the light chain in the anti-Trop 2 antibody RS7 is preferably shown as SEQ ID No.3 in the sequence table, and the amino acid sequence of the heavy chain is preferably shown as SEQ ID No.4 in the sequence table.
In a preferred embodiment of the invention, the b-segment of L 3 is preferably linked to the thiol group of the antibody as a thioether bond. To be used forIn the case of an example of this,The form of linkage to the cysteine residues in the antibodies is
In a preferred embodiment of the present invention, D may be a cytotoxic drug conventional in the ADC field, and the present invention is particularly preferably a hydroxyl-containing or amino-containing cytotoxic drug, more preferably a hydroxyl-containing or amino-containing topoisomerase inhibitor, still more preferably a hydroxyl-containing or amino-containing topoisomerase I inhibitor, still more preferably camptothecin or a derivative thereof, and most preferablyThe L 1 is preferably linked to the hydroxyl group of the cytotoxic drug in the form of an ether linkage or to the amino group of the cytotoxic drug. When said L 1 is linked to an amino group in said cytotoxic drug, the hydrogen on said amino group is not substituted or is substituted with one R 3; r 3 is C 1～C6 alkyl, C 3～C8 cycloalkyl, C 6～C14 aryl, 5-to 10-membered heteroaryl or-C (=O) R 3-1; the hetero atom in the 5-10 membered heteroaryl is selected from one or more of N, O and S, and the number of the hetero atom is 1, 2,3 or 4; r 3-1 is hydroxy-substituted C 1～C4 alkyl. When said L 1 is linked to said D, L 1 -D is preferablyWith L 2 asFor example, the L 1 -D may beWith L 2 as For example, the L 1 -D may be
In a preferred embodiment of the invention, m is preferably 7 to 8, more preferably 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7 or 7.8.
In a preferred embodiment of the present invention, when R 1 is substituted or unsubstituted C 1～C10 alkyl, the C 1～C10 alkyl is preferably C 1～C4 alkyl, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, most preferably methyl or ethyl.
In a preferred embodiment of the present invention, when R 1 is a substituted or unsubstituted C 6～C14 aryl, the C 6～C14 aryl is preferably phenyl, naphthyl or anthracenyl, more preferably phenyl.
In a preferred embodiment of the present invention, R 1-1、R1-2 and R 1-3 are independently preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, more preferably methyl.
In a preferred embodiment of the invention, in R 1, when the substituent in the substituted C 1～C10 alkyl is hydroxy-substituted C 1～C4 alkoxy, said C 1～C4 alkoxy is preferably methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy or tert-butoxy, more preferably ethoxy.
In a preferred embodiment of the present invention, when R 3 is-C (=o) R 3-1, R 3-1 is preferably hydroxy-substituted methyl, hydroxy-substituted ethyl, hydroxy-substituted n-propyl, hydroxy-substituted isopropyl, hydroxy-substituted n-butyl, hydroxy-substituted isobutyl, or hydroxy-substituted tert-butyl, and more preferably hydroxy-substituted methyl.
In a preferred embodiment of the present invention, said L 3 is preferably
In a preferred embodiment of the invention, said L is independently preferably a glycine residue or a phenylalanine residue; said p is preferably 4. The (L) p is preferablyWherein the g-terminus is attached to the carbonyl group in formula I.
In a preferred embodiment of the present invention, said R 1 is preferably substituted or unsubstituted C 1～C10 alkyl, or substituted or unsubstituted C 6～C14 aryl; the substituents in the substituted C 1～C10 alkyl and substituted C 6～C14 aryl are preferably one or more groups selected from the group consisting of, when multiple substituents are present, the substituents are the same or different: -NR 1- 1R1-2、-S(O)2R1-3 and hydroxy-substituted C 1～C4 alkoxy.
In a preferred embodiment of the present invention, R 2 is preferably hydrogen.
In a preferred embodiment of the present invention, n 1 is preferably 2.
In a preferred embodiment of the invention, said L' is independently preferably a valine residue, an alanine residue, a lysine residue, a phenylalanine residue or a citrulline residue; said p' is preferably 2. Said (L ') p' is preferablyWherein the h-terminus is attached to the carbonyl group in formula II.
In a preferred embodiment of the invention, thePreferably is
In a preferred embodiment of the invention, thePreferably is
In a preferred embodiment of the present invention, said L 2 is preferably
In a preferred embodiment of the present invention, n 2 is preferably 1.
In a preferred embodiment of the invention, when L 1 -D isWhen said L 2 is preferably
In a preferred embodiment of the present invention, said R 3 is preferably-C (=o) R 3-1.
In a preferred embodiment of the invention, when L 1 -D is In this case, the term (L ') p' is preferably
In a preferred embodiment of the invention, when D isWhen L 1 -D is preferably
In a preferred embodiment of the present invention, the antibody conjugated drug wherein the Ab is Trastuzumab, an anti-HER 2 antibody; the D is a cytotoxic drug; m is 2-8;
The structure of the L 1 is shown as a formula I or a formula II;
the L is independently a phenylalanine residue or a glycine residue; p is 0 to 4;
R 1 is substituted or unsubstituted C 1～C10 alkyl or substituted or unsubstituted C 6～C14 aryl; the substituents in the substituted C 1～C10 alkyl and substituted C 6～C14 aryl are one or more groups selected from the group consisting of, when multiple substituents are present, the substituents are the same or different: -NR 1-1R1-2、-S(O)2R1-3 and hydroxy-substituted C 1～C4 alkoxy; said R 1-1, said R 1-2 and said R 1-3 are independently C 1～C4 alkyl;
R 2 is hydrogen;
N 1 is 2;
The L' is independently a valine residue, an alanine residue, a lysine residue, a phenylalanine residue or a citrulline residue; the p' is 2-4;
When said L 1 is linked to an amino group in said cytotoxic drug, the hydrogen on said amino group is not substituted or is substituted with one R 3; r 3 is-C (=O) R 3-1;R3-1 is C 1～C4 alkyl substituted by hydroxy;
the L 2 is N 2 is 1-8;
The L 3 is
The amino acid sequence of the light chain in the anti-HER 2 antibody Trastuzumab is preferably shown as SEQ ID No.5 in a sequence table, and the amino acid sequence of the heavy chain is preferably shown as SEQ ID No.6 in the sequence table.
In a preferred embodiment of the present invention, the antibody conjugated drug wherein the Ab is Trastuzumab, an anti-HER 2 antibody; the D isM is 7-8;
The structure of the L 1 is shown as a formula I or a formula II;
the L is independently a phenylalanine residue or a glycine residue; p is 4;
R 1 is substituted or unsubstituted C 1～C4 alkyl or substituted or unsubstituted C 6～C14 aryl; the substituents in the substituted C 1～C4 alkyl and substituted C 6～C14 aryl are one or more groups selected from the group consisting of, when multiple substituents are present, the substituents are the same or different: -N (CH 3)2、-S(O)2CH3 and hydroxy-substituted ethoxy;
R 2 is hydrogen;
N 1 is 2;
The L' is independently a valine residue, an alanine residue, a lysine residue, a phenylalanine residue or a citrulline residue; the p' is 2-4;
When said L 1 is linked to an amino group in said cytotoxic drug, the hydrogen on said amino group is not substituted or is substituted with one R 3; r 3 is-C (=O) R 3-1;R3-1 is C 1～C4 alkyl substituted by hydroxy;
the L 2 is N 2 is 1-8;
The L 3 is
And when L 1 -D isWhen said L 2 is
The amino acid sequence of the light chain in the anti-HER 2 antibody Trastuzumab is preferably shown as SEQ ID No.5 in a sequence table, and the amino acid sequence of the heavy chain is preferably shown as SEQ ID No.6 in the sequence table.
In a preferred embodiment of the present invention, the antibody conjugated drug wherein the Ab is Trastuzumab, an anti-HER 2 antibody; l 1 -D isM is 7-8;
The structure of the L 1 is shown as a formula I or a formula II;
the L is independently a phenylalanine residue or a glycine residue; p is 4;
R 1 is substituted or unsubstituted C 1～C4 alkyl or substituted or unsubstituted C 6～C14 aryl; the substituents in the substituted C 1～C4 alkyl or substituted C 6～C14 aryl are one or more groups selected from the group consisting of, when multiple substituents are present, the substituents are the same or different: -N (CH 3)2、-S(O)2CH3 and hydroxy-substituted ethoxy;
R 2 is hydrogen;
N 1 is 2;
The L' is independently a valine residue, an alanine residue, a lysine residue, a phenylalanine residue or a citrulline residue; the p' is 2-4;
r 3 is-C (=O) R 3-1;R3-1 is methyl substituted by hydroxy;
the L 2 is N 2 is 1-8;
The L 3 is
And when L 1 -D isIn the case of (L ') p' isWhen L 1 -D isWhen said L 2 is
The amino acid sequence of the light chain in the anti-HER 2 antibody Trastuzumab is preferably shown as SEQ ID No.5 in a sequence table, and the amino acid sequence of the heavy chain is preferably shown as SEQ ID No.6 in the sequence table.
In a preferred embodiment of the present invention, the antibody-conjugated drug is preferably a compound shown in any one of the following:
Wherein Ab is anti-HER 2 antibody Trastuzumab, anti-B7-H3 antibody P2E5, anti-Claudin 18.2 antibody IMAB362 or anti-Trop 2 antibody RS7, and m is 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7 or 7.8; the amino acid sequence of the light chain in the anti-HER 2 antibody Trastuzumab is preferably shown as SEQ ID No.5 in a sequence table, and the amino acid sequence of the heavy chain is preferably shown as SEQ ID No.6 in the sequence table; the amino acid sequence of the light chain in the anti-B7-H3 antibody P2E5 is preferably shown as SEQ ID No.7 in a sequence table, and the amino acid sequence of the heavy chain is preferably shown as SEQ ID No.8 in the sequence table; the amino acid sequence of the light chain in the Claudin18.2 antibody IMAB362 is preferably shown as SEQ ID No.1 in a sequence table, and the amino acid sequence of the heavy chain is preferably shown as SEQ ID No.2 in the sequence table; the amino acid sequence of the light chain in the anti-Trop 2 antibody RS7 is preferably shown as SEQ ID No.3 in the sequence table, and the amino acid sequence of the heavy chain is preferably shown as SEQ ID No.4 in the sequence table.
In a preferred embodiment of the present invention, the antibody-conjugated drug is preferably a compound shown in any one of the following:
Further preferred is
More preferably
Wherein Ab is the anti-HER 2 antibody Trastuzumab; the amino acid sequence of the light chain in the anti-HER 2 antibody Trastuzumab is shown as SEQ ID No.5 in a sequence table, and the amino acid sequence of the heavy chain is shown as SEQ ID No.6 in the sequence table.
The invention also provides a linker-drug conjugate, which has the general structural formula of L 4-L2-L1 -D, wherein L 4 isL 2、L1 and D are as defined above, and the f-terminal of L 2 is connected to the D-terminal of L 4.
In a preferred embodiment of the invention, the linker-drug conjugate is preferably any one of the compounds shown below:
the invention provides a preparation method of the antibody coupling drug, which comprises the following steps of coupling the linker-drug conjugate with an antibody.
In the present invention, the conditions and operations of the coupling may be those conventional in the art.
The invention also provides a pharmaceutical composition comprising the antibody-conjugated drug and a pharmaceutically acceptable carrier.
The invention also provides an application of the antibody coupling medicament or the pharmaceutical composition in preparing medicaments for preventing or treating cancers. The cancer is preferably stomach cancer, breast cancer, non-small cell lung cancer, urothelial cancer or pancreatic cancer.
The invention also provides a pharmaceutical preparation comprising the antibody-conjugated drug.
In the present invention, m represents the molar ratio of cytotoxic drug molecule to Ab (also known as DAR, i.e. drug antibody coupling ratio), m may be an integer or a fraction, preferably understood as: the average molar ratio of the drug molecules in the antibody-conjugated drug obtained after the conjugation of the single monoclonal antibody molecule and the cytotoxic drug to the monoclonal antibody molecules can be generally determined by means of hydrophobic chromatography (Hydrophobic-Interaction Chromatography, HIC), polyacrylamide-SDS gel electrophoresis (SDS-PAGE, electrophoresis), liquid mass spectrometry (liquid chromatograph-mass spectrometer, LC-MS) and the like.
Antibodies of the invention may be prepared using techniques well known in the art, such as hybridoma methods, recombinant DNA techniques, phage display techniques, synthetic techniques, or combinations thereof, or other techniques known in the art.
Variants refer to mutants of the amino acid sequence of an antibody, as well as covalent derivatives of the native polypeptide, provided that the biological activity equivalent to the native polypeptide is retained. Amino acid sequence mutants generally differ from the native amino acid sequence in that one or more amino acids in the native amino acid sequence are replaced or one or more amino acids are deleted and/or inserted in the polypeptide sequence. Deletion mutants include fragments of the native polypeptide and N-and/or C-terminal truncation mutants. Typically, amino acid sequence mutants have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or more than 99% homology to the native sequence.
The term "treatment" or its equivalent, when used in reference to, for example, cancer, refers to a procedure or process used to reduce or eliminate the number of cancer cells in a patient or to alleviate symptoms of cancer. "treating" of cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorder will actually be eliminated, that the number of cells or disorders will actually be reduced or that the symptoms of the cancer or other disorder will actually be reduced. In general, methods of treating cancer are performed even with low likelihood of success, but are still considered to induce an overall beneficial course of action, given the patient's medical history and estimated survival expectancy.
The term "pharmaceutically acceptable carrier" refers to any formulation or carrier medium representative of a carrier capable of delivering an effective amount of the active agents of the present invention, which does not interfere with the biological activity of the active agents and which does not have toxic or side effects to the host or patient, including water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, and the like. Such matrices include suspending agents, viscosity enhancers, transdermal enhancers, and the like. Their formulations are well known to those skilled in the cosmetic or topical pharmaceutical arts.
The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that: the novel antibody coupling medicine can realize the wide application of cytotoxic medicines in the field of ADC and treat tumor patients with microtubule ADC drug resistance.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Preparation example 1 preparation of DS-8201a linker-drug conjugate GGFG-Dxd
Compounds GGFG-Dxd (structure below) were synthesized by known methods reported in WO2015146132A1 ,ESI-MS m/z：1034.5(M+H),1H-NMR(400MHz,DMSO-d6)δ8.61(t,J＝6.4Hz,1H),8.50(d,J＝8.5Hz,1H),8.28(t,J＝5.1Hz,1H),8.11(d,J＝7.5Hz,1H),8.05(t,J＝5.7Hz,1H),7.99(t,J＝5.9Hz,1H),7.77(d,J＝11.0Hz,1H),7.31(s,1H),7.25–7.16(m,5H),6.98(s,2H),6.51(s,1H),5.59(dt,J＝7.4,4.1Hz,1H),5.41(s,2H),5.20(s,2H),4.64(d,J＝6.1Hz,2H),4.53–4.40(m,1H),4.02(s,2H),3.74–3.37(m,8H),3.18–3.00(m,2H),3.04–2.97(m,1H),2.77(dd,J＝13.5,9.4Hz,1H),2.38(s,3H),2.19(dd,J＝14.9,8.5Hz,2H),2.11–2.05(m,2H),1.86(dd,J＝14.0,6.7Hz,2H),1.45(s,4H),1.20–1.14(m,2H),0.87(t,J＝7.1Hz,3H).
EXAMPLE 1 Synthesis of Compound DX01
Step 1 synthesis of compound IIIC:
Commercial compound IIIA (5.58 g,10 mmol) and commercial IIIB (3.16 g,10 mmol) were mixed in 100mL anhydrous N, N-dimethylformamide, HATU (1.14 g,3.0 mmol) was added, and triethylamine 10mL reacted at room temperature for 2h. After the reaction, the solvent is removed by reduced pressure distillation, and the crude product is subjected to silica gel column chromatography [ chloroform: methanol=10:1 (v/v) ] to afford the title compound IIIC (7.71 g, yield 90%), ESI-MS m/z:857.1 (M+H).
Step 2 synthesis of compound IIID:
Compound IIIC (7.71 g,9 mmol) was dissolved in dichloromethane and 9mL of trifluoroacetic acid was added and reacted at room temperature for 0.5h. After the reaction is finished, the solvent is removed by reduced pressure distillation, and the crude product is subjected to silica gel column chromatography [ dichloromethane: methanol=10:1 (v/v) ] to afford the trifluoroacetate salt of the title compound IIID (5.90 g, yield 90%), ESI-MS m/z:614.9 (M+H).
Step 3 synthesis of compound IIIE:
Tert-butyl 2-glycolate (2.5 g,18.9 mmol) and di (p-nitrophenyl) carbonate (6.3 g,20.8 mmol) were mixed and dissolved in 200mL anhydrous DMF, 25mL triethylamine was added and reacted at room temperature for 2 hours. After completion of the reaction of the starting materials by liquid chromatography, trifluoroacetic acid salt (7 g,9.6 mmol) of IIID was added thereto, and the reaction was continued for 1 hour. After the reaction, the solvent was removed by distillation under reduced pressure, 150mL of water was added, extraction was performed three times with ethyl acetate (100 mL each time), the organic phases were combined after separation, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography [ dichloromethane: ethyl acetate=10:1 (v/v) ] to afford the title compound IIIE (6.8 g, 92% yield), ESI-MS m/z:773.5 (M+H).
Step 4 synthesis of compound IIIF:
compound IIIE (7.73 g,10 mmol) was dissolved in 150mL dichloromethane and 9mL trifluoroacetic acid was added and reacted at room temperature for 0.5h. After the reaction is finished, the solvent is removed by reduced pressure distillation, and the crude product is subjected to silica gel column chromatography [ dichloromethane: methanol=10:1 (v/v) ] to afford the title compound IIIF (5.73 g, 80% yield), ESI-MS m/z:717.2 (M+H).
Step 5 synthesis of compound IIIG:
Compound IIIF (5.73 g,8 mmol) was mixed with commercially available Exatecan mesylate (4.5 g,8 mmol) in 30mL anhydrous DMF and HATU (3.8 g,10 mmol) was added, triethylamine 2mL and reacted at room temperature for 2h. After the reaction, the solvent is removed by reduced pressure distillation, and the crude product is subjected to silica gel column chromatography [ chloroform: methanol=10:1 (v/v) ] to afford the title compound IIIG (7.89 g, yield 87%), ESI-MS m/z:1134.1 (M+H).
Step 6 synthesis of compound DX 01:
compound IIIG (1 g,0.929 mmol) was dissolved in 20mL anhydrous DMF and 0.5mL 1, 8-diazabicyclo undec-7-ene was added and reacted at room temperature for 1 hour. After the reaction of the starting materials was completed, succinimidyl 6- (maleimide) hexanoate (428.5 mg,1.39 mmol) was directly added thereto and stirred at room temperature for 1 hour. The solvent was distilled off under reduced pressure, and the crude product was purified by silica gel column chromatography [ chloroform: methanol=8:1 (v/v) ] to afford the title compound (0.96 g, 73% yield), ESI-MS m/z:1105.3 (M+H).
Example 2
Synthesis of DX02, DX03, DX04
Referring to example 1, compound IIIA was reacted with a different commercially available amino fragment at step 1, and the subsequent procedure was the same as in example 1, to finally obtain compound DX02-DX04.
Compound DX02: pale yellow powder, ESI-MS m/z:1164.3 (m+h); compound DX03: pale yellow powder, ESI-MS m/z:1199.5 (m+h); compound DX04: pale yellow powder, ESI-MS m/z:1169.2 (M+H).
EXAMPLE 3 Synthesis of Compounds DX05, DX06
Step 1 Synthesis of Compound DXD-1
Commercial compound DXD (1 g,2.03 mmol) and t-butyldimethylchlorosilane (0.46 g,3.05 mmol) were dissolved in 20mL of anhydrous dichloromethane, 1mL of pyridine was added, and the mixture was stirred at room temperature for 2 hours. After the reaction, the solvent was evaporated under reduced pressure, and the crude product was purified by column chromatography [ dichloromethane: methanol=50:1 (v/v) ] to afford the title compound (1.11 g, yield 90%), ESI-MS m/z:608.1 (M+H).
Step 2 Synthesis of intermediate IX
1 Equivalent of compound DXD-1 was dissolved in anhydrous methylene chloride, 0.5 equivalent of triphosgene was added, and then 2 equivalents of p-dimethylaminopyridine was added to react at room temperature for 1 hour. Intermediate V (R represents different substituents corresponding to DX05 and DX06 respectively) was added and then reacted for 0.5 hour at room temperature, and 10% trifluoroacetic acid was added for 1 hour to remove the silicon protection. Then evaporating the solvent under reduced pressure, purifying the crude product by column chromatography to obtain an intermediate IX, and obtaining a target product (wherein R in DX05 is methyl; R in DX06 is R) by a subsequent reaction step according to example 1). Compound DX05: pale yellow solid, ESI-MS m/z:1107.3 (m+h); compound DX06: colorless oil, ESI-MS m/z:1181.2 (M+H).
EXAMPLE 4 Synthesis of Compounds DX07 and DX08
Intermediate VIII-5 and IX-5 were obtained as described in either preparation example 1 or example 2, and then treated with 1, 8-diazabicyclo undec-7-ene as described in step 6 of example 1 to react with commercially available starting material B to give the desired product. Compound DX07: colorless oil, ESI-MS m/z:1305.5 (m+h); compound DX08: pale yellow oil, ESI-MS m/z:1305.3 (M+H).
EXAMPLE 5 Synthesis of Compounds DX09, DX10
Step1 synthesis of compound 2:
commercial Compound 1 (10 g,26.4 mmol) was mixed with 4-azidobutyric acid (5.11 g,39.6 mmol) and dissolved in 100mL anhydrous DMF, EEDQ (13.1 g,52.8 mmol) was added and reacted for 5 hours at room temperature. After the reaction, the solvent was distilled off under reduced pressure, and then subjected to silica gel column chromatography [ dichloromethane: methanol=10:1 (v/v) ] to afford the title compound (11.8 g, 91% yield), ESI-MS m/z:491.3 (M+H).
Step 2 synthesis of compound 3:
Compound 2 (10 g,20.4 mmol) was dissolved in 100mL anhydrous DMF, cooled to 0℃and thionyl chloride (1.8 mL,24.5 mmol) was slowly added and the reaction continued at this temperature for 30min. After the reaction, ice water was slowly added to quench the reaction, and a solid was precipitated, filtered, and the cake was washed with water, methyl t-butyl ether, and dried under reduced pressure to give the title compound (8.6 g, yield 83%) ESI-MS m/z:509.1 (M+H).
Step3 synthesis of compound 4:
Compound 3 (1 g,1.97 mmol) was mixed with commercially available Exatecan mesylate (1.12 g,1.97 mmol) in 50mL anhydrous DMF and triethylamine (1 mL) was added and reacted at room temperature for 2 hours. After the reaction is finished, the solvent is removed by reduced pressure distillation, and the crude product is subjected to silica gel column chromatography [ dichloromethane: methanol=10:1 (v/v) ] to afford the title compound (1.1 g, 63% yield), ESI-MS m/z:908.1 (M+H).
Step4 synthesis of compound 5:
Compound 4 (1 g,1.10 mmol) was mixed with glycolic acid (83.7 mg,1.10 mmol) and dissolved in 20mL anhydrous DMF, HATU (836 mg,2.20 mmol) was added, triethylamine 1mL and reacted at room temperature for 1.5 h. After the reaction, the solvent was distilled off under reduced pressure, and the mixture was purified by silica gel column chromatography [ dichloromethane: methanol=10:1 (v/v) ] to afford the title compound (542 mg, yield 51%), ESI-MS m/z:966.4 (M+H).
Step 5 synthesis of compound DX 09:
Compound 5 (500 mg,0.518 mmol) and commercially available starting material C (128.6 mg,0.518 mmol) were dissolved in 10mL anhydrous DMF and a catalytic amount of cuprous bromide was added to react for 1 hour at room temperature. After the reaction, the solvent was distilled off under reduced pressure, and the mixture was purified by silica gel column chromatography [ chloroform: methanol=10:1 (v/v) ] to afford the title compound (546 mg, yield 87%), ESI-MS m/z:1214 (M+H).
Synthesis of Compound DX10 referring to the synthesis of DX09, the raw material compound 5 was changed to Compound 4 to give Compound DX10 as pale yellow solid, ESI-MS m/z:1116.3 (M+H).
EXAMPLE 6 Synthesis of Compounds DX11, DX12
Referring to example 5, the dipeptide starting material was replaced with the corresponding dipeptide starting material commercially available in the initial step, and the subsequent steps were the same as in example 5. Compound DX11: pale yellow solid, ESI-MS m/z:1088.1 (M+H). Compound DX12: pale yellow foamy solid, ESI-MS m/z:1193.3 (M+H).
Example 7 general procedure for ADC preparation
Anti-HER 2 antibody Trastuzumab (15 mg/ml concentration) was replaced with G25 desalting column to 50mM PB/1.0mM EDTA buffer (pH 7.0), 15 equivalents TECP was added, stirred at 37 ℃ for 2 hours to allow complete opening of the inter-chain disulfide bonds, then the reduced antibody solution pH was adjusted to 6.0 using phosphoric acid, and the water bath temperature was reduced to 25 ℃ for the coupling reaction. The linker-drug conjugates prepared in preparation example 1 and examples 1 to 6 were dissolved in DMSO, respectively, from which 12 equivalents of linker-drug conjugate linker were pipetted dropwise into the reduced antibody solution, and DMSO was added to a final concentration of 10% (v/v), and the reaction was stirred at 25 ℃ for 0.5 hour, after which the sample was filtered using a 0.22um membrane. Uncoupled small molecules were removed by purification using a tangential flow ultrafiltration system with buffer 50mM PB/1.0mM EDTA solution (pH 6.0), and after purification 6% sucrose was added and stored in a-20deg.C freezer. The absorbance values were measured at 280nm and 370nm, respectively, using the UV method, and DAR values were calculated, with the results shown in Table 1 below. The amino acid sequence of the light chain in the anti-HER 2 antibody Trastuzumab is shown as SEQ ID No.5 in the sequence table, and the amino acid sequence of the heavy chain is shown as SEQ ID No.6 in the sequence table.
TABLE 1 DAR values measured by different antibody conjugated drug (ADC) UV methods
ADC numbering Antibodies to Medicine connector DAR value
DS-8201a Trastuzumab GGFG-Dxd 7.6
ADC-1 Trastuzumab DX01 7.5
ADC-2 Trastuzumab DX02 7.4
ADC-3 Trastuzumab DX03 7.7
ADC-4 Trastuzumab DX04 7.2
ADC-5 Trastuzumab DX05 7.1
ADC-6 Trastuzumab DX06 7.3
ADC-7 Trastuzumab DX07 7.6
ADC-8 Trastuzumab DX08 7.5
ADC-9 Trastuzumab DX09 7.8
ADC-10 Trastuzumab DX10 7.5
ADC-11 Trastuzumab DX11 7.5
ADC-12 Trastuzumab DX12 7.4
Effect example 1: in vitro cytotoxic Activity assay
And selecting NCI-N87 cells and SK-BR-3 cells which are stably transfected and highly express Her2 as cell strains for detecting the in vitro activity of the experiment, and observing the quantitative effect of different antibody coupling drugs on cell killing. Seed density of each cell was initially selected: 2X 10 3 cells/well, and performing cytotoxicity activity measurement after 16-24 hours; then, the final concentration of the antibody-coupled drug prepared in test example 7 after sample addition was set to 5000nM as the initial concentration, 10 concentrations (4-10-fold dilution) of the designed series of 5000-0.006 nM were observed for 96 hours of killing (or inhibition) change,Luminescent Cell Viability Assay chemiluminescence staining, and IC50 was calculated after reading fluorescence data. From the results of the activity test, all ADCs showed some antitumor activity, part of the ADCs exceeded DS-8201a, part of the ADCs were equivalent to DS-8201a, and part of the ADCs were significantly weaker than DS-8201a, as shown in Table 2.
TABLE 2 in vitro cytotoxic Activity of different ADCs
Effect example 2: stability test
This example evaluates the stability of the antibody-conjugated drug of example 7 in a weakly acidic environment. Specifically, in this example, a part of the antibody-conjugated drug of example 7 was added to a buffer solution of pH 4.5, placed in a water bath at 37 ℃ for 1, 2, 3, 4 days with an internal standard (irinotecan as an internal standard substance) and then the release amount of the free drug was detected by high performance liquid chromatography, and the results are shown in table 3.
Table 3 evaluation of stability of partial ADC in weakly acidic environment
The stability results show that the ADC obtained by adopting the novel technical scheme has stronger stability than DS-8201a in a weak acid environment, and various normal tissues in a human body are weak acid, so that the ADC is expected to show better safety and similar effectiveness than DS-8201 a.
SEQUENCE LISTING
<110> Shanghai Fudan Zhangjiang biomedical Co., ltd
<120> Antibody-conjugated drug, intermediate thereof, preparation method and application
<130> P19011374C
<160> 8
<170> PatentIn version 3.5
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Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
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<223> IMAB362 heavy chain sequence
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Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
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Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
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Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile 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 Arg Tyr Thr Gly Val Pro Asp 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 Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu
85 90 95
Thr Phe Gly Ala 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> 4
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<223> RS7 heavy chain sequence
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Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly
100 105 110
Gln Gly Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Lys
450
<210> 5
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<213> Artificial Sequence
<220>
<223> Trastuzumab light chain sequence
<400> 5
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 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> 6
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Trastuzumab heavy chain 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 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 Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 7
<211> 217
<212> PRT
<213> Artificial Sequence
<220>
<223> P2E5 light chain sequence
<400> 7
Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly
1 5 10 15
Thr Val Thr Leu Thr Cys Gly Leu Asn Ser Gly Ser Val Ser Thr Ser
20 25 30
Tyr Phe Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Thr
35 40 45
Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala
65 70 75 80
Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Leu Leu Tyr Met Asp Ser
85 90 95
Gly Pro His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
130 135 140
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
145 150 155 160
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205
Lys Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 8
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> P2E5 heavy chain sequence
<400> 8
Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser
20 25 30
Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Met Gly Leu Glu Trp Val
35 40 45
Ala Ser Met Lys Pro Asp Gly Ser Val Lys His Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Ser Leu Asp
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Ser Tyr Asp Thr Arg Trp Gly Trp Phe Asp Pro Trp Gly Glu 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 Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met 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 Phe Leu Tyr 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 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys

Claims (19)

1. An antibody coupled drug has a structural general formula of Ab- (L 3-L2-L1-D)m);
Wherein Ab is anti-HER 2 antibody Trastuzumab;
D is a cytotoxic drug
M is 7-8;
the structure of L 1 is shown as a formula I; wherein the a end is connected with the cytotoxic drug, the e end is connected with the c end of the L 2,
L is independently a phenylalanine residue or a glycine residue; p is 4;
r 1 is substituted or unsubstituted C 1～C4 alkyl, or substituted or unsubstituted C 6～C14 aryl; the substituents in the substituted C 1～C4 alkyl and substituted C 6～C14 aryl are one or more groups selected from the group consisting of, when multiple substituents are present, the substituents are the same or different: -N (CH 3)2、-S(O)2CH3 and hydroxy-substituted ethoxy;
R 2 is independently hydrogen;
n 1 is 2;
l 2 is Wherein, the c end is connected with the e end of L 1, the f end is connected with the d end of L 3, and n 2 is independently 1-8;
L 3 is Wherein the b end is connected with the Ab, and the d end is connected with the f end of the L 2;
And when L 1 -D is When said L 2 is
2. The antibody-conjugated drug of claim 1, wherein,
The amino acid sequence of the light chain in the anti-HER 2 antibody Trastuzumab is shown as SEQ ID No.5 in a sequence table, and the amino acid sequence of the heavy chain is shown as SEQ ID No.6 in the sequence table;
and/or, the b segment of the L 3 is connected with the sulfhydryl group on the antibody in a thioether bond mode;
And/or m is 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7 or 7.8;
and/or, when said R 1 is substituted or unsubstituted C 1～C4 alkyl, said C 1～C4 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl;
And/or, when said R 1 is a substituted or unsubstituted C 6～C14 aryl, said C 6～C14 aryl is phenyl, naphthyl or anthracenyl;
And/or (L) p is Wherein the g-terminus is attached to the carbonyl group in formula I.
3. The antibody conjugated drug of claim 1, wherein when R 1 is substituted or is substituted C 1～C4 alkyl, R 1 is methyl or ethyl;
And/or, when said R 1 is C 6～C14 aryl, said R 1 is phenyl.
4. The antibody-conjugated drug of claim 1, wherein,
When D isWhen L 1 -D is
5. The antibody-conjugated drug of claim 1, wherein,
The Ab is an anti-HER 2 antibody Trastuzumab; l 1 -D is M is 7-8;
The structure of the L 1 is shown as a formula I;
the L is independently a phenylalanine residue or a glycine residue; p is 4;
R 1 is substituted or unsubstituted C 1～C4 alkyl or substituted or unsubstituted C 6～C14 aryl; the substituents in the substituted C 1～C4 alkyl or substituted C 6～C14 aryl are one or more groups selected from the group consisting of, when multiple substituents are present, the substituents are the same or different: -N (CH 3)2、-S(O)2CH3 and hydroxy-substituted ethoxy;
R 2 is hydrogen;
N 1 is 2;
the L 2 is N 2 is 1-8;
The L 3 is
6. The antibody-conjugated drug of claim 1, wherein the antibody-conjugated drug is a compound as shown in any one of the following:
Wherein Ab is anti-HER 2 antibody Trastuzumab and m is 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7 or 7.8.
7. The antibody-conjugated drug of claim 6, wherein the antibody-conjugated drug is a compound as shown in any one of the following:
Wherein the amino acid sequence of the light chain in the anti-HER 2 antibody Trastuzumab is shown as SEQ ID No.5 in a sequence table, and the amino acid sequence of the heavy chain is shown as SEQ ID No.6 in the sequence table.
8. The antibody-conjugated drug of claim 6, wherein the antibody-conjugated drug is a compound as shown in any one of the following:
or, wherein Ab is Trastuzumab, an anti-HER 2 antibody.
9. The antibody-conjugated drug of claim 8, wherein the antibody-conjugated drug is a compound as shown in any one of the following:
wherein Ab is the anti-HER 2 antibody Trastuzumab.
10. The antibody-conjugated drug of claim 8, wherein the antibody-conjugated drug is a compound as shown in any one of the following:
wherein Ab is the anti-HER 2 antibody Trastuzumab.
11. The antibody-conjugated drug of claim 8, wherein the antibody-conjugated drug is a compound as shown in any one of the following:
Or, wherein Ab is the anti-HER 2 antibody Trastuzumab; the amino acid sequence of the light chain in the anti-HER 2 antibody Trastuzumab is shown as SEQ ID No.5 in the sequence table, and the amino acid sequence of the heavy chain is shown as SEQ ID No.6 in the sequence table.
12. The antibody conjugated drug of claim 11, wherein the antibody conjugated drug is a compound as shown in any one of the following:
Wherein Ab is the anti-HER 2 antibody Trastuzumab; the amino acid sequence of the light chain in the anti-HER 2 antibody Trastuzumab is shown as SEQ ID No.5 in the sequence table, and the amino acid sequence of the heavy chain is shown as SEQ ID No.6 in the sequence table.
13. The antibody conjugated drug of claim 11, wherein the antibody conjugated drug is a compound as shown in any one of the following:
Wherein Ab is the anti-HER 2 antibody Trastuzumab; the amino acid sequence of the light chain in the anti-HER 2 antibody Trastuzumab is shown as SEQ ID No.5 in the sequence table, and the amino acid sequence of the heavy chain is shown as SEQ ID No.6 in the sequence table.
14. A linker-drug conjugate has the general structural formula L 4-L2-L1 -D, wherein L 4 isL 2、L1 and D are as defined in any one of claims 1 to 13, the f-terminus of L 2 being linked to the D-terminus of L 4.
15. The linker-drug conjugate of claim 14, it is characterized in that the method comprises the steps of, the linker-drug conjugate is any one of the compounds shown below:
16. a method of preparing an antibody-conjugated drug according to any one of claims 1 to 13, comprising the step of conjugating said antibody to a linker-drug conjugate according to claim 14 or 15.
17. A pharmaceutical composition comprising the antibody-conjugated drug of any one of claims 1-13 and a pharmaceutically acceptable carrier.
18. Use of an antibody-conjugated drug according to any one of claims 1 to 13, or a pharmaceutical composition according to claim 17, in the manufacture of a medicament for the prevention or treatment of cancer.
19. A pharmaceutical formulation comprising the antibody-conjugated drug of any one of claims 1-13.
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