WO2018006785A1 - Egfr antibody-drug conjugate and pharmaceutical use thereof - Google Patents

Abstract

Provided are an EGFR antibody-cytotoxic drug conjugate, or a pharmaceutically acceptable salt or solvent compound thereof, a pharmaceutical composition comprising the conjugate or the pharmaceutically acceptable salt or solvent compound thereof, and a use thereof as an anti-cancer drug, more particularly a use for preparing drugs for treating EGFR mediated diseases or symptoms.

Description

EGFR antibody-drug conjugate and its application in medicine Technical field

The present invention relates to a novel class of antibody-drug conjugates. In particular, the invention relates to EGFR antibody variant-cytotoxic drug conjugates, pharmaceutical compositions comprising the conjugates, and pharmaceutical uses of the conjugates or pharmaceutical compositions.

Background technique

The epidermal growth factor receptor (EGFR) is a large transmembrane glycoprotein with a molecular weight of approximately 170 kDa and is a member of the ErbB receptor family. The EGFR receptor itself is a tyrosine kinase that forms a dimer when bound to ligands such as EGF, TNF-a, etc., and activates downstream signals (such as MAPK, PI3K, Stat, etc.) by transmitting phosphorylation. Thereby maintaining cell growth and promoting cell division and proliferation. Due to the conservation of the ErbB family of receptors, EGFR can also form heterodimers with other family proteins (such as Her2, Her3, Her4), thereby regulating cell growth more broadly.

Mutations or overexpression of EGFR genes are common in a variety of malignant tumors of epidermal cell origin, such as head and neck cancer, colorectal cancer, lung cancer, pancreatic cancer, skin cancer, etc., and are considered to be the driving genes for these cancers. Therefore, the development of anticancer drugs targeting EGFR has been a hot topic in the medical field. So far, small molecule tyrosine kinase inhibitors of EGFR (such as Gefitinib, Erlotinib) and EGFR monoclonal antibodies (such as Cetuximab), Panidan Panitumumab nimotuzumab (Nimotuzumab, EP0699755, EP0712863), etc., has been marketed and has achieved significant clinical results. However, these EGFR-targeted drugs are not perfect and are only suitable for smaller patient populations. For example, small molecule inhibitors are usually only effective in lung cancer patients with specific EGFR mutations, and drug resistance often occurs after a period of use; EGFR monoclonal antibodies usually only have no mutations in downstream KRAS, BRAF, PIK3CA and other genes. Colorectal cancer, head and neck cancer and other patient groups are more effective. Not only that, these drugs also partially inhibit the normal physiological functions of EGFR in common cells, often accompanied by target-related side effects such as rash and diarrhea. Therefore, whether we can find a new anti-cancer drug against EGFR to overcome the drug resistance caused by the existing EGFR targeted therapy, expand the indications, and reduce the side-effects related to normal tissue targets, is urgently needed to overcome. A problem. In addition, the stability of the antibody and the activity of the drug are also important. It has been reported in the literature that the stability of the antibody or the activity of ADCC and CDC can be improved by the modification of the constant region and the framework region of the antibody, such as CN1237076C, EP2203180 and the like.

Antibody-Drug Conjugate or ADC, commonly known as "bio-missile", is a monoclonal antibody that accumulates ADC molecules into the tumor microenvironment and is mediated by antibody-tumor antigens. Endocytosis revolutionizes the technology of directing toxin molecules into cancer cells to kill cancer cells. Compared with traditional chemotherapy, the amount of toxin molecules used in ADC is greatly reduced, and more concentrated in the lesions, and the toxic side effects on normal tissues are smaller; in addition, ADC mainly uses direct binding of toxin molecules. The microtubule bundle inhibits the division and proliferation of cancer cells, and the action mechanism of monoclonal antibodies acting by inhibiting the receptor signaling pathway is different. Therefore, whether the downstream signaling protein has a gene mutation does not play a role in inhibiting the cancer cells by the ADC. Too much impact, so in theory, ADC can have a wider range of indications than traditional therapy, and the effect is more powerful. . The ADC related documents are WO2007008603, WO2013173393, WO2005081711, WO2013173391, WO2013173392, WO2013173393, and WO2012010287.

The inventor's prior application PCT/CN2016/072129 relates to a new class of toxin molecules and conjugates, but does not disclose specific conjugates of specific EGFR antibodies.

There is still a need to find antibody conjugates with significant pharmacological effects and small toxic side effects; at the same time, reducing the frequency of administration, improving compliance, and reducing treatment costs while achieving the same efficacy are also an important problem to be solved in the art.

Summary of the invention

In order to improve the coupling effect of the ligand, particularly the antibody and the drug, the present invention provides an antibody-drug conjugate of the formula (I) or a pharmaceutically acceptable salt or solvent compound thereof:

among them:

L ₁ , L ₂ are joint units;

Y is 1-8, preferably 2-5;

Ab is an anti-EGFR antibody or antigen-binding fragment thereof, and the anti-EGFR antibody or antigen-binding fragment thereof comprises the following CDR regions:

The Ab has three site mutations in the heavy chain of M258Y/S260T/T262E (YTE).

In a preferred embodiment of the present invention, the antibody-drug conjugate of the formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-EGFR antibody or antigen-binding fragment thereof A humanized antibody or fragment thereof.

In a preferred embodiment of the invention, an antibody-drug conjugate of the formula (I) or a medicament thereof A pharmaceutically acceptable salt or solvate compound, wherein said humanized antibody heavy chain variable region further comprises a heavy chain FR region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising a human IgG1 heavy chain The FR region, and/or the light chain variable region of the humanized antibody further comprises a light chain FR region of a human kappa chain or a human kappa chain variant, or a light chain of a human lambda chain or a human lambda chain variant FR area.

In a preferred embodiment of the present invention, the antibody-drug conjugate of the formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the humanized antibody has a constant heavy chain a region comprising human IgG1 or a variant thereof, human IgG2 or a variant thereof, human IgG3 or variant thereof or a constant region of human IgG4 or a variant thereof, preferably comprising a constant region of human IgG1 or a variant thereof, And/or the light chain of the humanized antibody further comprises a light chain constant region of a human kappa chain or a human kappa chain variant, or a light chain constant region of a human lambda chain or a human lambda chain variant.

In a preferred embodiment of the present invention, an antibody-drug conjugate of the formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-EGFR antibody comprises:

Light chain

Heavy chain

In a preferred embodiment of the present invention, the antibody-drug conjugate of the formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein L _{2 is} as defined in the following formula (L ₂ ) Show:

among them

X _{1 is} selected from a hydrogen atom, a halogen, a hydroxyl group, a cyano group, an alkyl group, an alkoxy group, and a cycloalkyl group;

X ₂ is selected from alkyl, cycloalkyl and heterocyclyl group;

m is 0-5, preferably 1-3; S is a sulfur atom.

In a preferred embodiment of the invention, the compound of formula (L ₂₎ is preferably the following structure:

In a preferred embodiment of the present invention, a compound of general formula or a pharmaceutically (I), a salt or solvate, wherein L1 (L ₁₎ a compound represented by the following formula:

among them

X ₃ is an alkyl group, and the alkyl group is optionally further taken up by a substituent of a halogen, a hydroxyl group, a cyano group or an alkyl group.

generation;

n is 0-5, preferably 1-3.

In a preferred embodiment of the invention, a compound of the formula (L ₁ ) preferably has the following structure:

among them

X ₃ is an alkyl group, and the alkyl group is optionally further taken up by a substituent of a halogen, a hydroxyl group, a cyano group or an alkyl group.

generation;

n is 0-5, preferably 1-3.

In a preferred embodiment of the present invention, an antibody-drug conjugate of the formula (I) or a pharmaceutically acceptable salt or solvate thereof, which is selected from the group consisting of the antibody of the formula (II) a drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof:

Wherein, Ab, L ₂ and y are as defined in the formula (I).

In a preferred embodiment of the present invention, an antibody-drug conjugate of the formula (I) or a pharmaceutically acceptable salt or solvate thereof, which is selected from the group consisting of the antibody of the formula (III) a drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof:

Wherein, Ab, L ₁ and y are as defined in the formula (I).

In a preferred embodiment of the present invention, an antibody-drug conjugate of the formula (I) or a pharmaceutically acceptable salt or solvate thereof having the following general structure:

Wherein mAb002 comprises a light chain of amino acid sequence such as SEQ ID NO: 3, and a heavy chain of SEQ ID NO: 4, y as defined in formula (I).

Another aspect of the invention relates to an EGFR antibody which can be used for the preparation of an antibody-drug conjugate as shown in the general formula (I) of the present invention or a pharmaceutically acceptable salt or solvate thereof, comprising the sequence SEQ ID NO : 7, SEQ ID NO: 8, SEQ ID NO: 9 and variants thereof, LCDR1, LCDR2, LCDR3 region, and SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and variations thereof The HCDR1, HCDR2, and HCDR3 regions shown in the body are characterized by having three site mutations of M258Y/S260T/T262E (YTE) on the heavy chain.

In a preferred embodiment of the present invention, there is provided an EGFR antibody which can be used for the preparation of an antibody-drug conjugate of the formula (I) of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, The EGFR antibody comprises a light chain having the amino acid sequence set forth in SEQ ID NO: 3 and a heavy chain as set forth in SEQ ID NO: 4.

Another aspect of the invention relates to a pharmaceutical composition comprising the antibody-drug conjugate of the formula (I) of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, and one or more A pharmaceutically acceptable excipient, diluent or carrier.

The present invention further provides an antibody-drug conjugate represented by the formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition of the present invention, for preparing a medicament for treating cancer Uses; preferably the cancer is a cancer that highly expresses EGFR, a cancer that has a mutation on the EGFR polypeptide, and a cancer that has a mutation in a gene downstream of the EGFR signaling pathway; more preferably, gastric cancer, pancreatic cancer, liver cancer, breast cancer, lung cancer , intestinal cancer, renal cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer; most preferably lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer.

The present invention further provides an antibody-drug conjugate of the formula (I) or a pharmaceutically acceptable salt thereof Or a solvate compound, or a pharmaceutical composition comprising the same as described above, for use in the preparation of a medicament for treating cancer in a mammal; preferably the cancer is a cancer which highly expresses EGFR, a cancer which has a mutation on the EGFR polypeptide, and an EGFR signaling pathway A cancer with a mutation in the downstream gene; more preferably gastric cancer, pancreatic cancer, liver cancer, breast cancer, lung cancer, intestinal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer Most preferred are lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer.

The invention further provides a method of treating cancer in a mammal, the method comprising administering to the mammal an effective amount of an antibody-drug conjugate of the formula (I) or a pharmaceutically acceptable salt or solvent compound thereof, Or a pharmaceutical composition comprising the same; wherein the mammal is preferably a human; the cancer is preferably a cancer that highly expresses EGFR, a cancer that has a mutation on the EGFR polypeptide, and a cancer that has a mutation in a gene downstream of the EGFR signaling pathway; more preferably For gastric cancer, pancreatic cancer, liver cancer, breast cancer, lung cancer, colon cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer; most preferred are lung cancer, colon cancer, rectum Cancer, colorectal cancer or head and neck cancer.

The present invention further provides an antibody-drug conjugate represented by the formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition of the present invention, for preparing a medicament for treating cancer Uses; preferably the cancer is a cancer having a mutation on an EGFR polypeptide comprising at least one EGFR mutation selected from the group consisting of L858R, L858R/T790M, L858R/T790M/C797S, Del19, Del19/T790M , Del19/T790M/C797S, T790M, G719X, L861Q, S768I, Exon 18indel/E709X, Exon 19insertion, Exon20insertion, A763_Y764insFQEA, Exon 18–25 duplication and EGFR-RAD51 rearrangement; more preferably EGFR polypeptide with mutations in gastric cancer, pancreatic cancer, Liver cancer, breast cancer, lung cancer, colon cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer; most preferably lung cancer, colon cancer, rectal cancer, colorectal cancer or Head and neck cancer.

The present invention further provides an antibody-drug conjugate represented by the formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the same as described above, in the preparation of a cancer for treating a mammal Use of the drug; preferably the cancer is a cancer having a mutation on the EGFR polypeptide, the EGFR mutant polypeptide comprising at least one EGFR mutation selected from the group consisting of L858R, L858R/T790M, L858R/T790M/C797S, Del19, Del19/T790M , Del19/T790M/C797S, T790M, G719X, L861Q, S768I, Exon 18indel/E709X, Exon 19insertion, Exon20insertion, A763_Y764insFQEA, Exon 18–25 duplication and EGFR-RAD51 rearrangement; more preferably EGFR polypeptide with mutations in gastric cancer, pancreatic cancer, Liver cancer, breast cancer, lung cancer, colon cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer; most preferably lung cancer, colon cancer, rectal cancer, colorectal cancer or Head and neck cancer.

The invention further provides a method of treating cancer in a mammal, the method comprising administering to the mammal an effective amount of an antibody-drug conjugate of the formula (I) or a pharmaceutically acceptable salt or solvent compound thereof, Or a pharmaceutical composition comprising the same; wherein the mammal is preferably a human; preferably the cancer is a cancer having a mutation on an EGFR polypeptide, the EGFR mutant polypeptide comprising at least one EGFR selected from the group consisting of Mutation: L858R, L858R/T790M, L858R/T790M/C797S, Del19, Del19/T790M, Del19/T790M/C797S, T790M, G719X, L861Q, S768I, Exon 18indel/E709X, Exon 19insertion, Exon20insertion, A763_Y764insFQEA, Exon 18–25duplication And EGFR-RAD51 rearrangement; more preferably EGFR polypeptide with mutations in gastric cancer, pancreatic cancer, liver cancer, breast cancer, lung cancer, intestinal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer Or head and neck cancer; most preferred is lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer.

The present invention further provides an antibody-drug conjugate represented by the formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition of the present invention, for preparing a medicament for treating cancer Wherein the cancer is a cancer having a mutation in a gene downstream of the EGFR signaling pathway, the downstream gene comprising at least one gene selected from the group consisting of Ras, B-Raf, PI3K, wherein the downstream gene mutation comprises at least one selected from the group consisting of The following mutations: K-Ras G12V, K-Ras G13D, N-Ras Q61K, H-Ras G12S; B-Raf V600E, B-Raf G468A; PIK3CA H1047R, PIK3CB E633K and p110γE1021K; more preferably downstream genes of the EGFR signaling pathway Mutant gastric cancer, pancreatic cancer, liver cancer, breast cancer, lung cancer, intestinal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer; most preferably lung cancer, colon cancer , rectal cancer, colorectal cancer or head and neck cancer.

The present invention further provides an antibody-drug conjugate represented by the formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the same as described above, in the preparation of a cancer for treating a mammal Use of the drug; preferably the cancer is a cancer having a mutation in a gene downstream of the EGFR signaling pathway, the downstream gene comprising at least one gene selected from the group consisting of Ras, B-Raf, PI3K, wherein the downstream gene mutation comprises at least one selected from the group consisting of The following mutations: K-Ras G12V, K-Ras G13D, N-Ras Q61K, H-Ras G12S; B-Raf V600E, B-Raf G468A; PIK3CA H1047R, PIK3CB E633K and p110γE1021K; more preferably downstream genes of the EGFR signaling pathway Mutant gastric cancer, pancreatic cancer, liver cancer, breast cancer, lung cancer, intestinal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer; most preferably lung cancer, colon cancer , rectal cancer, colorectal cancer or head and neck cancer.

The invention further provides a method of treating cancer in a mammal, the method comprising administering to the mammal an effective amount of an antibody-drug conjugate of the formula (I) or a pharmaceutically acceptable salt or solvent compound thereof, Or a pharmaceutical composition comprising the same; wherein the mammal is preferably a human; preferably the cancer is a cancer having a mutation in a gene downstream of the EGFR signaling pathway, the downstream gene comprising at least one gene selected from the group consisting of Ras, B-Raf, PI3K The downstream gene mutation comprises at least a mutation selected from the group consisting of K-Ras G12V, K-Ras G13D, N-Ras Q61K, H-Ras G12S; B-Raf V600E, B-Raf G468A; PIK3CA H1047R, PIK3CB E633K and p110γE1021K; more preferred are gastric cancer, pancreatic cancer, liver cancer, breast cancer, lung cancer, colon cancer, renal cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal with mutations in the downstream gene of the EGFR signaling pathway. Cancer or head and neck cancer; most preferred is lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer.

Detailed description of the invention

Unless defined otherwise, all technical and scientific terms used herein are consistent with the ordinary meaning Although the invention may be practiced or tested in any method or material similar or equivalent to those described herein, the preferred methods and materials are described herein. In describing and claiming the present invention, the following terms are used in accordance with the following definitions.

When a trade name is used in the present invention, the applicant intends to include a formulation of the trade name product, a generic drug of the trade name product, and an active drug moiety.

Terms used in the specification and claims have the following meanings unless stated to the contrary.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing from 1 to 20 carbon atoms, preferably an alkyl group having from 1 to 12 carbon atoms, more preferably from 1 to 10 carbons. The alkyl group of the atom is most preferably an alkyl group having 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 - dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2 -methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethyl Pentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2 , 5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-B Hexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-decyl, 2-methyl-2-ethylhexyl, 2-methyl-3-B Hexyl group, 2,2- Diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof. More preferred are lower alkyl groups having from 1 to 6 carbon atoms, non-limiting examples including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl Base, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethyl Butyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl Base, 2,3-dimethylbutyl and the like. The alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably one or more of the following groups independently selected from the group consisting of an alkane Base, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, naphthenic Oxyl, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably from 3 to 10 carbon atoms. One carbon atom, most preferably from 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene A polycycloalkyl group includes a spiro ring, a fused ring, and a cycloalkyl group.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms wherein one or more ring atoms are selected from nitrogen, oxygen or S(O). A hetero atom of _m (where m is an integer of 0 to 2), but excluding the ring moiety of -OO-, -OS- or -SS-, the remaining ring atoms being carbon. It preferably contains 3 to 12 ring atoms, of which 1 to 4 are hetero atoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl and the like. Polycyclic heterocyclic groups include spiro, fused, and bridged heterocyclic groups.

The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. The alkoxy group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio.

The term "bond" refers to a covalent bond represented by "-".

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

The term "halogen" means fluoro, chloro, bromo or iodo.

The term "pharmaceutical composition" means a mixture comprising one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable Carrier and excipients. The purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.

The term "pharmaceutically acceptable salt" refers to a salt of an antibody-drug conjugate of the present invention which is safe and effective for use in a mammal, and which has the desired biological activity, the antibody of the present invention - The drug conjugate contains at least one amino group and thus can form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate , citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, horse Acidate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, besylate, p-toluenesulfonate.

The term "solvent compound" means that the antibody-drug conjugate of the present invention forms a pharmaceutically acceptable solvent compound with one or more solvent molecules, and non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO. , ethyl acetate.

The term antibody-conjugate (ADC) refers to a monoclonal antibody or antibody fragment that is linked to a biologically active cytotoxin via a stable chemical linker compound.

The term "antigen or receptor" refers to a cell that is present on a target cell for recognition and binding to a target cell. Cell surface antigens or receptors expressed on target cells and/or tissues of proliferative diseases such as cancer are preferred in the present invention.

An "antibody" as used herein refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, full length antibodies, antibody binding fragments or derivatives. Sources of antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, genetically engineered antibodies (eg, bispecific) Sexual antibodies).

The term "full length antibody" refers to an immunoglobulin molecule (eg, IgM) comprising four polypeptide chains, ie, two heavy chains and two light chains, cross-linked to each other by disulfide bonds to form a multimer. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region, and the heavy chain constant region comprises three domains: CH1, CH2 and CH3. Each light chain comprises a light chain variable region (VL) and a light chain constant region, and the light chain constant region comprises one domain (CL1). The VH region and the VL region can be further divided into hypervariable regions, the term is a complementarity determining region (CDR), and a more conserved domain interspersed between each complementarity determining region, called a framework region (FR).

The term "antigen-binding fragment or derivative" includes any naturally occurring, enzymatically-derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex; typically includes a parent At least a portion of an antigen binding region or variable region (eg, one or more CDRs) of an antibody that retains at least some of the binding specificity of the parent antibody. An "antibody binding fragment or derivative" may be derived from an antibody, for example by appropriate standard techniques including proteolysis or recombinant genetic engineering techniques, including manipulation and expression of a DNA expressing an antibody variable region and a partial constant region. The full length is modified. "Antigen-binding fragments or derivatives" include, but are not limited to, (i) Fab fragments; (ii) F(ab') ₂ fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) (vi) a dAb fragment; and (vii) a minimal recognition unit (eg, an isolated complementarity determining region (CDR)) that mimics the amino acid residue of the hypervariable region of the antibody. Other engineering molecules such as bivalent antibodies, trivalent antibodies, tetravalent antibodies, and minibodies are also within the scope of "antigen-binding fragments or derivatives."

The term "EGFR" according to the invention refers in particular to human epidermal growth factor receptor 1, also known as ErbB-1 or HER1. EGFR is a receptor tyrosine kinase comprising an extracellular ligand binding domain, a membrane-cross domain and an intracellular kinase domain. After binding to its ligands (such as epidermal growth factor (EGF) and transforming growth factor alpha (TGFα)), EGFR forms homodimers or forms heterodimers with other ErbB receptors, and its kinase function is activated, resulting in Autophosphorylation of several tyrosines in the intracellular domain. An anti-EGFR antibody is an antibody that specifically binds to EGFR. In a specific embodiment, the anti-EGFR antibody can interfere with or inhibit activation of EGFR, for example by preventing ligand binding and/or receptor dimerization. Upon activation of EGFR, signals can be passed to downstream effectors, including PI3-K, RAS-RAF-MAPK P44/P42 and protein kinase C signaling pathways, which ultimately signal to the nucleus and regulate cell proliferation.

The antibody of the present invention is preferably a specific antibody against a cell surface antigen on a target cell, and a non-limiting example is nimotuzumab (trade name Taixinsheng), which is an epidermal growth factor receptor ( EGFR) is a monoclonal antibody that targets humanized monoclonal antibodies for the treatment of malignant tumors. EGFR is overexpressed in a variety of solid tumors, such as head and neck cancer, lung cancer, and colorectal cancer.

The term "Ras gene" refers to the human Ras gene family, which includes three functional genes, namely H-ras, K-ras and N-ras genes ((Adjei, 2001, J. Nat. Cancer Instit. 93: 1062). -1073), each containing a 5' non-coding exon and 4 coding exons, encoding a product with a relative molecular mass of 21 000 G protein monomers, these proteins have an isosine triphosphate binding site, It plays an important role in many various intracellular signaling pathways.

The term "Raf gene" refers to the Raf family, including three cellular homologs, referred to as A-raf, B-raf, and C-raf (also known as raf-1), respectively. This gene encodes a highly conserved serine-threonine-specific protein kinase, which is one of the effector factors of Ras and plays an important regulatory role in the transduction of signals that regulate cell proliferation. Among them, B-Raf has the complete amino acid sequence as shown in Gene Accession No. NP_004324.

The term "PI3K gene" refers to a phosphoinositide-3-kinase (PI3K, also known as phosphatidylinositol-4,5-diphosphate 3-kinase), which belongs to the proto-oncogene. In the present invention, "PI3K" includes all members of the PI3K family including IA (eg, PI3Kα, β, and δ), IB (eg, PI3Kγ), Class II (eg, PI3KC2α, β, and γ), and III. Class (eg, Vps34 yeast homolog).

The term "cytotoxic drug" refers to a chemical molecule that has a strong disruption to its normal growth in tumor cells. In principle, cytotoxic drugs can kill tumor cells at a sufficiently high concentration, but due to lack of specificity, while killing tumor cells, it also causes apoptosis of normal cells, leading to serious side effects.

The term "linker unit" in the present invention is L1 and L2, and refers to a chemical structural fragment or bond which is covalently linked at one end to the antibody and to the cytotoxic drug at the other end.

The term "drug loading" refers to the average number of cytotoxic drugs loaded on each ligand in the molecule, and can also be expressed as the ratio of the amount of drug to the amount of antibody. The range of drug loading can be per ligand (Pc) linkage. 1-8 cytotoxic drugs, in the embodiment of the present invention, the drug loading amount is expressed as y, and the characteristics of each ADC molecule after the coupling reaction can be characterized by a conventional method such as UV/visible spectroscopy, mass spectrometry, ELISA test and HPLC. The average number of drugs.

In the present invention, y may be limited by the number of attachment sites. In one embodiment of the invention, the cytotoxic drug is coupled via a linker unit to the ε-amino group of the N-terminal amino and/or lysine residue of the ligand, typically in a coupling reaction that is conjugated to the antibody. The number of drug molecules will be less than the theoretical maximum.

The loading of antibody cytotoxic drug conjugates can be controlled by the following non-limiting methods, including:

(1) controlling the molar ratio of the linking reagent to the monoclonal antibody,

(2) Control reaction time and temperature,

(3) Select different reagents.

The term "carrier" as used in the present invention refers to a system which changes the manner in which the drug enters the body and the distribution in the body, controls the release rate of the drug, and delivers the drug to the targeted organ. Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and increase bioavailability. Polymeric surfactants, which can be used as carriers, can be self-assembled due to their unique amphiphilic structure to form aggregates of various forms, such as micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to entrap drug molecules while having good permeability to the membrane and can be used as an excellent drug carrier.

The term "excipient" is an addition to a pharmaceutical preparation other than the main drug, and may also be referred to as an excipient. Such as adhesives, fillers, disintegrants, lubricants in tablets; semi-solid preparation ointments, matrix parts in creams; preservatives, antioxidants, flavoring agents, fragrances, and liquids in liquid preparations Solvents, emulsifiers, solubilizers, An osmotic pressure adjusting agent, a coloring agent and the like can be referred to as an excipient.

The term "diluent" is also known as a filler and its primary use is to increase the weight and volume of the tablet. The addition of the diluent not only ensures a certain volume, but also reduces the dose deviation of the main component and improves the compression moldability of the drug. When the drug of the tablet contains an oily component, it is necessary to add an absorbent to absorb the oily substance so as to maintain a "dry" state to facilitate tableting. Such as starch, lactose, inorganic salts of calcium, microcrystalline cellulose and the like.

The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oily phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerin to form a microemulsion. The injection or microemulsion can be injected into the bloodstream of the patient by a local injection. Alternatively, the solution and microemulsion are preferably administered in a manner that maintains a constant circulating concentration of the compound of the invention. To maintain this constant concentration, a continuous intravenous delivery device can be used. An example of such a device is the Deltec CADD-PLUS.TM.5400 intravenous pump.

The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension for intramuscular and subcutaneous administration. The suspension may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension, such as a solution prepared in 1,3-butanediol, in a non-toxic parenterally acceptable diluent or solvent. In addition, sterile fixed oils may conveniently be employed as a solvent or suspension medium. For this purpose, any blended fixed oil including synthetic mono- or diglycerides can be used. In addition, fatty acids such as oleic acid can also be prepared as an injection.

DRAWINGS

Figure 1: Mean pharmacokinetic parameters of cynomolgus monkeys after a single intravenous injection of 5 mg/kg of nimotuzumab (mAb001) and IgG1-YTE variant of nimotuzumab (mAb002) .

Figure 2: Comparison of in vivo efficacy of ADC-9 of the invention relative to ADC-8 and naked anti-mAb002 in a nude mouse lung cancer xenograft HCC827 model.

detailed description

The invention is further described in the following examples, which are not intended to limit the scope of the invention.

The experimental methods in the examples of the present invention which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the raw material or the manufacturer of the commodity. Reagents without specific source are routine reagents purchased from the market.

Example

The structure of the compound is determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS). The NMR shift (δ) is given in units of 10 ^-6 (ppm). NMR was measured using a Bruker AVANCE-400 nuclear magnetic apparatus, and the solvent was deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ), deuterated methanol (CD ₃ OD), internal standard was four. Methyl silane (TMS).

The measurement of MS was performed using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan) LCQ advantage MAX).

The HPLC was measured using an Agilent 1200 DAD high pressure liquid chromatograph (Sunfire C18 150 x 4.6 mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150 x 4.6 mm column).

Chiral HPLC analysis assays were performed using LC-10A vp (Shimadzu) or SFC-analytical (Berger Instruments Inc.).

Thin layer chromatography silica gel plate uses Yantai Yellow Sea HSGF254 or Qingdao GF254 silica gel plate. The specification of silica gel plate used for thin layer chromatography (TLC) is 0.15mm~0.2mm. The specification for thin layer chromatography separation and purification is 0.4mm. ~0.5mm.

Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as a carrier.

Chiral preparative column chromatography was performed using Prep Star SD-1 (Varian Instruments Inc.) or SFC-multigram (Berger Instruments Inc.).

The average value of ₅₀ measured kinase inhibition rate and IC NovoStar using a microplate reader (BMG, Germany).

The known starting materials of the present invention may be synthesized by or according to methods known in the art, or may be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Dari Companies such as chemicals.

Unless otherwise specified in the examples, the reactions can all be carried out under an argon atmosphere or a nitrogen atmosphere.

An argon atmosphere or a nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon having a volume of about 1 L.

The hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon of about 1 L volume.

The pressurized hydrogenation reaction was carried out using a Parr Model 3916EKX hydrogenation apparatus and a clear blue QL-500 type hydrogen generator or a HC2-SS type hydrogenation apparatus.

The hydrogenation reaction is usually evacuated, charged with hydrogen, and operated three times.

The microwave reaction used a CEM Discover-S Model 908860 microwave reactor.

Unless otherwise stated in the examples, the solution means an aqueous solution.

There is no particular description in the examples, and the reaction temperature is room temperature and is 20 ° C to 30 ° C.

In the example, the preparation of PBS buffer with pH=6.5: KH ₂ PO ₄ 8.5 g, K ₂ HPO ₄ .3H ₂ O 8.56 g, NaCl 5.85 g, EDTA 1.5 g in a bottle, to a volume of 2 L, ultrasonic Allow it to dissolve completely and shake it.

In the example, the preparation of acetic acid/sodium acetate buffer with pH=4.5: 9 g of anhydrous sodium acetate was placed in a bottle, purified water was added, and the volume was adjusted to 2 L. After shaking, 4.9 mL of sodium acetate was added, and the mixture was shaken. .

Preparation of Phosphate Buffer (pH=7.0) in the Example: 0.2 M Na ₂ HPO ₄ 61 mL was added with 0.2 M NaH ₂ PO ₄ 39 mL and shaken to obtain 0.2 M pH=7 buffer.

The progress of the reaction in the examples was monitored by thin layer chromatography (TLC). The system used for the reaction was: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: petroleum ether And the ethyl acetate system, D: acetone, the volume ratio of the solvent is adjusted depending on the polarity of the compound.

Purification compounds using column chromatography eluent systems and thin layer chromatography developer systems include: A: dichloromethane and methanol systems, B: n-hexane and ethyl acetate systems, C: dichloromethane and acetone System, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine and vinegar may also be added. Adjustment with an alkaline or acidic reagent such as an acid.

Some of the compounds of the invention were characterized by Q-TOF LC/MS. The Q-TOF LC/MS uses the Agilent 6530 Accurate Mass Quadrupole-Time of Flight Mass Spectrometer and the Agilent 1290-Infinity Ultra Performance Liquid Chromatograph (Agilent Poroshell 300SB-C8 5μm, 2.1 x 75mm column).

The known starting materials of the present invention can be synthesized by or according to methods known in the art, and the experimental methods in which no specific conditions are specified in the examples are usually carried out according to conventional conditions or according to the conditions recommended by the raw material or commodity manufacturer. . Reagents without specific source are routine reagents purchased from the market.

Example 1. Construction and expression of mAb001 antibody

The mAb001 antibody, which is nimotuzumab (EGFR antibody), binds specifically to the EGFR target. The preparation can be carried out according to the conventional method of the antibody: if the vector can be constructed, the eukaryotic cells such as HEK293 cells (Life Technologies Cat. No. 11625019) are transfected, and the expression is purified.

The sequence is as follows:

Light chain amino acid sequence of mAb001:

Heavy chain amino acid sequence of mAb001:

The sequence of its CDR regions is as follows:

Example 2. Construction and expression of mAb002 antibody

The mAb002 antibody is a variant of IgG1-YTE modified by nimotuzumab obtained by point mutation of nimotuzumab.

Primer PCR was designed to construct the antibody VH/VK gene fragment, and then homologously recombined with the expression vector pHr (with signal peptide and constant region gene (CH1-FC/CL) fragment) to construct the full-length antibody expression vector VH-CH1-FC- pHr/VK-CL-pHr. The original form of the plasmid was IgG1, and three site mutations of the IgG1-YTE antibody form, M258Y/S260T/T262E (YTE), were obtained by point mutation. The final mAb002 antibody sequence is set forth in SEQ ID NO: 3 and SEQ ID NO: 4. After the plasmid is verified by sequencing, the culture supernatant containing the antibody protein of interest is obtained by extraction and 293 cell transient expression by a well-known method in the field for isolation and purification.

Antibody sequence of mAb002:

mAb002 light chain amino acid sequence:

mAb002 heavy chain amino acid sequence:

Note: Double underlined represents the YTE mutation site.

The light and heavy chain DNA sequences designed in the examples are as follows:

mAb002 light chain DNA sequence:

Note: The underlined part is the signal peptide

mAb002 heavy chain DNA sequence:

Note: The underlined part is the signal peptide

mAb002 antibody purification analysis:

The above cell culture supernatant was centrifuged at high speed to remove impurities, and then subjected to Protein A column affinity chromatography. Rinse the column with PBS until the A280 reading drops to baseline. The protein of interest was eluted with 100 mM sodium acetate pH 3.0 and neutralized with 1 M Tris-HCl. The eluted sample was appropriately concentrated, and further purified by molecular sieve purification using a PBS-balanced gel chromatography column Superdex 200 (GE), and the absorption peak samples of the antibody monomers were collected. The sample can be concentrated or buffer replaced by ultrafiltration methods well known in the art to obtain a sample of the final suitable concentration.

Example 3

(S)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-N,3-dimethyl-2-((())) S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2- Methylpropionamide)-3-phenylpropionic acid

The compound of this example was prepared by the method disclosed in the patent application "WO2005081711".

MS m/z (ESI): 732.8 [M+1]

^{1 H NMR (400MHz, DMSO)} δ7.30-7.19 (m, 5H), 4.71-4.69 (m, 2H), 4.16-4.15 (m, 1H), 4.07-4.06 (m, 1H), 3.86-3.84 ( m, 1H), 3.70-3.66 (m, 3H), 3.52-3.48 (m, 1H), 3.41-3.40 (m, 1H), 3.34 (s, 4H), 3.28 (s, 1H), 3.27-3.26 ( m,1H), 3.22 (s, 2H), 3.13 (s, 1H), 295-2.88 (m, 2H), 2.67-2.65 (m, 3H), 2.47-2.45 (m, 2H), 2.33-2.31 ( m, 1H), 2.19-2.18 (m, 2H), 2.08-2.05 (m, 1H), 1.89-1.86 (m, 2H), 1.78-1.76 (m, 2H), 1.57-1.52 (m, 2H), 1.43-1.38 (m, 2H), 1.30-1.261 (m, 1H), 1.21-1.19 (dd, 2H), 1.15.11.11 (m, 3H), 1.07-0.97 (m, 13H), 0.88-0.85 (m) , 3H).

Example 4

(S)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-2-((S)-2-(6-) (2,5-Dioxy-2,5-dihydro-1H-pyrrol-1-yl)-N-methylcaproamide)-3-methylbutanamide)-N,3-dimethylbutanamide 3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamide)-3-phenylpropionic acid

The compound of this example was prepared by the method disclosed in the patent application "WO2005081711".

MS m/z (ESI): 925.8 [M+1]

¹ H NMR (400 MHz, DMSO) δ 8.54 - 8.51 (m, 1H), 8.35 - 8.34 (m, 1H), 8.13 - 8.11 (m, 1H), 7.22 - 7.19 (m, 5H), 7.00 - 6.99 ( m, 2H), 4.73-4.68 (m, 1H), 4.62-4.58 (m, 2H), 4.45-4.40 (m, 1H), 3.98-3.94 (m, 2H), 3.74-3.72 (m, 1H), 3.62-3.59 (m, 1H), 3.45-3.43 (m, 1H), 3.38-3.35 (m, 2H), 3.27-3.26 (m, 1H), 3.23 (s, 1H), 3.18-3.14 (m, 4H) ), 3.05-3.03 (m, 2H), 2.96-2.94 (m, 1H), 2.90-2.89 (m, 1H), 2.83-2.81 (m, 3H), 2.35-2.28 (m, 2H), 2.24-2.19 (m, 2H), 2.00-1.98 (m, 2H), 1.78-1.72 (m, 2H), 1.51-1.43 (m, 5H), 1.25-1.21 (m, 11H), 0.92-0.70 (m, 18H) .

Example 5

(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-dimethyl- 2-((S)-3-Methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0] Hex-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluorophenyl)propionic acid

first step

(1S,3S,5S)-tert-butyl ester 3-((1R,2R)-1-hydroxy-2-methyl-3-((4R,5S)-4-methyl-2-carbonyl-5-benzene Roxazol-3-yl)-3-carbonylpropyl)-2-azabicyclo[3.1.0]hexane-2-carboxylic acid 5c

Starting material (4R,5S)-4-methyl-5-phenyl-3-propionyloxazolidinone 5b (1.96 g, 9.26 mmol, using a known method "Journal of the American Chemical Society, 2003, 125 ( 50), 15512-15520 "prepared" dissolved in 25 mL of dichloromethane, under an argon atmosphere, cooled to 0 °C. The reaction solution was added dropwise triethylamine (1.49 mL, 10.93 mmol) at 0 ° C, then dibutylboron trifluoromethanesulfonate (9.7 mL, 9.72 mmol) was added dropwise, and the mixture was stirred at 0 ° C for 50 min. The reaction solution was cooled to -75 ° C, and (1S,3S,5S)-tert-butyl ester 3-formyl-2-azabicyclo[3.1.0]hexane-2-carboxylic acid 5a (2.16 g, 9.26 mmol) was added. A solution dissolved in 7 mL of dichloromethane was prepared by the method disclosed in the patent application "US20100249190", stirred at -75 ° C for 1.5 hours, stirred at 0 ° C for 2 hours, and stirred at room temperature for 1 hour. After the reaction was completed, a mixture of 36 mL of phosphate buffer (pH = 7.0) and methanol (V/V = 1:3) was added. A mixture of 36 mL of methanol and hydrogen peroxide (30%) (V/V = 2:1) was added at 0 ° C and stirred at room temperature for 1 hour. The organic phase was concentrated under reduced pressure. EtOAc (EtOAc) (EtOAc)EtOAc. The residue was purified by EtOAc EtOAcjjjjjjj

MS m/z (ESI): 345.1 [M-100+1]

Second step

(1S,3S,5S)-tert-butyl ester 3-((1R,2R)-1-methoxy-2-methyl-3-((4R,5S)-4-methyl-2-carbonyl-5 -Phenyloxazol-3-yl)-3-carbonylpropyl)-2-azabicyclo[3.1.0]hexane-2-carboxylic acid 5d

Starting material 5c (1.4 g, 3.15 mmol) was dissolved in 20 mL of dichloromethane, 1.4 g of crushed molecular sieves were added, and 1,8-bisdimethylaminonaphthalene (1.75 g, 8.19 mmol) was added at 0 ° C under an argon atmosphere. Trimethyloxonium tetrafluoroborate (1.16 g, 7.87 mmol) was reacted in the dark and stirred at room temperature for 40 hours. After completion of the reaction, the mixture was filtered, and the filter cake was washed with dichloromethane. The filtrate was washed with saturated ammonium chloride solution (50 mL×4) to remove excess 1,8-bisdimethylaminonaphthalene, and washed with saturated sodium chloride solution (120 mL). After drying over anhydrous sodium sulfate, EtOAc (EtOAc)EtOAc.

MS m/z (ESI): 459.4 [M + 1].

third step

(2R,3R)-3-((1S,3S,5S)-2-(tert-Butoxycarbonyl)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy- 2-methylpropionic acid 5e

The raw material 5d (400 mg, 0.87 mmol) was dissolved in 24 mL of tetrahydrofuran under an argon atmosphere, and the temperature was lowered to 0 ° C. 30% hydrogen peroxide (0.34 mL / 0.38 g, 3.31 mmol) was slowly added dropwise, and then lithium hydroxide monohydrate (62 mg) was added. , 1.48 mmol), and the reaction system was reacted at room temperature for 20 hours. After completion of the reaction, a solid sodium sulfite solid (440 mg, 3.48 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 1 hr. 2N Hydrochloric acid was added dropwise to the aqueous solution to pH 3-4, and the mixture was extracted with ethyl acetate (25 mL×3). The ethyl acetate layer was washed with water (50 mL) and saturated sodium chloride solution (50 mL). The residue was dried over anhydrous sodium

MS m/z (ESI): 200.1 [M - 100 + 1].

the fourth step

(S)-tert-butyl ester 2-amino-3-(2-fluorophenyl)propionic acid 5g

The starting material ((S)-2-amino-3-(2-fluorophenyl)propionic acid 5f (400 mg, 2.18 mmol) was prepared by a known method "Advanced Synthesis & Catalysis, 2012, 354 (17), 3327-3332". It is dissolved in 10 mL of tert-butyl acetate, added with perchloric acid (300 mg (70%), 3.3 mmol), and stirred at room temperature for 16 hours. After the reaction is completed, 6 mL of water is added, and the organic phase is saturated with sodium hydrogencarbonate solution. (5 mL) Washing. The aqueous phase was adjusted to pH=8 with saturated sodium hydrogen carbonate solution, dichloromethane (5 mL×3), and the organic phase was combined and washed sequentially with water (3mL) and saturated sodium chloride solution (5mL) The aqueous solution was dried (MgSO4).

the fifth step

(1S,3S,5S)-tert-butyl ester 3-((1R,2R)-3-(((S)-1-(tert-butoxy)-3-(2-fluorophenyl)-1-carbonyl) Propyl-2-yl)amino)-1-methoxy-2-methyl-3-carbonylpropyl)-2-azabicyclo[3.1.0]hexane-2-carboxylic acid 5h

Starting material 5e (100 mg, 0.334 mmol) was dissolved in 6 mL of dichloromethane and dimethylformamide (V/V = 5:1) mixture, and 5 g (80 mg, 0.334 mmol) of crude material was added. Additional N,N-diisopropylethylamine (0.29 mL, 1.67 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea Fluorophosphate (152.3 mg, 0.40 mmol). The reaction system was stirred at room temperature for 1 hour under an argon atmosphere. After the reaction, The mixture was stirred with EtOAc (3 mL). The resulting residue was purified to silica gel elut elut elut elut elut elut elut elut elut

MS m/z (ESI): 5221.

Step 6

(S)-tert-Butyl ester 2-((2R,3R)-3-((1S,3S,5S)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy -2-methylpropionamide)-3-(2-fluorophenyl)propionic acid 5i

The raw material 5h (173mg, 0.33mmol) was dissolved in 2mL dioxane, 5.6M hydrogen chloride dioxane solution (0.21mL, 1.16mmol) was added, stirred under argon atmosphere for 1 hour at room temperature, placed at 0 ° C 12 hours in the refrigerator. After completion of the reaction, the reaction solution was concentrated under reduced pressure, diluted with 5 mL of dichloromethane, and then 10mL of saturated sodium hydrogen carbonate solution was added and stirred for 10 minutes. The system was layered and the aqueous layer was extracted with dichloromethane (5 mL×3). The combined dichloromethane layers were washed with aq. EtOAc (EtOAc) Filtration and concentration of the filtrate under reduced pressure afforded crude title product 5i (77 mg, yellow liquid).

MS m/z (ESI): 421.2 [M + 1].

Seventh step

(S)-tert-Butyl ester 2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-sec-butyl )-1-(9H-fluoren-9-yl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-tricarbonyl-2-oxo- 4,7,10-triazatetradecyl-14-acyl)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methylpropanamide) -3-(2-fluorophenyl)propionic acid 5k

Crude 5i (77 mg, 0.183 mmol), (5S, 8S, 11S, 12R)-11-((S)-sec-butyl)-1-(9H-indol-9-yl)-5,8-diiso Propyl-12-methoxy-4,10-dimethyl-3,6,9-tricarbonyl-2-oxa-4,7,10-triazatetradecane-14-carboxylic acid 5j ( 116.8 mg, 0.183 mmol, prepared by the method disclosed in the patent application "WO 2013072813") dissolved in 6 mL of a mixed solvent of dichloromethane and dimethylformamide (V/V=5:1), and added N,N- Diisopropylethylamine (0.16 mL, 0.915 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (84 mg, 0.22 mmol). The reaction system was stirred at room temperature for 1 hour under an argon atmosphere. After the reaction was completed, 10 mL of water was added and stirred, and the layers were separated. The dichloromethane layer was washed with a saturated sodium chloride solution (10 mL) and dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure. The residue was purified by silica gel column chromatography elut elut elut elut elut elut

MS m/z (ESI): 1040.6 [M+1].

Eighth step

(S)-tert-Butyl ester 2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-di Methyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1 .0]Hex-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl)propionic acid 5l

The starting material 5k (190.5 mg, 0.183 mmol) was dissolved in 1.5 mL dichloromethane and 2 mL diethylamine was added. The reaction system was stirred at room temperature for 3 hours under an argon atmosphere. After the reaction is completed, the reaction solution is concentrated under reduced pressure to give To the crude title product 5l (150 mg, yellow viscous), the product was taken to the next step without purification.

MS m/z (ESI): 818.5 [M+1].

Step 9

(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-dimethyl- 2-((S)-3-Methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0] Hex-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluorophenyl)propanoic acid 5

The crude product 5l (150 mg, 0.183 mmol) was dissolved in 1 mL of dioxane, and 5.6 M hydrogen chloride dioxane solution 3 mL was added, and the mixture was stirred at room temperature for 12 hours under an argon atmosphere. After completion of the reaction, the reaction mixture was concentrated under reduced vacuo. The residue was purified by high-purpur chromatography to afford titled product 5 (28 mg, white powder solid).

MS m/z (ESI): 762.7 [M+1]

¹ H NMR (400 MHz, CD ₃ OD): δ 7.38-7.18 (m, 2H), 7.13-7.01 (m, 2H), 4.80-4.67 (m, 2H), 4.30-4.15 (m, 1H), 4.13 -4.01 (m, 1H), 3.96-3.83 (m, 2H), 3.75-3.60 (m, 2H), 3.42-3.11 (m, 9H), 3.06-2.95 (m, 1H), 2.70-2.58 (m, 4H), 2.28-2.01 (m, 4H), 1.88-1.70 (m, 3H), 1.57-1.25 (m, 4H), 1.22-0.95 (m, 18H), 0.92-0.80 (m, 4H), 0.78- 0.65 (m, 1H).

Example 6

(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2) -(6-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)-N-methylcaproamide)-3-methylbutyramide)-N,3-dimethyl Butyramide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methylpropanamide) -3-(2-fluorophenyl)propionic acid

first step

6-(2,5-Dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl chloride 6b

In the starting material 6-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid 6a (1.5 g, 7.10 mmol, using a known method "Journal of Medcinal Chemistry, 2013, 56 (24), 9955-9968 "prepared", drop a drop of N, N-dimethylformamide, under an argon atmosphere, after cooling in a dry ice bath, slowly add 15 mL of oxalyl chloride, stir vigorously while dropping, and drip off. The reaction was carried out for 1 hour at room temperature. After completion of the reaction, the reaction mixture was evaporated.

Second step

(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2) -(6-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)-N-methylcaproamide)-3-methylbutyramide)-N,3-dimethyl Butyramide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methylpropanamide) -3-(2-fluorophenyl)propionic acid 6

The starting material 5 (25 mg, 0.033 mmol) was dissolved in 3 mL of dichloromethane, and N,N-diisopropylethylamine (0.029 mL, 0.164 mmol) was added, and the reaction system was pre-formed under an argon atmosphere under ice bath. 6b (11.3 mg, 0.049 mmol) in dichloromethane was reacted at room temperature for 3 hours. After the reaction was completed, 5 mL of water was added, and the mixture was stirred for 20 minutes. The organic layer was dried over anhydrous sodium sulfate. ()), the yield was 22.4%.

MS m/z (ESI): 955.4 [M+1]

¹ H NMR (400 MHz, CD ₃ OD): δ 7.36-7.30 (m, 1H), 7.29-7.21 (m, 1H), 7.17-7.02 (m, 2H), 6.83-6.79 (m, 2H), 4.81 -4.71(m,2H),4.69-4.55(m,2H), 4.25-4.15(m,1H),4.13-4.04(m,1H),3.96-3.85(m,2H), 3.70-3.61(m, 1H), 3.55-3.46 (m, 3H), 3.40-3.21 (m, 4H), 3.18-3.10 (m, 2H), 3.07-2.96 (m, 4H), 2.67-2.56 (m, 2H), 2.54- 2.34 (m, 3H), 2.29-2.17 (m, 2H), 2.10 - 1.99 (m, 1H), 1.89-1.57 (m, 7H), 1.52-1.28 (m, 6H), 1.21-1.11 (m, 4H) ), 1.07-0.96 (m, 6H), 0.95-0.81 (m, 12H), 0.80-0.69 (m, 1H).

Example 7 Preparation of ADC-7

first step

S-(3-carbonylpropyl) thioacetate (1.61 mg, 12.2 μmol) was dissolved in 3.0 mL of acetonitrile solution, and was used; acetic acid/sodium acetate buffer (10.22 mg/) at pH=4.3 containing antibody mAb001. Ml, 30 mL, 2.04 mmol) was added to the above-prepared acetonitrile solution of S-(3-carbonylpropyl)thioacetate, followed by dropwise addition of 1.2 mL of an aqueous solution of sodium cyanoborohydride (49.86 mg, 793 μmol). The reaction was shaken at ° C for 2 hours. The reaction solution was purified by desalting on a Sephadex G25 gel column (elution phase: 0.05 M in PBS with a pH of 6.5) to remove unreacted S-(3-carbonylpropyl)thioacetate and sodium cyanoborohydride. , and then concentrated to a concentration of about

At 10 mg/ml, a PBS buffer solution (about 35 mL) of the title product 7a was obtained, and the next reaction was directly carried out.

Second step

About 0.4 mL of a 2.0 M hydroxylamine hydrochloride solution was added to 7a PBS buffer solution (35.0 mL), added, placed in a water bath shaker, and shaken at 25 ° C for 30 minutes to stop the reaction. The reaction solution was purified with a Sephadex G25 gel column to give the title product mAb001-propylthiol 7b in PBS buffer (concentration 5.38 mg/ml, 55 mL).

third step

Compound 6 (4.32 mg, 4.52 μmol) was dissolved in 1.1 mL of acetonitrile, added to mAb001-propanol PBS buffer solution 7b (5.38 mg/mL, 11 mL), placed in a water bath shaker, and shaken at 25 ° C. The reaction was stopped after 4 hours.

The reaction solution was purified by desalting with a Sephadex G25 gel column (eluting phase: 0.05 M in PBS, pH 6.5) to give crude title product 7 in PBS buffer (2.92 mg/mL, 20 mL). The residue was purified by desalting with a Sephadex G25 gel column (elution phase: 0.05 M in PBS with a pH of 6.5) to obtain a PBS buffer (4.25 mg/mL, 11.6 mL) of the product of the title structure 7 at 4 ° C. Store frozen.

Q-TOF LC/MS: Characteristic peaks: 150186.98 (M _Ab +0D), 151374.09 (M _Ab +1D), 152287.22 (M _Ab +2D), 153353.26 (M _Ab +3D), 154501.80 (M _Ab +4D), 155575.57 (M _Ab +5D).

Average: y = 2.2.

Example 8 Preparation of ADC-8

first step

S-(3-carbonylpropyl) thioacetate (0.7 mg, 5.3 μmol) was dissolved in 0.9 mL of acetonitrile solution, and used; acetic acid/sodium acetate buffer (10.13 mg/) at pH=4.3 containing antibody mAb002. Ml, 9.0 mL, 0.95 mmol) was added to the above-prepared acetonitrile solution of S-(3-carbonylpropyl) thioacetate, and then 1.0 mL of an aqueous solution of sodium cyanoborohydride (14.1 mg, 224 μmol) was added dropwise. The reaction was shaken at 25 ° C for 2 hours. After completion of the reaction, it was subjected to desalting purification on a Sephadex G25 gel column (elution phase: 0.05 M PBS solution having a pH of 6.5) to obtain a title product 8a solution, which was concentrated to about 10 mg/ml and directly subjected to the next reaction.

Second step

To 8a solution (10.0 mL), 0.3 mL of a 2.0 M hydroxylamine hydrochloride solution was added, and the reaction was shaken at 25 ° C for 30 minutes, and then the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: 0.05 M at pH 6.5). The title product mAb002-propanethiol 8b solution (concentration 6.2 mg/ml, 14 mL) was obtained after PBS solution.

third step

Raw material 4 (1.1 mg, 1.2 μmol) was dissolved in 0.3 mL of acetonitrile, added to 8b solution (6.2 mg/mL, 3.0 mL), and shaken at 25 ° C for 4 hours. The reaction solution was desalted on a Sephadex G25 gel column. Purification (elution phase: 0.05 M PBS solution with a pH of 6.5), and filtration through a 0.2 μm filter under sterile conditions to obtain the title structure product 8 in PBS buffer (3.5 mg/mL, 4.8 mL) at 4 Store at °C for free.

Q-TOF LC/MS: Characteristic peaks: 150332.9 (M _Ab +0D), 1514911.2 (M _Ab +1D), 152374.3 (M _Ab +2D), 153530.1 (M _Ab +3D), 154450.7 (M _Ab +4D).

Average: y = 2.0.

Example 9 Preparation of ADC-9

Raw material 6 (1.1 mg, 1.2 μmol) was dissolved in 0.3 mL of acetonitrile, added to 8b solution (6.2 mg/mL, 3.0 mL), and shaken at 25 ° C for 4 hours. The reaction solution was desalted on a Sephadex G25 gel column. Purification (elution phase: 0.05 M PBS solution with a pH of 6.5), and filtration through a 0.2 μm filter under sterile conditions to obtain the title structure product 9 in PBS buffer (3.4 mg/mL, 4.7 mL) at 4 Store at °C for free.

Q-TOF LC/MS: Characteristic peaks: 150336.6 (M _Ab +0D), 151530.5 (M _Ab +1D), 152436.1 (M _Ab +2D), 153625.1 (M _Ab +3D), 154607.5 (M _Ab +4D).

Average: y = 2.0.

Biological evaluation

Test Example 1: HCC827 Cell Proliferation Experiment

First, the purpose of the test:

The inhibitory effect of the sample of the invention on the proliferation of HCC827 cells was examined.

Second, the test materials:

Sample of the invention: ADC-9

Positive control drug: ADC-8

HCC827 cells: Chinese Academy of Sciences cell bank, article #TCHu153;

CCK-8: Cell Counting Kit-8, purchased from Dojindo, article number: CK04;

FBS: Fetal Bovine Serum, purchased from Gibco, article number 10099-141;

RPMI1640: purchased from Hyclone, article number: SH30809.01B;

VICTOR 3 multi-function microplate reader (PerkinElmer).

Third, the test method:

In a 1.96-well plate, 100 μl of RPMI1640 medium containing 5,000 HCC827 cells in 10% FBS was added to each well, and the plate was incubated at 37 ° C in a 5% CO ₂ incubator for 16 h.

2. The sample was diluted three-fold with RPMI1640 medium containing 10% FBS, and a total of 10 points were diluted at a starting dilution of 10 μg/ml.

3. The HCC827 cell culture plate laid the day before was taken out from the incubator, the upper layer culture solution was discarded, and 100 μl/well of the medium containing the diluted sample was added. Two duplicate wells were set for each concentration, and control wells without any drug were set. The cells were continuously cultured at 37 ° C under 5% CO ₂ .

After 4.72 hours, add 10 μl of CCK-8 solution to each well for color development, incubate in a 37 ° C, 5% CO ₂ incubator for 2 hours, read OD ₄₅₀ on the microplate reader, and treat it with Graphpad Prism 5 software. You can get the IC ₅₀ .

Fourth, the test results:

The biological activity of the samples of the present invention was obtained from the above analysis, and the calculated IC ₅₀ values are listed in Table 1 below:

Table IC ₅₀ of Samples invention HCC827 cell proliferation inhibition 1. This

Sample serial number	IC 50 (HCC827)/ng/ml
ADC-9	92
ADC-8	148.7

Conclusion: The preferred sample of the invention has significant proliferation inhibitory activity against HCC827 cells.

Test Example 2: Lovo cell proliferation assay

First, the purpose of the test:

The inhibitory effect of the sample of the invention on the proliferation of Lovo cells was examined.

Second, the test materials:

Sample of the invention: ADC-9

Positive control drug: ADC-8

Lovo cells: Chinese Academy of Sciences cell bank, article #TCHu82;

CCK-8: Cell Counting Kit-8, purchased from Dojindo, article number: CK04;

FBS: Fetal Bovine Serum, purchased from Gibco, article number 10099-141;

DMEM/F12: purchased from Hyclone, article number: SH30023.01;

VICTOR 3 multi-function microplate reader (PerkinElmer).

Third, the test method:

In a 1.96-well plate, 100 μl of 10% FBS DMEM/F12 medium containing 4000 Lovo cells was added to each well, and the plate was incubated at 37 ° C in a 5% CO ₂ incubator for 16 h.

2. The sample was diluted three-fold with DMEM/F12 medium containing 10% FBS, and a total of 10 points were diluted at a starting dilution of 100 μg/ml.

3. The Lovo cell culture plate laid the day before was taken out from the incubator, the upper layer culture solution was discarded, and 100 μl/well of the medium containing the diluted sample was added. Two duplicate wells were set for each concentration, and control wells without any drug were set. The cells were continuously cultured at 37 ° C under 5% CO ₂ .

After 4.72 hours, add 10 μl of CCK-8 solution to each well for color development, incubate in a 37 ° C, 5% CO ₂ incubator for 2 hours, read OD ₄₅₀ on the microplate reader, and treat it with Graphpad Prism 5 software. You can get the IC ₅₀ .

Fourth, the test results:

The biological activity of the samples of the present invention was obtained from the above analysis, and the calculated IC ₅₀ values are listed in Table 2 below:

Table IC ₅₀ of Samples invention inhibit the proliferation of Lovo cells 2. This

Sample serial number	IC 50 (Lovo)/ng/ml
ADC-9	934
ADC-8	2202

Conclusion: Preferred samples of the invention have significant proliferation inhibitory activity against Lovo cells.

Test Example 3: HCC827-Del19/T790M/C797S (DTC) cell proliferation assay

First, the purpose of the test:

The inhibitory effect of the sample of the invention on the proliferation of HCC827-DTC cells was examined.

Second, the test materials:

Sample of the invention: ADC-9

Control drug: mAb002, AZD-9291

HCC827 cells: Chinese Academy of Sciences cell bank, article #TCHu153;

HCC827-DTC cells: transfected with pCDH-EGFR Del19/T790M/C797S (DTC), ΔR8.9 and VSVG in 293T cells (ATCC, CRL-3216), virus supernatant was collected 48 hours later, filtered by 0.45 μm membrane The cell debris was discarded, resuspended by centrifugation at 50000 g for 2 hours, and the virus was concentrated 10 times. The concentrated virus was added to the plated HCC827 cells the day before, while 8 μg/ml of polybrene was added, and fresh medium was replaced after 24 hours, and after 2 hours, 2 μg/ml of puromycin was added for screening to obtain HCC827-DTC cells.

CCK-8: Cell Counting Kit-8, purchased from Dojindo, article number: CK04;

FBS: Fetal Bovine Serum, purchased from Gibco, article number 10099-141;

RPMI1640: purchased from Hyclone, article number: SH30809.01B;

VICTOR 3 multi-function microplate reader (PerkinElmer).

Third, the test method:

In a 1.96-well plate, 100 μl of 10% FBS RPMI1640 medium (puromycin 2 μg/ml) containing 5000 HCC827-DTC cells was added to each well, and the plate was incubated at 37 ° C in a 5% CO ₂ incubator for 16 hours.

2. The samples were serially diluted with RPMI 1640 medium containing 10% FBS and diluted for 10 points. The antibody and ADC were initially diluted to a concentration of 10 ug/ml (66.7 nM) and diluted 3-fold. AZD-9291 was initially diluted at 2500 nM and diluted 4 fold.

3. The HCC827-DTC cell culture plate laid the day before was taken out from the incubator, the upper layer culture solution was discarded, and 100 μl/well of the medium containing the diluted sample was added. Two duplicate wells were set for each concentration, and control wells without any drug were set. The cells were continuously cultured at 37 ° C under 5% CO ₂ .

After 4.72 hours, add 10 μl of CCK-8 solution to each well for color development, incubate in a 37 ° C, 5% CO ₂ incubator for 2 hours, read OD ₄₅₀ on the microplate reader, and treat it with Graphpad Prism 5 software. You can get the IC ₅₀ .

Fourth, the test results:

The biological activity of the samples of the present invention was obtained from the above analysis, and the calculated IC ₅₀ values are listed in Table 3 below:

TABLE Sample IC ₅₀ HCC827-DTC invention on proliferation inhibition of cell 3. This

Conclusion: Non-small cell lung cancer HCC827 cells have obvious resistance to AZD-9291 after expressing EGFR-DTC gene; naked anti-mAb002 has only partial inhibition effect on this cell. However, the sample ADC-9 of the present invention has a strong proliferation inhibitory activity against the drug-resistant strain HCC827-DTC.

Test Example 4: H1975-L858R/T790M/C797S (LTC) cell proliferation assay

First, the purpose of the test:

The inhibition of proliferation of H1975-LTC cells by the samples of the present invention was examined.

Second, the test materials:

Sample of the invention: ADC-9

Control drug: mAb002, AZD-9291

H1975 cells: Chinese Academy of Sciences cell bank, article #TCHu193;

H1975-LTC cells: 293T cells (ATCC, CRL-3216) were transfected with pCDH-EGFR L858R/T790M/C797S (LTC), ΔR8.9 and VSVG. After 48 hours, the virus supernatant was collected and discarded by a 0.45 μm filter. The cell debris was resuspended by centrifugation at 50000 g for 2 hours, and the virus was concentrated 10 times. The concentrated virus was added to the H1975 cells plated the day before, while 8 μg/ml of polybrene was added, and fresh medium was replaced after 24 hours, and after 2 hours, 2 μg/ml of puromycin was added for screening to obtain H1975-LTC cells.

CCK-8: Cell Counting Kit-8, purchased from Dojindo, article number: CK04;

FBS: Fetal Bovine Serum, purchased from Gibco, article number 10099-141;

RPMI1640: purchased from Hyclone, article number: SH30809.01B;

VICTOR 3 multi-function microplate reader (PerkinElmer).

Third, the test method:

In a 1.96-well plate, 100 μl of RPMI1640 medium (puromycin 2 μg/ml) containing 5,000 H1975-LTC cells in 10% FBS was added to each well, and the plate was incubated at 37 ° C in a 5% CO ₂ incubator for 16 hours.

2. The samples were serially diluted with RPMI 1640 medium containing 10% FBS and diluted for 10 points. The antibody and ADC were initially diluted to a concentration of 10 ug/ml (66.7 nM) and diluted 3-fold. AZD-9291 was initially diluted at 2500 nM and diluted 4 fold.

3. The H1975-LTC cell culture plate laid the day before was taken out from the incubator, the upper layer culture solution was discarded, and 100 μl/well of the medium containing the diluted sample was added. Two duplicate wells were set for each concentration, and control wells without any drug were set. The cells were continuously cultured at 37 ° C under 5% CO ₂ .

After 4.72 hours, add 10 μl of CCK-8 solution to each well for color development, incubate in a 37 ° C, 5% CO ₂ incubator for 2 hours, read OD ₄₅₀ on the microplate reader, and treat it with Graphpad Prism 5 software. You can get the IC ₅₀ .

Fourth, the test results:

The biological activity of the samples of the present invention was obtained from the above analysis, and the calculated IC ₅₀ values are listed in Table 4 below:

Table IC ₅₀ of Samples invention H1975-LTC cell proliferation inhibition 4. The

Sample serial number	IC 50 (H1975-LTC)/nM
ADC-9	5.96
mAb002	No inhibition
AZD-9291	>2500

Conclusion: Non-small cell lung cancer H1975 cells have obvious resistance to AZD-9291 after expressing EGFR-LTC gene; naked anti-mAb002 has almost no inhibitory effect on this cell. However, the sample ADC-9 of the present invention has a strong proliferation inhibitory activity against the drug-resistant strain H1975-LTC.

Test Example 5: EGFR antibody cynomolgus monkey pharmacokinetic test

First, the purpose of the test:

The pharmacokinetic changes of mAb002 relative to nimotuzumab (mAb001) in cynomolgus monkeys were compared.

Second, the test materials:

Sample of the invention: mAb002 (IgG1-YTE variant of nimotuzumab)

Positive control drug: mAb001, nimotuzumab injection, purchased from Baitai Biological, article number 0120131240

PBS: purchased from bio-organisms, item number PD0100

BSA: purchased from Amresco, article number 0332

Proclin300: purchased from Supelco, article number 48912-U

EGFR: purchased from Sino Biological, article number 10001-H08H

Anti-Human IgG (Fc)-peroxidase: purchased from Sigma, article number A0170

Human IgG: purchased from R&D; article number 1-001-A

Microplate reader: purchased from Thermo Scientific, model Multiskan FC, article number EQP-LI-030

Third, the test method:

ELISA is a method commonly used to detect pharmacokinetics in biological serum samples. In this study, the concentration of the antibody to be tested in the serum of cynomolgus monkeys was determined by ELISA.

Two groups of cynomolgus monkeys (3 in each group) were injected with 5 mg/kg of mAb001 and mAb002 antibody by constant-flow pump, before administration (0h) and 5min (±10s) and 2h (±2min) after the end of administration. ), 4h (±5min), 8h (±5min), 1d (±10min), 2d (±10min), 3d (±10min), 5d (±10min), 7d (±10min), 10d (±10min), 13d (±10min), 17d (±10min), 21d (±10min), 28d (±10min), about 1mL whole blood was collected from the saphenous vein of the lower extremity, and the blood sample was allowed to stand at room temperature for 1h~2h until fully coagulated, 4°C The serum was separated by centrifugation at 4000 g for 5 minutes.

A microplate is coated with EGFR to capture the antibody to be tested in the diluted serum sample. The detection antibody was an HRP-labeled goat anti-human IgG antibody (for the Fc segment). HRP catalyzes the TMB substrate to produce a soluble blue compound, the hydrochloric acid solution terminates the reaction, and absorbs light at a working wavelength of 450 nm and a reference wavelength of 620 nm. The value, the magnitude of the OD value, is positively correlated with the concentration of the antibody to be tested in the sample. Using SoftMax Pro v5.4.1 software, the standard curve of the measured antibody concentration (X-axis) and OD value (Y-axis) was drawn, and the four-parameter Logistic model was fitted. The weighting method of weight 1/y was used to obtain the curve parameters. . The unknown sample was calculated by regression of its OD value to the standard curve to obtain the sample blood concentration.

Fourth, the experimental results:

After a single intravenous injection of 5 mg/kg of mAb001 and mAb002 in cynomolgus monkeys, the average pharmacokinetic parameters of the animals in the two dose groups are shown in Table 5 below and Figure 1.

table 5

The mean C _{max of the} mAb002 group was basically the same as that of the mAb001 group, indicating that the amount of drug entered into the animal was basically the same, but the pharmacokinetic characteristics of the mAb002 group in the mAb001 group were significantly different in the cynomolgus monkey, which was expressed as AUC _(0-28d). High, terminal elimination half-life is long, clearance rate is low, and average residence time is long, indicating that the monkey drug-induced properties of the mutant antibody mAb002 (IgG1-YTE variant of nimotuzumab) have been significantly improved.

Test Example 6: EGFR-ADC in vivo drug effect test

First, the purpose of the test:

The in vivo efficacy of ADC-9 of the invention relative to ADC-8 and naked anti-mAb002 in a nude mouse lung cancer xenograft HCC827 model was compared.

Second, the test materials:

Sample of the invention: ADC-9

ADC-8

mAb002

HCC827 cells: Chinese Academy of Sciences cell bank, article #TCHu153;

Test animals: nude mice, SPF, 16-20 g, sputum, Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

Third, the test method:

1 The nude mouse laboratory environment was adapted for three days and randomly divided into 5 groups, 9 in each group.

2 tumor cell transplantation

HCC827 cells (5×10 ⁶ +50% matrigel/mouse) were inoculated subcutaneously into the right flank of nude mice, and on the 7th day after inoculation, the tumor was started to grow at 202.07±6.22 mm3 (d1).

3 dosage and method of administration

The route of administration was intraperitoneal (ip), once a week for 3 times. The specific dosage regimen is shown in Table 1.

4 transplanted tumor volume and body weight of nude mice

Tumor volume was measured twice a week, body weight was weighed and data was recorded.

5 data statistics

Excel statistical software was used: the average was calculated as avg; the SD value was calculated as STDEV; the SEM value was calculated as STDEV/SQRT; the difference between the groups was calculated as TTEST.

The calculation formula of tumor volume (V) is: V = 1/2 × L _length × L _short ²

Relative volume (RTV) = V _T /V ₀

Tumor inhibition rate (%) = (C _RTV -T _RTV ) / C _RTV (%)

Among them, V ₀ and V _T are the tumor volume at the beginning of the experiment and at the end of the experiment, respectively. C _RTV and T _RTV are the blank control group (Blank) at the end of the experiment and the relative tumor volume of the experimental group.

Fourth, the experimental results:

The anti-tumor effect is shown in Table 6 below and Figure 2

Table 6

The results of this experiment show that mAb002 (IgG1-YTE variant of nimotuzumab, see Example 2), ADC-8 (see Example 8) and ADC-9 (see Example 9) on mouse lung cancer xenografts HCC827 has a significant anti-tumor effect. Among them, ADC-9 showed more obvious anti-tumor effect than naked anti-mAb002 (0.1mg/head) at the same dose (0.1mg/only) or lower dose (0.03mg/head), indicating ADC-9 phase The advantage over the naked anti-drug effect. In addition, different ADCs were compared at the same dose (0.03 mg/mouse), and ADC-9 showed better efficacy than ADC-8. The tumor inhibition rate at 34 days of administration was 65.33% vs. 48.96%, respectively. , indicating that the ADC with toxin compound 6 (formulaly compound 5) showed stronger antitumor activity than the ADC with toxin compound 4 (MC-MMAF, which was originally compound 3).

Claims (16)

1. An antibody-drug conjugate of the formula (I) or a pharmaceutically acceptable salt or solvent compound thereof:

   

   among them:

   L ₁ , L ₂ are joint units;

   Y is 1-8, preferably 2-5;

   Ab is an anti-EGFR antibody or antigen-binding fragment thereof, and the anti-EGFR antibody or antigen-binding fragment thereof comprises the following CDR regions:

   

   Among them, the Ab has three site mutations of M258Y/S260T/T262E (YTE) on the heavy chain.
2. The antibody-drug conjugate of the formula (I), or a pharmaceutically acceptable salt or solvate thereof, according to claim 1, wherein the anti-EGFR antibody or antigen-binding fragment thereof is a humanized antibody or Its fragment.
3. The antibody-drug conjugate of the formula (I), or a pharmaceutically acceptable salt or solvate thereof, according to claim 1, wherein the heavy chain variable region of the humanized antibody further comprises a human source The heavy chain FR region of IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising the heavy chain FR region of human IgG1, and/or the light chain variable region of the humanized antibody further comprising a human kappa chain or human source The light chain FR region of the kappa chain variant, or the light chain FR region of the human lambda chain or human lambda chain variant.
4. The antibody-drug conjugate of the formula (I), or a pharmaceutically acceptable salt or solvate thereof, according to claim 1, wherein the heavy chain of the humanized antibody comprises human IgG1 or a variant thereof a constant region of a human, human IgG2 or variant thereof, human IgG3 or variant thereof or human IgG4 or a variant thereof, preferably comprising a constant region of human IgG1 or a variant thereof, and/or said humanization The light chain of the antibody further comprises a light chain constant region of a human kappa chain or a human kappa chain variant, or a light chain constant region of a human lambda chain or a human lambda chain variant.
5. The antibody-drug conjugate of the formula (I) or a pharmaceutically acceptable salt or solvate thereof, according to claim 1, wherein the anti-EGFR antibody comprises an amino acid sequence as shown in SEQ ID NO: A chain, and a heavy chain as shown in SEQ ID NO: 4 of the amino acid sequence.
6. The antibody-drug conjugate of the formula (I), or a pharmaceutically acceptable salt or solvate thereof, according to any one of claims 1 to 5, wherein L _{2 is} as defined by the following formula (L ₂ ) Show:

   

   among them

   X _{1 is} selected from a hydrogen atom, a halogen, a hydroxyl group, a cyano group, an alkyl group, an alkoxy group, and a cycloalkyl group;

   X _{2 is} selected from the group consisting of alkyl, cycloalkyl and heterocyclic;

   m is 0-5, preferably 1-3; S is a sulfur atom.
7. The antibody-drug conjugate of the formula (I), or a pharmaceutically acceptable salt or solvate thereof, according to any one of claims 1 to 5, wherein L ₁ is represented by the following formula (L ₁ ) Compound shown:

   

   among them

   X ₃ is an alkyl group, and the alkyl group is optionally further substituted with a substituent of a halogen, a hydroxyl group, a cyano group or an alkyl group;

   n is 0-5, preferably 1-3.
8. The antibody-drug conjugate of the formula (I), or a pharmaceutically acceptable salt or solvate thereof, according to any one of claims 1 to 5, which is selected from the group consisting of the antibody of the formula (II) a drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof:

   

   Wherein, Ab, L ₂ , y are as defined in claim 1.
9. The antibody-drug conjugate of the formula (I) according to any one of claims 1 to 5, or a pharmaceutically acceptable compound thereof An acceptable salt or solvent compound selected from the group consisting of an antibody-drug conjugate of the formula (III) or a pharmaceutically acceptable salt or solvent compound thereof:

   

   Wherein, Ab, L ₁ , y are as defined in claim 1.
10. The antibody-drug conjugate of the formula (I), or a pharmaceutically acceptable salt or solvate thereof, according to any one of claims 1 to 5, which is a compound represented by the following formula:

    

    Wherein mAb002 comprises a light chain of amino acid sequence such as SEQ ID NO: 3, and a heavy chain of SEQ ID NO: 4, y as defined in claim 1.
11. An EGFR antibody comprising an LCDR1, LCDR2, LCDR3 region of the sequence SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and variants thereof, and SEQ ID NO: 10, SEQ ID NO The HCDR1, HCDR2, HCDR3 region of SEQ ID NO: 12 and variants thereof, characterized in that it has three site mutations of M258Y/S260T/T262E (YTE) on the heavy chain; preferably, said The EGFR antibody comprises a light chain having the amino acid sequence set forth in SEQ ID NO: 3 and a heavy chain as set forth in SEQ ID NO: 4.
12. A pharmaceutical composition comprising the antibody-drug conjugate of the formula (I) according to claim 1 or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable Excipient, diluent or carrier.
13. The antibody-drug conjugate of the formula (I) according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition according to claim 12, prepared for treatment Use in a medicament for cancer; the cancer includes a cancer that highly expresses EGFR, a cancer that has a mutation on the EGFR polypeptide, and a cancer that has a mutation in a gene downstream of the EGFR signaling pathway; the cancer is preferably gastric cancer, pancreatic cancer, liver cancer, Breast cancer, lung cancer, colon cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer; more preferably lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer .
14. A method for treating cancer comprising administering to a subject a therapeutically effective amount of the antibody-drug conjugate of the formula (I) according to claim 1 or a pharmaceutically acceptable salt or solvent compound thereof, or The pharmaceutical composition according to claim 12; wherein the cancer is preferably a cancer that highly expresses EGFR, a cancer that has a mutation on the EGFR polypeptide, and a cancer that has a mutation in a gene downstream of the EGFR signaling pathway; more preferably, gastric cancer, pancreatic cancer , liver cancer, breast cancer, lung cancer, colon cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer; most preferably lung cancer, colon cancer, rectal cancer or colorectal cancer Head and neck cancer.
15. A method according to claim 14, wherein said cancer is preferably a cancer having a mutation on an EGFR polypeptide, said EGFR mutant polypeptide comprising at least one EGFR mutation selected from the group consisting of L858R, L858R/T790M, L858R/ T790M/C797S, Del19, Del19/T790M, Del19/T790M/C797S, T790M, G719X, L861Q, S768I, Exon 18indel/E709X, Exon 19insertion, Exon20insertion, A763_Y764insFQEA, Exon 18–25 duplication and EGFR-RAD51 rearrangement; more preferably EGFR polypeptide Mutant gastric cancer, pancreatic cancer, liver cancer, breast cancer, lung cancer, colon cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer; most preferred is lung cancer, colon Cancer, rectal cancer, colorectal cancer or head and neck cancer.
16. A method according to claim 14, wherein said cancer is preferably a cancer having a mutation in a gene downstream of the EGFR signaling pathway, said downstream gene comprising at least one gene selected from the group consisting of Ras, B-Raf, PI3K, wherein The downstream gene mutation comprises at least a mutation selected from the group consisting of K-Ras G12V, K-Ras G13D, N-Ras Q61K, H-Ras G12S; B-Raf V600E, B-Raf G468A; PIK3CA H1047R, PIK3CB E633K and p110γE1021K More preferably, gastric cancer, pancreatic cancer, liver cancer, breast cancer, lung cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck with mutations in the downstream gene of the EGFR signaling pathway Cancer; most preferred is lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer.

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