CN108811499A - The specificity coupling of cell-binding molecules - Google Patents

The specificity coupling of cell-binding molecules Download PDF

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CN108811499A
CN108811499A CN201580082141.5A CN201580082141A CN108811499A CN 108811499 A CN108811499 A CN 108811499A CN 201580082141 A CN201580082141 A CN 201580082141A CN 108811499 A CN108811499 A CN 108811499A
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acid
cell
drug
antibody
receptor
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CN108811499B (en
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赵珞博永新
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Hangzhou Dac Biotech Co Ltd
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Suzhou Mei Kang Jia Biological Technology Co Ltd
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    • C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C07C233/17—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
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    • A61K47/6851—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 a determinant of a tumour cell
    • A61K47/6869—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 a determinant of a tumour cell the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
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    • 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
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    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Abstract

The present invention describes the application of a kind of Cell binding agent-drug conjugates titer comprising bridging junctor and connector and conjugate.

Description

The specificity coupling of cell-binding molecules
Technical field
The present invention describes one kind and can be used for specificity connection compound, especially cytotoxic agent and biomolecule (carefully Born of the same parents' bonding agent) novel connecting body.Present invention comprises prepare cell-binding molecules drug (cytotoxic agent) to be conjugated conjugate Method：Or first with this kind of connector modified medicaments, then react with cell binding agent, or is first modified with this kind of connector thin Born of the same parents' bonding agent, then reacted with drug molecule.
Technical background
Albumen, especially antibody, as the investigational agent of experiment in vitro and the diagnostic tool of experiment in vivo or treatment Drug is widely used in medical and health industry (Gad, S.C.Drug discovery handbook, Wiley- Interscience,2005).In the application of albumen many, the base group modification of the albumen Chang Yaoyong meanings, such as cytotoxicity Molecule, radioactive element label or the molecule with chromatographic characterization, the needs detected with satisfaction treatment or diagnosis (Teicher, B.A.et al.Clin.Cancer Res.2011,17,6389-97；Elsadek,B.et al.,J.Control Release, 2012,157,4~28).One of these applications, antibody-drug are conjugated conjugate (ADCs), are obtained as hot spot in nearly 20 years Deep exploitation, it combines the cytotoxicity of antibody accurate targeting and anti-tumor agent comprising salmosin so that drug is passed by targeting It is fed into cancer cell, and normal cell is not had an impact.Especially, since U.S. FDA had approved in 2011 Adcetris (brentuximab vedotin) has approved Kadcyla (ado-trastuzumab emtansine) for 2013 Since, the means using antibody-drug conjugation conjugate as targeted therapy of cancer are public by international almost all of pharmacy Department and biotechnology company apply (Chari, R.et al, Angew.Chem., Int.Ed.2014,53,3796-3827； Sievers,E.L.et al.Annu Rev Med.2013,64,15-29；Mehrling,T.Future Oncol,2015,11, 549).There is more than 50 in the ADC drugs of clinical experimental stage at present according to the statistics of website www.clinictrails.gov..
The ADCs drugs of the first generation, including Kadcyla and Adcetris, be all respectively by cytotoxic drug molecule and On antibody made from natural amine groups or the non-selective coupling of intrachain cysteine sulfydryl.Because being had more than on IgG1 antibody 50 lysines for being exposed to surface and 8 cysteines, this non-selective coupling mode can cause cytotoxic molecule and The generation of antibody surface all areas is random cross-linked, generates varied, the wide in range ADC of DAR values (drug antibody ratio) distribution Group (Wang, L., et al.2005Protein Sci.14,2436；Hamblett,K.J.,et al.2004Clin.Cancer Res.10,7063).Part be not intended to generate ADC subgroups have circulating half-life shorten, it is inefficient, potential miss the target toxicity height with And in vivo the defects of pharmacokinetics (PK) property is uncertain (Hamblett, K.J.et al, Clin.Cancer Res.2004, 10,7063-7070；Adem,Y.T.et al,Bioconjugate Chem.2014,25,656-664；Boylan, N.J.Bioconjugate Chem.,2013,24,1008–1016；Strop,P.,et al Chem.Biol.2013,20, 161-167).In addition, the ADC of this traditional coupling mode production, consistency is very challenging between holding batch, needs more High production capacity (Wakankar, A.mAbs, 2011,3,161-172).
Therefore, numerous biotech companies and research institution are devoted to develop the fixed point conjugated pair of novel ADC drugs Linked method.In recent years there are also the ADC process for preparing medicine of fixed point (Panowski, S, 2014, mAbs 6,34), these Method includes：On antibody introduce non-matching cysteine, such as Genentech THIOMAB antibody (Junutula, J.R., et al Clin.Cancer Res.2010,16,4769；Junutula,J.R.,et al Nat Biotechnol.2008 26,925-32；US patents 8,309,300；7,855,275；7,521,541；7,723,485； WO2008/141044)；Or glutamine label is introduced, it can be by the glutamine transaminage with luxuriant former wheel Streptothrix amplification (mTG)(Strop,P.,Bioconjugate Chem.,2014,25,855–862；Strop,P.,et al., Chem.Biol.2013,20,161–167；The United States Patent (USP) 8,871,908 of Rinat companies of Pfizer) or by bacterial origin paddy ammonia Amide transaminase (MTGase) (Dennler, P., et al, Bioconjug.Chem.2014,25,569-578；Innate drugs Company's U.S. Patent application 20130189287；The United States Patent (USP) 7,893,019 of Italian Bio-Ker S.r.l. companies) known Not；Introduce sulfydryl-L-fucose (Dennler, P., et al, Bioconjugate Chemistry 2014,25,569； Okeley,N.M.,et alBioconjugate Chem.2013,24,1650)；Non-natural amino acid is introduced by mutagenesis (Axup,J.Y.,et al.,Proc.Natl.Acad.Sci.2012,109,16101–16106；Zimmerman,E.S.,et al.,Bioconjug.Chem.2014,25,351–361；Wu,P.,et al,Proc.Natl.Acad.Sci.2009,106, 3000-3005；Rabuka,D.,et al,Nat.Protoc.2012,7,1052-67；Sutro bio-pharmaceuticals company United States Patent (USP) 8,778,631 and U.S. Patent application 20100184135, World Intellectual Property Organization WIPO patent application WO2010/081110；Ambrx is public Take charge of patent WO2006/069246,2007/059312, United States Patent (USP) 7,332,571,7,696,312；With 7,638,299； Allozyne house journals WO2007/130453 and United States Patent (USP) 7,632,492；7,829,659)；Introduce selenocysteine (Hofer,T.,et al Biochemistry 2009,48,12047–12057；The graduate United States Patent (USP) of National Cancer 8,916,159)；It is formylglycine to generate enzyme (FGE) by the transforms cysteine on CXPXR consensus sequences with formylglycine (FGly) (Drake, P.M., et al., Bioconjug.Chem.2014,25,1331-1341. United States Patent (USP) 7,985,783； The United States Patent (USP) 8,097,701 of Redwood Life Sciences；8,349,910WithUnited States Patent (USP)Application20140141025, 20100210543)；Or sialic acid (Zhou, Q., et al is introduced by sugar engineering with galactolipin or sialyltransferase Bioconjug.Chem., 2014,25,510-520, the United States Patent (USP) 20140294867 of Sai Nuofei-Genzyme Corp.).With above-mentioned Method can produce uniform product, but they are required for antibody engineering and re-optimization cell culture condition. In addition, the expression of non-natural amino acid gene code usually not it is desired it is so high (Tian, F., et al, 2014, Proc.Natl.Acad.Sci.U.S.A.111,1766-71), this point has the cost of ADC products important influence.In addition, Stability of the ADC drugs obtained by cysteine side chain coupling usually in the circulatory system is limited, results in swollen in arrival Before oncocyte, the premature fracture of toxic small molecule load (Junutula, J.R., et al Nat.Biotechnol.2008,26, 925-32)。
Disulfide bond pattern in four kinds of hypotypes of IgG antibody has just been well known (Milstein in the sixties in last century C.Biochem J 1966,101:338-351；Pink JR,Milstein C.Nature 1967,214:92-94； Frangione B,Milstein C.Nature 1967,216:939-941；Pink JR,Milstein C.Nature 1967,216:941-942；Frangione B,et al.Biochem J 1968,106,15–21；Frangione B, Milstein C.J Mol Biol1968；33:893–906；Edelman GM,et al.Proc Natl Acad Sci USA 1969；63:78-85；Frangione B,et al.Nature 196,221:145-148,Spiegelberg,H.L.et al Biochemistry,1975,10,2157-63).Disulfide bond has very the structure, stability and biological function of IgG molecules Important role.In four kinds of hypotype IgG of IgG antibody1,IgG2,IgG3And IgG4In, each IgG molecules contain 12 chains in total Interior disulfide bond；Each disulfide bond is associated with an independent domain IgG.Two heavy chains pass through different number of two sulphur in hinge area Key is connected：IgG1And IgG4It is 2, IgG24, IgG311.In IgG1On, the last one cysteine and heavy chain on light chain Upper 5th cysteine forms disulfide bond and is connected.In IgG2,IgG3And IgG4On, on light chain the last one cysteine with again The 3rd cysteine forms disulfide bond (Liu, H.and May, K., 2012, mAbs 4,17-23) on chain.By reduction experiment, Alkylation and LC-MS analyses, it is known that humanized IgG1Characteristic (Liu, H, the et al of disulfide bonds difficulty or ease on antibody Anal.Chem.,2010,82,5219–5226).Interchain disulfide bond is more easy to be reduced fracture, and light and weight chain than intrachain disulfide bond Between than two heavy chains of disulfide bond between disulfide bond be more easy to be reduced fracture.The interchain disulfide bond on two heavy chain top compares lower part That more easy fracture.In addition, the disulfide bond on the domains CH2 is easiest to be reduced.Two sulphur in the domain VL, CL, VH, and CH1 Key have it is similar it is moderate can breaking property, and the disulfide bond in the domains CH3 is (Liu, H, the et al for being most not easy to be reduced Anal.Chem.,2010,82,5219–5226)。
Based on humanized IgG1The characteristics of antibody interchain disulfide bond is more easily broken off, many research institutes and company are fixed using chemistry The strategy of point connection, will natural antibody interchain disulfide bond restore after put up a bridge again and again crosslinked together, such as using being claimed For follow-on maleimide compound (NGMs), i.e., bromo-derivative or dibromo-maleimide class compound (Schumacher, F.F., et al Org.Biomol.Chem.2014,12,7261-7269), a three carbon bridges are formed using dialkyl reagent Key (Badescu, G., et al., Bioconjug.Chem.2014,25,1124-1136, PolyTherics Co., Ltds it is special Sharp WO2013/190272, WO2014/064424), using disubstituted heteroaryl bridged bond (Concortis Biosys Corp. United States Patent (USP)Application2015/0105539), or by bismaleimide it is used as bridged bond (WO2014/114207).We are same Come using bromo maleimide and dibromo-maleimide as connector coupling drug and antibody (WO2014/009774, PCT/IB2012/053554) for a long time.But the design of above-mentioned bridging junctor is by a cytotoxic molecule It is coupled with a pair of of disulfide bond, by being limited (about 2 pairs), most of feelings by can be used for being conjugated the cystine linkage number being coupled on antibody The ADCs drug DAR values produced under condition are less than 2.
Realize that its curative effect is limited to eventually arrive at the toxic small molecule number of tumour cell in view of ADCs drugs, to improve ADC therapeutic indexs, DAR values more preferably greater than 3 (Epenetos, A.A.et al, Cancer Res., 1986,46,3183- 3191；Chari,R.V.Acc.Chem.Res.,2008,41,98-107,Zhao,R.Y.2011J.Med.Chem.54,3606- 3623).Novel disulfide bridge connector in the present invention can not only connect 2 or more small molecules on each connector Drug realizes higher DAR (>=4), and in order to the interchain disulfide bond of selectively bridging antibody surface.This is because What the natural characteristic that three keys stretch in acetylene dicarbapentaborane determined, when two toxic small molecules are connected to the bridging junctor both ends of stretching, extension When, can be formed macromolecular (Spacing), other disulfide groups, as heavy chain interior lower end disulfide group is difficult to approach.Cause The disulfide bridge connector of this present invention, can antibody interchain free sulfhydryl group pair that selectively bridging is broken by TCEP or DTT, To generate the ADC that DAR is more than 4.It is other to be reduced excessive disulfide bond due to being difficult to by bridging junctor, especially for containing 2 The bridging junctor of the stretching, extension of a toxic small molecule approaches, can be after the completion of coupling with oxide such as hydroascorbic acid (DHAA) or Cu (II) is reconnected.In short, these bridging junctors in the present invention can be generated together in a manner of a kind of simplicity The uniform ADC drugs of matter.
Abstract of invention
Connector contains there are one acetylene dicarbapentaborane functional group in the present invention, 2 small-molecule drugs is connected, with cell binding agent (such as antibody) is combined.Cell-binding molecules-connector-drug conjugate can be expressed as：Wherein Cb is cell binding agent, and L is connector, and Drug1 and Drug2 are Medicine small molecules, and n is One 1 to 20 integer, and 2 element sulphur bridging Cb are covalently attached 2 or more drugs point in L, each bridging junctor L Son.Being applied in cell small molecule-drug conjugates has the advantages of this kind of connector：A) covalent cross-linkings (bridging again) cell The mode that open disulfide bond sulfydryl is restored on bonding agent (such as antibody) is conducive to the stability for keeping conjugate；B) can make Toxic small molecule/drug is connected to the specific position of cell binding agent, such as the interchain site of IgG antibody, uniform to generate ADC drugs.
On the one hand, the connector structure in the present invention can be expressed as public formula (I)
Acetylene ethanedioic acid structure wherein on connector can be reacted with a pair of of sulphur atom in cell binding agent；
Z1And Z2Be it is same or different can and drug toxicity reaction functional group, can by disulfide bond, ehter bond, ester bond, It is thioether bond, thioester bond, peptide bond, hydrazone bond, urethane bond, carbonic acid ester bond, amine key (two level, three-level or level Four), imine linkage, miscellaneous Naphthenic base, heteroaryl, alcoxyl oxime key or amido bond are combined with toxic small molecule drug；
R1And R2It is identical, the different or default straight chained alkyl containing 1~6 carbon atom, the branch of 3 to 6 carbon atoms Chain or naphthenic base, straight chain, branch or cycloalkenyl group or the ester group of alkynyl or 1~6 carbon atom, ether, amide groups or polyethoxy (OCH2CH2)p, wherein p is the combination of 0 to about 1000 integer or these groups；
In addition, R1And R2It includes C, N, O to be, S, the chain structure of Si and P atoms is optimal to contain 0~500 atom, covalently It is connected to X1Or X2And Z1Or Z2；R1And R2In each atom combined with all possible chemical mode, such as formed alkyl, Asia Alkyl, alkenylene, alkynylene, ether, polyoxy alkyl, ester, amine, imines, polyamine, hydrazine, hydrazone, amide, urea, semicarbazides, carbonohydrazides, alkane The combination of oxygen amine, polyurethane, amino acid, polypeptide, acyl-oxygen amine, hydroxamic acid or these groups；
X1And X2It is independently selected from NH, N (R3), O, S or CH2；R3It is H, the straight chained alkyl of 1~6 carbon atom, 3 to 6 carbon originals The branch or cyclic alkyl of son, straight chain, branch or the ester of cricoid alkenyl or alkynyl or 1~6 carbon atom, ether, amide or poly- Ethoxy units (OCH2CH2)p, wherein p is the combination of 0 to 1000 integer or these groups.
On the other hand, Cell binding agent-drug conjugates titer is represented by public formula (II), wherein cell binding agent in the present invention Cb, drug Drug1 and Drug2 react with bridging junctor tail end respectively：
Wherein：
Cb is cell binding agent, and optimal is antibody；
It is connector-drug component by a pair of of sulphur atom and cell-binding molecules coupling in bracket；
Drug1And Drug2For same or different cytotoxic agent, they are with disulfide bond, thioether bond, thioester bond, peptide Key, hydrazone bond, ehter bond, ester bond, urethane bond, carbonic acid ester bond, cycloheteroalkyl, heteroaryl, alcoxyl oxime key or amido bond with it is thin Born of the same parents' bonding agent links；
N is 1~20；R1,R2,X1And X2Definition with public formula (I).
On the other hand, the present invention includes a kind of cell binding agent of modification, is represented by public formula (III), wherein cell knot Mixture Cb is reacted with bridging junctor, includes the functional group Z that can be further reacted with Medicine small molecule on connector1And Z2：
Wherein Cb, Z1,Z2,n,R1,R2,X1, and X2Definition with public formula (I) and (II).
Further, the present invention includes a kind of drug molecule of modification, is represented by public formula (IV), wherein drug Drug1 And Drug2With the connection precursor reactant in public formula (I), still remaining with can be with the sulphur atom on cell binding agent to reaction Acetylene ethanedioic acid structure：
Wherein Drug1,Drug2,R1,R2,X1, and X2Definition with public formula (I) and (II).
The present invention also includes a kind of side preparing the cell-binding molecules as shown in public formula (II)-drug conjugation conjugate Method, wherein drug Drug1And Drug2It is connected with cell binding agent by bridging junctor.
The present invention also includes a kind of method preparing the cell-binding molecules modified as shown in public formula (III), wherein carefully Born of the same parents' binding molecule is reacted with the bridging junctor in public formula (I).
The invention also includes a kind of methods for the Medicine small molecule for preparing and being modified as shown in public formula (IV), wherein drug point Son is reacted with the bridging junctor in public formula (I).
Marginal data
A kind of bridging junctors containing polyethylene glycol functional group of Fig. 1 synthesize and its in antibody and answering in drug coupling With.
A kind of synthetic route of bridging junctors of Fig. 2 and pass through oxime key coupled antibody and drug.
A kind of synthesis of bridging junctors containing polypeptide of Fig. 3 and pass through amido bond coupled antibody and drug.
A kind of synthesis of the bridging junctor containing polyethylene glycol and polypeptide of Fig. 4.
A kind of synthesis of bridging junctors containing polyethylene glycol and polypeptide of Fig. 5 and connected in each connector by amido bond Connect 2 to 4 small-molecule drugs.
The synthesis for the tubulysin homologues that Fig. 6 is modified with the bridging junctor containing polypeptide and polyethylene glycol.
Bridging junctor coupling tubulysin homologue and cell-binding molecules of the Fig. 7 containing polyethylene glycol.
The synthesis for the cell-binding molecules-maytansinoids compounds that Fig. 8 is coupled by bridging junctor.
The synthesis for the cell-binding molecules-MMAF homologues that Fig. 9 is coupled by bridging junctor.
The synthesis for the cell-binding molecules-tubulysin homologues that Figure 10 is coupled by bridging junctor.
Detailed description of the invention
Definition
" alkyl " refers to the aliphatic hydrocarbon in the linear chain or branched chain containing 1 to 8 carbon atoms." branch " refers to one or more Alkyl such as methyl, ethyl or propyl compared with low carbon number are connected on a linear alkyl chain.The example of alkyl includes methyl, second Base, n-propyl, isopropyl, normal-butyl, tertiary butyl, n-pentyl, 3- amyls, octyl, nonyl, decyl, cyclopenta, cyclohexyl, 2, 2- dimethylbutyls, 2,3- dimethylbutyls, 2,2- dimethyl amyl groups, 2,3- dimethyl amyl groups, 3,3- dimethyl amyl groups, 2,3, 4-tri-methyl-amyls, 3- methylhexyls, 2,2- dimethylhexanyls, 2,4- dimethylhexanyls, 2,5- dimethylhexanyls, 3,5- diformazans Base hexyl, 2,4- dimethyl amyl groups, 2- methylheptyls, 3- methylheptyls, n-heptyl, different heptyl, n-octyl, iso-octyl.One C1-C8Alkyl can be unsubstituted or be replaced by one or more following groups, including but not limited to-C1-C8Alkyl ,- O-(C1-C8Alkyl), aryl ,-C (O) R' ,-OC (O) R' ,-C (O) OR' ,-C (O) NH2,-C(O)NHR',-C(O)N(R')2,- NHC(O)R',-SR',-S(O)2R' ,-S (O) R' ,-OH, halogen ,-N3,-NH2,-NH(R'),-N(R')2With-CN；It is wherein each R' is independently selected from-C1-C8Alkyl and aryl." halogen " refers to fluorine, chlorine, bromine, iodine atom, preferably fluorine and chlorine atom.
" miscellaneous alkyl " refers to as C2-C8Alkyl, wherein one is independent by O to four carbon atom, S and N atoms are substituted.
" carbocyclic ring " refers to a saturation or unsaturation ring, monocycle containing 3 to 8 carbon atoms or 7 to 13 carbon atoms It is bicyclic.Monocycle carbocyclic ring contains 3 to 6 atoms, typically 5 to 6 atom.Bicyclic carbocyclic contains 7 to 12 atoms, is formed [4,5], [5,5], [5,6] or [6,6] bicyclic system or 9 to 10 atoms form [5,6] or [6,6] bicyclic system.Typical C3- C8Include, but are not limited to cyclopropyl, cyclobutyl, cyclopenta, cyclopentadienyl group, cyclohexyl, cyclohexenyl group, 1,3- cyclohexadiene Base, Isosorbide-5-Nitrae-cyclohexadienyl, suberyl, 1,3- cycloheptadiene base, 1,3,5- cycloheptatriene base, cyclooctyl and cyclo-octadiene base.
“C3-C8Carbocyclic ring " refers to that 3,4,5,6,7 or 8 yuan contain saturated or unsaturated non-aromatic carbocyclic ring.One C3-C8 Carbocyclic ring can be unsubstituted or be replaced by one or more groups, including but not limited to-C1-C8Alkyl ,-O- (C1-C8 Alkyl), aryl ,-C (O) R' ,-OC (O) R' ,-C (O) OR' ,-C (O) NH2,-C(O)NHR',-C(O)N(R')2,-NHC(O) R',-SR',-S(O)R',-S(O)2R' ,-OH, halogen ,-N3,-NH2,-NH(R'),-N(R')2With-CN, wherein each R' is independent Selected from-C1-C8Alkyl and aryl.
" alkenyl " is referred to containing carbon-carbon double bond, the linear chain or branched chain aliphatic hydrocarbon of 2 to 8 carbon atoms.The example packet of alkenyl Include vinyl, acrylic, n-butene base, isobutenyl, 3- methyl-2-butene bases, n-pentene base, hexenyl, heptenyl, octene Base.
" alkynyl " is referred to containing triple carbon-carbon bonds, the linear chain or branched chain aliphatic hydrocarbon of 2 to 8 carbon atoms.The example packet of alkenyl Include acetenyl, propinyl, positive butynyl, 2- butynyls, 3- methylbutynyls, 5- pentynyls, n-pentene base, hexin base, heptyne Base, octynyl.
" alkylidene " refers to saturation, the branch of 1-18 carbon atom or straight chain or cyclic hydrocarbon free radical, including mother It is two obtained the monovalent radical center that two hydrogen is removed on the same carbon of alkane or on different carbon.Typical alkylidene packet It includes, but is not limited to methylene (- CH2), 1,2- ethyl (- CH2CH2), 1,3- propyl (- CH2CH2CH2), Isosorbide-5-Nitrae-butyl (- CH2CH2CH2CH2) etc..
" alkenylene " refer to it is undersaturated, the branch of 2-18 carbon atom or straight chain or cyclic hydrocarbon free radical, including Two obtained the monovalent radical center of two hydrogen is removed on the same carbon of maternal alkene or on different carbon.Typical alkenylene Include, but are not limited to 1,2- vinyl (- CH=CH-).
" alkynylene " refer to it is undersaturated, the branch of 2-18 carbon atom or straight chain or cyclic hydrocarbon free radical, including Two obtained the monovalent radical center of two hydrogen is removed on the same carbon of maternal alkynes or on different carbon.Typical alkynylene Include, but are not limited to acetenyl, propinyl and 4- pentynyls.
" aryl " or Ar refer to aromatic series or heteroaryl, are made of one or several rings, including 3 to 14 carbon atoms, preferential 6 To 10 carbon atoms.Heteroaryl refers to one or more carbon atoms (preferably 1,2,3 or 4 carbon atom) on aromatic ring by O, N, Si, Se, P or S replace (preferably O, S and N)." aryl " or Ar also refer to one or more hydrogen atoms on aromatic ring each independently by- R ', halogen ,-OR ', or-SR ' ,-NR ' R " ,-N=NR ' ,-N=R ' ,-NR ' R " ,-NO2,-S(O)R’,-S(O)2R’,-S(O)2OR’,-OS(O)2OR ' ,-PR ' R " ,-P (O) R ' R " ,-P (OR ') (OR ") ,-P (O) (OR ') (OR ") or-OP (O) (OR ') (OR ") is replaced, wherein R ', and R " is independent hydrogen, alkyl, alkenyl, alkynyl, miscellaneous alkyl, aryl, aralkyl, carbonyl or medicinal Salt.
" heterocycle " is a loop system, wherein 1 to 4 atom is taken by hetero atoms such as O, N, S, Se and P each independently Generation, preferably O, N and S.The definition of " heterocycle " also can refer to document The Handbook of Chemistry and Physics,78th Edition,CRC Press,Inc.,1997-1998,p.225to 226.Preferred non-aromatic class heterocycle Include, but are not limited to epoxy, aziridinyl, pyrrolidines, pyrazolidinyl, alkyl imidazole, Oxyranyle, tetrahydrofuran, two Butyl oxide link, Perarubicin, dioxanes base, dioxolanes, croak, piperazine, morpholine, pyrans, imidazoline, pyrrolinyl, pyrazoline Base, thiazolidinyl, tetrahydric thiapyran, dithiane, thiomorpholine, dihydropyran, Perarubicin, tetrahydropyridine, dihydropyridine, tetrahydrochysene Pyrimidine, dihydro thiapyran, hexamethylene imine and they are condensed with phenyl and the structure that generates.
" heteroaryl " or heteroaromatic refer to 5 to 14 yuan, and preferably 5 to 10 yuan of armaticity is miscellaneous, single, double or polycyclic, including Pyrroles, pyridine, pyrazoles, pyrimidine, pyrazine, tetrazole radical thienyl, indoles, quinoline, purine, imidazole radicals, thiophene, thiazole, benzo thiophene Azoles, furans, benzofuran, 1,2,4- triazoles, isothiazole, triazole, tetrazolium, isoquinolin, benzothiophene, isobenzofuran, pyrrole Azoles, carbazole, benzimidazole, isoxazole, pyridine nitric oxide and the ring structure that they are generated with phenyl condensation.
" alkyl ", " naphthenic base ", " alkenyl ", " alkynyl ", " aryl ", " heteroaryl ", " heterocycle " etc. also refer to corresponding " alkylene Base ", " cycloalkylidene ", " alkenylene ", " alkynylene ", " arlydene ", " inferior heteroaryl " and " sub- heterocycle " etc. remove two hydrogen atoms Group.
" aralkyl " refers to an acyclic alkanes base free radical, one of them and carbon atom, a typically end Or sp3The carbon atom of hydridization, connected hydrogen atom are substituted with aryl.Typical aryl alkyl includes, but are not limited to phenyl, 2- benzene Base -1- ethyl bases, 2- phenyl -1- vinyl, menaphthyl, 2- naphthalene -1- ethyl bases, 2- naphthalene -1- vinyl, naphthalene phenyl, 2- Naphthalene benzene -1- ethyls etc..
" heteroarylalkyl " refers to an acyclic alkanes base free radical, one of them and carbon atom, a typically end or sp3The carbon atom of hydridization, connected hydrogen atom are substituted by heteroaryl.Typical heteroarylalkyl includes, but are not limited to 2- benzo miaows Azoles methyl base, 2- furylethyls etc..
The example of " hydroxy-protective group " includes, but are not limited to methoxyl methyl ether, 2- methoxy (ethoxy) methyl ethers, oxinane Ether, benzylic ether, to methoxy-benzyl ether, trimethylsilyl ethers, triethyl group silicon ether, triisopropylsilyl ether, tert-butyldimethyl silyl Ether, trityl group silicon ether, acetonyl ester replace acetonyl ester, 2,2-Dimethylpropionic acid ester, benzoate, benzoic ether, methyl Sulphonic acid ester and p-methyl benzenesulfonic acid ester.
" leaving group " refers to the functional group that can be replaced by another group.For leaving group packet known in the art It includes, but is not limited to halogen (chlorine, bromine, iodine), mesyl, p-toluenesulfonyl, trifyl, triflate.
It is used, is defined as hereafter abbreviated with the present invention：Boc, tertbutyloxycarbonyl；BroP, three (dimethylamino of bromination Base) phosphine hexafluorophosphoric acid；CDI, carbonyl dimidazoles；DCC, dicyclohexylcarbodiimide；DCM, dichloromethane；DIAD, azo diformazan Sour diisopropyl ester；DIBAL-H, diisobutyl aluminium hydride；DIPEA, diisopropylethylamine；DEPC, pyrocarbonic acid diethyl ester；DMA, N, N- dimethylacetylamides；DMAP, p dimethylamino pyridine；DMF, n,N-Dimethylformamide；DMSO, dimethyl sulfoxide (DMSO)；DTT, Dithiothreitol (DTT)；EDC, 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides；ESI-MS, electron spray mass spectrometry； HATU, 2- (7- aoxidizes benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid esters；HOBt, I-hydroxybenzotriazole； HPLC, high performance liquid chromatography；NHS, n-hydroxysuccinimide；MMP, 4- methyl morpholine；PAB, p-aminophenyl；PBS, phosphoric acid Buffer solution (pH 7.0~7.5)；PEG, polyethylene glycol；SEC, molecular-exclusion chromatography；TCEP, trichloroethyl phosphate；TFA, trifluoro Acetic acid；THF, tetrahydrofuran；Val, valine.
" pharmaceutically " or " pharmaceutically acceptable " refer in appropriate circumstances to animals or humans apply after, will not Generate harmful, allergy or the molecular entity and composition of other adverse reactions.
" pharmaceutically acceptable solvate " or " solvate " refer to disclosed compound and combined with it One or more solvent molecule.Solvent in pharmaceutically acceptable solvate includes, but are not limited to water, isopropyl Alcohol, ethyl alcohol, methanol, DMSO, ethyl acetate, acetic acid and ethanol amine.
" pharmaceutic adjuvant " includes any carrier, diluent, auxiliary agent or excipient, and such as protection or antioxidant, filler collapse Solution agent, wetting agent, emulsifier, suspending agent, solvent, decentralized medium, coating, antibacterial and antifungal agent, etc. blend absorption delayer Deng.It is well-known in the art using these media and reagent in medicine activity component.Any conventional media or examination Agent, unless it is incompatible with active constituent, it can consider to be used in therapeutic combination.Supplementary active ingredients can also be added, and become Suitable therapeutic combination.
" pharmaceutical salts " refer to the derivative of disclosed compound, are acted on by parent compound and acid or alkali Salt.Pharmaceutically acceptable salt include conventional non-toxic salts or and parent compound, such as nontoxic inorganic or organic acid formed Quaternary ammonium salt.For example, conventional nontoxic salts include the derivative of inorganic acid, as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, Nitric acid etc.；With the salt prepared with organic acid, such as acetic acid, propionic acid, succinic acid, tartaric acid, citric acid, sulfonic acid, benzene sulfonic acid, grape Sugar, glutamic acid, benzoic acid, salicylic acid, p-methyl benzenesulfonic acid, oxalic acid, succinic acid, Malaysia organic acid, lactic acid etc..Other salt include ammonium Salt, such as trimethylamine, meglumine, epolamine salt etc. and metal salt, such as sodium, potassium, calcium, zinc and magnesium salts.
Pharmaceutical salts in this patent can be chemically synthesized from the parent for including acid or alkali with conventional.In general, This salt can by water or in organic solvent or in the mixture of two kinds of solvents, to parent compound free acid or In alkali, be added equivalent suitable alkali or acid and formed.In general, it is preferred to non-aqueous media such as ether, ethyl acetate, second Alcohol, isopropanol or acetonitrile.The inventory of suitable salt is found in Remington's Pharmaceutical Sciences, and 17th Ed., Mack Publishing Company, Easton, PA, 1985, p.1418, row refer to herein.
Novel conjugation conjugate disclosed in the present invention has used bridging junctor.Part connector and its synthesis such as Fig. 1 are extremely Shown in 10.
Bridging junctor
The synthesis of bridging junctor in this patent and preparation such as Fig. 1-9 institutes of agent-cell binding molecule conjugate Show.Bridging junctor has two elements：A) can substituted acetylene dicarbonyl compound, it is total that thioether can be formed with a pair of of thiol reaction Valence link；B) one can with the functional group of drug response, it include but not limited to disulfide, maleimide, halogen acetyl group, aldehyde, Ketone, nitrine, amine, alkoxyamine and hydrazine.Acetylene dicarbapentaborane can pass through acetylene diacid and amino, hydroxyl or sulfydryl at bridge substitution It reacts and obtains amide, ester or thiol esters.The synthesis example of bridging junctor is shown in Fig. 1,3,4,5,6,7,8 and 9.Acetylene dicarbapentaborane Bridge substitution can also obtain by acetylene and carboxylic acid halides or anhydride reaction form carbon-carbon bond.The synthesis example of these bridging junctors can See Fig. 2 and 10.
Preferably, the structure of bridging junctor compound such as (I) is shown：
Wherein acetylene dicarbonyl compound connector can be reacted with a pair of sulphur atom on cell-binding molecules.
Z1And Z2It is the same or different functional group that can be reacted with cytotoxic drug, generates disulfide bond, ehter bond, ester Key, thioether bond, thioester bond, peptide bond, hydrazone bond, urethane bond, carbonic acid ester bond, amine key (two level, three-level or level Four), imines Key, Heterocyclylalkyl, heteroaryl, alcoxyl oxime key or amido bond；
R1And R2Same or different H, the straight chained alkyl containing 1~6 carbon atom, the branch of 3 to 6 carbon atoms or Naphthenic base, straight chain, branch or cycloalkenyl group or the ester group of alkynyl or 1~6 carbon atom, ether, amide groups or polyethoxy unit (OCH2CH2)pOr poly- propoxy unit (OCH2(CH3)CH2)pUnit, wherein p are 0 to about 1000 integers or these groups Combination；
In addition, R1And R2It includes C, N, O to be, S, the chain structure of Si and P atoms is optimal to contain 0~500 atom, covalently It is connected to X1Or X2And Z1Or Z2；R1And R2In each atom combined with all possible chemical mode, such as formed alkyl, Asia Alkyl, alkenylene, alkynylene, ether, polyoxy alkyl, ester, amine, imines, polyamine, hydrazine, hydrazone, amide, urea, semicarbazides, carbonohydrazides, alkane The combination of oxygen amine, polyurethane, amino acid, polypeptide, acyl-oxygen amine, hydroxamic acid or these groups；
X1And X2It is independently selected from N (R3), O, S or CH2；R3It is H, the straight chained alkyl containing 1~6 carbon atom, 3 to 6 carbon originals The branch or cyclic alkyl of son, straight chain, branch or the ester of cricoid alkenyl or alkynyl or 1~6 carbon atom, ether, amide or poly- Ethoxy units (OCH2CH2)p, wherein p is the combination of 0 to 1000 integer or these groups.
In another embodiment, R1、R2And R3Can be respectively include C, N, O, S, the chain structure of Si and P atoms, It is covalently attached cell surface binding molecule and conjugation drug.The each atom of connector is used to form with all possible chemical mode In conjunction with, such as formed alkyl, alkylidene, alkenylene, alkynylene, ether, polyethylene glycol, ester, amine, imines, polyamine, hydrazine, hydrazone, amide, Urea, semicarbazides, carbonohydrazides, alcoxyl amine, polyurethane, amino acid, polypeptide, acetyl oxygen amine, hydroxamic acid or other structures；In addition, It should be appreciated that the atom for forming connector (L) can be saturated or unsaturated, it can be free radical, can mutually circularize At divalent ring structure, including cycloalkane, cyclic ethers, cyclammonium, arlydene, the structures such as heteroarylidene.
Functional group Z1And Z2Cytotoxic drug can be connected, can generate including disulfide bond, thioether, thiol esters, peptide, hydrazine, Ether, ester, carbamate, carbonic ester, oxime or amido bond.Such functional group includes, but are not limited to sulfydryl, disulfide group, amino, carboxylic Base, aldehyde radical, carbonyl, dimaleoyl imino, haloacetyl, diazanyl, alkoxyamino and hydroxyl.
The functional group Z that can be reacted with drug or cytotoxic molecule terminal amino group1And Z2It may be, but not limited to, N- hydroxyls Succinimide ester, p-nitrophenyl phenolic ester, dinitrobenzene phenolic ester, phenyl-pentafluoride phenolic ester；Can and the function of terminal sulfhydryl group reaction can be with It is to be also not necessarily limited to pyridyldithiol, nitropyridine disulfide group, dimaleoyl imino, halogenated acetic acids ester, carbonate chloride；Energy and end It can be also not necessarily limited to amino to hold the functional group of carbonyl or aldehyde radical reaction, alkoxyamino, hydrazine, acetyl oxygen amino；With end nitrine The function of base reaction can be also not necessarily limited to alkynyl.
In a preferred embodiment, R1、R2And R3For straight chained alkyl or polyethoxy unit containing 1 to 6 carbon atom (OCH2CH2)P, p=1~100.
Containing acetylene dicarbapentaborane class bridging junctor synthesis committed step be, acetylene dicarbapentaborane or derivatives thereof and other The condensation of Component Terminal amine (level-one or secondary amine), alcohol or mercaptan, such as shown in following (Ia)：
Wherein, the X in formula (Ia) is the X in structure formula (I)1Or X2, refer to N (R3), O, or S；R is R1And/or R2, R1、R2 And R3Define same formula (I).
Lv1 and Lv2 is identical or separate OH, F, Cl, Br, I, nitrophenol, n-hydroxysuccinimide (NHS), phenol, dinitrophenol, Pentafluorophenol, polytetrafluoroethylene phenol, difluorophenol, single fluorophenol, pentachlorophenol, trifluoromethyl sulphur Acid, imidazoles, chlorophenesic acid, tetrachlorophenol, 1- hydroxy benzo triazoles, p-methyl benzenesulfonic acid, methanesulfonic acid, 2- ethyl -5- phenyl are different Oxazole -3'- sulfonic acid, self acid anhydrides or the acid anhydrides formed with other acid anhydrides such as acetyl acid anhydride, formic anhydride；Or among polypeptide condensation reaction Body or Mitsunobu reaction intermediates；Condensation reagent includes：1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC), dicyclohexylcarbodiimide (DCC), N, N'- diisopropylcarbodiimides (DIC), 1- cyclohexyl -2- morpholine ethyls Carbodiimide tosilate (CMC or CME-CDI), carbonyl dimidazoles (CDI), TBTU (O- benzotriazole-N, N, N', N'- tetramethylureas tetrafluoro boric acid), O- benzotriazole-tetramethylurea hexafluorophosphoric acid ester (HBTU), benzotriazole -1- base oxygen (dimethylamino) the phosphorus hexafluorophosphate of base three (BOP), hexafluorophosphoric acid benzotriazole -1- bases-oxygroup tripyrrole alkyl phosphorus (PyBOP), pyrocarbonic acid diethyl ester (DEPC), N, N, N', N'- tetramethyl chloromethane amidine hexafluorophosphates, (7- aoxidizes three nitrogen of benzo to 2- Azoles)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid esters (HATU), 1- [(dimethylamine) (morpholinyl) methylene] -1 [1,2,3] three Azoles simultaneously [4,5-b] 1- pyridine -3- oxygen hexafluorophosphates (HDMA), chloro- 1, the 3- methylimidazoles hexafluorophosphates (CIP) of 2-, Chloro tripyrrole Wan Ji Phosphonium hexafluorophosphates (PyCloP), bis- (tetramethylene) Fluoro-formamides (BTFFH), N, N, N', N'- tetra- Methyl-sulphur-(1- oxo -2- pyridyl groups) thiocarbamide hexafluorophosphate, 2- (2- pyridone -1- bases) -1,1,3,3- tetramethylureas Tetrafluoroborate (TPTU), sulphur-(1- oxo -2- pyridyl groups)-N, N, N', N'- tetramethyl thiourea hexafluorophosphates, O- [(ethoxies Base carbonyl) cyano methylamine]-N, N, N', N'- tetramethyl thioureas hexafluorophosphate (HOTU), (1- cyano -2- ethyoxyl -2- oxos Ethyleneimino oxygroup) dimethylamino-morpholine-carbon hexafluorophosphate (COMU), (benzotriazole -1- bases oxygroup) two pyrroles Cough up alkane carbon hexafluorophosphate (HBPyU), N- benzyls-N '-carbodicyclo hexylimide (or load is on polymer), bihyrrolidinyl (N- succinimides oxygroup) carbon hexafluorophosphate (HSPyU), 1- (chloro- 1- pyrrolidinyls methylene) pyrrolidines hexafluorophosphoric acid Salt (PyClU), chloro- 1, the 3- methylimidazoles tetrafluoroborates (CIB) of 2-, (benzotriazole -1- bases oxygroup) two piperidines carbon six Fluorophosphate (HBPipU), 6- Chloro-Benzotriazoles -1,1,3,3- tetramethylurea tetrafluoro boric acid esters (TCTU), three (diformazan of bromination Base amino) phosphine hexafluorophosphoric acid (BroP), 1- n-propyls phosphoric anhydride (PPACA,), 2- isocyano groups ethyl morpholine (MEI), N, N, N', N'- tetramethylurea-oxygen-(N- succinimides base) hexafluorophosphate (HSTU), the bromo- 1- ethylpyridines tetrafluoro boric acids of 2- Salt (BEP), oxygen-[(ethoxy carbonyl) cyano methylamine]-N, N, N', N'- tetramethyl sulphur urine tetrafluoroborates (TOTU), 4- (4, 6- dimethoxy-triazine -2- bases) -4- methyl morpholine hydrochlorides (MMTM, DMTMM), 2- succinimidos -1,1,3,3- tetramethyls Base urea tetrafluoro boric acid ester (TSTU), N, N, N', N'- tetramethyls-O- (3,4- dihydro -4- oxos -1,2,3- phentriazine -3- bases) Urea tetrafluoroborate (TDBTU), azodicarbonyldipiperidine (ADD), bis- (4- chlorobenzyls) azodiformates (DCAD), azo Dicarboxylate (DBAD), diisopropyl azodiformate (DIAD), diethyl azodiformate (DEAD).
When X is CH2When, acetylene dicarbapentaborane is keyed other functional groups by C-C on bridging junctor, synthesizes key step at this time Suddenly it is two (trimethyl silicon substrate) acetylene, two metal salt of acetylene dibrominated magnesium (Grignard Reagent), acetylene dilithium salt or other acetylene, and The reaction of carboxylic acid halides or acid anhydrides, it is illustrated that such as (Ib), (Ic), (Id), (Ie), (If), (Ig) and (Ih)：
Here M is Na, K, Li, Cu, CuLi, Sn, Ti, Ca, Mg or Zn.
The specific example of bridging junctor synthesis is as Figure 1-10 shows.Usual acetylene dicarbapentaborane connector all include one can be with The functional group reacted with small-molecule drug on conjugate.
Cell-binding molecules-drug conjugates
The conjugate of the present invention can be indicated with formula below：
Wherein Cb is cell binding agent, and L is connector, and Drug1 and Drug2 are Medicine small molecules, and n is one 1 to 20 Integer, and 2 element sulphur bridging Cb are covalently attached 2 or more drug molecules in L, each bridging junctor L.
This bridging junctor L may be made of one or more connector components.Typically connector component includes： 6- maleimidohexanoic acids (" MC "), 3- maleimidoproprionic acids (" MP "), valine-citrulline (" val-cit " or " Vc "), alanine-phenylalanine (" ala-phe " or " af "), to aminobenzyloxycarbonyl (" PAB "), the thio valeric acids of 4- (" SPP "), 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acids (" MCC "), (4- acetyl group) aminobenzoic acid (" SIAB "), 4- Thiobutyric acids (SPDB), the thio -2- weight ratios butyric acid (2-Sulfo-SPDB) of 4-, one or more ethyoxyl - CH2CH2O- units (" EO " or " PEO "), further include the connector that other are well known in the art, and part is listed herein.
Including the example of the connector of these components has：
(including 6- maleimidohexanoic acid MC)
(including 3- maleimidoproprionic acid MP)
(comprising to aminobenzyloxycarbonyl PAB)
(include ethyl Maleimide ME)
(including valine-citrulline)
(including 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid MCC)
(including (4- acetyl group) aminobenzoic acid)
(including the thio -2- weight ratios butyric acid of 4-, the thio-SPDB of 2-)
Such as public formula (II) of the structure of preferred conjugate：
Wherein：
Cb is cell binding agent, and optimal is antibody；
Drug1And Drug2For same or different cytotoxic agent, they by bridging junctor with alkyl, alkylidene, Alkenylene, alkynylene, ether, poly-alkoxyl, ester, amine, imines, polyamine, hydrazine, hydrazone, amide, urea, semicarbazides, carbonohydrazides, alkoxy It is amine, carbamate, amino acid, peptide, acyl-oxygen amine, hydroxamic acid, disulfide bond, thioether, thioesters, carbamate, carbonic ester, miscellaneous Ring, miscellaneous alkyl, heteroaryl or alcoxyl oxime key and combinations thereof are linked with cell binding agent；
N is 1~20；R1,R2,X1And X2Definition with public formula (I).
See below more detailed description, and Drug1 and Drug2 can be any small-molecule drugs, including but not limited to, Tubulysin, calicheamicin, the auspicious statin of Australia, maytansine, CC-1065 analogs, morpholino, adriamycin, taxanes, Cryptophycins, Epothilones, Benzodiazepine series dimer is (for example, pyrroles's Benzodiazepine (PBD) or support horse are mould Element, indoles Benzodiazepine, imidazoles Benzodiazepine Huo oxazole Benzodiazepines dimer).
With synthesis of conjugate conjugate, the disulfide bond on cell binding agent is reduced first, generates a pair of of free sulfhydryl group, then It is reacted in the water phase of pH 5-9 with the bridging junctor in public formula (I), wherein the energy and water of 0-30% can be added or be added without Miscible organic solvent, such as：DMAC N,N' dimethyl acetamide, N,N-dimethylformamide, ethyl alcohol, methanol, acetone, acetonitrile, tetrahydrochysene Furans, isopropanol, dioxane, propylene glycol or ethylene glycol, to introduce Z1And Z2Reaction active groups, this reactive group can To be disulfide bond, dimaleoyl imino, halogen acetyl group, azide, 1- alkynes, ketone, aldehyde, alkoxy amino or hydrazides.Then, carefully The reactive group of cytotoxic molecule is reacted with the corresponding cell binding agent being modified.For example, the cell connected by disulfide bond The synthesis of bonding agent-drug conjugates is the disulfide bond in the cell binding agent by modification and the drug containing free sulfhydryl groups Between disulfide bond exchange to realize；By thioether bond combine cell combination-drug conjugates synthesis, be pass through through The cell binding agent and drug response containing free sulfhydryl groups of dimaleoyl imino or the modification of halogen acetyl group or ethyl sulfone is realized；It closes The conjugate of sour unstable hydrazone is included at molecule, can by a connector hydrazides group and carbonyl reaction realize to have Body method also be it is well known in the art (refer to P.Hamann et al., Hinman, L.M., et al, Cancer Res.1993, 53,3336-334；B.Laguzza et al.,J.Med.Chem.,1959,32；548-555；P.Trail et al., Cancer Res.,1997,57；100-105)；The conjugate of synthesis group containing triazole can pass through the 1- alkynes on a connector (dipole-diople interaction) is reacted to realize (Lutz, J-F.et al, Adv.Drug with click chemistry with the drug containing azido group Del.Rev.2008,60,958–970；Sletten,E.M.et al Acc.Chem.Research2011,44,666–676).
Alternatively, Medicine small molecule can with cell binding agent be coupled, containing reactive functionality, such as structural formula (III) cell-binding molecules modified shown in-connector structural response.For example, the drug comprising sulfydryl can and formula (III) maleimide, halogen acetyl group on connector or ethylsulfonyl, react in the buffer solution of pH 5.5-9, generate The conjugation conjugate of thioether bond link.Drug containing sulfydryl can with the two sulphur segment of pyridine in public formula (III) on connector into Row disulfide bond exchanges, and generates the conjugation conjugate of disulfide bond link.Drug containing hydroxyl or sulfydryl can connect with public formula (III) Halogen on halogen on junctor, especially alphahalogenated carboxylic acids, under the conditions of weak base, such as pH value 8.0-9.5, be obtained by the reaction ether or The conjugation conjugate of thioether bond link.The drug of hydroxyl, can also be under the action of the condensing agent of EDC or DCC and formula (I) carboxyl on connector is condensed into ester.Bridging junctor through drug modification is coupled with cell-binding molecules again.Contain The drug of amino can on connector in public formula (III) NHS, imidazoles, nitrophenol, N- hydroxysuccinimides, phenol, Dinitrophenol, Pentafluorophenol, 2,3,5,6- polytetrafluoroethylene phenols, difluorophenol, single fluorophenol, pentachlorophenol, trifluoromethayl sulfonic acid, Chlorophenesic acid, tetrachlorophenol, I-hydroxybenzotriazole, toluenesulfonic acid, methanesulfonic acid, 2- ethyl -5- phenyl isoxazole -3'- sulfonic acid Carboxylate reaction, obtain amido bond link conjugation conjugate.
Conjugate can be purified by the biochemical method of standard, such as be carried out with Sephadex G25 or Sephacryl S300 Gel filtration, adsorption chromatography, ion exchange or dialysis.In certain situations, such as small molecular cell bonding agent, (such as folic acid, melanocyte is thin Born of the same parents' stimulin, EGF etc.) combined with small-molecule drug after, the chromatographies such as HPLC, middle compression leg chromatography or ion-exchange chromatography can be passed through Method purifies.
Cell binding agent/molecule of modification
Preferably, such as public formula (III) of the structure for the cell binding agent modified through connector in the present invention：
Wherein Cb, Z1,Z2,n,R1,R2,X1, and X2It is identical as defined in public formula (I) and (II).
In a preferred embodiment, Z1And Z2Be disulfide group, dimaleoyl imino, halogen acetyl group, alkoxyamine, nitrine, ketone, Aldehyde, hydrazine, N-hydroxy-succinamide ester or phenol, dinitrophenol dinitrophenolate, Pentafluorophenol, polytetrafluoroethylene phenol, difluorophenol, single fluorobenzene Phenol, pentachlorophenol, trifluoromethayl sulfonic acid, imidazoles, chlorophenesic acid, tetrachlorophenol, I-hydroxybenzotriazole, toluenesulfonic acid, methylsulphur The carboxylate of acid, 2- ethyl -5- phenyl isoxazole -3'- sulfonic acid.Z1And Z2Can be reacted into cytotoxic drug thioether, hydrazone, Amide, alkane oxime, carbamate, ester, ether or disulfide bond.The cell binding agent of modification can pass through cell binding agent and formula (I) the bridging junctor of the description in carries out reaction and prepares as described in public formula (II) above.
In order to realize efficiently connecting for a pair of of the sulfydryl to dissociate in alkynyl and cell binding agent, especially antibody on bridging junctor It connects, needs that in the reaction system or after the completion of reaction, a small amount of organic solvent is added so that the structure in formula (III) is good in water Good dissolving.In modified cells bonding agent, the crosslinking agent (bridging connects) in public formula (I) is dissolved in first can be miscible with water In polar organic solvent, such as：Various alcohol such as methanol, ethyl alcohol and propyl alcohol, acetone, acetonitrile, tetrahydrofuran (THF), Isosorbide-5-Nitrae-dioxy Six rings, dimethylformamide (DMF), dimethylacetylamide (DMA) or dimethyl sulfoxide (DMSO) (DMSO)；Solution concentration is slightly higher, such as 1- 500mM.Simultaneously by cell binding agent, if antibody is dissolved in pH 5~9.5 with the concentration of 1~35mg/ml, best 6~8.5 it is slow In fliud flushing, reacted with the TCEP of 1~20 equivalent or DTT 20 minutes to 12 hours.After reduction, removed with SEC chromatographies DTT.TCEP can be directly entered in next step with SEC chromatographies or do not purify.In addition, with TCEP reduction antibody or its He can carry out at cell binding agent in the presence of public formula (I) bridging junctor, at this time cell binding agent crosslinking conjugation coupling just and TCEP reduction is synchronous to be carried out.
The reaction of modified cells bonding agent generally carries out in pH6 to 9, preferably 6.5 to 7.5 buffer solution, can be this The buffer salt system of any not nucleophilicity within the scope of pH.Typical buffer solution includes phosphate, triethanolamine hydrochloride, HEPES and MOPS buffer solutions can include other ingredients, such as cyclodextrin, sucrose and salt, such as sodium chloride and potassium chloride.By formula (I) the bridging junctor solution in is added in the cell combination agent solution of reduction, and at 4 to 45 DEG C, is preferably incubated at room temperature, and By measuring solution in 254nm or other suitable wavelength absorbances, to monitor the process of reaction.After completion of the reaction, may be used Conventionally to purify the cell binding agent of modification, such as use gel permeation chromatography or adsorption charomatography.
Nitropyridine thioketones, di nitryl pyridine dithione, pyrithione, the carboxamide pyridine two generated by measuring reaction The UV absorption of thioketones and diformamide pyridine dithione group, it may be determined that the degree that cell binding agent is modified.If Conjugate does not have chromophoric group, can use LC-MS or preferred UPLC-QTOF mass spectrums and capillary electrophoresis interfaced with mass spectrometry (CEMS) determination is analyzed.Different types of functional group can be contained on bridging junctor in the present invention, it can be anti-with various drugs It answers, especially the cytotoxic drug with suitable functional group.For example, the cell knot containing amino or hydroxyl substituent after modification Mixture can with the drug response containing n-hydroxysuccinimide (NHS) ester, after modification the cell binding agent containing sulfydryl can with containing horse Carry out the drug response of imide or halogenacetyl group.In addition, the cell binding agent containing carbonyl (aldehydes or ketones base) can be with after modification Drug response containing hydrazides or alkoxyamine.Those skilled in the art can easily live according to the reaction of wherein functional group Property determines what kind of connector used.
The cytotoxic drug of modification
It is preferably (IV) with the cytotoxic drug structure that the bridging junctor in the present invention is modified：
Wherein Drug1,Drug2,Z1,Z2,n,R1,R2,X1, and X2Definition it is identical as public formula (I) and (II).
The drug (IV) of modification, acetylene dicarbapentaborane therein is obtained by the reaction with drug molecule with the connector in public formula (I) Functional group can react with a pair of of sulfydryl on cell binding agent.Acetylene dicarbapentaborane is obtained by the condensation reaction of acetylene, Method has been described in reaction equation (Ia), (Ib), (Ic), (Id), (Ie), (If) in (Ig) and (Ih).
But to the drug containing sulfydryl, or pass through bridging junctor thioether, thioesters or disulfide bond and combination cell bonding agent chain The drug connect, preferably Drug1 or Drug2 R is connected to by thioether, thioesters or disulfide bond first1Or R2, generation R1-Drug1Or R2-Drug2Segment is re-incorporated on acetylene dicarbapentaborane, forms the medicine modified such as the bridging junctor of structure formula (IV) Object.
For example, the drug containing sulfydryl can in the buffer solution of neutral pH with R1Or R2On maleimide reaction, formed with The R that thioether bond combines1-Drug1Or R2-Drug2, then it is condensed, is formed as included thioether bond in public formula (IV) with acetylene dicarbapentaborane Modified medicaments.The drug of hydroxyl can be with the R containing halogen, p-methyl benzenesulfonic acid or methanesulfonates under the conditions of weak base1Or R2 Reaction, obtains with ehter bond combination R1-Drug1Or R2-Drug2, then it is condensed, is formed as wrapped in public formula (IV) with acetylene dicarbapentaborane The modified medicaments of sulfur-bearing ehter bond.Drug containing hydroxyl can also in structure (I) carry carboxyl connector, dehydrating agent such as Under EDC or DCC effects, it is condensed to yield such as the modified medicaments containing ester bond in public formula (IV).Drug containing sulfydryl can be with R1Or R2On Maleimide, ethylene sulfuryl or haloacetyl reaction, generate the R combined with thioether bond1-Drug1Or R2-Drug2, then It is condensed with acetylene dicarbapentaborane, forms the modified medicaments as included thioether bond in public formula (IV).Similarly, amino-containing drug also may be used To be condensed with the carboxyl on bridging junctor (I), the modified medicaments such as amide bond in public formula (IV) are obtained.The drug of modification Can be purified with the method for standard, such as silica gel or alumina column chromatography, recrystallize, prepare thin-layer chromatography, ion-exchange chromatography or High performance liquid chromatography.
Cell binding agent
Cell binding agent in the present invention, including can be currently known in conjugate and the cell binding agent that is modified Or any species for being shortly disclosed, can be combined with therapeutic potential or by the cell fragment that biology is modified, Compound or reaction.
Cell binding agent include but are not limited to macromolecular weight protein, such as complete antibody (polyclonal antibody, Dan Ke Grand antibody, dimer, polymer, multi-specificity antibody, such as bispecific antibody)；Single-chain antibody；Antibody fragment such as Fab, Fab',F(ab')2,Fv(Parham,J.Immunol.1983,131,2895-2902)；The segment generated by Fab expression libraries resists Idiotype (anti-Id) antibody；CDR；Bivalent antibody；Trivalent antibodies and any of the above described of immunologic specificity combination cancer cell antigen resist The epitope binding fragments of body；Viral antigen；Microbial antigen or the protein generated by immune system, can identify, in conjunction with spy Determine antigen or there is desired biological activity (Miller et al J.of Immunology 2003,170,4854- 4861)；Interferon (such as I, II, type III)；Polypeptide；Lymphokine such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, GM- CSF, interferon-γ (IFN-γ)；Hormone such as insulin, TRH (thyrotropin-releasing hormone (TRH)), MSH (melanophores Hormone), steroid hormone such as androgen and estrogen；Growth factor and colony stimulating factor, such as epidermal growth factor (EGF), Granulocyte macrophage colony stimulating factor (GM-CSF), transforming growth factor (TGF) such as TGFα, TGF β, insulin and pancreas islet Plain like growth factor (IGF-I, IGF-II), G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 1984, 5,155-158)；Vaccinia growth factor (VGF)；Fibroblast growth factor (FGF)；The protein of small-molecular-weight；Polypeptide；Peptide And peptide hormone, such as bombesin, gastrin, gastrin releasing peptide；Platelet-derived growth factor；Interleukins and cell because Son, for example, interleukin 2 (IL-2), interleukin-6 (IL-6), LIF ELISA, granular leukocyte macrophage collection G-CSF (GM-CSF)；Vitamin, such as folic acid；Apoprotein and glycoprotein, as transferrins (O'Keefe et al, J.Biol.Chem.1985 260932-937)；Carbohydrate-binding protein or lipoprotein, such as agglutinin；Cytotrophy transmits molecule；It is small Molecule inhibitor, it is non-peptide such as prostate-specific membrane antigen (PSMA) inhibitor and small molecule tyrosine kinase inhibitors (TKI) Or any other cell-binding molecules or substance, as bioactive polymer (Dhar, et al, Proc.Natl.Acad.Sci.2008,105,17356-61), bioactivity dendritic macromole (Lee, et al, Nat.Biotechnol.2005,23,1517-26；Almutairi,et al；Proc.Natl.Acad.Sci.2009,106, 685-90), nano-particle (Liong, et al, ACS Nano, 2008,19,1309-12；Medarova,et al, Nat.Med.2007,13,372-7；Javier, et al, Bioconjugate Chem.2008,19,1309-12), liposome (Medinai, et al, Curr.Phar.Des.2004,10,2981-9) and virus coat (Flenniken, et al, Viruses Nanotechnol.2009,327,71-93)。
In general, if monoclonal antibody appropriate is available, preferred monoclonal antibody is as cell surface knot Mixture.Antibody can be mouse source, people source, humanization, be fitted into or be derived from other species.
Production for the antibody in the present invention includes in vivo or in vitro method or combinations thereof.Produce Anti-TNF-α receptor peptide The method of antibody is well known in the art, such as United States Patent (USP) 4, described in 493,795.Typically by the way that myeloma is thin Born of the same parents are merged with the splenocyte of mouse being immunized with required antigen, come prepare monoclonal antibody (G.；Milstein, C.Nature 1975,256:495-497).Detailed process " Antibodies--A Laboratory Manual, Have in Harlow and Lane, eds., Cold Spring Harbor Laboratory Press, New York (1988) " Description, it is incorporated herein by reference.Specifically, target antigen, such as complete target cell can be used, the antigen detached from target cell is complete Mouse, rat, hamster or any other mammal is immunized in whole virus, the totivirus and virus protein of inactivation.Usually make Splenocyte is merged with myeloma cell with polyethylene glycol (PEG) 6000.By to HAT (hypoxanthine-aminopterin-thymus gland Pyrimidine) sensibility screen fused cell.By on the ability of their immune response specific receptors or inhibition target cell The ability of receptor active, it may be determined that implement the hybridoma of the monoclonal antibody of the present invention.
Production for the monoclonal antibody in the present invention carries out in Monoclonal hybridomas culture, wherein including nutrition Culture medium and the hybridoma that the antibody molecule with suitable antigen specificity can be secreted.Culture keeps filling under suitable conditions A period of time of foot, so that antibody molecule is secreted into culture medium by hybridoma.Then the culture medium containing antibody is collected.Using logical The further isolated antibody of well known technology is crossed, such as a-protein affinity chromatography, anion, cation, hydrophobic or volume exclusion Chromatography (especially by a-protein affinity chromatography and size exclusion chromatography), centrifugation, differential solubility or any other is pure Change the standard technique of protein.
The culture medium that can be used for preparing these compositions is well known in the present art, and available commercial, also includes Synthetic media.The example of the culture medium of one synthesis is the minimum dulbecco minimum essential medium Dulbecco (DMEM of Dulbecco's；Dulbecco et Al., Virol.1959,8,396) it is supplemented with 4.5g/ml glucose, 0-20mM glutamine, 0-20% fetal calf serums are several The heavy metals such as Cu, Mn, Fe or the Zn of ppm or/and such as PULLRONIC F68 block copolymerization of heavy metallic salt and antifoaming agent Object.
In addition, the cell line of production antibody can also be obtained by the technology other than fusion, such as tumor formation DNA is converted To bone-marrow-derived lymphocyte or tumor formation virus transfection, such as epstein-Barr virus (EBV, also referred to as 4 (HHV- of nerpes vinrus hominis 4)) or kaposi sarcoma-associate herpesvirus (KSHV), United States Patent (USP) 4,341,761 is seen；4,399,121；4,427,783； 4,444,887；4,451,570；4,466,917；4,472,500；4,491,632；4,493,890.Monoclonal antibody can also It is prepared by anti-receptor peptide containing terminal carboxyl group or peptide, these are all as it is known in the art, can refer to document Niman et al.,Proc.Natl.Acad.Sci.USA,1983,80:4949-4953；Geysen et al., Proc.Natl.Acad.Sci.USA,1985,82:178-182；Lei et al.Biochemistry 1995,34(20): 6675-6688.In general, anti-receptor peptide or peptide analogues are former as anti-receptor peptide monoclonal antibody immunity is generated, can individually make It is connect with or with immunogenic carrier.
Monoclonal antibody as binding molecule in the present invention can also be obtained by other techniques known in the art.It is special The method for not manufacturing complete human antibody usefully.A kind of method is phage display technology, it uses the side of affine enrichment Formula, the human antibody that can be used for selecting to be combined with antigentic specificity.Display technique of bacteriophage also has in the literature to be retouched in detail State, the construction and screening of phage display library in this field be also it is well known that can refer to document Dente et al, Gene.1994,148(1):7-13；Little et al,Biotechnol Adv.1994,12(3):539-55；Clackson et al.,Nature 1991,352:264-628；Huse et al.,Science 1989,246:1275-1281.
By generating monoclonal antibody with the inhuman hybridoma merged such as mouse cell, can be humanized to avoid generation Human anti-mouse antibody.Common antibody humanization's method is complementary determining region implantation technique, these methods are also by detail Description, such as United States Patent (USP) 5,859,205 and 6,797,492；Liu et al,Immunol Rev.2008,222:9-27； Almagro et al,Front Biosci.2008,13:1619-33；Lazar et al,MolImmunol.2007,44(8): 1986-98；Li et al,Proc.Natl.Acad.Sci.U S A.2006,103(10):3557-62 is incorporated by reference herein. Complete human antibody can also be by carrying transgenic mice, the rabbit of most human globulin's heavy and light chain with immunogen immune It is prepared by son, monkey or other mammals.The example of these mouse has：Xenomouse (Abgenix/Amgen), HuMAb- Mouse (Medarex/BMS) and VelociMouse (Regeneron), with reference to United States Patent (USP) 6,596,541,6,207,418,6, 150,584,6,111,166,6,075,181,5,922,545,5,661,016,5,545,80 6,5,436,149 and 5,569, 825.When for human treatment, the Variable Area of mouse and the constant region domains of people can also be fused, and become " chimeric antibody ", it Immunogenicity on mankind is substantially less than mouse monoclonal (Kipriyanov et al, MolBiotechnol.2004,26: 39-60；Houdebine,CurrOpinBiotechnol.2002,13:625-9).In addition, the fixed point in Antibody variable domains lures Change can cause antibody to have higher compatibility and specificity (Brannigan et al, Nat Rev Mol Cell Biol.2002,3:964-70；Adams et al,J Immunol Methods.1999,231:249-60), antibody constant region The change in domain can improve it and mediate the effector function combined with cytotoxicity.
The antibodies immunospecific of malignant cell antigen can also be obtained or be given birth to by any known method from commercial channels Production, such as chemical synthesis or recombination and expression techniques.There is the nucleotide sequence of the antibody of immunologic specificity to malignant cell antigen Coding can be commercially-available, such as from GenBank databases or similar database, Literature publication object obtains, or passes through conventional gram Grand and sequencing obtains.
Other than antibody, on target cell epitope or corresponding acceptor interaction (in conjunction with, block, targeting or its His type action) a kind of peptide or protein matter can also be used as binding molecule.These peptide or protein matter may be any random Peptide or protein matter, they have affinity to epitope or corresponding receptor, have to be not necessarily immunoglobulin class members.These Peptide can show that the technology of antibody separates (Szardenings, J Recept Signal by similar bacteriophage Transduct Res.2003；23(4):307-49).The peptide obtained from random peptide library can be similar with antibody and antibody fragment It is used.Peptide or protein matter binding molecule can be coupled or link to macromolecular or other substances, including but not limited to albumin, Polymer, liposome, nano-particle, dendrimer, so long as link can retain peptide or protein matter antigen binding it is special Property.
For treating cancer, autoimmune disease and/or communicable disease conjugate and drug molecule pass through The example of the antibody of the bridging junctor link of this patent includes, but are not limited to 3F8 (anti-GD2), Ah bar's monoclonal antibody (anti-CA-125), Ah Former times monoclonal antibody (anti-CD41 (beta 2 integrin alpha-IIb), adalimumab (anti-tnf-alpha), Adecatumumab (anti-EpCAM, CD326), Afelimomab (anti-tnf-alpha), Afutuzumab (anti-CD20), Alacizumab monoclonal antibodies (anti-vegf R2), ALD518 (anti-IL- 6), Alemtuzumab (Campath, MabCampath, anti-CD52), Altumomab (anti-CEA), Anatumomab (anti-TAG- 72), Anrukinzumab (IMA-638, anti-IL-13), Apolizumab (anti-HLA-DR), Archie monoclonal antibody (anti-CEA), Ah Fill in pearl monoclonal antibody (anti-L- selects PROTEIN C D62L), Atlizumab (tocilizumab, Actemra, RoActemra, anti-IL-6 Receptor), Atorolimumab (the anti-Rhesus factors), Bapineuzumab (anti-beta amyloid protein), Basiliximab (Simulect, anti-CD25 (the α chains of IL-2 receptors)), Bavituximab (anti-phosphatidylserine), Bectumomab (LymphoScan, anti-CD22), Baily monoclonal antibody (Benlysta, LymphoStat-B, anti-BAFF), Benralizumab are (anti- CD125), Bertilimumab (anti-CCL11 (eotaxin-1)), Besilesomab (Scintimun, anti-CEA related antigens), Bevacizumab (Avastin, anti-vegf-A), Biciromab (FibriScint, antifibrin II β chain), Bivatuzumab (anti-CD44v6), Blinatumomab (BiTE, anti-CD19), Brentuximab (cAC10, anti-CD30TNRSF8), Briakinumab (anti-IL-12, IL-23), Canakinumab (Ilaris, anti-IL-1), (C242 resists Cantuzumab CanAg), Capromab, Catumaxomab (Removab, anti-EpCAM, AntiCD3 McAb), CC49 (anti-TAG-72), Cedelizumab (anti-CD4), Certolizumab monoclonal antibodies (Cimzia anti-tnf-alpha), Cetuximab (Erbitux, IMC-C225, anti-EGFR), Citatuzumab bogatox (anti-EpCAM), Cixutumumab (anti-IGF-1), Clenoliximab (anti-CD4), Clivatuzu-mab (anti-MUC1), Conatumumab (anti-TRAIL-R 2), CR6261 (anti influenza A hemagglutinin), Dacetuzumab (anti-CD40), Daclizumab (Zenapax, anti-CD25 (IL-2 receptor alpha chains)), Daratumumab are (anti- CD38 (ring ADP ribose hydrolase), Denosumab (Prolia, anti-RANKL), Detumomab (anti-B lymphoma cells), Dorlimomab, Dorlixizumab, Ecromeximab (anti-GD3 gangliosides), and Eculizumab (Soliris, it is anti- C5), Edobacomab (antiendotoxin), Edrecolomab (Panorex, MAb17-1A, anti-EpCAM), Efalizumab (Raptiva, anti-LFA-1 (CD11a)), Efungumab (Mycograb, anti-Hsp90), Elotuzumab (anti-SLAMF7), Elsilimomab (anti-IL-6), Enlimomab monoclonal antibodies (anti-ICAM-1 (CD54)), Epitumomab (anti-episialin), according to His pearl monoclonal antibody (anti-CD22), Erlizumab (anti-ITGB2 (CD18)), Ertumaxomab (Rexomun, anti-HER2/neu, CD3), Yi Tala monoclonal antibodies (Abegrin, anti-alpha 2 integrin α v β 3), Exbivirumab (anti-hepatitis B surface antigen), Fanolesomab (NeutroSpec, anti-CD15), Faralimomab (anti-interferon receptor), Farletuzumab (anti-folic acid Receptor 1), Felvizumab (anti respiratory syncytial virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF- 1 receptor), Fontolizumab (anti-IFN-γ), Foravirumab (anti-rabies virus glycoprotein), Fresolimumab (anti-TGF-beta), Galiximab (anti-CD80), Gantenerumab (anti-beta amyloid), Gavilimomab (anti-CD147 (basigin)), Gemtuzumab (anti-CD 33), Girentuximab (anti-carbonic anhydrase 9), Glembatumumab (CR011, Anti- GPNMB), Golimumab (Simponi, anti-TNF-α), Gomiliximab (anti-CD23 (IgE receptors)), Ibalizumab (anti-CD4), Ibritumomab (anti-CD20), Igovomab (Indimacis-125, anti-CA-125), Imciromab (Myoscint, anti-cardiac myosin), Infliximab (Remicade, anti-tnf-alpha), Intetumumab (anti-CD51), Inolimomab (anti-CD25 (IL-2 receptor alpha chains), Yi Zhu monoclonal antibodies (anti-CD22), Ipilimumab (anti-CD152), Iratumumab (AntiCD3 McAb 0 (TNFRSF8)), Keliximab (anti-CD4), Labetuzumab (CEA-Cide, anti-CEA), Lebrikizumab (anti-il-13), Lemalesomab (anti-NCA-90 (G-Ag)), Lerdelimumab (anti-TGF β 2), Lexatumumab (anti-TRAIL-R 2), Libivirumab (anti-hepatitis B surface antigen), Lintuzumab (anti-CD 33), Shandong The wooden monoclonal antibody (anti-CD40) of rice, Rumi monoclonal antibody (anti-CD23 (IgE receptors), Mapatumumab (anti-TRAIL-R 1), Ma Ximo monoclonal antibodies (anti-T-cell receptors), matuzumab (anti-EGFR), Mepolizumab (Bosatria, anti-IL-5), Metelimumab are (anti- TGF β 1), Milatuzumab (anti-CD74), Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3 neuromeres Glycosides fat), Morolimumab (anti-rh factor), Motavizumab (Numax, anti respiratory syncytial virus), Muromonab-CD3 (Orthoclone OKT3, AntiCD3 McAb), Nacolomab (anti-C242), Naptumomab (anti-5T4), that His pearl monoclonal antibody (Tysabri, anti-alpha 2 integrin α 4), how Ba Dankang (anti-endotoxin), Necitumumab (anti-EGFR), Nerelimomab (anti-TNF-α), Nimotuzumab (Theracim, Theraloc, anti-EGFR), Nofetumomab, Ocrelizumab (anti-CD 20), the wooden monoclonal antibody (Afolimomab, anti-LFA-1 (CD11a)) of profit difficult to understand, Ofatumumab (Arzerra, anti-CD20), Olaratumab (anti-PDGF-R α), Omalizumab (area Xolair, anti-IgE Fc) Oportuzumab (anti-EpCAM), Oregovomab (OvaRex, anti-CA-125), Otelixizumab (AntiCD3 McAb), Pagibaximab (anti lipoteichoic acid), Palivizumab (Synagis, Abbosynagis, anti respiratory syncytial virus), pa Buddhist nun's monoclonal antibody (Vectibix, ABX-EGF, anti-EGFR), Panobacumab (resisting pseudomonas aeruginosa (Pseudomonas Aeruginosa)), pa examines pearl monoclonal antibody (anti-IL-4), Pemtumomab (Theragyn, anti-MUC1), Pertuzumab (Omnitarg, 2C4, anti-HER2/neu), Pexelizumab (anti-c5), Pintumomab (anti-gland cancer antigen), Priliximab (anti-CD4), Pritumumab (anti-vimentin), and PRO140 (anti-CCR5) Racotumomab (1E10, it is anti- NeuGc ALPHA2-3Gal (NeuGc, NGNA)-Ganglioside GM3)), Rafivirumab (anti-rabies virus glycoprotein), Ramucirumab (anti-vegf R2), Ranibizumab (Lucentis, anti-vegf-A), Raxibacumab (anti-anthrax toxin, Protective antigens), Regavirumab (anti-cytomegalovirus Glycoprotein B), Reslizumab (anti-IL-5), Rilotumumab (anti-HGF), Rituximab (MabThera, Rituxanmab, anti-CD 20), Robatumumab (anti-IGF-1 receptors), Rontalizumab (anti-IFN-α), Rovelizumab (LeukAr-rest, anti-CD11, CD18), Ruplizumab (Antova, Anti- CD154 (CD40L)), Satumomab (anti-TAG-72), Sevirumab (anti-cytomegalovirus), Sibrotuzumab are (anti- FAP), Western method wood monoclonal antibody (anti-IFN-α), Siltuximab (anti-IL-6), Siplizumab (anti-CD2), Smart MI95 are (anti- CD33), Solanezumab (anti-beta amyloid albumen), Sonepcizumab (anti-sphingosine-1-phosphate), Sontuzumab is (anti- Episialin), (LeukoScan, (granulocyte is anti-by anti-NCA-90 by Stamulumab (anti-myostatin), Sulesomab It is former)), Tacatuzumab (anti alpha alpha-fetoprotein), Tadocizumab (anti-alpha 2 integrin α IIb β 3), Talizumab (anti-IgE), Tanezumab (anti-NGF), Taplitumomab (anti-CD19), Tefibazumab (Aurexis, (anti-coagulation factors A)), Telimomab, Tenatumomab (anti-tenascin C), Teneliximab (anti-CD40), Teplizumab (AntiCD3 McAb), TGN1412 (anti-CD28), Ticilimumab (Tremelimumab, anti-CTLA-4), Tigatuzumab (anti-TRAIL-R 2), TNX-650 (anti-il-13), Tocilizumab (Atlizumab, Actemra, RoActemra, IL-6 receptor), Toralizumab (anti-CD154 (CD40L)), Tositumomab (anti-CD20), Herceptin (Trastuzumab, anti-HER2/ Neu), Tremelimumab (anti-CTLA-4), Tucotuzumab celmoleukin (anti-EpCAM), Tuvirumab are (anti-B-mode Hepatitis virus), Urtoxazumab (anti-Escherichia coli), Ustekinumab (Stelara, anti-IL-12, IL-23), Vapaliximab (anti-AOC3 (VAP-1)), tie up many pearls monoclonal antibody (4 β 7 of anti-alpha 2 integrin α), dimension trastuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1)), Visilizumab (Nuvion, AntiCD3 McAb), Vitaxin (anti-angiogenic integrins Avb3), Volociximab (5 β 1 of anti-alpha 2 integrin α), Votumumab (HumaSPECT, antitumor antigens CTAA16.88), Zalutumumab (HuMax-EGFR, Zanolimumab (HuMax-CD4, anti-CD4), Ziralimumab (anti-CD14s 7 (basigin)), Zolimomab (anti-CD5), EtanerceptAlefacept AbataceptRilonacept (Arcalyst), 14F7 (anti-IRP-2 (Fe regulatory protein 2)), 14G2a are (anti- GD2 gangliosides are derived from Nat.Cancer Inst., treat melanoma and solid tumor), (anti-PSMA is derived from Weill to J591 Prostate cancer is treated by Cornell medical colleges), 225.28S (anti-HMW-MAA (high molecular weight melanoma associated antigen), Sorin Radiofarmaci SRL (are derived from Milan, ITA, treat melanoma), and (anti-CEACAM3, CGM1 are derived from Nat to COL-1 Cancer Inst. treatment colorectal cancers and gastric cancer), CYT-356 (Treat prostate cancer), HNK20 (OraVax Inc. respiratory syncytial virus infection is treated), ImmuRAIT (is derived from Immunomedics, treat NHL), Lym-1 (anti-HLA- DR10, Peregrine Pharm), ((tumor necrosis factor, TNFA, TNF-α, TNFSF2 are derived from anti-TNF MAK-195F Abbott/Knoll, treatment pyemia toxic shock), MEDI-500 (T10B9, AntiCD3 McAb, TR α β (T cell receptor α/β), source In MedImmune Inc, for graft versus host disease disease), RING SCAN (anti-TAG 72 (tumor-associated glycoprotein 72), Derived from Neoprobe Corp., it to be used for breast cancer, colon cancer and the carcinoma of the rectum), Avicidin (anti-EPCAM (Epithelial Cell Adhesions point Son)), anti-TACSTD1 (tumour correlation Ca2+ oscillations tranducin 11), anti-GA733-2 (stomach and intestine tumor GAP-associated protein GAP 2), anti-EGP-2 (on Endoglin 2), anti-KSA, KS1/4 antigens, M4S, tumour antigen 17-1A, CD326 (NeoRx companies are derived from, colon cancer is treated, Oophoroma, prostate cancer and NHL), LymphoCide (is derived from Immunomedics), and Smart ID10 (are derived from Protein Design Labs), Oncolym (is derived from Techniclone Inc), and Allomune (is derived from BioTransplant), anti-vegf (being derived from Genentech)；CEAcide (be derived from Immunomedics), IMC-1C11 (being derived from ImClone Systems) and Cetuximab (is derived from ImClone).
Other can be used as the antibody of cell-binding molecules/ligand, including but not limited to, the antibody of following antigen：Aminopeptidase N (CD13), Annexin A1, B7-H3 (CD276, various cancers), CA125 (oophoroma), CA15-3 (various cancers), CA19-9 (various cancers), L6 (various cancers), Louis Y (various cancers), lewis X (various cancers), alpha-fetoprotein (various cancers Disease), CA242 (colorectal cancer), P-ALP (various cancers), prostate specific antigen (prostate cancer), prostate Acid phosphoric acid enzyme (prostate cancer), epidermal growth factor (various cancers), CD2 (Huo Qijin diseases, NHL lymthomas, multiple marrow Tumor), CD3 ε (t cell lymphoma, lung cancer, breast cancer, gastric cancer, oophoroma, autoimmune disease, malignant ascite), CD19 (B Cell malignancies), CD20 (non-Hodgkin lymphoma), CD22 (leukaemia, lymthoma, Huppert's disease, SLE), CD30 (Hodgkin lymphoma), CD33 (leukaemia, autoimmune disease), CD38 (Huppert's disease), CD40 (lymthoma, it is more Hair property myeloma, leukaemia (CLL)), CD51 (metastasis melanin tumor, sarcoma), CD52 (leukaemia), CD56 (cellule lungs Cancer, oophoroma, plum gram cell cancer and liquid tumors, Huppert's disease), CD66e (various cancers), CD70 (metastatic kidneys Cell cancer and non-Hodgkin lymphoma), CD74 (Huppert's disease), CD80 (lymthoma), CD98 (various cancers), mucoitin (various cancers), CD221 (entity tumor), CD227 (breast cancer, oophoroma), CD262 (non-small cell lung cancer and other cancers Disease), CD309 (oophoroma), CD326 (entity tumor), CEACAM3 (colorectal cancer, gastric cancer), CEACAM5 (carcinomebryonic antigen, CEA, CD66e) (mammary gland, colorectal cancer and lung cancer), DLL4, EGFR (EGF-R ELISA, various cancers), CTLA4 is (black Melanoma), CXCR4 (CD184, neoplastic hematologic disorder, entity tumor), Endoglin (CD105, solid tumor), EPCAM (epithelial cells Adhesion molecule, carcinoma of urinary bladder, head and neck cancer, colon cancer, NHL prostate cancers, oophoroma), ERBB2 (epidermal growth factor acceptor 2, lung Cancer, breast cancer, prostate cancer), FCGR1 (autoimmune disease), FOLR (folacin receptor, oophoroma), GD2 gangliosides (various cancers), G-28 (cell surface antigen glycolipid matter, melanoma), GD3 idiotypes (respective cancer), heat shock protein (various cancers), HER1 (lung cancer, gastric cancer), HER2 (breast cancer, lung cancer and oophoroma), HLA-DR10 (NHL), HLA-DRB (NHL, B cell leukemia), human chorionic gonadotrophin (various cancers), IGF1R (1 receptor of insulin-like growth factor, it is real Body tumor, leukemia), IL-2 receptors (IL-2R, T cell leukaemia and lymthoma), IL-6R (Interleukin-6 receptor, it is multiple Myeloma, rheumatic arthritis, Castleman disease, interleukin-6 rely on tumour), integral protein (α v β 3,5 β 1 of α, alpha 6 beta 4, α ll β 3,5 β 5 of α, α v β 5, various cancers), MAGE-1 (various cancers), MAGE-2 (various cancers), MAGE-3 (various cancers), MAGE 4 (various cancers), anti-rotation Human Placental Ferritin Receptor (various cancers), p97 (melanoma), MS4A1 (4 structural domain Asia man of cross-film Race A member 1, non-Hodgkin's B cell lymphoma, leukaemia), MUC1 or MUC1-KLH (breast cancer, oophoroma, cervix cancer, branch Tracheocarcinoma and α gastrointestinal cancers), MUC16 (CA125) (oophoroma), CEA (colorectal cancer), gp100 (melanoma), MART1 (melanoma), MPG (melanoma), MS4A1 (4 structural domain subfamily A member 1 of cross-film, Small Cell Lung Cancer, NHL), Nucleolin, Neu oncoprotein (respective cancer), P21 (various cancers) resist (NeuGc ALPHA2-3Gal) antibody engaging portion Position (breast cancer, melanoma), class PLAP testis alkaline phosphatase (oophoroma, carcinoma of testis), PSMA (prostate tumor), PSA is (preceding Row gland cancer), ROBO4, TAG 72 (tumor-associated glycoprotein 72, AML, gastric cancer, colorectal cancer, oophoroma), T cell cross-film egg (various cancers) in vain, Tie (CD202b), TNFRSF10B (A member of the TNF receptor family 10B, various cancers), TNFRSF13B (A member of the TNF receptor family 13B, Huppert's disease, NHL, other cancers, RA and SLE), TPBG (trophocyte glycoprotein, clear-cell carcinoma), TRAIL-R1 (TNF correlation necrosis inductions ligand receptor 1, lymthoma, NHL, knot The carcinoma of the rectum, lung cancer), VCAM-1 (CD106, melanoma), VEGF, VEGF-a, VEGF-2 (CD309) (various cancers).It is other Tumour is related, can be summarised and comment (Gerber, et al, mAbs 2009,1 by the antigen that antibody identifies:3,247-253； Novellino et al, Cancer ImmunolImmunother.2005,54 (3), 187-207；Franke,et al, Cancer BiotherRadiopharm.2000,15,459-76)。
Cell binding agent, preferably antibody, can be it is any can be to antitumor cell, virus infected cell, microorganism Infection cell, parasite infected cells, autoimmunity cell, the cell of activation, bone marrow cell activate T cell, B cell or black Chromatophore.More specifically, cell binding agent can be any drug that can fight one of following further antigens or receptor/point Son：CD3,CD4,CD5,CD6,CD7,CD8,CD9,CD10,CD11a,CD11b,CD11c,CD12w,CD14,CD15,CD16, CDw17,CD18,CD19,CD20,CD21,CD22,CD23,CD24,CD25,CD26,CD27,CD28,CD29,CD30,CD31, CD32,CD33,CD34,CD35,CD36,CD37,CD38,CD39,CD40,CD41,CD42,CD43,CD44,CD45,CD46, CD47,CD48,CD49b,CD49c,CD51,CD52,CD53,CD54,CD55,CD56,CD58,CD59,CD61,CD62E, CD62L,CD62P,CD63,CD66,CD68,CD69,CD70,CD72,CD74,CD79,CD79a,CD79b,CD80,CD81, CD82,CD83,CD86,CD87,CD88,CD89,CD90,CD91,CD95,CD96,CD98,CD100,CD103,CD105, CD106,CD109,CD117,CD120,CD125,CD126,CD127,CD133,CD134,CD135,CD138,CD141, CD142,CD143,CD144,CD147,CD151,CD147,CD152,CD154,CD156,CD158,CD163,CD166, .CD168,CD174,CD180,CD184,CDw186,CD194,CD195,CD200,CD200a,CD200b,CD209,CD221, CD227,CD235a,CD240,CD262,CD271,CD274,CD276(B7-H3),CD303,CD304,CD309,CD326,4- 1BB, 5AC, 5T4 (Trophoblast glycoprotein, TPBG, 5T4, WNT- activate inhibiting factor 1 or WAIF1), gland cancer antigen, AGS-5, AGS-22M6, activin receptor kinases 1, AFP, AKAP-4, ALK, α integrins, α v β 6, aminopeptidase N, amyloid egg White β, androgen receptor, angiogenesis promoting protein factor 2, angiogenesis promoting protein factor 3, Annexin A1, anthrax toxin are protected Shield property antigen, anti-rotation move protein receptor, AOC3 (VAP-1), B7-H3, bacillus anthracis, BAFF (B cell activity factor), B lymphs Oncocyte, bcr-abl, Magainin, BORIS, C5, C242 antigens, CA125 (sugar antigens 125, MUC16), CA-IX (or CAIX, Carbonic anhydrase 9), CALLA, CanAg, dog lupus erythematosus IL31, carbonic anhydrase IX, cardiac myosin, (C-C segments become CCL11 Change the factor 11), CCR4 (C-C Chemokine receptor4s, CD194), CCR5, CD3E (ε), CEA (carcinomebryonic antigen), CEACAM3, CEACAM5 (carcinomebryonic antigen), CFD (factor D), Ch4D5, cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18) are grown thickly Factors A, CRIPTO, FCSF1R (colony-stimulating factor 1 receptor, CD115), (colony stimulating factor 2, granulocyte-macrophage are thin by CSF2 Born of the same parents' colony stimulating factor (GM-CSF)), CTLA4 (cytotoxic T lymphocyte GAP-associated protein GAP 4), CTAA16.88 tumour antigens, CXCR4 (CD184), C-X-C Chemokine receptor4s, cyclic annular ADP ribalgilases, cell periodic protein B 1, CYP1B1 are big and small Cellular virus, cytomegalovirus Glycoprotein B, Dabigatran, DLL4 (class Δ ligand 4), DPP4 (double peptide-peptases 4), DR5 are (dead Receptor 5), Type of toxin -1 Escherichia coli shiga, Escherichia coli shiga Type of toxin -2, ED-B, EGFL7 (class EGF structural domains Albumen 7), EGFR, EGFRII, EGFRvIII, endothelial factor (CD105), Endothelin B receptor, endotoxin, EpCAM (epithelial cells Adhesion molecule), EphA2, Episialin, ERBB2 (epidermal growth factor acceptor 2), (TMPRSS2ETS merges base by ERBB3, ERG Cause), Escherichia coli, ETV6-AML, FAP (fibroblast activation protein alpha), FCGR1, alpha-fetoprotein, fibrin II β chains, Fibronectin extra domain-B, FOLR (folacin receptor), folacin receptor α, folic acid hydrolase, Fos related antigens 1, respiratory tract The F protein of syncytial virus, the receptor of curling, fucose GM1, GD2 gangliosides, G-28 (cell surface antigen glycolipid), GD3 Idiotype, GloboH, Glypican 3, NeuGc ALPHA2-3Gal, GM3, GMCSF receptor alpha chains, Growth differentiation factor 8, GP100, GPNMB (transmembrane glycoprotein NMB), GUCY2C (guanylate cyclase 2C), guanosine cyclic mono-phosphate (GC-C), intestines guanosine Cyclase of acid, guanosine cyclic mono-phosphate receptor, Thermostable α-amylase receptor (hSTAR), heat shock protein, hemagglutinin, hepatitis B surface Antigen, hepatitis type B virus, HER1 (human epidermal growth factor acceptor 1), HER2, HER2/neu, HER3 (ERBB-3), IgG4, HGF/SF (hepatocyte growth factor/dispersion factor), HHGFR, HIV-1, histone complexes, (mankind are thin in vain by HLA-DR Extracellular antigen), HLA-DR10, HLA-DRB, HMWMAA, human chorionic gonadotropin, HNGF, mankind's dispersion factor receptor swash Enzyme, HPV E6/E7, Hsp90, hTERT, ICAM-1 (Intercellular Adhesion Molecule 1), idiotype, IGF1R (IGF -1, para-insulin 1 receptor of growth factor), IGHE, IFN-γ, influenza hemagglutinin, IgE, the areas IgE Fc, IGHE, IL -1, IL-2R (interleukin-22 by Body), IL -4, IL-5, IL -6, IL-6R (Interleukin-6 receptor), IL-9, IL -10, IL -12, IL-13, IL-17, IL-17A, IL- 20, IL-22, IL-23, IL31RA, ILGF2 (insulin-like growth factor 2), integral protein (α 4, α IIb β 3, α v β 3,4 β 7 of α, α 5 β 1, alpha 6 beta 4,7 β 7 of α, α ll β 3,5 β 5 of α, α v β 5), interferon gamma inducible protein matter, ITGA2, ITGB2, KIR2D, LCK, Le, Legumain, Lewis-Y antigen, LFA-1 (Lymphatic diseases, CD11a), LHRH, LINGO-1, fat phosphorus wall Acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE A1, MAGE A3, MAGE 4, MART1, MCP-1, MIF (macrophage migration inhibitory factor or the glycosyl inhibition factor (GIF)), MS4A1 (4 structural domains of cross-film Subfamily A member 1), MSLN (mesothelin), MUC1 (mucin 1, cell surface correlation (MUC1) or Polymorphic epithelial mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCP1 (MCP 1), MelanA/MART1, ML-IAP, MPG, MS4A1, MYCN, Myelin-associated glycoprotein, Myostatin, NA17, NARP-1, NCA-90 (G-Ag), Nectin-4 (ASG-22ME), NGF, nerve cell apoptosis modulin enzyme 1, NOGO-A, Notch receptor, paranuclein, the production of Neu oncogene Object, NY-BR-1, NY-ESO-1, OX-40, OxLDL (OxLDL ELISA), OY-TES1, P21, p53 non-mutants, P97, PAP, resist (NeuGc ALPHA2-3Gal) paratope, PAX3, PAX5, and PCSK9, PDCD1 are (PD-1, procedural thin Born of the same parents' death protein 1, CD279), PDGF-R α (α platelet-derived growth factor receptors), PDGFR- β, PDL-1, PLAC1, class PLAP Testis alkaline phosphatase, platelet derived growth factor receptor β, sodium phosphate combine transporter, PMEL 17, poly sialic acid, albumen Enzyme 3 (PR1), prostate cancer, PS (phosphatidylserine), prostate gland cancer cell, pseudomonas aeruginosa, PSMA, PSA, PSCA are mad Dog disease viral glycoprotein, RHD (Rh polypeptides 1 (RhPI), CD240), the Rhesus factors, RANKL, RhoC, Ras mutation, RGS5, ROBO4, Respiratory Syncytial Virus(RSV), RON, sarcoma translocation breakpoint, SART3, Sclerostin, SLAMF7 (SLAM member 7), Selectin P, SDC1 (syndecan 1), systemic loupus erythematosus (a), somatomedin C, SIP (1- phosphoric acid sheath ammonia Alcohol), Somat, Human sperm protein 17, SSX2, STEAP1 (6- cross-film epitheliums prostate antigen 1), STEAP2, STn, TAG-72 (tumor-associated glycoprotein), survivin, T cell receptor, T cell transmembrane protein, (tumor vascular endothelium marks TEM1 1), TENB2, Tenascin C (TN-C), TGF- α, TGF-β (transforming growth factor β), TGF-β 1, TGF-β 2 (conversion growth because Son 2), Tie (CD202b), Tie2, TIM-1 (CDX-014), Tn, TNF, TNF-α, TNFRSF8, TNFRSF10B (neoplasm necrosis Factor acceptor superfamily member 10B), TNFRSF13B (A member of the TNF receptor family 13B), TPBG (trophocytes Glycoprotein), TRAIL-R1 (TNF correlation necrosis inductions ligand receptor 1), TRAILR2 (death receptor 5 (DR5)), tumour is relevant Ca2+ oscillations sensor 2, the glycosylated MUC1 of tomour specific, tweak receptor, TYRP1 (glycoprotein 75), TRP-2, tyrosinase, VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, vimentin, WT1, XAGE 1, table Cell up to any insulin growth factor receptor or any EGF-R ELISA.
In another specific embodiment, pass through cell binding agent-drug coupling of the bridging junctor link of this patent Object can be used for targeting cancer therapy.Target cancers include but not limited to that adrenocortical carcinoma, cancer of anus, carcinoma of urinary bladder, brain are swollen Tumor is (brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, former on curtain Beginning neuroderm and Pinealoma, pathways for vision and hypothalamic gliomas), breast cancer, carcinoid tumor, gastrointestinal cancer, Unknown small cell carcinoma, cervical carcinoma, colon cancer, carcinoma of endometrium, cancer of the esophagus, cholangiocarcinoma, especially because of family tumor (PNET), cranium Interior germinoma, cancer eye, intraocular melanoma, gallbladder cancer, gastric cancer (gastric cancer), Extaagonactal perm celi tumors, pregnant week are nourished Cytoma, head and neck cancer, hypopharyngeal cancer, islet-cell carcinoma, kidney (clear-cell carcinoma), laryngocarcinoma, leukaemia (acute lymphoblastic, it is acute Medullary system, chronic lymphocytic, chronic granulocyte, hair cell), lip and mouth cancers, liver cancer, lung cancer (non-small cell, it is small thin Born of the same parents), lymthoma (AIDS related, central nervous system, cutaneous T-cell, Hodgkin's disease, non-Hodgkin lymphoma), malignant mesothelioma, Melanoma, Merkel cell cancer, metastatic squamous neck cancer and invisible primary cancer, Huppert's disease and other thick liquid cells Tumour, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorder, nasopharyngeal carcinoma, neuroblastoma, carcinoma of mouth, mouth Pharynx cancer, osteosarcoma, oophoroma (epithelium, gonioma, low malignant tumour), cancer of pancreas (outer secretion, islet-cell carcinoma), secondary nose Sinus and CARCINOMA OF THE NASAL CAVITY, parathyroid carcinoma, carcinoma of penis, pheochromocytoma, pituitary tumor, plasma cell tumor, prostate cancer band muscle Tumor, the carcinoma of the rectum, clear-cell carcinoma (kidney), renal plevis and ureter (migratory cell), glandula cancer, Sai Saili syndromes, cutaneum carcinoma (skin Skin t cell lymphoma, Kaposi sarcoma, melanoma), intestinal tumor, soft tissue sarcoma, gastric cancer, carcinoma of testis, thymoma (pernicious), thyroid cancer, urethral cancer, uterine cancer, uncommon juvenile cancer, vaginal tumor, tumor of vulva and Willms Tumor.
In another specific embodiment, pass through cell binding agent-drug coupling of the bridging junctor link of this patent Object can be used as the ingredient and method that treat or prevent autoimmune disease.Autoimmune disease includes but not limited to, Achlorhydra autoimmune active chronic hepatitis, acute diseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Chinese mugwort Enlightening is sick, azoospermatism, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-GBM/TBM ephritis, antiphospholipid syndrome, Anti- exception enzyme syndrome, arthritis, atopic hypersensitivity, atopic dermatitis, autoimmune alpastic anemia, autoimmunity Property cardiomyopathy, autoimmune hemolytic anemia, oneself immunity hepatitis, autoimmune inner ear disease, autoimmune leaching Bar hyperblastosis syndrome, autoimmune peripheral nerve disease, autoimmune pancreatitis, the more endocrines synthesis of autoimmune Levy I, II and type III, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, Autoimmune uveitis Film is scorching, Balo diseases/Balo concentric sclerosis diseases, Bechets syndromes, BergerShi diseases, Bickerstaff encephalitis, Blau synthesis Sign, bullous pemphigoid, Castleman diseases, Chagas diseases, chronic fatigue immune dysfunction syndrome, chronic inflammatory are de- Myelin polyneuropathy, the multifocal osteomyelitis of chronic recurrent, chronic Lyme disease, chronic obstructive pulmonary disease, Churg- Strauss syndromes, cicatricial pemphigoid, coeliac disease, Cogan syndromes, cold coagulation disease, 2 deficiency disease of complement component, cranium Bone arteritis, CREST syndromes, Crohns diseases (idiopathic inflammatory bowel diseases), Cushing syndrome, skin leukocytosis blood Guan Yan, moral Ge Shi diseases, DercumShi diseases, dermatitis herpetiformis, dermatomyositis, type 1 diabetes, diffusivity cutaneous systemic sclerosis, Dressler syndromes, lupus erythematosus discoides, eczema, endometriosis, the relevant arthritis of Enthesopathy, Eosinophilic fascitises, epidermolysis bollosa, erythema nodosum, essential mixed cryoglobulinemia, Yi Wen Cotard, fibrodysplasia ossify disease, fibromyalgia, fibrosis myositis, fibrosing alveolitis, gastritis, stomach and intestine class Pemphigus, giant cell arteritis, glomerulonephritis, Goodpasture that syndrome, Graves disease, actue infectious polyradiculoneuritis, Hashimoto encephalitis, Hashimoto's thyroiditis, hemolytic anemia, anaphylactoid purpura, gestational hepatitis, suppurative hidradenitis, Hughes Syndrome (antiphospholipid syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis, spy's hair Property thrombocytopenic purpura (autoimmune thrombocytopenic purpura), IgA nephrosis (primary Jie Shi disease), inclusion body myositis, Flammatory demyelinating multiple neuritis, interstitial cystitis, irritable bowel syndrome, juvenile idiopathic arthritis, teenager's class wind Wet arthritis, Kawasaki disease, lambert-Eton Eaton-Lambert myasthenic syndrome, leucocyte clasticity vasculitis, lichen planus, firmly The property changed sclerosis, linear IgA diseases (LAD), Lou Gehrig diseases (also referred to as amyotrophic lateral sclerosis), lupus hepatitis is red Yabbi sore, Majeed syndromes, Meniere's disease, panarteritis under microscope, Miller-Fisher syndrome, Combination connective Tissue disease, morphoea, Mohammed, founder of Islam's-Ha Beiman diseases, Macaulay's syndrome, Huppert's disease, multiple sclerosis, severe flesh It is powerless, myositis, hypnosia, neuromyelitis optica (Devic diseases), neuromyotonia, scar of eyelid pemphigoid, Opsoclonus myoclonus syndromes, Ord thyroiditis, palindrome rheumatism, PANDAS is (certainly with the relevant children of streptococcus Body immunity neuropsychopathy), Paraneoplastic cerebellar degenerations, paraoxysmal nocturnal hemoglobinuria, Parry Romberg syndromes, Parsonnage-Turner syndromes, pars planitis, pemphigus, pemphigus vulgaris, anaemia, Surrounding encephalomyelitis, POEMS syndromes, nodular polyarteritis, polymyalgia rheumatica, polymyositis, primary biliary Hepatic sclerosis, primary sclerotic cholangitis, progressive neuro-inflammatory lesion, psoriasis, arthritic psoriasis, gangraenosum skin Skin is scorching, pure red cell aplasia, Rasmussen encephalitis, Raynaud's phenomenon, relapsing polychondritis, Reiter syndrome, not peaceful leg Syndrome, rear neurofibrillary, rheumatoid arthritis, rheumatoid heat, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler syndromes, Schnitzler syndrome, sclerotitis, chorionitis, Sjogren syndrome, spondyloarthropathy, sticky mass formed by blood stasis, Still diseases, stiff man syndrome, subacute bacterial endocarditis, Su Sake syndromes, Sweet syndromes, chorea minor are handed over Feel nerve anaemia, Takayasu arteritis, temporal arteritis (giant cell arteritis), Tolosa-Hunt syndromes, transverse ridge Marrow is scorching, ulcerative colitis (idiopathic inflammatory enteropathy), undifferentiated connective tissue disease, undeveloped ovules, vasculitis, in vain Purplish or white patches on the skin wind, Wegner's granulomatosis, Wilson's syndrome, Wiskott-Aldrich syndrome.
In another specific embodiment, in the conjugate and medicine for treating or preventing autoimmune disease The example for the binding molecule that object molecule is linked by the bridging junctor of this patent, including but not limited to, elastoresistance protein antibodies, The anti-epithelial cell antibody of Abys, anti-basement film IV collagen type antibody, antinuclear antibodies, anti-ds DNA, anti-ss DNA, the anti-heart Phospholipid antibody IgM, IgG, anti-chylous diarrhea antibody, anti-phospholipid antibody IgK, IgG, Anti SM antibody, anti-mitochondrial antibody, thyroid gland are anti- Body, microsomal antibody, T cell antibody, thyroglobulin antibody, anti-SCL-70, anti-Jo, anti-U.sub.1RNP, anti-La/SSB, Anti- SSA, anti-SSB, anti-parietal cell anti-body, anti-histone, anti-RNP, C-ANCA, P-ANCA, anti-centromere, antifibrin is former, Anti-glomerular basement membrane antibodies, Barrier pressure, 3 antibody of anti-Desmogein, anti-p62 antibody, anti-sp100 antibody, resist mitochondria (M2) antibody, rheumatoid factor antibodies, anti-MCV antibody, anti-topoisomerase enzyme antibody, anti-neutrophil leucocyte cytoplasm (cANCA) are anti- Body.
In certain preferred embodiments, the binding molecule in this patent on conjugate can be with autoimmune disease The receptor or receptor complex expressed on relevant activated lymphocyte are combined.Receptor or receptor complex include that ball is immunized Protein gene superfamily member (such as CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD56, CD70, CD79, CD79b, CD90, CD125, CD147, CD152/CTLA-4, PD-1 or ICOS), TNF receptors are super Family member (such as CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, INF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4 and APO-3), integral protein, Cytokine receptor, chemokine receptors, ajor histocompatibility albumen, agglutinin (c-type, S types or I types) or complement control egg In vain.
In another specific embodiment, the available cell combination to virus or microbial antigen with immunologic specificity Ligand is humanization or human monoclonal antibodies." viral antigen " includes but not limited to, any virus that can cause immune response Peptide, polypeptide protein (such as HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase glycosides enzyme, influenza virus blood Solidifying element, HTLV Tax, herpes simplex Simplex virus glycoprotein (such as gB, gC, gD and gE) and hepatitis B surface antibody).It is " micro- Biological antigens " include but not limited to any microbial polypeptide that can cause immune response, polypeptide, protein, sugar, polysaccharide or fat Matter molecule (such as bacterium, fungi, pathogenic protozoa or yeast polypeptides, including such as LPS and capsular polysaccharide).It can be used for treating disease The example of poison or the antibody of microorganism infection, including but not limited to：Palivizumab, it is for treating rsv infection, people source Change anti respiratory syncytial virus monoclonal antibody；PRO542 is a kind of CD4 fusion antibodies, for treating HIV infection；Ao Sita Wei is a kind of human antibody for treating hepatitis type B virus；PROTVIR is a kind of humanization IgG1 antibody, huge for treating Cell virus and anti-LPS antibody.
Cell-binding molecules-drug conjugates made from bridging junctor by this patent can be used for treating infectious disease Disease.These infectious diseases include but not limited to acinetobacter infection, lumpy jawl clams, lethargus (African typanosomiasis nagana), AIDS (Immune Deficiency Syndrome), amcbiasis, anaplasmosis, anthrax, hemolytic Yersinia infections, Argentina Hemorrhagic fever, roundworm disease, aspergillosis, astrovirus infection, Babesia Gibsoni, Bacillus cercus infection, bacterial pneumonia, bacterium Property vaginitis, bacteroides infection, balantidiasis, roundworm infection, BK virus infection, black piedra, infection with Blastocystis hominis, Blastomycosis, Bolivian hemorrhagic fever, borrelia infection, botulismus (and baby's botulismus), Brazilian hemorrhagic fever, Bu Lu Family name's bacillosis, bulkholderia cepasea infection, Bu Luli ulcer, calicivirus infection (norovirus and bed ripples virus), bending Bacillosis, candidiasis (candidiasis, thrush), cat scratch disease, cellulitis, Chagas diseases (American trypanosomiasis), ascus, Varicella, Chlamydia, infection involving chlamydia pneumoniae, cholera, chromoplast tumor, clonorchis sinensis, C. difficile infection, Coccidioidomycosis, colorado tick fever disease, common cold (acute viral nasopharyngitis, acute rhinitis), creutzfeldt-Jacob disease, Ke Li meter Asia-Crimean-Congo Hemorrhagic Fever, cryptococcosis, Cryptosporidiosis, cutaneous larva are migrated, cyclosporiasis, Enterobacteriaceae infections, enterovirus Infection, epidemic typhus, erythema infectioum (the 5th kind of disease), anxious rash, fasciolopsiasis, fascioliasis, mortality man Race's property is had a sleepless night, filariasis, C.perfringens food poisoning, free live body amoeba infection, fusobacterium infections, emphysematous gangrene (clostridial myonecrosis), geotrichosis, lattice Stedman-this Trusler-shekel disease syndrome, giardiasis, horse nose Subcutaneous ulcer, gonorrhoea, granulomatous diarrhea (fifth venereal disease), the infection of A group streptococcus, the infection of B group streptococcus, acute Haemophilus influenzae infection, Hand-foot-and-mouth disease (HFMD), Hantavirus pulmonary syndrome, helicobacter pylori infections, hemolytic uremic syndrome, hemorrhagic fever renal syndrome Heat, hepatitis A, hepatitis B, hepatitis C, hepatitis D, Hepatitis E, herpes simplex, histoplasmosis, hookworm Infection, Human bocavirus infection, mankind's ewingii Ehrlichiosis, human granulocyte's anaplasmosis, human metapneumovirus sense Dye, human monocyte's Ehrlichiosis, human papilloma virus infection, human parainfluenza viruses' infection, hymenolepiasis, end Pasteur Viral infectivity monocytosis,mononucleosis (list), influenza, etc. sporidiosis, Kawasaki disease, keratitis, golden lattice bacillus sense Dye, kuru, Lassa fever, legionaires' disease (veteran's disease), legionaires' disease (Pontiac fever), leishmaniasis, Lyme disease, lymph silk Parasitosis (elephantiasis), lymphocytic choriomeningitis, malaria, marburg hemorrhagic fever, measles, research of melioidosis (Hui Shi diseases), Meningitis, meningococcosis, metagonimiasis, microsporidiosis, molluscum contagiosum, parotitis, mouse typhus (place Property typhus), Eaton agent pneumonia, madura diseasemadura foot, fly-blown, neonatal ophthalmia (newborn's eye disease), anomaly creutzfeldt-Jacob disease (vCJD, nvCJD), nocardiasis, onchocercosis (river blindness), paracoccidioidomycosis (Paracoccidioidomycosis), lung fluke Disease, pasteurellosis, head louse, body louse, crab louse, pelvic infecton, pertussis, the plague, pneumococcal infection, Pneumocystis Carinii Pneumonia, Pneumonia, polio, general Salmonella infection, primary amebic meningo encephalitis, progressive multifocal leukoencephalopathy, psittacosis, Q Heat, rabies, rat-bite fever, respiratory syncytial virus infection, rhinosporidiosis, rhinovirus infection, rickettsial infection, rickettsia Body acne, Rift Valley fever, American spotted fever, rotavirus infection, rubella, salmonellosis, (serious acute respiratory integrates SARS Sign), scabies, snail fever, septicemia, shigellosis (Bacillary dysentery), herpes zoster (herpes zoster), smallpox (day Flower), sporothrix, staphylococcal food poisoning, infect staphylococcus aureus, strongyloidosis, syphilis, taeniasis, break Cold, barber's itch (Barber itches), the ringworm of scalp, ringworm of the body, jock itch, the tinea manuum, tinea manuum nigra, tinea pedis (athlete's foot), onychomycosis (onychomycosis), flower Spot tinea, toxocarasis (larva migrans,ocular), toxocarasis (visceral larva migrans), toxoplasmosis, trichinosis, trichomonad Disease, trichuriasis (whipworm infection), pulmonary tuberculosis, yatobyo, Ureaplasma Urealyticum Infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic Heat, viral pneumonia, West Nile fever, white piedra (tinea alba), artificial tuberculosis yersinia genus, Yersinia intestines Road disease, yellow fever, zygomycosis.
The cell binding agent of the present invention, more preferably antibody, the pathogenic strains of confrontation include but not limited to, Boydii not lever Bacterium, actinomyces Israeli, actinomyces and Propionibacterium, trypanosoma bocagei, HIV (Human immunodeficiency virus disease poison), in histolytica Ah Meter Ba, Anaplasma, Bacillus anthracis, hemolysis vibrion, Junin virus, Ascaris, aspergillus, Astroviridae, babesia Belong to, Bacillus cercus, various bacteria, Bacteroides, Escherichia coli, Ascaris, BK virus, tubercle bacterium, blastocystis hominis, skin Scorching blastomycete, machupo virus, Borrelia, clostridium botulinum, fresh breeze Calamus, Brucella, usually onion Bai Kehuo That moral bacterium and other bulkholderia cepasea kinds, mycobacterium buruli, Caliciviridae, campylobacter, usually white is false Silk yeast and other candidas, bartonella henselae, A group streptococcus and staphylococcus, schizotrypanum cruzi, the bloodthirsty bar of Roger Ducret Bacterium, VZV, chlamydia trachomatis, colorado tick fever virus, rhinovirus, coronavirus, CJD prions, Crimean Congo go out Fever virus, Cryptococcus neoformans, Cryptosporidium, ancylostoma braziliense, a variety of parasites, Cyclospora, band-like tapeworm, giant cell disease Poison, dengue fever virus (DEN-1, DEN-2, DEN-3 and DEN-4)-flavivirus, fragile Bifidobacterium, Bacterium diphtheriae split head Tapeworm, Guinea worm, Ebola virus, Echinococcus, Ehrlichia enterococcus spp, enterovirus genus, Pu Shi Garricks Secondary body, assays for parvovirus B 19, Human herpesviryus 6 and herpes virus hominis 7, Fasciolopsis buski, Fasciola hepatica and huge fasciola gigantica, FFI Prion, Filarioidea superfamily, C.perfringens, Fusobacterium, other clostridium, geotrichum candidum, GSS prions, Enteron aisle giardia lamblia, bulkholderia cepasea pierce spore bacillus pumilus and gram Candida, gonococcus, granuloma Cray primary Salmonella, micrococcus scarlatinae, Streptococcusagalactiae, haemophilus influenzae, enterovirus, mainly Coxsackie A disease poison and enteron aisle disease Poison 71, unknown virus, helicobacter pylori, Escherichia coli O 157：H7, bunyaviridae, hepatitis A virus, hepatitis B Virus, Hepatitis C Virus, Hepatitis D virus, Hepatitis E virus, herpes simplex virus 1, herpes simplex virus 2, pod membrane Histoplasma capsulatum, duodenal adenoma and carcinoma of ampulla haemophilus influenzae, human bocavirus, Ehrlichia, thermophilic phagocyte without Haemophilus, human metapneumovirus look into luxuriant and rich with fragrance Ehrlichia, human papilloma virus, human parainfluenza viruses, Hymenolepis nana and diminution Hymenolepis, Chinese mugwort Pasteur's virus, family of orthomyxoviridae family, bayesian isospora, golden lattice bacillus, Klebsiella Pneumoniae, Cray primary Salmonella, legionella pneumophilia, legionella pneumophilia, legionella pneumophilia, leishmania, Mycobacterium leprae and tuberculosis branch bar Bacterium, Leptospira, Listeria monocytogenes, Borrelia burgdoyferi and other Borrelia species, class Family name trichina and Wuchereria malayi, Lymphocyte function-associated antigen-1 (LCMV) Plasmodium, Marburg virus, measles Poison, Burkholderia pseudomallei, Neisseria meningitidis, Metagonimus Yokogawai, Microsporidia door, molluscum contagiosum Viral (MCV), mumps virus, typhoid fever rickettsia, mycoplasma pneumoniae, various bacteria and fungi autoeciousness dipteron fly larvae, Chlamydia trachomatis and NEISSERIA GONORRHOEAE, vCJD prions, Nocard's bacillus and other Nocardias, Onchocerca, disk Bao are quasi- Subfamily, secondary dragon belongs to Xi Mani and other pairs belong to, pasteurella, head louse, pediculus humanus corporis, Bordetella pertussis plague Yale Gloomy Salmonella, streptococcus pneumonia, pneumococcus, poliovirus, prevotella, Nai Shi grignard bacillus, JC viruses, parrot The hot Chlamydia of nautilus, Coxiella burnetii, hydrophobin, single-stranded coccus and spirillum, Respiratory Syncytial Virus(RSV), rhinosporidium seeberi, Rhinovirus, rickettsiae, by small strain Richettsia, Rift Valley fever virus, Richettsia Richettsia, rotavirus, rubella, sand Door Bordetella, sars coronavirus, Sarcoptes scabiei hominis, Schistosoma, body cell category, Shigella, varicellazoster virus, smallpox Major or smallpox are small, Sporothrix schenckii, staphylococcus aureus category, staphylococcus aureus, and streptococcus is suppurated, strongylid, Microspironema pallidum, tapeworm category, lockjaw, tinea category tinea sound uranium, tinea category, acrothesium floccosum, Trichophyton rubrum, Trichophyton mentagrophytes are red Color trichophyta, the Wai Ping Mildew in Vinnie gram, trichophyton category, cell death category, bow and arrow poison or bow and arrow poison, toxoplasma gondii, trichina, Trichomonas vaginalis, three kinds in three mounds, mycobacterium tuberculosis, not bright hila Tula bacterium, urea and equine encephalitis virus, Venezuela horse Encephalitis viruses, comma bacillus, Gua Nali plead illness poison, West Nile Virus, beigelii spores, and artificial tuberculosis yersinia genus is small Yersinia enterocolitica, flavivirus, Mucorales rank (mucormycosis) and Entomophthorales rank (entomophthora category nosomycosis), hair Mould mesh Pseudomonas aeruginosa, campylobacter (vibrios), Aeromonas, Emhorn bacterium, Yersinia ruckeri, shigella, shiga Bacillus, Shigella, salmonella, salmonella typhi, Treponema pertenue, Borrelia vincentii, Borrelia burgdoyferi, thin spiral shell Revolve body, Pneumocystis carinii, Bacillus abortus, Brucella, brucella, Mycoplasma, Rickettsia prowazeki, yochubio Rickettsia, chlamydiaceae, pathogenic fungus (aspergillus fumigatus, Candida albicans, Histoplasma capsulatum), protozoan is (molten Organize entamoeba, Tenas trichmonads, Hominis trichmonads, castellanella gambiense, Trypanosoma rhodesiense, Roche Li Shiman originals Worm, crithidia cunninghami, leishmania brasiliensis, Pneumocystis Carinii Pneumonia, Plasmodium vivax, plasmodium falciparum, plasmodium malaria Disease) or Helminiths (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium and hookworm).
Other are used as this patent cell binding agent, treat the antibody of viral disease, including but not limited to, cause a disease to following The antibody of property viral antigen：Poxvirus；Herpesviral；Adenovirus；Small flavivirus；Enterovirus；Picornavirus；Tiny disease Poison；Reovirus；Retrovirus；Influenza virus；Parainfluenza virus；Parotitis；Measles；Respiratory Syncytial Virus(RSV)；Rubella；Worm Matchmaker's virus；Rhabdovirus；Salmonella；The non-non- b Hepatitis virus of a/；Rhinovirus；Coronavirus；Rothau virus；Oncogenic virus, Such as HBV (hepatocellular carcinoma), human papilloma virus (cervical carcinoma, cancer of anus), Kaposi's sarcoma relevant herpesviral (card wave Help sarcoma sarcoma), nerpes vinrus hominis the 4th type (nasopharyngeal carcinoma, Burkitt lymphoma, primary central nervous system lymph Tumor), tumor virus (merkel's cells cancer), SV40 (simian virus 40), HCV (hepatocellular carcinoma), HTLV-1 (the white blood of human adult T cell Disease/lymthoma)；Immunologic derangement leads to virus, such as human immunodeficiency virus (AIDS)；Central nervous system virus, such as JCV (the multifocal leukoencephalopathy of progressive), Hepatitis C Virus (subacute sclerosing panencephalitis), LCV (lymphatic choroid plexus Meningitis), the rich Encephalitides in Asia, orthomyxovirus (brain inflammatory encephalitis), RV (rabies), long rhinovirus, herpesviral meningitis, Ramsay Hunter syndrome II types, poliovirus (poliovirus, rear polio syndrome), HTLV-1 (tropical paralytic paralysis))；Cytomegalovirus (cytomegalovirus retinitis, HSV (herpetic keratitis)；Cardiovascular virus, Such as CBV (pericarditis, myocarditis)；Intranasal inflammation/the viral pneumonia of respiratory system/acute viral, such as epstein-Barr virus (EBV infection/infectiousness monokaryon disease), cytomegalovirus, SARS coronavirus (serious acute respiratory syndrome) or positive mucus disease Poison, influenza virus a/b/c (influenza/bird flu), paramyxovirus, human parainfluenza virus, (human airway closes born of the same parents' disease to RSV Poison), hMPV；Digestive system virus (mumps virus, cytomegalovirus (cytomegalovirus esophagitis), adenovirus (adenovirus sense Dye), rotavirus, norwalk virus, astrovirus, coronavirus, hepatitis type B virus, CBV, hepatitis A virus, the third type Hepatitis virus, Hepatitis D virus, Hepatitis E virus, HGV)；Urogenital virus, such as BK virus, MuV (parotitis).
Further, the present invention also includes the conjugation conjugate connected with bridge connector and acceptable carrier, diluent or The composition that auxiliary material is constituted, with treating cancer, infection or autoimmune disease.Treating cancer, infection and autoimmune disease The method of disease can in vitro, in vivo or in vitro implementation.The example of in vitro use includes handling cell culture with it, to kill All cells other than not expressing the variant of target antigen；Or kill the variant for expressing unwanted antigen.It is in vitro to use Example be included in be transplanted and candidate stem cell (HSC) handled before (HSCT), to kill illness or malignant cell. For example, removing tumour cell from marrow before autotransplantation in treatment of cancer or in the treatment of autoimmune disease Or lymphocyte, or that T is removed from allogeneic bone marrow or tissue is thin for graft versus host disease in order to prevent before the transplant Born of the same parents and other lymphocytes.Such clinic vitro treatment can carry out as follows：From patient or other individual harvest bones Then marrow is incubated about 30 minutes to about 48 hours in the culture medium containing serum at about 37 DEG C, this is added in the culture medium The conjugate of invention, concentration range is from about 1pM to 0.1mM.Specific drug concentration and incubation time should be cured by professional clinical Teacher determines.After incubation, bone marrow cell is washed with the culture medium containing serum, and in accordance with known methods by intravenous injection to trouble Person.If patient between bone marrow collection and again infusion of therapeutic cell, also receives other treatments (such as ablation chemotherapy or total body radiation The course for the treatment of) in the case of, it should use standard armamentarium will treated bone marrow cell stored frozen in liquid nitrogen.
Clinical internal in use, can be using the conjugate that this patent connector links as solution or lyophilized solid confession It answers, solid can be re-dissolved in Injectable sterile water.The example of conjugate application program is as follows：Conjugate is once a week Property intravenous injection, continuously give 8 to 20 week.Bolus dose is given in 50-500ml physiological saline, people can be added thereto Seralbumin (such as concentrate solution of 0.5 to 1mL human serum albumins, 100mg/mL).It will be about to be injected intravenously dosage 50 μ g to 20mg/kg (weight) daily, or weekly, two weeks, three weeks or (10 μ g of per injection to 200mg/kg agent every month Amount).After treatmentWeek, patient can receive the second course of therapy.Related administration method, excipient, diluent, dosage are secondary The specific clinical protocol of number etc. can be determined by specialized clinician.
The example for the medical conditions that can be treated in vivo or in vitro method, includes the malignant tumour of any types cancer, from Body immunological diseases, graft rejection and infection (virus, bacterium or parasite).
Amount to reach the conjugate needed for desired biological effect will change with many factors, and the factor includes even Join the chemical characteristic of object, effect and bioavilability, the type of disease, patient species, the disease state of development of patient, administration way Diameter, all factors determine required dosage, administering mode and dosage regimen.
In general, can the aqueous physiological buffer containing 0.1 to 10%w/v conjugates be configured to the conjugate of the present invention In solution, used for injection.Typical dosage range is from 1 μ g/kg to 1g/kg (weight), 1 time a day.Preferred dosage model Enclose is daily 0.01mg/kg to 20mg/kg weight/daily or weekly or child's equivalent dose.Preferred dose of drug to be administered Amount is likely to be dependent on the type and extent of the progress of such as disease or illness, the general health of particular patient, selected chemical combination The relative biological efficacy of object, the formula of drug, administration route (intravenous, intramuscular or other), drug specify transport approach Pharmacokinetic property and injection speed (inject or continuous infusion) and dosage regimen (number of repetition in given time).
The conjugate of the present invention can also be administered in a unit, and wherein term " unit dose " is to refer to give The single dose of patient, and easily can dispose and pack, while active conjugate itself or medicine as described below Acceptable composition on keeps physics and chemically stable unit dose.Typical daily total dose range be from 0.01 to 100mg/kg weight.As general guide principle, the unit dose of the mankind in the range of 1mg to 3000mg daily or Either 3 weeks 2 weeks or every month weekly.Unit dosage ranges preferably 1 to 500mg, weekly to four times, more preferably 10mg to 500mg, weekly.Conjugate provided herein can by with one or more pharmaceutically acceptable excipient It mixes and is configured to pharmaceutical composition.Such units dosage composition can be administered orally, such as tablet, simple capsule or The drug of the form of soft gel capsule；Or intranasal administration, such as pulvis, the drug of nasal drop or aerosol；Or percutaneous drug delivery, such as make With topical ointment, emulsifiable paste, lotion, gel or spray or pass through transdermal patch delivery.
Drug/cytotoxic agent
The drug that can be coupled with the cell-binding molecules in the present invention is the small-molecule drug for including cytotoxic agent, can To be connected to, or it is connected on cell binding agent after being modified.It is 100 that " small-molecule drug " in the present invention, which refers to molecular weight, To 1800, more excellent 120 to 1400 organic and inorganic or metallo-organic compound.These small-molecule drugs are in the document of this field It has been be fully described that, it is incorporated herein by reference such as WO05058367A2 and United States Patent (USP) 4,956,303 etc..Small-molecule drug includes Known drug and drug i.e. to be disclosed.
Known drug includes but not limited to：
1) chemotherapeutics：A) alkylating agent, such as mustargen：That is general for chlorobenzene, chlorine Pune piperazine, cyclophosphamide, Dacarbazine, estradiol Mustargen, ifosfamide, mustargen, hydrochloride oxygen amine, nitrous oxide mustard, amlodipine hydrochloride, Mycophenolic Acid, dulcitol, piperazine Moor bromine alkane, novoembichin, phenesterin, prednimustine, thio-tepa, Trofosfamide pair, uracil；CC-1065 (including its Ah more comes Newly, Carzelesin and Bizelesin synthetic analogues)；Multi-kanamycin (including synthetic analogues KW-2189 and CBI-TMI)；Benzene And phenodiazine Zhuo dimer (such as pyrrolo- Benzodiazepine (PBD) or the U.S. mycin of support, indoles and Benzodiazepine, imidazo benzo The dimer of thiophene phenodiazine Zhuo or oxazolidine and Benzodiazepine)；Nitroso ureas (Carmustine, lomustine, chlorination clostridium element, Fotemustine, Nimustine, Rameau department spit of fland)；Alkylsulfonate (busulfan, 5a,6,9,9a-hexahydro-6,9-methano-2,4,5a,6,9,9a-hexahydro-6,9-methano-2,4 and sulphur)；Triazenes (Dacca bar Piperazine)；Compound containing platinum (carboplatin, cis-platinum, oxaliplatin)；Ethylene imine class, such as benzodihydropyrone, Carlow ketone, meturedepa With black thunder DOPA；Aziridine and methyl melamine, including hemel, diethylenetriamine, triethyl phosphine amide, Sanya Ethylenebis dithiocarbamate phosphamide and trihydroxy methyl methyl amine；B) plant alkaloid：As (vincristine, vincaleukoblastinum are long for vinca alkaloids Fields for spring sowing are pungent, vinorelbine, navelbine)；Taxoid (taxol, Docetaxel) and the like；Maytansine (DM1, DM2, DM3, DM4, maytansine and Ansamycin) and the like；Cryptophycin (especially 1 Hes of cryptophycin cryptophycin 8)；Epothilones, soft coral alcohol, discodermolide, bryostatin, aplysiatoxin, auspicious statin difficult to understand, Tubulysin, cephalostatin, pancratistatin, sarcodictyin, sponge inhibin；C) DNA topoisomerases Enzyme inhibitor, such as Etoposide replace Buddhist nun (9-aminocamptothecin, camptothecine, crisnatol, daunomycin, Etoposide, phosphoric acid Etoposide, Irinotecan, mitoxantrone, Novantrone, retinoic acid (retinol), Teniposide, topotecan, 9- nitro camplotheca acuminatas Alkali (RFS 2000))；Mitomycin (mitomycin C)；D) antimetabolite, such as antifol, DHFR inhibitor (first ammonia butterfly Purine, trimetrexate, denopterin, pteropterin, ammonia petrin (4-aminobenzoic acid) or other folacins)；IMP dehydrogenase presses down Preparation (Mycophenolic Acid, Tiazofurine, Ribavirin, EICAR)；Ribonucleotide reductase inhibitors (hydroxycarbamide, Deferoxamine)； Pyrimidine analogue, uracil analogues (ancitabine, azacitidine, 6- azauracils, capecitabine (Xeloda), Carmofur, Cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5 FU 5 fluorouracil, floxuridine, ratitrexed (Tomudex)；Analogue of cytosine (cytarabine, cytarabin, fludarabine)；(sulphur azoles is fast for purine analogue Purine, fludarabine, mercaptopurine, thiamine, thioguanine)；Folic acid supplement, such as Fu Luolin acid；E) hormonotherapy agent, such as receptor Antagonist, antiestrogenic (megestrol acetate, Raloxifene, tamoxifen), (his woods of Goss, acetic acid bright third are auspicious for LHRH excitants Woods)；(Bicalutamide, Flutamide, Ka Lusi ketone, his androsterone of propionic acid times, table Androstanediol, Goserelin, bright third is auspicious for antiandrogen Woods, U.S. Tilidine, Nilutamide, Testolactone, Trilostane and other inhibitor for androgen)；R8923, vitamine D3 Analog (CB1093, EB1089KH1060, Vitamin D3, ergocalciferol)；Photodynamic therapy agent (Verteporfin, phthalocyanine, Photosensitizer Pc4, de-methoxy-hypocrellin A)；Cell factor (interferon-' alpha ', interferon-γ, tumor necrosis factor (TNF), People's albumen containing TNF)；F) kinase inhibitor, such as BIBW 2992 (anti-EGFR/Erb2), Imatinib, Gefitinib, piperazine adds He is Buddhist nun, Sorafenib, Dasatinib, Sutent, Tarceva, nilotinib, Lapatinib, Axitinib, pazopanib, Vande Thani, E7080 (anti-vegf R2), mubritinib, Ponatinib (AP24534), bafetinib (INNO-406), Bosutinib (SKI-606), card is rich to replace Buddhist nun, vismodegib, iniparib, and Luso profit replaces Buddhist nun, CYT387, Axitinib, Tivozanib, Sorafenib, bevacizumab, Cetuximab, Herceptin, Lucentis, Victibix, Yi Sipingsi； G) antibiotic, such as Enediyne Antibiotic (Calicheamicin, especially Calicheamicin γ 1, δ 1, α 1 and β 1 (reference J.Med.Chem.1996,39 (11), 2103-2117；Angew Chem Intl.Ed.Engl.1994,33:183-186), it reaches Because of mycin, including dyne mycin A and deoxidation rice mycin, Ai Sipeila mycins, card reaches mycin, C-1027, maduropeptin with And neoearcinostain chromophore and related chromoprotein enediyne antibiotic chromophore), aclacinomysins, D actinomycin D, peace Aspergillin, azaserine, bleomycin, Cetocycline, kalamycin, carminomycin, carzinophillin, adriamycin, adriamycin, Quinoline is for adriamycin, 2- pyrrolinodoxorubicins and deoxydaunorubicin, epirubicin, Aclarubicin, idarubicin, marcomycin, Mycin, mycophenolic acid, Lip river mycin, Peplomycin, Peplomycin, puromycin, triferricdoxorubicin, adriamycin, streptozotocin, chain Urea helps rhzomorph, tubercidin, ubenimex, Zinostatin, zorubicin；H) other, special such as polyketide (kind litchi element) It is not bullatacin and bullatacinone；Gemcitabine, epoxidase element (as blocked luxuriant and rich with fragrance such rice cloth), bortezomib, Sha Lidu Amine, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, Allovectin-7, Xegeva, Provenge, Yervoy, isoprenylation inhibitor (such as Lovastatin), dopaminergic nerve Toxin (such as staurosporin), D actinomycin D (such as actinomycin D, dactinomycin D), bleomycin (such as Bleomycin A2, Bo Lai Mycin B2, Peplomycin), anthracycline antibiotic (such as daunorubicin), adriamycin (adriamycin), idarubicin, table is soft Than star, pirarubicin, zorubicin, mitoxantrone, MDR inhibitor (such as Verapamil), Ca2+Atpase inhibitor is (such as malicious Hu trailing plants Bu Su), inhibitors of histone deacetylase (Vorinostat, romidepsin, pabishta, valproic acid, Mocetinostat (MGCD0103), Belinostat, PCI-24781, grace replace Nuo Te, SB939, Resminostat, Givinostat, AR-42, CUDC-101, sulforaphen, Trichostatin A)；Celecoxib, glitazone, Epigallo-catechin gallate (EGCG), double sulphur Logical sequence, Salinosporamide A；Antiadrenergic drug object, such as aminoglutethimide, mitotane, Trilostane, aceglatone, aldehyde phosphorus Amide, amino-laevulic acid, amsacrine, Arabinoside, bestrabucil, bisantrene, edatraxate, defofamine, Mei Kexin, diaziquone, Eflornithine (DFMO), elfomithine, Elliptinium Acetate, ethyl gluconic acid, gallium nitrate, cytimidine, Hydroxycarbamide, ibandronate, lentinan, Lonidamine, mitoguazone, mitoxantrone, Mopidamol, C-283, spray Si Tading, Phenamet, pirarubicin, podophyllic acid, 2- ethyl hydrazines, procarbazine；Razoxane；Rhizomycin；West Assistant；Spiro germanium；Tenuazonic acid；Triethyleneiminobenzoquinone；Trichlorotriethylamine；Trichothecene (especially T-2 toxin, verrucarine A, Roridine A and anguidine), polyurethane, siRNA, antisense drug and nucleic acid decomposition enzyme.
2) autoimmune disease drug, including but not limited to cyclosporin, cyclosporin A, aminocaproic acid, imuran, Bromocriptine, Chlorambucil, chloroquine, cyclophosphamide, corticosteroid (such as Amcinonide, dexamethasone, Triamcinolone acetonide, third Sour beclomethasone, DHEA, Etanercept, hydroxychloroquine, infliximab, Meloxicam, methotrexate (MTX), Mycophenolate Mofetil, Prednisone, sirolimus, tacrolimus.
3) anti-infectious disease drug, including but not limited to a) aminoglycoside：Amikacin, astromicin, celebrating are big mould Plain (Netilmicin, sisomicin, Isepamicin), hygromycin B, kanamycins (amikacin, Arbekacin, amino deoxidation card That mycin, dibekacin, tobramycin), neomycin (framycetin, paromomycin, ribostamycin), Netilmicin is grand Mycin, streptomysin, tobramycin, verdamicin；B) amphenicols：Azidamfenicol, chloramphenicol, Florfenicol, methyl sulfone Mycin；C) Ansamycin：Geldanamycin, herbimycin；D) Carbapenems：Biapenem, doripenem, ertapenem, Imipenem/cilastatin, Meropenem, Panipenem；E) cephem：Carbacephem (loracarbef), Cefacetrile, chlorine ammonia Parasiticin, Cefradine, cefadroxil, cefalonium, cefaloridine, cefoxitin or cephalothin, cefalexin, cephalo Come star, Cefamandole, cefapirin, BL-S 640, fluorine azoles cephalosporin, cefazedone, ancef, cephalo drawing Ancestor, Cefcapene, Cefdaloxime, cephalo pyrrole, Cefixime, Cefoxitin, cefprozil, cefroxadine, cephalo replace Azoles, cefuroxime, Cefixime, Cefdinir, Cefditoren, cephalo pyrrole, cefetamet, Cefmenoxime, Cefodizime, cephalo Ni Xi, cefoperazone, ceforanide, cefotaxime, cefotiam, Cefozopran, cefalexin, Cefpimizole, Cefpiramide, Cefpirome, Cefpodoxime, cefprozil, Cefquinome, Cefsulodin, cefotaxime, Cefteram, cephalo Cloth is risen, Ceftiolene, Ceftizoxime, Ceftobiprole, ceftriaxone, cefuroxime, Cefuzonam, cephamycin (Cefoxitin, head Spore replaces smooth, cefmetazole), oxygen (carbon) cephem (Flomoxef, cephalo)；F) glycopeptide：Bleomycin, vancomycin (Ao Liwan Star, Te Lawan stars), teicoplanin (Dalbavancin), Ramoplanin, g) glycylcycline：Such as tigecycline, h) beta-lactamase Inhibitor：Penam (Sulbactam, Tazobactam Sodium), oxapenam (clavulanic acid)；I) lincosamide：Clindamycin, woods can be mould Element；J) lipopeptid：Daptomycin, A54145, Ca-dependent antibiotic (CDA)；K) macrolides：Azithromycin, Mycosporin, gram Drawing mycin, Dirithromycin, erythromycin, fluorine thunder mycin, josamycin, ketone lactone (Ketek, cethromycin), medecamycin, Mikamycin, oleandomycin, rifamycin (isoniazid, rifampin, Mycobutin, Rifapentine), sieve mycin, Luo Hong are mould Element, spectinomycin, spiramvcin, tacrolimus (FK506), troleandomycin, Ketek；L) monocycle amine：Aztreonam, for Ji Mo Nan；M) oxazolidinones：Linezolid；N) penicillins：Amoxicillin, ampicillin (Pivampicillin, extra large Lip river XiLin, Bacampicillin, ampicillin, adriamycin), Ah substitutes XiLin, azlocillin, parasiticin, tardocillin parasiticin, Tardocillin penicillin Vl phenoxymethylpenicillin, Crow XiLin, procaine penicillin (U.S. replaces XiLin), mezlocillin, methicillin, Naphthlazole, oxacillin, penamecillin, penicillin, pheneticillin, phenoxymethylpenicillin, Piperacillin, ammonia benzyl west Woods, sulphur benzene XiLin, temocillin, Ticarcillin；O) polypeptide：Bacitracin, colistin, polymyxin B, p) quinolones：I Qu Shaxing, Balofloxacin, Ciprofloxacin, crin is husky, Danofloxacin, Difloxacin, Enoxacin, Enrofloxacin, T-3811, Gatifloxacin, gemifloxacin, Grepafloxacin, Kano trovafloxacin, lavo-ofloxacin, Lomefloxacin, marbofloxacin, Mo Xisha Star, Nadifloxacin, Norfloxacin, Orbifloxacin, Ofloxacin, pefloxacin, trovafloxacin, Grepafloxacin, sitafloxacin, department Pa sand star, Temafloxacin hold in the palm husky star, trovafloxacin；Q) streptogramin：Pristinamycin, Quinupristin/Dalfopristin, r) sulphur Amine drug：Sulfa drugs：Sulfa drugs, sulphadiazine, sulphadiazine, sulphadiazine, salicylazosulfapyridine, sulfanilamide (SN) are different Oxazole, tamoxifen, methoxybenzyl aminopyrimidine-sulfamethoxazole (Compound New Nomin)；S) steroids antibacterials：Such as husband west Acid；T) Tetracyclines：Fortimicin, aureomycin, chlorine rice western ring element, demeclocycline, thunder not former times woods, U.S. western ring element, metacycline, Minocycline, terramycin, penimepicycline, pyrrolidinyl methyl tetracycline, tetracycline, glycylcycline (add ring as replaced Element)：U) other kinds of antibiotic：Annonacin, arsphenamine, bactoprenol inhibitor (bacillus), DANAL/AR inhibitor (seromycin), dictyostatin, circle suberite lactone, soft coral alcohol, Epothilones, ethambutol, Etoposide, method sieve Training south, Fusidic Acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, NAM synthetic inhibitors (such as Phosphonomycin), furantoin, taxol, the western mycin in Pulan, pyrazinamide, Quinupristin/Dalfopristin, rifampin (rifampin), Tazobactam Sodium Tinidazole, black chrysanthemum element.
4) antiviral drugs：A) entrance/fusion inhibitor：A Paweiluo, maraviro, vicriviroc, gp41 (En Fu Wei peptide), PRO 140, CD4 (Ai Bali pearls monoclonal antibody)；B) integrase inhibitor：Merck, elvitegravir, globoidnan A；C) ripe inhibitor：Bevirimat, vivecon；D) neuraminidase inhibitor：Oseltamivir pricks that Meter Wei, Peramivir；E) nucleosides and nucleotide：Abacavir, Ah former times's list Wei, adefovirdipivoxil, A Moxiwei, Abciximab, bromine Husband is fixed, and cidofovir, Clevudine, dexamethasone, Didanosine (ddI), elvucitabine, emtricitabine (FTC), grace is replaced Card Wei, famciclovir, fluorine draw XiLin (5-FU), 3 '-fluorine-substituted 2 ', 3 '-deoxynucleoside analogs (such as 3,3 '-fluoro- 2 ', 3 '- Stavudine (FLT) and 3 '-fluoro- 2 ', 3 '-dideoxyguanosines (FLG), Fomivirsen, 9-guanine, iodoxuridine, Lamivudine (3TC), 1- nucleosides (such as β -1- thymidines and β -1-2'- deoxycytidines), Penciclovir, racivir, Ribavirin, enlightening replace fourth, Stavudine (d4T), tower Ribavirin (viramidine), Sebivo, tenofovir, trifluridine Valaciclovir, figured silk fabrics is more VACV, zalcitabine (ddC), Zidovudine (AZT)；F) non-nucleoside：Amantadine, Ah 's pyridine, capravirine, two virtues Yl pyrimidines (etravirine, rilpivirine), delavirdine, tadenan, emivirine, efavirenz, phosphonic acid (phosphorus Acyl group formic acid), imiquimod, Peg-IFN alpha-2b, Loviride, lodenosine, methisazone, nevirapine, NOV-205, Long-acting interferon α, podophyllotoxin, rifampin, Rimantadine, resiquimod (R-848), tromantadine；G) protease inhibits Agent：Anpunave atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, Lopinavir, Nai Feinawei, Pleconaril, Ritonavir, inverase, telaprevir (VX-950), tipranavir；H) other types of antiviral agent Object：Antioxidase, Abiduoer, OK a karaoke club Nuo Laide, ceragenin, cyanogen Wei Lin-n, diaryl pyrimidine, epigallocatechin Gallate (EGCG), phosphonic acid, lattice Lifei is pungent, taribavirin (viramidine), hydroxycarbamide, KP-1461, and rice replaces Good fortune is new, pleconaril, blendes together inhibitor, Ribavirin, seliciclib.
5) drug of bridging body link through the invention also includes radioactive isotope.Radioactive isotope (radioactivity Nucleic) example have3H,11C,14C,18F,32P,35S,64Cu,68Ga,86Y,99Tc,111In,123I,124I, 125I,131I,133Xe ,177Lu,211At or213Bi.The antibody of labelled with radioisotope can be used for receptor target imaging experiment, or can be used for such as this Targeted therapy (the Wu et al Nature Biotechnology 2005,23 (9) of the antibody-drug conjugates of invention: 1137-1146).Cell-binding molecules, such as antibody can link ligand reagent by the connector of this patent, be marked. Ligand can use document (Current Protocols in Immunology, Volumes 1and 2, Coligen et al, Ed.Wiley-Interscience, New York, N.Y., Pubs. (1991)) described in method combined with radioactive metal, chela Close or generate compound.Can include DOTA, DOTP, DOTMA, DTPA and TETA with the cheland of complexation of metal ions (Macrocyclics, Dallas, TX) etc..
6) pharmaceutically acceptable salt of any of the above described drug, acid or derivative.
In another embodiment, the drug in structure formula (II) and (IV) can be chromonic molecule, and conjugate can be used for Detection, monitoring or the interaction of research cell-binding molecules and target cell.Chromonic molecule can absorb a kind of light, such as ultraviolet Light, fluorescence, infrared light, near infrared light or visible light；Chromonic molecule includes uranidin, red blood cell, iris pigment, and leucocyte is black One kind or a subclass of pigment and bluish-green pigment, one kind of fluorescent molecular (absorbing fluorescent chemical luminous again after light) or One subclass, one kind of visual light transduction molecule or a subclass, one kind of photon molecule or a subclass, the one of luminescent molecule One kind or a subclass of class or a subclass and Fluorescein compound.
Chromonic molecule may be selected from but not limited to, nonprotein organic fluorescence group, such as xanthene derivative (fluorescein, sieve Red bright, Oregon is green, Yihong and texas Red)；Cyanine derivative object (cyanine, indoles carbocyanine, oxa- cyanine, thiocyanine And merocyanine)；Squaric acid derivertives and cyclosubstituted side acid, including Seta, SeTau and Square dyestuff；Naphthalene derivatives (dansyl and Prodan derivative)；Coumarin derivative；(pyridyl group oxazole, nitro benzoxazoles and benzo dislike two to oxadiazole derivatives Azoles)；Anthracene derivant (Anthraquinones, including DRAQ5, DRAQ7 and CyTRAK oranges)；Pyrene derivatives (cascade blue etc.)；Oxazines derivative (Nile red, Nile blue, cresol-purple, oxazines 170 etc.)；Acridine derivatives (flavol flavine, acridine orange, acridine yellow etc.)；Aryl first Amine derivative (auramine, crystal violet, malachite green) and tetrapyrrole derivative (porphines, phthalocyanine, bilirubin).
Chromophores are selected from any analogs and derivatives of following fluorescent chemicals：CF dyestuffs (Biotium), DRAQ and CyTRAK probes (BioS-tatus), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLight Fluor (Thermo Scientific, Pierce), Atto and Tracy (Sigma Aldrich), FluoProbes (Interchim), Abberior dyestuffs (Abberior), DY and MegaStokes dyestuff (Dyomics), Sulfo Cy dyestuffs (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTau and Square dyestuffs (Biosearch Technologies), SureLight dyestuffs (APC, RPEPerCP, Phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech).
The widely used example that can connect precursor reactant or the fluorescent chemicals of coupling with the present invention has：Allophycocyanin (APC), amino kermes albumen, APC-Cy7 conjugates, BODIPY-FL, Cascade Blue, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, fluorescein, FluorX, Hydroxycoumarin, Sulforhodamine B, lucifer yellow, Me- methoxy coumarins, NBD, Pacific Blue, Pacific Orange, PE-Cy5 conjugate, PE-R- phycoerythrin (PE), Red 613, Seta- 555-Azide, Seta-555-DBCO, Seta-555-NHS, Seta-580-NHS, Seta-680-NHS, Seta-APC-780, Seta-PerCP-680, Seta-R-PE-670, SeTau-380-NHS, SeTau-405- maleimide, SeTau-405- NHS, SeTau-425-NHS, SeTau-647-NHS, Texas Red, TRITC, TruRed, X-Rhodamine.
It can be connected with the connector of the present invention, the fluorescent chemicals for studying nucleic acid or protein are selected from followingization Close object or derivatives thereof：7-AAD (7-aminoactinomycin D, CG- selectivity), acridine orange, chromomycin A3, CyTRAK oranges (Biostatus), DAPI, DRAQ5, DRAQ7, ethidium bromide, Hoechst33258, Hoechst33342, LDS 751, radiance Mycin, propidium iodide (PI), SYTOX is blue, and SYTOX is green, SYTOX oranges, thiazole orange, TO-PRO, cyanine dyes monomer, TOTO-1, TO- PRO-1, TOTO-3, TO-PRO-3, YOSeta-1, YOYO-1.It can be connected with the connector of the present invention, for studying cell Fluorescent chemicals, selected from following compounds or derivatives thereof：DCFH (2', 7'- dichlorofluorescin, oxidised form), DHR (dihydro Rhodamine 123, oxidised form, photochemical catalytic oxidation), Fluo-3 (AM esters, pH>6), Fluo-4 (AM esters, pH7.2), Indo-1 (AM esters, low high calcium (Ca 2+)), SNARF (pH 6/9).Preferred fluorescent chemicals are selected from：Allophycocyanin (APC), AmCyan1 (tetramer, Clontech), AsRed2 (tetramer, Clontech), Ji is green (monomer, MBL), Azurite, B- phycoerythrin (BPE), Cerulean, CyPet, DsRed monomers (Clontech), DsRed2 (" RFP ", Clontech), EBFP, EBFP2, ECFP, EGFP (weak dimer, Clontech), Emerald (weak dimer, Invitrogen), EYFP (weak dimer, Clontech), GFP (S65A mutation), GFP (S65C mutation), GFP (S65L mutation) GFP (Y66H mutation), GFP (Y66W mutation), GFPuv, HcRed1, J-Red, Katusha, Kusabira Orange are (single poly- Body, MBL), mCFP, mCherry (monomer, MBL), mKate (TagFP635, monomer, Evrogen), mKeima-Red (monomer, MBL), mKO, mOrange, mPlum, mRaspberry, mRFP1 (monomer, the laboratories Tsien), mStrawberry, mTFP1, MTurquoise2, P3 (phycobilisome compound), perdinin-Chlorophyll-protein complex (PerCP), R- Phycoerythrin (RPE), T-Sapphire, TagCFP (dimer, Evrogen), TagGFP (dimer, Evrogen), TagRFP (dimer, Evrogen), TagYFP (dimer, Evrogen), tdTomato (series connection dimer), Topaz, TurboFP602 (dimer, Evrogen), TurboFPP635 (dimer, Evrogen), TurboGFP (dimer, Evrogen), TurboRFP (dimer, Evrogen), TurboYFP (dimer, Evrogen), Venus, wild type GFP types, YPet, ZsGreen1 (tetramer, Clontech), ZsYellow1 (tetramer, Clontech).
In another embodiment it is preferred that the connector by this patent be connected to the cell toxicants of cell-binding molecules Agent is tubulysin, maytansine, taxanes, CC-1065 analogs, daunorubicin and doxorubicin compound, Benzodiazepine Dimer (such as pyrrolo- Benzodiazepine (PBD) or the U.S. mycin of support, indoles and Benzodiazepine, imidazo benzo thiophene phenodiazine Zhuo Or the dimer of oxazolidine and Benzodiazepine), block and found mycin class and Enediyne Antibiotic, D actinomycin D, nitrogen silk rhzomorph is won Bleomycin, epirubicin, tamoxifen, idarubicin, dolastatin, the auspicious statin of Australia (such as MMAE, MMAF, the auspicious statin of Australia PYE, the auspicious statin TP of Australia, the auspicious statin 2-AQ of Australia, 6-AQ, EB (AEB) and EFP (AEFP)), multi-kanamycin, thiotepa, Changchun is new Alkali, the Tallins Ban meter, nazumamide, microginin, radiosumin, alterobactin, microsclerominmin, Theonellamide, esperamicin, PNU-159682 and the like and derivative.
Tubulysin is the cytotoxic agent for being preferably used in conjugation coupling, this field document can be referred to, according to known Method is detached from natural origin or prepared by synthetic method, such as Balasubramanian, R.；et al.J.Med.Chem., 2009,52,238–240.Wipf,P.；et al.Org.Lett.,2004,6,4057–4060.Pando,O.；et al.J.Am.Chem.Soc.,2011,133,7692–7695.Reddy,J.A.；et al.Mol.Pharmaceutics,2009, 6,1518–1525.Raghavan,B.；et al.J.Med.Chem.,2008,51,1530–1533.Patterson,A.W.；et al.J.Org.Chem.,2008,73,4362–4369.Pando,O.；et al.Org.Lett.,2009,11(24),pp5567– 5569.Wipf,P.；et al.Org.Lett.,2007,9(8),1605–1607.Friestad,G.K.；Org.Lett., 2004,6,pp 3249–3252.Hillary M.Peltier,H.M.；et al.J.Am.Chem.Soc.,2006,128, 16018–16019.Chandrasekhar,S.；et al.J.Org.Chem.,2009,74,9531–9534.Liu,Y.；et al.Mol.Pharmaceutics,2012,9,168–175.Friestad,G.K.；et al.Org.Lett.,2009,11, 1095–1098.Kubicek,K.；et al.,Angew Chem Int Ed Engl,2010.49,4809-12.Chai,Y.；et al.,Chem Biol,2010,17:296-309.Ullrich,A.；et al.,Angew Chem Int Ed Engl,2009, 48,4422-5.Sani,M.；et al.Angew Chem Int Ed Engl,2007,46,3526-9.Domling,A.；et Al., Angew Chem Int Ed Engl, 2006.45,7235-9. patents：Zanda,M.；et al, Can.Pat.Appl.CA2710693(2011).Chai,Y.；et al.Eur.Pat.Appl.2174947(2010),PCT WO 2010034724.Leamon,C.；et al,PCT WO 2010033733,WO 2009002993.Ellman,J.；et al, PCT WO 2009134279；PCT WO 2009012958,US appl.20110263650,20110021568, Matschiner,G.；et al,PCT WO 2009095447.Vlahov,I.；et al,PCT WO 2009055562,WO 2008112873.Low,P.；et al,PCT WO 2009026177.Richter,W.,PCT WO 2008138561.Kjems, J.；et al,PCT WO 2008125116.Davis,M.；et al,PCT WO 2008076333.Diener,J.；et al, U.S.Pat.Appl.20070041901,WO 2006096754.Matschiner,G.；et al,PCT WO 2006056464.Vaghefi,F.；et al,5PCT WO 2006033913.Doemling,A.,Ger.Offen.DE 102004030227；PCT WO 2004005327；WO 2004005326；WO2004005269.Stanton,M.；et al, U.S.Pat.Appl.Publ.20040249130.Hoefle,G.；et al,Ger.Offen.DE 10254439；DE 10241152；DE 10008089.Leung,D.；et al,WO 2002077036.Reichenbach,H.；et al, Ger.Offen.DE 19638870；Wolfgang,R.；US 20120129779,Chen,H.,US appl.20110027274. The preferred structure for the tubulysin being connect with cell-binding molecules is described in patent PCT/IB2012/053554.
Pass through the example such as T01, T02, T03, T04, T05 of the antibody-tubulysin conjugate structures that bridging junctor connects And T06：
Wherein mAb is antibody；Z3It is H, OP (O) (OM1)(OM2), OCH2OP(O)(OM1)(OM2), OSO3M1Or O-, NH-, S- or CH2Glucosides (glucosides, galactoside, mannoside, glucoside, fructoside etc.)；M1And M2It independently is H, Na, K, Ca, Mg, NR1R2R3；N isX1,X2,R1,R2And R3It is identical as defined in formula (I).
Calicheamicin and relevant enediyne antibiotic are preferred cytotoxic agents, can refer to document：Nicolaou, K.C.et al,Science 1992,256,1172-1178；Proc.Natl.Acad.Sci USA.1993,90,5881- 5888 and United States Patent (USP) 4,970,198；5,053,394；5,108,912；5,264,586；5,384,412；5,606,040；5, 712,374；5,714,586；5,739,116；5,770,701；5,770,710；5,773,001；5,877,296；6,015, 562；6,124,310；8,153,768.The example of the structure of the antibody-calecheamicin analog connected by bridging junctor is such as C01：
Wherein mAb is antibody；N isX1, X2, R1, R2And R3It is identical as defined in formula (I).
Maytansine is preferred cytotoxic agent in this patent, and maytansine and its homologue are retouched in following United States Patent (USP) It states：4,256,746；4,361,650；4,307,016；4,294,757；4,294,757；4,371,533；4,424,219；4, 331,598；4,450,254；4,364,866；4,313,946；4,315,929；4,362,663；4,322,348；4,371, 533；4,424,219；5,208,020；5,416,064；5,208,020；5,416,064；6,333.410；6,441,163；6, 716,821,7,276,497；7,301,019；7,303,749；7,368,565；7,411,063；7,851,432 and 8,163, 888.The example such as M01 of one antibody-maytansine conjugate：
Wherein mAb is antibody；N isX1, X2, R1, R2And R3It is identical as defined in formula (I).
Taxane, including taxol (a kind of cytotoxicity natural products) and Docetaxel (a kind of semi-synthetic derivative) And the like, it is the preferred cytotoxic molecule of this patent, is described in the following literature：K C.Nicolaou et al.,J.Am.Chem.Soc.1995,117,2409-2420；Ojima et al,J.Med.Chem.1996,39:3889- 3896；1997,40,267-278；2002,45,5620-5623；Ojima et al.,Proc.Natl.Acad.Sci.,1999, 96:4256-4261；Kim et al.,Bull.Korean Chem.Soc.,1999,20,1389-1390；Miller,et al.J.Med.Chem.,2004,47,4802-4805；United States Patent (USP) 5,475,011 5,728,849,5,811,452；6,340, 701；6,372,738；6,391,913,6.436,931；6,589,979；6,596,757；6,706,708；7,008,942；7, 186,851；7,217,819；7,276,499；7,598,290 and 7,667,054.
Example such as Tx01, Tx02 and the Tx03 for the conjugate structure that antibody-taxane is connected via bridging junctor：
Wherein mAb is antibody；N isX1, X2, R1, R2And R3It is identical as defined in formula (I).
CC-1065 analogs and multi-kanamycin analog are also to be connect with the bridging junctor of this patent, preferred cell toxicant Property agent.The example and its synthesis of CC-1065 analogs and multi-kanamycin analog are found in：Warpehoski,et al, J.Med.Chem.31:590-603(1988),D.Boger et al.,J.Org.Chem；66；6654-6661,2001；The U.S. Patent 4169888,4391904,4671958,4816567,4912227,4923990,4952394,4975278,4978757, 4994578,5037993,5070092,5084468,5101038,5117006,5137877,5138059,5147786, 5187186,5223409,5225539,5288514,5324483,5332740,5332837,5334528,5403484, 5427908,5475092,5495009,5530101,5545806,5547667,5569825,5571698,5573922, 5580717,5585089,5585499,5587161,5595499,5606017,5622929,5625126,5629430, 5633425,5641780,5660829,5661016,5686237,5693762,5703080,5712374,5714586, 5739116,5739350,5770429,5773001,5773435,5786377 5786486,5789650,5814318, 5846545,5874299,5877296,5877397,5885793,5939598,5962216,5969108,5985908, 6060608,6066742,6075181,6103236,6114598,6130237,6132722,6143901,6150584, 6162963,6172197,6180370,6194612,6214345,6262271,6281354,6310209,6329497, 6342480,6486326,6512101,6521404,6534660,6544731,6548530,6555313,6555693, 6566336,6,586,618,6593081,6630579,6,756,397,6759509,6762179,6884869,6897034, 6946455,7,049,316,7087600,7091186,7115573,7129261,7214663,7223837,7304032, 7329507,7,329,760,7,388,026,7,655,660,7,655,661,7,906,54 5, and 8,012,978.Through bridging The example of the antibody-CC-1065 analog structures of junctor connection is as follows：
Wherein mAb is antibody；N isZ4It is H, PO (OM1)(OM2), SO3M1, CH2PO(OM1)(OM2), CH3N (CH2CH2)2NC (O)-, O (CH2CH2)2NC (O)-or glucosides；X3It is O, NH, NHC (O), OC (O), CO or default；X1、X2、R1、 R2、M1And M2It is identical as defined in formula (I).
Daunorubicin/doxorubicin analog is also preferably used for the coupling connected by the bridging junctor of this patent.Preferably Structure and its synthesis can refer to document：Hurwitz, E., et al., Cancer Res.1975,35,1175-1181.Yang, H.M.,and Reisfeld,R.A.,Proc.Natl.Acad.Sci.1988,85,1189-1193；Pietersz,C.A.,E., et al.,E.,et al.,Cancer Res.1988,48,926-9311；Trouet,et al.,1982,79,626-629； Z.Brich et al.,J.Controlled Release,1992,19,245-258；Chen et al.,Syn.Comm., 2003,33,2377-2390；King et al.,Bioconj.Chem.,1999,10,279-288；King et al., J.Med.Chem.,2002,45,4336-4343；Kratz et al.,J Med Chem.2002,45,5523-33；Kratz et al.,Biol Pharm Bull.Jan.1998,21,56-61；Lau et al.,Bioorg.Med.Chem.1995,3, 1305-1312；Scott et al.,Bioorg.Med.l Chem.Lett.1996 6,1491-1496；Watanabe et al.,Tokai J.Experimental Clin.Med.1990,15,327-334；Zhou et al., J.Am.Chem.Soc.2004,126,15656-7；WO 01/38318；United States Patent (USP) 5,106,951；5,122,368；5,146, 064；5,177,016；5,208,323；5,824,805；6,146,658；6,214,345；7569358；7,803,903；8, 084,586；8,053,205.The example of the antibody connected by bridging junctor-doxorubicin analog structure is as follows：
Wherein mAb is antibody；N isX3It is H, O, NH, NHC (O), NHC (O) NH, C (O) or OC (O)；X1、X2、 R1、R2、M1And M2It is identical as defined in formula (I).
The auspicious statin of Australia is the preferred cytotoxic agent connected with bridging junctor with dolastatin.The auspicious statin of Australia is (such as more The auspicious statin E (AE) of Australia, the auspicious statin EB (AEB) of Australia, the auspicious statin EFP (AEFP) of Australia, the auspicious statin E (MMAE) of monomethyl Australia, monomethyl The auspicious statin F (MMAF) of Australia, the phenylalanine variant of Australia auspicious statin F phenylenediamines (AFP) and MMAE) it is the similar of dolastatin Object is described in the following literature：Int.J.Oncol.1999,15,367-72；Molecular Cancer Therapeutics,2004,3(8),921-932；U.S. Patent application 11134826,20060074008,2006022925 is beautiful State's patent 4414205,4753894,4764368,4816444,4879278,4943628,4978744,5122368, 5165923,5169774,5286637,5410024,5521284,5530097,5554725,5585089,5599902, 5629197,5635483,5654399,5663149,5665860,5708146,5714586,5741892,5767236, 5767237,5780588,5821337,5840699,5965537,6004934,6033876,6034065,6048720, 6054297,6054561,6124431,6143721,6162930,6214345,6239104,6323315,6342219, 6342221,6407213,6569834,6620911,6639055,6884869,6913748,7090843,7091186, 7097840,7098305,7098308,7498298,7375078,7462352,7553816,7659241,7662387, 7745394,7754681,7829531,7837980,7837995,7902338,7964566,7964567,7851437, 7994135.The example for the conjugate structure that the auspicious statin of antibody-Australia is connected by bridging junctor such as AuO 1, AuO 2, AuO 3, AuO 4 and AuO 5：
Wherein mAb is antibody；N isX3For CH2, O, NH, NHC (O), NHC (O) NH, C (O), OC (O) or default； X4It is CH2, C (O), C (O) NH, C (O) N (R1) or C (O) O；X1, X2, R1, R2And R3It is identical as defined in formula (I).
Benzodiazepine dimer (such as pyrrolo- Benzodiazepine (PBD), hold in the palm U.S. mycin, indoles and Benzodiazepine, miaow The dimer of azoles and benzo thiophene phenodiazine Zhuo or oxazolidine and Benzodiazepine) it is preferred cytotoxic molecule in the present invention, It is also described in the document of this field：United States Patent (USP) 8,163,736；8,153,627；8,034,808；7,834,005；7,741, 319；7,704,924；7,691,848；7,678,787；7,612,062；7,608,615；7,557,099；7,528,128；7, 528,126；7,511,032；7,429,658；7,407,951；7,326,700；7,312,210；7,265,105；7,202, 239；7,189,710；7,173,026；7,109,193；7,067,511；7,064,120；7,056,913；7,049,311；7, 022,699；7,015,215；6,979,684；6,951,853；6,884,799；6,800,622；6,747,144；6,660, 856；6,608,192；6,562,806；6,977,254；6,951,853；6,909,006；6,344,451；5,880,122；4, 935,362；4,764,616；4,761,412；4,723,007；4,723,003；4,683,230；4,663,453；4,508, 647；4,464,467；4,427,587；4,000,304；U.S. Patent application 20100203007,20100316656, 20030195196.Example such as PB01, PB02, PB03, PB04, the PB05 of antibody-Benzodiazepine dimer conjugate structure, PB06, PB07 and PB08：
Wherein mAb is antibody；N isX3For CH2, O, NH, NHC (O), NHC (O) NH, C (O), OC (O) or default； X4It is CH2, C (O), C (O) NH, C (O) N (R1) or C (O) O；X1, X2, R1, R2And R3It is identical as defined in formula (I).In addition, R1With/ Or R2It can be default.
Drug/cytotoxic agent for being connected with the bridging junctor of the present invention, can be the drug/molecule being described above Any analog and/or derivative.It should be appreciated that each drug/cytotoxic agent as described herein can be modified, institute It obtains compound and still retains relative specific and/or activity.It should also be understood by those skilled in the art that many other compounds also can generation For drug/cytotoxic agent as described herein.Therefore, drug/cytotoxic agent of the invention further includes the similar of these compounds Object and derivative.
Whole documents in cited herein and the following examples are included in reference.
Embodiment
The present invention is further explained with the following examples, and the content of these embodiments is not intended to limit the present invention's Range.Cell line in embodiment, other than specified otherwise, be according to Unite States Standard culture collection (ATCC), The condition of German Culture Collection Center (DSMZ) or Chinese Academy of Sciences's Shanghai Cell Culture Center specification preserves.In addition to specified otherwise Outside, cell culture reagent comes from Invitrogen companies.All anhydrous reagents are obtained by commercial sources, and are stored in In Sure-Seal air-tight bottles.Other reagents and solvent are bought according to highest specification, without further handling when use. Varian Prostar HPLC carry out preparing HPLC purifying.NMR data obtains on Varian Mercury 400MHz, chemistry Displacement is as unit of ppm, and tetramethylsilane is to refer to (0.00ppm), and the unit of coupling constant (J) is Hz.Mass spectrometric data exists It is obtained on Waters XevoQTof mass spectrographs (connection Waters Acquity UPLC high performance liquid chromatographs and TUV detectors) ?.
1. 3- of example (2- (2- (2- hydroxyl-oxethyls) ethyoxyl) ethyoxyl) propanoic acid tert-butyl ester (34)
Be added under stiring into the anhydrous THF of 350mL 80mg (0.0025mol) metallic sodiums and triethylene glycol 2 (150.1g, 1.00mol).After sodium is completely dissolved, tert-butyl acrylate (24mL, 0.33mol) is added.The solution is stirred at room temperature 20 Hour, it is used in combination the 1.0M HCl of 8mL to neutralize.After vacuum revolving removes solvent, diluted with brine (250mL), ethyl acetate (3 × 125mL) extraction.The organic layer merged is washed with brine (100mL) and water (100mL), sodium sulphate drying removes solvent.It will Gained colorless oil is dried in vacuo, and obtains the product 34 of 69.78g (yield 76%).1H NMR:1.41(s,9H),2.49(t, 2H, J=6.4Hz), 3.59-3.72 (m, 14H) .ESI MS m/z-C13H25O6(M-H), calculated value .277.17, measured value 277.20.
2. 3- of example (2- (2- (2- (tosyloxy) ethyoxyl) ethyoxyl) ethyoxyl) propanoic acid tert-butyl ester (35)
34 (10.0g, 35.95mmol) are dissolved in acetonitrile (50.0mL), after pyridine (20.0mL) is added, at 30 minutes The interior 50mL acetonitrile solutions that toluene sulfochloride (7.12g, 37.3mmol) is added dropwise by charging hopper.After 5 hours, TLC analyses are aobvious Show that reaction is completed.The pyridine hydrochloride to be formed is filtered out, the solvent in filtrate is removed, by residue silica gel column purification, 20% second The hexane solution of acetoacetic ester obtains 11.2g (76% yield) compound 35 to neat ethyl acetate.1H NMR:1.40(s, 9H), 2.40 (s, 3H), 2.45 (t, 2H, J=6.4Hz), 3.52-3.68 (m, 14H), 4.11 (t, 2H, J=4.8Hz), 7.30 (d, 2H, J=8.0Hz), 7.75 (d, 2H, J=8.0Hz)；ESI MS m/z+C20H33O8S (M+H), calculated value 433.18, actual measurement Value 433.30.
3. 3- of example (2- (2- (2- nitrine base oxethyl) ethyoxyl) ethyoxyl) propanoic acid tert-butyl ester (36)
2- (2- (2- (2- (tosyloxy) ethyoxyl) ethyoxyl) ethoxies are added into DMF (50mL) under stiring Base)-propanoic acid tert-butyl ester 35 (4.0g, 9.25mmol) and sodium azide (0.737g, 11.3mmol).Reaction is heated to 80 DEG C, 4 After hour, TLC is analysis shows that reaction is completed.Reactant is cooled to room temperature, water (25mL) is used in combination to be quenched.With ethyl acetate (3 × It 35mL) extracts, combined organic layer is dried with anhydrous magnesium sulfate, filters, solvent is removed in vacuum.Crude product (about 90%TLC is pure) is no It is used through being further purified.1H NMR(CDCl3):1.40 (s, 9H), 2.45 (t, 2H, J=6.4Hz), 3.33 (t, 2H, J= 5.2Hz),3.53-3.66(m,12H).ESI MS m/z+C13H26N3O8(M+H), calculated value .304.18, measured value 304.20.
The 4. 13- amino -4,7,10- trioxa dodecanoic acid tert-butyl esters (37) of example
13- amino-bis- (4,7, the 10- trioxa dodecanoic acid tert-butyl ester), 38.
By crude product 36 (5.0g,) be dissolved in ethyl alcohol (80mL), 300mg 10%Pd/C are added.By the body System vacuumizes, and is passed through the hydrogen of 2atm with vigorous stirring, is then stirred at room temperature overnight hydrogenation reactor, and TLC is aobvious Show that starting material disappears.Coarse reactants filter over celite, and ethyl alcohol is used in combination to wash Celite pad.After removing solvent, in silicagel column The upper dichloromethane mixed liquor using methanol (5% to 15%) and 1% triethylamine is purified by flash, and obtains 13- amino -4,7,10- (1.83g, yield the 44%ESI MSm/z+C of trioxa dodecanoic acid tert-butyl ester butyl ester 3713H27NO5(M+H), calculated value 278.19, Measured value 278.30) and 13- amino-bis- (4,7, the 10- trioxa dodecanoic acid tert-butyl esters) 38 (2.58g, 32% yield, ESI MS m/z+C26H52NO10(M+H), calculated value 538.35, measured value 538.40)
5. 3- of example (2- (2- (2- amino ethoxies) ethyoxyl) ethyoxyl) propionate hydrochlorate (39)
Under stiring to 13- amino -4,7, the Isosorbide-5-Nitrae-of the 10- trioxa dodecanoic acids tert-butyl ester 37 (0.80g, 2.89mmol) 10ml HCl (36%) are added in dioxane (30ml) solution.After 0.5 hour, TLC is mixed by reaction analysis shows that reaction completion Close object concentration, and with EtOH and EtOH/ toluene azeotropic concentrations, form the HCl salt (purity of title product>90%, 0.640g, 86% yield), without being further purified.ESI MS m/z+C9H20NO5(M+H), calculated value 222.12, measured value 222.20.
6. 13- amino of example-bis- (4,7,10- trioxa dodecanoic acids hydrochloride) (40)
Under stiring, to 13- amino-bis- (4,7, the 10- trioxa dodecanoic acid tert-butyl esters) 38 (1.00g, 1.85mmol) Isosorbide-5-Nitrae-dioxane (30ml) solution in be added 10ml HCl (36%).After 0.5 hour, TLC is completed analysis shows that reacting, will Reaction mixture concentrate, and with EtOH and EtOH/ toluene azeotropic concentrations, form the hydrochloride (purity of title product>90%, 0.71g, 91% yield), without being further purified.ESI MS m/z+C18H36NO10(M+H), calculated value 426.22, measured value 426.20。
Bis- (2,5- dioxo pyrrolidin -1- bases) the butyl- 2- alkynes diacid salts (9) of example 7.
Into DMA (100ml) solution of butyl- 2- alkynes diacid 8 (2.0g, 17.54mmol) be added NHS (5.0g, 43.4mmol) and EDC (12.0g, 62.5mmol).Mixture is stirred overnight in the dark, concentrate and is purified on a silica gel column, is used EtOAc/DCM(1:10) it elutes, obtains title compound 9 (4.10g, 76% yield).ESI MS m/z+C12H9N2O8(M+H), Calculated value 309.03, measured value 309.20.
8. 4,7- dioxo -5- alkynes diacid (15) of example
At 0 DEG C, under stirring to containing bis- (trimethyl silyl) acetylene (5.0g, 29.34mmol) and iodine (0.37g, In dichloromethane (100mL) solution 1.45mmol), succinyl chloride (18.11g, 116.83mmol) is added dropwise.After adding, Mixture is stirred at room temperature (TLC is monitored, about 2 hours) until the reaction is complete.Reaction mixture is quenched with water (15mL), It is used in combination dichloromethane (3 × 70mL) to extract.Combined extract is washed with 15% hypo solution, uses anhydrous Na2SO4 It is dry, it is concentrated in vacuo.Products therefrom through silica gel column chromatography (100-200 mesh, 5% to 10%H2O/ acetonitriles) purifying, obtain title Product (5.50g, yield 85%).ESI MS m/z-C10H9O6(M-H), calculated value 226.05, measured value 226.10.
Example 9. (R, R, S, S, R, 4R, 4'R) -5,5'- (((4,7- dioxo -5- alkynes diyl) 3,1- phenylenes)) two (4- (2- ((1R, 3R) -1- acetoxy-3s-((2S, 3S)-N, 3- dimethyl -2- ((R) -1- methyl piperidine valeryls amino) -4- first Base amyl) thiazole -4- formamido groups) -2 methyl valeric acid) (79)
(4R) -4- (2- ((1R, 3R) -1- are added into THF (3.0ml) solution of compound 9 (25mg, 0.081mmol) Acetoxy-3-((2S, 3S)-N, 3 ((R)-1- methyl piperidine-2- formamido groups) valeryl amino)-4- methyl amyls) thiazole- 4- formamido groups) -5- (3- amino-4-hydroxylphenyls) -2 methyl valeric acid, 51 (Huang Y.et al, Med Chem.#44, 249thACS National Meeting, Denver, CO, Mar.22~26,2015；WO2014009774) (151mg, THF (4.0ml) solution 0.199mmol) and phosphate buffer (4mL, 100mM Na 2HPO 4, pH 7.0).At room temperature After stirring 4 hours, concentration of reaction solution simultaneously prepares HPLC (250mm × 20mm) purifying with C-18, with water/ethyl alcohol (90% to 50% Water, 55 minutes, flow velocity 15ml/ minutes).Merge the component containing product, concentrate and crystallized with EtOH/ n-hexanes, obtains title Compound (73mg, 53% yield).ESI MS m/z+C86H122N12NaO20S2(M+Na), calculated value 1729.83, measured value 1730.10.
10. 14,17- dioxos -4,7,10,21,24,27- of example, six oxa- -13,18- diazas, 33 carbon -15- alkynes - 1,30- diacid (86)
In 3- (2- (2- (2- amino ethoxies) ethyoxyl) ethyoxyl) propionate hydrochlorate 39 (601mg, 2.33mmol) In THF (6ml) solution, phosphate buffer (150mM NaH are added2PO 4, pH 7.2,4ml) and bis- (2,5- dioxo pyrroles Cough up alkane -1- bases) butyl- 2- alkynes diacid salt 9 (350mg, 1.13mmol).After dark place is stirred 4 hours at room temperature, mixture is concentrated SiO is used in combination2Column purification, with water/acetonitrile (1:9) it elutes.Merge the component containing product and concentration, obtains title compound (345mg, 59% yield).ESI MS m/z-C22H36N2O12(M-H), calculated value 519.22, measured value 519.30.
Example 11. (2- (2- (2- Carboxyethoxies) ethyoxyl) ethyoxyl) ethyl) dioxo-4,7,10,21-14,17-, 24,27- six oxa- -13,18- diaza pentatriacontane -15- alkynes -1,30- diacid (87)
Bis- (4,7,10- trioxa dodecanoic acid hydrochloride), the THF of 40 (650mg, 1.40mmol) in 13- amino- Phosphate buffer (150mM NaH are added in (6ml) solution2PO4, pH7.2,4ml) and bis- (2,5- dioxo pyrrolidin -1- Base) butyl- 2- alkynes diacid salt 9 (190mg, 0.61mmol).At room temperature after dark place stirring 4h, concentrated reaction mixture simultaneously uses C-18 HPLC (250mm × internal diameter 30mm) is prepared, is washed with water/ethyl alcohol (90% to 50% water, flow velocity 35ml/ minutes in 55 minutes) It is de-, merge the component containing product and concentration, obtains title compound (287 milligrams, yield 51%).ESI MS m/z- C40H67N2O22(M-H), calculated value 927.42, measured value 928.30.
Bis- six oxa- -13 (2,5- dioxo pyrrolidin -1- bases) 14,17- dioxos -4,7,10,21,24,27- of example 12., 15 alkynes -1,30- of 18- diaza tricyclics, two acid esters (88)
In six oxa- -13,18- diaza tricyclics of 1,4- dioxos -4,7,10,21,24,27-, ten alkynes -1 pentaene -15-, NHS (225mg, 1.96mmol) and EDC is added in DMA (6ml) solution of 86 (340mg, 0.653mmol) for 30- diacid (401mg, 2.08mmol).Mixture is stirred overnight in the dark, in SiO after concentration2It is purified on column, with EtOAc/DCM (5: 1) it elutes, obtains title compound 88 (330mg, 71% yield).ESI MS m/z+C30H43N4O16(M+H), calculated value 715.26 measured value 715.20.
(the 2- (2- (2- (3- ((2,5- dioxo pyrrolidin -1- bases) oxygroup) -3- oxopropoxies)) ethyoxyl) of example 13. Ethyoxyl) ethyl) six oxa- -13,18- diazas of -14,17- dioxos -4,7,10,21,24,27-, 33 carbon -15- alkynes - 1,30- diacid salts (89)
To bis- (2- (2- (2- (2- Carboxyethoxies) ethyoxyl) ethyoxyl) ethyl) dioxo-4-14,17- 13,18-, 7,10,21,24,27- six oxa- -13,18- diaza tricyclic butene-1 5- alkynes -1,30- diacid, 87 (280mg, 0.301mmol) DMA (6ml) solution in NHS (105.0mg, 0.913mmol) and EDC (200mg, 1.04mmol) is added.By mixture dark Place is stirred overnight, in SiO after concentration2It is purified on column, uses EtOH/DCM Elution, obtains title compound 89 (249mg, 63% yield).ESI MS m/z+C56H81N6O30(M+H), calculated value 1317.49, measured value 1317.80.
Example 14. (R, R, S, S, R, 4R, 4'R) -5,5'- (((14,17- dioxos -4,7,10,21,24,27- six oxa-s - 13,18-diazatriacont-15 alkynes, 1,30 diacyl) bis- (azane diyls)) bis- (4- hydroxyls -3,1- phenylenes)) two (4- (2- ((1R, 3R) -1- acetoxy-3s-((2S, 3S)-N, 3- dimethyl -2- ((R) -1- methyl piperidine -2- formamido groups) penta Base) -4- methyl amyls) thiazole -4- formamido groups) -2- methyl-acid) (90)
(4R) -4- (2- ((1R, 3R) -1- are added into THF (3.0ml) solution of compound 88 (30mg, 0.042mmol) Acetoxy-3-((2S, 3S)-N, 3- dimethyl-2- ((R)-1- methyl piperidine-2- formamido groups) valeryl amino)-4- methyl Amyl) thiazole -4- formamido groups) -5- (3- amino-4-hydroxylphenyls) -2 methyl valeric acid, 51 (Huang Y.et al, Med Chem.#44,249thACS National Meeting, Denver, CO, Mar.22~26,2015；WO2014009774) THF (4.0ml) solution and phosphate buffer (4ml, 100mM Na of (80mg, 0.107mmol)2HPO4,pH 7.0).In room After the lower stirring of temperature 4 hours, mixture is concentrated and is purified with C-18 preparation HPLC (250mm × internal diameter 20mm), with water/ethyl alcohol (95% to 50% water, flow velocity 15ml/ minutes).Merge the component containing product, concentrate and crystallized with EtOH/ n-hexanes, is obtained Title compound (48mg, 56% yield).ESI MS m/z-C98H147N14O26S2(M-H), calculated value 2000.01, measured value 2000.40.
15. conjugated compound 90 of example is combined with 91 antibody
To addition NaH in Trastuzumab (10mg/ml, 2.0mL, pH7.0-8.0)2PO4Buffer solution (100mM, pH6.5-7.5, 0.70-2.0mL), TCEP (20mM aqueous solutions, 28 μ L) and compound 90 (20mM DMA solution, 14 μ L).By mixture in room temperature It is incubated 2-16 hours, DHAA (135 μ L, 50mM) is then added.At room temperature after continuous overnight incubation, by mixture in G-25 columns Upper purifying, with 100mM NaH2PO4, 50mM NaCl pH Buffer solution elution, obtainConjugate 91 (about 87% yields) (In buffer solution).Drug/antibody ratio (DAR) passes through UPLC-Qtof mass spectroscopies It is 4.0.SEC HPLC (Tosoh Biosciences, Tskgel G3000SW, 7.8mm ID × 30cm, 0.5 ml/min, 100 minutes) analysis shows that content of monomer 96-99%, PAGE gel, which measures, shows single band.
16. compound 92 (4 tubulysin compounds are connected on each bridging junctor) of example
(4R) -4- (2- ((1R, 3R) -1- are added into THF (3.0ml) solution of compound 89 (35mg, 0.026mmol) Acetoxy-3-((2S, 3S)-N, 3 ((R)-1- methyl piperidine-2- formamido groups) valeryl amidos)-4- methyl amyls) thiazole- 4- formamido groups) -5- (3- amino-4-hydroxylphenyls) -2 methyl valeric acid, 51 (Huang Y.et al, Med Chem.#44, 249thACS National Meeting, Denver, CO, Mar.22~26,2015；WO2014009774) (100.6mg, THF (4.0ml) solution 0.132mmol) and phosphate buffer (4ml, 100mM Na2HPO4, pH7.0).It is stirred at room temperature After 4 hours, concentrated reaction mixture simultaneously with C-18 prepare HPLC (250mm × internal diameter 20mm) purify, with water/ethyl alcohol (95% to 50% water, in 50 minutes, flow velocity 15ml/ minutes) elution.Merge the component containing product, concentrates and with EtOH/ n-hexane knots Crystalline substance obtains title compound 92 (47.6mg, 47% yield).ESI MS m/z-C192H291N26O50S4(M-H), calculated value .3890.00, measured value 3890.30.
17. compound 92 of example prepares conjugate 93 with antibody coupling
To addition pH6.5-7.5 buffer solutions (0.70- in Trastuzumab solution (10mg/ml, 2.0mL, pH7.0-8.0) 2.0mL, 100mM NaH2PO4), TCEP (28 μ L, 20mM aqueous solution) and compound 92 (14 μ L, 20mM DMA solution).It will mixing Then DHAA (135 μ L, 50mM) was added in incubation at room temperature 2-16 hours in object.After being incubated overnight at room temperature, by mixture in G- It is purified on 25 columns, with 100mM NaH2PO4, 50mM NaCl pH Buffer solution elution, obtain's Conjugate 92 (In buffer solution,Yield).Drug/antibody ratio (DAR) passes through UPLC-Qtof mass spectrums It is measured as 8.0.SEC HPLC (Tosoh Biosciences, Tskgel G3000SW, 7.8mm ID × 30cm, 0.5 ml/min Clock, 100 minutes) analysis shows that content of monomer 96-99%, PAGE gel, which measures, shows single band.
The vitro cytotoxicity assessment (comparison T-DM1) of 18. conjugate 91 and 93 of example：
The cell line used in cytotoxicity experiment includes HL-60, a kind of mankind's promyelocytic leukemia cell line； NCI-N87, a kind of Human Gastric carcinoma's cell line；BT-474 is a kind of people's invasive ductal carcinoma cells system；And SKOV3, it is a kind of people Class ovarian cancer cell line.For HL-60, NCI-N87 and BT-474 cells, these cell growths are in the RPMI-containing 10%FBS 1640 culture mediums.For SKOV3 cells, cell growth is in the McCoy 5A culture mediums containing 10%FBS.When operation test, cell (180 μ l, 6000 cells) are added to 96 orifice plates and are incubated 24 hours in the environment of 37 DEG C and 5% carbon dioxide, later These cells (total volume 0.2mL) are handled with the compound (20 μ L) of various concentration.Control wells include cell and culture medium, nothing Test compound.After orifice plate is incubated 120 hours in the environment of 37 DEG C and 5% carbon dioxide, MTT (5mg/ are added thereto ML), hatch 1.5 hours at 37 DEG C.DMSO (180 μ L) is added after careful removal culture medium, shakes 15 minutes, in 490nm and 570nm measures absorbance, and 620nm is reference.Inhibiting rate is calculated according to following formula：Inhibiting rate %=[1- (assay values-blank Control value)/(controlling value-blank value)] × 100%
Compound Cytotoxicity result：
IC50(nM) N87 cells (Ag+) SK-OV-3 cells (Ag+) HL60 cells (Ag-)
Coupled object 91 0.108nM 0.089nM >20nM
Conjugate 93 0.037nM 0.029nM >10nM
T-DM1 0.270nM 0.191nM >15nM
Conjugate 91 is more than 185 (IC to the specificity of N87 cells50>20/IC50=0.108), super to SK-OV-3 cells Cross 225；Conjugate 93 is more than 270 (IC to the specificity of N87 cells50>10/IC50=0.037), it is more than to SK-OV-3 cells 344；Conjugate T-DM1 is more than 55 (IC to the specificity of N87 cells50>15/IC50=0.27), it is more than to SK-OV-3 cells 78。
Conjugate 91 and conjugate 93 are all more more effective than commercially available conjugate T-DM1.Conjugate 93DAR is 8, and activity is Three times of the conjugate 91 that DAR is 4.

Claims (29)

1. structural formula is the bridging junctor of (I)：
Acetylene ethanedioic acid structure wherein on connector can be reacted with a pair of of sulphur atom in cell binding agent；
Z1And Z2Be it is same or different can and cytotoxic drug reaction functional group, can by generate disulfide bond, ehter bond, Ester bond, thioether bond, thioester bond, peptide bond, hydrazone bond, urethane bond, carbonic acid ester bond, amine key (two level, three-level or level Four), imines Key, Heterocyclylalkyl, heteroaryl, alcoxyl oxime key or amido bond are combined with toxic small molecule drug；
R1And R2Identical, the different or default straight chained alkyl containing 1~6 carbon atom, the branch of 3 to 6 carbon atoms or Naphthenic base, straight chain, straight chain or cycloalkenyl group or the ester group of alkynyl or 1~6 carbon atom, ether, amide groups or polyethoxy (OCH2CH2)p, wherein p is the combination of 0 to about 1000 integer or these groups；
In addition, R1And R2It includes C, N, O to be, S, the chain structure of Si and P atoms is optimal to contain 0~500 atom, is covalently attached In X1Or X2And Z1Or Z2；R1And R2In each atom combined with all possible chemical mode, such as formed alkyl, alkylene Base, alkenylene, alkynylene, ether, polyoxy alkyl, ester, amine, imines, polyamine, hydrazine, hydrazone, amide, urea, semicarbazides, carbonohydrazides, alcoxyl The combination of amine, polyurethane, amino acid, polypeptide, acyl-oxygen amine, hydroxamic acid or these groups；
X1And X2It is independently selected from NH, N (R3), O, S or CH2；R3It is H, the straight chained alkyl of 1~6 carbon atom, 3 to 6 carbon atoms Branch or cyclic alkyl, straight chain, branch or cycloalkenyl group or the ester of alkynyl or 1~6 carbon atom, ether, amide or poly- ethoxy units (OCH2CH2)p, wherein p is the combination of 0 to 1000 integer or these groups.
2. structure is the Cell binding agent-drug conjugates titer of public formula (II)：
Wherein：
Cb is cell binding agent, and optimal is antibody；
It is connector-drug component by a pair of of sulphur atom and cell-binding molecules coupling in bracket；
Drug1And Drug2For same or different cytotoxic agent, they with alkyl, alkylidene, alkenylene, alkynylene, ether, Poly-alkoxyl, ester, amine, imines, polyamine, hydrazine, hydrazone, amide, urea, semicarbazides, carbonohydrazides, alkoxyamine, carbamate, amino Acid, peptide, acyl-oxygen amine, hydroxamic acid, disulfide bond, thioether, thioesters, carbamate, carbonic ester, heterocycle, miscellaneous alkyl, heteroaryl or Alcoxyl oxime key and combinations thereof is linked with cell binding agent；
N is 1~20；R1,R2,X1And X2Definition with claim 1.
3. the compound of structural formula such as (III)：
Wherein Cb, Z1,Z2,n,R1,R2,X1And X2Definition with claim 1 and 2.
4. the compound of structural formula such as (IV)：：
Wherein Drug1,Drug2,Z1,Z2,n,R1,R2,X1, and X2Definition with claim 1 and 2.
5. the synthesis in claim 1, containing acetylene dicarbapentaborane class bridging junctor is acetylene dicarbapentaborane or derivatives thereof, and The condensation of other components terminal amine (level-one or secondary amine), alcohol or mercaptan, such as shown in following (Ia)：
Wherein, X is X in claim 11Or X2Description, refer to N (R3), O, or S；R is R1And/or R2, R1、R2And R3Definition With claim 1.
Lv1 and Lv2 is identical or separate OH, F, Cl, Br, I, nitrophenol, n-hydroxysuccinimide (NHS), benzene Phenol, dinitrophenol, Pentafluorophenol, polytetrafluoroethylene phenol, difluorophenol, single fluorophenol, pentachlorophenol, trifluoromethane sulfonic acid, imidazoles, two Chlorophenol, tetrachlorophenol, 1- hydroxy benzo triazoles, p-methyl benzenesulfonic acid, methanesulfonic acid, 2- ethyl -5- phenyl isoxazole -3'- sulphurs Acid, acid anhydrides or the acid anhydrides formed with other acid anhydrides such as acetyl acid anhydride, formic anhydride；Or polypeptide condensation reaction intermediate or Mitsunobu Reaction intermediate；Condensation reagent includes：1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC), dicyclohexyl Carbodiimide (DCC), N, N'- diisopropylcarbodiimides (DIC), 1- cyclohexyl -2- morpholine ethyl carbodiimides are to toluene Sulfonate (CMC or CME-CDI), carbonyl dimidazoles (CDI), TBTU (O- benzotriazole-N, N, N', N'- tetramethylureas four Fluoboric acid), O- benzotriazole-tetramethylurea hexafluorophosphoric acid ester (HBTU), three (dimethylamino of benzotriazole -1- bases oxygroup Base) phosphorus hexafluorophosphate (BOP), hexafluorophosphoric acid benzotriazole -1- bases-oxygroup tripyrrole alkyl phosphorus (PyBOP), coke acid two Ethyl ester (DEPC), N, N, N', N'- tetramethyl chloromethane amidine hexafluorophosphates, 2- (7- aoxidizes benzotriazole)-N, N, N', N'- tetra- Methylurea hexafluorophosphoric acid ester (HATU), -1 [1,2,3] triazol [4,5-b] 1- pyrroles of 1- [(dimethylamine) (morpholinyl) methylene] Pyridine -3- oxygen hexafluorophosphate (HDMA), chloro- 1, the 3- methylimidazoles hexafluorophosphates (CIP) of 2-, chloro tripyrrole Wan Ji Phosphonium Hexafluorophosphate (PyCloP), bis- (tetramethylene) Fluoro-formamides (BTFFH), N, N, N', N'- tetramethyl-sulphur-(1- oxos- 2- pyridyl groups) thiocarbamide hexafluorophosphate, 2- (2- pyridone -1- bases) -1,1,3,3- tetramethylurea tetrafluoroborate (TPTU), Sulphur-(1- oxo -2- pyridyl groups)-N, N, N', N'- tetramethyl thiourea hexafluorophosphates, O- [(ethoxy carbonyl) cyano methylamine] - N, N, N', N'- tetramethyl thiourea hexafluorophosphate (HOTU), (1- cyano -2- ethyoxyl -2- oxo ethyleneiminos oxygroup) two Methylamino-morpholine-carbon hexafluorophosphate (COMU), (benzotriazole -1- bases oxygroup) two pyrrolidines carbon hexafluorophosphates (HBPyU), N- benzyls-N '-carbodicyclo hexylimide (or load is on polymer), bihyrrolidinyl (N- succinimide oxygen Base) carbon hexafluorophosphate (HSPyU), 1- (chloro- 1- pyrrolidinyls methylene) pyrrolidines hexafluorophosphate (PyClU), 2- is chloro- 1,3- methylimidazole tetrafluoroborate (CIB), (benzotriazole -1- bases oxygroup) two piperidines carbon hexafluorophosphates (HBPipU), 6- Chloro-Benzotriazoles -1,1,3,3- tetramethylurea tetrafluoro boric acid esters (TCTU), bromination three (dimethylamino) phosphine Hexafluorophosphoric acid (BroP), 1- n-propyls phosphoric anhydride (PPACA,), 2- isocyano groups ethyl morpholine (MEI), N, N, N', N'- tetra- Methylurea-oxygen-(N- succinimides base) hexafluorophosphate (HSTU), the bromo- 1- ethylpyridines tetrafluoroborates (BEP) of 2-, oxygen- [(ethoxy carbonyl) cyano methylamine]-N, N, N', N'- tetramethyl sulphur urine tetrafluoroborates (TOTU), 4- (4,6- dimethoxys three Piperazine -2- bases) -4- methyl morpholine hydrochlorides (MMTM, DMTMM), 2- succinimidos -1,1,3,3- tetramethylurea tetrafluoro boric acids Ester (TSTU), N, N, N', N'- tetramethyls-O- (3,4- dihydro -4- oxos -1,2,3- phentriazine -3- bases) urea tetrafluoroborate (TDBTU), azodicarbonyldipiperidine (ADD), bis- (4- chlorobenzyls) azodiformates (DCAD), two tertiary fourth of azoformic acid Ester (DBAD), diisopropyl azodiformate (DIAD), diethyl azodiformate (DEAD).
6. in claim 1, acetylene dicarbapentaborane is condensed with other functional groups on bridging junctor, extends carbochain, synthesis step two (trimethyl silicon substrate) acetylene, acetylene dibrominated magnesium (Grignard Reagent), acetylene dilithium salt or two metal salt of other acetylene and carboxylic acid halides or The reaction of acid anhydrides, it is illustrated that such as (Ib), (Ic), (Id), (Ie), (If), (Ig) and (Ih)：
Here M is Na, K, Li, Cu, CuLi, Sn, Ti, Ca, Mg or Zn.
7. in claims 2 and 4, public formula (II) is identical with the Drug1 and Drug2 that (IV) is inner or is each independently selected from：
1) chemotherapeutics：A) alkylating agent, such as mustargen：That is general for chlorobenzene, chlorine Pune piperazine, cyclophosphamide, Dacarbazine, estradiol nitrogen Mustard, ifosfamide, mustargen, hydrochloride oxygen amine, nitrous oxide mustard, amlodipine hydrochloride, Mycophenolic Acid, dulcitol, piperazine pool Bromine alkane, novoembichin, phenesterin, prednimustine, thio-tepa, Trofosfamide pair, uracil；CC-1065 (including its Ah more comes Newly, Carzelesin and Bizelesin synthesize homologue)；Multi-kanamycin (including synthesis homologue KW-2189 and CBI-TMI)；Benzene And phenodiazine Zhuo dimer (such as pyrrolo- Benzodiazepine (PBD) or the U.S. mycin of support, indoles and Benzodiazepine, imidazo benzo The dimer of thiophene phenodiazine Zhuo or oxazolidine and Benzodiazepine)；Nitroso ureas (Carmustine, lomustine, chlorination clostridium element, Fotemustine, Nimustine, Rameau department spit of fland)；Alkylsulfonate (busulfan, 5a,6,9,9a-hexahydro-6,9-methano-2,4,5a,6,9,9a-hexahydro-6,9-methano-2,4 and sulphur)；Triazenes (Dacca bar Piperazine)；Compound containing platinum (carboplatin, cis-platinum, oxaliplatin)；Ethylene imine class, such as benzodihydropyrone, Carlow ketone, meturedepa With black thunder DOPA；Aziridine and methyl melamine, including hemel, diethylenetriamine, triethyl phosphine amide, Sanya Ethylenebis dithiocarbamate phosphamide and trihydroxy methyl methyl amine；B) plant alkaloid, as (vincristine, vincaleukoblastinum are long for vinca alkaloids Fields for spring sowing are pungent, vinorelbine, navelbine)；Taxoid (taxol, Docetaxel) and its homologue；Maytansine biology Alkali (DM1, DM2, DM3, DM4,DM5, DM6, DM7, maytansine and Ansamycin) and its homologue；Cryptophycin is (especially It is cryptophycin 1 and cryptophycin8)；Epothilones, soft coral alcohol, discodermolide, bryostatin, sea hare poison Element, auspicious statin difficult to understand, tubulysin, cephalostatin, pancratistatin, sarcodictyin, sponge inhibin；c) DNA topoisomerase enzyme inhibitors, for example, Etoposide for Buddhist nun (9-aminocamptothecin, camptothecine, crisnatol, daunomycin, according to Support pool glycosides, etoposide phosphate, Irinotecan, mitoxantrone, Novantrone, retinoic acid (retinol), Teniposide, topology is replaced Health, 9-nitrocamptothecin (RFS 2000))；Mitomycin (mitomycin C)；D) antimetabolite, such as antifol, DHFR suppressions Preparation (methotrexate (MTX), trimetrexate, denopterin, pteropterin, ammonia petrin (4-aminobenzoic acid) or other folic acid homologues)； IMP dehydrogenase inhibitor (Mycophenolic Acid, Tiazofurine, Ribavirin, EICAR)；Ribonucleotide reductase inhibitors (hydroxyl Urea, Deferoxamine)；Pyrimidine homologue, uracil homologue (ancitabine, azacitidine, 6- azauracils, capecitabine (uncommon sieve Up to), Carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5 FU 5 fluorouracil, floxuridine, ratitrexed(Tomudex)；Cytimidine homologue (cytarabine, cytarabin, fludarabine)；Purine homology Object (imuran, fludarabine, mercaptopurine, thiamine, thioguanine)；Folic acid supplement, such as Fu Luolin acid；E) hormonotherapy Agent, such as receptor antagonist, antiestrogenic (megestrol acetate, Raloxifene, tamoxifen), LHRH excitants (his woods of Goss, acetic acid Leuprorelin)；Antiandrogen (Bicalutamide, Flutamide, Ka Lusi ketone, his androsterone of propionic acid times, table Androstanediol, Goserelin, Leuprorelin, U.S. Tilidine, Nilutamide, Testolactone, Trilostane and other inhibitor for androgen)；R8923, dimension Raw element D3 homologues (CB1093, EB1089KH1060, Vitamin D3, ergocalciferol)；(dimension is for pool for photodynamic therapy agent Sweet smell, phthalocyanine, photosensitizer Pc4, de-methoxy-hypocrellin A)；Cell factor (interferon-' alpha ', interferon-γ, tumor necrosis factor Sub (TNF), people's albumen containing TNF)；F) kinase inhibitor, such as BIBW 2992 (anti-EGFR/Erb2), Imatinib, Ji Fei is replaced Buddhist nun, piperazine Jia Tani, Sorafenib, Dasatinib, Sutent, Tarceva, nilotinib, Lapatinib, Axitinib, pa Azoles pa Buddhist nun, Vande Thani, E7080 (anti-vegf R2), mubritinib, Ponatinib (AP24534), bafetinib (INNO- 406), bosutinib (SKI-606), card is rich to replace Buddhist nun, and vismodegib, iniparib, for Buddhist nun, CYT387, A Xi are replaced Luso profit Buddhist nun, tivozanib, Sorafenib, bevacizumab, Cetuximab, Herceptin, Lucentis, Victibix, Yi Siping This；G) antibiotic, such as Enediyne Antibiotic (Calicheamicin, especially Calicheamicin γ 1, δ 1, α 1 and β 1 (reference J.Med.Chem.1996,39 (11), 2103-2117；Angew Chem Intl.Ed.Engl.1994,33:183-186), it reaches Because of mycin, including dyne mycin A and deoxidation rice mycin, Ai Sipeila mycins, card reaches mycin, C-1027, maduropeptin with And neoearcinostain chromophore and related chromoprotein enediyne antibiotic chromophore), aclacinomysins, D actinomycin D, peace Aspergillin, azaserine, bleomycin, Cetocycline, kalamycin, carminomycin, carzinophillin, adriamycin, adriamycin, Quinoline is for adriamycin, 2- pyrrolinodoxorubicins and deoxydaunorubicin, epirubicin, Aclarubicin, idarubicin, marcomycin, Mycin, mycophenolic acid, Lip river mycin, Peplomycin, Peplomycin, puromycin, triferricdoxorubicin, adriamycin, streptozotocin, chain Urea helps rhzomorph, tubercidin, ubenimex, Zinostatin, zorubicin；H) other, special such as polyketide (kind litchi element) It is not bullatacin and bullatacinone；Gemcitabine, epoxidase element (as blocked luxuriant and rich with fragrance such rice cloth), bortezomib, Sha Lidu Amine, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, Allovectin-7, Xegeva, Provenge, Yervoy, isoprenylation inhibitor (such as Lovastatin), dopaminergic nerve Toxin (such as staurosporin), D actinomycin D (such as actinomycin D, dactinomycin D), bleomycin (such as Bleomycin A2, Bo Lai Mycin B2, Peplomycin), anthracycline antibiotic (such as daunorubicin), adriamycin (adriamycin), idarubicin, table is soft Than star, pirarubicin, zorubicin, mitoxantrone, MDR inhibitor (such as Verapamil), Ca2+Atpase inhibitor is (such as malicious Hu trailing plants Bu Su), inhibitors of histone deacetylase (Vorinostat, romidepsin, pabishta, valproic acid, Mocetinostat (MGCD0103), Belinostat, PCI-24781, grace replace Nuo Te, SB939, Resminostat, Givinostat, AR-42, CUDC-101, sulforaphen, Trichostatin A)；Celecoxib, glitazone, Epigallo-catechin gallate (EGCG), double sulphur Logical sequence, Salinosporamide A；Antiadrenergic drug object, such as aminoglutethimide, mitotane, Trilostane, aceglatone, aldehyde phosphorus Amide, amino-laevulic acid, amsacrine, Arabinoside, bestrabucil, bisantrene, edatraxate, defofamine, Mei Kexin, diaziquone, Eflornithine (DFMO), elfomithine, Elliptinium Acetate, ethyl gluconic acid, gallium nitrate, cytimidine, Hydroxycarbamide, ibandronate, lentinan, Lonidamine, mitoguazone, mitoxantrone, Mopidamol, C-283, spray Si Tading, Phenamet, pirarubicin, podophyllic acid, 2- ethyl hydrazines, procarbazine；Razoxane；Rhizomycin；West Assistant；Spiro germanium；Tenuazonic acid；Triethyleneiminobenzoquinone；Trichlorotriethylamine；Trichothecene (especially T-2 toxin, verrucarine A, Roridine A and anguidine), polyurethane, siRNA, antisense drug and nucleic acid decomposition enzyme.
2) autoimmune disease drug, including but not limited to cyclosporin, cyclosporin A, aminocaproic acid, imuran, bromine are hidden Pavilion, Chlorambucil, chloroquine, cyclophosphamide, corticosteroid (such as Amcinonide, dexamethasone, Triamcinolone acetonide, propionic acid times Chlorine rice pine, DHEA, Etanercept, hydroxychloroquine, infliximab, Meloxicam, methotrexate (MTX), Mycophenolate Mofetil sprinkle Buddhist nun Pine, sirolimus, tacrolimus.
3) anti-infectious disease drug, including but not limited to a) aminoglycoside：Amikacin, astromicin, gentamicin (how For meter Xing, sisomicin, Isepamicin), hygromycin B, (amikacin, Arbekacin, it is mould that amino deoxidation blocks that kanamycins Element, dibekacin, tobramycin), neomycin (framycetin, paromomycin, ribostamycin), Netilmicin, spectinomycin, Streptomysin, tobramycin, verdamicin；B) amphenicols：Azidamfenicol, chloramphenicol, Florfenicol, Thiamphenicol； C) Ansamycin：Geldanamycin, herbimycin；D) Carbapenems：Biapenem, doripenem, ertapenem, imines training South/cilastatin, Meropenem, Panipenem；E) cephem：Carbacephem (loracarbef), Cefacetrile, chlorine ammonia benzyl mould Element, Cefradine, cefadroxil, cefalonium, cefaloridine, cefoxitin or cephalothin, cefalexin, cefaloglycin, Cefamandole, cefapirin, BL-S 640, fluorine azoles cephalosporin, cefazedone, ancef, cefbuperazone, head Spore card product, Cefdaloxime, cephalo pyrrole, Cefixime, Cefoxitin, cefprozil, cefroxadine, ceftezole, head Spore cefuroxime, Cefixime, Cefdinir, Cefditoren, cephalo pyrrole, cefetamet, Cefmenoxime, Cefodizime, cefonicid, Cefoperazone, ceforanide, cefotaxime, cefotiam, Cefozopran, cefalexin, Cefpimizole, cephalo Amine, Cefpirome, Cefpodoxime, cefprozil, Cefquinome, Cefsulodin, cefotaxime, Cefteram, ceftibuten, head Spore thiophene woods, Ceftizoxime, Ceftobiprole, ceftriaxone, cefuroxime, Cefuzonam, cephamycin (Cefoxitin, cefotetan, Cefmetazole), oxygen (carbon) cephem (Flomoxef, cephalo)；F) glycopeptide：Bleomycin, vancomycin (oritavancin, Te La Ten thousand stars), teicoplanin (Dalbavancin), Ramoplanin, g) glycylcycline：Such as tigecycline, h) beta-lactamase inhibitor： Penam (Sulbactam, Tazobactam Sodium), oxapenam (clavulanic acid)；I) lincosamide：Clindamycin, lincomycin；J) fat Peptide：Daptomycin, A54145, Ca-dependent antibiotic (CDA)；K) macrolides：Azithromycin, Mycosporin, clarithromycin, Dirithromycin, erythromycin, fluorine thunder mycin, josamycin, ketone lactone (Ketek, cethromycin), medecamycin, rice card are mould Element, oleandomycin, rifamycin (isoniazid, rifampin, Mycobutin, Rifapentine), sieve mycin, roxithromycin, grand sight Mycin, spiramvcin, tacrolimus (FK506), troleandomycin, Ketek；L) monocycle amine：Aztreonam, Tigemonam；m) Oxazolidinones：Linezolid；N) penicillins：Amoxicillin, ampicillin (Pivampicillin, extra large Lip river XiLin, Ba Anxi Woods, ampicillin, adriamycin), Ah substitutes XiLin, azlocillin, parasiticin, tardocillin parasiticin, benzyl star blueness Mycin penicillin Vl phenoxymethylpenicillin, Crow XiLin, procaine penicillin (U.S. replaces XiLin), mezlocillin, methicillin, naphthalene Fu Xi Woods, oxacillin, penamecillin, penicillin, pheneticillin, phenoxymethylpenicillin, Piperacillin, ampicillin, sulphur benzene XiLin, temocillin, Ticarcillin；O) polypeptide：Bacitracin, colistin, polymyxin B, p) quinolones：Alatrofloxacin, Balofloxacin, Ciprofloxacin, crin is husky, Danofloxacin, Difloxacin, Enoxacin, Enrofloxacin, T-3811, adds for sand Star, gemifloxacin, Grepafloxacin, Kano trovafloxacin, lavo-ofloxacin, Lomefloxacin, marbofloxacin, Moxifloxacin, that fluorine Sha Xing, Norfloxacin, Orbifloxacin, Ofloxacin, pefloxacin, trovafloxacin, Grepafloxacin, sitafloxacin, Sparfloxacin, Temafloxacin holds in the palm husky star, trovafloxacin；Q) streptogramin：Pristinamycin, Quinupristin/Dalfopristin, r) sulfonamides Object：Sulfa drugs：Sulfa drugs, sulphadiazine, sulphadiazine, sulphadiazine, salicylazosulfapyridine, bacteresulf, Tamoxifen, methoxybenzyl aminopyrimidine-sulfamethoxazole (Compound New Nomin)；S) steroids antibacterials：Such as Fusidic Acid；T) four Ring element class：Fortimicin, aureomycin, chlorine rice western ring element, demeclocycline, thunder not former times woods, U.S. western ring element, metacycline, minot ring Element, terramycin, penimepicycline, pyrrolidinyl methyl tetracycline, tetracycline, glycylcycline (such as tigecycline)：u) Other kinds of antibiotic：Annonacin, arsphenamine, bactoprenol inhibitor (bacillus), DANAL/AR inhibitor (circumfili ammonia Acid), dictyostatin, circle suberite lactone, soft coral alcohol, Epothilones, ethambutol, Etoposide, faropenem, husband Western ground acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, NAM synthetic inhibitors (such as phosphonomycin), Furantoin, taxol, the western mycin in Pulan, pyrazinamide, Quinupristin/Dalfopristin, rifampin (rifampin), Tazobactam Sodium Tinidazole, black chrysanthemum element.
4) antiviral drugs：A) entrance/fusion inhibitor：A Paweiluo, maraviro, vicriviroc, gp41 (En Fuwei Peptide), PRO 140, CD4 (Ai Bali pearls monoclonal antibody)；B) integrase inhibitor：Merck, elvite-gravir, globoidnan A；C) ripe inhibitor：Bevirimat, vivecon；D) neuraminidase inhibitor：Oseltamivir pricks that Meter Wei, Peramivir；E) nucleosides and nucleotide：Abacavir, Ah former times's list Wei, adefovirdipivoxil, A Moxiwei, Abciximab, bromine Husband is fixed, and cidofovir, Clevudine, dexamethasone, Didanosine (ddI), elvucitabine, emtricitabine (FTC), grace is replaced Card Wei, famciclovir, fluorine draw XiLin (5-FU), 3 '-fluorine-substituted 2 ', 3 '-deoxyribonucleoside homologues (such as 3,3 '-fluoro- 2 ', 3 '- Stavudine (FLT) and 3 '-fluoro- 2 ', 3 '-dideoxyguanosines (FLG), Fomivirsen, 9-guanine, iodoxuridine, Lamivudine (3TC), 1- nucleosides (such as β -1- thymidines and β -1-2'- deoxycytidines), Penciclovir, racivir, Ribavirin, enlightening replace fourth, Stavudine (d4T), tower Ribavirin (viramidine), Sebivo, tenofovir, trifluridine Valaciclovir, figured silk fabrics is more VACV, zalcitabine (ddC), Zidovudine (AZT)；F) non-nucleoside：Amantadine, Ah 's pyridine, capravirine, two virtues Yl pyrimidines (etravirine, rilpivirine), delavirdine, tadenan, emivirine, efavirenz, phosphonic acid (phosphorus Acyl group formic acid), imiquimod, Peg-IFN alpha-2b, Loviride, lodenosine, methisazone, nevirapine, NOV-205, Long-acting interferon α, podophyllotoxin, rifampin, Rimantadine, resiquimod (R-848), tromantadine；G) protease inhibits Agent：Anpunave atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, Lopinavir, Nai Feinawei, Pleconaril, Ritonavir, inverase, telaprevir (VX-950), tipranavir；H) other types of antiviral agent Object：Antioxidase, Abiduoer, OK a karaoke club Nuo Laide, ceragenin, cyanogen Wei Lin-n, diaryl pyrimidine, epigallocatechin Gallate (EGCG), phosphonic acid, lattice Lifei is pungent, taribavirin (viramidine), hydroxycarbamide, KP-1461, and rice replaces Good fortune is new, pleconaril, blendes together inhibitor, Ribavirin, seliciclib.
5) radioactive isotope (radionuclide), is selected from3H,11C,14C,18F,32P,35S,64Cu,68Ga,86Y,99Tc,111In,123I,124I, 125I,131I,133Xe,177Lu,211At or213Bi。
6) chromonic molecule can absorb a kind of light, such as ultraviolet light, fluorescence, infrared light, near infrared light or visible light；Uranidin, it is red One kind of cell, iris pigment, leucocyte, melanin and bluish-green pigment or a subclass, fluorescent molecular (shine again after absorbing light Fluorescent chemical) one kind or a subclass, visual light transduction molecule, photon molecule, luminescent molecule and fluorescein chemical combination Object；Nonprotein organic fluorescence group, such as (fluorescein, rhodamine, Oregon is green, Yihong and Texas for xanthene derivative It is red)；Cyanine derivative object (cyanine, indoles carbocyanine, oxa- cyanine, thiocyanine and merocyanine)；Squaric acid derivertives and ring substitution Side's acid, including Seta, SeTau and Square dyestuff；Naphthalene derivatives (dansyl and prodan derivative)；Cumarin derives Object；Oxadiazole derivatives (pyridyl group oxazole, nitro benzoxazoles and benzoxadiazole)；Anthracene derivant (Anthraquinones, including DRAQ5, DRAQ7 and CyTRAK orange)；Pyrene derivatives (cascade blue etc.)；Oxazines derivative (dislike by Nile red, Nile blue, cresol-purple Piperazine 170 etc.)；Acridine derivatives (flavol flavine, acridine orange, acridine yellow etc.)；Arylmethylamine derivative (auramine, crystal violet, peacock Malachite green) and tetrapyrrole derivative (porphines, phthalocyanine, bilirubin)；The homologue and derivative of following fluorescent chemicals：CF dyestuffs (Biotium), DRAQ and CyTRAK probes (BioS-tatus), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLight Fluor (Thermo Scientific, Pierce), Atto and Tracy (Sigma Aldrich), FluoProbes (Interchim), Abberior dyestuff (Abberior), DY and MegaStokes dyestuffs (Dyomics), Sulfo Cy dyestuffs (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTau and Square dyestuffs (Biosearch Technologies), SureLight dyestuffs (APC, RPEPerCP, Phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), allophycocyanin (APC), amino kermes albumen, APC-Cy7 conjugates, BODIPY-FL, Cascade Blue, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, fluorescein, FluorX, Hydroxycoumarin, Sulforhodamine B, lucifer yellow, Me- methoxy coumarins, NBD, Pacific Blue, Pacific Orange, PE-Cy5 conjugates, PE-R- phycoerythrin (PE), Red 613, Seta-555-Azide, Seta- 555-DBCO, Seta-555-NHS, Seta-580-NHS, Seta-680-NHS, Seta-APC-780, Seta-PerCP-680, Seta-R-PE-670, SeTau-380-NHS, SeTau-405- maleimide, SeTau-405-NHS, SeTau-425-NHS, SeTau-647-NHS, Texas Red, TRITC, TruRed, X-Rhodamine, 7-AAD (7-aminoactinomycin D, CG- choosings Selecting property), acridine orange, chromomycin A3, CyTRAK oranges (Biostatus), DAPI, DRAQ5, DRAQ7, ethidium bromide, Hoechst33258, Hoechst33342, LDS 751, mithramycin, propidium iodide (PI), SYTOX is blue, and SYTOX is green, SYTOX Orange, thiazole orange, TO-PRO, cyanine dyes monomer, TOTO-1, TO-PRO-1, TOTO-3, TO-PRO-3, YOSeta-1, YOYO-1. It can be connected with the connector of the present invention, the fluorescent chemicals for studying cell, selected from following compounds or derivatives thereof： DCFH (2', 7'- dichlorofluorescin, oxidised form), DHR (dihydro Rhodamine 123, oxidised form, photochemical catalytic oxidation), Fluo-3 (AM esters, pH>6), Fluo-4 (AM esters, pH7.2), Indo-1 (AM esters, low high calcium (Ca 2+)), SNARF (pH 6/ 9).Preferred fluorescent chemicals are selected from：Allophycocyanin (APC), (four is poly- by AmCyan1 (tetramer, Clontech), AsRed2 Body, Clontech), Ji is green (monomer, MBL), Azurite, B- phycoerythrin (BPE), Cerulean, CyPet, DsRed monomers (Clontech), DsRed2 (" RFP ", Clontech), EBFP, EBFP2, ECFP, EGFP (weak dimer, Clontech), Emerald (weak dimer, Invitrogen), EYFP (weak dimer, Clontech), GFP (S65A mutation), (S65C is prominent by GFP Become), GFP (S65L mutation) GFP (Y66H mutation), GFP (Y66W mutation), GFPuv, HcRed1, J-Red, Katusha, Kusabira Orange (single aggressiveness, MBL), mCFP, mCherry (monomer, MBL), mKate (TagFP635, monomer, Evrogen), mKeima-Red (monomer, MBL), mKO, mOrange, mPlum, mRaspberry, (monomer, Tsien are real by mRFP1 Test room), mStrawberry, mTFP1, mTurquoise2, P3 (phycobilisome compound), perdinin-chlorophyll-protein Compound (PerCP), R-phycoerythrin (RPE), T-Sapphire, TagCFP (dimer, Evrogen), TagGFP (dimer, Evrogen), TagRFP (dimer, Evrogen), TagYFP (dimer, Evrogen), tdTomato (series connection Dimer), Topaz, TurboFP602 (dimer, Evrogen), TurboFPP635 (dimer, Evrogen), TurboGFP (dimer, Evrogen), TurboRFP (dimer, Evrogen), TurboYFP (dimer, Evrogen), Venus is wild Type GFP types, YPet, ZsGreen1 (tetramer, Clontech), ZsYellow1 (tetramer, Clontech).
7) pharmaceutically acceptable salt, acid or derivative of said medicine.
8. in claims 2 and 4, public formula (II) and (IV) inner Drug1And Drug2For chromonic molecule, structural formula be (II) and (IV) conjugate can be used for detecting, and monitor or study the function of cell-binding molecules, the interaction with target cell, and/or The interaction of conjugate and object, especially target cell.
9. in claims 2 and 4, Drug1And Drug2It preferably is selected from tubulysin, calicheamicin, the auspicious statin of Australia, maytansine life Alkaloids, CC-1065 homologues, daunorubicin and doxorubicin compound, taxanes (taxanes), cryptophycin, angstrom Rich mycin, Benzodiazepine dimer (such as pyrrolo- Benzodiazepine (PBD), hold in the palm U.S. mycin, Anthramycin, indoles and benzo Phenodiazine is tall and erect, the dimer of imidazo benzo thiophene phenodiazine Zhuo or oxazolidine and Benzodiazepine), block vertical mycin class and Enediyne is anti- Raw element, D actinomycin D, nitrogen silk rhzomorph, bleomycin, epirubicin, tamoxifen, idarubicin, dolastatin/Australia it is auspicious he Spit of fland (such as MMAE, MMAF, the auspicious statin PYE of Australia, the auspicious statin TP of Australia, the auspicious statin 2-AQ of Australia, 6-AQ, EB (AEB) and EFP (AEFP)), multi-kanamycin, thiotepa, vincristine, the Tallins Ban meter, nazumamide, microginin, radiosumin, Alterobactin, microsclerominmin, theonellamide, esperamicin, siRNA, nucleolytic enzyme and/or its The homologue and derivative of pharmaceutically acceptable salt, acid and/or above-mentioned molecule.
10. cell binding agent/molecule in claim 2 and 3 is selected from antibody, albumen, vitamin (such as folic acid), peptide, polymer Pharmaceutical carrier, nanoparticulate drug carrier, dendrimer based on micella, liposome, lipoprotein and it is coated with cell combination The above-mentioned molecule of ligand or combinations thereof object.
11. cell binding agent/molecule in claim 2,3 and 10 is preferably antibody, complete antibody (polyclonal antibody, Dan Ke Grand antibody, dimer, polymer, multi-specificity antibody, such as bispecific antibody)；Single-chain antibody；In conjunction with the antibody of target cell Segment, monoclonal antibody, single monoclonal antibodies or the monoclonal antibody fragment for combining target cell are chimeric antibody, thin in conjunction with target The chimeric antibody fragment of born of the same parents, single domain antibody, in conjunction with the single domain antibody segment of target cell, surface modification antibody, single-stranded surface modification Antibody, or the surface modification antibody fragment of target cell is combined, humanized antibody, single chain humanized antibodies, or combine target cell Humanized antibody segment, anti-idiotype (anti-Id), CDR, bivalent antibody, trivalent antibodies, miniantibody, immune protein (SIP), lymphokine, hormone, vitamin, growth factor, a colony stimulating factor, Nutrient transport molecule, macromolecule Protein.
12. cell binding agent/molecule in claim 2,3 and 10 can be any reagent that can target following cell：It is swollen The cell of oncocyte, the cell of virus infection, microorganism infection, the cell of parasitic infection, the cell of autoimmune disease, The tumour cell of activation, bone marrow cell, the T cell of activation invade B cell or melanocyte.
13. cell binding agent/molecule in claim 2,3 and 10 can any can fight one of following further antigens or receptor Drug/molecule：CD3,CD4,CD5,CD6,CD7,CD8,CD9,CD10,CD11a,CD11b,CD11c,CD12w,CD14, CD15,CD16,CDw17,CD18,CD19,CD20,CD21,CD22,CD23,CD24,CD25,CD26,CD27,CD28,CD29, CD30,CD31,CD32,CD33,CD34,CD35,CD36,CD37,CD38,CD39,CD40,CD41,CD42,CD43,CD44, CD45,CD46,CD47,CD48,CD49b,CD49c,CD51,CD52,CD53,CD54,CD55,CD56,CD58,CD59,CD61, CD62E,CD62L,CD62P,CD63,CD66,CD68,CD69,CD70,CD72,CD74,CD79,CD79a,CD79b,CD80, CD81,CD82,CD83,CD86,CD87,CD88,CD89,CD90,CD91,CD95,CD96,CD98,CD100,CD103, CD105,CD106,CD109,CD117,CD120,CD125,CD126,CD127,CD133,CD134,CD135,CD138, CD141,CD142,CD143,CD144,CD147,CD151,CD147,CD152,CD154,CD156,CD158,CD163, CD166,.CD168,CD174,CD180,CD184,CDw186,CD194,CD195,CD200,CD200a,CD200b,CD209, CD221,CD227,CD235a,CD240,CD262,CD271,CD274,CD276(B7-H3),CD303,CD304,CD309, CD326,4-1BB, 5AC, 5T4 (Trophoblast glycoprotein, TPBG, 5T4, WNT- activate inhibiting factor 1 or WAIF1), gland cancer Antigen, AGS-5, AGS-22M6, activin receptor kinases 1, AFP, AKAP-4, ALK, α integrins, α v β 6, aminopeptidase N form sediment Powder sample albumen β, androgen receptor, angiogenesis promoting protein factor 2, angiogenesis promoting protein factor 3, Annexin A1, anthrax Toxin protective antigens, anti-rotation shifting protein receptor, AOC3 (VAP-1), B7-H3, bacillus anthracis, BAFF (B cell activity factor), B lymphoma cells, bcr-abl, Magainin, BORIS, C5, C242 antigens, CA125 (sugar antigens 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, dog lupus erythematosus IL31, carbonic anhydrase IX, cardiac myosin, CCL11 (C-C Segment chemotactic factor (CF) 11), CCR4 (C-C Chemokine receptor4s, CD194), CCR5, CD3E (ε), CEA (carcinomebryonic antigen), CEACAM3, CEACAM5 (carcinomebryonic antigen), CFD (factor D), Ch4D5, cholecystokinin 2 (CCK2R), CLDN18 (Claudin- 18) it, grows thickly factors A, CRIPTO, FCSF1R (colony-stimulating factor 1 receptor, CD115), (colony stimulating factor 2, grain is thin by CSF2 Born of the same parents-macrophage colony stimulating factor (GM-CSF)), CTLA4 (cytotoxic T lymphocyte GAP-associated protein GAP 4), CTAA16.88 Tumour antigen, CXCR4 (CD184), C-X-C Chemokine receptor4s, cyclic annular ADP ribalgilases, cell periodic protein B 1, CYP1B1, cytomegalovirus, cytomegalovirus Glycoprotein B, Dabigatran, DLL4 (class Δ ligand 4), DPP4 (double peptide-peptases 4), DR5 (death receptor 5), Type of toxin -1 Escherichia coli shiga, Escherichia coli shiga toxin type-2, ED-B, EGFL7 (class EGF domain proteins 7), EGFR, EGFRII, EGFRvIII, endothelial factor (CD105), Endothelin B receptor, endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2, Episialin, ERBB2 (epidermal growth factor acceptor 2), ERBB3, ERG (TMPRSS2ETS fusions), Escherichia coli, ETV6-AML, FAP (fibroblast activation protein alpha), FCGR1, first tire egg In vain, fibrin II β chains, fibronectin extra domain-B, FOLR (folacin receptor), folacin receptor α, folic acid hydrolase, Fos Related antigen 1, the F protein of Respiratory Syncytial Virus(RSV), the receptor of curling, fucose GM1, GD2 gangliosides, G-28 (cells Surface antigen glycolipid), GD3 idiotypes, GloboH, Glypican 3, NeuGc ALPHA2-3Gal, GM3, GMCSF receptor alpha chains, Growth differentiation factor 8, GP100, GPNMB (transmembrane glycoprotein NMB), GUCY2C (guanylate cyclase 2C), guanosine cyclic mono-phosphate (GC-C), intestines guanylate cyclase, guanosine cyclic mono-phosphate receptor, Thermostable α-amylase receptor (hSTAR), heat shock protein, blood Solidifying element, hepatitis B surface antigen, hepatitis type B virus, HER1 (human epidermal growth factor acceptor 1), HER2, HER2/neu, HER3 (ERBB-3), IgG4, HGF/SF (hepatocyte growth factor/dispersion factor), HHGFR, HIV-1, histone complexes, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, human chorionic gonadotropin, HNGF, the mankind dispersion because Sub- receptor kinase, HPV E6/E7, Hsp90, hTERT, ICAM-1 (Intercellular Adhesion Molecule 1), idiotype, IGF1R (IGF -1, 1 receptor of insulin-like growth factor), IGHE, IFN-γ, influenza hemagglutinin, IgE, the areas IgE Fc, IGHE, IL -1, IL-2R is (in vain 2 receptor of interleukin), IL -4, IL-5, IL -6, IL-6R (Interleukin-6 receptor), IL-9, IL -10, IL -12, IL-13, IL-17, IL- 17A, IL-20, IL-22, IL-23, IL31RA, ILGF2 (insulin-like growth factor 2), integral protein (α 4, α IIb β 3, α v β 3,4 β 7 of α, 5 β 1 of α, alpha 6 beta 4,7 β 7 of α, α ll β 3,5 β 5 of α, α v β 5), interferon gamma inducible protein matter, ITGA2, ITGB2, KIR2D, LCK, Le, Legumain, Lewis-Y antigen, LFA-1 (Lymphatic diseases, CD11a), LHRH, LINGO-1, Lipoteichoicacid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE A1, MAGE A3, MAGE 4, MART1, MCP-1, MIF (macrophage migration inhibitory factor or the glycosyl inhibition factor (GIF)), MS4A1 (across 4 structural domain subfamily A member 1 of film), MSLN (mesothelin), MUC1 (mucin 1, cell surface correlation (MUC1) or polymorphism on Skin mucoprotein (PEM)), MUC1-KLH, MUC16 (CA125), MCP1 (MCP 1), MelanA/MART1, ML- IAP, MPG, MS4A1, MYCN, Myelin-associated glycoprotein, Myostatin, NA17, NARP-1, NCA-90 (G-Ag), Nectin-4 (ASG-22ME), NGF, nerve cell apoptosis modulin enzyme 1, NOGO-A, Notch receptor, paranuclein, Neu are caused Oncoprotein, NY-BR-1, NY-ESO-1, OX-40, OxLDL (OxLDL ELISA), OY-TES1, P21, p53 are non-prominent Variant, P97, PAP resist (NeuGc ALPHA2-3Gal) paratope, PAX3, PAX5, PCSK9, PDCD1 (PD-1, program Property cell death albumen 1, CD279), PDGF-R α (α platelet-derived growth factor receptors), PDGFR- β, PDL-1, PLAC1, class PLAP testis alkaline phosphatases, platelet derived growth factor receptor β, sodium phosphate joint transporter, PMEL 17, poly sialic acid, Protease 3 (PR1), prostate cancer, PS (phosphatidylserine), prostate gland cancer cell, pseudomonas aeruginosa, PSMA, PSA, PSCA, rabies virus glucoprotein, RHD (Rh polypeptides 1 (RhPI), CD240), the Rhesus factors, RANKL, RhoC, Ras mutation, RGS5, ROBO4, Respiratory Syncytial Virus(RSV), RON, sarcoma translocation breakpoint, SART3, Sclerostin, SLAMF7 (SLAM members 7), Selectin P, SDC1 (syndecan 1), systemic loupus erythematosus (a), somatomedin C, SIP (1- phosphoric acid Sphingol), Somat, Human sperm protein 17, SSX2, STEAP1 (6- cross-film epitheliums prostate antigen 1), STEAP2, STn, TAG-72 (tumor-associated glycoprotein), survivin, T cell receptor, T cell transmembrane protein, TEM1 (tumor vascular endothelium marks 1), TENB2, Tenascin C (TN-C), TGF- α, TGF-β (transforming growth factor β), TGF-β 1, (conversion is grown TGF-β 2 note The factor 2), Tie (CD202b), Tie2, TIM-1 (CDX-014), Tn, TNF, TNF-α, (tumour is bad by TNFRSF8, TNFRSF10B Necrosis factor receptor superfamily member 10B), TNFRSF13B (A member of the TNF receptor family 13B), TPBG (are nourished thin Born of the same parents' glycoprotein), TRAIL-R1 (TNF correlation necrosis inductions ligand receptor 1), TRAILR2 (death receptor 5 (DR5)), tumour correlation Ca2+ oscillations sensor 2, the glycosylated MUC1 of tomour specific, tweak receptor, TYRP1 (glycoprotein 75), TRP-2, tyrosine Enzyme, VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, vimentin, WT1, XAGE 1, express the cell of any insulin growth factor receptor or any EGF-R ELISA.
14. the tumour cell in claim 12 is thin selected from lymphoma cell, myeloma cell, renal cell carcinoma cell, breast cancer Born of the same parents, prostate gland cancer cell, ovarian cancer cell, colon cancer cell, stomach cancer cell, squamous cell carcinoma, Small Cell Lung Cancer, non-small cell Lung carcinoma cell, testicular cancer cell or any uncontrolled, tachyauxesis, differentiation and carcinogenic cell.
15. claim 1, the connector component R in 2,3 and 41And R2, can be by one or more connector component structure At：6- maleimidohexanoic acids (MC), 3- maleimidoproprionic acids (MP), valine-citrulline (val-cit), alanine- Phenylalanine (ala-phe), to aminobenzyloxycarbonyl (PAB), the thio valeric acids of 4- (SPP), 4- (N- maleimidomehyls) ring Hexane -1- carboxylic acids (MCC), 4- Thiobutyric acids (SPDB), ethyl maleimide (ME), the thio -2- weight ratios butyric acid (2- of 4- Sulfo-SPDB), two mercaptan of pyridine (PySS), alkoxy amino (AOA), ethyleneoxy (EO), two sulphur of 4- methyl -4--valeric acid (MPDP), nitrine (N3), alkynes, two is thio, peptide, and/or (4- acetyl group) Aminobenzoate (SIAB).
16. in claim 2, Drug1And Drug2For Tubulysin homologues, the preferred coupling with general structure (II) Object T01, T02, T03, T04, T05 and T06：
Wherein mAb is antibody；Z3And Z3' it is H, OP (O) (OM1)(OM2), OCH2OP(O)(OM1)(OM2), OSO3M1, R1Or O- sugar Glycosides (glucosides, galactoside, mannoside, glucoside, fructoside etc.), NH- glucosides, S-glycosides or CH2Glucosides；M1And M2Solely It is on the spot H, Na, K, Ca, Mg, NR1R2R3；N is 1~20；X1, X2, R1, R2And R3It is identical as defined in claim 1.
17. in claim 2, Drug1And Drug2For Calicheamicin homologue, the preferred coupling with general structure (II) Object C01：
Wherein mAb is antibody；N is 1~20；X1, X2, R1And R2It is identical as defined in claim 1.
18. in claim 2, Drug1And Drug2For maytansine alkaloid homologue, the preferred idol with general structure (II) Join object M01：
Wherein mAb is antibody；N is 1~20；X1, X2, R1And R2It is identical as defined in claim 1.
19. in claim 2, Drug1And Drug2For taxane homologue, the preferred conjugate with general structure (II) Tx01, Tx02 and Tx03：
Wherein mAb is antibody；N is 1~20；X1, X2, R1And R2It is identical as defined in claim 1.
20. in claim 2, Drug1And Drug2For CC-1065 homologues, the preferred following idol with general structure (II) Join object CC01, CC02, CC03：
Wherein mAb is antibody；N is 1~20；Z4And Z4' it is HH, PO (OM1)(OM2), SO3M1, CH2PO(OM1)(OM2), CH3N (CH2CH2)2NC (O)-, O (CH2CH2)2NC (O)-, R1Or glucosides；X3And X3' it is O, NH, NHC (O), OC (O), C (O) O, R1Or It is default；X1、X2、R1、R2、M1And M2It is identical as defined in claim 1.
21. in claim 2, Drug1And Drug2It is preferred with general structure (II) for daunorubicin/adriamycin homologue Conjugate Da01, Da02, Da03 and Da04：
Wherein mAb is antibody；N is 1~20；X3And X3' be independent H, O, NH, NHC (O), NHC (O) NH, C (O), OC (O) or R1；X1、X2、R1And R2It is identical as defined in claim 1.
22. in claim 2, Drug1And Drug2For the auspicious statin homologue of Australia, the preferred conjugate with general structure (II) Au01, Au02, Au03, Au04 and Au05：
Wherein mAb is antibody；N is 1~20；X3And X3' it is independent CH2, O, NH, NHC (O), NHC (O) NH, C (O), OC (O) R1 Or it is default；X4And X4' it is independent CH2, C (O), C (O) NH, C (O) N (R1), R1, NHR1, NR1, C (O) R1Or C (O) O；Z3With Z3' it is independent H, R1, OP (O) (OM1)(OM2), NHR1, OCH2OP(O)(OM1)(OM2), OSO3M1Or O-glycosides (glucosides, half Lactoside, mannoside, glucoside, fructoside etc.), NH- glucosides, S-glycosides or CH2Glucosides；M1And M2H independently is, Na, K, Ca, Mg, NR1R2R3；X1, X2, R1, R2And R3It is identical as defined in claim 1.
23. in claim 2, Drug1And Drug2It is preferred with general structure (II) for Benzodiazepine dimer homologue Conjugate PB01, PB02, PB03, PB04, PB05, PB06, PB07 and PB08：
Wherein mAb is antibody；N is 1~20；X3And X3' it is independent CH2, O, NH, NHC (O), NHC (O) NH, C (O), OC (O), OC(O)(NR3), R1, NHR1, NR1, C (O) R1Or it is default；X4And X4' it is independent CH2, C (O), C (O) NH, C (O) N (R1), R1, NHR1, NR1, C (O) R1Or C (O) O；X1, X2, R1, R2And R3It is identical as defined in claim 1.In addition, R1And/or R2It can lack It saves.
24. the pharmaceutical composition of a kind for the treatment of or prevention for cancer, autoimmune disease or infectious disease, including treatment has The claim 2 for imitating dosage, conjugate and its pharmaceutically acceptable salt in 16,17,18,19,20,21,22 and/or 23, Carrier, diluent or excipient or combinations thereof.
25. claim 2, the conjugate in 16,17,18,19,20,21,22,23 or 24 has external, in vivo or in vitro work Property.
26. claim 2, the conjugate in 16,17,18,19,20,21,22 or 23, wherein connector component R1And/or R2Packet Peptide containing 1~20 natural or non-natural amino acids unit, amino benzyl unit, 6- maleimidocaproyl units, two sulphur Compound, thio-ether units, hydrazone unit, triazole unit or alcoxyl oxime unit.
27. claim 2, the connector in 16,17,18,19,20,21,22 or 23 can be cut off by protease.
28. a kind of pharmaceutical composition, include the claim 2 of therapeutically effective amount, 16,17,18,19,20,21,22,23 or/and Conjugate in 24, with other therapeutic agents such as chemotherapeutics, radiotherapy, immunotherapeutic agent, autoimmune disease drug resists Infection medicine or other conjugates use simultaneously, and collaboration effectively treats or prevents cancer, autoimmune disease or infectious disease.
29. the synergist in claim 28 preferably is selected from one or more of agents：Orencia, acetic acid Ah's bit Dragon, paracetamol/hydrocodone, adalimumab, Afatinib maleate, alemtuzumab, alitretinoin, toltrazuril Monoclonal antibody, amphetamine salt-mixture (amphetamine/Dexamfetamine) Anastrozole, Aripiprazole, atazanavir, Atezolizumab, Ah Atorvastatin, pazopanib, Belling, bevacizumab, Cabazitaxel, card is rich to be replaced, and bexarotene, blinatumomab, boron is for assistant Rice, posupini, brentuximab vedotin, budesonide, Budesonide/formoterol, buprenorphine, capecitabine, Carfilzomib, celecoxib, ceritinib, Cetuximab, cyclosporine, cinacalcet, (R)-3-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine, dabigatran reach La Feini, A Fadabeiting, darunavir, imatinib mesylate, Dasatinib, denileukin, Di Nuosaimai, double the third penta Sour sodium, R-lansoprazole, dexmethylphenidate, Dinutuximab, Doxycycline, Duloxetine, emtricitabine/rilpivirine/replace promise Good fortune Wei ground Suo Puxi fumarates, emtricitabine/tenofovir/efavirenz, Enoxaparin, the miscellaneous Shandong amine of grace, epoetin alfa, Tarceva, esomeprazole, Ezetimibe, Ezetimibe/Simvastatin, fenofibrate, Filgrastim, fingomode, third Sour fluticasone, fluticasone/salmeterol, fulvestrant, Gefitinib, copaxone, goserelin acetate, she replaces at horse Buddhist nun, ibritumomab tiuxetan replace Buddhist nun, insulin insulin aspart, insulin detemir, insulin glargine, insulin lispro, interference according to Shandong Plain β 1a, interferon beta 1b, Lapatinib, Ipilimumab, Ipratropium Bromide/Salbutamol, acetic acid lanreotide, lenalidomide, Joint xylenesulfonate, Letrozole, Levothyroxine, levothyrocine, lidocaine, Linezolid, Liraglutide, MEDI4736, Memantine, methylphenidate, metoprolol, modafinil, Mometasone, nilotinib, Nivolumab, method wood difficult to understand are single It is anti-, Ao Lita pearl monoclonal antibodies, pazopanib, pyridine aldoxime methyliodide (PAM) monoclonal antibody, pemetrexed, handkerchief trastuzumab, pneumococcal conjugated vaccine, pool Ma Du Amine, Pregabalin, Quetiapine, Rabeprazole radium chloride 223, Raloxifene draw for drawing Wei, Lei Morui monoclonal antibodies, and Lucentis is auspicious Ge Feini, Rituximab, razaxaban, romidepsin, rosuvastatin, Luso replace Buddhist nun's phosphate, salbutamol to take charge of Wella It is bright, silaenafil, siltuximab, sitagliptin, sitagliptin/melbine, solifenacin, Sorafenib, Sutent, Tadalafei, tamoxifen replace La Puwei, tesirolimus, tenofovir/emtricitabine, testosterone gel, Thalidomide (Talidex), Tiotropium Bromide, Toremifene, Trimetinib, Herceptin, Tretinoin, especially gram monoclonal antibody, Valsartan, It is Vande Thani, Wei Luofeini, Vorinostat, VEGF Trap, Zostavax and its homologue, derivative, pharmaceutically acceptable Salt, carrier, diluent or excipient, or combinations thereof.
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