CN108811499A - Specific conjugation of cell-binding molecules - Google Patents

Abstract

The present invention describes a class of cell-binding agent-drug conjugates comprising a bridge linker and the use of the linkers and conjugates.

Description

Specific conjugation of cell-binding molecules

technical field

The present invention describes a new class of linkers that can be used to specifically link compounds, in particular cytotoxic agents, and biomolecules (cell-binding agents). The present invention includes a method for preparing a cell-binding molecular drug (cytotoxic agent) conjugate: either first modifying the drug with such a linker, and then reacting with the cell-binding agent, or first modifying the cell-binding agent with this type of linker , and then react with drug molecules.

technical background

Proteins, especially antibodies, are widely used in the medical and health industry as research reagents for in vitro experiments and diagnostic tools or therapeutic drugs for in vivo experiments (Gad, S.C. Drug discovery handbook, Wiley-Interscience, 2005). In many applications of proteins, the protein is often modified with interesting groups, such as cytotoxic molecules, radioactive element labels or molecules with chromatographic properties, to meet the needs of therapeutic or diagnostic tests (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 conjugates (ADCs), has been intensively developed as a hot spot in the past 20 years, which combines the precise targeting of antibodies and the cytotoxicity of anti-tumor agents, allowing drugs to be targeted. Target delivery into cancer cells without affecting normal cells. In particular, since the U.S. FDA approved Adcetris (brentuximab vedotin) in 2011 and Kadcyla (ado-trastuzumab emtansine) in 2013, the use of antibody-drug conjugates as a targeted therapy for cancer has been internationally recognized. Applied by almost all pharmaceutical companies and biotechnology companies (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). According to the website www.clinictrails.gov., there are currently more than 50 ADC drugs in clinical trials.

The first generation of ADCs drugs, including Kadcyla and Adcetris, are prepared by non-selective coupling of cytotoxic drug molecules and natural lysine amine groups or interchain cysteine sulfhydryl groups on antibodies, respectively. Because IgG1 antibodies have more than 50 surface-exposed lysines and 8 cysteines, this non-selective conjugation leads to random cross-linking of cytotoxic molecules and all regions of the antibody surface, resulting in a variety of , ADC group with broad distribution of DAR value (drug antibody ratio) (Wang, L., et al. 2005 Protein Sci. 14, 2436; Hamblett, K. J., et al. 2004 Clin. Cancer Res. 10, 7063). Partially undesired subsets of ADCs have drawbacks such as shortened circulating half-life, low potency, high potential off-target toxicity, and uncertain in vivo pharmacokinetic (PK) properties (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, it is very challenging to maintain batch-to-batch consistency for ADCs produced by this traditional conjugation method, requiring higher production capacity (Wakankar, A. mAbs, 2011, 3, 161–172).

Therefore, many biotechnology companies and research institutions are devoting themselves to the development of novel site-directed conjugation methods for ADC drugs. In recent years, there have also been some site-specific ADC drug preparation methods (Panowski, S, 2014, mAbs 6, 34), these methods include: introducing unpaired cysteines on antibodies, such as Genentech's THIOMAB antibody (Junutula, JR, et al Clin.Cancer Res.2010,16,4769; Junutula, JR, et al NatBiotechnol.2008 26,925-32; US Patent 8,309,300; 7,855,275; 7,521,541; 7,723,485; WO2008/141044); Transglutaminase (mTG) amplified from Streptotrichum mobara (Strop, P., Bioconjugate Chem., 2014, 25, 855–862; Strop, P., et al., Chem. Biol. 2013, 20, 161 –167; U.S. Patent No. 8,871,908 to Pfizer Rinat Corporation) or transglutaminase (MTGase) derived from bacteria (Dennler, P., et al, Bioconjug. Chem. 2014, 25, 569–578; U.S. Patent Applied by Innate Pharmaceuticals Inc. 20130189287; U.S. Patent 7,893,019 of Bio-Ker Srl, Italy); the introduction of mercapto-L-fucose (Dennler, P., et al, Bioconjugate Chemistry 2014,25,569; Okeley, NM, et al Bioconjugate Chem.2013,24 , 1650); introduction of unnatural amino acids by mutagenesis (Axup, JY, et al., Proc.Natl.Acad.Sci.2012, 109, 16101–16106; Zimmerman, ES, et al., Bioconjug.Chem. –361; Wu, P., et al, Proc. Natl. Acad. Sci. 2009, 106, 3000-3005; Rabuka, D., et al, Nat. Protoc. 2012, 7, 1052-67; Sutro Biopharmaceuticals Company US Patent 8,778,631 and US Patent Application 20100184135, World Intellectual Property Patent Application WO2010/081110; Ambrx Company Patent WO2006/069246, 2007/059312, US Patent 7,332,571, 7,696,312; and 7,638,299; Allozyne Company Patent WO2007 /130453 and U.S. Patents 7,632,492; 7,829,659); introduction of selenocysteine (Hofer, T., et al Biochemistry 2009, 48, 12047–12057; U.S. Patent 8,916,159 to the National Cancer Institute); generated from formylglycine Enzyme (FGE) converts cysteine on the CXPXR consensus sequence to formylglycine (FGly) (Drake, PM, et al., Bioconjug. Chem. 2014, 25, 1331–1341. US Patent 7,985,783; Redwood Life Sciences 8,097,701; 8,349,910 and US patent applications 20140141025, 20100210543); or introduce sialic acid by glycoengineering with galactose or sialyltransferase (Zhou, Q., et alBioconjug.Chem., 2014, 25, 510-520, Nofi-Genzyme US Patent 20140294867). Uniform products can be produced using the methods described above, but they all require antibody engineering and re-optimization of cell culture conditions. Furthermore, the expression of genetic codes for unnatural amino acids is often not as high as hoped (Tian, F., et al, 2014, Proc. Natl. Acad. Sci. USA111, 1766-71), which has important implications for the cost of ADC products Impact. In addition, ADC drugs obtained by conjugating cysteine side chains often have limited stability in the circulation, leading to premature cleavage of the toxic small molecule payload before reaching tumor cells (Junutula, JR, et al Nat. Biotechnol. 2008, 26, 925-32).

The disulfide bond structures in the four subtypes of IgG antibodies have been known since the 1960s (Milstein 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 CJ Mol Biol 1968; 33:893–906; Edelman GM, et al. Proc Natl Acad Sci USA 1969; 63:78-85; Frangione B, et al. , 2157-63). Disulfide bonds play an important role in the structure, stability and biological function of IgG molecules. Of the four subtypes of IgG antibodies, IgG ₁ , IgG ₂ , IgG ₃ and IgG ₄ , each IgG molecule contains a total of 12 intrachain disulfide bonds; each disulfide bond is associated with a separate IgG domain. The two heavy chains are linked by different numbers of disulfide bonds at the hinge region: 2 for IgG ₁ and IgG ₄ , 4 for IgG ₂ , and 11 for IgG ₃ . In IgG ₁ , the last cysteine on the light chain forms a disulfide bond with the fifth cysteine on the heavy chain. In IgG ₂ , IgG ₃ and IgG ₄ , the last cysteine on the light chain forms a disulfide bond with the third cysteine on the heavy chain (Liu, H. and May, K., 2012, mAbs 4 , 17-23). Through reduction experiments, alkylation and LC-MS analysis, the characteristics of the difficulty of breaking disulfide bonds on human IgG ₁ antibodies can be known (Liu, H, et alAnal.Chem., 2010, 82, 5219–5226). Interchain disulfide bonds are more reductively cleaved than intrachain disulfide bonds, and disulfide bonds between light and heavy chains are more reductively cleaved than disulfide bonds between two heavy chains. The interchain disulfide bonds in the upper part of the two heavy chains are more prone to breaking than in the lower one. In addition, the disulfide bond on the CH2 domain is the easiest to be reduced. The disulfide bonds of the VL, CL, VH, and CH1 domains have similar moderate breakability, while the disulfide bonds of the CH3 domain are the least reducible (Liu, H, et alAnal.Chem., 2010, 82 , 5219–5226).

Based on the fact that the interchain disulfide bonds of human IgG ₁ antibodies are more likely to be broken, many research institutes and companies adopt the strategy of chemical site-specific ligation to reduce the interchain disulfide bonds of natural antibodies and then build bridges to recrosslink them, such as The use of so-called next-generation maleimides (NGMs), namely bromide or dibromomaleimides (Schumacher, FF, et al Org. Biomol. Chem. 2014, 12, 7261–7269 ), using a dialkylating agent to form a three-carbon bridge (Badescu, G., et al., Bioconjug. Chem. 2014, 25, 1124–1136, patents WO2013/190272, WO2014/064424 of PolyTherics Ltd.), Disubstituted heteroaryl bridges were used (US patent application 2015/0105539 to Concortis Biosystems), or via bismaleimides as bridges (WO2014/114207). We have also used bromomaleimide and dibromomaleimide as linkers for conjugating drugs and antibodies for a long time (WO2014/009774, PCT/IB2012/053554). However, the design of the above-mentioned bridge linker is to couple a cytotoxic molecule with a pair of disulfide bonds. Due to the limited number of disulfide bonds available for conjugation on the antibody (about 2 pairs), most Under the circumstances, the DAR value of the ADCs produced is lower than 2.

In view of the fact that the efficacy of ADCs drugs is limited by the number of toxic small molecules that finally reach tumor cells, in order to improve the therapeutic index of ADCs, the DAR value is preferably greater than 3 (Epenetos, AA et al, Cancer Res., 1986, 46, 3183–3191; Chari, RVAcc. Chem. Res., 2008, 41, 98-107, Zhao, RY2011 J. Med. Chem. 54, 3606-3623). The new disulfide bridge linker in the present invention can not only connect two or more small molecule drugs on each linker to achieve higher DAR (≥4), but also in order to selectively bridge the antibody Interchain disulfide bonds on the surface. This is due to the natural characteristics of the extended triple bond in the acetylene dicarbonyl group. When two toxic small molecules are connected to the two ends of the extended bridge linker, a macromolecule ( spacing), other disulfide groups, such as the lower disulfide group inside the heavy chain, are inaccessible. Therefore, the disulfide bridge linker of the present invention can selectively bridge the free sulfhydryl pairs between antibody chains broken by TCEP or DTT, thereby producing an ADC with a DAR greater than 4. Other disulfide bonds that have been reduced too much are difficult to access by bridges, especially extended bridges containing 2 toxic small molecules, and can be treated with oxides such as dehydroascorbic acid (DHAA) or Cu(II) reconnects. In conclusion, these bridging bodies in the present invention can generate homogeneous ADC drugs in a convenient manner.

Summary of the invention

In the present invention, the linker contains an acetylene dicarbonyl functional group, connects two small molecule drugs, and combines with a cell-binding agent (such as an antibody). Cell-binding molecule-linker-drug conjugates can be expressed as: Among them, Cb is a cell-binding agent, L is a linker, Drug1 and Drug2 are small drug molecules, n is an integer from 1 to 20, and two sulfur elements bridge Cb to L, and each bridge linker L is covalently connected to 2 one or more drug molecules. The advantages of using this type of linker in cell small molecule-drug conjugates are: a). The way of covalently cross-linking (re-bridging) the opened disulfide bond thiol on the cell-binding agent (such as an antibody) is beneficial Maintain the stability of the conjugate; b). The toxic small molecule/drug can be linked to a specific position of the cell-binding agent, such as the interchain site of an IgG antibody, thereby producing a uniform ADC drug.

In one aspect, the linker structure in the present invention can be expressed as formula (I)

Among them, the acetylene glycol structure on the linker can react with a pair of sulfur atoms in the cell-binding agent;

Z ₁ and Z ₂ are the same or different functional groups that can react with toxic drugs, and can pass disulfide bonds, ether bonds, ester bonds, thioether bonds, thioester bonds, peptide bonds, hydrazone bonds, and carbamate bonds , carbonate bond, amine bond (secondary, tertiary or quaternary), imine bond, heterocycloalkyl, heteroaryl, alkhydroxime bond or amide bond combined with toxic small molecule drugs;

R ₁ and R ₂ are the same, different or default straight chain alkyl containing 1 to 6 carbon atoms, branched or cycloalkyl with 3 to 6 carbon atoms, straight chain, branched or cycloalkene group or alkynyl group, or an ester group, ether group, amide group or polyethoxy group (OCH ₂ CH ₂ ) _p of 1 to 6 carbon atoms, wherein p is an integer from 0 to about 1000, or a combination of these groups ;

In addition, R ₁ and R ₂ are chain structures containing C, N, O, S, Si, and P atoms, optimally containing 0 to 500 atoms, covalently linked to X ₁ or X ₂ and Z ₁ or Z _{2 ;} each atom in R1 and R2 is _combined in all possible chemical ways, such as forming an alkyl group, an alkylene group, an alkenylene group, an alkynylene group, an ether, a polyoxyalkylene group, an ester, an amine, an imine, Polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, polyurethanes, amino acids, polypeptides, acyloxyamides, hydroxamic acids, or combinations of these groups;

X ₁ and X ₂ are independently selected from NH, N(R ₃ ), O, S or CH ₂ ; R ₃ is H, straight chain alkyl with 1 to 6 carbon atoms, branched chain with 3 to 6 carbon atoms or Cyclic alkyl, linear, branched or cyclic alkenyl or alkynyl, or ester, ether, amide or polyethoxy unit (OCH ₂ CH ₂ ) _p with 1 to 6 carbon atoms, where p is 0 Integers up to 1000, or combinations of these groups.

On the other hand, the cell-binding agent-drug conjugate of the present invention can be expressed as formula (II), wherein the cell-binding agent Cb, Drug1 and Drug2 have respectively reacted with the tail end of the bridge linker:

in:

Cb is a cell-binding agent, preferably an antibody;

In brackets is the linker-drug component coupled to the cell-binding molecule via a pair of sulfur atoms;

Drug ₁ and Drug ₂ are the same or different cytotoxic agents, which are represented by disulfide bonds, thioether bonds, thioester bonds, peptide bonds, hydrazone bonds, ether bonds, ester bonds, carbamate bonds, carbonate bonds , cycloheteroalkyl group, heteroaryl group, alkhydroxime bond or amide bond link with the cell-binding agent;

n is 1-20; the definitions of R ₁ , R ₂ , X ₁ and X ₂ are the same as formula (I).

In another aspect, the present invention includes a modified cell-binding agent, which can be expressed as formula (III), wherein the cell-binding agent Cb has reacted with a bridging linker, and the linker contains a functional group Z that can further react with a small drug molecule ₁ and Z2 _:

The definitions of Cb, Z ₁ , Z ₂ , n, R ₁ , R ₂ , X ₁ , and X ₂ are the same as formulas (I) and (II).

Furthermore, the present invention includes a modified drug molecule, which can be expressed as formula (IV), wherein the drugs Drug ₁ and Drug ₂ have reacted with the linker in formula (I), and still retain the ability to bind to the cell-binding agent. Sulfur atom pair reaction acetylene dioic acid structure:

The definitions of Drug ₁ , Drug ₂ , R ₁ , R ₂ , X ₁ , and X ₂ are the same as formulas (I) and (II).

The present invention also includes a method for preparing a cell-binding molecule-drug conjugate as shown in formula (II), wherein Drug ₁ and Drug ₂ are connected to a cell-binding agent through a bridge linker.

The present invention also includes a method of preparing a modified cell-binding molecule represented by formula (III), wherein the cell-binding molecule has been reacted with a bridge linker in formula (I).

The present invention also includes a method for preparing the modified small drug molecule shown in formula (IV), wherein the drug molecule has reacted with the bridge linker in formula (I).

illustration

Fig. 1 Synthesis of a bridge linker containing polyethylene glycol functional groups and its application in antibody-drug coupling.

Fig. 2 A synthetic route of a bridge linker and coupling antibody and drug through oxime bond.

Fig. 3 Synthesis of a bridge linker containing polypeptide and coupling antibody and drug through amide bond.

Fig. 4 Synthesis of a bridge linker containing polyethylene glycol and polypeptide.

Figure 5. Synthesis of a bridge linker containing polyethylene glycol and polypeptide and linking 2 to 4 small molecule drugs per linker via amide bonds.

Figure 6 Synthesis of tubulysin homologues modified with bridge linkers containing polypeptide and polyethylene glycol.

Figure 7. Conjugation of tubulysin homologues to cell-binding molecules using polyethylene glycol-containing bridging linkers.

Figure 8 Synthesis of cell-binding molecule-maytansinoids compounds coupled via bridge linkers.

Figure 9 Synthesis of a cell-binding molecule-MMAF homologue coupled via a bridging linker.

Figure 10 Synthesis of a cell-binding molecule-tubulysin homologue coupled via a bridging linker.

Detailed Description of the Invention

definition

"Alkyl" means a straight or branched chain aliphatic hydrocarbon containing from 1 to 8 carbon atoms. "Branched" means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclopentyl, Hexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl base, 2,3,4–trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5 -Dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, isooctyl. A C ₁ -C ₈ alkyl group may be unsubstituted or substituted by one or more of the following groups, including but not limited to -C ₁ -C ₈ alkyl, -O-(C ₁ -C ₈ alkyl ), aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C(O) N(R') ₂ ,-NHC(O)R',-SR',-S(O) ₂ R',-S(O)R',-OH,halogen,-N ₃ ,-NH ₂ ,- NH(R'), -N(R') ₂ and -CN; wherein each R' is independently selected from -C ₁ -C ₈ alkyl and aryl. "Halogen" refers to fluorine, chlorine, bromine, iodine atoms, preferably fluorine and chlorine atoms.

"Heteroalkyl" means a C ₂ -C ₈ alkyl group in which one to four carbon atoms are independently replaced by O, S and N atoms.

"Carbocycle" means a saturated or unsaturated ring, a monocyclic ring of 3 to 8 carbon atoms or a bicyclic ring of 7 to 13 carbon atoms. Monocyclic carbocycles contain 3 to 6 atoms, typically 5 to 6 atoms. Bicyclic carbocycles containing 7 to 12 atoms to form a [4,5], [5,5], [5,6] or [6,6] bicyclic ring system, or 9 to 10 atoms to form a [5,6] Or [6,6] bicyclic system. Typical C ₃ -C ₈ include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1, 4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cyclohexatrienyl, cyclooctyl and cyclooctadienyl.

"C ₃ -C ₈ carbocycle" refers to a 3, 4, 5, 6, 7 or 8 membered non-aromatic carbocycle containing saturation or unsaturation. A C ₃ -C ₈ carbocycle may be unsubstituted or substituted by one or more groups, including but not limited to -C ₁ -C ₈ alkyl, -O-(C ₁ -C ₈ alkyl) , aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C(O)N (R') ₂ , -NHC(O)R', -SR', -S(O)R', -S(O) ₂ R', -OH, Halogen, -N ₃ , -NH ₂ , -NH (R'), -N(R') ₂ and -CN, wherein each R' is independently selected from -C ₁ -C ₈ alkyl and aryl.

"Alkenyl" means a straight or branched chain aliphatic hydrocarbon containing carbon-carbon double bonds, 2 to 8 carbon atoms. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, 3-methyl-2-butenyl, n-pentenyl, hexenyl, heptenyl, octenyl.

"Alkynyl" means a straight or branched chain aliphatic hydrocarbon containing a carbon-carbon triple bond, of 2 to 8 carbon atoms. Examples of alkenyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, n-pentenyl, hexynyl, heptynyl, Octynyl.

"Alkylene" means a saturated, branched or straight chain or cyclic hydrocarbon radical of 1 to 18 carbon atoms, consisting of two hydrogens obtained by removal of two hydrogens from the same carbon or different carbons of a parent alkane. a monovalent free radical center. Typical alkylene groups include, but are not limited to, methylene (-CH ₂ -), 1,2-ethyl (-CH ₂ CH ₂ -), 1,3-propyl (-CH ₂ CH ₂ CH ₂ - ), 1,4-butyl (-CH ₂ CH ₂ CH ₂ CH ₂ -), etc.

"Alkenylene" means an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2 to 18 carbon atoms, obtained by removing two hydrogens from the same carbon or different carbons of a parent alkene Two monovalent free radical centers. Typical alkenylene groups include, but are not limited to, 1,2-ethenyl (-CH=CH-).

"Alkynylene" means an unsaturated, branched or straight-chain or cyclic hydrocarbon radical of 2 to 18 carbon atoms, comprising a parent alkyne derived by removal of two hydrogens on the same carbon or on different carbons. two monovalent free radical centers. Typical alkynylene groups include, but are not limited to, ethynyl, propynyl and 4-pentynyl.

"Aryl" or Ar means an aromatic or heteroaryl group consisting of one or several rings containing 3 to 14 carbon atoms, preferably 6 to 10 carbon atoms. Heteroaryl means that one or more carbon atoms (preferably 1, 2, 3 or 4 carbon atoms) on the aromatic ring are substituted by O, N, Si, Se, P or S (preferably O, S and N). "Aryl" or Ar also means that one or more hydrogen atoms on the aromatic ring are independently replaced by -R', halogen, -OR', or -SR', -NR'R", -N=NR', - N=R',-NR'R",-NO ₂ ,-S(O)R',-S(O) ₂ R',-S(O) ₂ OR',-OS(O) ₂ OR', -PR'R", -P(O)R'R", -P(OR')(OR"), -P(O)(OR')(OR"), or -OP(O)(OR') (OR"), where R', R" are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, aralkyl, carbonyl or a pharmaceutically acceptable salt.

A "heterocycle" is a ring system in which 1 to 4 atoms are each independently replaced by heteroatoms such as O, N, S, Se and P, preferably O, N and S. The definition of "heterocycle" can also refer to the literature The Handbook of Chemistry and Physics, 78th Edition, CRC Press, Inc., 1997-1998, p.225 to 226. Preferred non-aromatic heterocycles include, but are not limited to, epoxy, ethyleneimino, pyrrolidine, pyrazolidinyl, alkylimidazole, oxirane, tetrahydrofuran, dioxolane, pirarubicin, Dioxanyl, dioxolane, piperazine, morpholine, pyran, imidazoline, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyran, dithiane, thiomorpholine, Dihydropyran, pyranodoxorubicin, tetrahydropyridine, dihydropyridine, tetrahydropyrimidine, dihydrothiopyran, hexamethyleneimine and their structures resulting from condensation with phenyl.

"Heteroaryl" or aromatic heterocycle refers to 5 to 14 membered, preferably 5 to 10 membered, aromatic hetero, mono, bis or polycyclic rings, including pyrrole, pyridine, pyrazole, pyrimidine, pyrazine, tetrazolyl Thienyl, indole, quinoline, purine, imidazolyl, thiophene, thiazole, benzothiazole, furan, benzofuran, 1,2,4-triazole, isothiazole, triazole, tetrazole, isoquinoline , benzothiophene, isobenzofuran, pyrazole, carbazole, benzimidazole, isoxazole, pyridine nitroxide, and their ring structures produced by condensation with phenyl.

"Alkyl", "cycloalkyl", "alkenyl", "alkynyl", "aryl", "heteroaryl", "heterocycle", etc. Alkyl", "alkenylene", "alkynylene", "arylene", "heteroarylene" and "heterocycle" and other groups without two hydrogen atoms.

"Aralkyl" refers to an acyclic alkane free radical in which one ^of the carbon atoms, usually a terminal or sp hybridized carbon atom, is replaced by an aryl group. Typical arylalkyl groups include, but are not limited to, phenyl, 2-phenyl-1-ethyl, 2-phenyl-1-vinyl, naphthylmethyl, 2-naphthyl-1-ethyl, 2-naphthyl-1-vinyl, naphthalenephenyl, 2-naphthylbenzene-1-ethyl, etc.

"Heteroaralkyl" means an acyclic alkane free radical in which one ^of the hydrogen atoms attached to a carbon atom, usually a terminal or sp hybridized carbon atom, is replaced by a heteroaryl group. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl and the like.

Examples of "hydroxyl protecting groups" include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethyl Silicone ether, triethylsilyl ether, triisopropylsilyl ether, tert-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetyl ester, substituted acetyl ester, 2,2-dimethylpropionic acid Esters, Benzoates, Benzoates, Methanesulfonates and p-Toluenesulfonates.

"Leaving group" refers to a functional group that can be replaced by another group. Leaving groups well known in the art include, but are not limited to, halogen (chloro, bromo, iodo), methanesulfonyl, p-toluenesulfonyl, trifluoromethanesulfonyl, triflate.

The following abbreviations are used in the present invention and are defined as: Boc, tert-butoxycarbonyl; BroP, tris(dimethylamino)phosphine hexafluorophosphate bromide; CDI, carbonyldiimidazole; DCC, dicyclohexylcarbodiimide ; DCM, dichloromethane; DIAD, diisopropyl azodicarboxylate; DIBAL-H, diisobutylaluminum hydride; DIPEA, diisopropylethylamine; DEPC, diethyl pyrocarbonate; DMA, N, N-dimethylacetamide; DMAP, p-dimethylaminopyridine; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; DTT, dithiothreitol; EDC, 1-( 3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; ESI-MS, electrospray mass spectrometry; HATU, 2-(7-benzotriazole oxide)-N,N,N ',N'-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; NHS, N-hydroxysuccinimide; MMP, 4-methylmorpholine; PAB, p-aminophenyl; PBS, phosphate buffer (pH 7.0-7.5); PEG, polyethylene glycol; SEC, size-exclusion chromatography; TCEP, trichloroethyl phosphate; TFA, trifluoroacetic acid; THF, tetrahydrofuran ; Val, valine.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce deleterious, allergic or other adverse reactions when administered to animals or humans under appropriate circumstances.

"Pharmaceutically acceptable solvate" or "solvate" refers to a disclosed compound in association with one or more solvent molecules. Solvents in pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

"Pharmaceutical excipient" includes any carrier, diluent, adjuvant or vehicle, such as protective or antioxidant, filler, disintegrant, wetting agent, emulsifier, suspending agent, solvent, dispersion medium, coating, Antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The use of such media and agents in pharmaceutical active ingredients is well known in the art. Any conventional vehicles or agents are contemplated for use in the therapeutic compositions unless incompatible with the active ingredient. Supplementary active ingredients can also be incorporated to make suitable therapeutic compositions.

"Pharmaceutically acceptable salt" refers to derivatives of the disclosed compounds which are salts formed by the reaction of the parent compound with an acid or base. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts formed with the parent compound, such as non-toxic inorganic or organic acids. For example, conventional non-toxic salts include derivatives of inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.; and salts prepared with organic acids, such as acetic acid, propionic acid, succinic acid, Tartaric acid, citric acid, sulfonic acid, benzenesulfonic acid, glucose, glutamic acid, benzoic acid, salicylic acid, p-toluenesulfonic acid, oxalic acid, succinic acid, maleic organic acid, lactic acid, etc. Other salts include ammonium salts such as trimethylamine, meglumine, pyrrolethanol and the like, and metal salts such as sodium, potassium, calcium, zinc and magnesium salts.

The pharmaceutically acceptable salts in this patent can be synthesized from bases containing acids or bases by conventional chemical methods. Generally, such salts are formed by adding an equivalent amount of the appropriate base or acid to the free acid or base of the parent compound in water or an organic solvent or a mixture of both solvents. In general, non-aqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17 ^th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418, which is incorporated herein by reference.

The novel conjugated conjugates disclosed in the present invention use a bridging linker. Some linkers and their synthesis are shown in Figures 1 to 10.

bridging body

The synthesis of the bridge linker in this patent and the preparation of the drug-cell binding molecule conjugate are shown in Figures 1-9. The bridge linker has two elements: a) a substitutable acetylene dicarbonyl compound that can react with a pair of thiols to form a thioether covalent bond; b) a drug-reactive functional group, which includes but is not limited to disulfide , maleimides, haloacetyls, aldehydes, ketones, azides, amines, alkoxyamines, and hydrazines. The bridging substitution of acetylene dicarbonyl can be obtained by reacting acetylene diacid with amino, hydroxyl or mercapto groups to obtain amides, esters or mercapto esters. Examples of synthesis of bridge linkers are shown in Figures 1, 3, 4, 5, 6, 7, 8 and 9. The bridge substitution of acetylene dicarbonyl can also be obtained by reacting acetylene with acid halide or anhydride to form a carbon-carbon bond. Synthetic examples of these bridging linkers can be seen in Figures 2 and 10 .

Preferably, the structure of the bridging body compound is as shown in (I):

Wherein the acetylene dicarbonyl compound linker can react with a pair of sulfur atoms on the cell-binding molecule.

_Z1 and _Z2 are the same or different functional groups that can react with cytotoxic drugs to form disulfide bonds, ether bonds, ester bonds, thioether bonds, thioester bonds, peptide bonds, hydrazone bonds, and carbamate bonds , carbonate bond, amine bond (secondary, tertiary or quaternary), imine bond, heterocycloalkyl, heteroaryl, alkhydroxime bond or amide bond;

R1 and R2 are the same or different H, straight _- chain alkyl containing ₁ to 6 carbon atoms, branched or cycloalkyl with 3 to 6 carbon atoms, straight-chain, branched or cycloalkenyl or Alkynyl, or ester group, ether group, amide group or polyethoxy unit (OCH ₂ CH ₂ ) _p with 1 to 6 carbon atoms, or polypropoxy unit (OCH ₂ (CH ₃ ) CH ₂ ) _p unit , where p is an integer from 0 to about 1000, or a combination of these groups;

In addition, R ₁ and R ₂ are chain structures containing C, N, O, S, Si, and P atoms, optimally containing 0 to 500 atoms, covalently linked to X ₁ or X ₂ and Z ₁ or Z _{2 ;} each atom in R1 and R2 is _combined in all possible chemical ways, such as forming an alkyl group, an alkylene group, an alkenylene group, an alkynylene group, an ether, a polyoxyalkylene group, an ester, an amine, an imine, Polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, polyurethanes, amino acids, polypeptides, acyloxyamides, hydroxamic acids, or combinations of these groups;

X ₁ and X ₂ are independently selected from N(R ₃ ), O, S or CH ₂ ; R ₃ is H, straight chain alkyl with 1 to 6 carbon atoms, branched chain or ring with 3 to 6 carbon atoms like alkyl, linear, branched or cyclic alkenyl or alkynyl, or ester, ether, amide or polyethoxy unit (OCH ₂ CH ₂ ) _p with 1 to 6 carbon atoms, where p is 0 to An integer of 1000, or a combination of these groups.

In another embodiment, R ₁ , R ₂ and R ₃ can be chain structures including C, N, O, S, Si, and P atoms respectively, covalently linking the cell surface binding molecule and the conjugated drug. Atoms used to form linkers combine in all possible chemical ways, such as forming alkyl, alkylene, alkenylene, alkynylene, ether, polyethylene glycol, ester, amine, imine, polyamine, hydrazine , hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, polyurethane, amino acid, polypeptide, acetoxyamine, hydroxamic acid, or other structures; in addition, it should be understood that the atoms forming the linker (L) can be It is saturated or unsaturated, it can be a free radical, and it can form a bivalent ring structure with each other, including cycloalkane, cyclic ether, cyclic amine, arylene, heteroarylene and other structures.

Functional groups Z ₁ and Z ₂ can link cytotoxic drugs, and can generate bonds including disulfide bonds, thioethers, mercapto esters, peptides, hydrazines, ethers, esters, carbamates, carbonates, oximes or amide bonds. Such functional groups include, but are not limited to, mercapto, dithio, amino, carboxyl, aldehyde, carbonyl, maleimido, haloacetyl, hydrazino, alkoxyamino, and hydroxyl groups.

_The functional groups Z1 and Z2 that can react with the terminal amino groups of drugs or cytotoxic molecules can be, but not limited to, N _- hydroxysuccinimide esters, p-nitrophenol esters, dinitrophenol esters, and pentafluorophenol esters; Functional groups reactive with terminal thiols may be, but are not limited to, pyridyldithiol, nitropyridyldithiol, maleimido, haloacetate, carbonyl chloride; functional groups reactive with terminal carbonyl or aldehyde groups It can be, but is not limited to, amino group, alkoxyamino group, hydrazine, acetoxyamino group; the function reactive with terminal azido group can be, but is not limited to, alkynyl group.

In a preferred embodiment, R ₁ , R ₂ and R ₃ are linear alkyl groups containing 1 to 6 carbon atoms, or polyethoxy units (OCH ₂ CH ₂ ) _P , p=1-100.

The key step in the synthesis of acetylene dicarbonyl bridge linkers is, acetylene dicarbonyl or derivatives thereof, and the condensation of other component terminal amines (primary or secondary amines), alcohols or thiols, as shown in (Ia) below Show:

Wherein, X in formula (Ia) is X ₁ or X ₂ in structural formula (I), referring to N(R ₃ ), O, or S; R is R ₁ and/or R ₂ , R ₁ , R ₂ and R ₃ defines the same formula (I).

The same or independent OH, F, Cl, Br, I, nitrophenol, N-hydroxysuccinimide (NHS), phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, dinitrophenol, Lv1 and Lv2 Fluorophenol, monofluorophenol, pentachlorophenol, trifluoromethanesulfonic acid, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, p-toluenesulfonic acid, methanesulfonic acid, 2-ethyl -5-phenylisoxazole-3'-sulfonic acid, self anhydride or anhydride formed with other anhydrides such as acetyl anhydride, formic anhydride; or polypeptide condensation reaction intermediate or Mitsunobu reaction intermediate; condensation reagents include: 1-( 3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide ( DIC), 1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate (CMC, or CME-CDI), carbonyldiimidazole (CDI), TBTU (O-benzotriazole- N,N,N',N'-tetramethyluronium tetrafluoroboric acid), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), benzotriazol-1-yloxy Tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), diethylpyrocarbonate (DEPC), N ,N,N',N'-tetramethylchloroformamidine hexafluorophosphate, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate Ester (HATU), 1-[(dimethylamine)(morpholinyl)methylene]-1[1,2,3]triazolo[4,5-b]1-pyridine-3-oxafluoro Phosphate (HDMA), 2-chloro-1,3-dimethylimidazolium hexafluorophosphate (CIP), chlorotripyrrolidinylphosphonium hexafluorophosphate (PyCloP), bis(tetramethylene)fluoride Formamide (BTFFH), N,N,N',N'-tetramethyl-thio-(1-oxo-2-pyridyl)thiouronium hexafluorophosphate, 2-(2-pyridone- 1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TPTU), sulfur-(1-oxo-2-pyridyl)-N,N,N',N'- Tetramethylthiourea hexafluorophosphate, O-[(ethoxycarbonyl)cyanomethylamine]-N,N,N',N'-tetramethylthiourea hexafluorophosphate (HOTU), (1 -cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylamino-morpholine-carbenium hexafluorophosphate (COMU), (benzotriazol-1-yloxy base) dipyrrolidinyl carbon hexafluorophosphate (HBPyU), N-benzyl-N'-cyclohexylcarbodiimide (or loaded on a polymer), dipyrrolidinyl (N-succinimidyloxy ) carbonium hexafluorophosphate (HSPyU), 1-(chloro-1-pyrrolidinylmethylene)pyrrolidine hexafluorophosphate (PyClU), 2-chloro-1,3-dimethylimidazolium tetrafluoroboron (CIB), (Benzotriazol-1-yloxy)dipiperidinium hexafluorophosphate (HBPipU) , 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), brominated tris(dimethylamino)phosphine hexafluorophosphate (BroP), 1- N-Propyl Phosphoric Anhydride (PPACA, ), 2-isocyanoethylmorpholine (MEI), N,N,N',N'-tetramethylurea-oxygen-(N-succinimido)hexafluorophosphate (HSTU), 2 -Bromo-1-ethylpyridine tetrafluoroborate (BEP), oxy-[(ethoxycarbonyl)cyanomethylamine]-N,N,N',N'-tetramethylthiourea tetrafluoroboron Hydrochloride (TOTU), 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (MMTM, DMTMM), 2-succinimidyl-1, 1,3,3-tetramethyluronium tetrafluoroborate (TSTU), N,N,N',N'-tetramethyl-O-(3,4-dihydro-4-oxo-1, 2,3-Benzotriazin-3-yl)urea tetrafluoroborate (TDBTU), azodicarbonyl dipiperidine (ADD), bis(4-chlorobenzyl)azodicarboxylate ( DCAD), di-tert-butyl azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD), diethyl azodicarboxylate (DEAD).

When X is CH ₂ , the acetylene dicarbonyl group on the bridge linker is connected to other functional groups through a CC bond. At this time, the key steps of the synthesis are bis(trimethylsilyl)acetylene, acetylene magnesium dibromide (Grignard reagent), and acetylene dicarbonyl The reactions of lithium salts or other acetylene dimetallic salts with acyl halides or anhydrides are illustrated as (Ib), (Ic), (Id), (Ie), (If), (Ig) and (Ih):

Here M is Na, K, Li, Cu, CuLi, Sn, Ti, Ca, Mg or Zn.

Specific examples of bridge linker synthesis are shown in Figures 1-10. Usually, the acetylene dicarbonyl linker contains a functional group that can react with the small molecule drug on the conjugate.

Cell-binding molecule-drug conjugates

The conjugate of the present invention can be represented by the following formula:

Among them, Cb is a cell-binding agent, L is a linker, Drug1 and Drug2 are small drug molecules, n is an integer from 1 to 20, and two sulfur elements bridge Cb to L, and each bridge linker L is covalently connected to 2 one or more drug molecules.

This bridging linker L may consist of one or more linker components. Typical linker components include: 6-maleimidocaproic acid ("MC"), 3-maleimidopropionic acid ("MP"), valine-citrulline ("val- cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af"), p-aminobenzyloxycarbonyl ("PAB"), 4-thiopentanoic acid ("SPP") , 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid ("MCC"), (4-acetyl)aminobenzoic acid ("SIAB"), 4-thiobutyric acid ( SPDB), 4-thio-2-hydroxysulfobutanoic acid (2-Sulfo-SPDB), one or more ethoxy-CH ₂ CH ₂ O- units ("EO"or"PEO"), also including Other linkers are well known in the art, some of which are listed here.

Examples of linkers containing these components are:

(contains 6-maleimidocaproic acid MC)

(contains 3-maleimidopropionic acid MP)

(contains p-aminobenzyloxycarbonyl PAB)

(contains ethylmaleimide ME)

(contains valine-citrulline)

(contains 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid MCC)

(contains (4-acetyl)aminobenzoic acid)

(contains 4-thio-2-hydroxysulfobutanoic acid, 2-thio-SPDB)

The structure of the preferred conjugate is as formula (II):

in:

Cb is a cell-binding agent, preferably an antibody;

Drug ₁ and Drug ₂ are the same or different cytotoxic agents, which are linked by bridges in the form of alkyl, alkylene, alkenylene, alkynylene, ether, polyalkoxy, ester, amine, imine, Polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, carbamates, amino acids, peptides, acyloxyamides, hydroxamic acids, disulfide bonds, thioethers, thioesters, Carbamate, carbonate, heterocyclic, heteroalkyl, heteroaryl, or alkhydroxime linkages and combinations thereof linked to cell-binding agents;

n is 1-20; the definitions of R ₁ , R ₂ , X ₁ and X ₂ are the same as formula (I).

See below for a more detailed description, Drug1 and Drug2 can be any small molecule drug, including but not limited to, tubulysin, calicheamicin, auristatin, maytansine, CC-1065 analog, morpholino, doxorubicin , taxanes, cryptophycins, epothilones, benzodiazepine series dimers (for example, pyrrole benzodiazepines (PBD) or tomamycin, indole benzodiazepines, imidazole benzodiazepines diazepine or oxazole benzodiazepine dimer).

To synthesize conjugated conjugates, the disulfide bonds on the cell-binding agent are first reduced to generate a pair of free sulfhydryl groups, and then react with the bridge linker in the formula (I) in the aqueous phase of pH 5-9, where it can Add or not add 0-30% of water-miscible organic solvents, such as: N,N-dimethylacetamide, N,N-dimethylformamide, ethanol, methanol, acetone, acetonitrile, tetrahydrofuran, Isopropanol, dioxane, propylene glycol, or ethylene glycol to introduce Z ₁ and Z ₂ reactive groups, which can be disulfide bonds, maleimide groups, haloacetyl groups, azides Azide, 1-yne, ketone, aldehyde, alkoxyamino or hydrazide. The reactive group of the cytotoxic molecule then reacts with the corresponding modified cell-binding agent. For example, the synthesis of cell-binding agent-drug conjugates linked by disulfide bonds is achieved by disulfide bond exchange between the disulfide bond in the modified cell-binding agent and the drug containing a free sulfhydryl group; The synthesis of ether-bonded cell-binding sub-drug conjugates is achieved by reacting cell-binding agents modified with maleimide groups or haloacetyl groups or ethylsulfones with free sulfhydryl-containing drugs; synthetic intramolecular Conjugates of acid-labile hydrazones can be realized by the reaction of a hydrazide group and a carbonyl in a linker, and the specific method is also known in the art (referring to P.Hamann et al., Hinman, LM, 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 synthesis of conjugates containing triazole groups can be achieved by a click chemical reaction (dipolar cycloaddition) between 1-alkynes on a linker and drugs containing azide groups (Lutz, JF. et al, Adv. Drug Del. Rev. 2008, 60, 958–970; Sletten, EM et al Acc. Chem. Research 2011, 44, 666–676).

Alternatively, the drug small molecule can react with a modified cell-binding molecule-linker structure as shown in formula (III) that has been coupled with a cell-binding agent and contains a reactive functional group. For example, a drug containing a sulfhydryl group can be reacted with a maleimide, haloacetyl, or ethylsulfonyl group on a linker in formula (III) in a pH 5.5-9 buffer to generate a thioether-linked conjugate. Conjugates. The thiol-containing drug can perform disulfide bond exchange with the pyridyl disulfide fragment on the linker in formula (III) to generate a disulfide bond-linked conjugate. Drugs containing hydroxyl or mercapto groups can react with the halogen on the linker in formula (III), especially the halogen on the α-halogenated carboxylic acid, under weak base conditions, such as pH 8.0-9.5, to react to obtain ether or thioether bonds Linked Conjugates. Hydroxyl-containing drugs can also be condensed into esters with carboxyl groups on the linker in formula (I) under the action of a condensing agent of EDC or DCC. The drug-modified bridge linker is then coupled to a cell-binding molecule. Drugs containing amino groups can be combined with NHS, imidazole, nitrophenol, N-hydroxysuccinimide, phenol, dinitrophenol, pentafluorophenol, 2,3,5,6 - Tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, trifluoromethanesulfonic acid, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, toluenesulfonic acid, methanesulfonic acid, 2-ethyl Carboxylate reaction of 5-phenylisoxazole-3'-sulfonic acid to obtain a conjugate conjugate linked by amide bond.

The conjugate can be purified by standard biochemical methods, such as gel filtration with Sephadex G25 or Sephacryl S300, adsorption chromatography, ion exchange or dialysis. In some cases, such as small-molecule cell-binding agents (such as folic acid, melanocyte stimulating hormone, EGF, etc.) combined with small-molecule drugs, they can be purified by chromatography such as HPLC, medium-pressure column chromatography, or ion-exchange chromatography.

Modified cell-binding agents/molecules

Preferably, the structure of the cell-binding agent modified by the linker in the present invention is as formula (III):

wherein Cb, Z ₁ , Z ₂ , n, R ₁ , R ₂ , X ₁ , and X ₂ are the same as those defined in formulas (I) and (II).

_In preferred embodiments, Z and Z are _dithio , maleimide, haloacetyl, alkoxyamine, azide, ketone, aldehyde, hydrazine, N-hydroxysuccinimide ester or Phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, trifluoromethanesulfonic acid, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole , toluenesulfonic acid, methanesulfonic acid, 2-ethyl-5-phenylisoxazole-3'-sulfonic acid carboxylate. Z1 and _Z2 can react with cytotoxic drugs to form thioethers, hydrazones, amides, _alkoximes , carbamates, esters, ethers or disulfide bonds. Modified cell-binding agents can be prepared by reacting the cell-binding agent with the bridge linker described in formula (I) as described above in formula (II).

In order to realize the high-efficiency connection between the alkyne group on the bridging body and the cell-binding agent, especially the free pair of sulfhydryl groups in the antibody, it is necessary to add a small amount of organic solvent in the reaction system or after the reaction is completed so that the structure in formula (III) is Dissolves well in water. When modifying the cell-binding agent, first dissolve the cross-linking agent (bridge connection) in the formula (I) in a polar organic solvent that can be miscible with water, for example: various alcohols such as methanol, ethanol and propanol, acetone , acetonitrile, tetrahydrofuran (THF), 1,4-dioxane, dimethylformamide (DMF), dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO); the solution concentration is slightly higher, such as 1-500mM. At the same time, dissolve the cell-binding agent, such as an antibody, at a concentration of 1-35 mg/ml in a buffer solution with a pH of 5-9.5, preferably 6-8.5, and react with 1-20 equivalents of TCEP or DTT for 20 minutes to 12 hours. After reduction, DTT was removed by SEC column purification. TCEP can be purified by SEC column or directly into the next step without purification. In addition, the antibody or other cell-binding agent reduced by TCEP can be carried out in the presence of the bridge linker of formula (I), at this time the cross-linking conjugation of the cell-binding agent is carried out simultaneously with the reduction of TCEP.

The reaction of modifying the cell-binding agent is generally carried out in a buffer solution with a pH of 6 to 9, preferably 6.5 to 7.5, and any non-nucleophilic buffer salt system within this pH range can be used. Typical buffers include phosphate, triethanolamine hydrochloride, HEPES, and MOPS buffers, which may contain other components such as cyclodextrins, sucrose, and salts such as sodium chloride and potassium chloride. Add the bridging linker solution in formula (I) to the reduced cell-binding agent solution, and incubate at 4 to 45°C, preferably at room temperature, and monitor the reaction by measuring the absorbance of the solution at 254nm or other suitable wavelengths process. After the reaction is complete, the modified cell-binding agent can be purified according to conventional methods, for example, using gel filtration chromatography or adsorption chromatography.

By measuring the UV absorbance of the nitropyridinethione, dinitropyridinethione, pyrithione, carboxamide pyrithione, and dicarboxamide pyrithione groups produced by the reaction, it can be determined that the cell-binding agent is blocked. The degree of modification. If the conjugate has no chromophoric group, it can be analyzed and determined by LC-MS or more preferably UPLC-QTOF mass spectrometry, and capillary electrophoresis mass spectrometry (CEMS). The bridge linker in the present invention can contain different types of functional groups, which can react with various drugs, especially cytotoxic drugs with suitable functional groups. For example, modified cell-binding agents containing amino or hydroxyl substituents can react with drugs containing N-hydroxysuccinimide (NHS) esters, and modified cell-binding agents containing sulfhydryl groups can react with drugs containing maleimide or Drug reactions of haloacetyl groups. In addition, modified cell-binding agents containing carbonyl groups (aldehyde or ketone groups) can react with drugs containing hydrazides or alkoxyamines. Those skilled in the art can easily determine which linker to use according to the reactivity of the functional groups therein.

Modified Cytotoxic Drugs

The cytotoxic drug structure modified with the bridge linker in the present invention is preferably (IV):

The definitions of Drug ₁ , Drug ₂ , Z ₁ , Z ₂ , n, R ₁ , R ₂ , X ₁ , and X ₂ are the same as formulas (I) and (II).

The modified drug (IV) is obtained by reacting the linker in the formula (I) with the drug molecule, wherein the acetylene dicarbonyl functional group can react with a pair of sulfhydryl groups on the cell-binding agent. Acetylene dicarbonyl is obtained by the condensation reaction of acetylene, and its method is already in reaction formula (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig) and (Ih) describe.

However, for drugs containing sulfhydryl groups, or drugs linked to cell-binding agents through bridge linkers using thioether, thioester or disulfide bonds, the preferred way is that Drug1 or Drug2 is first linked by thioether, thioester or disulfide bonds To R ₁ or R ₂ , the generated R ₁ -Drug ₁ or R ₂ -Drug ₂ fragments are combined with acetylene dicarbonyl to form a drug modified by a bridge linker such as structural formula (IV).

For example, a drug containing a sulfhydryl group can react with maleimide on R ₁ or R ₂ in a neutral pH buffer to form R ₁ -Drug ₁ or R ₂ -Drug ₂ bonded by a thioether bond, and then Condensation with acetylene dicarbonyl forms a modified drug comprising a thioether linkage as in formula (IV). Hydroxyl-containing drugs can react with R ₁ or R ₂ containing halogen, p-toluenesulfonic acid or mesylate under weak base conditions to obtain R ₁ -Drug ₁ or R ₂ -Drug ₂ bonded by ether bonds, It is then condensed with an acetylene dicarbonyl to form a modified drug comprising a thioether linkage as in formula (IV). Drugs containing hydroxyl groups can also be condensed with carboxyl-containing linkers in structure (I) under the action of dehydrating agents such as EDC or DCC to obtain modified drugs containing ester bonds as in formula (IV). Drugs containing sulfhydryl groups can react with maleimide, vinylsulfone or haloacetyl groups on R ₁ or R ₂ to generate R ₁ -Drug ₁ or R ₂ -Drug ₂ bonded by thioether bonds, and then react with The acetylene dicarbonyl is condensed to form a modified drug comprising a thioether linkage as in formula (IV). Similarly, the amino group-containing drug can also be condensed with the carboxyl group on the bridge linker (I) to obtain a modified drug containing an amide bond as in formula (IV). The modified drug can be purified by standard methods such as silica gel or alumina column chromatography, recrystallization, preparative thin layer chromatography, ion exchange chromatography or high performance liquid chromatography.

cell binding agent

The cell-binding agents in the present invention, including conjugated and modified cell-binding agents, can be any type of molecules known or to be disclosed, capable of binding to cell fragments that have therapeutic significance or are biologically modified, compound or react.

Cell-binding agents include, but are not limited to, large molecular weight proteins such as whole antibodies (polyclonal, monoclonal, dimer, multimeric, multispecific, e.g. bispecific); single chain antibodies; antibody fragments Such as Fab, Fab', F(ab') ₂ , F _v (Parham, J. Immunol.1983, 131, 2895-2902); fragments produced by Fab expression library, anti-idiotype (anti-Id) antibody; CDR ; diabodies; trivalent antibodies and epitope-binding fragments of any of the above antibodies that immunospecifically bind cancer cell antigens; viral antigens; biological activity (Miller et al J.of Immunology 2003,170,4854-4861); interferon (such as type I, II, III); polypeptide; lymphokines such as IL-2, IL-3, IL-4, IL- 5. IL-6, IL-10, GM-CSF, interferon-γ (IFN-γ); hormones such as insulin, TRH (thyrotropin-releasing hormone), MSH (melanocyte-stimulating hormone), steroid hormones such as androgen Hormones and estrogens; growth factors and colony-stimulating factors such as epidermal growth factor (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factors (TGF) such as TGFα, TGFβ, insulin, and insulin-like growth Factors (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); Small molecular weight proteins; polypeptides; peptides and peptide hormones such as bombesin, gastrin, gastrin-releasing peptide; platelet-derived growth factors; interleukins and cytokines, e.g., interleukin-2 (IL- 2), interleukin-6 (IL-6), leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor (GM-CSF); vitamins, such as folic acid; apoproteins and glycoproteins, such as transferrin ( O'Keefe et al, J.Biol.Chem.1985 260932-937); carbohydrate-binding proteins or lipoproteins, such as lectins; cellular nutrient delivery molecules; small molecule inhibitors, such as prostate-specific membrane antigen (PSMA) inhibitors and small molecule tyrosine kinase inhibitors (TKI), non-peptide or any other cell-binding molecules or substances, such as bioactive polymers (Dhar, et al, Proc.Natl.Acad.Sci.2008,105,17356-61 ), biologically active dendrimers (Lee, et al, Nat.Biotechnol.2005, 23, 1517-26; Almutairi, et a l; Proc.Natl.Acad.Sci.2009,106,685-90), nanoparticles (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, monoclonal antibodies are preferred as cell surface binding agents if appropriate monoclonal antibodies are available. Antibodies can be murine, human, humanized, chimeric or derived from other species.

The production of antibodies for use in the present invention includes in vivo or in vitro methods or a combination thereof. Methods for producing polyclonal anti-receptor peptide antibodies are well known in the art, for example as described in US Patent 4,493,795. Monoclonal antibodies are typically produced by fusing myeloma cells with spleen cells from mice that have been immunized with the desired antigen ( G.; Milstein, C. Nature 1975, 256:495-497). Detailed procedures are described in "Antibodies--A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory Press, New York (1988)", which is incorporated herein by reference. Specifically, a mouse, rat, hamster or any other mammal can be immunized with an antigen of interest, such as whole target cells, antigens isolated from target cells, whole virus, inactivated whole virus and viral proteins. Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000. Fusion cells were screened for sensitivity to HAT (hypoxanthine-aminopterin-thymidine). Hybridomas carrying out the monoclonal antibodies of the invention can be identified by their ability to immunoreact with a specific receptor or to inhibit the activity of a receptor on a target cell.

The production of monoclonal antibodies for use in the present invention is carried out in monoclonal hybridoma cultures comprising a nutrient medium and hybridomas that secrete antibody molecules having the appropriate antigen specificity. The cultures are maintained under suitable conditions for a period of time sufficient to allow the hybridomas to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. Antibody molecules are further separated by well-known techniques, such as protein A affinity chromatography, anionic, cationic, hydrophobic or size exclusion chromatography (especially by protein A affinity chromatography and size exclusion chromatography), centrifugation, differential Solubility or any other standard technique for protein purification.

Useful media for the preparation of these compositions are well known in the art and are commercially available, including synthetic media. An example of a synthetic medium is Dulbecco's Minimal Essential Medium (DMEM; Dulbecco et al., Virol. 1959, 8, 396) supplemented with 4.5 g/ml glucose, 0-20 mM glutamine, 0-20% fetal bovine serum, several Heavy metals such as Cu, Mn, Fe or Zn or/and heavy metal salts in ppm, and defoamers such as polyoxyethylene-polyoxypropylene block copolymers.

Alternatively, antibody-producing cell lines can also be obtained by techniques other than fusion, such as transformation of oncogenic DNA into B lymphocytes, or transfection of oncogenic viruses, such as Epstein-Barr virus (EBV, also known as human herpesvirus 4 (HHV-4)) or Kaposi's sarcoma-associated herpes virus (KSHV), as found in US Patents 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; Monoclonal antibodies can also be prepared by anti-receptor peptides or peptides containing a terminal carboxyl group, which are well known in the art, for reference 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. Generally, anti-receptor peptides or peptide analogs are used as immunogens for generating anti-receptor peptide monoclonal antibodies, either alone or linked to immunogenic carriers.

Monoclonal antibodies used as binding molecules in the present invention can also be obtained by other techniques known in the art. Particularly useful are methods of making fully human antibodies. One method is phage display technology, which uses affinity enrichment and can be used to select human antibodies that specifically bind to an antigen. Phage display technology is also described in detail in the literature, and the construction and screening of phage display libraries are also well known in the art. References can be made to 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.

Monoclonal antibodies produced by hybridomas fused with non-human, eg mouse cells, can be humanized to avoid the production of human anti-mouse antibodies. Common antibody humanization methods are CDR grafting techniques, which have also been described in detail, such as US Patents 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, Mol Immunol.2007,44(8):1986-98; Li et al, Proc.Natl.Acad.Sci.US A.2006,103(10):3557 -62, incorporated herein by reference. Fully human antibodies can also be prepared by immunizing transgenic mice, rabbits, monkeys or other mammals carrying a majority of human globulin heavy and light chains with the immunogen.这些老鼠的例子有：Xenomouse(Abgenix/Amgen)，HuMAb-Mouse(Medarex/BMS)和VelociMouse(Regeneron)，参考美国专利6,596,541,6,207,418,6,150,584,6,111,166,6,075,181,5,922,545,5,661,016,5,545,806,5,436,149和5,569,825。 When used in human therapy, mouse variable regions and human constant regions can also be fused to become "chimeric antibodies", which are significantly less immunogenic in humans than mouse mAbs (Kipriyanov et al, MolBiotechnol . 2004, 26:39-60; Houdebine, Curr Opin Biotechnol. 2002, 13:625-9). In addition, site-directed mutagenesis in antibody variable regions can lead to antibodies with higher affinity and specificity (Brannigan et al, Nat Rev Mol Cell Biol. 2002, 3:964-70; Adams et al, J Immunol Methods. 1999,231:249-60), changes in the constant region of an antibody can improve its effector functions that mediate binding and cytotoxicity.

Antibodies immunospecific for malignant cell antigens can also be obtained commercially or produced by any known method, such as chemical synthesis or recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for malignant cell antigens can be obtained commercially, eg, from the GenBank database or similar databases, literature publications, or by conventional cloning and sequencing.

In addition to antibodies, a peptide or protein that interacts (binds, blocks, targets or otherwise) with an epitope or corresponding receptor on a target cell can also serve as a binding molecule. These peptides or proteins may be any random peptide or protein that has an affinity for the epitope or corresponding receptor and does not necessarily have to be a member of the immunoglobulin family. These peptides can be isolated by techniques similar to phage display antibodies (Szardenings, J Recept Signal Transduct Res. 2003; 23(4):307-49). Peptides obtained from random peptide libraries can be used similarly to antibodies and antibody fragments. Peptide or protein binding molecules can be coupled or linked to macromolecules or other substances, including but not limited to albumin, polymers, liposomes, nanoparticles, dendrimers, so long as such linkage retains antigen binding of the peptide or protein specificity.

Examples of antibodies linked to drug molecules via the bridge linker of this patent include, but are not limited to, 3F8 (anti-GD2), Abba Monoclonal antibody (anti-CA-125), abciximab (anti-CD41 (integrin α-IIb), adalimumab (anti-TNF-α), adecatumumab (anti-EpCAM, CD326), afelimomab ( Anti-TNF-α), Afutuzumab (anti-CD20), Alacizumab monoclonal antibody (anti-VEGFR2), 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), Azetizumab (anti-CEA), Atlizumab (anti-L-selectin CD62L), Atlizumab (tocilizumab, Actemra, RoActemra, anti-IL-6 receptor), Atorolimumab (anti-Rhesus factor), Bapineuzumab (anti-beta amyloid), Basiliximab (Simulect, anti-CD25 (alpha chain of IL-2 receptor) ), Bavituximab (anti-phosphatidylserine), Bectumomab (LymphoScan, anti-CD22), belimumab (Benlysta, LymphoStat-B, anti-BAFF), benralizumab (anti-CD125), Bertilimumab (anti-CCL11 (eotaxin-1)) , Besilesomab (Scintimun, anti-CEA-related antigen), Bevacizumab (Avastin, anti-VEGF-A), Biciromab (FibriScint, anti-fibrin II beta chain), Bivatuzumab (anti-CD44v6), Blinatumomab (BiTE, anti-CD19), Brentuximab (cAC10, anti-CD30TNRSF8), Briakinumab (anti-IL-12, IL-23), Canakinumab (Ilaris, anti-IL-1), Cantuzumab (C242, anti-CanAg), Capromab, Catumaxomab (Removab, anti-EpCAM, anti-CD3 ), CC49 (anti-TAG-72), Cedelizumab (anti-CD4), Certolizumab monoclonal antibody (Cimzia anti-TNF-α), cetuximab (Erbitux , IMC-C225, anti-EGFR), Citatuzumab bogatox (anti-EpCAM), Cixutumumab (anti-IGF-1), Clenoliximab (anti-CD4), Clivatuzu-mab (anti-MUC1), Conatumumab (anti-TRAIL-R2), CR6261 (anti-influenza A hemagglutinin), Dacetuzumab (anti-CD40), Daclizumab (Zenapax, anti-CD25 (IL-2 receptor alpha chain)), Daratumumab (anti-CD38 (cyclic ADP ribose hydrolase), Denosumab (Prolia, anti-RANKL), Detumomab (anti-B lymphoma cells), Dorlimomab, Dorlixizumab, Ecromeximab (anti-GD3 ganglioside), Eculizumab (Soliris, anti-C5), Edobacomab (anti-endotoxin), 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 antibody (anti-ICAM-1 (CD54)), Epitumomab (anti-episialin), Etruzumab (anti-CD22), Erlizumab (anti-ITGB2(CD18)), Ertumaxomab (Rexomun, anti-HER2/neu, CD3), Etalizumab (Abegrin, anti-integrin αvβ3), Exbivirumab (anti-HBsAg), Fanolesomab (NeutroSpec, anti-CD15), Faralimomab (anti-interferon receptor), Farletuzumab (anti-folate 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-β), Galiximab (anti-CD80), Gantenerumab ( anti-beta amyloid), Gavilimomab (anti-CD147 (basigin)), Gemtuzumab (anti-CD33), Girentuximab (anti-carbonic anhydrase 9), Glembatumumab (CR011, anti-G PNMB), Golimumab (Simponi, anti-TNF-α), Gomiliximab (anti-CD23 (IgE receptor)), Ibalizumab (anti-CD4), Ibritumomab (anti-CD20), Igovomab (Indimacis-125, anti-CA-125) , Imciromab (Myoscint, anti-cardiac myosin), Infliximab (Remicade, anti-TNF-α), Intetumumab (anti-CD51), Inolimomab (anti-CD25 (IL-2 receptor alpha chain), inotuzumab (anti-CD22 ), Ipilimumab (anti-CD152), Iratumumab (anti-CD30 (TNFRSF8)), Keliximab (anti-CD4), Labetuzumab (CEA-Cide, anti-CEA), Lebrikizumab (anti-IL-13), Lemalesomab (anti-NCA-90 (grain cell antigen)), Lerdelimumab (anti-TGFβ2), Lexatumumab (anti-TRAIL-R2), Libivirumab (anti-HBsAg), Lintuzumab (anti-CD33), Lumilimumab (anti-CD40), Lumilimumab (anti-CD23 (IgE receptor), Mapatumumab (anti-TRAIL-R1), Masimomab (anti-T-cell receptor), Matuzumab (anti-EGFR), Mepolizumab (Bosatria, anti-IL-5), Metelimumab ( Anti-TGFβ1), Milatuzumab (anti-CD74), Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3 ganglioside), Morolimumab (anti-rhesus factor), Motavizumab (Numax, anti-RSV ), Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolomab (anti-C242), Naptumomab (anti-5T4), Natalizumab (Tysabri, anti-integrin α4), Nebumab (anti-endotoxin ), Necitumumab (anti-EGFR), Nerelimomab (anti-TNF-α), Nimotuzumab (Theracim, Theraloc, anti-EGFR), Nofetumomab, Ocrelizumab (anti-CD20), Olimumab (Afolimomab, anti-LFA-1 ( CD11a)), Ofatumumab (Arzerra, anti-CD20), Olaratumab (anti-PD GF-Rα), Omalizumab (Xolair, anti-IgE Fc region) Oportuzumab (anti-EpCAM), Oregovomab (OvaRex, anti-CA-125), Otelixizumab (anti-CD3), Pagibaximab (anti-lipoteichoic acid), Palivizumab (Synagis, Abbosynagis , anti-respiratory syncytial virus), panitumumab (Vectibix, ABX-EGF, anti-EGFR), Panobacumab (anti-Pseudomonas aeruginosa (Pseudomonas aeruginosa)), Pacotuzumab (anti-IL-4), Pemtumomab ( Theragyn, anti-MUC1), Pertuzumab (Omnitarg, 2C4, anti-HER2/neu), Pexelizumab (anti-c5), Pintumomab (anti-adenocarcinoma antigen), Priliximab (anti-CD4), Pritumumab (anti-vimentin), PRO140 (anti- -CCR5) Racotumomab (1E10, anti-N-glycolylneuraminic acid (NeuGc, NGNA)-ganglioside GM3)), Rafivirumab (anti-rabies virus glycoprotein), Ramucirumab (anti-VEGFR2), Ranibizumab (Lucentis, anti- VEGF-A), Raxibacumab (anti-anthrax toxin, protective antigen), Regavirumab (anti-CMV glycoprotein B), Reslizumab (anti-IL-5), Rilotumumab (anti-HGF), Rituximab (MabThera, Rituxanmab, anti -CD20), Robatumumab (anti-IGF-1 receptor), Rontalizumab (anti-IFN-α), Rovelizumab (LeukAr-rest, anti-CD11, CD18), Ruplizumab (Antova, anti-CD154 (CD40L)), Satumomab (anti-TAG -72), Sevirumab (anti-CMV), Sibrotuzumab (anti-FAP), Sifalimumab (anti-IFN-α), Siltuximab (anti-IL-6), Siplizumab (anti-CD2), Smart MI95 (anti-CD33), Solanezumab (anti-amyloid-beta), Sonepcizumab (anti-sphingosine-1-phosphate), Sontuzumab (anti-episialin), Stamulumab (anti-myostatin), Sulesomab (LeukoScan, anti-NCA- 90 (granulocyte antigen)), Tacatuzumab (anti-α-fetoprotein), Tadocizumab (anti-integrin αIIbβ3), Talizumab (anti-IgE), Tanezumab (anti-NGF), Taplitumomab (anti-CD19), Tefibazumab (Aurexis, (anti-clotting factor A)), Telimomab, Tenatumomab (anti-tenascin C), Teneliximab (anti-CD40), Teplizumab (anti-CD3), TGN1412 (anti-CD28), Ticilimumab (Tremelimumab, anti-CTLA-4), Tigatuzumab ( Anti-TRAIL-R2), TNX-650 (anti-IL-13), Tocilizumab (Atlizumab, Actemra, RoActemra, IL-6 receptor), Toralizumab (anti-CD154 (CD40L)), Tositumomab (anti-CD20), trastuzumab Anti (Herceptin, anti-HER2/neu), Tremelimumab (anti-CTLA-4), Tucotuzumab celmoleukin (anti-EpCAM), Tuvirumab (anti-Hepatitis B virus), Urtoxazumab (anti-E. coli), Ustekinumab (Stelara, anti-IL -12, IL-23), Vapaliximab (anti-AOC3 (VAP-1)), Vedolizumab (anti-integrin α4β7), veltuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1) 1)), Visilizumab (Nuvion, anti-CD3), Vitaxin (anti-angiointegrin avb3), Volociximab (anti-integrin α5β1), Votumumab (HumaSPECT, anti-tumor antigen CTAA16.88), Zalutumumab (HuMax-EGFR, Zanolimumab ( HuMax-CD4, anti-CD4), Ziralimumab (anti-CD147(basigin)), Zolimomab (anti-CD5), etanercept Alefacept Abatacept Rilonacept (Arcalyst), 14F7 (anti-IRP-2 (iron regulatory protein 2)), 14G2a (anti-GD2 ganglioside, derived from Nat.Cancer Inst., for the treatment of melanoma and solid tumors), J591 (anti-PSMA , from Weill Cornell School of Medicine, for the treatment of prostate cancer), 225.28S (anti-HMW-MAA (high molecular weight melanoma-associated antigen), SorinRadiofarmaci SRL (from Milan, Italy, for the treatment of melanoma), COL-1 (anti-CEACAM3, CGM1 , derived from NatCancer Inst. for the treatment of colorectal and gastric cancer), CYT-356 ( Prostate cancer), HNK20 (OraVax Inc. for respiratory syncytial virus infection), ImmuRAIT (from Immunomedics, for NHL), Lym-1 (anti-HLA-DR10, Peregrine Pharm), MAK-195F (anti-TNF (tumor necrosis factor , TNFA, TNF-α, TNFSF2, derived from Abbott/Knoll, for the treatment of sepsis and toxic shock), MEDI-500 (T10B9, anti-CD3, TRαβ (T cell receptor α/β), derived from MedImmune Inc, used Graft-versus-host disease), RING SCAN (anti-TAG 72 (tumor-associated glycoprotein 72), from Neoprobe Corp., for breast, colon and rectal cancer), Avicidin (anti-EPCAM (epithelial cell adhesion molecule)), anti-TACSTD1 (tumor-associated calcium signaling 1), anti-GA733-2 (gastrointestinal tumor-associated protein 2), anti-EGP-2 (epithelial glycoprotein 2), anti-KSA, KS1/4 antigen, M4S , Tumor Antigen 17-1A, CD326 (from NeoRx, for the treatment of colon cancer, ovarian cancer, prostate cancer and NHL), LymphoCide (from Immunomedics), Smart ID10 (from ProteinDesign Labs), Oncolym (from Techniclone Inc) , Allomune (from BioTransplant), Anti-VEGF (from Genentech); CEAcide (from Immunomedics), IMC-1C11 (from ImClone Systems) and Cetuximab (from ImClone).

Other antibodies that may serve as cell-binding molecules/ligands include, but are not limited to, antibodies to the following antigens: Aminopeptidase N (CD13), Annexin A1, B7-H3 (CD276, various cancers), CA125 (ovarian cancer ), CA15-3 (various cancers), CA19-9 (various cancers), L6 (various cancers), Lewis Y (various cancers), Lewis X (various cancers), alpha-fetoprotein (various cancers) ), CA242 (colorectal cancer), placental alkaline phosphatase (various cancers), prostate specific antigen (prostate cancer), prostatic acid phosphatase (prostate cancer), epidermal growth factor (various cancers), CD2 (Hodgkin Gold disease, NHL lymphoma, multiple myeloma), CD3ε (T cell lymphoma, lung cancer, breast cancer, gastric cancer, ovarian cancer, autoimmune disease, malignant ascites), CD19 (B cell malignancies), CD20 (non Hodgkin lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, SLE), CD30 (Hodgkin lymphoma), CD33 (leukemia, autoimmune disease), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD51 (metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lung cancer, ovarian cancer, Merkel cell carcinoma, and liquid tumors, multiple myeloma), CD66e (various cancers), CD70 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (various cancers), mucin (various cancers), CD221 (solid tumors), CD227 (breast cancer, ovarian cancer), CD262 (non-small cell lung cancer and other cancers), CD309 (ovarian cancer), CD326 (solid tumors), CEACAM3 (colorectal cancer, gastric cancer), CEACAM5 (carcinoembryonic antigen, CEA, CD66e) (breast, colorectal and lung cancers), DLL4, EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD184, blood tumors , solid tumors), Endoglin (CD105, solid tumors), EPCAM (epithelial cell adhesion molecule, bladder cancer, head and neck cancer, colon cancer, NHL prostate cancer, ovarian cancer), ERBB2 (epidermal growth factor receptor 2, lung cancer, breast cancer carcinoma, prostate cancer), FCGR1 (autoimmune disease), FOLR (folate receptor, ovarian cancer), GD2 ganglioside (various cancers), G-28 (cell surface antigen glycolipid, melanoma), GD3 Idiotypes (respective cancers), heat shock proteins (various cancers), HER1 (lung, gastric), HER2 (breast, lung, and ovarian), HLA-DR10 (NHL), HLA-DRB (NHL, B-cell leukemia ), human chorionic gonadotropin (various cancers), IGF1R (insulin-like growth factor 1 receptor body, solid tumors, blood cancers), IL-2 receptor (interleukin 2 receptor, T cell leukemia and lymphoma), IL-6R (interleukin 6 receptor, multiple myeloma, rheumatoid arthritis, Castleman disease, leukocyte interleukin-6-dependent tumors), integrin (αvβ3, α5β1, α6β4, αllβ3, α5β5, αvβ5, various cancers), MAGE-1 (various cancers), MAGE-2 (various cancers), MAGE-3 (various cancers) cancers), MAGE 4 (various cancers), anti-transferrin receptor (various cancers), p97 (melanoma), MS4A1 (transmembrane 4 domain subfamily A member 1, non-Hodgkin B-cell lymphoid tumor, leukemia), MUC1 or MUC1-KLH (breast cancer, ovarian cancer, cervical cancer, bronchial cancer and alpha gastrointestinal cancer), MUC16 (CA125) (ovarian cancer), CEA (colorectal cancer), gp100 (melanin tumor), MART1 (melanoma), MPG (melanoma), MS4A1 (transmembrane 4 domain subfamily A member 1, small cell lung cancer, NHL), Nucleolin, Neu oncogene product (respective cancer), P21 (respective cancer), anti-(N-glycolylneuraminic acid) antibody binding site (breast cancer, melanoma), PLAP-like testicular alkaline phosphatase (ovarian cancer, testicular cancer), PSMA (prostate tumor), PSA (prostate cancer ), ROBO4, TAG 72 (tumor-associated glycoprotein 72, AML, gastric cancer, colorectal cancer, ovarian cancer), T cell transmembrane protein (various cancers), Tie (CD202b), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, various cancers), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, NHL, other cancers, RA and SLE), TPBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (TNF-related necrosis-inducing ligand receptor 1, lymphoma, NHL, colorectal cancer, lung cancer), VCAM-1 (CD106, melanoma), VEGF, VEGF-a, VEGF-2 (CD309) (various cancers) . Other tumor-associated antigens that can be recognized by antibodies have been summarized and reviewed (Gerber, et al, mAbs 2009, 1:3, 247-253; Novellino et al, Cancer ImmunolImmunother.2005, 54(3), 187-207; Franke, et al, Cancer Biother Radiopharm. 2000, 15, 459-76).

The cell-binding agent, preferably an antibody, can be any agent capable of combating tumor cells, virus-infected cells, microorganism-infected cells, parasite-infected cells, autoimmune cells, activated cells, bone marrow cells, activated T cells, B cells, or melanocytes . More specifically, the cell-binding agent can be any drug/molecule capable of acting against one of the following antigens or receptors: 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, 4 -1BB, 5AC, 5T4 (Trophoblast Glycoprotein, TPBG, 5T4, WNT-Activation Inhibitor 1 or WAIF1), Adenocarcinoma Antigen, AGS-5, AGS-22M6, Activin Receptor Kinase 1, AFP, AKAP-4, ALK, α integrin, αvβ6, aminopeptidase N, amyloid β, androgen receptor, pro-angiogenic protein factor 2, pro-angiogenic protein factor 3, annexin A1, anthrax toxin protective antigen, antimetastatic Protein receptors, AOC3(VAP-1), B7-H3, Bacillus anthracis, BAFF(B cell activating factor), B lymphoma cells, bcr-ab l, bombesin, BORIS, C5, C242 antigen, CA125 (sugar antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, canine lupus erythematosus IL31, carbonic anhydrase IX, myocardial Myosin, CCL11 (C-C fragment chemokine 11), CCR4 (C-C chemokine receptor 4, CD194), CCR5, CD3E (ε), CEA (carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (factor D), Ch4D5, cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18), clustering factor A, CRIPTO, FCSF1R (colony stimulating factor 1 receptor, CD115), CSF2 (colony stimulating factor 2, granulocyte - Macrophage colony stimulating factor (GM-CSF)), CTLA4 (cytotoxic T lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4 (CD184), C-X-C chemokine receptor 4, circular ADP ribonucleic acid Enzymes, Cyclin B1, CYP1B1, CMV, CMV Glycoprotein B, Dabigatran, DLL4 (Delta-like Ligand 4), DPP4 (Dipeptide-Peptidase 4), DR5 (Death Receptor 5), Large Intestine Bacillus shiga toxin type-1, E. coli shiga toxin type-2, ED-B, EGFL7 (EGF-like domain protein 7), EGFR, EGFRII, EGFRvIII, endoglin (CD105), endothelin B receptor, endotoxin, EpCAM (Epithelial Cell Adhesion Molecule), EphA2, Episialin, ERBB2 (Epidermal Growth Factor Receptor 2), ERBB3, ERG (TMPRSS2ETS Fusion Gene), Escherichia coli, ETV6-AML, FAP (Fibroblast Activation Protein α), FCGR1 , alpha-fetoprotein, fibrin II beta chain, fibronectin extra domain-B, FOLR (folate receptor), folate receptor alpha, folate hydrolase, Fos-related antigen 1, F protein of respiratory syncytial virus, frizzled Receptor, fucose GM1, GD2 ganglioside, G-28 (cell surface antigen glycolipid), GD3 idiotype, GloboH, Glypican 3, N-glycolylneuraminic acid, GM3, GMCSF receptor alpha chain, Growth differentiation factor 8, GP100, GPNMB (transmembrane glycoprotein NMB), GUCY2C (guanylate cyclase 2C), guanylate cyclase C (GC-C), intestinal guanylate cyclase, guanosine Acid cyclase C receptor, heat stable enterotoxin receptor (hSTAR), heat shock protein, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1 (human epidermal growth factor receptor 1), HER2, HER2/ neu, HER3 (ERBB-3), IgG4, HGF/SF ( Hepatocyte Growth Factor/Scatter Factor), HHGFR, HIV-1, Histone Complex, HLA-DR (Human Leukocyte Antigen), HLA-DR10, HLA-DRB, HMWMAA, Human Chorionic Gonadotropin, HNGF, Human Scatter Factor receptor kinase, HPV E6/E7, Hsp90, hTERT, ICAM-1 (intercellular adhesion molecule 1), idiotype, IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-γ, Influenza hemagglutinin, IgE, IgE Fc region, IGHE, IL–1, IL-2R (interleukin 2 receptor), 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), integrin (α4 , αIIbβ3, αvβ3, α4β7, α5β1, α6β4, α7β7, αllβ3, α5β5, αvβ5), interferon γ-induced protein, ITGA2, ITGB2, KIR2D, LCK, Le, Legumain, Lewis-Y antigen, LFA-1 (lymphocyte function Related antigen 1, CD11a), LHRH, LINGO-1, lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE A1 , MAGE A3, MAGE4, MART1, MCP-1, MIF (macrophage migration inhibitory factor, or glycosyl inhibitory factor (GIF)), MS4A1 (transmembrane 4 domain subfamily A member 1), MSLN (mesothelin ), MUC1 (Mucin 1, Cell Surface Associated (MUC1) or Polymorphic Epithelial Mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCP1 (Monocyte Chemotactic Protein 1), MelanA/MART1, ML-IAP, MPG, MS4A1, MYCN, myelin-associated glycoprotein, Myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), Nectin-4 (ASG-22ME), NGF, neural cell apoptosis regulatory protease 1, NOGO-A, Notch receptor, nucleolin, Neu oncogene product, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low-density lipoprotein), OY-TES1, P21, p53 non-mutant, P97, PAP, anti-(N-glycolylneuraminic acid) antibody binding site, PAX3, PAX5, PCSK9, PDCD1 (PD-1, programmed cell death protein 1, CD279), PDGF-Rα (α platelet-derived growth factor body), PDGFR-β, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived growth factor receptor β, sodium phosphate associated transporter, PMEL 17, polysialic acid, protease 3 (PR1), prostate cancer , PS (phosphatidylserine), prostate cancer cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD240), Rhesus factor, RANKL, RhoC, Ras mutation , RGS5, ROBO4, RSV, RON, sarcoma translocation breakpoint, SART3, Sclerostin, SLAMF7 (SLAM member 7), Selectin P, SDC1 (syndecan 1), systemic lupus erythematosus (a) , somatomodulin C, SIP (sphingosine-1-phosphate), somatostatin, sperm protein 17, SSX2, STEAP1 (6-transmembrane epithelial prostate antigen 1), STEAP2, STn, TAG-72 (tumor-associated carbohydrate protein), survivin, T cell receptor, T cell transmembrane protein, TEM1 (tumor vascular endothelial marker 1), TENB2, Tenascin C (TN-C), TGF-α, TGF-β (transforming growth factor β), TGF-β1, TGF-β2 (transforming growth factor 2), Tie (CD202b), Tie2, TIM-1 (CDX-014), Tn, TNF, TNF-α, TNFRSF8, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblast glycoprotein), TRAIL-R1 (TNF-related necrosis-inducing ligand receptor 1), TRAILR2 (death receptor 5 (DR5)), Tumor-associated calcium signal sensor 2, tumor-specific glycosylated MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, tyrosinase, VCAM-1 (CD106), VEGF, VEGF-A, VEGF -2(CD309), VEGFR-1, VEGFR2, vimentin, WT1, XAGE 1, cells expressing any insulin growth factor receptor, or any epidermal growth factor receptor.

In another specific embodiment, the cell-binding agent-drug conjugate linked by the bridge connector of this patent can be used for targeted cancer therapy. Target cancers include, but are not limited to, adrenocortical carcinoma, anal cancer, bladder cancer, brain tumors (brainstem glioma, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal and pineal tumors, visual pathway and hypothalamic gliomas), breast cancer, carcinoid tumors, gastrointestinal cancer, unknown small cell carcinoma, cervical cancer, colon cancer, endometrial cancer, Esophageal cancer, extrahepatic cholangiocarcinoma, Ewing family tumor (PNET), intracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer (gastric cancer), extragonadal germ cell tumor, gestational trophoblastic tumor, Head and neck cancer, hypopharyngeal cancer, pancreatic islet cell carcinoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia (acute lymphocyte, acute myeloid, chronic lymphocyte, chronic granulocyte, hair cell), lip and mouth cancer, liver cancer , lung cancer (non-small cell, small cell), lymphoma (AIDS-related, central nervous system, skin T cells, Hodgkin's disease, non-Hodgkin's disease), malignant mesothelioma, melanoma, Merkel cell carcinoma , metastatic squamous neck cancer with occult primary cancer, multiple myeloma and other plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myelodysplastic syndrome, nasopharyngeal carcinoma, neuroblastoma, oral cavity Carcinoma, oropharyngeal cancer, osteosarcoma, ovarian cancer (epithelial, germ cell tumors, low-grade tumors), pancreatic cancer (exocrine, islet cell cancer), paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, chromaffin cells tumors, pituitary tumors, plasma cell tumors, prostate cancer rhabdomyosarcoma, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter (transitional cell), salivary gland cancer, Sesely syndrome, skin cancer (cutaneous T-cell lymphoid Kaposi's sarcoma, melanoma), small bowel tumors, soft tissue sarcomas, gastric cancer, testicular cancer, thymoma (malignant), thyroid cancer, urinary tract cancer, uterine cancer, unusual juvenile cancers, vaginal tumors, vulvar tumors and Wilms tumor.

In another specific embodiment, the cell-binding agent-drug conjugate linked by the bridge linker of this patent can be used as a composition and method for treating or preventing autoimmune diseases. Autoimmune diseases include, but are not limited to, Achlorhydra autoimmune active chronic hepatitis, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison's disease, azoospermia, alopecia areata, amyotrophic lateral sclerosis, Ankylosing spondylitis, anti-GBM/TBM nephritis, antiphospholipid syndrome, anti-abnormal enzyme syndrome, arthritis, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune Immune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndromes I, II, and III type, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune uveitis, Balo's disease/Balo's concentric sclerosis, Bechets syndrome, Berger's disease, Bickerstaff encephalitis, Blau's syndrome, bullae Pemphigoid, Castleman's disease, Chagas' disease, chronic fatigue immunodeficiency syndrome, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal osteomyelitis, chronic Lyme disease, chronic obstructive pulmonary disease, Churg-Strauss syndrome, cicatricial pemphigoid, celiac disease, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, cranial arteritis, CREST syndrome, Crohns disease (idiopathic inflammatory bowel disease) , Cushing's syndrome, cutaneous leukocytosis vasculitis, DeGold's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes mellitus, diffuse cutaneous systemic sclerosis, Dressler syndrome, discoid lupus erythematosus , Eczema, Endometriosis, Enthesitis-Associated Arthritis, Eosinophilic Fasciitis, Epidermolysis Bullosa, Erythema Nodosum, Idiopathic Mixed Cryoglobulinemia, Evans Syndrome fibrous dysplastic ossification, fibromyalgia, fibromyositis, fibrous alveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, Goodpaschul Syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, pregnancy hepatitis, hidradenitis suppurativa, Hughes syndrome (anti Phospholipid syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (autoimmune thrombocytopenic purpura), IgA nephropathy (Primary Jayson's disease), inclusion body myositis, inflammatory demyelinating polyneuritis, interstitial cystitis, irritable bowel syndrome, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, Kawasaki's disease, Lambert-Eaton myasthenia gravis syndrome, leukocytoclastic vasculitis, lichen planus, sclerosclerosis, linear IgA disease (LAD), Lou Gehrig disease (also called amyotrophic lateral sclerosis), lupus Hepatitis, lupus erythematosus, Majeed syndrome, Meniere Miller's disease, microscopic polyarteritis, Miller-Fischer syndrome, mixed connective tissue disease, morphea, Mehmet-Habermann's disease, Macaulay's syndrome, multiple myeloma, multiple sclerosis , myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), neuromyotonia, cicatricial pemphigoid of the eyelids, Opsoclonus myoclonus syndrome, Ord's thyroiditis, palindromic rheumatism, PANDAS (with chain coccus-associated pediatric autoimmune neuropsychiatric disease), Paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry Romberg syndrome, Parsonnage-Turner syndrome, planar cyclopitis, pemphigus, pemphigus vulgaris , anemia, peripheral encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory Neuropathy, psoriasis, psoriatic arthritis, gangrenous dermatitis, pure red cell aplasia, Rasmussen's encephalitis, Raynaud's phenomenon, relapsing polychondritis, Reiter's syndrome, restless legs syndrome, posterior neurofibrosis, class Rheumatoid arthritis, rheumatoid fever, sarcoidosis, schizophrenia, Schmidt's syndrome, Schnitzler's syndrome, Schnitzler's syndrome, scleritis, scleroderma, Sjogren's syndrome, spondyloarthropathies, mucous Thick blood syndrome, Still's disease, stiff man's syndrome, subacute bacterial endocarditis, Susac's syndrome, Sweet's syndrome, chorea minor, sympathetic anemia, Takayasu arteritis, temporal arteritis (giant cell arteritis), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis (idiopathic inflammatory bowel disease), undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, vasculitis, vitiligo, Wegener's granulation Swelling disease, Wilson's syndrome, Westcott-Aldrich syndrome.

In another specific embodiment, on the conjugate used to treat or prevent autoimmune diseases, examples of binding molecules linked to drug molecules through the bridge linker of this patent include but are not limited to anti-elastin Antibody, Abys anti-epithelial cell antibody, anti-basement membrane collagen IV antibody, anti-nuclear antibody, anti-ds DNA, anti-ss DNA, anti-cardiolipin antibody IgM, IgG, anti-celiac antibody, anti-phospholipid antibody IgK, IgG, anti- SM antibody, anti-mitochondrial antibody, thyroid antibody, 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 antibody, anti-histone, anti-RNP, C-ANCA, P-ANCA, anti-centromere, anti-fibrinogen, anti-GBM antibody, anti-ganglioside antibody, anti-Desmogein 3 antibody, anti-p62 antibody, anti sp100 antibody, anti-mitochondrial (M2) antibody, rheumatoid factor antibody, anti-MCV antibody, anti-topoisomerase antibody, anti-neutrophil cytoplasmic (cANCA) antibody.

In some preferred embodiments, the binding molecules on the conjugates in this patent can bind to receptors or receptor complexes expressed on activated lymphocytes associated with autoimmune diseases. The receptor or receptor complex contains, members of the immunoglobulin gene superfamily (e.g., 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), members of the TNF receptor superfamily (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), integrins, cytokine receptors, chemokine receptors, Major histocompatibility proteins, lectins (type C, S, or type I) or complement control proteins.

In another embodiment, useful cell-binding partners immunospecific for viral or microbial antigens are humanized or human monoclonal antibodies. "Viral antigen" includes, but is not limited to, any viral peptide, polypeptide protein (such as HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV Tax, Herpes herpes simplex virus glycoproteins (such as gB, gC, gD and gE) and hepatitis B surface antigen). A "microbial antigen" includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide or lipid molecule (such as bacterial, fungal, pathogenic protozoan or yeast polypeptides, including such as LPS and capsular polysaccharides) capable of eliciting an immune response ). Examples of antibodies that can be used to treat viral or microbial infections include, but are not limited to: Palivizumab, which is a humanized anti-RSV monoclonal antibody used to treat RSV infection; PRO542, which is a CD4 Fusion antibody, for the treatment of HIV infection; oseltamivir, a human antibody for the treatment of hepatitis B virus; PROTVIR, a humanized IgG1 antibody for the treatment of cytomegalovirus, and anti-LPS antibody .

The cell-binding molecule-drug conjugate prepared through the bridge linker of this patent can be used to treat infectious diseases. These infectious diseases include, but are not limited to, Acinetobacter infection, actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (acquired immunodeficiency syndrome), amoebiasis, anaplasmosis, anthrax , Yersinia hemolytica infection, Argentinian hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterial vaginosis, Bacteroides infection, balanula disease, roundworm infection, BK virus infection, black hair sarcoidosis, Blastocystis hominis infection, blastomycosis, Bolivian hemorrhagic fever, Borrelia infection, botulism (and infant botulism), Brazilian hemorrhagic fever, Brucellosis Bacillosis, Burkholderia infection, Buruli ulcer, calicivirus infection (norovirus and sapovirus), campylobacteriosis, candidiasis (candidiasis, thrush), cat scratch disease , cellulitis, Chagas disease (Chagas disease), ascus, varicella, chlamydia, Chlamydia pneumoniae infection, cholera, melanoblastoma, Clonorchis sinensis, Clostridium difficile infection, coccidioidomycosis, Colorado tick Fever, common cold (acute viral nasopharyngitis, acute rhinitis), Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever, cryptococcosis, cryptosporidiosis, cutaneous larval migration, cyclosporidiosis, enterobacteriaceae Infection, Enterovirus infection, Epidemic typhus, Erythema infectious (fifth disease), Acute rash, Fascioliasis, Fascioliasis hepatica, Fatal familial insomnia, Filariasis, Capsule perfringens Clostridial food poisoning, free living amoeba infection, fusobacterial infection, gas gangrene (clostridial myonecrosis), geothriosis, Gerstmann-Strassler-Shekel disease syndrome, Giardiasis, equine gangrene, gonorrhea, granulomatous diarrhea (fifth STD), group A streptococcal infection, group B streptococcal infection, Haemophilus influenzae infection, hand, foot and mouth disease (HFMD), Hantavirus Pulmonary syndrome, Helicobacter pylori infection, hemolytic uremic syndrome, hemorrhagic fever with renal syndrome, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex, histoplasmosis , hookworm infection, human bocavirus infection, human ewingii ehrlichiosis, human granulocytic anaplasmosis, human metapneumovirus infection, human monocytic ehrlichiosis, human papillomavirus infection, human parainfluenza Viral infection, Hymenoleciasis, Epstein-Barr virus infectious mononucleosis (mono), influenza, Isosporidiosis, Kawasaki disease, keratitis, Kingella infection, Kuru disease, Lassa fever , Legionnaires' disease (Legion's disease), Legionnaires' disease (Pontiac fever), Leishmaniasis, Lyme disease, lymphatic filariasis (elephantiasis), lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever , measles, melioidosis (Wyeth's disease), meningitis, meningococcal disease, opistomiasis, microsporidiosis, molluscum contagiosum, mumps, mouse typhus (endemic typhus), mycoplasma Pneumonia, mycetoma, myiasis, neonatal conjunctivitis (neonatal eye disease), variant Creutzfeldt-Jakob disease (vCJD, nvCJD), nocardiosis, onchocerciasis (river blindness ), paracoccidioidomycosis (South American blastomycosis), paragonimiasis, pasteurillosis, head lice, body lice, pubic lice, pelvic inflammatory disease, pertussis, plague, pneumococcal infection, pneumocystis pneumonia, Pneumonia, poliomyelitis, Prevotella infection, primary amebic meningoencephalitis, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, rat-bite fever, respiratory syncytial virus infection, rhinosporidium disease, rhinovirus infection, rickettsial infection, rickettsial pox, Rift Valley fever, Rocky Mountain spotted fever, rotavirus infection, rubella, salmonellosis, SARS (severe acute respiratory syndrome), scabies, schistosomiasis disease, sepsis, shigellosis (Bacillary dysentery), herpes zoster (herpes zoster), smallpox (smallpox), sporothrix, staphylococcal food poisoning, infection with staphylococcus aureus, strongyloidiasis, syphilis , Taeniasis, Tetanus, Tinea barbae (Barber itch), Tinea scalp, Tinea corporis, Jock itch, Tinea manuum, Tinea palmis, Tinea pedis (Hong Kong athlete's foot), Onychomycosis (Onychomycosis), Tinea versicolor, Toxocara Disease (ocular larval migratory disease), toxocarasis (visceral larval migratory disease), toxoplasmosis, trichinellosis, trichomoniasis, trichuriasis (whipworm infection), tuberculosis, tularemia, Ureaplasma urealyticum Infections, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, albino sarcoidosis (tinea), Yersinia pseudotuberculosis, Yersinia pestis enteropathy, yellow fever , Zygomycosis.

The cell-binding agent of the present invention is more preferably an antibody, and the pathogenic strains against include but are not limited to, Acinetobacter baumannii, Actinomyces israeli, Actinomyces and Propionibacterium, Trypanosoma brucei, HIV (human immunodeficiency Viruses), Entamoeba histolytica, Anaplasma, Bacillus anthracis, Vibrio haemolyticus, Junin virus, Ascaris, Aspergillus, Astroviridae, Babesia, Bacillus cereus, various Bacteria, Bacteroides, Escherichia coli, Ascaris, BK virus, Nodularia, Blastocystis hominis, Blastomyces dermatitidis, Machupo virus, Borrelia, Clostridium botulinum, Borrelia, Brucella Genus, usually Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerans, Caliciviridae, Campylobacter, usually Candida albicans and other Candida species, Han Baltonella, Group A Streptococcus and Staphylococcus, Trypanosoma cruzi, Haemophilus ducrei, VZV, Chlamydia trachomatis, Colorado tick fever virus, rhinovirus, coronavirus, CJD prions, Crimean-Congo hemorrhagic fever Viruses, Cryptococcus neoformans, Cryptosporidium, Hookworm brasiliensis, Various parasites, Cyclospora, Taenia zoster, Cytomegalovirus, Dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4) - Flavivirus, Bifidobacterium fragilis, Corynebacterium diphtheriae, Schizophrenia, Guinea worm, Ebola virus, Echinococcus spp, Ehrlichia enterococcus, Enterovirus spp, Prussell's rickets Subbody, parvovirus B19, human herpesvirus 6 and 7, Fasciola brucei, Fasciola hepatica and Fasciola gigantea, FFI prions, Filaria superfamily, Clostridium perfringens, Fusobacterium Genus, other Clostridia, Geotrichum candidum, GSS prions, Giardia enterica, Burkholderia, Microbacillus thorns and Candida graminis, Neisseria gonorrhoeae, Klebsiella granuloma Bacteria, Streptococcus pyogenes, Streptococcus agalactiae, Haemophilus influenzae, Enteroviruses, mainly Coxsackie virus A and Enterovirus 71, Anonymous virus, Helicobacter pylori, Escherichia coli O157:H7, Bunney Subviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Herpes simplex virus 1, Herpes simplex virus 2, Histoplasma capsulatus, Duodenum Adenoma and ampullary carcinoma Haemophilus influenzae, human bocavirus, Ehrlichia, Haemophilus phagocytophilum, human metapneumovirus, Ehrlichia chaffae, human papillomavirus, human parainfluenza virus , Hymenonas minutiae and Hymenonas minutiae, Epstein-Barr virus, Orthomyxoviridae family, Isospora beineri, Kinga, Klebsiella pneumoniae, Klebsiella, Legionella pneumophila, Legionella pneumophila, Legionella pneumophila, Leishmania spp., Mycobacterium leprae and Mycobacterium tuberculosis, Leptospira spp., Listeria monocytogenes, Borrelia burgdorferi and other Borrelia spp. Species, Trichinella bancrofti and Filaria malayi, Lymphocytic choriomeningitis virus (LCMV), Plasmodium, Marburg virus, Measles virus, Burkholderia pseudomallei, Neisseria meningitidis, Yokokawa Regeneric trematodes, microspores Hymenophora, Molluscum contagiosum virus (MCV), Mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, Diptera larvae parasitic to various bacteria and fungi, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prions , Nocardia and other Nocardia spp., Onchocercia spp., Pythiaceae, Parasaurus simani and other subgenus, Pasteurella spp., head lice, human lice, pertussis Bordetella pestis, Yersinia pestis, Streptococcus pneumoniae, Pneumococcus pneumoniae, Poliovirus, Prevotella spp., Grassella nauizii, JC virus, Chlamydia psittaci, Coxella burgdorferi , rabies virus, monostreptococci and spirochetes, respiratory syncytial virus, rhinosporum, rhinoviruses, Rickettsia spp, Rickettsia parvus, Rift Valley fever virus, Rickettsia rickettsiae , Rotavirus, Rubella, Salmonella, SARS-CoV, Human Sarcoptes, Schistosoma, Soma, Shigella, Varicella Zoster Virus, Smallpox Major or Smallpox, Sporothrix schenckii, Golden Staphylococcus spp., Staphylococcus aureus, Streptococcus pyogenes, Strongyloides, Treponema pallidum, Taenia, Tetanus, Tinea spp., Tinea spp., Epidermophyton flocculus, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton rubrum, Exophyla wernikei, Trichophyton spp, Cell Death spp, Toxocara or Toxocara, Toxoplasma gondii, Trichinella spiralis, Trichomonas vaginalis, Trichomonas three, Mycobacterium tuberculosis , Latula francii, urea and equine encephalitis virus, Venezuelan equine encephalitis virus, Vibrio cholerae, Guanarito virus, West Nile virus, beigelii mycospora, Yersinia pseudotuberculosis, enterocolitica Yersinia inflammatoryis, yellow fever virus, Mucorales (mucormycosis) and Entomomyceses (Entomomyces fungal diseases), Mucorales Pseudomonas aeruginosa, Campylobacter (Vibrio), Aeromonas Bacillus, Escherichia, Yersinia, Shigella dysenteriae, Shigella, Shigella, Salmonella, Salmonella typhi, Yasporium, Borrelia kinsenia, Borrelia burgdorferi, Leptospirosis, Cardiac Pneumocystis mori, Brucella abortus, Brucella, Brucella, Mycoplasma, Rickettsia praustzii, Rickettsia tsutsugamushi, Chlamydia, pathogenic fungi (Aspergillus fumigatus, white Candida, Histoplasma capsulatus), protozoa (Entamoeba histolytica, Trichomonas Tenas, Trichomonas Hominis, Trypanosoma gambiae, Trypanosoma rhodesia, Leishmania rosenbergii, Leish tropicalis Manninia, Leishmania brasiliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria) or Helminiths (S. japonicum, S. mansoni, S. haematobium, and hookworms).

Other antibodies used as cell-binding agents of this patent to treat viral diseases include, but are not limited to, antibodies to the following pathogenic virus antigens: poxvirus; herpesvirus; adenovirus; small flavivirus; enterovirus; small ribonucleic acid Viruses; parvoviruses; reoviruses; retroviruses; influenza viruses; parainfluenza viruses; mumps; measles; respiratory syncytial virus; rubella; arboviruses; rhabdoviruses; salmonella; non-a/non-b hepatitis viruses ; rhinoviruses; coronaviruses; Rotoviruses; oncogenic viruses such as HBV (hepatocellular carcinoma), human papillomavirus (cervical, anal), Kaposi's sarcoma-associated herpesviruses (Kaposi's sarcoma sarcoma), human herpesvirus type IV (nasopharyngeal carcinoma, Burkitt lymphoma, primary central nervous system lymphoma), oncovirus (Merkel cell carcinoma), SV40 (simian virus 40), HCV (liver cell carcinoma), HTLV-1 (adult T-cell leukemia/lymphoma); immune disorders causing viruses, such as human immunodeficiency virus (AIDS); central nervous system viruses, such as JCV (progressive multifocal leukoencephalopathy), C Hepatitis virus (subacute sclerosing panencephalitis), LCV (lymphocytic choriomeningitis), Acau virus encephalitis, orthomyxovirus (encephalitic encephalitis), RV (rabies), proboscis virus, Herpesvirus meningitis, Ramsay Hunt syndrome type II, poliovirus (poliomyelitis virus, post-polio syndrome), HTLV-1 (tropical paralysis paralysis)); cytomegalovirus (cytomegalovirus Viral retinitis, HSV (herpetic keratitis); cardiovascular virus, such as CBV (pericarditis, myocarditis); respiratory/acute viral endonasal inflammation/viral pneumonia, such as Epstein-Barr virus (EBV infection/ Infectious mononucleosis), cytomegalovirus, SARS coronavirus (severe acute respiratory syndrome) or orthomyxovirus, influenza virus a/b/c (influenza/avian flu), paramyxovirus, human parainfluenza virus, RSV (human respiratory syncytial virus), hMPV; digestive virus (mumps virus, cytomegalovirus (CMV esophagitis), adenovirus (adenovirus infection), rotavirus, norwalk virus, astrovirus, Coronavirus, Hepatitis B virus, CBV, Hepatitis A virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, HGV); Urogenital virus, such as BK virus, MuV (mumps).

Furthermore, the present invention also includes a composition composed of a conjugated conjugate connected with a bridge body and an acceptable carrier, diluent or auxiliary material for the treatment of cancer, infection or autoimmune disease. Methods of treating cancer, infection and autoimmune diseases can be practiced in vitro, in vivo or ex vivo. Examples of in vitro use include treating cell cultures with it to kill all cells except a variant that does not express the target antigen; or to kill a variant that expresses an undesired antigen. Examples of ex vivo use include the treatment of hematopoietic stem cells (HSC) to kill diseased or malignant cells prior to transplantation (HSCT). For example, removal of tumor cells or lymphocytes from bone marrow prior to autologous transplantation in cancer therapy or in the treatment of autoimmune diseases, or removal of T cells from allogeneic bone marrow or tissue prior to transplantation to prevent graft-versus-host disease cells and other lymphocytes. Such clinical ex vivo treatment can be carried out as follows: harvest bone marrow from patients or other individuals, and then incubate at about 37° C. in serum-containing medium for about 30 minutes to about 48 hours, and add the present invention to the medium. Conjugates at concentrations ranging from about 1 pM to 0.1 mM. The specific drug concentration and incubation time should be determined by professional clinicians. After incubation, the bone marrow cells are washed with serum-containing medium and administered intravenously to the patient according to known methods. If the patient is receiving other treatments (such as ablative chemotherapy or a course of whole body radiation) between bone marrow harvesting and reinfusion of therapeutic cells, the processed bone marrow cells should be stored frozen in liquid nitrogen using standard medical equipment.

In clinical in vivo use, the patented linker-linked conjugates can be supplied as solutions or lyophilized solids, and the solids can be re-dissolved in sterile water for injection. An example of the administration regimen of the conjugate is as follows: the conjugate is injected intravenously once a week for 8 to 20 consecutive weeks. Bolus doses are administered in 50-500 ml of physiological saline, to which human serum albumin may be added (eg, 0.5 to 1 mL of a concentrated solution of human serum albumin, 100 mg/mL). The intravenous dose will be about 50 [mu]g to 20 mg/kg (body weight) daily, or weekly, two weeks, three weeks or monthly (10 [mu]g to 200 mg/kg dose per injection). After treatment Week, patients can receive the second course of treatment. Specific clinical protocols regarding routes of administration, excipients, diluents, doses, times, etc. can be determined by professional clinicians.

Examples of medical conditions that may be treated in vivo or ex vivo include malignancies of any type of cancer, autoimmune diseases, graft rejection and infections (viral, bacterial or parasitic).

The amount of conjugate required to achieve the desired biological effect will vary with a variety of factors including the chemical identity, potency and bioavailability of the conjugate, type of disease, patient race, disease progression in the patient Condition, route of administration, all factors determine the required dose, mode of administration and regimen.

In general, the conjugates of the invention can be formulated for injection in aqueous physiological buffer solution containing 0.1 to 10% w/v conjugate. Typical doses range from 1 μg/kg to 1 g/kg (body weight) once daily. A preferred dosage range is 0.01 mg/kg to 20 mg/kg body weight per day or week, or infant equivalent. The preferred dosage of the drug to be administered may depend upon such factors as the type and degree of progression of the disease or condition, the general health of the particular patient, the relative biological efficacy of the selected compound, the formulation of the drug, the route of administration (intravenous, intramuscular or other), the pharmacokinetic properties of the drug's assigned route of delivery, and the rate of administration (bolus or continuous infusion) and dosing regimen (number of repetitions over a given period of time).

The conjugates of the present invention can also be administered in unit dosage form, where the term "unit dosage" means a single dose that can be administered to a patient and that can be easily handled and packaged, while the active conjugate itself or as described below The pharmaceutically acceptable composition described above maintains a physically and chemically stable unit dosage. Typical total daily dosages range from 0.01 to 100 mg/kg body weight. As a general guideline, unit doses for humans range from 1 mg to 3000 mg daily or weekly, or 2 weeks, 3 weeks or monthly. The unit dosage range is preferably 1 to 500 mg once to four times a week, more preferably 10 mg to 500 mg once a week. The conjugates provided herein can be formulated into pharmaceutical compositions by mixing with one or more pharmaceutically acceptable excipients. Such unit dosage compositions may be administered orally, as in the form of tablets, simple capsules or soft gel capsules; or intranasally, as in powders, nasal drops or aerosols; or dermally. Medicines such as topical ointments, creams, lotions, gels, or sprays or by transdermal patches.

drug/cytotoxic agent

Drugs that can be coupled to the cell-binding molecules of the present invention are small-molecule drugs, including cytotoxic agents, that can be linked or modified to be linked to cell-binding agents. "Small molecule drug" in the present invention generally refers to organic, inorganic or metal organic compounds with a molecular weight of 100 to 1800, more preferably 120 to 1400. These small molecule drugs have been fully described in the literature in this field, such as WO05058367A2 and US Patent 4,956,303, etc., which are incorporated herein by reference. Small molecule drugs include known drugs and drugs to be disclosed.

Known medications include, but are not limited to:

1) Chemotherapeutic agents: a) Alkylating agents, such as nitrogen mustard: chlorpheniramine, chlorpronazine, cyclophosphamide, dacarbazine, estradiol mustard, ifosfamide, nitrogen mustard, dihydrochloride Methoxyamine, dinitrogen mustard, amlodipine hydrochloride, mycophenolic acid, dulcitol, pipobromide, new nitrogen mustard, benzene mustard cholesterol, pine mustard, thiotepa, trofosamide , uracil; CC-1065 (including its adolaisine, kizellesine and bizelesine synthetic analogs); duocamycin (including its synthetic analogs KW-2189 and CBI-TMI); Dimers of azepines (such as pyrrolobenzodiazepines (PBD) or tomemycin, indolobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinazepine dimers) polymers); nitrosoureas (carmustine, lomustine, clostocin, formustine, nimustine, lamustine); alkylsulfonates (busulfan , endosulfan, endosulfan and sulfur); triazenes (dacarbazine); platinum-containing compounds (carboplatin, cisplatin, oxaliplatin); aziridines such as chromanone, carboplatin Loxone, methutepa, and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenetriamine, triethylphosphoramide, triethylenethiophosphoramide, and trimethylol Methylamine; b) Plant alkaloids: such as vinca alkaloids (vincristine, vinblastine, vindesine, vinorelbine, norvinblastine); taxoids (paclitaxel, docetaxel) and their analogs ; maytansine (DM1, DM2, DM3, DM4, maytansine, and ansamycin) and their analogs; cryptophycin (particularly cryptophycin 1 and cryptophycin 8); epothilone, cortinol, dimolide , bryostatin, dolastatin, auristatin, tubulysin, cephalostatin, pancratistatin, sarcodictyn, spongatin; c) DNA topoisomerase inhibitors, such as etoposide (9-aminocamptothecin , camptothecin, clinatol, daunorubicin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, nodiposide, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptothecin (RFS 2000)); mitomycin (mitomycin C); d) antimetabolites such as antifolates, DHFR inhibitors (methotrexate, Mycophenolic acid, pteroxin, aminopterin (4-aminobenzoic acid) or other folic acid analogs); IMP dehydrogenase inhibitors (mycophenolic acid, thiazofurin, ribavirin, EICAR ); ribonucleotide reductase inhibitors (hydroxyurea, deferoxamine); pyrimidine analogs, uracil analogs (amcitabine, azacitidine, 6-azauracil, capecitabine (Shiro Da), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-fluorouracil, fluorouridine, ratitrexed (Tomudex); cytosine analogs (cytarabine , cytosine arabinoside, fludarabine); purines analogues (azathioprine, fludarabine, mercaptopurine, thiamine, thioguanine); folic acid supplements, such as florinic acid; e) hormone therapy agents, such as receptor antagonists, antiestrogens ( megestrol, raloxifene, tamoxifen), LHRH stimulants (gostaline, leuprolide acetate); antiandrogens (bicalutamide, flutamide, caruspristone , betandrosterone propionate, epiandrosterol, goserelin, leuprolide, metilidine, nilutamide, testolide, trolosteine and other androgen inhibitors); retinoid analogues of vitamin D3 (CB1093, EB1089KH1060, cholecalciferol, ergocalciferol); photodynamic therapy agents (verteporfin, phthalocyanine, photosensitizer Pc4, demethoxy-hypocretin A); Cytokines (Interferon-α, Interferon-γ, Tumor Necrosis Factor (TNF), TNF-containing human protein); f) Kinase inhibitors, such as BIBW 2992 (anti-EGFR/Erb2), imatinib, Gem Fitinib, Pegatanib, Sorafenib, Dasatinib, Sunitinib, Erlotinib, Nilotinib, Lapatinib, Axitinib, Pazopanib, Van Detanib, E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534), bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib, Vimodegil, iniparib, ruxolitinib Ni, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, ispins; g) Antibiotics, such as enediyne antibiotics (calicheamicin, especially calicheamicin γ1, δ1, α1 and β1 (refer to J.Med.Chem.1996,39(11), 2103-2117; Angew Chem. Intl.Ed.Engl.1994,33:183-186), dynemycin, including dynemycin A and deoxymethycin, esperamycin, cardamycin, C-1027, maduropeptin and Neocarcinogen chromophores and related chromoproteins enediyne antibiotic chromophores), aclacinomysins, actinomycins, antramycins, azaserine, bleomycin, kanomycin, karamycin, Carmine, carcinophilin, doxorubicin, doxorubicin, morpholino doxorubicin, 2-pyrroline doxorubicin and deoxydaunorubicin, epirubicin, arubicin, idarubicin Star, marcomycin, mycomycin, mycophenolic acid, lopicomycin, pelomycin, pelomycin, puromycin, triiron doxorubicin, doxorubicin, streptozotocin, streptozotocin Zotocin, tubercidin, ubenimex, netastatin, zorubicin; h) others, such as polyketides (annonacein), especially bullatacin and bullatacinone; gemcitabine, cyclooxygenase (such as carfilomib), bortezomib, thalidomide, lenalidomide, pomalidom ide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allowctin-7, Xegeva, Provenge, Yervoy, prenylation inhibitors (eg, lovastatin), dopaminergic neurotoxins (eg, staurosporine) , actinomycins (such as actinomycin D, dactinomycin), bleomycin (such as bleomycin A2, bleomycin B2, pelomycin), anthracyclines (such as daunomycin adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, MDR inhibitors (eg, verapamil) , Ca ²⁺ ATPase inhibitors (such as thapsigargin), histone deacetylase inhibitors (vorinostat, romidepsin, panobinostat, valproic acid, Mocetinostat (MGCD0103), Belinostat, PCI -24781, Entinostat, SB939, Resminostat, Givinostat, AR-42, CUDC-101, Sulforaphane, Trichostatin A); Celecoxib, Glitazones, Epigallocatechin Gallatin Esters, Disulfiram, Salinosporamide A; Anti-Adrenal Drugs such as Amglutethimide, Mitotane, Trilosteine, Aceglucuronolactone, Aldophosphamide, Aminolevulinic Acid, Amsacrine, Arabinoside , bestrabucil, bisantrene, edatraxate, defofamine, mexican, decquinone, eflornithine (DFMO), elfomithine, elixitium, ethyl gluconate, gallium nitrate, cytosine, hydroxyurea, i Bandronate, Lentinan, Lonidamine, Mitoguanidine Hydrazone, Mitoxantrone, Mopedadol, Diamine Niacridine, Pentostatin, Methionine, Pirarubicin, Podophyllum Acid, 2-ethylhydrazine, procarbazine; piperazine diketopane; gentamicin; sizole; spirogermanium; A, bacitracin A, and anguidine), polyurethanes, siRNA, antisense drugs, and nucleolytic enzymes.

2) Drugs for autoimmune diseases, including but not limited to cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (eg Amcinonide, dexamethasone, triamcinolone acetonide, beclomethasone dipropionate, DHEA, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mycophenolate mofetil, Prednisone, sirolimus, tacrolimus.

3) Drugs for anti-infective diseases, including but not limited to a) Aminoglycosides: Amikacin, Asemicin, Gentamicin (Netilmicin, Sisomicin, Isopamicin), tide Mycin B, kanamycin (amikacin, arbekacin, aminodeoxykanamycin, dibekacin, tobramycin), neomycin (framycetin, paromomycin, ribose Mycin), netilmicin, spectinomycin, streptomycin, tobramycin, methylzithramycin; b) amido alcohols: azidochloramphenicol, chloramphenicol, florfenicol, thiamphenicol; c) ansamycin: geldanamycin, herbimycin; d) carbapenems: biapenem, doripenem, ertapenem, imipenem/western Sistatin, meropenem, panipenem; e) Cephems: carbacephem (loracarbid), cefotrinitrile, ampicillin, cephradine, cefadroxine, cefuronine, cefotaxime, cephalothin Or cephalosporins, cephalexin, cephalosporins, cefamandole, cefapirin, oxamazole cephalosporins, fluxazole cephalosporins, forzidone, oxazoline cephalosporins, cefbuperazone, cefcapine, Cefotaxime, cefepime, cefixime, cefoxitin, cefprozil, cefmethoxam, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, cephalosporin Hemet, cefmenoxime, cefodizime, cefanixime, cefoperazone, cefreide, cefotaxime, cefazolin, cefazolan, cephalexin, cefmidazole, cefpiramide, cephalosporin Piro, cefpodoxime, cefprozil, cefquinol, cefsulodin, ceftazidime, cefditoren, ceftibuten, cefotaxime, ceftizoxime, ceftazidime, ceftriaxone, cefuroxime, cefuroxime Zonan, cephamycin (cefoxitin, cefotetan, ceftriaxone), oxygen (carbon) cephem (fluoxef, cephalosporin); f) glycopeptide: bleomycin, vancomycin (Ollie Wanxing, telavancin), teicoplanin (dalbavancin), ramoplanin, g) glycylcyclines: such as tigecycline, h) beta-lactamase inhibitors: green Mylan (sulbactam, tazobactam), oxypenicillane (clavulanic acid); i) lincosamide: clindamycin, lincomycin; j) lipopeptide: daptomycin, A54145 , calcium-dependent antibiotics (CDA); k) macrolides: azithromycin, clindamycin, clarithromycin, dirithromycin, erythromycin, fluremycin, josamycin, ketolides ( Telithromycin, erythromycin), midecamycin, mikamycin, oleandomycin, rifamycin (isoniazid, rifampicin, rifabutin, rifapentine), lopimycin roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandomycin, telithromycin; l) monocyclic amines: aztreonam, tigemonan; m) Oxazolidinones: Linezolid; n) Penicillins: Amoxicillin, Ampicillin (piamicillin, Hexacillin, Bahamicillin, Ampicillin, Adriamycin), Amoxicillin, Azlocillin, Benzyl Penicillin, Benzathine Penicillin Benzyl Penicillin, Benzathine Penicillin, Phenoxymethyl Penicillin, Cloxillin, Procaine Penicillin (Methenicillin), Mezlocillin, Methicillin , nafcillin, oxacillin, acemetacillin, penicillin, finecillin, phenoxymethylpenicillin, piperacillin, ampicillin, sulfbenzillin, temoxicillin, ticarcillin; o) Polypeptide: Bacillus Peptides, colistin, polymyxin B, p)quinolones: alatrafloxacin, baloxacin, ciprofloxacin, Clinza, danofloxacin, difloxacin, enoxacin , enrofloxacin, garefloxacin, gatifloxacin, gemifloxacin, gepafloxacin, canotrovafloxacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nafloxacin Star, Norfloxacin, Orbifloxacin, Ofloxacin, Pefloxacin, Trovafloxacin, Gepafloxacin, Sitafloxacin, Sparfloxacin, Temafloxacin, Torofloxacin, Trovafloxacin; q) Streptavicin: Prinamycin, Quinupristin/Dalfopristin, r) Sulfa Drugs: Sulfa Drugs: Sulfa Drugs, Sulfadiazine, Sulfadiazine, Sulfadiazine, sulfasalazine, sulfisoxazole, tamoxifen, trimethoprim-sulfamethoxazole (co-trimoxazole); s) steroid antimicrobials: such as fusidic acid; t) tetracyclines: doxycycline, Chlortetracycline, Clomicycline, Demeclocycline, Ramoxiline, Mexicycline, Metacycline, Minocycline, Oxytetracycline, Penicillin V, Potassium, Pipercycline, Pyrrolidinylmethyltetracycline , tetracycline, glycylcycline (eg tigecycline): u) other types of antibiotics: anemone, arsenamin, bacterial terpene alcohol inhibitors (Bacillus), DANAL/AR inhibitors (cycloserine) , dictyostatin, ecrospongerolactone, cortinol, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirol Star, NAM synthesis inhibitors (eg, fosfomycin), nitrofurantoin, paclitaxel, prancimycin, pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampicin), tazobactant Nitazole, black chrysanthemum.

4) Antiviral drugs: a) entry/fusion inhibitors: apavelor, maraviroc, vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab); b) integration Enzyme inhibitors: raltegravir, elvitegravir, globoidnan A; c) maturation inhibitors: bevirimat, vivecon; d) neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; E) Nucleosides and nucleotides: abacavir, aciximavir, adefovir, amoxivir, abciximab, brovudine, cidofovir, clavudine, dexamethasone, des Hydroxyinosine (ddI), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluracillin (5-FU), 3'-fluoro substituted 2',3'-deoxynucleoside analogs (such as 3 , 3'-fluoro-2', 3'-dideoxythymidine (FLT) and 3'-fluoro-2', 3'-dideoxyguanosine (FLG), fomivirsen, 9-guanine, iodine glycosides, lamivudine (3TC), 1-nucleosides (such as β-1-thymidine and β-1-2'-deoxycytidine), penciclovir, racivir, ribavirin, ditidine , stavudine (d4T), talibavirin (viramidine), telbivudine, tenofovir, trifluridine valacyclovir, valganciclovir, zalcitabine (ddC) , zidovudine (AZT); f) non-nucleosides: amantadine, atipyridine, capravirine, diarylpyrimidine (etravirine, rilpivirine), delavirdine, docosanol , emivirine, efavirenz, foscarnet (phosphoroformic acid), imiquimod, peginterferon, loviramide, lord adenosine, inthizone, nevirapine, NOV-205, peginterferon alfa, podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), acetamantane; g) protease inhibitors: amprenavir atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h) Other types of antiviral drugs: antioxidative enzymes, arbidol, calanolide, ceragenin, cyanverine-n, diarylpyrimidines, epigallocatechin gallate (EGCG), foscarnet , Griffithine, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, mixed inhibitors, ribavirin, seliciclib.

5) Drugs linked by the bridges of the present invention also include radioisotopes. Examples of radioactive isotopes (radionuclide) are ³ H, ¹¹ C, ¹⁴ C, ¹⁸ F, ³² P, ³⁵ S, ⁶⁴ Cu, ⁶⁸ Ga, ⁸⁶ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²³ I, ¹²⁴ I, 125I , ¹³¹ I, ¹³³ Xe, ¹⁷⁷ Lu, ²¹¹ At or ²¹³ Bi. Radioisotope-labeled antibodies can be used in receptor targeting imaging experiments, or can be used in targeted therapy as antibody-drug conjugates of the present invention (Wu et al Nature Biotechnology 2005, 23(9):1137-1146). Cell-binding molecules, such as antibodies, can be labeled by linking ligand reagents with linkers of this patent. Ligands can bind, chelate, or form complexes with radioactive metals as described in the literature (Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, New York, NY, Pubs. (1991)). thing. Chelating ligands that can complex metal ions include DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, TX) and the like.

6) A pharmaceutically acceptable salt, acid or derivative of any of the above drugs.

In another embodiment, the drugs in structural formulas (II) and (IV) can be chromogenic molecules, and the conjugates can be used to detect, monitor or study the interaction between cell-binding molecules and target cells. A chromophore that absorbs a type of light, such as ultraviolet, fluorescent, infrared, near-infrared, or visible light; includes a class or subclass of yellow, erythrocyte, iridescent, leukocyte, melanin, and cyan pigments , a class or a subclass of fluorescent molecules (fluorescent chemicals that absorb light and then emit light), a class or a subclass of visual phototransduction molecules, a class or a subclass of photonic molecules, and a class of luminescent molecules or a subclass and a class or a subclass of fluorescein compounds.

Chromogenic molecules can be selected from, but are not limited to, non-proteinaceous organic fluorophores such as xanthene derivatives (fluorescein, rhodamine, Oregon green, eosin and Texas red); cyanine derivatives (cyanine, indocarbocyanines, oxacyanines, thiocyanines, and merocyanines); squaraine derivatives and ring-substituted squaraines, including Seta, SeTau, and Square dyes; naphthalene derivatives (dansyl and fluorosilicic acid sodium derivatives); coumarin derivatives; oxadiazole derivatives (pyridyloxazole, nitrobenzoxazole and benzoxadiazole); anthracene derivatives (anthraquinones, including DRAQ5, DRAQ7 and CyTRAK orange); pyrene derivatives (cascade blue, etc.); oxazine derivatives (Nile red, Nile blue, cresyl violet, oxazine 170, etc.); acridine derivatives (flavonoids, acridine orange, acridine yellow, etc.); arylmethylamine derivatives (auramine, crystal violet, malachite green) and tetrapyrrole derivatives (porphine, phthalocyanine, bilirubin).

Chromogenic molecules were selected from any analogs and derivatives of the following fluorescent compounds: CF dyes (Biotium), DRAQ and CyTRAK probes (BioS-tatus), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLightFluor (Thermo Scientific, Pierce ), Atto and Tracy (Sigma Aldrich), FluoProbes (Interchim), Abberior dyes (Abberior), DY and MegaStokes dyes (Dyomics), Sulfo Cy dyes (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTau and Square dyes (BiosearchTechnologies ), SureLight dyes (APC, RPEPerCP, Phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech).

Examples of widely used fluorescent compounds that can be reacted or conjugated with the linkers of the invention are: allophycocyanin (APC), aminonopalin, APC-Cy7 conjugates, BODIPY-FL, Cascade Blue, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, Fluorescein, FluorX, Hydroxycoumarin, Lissamine Rhodamine B, Fluorescent Yellow, Me-Methoxycoumarin, 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.

Fluorescent compounds that can be linked to the linkers of the invention for the study of nucleic acids or proteins, selected from the following compounds or derivatives thereof: 7-AAD (7-aminoactinomycin D, CG-selective), acridine Orange, Chromomycin A3, CyTRAK Orange (Biostatus), DAPI, DRAQ5, DRAQ7, Ethidium Bromide, Hoechst33258, Hoechst33342, LDS 751, Miteromycin, Propidium Iodide (PI), SYTOX Blue, SYTOX Green, SYTOX Orange, Thiazole Orange, TO-PRO, Cyanine Dye Monomer, TOTO-1, TO-PRO-1, TOTO-3, TO-PRO-3, YOSeta-1, YOYO-1. Can be connected with linker of the present invention, be used for the fluorescent compound of studying cell, be selected from following compound or its derivative: DCFH (2 ', 7 '-dichlorodihydrofluorescein, oxidized form), DHR (dihydrorhodan Ming 123, oxidized form, photocatalytic oxidation), Fluo-3 (AM ester, pH>6), Fluo-4 (AM ester, pH 7.2), Indo-1 (AM ester, low/high calcium (Ca 2+ )), SNARF (pH 6/9). Preferred fluorescent compounds are selected from: Allophycocyanin (APC), AmCyan1 (tetramer, Clontech), AsRed2 (tetramer, Clontech), Thistle Green (monomer, MBL), Azurite, B-phycoerythrin ( BPE), Cerulean, CyPet, DsRed monomer (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 (monomer, MBL), mCFP, mCherry (monomer, MBL), mKate (TagFP635, monomer, Evrogen), mKeima-Red (monomer, MBL), mKO, mOrange, mPlum, mRaspberry, mRFP1 (monomer , Tsien laboratory), mStrawberry, mTFP1, mTurquoise2, P3 (phycobilisome complex), peridinoxanthin-chlorophyll-protein complex (PerCP), R-phycoerythrin (RPE), T-Sapphire, TagCFP (two Evrogen), TagGFP (dimer, Evrogen), TagRFP (dimer, Evrogen), TagYFP (dimer, Evrogen), tdTomato (tandem dimer), Topaz, TurboFP602 (dimer, Evrogen ), TurboFPP635 (dimer, Evrogen), TurboGFP (dimer, Evrogen), TurboRFP (dimer, Evrogen), TurboYFP (dimer, Evrogen), Venus, wild-type GFP type, YPet, ZsGreen1 (four Polymer, Clontech), ZsYellow1 (tetramer, Clontech).

In another embodiment, preferred cytotoxic agents linked to cell binding molecules via the patented linkers are tubulysin, maytansine, taxanes, CC-1065 analogs, daunorubicin and doxorubicin compounds , benzodiazepine dimers (such as pyrrolobenzodiazepines (PBD) or tomemycin, indolobenzodiazepines, imidazobenzothiadiazepines or oxazolidinbenzodiazepines Azepine dimer), calimycin and enediyne antibiotics, actinomycin, azothricin, bleomycin, epirubicin, tamoxifen, idarubicin, Dolastatin, auristatin (such as MMAE, MMAF, auristatin PYE, auristatin TP, auristatin 2-AQ, 6-AQ, EB(AEB) and EFP(AEFP)), duocarmycin , thiotepa, vincristine, semimetalin, nazumamide, microginin, radiosumin, alterobactin, microsclerominmin, theonellamide, esperamicin, PNU-159682 and their analogues and derivatives.

Tubulysin is a preferred cytotoxic agent for conjugation, which can be prepared by separating or synthesizing from natural sources according to known methods with reference to literature in the art, 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.;etal.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.;etal.,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. Patent: Zanda, M.; et al, Can.Pat.Appl.CA2710693 ( 2011). Chai, Y.; et al. Eur. Pat. Appl. 2174947 (2010), PCT WO2010034724. 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,WO2008112873.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 WO2006056464. Vaghefi, F.; et al, 5 PCT WO 2006033913. Doemling, A., Ger. Offen. DE102004030227; PCT WO 2004005327; et al, U.S.Pat.Appl.Publ.20040249130.Hoefle, G.; et al, Ger.Offen.DE 10254439; DE10241152; DE 10008089.Leung, D.; , Ger.Offen.DE 19638870; Wolfgang, R.; US 20120129779, Chen, H., US appl.20110027274. A preferred structure of tubulysin linked to a cell binding molecule is described in patent PCT/IB2012/053554.

Examples of antibody-tubulysin conjugate structures linked by bridge linkers are T01, T02, T03, T04, T05 and T06:

where mAb is an antibody; Z ₃ is H, OP(O)(OM ₁ )(OM ₂ ), OCH ₂ OP(O)(OM ₁ )(OM ₂ ), OSO ₃ M ₁ or O-, NH-, S- or CH ₂ -glycoside (glucoside, galactoside, mannoside, glucoside, fructoside, etc.); M ₁ and M ₂ are independently H, Na, K, Ca, Mg, NR ₁ R ₂ R ₃ ; n is X ₁ , X ₂ , R ₁ , R ₂ and R ₃ are as defined in formula (I).

Calicheamicin and related enediyne antibiotics are preferred cytotoxic agents, for reference: Nicolaou, K.C. et al, Science 1992, 256, 1172-1178; Proc. Natl. Acad. Sci USA. 1993, 90 ,5881-5888，和美国专利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。 An example of the structure of an antibody-calicheamicin analog linked by a bridging linker is C01:

where mAb is an antibody; n is X ₁ , X ₂ , R ₁ , R ₂ and R ₃ are the same as defined in formula (I).

Maytansine is the preferred cytotoxic agent in this patent, and maytansine and its homologues are described in the following U.S. patents: 4,256,746; 4,361,650; 4,307,016; 4,294,757; 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和8,163,888。 An example of an antibody-maytansinoid conjugate such as M01:

where mAb is an antibody; n is X ₁ , X ₂ , R ₁ , R ₂ and R ₃ are the same as defined in formula (I).

Taxanes, including paclitaxel (a cytotoxic natural product) and docetaxel (a semi-synthetic derivative) and their analogs, are the preferred cytotoxic molecules of this patent and are described in: 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；美国专利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和7,667,054 .

Examples of antibody-taxane conjugates connected via a bridge linker are Tx01, Tx02 and Tx03:

where mAb is an antibody; n is X ₁ , X ₂ , R ₁ , R ₂ and R ₃ are the same as defined in formula (I).

CC-1065 analogues and Duocamycin analogues are also linked to the bridge linker of this patent, preferred cytotoxic agents. Examples of CC-1065 analogs and duocamacin analogs and their synthesis can be found in: Warpehoski, et al, J. Med. Chem. 31:590-603 (1988), D. Boger et al., J. Org .Chem；66；6654-6661,2001；美国专利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, 049,316,7087600,7091186,7115573,7129261,7214663,7223837,7304032,7329507,7,329,760,7,388,026,7,655,660,7,655,661,7,906,542,7,088,98 An example of an antibody-CC-1065 analog structure linked via a bridge linker is as follows:

where mAb is an antibody; n is Z ₄ is H, PO(OM ₁ )(OM ₂ ), SO ₃ M ₁ , CH ₂ PO(OM ₁ )(OM ₂ ), CH ₃ N(CH ₂ CH ₂ ) ₂ NC(O)-, O( CH ₂ CH ₂ ) ₂ NC(O)- or glycoside; X ₃ is O, NH, NHC(O), OC(O), CO or default; X ₁ , X ₂ , R ₁ , R ₂ , M ₁ and M ₂ are the same as defined in formula (I).

Daunorubicin/doxorubicin analogues are also preferred for conjugation via the bridge linkers of this patent. The preferred structure and its synthesis can be referred to: 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; US Patents 5,106,951; 5,122,368; 5,146,064; ; 6,214,345; 7,569,358; 7,803,903; 8,084,586; 8,053,205. An example of an antibody-doxorubicin analog structure linked by a bridging linker is shown below:

where mAb is an antibody; n is X ₃ is H, O, NH, NHC(O), NHC(O)NH, C(O), or OC(O); X ₁ , X ₂ , R ₁ , R ₂ , M ₁ and M ₂ are related to the formula The definition is the same in (1).

Auristatin and dolastatin are preferred cytotoxic agents linked to the bridging body. Auristatin (such as dolostatin E (AE), auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF ), auristatin F phenylenediamine (AFP) and the phenylalanine variant of MMAE) are analogs of dolastatin, described in the following documents: Int.J.Oncol.1999,15,367-72; Molecular CancerTherapeutics,2004,3(8),921-932；美国专利申请11134826,20060074008,2006022925,美国专利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. Examples of antibody-aurestatin conjugate structures connected by a bridge linker such as AuO 1, AuO 2, AuO 3, AuO 4 and AuO 5:

where mAb is an antibody; n is X ₃ is CH ₂ , O, NH, NHC(O), NHC(O)NH, C(O), OC(O) or default; X ₄ is CH ₂ , C(O), C(O)NH , C(O)N(R ₁ ) or C(O)O; X ₁ , X ₂ , R ₁ , R ₂ and R ₃ are as defined in formula (I).

Benzodiazepine dimers (such as pyrrolobenzodiazepines (PBD), tomemycin, indolobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinbenzodiazepines 7,834,005; 7,741,319; 7,704,924; 7,691,848; 7,678,787; 7,612,062; ；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；美国专利申请20100203007,20100316656,20030195196。 Examples of antibody-benzodiazepine dimer conjugate structures are PB01, PB02, PB03, PB04, PB05, PB06, PB07 and PB08:

where mAb is an antibody; n is X ₃ is CH ₂ , O, NH, NHC(O), NHC(O)NH, C(O), OC(O) or default; X ₄ is CH ₂ , C(O), C(O)NH , C(O)N(R ₁ ) or C(O)O; X ₁ , X ₂ , R ₁ , R ₂ and R ₃ are as defined in formula (I). In addition, R ₁ and/or R ₂ can be defaulted.

The drug/cytotoxic agent used to connect to the bridge linker of the present invention can be any analogue and/or derivative of the drug/molecule described above. It should be understood that each of the drugs/cytotoxic agents described herein can be modified such that the resulting compound retains the relevant specificity and/or activity. Those skilled in the art will also appreciate that many other compounds may be substituted for the drugs/cytotoxic agents described herein. Therefore, the drugs/cytotoxic agents of the present invention also include analogs and derivatives of these compounds.

All documents cited herein and in the Examples below are incorporated by reference.

Example

The present invention is further illustrated by the following examples, the contents of which are not intended to limit the scope of the invention. In the examples, the cell lines, unless otherwise specified, were preserved according to the conditions specified by the American Culture Collection (ATCC), the German Culture Collection (DSMZ) or the Shanghai Cell Culture Center of the Chinese Academy of Sciences. Unless otherwise specified, cell culture reagents were from Invitrogen. All anhydrous reagents were obtained commercially and stored in Sure-Seal sealed bottles. All other reagents and solvents were purchased according to the highest specification and used without further treatment. Varian Prostar HPLC for preparative HPLC purification. NMR data were acquired on Varian Mercury 400 MHz, chemical shifts are in ppm, tetramethylsilane is referenced (0.00 ppm), and coupling constants (J) are in Hz. Mass spectral data were obtained on a Waters XevoQTof mass spectrometer (connected to a Waters Acquity UPLC high performance liquid chromatograph and a TUV detector).

Example 1. tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propionate (34)

To 350 mL of anhydrous THF was added 80 mg (0.0025 mol) of sodium metal and triethylene glycol 2 (150.1 g, 1.00 mol) with stirring. After the sodium was completely dissolved, tert-butyl acrylate (24 mL, 0.33 mol) was added. The solution was stirred at room temperature for 20 hours and neutralized with 8 mL of 1.0M HCl. The solvent was removed by rotary evaporation in vacuo, diluted with brine (250 mL), and extracted with ethyl acetate (3×125 mL). The combined organic layers were washed with brine (100 mL) and water (100 mL), dried over sodium sulfate, and the solvent was removed. The resulting colorless oil was dried in vacuo to afford 69.78 g (76% yield) of product 34. ¹ H NMR: 1.41 (s, 9H), 2.49 (t, 2H, J=6.4Hz), 3.59-3.72 (m, 14H). ESI MS m/z C ₁₃ H ₂₅ O ₆ (MH), calcd. 277.17 , the measured value is 277.20.

Example 2. tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propionate (35)

34 (10.0 g, 35.95 mmol) was dissolved in acetonitrile (50.0 mL), and after adding pyridine (20.0 mL), a solution of tosyl chloride (7.12 g, 37.3 mmol) in 50 mL of acetonitrile was added dropwise through an addition funnel within 30 minutes. After 5 hours, TLC analysis showed the reaction was complete. The formed pyridine hydrochloride was filtered off, the solvent in the filtrate was removed, and the residue was purified with a silica gel column, 20% ethyl acetate in n-hexane solution was eluted to pure ethyl acetate to obtain 11.2 g (76% yield) of the compound 35. ¹ H 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+C ₂₀ H ₃₃ O ₈ S(M+H), calculated value 433.18, found value 433.30.

Example 3. tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propionate (36)

To DMF (50 mL) was added tert-butyl 2-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)-propionate 35 (4.0 g , 9.25mmol) and sodium azide (0.737g, 11.3mmol). The reaction was heated to 80°C and after 4 hours, TLC analysis showed the reaction was complete. The reaction was cooled to room temperature and quenched with water (25 mL). Extracted with ethyl acetate (3 x 35 mL), the combined organic layers were dried over anhydrous magnesium sulfate, filtered, and the solvent was removed in vacuo. The crude product (about 90% TLC pure) was used without further purification. ¹ H NMR (CDCl ₃ ): 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+C ₁₃ H ₂₆ N ₃ O ₈ (M+H), calculated value. 304.18, found value 304.20.

Example 4. tert-butyl 13-amino-4,7,10-trioxadodecanoate (37)

13-Amino-bis(4,7,10-trioxadodecanoic acid tert-butyl ester), 38.

Crude product 36 (5.0g, ) was dissolved in ethanol (80 mL), and 300 mg of 10% Pd/C was added. The system was evacuated, and 2 atm of hydrogen gas was introduced under vigorous stirring, and then the hydrogenation reactor was stirred at room temperature overnight, and TLC showed that the starting material disappeared. The crude reaction was filtered on celite, and the celite pad was washed with ethanol. After removal of the solvent, purification on a silica gel column eluting with methanol (5% to 15%) and 1% triethylamine in dichloromethane gave 13-amino-4,7,10-trioxadodecane Butyl tert-butylate 37 (1.83 g, yield 44% ESI MS m/z+C ₁₃ H ₂₇ NO ₅ (M+H), calcd. 278.19, found 278.30) and 13-amino-bis(4,7 , tert-butyl 10-trioxadodecanoate) 38 (2.58 g, 32% yield, ESIMS m/z+C ₂₆ H ₅₂ NO ₁₀ (M+H), calcd. 538.35, found 538.40).

Example 5. 3-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)propionate hydrochloride (39)

To a stirred solution of tert-butyl 13-amino-4,7,10-trioxadodecanoate 37 (0.80 g, 2.89 mmol) in 1,4-dioxane (30 mL) was added 10 mL of HCl ( 36%). After 0.5 h, TLC analysis indicated that the reaction was complete, and the reaction mixture was concentrated and azeotroped with EtOH and EtOH/toluene to form the HCl salt of the title product (>90% purity, 0.640 g, 86% yield) without further purification . ESI MS m/z+C ₉ H ₂₀ NO ₅ (M+H), calcd. 222.12, found 222.20.

Example 6. 13-Amino-bis(4,7,10-trioxadodecane hydrochloride) (40)

Under stirring, to 13-amino-bis(4,7,10-trioxadodecanoic acid tert-butyl ester) 38 (1.00g, 1.85mmol) in 1,4-dioxane (30ml) solution 10 ml HCl (36%) was added. After 0.5 h, TLC analysis showed the reaction was complete, and the reaction mixture was concentrated and azeotroped with EtOH and EtOH/toluene to form the hydrochloride salt of the title product (>90% purity, 0.71 g, 91% yield) without Further purification. ESI MS m/z+ _C18H36NO10 (M+H), calcd. _426.22 , found _426.20 .

Example 7. Bis(2,5-dioxopyrrolidin-1-yl)but-2-ynedioate (9)

To a solution of but-2-ynedioic acid 8 (2.0 g, 17.54 mmol) in DMA (100 ml) was added NHS (5.0 g, 43.4 mmol) and EDC (12.0 g, 62.5 mmol). The mixture was stirred overnight in the dark, concentrated and purified on a silica gel column eluting with EtOAc/DCM (1:10) to afford the title compound 9 (4.10 g, 76% yield). ESI MS m/z+C ₁₂ H ₉ N ₂ O ₈ (M+H), calcd. 309.03, found 309.20.

Example 8. 4,7-dioxo-5-yndioic acid (15)

To a solution of bis(trimethylsilyl)acetylene (5.0 g, 29.34 mmol) and iodine (0.37 g, 1.45 mmol) in dichloromethane (100 mL) was added dropwise at 0°C with stirring, succinyl chloride ( 18.11 g, 116.83 mmol). After the addition was complete, the mixture was stirred at room temperature until the reaction was complete (monitored by TLC, about 2 hours). The reaction mixture was quenched with water (15 mL) and extracted with dichloromethane (3 x 70 mL). The combined extracts were washed with 15% sodium thiosulfate solution, dried _over anhydrous _Na2SO4 and concentrated in vacuo. The resulting product was purified by silica gel column chromatography (100-200 mesh, 5% to 10% H ₂ O/acetonitrile) to obtain the title product (5.50 g, 85% yield). ESI MS m/z C ₁₀ H ₉ O ₆ (MH), calculated 226.05, found 226.10.

Example 9. (R, R, S, S, R, 4R, 4'R)-5,5'-(((4,7-dioxo-5-ynediyl)3,1-phenylene )) bis(4-(2-((1R, 3R)-1-acetoxy-3-((2S, 3S)-N, 3-dimethyl-2-((R)-1-methyl Piperidine pentylamino)-4-methylpentyl)thiazole-4-carboxamido)-2-methylpentanoic acid) (79)

(4R)-4-(2-((1R,3R)-1-Acetoxy-3-((2S,3S)-N , 3((R)-1-methylpiperidine-2-formylamino)pentanoylamino)-4-methylpentyl)thiazole-4-carboxamido)-5-(3-amino-4- Hydroxyphenyl)-2-methylpentanoic acid, 51 (Huang Y. et al, Med Chem. #44, 249 ^th ACS National Meeting, Denver, CO, Mar. 22-26, 2015; WO2014009774) (151mg, 0.199mmol) THF (4.0ml) and phosphate buffer (4mL, 100mM Na 2 HPO 4 , pH 7.0). After stirring at room temperature for 4 hours, the reaction was concentrated and purified by C-18 preparative HPLC (250 mm x 20 mm), water/ethanol (90% to 50% water, 55 min, flow rate 15 ml/min). Fractions containing product were combined, concentrated and crystallized from EtOH/n-hexane to afford the title compound (73 mg, 53% yield). ESI MS m/ _z +C ₈₆ H ₁₂₂ N ₁₂ Na O ₂₀ S ₂ (M+Na), calculated 1729.83, found 1730.10.

Example 10. 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatristricarbon-15-yne-1,30-dioic acid ( 86)

To a solution of 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propionate hydrochloride 39 (601mg, 2.33mmol) in THF (6ml) was added phosphate buffer (150 mM NaH ₂ PO 4 , pH 7.2, 4 ml) and bis(2,5-dioxopyrrolidin-1-yl)but-2-ynedioate 9 (350 mg, 1.13 mmol). After stirring at room temperature in the dark for 4 hours, the mixture was concentrated and purified on a SiO ₂ column, eluting with water/acetonitrile (1:9). Fractions containing product were combined and concentrated to afford the title compound (345 mg, 59% yield). ESI MS m/z C ₂₂ H ₃₆ N ₂ O ₁₂ (MH), calcd. 519.22, found 519.30.

Example 11. (2-(2-(2-Carboxyethoxy)ethoxy)ethoxy)ethyl)-14,17-dioxo-4,7,10,21,24,27-hexa Oxa-13,18-diazapentacosane-15-yne-1,30-dioic acid (87)

Add phosphate buffer (150mM NaH ₂ PO ₄ , pH 7.2, 4ml) and bis(2,5-dioxopyrrolidin-1-yl)but-2-ynedioate 9 (190mg, 0.61mmol). After stirring in the dark at room temperature for 4 h, the reaction mixture was concentrated and preparative HPLC with C-18 (250 mm × inner diameter 30 mm), eluted with water/ethanol (90% to 50% water in 55 minutes, flow rate 35 ml/min), and combined The product was fractionated and concentrated to afford the title compound (287 mg, 51% yield). ESI MS m/z C ₄₀ H ₆₇ N ₂ O ₂₂ (MH), calcd. 927.42, found 928.30.

Example 12. Bis(2,5-dioxopyrrolidin-1-yl)14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazepine Tricyclopentadecyne-1,30-dioate (88)

In 1,4-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatricyclopentacene-15-yne-1,30-dioic acid, 86 (340mg, 0.653mmol) in DMA (6ml) was added NHS (225mg, 1.96mmol) and EDC (401mg, 2.08mmol). The mixture was stirred overnight in the dark, concentrated and purified on SiO ₂ column eluting with EtOAc/DCM (5:1) to afford the title compound 88 (330 mg, 71% yield). ESI MS m/z+C ₃₀ H ₄₃ N ₄ O ₁₆ (M+H), calculated 715.26, found 715.20.

Example 13. (2-(2-(2-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropoxy))ethoxy)ethoxy Base) ethyl) -14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatristricarbon-15-yne-1,30- Diacid salt(89)

To 13,18-bis(2-(2-(2-(2-carboxyethoxy)ethoxy)ethoxy)ethyl)-14,17-dioxo-4,7,10,21 , 24,27-hexaoxa-13,18-diazatricyclobutene-15-yne-1,30-dioic acid, 87 (280mg, 0.301mmol) in DMA (6ml) solution was added NHS (105.0 mg, 0.913 mmol) and EDC (200 mg, 1.04 mmol). The mixture was stirred overnight in the dark, concentrated and purified _on a SiO column with EtOH/DCM Elution afforded the title compound 89 (249 mg, 63% yield). ESI MS m/z+C ₅₆ H ₈₁ N ₆ O ₃₀ (M+H), calculated 1317.49, found 1317.80.

Example 14. (R, R, S, S, R, 4R, 4'R)-5,5'-(((14,17-dioxo-4,7,10,21,24,27-six Oxa-13,18-diazatriacont-15 alkyne 1,30 diacyl)bis(azanediyl))bis(4-hydroxy-3,1-phenylene))bis(4-(2-((1R ,3R)-1-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentyl) -4-methylpentyl)thiazole-4-carboxamido)-2-methyl-acid)(90)

To a solution of compound 88 (30mg, 0.042mmol) in THF (3.0ml) was added (4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)-N , 3-Dimethyl-2-((R)-1-methylpiperidine-2-formylamino)pentanoylamino)-4-methylpentyl)thiazole-4-formylamino)-5- (3-Amino-4-hydroxyphenyl)-2-methylpentanoic acid, 51 (Huang Y. et al, MedChem. #44, 249 ^th ACS National Meeting, Denver, CO, Mar. 22～26, 2015; WO2014009774) (80 mg, 0.107 mmol) in THF (4.0 ml) and phosphate buffer (4 ml, 100 mM Na ₂ HPO ₄ , pH 7.0). After stirring at room temperature for 4 hours, the mixture was concentrated and purified by C-18 preparative HPLC (250mm x 20mm id), water/ethanol (95% to 50% water, flow rate 15ml/min). Fractions containing product were combined, concentrated and crystallized from EtOH/n-hexane to afford the title compound (48 mg, 56% yield). ESI MS m/z C ₉₈ H ₁₄₇ N ₁₄ O ₂₆ S ₂ (MH), calculated 2000.01, found 2000.40.

Example 15. Antibody Binding of Conjugated Compounds 90 and 91

To Herceptin (10 mg/ml, 2.0 mL, pH 7.0-8.0) was added NaH ₂ PO ₄ buffer (100 mM, pH 6.5-7.5, 0.70-2.0 mL), TCEP (20 mM in water, 28 μL) and compound 90 (20 mM DMA solution, 14 μL). The mixture was incubated at room temperature for 2-16 hours, then DHAA (135 μL, 50 mM) was added. After continuous overnight incubation at room temperature, the mixture was purified on a G-25 column with 100 mM NaH ₂ PO ₄ , 50 mM NaCl pH eluted with buffer to obtain Conjugate 91 (about 87% yield) ( in the buffer). The drug/antibody ratio (DAR) was determined to be 4.0 by UPLC-Qtof mass spectrometry. SEC HPLC (Tosoh Biosciences, Tskgel G3000SW, 7.8 mm ID×30 cm, 0.5 ml/min, 100 min) analysis showed monomer content of 96-99%, and SDS-PAGE gel measurement showed a single band.

Example 16. Compound 92 (4 tubulysin compounds are connected to each bridge linker)

To a solution of compound 89 (35 mg, 0.026 mmol) in THF (3.0 ml) was added (4R)-4-(2-((1R, 3R)-1-acetoxy-3-((2S, 3S)-N , 3((R)-1-methylpiperidine-2-formylamino)pentanylamino)-4-methylpentyl)thiazole-4-carboxamido)-5-(3-amino-4- Hydroxyphenyl)-2-methylpentanoic acid, 51 (Huang Y. et al, Med Chem. #44, 249 ^th ACS National Meeting, Denver, CO, Mar. 22-26, 2015; WO2014009774) (100.6mg, 0.132mmol ) in THF (4.0 ml) and phosphate buffer (4 ml, 100 mM Na ₂ HPO ₄ , pH 7.0). After stirring at room temperature for 4 hours, the reaction mixture was concentrated and purified by C-18 preparative HPLC (250mm x 20mm id) eluting with water/ethanol (95% to 50% water over 50 minutes at a flow rate of 15ml/min). Fractions containing product were combined, concentrated and crystallized from EtOH/n-hexane to afford the title compound 92 (47.6 mg, 47% yield). ESI MS m/z C ₁₉₂ H ₂₉₁ N ₂₆ O ₅₀ S ₄ (MH), Calcd.3890.00, Found 3890.30.

Example 17. Preparation of Conjugate 93 by Conjugating Compound 92 and Antibody

Add pH 6.5-7.5 buffer (0.70-2.0 mL, 100 mM NaH ₂ PO ₄ ), TCEP (28 μL, 20 mM aqueous solution) and compound to Herceptin solution (10 mg/ml, 2.0 mL, pH 7.0-8.0) 92 (14 μL, 20 mM DMA solution). The mixture was incubated at room temperature for 2-16 hours, then DHAA (135 μL, 50 mM) was added. After overnight incubation at room temperature, the mixture was purified on a G-25 column with 100 mM NaH ₂ PO ₄ , 50 mM NaCl pH eluted with buffer to obtain The conjugate 92 ( buffer, Yield). The drug/antibody ratio (DAR) was determined to be 8.0 by UPLC-Qtof mass spectrometry. SEC HPLC (Tosoh Biosciences, Tskgel G3000SW, 7.8 mm ID×30 cm, 0.5 ml/min, 100 min) analysis showed monomer content of 96-99%, and SDS-PAGE gel measurement showed a single band.

Example 18. In vitro cytotoxicity assessment of conjugates 91 and 93 (compared to T-DM1):

Cell lines used in cytotoxicity assays included HL-60, a human promyelocytic leukemia cell line; NCI-N87, a human gastric cancer cell line; BT-474, a human invasive ductal carcinoma cell line; and SKOV3, a human ovarian cancer cell line. For HL-60, NCI-N87 and BT-474 cells, these cells were grown in RPMI-1640 medium with 10% FBS. For SKOV3 cells, cells were grown in McCoy's 5A medium with 10% FBS. To run the assay, cells (180 μl, 6000 cells) were added to 96-well plates and incubated at 37°C and 5% carbon dioxide for 24 hours, after which the cells were treated with different concentrations of compounds (20 μL) (total volume 0.2 mL ). Control wells contained cells and medium without test compound. After the well plate was incubated for 120 hours at 37°C in an environment of 5% carbon dioxide, MTT (5 mg/mL) was added thereto and incubated at 37°C for 1.5 hours. After carefully removing the medium, add DMSO (180 μL), shake for 15 minutes, and measure the absorbance at 490 nm and 570 nm, with 620 nm as the reference. Calculate the inhibition rate according to the following formula: inhibition rate%=[1-(analytical value-blank control value)/(control value-blank control value)]×100%

Compound Cytotoxicity Results:

IC ₅₀ (nM) N87 cells (Ag+) SK-OV-3 cells (Ag+) HL60 cells (Ag-) Conjugate 91 0.108nM 0.089nM >20nM Conjugate 93 0.037nM 0.029nM >10nM T-DM1 0.270nM 0.191nM >15nM

The specificity of conjugate 91 to N87 cells was over 185 (IC ₅₀ >20/IC ₅₀ =0.108), and over 225 to SK-OV-3 cells; the specificity of conjugate 93 to N87 cells was over 270 (IC ₅₀ > 10/IC ₅₀ =0.037), more than 344 for SK-OV-3 cells; the specificity of the conjugate T-DM1 for N87 cells was more than 55 (IC ₅₀ >15/IC ₅₀ =0.27), for SK-OV-3 Cells over 78.

Both conjugate 91 and conjugate 93 were more effective than the commercially available conjugate T-DM1. Conjugate 93 has a DAR of 8, and its activity is three times that of conjugate 91 with a DAR of 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.