CN113698335A - Specific conjugation of cell binding molecules - Google Patents

Specific conjugation of cell binding molecules Download PDF

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CN113698335A
CN113698335A CN202110978276.7A CN202110978276A CN113698335A CN 113698335 A CN113698335 A CN 113698335A CN 202110978276 A CN202110978276 A CN 202110978276A CN 113698335 A CN113698335 A CN 113698335A
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赵珞博永新
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Hangzhou Dac Biotech Co Ltd
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    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Abstract

The present invention describes 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
The present application is a divisional application filed on 2015, 07/04, application No. 201580082141.5, entitled "specific conjugation of cell binding molecules".
Technical Field
The present invention describes a novel class of linkers useful for specifically linking compounds, particularly cytotoxic agents, and biomolecules (cell-binding agents). The present invention includes methods of making cell-binding molecule drug (cytotoxic agent) conjugate: either the drug is first modified with such linkers and then reacted with the cell-binding agent, or the cell-binding agent is first modified with such linkers and then reacted with the drug molecule.
Technical Field
Proteins, particularly antibodies, are widely used in the health and medical industry as research reagents for in vitro experiments and diagnostic tools for in vivo experiments or therapeutic drugs (Gad, s.c. drug discovery handbook, Wiley-Interscience, 2005). In many applications of proteins, the proteins are often modified with groups of interest, such as cytotoxic molecules, radioactive element tags or molecules with chromatographic properties, to meet the needs of therapeutic or diagnostic assays (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 extensively developed in the last 20 years as a hotspot, combining the precise targeting of antibodies and the cytotoxicity of anti-tumor agents, allowing targeted delivery of drugs into cancer cells without affecting normal cells. In particular, since Adcetris (brentuximab vedotin) was approved by the U.S. FDA in 2011 and Kadcyl (ado-trastuzumab emtansine) was approved in 2013, the use of antibody-drug conjugate as a means for targeted cancer therapy has been applied by almost all pharmaceutical and biotech companies internationally (Chari, R.et al, Angew. chem., int.Ed.2014,53, 3796-3827; Sievers, E.L.Annu Rev Med.2013,64, 15-29; hrMeling, T.Future Oncol,2015,11, 549). There are currently more than 50 ADC drugs in the clinical trial phase according to the statistics of website www.clinictrails.gov..
The first generation of ADCs drugs, including Kadcylla and Adcetris, were prepared by non-selective conjugation of native lysine amino groups or interchain cysteine thiol groups on cytotoxic drug molecules and antibodies, respectively. Because there are more than 50 surface-exposed lysines and 8 cysteines on IgG1 antibodies, this non-selective coupling mode results in random cross-linking of cytotoxic molecules to all regions of the antibody surface, creating a diverse population of ADCs with a broad distribution of DAR values (drug-to-antibody ratios) (Wang, l., et al 2005protein sci.14, 2436; Hamblett, k.j., et al 2004clin.cancer res.10, 7063). Some undesirable subsets of ADCs have disadvantages of shortened circulating half-life, low efficiency, high potential off-target toxicity, and uncertain Pharmacokinetic (PK) profiles in vivo (Hamblett, k.j.et al, clin.cancer res.2004,10, 7063-. Furthermore, it is very challenging to maintain batch-to-batch consistency for ADCs produced in this traditional coupling format, requiring higher throughput (Wakankar, a. mabs,2011,3, 161-172).
Therefore, many biotechnology companies and research institutes have been working on the development of novel site-directed conjugation methods for ADC drugs. In recent years there have also been some site-directed ADC drug preparation methods (Panowski, S,2014, mAbs 6,34) including: introducing an unpaired cysteine on an antibody, such as Genentech's ThiMAB antibody (Junutula, J.R., et al Clin. cancer Res.2010, 16,4769; Junutula, J.R., et al Nat Biotechnol.200826, 925-32; US patent 8,309,300; 7,855,275; 7,521,541; 7,723,485; WO 2008/141044); or glutamine-tagged transglutaminase (mTG) which can be amplified by Streptomyces mobaraensis (Strop, P., Bioconjugate chem.,2014,25,855-862; Strop, P., et al., chem. biol.2013,20,161-167; U.S. Pat. No. 8,871,90,161-167 to Rinat, Perey Co., Ltd8) Or from bacterial-derived transglutaminase (MTGase) (Dennler, P., et al, bioconjugate. chem.2014,25, 569-578; innate drug company, U.S. patent application No. 20130189287; U.S. patent 7,893,019 to Bio-Ker S.r.l. company, Italy); introduction of thiol-L-fucose (Dennler, P., et al, Bioconjugate Chemistry 2014,25, 569; Okeley, N.M., et al Bioconjugate chem.2013,24,1650); introduction of unnatural amino acids by mutagenesis (Axup, J.Y., et al, Proc. Natl.Acad.Sci.2012,109,16101-16106; Zimmerman, E.S., et al, bioconjugateg.Chem.2014, 25,351-361; Wu, P.et al, Proc. Natl.Acad.Sci.2009,106,3000-3005; Rabuka, D.et al, nat. Protoc.2012,7,1052-67; Sutro biopharmaceutical U.S. Pat. No. 8,778,631 and U.S. Pat. No. 20100184135, world intellectual property patent application WO 2010/081110; Ambrx Co. WO2006/069246,2007/059312, U.S. Pat. No. 7,332,571,7,696,312; and 7,638,299; Allozyne Co. WO 2007/130453; Allozyn Co. Pat. WO 2007/130453/11; E.S. Pat. No. 25; Zi. Sci.S. Ser. 25; Zi.S. No. 25; Zimmerman.S. Ser. 3,3000, 7,3000-11; Rabub.Andus patent 7,632,492; 7,829,659); introduction of selenocysteine (Hofer, T., et al Biochemistry 2009,48, 12047-12057; U.S. Pat. No. 8,916,159 of the national cancer institute); conversion of cysteines on the CXPXR consensus sequence to formylglycine (FGly) with a Formylglycine Generating Enzyme (FGE) (Drake, P.M., et al, bioconjugate. chem.2014,25,1331-Andunited states patentApplication for20140141025,20100210543); or introduction of sialic acid by glycoengineering with galactose or sialyltransferase (Zhou, q., et al bioconjugate. chem.,2014,25,510-. Homogeneous products can be produced by the methods described above, but they all require antibody engineering and re-optimization of cell culture conditions. Furthermore, the expression encoded by the unnatural amino acid gene is often not as high as desired (Tian, f., et al,2014, proc. natl. acad. sci. u.s.a.111,1766-71), which has a significant impact on the cost of the ADC product. In addition, ADC drugs obtained by cysteine side chain conjugation are often of limited stability in the circulation, resulting in premature fragmentation of the toxic small molecule load before reaching the tumor cells (Junutula, j.r., et al nat. biotechnol.2008,26,925-32).
The disulfide bond structure of the four subtypes of IgG antibodies was well known in the sixties of the last century (Milstein C.biochem J1966,101: 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 J1968, 106, 15-21; Frangione B, Milstein C.J Mol Biol 1968; 33: 893-906; Edelman GM, Proc Natl Acad Sci USA 1969; 63: 78-85; Frangione B, et al. Nature 196,221:145-148, Spiegelberg, H.L.Biochemical USA 1965, 2157; 2157). Disulfide bonds play an important role in the structure, stability and biological function of IgG molecules. Four subclasses of IgG antibodies1,IgG2,IgG3And IgG4Contains a total of 12 intrachain disulfide bonds per IgG molecule; 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: IgG1And IgG4Is 2, IgG24, IgG 311 pieces of the feed. In IgG1In addition, the last cysteine in the light chain is disulfide bonded to the 5 th cysteine in the heavy chain. In IgG2,IgG3And IgG4Above, the last cysteine on the light chain forms a disulfide bond with the 3 rd cysteine on the heavy chain (Liu, h.and May, k.2012, mAbs 4, 17-23). Human IgG was known from reduction experiments, alkylation and LC-MS analysis1The ease of disulfide bond cleavage on antibodies (Liu, H, et al anal. chem.,2010,82, 5219-5226). The interchain disulfide bonds are more susceptible to reductive cleavage than the intrachain disulfide bonds, and the disulfide bonds between the light and heavy chains are more susceptible to reductive cleavage than the disulfide bonds between the two heavy chains. The interchain disulfide bond in the upper part of the two heavy chains is more easily cleaved than in the lower one. Furthermore, the disulfide bond in the CH2 domain is the most easily reduced. The disulfide bonds of the VL, CL, VH, and CH1 domains have similar moderate cleavability, while the disulfide bond of the CH3 domain is the least susceptible to reduction (Liu, H, et al anal. chem.,2010,82, 5219-.
Based on human IgG1The characteristic that disulfide bonds between antibody chains are easier to break, and a plurality of research institutes and companies adopt a chemical fixed-point connection strategy,reduction of the interchain disulfide bonds of natural antibodies followed by bridging and re-crosslinking may be accomplished, for example, using the so-called next generation maleimide compounds (NGMs), i.e., bromides or dibromomaleimides (Schumacher, F.F., et al org. Biomol. chem.2014,12,7261-7269), using a double alkylating agent to form a three-carbon bridge (Badescu, G.et al., bioconjugate. chem.2014,25,1124-1136, Polytherics Limited, WO2013/190272, WO2014/064424), using a double substituted heteroaryl bridge (Concortis biosystems, U.S. Patents)Application for2015/0105539) or via bismaleimides as bridges (WO 2014/114207). We have also used bromomaleimide and dibromomaleimide as linkers to couple drugs and antibodies (WO2014/009774, PCT/IB2012/053554) for a long time. However, the bridge linker described above is designed to couple a cytotoxic molecule to a pair of disulfide bonds, and since the number of disulfide bonds available for conjugate coupling on an antibody is limited (about 2 pairs), ADCs drugs with DAR values below 2 are produced in most cases.
In view of the limitations of the number of toxic small molecules that ultimately reach tumor cells for the ADCs drug to achieve its therapeutic effect, it is desirable that the DAR value be greater than 3 in order to increase the ADC therapeutic index (Epenetos, a.a. et al, Cancer res.,1986,46, 3183-. The novel disulfide bridge linker can be used for connecting 2 or more small-molecule drugs on each linker, realizing higher DAR (not less than 4), and selectively bridging interchain disulfide bonds on the surface of an antibody. This is due to the natural nature of the triple bond extension in acetylene dicarbonyl, which forms a macromolecule when two toxic small molecules are attached to either end of an extended bridge linker: (
Figure BDA0003226420300000043
Spacing), other disulfide groups, such as the lower disulfide group inside the heavy chain, are difficult to access. Thus, the disulfide bridge linkers of the invention can selectively bridge free sulfhydryl pairs between antibody chains cleaved by TCEP or DTT, thereby producing ADCs with DAR greater than 4. Other reduced overhead disulfide bonds due to difficultyAccessible by a bridge linker, particularly an extended bridge linker containing 2 toxic small molecules, can be reconnected after coupling is complete with an oxide such as dehydroascorbic acid (DHAA) or cu (ii). In summary, these bridge linkers of the present invention can produce homogeneous and homogeneous ADC drugs in a simple manner.
Summary of the invention
The linker of the invention contains an acetylene dicarbonyl functional group, and 2 small molecule drugs are connected to the linker and combined with a cell binding agent (such as an antibody). The cell binding molecule-linker-drug conjugate can be represented as:
Figure BDA0003226420300000041
where Cb is a cell binding agent, L is a linker, Drug 1and Drug2 are small Drug molecules, n is an integer from 1 to 20, and 2 sulfur bridges Cb to L, each bridge linker L covalently linking 2 or more Drug molecules. The advantages of using such linkers in small cell molecule-drug conjugates are: a) covalently cross-linking (re-bridging) the thiol groups of the opened disulfide bonds on the cell binding agent (e.g. antibody) in a manner that facilitates the maintenance of the stability of the conjugate; b) toxic small molecules/drugs can be attached at specific locations of the cell binding agent, such as interchain sites of IgG antibodies, resulting in a homogeneous ADC drug.
In one aspect, the linker structure of the invention can be represented by formula (I)
Figure BDA0003226420300000042
Wherein the acetylenedicarboxylic acid structure on the linker is capable of reacting with a pair of sulfur atoms in a cell binding agent;
Z1and Z2Is the same or different functional group capable of reacting with toxic drug, and can be combined with toxic small molecule drug via disulfide bond, ether bond, ester bond, thioether bond, thioester bond, peptide bond, hydrazone bond, carbamate bond, carbonate bond, amine bond (secondary, tertiary or quaternary), imine bond, heterocycloalkyl, heteroaryl, alkoxyoxime bond or amide bond；
R1And R2Is the same, different or default straight-chain alkyl group with 1 to 6 carbon atoms, branched chain or cycloalkyl group with 3 to 6 carbon atoms, straight chain, branched chain or cycloalkenyl or alkynyl, or ester group, ether group, amide group or polyethoxy (OCH) group with 1 to 6 carbon atoms2CH2)pWherein p is an integer from 0 to about 1000, or a combination of these groups;
in addition, R1And R2Is a chain structure containing C, N, O, S, Si and P atoms, optimally contains 0-500 atoms, and is covalently connected with X1Or X2And Z1Or Z2；R1And R2Are combined in all possible chemical ways, such as to form an alkyl, alkylene, alkenylene, alkynylene, ether, polyoxyalkyl, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, polyurethane, amino acid, polypeptide, acyloxyamine, hydroxamic acid, or a combination of these groups;
X1and X2Independently selected from NH, N (R)3) O, S or CH2；R3Is H, a straight chain alkyl group of 1 to 6 carbon atoms, a branched or cyclic alkyl group of 3 to 6 carbon atoms, a straight chain, branched or cyclic alkenyl or alkynyl group, or an ester, ether, amide or polyethoxy unit (OCH) of 1 to 6 carbon atoms2CH2)pWherein p is an integer from 0 to 1000, or a combination of these groups.
Alternatively, the cell-binding agent-Drug conjugate of the present invention can be represented by formula (II), wherein the cell-binding agent Cb, the drugs Drug 1and Drug2 have reacted with the bridge linker tails:
Figure BDA0003226420300000051
wherein:
cb is a cell binding agent, most preferably an antibody;
within the brackets is a linker-drug component coupled to the cell binding molecule via a pair of sulfur atoms;
Drug1and Drug2Are the same or different cytotoxic agents linked to the cell binding agent by a disulfide bond, thioether bond, thioester bond, peptide bond, hydrazone bond, ether bond, ester bond, carbamate bond, carbonate bond, cycloheteroalkyl, heteroaryl, alkoxyoxime bond, or amide bond;
n is 1 to 20; r1,R2,X1And X2Is as defined in formula (I).
In another aspect, the invention includes a modified cell-binding agent, represented by formula (III), wherein the cell-binding agent Cb has been reacted with a bridge linker comprising a functional group Z capable of further reaction with a small drug molecule1And Z2：
Figure BDA0003226420300000061
Wherein Cb, Z1,Z2,n,R1,R2,X1And X2Are as defined in formulae (I) and (II).
Still further, the present invention includes a modified Drug molecule, which can be represented by formula (IV), wherein the Drug is Drug1And Drug2Having reacted with the linker in formula (I), still retains the acetylenedicarboxylic acid structure capable of reacting with the sulfur atom pairs on the cell-binding agent:
Figure BDA0003226420300000062
wherein the Drug1,Drug2,R1,R2,X1And X2Are as defined in formulae (I) and (II).
The invention also includes a method of preparing a cell binding molecule-Drug conjugate of formula (II) wherein the Drug1And Drug2Linked by a bridge linker and a cell binding agent.
The invention also includes a method of making a modified cell binding molecule as shown in formula (III) wherein the cell binding molecule has been reacted with the bridge linker in formula (I).
The invention also includes a method of making a modified drug small molecule as shown in formula (IV) wherein the drug molecule has been reacted with the bridge linker in formula (I).
Description of the figures
FIG. 1 is a synthesis of a bridge linker containing polyethylene glycol functional groups and its application in antibody and drug coupling.
FIG. 2 is a synthetic route for a bridge linker and coupling of antibodies and drugs via oxime linkages.
FIG. 3 synthesis of a bridge linker containing a polypeptide and coupling of antibodies to drugs via amide bonds.
FIG. 4 is a synthesis of a bridge linker comprising polyethylene glycol and a polypeptide.
FIG. 5 Synthesis of a bridge linker containing polyethylene glycol and a polypeptide and 2 to 4 small molecule drugs attached to each linker by amide bonds.
FIG. 6 Synthesis of tubulysin homologues modified with a bridge linker comprising a polypeptide and polyethylene glycol.
FIG. 7 coupling of tubulysin homologues to cell binding molecules using a bridging linker comprising polyethylene glycol.
FIG. 8 Synthesis of a cell binding molecule-maytansinoids compound coupled via a bridging linker.
FIG. 9 Synthesis of cell binding molecule-MMAF homologs coupled by bridge linkers.
FIG. 10 Synthesis of cell binding molecule-tubulysin homolog coupled by a bridge linker.
Detailed description of the invention
Definition of
"alkyl" refers to straight or branched chain aliphatic hydrocarbons containing from 1 to 8 carbon atoms. "branched" means that one or more lower carbon number 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, cyclopentylHexyl, 2, 2-dimethylbutyl, 2, 3-dimethylbutyl, 2, 2-dimethylpentyl, 2, 3-dimethylpentyl, 3, 3-dimethylpentyl, 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 C1-C8Alkyl groups may be unsubstituted or substituted with one or more groups including, but not limited to, -C1-C8Alkyl, -O- (C)1-C8Alkyl), aryl, -C (O) R ', -OC (O) R ', -C (O) OR ', -C (O) NH2,-C(O)NHR',-C(O)N(R')2,-NHC(O)R',-SR',-S(O)2R ', -S (O) R', -OH, halogen, -N3,-NH2,-NH(R'),-N(R')2and-CN; wherein each R' is independently selected from-C1-C8Alkyl groups and aryl groups. "halogen" means a fluorine, chlorine, bromine, iodine atom, preferably a fluorine and chlorine atom.
"heteroalkyl" means C2-C8Alkyl, wherein one to four carbon atoms are independently replaced by O, S and N atoms.
"carbocycle" refers to a saturated or unsaturated ring, monocyclic having 3 to 8 carbon atoms or bicyclic having 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 [4,5 ]],[5,5],[5,6]Or [6,6 ]]Bicyclic systems, or 9 to 10 atoms, forming [5,6 ]]Or [6,6 ]]A bicyclic ring system. Typical C3-C8Including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1, 3-cyclohexadienyl, 1, 4-cyclohexadienyl, cycloheptyl, 1, 3-cycloheptadienyl, 1,3, 5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl.
“C3-C8Carbocycle "means a3, 4,5,6,7 or 8 membered non-aromatic carbocyclic ring containing either saturation or unsaturation. A C3-C8Carbocycle may be unsubstituted or substituted with one or more groups including, but not limited to-C1-C8Alkyl, -O- (C)1-C8Alkyl), aryl-C(O)R',-OC(O)R',-C(O)OR',-C(O)NH2,-C(O)NHR',-C(O)N(R')2,-NHC(O)R',-SR',-S(O)R',-S(O)2R', -OH, halogen, -N3,-NH2,-NH(R'),-N(R')2and-CN, wherein each R' is independently selected from-C1-C8Alkyl groups and aryl groups.
"alkenyl" refers to straight or branched chain aliphatic hydrocarbons containing 2 to 8 carbon atoms with a carbon-carbon double bond. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, 3-methyl-2-butenyl, n-pentenyl, hexenyl, heptenyl, octenyl.
"alkynyl" refers to a straight or branched chain aliphatic hydrocarbon containing from 2 to 8 carbon atoms with a carbon-carbon triple bond. Examples of alkenyl include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, n-pentenyl, hexynyl, heptynyl, octynyl.
"alkylene" refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of 1 to 18 carbon atoms containing two monovalent radical centers derived from the removal of two hydrogens on the same or different carbons of a parent alkane. Typical alkylene groups include, but are not limited to, methylene (-CH)2-, 1, 2-Ethyl (-CH)2CH2-, 1, 3-propyl (-CH)2CH2CH2-, 1, 4-butyl (-CH)2CH2CH2CH2-) and the like.
"alkenylene" refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2 to 18 carbon atoms containing two monovalent radical centers derived from a parent olefin by the removal of two hydrogens on the same or different carbons. Typical alkenylene groups include, but are not limited to, 1, 2-vinyl (-CH ═ CH-).
"alkynylene" refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2 to 18 carbon atoms containing two monovalent radical centers derived from the removal of two hydrogens on the same or different carbons of a parent alkyne. Typical alkynylene groups include, but are not limited to, ethynyl, propynyl, and 4-pentynyl.
"aryl" or Ar refers to aromatic or heteroaromatic groups, consisting of one or more ringsAnd, contain 3 to 14 carbon atoms, preferably 6 to 10 carbon atoms. Heteroaryl refers to the substitution of one or more carbon atoms (preferably 1,2,3 or 4 carbon atoms) on the aromatic ring with 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 each independently replaced by-R ', halogen, -OR ', OR-SR ', -NR ' R ", -N ═ NR ', -N ═ R ', -NR ' R", -NO2,-S(O)R’,-S(O)2R’,-S(O)2OR’,-OS(O)2OR ', -PR ' R ', -P (O) R ', -P (OR ') (OR '), -P (O) (OR ') OR-OP (O ') (OR ') "), wherein R ', R ' are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, aralkyl, carbonyl OR pharmaceutically acceptable salts.
"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. "heterocyclic" is also defined in 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, vinylimino, pyrrolidine, pyrazolidinyl, alkylimidazole, oxiranyl, tetrahydrofuran, dioxolane, doxorubicin, dioxanyl, dioxolane, gua, piperazine, morpholine, pyran, imidazoline, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyran, dithiane, thiomorpholine, dihydropyran, pyraclostrobin, tetrahydropyridine, dihydropyridine, tetrahydropyrimidine, dihydrothiopyran, hexamethyleneimine and the structures resulting from their condensation with phenyl.
"heteroaryl" or heteroaromatic refers to 5 to 14, preferably 5 to 10, aromatic hetero-, mono-, bi-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 ring structures resulting from their condensation with phenyl.
"alkyl", "cycloalkyl", "alkenyl", "alkynyl", "aryl", "heteroaryl", "heterocycle" and the like also refer to the corresponding "alkylene", "cycloalkylene", "alkenylene", "alkynylene", "arylene", "heteroarylene", and "heterocyclylene" and the like, free of two hydrogen atoms.
"aralkyl" refers to an acyclic alkane radical wherein one is joined to a carbon atom, usually a terminal or sp3The carbon atom to which the hydrogen atom is attached 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, naphthylphenyl, 2-naphthylbenzene-1-ethyl, and the like.
"Heteroaralkyl" means an acyclic alkyl radical in which one is terminal or sp with a carbon atom3The carbon atom to which the hydrogen atom is attached is replaced by a heteroaryl group. Typical heteroaralkyl groups include, but are not limited to, 2-benzimidazolyl, 2-furoethyl and the like.
Examples of "hydroxy protecting groups" include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilylether, triethylsilyl ether, triisopropylsilyl ether, tert-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetyl ester, substituted acetyl ester, 2, 2-dimethylpropionate, benzoate ester, methylsulfonate ester, and p-toluenesulfonate ester.
"leaving group" means a functional group that can be substituted with another group. Leaving groups well known in the art include, but are not limited to, halo (chloro, bromo, iodo), methanesulfonyl, p-toluenesulfonyl, trifluoromethanesulfonyl, trifluoromethanesulfonate.
The following abbreviations are used in the present invention and are defined as: boc, tert-butoxycarbonyl; BroP, tris (dimethylamino) phosphine bromide hexafluorophosphate; CDI, carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCM, dichloromethane; DIAD, diisopropyl azodicarboxylate; DIBAL-H, diisobutylaluminum hydride; DIPEA, diisopropylethylamine; DEPC, diethylpyrocarbonate; DMA, N-dimethylacetamide; DMAP, p-dimethylaminopyridine; DMF, N-dimethylformamide; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EDC, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; ESI-MS, electrospray mass spectrometry; HATU, 2- (7-benzotriazol oxide) -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" refer to molecular entities and compositions that do not produce deleterious, allergic, or other untoward effects when administered to an animal or human, as appropriate.
"pharmaceutically acceptable solvate" or "solvate" refers to the disclosed compound and one or more solvent molecules associated therewith. Solvents in pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
"pharmaceutical excipients" include any carrier, diluent, adjuvant or excipient, such as protective or antioxidant agents, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents in pharmaceutically active compositions is well known in the art. Any conventional vehicle or agent, except those incompatible with the active ingredient, is contemplated for use in the therapeutic compositions. Auxiliary active ingredients may also be added to make suitable therapeutic compositions.
"pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds that form salts by reacting the parent compound with an acid or a base. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts formed with the parent compound, e.g., 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, and the like; 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 acids, lactic acid, and the like. Other salts include ammonium salts such as trimethylamine, meglumine, pyrrolethoxide and the like, and metal salts such as sodium, potassium, calcium, zinc and magnesium salts.
The pharmaceutically acceptable salts of this patent can be synthesized from the parent, which contains an acid or base, by conventional chemical methods. In general, such salts can be 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 two solvents. In general, nonaqueous 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,17thed., Mack Publishing Company, Easton, PA,1985, p.1418, incorporated herein by reference.
The novel conjugate disclosed in the present invention uses a bridge linker. Part of the linker and its synthesis are shown in FIGS. 1 to 10.
Bridge connector
The synthesis of the bridge linker, and the preparation of the drug-cell binding molecule conjugate in this patent are shown in FIGS. 1-9. The bridge linker has two elements: a) an acetylene dicarbonyl compound which may be substituted and which may react with a pair of thiols to form a thioether covalent bond; b) a functional group reactive with the drug, which includes, but is not limited to, disulfide, maleimide, haloacetyl, aldehyde, ketone, azide, amine, alkoxyamine, and hydrazine. Bridged substitution of acetylene dicarbonyl can be by reaction of acetylene diacid with amino, hydroxyl or sulfhydryl groups to give amides, esters or sulfhydryl esters. Examples of bridge linker syntheses are shown in figures 1,3,4,5,6,7,8 and 9. The bridged substitution of acetylene dicarbonyl can also be obtained by reaction of acetylene with an acid halide or anhydride to form a carbon-carbon bond. Examples of the synthesis of these bridge connectors can be seen in fig. 2 and 10.
Preferably, the bridge linker compound has the structure shown in (I):
Figure BDA0003226420300000111
wherein the acetylene dicarbonyl compound linker is capable of reacting with a pair of sulfur atoms on a cell-binding molecule.
Z1And Z2Are the same or different functional groups capable of reacting with a cytotoxic drug to form a disulfide bond, an ether bond, an ester bond, a thioether bond, a thioester bond, a peptide bond, a hydrazone bond, a carbamate bond, a carbonate bond, an amine bond (secondary, tertiary or quaternary), an imine bond, a heterocycloalkyl, a heteroaryl, an alkoxyoxime bond or an amide bond;
R1and R2Is the same or different H, straight-chain alkyl containing 1 to 6 carbon atoms, branched chain or cycloalkyl containing 3 to 6 carbon atoms, straight chain, branched chain or cycloalkenyl or alkynyl, or ester, ether, amide or polyethoxy unit (OCH) containing 1 to 6 carbon atoms2CH2)pOr polypropoxy units (OCH)2(CH3)CH2)pUnits wherein p is an integer from 0 to about 1000, or a combination of these groups;
in addition, R1And R2Is a chain structure containing C, N, O, S, Si and P atoms, optimally contains 0-500 atoms, and is covalently connected with X1Or X2And Z1Or Z2；R1And R2Are combined in all possible chemical ways, such as to form an alkyl, alkylene, alkenylene, alkynylene, ether, polyoxyalkyl, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, polyurethane, amino acid, polypeptide, acyloxyamine, hydroxamic acid, or a combination of these groups;
X1and X2Is independently selected from N (R)3) O, S or CH2；R3Is H, a straight chain alkyl group having 1 to 6 carbon atoms, a branched or cyclic alkyl group having 3 to 6 carbon atoms, a straight chain, branched or cyclic alkenyl or alkynyl group, or an ester, ether, amide or polyethoxy unit (OCH) having 1 to 6 carbon atoms2CH2)pWherein p is an integer from 0 to 1000, or a combination of these groups.
In another embodiment, R1、R2And R3Can be respectively a chain structure containing C, N, O, S, Si and P atoms, and is covalently connected with the cell surface binding molecule and the conjugated drug. The various atoms used to form the linker are joined 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; further, it is to be understood that the atoms forming the linker (L) may be saturated or unsaturated, may be free radicals, and may be cyclized with each other to form a divalent ring structure, including cycloalkane, cyclic ether, cyclic amine, arylene, heteroarylene, and the like.
Functional group Z1And Z2Cytotoxic drugs can be linked and can form disulfide bonds, thioethers, sulfhydryl esters, peptides, hydrazines, ethers, esters, carbamates, carbonates, oximes, or amide bonds. Such functional groups include, but are not limited to, mercapto, disulfide, amino, carboxyl, aldehyde, carbonyl, maleimide, haloacetyl, hydrazine, alkoxyamino, and hydroxyl.
Functional group Z capable of reacting with terminal amino group of drug or cytotoxic molecule1And Z2May be, but is not limited to, N-hydroxysuccinimide ester, p-nitrophenol ester, dinitrophenol ester, pentafluorophenol ester; the functional group capable of reacting with a terminal thiol group may be, but is not limited to, pyridyl disulfide group, nitropyridine disulfide group, maleimido group, haloacetate, carbonic acid chloride; functional groups capable of reacting with a terminal carbonyl or aldehyde group may be, but are not limited to, amino, alkoxyamino, hydrazine, acetoxyamino; the functional group that reacts with the terminal azide group may be, but is not limited to, an alkynyl group.
In a preferred embodiment, R1、R2And R3Is a linear alkyl radical having from 1 to 6 carbon atoms, or a polyethoxy unit (OCH)2CH2)P，p＝1～100。
The key step in the synthesis of bridge linkers containing acetylenedicarbonyl groups is the condensation of acetylenedicarbonyl or its derivatives with other components, terminal amines (primary or secondary), alcohols or thiols, as shown in (Ia) below:
Figure BDA0003226420300000121
wherein X in formula (Ia) is X in formula (I)1Or X2Is denoted by N (R)3) O, or S; r is R1And/or R2，R1、R2And R3The same formula (I) is defined.
Lv 1and Lv2 are identical or independent of each other and are OH, F, Cl, Br, I, nitrophenol, N-hydroxysuccinimide (NHS), phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, trifluoromethylsulfonic acid, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, p-toluenesulfonic acid, methanesulfonic acid, 2-ethyl-5-phenylisoxazole-3' -sulfonic acid, an anhydride formed from an anhydride or anhydride with another anhydride such as acetic anhydride or formic anhydride; or a polypeptide condensation reaction intermediate or a Mitsunobu reaction intermediate; the condensing agent comprises: 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-benzotriazol-N, N, N', N '-tetramethyluronium tetrafluoroborate), O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU), benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinylphosphates (PyBOP), Diethylpyrocarbonate (DEPC), N, N, N', n ' -Tetramethylchloroformamidine hexafluorophosphate, 2- (7-benzotriazol oxide) -N, N, N ', N ' -tetramethyluronium Hexafluorophosphate (HATU), 1- [ (dimethylamine) (morpholinyl) methylene]-1[1,2,3]Triazolo [4,5-b]1-pyridine-3-oxidophorophosphate (HDMA), 2-chloro-1, 3-dimethylimidazolium hexafluorophosphate (CIP), chlorotriazolylphosphonium hexafluorophosphate (PyCloP), bis (tetramethylene) fluorocarboxamide (BTFFH), N, N, N ', N' -tetramethyl-thio- (1-oxo-2-pyridinyl) thiouroniumHexafluorophosphate, 2- (2-pyridon-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate (TPTU), thio- (1-oxo-2-pyridyl) -N, N, N ', N' -tetramethylthiouronium hexafluorophosphate, O- [ (ethoxycarbonyl) cyanomethylamine]-N, N, N ', N ' -tetramethylthiourea Hexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylamino-morpholine-carbonium hexafluorophosphate (COMU), (benzotriazol-1-yloxy) dipyrrolidine carbohexafluorophosphate (HBPyU), N-benzyl-N ' -cyclohexylcarbodiimide (or supported on a polymer), dipyrrolidinyl (N-succinimidyloxy) carbonium hexafluorophosphate (HSPyU), 1- (chloro-1-pyrrolidinylmethylene) pyrrolidine hexafluorophosphate (PyClU), 2-chloro-1, 3-dimethylimidazole tetrafluoroborate (CIB), (benzotriazol-1-yloxy) dipiperidine carbohexafluorophosphate (HBPipU), 6-chlorobenzotriazole-1, 1,3, 3-tetramethyluronium tetrafluoroborate (TCTU), tris (dimethylamino) phosphonium bromide hexafluorophosphate (BroP), 1-n-propylphosphoric anhydride (PPACA,
Figure BDA0003226420300000132
) 2-isocyanoethylmorpholine (MEI), N' -tetramethylurea-oxy- (N-succinimidyl) Hexafluorophosphate (HSTU), 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP), oxy- [ (ethoxycarbonyl) cyanomethylamine]-N, N '-tetramethylthionurebetrafluoroborate (TOTU), 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (MMTM, DMTMM), 2-succinimidyl-1, 1,3, 3-tetramethyluronium tetrafluoroborate (TSTU), 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 CH2When the acetylenedicarbonyl group on the bridge linker is attached to another functional group through a C — C bond, the key step in the synthesis is the reaction of bis (trimethylsilyl) acetylene, acetylene dibromide (grignard reagent), acetylene dilithium salt or other acetylene bimetallic salt, and an acid halide or anhydride, as shown in (Ib), (Ic), (Id), (Ie), (If), (Ig) and (Ih):
Figure BDA0003226420300000131
Figure BDA0003226420300000141
where M is Na, K, Li, Cu, CuLi, Sn, Ti, Ca, Mg or Zn.
Specific examples of bridge linker syntheses are shown in FIGS. 1-10. Typically, the acetylene dicarbonyl linker comprises a functional group that can react with a small molecule drug on the conjugate.
Cell binding molecule-drug conjugates
The conjugates of the invention can be represented by the following formula:
Figure BDA0003226420300000142
where Cb is a cell binding agent, L is a linker, Drug 1and Drug2 are small Drug molecules, n is an integer from 1 to 20, and 2 sulfur bridges Cb to L, each bridge linker L covalently linking 2 or more Drug molecules.
This bridge linker L may be composed of one or more linker components. Typical linker components include: 6-Maleimidohexanoic 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-thiobutanoic acid (SPDB), 4-thio-2-hydroxysulfobutanoic acid (2-Sulfo-SPDB), one or more ethoxy-CH2CH2The O-unit ("EO" or "PEO"), also includes other linkers well known in the art, some of which are listed herein.
Examples of linkers comprising these components are:
Figure BDA0003226420300000143
(containing 6-Maleimidohexanoic acid MC)
Figure BDA0003226420300000144
(including 3-Maleimidopropionic acid MP)
Figure BDA0003226420300000151
(including p-aminobenzyloxycarbonyl group PAB)
Figure BDA0003226420300000152
Figure BDA0003226420300000153
(containing Ethylmaleimide ME)
Figure BDA0003226420300000154
(including valine-citrulline)
Figure BDA0003226420300000155
(comprising 4- (N-Maleimidomethyl) cyclohexane-1-carboxylic acid MCC)
Figure BDA0003226420300000156
(comprising (4-acetyl) aminobenzoic acid)
Figure BDA0003226420300000157
(comprising 4-thio-2-hydroxysulfobutyric acid, 2-thio-SPDB)
Preferred conjugates are of the formula (II):
Figure BDA0003226420300000161
wherein:
cb is a cell binding agent, most preferably an antibody;
Drug1and Drug2(ii) are the same or different cytotoxic agents linked to the cell binding agent by a bridging linker with alkyl, alkylene, alkenylene, alkynylene, ether, polyalkoxy, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, carbamate, amino acid, peptide, acyloxyamine, hydroxamic acid, disulfide bond, thioether, thioester, carbamate, carbonate, heterocycle, heteroalkyl, heteroaryl or alkoxyoxime bonds, and combinations thereof;
n is 1 to 20; r1,R2,X1And X2Is as defined in formula (I).
As described in more detail below, Drug 1and Drug2 can be any small molecule Drug, including, but not limited to, tubulysin, calicheamicin, auristatin, maytansine, CC-1065 analogs, morpholinos, doxorubicin, taxanes, cryptophycins, epothilones, benzodiazepine series dimers (e.g., Pyrrole Benzodiazepine (PBD) or tomaymycin, indole benzodiazepine, imidazole benzodiazepine, or oxazole benzodiazepine dimer).
To synthesize conjugate conjugates, the disulfide bond on the cell-binding agent is first reduced to produce a pair of free thiols, which are then reacted with the bridging linker of formula (I) in an aqueous phase at pH 5-9, with or without the addition of 0-30% of a water-miscible organic solvent, such as: n, N-dimethylacetamide, N-dimethylformamide, ethanol, methanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, dioxane, propylene glycol, or ethylene glycol to introduce Z1And Z2A reactive group which may be a disulfide bond, a maleimide group, a haloacetyl group, an azide, a 1-alkyne, a ketone, an aldehyde, an alkaneAn oxyamino group or a hydrazide. The reactive group of the cytotoxic molecule then reacts with the corresponding modified cell-binding agent. For example, the synthesis of disulfide-linked cell-binding agent-drug conjugates is achieved by disulfide bond exchange between disulfide bonds in the modified cell-binding agent and a drug containing a free thiol group; the synthesis of the cell bonder-drug conjugate combined by thioether bond is realized by the reaction of cell bonder modified by maleimide group or halogen acetyl group or ethyl sulfone and drug containing free sulfhydryl; synthesis of intramolecular acid-labile hydrazone conjugates by reaction of a hydrazide group and a carbonyl group in a linker is also well known in the art (see P.Hamann et al, Hinman, L.M., et al, Cancer Res.1993,53, 3336-334; B.Laguzza et al, J.Med.Chem.,1959, 32; 548-555; P.Trail et al, Cancer Res.,1997, 57; 100-105); synthesis of conjugates containing triazole groups can be achieved by click chemistry (dipolar cycloaddition) of 1-alkynes on one linker with drugs containing azide groups (Lutz, J-F.et al, adv. drug Del. Rev.2008,60,958-.
Alternatively, the drug small molecule can be reacted with a modified cell binding molecule-linker structure as shown in structural formula (III) that has been coupled to a cell binding agent and contains a reactive functional group. For example, a thiol-containing drug can be reacted with maleimide, haloacetyl, or ethylsulfonyl on the linker of formula (III) in a buffer at pH 5.5-9 to form thioether-linked conjugate conjugates. The sulfhydryl-containing drug may undergo disulfide bond exchange with a pyridine disulfide fragment on the linker of formula (III) to produce a disulfide-linked conjugate. Drugs containing a hydroxyl or thiol group can be reacted with a halogen on the linker of formula (III), especially a halogen on an alpha halocarboxylic acid, under mild base conditions, such as pH 8.0-9.5, to give ether or thioether linked conjugate. The hydroxyl-containing drug can also be condensed into ester with carboxyl on the linker in formula (I) under the action of EDC or DCC condensing agent. The drug-modified bridge linker is then coupled to a cell binding molecule. The drug containing amino group can react with carboxylic acid ester of 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-5-phenylisoxazole-3' -sulfonic acid on the linker in formula (III) to obtain the amide bond linked conjugate.
The conjugate may be purified by standard biochemical methods, such as gel filtration, adsorption chromatography, ion exchange or dialysis with Sephadex G25 or Sephacryl S300. In some cases, for example, small molecule cell binding agents (e.g., folic acid, melanocyte stimulating hormone, EGF, etc.) can be purified by HPLC, medium pressure column chromatography, ion exchange chromatography, or other chromatographic methods after binding to the small molecule drug.
Modified cell binding agents/molecules
Preferably, the structure of the cell binding agent modified with a linker of the invention is according to formula (III):
Figure BDA0003226420300000171
wherein Cb, Z1,Z2,n,R1,R2,X1And X2The same as defined in formulas (I) and (II).
In a preferred embodiment, Z1And Z2Is dithio, maleimido, haloacetyl, alkoxyamine, azide, ketone, aldehyde, hydrazine, N-hydroxysuccinimide ester or a carboxylic ester of 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. Z1And Z2Can react with cytotoxic drugs to form thioethers, hydrazones, amides, alkyloximes, carbamates, esters, ethers or disulfide bonds. The modified cell-binding agents can be prepared by reacting the cell-binding agent with a bridge linker as described in formula (I) as disclosed aboveThe formula (II).
In order to achieve efficient connection of the alkynyl group on the bridge linker to the cell binding agent, especially to a pair of free thiol 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 to make the structure in formula (III) well soluble in water. In modifying the cell binding agent, the cross-linking agent (bridge linkage) of formula (I) is first dissolved in a polar organic solvent that is 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 Dimethylsulfoxide (DMSO); the solution concentration is slightly higher, e.g., 1-500 mM. Meanwhile, a cell binding agent, such as an antibody, is dissolved in a buffer solution with pH of 5-9.5, preferably 6-8.5 at a concentration of 1-35 mg/ml, and reacted with 1-20 equivalents of TCEP or DTT for 20 minutes to 12 hours. After reduction, the DTT was removed by SEC column purification. TCEP can be purified on SEC columns or be carried directly to the next step without purification. Furthermore, antibodies or other cell-binding agents reduced with TCEP can be carried out in the presence of the bridge linker of formula (I), in which case the cross-linking conjugation of the cell-binding agent is carried out simultaneously with the reduction of TCEP.
The reaction to modify the cell binding agent is generally carried out in a buffer at a pH of 6 to 9, preferably 6.5 to 7.5, and may be any buffer salt system without nucleophilicity in this pH range. Typical buffers include phosphate, triethanolamine hydrochloride, HEPES, and MOPS buffers, and may contain other ingredients such as cyclodextrins, sucrose, and salts such as sodium chloride and potassium chloride. The solution of the bridge linker of formula (I) is added to the reduced cell-binding agent solution and incubated at 4 to 45 deg.C, preferably room temperature, and the progress of the reaction is monitored by measuring the absorbance of the solution at 254nm or other suitable wavelength. After completion of the reaction, the modified cell-binding agent may be purified according to conventional methods, for example using gel filtration chromatography or adsorption chromatography.
The degree to which the cell-binding agent is modified can be determined by measuring the uv absorbance of the nitro-pyrithione, dinitro-pyridine-disulfide-ketone, pyrithione, formamide pyridine-disulfide-ketone, and dimethylamide pyridine-disulfide-ketone groups produced by the reaction. If the conjugate has no chromophore, it can be determined analytically by LC-MS or more preferably UPLC-QTOF mass spectrometry, and Capillary Electrophoresis Mass Spectrometry (CEMS). The bridge linker of the present invention may contain various kinds of functional groups thereon, and may react with various drugs, particularly cytotoxic drugs having suitable functional groups. For example, a modified cell-binding agent containing an amino or hydroxyl substituent can be reacted with a drug containing an N-hydroxysuccinimide (NHS) ester, and a modified thiol-containing cell-binding agent can be reacted with a drug containing a maleimide or haloacetyl group. In addition, cell-binding agents modified to contain a carbonyl group (aldehyde or ketone group) can react with drugs containing hydrazide or alkoxyamine. Those skilled in the art can readily determine what linker to use based on the reactivity of the functional groups therein.
Modified cytotoxic drugs
The cytotoxic drug structure modified with the bridge linker of the invention is preferably (IV):
Figure BDA0003226420300000191
wherein the Drug1,Drug2,Z1,Z2,n,R1,R2,X1And X2Are as defined for formulae (I) and (II).
The modified drug (IV) is obtained by reacting the linker of formula (I) with a drug molecule, wherein the acetylene dicarbonyl functional group is reactive with a pair of sulfhydryl groups on a cell binding agent. Acetylene dicarbonyl is obtained by condensation of acetylene, which is described in the reaction schemes (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig) and (Ih).
However, for sulfhydryl-containing drugs, or drugs linked to cell-binding agents via bridging linkers using a thioether, thioester, or disulfide bond, it is preferred that Drug1 or Drug2 be linked first to R via a thioether, thioester, or disulfide bond1Or R2R produced1-Drug1Or R2-Drug2The fragment is then bound to an acetylene dicarbonyl to form a bridge linker of formula (IV)Decorative medicines.
For example, a thiol-containing drug can be reacted with R in a buffer at neutral pH1Or R2Reaction of the above maleimide to form a thioether-bonded R1-Drug1Or R2-Drug2And then condensed with acetylene dicarbonyl to form a modified drug comprising a thioether bond as in formula (IV). The hydroxyl-containing drug can be reacted with R containing halogen, p-toluenesulfonic acid or mesylate under the weak base condition1Or R2Reaction to give the ether bond R1-Drug1Or R2-Drug2And then condensed with acetylene dicarbonyl to form a modified drug comprising a thioether bond as in formula (IV). The drug containing hydroxyl can be condensed with a linker having carboxyl in the structure (I) under the action of a dehydrating agent such as EDC or DCC to obtain the modified drug containing ester bond in the formula (IV). The sulfhydryl-containing drug may be reacted with R1Or R2The maleimide, vinylsulfone or haloacetyl on the compound reacts to generate the R combined by thioether bond1-Drug1Or R2-Drug2And then condensed with acetylene dicarbonyl to form a modified drug comprising a thioether bond as in formula (IV). Similarly, amino group-containing drugs can also be condensed with the carboxyl groups on the bridge linker (I) to give modified drugs containing amide bonds as in formula (IV). The modified drug may 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 agents
The cell-binding agents of the invention, including conjugates and modified cell-binding agents, can be any of a variety of molecules now known or later disclosed that are capable of binding, complexing or otherwise reacting with a cell fragment of therapeutic interest or that have been biologically modified.
Cell-binding agents include, but are not limited to, large molecular weight proteins, such as whole antibodies (polyclonal antibodies, monoclonal antibodies, dimers, multimers, multispecific antibodies, e.g., bispecific antibodies); a single chain antibody; antibody fragments such as Fab, Fab ', F (ab')2,Fv(Parham,J.Immunol.1983,131, 2895-2902); fragments produced by the Fab expression library, anti-idiotype (anti-Id) antibodies; a CDR; a bivalent antibody; trivalent antibodies and epitope-binding fragments of any of the above antibodies that immunospecifically bind to a cancer cell antigen; a viral antigen; microbial antigens or proteins produced by the immune system, capable of recognizing, binding to specific antigens or having a desired biological activity (Miller et al J. of Immunology 2003,170, 4854-4861); interferons (e.g., type I, II, III); a 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 (thyroid stimulating hormone releasing hormone), MSH (melanocyte stimulating hormone), steroid hormones such as androgen and estrogen; 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 TGF α, TGF β, insulin and insulin-like growth factors (IGF-I, IGF-II), G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 1984,5, 155-); vaccinia Growth Factor (VGF); fibroblast Growth Factor (FGF); a small molecular weight protein; a polypeptide; peptides and peptide hormones, such as bombesin, gastrin-releasing peptide; platelet-derived growth factors; interleukins and cytokines, for example, 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.1985260932-937); carbohydrate binding proteins or lipoproteins, such as lectins; a cellular nutrient-delivery molecule; small molecule inhibitors such as Prostate Specific Membrane Antigen (PSMA) inhibitors and small molecule Tyrosine Kinase Inhibitors (TKI), non-peptides or any other cell binding molecule or substance such as bioactive polymers (Dhar, et al, Proc. Natl. Acad. Sci.2008,105,17356-61), bioactive dendrimers (Lee, et al, Nat. Biotechnol.2005,23,1517-26; Almutairi, et al; Proc. Natl. Acad. Sci.2009,106,685-90), nanoparticles (Liong, et al, ACS Nano,2008,19, 1309-12; Metarova, et al, Nat. Med.2007,13,372-7; Javier, et al, Bioconjugate Chem.2008,19,1309-12), liposomes (et al, Curr. Drr. Phar. Des. 10,2981-9) and any other cell binding molecule or substance, such as bioactive polymers (Dhar, et al, Natc. Natl. Acad. Acad.Sci.2008, et al., Bioconugate Chem.2008,19,1309-12), liposomes (Liposome, et al, Inc. Phar. diseasesToxic shells (Flenniken, et al, Virus Nanotechnol.2009,327, 71-93).
In general, monoclonal antibodies are preferred as cell surface binding agents if appropriate monoclonal antibodies are available. The antibody may 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 combinations thereof. Methods for producing polyclonal anti-receptor peptide antibodies are well known in the art, for example, as described in U.S. patent 4,493,795. Monoclonal antibodies are typically prepared by fusing myeloma cells with spleen cells of mice that have been immunized with the desired antigen(s) (ii)
Figure BDA0003226420300000211
G; milstein, C.Nature 1975,256: 495-497). The detailed procedure is 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 may be immunized with an antigen of interest, such as an intact target cell, an antigen isolated from a target cell, an intact virus, an inactivated whole virus, and viral proteins. Spleen cells are typically fused with myeloma cells using polyethylene glycol (PEG) 6000. Fused cells were screened for sensitivity to HAT (hypoxanthine-aminopterin-thymidine). Hybridomas that embody the monoclonal antibodies of the invention can be determined by their ability to immunoreact with specific receptors or to inhibit the activity of receptors on target cells.
The production of monoclonal antibodies for use in the present invention is carried out in monoclonal hybridoma cultures comprising a nutrient medium and hybridomas secreting antibody molecules with the appropriate antigen specificity. The culture is maintained under suitable conditions for a period of time sufficient for the hybridoma to secrete the antibody molecule into the culture medium. The antibody-containing medium is then collected. Antibody molecules are further separated using techniques well known in the art, such as protein A affinity chromatography, anionic, cationic, hydrophobic or size exclusion chromatography (particularly by protein A affinity chromatography and size exclusion chromatography), centrifugation, differential solubility or any other standard technique for purifying proteins.
Media useful for preparing 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 4.5g/ml glucose, 0-20mM glutamine, 0-20% fetal bovine serum, several ppm of heavy metals or/and heavy metal salts such as Cu, Mn, Fe or Zn, and antifoams such as polyoxyethylene-polyoxypropylene block copolymers.
Alternatively, antibody-producing cell lines can be obtained by techniques other than fusion, such as transformation of neoplastic DNA into B lymphocytes, or transfection of a neoplastic virus, such as epstein-barr virus (EBV, also known as human herpesvirus 4(HHV-4)) or kaposi's sarcoma-associated herpesvirus (KSHV), see U.S. patent 4,341,761; 4,399,121, respectively; 4,427,783, respectively; 4,444,887; 4,451,570, respectively; 4,466,917, respectively; 4,472,500, respectively; 4,491,632, respectively; 4,493,890. Monoclonal antibodies can also be prepared by anti-receptor peptides or peptides containing terminal carboxyl groups, as is well known in the art, for example, see the Niman et al, Proc. Natl. Acad. Sci. USA,1983,80: 4949-4953; geysen et al, Proc. Natl. Acad. Sci. USA,1985,82: 178-; lei et al biochemistry 1995,34(20): 6675-6688. In general, anti-receptor peptides or peptide analogs are used alone or in conjunction with an immunogenic carrier as immunogens to generate monoclonal antibodies against receptor peptides.
Monoclonal antibodies useful as binding molecules in the present invention may 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 an affinity enrichment format and can be used to select for human antibodies that specifically bind to an antigen. Phage display techniques are also described in detail in the literature, and the construction and screening of phage display libraries is also well known in the art, as described in Dente et al, Gene.1994,148(1): 7-13; littlet et al, Biotechnol adv.1994,12(3): 539-55; clackson et al, Nature 1991,352: 264-; huse et al, Science 1989,246: 1275-.
Monoclonal antibodies are produced by hybridomas fused to non-human, e.g., mouse, cells and can be humanized to avoid production of human anti-mouse antibodies. Common antibody humanization methods are complementarity determining region grafting techniques, which have also been described in detail, e.g., U.S. Pat. nos. 5,859,205 and 6,797,492; liu et al, Immunol Rev.2008,222: 9-27; almagro et al, Front biosci.2008,13: 1619-33; lazar et al, MolImmunol.2007,44(8): 1986-98; li et al, Proc. Natl. Acad. Sci. U S A.2006,103(10):3557-62, incorporated herein by reference. Whole human antibodies can also be prepared by immunizing a transgenic mouse, rabbit, monkey or other mammal carrying a large portion of the heavy light chain of human globin with an immunogen. Examples of such mice are: xenomouse (Abgenix/Amgen), HuMAb-Mouse (Metarex/BMS) and VelociMouse (Regeneron), see U.S. Pat. Nos. 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 and 5,569,825. For human therapy, the mouse variable region and the human constant region can also be fused to form a "chimeric antibody" that is significantly less immunogenic in humans than the mouse monoclonal antibody (Kipriyanov et al, MolBiotechnol.2004,26: 39-60; Houdbine, CurropinBiotechnol.2002,13: 625-9). In addition, site-directed mutagenesis of the variable region of an antibody can result in 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), and alterations in the constant region of an antibody can enhance its effector functions mediating binding and cytotoxicity.
Immunospecific antibodies to malignant cell antigens may also be obtained commercially or produced by any known method, such as chemical synthesis or recombinant expression techniques. Nucleotide sequence encoding antibodies immunospecific for malignant cell antigens are commercially available, for example from GenBank databases or similar databases, literature publications, or by routine 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 the target cell may also serve as a binding molecule. These peptides or proteins may be any random peptides or proteins that have an affinity for an epitope or a corresponding receptor and are not necessarily immunoglobulin family members. These peptides can be isolated by techniques similar to phage display antibodies (Szardnings, J Recept Signal Transmission Res.2003; 23(4): 307-49). Peptides obtained from random peptide libraries can be used similarly to antibodies and antibody fragments. The peptide or protein binding molecule may be coupled or linked to a macromolecule or other substance, including but not limited to albumin, polymers, liposomes, nanoparticles, dendrimers, so long as such linkage retains the antigen binding specificity of the peptide or protein.
Examples of antibodies on conjugates for the treatment of cancer, autoimmune and/or infectious diseases, and drug molecules linked by the bridging linker of this patent include, but are not limited to, 3F8 (anti-GD 2), abazumab (anti-CA-125), abciximab (anti-CD 41 (integrin α -IIb), adalimumab (anti-TNF- α), Adecatumumab (anti-EpCAM, CD326), aphidimab (anti-TNF- α), afutumumab (anti-CD 20), alezizumab (anti-VEGFR 2), ALD518 (anti-IL-6), Alemtuzumab (Campath, mabcapampath, anti-CD 52), altumumab (anti-CEA), antatumomab (anti-TAG-72), ankuzumab (anti-imab-638, anti-IL-13), Apolizumab (anti-HLA-CEA), azithromycin-DR-CEA), azithromycin-6762 (anti-attuzumab), azithromycin-CD 62L (anti-CD L), RoActemra, anti-IL-6 receptor), Atorolimumab (anti-Rheumas factor), Bapineuzumab (anti-amyloid beta protein), Basiliximab (Simulect, anti-CD 25 (alpha chain of IL-2 receptor)), Bavituximab (anti-phosphatidylserine), Bectumomab (Lymphoscan, anti-CD 22), Bellinumab (Benlysta, Lymphoststat-B, anti-BAFF), Benralizumab (anti-CD 125), Bertilimumab (anti-CCL 11(eotaxin-1)), Besilesomab (Scintimun, anti-CEA-related antigen), Bevacizumab (Avastin, anti-VEGF-A), Biciomab (Fibri Scirumab, anti-fibrin II beta chain), Bivazumab (anti-CD-7), Blinizumab (Avastin-3544), anti-Catualab (anti-CD 3523), anti-Catualab (anti-CD cAC 10), anti-Catualab (anti-VEGF-A), Bivatuzumab (Bivazumab, anti-C3528, anti-Cmax (anti-CD 3655-CD 3523, anti-CA-C3528), anti-CA-CT-C-36493, anti-CAvituzumab (anti-CCLvE-CT-C-CT-1, anti-CT-2, anti-CT-2, anti-CT-2, anti-CT-C-CT-2, anti-C-CT-2, anti-CT-C-2, anti-C-CT (anti-CT-C-2-C-E-2, anti-E-C-E-2, anti-C-2, anti-C-E-C-E-2, anti-C-2, anti-C-2, anti-C-E-C-2, anti-2, anti-C-2, anti-C-2, anti-E-C-2, anti-C-E-C-E, anti-2, anti-E-2, anti-C-E-C-E-2, anti-E-C-E-2, anti-E-C-2, anti-C-E-C-2, anti-C-E-2, anti-E, anti-rituximab (Cimzia anti-TNF-. alpha.), cetuximab (erbitux, IMC-C225, anti-EGFR), Citatuzumab bogatox (anti-EpCAM), Cixuumumab (anti-IGF-1), Clenoliximab (anti-CD 4), Clivazumab-MAb (anti-MUC 1), Conatumumab (anti-TRAIL-R2), CR6261 (anti-influenza A hemagglutinin), Dacetuzumab (anti-CD 40), Daclizumab (Zenapax, anti-CD 25(IL-2 receptor. alpha. chain)), Daratumumab (anti-CD 38 (cyclic ADP ribohydrolase), Denosumab (Prolia, anti-RANKL), Detumomab (anti-B lymphoma cell), Dorlomab, Doximab, Eclipzumab, Ecromeximab (anti-TNF-3), Enliozumab (anti-Lipolab, anti-Epilozumab) and anti-Epilozumab (anti-Epilozumab-Epsilomab) (anti-EPO-11), anti-Epilozumab (anti-Epilozumab-EPO-11), anti-Epilozumab (anti-Epilozumab), anti-Epilozumab-EPO-11), anti-Epilozumab (anti-EPO-11), anti-EPO 86523), Dolomab), epitazumab (anti-CD 22), Erlizumab (anti-ITGB 2(CD18)), Ertumaxomab (Rexomum, anti-HER 2/neu, CD3), Epitazumab (Abegrin, anti-integrin α v β 3), Exbivirumab (anti-hepatitis B surface antigen), Fanolisomab (NeutroSpec, anti-CD 15), Faralimomab (anti-interferon receptor), Farleuzumab (anti-folate receptor 1), Felvizumab (anti-respiratory syncytial virus), Fezakinumab (anti-IL-22), Figituzumab (anti-IGF-1 receptor), Fontolizumab (anti-IFN- γ), Foravizumab (anti-rabies virus glycoprotein), Fresolizumab (anti-TGF- β), Galiximab (anti-CD 7), Gantenerimumab (anti-IFN- β), Govatimumab (anti-Geimumab 23), anti-Geimumab (anti-Gernizumab), anti-Gernizumab (anti-TNF-IgG 33), anti-GPibrinomab (anti-GPE β), anti-TGF-IgG 23), anti-IgG receptor (anti-IgG-35011), ibritumomab (anti-CD 20), Igomoab (Indumcia-125, anti-CA-125), Imciromab (Myoscint, anti-cardiac myosin), Infliximab (Remicade, anti-TNF-. alpha.), Intetumumab (anti-CD 51), Indomomab (anti-CD 25(IL-2 receptor. alpha. -chain), Ebizumab (anti-CD 22), Iplimumab (anti-CD 152), Iratumumab (anti-CD 30(TNFRSF8)), Keliximab (anti-CD 4), Labetuzumab (CEA-Cide, anti-CEA), Lebrizumab (anti-IL-13), Lemallesomab (anti-NCA-90 (granulocyte antigen)), Letudelumumab (anti-TGF-. beta.2), Lexamumab (anti-TRAIL-R2), Libivumab (anti-HBsAb), anti-HBsAb (anti-TNF-. beta.25), anti-liver antigen 33 (anti-CD 33)), LetudelumumabMitsumumab (anti-CD 40), lumimab (anti-CD 23(IgE receptor), Mapatumumab (anti-TRAIL-R1), macimomab (anti-T-cell receptor), matuzumab (anti-EGFR), Mepolizumab (Bosatria, anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab (anti-CD 74), Minretumumab (anti-TAG-72), Mitumomab (BEC-2, anti-GD 3 ganglioside), Morolimumab (anti-rhesus factor), Motavivimab (Numax, anti-respiratory syncytial virus), Murooab-CD 3(Orthoclone OKT3, anti-CD 3), Nacolomomab (anti-C242), Naptolimumab (anti-5T 4), natalizumab (sabbri, anti-Typeim alpha 4), Netuzumab (anti-Thermomab) (anti-EGFR), Netuzumab (anti-TNF-C20), anti-TNF-gamma-TNF-E-D11 a, anti-TNF-gamma-TNF-E factor, and E-E, wherein, ofatumumab (Arzerra, anti-CD 20), Olaratumab (anti-PDGF-R alpha), Omalizumab (Xolair, anti-IgE Fc region) Oportuzumab (anti-EpCAM), Oregomoub (Ovarex, anti-CA-125), Otelixizumab (anti-CD 3), Pagibaximab (anti-lipoteichoic acid), Palivizumab (Synagis, Abbossynagis, anti-respiratory syncytial virus), panitumumab (Vebix, ABX-EGF, anti-EGFR), Panobazumab (anti-Pseudomonas aeruginosa), Pacoruzumab (anti-IL-4), Pemtumumab (Theragryn, anti-MUC 1), Pertuzumab (Omnitarg, 2C 7, anti-HER 2/neferi), Pemtumumab (anti-Theragryn, anti-CCR 3668), anti-VEGF-neuropeptide Ab (anti-VEGF-Pro-2C 11), anti-VEGF-2C 5631, anti-VEGF-2C 5634, anti-VEGF-2C-5634, anti-VEGF-2-VEGF-3, anti-VEGF-2-VEGF-2-C-pregabane, anti-pregabalonoma-pregabane, anti-pregabane-pregabalonone, anti-pregabalononema-VEGF-4, anti-VEGF-pregabalonoma-4, anti-pregabalononema-pregabane-pregabalonol, anti-pregabane-4, anti-pregabane-pregabalonol, anti-pregabalin (anti-pregabalin, anti-pregabalin, anti-VEGF-pregabalin, anti-pregabalin (anti-pregabalin, anti, raxibacumab (anti-anthrax toxin, protective antigen), Regavirumab (anti-cytomegalovirus glycoprotein B), Reslizumab (anti-IL-5), Rilutummab (anti-HGF), Rituximab (MabThera, Rituxanmab, anti-CD 20), Robatalimumab (anti-IGF-1 receptor), Rontalizumab (anti-IFN-. alpha.), Rovelizumab (LeukAr-rest, anti-CD 11, CD18), Ruplizumab (antva, anti-CD 154(CD40L)), Satumomab (anti-TAG-72), Sevirumab (anti-cytomegalovirus), Sibrotuzumab (anti-FAP), Sifarumab (anti-IFN-. alpha.), Sirtuximab (anti-IL-6), Siplizumab (anti-CD 2), CD95 (anti-pcrt), Regavirumab (anti-33), anti-amyloid beta protein), and amyloidizumab (anti-sphingosine-1-phosphate), Sontuzumab (anti-epidialin), Stamulumab (anti-myostatin), Sulesomab (Leukoscan, anti-NCA-90 (granulocyte antigen)), Tacatuzumab (anti-alpha fetoprotein), Tadocuzumab (anti-integrin. alpha. IIb. beta.3), Talizumab (anti-IgE), Tanezumab (anti-NGF), Taplituzumab (anti-CD 19), Tefibuzumab (Aurexis, (anti-coagulation factor A)), Telimomab, Tenatumumab (anti-tenascin C), Tenelizumab (anti-CD 40), Teplizumab (anti-CD 3), TGN1412 (anti-CD 28), Ticilizumab (Tremelizumab, anti-CTLA-4), Tigatituzumab (anti-CD 40), Treplizumab (anti-CD 3), TGN1412 (anti-CD 28), Ticilizumab (Tremella Tortuzumab, anti-CTLA-4), Tigatizumab (anti-CTLA-650), anti-CTLA-13), anti-CTLA-13 (anti-CTLA-13), anti-CTLA-13, anti-CTLA-2 (anti-CTLA-13), anti-CTLA-II (anti-13, anti-CTX-CTLA-2, anti-TNF-2, anti-TNF-6, anti-TNF-2, anti-TNF-2, anti-TNF-6, anti-TNF-2, anti-TNF-2, anti-TNF-2, anti-TNF-2, or fragment (anti-TNF-2, anti-TNF-2, anti-TNF-2, or fragment, or a (anti-TNF-2, or fragment (anti-TNF-2, or fragment (anti-TNF-2, or fragment of a (anti-TNF-2, or fragment of a (anti-TNF-2, or fragment of a-TNF-type (anti-TNF-2, or fragment of a-2, or fragment of a-TNF-, urtoxazumab (anti-E.coli), Ustekinumab (Stelara, anti-IL-12, IL-23), Vapaliximab (anti-AOC 3(VAP-1)), Vidolizumab (anti-integrin. alpha.4. beta.7), Vetuzumab (anti-AOC 3(VAP-1)), Viilizumab (Nuvion, anti-CD 3), Vitaxin (anti-angiointegrin avb3), Volociximab (anti-integrin. alpha.5. beta.1), Votumumab (HumaSPECT, anti-tumor antigen CTAA16.88), Zutumumab (HuMax-EGFR, Zolanmumab (HuMax-CD4, anti-CD 4), Ziralimumab (anti-CD 147 (baisin)), anti-CD 39147 (anti-Zolinoma (anti-CD 64), Easter @
Figure BDA0003226420300000261
Alefacept
Figure BDA0003226420300000262
Abatacept
Figure BDA0003226420300000263
Rilonacept (Arcalalyst), 14F7 (anti-IRP-2 (IRP-2), 14G2a (anti-GD 2 ganglioside, Nat. Cancer Inst., treatment of melanoma and solid tumors), J591 (anti-PSMA, west Cornell institute of medicine, treatment of prostate Cancer), 225.28S (anti-HMW-MAA (high molecular weight melanoma associated antigen), Sorin radiofacci SRL (Nat. origin, treatment of melanoma), COL-1 (anti-CEACAM 3, CGM1, Nat Cancer Ins)t. treatment of colorectal and gastric cancer), CYT-356(
Figure BDA0003226420300000264
For the treatment of prostate cancer), HNK20(OraVax Inc. for the treatment of respiratory syncytial virus infection), ImmuRAIT (derived from Immunomedics, for the treatment of NHL), Lym-1 (anti-HLA-DR 10, Peregrine Pharm), MAK-195F (anti-TNF (tumor necrosis factor, TNFA, TNF- α, TNFSF2, derived from Abbott/Knell, for the treatment of septic shock), MEDI-500(T10B9, anti-CD 3, TR α β (T cell receptor α/β), derived from Medmene Inc, for graft-versus-host disease), RING SCAN (anti-TAG 72 (tumor-associated glycoprotein 72), derived from Neoprene Corp, for breast, colon and rectal cancers), Avicidin (anti-EPGA (epithelial cell adhesion molecule)), anti-TACSTD 1 (tumor-associated calcium signal transduction glycoprotein 1), anti-733-2 (gastrointestinal tumor-associated protein 2), anti-epithelial-EGP 2 (EGA 2), anti-epithelial-A2, KS1/4 antigen, M4S, tumor antigen 17-1A, CD326 (from NeoRx for treatment of colon cancer, ovarian cancer, prostate cancer and NHL), LymphoCide (from immunology), Smart ID10 (from Protein Design Labs), Oncolom (from Techniclone Inc), Allomne (from BioTransplant), anti-VEGF (from Genentech); CEAcide (from Immunodics), IMC-1C11 (from ImClone Systems) and Cetuximab (from ImClone).
Other antibodies that may act 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's disease, NHL lymphoma, multiple myeloma), CD3 epsilon (T-cell lymphoma, lung cancer, breast cancer, stomach cancer, ovarian cancer, autoimmune disease, malignant ascites), CD19 (B-cell malignancy), CD20 (non-Hodgkin's 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, merk cell carcinoma, as well as liquid tumors, multiple myeloma), CD66e (various cancers), CD70 (metastatic renal cell carcinoma and non-Hodgkin lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (various cancers), mucin (various cancers), CD221 (solid tumor), CD227 (breast cancer, ovarian cancer), CD262 (non-small cell and other lung cancers), CD309 (ovarian cancer), CD326 (solid tumor), CEACAM3 (colorectal cancer, gastric cancer), CEM 5 (carcinoembryonic antigen, CEA, CD66e) (breast, colorectal cancer and lung cancer), DLL4, EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4(CD184, hematological 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, prostate cancer), FCGR1 (autoimmune disease), FOLR (folate receptor, ovarian cancer), GD2 ganglioside (various cancers), G-28 (cell surface antigen lipids, melanoma), GD3 idiotype (respective cancers), heat shock proteins (various cancers), HER1 (lung cancer, stomach cancer), HER2 (breast cancer, lung cancer and ovarian cancer), HLA-DR10(NHL), HLA-DRB (NHL, B cell leukemia), human chorionic gonadotropin (various cancers), IGF1R (insulin-like growth factor 1 receptor, solid tumors, blood cancers), IL-2 receptors (interleukin 2 receptors, T cell leukemia and lymphoma), IL-6R (interleukin 6 receptor, multiple myeloma, rheumatoid arthritis, Castleman's disease, interleukin 6 dependent tumors), integrins (α v β 3, α 5 β 1, α 6 β 4, α ll β 3, α 5 β 5, α v β 5, various cancers), MAGE-1 (various cancers), MAGE-2 (various cancers), MAGE-3 (various cancers), MAGE 4 (various cancers), anti-transferrin receptor (various cancers), p97 (melanoma), MS4A1 (transmembrane 4 domain subfamily A member 1, non-Hodgkin B cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast cancer, ovarian cancer, cervical cancer, bronchial cancer and alpha gastrointestinal tract cancer), MUC16 CA (125) (ovarian cancer), CEA (colorectal cancer), gp100 (melanoma), MART1 (melanoma), MPG (melanoma), MS4a1 (transmembrane 4 domain subfamily a member 1, small cell lung cancer, NHL), Nucleolin, Neu oncogene product (respective cancers), P21 (various cancers), anti- (N-glycolylneuraminic acid) antibody binding site (breast cancer, melanoma), PLAP testicular alkaline phosphatase (ovarian cancer, testicular cancer), PSMA (prostate cancer), 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 (trophoblastic cell 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 recognized by antibodies have been summarized and reviewed (Gerber, et al, mAbs 2009, 1:3, 247-.
The cell binding agent, preferably an antibody, can be any agent that is capable of antagonizing tumor cells, virally infected cells, microbially infected cells, parasitically infected cells, autoimmune cells, activated cells, bone marrow cells, activated T cells, B cells, or melanocytes. More specifically, the cell binding agent may 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, CD, CD274, CD276(B7-H3), CD303, CD304, CD309, CD326,4-1BB,5AC,5T4(Trophoblast glycoprotein, TPBG, 5T4, WNT-activated inhibitor 1 or WAIF1), adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor kinase 1, AFP, AKAP-4, ALK, α integrin, α v β 6, aminopeptidase N, amyloid β, androgen receptor, angiogenesis promoting protein factor 2, angiogenesis promoting protein factor 3, annexin A1, anthrax toxin protective antigen, anti-transfer protein receptor, AOC3(VAP-1), B7-H3, Bacillus anthracis, BAAOFF (B cell activating factor), B lymphoma cells, bcr-abl, bombesin, RIS, C5, C242 antigen, CA125 (carbohydrate antigen, MUC 125-52), CAIX 16-BOCA, CALA 31, CALA 9, CALA 6326, carbonic anhydrase IX, cardiac myoglobin, CCL11(C-C fragment chemokine 11), CCR4(C-C chemokine receptor 4, CD194), CCR5, CD3E (epsilon), CEA (carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (factor D), Ch4D5, cholecystokinin 2(CCK2R), CLDN18(Claudin-18), clusterin 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, cyclic ribonuclease, cyclin B1, CYP1B1, Cytomegalovirus B, Dabigan (DPP-like peptide-4), DPP-364-delta-peptide (DPP-4), DR5 (death receptor 5), Escherichia coli shiga toxin type-1, Escherichia 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 (TMPRSS2 ETS fusion gene), Escherichia coli, ETV6-AML, FAP (fibroblast activation protein alpha), FCGR1, alpha fetoprotein, fibrin II beta chain, fibronectin extra domain-B, FOLR (folate receptor), folate receptor alpha, folate hydrolase, Fos-related antigen 1, respiratory syncytial virus F protein, crimped receptor, fucosyl 1, GD2 ganglioside, G-28 (cell surface antigen glycolipid), GD3, Globyphon 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, guanylate cyclase C receptor, thermostable enterotoxin receptor (hSTAR), heat shock proteins, 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 E2/E7, Hsp90, hTERT, ICAM-1 (intercellular adhesion molecule 1), idiotype, IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-. gamma., 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 (α 4, α IIb β 3, α v β 3, α 4 β 7, α 5 β 1, α 6 β 4, α 7 β 7, α ll β 3, α 5 β 5, α v β 5), interferon-. gamma-inducible protein, ITGA2, ITGB2, KIR2D, LCK, Leman-Y antigen, LFA-1 (lymphocyte function-associated 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, MAGE 4, MART1, MCP-1, MIF (macrophage migration inhibitory factor, or Glycosyl Inhibitory Factor (GIF)), MS4A1 (transmembrane 4 domain subfamily A member 1), MSMSMesothelin, MUC1 (mucin 1, cell surface-associated (MUC1) or Polymorphic Epithelial Mucin (PEM)), MUC1-KLH, MUC16 CA125), 1 (monocyte protein 1), Melana/MART 3, IAP, MPG, MS4A1, CN, myelos associated antigen, Myostatin-84, Netas-84, phospholipid 7374, Nectin-22, ASNA-7374, My-11, Mylar-S-11, My-S, NGF, neuronal apoptosis-regulating protease 1, NOGO-A, Notch receptors, nucleolin, Neu oncogene products, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low density lipoprotein), OY-TES1, P21, A 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-R alphA (alphA platelet-derived growth factor receptor), PDGFR-betA, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived growth factor receptor betA, sodium phosphate cotransporter, PMEL 17, poly, protease 3(PR1), prostate cancer, PS (phosphatidylserine), prostate cancer cells, Aerugo, PSMA, PSA, PSCA, rabies glycoprotein, RHD (Rh polypeptide 1(RhPI), CD240), Rhesuus factor, RANKL, RhoC, Ras mutation, RGS5, ROBO4, respiratory syncytial virus, RON, sarcoma translocation breakpoint, SART3, Sclerostin, SLAMF7(SLAM member 7), Selectin P, SDC1 (syndecan 1), systemic lupus erythematosus (a), somatomedin C, SIP (sphingosine-1-phosphate), somatostatin, sperm protein 17, SSX2, STEAP1 (6-transmembrane epithelial prostate antigen 1), STEAP2, STn, TAG-72 (tumor-associated glycoprotein), survivin, T cell receptor, T cell protein, TEM1 (tumor vascular endothelial marker 1), TENB2, Tenascin C (TN-C), TGF-alpha, TGF-beta (transforming growth factor beta), TGF-beta 1, TGF-beta 2 (transforming growth factor beta 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-specifically 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 example, cell-binding agent-drug conjugates linked by the bridge linker of this patent are useful for cancer targeting therapy. Target cancers include, but are not limited to, adrenocortical carcinoma, anal carcinoma, bladder carcinoma, brain tumors (brain stem glioma, cerebellar astrocytoma, brain astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal and pineal tumors, visual pathway and hypothalamic glioma), breast carcinoma, carcinoid tumors, gastrointestinal cancer, unknown small cell carcinoma, cervical carcinoma, colon carcinoma, endometrial carcinoma, esophageal carcinoma, extrahepatic bile duct carcinoma, ewing family tumor (PNET), intracranial germ cell tumors, eye carcinoma, intraocular melanoma, gallbladder carcinoma, gastric carcinoma (stomach carcinoma), extragonadal germ cell tumors, peritrophoblastoma, head and neck carcinoma, hypopharynx carcinoma, islet cell carcinoma, renal carcinoma (renal cell carcinoma), leukemia (acute lymphocyte, acute myeloid, chronic lymphocyte, chronic granulocyte, hair cell), colon carcinoma, bladder carcinoma, and other cell, Lip and oral cancers, liver cancer, lung cancer (non-small cell, small cell), lymphoma (aids-related, central nervous system, cutaneous T-cell, hodgkin's disease, non-hodgkin's disease), malignant mesothelioma, melanoma, merkel cell carcinoma, metastatic squamous neck cancer and occult primary cancer, multiple myeloma and other plasma cell tumors, mycosis fungoides, myelodysplastic syndrome, myelodysplastic disorders, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer (epithelial, germ cell tumor, low malignancy), pancreatic cancer (exocrine, islet cell carcinoma), paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, plasma cell tumor, prostate rhabdomyosarcoma, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter (transitional cell), colon cancer, Salivary gland cancer, seiili syndrome, skin cancer (cutaneous T cell lymphoma, kaposi's sarcoma, melanoma), small intestine tumor, soft tissue sarcoma, gastric cancer, testicular cancer, thymoma (malignant), thyroid cancer, urinary tract cancer, uterine cancer, unusual juvenile cancer, vaginal tumor, vulval tumor, and wilms tumor.
In another specific embodiment, cell-binding agent-drug conjugates linked by the bridge linkers of this patent are useful as compositions and methods for treating or preventing autoimmune diseases. Autoimmune diseases include, but are not limited to, Achlorhydradra autoimmune active chronic hepatitis, acute disseminated encephalomyelitis, acute hemorrhagic leukocytitis, Addison's disease, azoospermia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-GBM/TBM nephritis, antiphospholipid syndrome, anti-dysenzymic syndrome, arthritis, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune multiple endocrine syndrome types I, II and III, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune uveitis, Balo disease/Balo homosclerosis, bechets syndrome, Berger's disease, Bickerstaff encephalitis, Blau syndrome, bullous pemphigoid, Castleman's disease, Chagas disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal osteomyelitis, chronic Lyme disease, chronic obstructive pulmonary disease, Churg-Strauss syndrome, cicatricial pemphigoid, coeliac disease, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, Creutzfeldt-Jakob disease, ST syndrome, Crohn's disease (idiopathic inflammatory bowel disease), Cushing's syndrome, cutaneous leukocytosis vasculitis, Degoid's disease, Dercuum's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes mellitus, diffuse cutaneous systemic sclerosis, Dressler syndrome, discoid lupus erythematosus, eczema, endometriosis, anchorage-dependent arthritis, Eosinopophilus fasciitis, epidermolysis pilosus, nodular erythema, idiopathic mixed cryoglobulinemia, Erwinian syndrome, fibrodysplastic ossification, fibromyalgia, fibrotic myositis, fibrotic alveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch's purpura, hepatitis of pregnancy, hidradenitis suppurativa, Huss syndrome (antiphospholipid syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (autoimmune thrombocytopenic purpura), IgA nephropathy (Bergey's disease), inclusion body myositis, inflammatory demyelinating polyneuritis, interstitial cystitis, irritable bowel syndrome, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, Kawasaki disease, Lambertian-Eton myasthenia gravis syndrome, leukocyte clastic vasculitis, lichen planus, sclerosclerosis, Linear IgA disease (LAD), Lou Gehrig's disease (also known as amyotrophic lateral sclerosis), lupus hepatitis, lupus erythematosus, Majeed's syndrome, Meniere's disease, microscopic polyarteritis, Miller-Fisher syndrome, mixed connective tissue disease, morphosis, Mohammerd-Huberman disease, Mkocurie syndrome, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, lethargy, neuromyelitis optica (Devic's disease), neuromuscular sclerosis, eyelid cicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord thyroiditis, Hui rheumatism, PANDAS (a pediatric autoimmune neuropsychiatric disease associated with streptococci), paraneal cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry Romberg syndrome, Parsonnage-Turner syndrome, parsonage planitis, 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 encephalitis, Raynaud's phenomenon, recurrent polychondritis, Reiter's syndrome, restless leg syndrome, posterior neurofibrosis, rheumatoid arthritis, rheumatoid fever, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler's syndrome, Schnierler syndrome, Schniemann-strander syndrome, scleritis, scleroderma, Sjogren's syndrome, spondyloarthropathy, mucoviscidosis, Still disease, stiff person syndrome, subacute endocarditis disease, suzak syndrome, Sweet 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 granulomatosis, wilson's syndrome, wiskott-aldrich syndrome.
In another specific embodiment, examples of binding molecules linked to drug molecules through the bridging linker of the present invention on conjugates for the treatment or prevention of autoimmune diseases include, but are not limited to, anti-elastin antibodies, Abys anti-epithelial cell antibodies, anti-basement membrane type IV collagen antibodies, antinuclear antibodies, anti-ds DNA, anti-ss DNA, anti-cardiolipin antibodies IgM, IgG, anti-celiac disease antibodies, anti-phospholipid antibodies IgK, IgG, anti-SM antibodies, anti-mitochondrial antibodies, thyroid antibodies, microsomal antibodies, T cell antibodies, thyroglobulin antibodies, anti-SCL-70, anti-Jo, anti-u.sub.1rnp, anti-La/SSB, anti-SSA, anti-SSB, anti-parietal cell antibodies, anti-histone, anti-RNP, C-ANCA, P-ANCA, anti-centromere, anti-fibrinogen, anti-GBM antibodies, anti-ganglioside antibodies, anti-desmogen 3 antibodies, anti-p 62 antibody, anti-sp 100 antibody, anti-mitochondrial (M2) antibody, rheumatoid factor antibody, anti-MCV antibody, anti-topoisomerase antibody, anti-neutrophil cytoplasmic (cANCA) antibody.
In certain preferred embodiments, the binding molecules on the conjugates of the present invention bind to a receptor or receptor complex expressed on activated lymphocytes associated with autoimmune diseases. The receptor or receptor complex comprises, a member of the immunoglobulin 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), a member of the TNF receptor superfamily (e.g., 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), an integrin, a cytokine receptor, a major histocompatibility factor receptor, a lectin (C, S-I) or a complement control protein.
In another embodiment, useful cell binding ligands 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 (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV Tax, herpes simplex virus glycoproteins (e.g., gB, gC, gD and gE) and hepatitis B surface antigen) capable of eliciting an immune response. "microbial antigens" include, but are not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide or lipid molecule capable of eliciting an immune response (e.g., bacterial, fungal, pathogenic protozoan or yeast polypeptides, including, e.g., LPS and capsular polysaccharides). Examples of antibodies that may be used to treat viral or microbial infections include, but are not limited to: palivizumab, which is a humanized anti-respiratory syncytial virus monoclonal antibody for the treatment of RSV infection; PRO542, a CD4 fusion antibody, used to treat HIV infection; ostevir, a human antibody used in the treatment of hepatitis B virus; PROTVIR, a humanized IgG1 antibody for the treatment of cytomegalovirus, and anti-LPS antibodies.
The cell binding molecule-drug conjugates made by the bridge linkers of this patent are useful for treating infectious diseases. These infectious diseases include, but are not limited to, acinetobacter infection, actinomycosis, african narcolepsy (african trypanosomiasis), aids (acquired immunodeficiency syndrome), amebiasis, anaplasmosis, anthrax, yersinia haemolytica infection, argentina hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, bacillus cereus infection, bacterial pneumonia, bacterial vaginitis, bacteroidal infection, venosasis, ascaris infection, BK viral infection, black birth control disease, human blastocyst protozoa infection, blastomycosis, viia hemorrhagic fever, borrelia infection, botulism (and infantile botulism), brazilian hemorrhagic fever, brucellosis, burkholderia infection, bruxiella ulcer, calicivirus infection (norovirus and saporovirus), campylobacteriosis, candidiasis (candidiasis, thrush), cat scratch disease, cellulitis, Chagas disease (trypanosomiasis americana), ascomycetes, chicken pox, chlamydia pneumoniae infection, cholera, glioblastoma, clonorchiasis 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 larva migratory, cyclosporinosis, enterobacter infection, enterovirus infection, epidemic typhus, erythema infectioum (fifth disease), acute eruption, fascioliasis, fatal familial insomnia, filariasis, clostridium capsulatum food poisoning, free living amoeba infection, clostridium infection, aeronevus necrotica (fusobacterial myonecrosis), filariasis, germann-straussler-scherrella-scherreri syndrome, giardiasis, melioidosis, gonorrhea, granulomatous diarrhea (fifth disease), 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, renal syndrome hemorrhagic fever, 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 papilloma virus infection, human parainfluenza virus infection, membranous taenia disease, epstein-barr virus infectious mononucleosis (mononucleosis), influenza, isospora, kawasaki disease, keratitis, gigerbilosis, kuru, lassa fever, Legionella disease (Backward legionnaires 'disease), Legionella disease (Pontiake fever), Leishmaniasis, Lyme disease, lymphofilariasis (elephantiasis), lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever, measles, melioidomycosis (Whitman's disease), meningitis, meningococcosis, posterior genital trematosis, microsporosis, molluscum contagiosum, parotitis, mouse typhus (endemic typhus), mycoplasmal pneumonia, foot edema, myiasis, neonatal conjunctivitis (neonatal eye disease), variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardia disease, onchocerciasis (Heanopheles), paracoccidioidomycosis (southern Eimeria), paragonimiasis, paragonitis, Pasteuresis, head lice, body lice, pubic louse, pelvic inflammatory disease, pertussis, pneumonic infection, pneumococcal disease, poliomyelitis, previtamin infection, primary amebic meningoencephalitis, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, rat bite heat, respiratory syncytial virus infection, nosemosis, rhinovirus infection, rickettsia pox, rift valley heat, rocky mountain spotted fever, rotavirus infection, rubella, salmonellosis, SARS (severe acute respiratory syndrome), scabies, schistosomiasis, septicemia, shigellasis (Bacillary dysentery), herpes zoster (shingles), smallpox (smallpox), sporothrix, staphylococcal food poisoning, staphylococcus aureus infection, strongylosis, syphilis, taeniasis, tetanus, tinea barbarum (Barber itch), scalp tinea, tinea corporis, tinea cruris, tinea manuum, harbourne, tinea pedis (tinea pedis), onychomycosis (onycis), tinea versicolor, ascariasis (eye larva migration), toxocariasis (visceral larval transmigration), toxoplasmosis, trichinosis, trichomoniasis, trichiasis (whipworm infection), tuberculosis, tularemia, ureaplasma urealyticum infection, venezuelan equine encephalitis, venezuelan hemorrhagic fever, viral pneumonia, west nile fever, leukosarcoidosis (tinea alba), yersinia pseudotuberculosis, yersinia pestis enteropathy, yellow fever, zygomycosis.
The cell binding agents of the invention, more preferably antibodies, are directed against pathogenic strains including, but not limited to, Acinetobacter baumannii, Actinomyces israelii, Actinomyces and Propionibacterium, Trypanosoma brucei, HIV (human immunodeficiency virus), entamoeba histolytica, Anaplasmacytes, Bacillus anthracis, Vibrio haemolyticus, Hunnins, ascaris, Aspergillus, Astroviridae, Babesia, Bacillus cereus, various bacteria, Bacteroides, Escherichia coli, ascaris, BK virus, Oesophaga, Protozoa hominis, Blastomyces dermatitidis, Marulovirus, Borrelia, Clostridium botulinum, Sinomenii, Brucella, typically Burkholderia cepacis and other Burkholderia species, Mycobacteria ulceraceae, Calicidae, Campylobacter, typically Candida albicans and other Candida species, Burmannia, group A streptococci and staphylococci, Trypanosoma cruzi, Haemophilus ducreyi, VZV, Chlamydia trachomatis, Colorado tick fever virus, rhinovirus, coronavirus, CJD prion, Climiya-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium, hookeria brasiliensis, various parasites, Cyclosporidium, Taenia ribbon, Cytomegalovirus, dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4) -flavivirus, Bifidobacterium fragilis, Corynebacterium diphtheriae, cestode, Melilonella, Ebola, Echinococcus, Enterococcus, Enterovirus, Rickettsia prosii, Brucella parvovirus B19, human herpesvirus 6 and human herpesvirus 7, fasciola gingivalis, Pediobolus hepatica and Pectinopsis megafasciola, FFI virus, Hyperperfringens, Clostridium prions, clostridium, other clostridia, geotrichum candidum, GSS prion, giardia enterica, burkholderia, bacillus spinosus and candida, gonococcus, klebsiella granulomatosa, streptococcus pyogenes, streptococcus agalactiae, haemophilus influenzae, enteroviruses, mainly coxsackie a and enterovirus 71, innominate virus, helicobacter pylori, escherichia coli O157: h7, Bunyaviridae, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, herpes simplex virus 1, herpes simplex virus 2, histoplasma capsulatum, duodenal adenoma and Chlamydomonas ampullatus, human bocavirus, ehrlichia, phagocytophile haemophilus, human metapneumovirus, Ehrlichia chalcone, human papilloma virus, human parainfluenza virus, Taenia miniata and Thymenia amesii, Epidera virus, Orthomyxoviridae family, Isospora beijerinckii, Chryseobacterium, Klebsiella pneumoniae, Legionella pneumophila, Leishmania, Mycobacterium leprae and Mycobacterium tuberculosis, Leptospira, monocytogenes, Listeria borrelia, Borrelia borrelia and other species of the genera, spanish and Malathia, lymphocytic choriomeningitis virus (LCMV), Plasmodium, Marburg, measles, Burkholderia pseudomallei, Neisseria meningitidis, retrograduate schistosomiasis, Microsporozoales, Molluscum Contagiosum (MCV), mumps, Rickettsia typhi, Mycoplasma pneumoniae, multiple bacterial and fungal parasitic dipteran larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prions, Nocardia and other Nocardia genera, Spanish, Paeoniaceae, Simania para and other subgenericosida genera, Pasteurella, head lice, human pediculus humanus, Bordetella pertussis Yersinia pestis, Streptococcus pneumoniae, pneumococci, poliovirus, Prevotella, Neisseria, JC virus, Chlamydia psittaci, Coxiella pneumoniae, rabies virus, S.unicus and Spirosoma, respiratory syncytial virus, nosema, rhinovirus, Rickettsia, Leptosphaeria, Rickettsia, rotavirus, rubella, Salmonella, SARS coronavirus, human scabies, Haemophilus, somatic cell, Shigella, varicella zoster virus, Notopterygium smallpox or smallpox, Trichosporon aureus, Staphylococcus aureus, Streptococcus pyogenes, Torulopsis, Treponema pallidum, tapeworm, tetanus, ringworm, uranium, tinea genus, Epidermophyton floccosum, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton rubrum, Venezon venenum , Trichophyton genus, Toxophyte or Toxolodon, Toxoplasma gondii, Trichophyton vaginalis, Mycobacterium tuberculosis, France Langerella, urea and equine encephalitis viruses, venezuelan equine encephalitis viruses, vibrio cholerae, melon naipoto viruses, west nile virus, beigelii filariosis, yersinia pseudotuberculosis, yersinia enterocolitica, yellow fever viruses, mucorales order (mucormycosis) and entomomycetales order (entomomycetous mycosis), mucorales order pseudomonas aeruginosa, campylobacter (vibrio), aeromonas, ehrlichia, yersinia, shigella, salmonella typhi, sargassum, treponema pernici, borrelia burgdorferi, spirochete, pneumocystis carinii, brucella abortus, brucella, mycoplasma, rickettsia pusturti, rickettsia, chlamydia, pathogenic fungi (aspergillus fumigatus, candida albicans, histoplasma capsulatus), protozoa (amoeba immaturus, tenas Trichomonas, Hominis Trichomonas, Trypanosoma gambiense, Trypanosoma rhodesiense, Leishmania rosenbergii, Leishmania tropicalis, Leishmania brasiliensis, Pneumocystis pneumoniae, Plasmodium vivax, Plasmodium falciparum, or Helminiththths (Schistosoma japonicum, Schistosoma mansoni, Schistosoma Egypti and hookworm).
Other antibodies useful as cell-binding agents in the present patent for the treatment of viral diseases include, but are not limited to, antibodies to the following pathogenic viral antigens: poxviruses; herpes virus; an adenovirus; a small yellow virus; enteroviruses; picornavirus; parvovirus; reovirus; a retrovirus; an influenza virus; a parainfluenza virus; parotitis; measles; respiratory syncytial virus; rubella; arbovirus virus; a rhabdovirus; salmonella; non-a/non-b hepatitis virus; a rhinovirus; a coronavirus; a rotordo virus; oncogenic viruses, such as HBV (hepatocellular carcinoma), human papilloma virus (cervical cancer, anal carcinoma), kaposi's sarcoma-associated herpes virus (kaposi's sarcoma), human herpes virus type four (nasopharyngeal carcinoma, burkitt's lymphoma, primary central nervous system lymphoma), virus (merkel cell carcinoma), SV40 (simian virus 40), HCV (hepatocellular carcinoma), HTLV-1 (adult T-cell leukemia/lymphoma); immune disorders result in viruses such as human immunodeficiency virus (aids); central nervous system viruses, such as JCV (progressive multifocal leukoencephalopathy), hepatitis c virus (subacute sclerosing panencephalitis), LCV (lymphocytic choriomeningitis), subacroviral encephalitis, orthomyxovirus (encephalitis), RV (rabies), probovirus, herpesvirus meningitis, ramusch hunter syndrome type II, poliovirus (poliovirus, post-polio syndrome), HTLV-1 (tropical palsy)); cytomegalovirus (cytomegalovirus retinitis, HSV (herpetic keratitis), cardiovascular viruses, such as CBV (pericarditis, myocarditis), respiratory/acute viral intranasal inflammation/viral pneumonia, such as Epstein-Barr virus (EBV infection/infectious mononucleosis), cytomegalovirus, Severe coronavirus (severe acute respiratory syndrome) or orthomyxovirus, influenza a/b/c (influenza/avian influenza), paramyxovirus, human parainfluenza virus, RSV (human respiratory syncytial virus), hMPV, digestive system viruses (mumps virus, cytomegalovirus (cytomegalovirus 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 viruses, such as BK virus, MuV (mumps).
Still further, the invention also includes a conjugate coupled to a bridge and an acceptable carrier, diluent or excipient for treating cancer, infection or autoimmune disease. Methods of treating cancer, infections and autoimmune diseases can be performed in vitro, in vivo or ex vivo. Examples of in vitro uses include treating a cell culture with it to kill all cells except for variants that do not express the target antigen; or to kill variants that express the undesired antigen. Examples of ex vivo use include treatment of Hematopoietic Stem Cells (HSCs) to kill diseased or malignant cells prior to transplantation (HSCT). For example, tumor cells or lymphocytes are removed from bone marrow prior to autologous transplantation in the treatment of cancer or in the treatment of autoimmune diseases, or T cells and other lymphocytes are removed from allogeneic bone marrow or tissue prior to transplantation in order to prevent graft versus host disease. Such clinical ex vivo treatment may be carried out as follows: bone marrow is harvested from a patient or other individual and then incubated in serum-containing medium at about 37 ℃ for about 30 minutes to about 48 hours, to which medium the conjugate of the invention is added at a concentration ranging from about 1pM to 0.1 mM. The specific drug concentration and incubation time should be determined by a skilled clinician. After incubation, the bone marrow cells are washed with serum-containing medium and administered to the patient intravenously according to known methods. In the case of patients who have received additional treatment (e.g., ablative chemotherapy or whole body radiation therapy) between bone marrow harvest and reinfusion of the treated cells, the treated bone marrow cells should be cryopreserved in liquid nitrogen using standard medical equipment.
In clinical in vivo use, canThe conjugates linked to the connectors of this patent are supplied as solutions or lyophilized solids that can be re-dissolved in sterile water for injection. Examples of conjugate administration regimens are as follows: the conjugate was administered intravenously once a week for 8 to 20 weeks. A bolus dose is given in 50-500mL of physiological saline to which human serum albumin (e.g., 0.5 to 1mL of a concentrated solution of human serum albumin, 100mg/mL) can be added. The intravenous dose will be about 50 μ g to 20mg/kg (body weight) per day, or weekly, bi-weekly, tri-weekly or monthly (10 μ g to 200mg/kg dose per injection). After treatment
Figure BDA0003226420300000371
Weekly, the patient may receive a second course of treatment. The specific clinical regimen for the route of administration, excipients, diluents, dosage, number of times, etc., can be determined by a skilled clinician.
Examples of medical conditions that can be treated with in vivo or ex vivo methods 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 depending upon a variety of factors including the chemical identity, potency and bioavailability of the conjugate, the type of disease, the patient's ethnicity, the patient's disease progression, the route of administration, all factors determining the required dose, mode of administration and dosage regimen.
In general, the conjugates of the invention can be formulated for injection in an aqueous physiological buffer solution containing 0.1 to 10% w/v conjugate. Typical doses range from 1. mu.g/kg to 1g/kg (body weight) 1 time per day. Preferred dosage ranges are from 0.01mg/kg to 20mg/kg body weight per day or weekly, or infant equivalent. The preferred dosage of the drug to be administered may depend on such factors as the type and extent of progression of the disease or condition, the overall health status 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-designated delivery route, as well as the rate of administration (bolus or continuous infusion) and the dosage regimen (number of repetitions in a given time period).
The conjugates of the invention can also be administered in unit dosage form, where the term "unit dose" refers to a single dose that can be administered to a patient and can be readily handled and packaged while the active conjugate itself, or a pharmaceutically acceptable composition as described below, remains a physically and chemically stable unit dose. Typical total daily doses range from 0.01 to 100mg/kg body weight. As a general guideline, the unit dosage for humans ranges from 1mg to 3000mg daily or weekly, or 2 weeks, 3 weeks or monthly. The unit dosage range is preferably 1 to 500mg, once to four times a week, more preferably 10mg to 500mg, once a week. The conjugates provided herein can be formulated into pharmaceutical compositions by mixing with one or more pharmaceutically acceptable excipients. Such unit dose compositions may be administered orally, e.g., as a medicament in the form of a tablet, simple capsule or soft gel capsule; or intranasally, such as powders, nasal drops or aerosols; or dermally, such as with a topical ointment, cream, lotion, gel or spray or via a transdermal patch.
Drug/cytotoxic agent
Drugs that can be conjugated to the cell binding molecules of the present invention are small molecule drugs that include cytotoxic agents, and can be attached, or modified, to the cell binding agent. The "small molecule drug" in the present invention broadly refers to an organic, inorganic or organometallic compound having a molecular weight of 100 to 1800, more preferably 120 to 1400. Such small molecule drugs are well described in the literature, such as WO05058367A2 and U.S. Pat. No. 3,4,956,303, etc., which are incorporated herein by reference. Small molecule drugs include known drugs and drugs to be disclosed.
Known drugs include, but are not limited to:
1) chemotherapeutic agents: a) alkylating agents, such as nitrogen mustards: chlorpheniramine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine hydrochloride, dimethoxyamine hydrochloride, mechlorethamine oxide, amlodipine hydrochloride, mycophenolic acid, dulcitol, pipobroman, neomechlorethamine, benzeneMustard cholesterol, prednimine, thiotepa, triamcinolone pair, uracil; CC-1065 (including its aldorexin, kazelaixin and bizelaixin synthetic analogs); duocarmycins (including the synthetic analogs KW-2189 and CBI-TMI); benzodiazepine dimers (e.g., dimers of Pyrrolobenzodiazepine (PBD) or tolmetin, indolophenyldiazepine, imidazobenzothiadiazole, or oxazolidobenzodiazepine); nitrosoureas (carmustine, lomustine, fustin chloride, fotemustine, nimustine, lamustine); alkyl sulfonates (busulfan, endosulfan and sulfur); triazenes (dacarbazine); platinum-containing compounds (carboplatin, cisplatin, oxaliplatin); aziridines, such as chromanone, carotenone, metoclopramide and lindopa; ethyleneimine and methyl melamine, including hexamethylmelamine, triethylenetriamine, triethylphosphoramide, triethylenethiophosphoramide and trimethylolmethylamine; b) plant alkaloid: such as vinca alkaloids (vincristine, vinblastine, vindesine, vinorelbine, catharanthine); taxoids (paclitaxel, docetaxel) and their analogs; maytansine (DM1, DM2, DM3, DM4, maytansine and ansamycin) and analogues thereof; cryptophycin (especially cryptophycin 1and cryptophycin 8); epothilone, juncecrogol, discodermolide, bryozoalactone, dolastatin, auristatin, tubulysin, cephalostatin, pancratistatin, sarcodictyin, spongistatin; c) DNA topoisomerase inhibitors, such as etoposide tinib (9-aminocamptothecin, camptothecin, clinatot, daunomycin, etoposide phosphate, irinotecan, mitoxantrone, nosaline, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptothecin (RFS 2000)); mitomycin (mitomycin C); d) antimetabolites, such as antifolates, DHFR inhibitors (methotrexate, trametet, dimethylfolic acid, pteropterin, aminopterin (4-aminobenzoic acid) or other folic acid analogs); IMP dehydrogenase inhibitors (mycophenolic acid, thiazolofuran, ribavirin, EICAR); ribonucleotide reductase inhibitors (hydroxyurea, deferoxamine); pyrimidine analogs, uracil analogs (ancitabine, azacitidine, 6-azauracil, capecitabine (Hirocine)Dara), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-fluorouracil, floxuridine, ratitrexed (tomudex); cytosine analogs (cytarabine, cytosine arabinoside, fludarabine); purine analogs (azathioprine, fludarabine, mercaptopurine, thiamine, thioguanine); folic acid supplements such as florolinic acid; e) hormonal therapy agents such as receptor antagonists, antiestrogens (megestrol, raloxifene, tamoxifen), LHRH agonists (gostadrine, leuprolide acetate); anti-androgens (bicalutamide, flutamide, carrousel, betaandrosterone propionate, epiandrosterone, goserelin, leuprorelin, metulidine, nilutamide, testolactone, trilostane and other androgen inhibitors); retinoids, vitamin D3 analogues (CB1093, EB1089KH1060, cholecalciferol, ergocalciferol); photodynamic therapy agents (verteporfin, phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin a); cytokines (interferon- α, interferon- γ, Tumor Necrosis Factor (TNF), TNF-containing human proteins); f) kinase inhibitors, such as BIBW 2992 (anti-EGFR/Erb 2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080 (anti-VEGFR 2), mubritinib, ponatinib (AP 34), bafetinib (INNO-406), bosutinib (sk24ni-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, felinib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, istussin; g) antibiotics, such as enediynes (calicheamicins, in particular calicheamicin gamma 1, delta 1, alpha 1and beta 1 (see J.Med.chem.1996,39 (11)), 2103-Morpholino doxorubicin, 2-pyrroline doxorubicin and deoxydaunorubicin, epirubicin, doxorubicin, idarubicin, marcfortine, mycin, mycophenolic acid, lopithromycin, pellomycin, puromycin, triiron doxorubicin, streptozotocin, tubercidin, ubenimex, setastin, zorubicin; h) others, such as polyketides (annonaceous acetogenins), in particular bullatacin and bullatacinone; gemcitabine, epoxygenases (e.g., capeline), bortezomib, thalidomide, lenalidomide, pomidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allovivin-7, Xegeva, Provenge, Yervoy, prenylation inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., staurosporins), actinomycins (e.g., actinomycin D, dactinomycin), bleomycin (e.g., bleomycin A2, bleomycin B2, pelomycin), anthracyclines (e.g., daunorubicin), doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, mitorubicin, MDR inhibitors (e.g., verapamil), Ca2+Inhibitors of ATPase (e.g., thapsigargin), inhibitors of histone deacetylase (vorinostat, romidepsin, panobinostat, valproic acid, Mocetinostat (MGCD0103), Belinostat, PCI-24781, entinostat, SB939, remininostat, Givinostat, AR-42, CUDC-101, sulforaphane, trichostatin A); celecoxib, glitazones, epigallocatechin gallate, disulfiram, Salinosporamide a; anti-adrenal agents, such as aminoglutethimide, mitotane, trilostane, acetoglucuronolactone, aldphosphoramide, aminolevulinic acid, amsacrine, arabinoside, bestraucil, bison, edatraxate, defofamine, meclocine, disazoquinone, eflornithine (DFMO), efamitine, etiloamine, ethylgluconic acid, gallium nitrate, cytosine, hydroxyurea, ibandronate, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidanol, diaminenitracridine, pentostatin, mechlorethamine, pirarubicin, podophyllic acid, 2-ethylhydrazine, procarbazine;
Figure BDA0003226420300000411
piperazinedione propane; rhizomycin; (iv) Wenzuo; spiro germanium; geobacillus azavor; a tri-imine quinone; trichlorotriethylamine; trichothecenes (in particular T-2 toxin, verrucomicin A, bacillocin A and anguidine), polyurethanes, siRNAs, antisense drugs and nucleolytic enzymes.
2) Autoimmune disease agents, including but not limited to cyclosporine, cyclosporin a, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g., amcinonide, dexamethasone, triamcinolone acetonide, beclomethasone propionate, DHEA, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mycophenolate mofetil, prednisone, sirolimus, tacrolimus.
3) Anti-infectious disease agents, including but not limited to a) aminoglycosides: amikacin, astemicin, gentamicin (netilmicin, sisomicin, isepamicin), hygromycin B, kanamycin (amikacin, arbekacin, aminoxykanamycin, dibekacin, tobramycin), neomycin (framycetin, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, methylgestomycin; b) amide alcohols: azidochloramphenicol, chloramphenicol, florfenicol, thiamphenicol; c) ansamycin: geldanamycin, herbimycin; d) carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem; e) cephem: cephem (loracarbef), cephalosporacetonitrile, ampicillin, cephradine, cefadroxil, cephalonine, ceftazidime, cephalothin or cephalotaxin, cephalexin, cephramycin, cefamandole, cefapirin, azapiromidin, fluxazole cephalosporin, sporocetone, azolin cephalosporin, cefbuperazone, cefcapene, cefixime, cefpodoxime, cefixime, cefoxitin, cefprozil, cefetaxeme, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefpira, cefetamet, cefepime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, thienam, cefotaxime, cefozopran, cefazolin, cephalexin, cefepime, cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram, ceftibuten, cefotiarin, ceftizoxime, cefprozil, ceftriaxone, cefuroxime, ceftizoxime, cephamycins (cefoxitin, cefotetan, cefcyanazole), oxacephems (flomoxef, cephalosporins); f) glycopeptide: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin, g) glycylcyclin: such as tigecycline, h) beta-lactamase inhibitors: penicillane (sulbactam, tazobactam), oxapenem (clavulanic acid); i) lincosamide: clindamycin, lincomycin; j) lipopeptides: daptomycin, a54145, Calcium Dependent Antibiotic (CDA); k) macrolides: azithromycin, clarithromycin, dirithromycin, erythromycin, fluramycin, josamycin, ketolide (telithromycin, sequoyimycin), midecamycin, mickamycin, oleandomycin, rifamycin (isoniazid, rifampin, rifabutin, rifapentine), ropiniromycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), oleandomycin acetate, telithromycin; l) monocyclic amines: aztreonam, tigemonam; m) oxazolidinones: linezolid; n) penicillins: amoxicillin, ampicillin (pivampicillin, silocillin, bacampicillin, ampicillin, doxorubicin), azlocillin, benzylpenicillin, benzathine phenoxymethylpenicillin, cloxacillin, procaine penicillin (metilin), mezlocillin, methicillin, nafcillin, oxacillin, acemethicillin, penicillin, nafcillin, piperacillin, ampicillin, sulfonicillin, temocillin, ticarcillin; o) a polypeptide: bacitracin, colistin, polymyxin B, p) quinolones: alatrefloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, gatifloxacin, gemifloxacin, grepafloxacin, carnotrexacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q) streptogramins: pristinamycin, quinupristin/dalfopristin, r) sulfonamides: sulfonamides: sulfonamides, sulfadiazine, sulfasalazine, sulfisoxazole, tamoxifen, trimethoprim-sulfamethoxazole (sulfamethoxazole); s) steroid antibacterial drugs: such as fusidic acid; t) tetracyclines: doxycycline, chlortetracycline, cimeticycline, demeclocycline, ramoxiline, mecycline, methacycline, minocycline, oxytetracycline, penicillin V kalipecycline, pyrrolidinemethyltetracycline, tetracycline, glycylcycline (such as tigecycline): u) other types of antibiotics: annonaceous acetogenins, arsine, bacteroidal terpineol inhibitors (bacilli), DANAL/AR inhibitors (cycloserine), dictyostatin, discodermolide, saxifragol, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalitide, metronidazole, mupirocin, NAM synthesis inhibitors (e.g. fosfomycin), nitrofurantoin, paclitaxel, pratensomycin, pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampin), tazobactam tinidazole, echinacon.
4) Antiviral drugs: a) entry/fusion inhibitors: apaviralo, maraviroc, vicrivroc, gp41 (enfuvirtide), PRO140, CD4 (abalizumab); b) integrase inhibitors: raltegravir, elvitegravir, globoid dnan a; c) maturation inhibitors: bevirimat, vivocon; d) neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; E) nucleosides and nucleotides: abacavir, adefovir, armocivir, abciximab, brivudine, cidofovir, cladribine, dexamethasone, didanosine (ddI), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluvalillin (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, idoxuridine, lamivudine (3TC), 1-nucleosides (such as β -1-thymidine and β -1-2' -deoxycytidine), penciclovir, racivir, ribavirin, dilastatin, vudine (d4T), talibavirine (viramidine), telbivudine, tenofovir, trifluridine valacyclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f) non-nucleoside: amantadine, atitidine, carboprvirine, diarylpyrimidine (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphoryl formic acid), imiquimod, pegylated interferon, lovirine, lodenosine, methidathiozone, nevirapine, NOV-205, long-acting interferon alpha, podophyllotoxin, rifampin, rimantadine, resiquimod (R-848), acetimidamantadine; g) protease inhibitors: amprenavir atazanavir, boceprevir, daronavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h) other types of antiviral drugs: antioxidase, arbidol, kalarotide, ceragenin, cyanovirin-n, diarylpyrimidine, epigallocatechin gallate (EGCG), foscarnet, griffine, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, anabolic inhibitor, ribavirin, seliciclib.
5) Drugs linked through the bridges of the present invention also include radioisotopes. Examples of radioactive isotopes (radionuclides) are3H，11C，14C，18F，32P，35S，64Cu，68Ga，86Y，99Tc，111In，123I，124I，125I，131I，133Xe，177Lu，211At or213And (4) Bi. The radioisotope labeled antibodies may be used in receptor targeted imaging experiments, or may be used in targeted therapy as antibody-drug conjugates of the invention (Wu et al Nature Biotechnology 2005,23(9): 1137-1146). Cell binding molecules, such as antibodies, may be labeled by linking the ligand reagent to a linker of the present patent. The ligands can be used in the literature (Current Protocols in Immunology, Vo)Lumes 1and 2, Coligen et al, Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991)) bind to, chelate or form a complex with radioactive metals. Chelating ligands that can complex metal ions include DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, TX), among others.
6) A pharmaceutically acceptable salt, acid or derivative of any of the above.
In another example, the drugs of structural formulae (II) and (IV) may be chromogenic molecules, and the conjugates may be used to detect, monitor or study the interaction of cell-binding molecules with target cells. The chromonic molecule can absorb a light, such as ultraviolet, fluorescent, infrared, near infrared, or visible light; the chromophoric molecules include yellow pigment, red blood cell, iridescent pigment, white blood cell, melanin and blue-green pigment, fluorescent molecule (fluorescent chemical substance capable of absorbing light and emitting light), visual light transduction molecule, photon molecule, luminescent molecule and fluorescein compound.
The chromonic molecule can be selected from, but is not limited to, non-protein organic fluorophores such as xanthene derivatives (fluorescein, rhodamine, Oregon Green, eosin, and Texas Red); cyanine derivatives (cyanines, indocarbocyanines, oxacyanines, thiacyanines, and merocyanines); squaric acid derivatives and ring-substituted squaric acids, including Seta, SeTau and Square dyes; naphthalene derivatives (dansyl and sodium fluorosilicate derivatives); coumarin derivatives; oxadiazole derivatives (pyridyloxazole, nitrobenzoxazole and benzooxadiazole); 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 (flavonol flavin, acridine orange, acridine yellow, etc.); arylmethylamine derivatives (auramine, crystal violet, malachite green) and tetrapyrrole derivatives (porphine, phthalocyanine, bilirubin).
The chromogenic molecule is selected from any analogues and derivatives of the following fluorescent compounds: CF dyes (Biotium), DRAQ and CyTRAK probes (BioS-tatus), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLight Fluor (Thermo Scientific, Pierce), Atto and Tracy (Sigma Aldrich), FluProbes (Interchim), Abberior dyes (Abberior), DY and MegaStokes dyes (Dyomics), Sulfo Cy dyes (Cyandy), HiLyte Fluor (Anaspec), Seta, Setau and Square dyes (Biosearch Technologies), SureLight dyes (APC, RPEPercP, Phyobilisomes) (Columbia Biosciences), APCXL, RPE, BPE (Phoco-Biotech).
Examples of widely used fluorescent compounds that can be reacted or coupled to the linkers of the present invention are: allophycocyanin (APC), annatto, APC-Cy7 conjugate, BODIPY-FL, Cascade Blue, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, fluorescein, FluorX, hydroxycoumarin, lissamine rhodamine B, lucifer 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, Seu-380-NHS, Seta-405-maleimide, Seta-NHS 405-NHS 425-Seu-NHS, Seta-647-S, texas Red, TRITC, TruRed, X-Rhodamine.
Fluorescent compounds which can be linked to the linker of the invention for the study of nucleic acids or proteins are 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, mithramycin, Propidium Iodide (PI), SYTOX blue, SYTOX green, SYTOX orange, thiazole orange, TO-PRO, cyanine dye monomers, TOTO-1, TO-PRO-1, TOTO-3, TO-PRO-3, YOSeta-1, YOYO-1. Fluorescent compounds which can be linked to the linker of the invention for the study of cells are selected from the following compounds or derivatives thereof: DCFH (2', 7' -dichlorodihydrofluorescein, oxidized form), DHR (dihydrorhodamine 123, oxidized form, photocatalytic oxidation), Fluo-3(AM ester, pH >6), Fluo-4(AM ester, pH7.2), 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), Cirsium 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 (S65 mutation), GFP (S65C mutation), GFP (S65L mutation) GFP (Y66H mutation), GFP (Y66W mutation), GFPuv, cRed1, HJ-Red, Katusha, Kusarara Orange (monomer, MBoimer, mCopofp, mColophor 63L, monomer, MBherl 635, Khryse 635, KmRed, Kyurn-5, TsRed monomer, Skumura-M2, Tahryser-5, Tamcorph, Tamcopic, Tahryse, Tamcopic, Tahryse, Tamciror-5, Tamcopic complex, Tamciro-5, Tamciro, Tahryse, Tamciro, Tachy-5, Tachy, Tachytrix-5, Tachy, Tachytrix-5, Tachy, Tachytrix-5, Tachytrix, Tachy, Tachytrix, Tachy, Tachytrix, Tachy, Tachytrix, and Tachytri, T-Sapphire, TagCFP (dimer, Evrogen), TagGFP (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 (tetramer, Clontech), zsyollow 1 (tetramer, Clontech).
In another embodiment, preferred cytotoxic agents that are linked to the cell binding molecule via the linkers of this patent are tubulysin, maytansine, taxanes, CC-1065 analogs, daunorubicin and doxorubicin compounds, benzodiazepine dimers (e.g., Pyrrolobenzodiazepine (PBD) or tomomycin, indolophenyldiazepine, imidazobenzothiadiazole or dimers of oxazolidobenzodiazepine), calicheamicin and enediyne antibiotics, actinomycin, azaserine, bleomycin, epirubicin, tamoxifen, idarubicin, dolastatin, auristatin (e.g., MMAE, MMAF, auristatin E, auristatin TP, auristatin 2-AQ, 6-AQ, EB (AEB) and EFP (AEFP)), dactinomycin, thiotepa, vincristine, hemistaline, nazumab, genistinin, allopurin, microsclerominin, theonellamide, esperamicin, PNU-159682 and analogs and derivatives thereof.
Tubulysin is a preferred cytotoxic agent for conjugate coupling and may be prepared by methods known in the art, isolated or synthesized from natural sources, 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.; pharmaceutical, 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), pp 5567-5569. 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.; pharmaceutical, 2012,9, 168-175 Friestad, g.k., al.mol.pharmaceuticals, g.k.; et al org.lett.,2009,11, 1095-1098. Kubicek, k.; et al, Angew Chem Int Ed Engl,2010.49,4809-12.Chai, Y.; et al, Chem Biol,2010,17:296-309 Ullrich, A.; et al, Angew Chem Int Ed Engl,2009,48,4422-5.Sani, M.; et al, Angew Chem Int Ed Engl,2007,46,3526-9.Domling, A.; et al, Angew Chem Int Ed Engl,2006.45,7235-9. patent: zanda, m.; et al, can.pat.appl.ca 2710693(2011) Chai, y; eur.pat.appl.2174947(2010), PCT WO 2010034724, Leamon, c.; et al, PCT WO 2010033733, WO 2009002993, Ellman, J.; et al, PCT WO 2009134279; PCT WO 2009012958, US appl.20110263650,20110021568, Matschiner, g.; et al, PCT WO 2009095447.Vlahov, I.; et al, PCT WO 2009055562, WO 2008112873.Low, P.; et al, PCT WO 2009026177.Richter, W., PCT WO 2008138561.Kjems, J.; et al, PCT WO 2008125116.Davis, M.; et al, PCT WO 2008076333.diene, j.; et al, U.S. Pat. appl.20070041901, WO 2006096754, Matschiner, g.; et al, PCT WO 2006056464.Vaghefi, F.; et al,5PCT WO 2006033913.Doemling, a., ger. offen.de 102004030227; PCT WO 2004005327; WO 2004005326; wo2004005269.stanton, m.; et al, U.S. Pat. appl.pub.20040249130. hoefle, g.; et al, Ger.Offen.DE 10254439; DE 10241152; DE 10008089.Leung, d.; et al, WO 2002077036.Reichenbach, H.; et al, Ger.Offen.DE 19638870; wolfgang, r.; US 20120129779, Chen, h, US appl.20110027274. A preferred structure of tubulysin linked to a cell binding molecule is described in patent PCT/IB 2012/053554.
Examples of antibody-tubulysin conjugate structures linked by a bridging linker are T01, T02, T03, T04, T05 and T06:
Figure BDA0003226420300000471
Figure BDA0003226420300000481
wherein the mAb is an antibody; z3Is H, OP (O) (OM)1)(OM2),，OCH2OP(O)(OM1)(OM2)，OSO3M1Or O-, NH-, S-or CH2Glycosides (glycosides, galactosides, mannosides, glucosides, fructosides, etc.); m1And M2Independently H, Na, K, Ca, Mg, NR1R2R3(ii) a n is
Figure BDA0003226420300000482
X1,X2,R1,R2And R3The same as defined in formula (I).
Calicheamicin and related enediyne antibiotics are preferred cytotoxic agents, and reference may be made to: nicolaou, K.C. et al, Science 1992,256, 1172-; proc.Natl.Acad.Sci USA.1993,90,5881-; 5,053,394; 5,108,912, respectively; 5,264,586, respectively; 5,384,412, respectively; 5,606,040, respectively; 5,712,374; 5,714,586; 5,739,116; 5,770,701; 5,770,710; 5,773,001; 5,877,296; 6,015,562, respectively; 6,124,310, respectively; 8,153,768. Examples of structures of antibody-calicheamicin analogs linked by a bridge linker are C01:
Figure BDA0003226420300000491
wherein the mAb is an antibody; n is
Figure BDA0003226420300000493
X1，X2，R1，R2And R3The same as defined in formula (I).
Maytansine is the preferred cytotoxic agent in this patent, and maytansine and its homologs are described in the following U.S. patents: 4,256,746, respectively; 4,361,650, respectively; 4,307,016, respectively; 4,294,757, respectively; 4,294,757, respectively; 4,371,533, respectively; 4,424,219, respectively; 4,331,598, respectively; 4,450,254, respectively; 4,364,866, respectively; 4,313,946, respectively; 4,315,929, respectively; 4,362,663, respectively; 4,322,348, respectively; 4,371,533, respectively; 4,424,219, respectively; 5,208,020; 5,416,064; 5,208,020; 5,416,064; 6,333.410, respectively; 6,441,163; 6,716,821,7,276,497; 7,301,019, respectively; 7,303,749, respectively; 7,368,565; 7,411,063, respectively; 7,851,432, and 8,163,888. An example of an antibody-maytansine conjugate is M01:
Figure BDA0003226420300000492
wherein the mAb is an antibody; n is
Figure BDA0003226420300000494
X1，X2，R1，R2And R3The same as defined in formula (I).
Taxanes, including paclitaxel (a cytotoxic natural product) and docetaxel (a semi-synthetic derivative) and analogs thereof, are the preferred cytotoxic molecules of this patent, and are described in the following references: k c. nicolaou et al, j.am. chem.soc.1995,117, 2409-2420; ojima et al, J.Med.chem.1996,39: 3889-; 1997,40, 267-278; 2002,45, 5620-5623; ojima et al, Proc.Natl.Acad.Sci.,1999,96: 4256-; kim et al, bull chem.soc, 1999,20, 1389-; miller, et al.J.Med.chem.,2004,47, 4802-; U.S. Pat. nos. 5,475,0115,728,849, 5,811,452; 6,340,701; 6,372,738; 6,391,913,6.436,931; 6,589,979, respectively; 6,596,757, respectively; 6,706,708, respectively; 7,008,942, respectively; 7,186,851, respectively; 7,217,819, respectively; 7,276,499, respectively; 7,598,290, and 7,667,054.
Examples of conjugate structures where the antibody-taxane is linked via a bridging linker are as Tx01, Tx02 and Tx 03:
Figure BDA0003226420300000501
wherein the mAb is an antibody; n is
Figure BDA0003226420300000502
X1，X2，R1，R2And R3The same as defined in formula (I).
CC-1065 analogs and duocarmycin analogs are also preferred cytotoxic agents for attachment to the bridge linkers of this patent. Examples of CC-1065 analogs and duocarmycin 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 and 6661, 2001; U.S. Pat. Nos. 4169888,4391904,4671958,4816567,4912227,4923990,4952394,4975278,4978757,4994578 578,5037993,5070092,5084468,5101038,5117006,5137877,5138059,5147786,5187186,5223409,5225539,5288514, 5324483483483, 5332740,5332837, 533434528, 5403484, 27908,5475092,5495009 009 92009, 554501, 5545806,5547667, 55825, 5573922, 5585089,5585499,87161, 55956017, 56229222929, 56565656565634575639, 41780,60575679575639,5639,5639,5639,92574354779, 605743547760574354775660566056605660566056605660566056605639,5660566056605639,566057479, 605660566056375660566056605660566056605660566056605660566056605660567, 6056605660566056605660566056605660566056605660566056605660566056605660566056605630, 566056605660566056605660566056605660566056605660566056605660566056605660566056605660567, 64566056605660566056605660566056605660566056605660566056605660566056605660566056605660566056605637566056605660567, 5660566056605637566056605660566056605660566056605660567, 56605660566056605660566056605660566056605660566056605660566056605660566056605660566056605660566056605660567, 566056605660566056605660566056605660566056605660566056605660566056605660566056605660566056605660567, 566056605660566056605660566056605660567, 5660566056605660567, 566056605660566056605660566056605660566056605660566056605660566056605660566056605660566056605660567, 56605660567, 566056605660566056605660566056605660566056605660566056605630, 566056605660566056605660566056605660566056605660566056605660567, 5660566056605660567, 56605660566056605660567, 566056605660566056605660566056605660566056605660566056375660566056605660566056605660566056605660567, 56605660566056605660566056605660566056605660566056375637566056605637563756605660563756605660563756605660567, 567, 5637567, 567, 56605660567, 566056605660566056605660567, 5660567, 5660566056605660566056605660566056605660566056605660566056605660566056605660566056605660566056605660566056375637566056605637563756605660567, 567, 56605660567, 56605660563756605660563756375637563756375637566056605660567, 56605660566056605660567, 56605660566056605660. Examples of antibody-CC-1065 analog structures linked via a bridge linker are as follows:
Figure BDA0003226420300000511
Figure BDA0003226420300000521
wherein the mAb is an antibody; n is
Figure BDA0003226420300000522
Z4Is H, PO (OM)1)(OM2)，SO3M1，CH2PO(OM1)(OM2)，CH3N(CH2CH2)2NC(O)-，O(CH2CH2)2NC (O) -or a glycoside; x3Is O, NH, NHC (O), OC (O), CO or default; x1、X2、R1、R2、M1And M2The same as defined in formula (I).
Daunorubicin/doxorubicin analogs are also preferred for coupling via the bridge linker connection of this patent. Preferred structures and their synthesis are referred to in the literature: 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., Cancer res.1988,48, 926-one 9311; trouet, et al, 1982,79, 626-629; Z.Brich et al, J.controlled Release,1992,19, 245-; chen et al, syn.comm.,2003,33, 2377-; king et al, bioconj. chem.,1999,10, 279-one 288; king et al, j.med.chem.,2002,45, 4336-; 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-; scott et al, bioorg.med.l chem.lett.19966, 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 patent 5,106,951; 5,122,368, respectively; 5,146,064, respectively; 5,177,016, respectively; 5,208,323, respectively; 5,824,805, respectively; 6,146,658; 6,214,345; 7569358, respectively; 7,803,903, respectively; 8,084,586, respectively; 8,053,205. Examples of antibody-doxorubicin analogue structures linked by a bridge linker are shown below:
Figure BDA0003226420300000523
Figure BDA0003226420300000531
wherein the mAb is an antibody; n is
Figure BDA0003226420300000532
X3Is H, O, NH, NHC (O) NH, C (O), or OC (O); x1、X2、R1、R2、M1And M2The same as defined in formula (I).
Auristatins and dolastatins are preferred cytotoxic agents linked to a bridge linker. Auristatins (e.g., dolauristatin e (ae), auristatin eb (aeb), auristatin efp (aefp), monomethyl auristatin e (MMAE), monomethyl auristatin F (mmaf), Auristatin F Phenylenediamine (AFP) and phenylalanine variants of MMAE) are analogs of dolastatin, as described in: int.j.oncol.1999,15,367-72; molecular Cancer Therapeutics,2004,3(8), 921-932; us patent application 11134826,20060074008,2006022925, us patent 4414205,4753894,4764368,4816444,4879278,4943628,4978744,5122368,5165923,5169774,5286637,5410024,5521284,5530097,5554725,5585089,5599902,5629197,5635483,5654399,5663149,5665860,5708146,5714586,5741892,5767236,5767237,5780588,5821337,5840699,5965537,6004934,6033876,6034065,6048720,6054297,6054561,6124431,6143721,6162930,6214345,6239104,6323315,6342219,6342221,6407213,6569834,6620911,6639055,6884869,6913748,7090843,7091186,7097840,7098305,7098308,7498298,7375078,7462352,7553816,7659241,7662387,7745394,7754681,7829531,7837980,7837995,7902338,7964566,7964567,7851437,7994135. Examples of conjugate structures in which antibodies-auristatins are linked by a bridging linker are as in AuO 1, AuO 2, AuO 3, AuO 4 and AuO 5:
Figure BDA0003226420300000541
Figure BDA0003226420300000551
wherein the mAb is an antibody; n is
Figure BDA0003226420300000552
X3Is CH2O, NH, NHC (O) NH, C (O), OC (O) or Default; x4Is CH2，C(O)，C(O)NH，C(O)N(R1) Or C (O) O; x1，X2，R1，R2And R3The same as defined in formula (I).
Benzodiazepine dimers (e.g., dimers of Pyrrolobenzodiazepine (PBD), tolmetin, indolophenyldiazepine, imidazobenzothiadiazole, or oxazolidinobenzodiazepine) are preferred cytotoxic molecules of the present invention and are also described in the literature in the art: us patent 8,163,736; 8,153,627, respectively; 8,034,808, respectively; 7,834,005, respectively; 7,741,319, respectively; 7,704,924, respectively; 7,691,848, respectively; 7,678,787, respectively; 7,612,062, respectively; 7,608,615, respectively; 7,557,099, respectively; 7,528,128, respectively; 7,528,126, respectively; 7,511,032, respectively; 7,429,658, respectively; 7,407,951, respectively; 7,326,700, respectively; 7,312,210, respectively; 7,265,105, respectively; 7,202,239, respectively; 7,189,710, respectively; 7,173,026, respectively; 7,109,193, respectively; 7,067,511, respectively; 7,064,120, respectively; 7,056,913, respectively; 7,049,311, respectively; 7,022,699, respectively; 7,015,215, respectively; 6,979,684, respectively; 6,951,853, respectively; 6,884,799, respectively; 6,800,622, respectively; 6,747,144, respectively; 6,660,856, respectively; 6,608,192, respectively; 6,562,806, respectively; 6,977,254, respectively; 6,951,853, respectively; 6,909,006, respectively; 6,344,451, respectively; 5,880,122, respectively; 4,935,362, respectively; 4,764,616, respectively; 4,761,412, respectively; 4,723,007, respectively; 4,723,003, respectively; 4,683,230, respectively; 4,663,453, respectively; 4,508,647, respectively; 4,464,467, respectively; 4,427,587, respectively; 4,000,304, respectively; U.S. patent application 20100203007,20100316656,20030195196. Examples of antibody-benzodiazepine dimer conjugate structures are PB01, PB02, PB03, PB04, PB05, PB06, PB07 and PB 08:
Figure BDA0003226420300000553
Figure BDA0003226420300000561
Figure BDA0003226420300000571
wherein the mAb is an antibody; n is
Figure BDA0003226420300000572
X3Is CH2O, NH, NHC (O) NH, C (O), OC (O) or Default; x4Is CH2，C(O)，C(O)NH，C(O)N(R1) Or C (O) O; x1，X2，R1，R2And R3The same as defined in formula (I). In addition, R1And/or R2A default may be used.
The drug/cytotoxic agent used in conjunction with the bridge linkers of the present invention may be any analog and/or derivative of the drug/molecule described previously. It is understood that each of the drugs/cytotoxic agents described herein may be modified and the resulting compounds still retain the relevant specificity and/or activity. It will also be appreciated by those skilled in the art that many other compounds may be substituted for the drug/cytotoxic agent described herein. Thus, the drug/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.
Examples
The 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 were stored under the conditions specified in the American Standard culture Collection (ATCC), German culture Collection (DSMZ) or Shanghai cell culture Collection of Chinese academy of sciences, except for the specific instructions. Cell culture reagents were obtained from Invitrogen, unless otherwise specified. All anhydrous reagents were obtained commercially and stored in Sure-Seal bottles. Other reagents and solvents were purchased according to the highest specifications and used without further treatment. The Varian Prostar HPLC was subjected to preparative HPLC purification. NMR data were obtained at Varian Mercury 400MHz with chemical shifts in ppm, tetramethylsilane as reference (0.00ppm) and coupling constants in Hz (J). 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 1.3- (2- (2- (2-hydroxyethoxy) ethoxy) propionic acid tert-butyl ester (34)
Figure BDA0003226420300000581
To 350mL of anhydrous THF were added, with stirring, 80mg (0.0025mol) of sodium metal and triethylene glycol 2(150.1g, 1.00 mol). After complete dissolution of sodium, tert-butyl acrylate (24mL, 0.33mol) was added. The solution was stirred at room temperature for 20 hours and neutralized with 8mL of 1.0M HCl. The solvent was removed by rotary evaporation in vacuo, diluted with brine (250mL) and extracted with ethyl acetate (3X 125 mL). The combined organic layers were washed with brine (100mL) and water (100mL), dried over sodium sulfate, and the solvent was removed. The resulting colorless oil was dried in vacuo to yield 69.78g (76% yield) of product 34.1H NMR:1.41(s,9H),2.49(t,2H,J＝6.4Hz),3.59-3.72(m,14H).ESI MS m/z-C13H25O6(M-H), calculated 277.17, found 277.20.
Example 2.3- (2- (2- (2- (tosyloxy) ethoxy) propionic acid tert-butyl ester (35)
Figure BDA0003226420300000582
34(10.0g, 35.95mmol) was dissolved in acetonitrile (50.0mL), pyridine (20.0mL) was added, and then tosyl chloride (7.12g, 37.3mmol) in 50mL acetonitrile was added dropwise over 30 minutes through an addition funnel. After 5 hours, TLC analysis indicated reactionAnd (4) finishing. The pyridine hydrochloride formed is filtered off, the solvent is removed from the filtrate and the residue is purified on a silica gel column, eluting with 20% ethyl acetate in n-hexane to pure ethyl acetate, to give 11.2g (76% yield) of compound 35.1H NMR:1.40(s,9H),2.40(s,3H),2.45(t,2H,J＝6.4Hz),3.52-3.68(m,14H),4.11(t,2H,J＝4.8Hz),7.30(d,2H,J＝8.0Hz),7.75(d,2H,J＝8.0Hz)；ESI MS m/z+C20H33O8S (M + H), calcd for 433.18, found 433.30.
Example 3.3- (2- (2- (2-azidoethoxy) ethoxy) propionic acid tert-butyl ester (36)
Figure BDA0003226420300000591
To DMF (50mL) was added 2- (2- (2- (2- (tosyloxy) ethoxy) -propionic acid tert-butyl ester 35(4.0g, 9.25mmol) and sodium azide (0.737g, 11.3mmol) with stirring. The reaction was heated to 80 ℃ and after 4 hours, TLC analysis showed the reaction was complete. The reaction was cooled to room temperature and quenched with water (25 mL). Extraction was performed with ethyl acetate (3X 35mL), and 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.1H NMR(CDCl3):1.40(s,9H),2.45(t,2H,J＝6.4Hz),3.33(t,2H,J＝5.2Hz),3.53-3.66(m,12H).ESI MS m/z+C13H26N3O8(M + H), calcd. 304.18, found 304.20.
Example 4.13 tert-butyl amino-4, 7, 10-trioxadecanoate (37)
13-amino-bis (tert-butyl 4,7, 10-trioxadecanoate), 38.
Figure BDA0003226420300000592
Crude product 36 is added
Figure BDA0003226420300000593
Dissolved in ethanol (80mL) and 300mg of 10% Pd/C was added. The system isVacuum was applied and 2atm of hydrogen was passed through under vigorous stirring, then the hydrogenation reactor was stirred at room temperature overnight and TLC showed disappearance of the starting material. The crude reaction was filtered over celite and the celite pad was washed with ethanol. After removal of the solvent, purification was carried out on a silica gel column eluting with a mixture of methanol (5% to 15%) and 1% triethylamine in dichloromethane to give tert-butyl 13-amino-4, 7, 10-trioxadecanoate 37(1.83g, yield 44% ESI MS m/z + C)13H27NO5(M + H), calculated 278.19, found 278.30) and 13-amino-bis (tert-butyl 4,7, 10-trioxadecanoate) 38(2.58g, 32% yield, ESI MS M/z + C26H52NO10(M + H), calculated 538.35, found 538.40).
Example 5.3- (2- (2- (2-aminoethoxy) ethoxy) propionic acid hydrochloride (39)
Figure BDA0003226420300000594
To a solution of tert-butyl 13-amino-4, 7, 10-trioxadecanoate 37(0.80g, 2.89mmol) in 1, 4-dioxane (30ml) was added 10ml HCl (36%) with stirring. After 0.5 h, TLC analysis showed the reaction was complete, the reaction mixture was concentrated and azeotropically concentrated with EtOH and EtOH/toluene to form the HCl salt of the title product (purity)>90%, 0.640g, 86% yield) without further purification. ESI MS m/z + C9H20NO5(M + H), calculated 222.12, found 222.20.
Example 6.13-amino-bis (4,7, 10-trioxadecanoic acid hydrochloride) (40)
Figure BDA0003226420300000601
To a solution of 13-amino-bis (tert-butyl 4,7, 10-trioxadecanoate) 38(1.00g, 1.85mmol) in 1, 4-dioxane (30ml) was added 10ml HCl (36%) with stirring. After 0.5 h, TLC analysis showed the reaction was complete, the reaction mixture was concentrated and azeotropically concentrated with EtOH and EtOH/toluene to form the hydrochloride salt of the title product: (Purity of>90%, 0.71g, 91% yield) without further purification. ESI MS m/z + C18H36NO10(M + H), calculated 426.22, found 426.20.
EXAMPLE 7 bis (2, 5-dioxopyrrolidin-1-yl) but-2-ynedioate (9)
Figure BDA0003226420300000602
To a solution of but-2-ynedioic acid 8(2.0g, 17.54mmol) in DMA (100ml) were added NHS (5.0g, 43.4mmol) and EDC (12.0g, 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 give the title compound 9(4.10g, 76% yield). ESI MS m/z + C12H9N2O8(M + H), calculated 309.03, found 309.20.
Example 8, 7-dioxo-5-alkynedioic acid (15)
Figure BDA0003226420300000603
To a solution of bis (trimethylsilyl) acetylene (5.0g, 29.34mmol) and iodine (0.37g, 1.45mmol) in dichloromethane (100mL) was added succinyl chloride (18.11g, 116.83mmol) dropwise with stirring at 0 deg.C. After the addition was complete, the mixture was stirred at room temperature until the reaction was complete (TLC monitoring, about 2 hours). The reaction mixture was quenched with water (15mL) and extracted with dichloromethane (3X 70 mL). The combined extracts were washed with 15% sodium thiosulfate solution and dried over anhydrous Na2SO4Drying and vacuum concentrating. Subjecting the obtained product to silica gel column chromatography (100-200 mesh, 5-10% H)2O/acetonitrile) to yield the title product (5.50g, 85% yield). ESI MS m/z-C10H9O6(M-H), calculated 226.05, found 226.10.
EXAMPLE 9 (R, R, S, S, R, 4R, 4'R) -5,5' - (((4, 7-dioxo-5-alkynediyl) 3, 1-phenylene)) bis (4- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine pentanoylamino) -4-methylpentyl) thiazole-4-carboxamido) -2-methylpentanoic acid) (79)
Figure BDA0003226420300000611
To a solution of compound 9(25mg,0.081mmol) in THF (3.0ml) was added (4R) -4- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3((R) -1-methylpiperidine-2-carboxamido) pentanoylamino) -4-methylpentyl) thiazole-4-carboxamido) -5- (3-amino-4-hydroxyphenyl) -2-methylpentanoic acid, 51(Huang y.et al, Med Chem. #44,249thACS National Meeting, Denver, CO, Mar.22-26,2015; WO2014009774) (151mg, 0.199mmol) in THF (4.0mL) and phosphate buffer (4mL,100mM Na 2HPO 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. times.20 mm) using water/ethanol (90% to 50% water, 55 min, flow rate 15 ml/min). The product-containing fractions were combined, concentrated and crystallized from EtOH/n-hexane to give the title compound (73mg, 53% yield). ESI MS m/z + C86H122N12NaO20S2(M + Na), calculated 1729.83, found 1730.10.
Example 10.14, 17-dioxo-4, 7,10,21,24, 27-hexaoxa-13, 18-diazatritridec-15-yne-1, 30-dioic acid (86)
Figure BDA0003226420300000612
To a solution of 3- (2- (2- (2-aminoethoxy) ethoxy) propionic acid hydrochloride 39(601mg, 2.33mmol) in THF (6ml) was added phosphate buffer (150mM NaH)2PO 4, pH7.2,4ml) and bis (2, 5-dioxopyrrolidin-1-yl) but-2-ynedioic acid salt 9(350mg, 1.13 mmol). After stirring at room temperature for 4 hours in the dark, the mixture is concentrated and SiO is used2Column purification, eluting with water/acetonitrile (1: 9). The product-containing fractions were combined and concentrated to give the title compound (345mg, 59% yield). ESI MS m/z-C22H36N2O12(M-H), calculated 519.22, found 519.30.
EXAMPLE 11 (2- (2- (2-carboxyethoxy) ethoxy) ethyl) -14, 17-dioxo-4, 7,10,21,24, 27-hexaoxa-13, 18-diazatripentadecan-15-yne-1, 30-dioic acid (87)
Figure BDA0003226420300000621
To a solution of 13-amino-bis (4,7, 10-trioxadecanoic acid hydrochloride), 40(650mg, 1.40mmol) in THF (6ml) was added phosphate buffer (150mM NaH)2 PO4pH7.2,4ml) and bis (2, 5-dioxopyrrolidin-1-yl) but-2-ynedioic acid salt 9(190mg, 0.61 mmol). After stirring at room temperature in the dark for 4h, the reaction mixture was concentrated and eluted with C-18 preparative HPLC (250 mm. times.30 mm ID), eluting with water/ethanol (90% to 50% water over 55 min., flow rate 35 ml/min), the fractions containing the product were combined and concentrated to give the title compound (287 mg, 51% yield). ESI MS m/z-C40H67N2O22(M-H), calculated 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-diazacyclopentadecyne-1, 30-dioate (88)
Figure BDA0003226420300000622
To a solution of 1, 4-dioxo-4, 7,10,21,24, 27-hexaoxa-13, 18-diazatricyclopentadecene-15-yne-1, 30-dioic acid, 86(340mg, 0.653mmol) in DMA (6ml) were added NHS (225mg, 1.96mmol) and EDC (401mg, 2.08 mmol). The mixture was stirred overnight in the dark, concentrated and then on SiO2Purify on column eluting with EtOAc/DCM (5:1) to give the title compound 88(330mg, 71% yield). ESI MS m/z + C30H43N4O16(M + H), calculated 715.26, found 715.20.
EXAMPLE 13 (2- (2- (2- (3- ((2, 5-dioxopyrrolidin-1-yl) oxy) -3-oxopropoxy)) ethoxy) ethyl) -14, 17-dioxo-4, 7,10,21,24, 27-hexaoxa-13, 18-diazatritridec-15-yne-1, 30-dioate (89)
Figure BDA0003226420300000631
To a solution of 13, 18-bis (2- (2- (2- (2-carboxyethoxy) ethoxy) ethyl) -14, 17-dioxo-4, 7,10,21,24, 27-hexaoxa-13, 18-diazacyclobuten-15-yne-1, 30-dioic acid, 87(280mg, 0.301mmol) in DMA (6ml) were added NHS (105.0mg, 0.913mmol) and EDC (200mg, 1.04 mmol). The mixture was stirred overnight in the dark, concentrated and then on SiO2Purify on column with EtOH/DCM
Figure BDA0003226420300000632
Figure BDA0003226420300000633
Elution afforded the title compound 89(249mg, 63% yield). ESI MS m/z + C56H81N6O30(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-hexaoxa-13, 18-diazatriont-15-yne 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)
Figure BDA0003226420300000634
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-carboxamido) pentanoylamino) -4-methylpentyl) thiazole-4-carboxamido) -5- (3-amino-4-hydroxyphenyl) -2-methylpentanoic acid, 51(Huang Y.et al, Med Chem. #44,249thACS National Meeting, Denver, CO, Mar.22-26,2015; WO2014009774) (80mg, 0.107mmol) in THF (4.0ml) and phosphoric acidSalt buffer (4ml,100mM Na)2HPO4pH 7.0). After stirring at room temperature for 4 hours, the mixture was concentrated and purified by C-18 preparative HPLC (250 mm. times.20 mm. ID.) using water/ethanol (95% to 50% water, flow rate 15 ml/min). The product-containing fractions were combined, concentrated and crystallized from EtOH/n-hexane to give the title compound (48mg, 56% yield). ESI MS m/z-C98H147N14O26S2(M-H), calculated 2000.01, found 2000.40.
Example 15 antibody binding of conjugated Compounds 90 and 91
Figure BDA0003226420300000641
Adding NaH to herceptin (10mg/mL, 2.0mL, pH7.0-8.0)2PO4Buffer (100mM, pH6.5-7.5, 0.70-2.0mL), TCEP (20mM aqueous solution, 28. mu.L) and Compound 90(20mM DMA solution, 14. mu.L). The mixture was incubated at room temperature for 2-16 hours, followed by the addition of DHAA (135. mu.L, 50 mM). After continuous overnight incubation at room temperature, the mixture was purified on a G-25 column using 100mM NaH2PO4，50mM NaCl pH
Figure BDA0003226420300000642
Figure BDA0003226420300000643
Eluting with a buffer solution to obtain
Figure BDA0003226420300000644
Conjugate 91 (about 87% yield) (ii)
Figure BDA0003226420300000645
In a buffer). The drug/antibody ratio (DAR) was determined to be 4.0 by UPLC-Qtof mass spectrometry. SEC HPLC (Tosoh Biosciences, Tskgel G3000SW, 7.8mm ID. times.30 cm, 0.5 ml/min, 100 min) analysis showed a monomer content of 96-99%, SDS-PAGE gel measurement showed a single band.
Example 16 Compound 92(4 tubulysin Compounds attached to Each bridge linker)
Figure BDA0003226420300000646
To a solution of compound 89(35mg,0.026mmol) in THF (3.0ml) was added (4R) -4- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3((R) -1-methylpiperidine-2-carboxamido) pentanamido) -4-methylpentyl) thiazole-4-carboxamido) -5- (3-amino-4-hydroxyphenyl) -2-methylpentanoic acid, 51(Huang Y.et al, Med Chem. #44,249thACS National Meeting, Denver, CO, Mar.22-26,2015; WO2014009774) (100.6mg, 0.132mmol) in THF (4.0ml) and phosphate buffer (4ml,100mM Na)2HPO4pH 7.0). After stirring at room temperature for 4 hours, the reaction mixture was concentrated and purified by C-18 preparative HPLC (250 mm. times.20 mm ID) eluting with water/ethanol (95% to 50% water over 50 min, flow rate 15 ml/min). The product-containing fractions were combined, concentrated and crystallized from EtOH/n-hexane to give the title compound 92(47.6mg, 47% yield). ESI MS m/z-C192H291N26O50S4(M-H), calculated 3890.00, found 3890.30.
EXAMPLE 17 conjugation of Compound 92 to an antibody preparation of conjugate 93
Figure BDA0003226420300000651
To a herceptin solution (10mg/mL, 2.0mL, pH7.0-8.0) was added a buffer (0.70-2.0mL, 100mM NaH) of pH6.5-7.52PO4) TCEP (28. mu.L, 20mM in water) and Compound 92 (14. mu.L, 20mM in DMA). The mixture was incubated at room temperature for 2-16 hours, followed by the addition of DHAA (135. mu.L, 50 mM). After overnight incubation at room temperature, the mixture was purified on a G-25 column using 100mM NaH2PO4，50mM NaCl pH
Figure BDA0003226420300000652
Figure BDA0003226420300000653
Eluting with a buffer solution to obtain
Figure BDA0003226420300000654
The conjugate 92 (b) ((
Figure BDA0003226420300000655
In the buffer solution, the buffer solution is added,
Figure BDA0003226420300000656
yield). The drug/antibody ratio (DAR) was determined to be 8.0 by UPLC-Qtof mass spectrometry. SEC HPLC (Tosoh Biosciences, Tskgel G3000SW, 7.8mm ID. times.30 cm, 0.5 ml/min, 100 min) analysis showed a monomer content of 96-99%, SDS-PAGE gel measurement showed a single band.
Example 18 in vitro cytotoxicity assessment of conjugates 91 and 93 (comparative T-DM 1):
cell lines used in cytotoxicity experiments 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 5A medium containing 10% FBS. To run the assay, cells (180 μ Ι, 6000 cells) were added to 96-well plates and incubated at 37 ℃ in an environment of 5% carbon dioxide for 24 hours, after which the cells were treated with different concentrations of compound (20 μ Ι) in a total volume of 0.2 mL. Control wells contained cells and media, with no test compound. After the plate was incubated at 37 ℃ for 120 hours in an atmosphere of 5% carbon dioxide, MTT (5mg/mL) was added thereto and incubated at 37 ℃ for 1.5 hours. After careful removal of the medium DMSO (180. mu.L) was added, shaken for 15 min and the absorbance measured at 490nm and 570nm, 620nm as reference. The inhibition rate was calculated according to the following formula: inhibition [% 1- (assay value-blank value)/(control value-blank value) ] × 100%
Compound cytotoxicity results:
IC50(nM) n87 cells (Ag +) SK-OV-3 cells (Ag +) HL60 cell (Ag-)
Coupling 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 exceeded 185 (IC)50>20/IC500.108), for SK-OV-3 cells over 225; conjugate 93 has specificity over 270 (IC) for N87 cells50>10/IC500.037), greater than 344 for SK-OV-3 cells; the specificity of the conjugate T-DM1 on N87 cells exceeded 55 (IC)50>15/IC500.27), over 78 for SK-OV-3 cells.
Both conjugate 91 and conjugate 93 were more effective than the commercially available conjugate T-DM 1. Conjugate 93DAR was 8, which was three times more active than conjugate 91 with DAR 4.

Claims (29)

1. A bridge connector of formula (I):
Figure FDA0003226420290000011
wherein the acetylenedicarboxylic acid structure on the linker is capable of reacting with a pair of sulfur atoms in a cell binding agent;
Z1and Z2Are the same or different functional groups capable of reacting with the cytotoxic drug,can pass throughGenerating a disulfide bond, an ether bond, an ester bond, a thioether bond, a thioester bond, a peptide bond, a hydrazone bond, a carbamate bond, a carbonate bond, an amine bond (secondary, tertiary or quaternary), an imine bond, a heterocycloalkyl group, a heteroaryl group, an oxime alkoxy bond or an amide bond to be combined with the toxic small molecule drug;
R1and R2Is the same, different or default straight-chain alkyl group with 1 to 6 carbon atoms, branched or cyclic alkyl group with 3 to 6 carbon atoms, straight-chain or cyclic alkenyl or alkynyl group, or ester group, ether group, amide group or polyethoxy (OCH) group with 1 to 6 carbon atoms2CH2)pWherein p is an integer from 0 to about 1000, or a combination of these groups;
in addition, R1And R2Is a chain structure containing C, N, O, S, Si and P atoms, optimally contains 0-500 atoms, and is covalently connected with X1Or X2And Z1Or Z2；R1And R2Are combined in all possible chemical ways, such as to form an alkyl, alkylene, alkenylene, alkynylene, ether, polyoxyalkyl, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, polyurethane, amino acid, polypeptide, acyloxyamine, hydroxamic acid, or a combination of these groups;
X1and X2Independently selected from NH, N (R)3) O, S or CH2；R3Is H, a straight chain alkyl group of 1 to 6 carbon atoms, a C3 to C6Branched or cyclic alkyl, straight-chain, branched or cyclic alkenyl or alkynyl, or an ester, ether, amide or polyethoxy unit (OCH) having 1 to 6 carbon atoms2CH2)pWherein p is an integer from 0 to 1000, or a combination of these groups.
2. A cell-binding agent-drug conjugate having the structure of formula (II):
Figure FDA0003226420290000012
wherein:
cb is a cell binding agent, most preferably an antibody;
within the brackets is a linker-drug component coupled to the cell binding molecule via a pair of sulfur atoms;
Drug1and Drug2(ii) are the same or different cytotoxic agents linked to the cell binding agent with alkyl, alkylene, alkenylene, alkynylene, ether, polyalkoxy, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, carbamate, amino acid, peptide, acyloxyamine, hydroxamic acid, disulfide bond, thioether, thioester, carbamate, carbonate, heterocycle, heteroalkyl, heteroaryl, or alkoxyoxime bonds, and combinations thereof;
n is 1 to 20; r1,R2,X1And X2Is as defined in claim 1.
3. A compound of formula (III):
Figure FDA0003226420290000021
wherein Cb, Z1,Z2,n,R1,R2,X1And X2As defined in claims 1and 2.
4. A compound of formula (IV): :
Figure FDA0003226420290000022
wherein the Drug1,Drug2,Z1,Z2,n,R1,R2,X1And X2As defined in claims 1and 2.
5. In claim 1, the bridged linker containing acetylene dicarbonyl is synthesized by condensation of acetylene dicarbonyl or its derivatives with other components, terminal amines (primary or secondary amines), alcohols or thiols, as shown in (Ia) below:
Figure FDA0003226420290000023
wherein X is X in claim 11Or X2Description of (1) refers to N (R)3) O, or S; r is R1And/or R2，R1、R2And R3The definition is the same as claim 1.
Lv 1and Lv2 are identical or independent of each other and are OH, F, Cl, Br, I, nitrophenol, N-hydroxysuccinimide (NHS), phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, trifluoromethylsulfonic acid, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, p-toluenesulfonic acid, methanesulfonic acid, 2-ethyl-5-phenylisoxazole-3' -sulfonic acid, acid anhydride or acid anhydride with other acid anhydrides such as acetic anhydride, formic anhydride; or a polypeptide condensation reaction intermediate or a Mitsunobu reaction intermediate; the condensing agent comprises: 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-benzotriazol-N, N, N ', N ' -tetramethyluronium tetrafluoroborate), O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU), benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), diethyl pyrocarbonate (DEPC), N, N, N ', N' -tetramethylchloroformamidine hexafluorophosphate, 2- (7-oxybenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), 1- [ (dimethylamine) (morpholinyl) methylene chloride]-1[1,2,3]Triazolo [4,5-b]1-pyridine-3-oxidophorophosphate (HDMA), 2-chloro-1, 3-dimethylimidazolium hexafluorophosphate (CIP), chlorotriazolylphosphonium hexafluorophosphate (PyCloP), bis (tetramethylene) fluorocarboxamide (BTFFH), N, N, N ', N' -tetramethyl-thio- (1-oxo-2-pyridyl) thiouronium hexafluorophosphate, 2- (2-pyridon-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate (TPTU), thio- (1-oxo-2-pyridyl) -N, N, N ', N' -tetramethylthiouronium hexafluorophosphate, O- [ (ethoxycarbonyl) cyanomethylamine]-N, N, N ', N ' -tetramethylthiourea Hexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylamino-morpholine-carbonium hexafluorophosphate (COMU), (benzotriazol-1-yloxy) dipyrrolidine carbohexafluorophosphate (HBPyU), N-benzyl-N ' -cyclohexylcarbodiimide (or supported on a polymer), dipyrrolidinyl (N-succinimidyloxy) carbonium hexafluorophosphate (HSPyU), 1- (chloro-1-pyrrolidinylmethylene) pyrrolidine hexafluorophosphate (PyClU), 2-chloro-1, 3-dimethylimidazole tetrafluoroborate (CIB), (benzotriazol-1-yloxy) dipiperidine carbohexafluorophosphate (HBPipU), 6-chlorobenzotriazole-1, 1,3, 3-tetramethyluronium tetrafluoroborate (TCTU), tris (dimethylamino) phosphonium bromide hexafluorophosphate (BroP), 1-n-propylphosphoric anhydride (PPACA,
Figure FDA0003226420290000032
) 2-isocyanoethylmorpholine (MEI), N' -tetramethylurea-oxy- (N-succinimidyl) Hexafluorophosphate (HSTU), 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP), oxy- [ (ethoxycarbonyl) cyanomethylamine]-N, N, N ', N' -tetramethylthionurebetrafluoroborate (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)Azobisformyldipiperidine (ADD), bis (4-chlorobenzyl) azodicarboxylate (DCAD), di-tert-butyl azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD), diethyl azodicarboxylate (DEAD).
6. In claim 1, the carbon chain is extended by condensation of acetylene dicarbonyl groups on the bridge linker with other functional groups, and the synthetic step is the reaction of bis (trimethylsilyl) acetylene, magnesium acetylene dibromide (Grignard reagent), acetylene dilithium salt or other acetylene dimetallic salt, and an acid halide or anhydride, as shown in (Ib), (Ic), (Id), (Ie), (If), (Ig) and (Ih):
Figure FDA0003226420290000031
Figure FDA0003226420290000041
where M is Na, K, Li, Cu, CuLi, Sn, Ti, Ca, Mg or Zn.
7. In claims 2 and 4, Drug 1and Drug2 in formulas (II) and (IV) are the same or each independently selected from:
1) chemotherapeutic agents: a) alkylating agents, such as nitrogen mustards: chlorpheniramine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, dimethoxyamine hydrochloride, mechlorethamine oxide, amlodipine hydrochloride, mycophenolic acid, dulcitol, pipobroman, neomechlorethamine, benzene mustard cholesterol, prednimustine, thiotepa, triamcinolone pair, uracil; CC-1065 (including its aldorexin, kazelaixin and bizelaixin synthetic homologs); duocarmycin (including the synthetic homologs KW-2189 and CBI-TMI); benzodiazepine dimers (e.g., dimers of Pyrrolobenzodiazepine (PBD) or tolmetin, indolophenyldiazepine, imidazobenzothiadiazole, or oxazolidobenzodiazepine); nitrosoureas (carmustine, lomustine, fustin chloride, fotemustine, nimustine, lamustine); the alkyl sulfonate (busulfan,endosulfan, and sulfur); triazenes (dacarbazine); platinum-containing compounds (carboplatin, cisplatin, oxaliplatin); aziridines, such as chromanone, carotenone, metoclopramide and lindopa; ethyleneimine and methyl melamine, including hexamethylmelamine, triethylenetriamine, triethylphosphoramide, triethylenethiophosphoramide and trimethylolmethylamine; b) plant alkaloids, such as vinca alkaloids (vincristine, vinblastine, vindesine, vinorelbine, catharanthine); taxoids (paclitaxel, docetaxel) and homologs thereof; maytansine alkaloids (DM1, DM2, DM3, DM4,DM5，DM6，DM7maytansine and ansamycin) and homologs thereof; cryptophycin (especially cryptophycin 1and cryptophycin 8); epothilone, juncecrogol, discodermolide, bryozoalactone, dolastatin, auristatin, tubulysin, cephalostatin, pancratistatin, sarcodictyin, spongistatin; c) DNA topoisomerase inhibitors, such as etoposide tinib (9-aminocamptothecin, camptothecin, clinatot, daunomycin, etoposide phosphate, irinotecan, mitoxantrone, nosaline, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptothecin (RFS 2000)); mitomycin (mitomycin C); d) antimetabolites, such as antifolates, DHFR inhibitors (methotrexate, trexate, denoxate, pteropterin, aminopterin (4-aminobenzoic acid), or other folate homologs); IMP dehydrogenase inhibitors (mycophenolic acid, thiazolofuran, ribavirin, EICAR); ribonucleotide reductase inhibitors (hydroxyurea, deferoxamine); pyrimidine homologues, uracil homologues (ancitabine, azacitidine, 6-azauracil, capecitabine (hiloda), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-fluorouracil, floxuridine, ratitrexed (Tomudex), cytosine homologues (cytarabine, cytosine arabinoside, fludarabine), purine homologues (azathioprine, fludarabine, mercaptopurine, thiamine, thioguanine), folic acid supplements, such as lozolirtin acid, e) hormone therapy agents, such as receptor antagonists, antiestrogens (megestrol, raloxifene, tamoxifen), LHRH agonists (Gostalin, leuprolide acetate)Forest); anti-androgens (bicalutamide, flutamide, carrousel, betaandrosterone propionate, epiandrosterone, goserelin, leuprorelin, metulidine, nilutamide, testolactone, trilostane and other androgen inhibitors); retinoids, vitamin D3 homologues (CB1093, EB1089KH1060, cholecalciferol, ergocalciferol); photodynamic therapy agents (verteporfin, phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin a); cytokines (interferon- α, interferon- γ, Tumor Necrosis Factor (TNF), TNF-containing human proteins); f) kinase inhibitors, such as BIBW 2992 (anti-EGFR/Erb 2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080 (anti-VEGFR 2), mubritinib, ponatinib (AP 34), bafetinib (INNO-406), bosutinib (sk24ni-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, felinib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, istussin; g) antibiotics, such as enediynes antibiotics (calicheamicin, particularly calicheamicin γ 1, δ 1, α 1and β 1 (see J.Med. Chem.1996,39 (11)), 2103-, triiron doxorubicin, adriamycin, streptozotocin, tubercidin, ubenimex, setastatin, zorubicin; h) others, such as polyketides (annonaceous acetogenins), in particular bullatacin and bullatacinone; gemcitabine, epoxygenases (e.g., capeline), bortezomibThalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, alloviten-7, Xegeva, Provenge, Yervoy, prenylation inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., staurosporine), actinomycins (e.g., actinomycin D, dactinomycin), bleomycin (e.g., bleomycin A2, bleomycin B2, pelomycin), anthracyclines (e.g., daunorubicin), doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, MDR inhibitors (e.g., verapamil), Ca22+Inhibitors of ATPase (e.g., thapsigargin), inhibitors of histone deacetylase (vorinostat, romidepsin, panobinostat, valproic acid, Mocetinostat (MGCD0103), Belinostat, PCI-24781, entinostat, SB939, remininostat, Givinostat, AR-42, CUDC-101, sulforaphane, trichostatin A); celecoxib, glitazones, epigallocatechin gallate, disulfiram, Salinosporamide a; anti-adrenal agents, such as aminoglutethimide, mitotane, trilostane, acetoglucuronolactone, aldphosphoramide, aminolevulinic acid, amsacrine, arabinoside, bestraucil, bison, edatraxate, defofamine, meclocine, disazoquinone, eflornithine (DFMO), efamitine, etiloamine, ethylgluconic acid, gallium nitrate, cytosine, hydroxyurea, ibandronate, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidanol, diaminenitracridine, pentostatin, mechlorethamine, pirarubicin, podophyllic acid, 2-ethylhydrazine, procarbazine;
Figure FDA0003226420290000061
piperazinedione propane; rhizomycin; (iv) Wenzuo; spiro germanium; geobacillus azavor; a tri-imine quinone; trichlorotriethylamine; trichothecenes (in particular T-2 toxin, verrucomicin A, bacillocin A and anguidine), polyurethanes, siRNAs, antisense drugs and nucleolytic enzymes.
2) Autoimmune disease agents, including but not limited to cyclosporine, cyclosporin a, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g., amcinonide, dexamethasone, triamcinolone acetonide, beclomethasone propionate, DHEA, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mycophenolate mofetil, prednisone, sirolimus, tacrolimus.
3) Anti-infectious disease agents, including but not limited to a) aminoglycosides: amikacin, astemicin, gentamicin (netilmicin, sisomicin, isepamicin), hygromycin B, kanamycin (amikacin, arbekacin, aminoxykanamycin, dibekacin, tobramycin), neomycin (framycetin, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, methylgestomycin; b) amide alcohols: azidochloramphenicol, chloramphenicol, florfenicol, thiamphenicol; c) ansamycin: geldanamycin, herbimycin; d) carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem; e) cephem: cephem (loracarbef), cephalosporacetonitrile, ampicillin, cephradine, cefadroxil, cephalonine, ceftazidime, cephalothin or cephalotaxin, cephalexin, cephramycin, cefamandole, cefapirin, azapiromidin, fluxazole cephalosporin, sporocetone, azolin cephalosporin, cefbuperazone, cefcapene, cefixime, cefpodoxime, cefixime, cefoxitin, cefprozil, cefetaxeme, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefpira, cefetamet, cefepime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, thienam, cefotaxime, cefozopran, cefazolin, cephalexin, cefepime, cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram, ceftibuten, cefotiarin, ceftizoxime, cefprozil, ceftriaxone, cefuroxime, ceftizoxime, cephamycins (cefoxitin, cefotetan, cefcyanazole), oxacephems (flomoxef, cephalosporins); f) glycopeptide: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin, g) glycylcyclin: such as tigecycline, h) beta-lactamase inhibitors: penicillane (sulbactam, tazobactam), oxapenem (clavulanic acid); i) lincosamide: clindamycin, lincomycin; j) lipopeptides: daptomycin, a54145, Calcium Dependent Antibiotic (CDA); k) macrolides: azithromycin, clarithromycin, dirithromycin, erythromycin, fluramycin, josamycin, ketolide (telithromycin, sequoyimycin), midecamycin, mickamycin, oleandomycin, rifamycin (isoniazid, rifampin, rifabutin, rifapentine), ropiniromycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), oleandomycin acetate, telithromycin; l) monocyclic amines: aztreonam, tigemonam; m) oxazolidinones: linezolid; n) penicillins: amoxicillin, ampicillin (pivampicillin, silocillin, bacampicillin, ampicillin, doxorubicin), azlocillin, benzylpenicillin, benzathine phenoxymethylpenicillin, cloxacillin, procaine penicillin (metilin), mezlocillin, methicillin, nafcillin, oxacillin, acemethicillin, penicillin, nafcillin, piperacillin, ampicillin, sulfonicillin, temocillin, ticarcillin; o) a polypeptide: bacitracin, colistin, polymyxin B, p) quinolones: alatrefloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, gatifloxacin, gemifloxacin, grepafloxacin, carnotrexacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q) streptogramins: pristinamycin, quinupristin/dalfopristin, r) sulfonamides: sulfonamides: sulfonamides, sulfadiazine, sulfasalazine, sulfisoxazole, tamoxifen, trimethoprim-sulfamethoxazole (sulfamethoxazole); s) steroid antibacterial drugs: such as fusidic acid; t) tetracyclines: doxycycline, chlortetracycline, cimeticycline, demeclocycline, ramoxiline, mecycline, methacycline, minocycline, oxytetracycline, penicillin V kalipecycline, pyrrolidinemethyltetracycline, tetracycline, glycylcycline (such as tigecycline): u) other types of antibiotics: annonaceous acetogenins, arsine, bacteroidal terpineol inhibitors (bacilli), DANAL/AR inhibitors (cycloserine), dictyostatin, discodermolide, saxifragol, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalitide, metronidazole, mupirocin, NAM synthesis inhibitors (e.g. fosfomycin), nitrofurantoin, paclitaxel, pratensomycin, pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampin), tazobactam tinidazole, echinacon.
4) Antiviral drugs: a) entry/fusion inhibitors: apaviralo, maraviroc, vicrivroc, gp41 (enfuvirtide), PRO140, CD4 (abalizumab); b) integrase inhibitors: raltegravir, elvite-gravir, globoid dna a; c) maturation inhibitors: bevirimat, vivocon; d) neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; E) nucleosides and nucleotides: abacavir, adefovir, armocivir, abciximab, brivudine, cidofovir, cladribine, dexamethasone, didanosine (ddI), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluxacillin (5-FU),3 '-fluoro-substituted 2', 3 '-deoxynucleoside homologs (such as 3, 3' -fluoro-2 ', 3' -dideoxythymidine (FLT) and 3 '-fluoro-2', 3 '-dideoxyguanosine (FLG), fomivirsen, 9-guanine, idoxuridine, lamivudine (3TC), 1-nucleosides (such as β -1-thymidine and β -1-2' -deoxycytidine), penciclovir, racivir, ribavirin, dilastatin, vudine (d4T), talivirin (viramidine), telbivudine, tenofovir, trifluridine valacyclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f) non-nucleoside: amantadine, atitidine, carboprvirine, diarylpyrimidine (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphoryl formic acid), imiquimod, pegylated interferon, lovirine, lodenosine, methidathiozone, nevirapine, NOV-205, long-acting interferon alpha, podophyllotoxin, rifampin, rimantadine, resiquimod (R-848), acetimidamantadine; g) protease inhibitors: amprenavir atazanavir, boceprevir, daronavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h) other types of antiviral drugs: antioxidase, arbidol, kalarotide, ceragenin, cyanovirin-n, diarylpyrimidine, epigallocatechin gallate (EGCG), foscarnet, griffine, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, anabolic inhibitor, ribavirin, seliciclib.
5) A radioisotope (radionuclide) selected from3H，11C，14C，18F，32P，35S，64Cu，68Ga，86Y，99Tc，111In，123I，124I，125I，131I，133Xe，177Lu，211At or213Bi。
6) A chromonic molecule capable of absorbing a light, such as ultraviolet, fluorescent, infrared, near infrared, or visible light; yellow pigment, red blood cell, iridescent pigment, white blood cell, melanin and blue-green pigment, fluorescent molecule (fluorescent chemical substance which absorbs light and then emits light), visual light transduction molecule, photon molecule, luminescence molecule and fluorescein compound; non-protein organic fluorophores such as xanthene derivatives (fluorescein, rhodamine, oregon green, eosin and texas red); cyanine derivatives (cyanines, indocarbocyanines, oxacyanines, thiacyanines, and merocyanines); squaric acid derivatives and ring-substituted squaric acids, including Seta, SeTau and Square dyes; naphthalene derivatives (dansyl and sodium fluorosilicate derivatives); coumarin derivatives; oxadiazole derivatives (pyridyloxazole, nitrobenzoxazole and benzooxadiazole); 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 (flavonol flavin, acridine orange, acridine yellow, etc.); arylmethylamine derivatives (auramine, crystal violet, malachite green) and tetrapyrrole derivatives (porphine, phthalocyanine, bilirubin); homologs and derivatives of the following fluorescent compounds: CF dyes (Biotium), DRAQ and CyTRAK probes (BioS-tatus), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLight Fluor (Thermo Scientific, Pierce), Atto and Tracy (Sigma Aldrich), FluProbes (Interchim), Abberior dyes (Abberior), DY and MegaStokes dyes (Dyomics), Sulfo Cy dyes (Cyandye), HiLyte Fluor (Anaspec), Seta, Setau and Square dyes (Biosearch Technologies), SureLight dyes (APC, RPEPerCP, Phytobimes) (Columbia Biosciences), APCXL, RPE, BPE (Phyco-Bioh), allophycocyanin (Biotecn), BODIPC-APC, BioProch 3, BioCydipta-3, BioCy-3, Biophycin, Cydipta-Cyrpe-3, Cydipta-Cydipy-3, Cydipy-Cyindamine Cyindac (Biophyte dyes, Cyindac) 3, Cyindac-19, Cyindac-19, Cyindamine-Cyindamine Fluorose (Biocide, Cyindamine, Cyindac, Cyindamine, Cy, 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-amino actinomycin D, CG-selective), acridine orange, chromomycin A3, CyTRAK orange (Biostatus), DAPI, Q DRAQ5, DRAQ7, ethidium bromide, Hoechst33258, hohsett 33342, LDS 751, mithramycin, Propidium Iodide (PI), SYTOX blue, TOX green, TOX 3526, TO-1 thiazole-orange, PROTO-1, Proto-1, TOTO-3, TO-PRO-3, YOSeta-1, YOYO-1. Fluorescent compounds which can be linked to the linker of the invention for the study of cells are selected from the following compounds or derivatives thereof: DCFH (2', 7' -dichlorodihydrofluorescein, oxidized form), DHR (dihydrorhodamine 123, oxidized form, photocatalytic oxidation), Fluo-3(AM ester, pH >6), Fluo-4(AM ester, pH7.2), 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), Cirsium 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 (S65 mutation), GFP (S65C mutation), GFP (S65L mutation) GFP (Y66H mutation), GFP (Y66W mutation), GFPuv, cRed1, HJ-Red, Katusha, Kusarara Orange (monomer, MBoimer, mCopofp, mColophor 63L, monomer, MBherl 635, Khryse 635, KmRed, Kyurn-5, TsRed monomer, Skumura-M2, Tahryser-5, Tamcorph, Tamcopic, Tahryse, Tamcopic, Tahryse, Tamciror-5, Tamcopic complex, Tamciro-5, Tamciro, Tahryse, Tamciro, Tachy-5, Tachy, Tachytrix-5, Tachy, Tachytrix-5, Tachy, Tachytrix-5, Tachytrix, Tachy, Tachytrix, Tachy, Tachytrix, Tachy, Tachytrix, and Tachytri, T-Sapphire, TagCFP (dimer, Evrogen), TagGFP (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 (tetramer, Clontech), zsyollow 1 (tetramer, Clontech).
7) Pharmaceutically acceptable salts, acids or derivatives of the above drugs.
8. In claims 2 and 4, Drug in formulas (II) and (IV)1And Drug2Conjugates of formula (II) and (IV) are useful for detecting, monitoring or studying the function of a cell-binding molecule, its interaction with a target cell, and/or its interaction with a target, particularly a target cell, for chromogenic molecules.
9. In claims 2 and 4, Drug1And Drug2Preferably selected from tubulysin, calicheamicin, auristatin, maytansinoids, CC-1065 homologues, daunorubicin and doxorubicin compounds, taxanes (taxanes), cryptophycin,epothilone, benzodiazepine dimers (e.g. Pyrrolobenzodiazepine (PBD), tomomycin, antromycin, indolophenyldiazepine, imidazobenzothiadiazole or oxazolidobenzodiazepine dimers), calicheamicin and enediyne antibiotics, actinomycin, azaserine, bleomycin, epirubicin, tamoxifen, idarubicin, dolastatin/auristatin (e.g. MMAE, MMAF, auristatin PYE, auristatin TP, auristatin 2-AQ, 6-AQ, EB (AEB) and efp (aefp)), duocarmycin, thiotepa, vincristine, hemimitadine, namide, microgenin, radiosumin, alterobin, roscleromin, theonelamide, esperamicamicin, siRNA, a pharmaceutically acceptable salt, acid and/or a derivative thereof and/or homologue thereof.
10. The cell binding agent/molecule of claims 2 and 3 selected from the group consisting of antibodies, proteins, vitamins (e.g., folic acid), peptides, polymeric micelles, liposomes, lipoprotein-based drug carriers, nanoparticle drug carriers, dendrimers, and coated with cell binding ligands or combinations thereof.
11. The cell binding agent/molecule of claims 2,3 and 10 is preferably an antibody, a whole antibody (polyclonal antibody, monoclonal antibody, dimer, multimer, multispecific antibody, e.g. bispecific antibody); a single chain antibody; an antibody fragment that binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody or a monoclonal antibody fragment that binds to a target cell, a chimeric antibody fragment that binds to a target cell, a single domain antibody fragment that binds to a target cell, a surface modified antibody, a single chain surface modified antibody, or a surface modified antibody fragment that binds to a target cell, a humanized antibody, a single chain humanized antibody, or a humanized antibody fragment that binds to a target cell, an anti-idiotypic antibody (anti-Id), a CDR, a diabody, a trivalent antibody, a minibody, an immune protein (SIP), a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a trophic transport molecule, a high molecular weight protein.
12. The cell-binding agent/molecule of claims 2,3 and 10 may be any agent capable of targeting the following cells: tumor cells, virus-infected cells, microorganism-infected cells, parasite-infected cells, cells of autoimmune diseases, activated tumor cells, bone marrow cells, activated T cells, invading B cells, or melanocytes.
13. The cell binding agent/molecule of claims 2,3 and 10 may 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, CD, CD274, CD276(B7-H3), CD303, CD304, CD309, CD326,4-1BB,5AC,5T4(Trophoblast glycoprotein, TPBG, 5T4, WNT-activated inhibitor 1 or WAIF1), adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor kinase 1, AFP, AKAP-4, ALK, α integrin, α v β 6, aminopeptidase N, amyloid β, androgen receptor, angiogenesis promoting protein factor 2, angiogenesis promoting protein factor 3, annexin A1, anthrax toxin protective antigen, anti-transfer protein receptor, AOC3(VAP-1), B7-H3, Bacillus anthracis, BAAOFF (B cell activating factor), B lymphoma cells, bcr-abl, bombesin, RIS, C5, C242 antigen, CA125 (carbohydrate antigen, MUC 125-52), CAIX 16-BOCA, CALA 31, CALA 9, CALA 6326, carbonic anhydrase IX, cardiac myoglobin, CCL11(C-C fragment chemokine 11), CCR4(C-C chemokine receptor 4, CD194), CCR5, CD3E (epsilon), CEA (carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (factor D), Ch4D5, cholecystokinin 2(CCK2R), CLDN18(Claudin-18), clusterin 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, cyclic ribonuclease, cyclin B1, CYP1B1, Cytomegalovirus B, Dabigan (DPP-like peptide-4), DPP-364-delta-peptide (DPP-4), DR5 (death receptor 5), Escherichia coli shiga toxin type-1, Escherichia 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 (TMPRSS2 ETS fusion gene), Escherichia coli, ETV6-AML, FAP (fibroblast activation protein alpha), FCGR1, alpha fetoprotein, fibrin II beta chain, fibronectin extra domain-B, FOLR (folate receptor), folate receptor alpha, folate hydrolase, Fos-related antigen 1, respiratory syncytial virus F protein, crimped receptor, fucosyl GM1, GD2 ganglioside, G-28 (cell surface antigen), Globn 3, Glyphon 3, Glyphytal 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, guanylate cyclase C receptor, thermostable enterotoxin receptor (hSTAR), heat shock proteins, 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 E2/E7, Hsp90, hTERT, ICAM-1 (intercellular adhesion molecule 1), idiotype, IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-. gamma., 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 (α 4, α IIb β 3, α v β 3, α 4 β 7, α 5 β 1, α 6 β 4, α 7 β 7, α ll β 3, α 5 β 5, α v β 5), interferon-. gamma-inducible protein, ITGA2, ITGB2, KIR2D, LCK, Leman-Y antigen, LFA-1 (lymphocyte function-associated 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, MAGE 4, MART1, MCP-1, MIF (macrophage migration inhibitory factor, or Glycosyl Inhibitory Factor (GIF)), MS4A1 (transmembrane 4 domain subfamily A member 1), MSMSMesothelin, MUC1 (mucin 1, cell surface-associated (MUC1) or Polymorphic Epithelial Mucin (PEM)), MUC1-KLH, MUC16 CA125), 1 (monocyte protein 1), Melana/MART 3, IAP, MPG, MS4A1, CN, myelos associated antigen, Myostatin-84, Netas-84, phospholipid 7374, Nectin-22, ASNA-7374, My-11, Mylar-S-11, My-S, NGF, neuronal apoptosis-regulating protease 1, NOGO-A, Notch receptors, nucleolin, Neu oncogene products, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low density lipoprotein), OY-TES1, P21, A 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-R alphA (alphA platelet-derived growth factor receptor), PDGFR-betA, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived growth factor receptor betA, sodium phosphate cotransporter, PMEL 17, poly, protease 3(PR1), prostate cancer, PS (phosphatidylserine), prostate cancer cells, Aerugo, PSMA, PSA, PSCA, rabies glycoprotein, RHD (Rh polypeptide 1(RhPI), CD240), Rhesuus factor, RANKL, RhoC, Ras mutation, RGS5, ROBO4, respiratory syncytial virus, RON, sarcoma translocation breakpoint, SART3, Sclerostin, SLAMF7(SLAM member 7), Selectin P, SDC1 (syndecan 1), systemic lupus erythematosus (a), somatomedin C, SIP (sphingosine-1-phosphate), somatostatin, sperm protein 17, SSX2, STEAP1 (6-transmembrane epithelial prostate antigen 1), STEAP2, STn, TAG-72 (tumor-associated glycoprotein), survivin, T cell receptor, T cell protein, TEM1 (tumor vascular endothelial marker 1), TENB2, Tenascin C (TN-C), TGF-alpha, TGF-beta (transforming growth factor beta), TGF-beta 1, TGF-beta 2 (transforming growth factor beta 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-specifically 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.
14. The tumor cell of claim 12, wherein the tumor cell is selected from the group consisting of lymphoma cells, myeloma cells, renal cell carcinoma cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colon cancer cells, gastric cancer cells, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer cells, testicular cancer cells, and any uncontrolled, accelerated growth, differentiation carcinogenic cell.
15. Linker component R according to claims 1,2,3 and 41And R2May be comprised of one or more linker components: 6-Maleimidohexanoic acid (MC), 3-Maleimidopropionic acid (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), 4-Thiooctanoic acid (SPP), 4- (N-Maleimidomethyl) cyclohexane-1-carboxylic acid (MCC), 4-Thiobutanoic acid (SPDB), Ethylmaleimide (ME), 4-thio-2-hydroxysulfobutanoic acid (2-Sulfo-SPDB), Pyridinethiol (PySS), alkoxyamino (AOA), Ethyleneoxy (EO), 4-methyl-4-dithio-pentanoic acid (MPDP), Azide (N-Maleimidopropanoic acid (MP), Val-citrulline-1-carboxylic acid (MCC), 4-Thiobutanoic acid (SPDB), 4-Thiobutanoic acid (MED), 4-Thiobutanoic acid (MPDP), and N-Thiooctane (N-L-D), and its salts3) An alkyne, a dithio, a peptide, and/or a (4-acetyl) aminobenzoate (SIAB).
16. The method of claim 2, wherein the Drug1And Drug2Preferred conjugates of the general structural formula (II), which are tubulisin homologs, T01, T02, T03, T04, T05 and T06:
Figure FDA0003226420290000131
Figure FDA0003226420290000141
Figure FDA0003226420290000151
wherein the mAb is an antibody; z3And Z3' is H, OP (O) (OM)1)(OM2)，OCH2OP(O)(OM1)(OM2)，OSO3M1，R1Or O-glycoside (glycoside, galactoside, mannoside, glucoside, fructoside, etc.), NH-glycoside, S-glycoside or CH2-a glycoside; m1And M2Independently H, Na, K, Ca, Mg, NR1R2R3(ii) a n is
Figure FDA0003226420290000152
X1，X2，R1，R2And R3As defined in claim 1.
17. The method of claim 2, wherein the Drug1And Drug2A preferred conjugate, C01, which is a calicheamicin homolog, has the general structural formula (II):
Figure FDA0003226420290000153
wherein m isAb is an antibody; n is
Figure FDA0003226420290000154
X1，X2，R1And R2As defined in claim 1.
18. The method of claim 2, wherein the Drug1And Drug2A preferred conjugate, M01, which is a maytansinoid homologue, having the general structural formula (II):
Figure FDA0003226420290000161
wherein the mAb is an antibody; n is
Figure FDA0003226420290000162
X1，X2，R1And R2As defined in claim 1.
19. The method of claim 2, wherein the Drug1And Drug2Preferred conjugates having the general structural formula (II) Tx01, Tx02 and Tx03 are taxane homologs:
Figure FDA0003226420290000163
Figure FDA0003226420290000171
wherein the mAb is an antibody; n is
Figure FDA0003226420290000172
X1，X2，R1And R2As defined in claim 1.
20. The method of claim 2, wherein the Drug1And Drug2Preferred conjugates having the general structural formula (II) are CC01, CC02, CC03, which are CC-1065 homologues:
Figure FDA0003226420290000173
wherein the mAb is an antibody; n is
Figure FDA0003226420290000182
Z4And Z4Is HH, PO (OM)1)(OM2)，SO3M1，CH2PO(OM1)(OM2)，CH3N(CH2CH2)2NC(O)-，O(CH2CH2)2NC(O)-，R1Or a glycoside; x3And X3' is O, NH, NHC (O), OC (O), C (O) O, R1Or by default; x1、X2、R1、R2、M1And M2As defined in claim 1.
21. The method of claim 2, wherein the Drug1And Drug2Preferred conjugates Da01, Da02, Da03 and Da04 having the general structural formula (II) are daunorubicin/doxorubicin homologues:
Figure FDA0003226420290000181
Figure FDA0003226420290000191
wherein the mAb is an antibody; n is
Figure FDA0003226420290000192
X3And X3' is independently H, O, NH, NHC (O) NH, C (O), OC (O) or R1；X1、X2、R1And R2As defined in claim 1.
22. The method of claim 2, wherein the Drug1And Drug2Preferred conjugates having the general structural formula (II) are auristatin homologs Au01, Au02, Au03, Au04, and Au 05:
Figure FDA0003226420290000193
Figure FDA0003226420290000201
wherein the mAb is an antibody; n is
Figure FDA0003226420290000202
X3And X3' is independent CH2，O，NH，NHC(O)，NHC(O)NH，C(O)，OC(O)R1Or by default; x4And X4' is independent CH2，C(O)，C(O)NH，C(O)N(R1)，R1，NHR1，NR1，C(O)R1Or C (O) O; z3And Z3' is independently H, R1，OP(O)(OM1)(OM2)，NHR1，OCH2OP(O)(OM1)(OM2)，OSO3M1Or O-glycoside (glycoside, galactoside, mannoside, glucoside, fructoside, etc.), NH-glycoside, S-glycoside or CH2-a glycoside; m1And M2Independently H, Na, K, Ca, Mg, NR1R2R3；X1，X2，R1，R2And R3As defined in claim 1.
23. The method of claim 2, wherein the Drug1And Drug2Preferred conjugates of benzodiazepine dimer homologues having the general structural formula (II) PB01, PB02,PB03, PB04, PB05, PB06, PB07, and PB 08:
Figure FDA0003226420290000203
Figure FDA0003226420290000211
Figure FDA0003226420290000221
wherein the mAb is an antibody; n is
Figure FDA0003226420290000222
X3And X3' is independent CH2，O，NH，NHC(O)，NHC(O)NH，C(O)，OC(O)，OC(O)(NR3)，R1，NHR1，NR1，C(O)R1Or by default; x4And X4' 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 R3As defined in claim 1. In addition, R1And/or R2A default may be used.
24. A pharmaceutical composition for the treatment or prevention of cancer, autoimmune disease or infectious disease comprising a therapeutically effective amount of the conjugate of claims 2, 16, 17, 18, 19, 20, 21, 22, and/or 23, and a pharmaceutically acceptable salt, carrier, diluent, or excipient thereof, or a combination thereof.
25. The conjugate of claim 2, 16, 17, 18, 19, 20, 21, 22, 23 or 24, having in vitro, in vivo or ex vivo activity.
26. The conjugate of claim 2, 16, 17, 18, 19, 20, 21, 22, or 23, wherein linker component R1And/or R2A peptide comprising 1-20 natural or unnatural amino acid units, aminobenzyl units, 6-maleimidocaproyl units, disulfides, thioether units, hydrazone units, triazole units, or alkoxyoxime units.
27. The linker of claim 2, 16, 17, 18, 19, 20, 21, 22, or 23 which is cleavable by a protease.
28. A pharmaceutical composition comprising a therapeutically effective amount of a conjugate of claims 2, 16, 17, 18, 19, 20, 21, 22, 23 or/and 24 for simultaneous use with other therapeutic agents such as chemotherapeutic agents, radiation therapy, immunotherapeutic agents, autoimmune disease agents, anti-infective agents or other conjugates, synergistically effective to treat or prevent cancer, autoimmune disease or infectious disease.
29. The synergist of claim 28 is preferably selected from one or more of the following drugs: (iii) abatacept, abiraterone acetate, acetaminophen/hydrocodone, adalimumab, afatinib maleate, alemtuzumab, alitretinoin, trastuzumab, amphetamine mixed salt (amphetamine/dextroamphetamine) anastrozole, aripiprazole, atazanavir, Atezolizumab, atorvastatin, axitinib, beline, bevacizumab, cabazitaxel, cabozotene, bexarotene, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, budesonide/formoterol, buprenorphine, capecitabine, carfilzomib, celecoxib, certiniib, cetuximab, ciclosporin, cinacalcetrizininib, dabigatran, dabrafenib, daraflavine, darunavir, imatinib mesylate, valacitinib, dexlansoprazole, valacil, dexmethylphenidate, Dinutuximab, doxycycline, duloxetine, emtricitabine/rilpivirine/tenofovir disoproxil fumarate, emtricitabine/tenofovir/efavirenz, enoxaparin, enzalutamide, alfapentine, erlotinib, esomeprazole, ezetimibe/simvastatin, fenofibrate, filgrastim, fingolimod, fluticasone propionate, fluticasone/salmeterol, fulvestrant, gefitinib, glatiramer, goserelin acetate, imatinib, temozolob, ibrutinib, insulin aspart, insulin detemir, insulin glargine, interferon beta 1a, interferon beta 1b, lapatinib, Iplilimumab, ipratropium bromide/salbutamol, lanetamol acetate, lefludarabine, xylene sulfonate, letrozole, levothyroxine, lidocaine, linezolid, liraglutide, MEDI4736, memantine, methylphenidate, metoprolol, modafinil, mometasone, nilotinib, Nivolumab, ofatumumab, aurilizumab, Pazopanib, pemetrexed, pertuzumab, pneumococcal conjugate vaccine, pomalidomide, pregabalin, quetiapine, rabeprinozide 223 chloride, raloxifene, latilatavir, ramucirumab, ranibizumab, Rigefinitib, rituximab, rivaroxaban, romidepsin, rosuvastatin, ruxotinib phosphate, albuterol, sevelamine, sildenafil, siultximab, sitagliptin/metformin, solifenacin, sorafenib, sunitinib, tematinib, ritabine, rituximab, temsirolimumab, and/temsirolimumab, testosterone gel, thalidomide (Talidex), tiotropium bromide, toremifene, tremetinib, trastuzumab, Tretinoin, ecutezumab, valsartan, vandetanib, vemurafenib, vorinostat, aflibercept, Zostavax and its homologues, derivatives, pharmaceutically acceptable salts, carriers, diluents or excipients, or combinations thereof.
CN202110978276.7A 2015-07-04 2015-07-04 Specific conjugation of cell binding molecules Pending CN113698335A (en)

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