CN108811499B - Specific conjugation of cell binding molecules - Google Patents

Specific conjugation of cell binding molecules Download PDF

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CN108811499B
CN108811499B CN201580082141.5A CN201580082141A CN108811499B CN 108811499 B CN108811499 B CN 108811499B CN 201580082141 A CN201580082141 A CN 201580082141A CN 108811499 B CN108811499 B CN 108811499B
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hexafluorophosphate
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CN108811499A (en
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赵珞博永新
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Hangzhou Dac Biotech Co Ltd
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Abstract

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

Description

Specific conjugation of cell binding molecules
Technical Field
The present invention describes a 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.et al, 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 widely distributed DAR values (drug-to-antibody ratios) (Wang, l., et al 2005protein sci.14, 2436; Hamblett, k.j., et al 2004clin.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 consistency for ADCs produced by this traditional coupling method, requiring higher throughput (Wakankar, a. mabs,2011,3,161-.
Therefore, numerous biotechnology companies and research institutes have been working on the development of novel site-directed conjugate coupling 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 Trans-Amylase (mTG) which can be amplified with Streptomyces mobaraensis (Strop, P., Bioconjugate chem.,2014,25,855-862; Strop, P., et al., chem. biol.2013,20,161-167; U.S. Pat. No. 8,871,908 of Rinat Co., Perey) or from Streptomyces gramicusBacterial-derived glutamyltransferases (MTGases) (Dennler, P., et al, bioconjugate. chem.2014,25,569-; 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, bioconjugate. 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. application 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, WO 2007/130453; U.S. Pat. 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 circulating system, 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 have important roles 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). Interchain disulfide bonds are more susceptible to reductive cleavage than intrachain disulfide bonds, and disulfide bonds between light and heavy chains are more susceptible to reductive cleavage than disulfide bonds between 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 cleavable properties, whereas 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 the disulfide bond between antibody chains is easier to break is adopted by many research institutes and companiesThe strategy of ligation, in which the interchain disulfide bonds of natural antibodies are reduced and then re-cross-linked by bridging, is for example the use of maleimide compounds (NGMs) known as next generation, i.e., bromides or dibromomaleimides (Schumacher, F.F., et al org. biomol. chem.2014,12,7261-7269), the use of double alkylating agents to form a three-carbon bridge (Badescu, G., et al., bioconjugate. chem.2014,25,1124-1136, Polytherics Limited patents WO2013/190272, WO2014/064424), the use of double substituted heteroaryl bridges (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 as limited by the number of disulfide bonds available for conjugate coupling on the antibody (approximately 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 drugs to achieve their therapeutic effect, it is desirable that their 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 of the invention can not only link 2 or more small molecule drugs on each linker to realize higher DAR (not less than 4), but also selectively bridge 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 GDA0001743837240000043
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. OthersThe reduced headless disulfide bond, which is difficult to access by the 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 GDA0001743837240000041
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 elements bridge Cb to L, each bridging 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) is advantageous for maintaining 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 GDA0001743837240000042
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 a toxic drug and can be selected from the group consisting of disulfide bonds, ether bonds, ester bonds, thioether bonds, thioester bonds, peptide bonds, hydrazone bonds, carbamate bonds, carbonate bonds, amine bonds (secondary, tertiary or quaternary), imine bonds, heterocycloalkyl groups, heteroaryl groups, alkoxyoxime groupsBonding the bond or amide bond to a toxic small molecule drug;
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 R2The individual atoms in (a) are combined in all possible chemical ways, such as to form 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 combinations 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 GDA0001743837240000051
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 GDA0001743837240000061
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 of formula (I), still retains the acetylenedicarboxylic acid structure capable of reacting with the sulfur atom of the cell-binding agent:
Figure GDA0001743837240000062
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 a 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 the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl,n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 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 5to 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 two-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 one 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 an aromatic or heteroaromatic group, consisting of one or several rings, containing from 3 to 14 carbon atoms, preferably from 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.225to 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 5to 14, preferably 5to 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, removed from 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 "hydroxyl protecting groups" include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyran ether, benzyl ether, p-methoxybenzyl ether, trimethylsilylether, triethylsilyl ether, triisopropylsilyl ether, tert-butyldimethylsilylether, triphenylmethylsilicone, acetyl ester, substituted acetyl ester, 2, 2-dimethylpropionate, benzoate, methylsulfonate and p-toluenesulfonate.
"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-oxabenzotriazol) -N, N' -tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; NHS, N-hydroxysuccinimide; MMP, 4-methylmorpholine; PAB, p-aminophenyl; PBS, phosphoric acid buffer solution (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 GDA0001743837240000111
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 R2The individual atoms in (a) are combined in all possible chemical ways, such as to form 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 combinations of these groups;
X1and X2Is independently selected from N (R)3) O, S or CH2；R3Is H, straight-chain alkyl having 1 to 6 carbon atoms, branched or cyclic alkyl having 3 to 6 carbon atoms, straight-chain, branched or cyclic alkenyl or alkynyl, or 1 to EEster, ether, amide or polyethoxy units (OCH) of 6 carbon atoms2CH2)pWherein p is an integer from 0 to 1000, or a combination of these groups.
In another embodiment, R1、R2And R3May be a chain structure comprising C, N, O, S, Si, and P atoms, respectively, covalently linking 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 to form 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 to 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, alkoxylamine 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 straight chain alkyl group having 1 to 6 carbon atoms,or polyethoxy units (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 GDA0001743837240000121
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 of 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-oxytrialkyl phosphate (PyBOP), Diethylpyrocarbonate (DEPC), N, n, N ', N' -Tetramethylchloroformamidine hexafluorophosphate, 2- (7-benzotriazol oxide) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), 1- [ (dimethylamine) (morpholinyl) methylene) phosphate]-1[1,2,3]Triazolo [4,5-b]1-pyridine-3-oxidophorophosphate (HDMA), 2-chloro-1, 3-dimethylImidazolium hexafluorophosphate (CIP), chlorotriazolyl alkylphosphonium 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) pyrrolidinehexafluorophosphate (PyClU), 2-chloro-1, 3-dimethylimidazolium 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 GDA0001743837240000132
) 2-isocyanoethylmorpholine (MEI), N' -tetramethylurea-oxy- (N-succinimidyl) Hexafluorophosphate (HSTU), 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP), oxy- [ (ethoxycarbonyl) cyanomethylamine]N, N, N ', N' -tetramethylthiouretetrafluoroborate (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), Azodicarbonyldipiperidine (ADD), bis (4-chlorobenzyl) azodicarboxylate (DCAD), di-tert-butyl azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD), diethyl azodicarboxylate (DEAD).
When X is CH2When the acetylene dicarbonyl on the bridge connector is connected with other functional groups through C-C bonds, the key steps for synthesizing the acetylene are bis (trimethylsilyl) acetylene and acetylene magnesium dibromide(Grignard reagent), acetylene dilithium salt or other acetylene bimetallic salt, and acid halides or anhydrides as shown in (Ib), (Ic), (Id), (Ie), (If), (Ig) and (Ih):
Figure GDA0001743837240000131
Figure GDA0001743837240000141
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 GDA0001743837240000142
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-maleimidocaproic acid ("MC"), 3-maleimidopropionic acid ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af"), p-aminobenzyloxycarbonyl ("PAB"), 4-thiovaleric acid ("SPP"), 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid ("MCC"), 4- (acetyl) aminobenzoic acid ("SIAB"), 4-thiobutyric acid (SPDB), 4-thio-2-hydroxysulfobutyric acid (2-Sulfo-SPDB), one or more ethoxy-CH-caproic acid ("MC"), and one or more of 3-maleimidopropionic acid ("MP")2CH2O-units ("EO" or "PEO"), also includingHis connecting body well known in the art, some of which are listed here.
Examples of linkers comprising these components are:
Figure GDA0001743837240000143
(containing 6-Maleimidohexanoic acid MC)
Figure GDA0001743837240000144
(including 3-Maleimidopropionic acid MP)
Figure GDA0001743837240000151
(including p-aminobenzyloxycarbonyl group PAB)
Figure GDA0001743837240000152
Figure GDA0001743837240000153
(containing Ethyl maleimide ME)
Figure GDA0001743837240000154
(including valine-citrulline)
Figure GDA0001743837240000155
(comprising 4- (N-Maleimidomethyl) cyclohexane-1-carboxylic acid MCC)
Figure GDA0001743837240000156
(comprising (4-acetyl) aminobenzoic acid)
Figure GDA0001743837240000157
(comprising 4-thio-2-hydroxysulfobutyric acid, 2-thio-SPDB)
Preferred conjugates are of the formula (II):
Figure GDA0001743837240000161
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, aminourea, 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 sulfhydryl groups, and 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 alkoxyamino 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 free thiol-containing drug; the synthesis of the cell bonder-drug conjugate combined by thioether bond is realized by the reaction of cell bonder modified by maleimide group, halogen acetyl group or ethyl sulfone and drug containing free sulfhydryl; synthesis of conjugates containing acid labile hydrazones in the molecule can be achieved by reaction of a hydrazide group and a carbonyl group in a linker, and the specific methods are also well known in the art (see P.Hamann et al, Hinman, L.M., et al, Cancer Res.1993,53, 3336-; 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. 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 GDA0001743837240000171
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, azido, ketone, aldehyde, hydrazine, N-hydroxysuccinimide ester or phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, trifluoromethanesulfonic acid, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, toluenesulfonic acid, methanesulfonic acid, 2-ethyl-5-phenylisoxazoleCarboxylic acid esters of oxazole-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 agent may be prepared as described in formula (II) above by reacting the cell-binding agent with a bridge linker as described in formula (I).
In order to achieve efficient linkage 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 cyclodextrin, 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 chromophoric groups, 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 GDA0001743837240000191
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).
But for drugs containing sulfhydryl groups, or by thioether, sulfur, via bridging linkagesThe ester or disulfide linkage to the Drug bound to the cell binding agent is preferably by Drug1 or Drug2 first linked to R by a thioether, thioester or disulfide linkage1Or R2R produced1-Drug1Or R2-Drug2The fragment is then bound to an acetylene dicarbonyl to form a bridge linker modified drug as shown in structural formula (IV).
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 present 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, e.g., interleukin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory factor, granulocyte macrophage colony stimulating factor (GM-CSF); vitamins, such as folic acid; apoproteins and glycoproteins, such as transferrin (O' Keefe et al, J.biol.chem.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 (TKIs), non-peptides or any other cell binding molecule or substance, such as bioactive polymers (Dhar, et al, proc2008,105,17356-61), 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; medarova, et al, nat. med.2007,13,372-7; javier, et al, Bioconjugate chem.2008,19,1309-12), liposomes (Medinai, et al, curr. phar. des.2004,10, 2981-9) and viral coat (flinniken, et al, Viruses nanotechnol.2009,327, 71-93).
In general, monoclonal antibodies are preferred as cell surface binding agents if appropriate monoclonal antibodies are available. 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 GDA0001743837240000211
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 implement monoclonal antibodies of the invention can be identified by their ability to immunoreact with specific receptors or to inhibit receptor activity 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 herpes virus 4(HHV-4)) or kaposi's sarcoma-associated herpes virus (KSHV), as described in 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 approach to select 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 and light chains of human globulin 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; Houdebine, 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 members of the immunoglobulin family. 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 alpha-IIb), adalimumab (anti-TNF-alpha), Adecatuzumab (anti-EpCAM, CD326), Aframumab (anti-TNF-alpha), Afuzumab (anti-CD 20), Alacizumab (anti-VEGFR 2), ALD518 (anti-IL-6), Altuzumab (Campath, MabPaath, anti-CD 52), Altumomab (anti-CEA), Anatumomab (anti-TAG-72), Ankinzumab (IMA-638, anti-IL-13), Apolizumab (anti-HLA-CEA), Azimumab (anti-CD-62L), Azimumab (anti-CD-62), atlizumab (tocilizumab, Actemra, Roactetramra, anti-IL-6 receptor), Atorlimumab (anti-Rhesus factor), Bapineuzumab (anti-beta amyloid), Basiliximab (Simulect, anti-CD 25 (alpha chain of IL-2 receptor)), Bavituximab (anti-phosphatidylserine), Bectumomab (LymphosScan, anti-CD 22), Bellinumab (Benlysta, Lymphostat-B, anti-BAFF), Benralizumab (anti-CD 125), Bertilimumab (anti-CCL 11(eotaxin-1)),besilesomab (Scientimun, anti-CEA associated antigen), bevacizumab (Avastin, anti-VEGF-A), Biciromab (FibriScint, anti-fibrin II beta chain), Bivatuzumab (anti-CD 44v6), Blinatumomab (BiTE, anti-CD 19), Brentuximab (cAC10, anti-CD 30TNRSF8), Briakumumab (anti-IL-12, IL-23), Canakumumab (Ilaris, anti-IL-1), Cantuzumab (C242, anti-CanAg), Camaprobab, Catuzomab (Removab, anti-EpCAM, anti-CD 3), CC-72), Cedeluzumab (anti-CD 4), Certololizumab (Cimzira anti-TNF-alpha), Cetuzumab (Cituzumab), anti-TNF-alpha), Cetuzumab (Cituzumab), anti-EGFR-C-4835 (anti-TAG-CT-3625), anti-TNF-CR 2, anti-Datuzumab (anti-TNF-CR 24), anti-TNF-CT-11), anti-CT-2, anti-CT (anti-CT-2), anti-CT-2), anti-CT-2, anti-CT (anti-CT-2, anti-CT (CT-CT (CT-CT, denosumab (Prolia, anti-RANKL), Detumomab (anti-B lymphoma cells), Dorlimomab, Dorlixizumab, Ecromeximab (anti-GD 3 ganglioside), Eculizumab (Soliris, anti-C5), Edibaombomab (anti-endotoxin), Edromomab (Panorex, MAb17-1A, anti-EpCAM), Efalizumab (Raptiva, anti-LFA-1 (CD11A)), Efungumab (Mycogarb, anti-Hsp 90), Elotuzumab (anti-SLAMF 7), Elsimomab (anti-IL-6), Enlimomab (anti-ICAM-1 (CD54)), Epitumab (anti-epidialin), Epitazumab (anti-CD 22), Erzuomab (anti-Fezuomab-2), anti-Epitumab (anti-interferon-Epalutab 3), Epitumab (anti-Epitomab 3), Epitamin-Epitumab (anti-Fevizumab), Epitumab), Epitamin (anti-Fevizumab, Epitumab), Epitumab (anti-Fevix 18), Epitumab), Epalutab (anti-Epalutab) (anti-Feviutab) (Fevit 3), Epalutab) (anti-Feviutab) (anti-Epalutab) (anti-Feviutab) (Feviutab), Epalutab-Epalutab) (anti-2), Epalutab-Epalutab) (anti-2), Epalutab) (anti-2), Epalutab) (anti-2), Epalutab) (anti-2), Epalutab) (anti-Epalutab) (anti-2), Epalutab) (anti-1 (anti-2), Epalutab) (anti-Epalutab, figitumumab (anti-IGF-1 receptor), Fontolizumab (anti-IFN-. gamma.), Foravirumab (anti-rabies glycoprotein), Fresolimumab (anti-TGF-. beta.), Galiximab (anti-CD 80), Gantenerumab (anti-beta amyloid), Gavilimomab (anti-CD 147 (baigin)), Gemtuzumab (anti-CD 33), Girentuzumab (anti-carbonic anhydrase 9), Glembuumumab (CR011, anti-GPB), Golomimab (Simmoni, anti-TNF-. alpha.), Gomiliximab (anti-CD 23(IgE receptor)), Ibalizumab (anti-CD 4), Ibrolimumab (anti-CD 20), Igimomab (Indomaine-125, anti-CA-125), and Myocinomab (Myoco (anti-IFN-125)scint, anti-myocardial myosin), Infliximab (Remicade, anti-TNF-. alpha.), Intetumumab (anti-CD 51), Inolimomab (anti-CD 25(IL-2 receptor. alpha. chain), eculizumab (anti-CD 22), Ipiumumab (anti-CD 152), Iratumumab (anti-CD 30(TNFRSF8)), Keliximab (anti-CD 4), Labetuzumab (CEA-Cide, anti-CEA), Lebrikizumab (anti-IL-13), Lemalesomab (anti-NCA-90 (granulocyte antigen)), Lerdelimumab (anti-TGF. beta.2), Lexatuzumab (anti-TRAIL-R2), Libivirumab (anti-HBsAg), Lintuzumab (anti-CD 33), Lumiumab (anti-CD 465), anti-CD 23 (anti-CD 23), TRAIL-R1), anti-TRAIL-Mettuzumab (anti-Mettuzumab), anti-Mettuzumab (anti-EGFR-Mettuzumab) and Mettuzumab (anti-TGF-TNF-13), mitumomab (BEC-2, anti-GD 3 ganglioside), morrolimumab (anti-rhesus factor), Motavizumab (Numax, anti-respiratory syncytial virus), Muromonab-CD3(Orthoclone OKT3, anti-CD 3), Nacolomab (anti-C242), naptumumab (anti-5T 4), natalizumab (Tysabri, anti-integrin α 4), nebumumab (anti-endotoxin), Necitumumab (anti-EGFR), neremolimumab (anti-TNF- α), Nimotuzumab (theram, Theraloc, anti-EGFR), nofetumumab, Ocrelizumab (anti-CD 20), orimumab (afolimab, anti-LFA-1 (CD 11), ofzerumumab (arva, anti-CD 20), anti-respiratory syncytial virus (anti-PDGF), anti-paulomab (anti-paulomab), anti-paxil (anti-PDGF), anti-paxil (anti-EGFR), norgestimatamumab (anti-EGFR), netuzumab (anti-LFA), anti-LFA-CD a), afovazumab (afovariac, anti-PDGF), anti-PDGF-pauci (anti-pauci), PDGF, anti-pauci, antibody (anti-C), PDGF-pauci region (anti-C), PDGF-C, antibody (anti-C), antibody (anti-TNF-C), antibody (antibody), antibody (antibody, antibody, ABX-EGF, anti-EGFR), Panobazumab (anti-Pseudomonas aeruginosa), Parkinsonizumab (anti-IL-4), Pemtumomab (Theragyn, anti-MUC 1), Pertuzumab (Omnitarg, 2C4, anti-HER 2/neu), Pexelizumab (anti-C5), Pintumomab (anti-adenocarcinoma antigen), Priliximab (anti-CD 4), Pritumumab (anti-vimentin), PRO140 (anti-CCR 5), Racotumomab (1E10, anti-N-glycolyl neuraminic acid (Neugc, NGNA) -ganglioside GM3), Rafivirumab (anti-rabies virus glycoprotein), Ramuirumab (anti-2), Ranicizumab (Lunicizumab, anti-VEGF A), anti-protective antigen (anti-VEGF), anti-protective antigen), anti-VEGF antigen (anti-VEGF), anti-protective antigen (anti-VEGF)Cytovirus glycoprotein B), Reslizumab (anti-IL-5), Rilotuzumab (anti-HGF), Rituximab (MabThera, Rituxanmab, anti-CD 20), Robatimumab (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), Seviruzumab (anti-cytomegalovirus), Sibrotuzumab (anti-FAP), Siflumumab (anti-IFN-. alpha.), Sirtuzumab (anti-IL-6), Siplizumab (anti-CD 2), Siplizumab (anti-CD 95 (anti-CD 33), Solanazemab (anti-beta-amyloid), Sonepucinol-1-IL-6), Siplizumab (anti-CD 2), anti-Tatuzumab (anti-Tavacizumab), anti-S-TNF-gamma-2), anti-TNF 95 (anti-CD 33), Solanazezumab (anti-amyloid-beta-amyloid), Sonecozumab (anti-beta-amyloid), Sonexidin-1-2), anti-beta-TNF-gamma-2), anti-gamma-3, anti-beta-gamma-beta-amyloid, anti-beta-gamma-beta-gamma-protein, anti-gamma-3, beta-gamma-, tanezumab (anti-NGF), Tapliumomab (anti-CD 19), Tefibuzumab (Aurexis, (anti-aggregatory factor A)), Telimomab, Tenitumomab (anti-tenascin C), Teneliximab (anti-CD 40), Teplizumab (anti-CD 3), TGN1412 (anti-CD 28), Ticilimumab (Tremelimumab, anti-CTLA-4), Tigatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13), Tocilimab (Atlizumab, Actemra, RoActemra, IL-6 receptor), Tolizumab (anti-CD 154(CD40L)), Tosituzumab (anti-CD 20), trastuzumab (rivumab, anti-HER 2/neuzumab), Tremellalizumab-4), anti-CTLA-CD 154 (anti-CTLA-11 3668)), Tosituzumab (anti-CD 5634), Trastuzumab (anti-TNF-II-11-II), anti-hepatitis B (anti-TUzumab), anti-TUMAb (anti-TUMAb-11), anti-TUMAb (anti-CTLA-11), anti-TUMAb-11), anti-hepatitis C (anti-11-TUMAb), anti-TUMAb-hepatitis-11-E-7), vepalimomab (anti-AOC 3(VAP-1)), Visilizumab (Nuvion, anti-CD 3), Vitaxin (anti-angiointegrin avb3), Volociximab (anti-integrin. alpha.5. beta.1), Votumumab (HumaSPECT, anti-tumor antigen CTAA16.88), Zaluteumab (HuMax-EGFR, Zanolimumab (HuMax-CD4, anti-CD 4), Ziralimumab (anti-CD 147 (baigin)), Zolinmomab (anti-CD 5), Enasicept
Figure GDA0001743837240000261
Alefacept
Figure GDA0001743837240000262
Abatacept
Figure GDA0001743837240000263
Rilonacept (Arcalalyst), 14F7 (anti-IRP-2 (Ferro regulatory protein 2)), 14G2a (anti-GD 2 ganglioside, Nat. Cancer Inst., treatment of melanoma and solid tumors), J591 (anti-PSMA, wei Cornell institute of medicine, treatment of prostate Cancer), 225.28S (anti-HMW-MAA (high molecular weight melanoma associated antigen), Sorin Radiofarci SRL (Nat. derived from Milan Italy, treatment of melanoma), COL-1 (anti-CEACAM 3, CGM1, Nat Cancer Inst., treatment of colorectal Cancer and gastric Cancer), CYT-356 (anti-IRP-2 (IRP-2)), 14G2a (anti-GD 2 ganglioside, Nat. Cancer Inst., treatment of melanoma and prostate Cancer), and/or (anti-HMW) and/S (anti-HMW)
Figure GDA0001743837240000264
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/Knoll, for the treatment of septic shock), MEDI-500(T10B9, anti-CD 3, TR α β (T cell receptor α/β), derived from Medmenone Inc, for graft-host disease, NG RISCAN (anti-TAG 72 (tumor-associated glycoprotein 72), derived from Neoprene Corp, for breast, colon and rectal cancers), Avicidin (anti-EPCAM (epithelial cell adhesion molecule)), anti-CSTTAD 1 (tumor-associated calcium glycoprotein 733 transduction, anti-gastrointestinal GA-2 (tumor-associated protein 2), anti-epithelial-2 (EGP 2), anti-KSA, KS1/4 antigen, M4S, tumor antigen 17-1A, CD326 (from NeoRx for treatment of colon, ovarian, prostate and NHL), LymphoCide (from immunology), Smart ID10 (from Protein Design Labs), Oncolym (from Techniclone Inc), Allomum (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 lung cancer and other cancers), CD309 (ovarian cancer), CD326 (solid tumor), CEACAM3 (colorectal cancer, gastric cancer), CEACAM5 (carcinoembryonic antigen), CEA, CD66e) (breast, colorectal and lung cancers), DLL4, EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4(CD184, hematological tumors, solid tumors), Endoglin (CD105, solid tumors), EPCAM (epithelial cell adhesion molecules, 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 diseases), FOLR (folate receptor, ovarian cancer), GD2 gangliosides (various cancers), G-28 (cell surface antigen lipids, melanoma), GD3 idiotype (respective cancers), heat shock proteins (various cancers), HER1 (lung cancer, stomach cancer), 2 (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 receptor (interleukin 2 receptor, 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 a1, non-hodgkin B cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast cancer), Ovarian cancer, cervical cancer, bronchial cancer, and α gastrointestinal tract cancer), MUC16(CA125) (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 products (respective cancers), P21 (various cancers), anti- (N-glycolylneuraminic acid) antibody binding sites (breast cancer, melanoma), PLAP-like 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 proteins (various cancers), Tie (CD202B), TNFRSF10B (tumor necrosis factor receptor superfamily 10B, various cancers), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, NHL, other cancers, RA and SLE), TPBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (TNF-related necrosis-inducing ligand receptor 1, lymphoma, NHL, colorectal cancer, lung cancer), VCAM-1(CD106, melanoma), VEGF, VEGF-a, VEGF-2(CD309) (various cancers). Other tumor-associated, antigens 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, CD15, CD227, CD235a, CD240, CD262, CD271, CD274, CD276(B7-H3), CD303, CD304, CD309, CD326,4-1BB,5AC,5T4(Trophoblast glycoprotein, TPBG, 5T4, WNT-activation inhibitor 1 or WAIF1), adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor kinase 1, AFP, AKAP-4, ALK, alpha integrin, alpha v beta 6, aminopeptidase N, amyloid beta, androgen receptor, angiopoietin factor 2, angiopoietin factor 3, annexin A1, anthrax toxin protective antigen, anti-metastasis protein receptor, AOC3(VAP-1), B7-H3, Bacillus, BAFF (B cell activating factor), B lymphoma cell, bcr-abl, bombesin, BOC 23, MUC 125, RISC 125 antigen (36CA 242), anthrax-36IX antigen (36125 CA242, 36CA-36IX, carbonic anhydrase 9), CALLA, CanAg, canine lupus erythematosus IL31, 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, Cycleoplast-macrophage colony stimulating factor 1, Cytomegalovirus virus B1, Cytomegalovirus 1, dabigatran, DLL4 (DeltaLigh-like 4), DPP4 (dipeptidyl-peptidase 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 (TMPRSS2ETS 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 receptor, folate hydrolase, Fos-related antigen 1, respiratory syncytial virus F protein, frizzled receptor, GM 23, GD 734, g-28 (cell surface antigen glycolipid), GD3 idiotype, GloboH, Glypian 3, N-glycolylneuraminic acid, GM3, GMCSF receptor alpha chain, growth differentiation factor 8, GP100, GPNMB (transmembrane glycoprotein NMB), GUCY2C (ornithine cyclase 2C), guanylate cyclase C (GC-C), intestinal guanylate cyclase, guanylate cyclase C receptor, heat stable 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/scatter factor, HHR, HIV-1, histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, DRHLA-B, HMAA, human chorionic gonadotropin WMAA, HNGF, human scatter factor receptor kinase, HPV E6/E7, Hsp90, hTERT, ICAM-1 (intercellular adhesion molecule 1), idiotype, IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN- γ, influenza hemagglutinin, IgE, IgE Fc region, IGHE, IL-1, IL-2R (interleukin 2 receptor), IL-4, IL-5, IL-6, IL-6R (interleukin 6 receptor), IL-9, IL-10, IL-12, IL-13, IL-17, IL-17A, IL-20, IL-22, IL-23, IL31RA, ILGF2 (insulin-like growth factor 2 integrin), alpha 4, alpha IIb β 3, alpha v β 3, alpha 4 β 7, alpha 5 β 1, alpha 6 β 4, alpha 7 β 7, alpha 5 β 5, alpha v β 5, interferon gamma-inducing protein, ITGA2, ITGB2, KIR2D, LCK, Le, Legumain, Lewis-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 inhibitor, or Glycosyl Inhibitor (GIF)), MS4A1 (transmembrane 4 domain subfamily A member 1), MSLN (mesothelin), MUC1 (mucin 1, cell surface-associated (epithelial 1) or polymorphic mucin (PEM), MUC 1-PEMH, MUC 8937 (MCP) or MULN I19), MULN 9611, MUCA 9634, MAG-associated glycoprotein, MAR-11, MAG-associated glycoprotein, MAG-7, MAG-11, MAG-7, myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), Nectin-4(ASG-22ME), NGF, neuronal apoptosis-regulating protease 1, NOGO-A, Notch receptor, nucleolin, Neu oncogene product, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low density lipoprotein), OY-TES1, P21, P53 non-mutant, P97, PAP, anti- (N-glycolylneuraminic acid) antibody binding site, PAX3, PAX5, PCSK9, PDCD1(PD-1, programmed cell death protein 1, CD279), PDGF-R alphA (alphA platelet-derived growth factor receptor), PDGFR-betA, PDL-1, PLAC1, PLAP-like testis alkaline phosphatase, platelet-derived growth factor receptor betA, combined transporter, PMEL 17, polysialic acid, protease 3(PR1), prostate cancer, PS (phosphatidylserine), prostate cancer cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, rabies glycoprotein, RHD (Rh polypeptide 1(RhPI), CD240), Rhesus factor, RANKL, RhoC, Ras mutations, RGS5, ROBO4, respiratory syncytial virus, RON, sarcoma translocation breakpoint, SART3, Sclerostin, SLAMF7(SLAM member 7), Selectin P, SDC1 (syndecan 1), systemic lupus erythematosus (a), growth regulator C, SIP (sphingosine-1-phosphate), somatostatin, sperm protein 17, SSX2, STEAP1 (6-epithelial prostate antigen 1), STEAP2, STn, TAG-72 (tumor-associated), survivin, T cell receptor, T cell transmembrane protein, 1 (tumor vessel endothelial marker 1), TENB-associated glycoprotein, TENB-32 (TNC-TN-2), TGF- α, TGF- β (transforming growth factor β), TGF- β 1, TGF- β 2 (transforming growth factor 2), Tie (CD202B), Tie2, TIM-1(CDX-014), Tn, TNF, TNF- α, TNFRSF8, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblast glycoprotein), TRAIL-R1 (TNF-related necrosis inducing ligand receptor 1), TRAILR2 (death receptor 5(DR5)), tumor-associated calcium signaling sensor 2, tumor-specific glycosylated MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, tyrosinase, VCAM-1(CD106), VEGF, VEGF-A, VEGF-2(CD309), VEGFR-1, 2, vimentin, WT1, XAGE 1, VEGFR growth factor receptors expressing any insulin, 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 targeted therapy. Target cancers include, but are not limited to, adrenocortical carcinoma, anal carcinoma, bladder carcinoma, large 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, perigonadal cell tumors, head and neck carcinoma, hypopharynx carcinoma, islet cell carcinoma, renal carcinoma (renal cell carcinoma), laryngeal carcinoma, leukemia (acute lymphocytes, acute myeloid lineage, chronic lymphocytes, chronic granulocytic tumors, hair cells), lip and oral cancers, liver cancer, lung cancer (non-small cell, small cell), lymphoma (aids-associated, 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, nasopharyngeal, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer (epithelial, germ cell tumor, low malignancy), pancreatic cancer (exocrine, islet cell cancer), paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, plasmacytoma, prostate rhabdomyosarcoma, rectal cancer, renal cell cancer (kidney cancer), Renal pelvis and ureter (migrating cells), salivary gland cancer, seiri 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, Achlorhydra autoimmune active chronic hepatitis, acute disseminated encephalomyelitis, acute hemorrhagic leukolitis, Addison's disease, azoospermia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-GBM/TBM nephritis, anti-phospholipid syndrome, anti-dyszyme 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, scarring bullous pemphigoid, coeliac disease, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, cranial arteritis, CREST syndrome, Crohn's disease (idiopathic inflammatory bowel disease), Cushing syndrome, cutaneous leukocytosis, dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes mellitus, diffuse cutaneous systemic sclerosis, Dressler syndrome, discoid erythema, lupus, endometriosis, adnexitis-related arthritis, eosinophilic fasciitis, epidermolysis bullosa, erythema nodosum, idiopathic mixed cryoglobulinemia, Erwinia syndrome, fibrodysplastic ossification, fibromyalgia, fibromyositis, fibrositis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, Goodpasture's syndrome, Graves ' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, allergic purpura, hepatitis of pregnancy, hidradenitis suppurativa, Houston syndrome (antiphospholipid syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (autoimmune thrombocytopenic purpura), nephropathy (Bergey's IgA disease), inclusion body myositis, inflammatory demyelinating polyneuritis, interstitial cystitis, irritable bowel syndrome, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, kawasaki disease, lamber-eaton 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 syndrome, meniere's disease, microscopic polyarteritis, miller-fisher syndrome, mixed connective tissue disease, hard spot, muhimeder-habeneman disease, maculi syndrome, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), neuromuscular neurosis, blepharamalis-cicatricial pemphigoid, opsis myoclonus syndrome, Ord thyroiditis, whisperinus-whispervoyas disease, PANDAS (Streptococcus-related pediatric autoimmune neuropsychiatric disease), paraenoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry Romberg syndrome, Parsonnage-Turner syndrome, parsley, pemphigus vulgaris, anemia, peripheral encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, gangrene dermatitis, pure red cell aplasia, Rasmussen encephalitis, Rasmussen's phenomenon, relapsing polychondritis, Reiter's syndrome, restless leg syndrome, posterior neurofibrosis, rheumatoid arthritis, rheumatic sarcoidosis, schizophrenia, Schmitt's syndrome, Schnitz syndrome, scleritis, scleroderma, sjogren's syndrome, spondyloarthropathies, hyperviscosity, Still's disease, stiff person's syndrome, subacute bacterial endocarditis, suzak's 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-Desmogein 3 antibody, 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, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, Babesia disease, Bacillus cereus infection, bacterial pneumonia, bacterial vaginitis, Bacteroides infection, Saccharomycosis, ascariasis, BK virus infection, black knot disease, human blastocyst protozoa infection, blastomycosis, Borliviana hemorrhagic fever, Borrelia infection, botulism (and botulism), Brazilian hemorrhagic fever, Brucella, Burkholderia infection, Brucella ulcer, calicivirus infection (norovirus and Sabovirus), Campylobacter disease, candidiasis (candidiasis, thrush), cat scratch disease, cellulitis, Chagas disease (trypanosomiasis americana), ascomycetes, chickenpox, chlamydia pneumoniae infection, cholera, phaeoblastoma, clonorchis sinensis, clostridium difficile infection, coccidioidomycosis, colorado tick fever, common cold (acute viral nasopharyngitis, acute rhinitis), creutzfeldt-jakob disease, crimean-congo hemorrhagic fever, cryptococcosis, cryptosporidiosis, cutaneous larva migration, cyclosporinosis, enterobacter infection, enterovirus infection, epidemic typhus, infectious erythema (fifth disease), rash, fascioliasis, filariasis, clostridium perfringens food poisoning, free living amoeba infection, clostridium infection, aeronecrocarpus (fuscinosis), filariasis, gerstman-straussler-scheinker disease 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 lung syndrome, helicobacter pylori infection, hemolytic uremic syndrome, nephrotic 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 papillomavirus infection, human parainfluenza virus infection, membranous taenia, infectious viral mononucleosis (mononucleosis), influenza, isosporosis, kawasaki disease, keratitis, a King's infection, Kuru, lassa fever, Legionella disease (retrograde army-legionnaires ' disease), Legionella disease (Pontiacre fever), leishmaniasis, Lyme disease, lymphofilariasis (elephantiasis), lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever, measles, melioidosis (Huygur's disease), meningitis, meningococcosis, posterior schistosomiasis, microsporidiosis, molluscum contagiosum, parotitis, mouse typhus (endemic typhus), mycoplasmal pneumonia, foot edema, myiasis, neonatal conjunctivitis (neonatal eye disease), variant Creutzfeldt-Jakod, nvCJD, Nocardia disease, onchocerciasis (Heisy), paracoccidioidomycosis (southern American mycosis), parageminiasis, paragonimiasis, pneumoconiosis, pasteurellosis, and pediculosis, pubic lice, pelvic inflammatory disease, pertussis, plague, pneumococcal infections, pneumococcal pneumonia, poliomyelitis, prevotella infection, primary amebic meningoencephalitis, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, rat bite fever, respiratory syncytial virus infection, nosesporidiosis, rhinovirus infection, rickettsialism infection, rickettsialpox, rift valley fever, rocky mountain spotted fever, rotavirus infection, rubella, salmonellosis, SARS (severe acute respiratory syndrome), scabies, schistosomiasis, septicemia, shigellasis (Bacillary dysentery), herpes zoster (shingles), smallpox (smallpox), sporotrichosis, staphylococcal food poisoning, staphylococcus aureus infection, strongyloides stercoralis, syphilis, taeniasis, tetanus, tinea barbarum (Barber itch), tinea capitis, tinea corporis, tinea cruris, tinea manuum, tinea metacarpus, tinea pedis, tinea unguium (onychomycosis), tinea versicolor, toxocariasis (eye larva migration disorder), toxocariasis (visceral larva migration disorder), toxoplasmosis, trichinosis, trichomoniasis (trichuris infection), tuberculosis, tularemia, ureaplasma urealyticum infection, venezuelan equine encephalitis, venezuneri hemorrhagic fever, viral pneumonia, west nile fever, leukotrichia (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, Anaplasma, Bacillus anthracis, Vibrio haemolyticus, Hunningvirus, ascaris, Aspergillus, Astroviridae, Babesia, Bacillus cereus, various bacteria, Bacteroides, Escherichia coli, ascaris, BK virus, Oesophaga, Protozoa, Blastomyces dermatitidis, Marjopsis, Borrelia, Clostridium botulinum, Sinomenii, Brucella, typically Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerosa, Caliciviridae, Campylobacter, typically Candida albicans and other Candida species, henkel, group A streptococci and staphylococci, trypanosoma cruzi, Leptophilus ducreyi, VZV, Chlamydia trachomatis, Colorado tick fever virus, rhinovirus, coronavirus, CJD prion, Climia-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium, Harpagophyta, various parasites, Cyclosporidium, Taenia ribbon, Cytomegalovirus, dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4) -flavivirus, Bifidobacterium fragilis, Corynebacterium diphtheriae, Schistosoma, Meldonolone nematode, Ebola, Echinococcus, Enterococcus, Enterovirus, Rickettsia, parvovirus B19, human herpesvirus 6 and human herpesvirus 7, Brucella, Pediculus hepatica and Megaster megalosa, FFI prions, filariales superfamily, clostridium perfringens, clostridium, other clostridia, geotrichum candidum, GSS prions, giardia enterica, burkholderia, bacillus spinosus and candida albicans, gonococci, klebsiella granuloma, streptococcus pyogenes, streptococcus agalactiae, haemophilus influenzae, enteroviruses, mainly coxsackie a virus 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 haemophilus thermoforniae, human bocavirus, ehrlichia, phagocytophile haemophilus, human metapneumovirus, Ehrlichia chaffeensis, human papilloma virus, human parainfluenza virus, Micromembrana tenuis and Micromembrana minitans, Epiderivirus, family Orthomyxoviridae, Isospora beijerinckii, Chryseobacterium, Klebsiella pneumoniae, Legionella pneumophila, Leishmania, Mycobacterium leprae and Mycobacterium tuberculosis, Leptospira, Listeria monocytogenes, Borrelia burgdorferiae and other species of the genus Borrelia, trichina and malaysia, lymphocytic choriomeningitis virus (LCMV), plasmodium, marburg virus, measles virus, burkholderia pseudomallei, neisseria meningitidis, retrograduate schistosomiasis, microsporida, Molluscum Contagiosum Virus (MCV), parotitis virus, rickettsia typhi, mycoplasma pneumoniae, multiple bacterial and fungal parasitic diptera larvae, chlamydia trachomatis and neisseria gonorrhoeae, vCJD prion, nocardia and other nocardia species, filaria discoideae, parabolaceae, simania and other subgenera species, pasteurella, head lice, human pediculus humanus, bordetella pertussis Yersinia pestis, streptococcus pneumoniae, pneumococcus, poliovirus, prevotella, grignard, JC fever chlamydia virus, coxococcus bokei, rabies virus, unicellular and spirochete bacteria, respiratory syncytial virus, nosema bacteria, rhinovirus, rickettsia, Leptosia, Rickettsia, rotavirus, rubella, Salmonella, SARS coronavirus, human sarcoptides, Haemonchus, somatocyte, Shigella, varicella zoster virus, Gossda or smallpox, Sphaerotheca schericola, Staphylococcus aureus, Streptococcus pyogenes, Strongyloides, Treponema pallidus, Taenia, tetanus, Trichophyton ringtonia, Xylophyrophyton, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton rubrum, Venezanum outer bottle , Trichophyton genus, Toxophyrus or toxoplasma, Toxoplasma gondii, Trichomonas vaginalis, Trichosporon trichomonas, Sanqiu, mycobacterium tuberculosis, Tulipa fraserra, Urea and equine encephalitis viruses, Venezuelan equine encephalitis virus, Vibrio cholerae, Guaranritot virus, West Nile virus, Beigelii filariosis, Yersinia pseudotuberculosis, Yersinia enterocolitica, yellow fever virus, Mucor order (mucormycosis) and Entomophthora order (Entomophthora mycosis), Pseudomonas aeruginosa of Mucor order, Campylobacter (Vibrio), Aeromonas, Eisenia, Yersinia dysenteriae, Shigella, Salmonella typhi, Spira japonicam, Pegasseudon perna, Borrelia burgdorferi, Microspira, Pneumocystis, Brucella abortus, Brucella, Mycoplasma, Rickettsia, tsutsutsutsugamsii, Chlam, and pathogenic fungi (Aspergillus fumigatus, candida albicans, histoplasma capsulata), protozoa (entamoeba histolytica, Tenas trichomonas, Hominis trichomonas, trypanosoma gambiae, trypanosoma rhodesiense, leishmania rozei, leishmania tropical, leishmania brasiliensis, pneumocystis pneumonia, plasmodium vivax, plasmodium falciparum, malaria plasmodium malaria) or helminthis (schistosoma japonicum, schistosoma mansoni, schistosoma japonicum 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 carcinoma, anal carcinoma), kaposi's sarcoma-associated herpes virus (kaposi's sarcoma), human herpes virus type four (nasopharyngeal carcinoma, burkitt 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 lead to 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 harvesting 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, the conjugates linked to the connectors of the present patent can be supplied as solutions or lyophilized solids that can be reconstituted 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 GDA0001743837240000371
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 on a number of factors, including the chemical identity, potency and bioavailability of the conjugate, the type of disease, the patient's race, the patient's disease progression, the route of administration, all factors determining the required dose, mode of administration and 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 disorder, 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 dosage ranges are 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 term "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, 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 analogs); duocarmycin (including the synthetic analogs KW-2189 and CBI-TMI); benzodiazepine dimers (e.g., dimers of Pyrrolobenzodiazepine (PBD) or tolmetin, indolophenyldiazepine, imidazobenzothiadiazole, or oxazolidinobenzodiazepine); 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); epothilones, soft-gridsSarcandra alcohol, dimolide, bryolactone, 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 (hiloda), carmofur, cytarabine, dideoxyuridine, doxifluridine, inotabine, 5-fluorouracil, floxuridine, ratitrexed (tomudex), cytosine analogs (cytarabine, cytosine arabinoside, fludarabine), purine analogs (azathioprine, fludarabine, mercaptopurine, thiamine, thioguanine), folic acid supplements, such as flurolinic acid, e) hormonal therapy agents, such as receptor antagonists, antiestrogens (megestrol, raloxifene, tamoxifen), LHRH agonists (gostaline, leuprolide acetate); anti-androgens (bicalutamide, flutamide, carrousel, betaandrosterone propionate, epiandrosterone, goserelin, leuprorelin, metulidine, nilutamide, testolactone, trilostane and other androgen inhibitors); retinoids, vitamin D3 analogs (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, and apatinibCetinib, pazopanib, vandetanib, E7080 (anti-VEGFR 2), mubritinib, ponatinib (AP 245634), bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib, wibikinimod, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, ispingus; g) antibiotics, such as enediynes antibiotics (calicheamicin, in particular calicheamicin γ 1, δ 1, α 1and β 1 (cf. J.Med. Chem.1996,39 (11)), 2103-, puromycin, triiron doxorubicin, streptozotocin, tubercidin, ubenimex, setastatin, zorubicin; h) others, such as polyketides (annonacins), in particular bullatacin and bullatacinone; gemcitabine, epoxygenases (e.g., Carborundum), 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, pirorubicin, zorubicin, mitoxantrone, MDR inhibitors (e.g., verapamil), Ca2+ATPase inhibitors (e.g. thapsigargin), histone deacetylase inhibitors (vorinostat, romidepsin, panobinostat, propylpentostatinAcid, 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, etioammonium, ethylgluconic acid, gallium nitrate, cytosine, hydroxyurea, ibandronate, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidanol, diaminenitracridine, pentostatin, methionine ammonia nitrogen mustard, pirarubicin, podophyllic acid, 2-ethylhydrazine, procarbazine;
Figure GDA0001743837240000411
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, dessamson, 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 (framycin, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, tretinomycin; b) amide alcohols: azidochloramphenicol, chloramphenicol, florfenicol, thiamphenicol; c) ansamycin: geldanamycin, herbimycin; d) carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem; e) cephem: cephem (loracarbef), cephalosporins, chlorammonicillin, cephradine, cefadroxil, cefalonium, ceftiofur or cephalotaxin, cephalexin, cefalexin, cefamandole, cefapirin, hydroxylamine-azole cephalosporin, fluxazole-cephalosporin, sporocetone, oxazoline-cephalosporin, cefbuperazone, cefcapene, cefixime, ceftizoxime, cefixime, cefoxitin, cefprozil, cefmetazole, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefetamet, cefepime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, thienam, thiethylamine-cephalosporins, cefazolin, cefepime, cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram, ceftibuten, cefoselin, ceftizoxime, cefpiramide, ceftriaxone, cefuroxime, ceftizoxime, cephamycin (cefoxitin, cefotetan, cefcyanzole), oxacephem (flomoxef, cepham); 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, framycetin, josamycin, ketolide (telithromycin, seythromycin), 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), altoxicillin, azlocillin, benzylpenicillin, benzathine phenoxymethyl penicillin, cloxacillin, procaine penicillin (mecillin), mezlocillin, methicillin, nafcillin, oxacillin, acemethicillin, penicillin, phenacetin, phenoxymethyl penicillin, 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, carnotrafloxacin, 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, penicillins 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, epothilones, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalite, 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, dithiin, stavudine (d4T), talmivir (viramidine), telbivudine, tenofovir, trifluridine valacyclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f) non-nucleoside: amantadine, acipiridine, carpivirine, diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol, ethmivirine, efavirenz, foscarnet (phosphoryl formic acid), imiquimod, peginterferon, lovirine, vidarabine, methidadone, nevirapine, NOV-205, long-acting interferon alpha, podophyllotoxin, rifampin, rimantadine, resiquimod (R-848), aceticadamantane; 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. Ligands can be bound, chelated, or complexed to radioactive metals using methods described in the literature (Current Protocols in Immunology, Volumes 1and 2, origin et al, ed. wiley-Interscience, New York, n.y., Pubs. (1991)). 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, thiocyanines 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, luciferin, Me-methoxycoumarin, NBD, Pacific Blue, Pacific Orange, PE-Cy5 conjugate, PE-R-Phycoerythrin (PE), Red 613, Seta-555-Azide, Seta-555-DBCO, Seta-555-NHS, Seta-580-NHS, Seta 680-NHS, Seta-APC-780, Seta-PerCP-680, Seta-R-PE-670, Setau-380-NHS, SeTau-405-maleimide, Seta-NHS 405-425-Seu-NHS, Setau-647-NHS, 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 investigation 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 (single, mCopoL, mCoiMcFP, monomer, MBherry 635, KmSkumura Orange 635, KmSa-5, McRed monomer, TsMa-5, McRubri, McClont, McFP, McClont, McClontech, TsMa-5, TsMa, McSa, McTamSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSmSfP, r-phycerythrin (rpe), T-Sapphire, TagCFP (dimer, Evrogen), TagGFP (dimer, Evrogen), TagRFP (dimer, Evrogen), TagYFP (dimer, Evrogen), tdTomato (tandem dimer), Topaz, TurboFP602 (dimer, Evrogen), TurboFPP635 (dimer, Evrogen), TurboGFP (dimer, Evrogen), TurboRFP (dimer, Evrogen), TurboYFP (dimer, Evrogen), Venus, wild-type GFP type, YPet, ZsGreen1 (tetramer, Clontech), zschellow 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 tomaymycin, indolophenyldiazepine, imidazobenzothiadiazine or dimers of oxazolidobenzodiazepine), calicheamicins and enediynes 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)), dactinomycin, thiotepa, vincristine, hemimitataline, nazumide, microginin, radiosubin, alternactin, microsclerominin, theonellamide, esperamicin, PNU-159682 and analogues 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., mol.pharmaceutical, 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 GDA0001743837240000471
Figure GDA0001743837240000481
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, fruits)Glycosides, etc.); m1And M2Independently H, Na, K, Ca, Mg, NR1R2R3(ii) a n is
Figure GDA0001743837240000482
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 GDA0001743837240000491
wherein the mAb is an antibody; n is
Figure GDA0001743837240000494
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 GDA0001743837240000492
wherein the mAb is an antibody; n is
Figure GDA0001743837240000493
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 GDA0001743837240000501
wherein the mAb is an antibody; n is
Figure GDA0001743837240000502
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, 5332740,5332837,5334528, 5403484, 27908,5475092,5495009 009, 5501, 5545806,5547667, 55825, 557 98, 5573922, 5585089,5585499,87161, 55956017, 5622929, 565656563425, 41780,605639,829, 417957435743574354779, 605756375637563756375637563756375637563756375637563756375637567, 60574354779, 6056375637563756375637563756375637563756375637563756375637563756375637567, 605637563756375637563756375637563756375637567, 64049, 54605637563756375637563756375637563756375637563756375637563756375637563756375637567, 54779, 54605637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637567, 544759, 544708, 54435443544759, 54605637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637567, 544354435443544759, 5443544759, 544759, 5443544354435443544759, 544354435443544759, 544354435443544354435443544354435443544354435443544354435443544759, 60563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637567, 605637567, 60563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637567, 6056375637569, 6056375637563756375637569, 605660567, 60567, 6056375660563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637567, 60567, and 605637567, 6056375637563756375637567, 6056375637563756375660566056375637563756375637563756375637567, 60563756375637. Examples of antibody-CC-1065 analog structures linked via a bridge linker are as follows:
Figure GDA0001743837240000511
Figure GDA0001743837240000521
wherein the mAb is an antibody; n is
Figure GDA0001743837240000522
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 bridging linker are shown below:
Figure GDA0001743837240000523
Figure GDA0001743837240000531
wherein the mAb is an antibody; n is
Figure GDA0001743837240000532
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; U.S. patent application 11134826,20060074008,2006022925, U.S. patent nos. 4414205,4753894,4764368,4816444, 4879278,4943628,4978744,5122368,5165923,5169774,5286637,5410024,5521284, 5530097,5554725,5585089,5599902,5629197, 565483, 5654399,5663149,5665860, 0815746, 5714586,5741892,5767236,5767237,5780588, 5837, 5840699,5965537, 6004934,6033876,6034065,6048720,6054297,6054561,6124431,6143721, 62930, 6214345,6239104,6323315,6342219, 4263426342634213, 6569834, 60396911, 6090561, 605637563756374156989, 7041924192989, 70417941794192989, 70989, 709841924192989, 70989, 79417997989, 70989, 7941989, 70989, 79417941989, 70989, 6433989, 6056989, 60644156989, 6056989, 60644156300, 6056989, 606433989, 6056300, 6056989, 6056300, 6056989, 6056300. 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 GDA0001743837240000541
Figure GDA0001743837240000551
wherein the mAb is an antibody; n is
Figure GDA0001743837240000552
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), tolamectin, indolophenyldiazepine, imidazobenzothiadiazole, or oxazolidinobenzodiazepines) 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 GDA0001743837240000553
Figure GDA0001743837240000561
Figure GDA0001743837240000571
wherein the mAb is an antibody; n is
Figure GDA0001743837240000572
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 maintained 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 where otherwise specified. 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 (J) in Hz. Mass spectral data were obtained on a Waters XevoQTof mass spectrometer (connected to a Waters Acquity UPLC high performance liquid chromatograph and a TUV detector).
EXAMPLE 1.3- (2- (2- (2-hydroxyethoxy) ethoxy) propionic acid tert-butyl ester (34)
Figure GDA0001743837240000581
80mg (0) was added to 350mL of anhydrous THF with stirring0025mol) metallic sodium 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 GDA0001743837240000582
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 showed the reaction was complete. 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 GDA0001743837240000591
To DMF (50mL) was added 2- (2- (2- (2- (tosyloxy) ethoxy) -propionic acid tert-butyl ester with stirringButyl ester 35(4.0g, 9.25mmol) and sodium azide (0.737g, 11.3 mmol). 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 GDA0001743837240000592
The crude 36(5.0g,
Figure GDA0001743837240000593
) Dissolved in ethanol (80mL) and 300mg of 10% Pd/C was added. The system was evacuated and purged with 2atm of hydrogen 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, it was purified on a silica gel column using 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 GDA0001743837240000594
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 GDA0001743837240000601
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)>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 GDA0001743837240000602
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 GDA0001743837240000603
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 GDA0001743837240000611
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 mm)% 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 GDA0001743837240000612
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 GDA0001743837240000621
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)2PO4pH7.2,4ml) 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 washed with C-18 preparative HPLC (250 mm. times.30 mm ID), water/ethanol (90% to 50% water in 55 min, flow rate 35 ml/min)Fractions containing 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 GDA0001743837240000622
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 GDA0001743837240000631
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 GDA0001743837240000632
Figure GDA0001743837240000633
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 GDA0001743837240000634
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 phosphate 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 GDA0001743837240000641
To herceptin (10mg/mL, 2.0mL, pH)7.0-8.0) adding NaH2PO4Buffer (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 GDA0001743837240000642
Figure GDA0001743837240000643
Eluting with a buffer solution to obtain
Figure GDA0001743837240000644
Conjugate 91 (about 87% yield) (ii)
Figure GDA0001743837240000645
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 GDA0001743837240000646
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 andpurification was performed 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 GDA0001743837240000651
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 incubation overnight at room temperature, the mixture was purified on a G-25 column using 100mM NaH2PO4，50mM NaCl pH
Figure GDA0001743837240000652
Figure GDA0001743837240000653
Eluting with a buffer solution to obtain
Figure GDA0001743837240000654
The conjugate 92 (b) ((
Figure GDA0001743837240000655
In the buffer solution, the buffer solution is added,
Figure GDA0001743837240000656
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 (18)

1. A bridge linker of formula (I) for use in the coupling of an antibody to a cytotoxic drug:
Figure FDA0003164261390000011
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 identical or different functional groups capable of reacting with cytotoxic drugs by forming ester or amide bonds with a small toxic moietyA sub-drug combination selected from: dithio, maleimido, haloacetyl, alkoxyamine, azide, ketone, aldehyde, hydrazine, N-hydroxysuccinimide ester, phenol, nitrophenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, trifluoromethanesulfonic acid, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, toluenesulfonic acid, methanesulfonic acid or a carboxylic ester of 2-ethyl-5-phenylisoxazole-3' -sulfonic acid;
R1and R2Is the same or different straight-chain alkyl group with 1-6 carbon atoms, branched-chain alkyl group with 3-6 carbon atoms, or ester group, amide group or polyethoxy (OCH) group with 1-6 carbon atoms2CH2)pWherein p is an integer from 0 to 100, or a combination of these groups;
X1and X2Independently selected from NH, N (R)3) O, S or CH2；R3Is a straight chain alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, or an ester, amide or polyethoxy unit (OCH) of 1 to 6 carbon atoms2CH2)pWherein p is an integer from 0 to 100, or a combination of these groups;
or R1And R2Is default, and X1And X2Independently selected from NH, N (R)3) Or CH2；R3Is a straight chain alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, or an ester, amide or polyethoxy unit (OCH) of 1 to 6 carbon atoms2CH2)pWherein p is an integer from 0 to 100, or a combination of these groups.
2. A compound of structural formula (IV):
Figure FDA0003164261390000012
wherein R is1、R2、X1And X2Is as defined in claim 1, Drug1And Drug2Are the same or different cytotoxic agents, wherein R1And Drug1Linked by ester or amide linkages, and R2And Drug2Linked by ester or amide linkages.
3. A method of making the bridge connector of claim 1, comprising: condensation of acetylene dicarbonyl or derivatives thereof with a terminal amine, alcohol or thiol of the other component as shown in (Ia) below:
Figure FDA0003164261390000021
wherein X is N (R)3) O, or S; r is R1Or R2，R1、R2And R3The definition of the method is the same as that of claim 1,
lv 1and Lv2 are the same or are respectively and independently selected from: OH, F, Cl, Br, I, nitrophenol, N-hydroxysuccinimide, phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, trifluoromethanesulfonic acid, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, p-toluenesulfonic acid, methanesulfonic acid, 2-ethyl-5-phenylisoxazole-3' -sulfonic acid, acid anhydride; or a polypeptide condensation reaction intermediate or a Mitsunobu reaction intermediate; the condensing agent comprises: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, N, N ' -diisopropylcarbodiimide, 1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate, carbonyldiimidazole, O-benzotriazol-N, N, N ', N ' -tetramethyluronium tetrafluoroborate, O-benzotriazol-tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate, benzotriazol-1-yl-oxytripyrrolidinophosphate hexafluorophosphate, diethyl pyrocarbonate, N, N ', N ' -tetramethylformamidinium hexafluorophosphate, 2- (7-oxybenzotriazole) -N, n, N ', N' -tetramethyluronium hexafluorophosphate, 1- [ (dimethylamine) (morpholinyl) methylene ] -1[1,2,3] triazolo [4,5-b ] 1-pyridine-3-oxohexafluorophosphate, 2-chloro-1, 3-dimethylimidazolium hexafluorophosphate, chlorotriazolylphosphonium hexafluorophosphate, bis (tetramethylene) fluorocarboxamide, N, N, N ', N' -tetramethyl-thio- (1-oxo-2-pyridyl) thiouronium hexafluorophosphate, 2- (2-pyridone-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate, thio- (1-oxo-2-pyridyl) -N, N, n ', N ' -tetramethylthiourea hexafluorophosphate, O- [ (ethoxycarbonyl) cyanomethylamine ] -N, N, N ', N ' -tetramethylthiourea hexafluorophosphate, (1-cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylamino-morpholine-carbonium hexafluorophosphate, (benzotriazol-1-yloxy) dipyrrolidinecarbohexafluorophosphate, N-benzyl-N ' -cyclohexylcarbodiimide, dipyrrolidinyl (N-succinimidyloxy) carbonium hexafluorophosphate, 1- (chloro-1-pyrrolidinylmethylene) pyrrolidinium hexafluorophosphate, 2-chloro-1, 3-dimethylimidazolium tetrafluoroborate, (benzotriazol-1-yloxy) dipiperidinecarbohexafluorophosphate, 6-chlorobenzotriazole-1, 1,3, 3-tetramethyluronium tetrafluoroborate, tris (dimethylamino) phosphine hexafluorophosphate bromide, 1-N-propylphosphoric anhydride, 2-isocyanoethylmorpholine, N, N, N ', N ' -tetramethyluronium-oxy- (N-succinimidyl) hexafluorophosphate, 2-bromo-1-ethylpyridinium tetrafluoroborate, oxy- [ (ethoxycarbonyl) cyanomethylamine ] -N, N, N ', N ' -tetramethylthiouretetrafluoroborate, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholinium hydrochloride, 2-succinimidyl-1, 1,3, 3-tetramethyluronium tetrafluoroborate, N, N, N ', n' -tetramethyl-O- (3, 4-dihydro-4-oxo-1, 2, 3-benzotriazin-3-yl) urea tetrafluoroborate, azodicarbonyl dipiperidine, bis (4-chlorobenzyl) azodicarboxylate, di-tert-butyl azodicarboxylate, diisopropyl azodicarboxylate, diethyl azodicarboxylate.
4. A method of making the bridge connector of claim 1, comprising: bis (trimethylsilyl) acetylene, magnesium acetylene dibromide, acetylene dimetallic salts, and acid halides or anhydrides as shown in (Ib), (Ic), (Ie), (If), (Ig) and (Ih):
Figure FDA0003164261390000031
where M is Na, K, Li, Cu, CuLi, Sn, Ti, Ca, Mg or Zn.
5. The compound of claim 2, wherein the Drug1And Drug2For chromogenic molecules, the compounds of formula (IV) can be used to detect, monitor or study the function of cell-binding molecules, the interaction of cell-binding molecules with target cells, and/or the interaction of conjugates with target cells.
6. The compound of claim 2, wherein the Drug1And Drug2Selected from: tubuyysin, calicheamicin, maytansinoids, CC-1065 homologues, daunorubicin or doxorubicin compounds, taxanes, cryptophycin, epothilones, benzodiazepine dimers, calicheamicins and enediynes antibiotics, actinomycin, azaserine, bleomycin, epirubicin, tamoxifen, idarubicin, dolastatin, auristatin, duocarmycin, thiotepa, vincristine, hemistalin, azumamide, microogin, radiosubmin, alterobin, microsclerominin, theonelamide, esperamicin, siRNA, ribozymes and/or pharmaceutically acceptable salts thereof and/or homologues of the above molecules.
7. The compound of claim 2, wherein the Drug1And Drug2Selected from: pyrrolobenzodiazepines, tolmetins, antrocin, indolophenyldiazepines, imidazobenzothiadiazolazepinoids or oxazolidinebenzodiazepine dimers, MMAE, MMAF and auristatins.
8. The compound of claim 2, wherein linker component R1And R2Consisting of one or more of the following linker components: 6-maleimidocaproic acid, 3-maleimidopropionic acid, valine-citrulline, alanine-phenylalanine, p-aminobenzyloxycarbonyl, 4-thiovaleric acid, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid, 4-thiobutyric acid, ethylmaleimide, 4-thio-2-hydroxysulfobutyric acid, pyridinedithiol, alkoxyamino, vinyloxy, 4-methyl-4-dithio-valeric acid, azide, alkyne, dithio, peptide, andor (4-acetyl) aminobenzoate.
9. The compound of claim 2, wherein Drug1And Drug2Is Tubulysin homologue.
10. The compound of claim 2, wherein Drug1And Drug2Is a calicheamicin homolog.
11. The compound of claim 2, wherein Drug1And Drug2Is maytansine alkaloid homolog.
12. The compound of claim 2, wherein Drug1And Drug2Is taxane homologue.
13. The compound of claim 2, wherein Drug1And Drug2Is CC-1065 homologue.
14. The compound of claim 2, wherein Drug1And Drug2Is daunorubicin or doxorubicin homolog.
15. The compound of claim 2, wherein Drug1And Drug2Is an auristatin homolog.
16. The compound of claim 2, wherein Drug1And Drug2Is a benzodiazepine dimer homologue.
17. A compound selected from:
Figure FDA0003164261390000041
Figure FDA0003164261390000051
。
18. a compound selected from:
Figure FDA0003164261390000052
。
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