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WO2015155753A2 - Novel linkers and their uses in specific conjugation of drugs to a biological molecule - Google Patents

Novel linkers and their uses in specific conjugation of drugs to a biological molecule Download PDF

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Publication number
WO2015155753A2
WO2015155753A2 PCT/IB2015/056083 IB2015056083W WO2015155753A2 WO 2015155753 A2 WO2015155753 A2 WO 2015155753A2 IB 2015056083 W IB2015056083 W IB 2015056083W WO 2015155753 A2 WO2015155753 A2 WO 2015155753A2
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cell
drug
receptor
conjugate
antibody
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WO2015155753A3 (en
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Robert Yongxin Zhao
Robert Zhao
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Sushou M conj Biotech Co Ltd
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Sushou M conj Biotech Co Ltd
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Priority to PCT/IB2015/056083 priority Critical patent/WO2015155753A2/en
Priority to CN202210788010.0A priority patent/CN115300640A/en
Priority to CA2991975A priority patent/CA2991975C/en
Priority to CN201580082582.5A priority patent/CN108289964B/en
Priority to JP2018506984A priority patent/JP6817288B2/en
Publication of WO2015155753A2 publication Critical patent/WO2015155753A2/en
Publication of WO2015155753A3 publication Critical patent/WO2015155753A3/en
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/08—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon radicals, substituted by hetero atoms, attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
    • C07D263/08—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D263/16—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D263/18—Oxygen atoms
    • C07D263/20—Oxygen atoms attached in position 2
    • C07D263/26—Oxygen atoms attached in position 2 with hetero atoms or acyl radicals directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/60—Two oxygen atoms, e.g. succinic anhydride
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/06—Peri-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes

Definitions

  • the present invention relates to linkers used for the specific conjugation of compounds, in particular, cytotoxic agents to pairs of sulfur atoms of a biological molecule at two drugs per linker.
  • the present invention also relates to methods of making cell-binding agent-drug (cytotoxic agent) conjugates in a specific manner comprising either modification of drugs with these linkers first, followed by reaction with prepared cell-binding agents; or modification of cell-binding agents with these linkers first, followed by reaction with drugs.
  • chemotherapeutic drugs are their narrow therapeutic windows due to they normally cannot discriminate between normal and malignant cells, thus causes side effects which limit the tolerated doses below the clinically effective ones.
  • immunotherapy normally in the form of monoclonal antibodies (mAb) can specifically bind to certain proteins or molecules of malignant cells, leaving normal cells unharmed, and thus has less side effects and bigger therapeutic windows than chemotherapy.
  • Monoclonal antibodies (mAb) can target against malignant cells by several mechanisms, such as, 1). Making the cancer cell more visible to the immune system (Villaruz, L. C. et al, 2014, Transl Lung Cancer Res, 3, 2-14; Camacho, L. H. 2015 Cancer Med 4, 661-72); 2).
  • Blocking growth signals (Dillman, R. O. 2011, Cancer Biother Radiopharm, 26, 1-64; Ferris, R. L. et al 2010, J Clin Oncol, 28, 4390-9); 3). Stopping new blood vessels from forming (Arrillaga-Romany, I., et al, 2014, Expert Opin Investig Drugs, 23, 199-210); 4). Delivering radiation to cancer cells (Chapuy, B. et al, 2007, Biotechnol J. 2, 1435-43); 5). Delivering chemotherapy drug to cancer cells (Chari R. J. 2008 Acc Chem Res. 41, 98-107;
  • the first-generation ADCs including Kadcyla and Adcetris, are produced through nonselective conjugation of native lysine amines or interchain cysteine thiols on an antibody respectively to a cytotoxic drug. Since there are over 50 surface-exposed lysines and 8 hinge cysteine residues in IgGl antibodies, this nonselective conjugation results in randomly cross-linkage of cytotoxic drugs to practically all areas of the antibody molecule, particularly having a diverse population of ADCs with a wide distribution of drugs per antibody (DAR) (Wang, L., et al. 2005 Protein Sci. 14, 2436; Hamblett, K. J., et al. 2004 Clin. Cancer Res. 10, 7063).
  • DAR drugs per antibody
  • Disulfide bond structure is critical for the structure, stability, and biological functions of IgG molecules.
  • each IgG contains a total of 12 intra-chain disulfide bonds; each disulfide bond is associated with an individual IgG domain.
  • the two heavy chains are connected in the hinge region by a variable number of disulfide bonds: 2 for IgGi and IgG 4 , 4 for IgG 2 and 11 for IgG 3 .
  • the light chain of the IgGi is connected to the heavy chain by a disulfide bond between the last cysteine residue of the light chain and the fifth cysteine residue of the heavy chain.
  • the light chain is linked to the heavy chain by a disulfide bond between the last cysteine residue of the light chain and the third cysteine residue of the heavy chain (Liu, H. and May, K., 2012, mAbs
  • the upper disulfide bond of the two inter heavy chain disulfide bonds was more susceptible than the lower one. Furthermore, disulfide bonds in the CH2 domain were the most susceptible to reduction. Disulfide bonds in VL, CL, VH, and CHI domains had similar and moderate susceptibility, while disulfide bonds in the CH3 domain were the least susceptible to reduction (Liu, H, et al Anal. Chem., 2010, 82, 5219-5226).
  • next generation maleimides (NGMs) (Schumacher, F.F., et al 2014, Org. Biomol. Chem. 12, 7261-7269; UCL Cancer Institute), applying bis-alkylating reagents via a three-carbon bridge (Badescu, G., et al., 2014, Bioconjug. Chem.
  • bridge linkers were designed in the way to conjugate only one cytotoxic agents to a pair of disulfide bonds, and therefore at most of time they only produced ADCs at DAR less than 2 (drugs per antibody), due to limited numbers (about two pairs) of reduced disulfide bonds are more accessible for conjugation.
  • novel disulfur bridge linkers of this invention that not only are able to conjugate two or more drugs per linker for achieving higher DARs (>4), but also can selectively rebridge pairs of reduced inter chain disulfide bonds on surface of antibody, which are generated by overloaded TCEP or DTT reduction agents. And the over reduced pairs of thiol groups that are inaccessibly reached by the bridge linkers can be recoupled (regenerated) by an oxide, e.g. dehydroascorbic acid (DHAA) or Cu(II), to form back disulfide bonds at the end of conjugation.
  • DHAA dehydroascorbic acid
  • Cu(II) Cu(II
  • the bridge linkers of this invention containing a 2,3-disubstituted succinic group, or 2- monosubstituted, or 2,3-disubstituted fumaric or maleic (trans (E) ⁇ or cis (Zj-butenedioic) group have less payload loss as compared to their nonhydrolyzed bromo or dibromo- maleimide linkers which were tested in our lab.
  • the methods of the instant invention can be used to for the immunoconjugates that carry a combination of drugs, in particular different drugs, which can be delivered simultaneously and specifically to a particular target site, where the therapeutic molecules in the medicament are highly homogeneous, with lot-to-lot consistency.
  • immunoconjugates include: simultaneous targeted delivery of multiple drugs that act synergistically in target- ing malignant cells; combining drugs that act in different phases of the cell cycle to increase the number of target cells exposed to a particular pharmaceutical drug or effect; minimized exposure to non-target cells, tissues or organs; precise control over drug pay- loads and drug ratios leading to homogenous final products.
  • the bridge linkers of the invention can make homogeneous production of specific ADCs in a simple manner.
  • the present invention provides linkers containing a 2,3-disubstituted succinic group, or 2-monosubstituted, or 2,3-disubstituted fumaric or maleic (trans (E)- or cis (Zj- butenedioic) group to link two drugs to a cell-binding agent (e.g., an antibody).
  • a cell-binding agent e.g., an antibody
  • the preferred formula of the cell-binding molecule-linker-drug conjugates can be represented as: , wherein Cb is a cell-binding agent, L Cb is a cell-binding agent, L is a linker containing succinic, fumaric or maleic group; Drugl and Drug2 are a drug molecule; n is an integer from 1 to 30; and two S (sulfur) elements from Cb bridgely link to L, which covalently connects two or more drugs.
  • the advantages in applying the linker in the cell molecule-drug conjugate are: a).
  • the linker is represented by Formula (I)
  • U and LP represent the same or different leaving group that can be substituted by a thiol.
  • Such leaving groups are, but are not limited to, a halide (e.g., fluoride, chloride, bromide, and iodide), methanesulfonyl (mesyl), /?-toluenesulfonyl (tosyl),
  • triflate trifluoromethylsulfonyl
  • trifluoromethylsulfonate nitrophenol, N- hydroxysuccinimide (NHS)
  • phenol dinitrophenol; pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, 2-ethyl-5-phenylisoxazolium-3'-sulfonate, or an intermediate molecule generated with a condensation reagent for Mitsunobu reactions.
  • both U and LP are not H; when represents a double bond, either U or LP can be H, but are not H at the same time.
  • Z 1 and Z 2 are the same or different a function group that enables to react with a cytotoxic drug, to form a disulfide, ether, ester, thioether, thioester, peptide, hydrazone, carbamate, carbonate, amine (secondary, tertiary, or quarter), imine, cycloheteroalkyane, heteroaromatic, alkyloxime or amide bond;
  • R 1 and R 2 are the same or different, and are absent, linear alkyl having from 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl or alkynyl, or 1-6 carbon atoms of esters, ether, amide, or polyethyleneoxy unit of formula (OCH 2 CH 2 ) p , wherein p is an integer from 0 to about 1000, or combination thereof.
  • R 1 and R 2 are respectively a chain of atoms selected from C, N, O, S, Si, and P, preferably having 0-500 atoms, which covalently connects to X 1 or X 2 and Z 1 or Z 2 .
  • the atoms used in forming the R 1 and R 2 may be combined in all chemically relevant ways, such as forming alkylene, alkenylene, and alkynylene, ethers, polyoxyalkylene, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas,
  • semicarbazides carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, peptides, acyloxylamines, hydroxamic acids, or combination thereof.
  • X 1 and X 2 are independently selected from NH, N(R 3 ), O, S or C H 2 ;
  • R 3 is H, linear al- kyl having from 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl or alkynyl, or 1-6 carbon atoms of esters, ether, amide, or polyethyleneoxy unit of formula H 2 H 2 )p, wherein p is an integer from 0 to about 1000, or combination thereof.
  • this invention provides a cell-binding agent-drug conjugate of Formula (II), in which the cell-binding agent, Cb, and the drug, Drugl and Drug2, have reacted at the ends of the bridge linker:
  • Cb represents a cell-binding agent, preferred an antibody
  • the linker-drug components that are conjugated to pairs of sulfur atoms of the cell-binding molecule.
  • the sulfur atoms are preferred pairs of thiols reduced from the interchain disulfide bonds of the cell-binding agent by a reduction agent, such as DTT and/or TCEP;
  • Drug 1 and Drug 2 represent the same or different cytotoxic agents, which linked to the cell-binding agent via the bridge linker by a disulfide, thioether, thioester, peptide, hydra- zone, ether, ester, carbamate, carbonate, cycloheteroalkyane, heteroaromatic, alkoxime or amide bond; are described the same previously in
  • the present invention provides a modified cell-binding agent of Formula (III), in which the cell-binding agent, Cb, through its pair of thiols generated with reduction of disulfide bonds, has reacted with the bridge linker, which has Z 1 and Z 2 , the function groups capable of reacting with a drug:
  • the present invention provides a modified drug of Formula
  • Drug 1 , Drug 2 , U, U', R 1 , R 2 , X 1 , and X 2 are defined the same as in Formula (I) and (II).
  • the present invention further relates to a method of making a cell-binding molecule- drug conjugate of Formula (II), wherein the drugs, Drug 1 and Drug 2 are linked to a cell- binding agent via the bridge linker.
  • the present invention also relates to a method of making a modified cell-binding molecule of Formula (III), wherein the cell-binding molecule is reacted with the bridge linker of Formula (I).
  • the present invention also relates to a method of making a modified drug of formula (IV), wherein the drug is reacted with the bridge linker of Formula (I).
  • Figure 1 shows the synthesis of a bridge linker containing polyethylene glycols and the application of this linker in the conjugation of an antibody with drugs via amide bonds.
  • Figure 2 shows the synthesis of a bridge linker containing polyethylene glycols and the application of this linker in the conjugation of drugs to an antibody via amide bonds.
  • Figure 3 shows the synthesis of a bridge linker containing polyethylene glycols and the application of this linker in the conjugation of drugs to an antibody via oxime linkage.
  • Figure 4 shows the synthesis of a bridge linker containing polyethylene glycols and the application of this linker in the conjugation of two drugs to an antibody via hydrazone linkage.
  • Figure 5 shows the synthesis of bridge linkers containing polyethylene glycols and the application in the conjugation of two different drugs per linker to an antibody via amide linkage.
  • Figure 6 shows the synthesis of bridge linkers and the application in the conjugation of two different drugs per linker to an antibody via hinder amide linkage.
  • Figure 7 shows the synthesis of bridge linkers containing peptides or polyethylene glycols and the application of these linkers in the conjugation of two (different) drugs to an antibody via hydrazone linkage.
  • Figure 8 shows the synthesis of the conjugatable analogs of MMAE, Tubulysin and PBD cytotoxic drugs.
  • Figure 9 shows the synthesis of the conjugatable analogs of PBD, MMAF, and Tubulysin D cytotoxic drugs.
  • Figure 10 shows the synthesis of the conjugates of cell-binding molecule-tubulysin analogs via the bridge-linker.
  • Figure 11 shows the synthesis of the conjugates of both PBD dimer analog and Tubulysin B analog per linker, or both MMAE and Tubulysin D analog per linker, to an antibody.
  • Figure 12 shows the synthesis of the conjugates of both PBD dimer analog
  • MMAF analog per linker or both PBD dimer and Tubulysin B analog per linker, to an antibody.
  • Figure 13 shows the synthesis of the conjugates of both Maytansinoid analog and Tubulysin B analog per linker, to an antibody.
  • Figure 14 shows the synthesis of the conjugates of both Maytansinoid analog
  • PBD dimer analog per linker or two Tubulysin B analogs per linker, to an antibody.
  • Figure 15 shows the synthesis of the conjugates of both two MMAF analogs per linker, or two Tubulysin B analogs per linker, containing polyethylene glycols, to an antibody
  • Figure 16 shows the comparison of the anti-tumor effect of conjugate compounds 127, 129 and 142 with T-DM1 using human gastric tumor N87 cell model at dosing, 3 mg/kg, i.v., one injection. All the four conjugates did not cause the animal body weight loss (top figure). The animals at control group were sacrificed at day 37 due to the tumor volume larger than 1500 mm and they were too sick.
  • the three compounds 127, 129 and 142 were better than T-DM1: All 6/6 animals at the groups of compound 127 and 129 had completely no tumor measurable at day 13 till day 60 (the end of experiment). All 6/6 animals at the group of Compound 142 group had no tumor measurable at day 21 and 2/6 animals had tumor growth (measurable) back at days 48, which still inhibited the tumor growth for over 55 days. In contrast T-DM1 at dose of 3 mg/Kg was not able to eradicate the tumors completely although it had inhibited the tumor growth for about 28 days.
  • Alkyl refers to an aliphatic hydrocarbon group which may be straight or branched having 1 to 8 carbon atoms in the chain. "Branched” means that one or more lower C numbers of alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain.
  • alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-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-methyl-hexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5- dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n- heptyl, isoheptyl, n-octyl, and isooct
  • a C 1 -C 8 alkyl group can be unsubstituted or substituted with one or more groups including, but not limited to, -C 1 -C 8 alkyl,-O-(C 1 -C 8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH 2 , -C(O)NHR', -C(O)N(R') 2 , -NHC(O)R', - SR', -S(O) 2 R', -S(O)R', -OH, -halogen, -N 3 , -NH 2 , -NH(R'), -N(R') 2 and -CN; where each
  • R' is independently selected from -C 1 -C 8 alkyl and aryl.
  • Halogen refers to fluorine, chlorine, bromine or iodine atom; preferably fluorine and chlorine atom.
  • Heteroalkyl refers to C 2 -C 8 alkyl in which one to four carbon atoms are inde- pendently replaced with a heteroatom from the group consisting of O, S and N.
  • Carbocycle refers to a saturated or unsaturated ring having 3 to 8 carbon atoms as a monocycle or 7 to 13 carbon atoms as a bicycle.
  • Monocyclic carbocycles have 3 to 6 ring atoms, more typically 5 or 6 ring atoms.
  • Bicyclic carbocycles have 7 to 12 ring atoms, arranged as a bicycle [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms arranged as a bicycle [5,6] or [6,6] system.
  • Representative C 3 -C 8 carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5- cycloheptatrienyl, -cyclooctyl, and -cyclooctadienyl.
  • a "C 3 -C 8 carbocycle” refers to a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated nonaromatic carbocyclic ring.
  • a C 3 -C 8 carbocycle group can be unsubstituted or substituted with one or more groups including, but not limited to, -C 1 -C 8 alkyl,-O-(C 1 -C 8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH 2 , -C(O)NHR', -C(O)N(R') 2 , - NHC(O)R', -SR', -S(O)R',-S(O) 2 R', -OH, -halogen, -N 3 , -NH 2 , -NH(R'), -N(R') 2 and -CN; where each R
  • alkenyl refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond which may be straight or branched having 2 to 8 carbon atoms in the chain.
  • alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, hexylenyl, heptenyl, octenyl.
  • Alkynyl refers to an aliphatic hydrocarbon group containing a carbon-carbon triple bond which may be straight or branched having 2 to 8 carbon atoms in the chain.
  • exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5- pentynyl, n-pentynyl, hexylynyl, heptynyl, and octynyl.
  • Alkylene refers to a saturated, branched or straight chain or cyclic hydrocarbon rad- ical of 1-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane.
  • Typical alkylene radicals include, but are not limited to: methylene (-CH 2 -), 1,2- ethyl (-CH 2 CH 2 -), 1,3-propyl (-CH 2 CH 2 CH 2 -), 1,4-butyl (-CH 2 CH 2 CH 2 CH 2 -), and the like.
  • Alkenylene refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkene.
  • Alkynylene refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkyne.
  • Typical alkynylene radicals include, but are not limited to: acetylene, propargyl and 4-pentynyl.
  • Aryl or Ar refers to an aromatic or hetero aromatic group, composed of one or several rings, comprising three to fourteen carbon atoms, preferentially six to ten carbon atoms.
  • hetero aromatic group refers one or several carbon on aromatic group, preferentially one, two, three or four carbon atoms are replaced by O, N, Si, Se, P or S, preferentially by O, S, and N.
  • Heterocycle refers to a ring system in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group of O, N, S, Se, B, Si and P. Preferable heteroatoms are O, N and S. Heterocycles are also described in The Handbook of Chemistry and Physics, 78th Edition, CRC Press, Inc., 1997-1998, p. 225 to 226, the disclosure of which is hereby incorporated by reference.
  • Preferred nonaromatic heterocyclic include, but are not limited to epoxy, aziridinyl, thiiranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxolanyl, piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl,
  • dihydropyranyl tetrahydropyranyl, dihydropyranyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl, as well as the fused systems resulting from the condensation with a phenyl group.
  • heteroaryl refers to a 5 to 14, preferably 5 to 10 membered aromatic hetero, mono-, bi- or multicyclic ring.
  • examples include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1,2,4-thiadiazolyl, isothiazolyl, triazoyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, pyridyl-N-oxide, as well as the fused systems resulting from the condensation with a phenyl group.
  • Alkyl refers also to the corresponding "alkylene”, “cycloalkylene”, “alkenylene”, “alkynylene”, “arylene”, “heteroarylene”, “heterocyclene” and the likes which are formed by the removal of two hydrogen atoms.
  • Arylalkyl refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp carbon atom, is replaced with an aryl radical.
  • Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-l- yl, 2-phenylethen-l-yl, naphthylmethyl, 2-naphthylethan-l-yl, 2-naphthylethen-l-yl, naphthobenzyl, 2-naphthophenylethan-l-yl and the like.
  • Heteroarylalkyl refers to an acyclic alkyl radical in which one of the hydrogen at- oms bonded to a carbon atom, typically a terminal or sp carbon atom, is replaced with a heteroaryl radical.
  • Typical heteroarylalkyl groups include, but are not limited to, 2- benzimidazolylmethyl, 2-furylethyl and the like.
  • hydroxyl protecting group examples include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, /?-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, i-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetate ester, substituted acetate esters, pivaloate, benzoate, methanesulfonate and /?-toluenesulfonate.
  • leaving group refers to a functional group that can be substituted by another func- tional group. Such leaving groups are well known in the art, and examples include, but are not limited to, a halide (e.g., chloride, bromide, and iodide), methanesulfonyl (mesyl), /?- toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), and trifluoromethyl sulfonate.
  • a halide e.g., chloride, bromide, and iodide
  • methanesulfonyl meyl
  • /?- toluenesulfonyl tosyl
  • triflate trifluoromethylsulfonate
  • Boc ie/ -butoxy carbonyl
  • BroP bromotrispyrrolidinophosphonium hexafluorophosphate
  • CDI ⁇ , ⁇ -carbonyldiimidazole
  • DCC dicyclohexylcarbodiimide
  • DCE dichloroethane
  • DCM dichloromethane
  • DIAD diisopropylazodicarboxylate
  • DIBAL-H diisobutyl- aluminium hydride
  • DIPEA diisopropylethylamine
  • DEPC diethyl phosphorocyanidate
  • DMA ⁇ , ⁇ -dimethyl acetamide
  • DMAP 4-(7V, N-dimethylamino)pyridine
  • DMF N,N- dimethylformamide
  • DMSO dimethylsulfoxide
  • DTT dithiothreitol
  • EDC l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • ESI-MS electrospray mass spectrometry
  • HATU O-(7-azabenzotriazol-l-yl)-N, N, N' , N'-tetramethyluronium hexafluorophosphate
  • HOBt 1-hydroxybenzotriazole
  • “Pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
  • “Pharmaceutically acceptable solvate” or “solvate” refer to an association of one or more solvent molecules and a disclosed compound. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, etha- nol, methanol, DMSO, ethyl acetate, acetic acid and ethanolamine.
  • “Pharmaceutically acceptable excipient” includes any carriers, diluents, adjuvants, or vehicles, such as preserving or antioxidant agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • preserving or antioxidant agents such as preserving or antioxidant agents, fillers, disintegrating agents, wetting agents, emulsifying agents, 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 for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions as suitable therapeutic combinations.
  • pharmaceutically acceptable salts refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof.
  • the pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, tartaric, citric, methanesulfonic, benzenesulfonic, glucuronic, glutamic, benzoic, salicylic, toluenesulfonic, oxalic, fumaric, maleic, lactic and the like.
  • Further addition salts include ammonium salts such as tromethamine, meglumine, epolamine, etc., metal salts such as sodium, potassium, calcium, zinc or magnesium.
  • the pharmaceutical salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
  • such salts can be prepared via reaction the free acidic or basic forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.
  • non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17 th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418, the disclosure of which is hereby incorporated by reference.
  • administering refers to any mode of transferring, delivering, introducing or transporting a pharmaceutical drug or other agent to a subject. Such modes include oral administration, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous or intrathecal administration. Also contemplated by the present invention is utilization of a device or instrument in administering an agent. Such device may utilize active or passive transport and may be slow-release or fast-release delivery device.
  • novel conjugates disclosed herein use the bridge linkers. Examples of some suitable linkers and their synthesis are shown in Figures 1 to 15.
  • the synthetic routes to produce bridge linkers as well as the preparation of the conjugates of drugs to a cell binding molecules of the present invention are shown in Figures 1- 15.
  • the bridge linkers possess two elements: a) A Substituent that is a 2,3-disubstituted succinic group; or 2-monosubstituted, or 2,3-disubstituted fumaric group; or 2- monosubstituted, or 2,3-disubstituted maleic group; which can react to a pair of thiols to form covalent thioether bonds, and b) A group, such as but not limited to, a disulfide, maleimide, haloacetyl, aldehyde, ketone, azide, amine, alkoxyamine, hydrazide, ethenesulfonyl, acyl halide (acid halide), acryl (acryloyl), and/or acid anhydride group, capable of reaction with a drug
  • the bridge substituents of 2,3-disubstituted succinic group; or 2-monosubstituted, or 2,3-disubstituted fumaric group; or 2-monosubstituted, or 2,3- disubstituted maleic group can be introduced by direct condensation of these 2,3- disubstituted succinic acid, or 2-monosubstituted or 2,3-disubstituted fumaric or maleic with an amine, an alcohol, or a thiol group to form amide, ester or thioester bonds.
  • the synthesis of these bridge linkers is exampled in the Figures 1, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14 and 15.
  • the bridge linkers are compounds of the Formula (I) below:
  • both U and LP are not H; when represents a double bond, either U or LP can be H, but are not H at the same time.
  • the component: which can be 2,3-disubstituted succinic group, or 2-monosubstituted or 2,3-disubstituted fumaric group, or 2- monosubstituted or 2,3-disubstituted maleic group, is capable of reacting with a pair of sulfur atoms of the cell-binding agent;
  • the sulfur atoms are preferred pairs of thiols re- prised from the interchain disulfide bonds of the cell-binding agent by a reducing agent, such as dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH) and tris (2- carboxyethyl) phosphine (TCEP), or/and beta mercaptoethanol ( ⁇ - ⁇ , 2-ME
  • U and U' represent the same or different leaving group that can be substituted by a thiol.
  • Such leaving groups are, but are not limited to, a halide (e.g., fluoride, chloride, bromide, and iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethyl- sulfonyl (triflate), trifluoromethylsulfonate, nitrophenol, N-hydroxysuccinimide (NHS), phenol; dinitrophenol; pentafluorophenol, tetrafluorophenol, difluorophenol,
  • a halide e.g., fluoride, chloride, bromide, and iodide
  • methanesulfonyl methanesulfonyl
  • p-toluenesulfonyl tosyl
  • triflate trifluoro
  • Z 1 and Z 2 are the same or different a function group that enables to react with a cytotoxic drug, to form a disulfide, thioether, thioester, peptide, hydrazone, ether, ester, carbamate, carbonate, amine (secondary, tertiary, or quarter), imine, cycloheteroalkyane, heteroaromatic, alkoxime or amide bond;
  • R 1 and R 2 are the same or different, and are absent, linear alkyl having from 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl or alkynyl, or 1-6 carbon atoms of esters, ether, amide, or polyethyleneoxy unit of formula (OCH 2 CH 2 ) p , or polypropyleneoxy unit of formula (OCH 2 (CH3)CH 2 ) p wherein p is an integer from 0 to about 1000, or combination thereof.
  • R 1 and R 2 are respectively a chain of atoms selected from C, N, O, S, Si, and P, preferably having 0-500 atoms, which covalently connects to X 1 or X 2 and Z 1 or Z 2 .
  • the atoms used in forming the R 1 and R 2 may be combined in all chemically relevant ways, such as forming alkylene, alkenylene, and alkynylene, ethers, polyoxyalkylene, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas,
  • X 1 and X 2 are independently selected from NH, N(R 3 ), O, S or CH 2 ; Wherein R 3 is H, linear alkyl having from 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl or alkynyl, or 1-6 carbon atoms of esters, ether, amide, or polyethyleneoxy unit of formula (OCH 2 CH 2 ) p , wherein p is an integer from 0 to about 1000, or combination thereof.
  • R 1 , R 2 , and R 3 can be respectively a chain of atoms selected from C, N, O, S, Si, and P which covalently connects the cell-surface binding molecule and/or the conjugated drug.
  • the atoms used in forming the bridge linker may be combined in all chemically relevant ways, such as forming alkylene, alkenylene, and alkynylene, ethers, polyoxyalkylene, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, acyloxylamines, hydroxamic acids, and many others.
  • the atoms forming the linker (L) may be either saturated or unsaturated, or may be radicals, or may be cyclized upon each other to form divalent cyclic structures, including cyclo alkanes, cyclic ethers, cyclic amines, arylenes, heteroarylenes, and the like in the linker.
  • Examples of the functional groups, Z 1 and Z 2 which enable linkage of a cytotoxic drug, include groups that enable linkage via a disulfide, thioether, thioester, peptide, hydrazone, ester, carbamate, carbonate, alkoxime or an amide bond.
  • Such functional groups include, but are not limited to, thiol, disulfide, amino, carboxy, aldehydes, ketone, maleimido, haloacetyl, hydrazines, alkoxyamino, and/or hydroxy.
  • Examples of the functional groups, Z 1 and Z 2 , that enable reaction with the terminal of amine of a drug/ cytotoxic agent can be, but not limited to, N-hydroxysuccinimide esters, p-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl esters, carboxylic acid chlorides or carboxylic acid anhydride;
  • Examples of thiol can be, as but not limited to, pyridyldisulfides, nitropyridyldisulfides, maleimides, haloacetates,
  • ketone or aldehyde can be, as but not limited to, amines, alkoxyamines, hydrazines, acyloxylamine, or hydrazide;
  • azide can be, as but not limited to, alkyne. Examples of these function groups are displayed below:
  • X 1 is F, CI, Br, I or Lv 3
  • X 2 is O, NH, N(R 1 ), or CH 2
  • R 5 and R 3 are H, R 1 , aromatic, heteroaromatic, or aromatic group wherein one or several H atoms are replaced independently by -R 1 , -halogen, -OR 1 , -SR 1 , -NR1R2, - NO 2 , -S(O)R 1 , - S(O) 2 R 1, or -COOR 1
  • Lv 3 is a leaving group selected from nitrophenol; N-hydroxysuc- cinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol;
  • difluorophenol difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5- phenylisoxazolium-3 '-sulfonate, anhydrides formed its self, or formed with the other anhydride, e.g. acetyl anhydride, formyl anhydride; or an intermediate molecule generated with a condensation reagent for peptide coupling reactions, or for Mitsunobu reactions.
  • anhydrides formed its self, or formed with the other anhydride, e.g. acetyl anhydride, formyl anhydride; or an intermediate molecule generated with a condensation reagent for peptide coupling reactions, or for Mitsunobu reactions.
  • the key step of synthesis of the bridge linker containing 2,3-disubstituted succinic group, or 2-monosubstituted or 2,3-disubstituted fumaric group, or 2-monosubstituted or 2,3-disubstituted maleic group is the condensation of the 2,3-disubstituted succinic acid, or 2-monosubstituted or 2,3-disubstituted fumaric acid, or 2-monosubstituted or 2,3- disubstituted maleic acid, or its acid derivatives, with the other components containing an amine (1° or 2° amines), alcohol, or thiol on their terminal, as shown in the following scheme (la):
  • X is X 1 or X 2 described in Formula (I) as NH, N(R 3 ), O, or S; R is R 1 and/or R 2 that described in Formula (I); R 3 is the same defined in Formula (I).
  • Lvi and Lv 2 are the same or independently OH; F; CI; Br; I; nitrophenol; N-hydroxy- succinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol;
  • difluorophenol difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5- phenylisoxazolium-3 '-sulfonate, anhydrides formed its self, or formed with the other anhydride, e.g. acetyl anhydride, formyl anhydride; or an intermediate molecule generated with a condensation reagent for peptide coupling reactions, or for Mitsunobu reactions, e.g.
  • condensation reagents are: EDC (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide), DCC (Dicyclohexyl-carbodiimide), ⁇ , ⁇ '-Diisopropylcarbodiimide (DIC), N-Cyclohexyl- N'-(2-morpholino-ethyl)carbodiimide metho-p-toluenesulfonate (CMC,or CME-CDI), 1,1 '- Carbonyldiimi-dazole (CDI),TBTU (O-(Benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), N,N,N',N'-Tetramethyl-O-(lH-benzotriazol-l-yl)uronium
  • HBTU hexafluorophosphate
  • BOP Benzotriazol- l-yloxytris(dimethylamino)phosphonium hexafluorophosphate
  • BOP Benzotriazol- l-yloxytripyrrolidinophosphonium
  • hexafluorophosphate PyBOP
  • Diethyl cyanophosphonate DEPC
  • Chloro- ⁇ , ⁇ , ⁇ ', ⁇ '- tetramethylformamidinium hexafluorophosphate l-[Bis(dimethylamino)methylene]-lH- l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), l-[(Dimethylami- no)(morpholino) methylene]- lH-[l,2,3]triazolo[4,5-b]pyridine-l-ium 3-oxide hexafluorophosphate (HDMA), 2-Chloro-l,3-dimethylimidazolidinium hexafluorophosphate (CIP), Chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), Fluoro-N,N,N',N'-bis
  • Oxido-2-pyridyl)-N,N,N',N'-tetramethylthiuronium tetrafluoroborate O-[(Ethoxycarbonyl) cyano-methylenamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate (HOTU), (1- Cyano-2-ethoxy-2-oxoethylidenaminooxy) dimethylamino-morpholino-carbenium hexafluorophosphate(COMU), O-(Benzotriazol-l-yl)-N,N,N',N'-bis(tetramethylene) uronium hexafluorophosphate (HBPyU), N-Benzyl-N'-cyclohexylcarbodiimide (with, or without polymer-bound), Dipyrrolidino(N-succinimidyl-oxy)carbenium hexafluorophosphat
  • DMTMM N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), O- (3,4-Dihydro-4-oxo-l,2,3-benzotriazin-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TDBTU), l, -(Azodicarbonyl)dipiperidine (ADD), Di-(4-chlorobenzyl) azodicar- boxylate (DCAD), Di-tert-butyl azodicarboxylate (DBAD), Diisopropyl azodicarboxylate (DIAD), Diethyl azodicarboxylate (DEAD).
  • ADD Di-(4-chlorobenzyl) azodicar- boxylate
  • DAD Di-tert-butyl azodicarboxylate
  • DIAD Diis
  • bridge substituents of 2,3-disubstituted succinic group, or 2- monosubstituted or 2,3-disubstituted fumaric group, or 2-monosubstituted or 2,3- disubstituted maleic group can be condensated with linker components containing function groups capable to react to drugs of desired conjugation.
  • Cb is a cell-binding agent
  • L is linker containing succinic, fumaric or maleic group
  • Drug 1 and Drug 2 are a drug molecule
  • n is an integer from 1 to 30
  • S (sulfur) elements from Cb bridgely link to L which covalently connects two or more drugs (per bridge linker L).
  • the bridge linker L may be composed of one or more linker components.
  • exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe” or “af”), p- aminobenzyloxycarbonyl (“PAB”), 4-thiopentanoate (“SPP”), 4-(N-maleimidomethyl)- cyclohexane- 1 carboxylate (“MCC”), (4-acetyl)aminobenzoate (“SIAB”), 4-thio-butyrate (SPDB), 4-thio-2-hydroxysulfonyl-butyrate (2-Sulfo-SPDB), ethyleneoxy -CH 2 CH 2 O- as one or more repeating units (“EO” or “PEO”). Additional linker components are known in the art and some are described herein.
  • the conjugates have the following Formula ( ⁇ ):
  • Cb represents a cell-binding agent, preferably an antibody, which conjugates to Drug 1 and Drug 2 via a pair of sulfur atoms (thiols).
  • the conjugatable thiol groups can generally be generated from dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH) and tris (2-carboxyethyl) phosphine (TCEP), or/and beta mercaptoethanol ( ⁇ - ⁇ , 2-ME) reduction of pairs of disulfide bonds on the surface of cell-binding molecule.
  • Drug 1 and Drug 2 represent the same or different cytotoxic agents, linked to the cell- binding agent via the bridge linker through an alkyl, alkylene, alkenylene, alkynylene, ether, polyoxyalkylene, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, urethanes, amino acid, peptide, acyloxylamine, hydroxamic acid, disulfide, thioether, thioester, carbamate, carbonate, heterocyclic ring, heteroalkyl, heteroaromatic, or alkoxime bond, or combination thereof.
  • Drug 1 and Drug 2 can be any of many small molecule drugs, including, but not limited to, tubulysins, calicheamicins, auristatins,
  • benzodiazepine dimers e.g., dimmers of pyrrolobenzodiazepine (PBD) or tomaymycin
  • indolinobenzodiazepines imidazobenzothiadiazepines, or
  • the cell-binding agent can be first modified with the bridge linkers of the present invention through reduction of disulfide bonds of the cell- binding molecule.
  • the yielded a pair of free thiols can react to the bridge linker of Formula (I) at pH 5-9 aqueous media with or without addition of 0-30% of water mixable (miscible) organic solvents, such as DMA, DMF, ethanol, methanol, acetone, acetonitrile, THF, isopropanol, dioxane, propylene glycol, or ethylene diol, to introduce the reactive groups of Z 1 and Z 2 containing disulfide, maleimido, haloacetyl, azide, 1-yne, ketone, aldehyde, alkoxyamino, or hydrazide groups.
  • a reactive group of a cytotoxic agent reacts to the modified cell-binding molecule accordingly.
  • synthesis of the cell-binding agent-drug conjugates linked via disulfide bonds is achieved by a disulfide exchange between the disulfide bond in the modified cell-binding agent and a drug containing a free thiol group.
  • Synthesis of the cell-binding agent-drug conjugates linked via thioether is achieved by reaction of the maleimido or haloacetyl or ethylsulfonyl modified cell-binding agent and a drug containing a free thiol group.
  • Synthesis of conjugates bearing an acid labile hydrazone can be achieved by reaction of a carbonyl group with the hydrazide moiety in the linker, by methods known in the art (see, for example, P. Hamann et al., Cancer Res. 53, 3336-334, 1993; B. Laguzza et al., J. Med. Chem., 32; 548-555, 1959; P. Trail et al., Cancer Res., 57; 100-105, 1997).
  • Synthesis of conjugates bearing triazole linkage can be achieved by reaction of a 1-yne group of the drug with the azido moiety in the linker, through the click chemistry (Huisgen cycloaddition) (Lutz, J-F. et al, 2008, Adv.
  • the drug can react with the bridge linkers of the present invention that have conjugated to a cell-binding molecule to give a modified cell-binding molecule linker of Formula (III) bearing functionalities.
  • a thiol-containing drug can be reached with the modified cell-binding molecule bridge linker of Formula (III) bearing a maleimdo, or a haloacetyl, or an ethylsulfonyl substituent at pH 5.5-9.0 in aqueous buffer to give a cell-binding molecule-drug conjugate via a thioether linkage.
  • a thiol-containing drug can undergo disulfide exchange with a modified bridge linker of Formula (III) bear- ing a pyridyldithio moiety to give a conjugate a disulfide bond linkage.
  • a drug bearing a hydroxyl group or a thiol group can be reacted with a modified bridge linker of Formula (III) bearing a halogen, particularly the alpha halide of carboxylates, in the presence of a mild base, e.g. pH 8.0-9.5, to give a modified drug bearing an ether or thiol ether link.
  • a hydroxyl group containing drug can be condensed with a bridge cross linker of Formula (I) bearing a carboxyl group, in the presence of a dehydrating agent, such as EDC or DCC, to give ester linkage, then the subject drug modified bridge linker undergoes the conjugation with a cell-binding molecule.
  • a dehydrating agent such as EDC or DCC
  • a drug containing an amino group can condensate with a carboxyl ester of NHS, imidazole, nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1- hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3 '-sulfonate on the cell-binding molecule-bridge linker of Formula (III) to give a conjugate via amide bond linkage.
  • NHS N-hydroxysuccinimide
  • the conjugate may be purified by standard biochemical means, such as gel filtration on a Sephadex G25 or Sephacryl S300 column, adsorption chromatography, and ion exchange or by dialysis.
  • a small molecule as a cell-binding agent e.g. folic acid, melanocyte stimulating hormone, EGF etc
  • a small molecular drugs can be purified by chromatography such as by HPLC, medium pressure column chromatography or ion exchange chromatography.
  • the cell-binding agent modified by reaction with linkers of the present invention are preferably represented by the Formula (III)
  • Cb, Z 1 , Z 2 , n, R 1 , R 2 , Xi, and X 2 are defined the same as in
  • Z 1 and Z 2 are a disulfide substituent, maleimido, haloacetyl, alkoxyamine, azido, ketone, aldehyde, hydrazine, alkyne, an N-hydroxysuccinimide ester, or a carboxyl ester formed with phenol; dinitrophenol; pentafluorophenol; tetrafluoro- phenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole;
  • the modified cell-binding agent can be prepared via a reaction of the cell- binding agent with the bridge linkers of Formula (I) as described in Formula (II) above.
  • a small percentage of organic co- solvent may be required to add to the reaction mixture, as well in the solution after the reaction to maintain solubility of the Formula (III) in aqueous solution.
  • the cross-linking reagent (bridge linker) of Formula (I) can be first dissolved in a polar organic solvent that is miscible with water, for example different alcohols, such as methanol, ethanol, and propanol, acetone, acetonitrile, tetrahydrofuran (THF), 1,4-dioxane, dimethyl formamide (DMF), dimethyl acetamide (DMA), or dimethylsulfoxide (DMSO) at a high concentration, for example 1-500 mM.
  • a polar organic solvent that is miscible with water
  • a polar organic solvent that is miscible with water
  • alcohols such as methanol, ethanol, and propanol
  • acetone acetonitrile
  • THF tetrahydrofuran
  • DMF dimethyl formamide
  • DMA dimethyl acetamide
  • DMSO dimethylsulfoxide
  • the cell-binding molecule such as antibody dissolved in an aqueous buffer pH 5-9.5, preferably pH 6-8.5, at 1-35 mg/ml concentration was treated with 1-20 equivalent of TCEP or DTT for 20 min to 12 hour. After the reduction, DTT can be removed by SEC chromatographic purification. TCEP can be optionally removed by SEC chromatography too, or staying in the reaction mixture for the next step reaction without purification.
  • the reduction of antibodies or the other cell-binding agents with TCEP can be performed with a bridge linker of Formula (I), for which the cross-linking conjugation for the cell-binding molecules can be achieved simultaneously along with the TCEP reduction.
  • aqueous solutions for the modification of cell-binding agents are buffered between pH 6 and 9, preferably between 6.5 and 7.5 and can contain any non-nucleophilic buffer salts useful for these pH ranges.
  • Typical buffers include phosphate, triethanolamine HC1, HEPES, and MOPS buffers, which can contain additional components, such as cyclodextrins, sucrose and salts, for examples, NaCl and KC1.
  • the progress of the reaction can be monitored by measuring the decrease in the absorption at 254 nm, or increase in the absorption at 280 nm, or the other appropriate wavelength.
  • isolation of the modified cell- binding agent can be performed in a routine way, using for example gel filtration chromatography, or adsorptive chromatography.
  • the extent of modification can be assessed by measuring the absorbance of the nitropyridine thione, dinitropyridine dithione, pyridine thione, carboxamidopyridine dithione and dicarboxamidopyridine dithione group released via UV spectra.
  • the modification or conjugation reaction can be monitored by LC-MS, preferably by UPLC-QTOF mass spectrometry, or Capil- lary electrophoresis-mass spectrometry (CE-MS).
  • the bridge cross-linkers described herein have diverse functional groups that can react with any drugs, preferably cytotoxic agents that possess a suitable substituent.
  • the modified cell-binding molecules bearing an amino or hydroxyl substituent can react with drugs bearing an
  • the modified cell-binding molecules bearing a thiol substituent can react with drugs bearing a maleimido or haloacetyl group.
  • the modified cell-binding molecules bearing a carbonyl (ketone or aldehyde) substituent can react with drugs bearing a hydrazide or an alkoxyamine.
  • cytotoxic drugs modified by reaction with cross-linkers of the present invention are preferably represented by the Formula (IV):
  • the modified drugs can be prepared via reaction of the drug with the linkers of the Formula (I) to give a modified drug of Formula (IV) bearing functionality of an 2,3- disubstituted succinic group, or 2-monosubstituted or 2,3-disubstituted fumaric group, or 2-monosubstituted or 2,3-disubstituted maleic group.
  • the Drug 1 or Drug 2 may be synthesized to connect to R 1 , or R 2 in a piece of components via the linkage of thioether, thioester or disulfide bond first.
  • R 1 -Drug 1 or R 2 -Drug 2 component is assembled to 2,3-disubstituted succinic acid, or 2-monosubstituted or 2,3-disubstituted fumaric acid, or 2-monosubstituted or 2,3-disubstituted maleic acid to form the bridge linker modified drugs of Formula (IV).
  • a thiol-containing drug can be reacted with the linker of components R 1 or R 2 bearing a maleimdo substituent at neutral pH in aqueous buffer to give a R 1 -Drug 1 or R 2 -Drug 2 compartment bearing thioether linkage, and following by condensation with either 2,3-disubstituted succinic acid, or 2-monosubstituted or 2,3- disubstituted fumaric acid, or 2-monosubstituted or 2,3-disubstituted maleic acid to give a modified drug of Formula (IV) bearing thioether linkage.
  • a drug bearing a hydroxyl group can be reacted with a linker component R 1 or R 2 bearing a halogen, or a tosylate, or a mesylate, in the presence of a mild base, to give a R 1 -Drug 1 or R 2 -Drug 2 compartment bearing ether linkage, and following by condensation with 2,3-disubstituted succinic acid, or 2-monosubstituted or 2,3-disubstituted fumaric acid, or 2-monosubstituted or 2,3- disubstituted maleic acid to give a modified drug of Formula (IV) bearing thioether linkage.
  • a hydroxyl group containing drug can be condensed with a linker of Formula (I) bearing a carboxyl group, in the presence of a dehydrating agent, such as EDC or dicyclohexylcarbodiimide (DCC), to give a modified drug of Formula (IV) via ester linkage.
  • a dehydrating agent such as EDC or dicyclohexylcarbodiimide (DCC)
  • a drug bearing a thiol group can also react the linker of components R 1 or R 2 bearing a maleimido or a vinylsulfonyl, or a haloacetyl group, give a R 1 -Drug 1 or R 2 -Drug 2 compartment bearing thioether linkage, and following by condensation with a compartment of 2,3-disubstituted succinic acid, or 2-monosubstituted or 2,3-disubstituted fumaric acid, or 2-monosubstituted or 2,3-disubstituted maleic acid to give a modified drug of Formula (IV) bearing thioether linkage.
  • An amino group containing drug can similarly undergo condensation with a carboxyl group on the bridge linker of Formula (I) to give a modified drug of Formula (IV) bearing amide bonds.
  • the modified drug can be purified by standard methods such as column chromatography over silica gel or alumina, crystallization, preparatory thin layer chromatography, ion exchange chromatography, or HPLC.
  • the cell-binding molecule that comprises the conjugates and the modified cell-binding agents of the present invention may be of any kind presently known, or that become known, molecule that binds to, complexes with, or reacts with a moiety of a cell population sought to be therapeutically or otherwise biologically modified.
  • the cell binding agents include, but are not limited to, large molecular weight proteins such as, for example, full-length antibodies (polyclonal antibodies, monoclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies); single chain antibodies; fragments of antibodies such as Fab, Fab', F(ab') 2 , F v [Parham, J. Immunol. 131, 2895-2902 (1983)], fragments produced by a Fab expression library, anti-idiotypic
  • anti-Id antibodies antibodies, CDR's, diabody, triabody, tetrabody, miniantibody, small immune proteins (SIP), and epitope-binding fragments of any of the above which immuno- specifically bind to cancer cell antigens, viral antigens, microbial antigens or a protein generated by the immune system that is capable of recognizing, binding to a specific antigen or exhibiting the desired biological activity (Miller et al (2003) J. of Immunology
  • interferons such as type I, II, III
  • peptides such as lymphokines such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, GM-CSF, interferon-gamma (IFN- ⁇ ); hormones such as insulin, TRH (thyrotropin releasing hormones), MSH (melanocyte- stimulating hormone), steroid hormones, such as androgens and estrogens, melanocyte- stimulating hormone (MSH); growth factors and colony-stimulating factors such as epidermal growth factors
  • EGF granulocyte-macrophage colony-stimulating factor
  • TGF transforming growth factors
  • IGF-I, IGF-II insulin and insulin like growth factors
  • GEF vaccinia growth factors
  • FGFs fibroblast growth factors
  • platelet-derived growth factors such as interleukin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory factors, granulocyte- macrophage colony- stimulating factor (GM-CSF); vitamins, such as folate; apoproteins and glycoproteins, such as transferrin [O'Keefe
  • a monoclonal antibody is preferred as a cell-surface binding agent if an appropriate one is available.
  • the antibody may be murine, human, humanized, chimeric, or derived from other species.
  • a monoclonal antibody is typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen (Kohler, G.; Milstein, C.
  • monoclonal antibodies are produced by immunizing mice, rats, hamsters or any other mammal with the antigen of interest such as the intact target cell, antigens isolated from the target cell, whole virus, attenuated whole virus, and viral proteins.
  • Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000.
  • Fused hybrids are selected by their sensitivity to HAT (hypoxanthine-aminopterin-thymine).
  • Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact specified receptors or inhibit receptor activity on target cells.
  • a monoclonal antibody used in the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity.
  • the culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium.
  • the antibody-containing medium is then collected.
  • the antibody molecules can then be further isolated by well-known techniques, such as using protein-A affinity chromatography; anion, cation, hydrophobic, or size exclusive chromatographies (particularly by affinity for the specific antigen after protein A, and sizing column chromatography); centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol. 8, 396 (1959)) supplemented with 4.5 gm/1 glucose, 0-20 mM glutamine, 0-20% fetal calf serum, several ppm amount of heavy metals, such as Cu, Mn, Fe, or Zn, etc, or/and the heavy metals added in their salt forms, and with an anti-foaming agent, such as polyoxyethylene- polyoxypropylene block copolymer.
  • DMEM Dulbecco's minimal essential medium
  • DMEM Dulbecco's minimal essential medium
  • heavy metals such as Cu, Mn, Fe, or Zn, etc
  • an anti-foaming agent such as polyoxyethylene- polyoxypropylene block copolymer.
  • antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfec- tion with an oncovirus, such as Epstein-Barr virus (EBV, also called human herpesvirus 4
  • EBV Epstein-Barr virus
  • HHV-4 Kaposi's sarcoma-associated herpesvirus
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • a monoclonal antibody may also be produced via an anti-receptor peptide or peptides containing the carboxyl terminal as described well-known in the art. See Niman et al., Proc. Natl. Acad. Sci. USA, 80: 4949-4953 (1983); Geysen et al., Proc. Natl. Acad. Sci.
  • the anti-receptor peptide or a peptide analog is used either alone or conjugated to an immunogenic carrier, as the immunogen for producing anti-receptor peptide monoclonal antibodies.
  • phage display technology which can be used to select a range of human antibodies binding specifically to the antigen using methods of affinity enrichment. Phage display has been thoroughly described in the literature and the construction and screening of phage display libraries are well known in the art, see, e.g.,
  • Monoclonal antibodies derived by hybridoma technique from another species than human, such as mouse, can be humanized to avoid human anti-mouse antibodies when infused into humans.
  • humanization of antibodies are complementarity-determining region grafting and resurfacing. These methods have been extensively described, see e.g. U.S. Pat. Nos. 5,859,205 and 6,797,492; Liu et al, Immunol Rev. 222:9-27 (2008); Almagro et al, Front Biosci. 13: 1619-33 (2008); Lazar et al, Mol Immunol. 44(8): 1986-98 (2007); Li et al, Proc. Natl. Acad. Sci. U S A. 103(10):3557-62 (2006) each incorporated herein by reference.
  • Fully human antibodies can also be prepared by immunizing transgenic mice, rabbits, monkeys, or other mammals, carrying large portions of the human immunoglobulin heavy and light chains, with an immunogen.
  • mice examples include the Xenomouse. (Abgenix/Amgen), the HuMAb-Mouse (Medarex/BMS), the VelociMouse (Regeneron), see also U.S. Pat. No. 6,596,541 , 6,207,418, No. 6,150,584, No. 6,111,166, No. 6,075,181, No. 5,922,545, Nos. 5,661,016, 5,545,806, 5,436,149 and 5,569,825.
  • murine variable regions and human constant regions can also be fused to construct called "chimeric antibodies" that are considerably less immunogenic in man than murine mAbs (Kipriyanov et al, Molarcomas).
  • Antibodies immuno specific for a malignant cell antigen can also be obtained commercially or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques.
  • the nucleotide sequence encoding antibodies immuno specific for a malignant cell antigen can be obtained commercially, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
  • a peptide or protein that bind/block/target or in some other way interact with the epitopes or corresponding receptors on a targeted cell can be used as a binding molecule.
  • These peptides or proteins could be any random peptide or proteins that have an affinity for the epitopes or corresponding receptors and they don't necessarily have to be of the immunoglobulin family.
  • These peptides can be isolated by similar techniques as for phage display antibodies (Szardenings, J Recept Signal Transduct Res. 2003; 23(4):307-49). The use of peptides from such random peptide libraries can be similar to antibodies and antibody fragments.
  • binding molecules of peptides or proteins may be conjugated on or linked to a large molecules or materials, such as, but is not limited, an albumin, a polymer, a liposome, a nano particle, a dendrimer, as long as such attachment permits the peptide or protein to retain its antigen binding specificity.
  • a large molecules or materials such as, but is not limited, an albumin, a polymer, a liposome, a nano particle, a dendrimer, as long as such attachment permits the peptide or protein to retain its antigen binding specificity.
  • antibodies used for conjugation of drugs via the bridge linkers of this prevention for treating cancer, autoimmune disease, and/or infectious disease include, but are not limited to, 3F8 (anti-GD2), Abagovomab (anti CA-125), Abciximab (anti CD41
  • Adalimumab anti-TNF-a
  • Adecatumumab anti-EpCAM, CD326)
  • Afelimomab anti-TNF-a
  • Afutuzumab anti-CD20
  • Alacizumab pegol anti-VEGFR2
  • ALD518 anti-IL-6
  • Alemtuzumab Campath, MabCampath, anti- CD52
  • Altumomab anti-CEA
  • Anatumomab anti-TAG-72
  • Anrukinzumab IMA-638, anti-IL-13
  • Apolizumab anti-HLA-DR
  • Arcitumomab anti-CEA
  • Aselizumab anti-L-selectin (CD62L)
  • Atlizumab tocilizumab, Actemra, RoActemra, anti-IL-6 receptor
  • Atorolimumab anti-Rhesus factor
  • Bapineuzumab anti-beta amyloid
  • Basiliximab Simulect, antiCD25 (a chain of IL-2 receptor)
  • Bavituximab anti-phosphatidylserine
  • Bectumomab LymphoScan, anti-CD22
  • Belimumab Benlysta, LymphoStat-B, anti-
  • Besilesomab (Scintimun, anti-CEA-related antigen), Bevacizumab (Avastin, anti-VEGF- A), Biciromab (FibriScint, anti-fibrin II beta chain), Bivatuzumab (anti-CD44 v6), Blinatumomab (BiTE, anti-CD19), Brentuximab (cACIO, anti-CD30 TNFRSF8),
  • Briakinumab (anti-IL-12, IL-23) Canakinumab (Ilaris, anti-IL-1), Cantuzumab (C242, anti-IL-12, IL-23) Canakinumab (Ilaris, anti-IL-1), Cantuzumab (C242, anti-IL-12, IL-23) Canakinumab (Ilaris, anti-IL-1), Cantuzumab (C242, anti-
  • CanAg Capromab, Catumaxomab (Removab, anti-EpCAM, anti-CD3), CC49 (anti-TAG- 72), Cedelizumab (anti-CD4), Certolizumab pegol (Cimzia anti-TNF-a), Cetuximab (Er- bitux, IMC-C225, anti-EGFR), Citatuzumab collectingox (anti-EpCAM), Cixutumumab (anti- IGF-1), Clenoliximab (anti-CD4), Clivatuzumab (anti-MUCl), Conatumumab (anti- TRAIL-R2), CR6261 (anti-Influenza A hemagglutinin), Dacetuzumab (anti-CD40),
  • Daclizumab (Zenapax, anti-CD25 (a chain of IL-2 receptor)), Daratumumab (anti-CD38 (cyclic ADP ribose hydrolase), Denosumab (Prolia, anti-RANKL), Detumomab (anti-B- lymphoma cell), Dorlimomab, Dorlixizumab, Ecromeximab (anti-GD3 ganglioside), Eculizumab (Soliris, anti-C5), Edobacomab (anti-endotoxin), Edrecolomab (Panorex, MAbl7-lA, anti-EpCAM), Efalizumab (Raptiva, anti-LFA-1 (CD1 la), Efungumab
  • Enlimomab pegol (anti-IC AM- 1 (CD54)), Epitumomab (anti-episialin), Epratuzumab (anti-CD22), Erlizumab (anti-ITGB2 (CD 18)), Ertumaxomab (Rexomun, anti-HER2/neu, CD3), Etaracizumab (Abegrin, anti-integrin ⁇ ⁇ ⁇ 3 ), Exbivirumab ( anti-hepatitis B surface antigen), Fanolesomab (NeutroSpec, anti-CD15), Faralimomab (anti-interferon receptor),
  • Farletuzumab (anti-folate receptor 1), Felvizumab (anti-respiratory syncytial virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF-1 receptor), Fontolizumab (anti-IFN- ⁇ ), Foravirumab (anti-rabies virus glycoprotein), Fresolimumab (anti-TGF- ⁇ ), Galiximab (anti-CD80), Gantenerumab (anti- beta amyloid), Gavilimomab (anti-CD 147 (basigin)), Gemtuzumab (anti-CD33), Girentuximab (anti-carbonic anhydrase 9), Glembatumumab
  • IgE receptor Mapatumumab (anti-TRAIL-Rl), Maslimomab (anti- T-cell receptor), Matuzumab (anti-EGFR), Mepolizumab (Bosatria, anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab (anti-CD74), Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3 ganglioside), Morolimumab (anti-Rhesus factor), Motavizumab (Numax, anti- respiratory syncytial virus), Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolomab
  • Oregovomab (OvaRex, anti-CA-125), Otelixizumab (anti-CD3), Pagibaximab (anti- lipoteichoic acid), Palivizumab (Synagis, Abbosynagis, anti-respiratory syncytial virus), Panitumumab (Vectibix, ABX-EGF, anti-EGFR), Panobacumab (anti- Pseudomonas aeruginosa), Pascolizumab (anti-IL-4), Pemtumomab (Theragyn, anti-MUCl), Pertuzumab (Omnitarg, 2C4, anti-HER2/neu), Pexelizumab (anti-C5), Pintumomab (anti- adenocarcinoma antigen), Priliximab (anti-CD4), Pritumumab (anti-vimentin), PRO 140 (anti-CCR5), Ra
  • Solanezumab anti-beta amyloid
  • Sonepcizumab anti-sphingosine-1 -phosphate
  • Sontuzumab (anti-episialin), Stamulumab (anti-myo statin), Sulesomab (LeukoScan, (anti- NCA-90 (granulocyte antigen), Tacatuzumab (anti-alpha-fetoprotein), Tadocizumab (anti- integrin ⁇ 3 ⁇ 4 ⁇ 3 ), Talizumab (anti-IgE), Tanezumab (anti-NGF), Taplitumomab (anti-CD 19), Tefibazumab (Aurexis, (anti-clumping factor A), Telimomab, Tenatumomab (anti-tenascin C), Teneliximab (anti-CD40), Teplizumab (anti-CD3), TGN1412 (anti-CD28),
  • Ticilimumab (Tremelimumab, (anti-CTLA-4), Tigatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13), Tocilizumab (Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor),
  • Toralizumab anti-CD154 (CD40L)
  • Tositumomab anti-CD20
  • Trastuzumab trastuzumab
  • Vedolizumab (anti-integrin ⁇ 4 ⁇ 7 ), Veltuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1), Visilizumab (Nuvion, anti-CD3), Vitaxin (anti-vascular integrin avb3),
  • Volociximab anti-integrin ⁇ 5 ⁇ 1
  • Votumumab (HumaSPECT, anti-tumor antigen
  • CTAA16.88 Zalutumumab (HuMax-EGFr, (anti-EGFR), Zanolimumab (HuMax-CD4, anti-CD4), Ziralimumab (anti-CD 147 (basigin)), Zolimomab (anti-CD5), Etanercept (Enbrel®), Alefacept (Amevive®), Abatacept (Orencia®), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (Iron Regulatory Protein 2)], 14G2a (anti-GD2 ganglioside, from Nat. Cancer
  • J591 anti-PSMA, Weill Cornell Medical School for prostate cancers
  • 225.28S anti-HMW-MAA (High molecular weight-melanoma- associated antigen)
  • Sorin Radiofarmaci S.R.L. Med, Italy
  • COL-1 anti- CEACAM3, CGMl, from Nat. Cancer Inst. USA for colorectal and gastric cancers
  • CYT- 356 Oncoltad®, for prostate cancers
  • HNK20 OraVax Inc.
  • ImmuRAIT from Immunomedics for NHL
  • Lym-1 anti-HLA-DRlO, Peregrine Pharm. for Cancers
  • MAK-195F anti-TNF (tumor necrosis factor; TNFA, TNF-alpha; TNFSF2), from Abbott / Knoll for Sepsis toxic shock
  • MEDI-500 [T10B9, anti-CD3, TRaP (T cell receptor alpha/beta), complex, from Medlmmune Inc for Graft-versus-host disease]
  • RING SCAN anti-TAG 72 (tumour associated glycoprotein 72), from Neoprobe
  • antibodies as cell binding molecules/ligands include, but are not limited to, are antibodies against the following antigens: Aminopeptidase N (CD13), Annexin Al, B7-H3 (CD276, various cancers), CA125 (ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carci- nomas), CA242 (colorectal), placental alkaline phosphatase (carcinomas), prostate specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma), CD3 epsilon (T cell lymphoma, lung, breast, gastric, ovarian cancers, autoimmune diseases, malignant ascites), CD 19 (B cell malignancies), CD20 (non-Hodgkin'
  • Endoglin CD 105, solid tumors
  • EPCAM epidermal cell adhesion molecule
  • bladder head, neck, colon, NHL prostate, and ovarian cancers
  • ERBB2 Epidermal Growth Factor Receptor 2; lung, breast, prostate cancers
  • FCGR1 autoimmune diseases
  • FOLR farnesoid receptor
  • GD2 ganglioside cancers
  • G-28 a cell surface antigen glyvolipid, melanoma
  • GD3 idiotype cancers
  • Heat shock proteins cancers
  • HLA-DR10 (NHL), HLA-DRB (NHL, B cell leukemia), human chorionic gonadotropin (carcinoma), IGF1R (insulin-like growth factor 1 receptor, solid tumors, blood cancers), IL-2 receptor (interleu- kin 2 receptor, T-cell leukemia and lymphomas), IL-6R (interleukin 6 receptor, multiple myeloma, RA, Castleman's disease, IL6 dependent tumors), Integrins ( ⁇ 3, ⁇ 5 ⁇ 1, ⁇ 6 ⁇ 4, ⁇ 11 ⁇ 3, ⁇ 5 ⁇ 5, ⁇ 5, for various cancers), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE 4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma), MS4A1 (membrane-spanning 4-domains subfamily A member 1, Non- Ho
  • TRAIL-R1 Tumor necrosis apoprosis Inducing ligand Receptor 1, lymphoma, NHL, colorectal, lung cancers
  • VCAM-1 CD 106, Melanoma
  • VEGF VEGF-A
  • VEGF-2 CD309
  • Some other tumor associated antigens recognized by antibodies have been reviewed (Gerber, et al, mAbs 1:3, 247-253 (2009); Novellino et al, Cancer Immunol Immunother. 54(3), 187-207 (2005). Franke, et al, Cancer
  • the cell-binding agents can be any agents that are able to against tumor cells, virus infected cells, microorganism infected cells, parasite infected cells, autoimmune cells, activated cells, myeloid cells, activated T-cells, B cells, or mela- nocytes.
  • the cell binding agents can be any agent/molecule that is able to against any one of the following antigens or receptors: CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CDl la, CDl lb, CDl lc, CD12w, CD14, CD15, CD16, CDwl7, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD51, CD52, CD53, CD54,
  • Angiopoietin 2 Angiopoietin 3, Annexin Al, Anthrax toxin protective antigen, Anti- transferrin receptor, AOC3 (VAP-1), B7-H3, Bacillus anthracis anthrax, BAFF (B-cell activating factor), B-lymphoma cell, bcr-abl, Bombesin, BORIS, C5, C242 antigen, CA125
  • Clumping factor A CRIPTO, FCSF1R (Colony stimulating factor 1 receptor, CD115), CSF2 (colony stimulating factor 2, Granulocyte-macrophage colony- stimulating factor (GM-CSF)), CTLA4 (cytotoxic T-lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4 (CD 184), C-X-C chemokine receptor type 4, cyclic ADP ribose hydrolase, Cyclin B l, CYP1B 1, Cytomegalovirus, Cytomegalovirus glycoprotein B, Dabigatran,
  • DLL4 delta-like-ligand 4
  • DPP4 Dipeptidyl-peptidase 4
  • DR5 Death receptor 5
  • E. coli shiga toxin type-1 E. coli shiga toxin type-2, ED-B, EGFL7 (EGF-like domain- containing protein 7), EGFR, EGFRII, EGFRvIII, Endoglin (CD 105), Endothelin B receptor, Endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2, Episialin, ERBB2 (Epidermal Growth Factor Receptor 2), ERBB3, ERG (TMPRSS2 ETS fusion gene), Escherichia coli, ETV6-AML, FAP (Fibroblast activation protein alpha), FCGR1, alpha-Fetoprotein, Fibrin II, beta chain, Fibronectin extra domain-B, FOLR (folate receptor), Folate receptor alpha, Folate hydrolase, Fo
  • GMCSF receptor a-chain Growth differentiation factor 8
  • GP100 GPNMB
  • GUCY2C (Guanylate cyclase 2C, guanylyl cyclase C(GC-C), intestinal Guanylate cyclase, Guanylate cyclase-C receptor, Heat-stable entero- toxin receptor (hSTAR)), Heat shock proteins, Hemagglutinin, Hepatitis B surface antigen, Hepatitis B virus, HER1 (human epidermal growth factor receptor 1), HER2, HER2/neu,
  • HER3 (ERBB-3), IgG4, HGF/SF (Hepatocyte growth factor/scatter factor), HHGFR, HIV- 1, Histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB , HMWMAA, Human chorionic gonadotropin, HNGF, Human scatter factor receptor kinase, HPV E6/E7, Hsp90, hTERT, ICAM-1 (Intercellular Adhesion Molecule 1), Idiotype, IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN- ⁇ , Influeza hemagglutinin, IgE, IgE Fc region, IGHE, IL-1, IL-2 receptor (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,
  • KIR2D KIR2D, LCK, Le, Legumain, Lewis- Y antigen, LFA-1 (Lymphocyte function-associated antigen 1, CDl la), LHRH, LINGO-1, Lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT- 1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE Al, MAGE A3, MAGE 4, MARTI, MCP-1, MIF (Macrophage migration inhibitory factor, or glycosylation- inhibiting factor (GIF)), MS4A1 (membrane- spanning 4-domains subfamily A member 1),
  • MSLN mesothelin
  • MUCl Mucin 1, cell surface associated (MUC1) or polymorphic epithelial mucin (PEM)
  • MUC1-KLH MUC16 (CA125), MCPl(monocyte chemotactic protein 1)
  • MelanA/MARTl MEG1
  • MPG MEG1
  • MS4A1 membrane-spanning 4-domains subfamily A
  • MYCN Myelin- associated glycoprotein
  • Myostatin Myostatin
  • NA17 NARP-1
  • NCA- 90 granulocyte antigen
  • Nectin-4 ASG-22ME
  • NGF Neural apoptosis-regulated proteinase 1, NOGO-A, Notch receptor, Nucleolin, Neu oncogene product, NY-BR-1, NY- ESO-1, OX-40, OxLDL (Oxidized low-density lipoprotein), OY-TES 1, P21, p53 nonmutant, P97, Page4, PAP, Paratope
  • Phosphatidylserine Prostatic carcinoma cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, Rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD240), Rhesus factor, RANKL, RhoC, Ras mutant, RGS5, ROBO4, Respiratory syncytial virus, RON, Sarcoma translocation breakpoints, SART3, Sclerostin, SLAMF7 (SLAM family member
  • TGF-C Tenascin C
  • TGF-a TGF- ⁇
  • TGF- ⁇ Transforming growth factor beta
  • TGF- ⁇ TGF ⁇ 2
  • Tie CD202b
  • Tie2 Tie2
  • TIM-1 CDX-014
  • Tn TNF, TNF-a
  • TNFRSF8 TNFRSF10B
  • TNFRSF13B tumor necrosis factor receptor superfamily member 13B
  • TPBG trophoblast glycoprotein
  • TRAIL-R1 Tuor necrosis apoprosis Inducing ligand Receptor 1
  • TRAILR2 Death receptor 5 (DR5)
  • tumor specific glycosylation of MUC1, TWEAK receptor TRP-2
  • Tyrosinase VCAM-1 (CD 106)
  • the cell-binding ligand-drug conjugates via the bridge linkers of this invention are used for the targeted treatment of cancers.
  • the targeted cancers include, but are not limited, Adrenocortical Carcinoma, Anal Cancer, Bladder Cancer, Brain Tumor (Adult, Brain Stem Glioma, Childhood, Cerebellar Astrocytoma, Cerebral
  • Gallbladder Cancer Gastric Cancer (Stomach), Germ Cell Tumor, Extragonadal, Gestational Trophoblastic Tumor, Head and Neck Cancer, Hypopharyngeal Cancer, Islet Cell Carcinoma, Kidney Cancer (renal cell cancer), Laryngeal Cancer, Leukemia (Acute Lymphoblastic, Acute Myeloid, Chronic Lymphocytic, Chronic Myelogenous, Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer (Non-Small Cell, Small Cell, Lymphoma (AIDS -Related, Central Nervous System, Cutaneous T-Cell, Hodgkin's Disease, Non-Hodgkin's Disease, Malignant Mesothelioma, Melanoma, Merkel Cell Carcinoma, Metasatic Squamous Neck Cancer with Occult Primary, Multiple Myeloma, and Other Plasma Cell Neoplasms, Mycosis Fungoides, Myelodysplastic Syndrome, Myeloproli-
  • Osteosarcoma Osteosarcoma, Ovarian Cancer (Epithelial, Germ Cell Tumor, Low Malignant Potential Tumor), Pancreatic Cancer (Exocrine, Islet Cell Carcinoma), Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pheochromocytoma Cancer, Pituitary Cancer, Plasma Cell Neoplasm, Prostate Cancer Rhabdomyosarcoma, Rectal Cancer, Renal Cell Cancer (kidney cancer), Renal Pelvis and Ureter (Transitional Cell),
  • Salivary Gland Cancer Sezary Syndrome, Skin Cancer, Skin Cancer (Cutaneous T-Cell Lymphoma, Kaposi's Sarcoma, Melanoma), Small Intestine Cancer, Soft Tissue Sarcoma, Stomach Cancer, Testicular Cancer, Thymoma (Malignant), Thyroid Cancer, Urethral Cancer, Uterine Cancer (Sarcoma), Unusual Cancer of Childhood, Vaginal Cancer, Vulvar Cancer, Wilms' Tumor.
  • the cell-binding-drug conjugates via the bridge likers of this invention are used in accordance with the compositions and methods for the treat- ment or prevention of an autoimmune disease.
  • the autoimmune diseases include, but are not limited, Achlorhydra Autoimmune Active Chronic Hepatitis, Acute Disseminated Encephalomyelitis, Acute hemorrhagic leukoencephalitis, Addison's Disease,
  • Haemolytic anaemia Henoch- Schonlein purpura, Herpes gestationis, Hidradenitis suppurativa, Hughes syndrome (See Antiphospholipid syndrome), Hypogammaglobulinemia, Idiopathic Inflammatory Demyelinating Diseases, Idiopathic pulmonary fibrosis, Idiopathic thrombocytopenic purpura (See Autoimmune thrombocytopenic purpura), IgA nephropathy (Also Berger's disease), Inclusion body myositis, Inflammatory demyelinating polyneuopathy, Interstitial cystitis, Irritable Bowel Syndrome , Juvenile idiopathic arthritis, Juvenile rheumatoid arthritis, Kawasaki's Disease, Lambert-Eaton myasthenic syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Linear IgA disease (LAD), Lou Gehrig's Disease (Als
  • Neuromyotonia Occular cicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord thyroiditis, Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria, Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis, Pemphigus, Pemphigus vulgaris, Pernicious anaemia, Perivenous encephalo- myelitis, POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica, Polymyositis,
  • a binding molecule used for the conjugate via the bridge linkers of this invention for the treatment or prevention of an autoimmune disease can be, but are not limited to, anti-elastin antibody; Abys against epithelial cells antibody;
  • Anti-Basement Membrane Collagen Type IV Protein antibody Anti-Nuclear Antibody; Anti ds DNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG; anti-celiac antibody; Anti Phospholipid Antibody IgK, IgG; Anti SM Antibody; Anti Mitochondrial Antibody; Thyroid Antibody; Microsomal Antibody, T-cells antibody; Thyroglobulin Antibody, Anti SCL-70; Anti- Jo; Anti-U.sub.lRNP; Anti-La/SSB; Anti SSA; Anti SSB; Anti Perital Cells Antibody; Anti Histones; Anti RNP; C-ANCA; P-ANCA; Anti centromere; Anti- Fibrillarin, and Anti GBM Antibody, Anti-ganglioside antibody; Anti-Desmogein 3 antibody; Anti-p62 antibody; Anti-s lOO antibody; Anti-Mitochondrial(M2) antibody; Rheu- matoid factor antibody; Anti-MCV antibody; Anti
  • the binding molecule for the conjugate in the present invention can bind to both a receptor and a receptor complex expressed on an activated lymphocyte which is associated with an autoimmune disease.
  • the receptor or receptor complex can comprise an immunoglobulin gene superfamily member (e.g. CD2, CD3,
  • useful cell binding ligands that are immuno specific for a viral or a microbial antigen are humanized or human monoclonal antibodies.
  • viral antigen includes, but is not limited to, any viral peptide, polypeptide protein (e.g. HIV gpl20, HIV nef, RSV F glycoprotein, influenza virus neuramimi- dase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g. gB, gC, gD, and gE) and hepatitis B surface antigen) that is capable of eliciting an immune response.
  • polypeptide protein e.g. HIV gpl20, HIV nef, RSV F glycoprotein
  • influenza virus neuramimi- dase influenza virus hemagglutinin
  • HTLV tax herpes simplex virus glycoprotein
  • herpes simplex virus glycoprotein e.g. gB, gC, gD, and gE
  • microbial antigen includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., a bacteria, fungi, pathogenic protozoa, or yeast polypeptides including, e.g., LPS and capsular polysaccharide 5/8) that is capable of eliciting an immune response.
  • microbial antigen includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., a bacteria, fungi, pathogenic protozoa, or yeast polypeptides including, e.g., LPS and capsular polysaccharide 5/8) that is capable of eliciting an immune response.
  • antibodies available 1 for the viral or microbial infection include, but are not limited to, Palivizumab which is a humanized anti-respiratory syncytial virus monoclonal antibody for the treatment of RSV infection; PRO542 which is a CD4 fusion antibody for the treatment of HIV infection; Ostavir which is a human antibody for the treatment of hepatitis B virus; PROTVIR which is a humanized IgG.sub. l antibody for the treatment of cytomegalovirus; and anti-LPS antibodies.
  • Palivizumab which is a humanized anti-respiratory syncytial virus monoclonal antibody for the treatment of RSV infection
  • PRO542 which is a CD4 fusion antibody for the treatment of HIV infection
  • Ostavir which is a human antibody for the treatment of hepatitis B virus
  • PROTVIR which is a humanized IgG.sub. l antibody for the treatment of cytomegalovirus
  • anti-LPS antibodies anti-LPS antibodies.
  • infectious diseases include, but are not limited to, Acinetobacter infections, Actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired immune deficiency syndrome), Amebiasis, Anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereus infection, Bacterial pneumonia, Bacterial vaginosis, Bacteroides infection, Balantidiasis,
  • Baylisascaris infection BK virus infection, Black piedra, Blastocystis hominis infection, Blastomycosis, Venezuelan hemorrhagic fever, Borrelia infection, Botulism (and Infant botulism), Brazilian hemorrhagic fever, Brucellosis, Burkholderia infection, Buruli ulcer,
  • Calicivirus infection (Norovirus and Sapovirus), Campylobacteriosis, Candidiasis
  • Acute coryza Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever, Cryptococcosis, Cryptosporidiosis, Cutaneous larva migrans, Cyclosporiasis, Cysticercosis, Cytomegalovirus infection, Dengue fever, Dientamoebiasis, Diphtheria, Diphyllobothriasis, Dracunculiasis, Ebola hemorrhagic fever, Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworm infection), Enterococcus infection, Enterovirus infection, Epidemic typhus,
  • Erythema infectiosum (Fifth disease), Exanthem subitum, Fasciolopsiasis, Fasciolosis, Fatal familial insomnia, Filariasis, Food poisoning by Clostridium perfringens, Free-living amebic infection, Fusobacterium infection, Gas gangrene (Clostridial myonecrosis), Geotrichosis, Gerstmann-Straussler-Scheinker syndrome, Giardiasis, Glanders, Gnathosto- miasis, Gonorrhea, Granuloma inguinale (Donovanosis), Group A streptococcal infection,
  • Group B streptococcal infection Haemophilus influenzae infection, Hand, foot and mouth disease (HFMD), Hantavirus Pulmonary Syndrome, Helicobacter pylori infection, Hemolytic-uremic syndrome, Hemorrhagic fever with renal syndrome, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infec- tion, Human bocavirus infection, Human ewingii ehrlichiosis, Human granulocytic anaplasmosis, Human metapneumo virus infection, Human monocytic ehrlichiosis, Human papillomavirus infection, Human parainfluenza virus infection, Hymenolepiasis, Epstein- Barr Virus Infectious Mononucleosis (Mono), Influenza, Isosporiasis, Kawasaki disease, Keratitis, Kingella kingae infection, Kuru
  • Molluscum contagiosum Mumps, Murine typhus (Endemic typhus), Mycoplasma pneumonia, Mycetoma, Myiasis, Neonatal conjunctivitis (Ophthalmia neonatorum), (New) Variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardiosis, Onchocerciasis (River blindness), Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis, Pasteurellosis, Pediculosis capitis (Head lice), Pediculosis corporis (Body lice), Pediculosis pubis (Pubic lice, Crab lice), Pelvic inflammatory disease, Pertussis (Whooping cough),
  • Tinea manuum (Ringworm of the Hand), Tinea nigra, Tinea pedis (Athlete's foot), Tinea unguium (Onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxocariasis (Ocular Larva Migrans), Toxocariasis (Visceral Larva Migrans), Toxoplasmosis, Trichinellosis, Trichomoniasis, Trichuriasis (Whipworm infection), Tuberculosis, Tularemia, Ureaplasma urealyticum infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever,
  • Viral pneumonia West Nile Fever, White piedra (Tinea blanca), Yersinia pseudotuberculosis infection, Yersiniosis, Yellow fever, Zygomycosis.
  • the cell binding molecule which is more preferred to be an antibody described in this patent that are against pathogenic strains include, but are not limit, Acinetobacter baumannii, Actinomyces israelii, Actinomyces gerencseriae and Propionibacterium propionicus, Trypanosoma brucei, HIV (Human immunodeficiency virus), Entamoeba histolytica, Anaplasma genus, Bacillus anthracis, Arcanobacterium haemolyticum, Junin virus, Ascaris lumbricoides, Aspergillus genus, Astroviridae family, Babesia genus, Bacillus cereus, multiple bacteria, Bacteroides genus, Balantidium coli, Baylisascaris genus, BK virus, Piedraia hortae, Blastocystis hominis, Blastomyces dermatitides, Machupo virus, Borrelia genus, Clostridium bot
  • Cyclospora cayetanensis Taenia solium, Cytomegalovirus, Dengue viruses (DEN-1, DEN- 2, DEN-3 and DEN-4) - Flaviviruses, Dientamoeba fragilis, Corynebacterium diphtheriae, Diphyllobothrium, Dracunculus medinensis, Ebolavirus, Echinococcus genus, Ehrlichia genus, Enterobius vermicularis, Enterococcus genus, Enterovirus genus, Rickettsia prowazekii, Parvovirus B 19, Human herpesvirus 6 and Human herpesvirus 7, Fasciolopsis buski, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Clostridium perfringens, Fusobacterium genus, Clostridium perfringens; other Clostridium species, Geotrichum candidum, GSS pri
  • Enteroviruses mainly Coxsackie A virus and Enterovirus 71, Sin Nombre virus, Helicobacter pylori, Escherichia coli O157:H7, Bunyaviridae family, 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, Ancylostoma duodenale and Necator americanus, Hemophilus influenzae, Human bocavirus, Ehrlichia ewingii, Anaplasma phagocytophilum, Human metapneumovirus, Ehrlichia chaffeensis, Human papillomavirus, Human parainfluenza viruses, Hymenolepis nana and Hymenolepis diminuta, Ep- stein-Barr Virus, Orthomy-xoviridae family, Isospora belli
  • Trichophyton genus Trichophyton tonsurans, Trichophyton genus, Epidermophyton floccosum, Trichophyton rubrum, and Trichophyton mentagrophytes, Trichophyton rubrum, Hortaea wasneckii, Trichophyton genus, Malassezia genus, Toxocara canis or Toxocara cati, Toxoplasma gondii, Trichinella spiralis, Trichomonas vaginalis, Trichuris trichiura, Mycobacterium tuberculosis, Francisella tularensis, Ureaplasma urealyticum,
  • Venezuelan equine encephalitis virus Vibrio colerae, Guanarito virus, West Nile virus, Trichosporon beigelii, Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fever virus, Mucorales order (Mucormycosis) and Entomophthorales order (Entomophthora- mycosis), Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Edwardsiella tarda, Yersinia pestis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei,
  • Salmonella typhimurium, Treponema per pneumonia, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Pneumocystis carinii, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Clamydia spp.; pathogenic fungi (Aspergillus fumigatus, Candida albicans, Histoplasma capsulatum); protozoa (Entomoeba histolytica, Trichomonas tenas,
  • Trichomonas hominis Tryoanosoma gambiense, Trypanosoma rhodesiense, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms).
  • antibodies as cell binding ligands used in this invention for treatment of viral disease include, but are not limited to, antibodies against antigens of pathogenic viruses, including as examples and not by limitation: Poxyiridae, Herpesviridae, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, Oncovirus [such as, HBV (Hepatocellular carcinoma), HPV (Cervical cancer, Anal cancer), Kaposi's sarcoma-associated herpesvirus (Kaposi's sarcoma), Epstein-Barr virus (Nas
  • the present invention also concerns pharmaceutical compositions comprising the conjugate via the bridge linkers of the invention together with a pharmaceutically acceptable carrier, diluent, or excipient for treatment of cancers, infections or autoimmune disorders.
  • a pharmaceutically acceptable carrier diluent, or excipient for treatment of cancers, infections or autoimmune disorders.
  • the method for treatment of cancers, infections and autoimmune disorders can be practiced in vitro, in vivo, or ex vivo.
  • in vitro uses include treatments of cell cultures in order to kill all cells except for desired variants that do not express the target antigen; or to kill variants that express undesired antigen.
  • ex vivo uses include treatments of hematopoietic stem cells (HSC) prior to the performance of the transplantation (HSCT) into the same patient in order to kill diseased or malignant cells.
  • HSC hematopoietic stem cells
  • the bone marrow cells are washed with medium containing serum and returned to the patient by i.v. infusion according to known methods.
  • the treated marrow cells are stored frozen in liquid nitrogen using standard medical equipment.
  • the conjugate via the linkers of the invention will be supplied as solutions or as a lyophilized solid that can be redissolved in sterile water for injection.
  • suitable protocols of conjugate administration are as follows. Conjugates are given weekly for 8-20 weeks as an i.v. bolus. Bolus doses are given in 50 to 500 ml of normal saline to which human serum albumin (e.g. 0.5 to 1 mL of a concentrated solution of human serum albumin, 100 mg/mL) can be added. Dosages will be about 50 ⁇ g to 20 mg/kg of body weight per week, i.v. (range of 10 ⁇ g to 200 mg/kg per injection). 4-20 weeks after treatment, the patient may receive a second course of treatment. Specific clinical protocols with regard to route of administration, excipients, diluents, dosages, times, etc., can be determined by the skilled clinicians.
  • Examples of medical conditions that can be treated according to the in vivo or ex vivo methods of killing selected cell populations include malignancy of any types of cancer, autoimmune diseases, graft rejections, and infections (viral, bacterial or parasite).
  • the amount of a conjugate which is required to achieve the desired biological effect will vary depending upon a number of factors, including the chemical characteristics, the potency, and the bioavailability of the conjugates, the type of disease, the species to which the patient belongs, the diseased state of the patient, the route of administration, all factors which dictate the required dose amounts, delivery and regimen to be administered.
  • the conjugates via the linkers of this invention may be provided in an aqueous physiological buffer solution containing 0.1 to 10% w/v conjugates for parenteral administration.
  • Typical dose ranges are from 1 g/kg to 0.1 g/kg of body weight per day; a preferred dose range is from 0.01 mg/kg to 20 mg/kg of body weight per day, or per week, or an equivalent dose in a human child.
  • the preferred dosage of drug to be administered is likely to depend on such variables 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 compound selected, the formulation of the compound, the route of administration
  • the conjugates via the linkers of the present invention are also capable of being ad- ministered in unit dose forms, wherein the term "unit dose” means a single dose which is capable of being administered to a patient, and which can be readily handled and packaged, remaining as a physically and chemically stable unit dose comprising either the active conjugate itself, or as a pharmaceutically acceptable composition, as described hereinafter.
  • unit doses for humans range from 1 mg to 3000 mg per day, or per week, per two week or per month.
  • the unit dose range is from 1 to 500 mg administered one to four times a week, and even more preferably from 1 mg to 100 mg, once a week.
  • Conjugates provided herein can be formulated into pharmaceutical compositions by admixture with one or more pharmaceutically acceptable excipients.
  • Such unit dose compositions may be prepared for use by oral administration, particularly in the form of tablets, simple capsules or soft gel capsules; or intranasal, particularly in the form of powders, nasal drops, or aerosols; or dermally, for example, topically in ointments, creams, lotions, gels or sprays, or via trans-dermal patches.
  • Drugs that can be conjugated to a cell-binding molecule in the present invention are small molecule drugs including cytotoxic agents, which can be linked to or after they are modified for linkage to the cell-binding agent.
  • a "small molecule drug” is broadly used herein to refer to an organic, inorganic, or organometallic compound that may have a molecular weight of for example 100 to 1800, more suitably from 120 to 1400.
  • Small molecule drugs are well characterized in the art, such as in WO05058367A2, and in U.S. Patent No. 4,956,303, among others and are incorporated in their entirety by reference.
  • the drugs include known drugs and those that may become known drugs.
  • Drugs that are known include, but not limited to,
  • Chemotherapeutic agents a).
  • Alkylating agents such as Nitrogen mustards:
  • Plant Alkaloids such as Vinca alkaloids: (vincristine, vinblastine, vindesine, vinorelbine, navelbin); Taxoids: (paclitaxel, docetaxol) and their analogs, Maytansinoids (DM1, DM2, DM3, DM4, maytansine and ansamitocins) and their analogs, cryptophycins (particularly cryptophycin 1 and cryptophycin 8); epothilones, eleutherobin, discodermo- lide, bryostatins, dolostatins, auristatins, tubulysins, cephalostatins; pancratistatin; a sarcodictyin; spongistatin; c).
  • DNA Topoisomerase Inhibitors such as [Epipodophyllins:
  • Anti-metabolites such as ⁇ [Anti-folate: DHFR inhibitors: (methotrexate, trimetrexate, denopterin, pteropterin, aminopterin (4-aminopteroic acid) or the other folic acid analogues); IMP dehydrogenase
  • Inhibitors (mycophenolic acid, tiazofurin, ribavirin, EICAR); Ribonucleotide reductase Inhibitors: (hydroxyurea, deferoxamine)]; [Pyrimidine analogs: Uracil analogs:
  • Cytosine analogs (cytarabine, cytosine arabinoside, fludarabine); Purine analogs: (azathioprine, fludarabine, mercaptopurine, thiamiprine, thioguanine)]; folic acid replenisher, such as frolinic acid ⁇ ; e).
  • Hormonal therapies such as ⁇ Receptor antagonists: [Anti-estrogen: (megestrol, raloxifene, tamoxifen); LHRH agonists: (goscrclin, leuprolide acetate); Anti-androgens: (bicalutamide, flutamide, calusterone, dromostanolone propionate, epitiostanol, goserelin, leuprolide, mepitiostane, nilutamide, testolactone, trilostane and other androgens inhibitors)]; Retinoids/Deltoids: [Vitamin D3 analogs: (CB 1093, EB 1089 KH 1060, cholecalciferol, ergocalciferol); Photodynamic therapies: (verteporfin, phthalocyanine, photo sensitizer Pc4, demethoxy-hypocrellin A); Cytokines: (Interferon- alpha, Interfer
  • Kinase inhibitors such as BIBW 2992 (anti-EGFR/Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib.
  • vandetanib E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534), bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, Trastuzumab,
  • antibiotics such as the enediyne antibiotics (e.g. calicheamicins, especially calicheamicin ⁇ , ⁇ , ⁇ 1 and ⁇ 1, see, e.g., J. Med. Chem., 39 (11), 2103-2117 (1996), Angew Chem Intl. Ed. Engl.
  • enediyne antibiotics e.g. calicheamicins, especially calicheamicin ⁇ , ⁇ , ⁇ 1 and ⁇ 1, see, e.g., J. Med. Chem., 39 (11), 2103-2117 (1996), Angew Chem Intl. Ed. Engl.
  • dynemicin including dynemicin A and deoxydynemicin; esperamicin, kedarcidin, C-1027, maduropeptin, as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin;
  • chromomycins dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, nitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; f).
  • Celecoxib glitazones, epigallocatechin gallate, Disulfiram, Salinosporamide A.; Anti- adrenals, such as aminoglutethimide, mitotane, trilostane; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; arabinoside, bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; eflornithine (DFMO), elfomithine; elliptinium acetate, etoglucid; gallium nitrate; gacytosine, hydroxyurea; ibandronate, lentinan;
  • Anti- adrenals such as aminoglutethimide, mitotane, trilostane; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; arabinoside, bestrabucil;
  • lonidamine mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK ® ; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucarin A, roridin A and anguidine); urethane, siRNA, antisense drugs, and a nucleolytic enzyme.
  • An anti- autoimmune disease agent includes, but is not limited to, cyclosporine, cyclosporin A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g. amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, f uocortolone danazol, dexamethasone, Triamcinolone acetonide, beclometasone dipropionate), DHEA, enanercept,
  • corticosteroids e.g. amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, f uocortolone danazol, dexamethasone, Triamcinolone acetonide, beclometasone diprop
  • An anti-infectious disease agent includes, but is not limited to, a).
  • Aminoglycosides amikacin, astromicin, gentamicin (netilmicin, sisomicin, isepamicin), hygromycin B, kanamycin (amikacin, arbekacin, bekanamycin, dibekacin, tobramycin), neomycin
  • carbacephem (loracarbef), cefacetrile, cefaclor, cefradine, cefadroxil, cefalonium, cefaloridine, cefalotin or cefalothin, cefalexin, cefaloglycin, cefamandole, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin, cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin, cefp
  • Glycopeptides bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin; g).
  • Glycylcyc lines e. g. tigecycline; g).
  • ⁇ - Lactamase inhibitors penam (sulbactam, tazobactam), clavam (clavulanic acid); i).
  • Lincosamides clindamycin, lincomycin; j). Lipopeptides: daptomycin, A54145, calcium-dependent antibiotics (CDA); k). Macrolides: azithromycin, cethromycin, clarithromycin, dirithromycin, erythromycin, flurithromycin, josamycin, ketolide (telithromycin, cethromycin), midecamycin, miocamycin, oleandomycin, rifamycins (rifampicin, rifampin, rifabutin, rifapentine), rokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandomycin, telithromycin; 1).
  • Monobactams aztreonam, tigemonam; m).
  • Oxazolidinones linezolid; n).
  • Penicillins amoxicillin, ampicillin (pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin), azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethyl-penicillin, clometocillin, procaine benzylpenicillin, carbenicillin (carindacillin), cloxacillin, dicloxacillin, epicillin, flucloxacillin, mecillinam (pivmecillinam), mezlocillin, meticillin, nafcillin, oxacillin, penamecillin, penicillin, pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin, sulbeniciUin, temocillin
  • Streptogramins pristinamycin, quinupristin/dalfopristin); r).
  • Sulfonamides mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim- sulfamethoxazole (co-trimoxazole); s).
  • Steroid antibacterials e.g. fusidic acid; t).
  • Tetracyclines doxycycline, chlortetracycline, clomocycline,
  • demeclocycline demeclocycline, lymecycline, meclocycline, metacycline, minocycline, oxytetracycline, penimepicycline, rolitetracycline, tetracycline, glycylcyclines (e.g. tigecycline); u).
  • Other types of antibiotics annonacin, arsphenamine, bactoprenol inhibitors (Bacitracin),
  • DADAL/AR inhibitors cycloserine
  • dictyostatin discodermolide
  • eleutherobin epothilone
  • ethambutol etoposide
  • faropenem fusidic acid
  • furazolidone isoniazid
  • laulimalide metronidazole
  • mupirocin mycolactone
  • NAM synthesis inhibitors e. g.
  • quinupristin/dalfopristin quinupristin/dalfopristin, rifampicin (rifampin), tazobactam tinidazole, uvaricin;
  • Anti-viral drugs a). Entry/fusion inhibitors: aplaviroc, maraviroc, vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab); b). Integrase inhibitors: raltegravir,
  • elvitegravir globoidnan A
  • Maturation inhibitors bevirimat, becon
  • Neuraminidase inhibitors oseltamivir, zanamivir, peramivir
  • Nucleosides &_nucleotides abacavir, aciclovir, adefovir, amdoxovir, apricitabine, brivudine, cidofovir, clevudine,
  • dexelvucitabine didanosine (ddl), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU), 3 '-fluoro-substituted 2', 3 '-dideoxynucleoside analogues
  • fLT 3'-fluoro-2',3'-dideoxythymidine
  • FLG 3'-fluoro-2',3'-dideoxyguanosine
  • fomivirsen e.g. 3'-fluoro-2',3'-dideoxythymidine (FLT) and 3'-fluoro-2',3'-dideoxyguanosine (FLG)
  • fomivirsen e.g. 3'-fluoro-2',3'-dideoxythymidine (FLT) and 3'-fluoro-2',3'-dideoxyguanosine (FLG)
  • fomivirsen e.g. 3'-fluoro-2',3'-dideoxythymidine (FLT) and 3'-fluoro-2',3'-dideoxyguanosine (FLG)
  • fomivirsen e.g. 3'-fluoro-2',3'-dideoxyth
  • Non-nucleosides amantadine, ateviridine, capravirine, diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphonoformic acid), imiquimod, interferon alfa, loviride, lodenosine, methisazone, nevirapine, NOV-205, peginterferon alfa,
  • Protease inhibitors amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h).
  • anti- virus drugs abzyme, arbidol, calanolide a, ceragenin, cyanovirin-n, diarylpyrimidines, epigallocatechin gallate (EGCG), foscarnet, griffithsin, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, portmanteau inhibitors, ribavirin, seliciclib.
  • the drugs used for conjugates via a bridge linker of the present invention also in- elude radioisotopes.
  • radioisotopes radioisotopes
  • Rad ⁇ i ⁇ oi ⁇ sotope labeled antibodies are useful in receptor targeted imaging experiments or can be for targeted treatment such as with the antibody-drug conjugates of the invention (Wu et al
  • the cell binding molecules e.g. an antibody can be labeled with ligand reagents through the bridge linkers of the present patent that bind, chelate or otherwise complex a radioisotope metal, using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley- Interscience, New York, Pubs. (1991).
  • Chelating ligands which may complex a metal ion include DOTA, DOTP, DOTMA, DTP A and TETA (Macrocyclics, Dallas, Tex. USA).
  • the drug in the Formula (II) and (IV) can a chromophore molecule, for which the conjugate can be used for detection, monitoring, or study the interaction of the cell binding molecule with a target cell.
  • Chromophore molecules are a compound that have the ability to absorb a kind of light, such as UV light, florescent light, IR light, near IR light, visual light;
  • a chromatophore molecule includes a class or subclass of xanthophores, erythrophores, iridophores, leucophores, melanophores, and cyanophores; a class or subclass of fluorophore molecules which are fluorescent chemical compounds re- emitting light upon light; a class or subclass of visual phototransduction molecules; a class or subclass of photophore molecules; a class or subclass of luminescence molecules; and a class or subclass of luciferin compounds.
  • the chromophore molecule can be selected from, but not limited, Non-protein organic fluorophores, such as: Xanthene derivatives (fluorescein, rhodamine, Oregon green, eosin, and Texas red); Cyanine derivatives: (cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine); Squaraine derivatives and ring-substituted squaraines, including Seta, SeTau, and Square dyes; Naphthalene derivatives (dansyl and prodan derivatives); Coumarin derivatives; Oxadiazole derivatives (pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole); Anthracene derivatives (anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange); Pyrene derivatives (cascade blue, etc); Oxazine derivatives (Nile red, Nile blue, cres
  • Acridine derivatives proflavin, acridine orange, acridine yellow etc.
  • Arylmethine derivatives auramine, crystal violet, malachite green).
  • Tetrapyrrole derivatives porphin, phthalocyanine, bilirubin.
  • a chromophore molecule can be selected from any analogs and derivatives of the following fluorophore compounds: CF dye (Biotium), DRAQ and CyTRAK probes (BioStatus), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLight Fluor (Thermo Scientific, Pierce), Atto and Tracy (Sigma Aldrich), FluoProbes (Interchim), Abberior
  • Dyes (Abberior), DY and MegaStokes Dyes (Dyomics), Sulfo Cy dyes (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTau and Square Dyes (SETA BioMedicals), Quasar and Cal Fluor dyes (Biosearch Technologies), SureLight Dyes (APC, RPEPerCP,
  • the fluorophore compounds that can be linked to the linkers of the invention for study of nucleic acids or proteins are selected from the following compounds or their derivatives: 7-AAD (7-aminoactinomycin D, CG-selective), Acridine Orange, Chromomycin A3, CyTRAK Orange (Biostatus, red excitation dark), DAPI, DRAQ5, DRAQ7, Ethidium Bromide, Hoechst33258, Hoechst33342, LDS 751, Mithramycin, Propidiumlodide (PI), SYTOX Blue, SYTOX Green, SYTOX Orange, Thiazole Orange, TO-PRO: Cyanine
  • the fluorophore compounds that can be linked to the linkers of the invention for study cells are selected from the following compounds or their derivatives: DCFH (2'7'Dichorodihydro- fluorescein, oxidized form), DHR (Dihydrorhodamine 123, oxidized form, light catalyzes oxidation), Fluo-3 (AM ester. pH > 6), Fluo-4 (AM ester. pH 7.2), Indo-1 (AM ester, low/high calcium (Ca2+)), SNARF(pH 6/9).
  • the preferred fluorophore compounds that can be linked to the linkers of the invention for study proteins/antibodies are selected from the following compounds or their derivatives: Allophycocyanin(APC), AmCyanl (tetram- er, Clontech), AsRed2 (tetramer, Clontech), Azami Green (monomer, MBL), Azurite, B- phycoerythrin(BPE), Cerulean, CyPet, DsRed monomer (Clontech), DsRed2 ("RFP",
  • the drug in the Formula (II) and (IV) can be a polyalkylene glycols that are used for extending the half-life of the cell-binding molecule when administered to a mammal.
  • Polyalkylene glycols include, but are not limited to, poly(ethylene glycols) (PEGs), poly(propylene glycol) and copolymers of ethylene oxide and propylene oxide; particularly preferred are PEGs, and more particularly preferred are
  • hydroxyPEGs e.g., hydroxyPEGs activated at a single terminus, including reactive esters of hydroxyPEG-monocarboxylic acids, hydroxyPEG- monoaldehydes, hydroxyPEG-monoamines, hydroxyPEG-monohydrazides, hydroxyPEG- monocarbazates, hydroxyPEG-monoiodoacetamides, hydroxyPEG-monomaleimides, hydroxyPEG-monoorthopyridyl disulfides, hydroxyPEG-monooximes, hydroxyPEG- monophenyl carbonates, hydroxyPEG-monophenyl glyoxals, hydroxyPEG- monothiazolidine-2-thiones, hydroxyPEG-monothioesters, hydroxyPEG-monothiols, hydroxyPEG-monotriazines and hydroxyPEG-monovinylsulfones).
  • the polyalkylene glycol has a molecular weight of from about 10 Daltons to about 200 kDa, preferably about 88 Da to about 40 kDa; two branches each with a molecular weight of about 88 Da to about 40 kDa; and more preferably two branches, each of about 88 Da to about 20 kDa.
  • the polyalkylene glycol is poly(ethylene) glycol and has a molecular weight of about 10 kDa; about 20 kDa, or about 40 kDa.
  • the PEG is a PEG 10 kDa (linear or branched), a PEG 20 kDa (linear or branched), or a PEG 40 kDa (linear or branched).
  • a number of US patents have disclosed the preparation of linear or branched "non- antigenic" PEG polymers and derivatives or conjugates thereof, e.g., U.S. Pat. Nos. 5,428,128;
  • mAb is an antibody; n is 1-30; R' and R" are independently H or CH 3 ; m 3 and m4 are independently 0-5000; " X 1, X 2 , R 1 , R 2 , and R 3 are the same defined
  • R 4 is OH, H, or R 1, or R 3 that is defined as in Formula (I).
  • the preferred cytotoxic agents that conjugated to a cell- binding molecule via a bridge linker of this patent are tubulysins, maytansinoids, taxanoids (taxanes), CC-1065 analogs, daunorubicin and doxorubicin compounds, benzodiazepine dimers (e.g., dimers of pyrrolobenzodiazepine (PBD), tomaymycin, anthramycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidino-benzodiazepines), calicheamicins and the enediyne antibiotics, actinomycin, azaserines, bleomycins, epirubicin, tamoxifen, idarubicin, dolastatins, auristatins (e.g.
  • auristatin E monomethyl auristatin E, MMAE , MMAF, auristatin PYE, auristatin TP, Auristatins 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)), duocarmycins, thiotepa, vincristines, hemiasterlins, convoumamides, microginins, radiosumins,reterobactins, microsclerodermins, theonellamides,
  • Tubulysins that are preferred for conjugation in the present invention are well known in the art and can be isolated from natural sources according to known methods or prepared synthetically according to known methods (e. g. Balasubramanian, R.; et al. J. Med.
  • T01, T02, T03, T04, T05, T06 and T07 are T01, T02, T03, T04, T05, T06 and T07 as following:
  • mAb is an antibody
  • Z 3 and Z' 3 are independently H, OP(O)(OMi)(OM 2 ), OCH 2 OP(O)(OMi)(OM 2 ), OSO 3 M 1 , R 1 , or O-glycoside (glucoside, galactoside, mannoside, glucuronoside, alloside, fructoside, etc), NH-glycoside, S-glycoside or CH 2 - glycoside;
  • M 1 and M 2 are independently H, Na, K, Ca, Mg, NH 4 , NR 1 R 2 R 3 ; n is 1-30;
  • X 1 , X 2 , R 1 , R 2 and R 3 are the same defined in Formula (I) and (II).
  • mAb is an antibody; n is 1-30; X 1 , X 2 , R 1 , R 2 and R 3 are the same defined in Formula (I) and (II).
  • Maytansinoids that are preferred to be used in the present invention including maytansinol and its analogues are described in U.S. Patent Nos. 4,256,746, 4,361,650, 4,307,016, 4,294,757, 4,294,757, 4,371,533, 4,424,219, 4,331,598, 4,450,254, 4,364,866, 4,313,946, 4,315,929 4,362,663, 4,322,348, 4,371,533, 4,424,219, 5,208,020, 5,416,064, 5,208,020; 5,416,064; 6,333.410; 6,441,163; 6,716,821, 7,276,497, 7,301,019, 7,303,749, 7,368,565, 7,411,063, 7,851,432, and 8,163,888 .
  • An example of the structure of the conjugate of the antibody- Maytansinoids via the bridge linker is as the following M01:
  • mAb is an antibody
  • n is 1 ⁇ 30
  • X 1 , X 2 , R 1 , R 2 and R 3 are the same
  • Taxanes which includes Paclitaxel (Taxol), a cytotoxic natural product, and docetaxel (Taxotere), a semi-synthetic derivative, and their analogs which are preferred for conjugation via the bridge linkers of the present patent are exampled in:. K C. Nicolaou et al., J. Am. Chem. Soc. 117, 2409-2420, (1995); Ojima et al, J. Med. Chem. 39:3889-3896 (1996); 40:267-278 (1997); 45, 5620-5623 (2002); Ojima et al., Proc. Natl. Acad.
  • Examples of the structures of the conjugate of the antibody- taxanes via the bridge linker are as the following Tx01, Tx02 and Tx03.
  • mAb is an antibody; n is 1 ⁇ 30; X 1 , X 2 , R 1 and R 2 are the same defined in Formula (I) and (II).
  • CC-1065 analogues and doucarmycin analogs are also preferred to be used for a conjugate with the bridge linkers of the present patent.
  • the examples of the CC-1065 analogues and doucarmycin analogs as well as their synthesis are described in: e.g.
  • Daunorubicin/Doxorubicin Analogues are also preferred for conjugation via the bridge linkers of the present patent.
  • the preferred structures and their synthesis are exam- pled in: Hurwitz, E., et al., Cancer Res. 35, 1175-1181 (1975). Yang, H. M., and Reisfeld, R. A., Proc. Natl. Acad. Sci. 85, 1189-1193 (1988); Pietersz, C. A., E., et al., E., et al.," Cancer Res. 48, 926-9311 (1988); Trouet, et al., 79, 626-629 (1982); Z. Brich et al., J.
  • mAb is an antibody
  • n is 1 ⁇ 30
  • X 3 and X' 3 are independently H, O, NH, NHC(O),
  • Auristatins and dolastatins are preferred in conjugation via the bridge linkers of this patent.
  • the auristatins e. g. auristain E (AE) auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), Monomethylauristatin (MMAF), Auristatin F phenylene diamine (AFP) and a phenylalanine variant of MMAE
  • AE auristain E
  • AEB auristatin EFP
  • MMAE monomethyl auristatin E
  • MMAF Monomethylauristatin
  • AFP Auristatin F phenylene diamine
  • AFP phenylalanine variant of MMAE
  • benzodiazepine dimers e. g. dimmers of pyrrolobenzodiazepine (PBD) or (tomaymycin), indolinobenzodiazepines, imidazobenzothiadiazepines, or
  • oxazolidinobenzodiazepines which are preferred cytotoxic agents according to the present invention are exampled in the art: US Patent Nos . 8,163,736; 8,153,627; 8,034,808;
  • X 1 , X 2 , R 1, R 2 and R 3 are the same defined in Formula (I) and (II).
  • R 1 and/or R 2 can be absent.
  • two or more different cytotoxic agents are preferred conjugated to a cell-binding molecule via a bridge linker of this patent.
  • the two or more different cytotoxic agents can be selected from any combinations of tubulysins, maytansinoids, taxanoids (taxanes), CC-1065 analogs, daunorubicin and doxorubicin compounds, benzodiazepine dimers (e.g., dimers of pyrrolobenzodiazepine (PBD), tomaymycin, anthramycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidino-benzodiazepines), calicheamicins and the enediyne antibiotics, actinomycin, azaserines, bleomycins, epirubicin, tamoxifen, idarubicin, dolastatins, auristatins (e.g.
  • auristatin E monomethyl auristatin E, MMAE , MMAF, auristatin PYE, auristatin TP, Auristatins 2- AQ, 6-AQ, EB (AEB), and EFP (AEFP)), duocarmycins, thiotepa, vincristines, hemiasterlins, soloumamides, microginins, radiosumins,reterobactins, microsclerodermins, theonellamides, esperamicins, PNU- 159682, and their analogues and derivatives above thereof.
  • Examples of the structures of the conjugates containing two or more different cytotoxic agents via the bridge linker are as the following Z01, Z02, Z02, Z04, Z05, Z06,
  • R 1 and/or R 2 are the same defined in Formula (I) and (II).
  • R 1 and/or R 2 can be absent.
  • cell-binding ligands or receptors can be conjugated to a cell-binding molecule via a bridge linker of this patent.
  • These conjugated cell-binding ligands or receptors in particular, antibody-receptor conjugates, can be not only to work as a targeting conductor/director to deliver the conjugate to malignant cells, but also be used to modulate or co-stimulate a desired immune response or altering signaling pathways.
  • the cell-binding ligands or receptors are preferred to conjugate to an antibody of TCR (T cell receptors) T cell, or of CARs (chimeric antigen receptors) T cells, or of B cell receptor (BCR), or the cytotoxic cells.
  • the cell-binding ligands or receptors are selected, but not limited, from: Folate derivatives (binding to the folate receptor, a protein over-expressed in ovarian cancer and in other malignancies) (Low, P. S. et al 2008, Acc. Chem. Res. 41, 120-129); Glutamic acid urea derivatives (binding to the prostate specific membrane antigen, a surface marker of prostate cancer cells) (Hillier, S. M.et al, 2009, Cancer Res.
  • Somatostatin also known as growth hormone-inhibiting hormone (GHIH) or somatotropin release-inhibiting factor (SRIF)) or somatotropin re- lease-inhibiting hormone
  • GKIH growth hormone-inhibiting hormone
  • SRIF somatotropin release-inhibiting factor
  • somatotropin re- lease-inhibiting hormone and its analogues such as octreotide (Sandostatin) and lanreotide
  • Asn-Gln-Trp-Ala-Val-Gly-H is-Leu-Met-NH 2 )/gastrin-releasing peptide (GRP) (BB 1, GRP receptor subtype (BB2), the BB3 and BB4) for renal cell, breast, lung, gastric and prostate carcinomas, and neuroblastoma (and neuroblastoma (Ohlsson, B., et al, 1999, Scand. J, Gastroenterology 34 (12): 1224-9; Weber, H. C, 2009, Cur. Opin. Endocri. Diab. Obesity 16(1): 66-71, Gonzalez N, et al, 2008, Cur. Opin. Endocri. Diab. Obesity
  • Neurotensin (NTR1, NTR2, NTR3) for small cell lung cancer
  • Substance P NK1 receptor
  • Neuropeptide Y Y1-Y6
  • Homing Peptides include RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), the dimeric and multimeric cyclic RGD peptides (e.g. cRGDfV) that recognize receptors (integrins) on tumor surfaces (Laakkonen P,
  • TAASGVRSMH and LTLRWVGLMS chondroitin sulfate proteoglycan NG2 receptor
  • F3 peptide 31 amino acid peptide that binds to cell surface-expressed nucleolin receptor
  • Peptide Hormones such as luteinizing hormone-releasing hormone (LHRH) agonists and antagonists, and gonadotropin-releasing hormone (GnRH) agonist, acts by targeting follicle stimulating hormone (FSH) and luteinising hormone (LH), as well as testosterone production, e.g. buserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-
  • LHRH luteinizing hormone-releasing hormone
  • GnRH gonadotropin-releasing hormone
  • FSH follicle stimulating hormone
  • LH gonadotropin-releasing hormone
  • testosterone production e.g. buserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-
  • the cell-binding ligands or receptors can be Ig-based and non-Ig-based protein scaf- fold molecules.
  • the Ig-Based scaffolds can be selected, but not limited, from Nanobody (a derivative of VHH (camelid Ig)) (Muyldermans S., 2013 Annu Rev Biochem. 82, 775- 797); Domain antibodies (dAb, a derivative of VH or VL domain) (Holt, L. J, et al, 2003, Trends Biotechnol. 21, 484-490); Bispecific T cell Engager (BiTE, a bispecific diabody) (Baeuerle, P. A, et al, 2009, Curr. Opin. Mol. Ther.
  • Non-Ig scaffolds can be selected, but not limited, from Anticalin (a derivative of Lipocalins) (Skerra A. 2008, FEBS J., 275(11): 2677-2683; Beste G, et al, 1999 Proc. Nat. Acad. USA. 96(5): 1898-1903; Skerra, A. 2000
  • DARPins a derivative of ankrin repeat (AR) proteins
  • AR krin repeat
  • DARPin C9 a derivative of ankrin repeat (AR) proteins
  • DARPin Ec4 a derivative of ankrin repeat (AR) proteins
  • E69_LZ3_E01 a derivative of ankrin repeat (AR) proteins
  • DARPin C9 a derivative of ankrin repeat (AR) proteins
  • DARPin Ec4 DARPin E69_LZ3_E01
  • Winkler J et al, 2009 Mol Cancer Ther. 8(9), 2674-2683
  • Patricia M-K. M. et al, Clin Cancer Res. 2011; 17(1): 100-110
  • Boersma Y. L et al, 2011 J. Biol. Chem. 286(48),41273-41285
  • Avimers a domain A/low-density lipoprotein (LDL) receptor
  • Examples of the structures of the conjugate of the antibody-cell-binding ligands or receptors via the bridge linker are as the following: LB01 (PMSA ligand conjugate), LB02 (Folate receptor conjugate), LB03 (Somatostatin receptor conjugate), LB04 (Octreotide, a
  • Somatostatin analog receptor conjugate LB05 (Lanreoiide, a Somatostatin analog receptor conjugate), LB06 (CAIX receptor conjugate), LB07(CAIX receptor conjugate), LB08 (luteinizing hormone-releasing hormone (LH-RH) ligand and GnRH conjugate), LB09 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand conjugate), LB 10 (GnRH antagonist, Abarelix conjugate), LB 11 (cobalamin, VB 12 analog conjugate), LB 12
  • GPPr Gastrin releasing peptide receptor
  • MBA conjugate LB 13 ( ⁇ ⁇ ⁇ 3 integrin receptor, cyclic RGD pentapeptide conjugate), LB 14 (hetero-bivalent peptide ligand for VEGF receptor conjugate), LB 15 (Neuromedin B conjugate), LB 16 (a G-protein coupled receptor, bombesin conjugate) and LB 17 (a Toll-like receptor, TLR 2 conjugate).
  • LB 13 ⁇ ⁇ ⁇ 3 integrin receptor, cyclic RGD pentapeptide conjugate
  • LB 14 hetero-bivalent peptide ligand for VEGF receptor conjugate
  • LB 15 Neuroromedin B conjugate
  • LB 16 a G-protein coupled receptor, bombesin conjugate
  • LB 17 a Toll-like receptor, TLR 2 conjugate.
  • the drugs/ cytotoxic agents used for conjugation via a bridge linker of the present patent can be any analogues and/or derivatives of drugs/molecules described in the present patent.
  • drugs/cytotoxic agents will readily understand that each of the drugs/cytotoxic agents described herein can be modified in such a manner that the resulting compound still retains the specificity and/or activity of the starting compound.
  • the skilled artisan will also understand that many of these compounds can be used in place of the drugs/cytotoxic agents described herein.
  • the drugs/cytotoxic agents of the present invention include analogues and derivatives of the compounds described herein.
  • the azide compound 86 (2.51 g, 9.68 mmol) dissolved in 1,4-dioxane (30 mL) and was added 10 ml of HC1 (conc). The mixture was stirred for 35 min, diluted with EtOH (30 ml) and toluene (30 ml) and evacuated under vacuum. The crude mixture was purified on silica gel using a mixture of methanol (from 5% to 10%) and 1% formic acid in methylene chloride as the eluant to give title compound 87 (1.63 g, 83% yield), ESI MS m/z- C 7 H 12 N 3 O 4 (M-H), cacld. 202.06, found 202.30.
  • Example 7 (4R)-4-(2-((lR,3R)-l-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-l-methyl- piperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(3-(3- (2-( -aminoethoxy)ethoxy)propanamido)-4-hydroxyphenyl)-2-methylpentanoic acid (95).
  • Example 8 4-(benzyloxy)-3-methoxybenzoic acid 4-Hydroxy-3-methoxybenzoic acid (50.0 g, 297.5 mmol) in the mixture of ethanol (350 ml) and NaOH solution (2.0 ML 350 ml) was added BnBr (140.0 g. 823.5 mmol ). The mixture was stirred at 65 °C for 8 h, concentrated, co-evaporated with water (2 x 400 ml) to -400 ml, acidified with 6 M HC1 to pH 3.0, filtered the solid, crystallized with EtOH, dried over the oven at 45 °C with vacuum to afford the title compound (63.6 g, 83% yield). ESI MS m/z+ 281.2 (M + Na).
  • Example 22 (S)-8-((5-(((S)-10-(3-(2-(2-aminoethoxy)ethoxy)propanoyl)-7-methoxy- 2-methylene-5-oxo-2,3 ,5.10.1 1.1 1 a-hexahydro- 1 H-benzo[e]pyrrolo[ 1 ,2-a] [ 1 ,4]diazepin-8- yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-2,3-dihydro- 1 H-benzo[e]pyrrolo[ 1 ,2- a][l,4]diazepin-5(l laH)-one, (100).
  • Boc-L-proline (10.0 g, 46.4 mmol) dissolved in 50 mL THF was cooled to 0 °C, to which BH 3 in THF (1.0 M, 46.4 mL) was added carefully. The mixture was stirred at 0 °C for 1.5 h then poured onto ice water and extracted with ethyl acetate. The organic layer was washed with brine (50 mL), dried over anhydrous Na 2 SO 4 , and concentrated under reduced pressure to give the title compound (8.50 g, 91% yield) as a white solid.
  • n-Butyllithium in hexane (21.6 mL, 2.2 M, 47.43 mmol) was added dropwise at -78 °C to a stirred solution of 4-methyl-5-phenyloxazolidin-2-one (8.0 g, 45.17 mmol) in THF (100 mL) under N 2 .
  • the solution was maintained at -78 ° C for 1 h then propionyl chloride (4.4 mL, 50.59 mmol) was added slowly.
  • the reaction mixture was warmed to -50 °C, stirred for 2 h then quenched by addition of a saturated solution of ammonium chloride (100 mL).
  • oxazoiidinone auxiliary The aqueous phase was acidified to pH 3 with 1N HCl and extracted with ethyl acetate (3 x 50 mL). The organic layer was washed with brine (50 mL), dried over Na 2 SO 4 , filtered and concentrated in vacuo to afford the title compound as colorless oil (1.15 g. 98% yield).
  • the reaction mixture was quenched by addition of 10% aqueous citric acid (5 mL), and acidified to pH 3 with an additional 10% aqueous citric acid (110 mL).
  • the mixture was extracted with ethyl acetate (150 mL x 3).
  • the organic extracts were washed with water (50 mL), saturated aqueous sodium hydrogen carbonate (50 mL), and saturated aqueous sodium chloride (50 mL), dried over Na 2 SO 4 , and concen- trated in vacuo.
  • the residue was purified by column chromatography on silica gel using ethyl acetate/hexane (1:4) as an eluent to give title compound (5.50 g, 93% yield).
  • reaction mixture was then diluted with ethyl acetate (80 mL), washed with 1 N aqueous potassium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium hydrogen carbonate (40 mL), and saturated aqueous sodium chloride (40 mL), dried over Na 2 SO 4 , and concentrated in vacuo.
  • the residue was purified by column chromatography (15-75% ethyl ace- tate/hexanes) to afford the title compound (130 mg, 83% yield) as a white solid.
  • Example 36 (S)-methyl 2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-2-((tert- butoxycarbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)- pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate
  • Boc-Val-OH (51.8 mg, 0.236 mmol) in DCM (5 mL) at 0 °C was added
  • Example 42 (2S)-2-((2R,3R)-3-((2S)-l-((l lS,14S,17S)-l-amino-17-((R)-sec-butyl)- 11 , 14-diisopropyl- 18-methoxy- 10, 16-dimethyl-9, 12, 15-trioxo-3 ,6-dioxa- 10, 13,16- triazaicosan-20-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (103).
  • Example 47 Conjugated compound 126 to an antibody for 127.
  • the drug/antibody ratio (DAR) (the combination of PBD dimer and MMAF per antibody) was 3.85, which was determined via UPLC- Qtof mass spectrum. It was 96-99% monomer analyzed by SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID x 30 cm, 0.5 ml/min, 100 min) and a single band measured by SDS-PAGE gel.
  • Example 49 Conjugated compound 128 to an antibody for 129.
  • the drug/antibody ratio (DAR) (the combination of PBD dimer and Tubulysin analog per antibody) was 3.9, which was determined via UPLC-Qtof mass spectrum. It was 96-99% monomer analyzed by SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID x 30 cm, 0.5 ml/min, 100 min) and a single band measured by SDS-PAGE gel. -dioxopyrrolidin-l-yl) 2,3-dibromosuccinate (9)
  • Example 52 Conjugated compound 141 to an antibody for 142.
  • the mixture was purified on G-25 column eluted with 100 mM NaH 2 PO 4 , 50 mM NaCl pH 6.0-7.5 buffer to afford 16.4-17.6 mg of the conjugate compound 92 (82% ⁇ 88% yield) in 13.1-15.2 ml buffer.
  • the drug/antibody ratio (DAR) was 3.9, which was determined via UPLC-Qtof mass spectrum. It was 96-99% monomer analyzed by SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID x 30 cm, 0.5 ml/min, 100 min) and a single band measured by SDS-PAGE gel.
  • Example 53 In vitro cytotoxicity evaluation of conjugates 127, 129 and 142 in comparison with T-DM1:
  • the cell lines used in the cytotoxicity assays were HL-60, a human promyelocytic leukemia cell line; NCI-N87, a human gastric carcinoma cell line; BT-474, a human invasive ductal carcinoma cell line; and SKOV3, a human ovarian carcinoma cell line.
  • HL-60, NCI-N87, and BT-474 cells the cells were grown in RPMI-1640 with 10% FBS.
  • SKOV3 cells the cells were grown in McCoy's 5 A Medium with 10% FBS.
  • the cells 180 ⁇ , 6000 cells
  • the cells were treated with test compounds (20 ⁇ ) at various concentrations in appropriate cell culture medium (total volume, 0.2 mL).
  • the control wells contain cells and the medium but lack the test compounds.
  • the plates were incubated for 120 hours at 37°C with 5% CO 2 .
  • MTT (5 mg/ml) was then added to the wells (20 ⁇ ) and the plates were incubated for 1.5hr at 37°C.
  • the medium was carefully removed and DMSO (180 ⁇ ) was added afterward. After it was shaken for
  • inhibition% [1-(assay-blank)/(control-blank)] x 100.
  • the three new conjugates 127, 129 and 142 were extremely more potent than the commercial conjugate T-DM1.
  • Example 54 Antitumor Activity In vivo.
  • the in vivo efficacy of conjugates 127, 129 and 142 along with T-DM1 were evaluated in a human gastric carcinoma N-87 cell line tumor xenograft models.
  • Five- week-old female BALB/c Nude mice (30 animals) were inoculated subcutaneously in the area under the right shoulder with N-87 carcinoma cells (5 x 10 6 cells/mouse) in 0.1 mL of serum- free medium.
  • the tumors were grown for 8 days to an average size of 133 mm 3 .
  • the animals were then randomly divided into 5 groups (6 animals per group). The first group of mice served as the control group and was treated with the phosphate-buffered saline vehicle.
  • the remaining three groups were treated with conjugates 127, 129, 142 and T- DM1 respectively at dose of 3 mg/Kg administered intravenously.
  • the weight of the animals was also measured at the same time.
  • a mouse was sacrificed when any one of the following criteria was met: (1) loss of body weight of more than 20% from pretreatment weight, (2) tumor volume larger than 1500 mm , (3) too sick to reach food and water, or (4) skin necrosis.
  • a mouse was considered to be tumor-free if no tumor was palpable. The results were plotted in Figure 16. All the four conjugates did not cause the animal body weight loss.

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Abstract

The present invention relates to novel linkers containing a 2,3-disubstituted succinic group, or 2-monosubstituted, or 2,3-disubstituted fumaric or maleic (trans (E)- or cis (Z)- butenedioic) group for conjugation of two or more compounds/cytotoxic agents per linker to a cell-binding molecule, through bridge linking pairs of thiols on the cell-binding molecule specifically. The invention also relates to methods of making such linkers, and of using such linkers in making homogeneous conjugates, as well as of application of the conjugates in treatment of cancers, infections and autoimmune disorders.

Description

NOVEL LINKERS AND THEIR USES IN SPECIFIC CONJUGATION OF DRUGS TO A BIOLOGICAL MOLECULE
FIELD OF THE INVENTION
The present invention relates to linkers used for the specific conjugation of compounds, in particular, cytotoxic agents to pairs of sulfur atoms of a biological molecule at two drugs per linker. The present invention also relates to methods of making cell-binding agent-drug (cytotoxic agent) conjugates in a specific manner comprising either modification of drugs with these linkers first, followed by reaction with prepared cell-binding agents; or modification of cell-binding agents with these linkers first, followed by reaction with drugs.
BACKGROUND OF THE INVENTION
The big challenge of chemotherapeutic drugs is their narrow therapeutic windows due to they normally cannot discriminate between normal and malignant cells, thus causes side effects which limit the tolerated doses below the clinically effective ones. In contrast, immunotherapy, normally in the form of monoclonal antibodies (mAb) can specifically bind to certain proteins or molecules of malignant cells, leaving normal cells unharmed, and thus has less side effects and bigger therapeutic windows than chemotherapy. Monoclonal antibodies (mAb) can target against malignant cells by several mechanisms, such as, 1). Making the cancer cell more visible to the immune system (Villaruz, L. C. et al, 2014, Transl Lung Cancer Res, 3, 2-14; Camacho, L. H. 2015 Cancer Med 4, 661-72); 2). Blocking growth signals (Dillman, R. O. 2011, Cancer Biother Radiopharm, 26, 1-64; Ferris, R. L. et al 2010, J Clin Oncol, 28, 4390-9); 3). Stopping new blood vessels from forming (Arrillaga-Romany, I., et al, 2014, Expert Opin Investig Drugs, 23, 199-210); 4). Delivering radiation to cancer cells (Chapuy, B. et al, 2007, Biotechnol J. 2, 1435-43); 5). Delivering chemotherapy drug to cancer cells (Chari R. J. 2008 Acc Chem Res. 41, 98-107;
Mullard A. 2013, Nature Reviews Drug Discovery 12, 329-332; Zhao, R. J. 2012, J. Med. Chem., 55, 766-782 ); 6). Delivering enzyme to cancer cells (Francis R. J. et al, 2002, Br. J Cancer 87, 600-7). One of these above strategies, delivering chemotherapy to cancer cells called antibody-drug conjugates (ADCs), which enables to target and deliver drugs to cancer cells leaving normal cells largely unaffected by the exquisite targeting ability of antibodies, has been intensely exploitation in the last two decades. In particular, since US FDA approvals of Adcetris (brentuximab vedotin) in 2011 and Kadcyla (ado-trastuzumab emtansine) in 2013, the applications of antibody-drug conjugate (ADC) as a promise targeted treatment of cancers have been exploded and almost every major pharmaceutical and biotech company has adopted this approach (Chari, R. et al, Angew. Chem., Int. Ed. 2014, 53, 3796-3827; Sievers, E. L. et al. Annu Rev Med. 2013, 64, 15-29; Mehrling, T. Future Oncol, 2015, 11, 549). Currently there are more than 50 ADC drugs in the clinic trials according to www.clinictrails.gov.
The first-generation ADCs, including Kadcyla and Adcetris, are produced through nonselective conjugation of native lysine amines or interchain cysteine thiols on an antibody respectively to a cytotoxic drug. Since there are over 50 surface-exposed lysines and 8 hinge cysteine residues in IgGl antibodies, this nonselective conjugation results in randomly cross-linkage of cytotoxic drugs to practically all areas of the antibody molecule, particularly having a diverse population of ADCs with a wide distribution of drugs per antibody (DAR) (Wang, L., et al. 2005 Protein Sci. 14, 2436; Hamblett, K. J., et al. 2004 Clin. Cancer Res. 10, 7063). Thus some of the undesired ADC subpopulation could lead to shorter circulation half-life, lower efficacy, potentially increased off-target toxicity and a wide range of in vivo pharmacokinetic (PK) properties (Hamblett, K. J. et al, Clin.
Cancer Res. 2004, 10, 7063-7070; Adem, Y. T. et al, Bioconjugate Chem. 2014, 25, 656-664; Boylan, N. J. Bioconjugate Chem., 2013, 24, 1008-1016; Strop, P., et al 2013 Chem. Biol. 20, 161-167). In addition, with this classical conjugation, the batch-to-batch consistency in ADC production can be challenging and may require diligent manufacturing capabilities (Wakankar, A. mAbs, 2011, 3, 161-172).
Therefore, biotechnology companies and academic institutions are highly focusing on establishing novel reliable methods for site-specific ADC conjugation. So far, there are several approaches developed in recent years for site selective ADC preparation
(Panowski, S, 2014, mAbs 6, 34). They include incorporation of unpaired cysteines, e.g. engineered reactive cysteine residues, called THIOMAB from Genentech (Junutula, J. R., et al 2010 Clin. Cancer Res. 16, 4769; Junutula, J. R., et al 2008 Nat Biotechnol. 26, 925- 32; US Patents 8,309,300; 7,855,275; 7,521,541; 7,723,485, WO2008/141044), genetically introduced glutamine tag with Strep to verticillium mobaraense transglutaminase (mTG) (Strop, P., Bioconjugate Chem., 2014, 25, 855-862; Strop, P., et al., 2013, Chem. Biol. 20,
161-167; US Patent 8,871,908 for Rinat-Pfizer) or with Microbial transglutaminase (MTGase) (Dennler, P., et al, 2014, Bioconjug. Chem. 25, 569-578. US pat appl
20130189287 for Innate Pharma; US Pat 7,893,019 for Bio-Ker S.r.l. (IT)), incorporation of thiolfucose (Okeley, N. M., et al 2013 Bioconjugate Chem. 24, 1650), incorporation of unnatural amino acids through mutagenesis (Axup, J.Y., et al., 2012, Proc. Natl. Acad. Sci. 109, 16101-16106; Zimmerman, E.S., et al., 2014, Bioconjug. Chem. 25, 351-361; Wu, P., et al, 2009 Proc. Natl. Acad. Sci. 106, 3000-3005; Rabuka, D., et al, 2012 Nat. Protoc. 7, 1052-67; US Pattent 8,778,631 and US Pat Appl. 20100184135,
WO2010/081110 for Sutro Biopharma; WO2006/069246, 2007/059312, US Patents
7,332,571, 7,696,312, and 7,638,299 for Ambrx; WO2007/130453, US patents 7,632,492 and 7,829,659 for Allozyne), Incorporation of selenocysteine into antibodies (Hofer, T., et al 2009, Biochemistry 48, 12047-12057; US Patent 8,916,159 for US National Cancer Institute), Convertion of cysteines located in the CXPXR consensus sequence to
formylglycine (FGly) with formylglycine generating enzyme (FGE) (Drake, P.M., et al.,
2014, Bioconjug. Chem. 25, 1331-1341. Carrico, I. S. et al 7,985,783; 8,097,701;
8,349,910, and US Pat Appl 20140141025, 20100210543 for Redwood Bioscience), and through glycoengineeringly introduction of sialic acid with the use of galactosyl- and sialytransferases (Zhou, Q., et al 2014, Bioconjug. Chem., 25, 510-520, US Pat Appl 20140294867 for Sanofi-Genzyme). These above methods have produced nearly homogeneous product profiles, but they are required antibody-engineering processes and reoptimization of cell culture conditions. Moreover, expression yields for genetic encoding of an unnatural amino acid were typically not promisingly high enough (Tian, F., et al, 2014, Proc. Natl. Acad. Sci. U. S. A. I l l, 1766-71) which has a significant impact on the cost of goods of the ADC. In addition, it has been known that ADCs obtained by conjugation to cysteine side chains often display limited stability in circulation, leading to premature disconnection of the cytotoxic pay load before the tumor site is reached (Junutula, J. R., et al 2008, Nat. Biotechnol. 26, 925-32).
The disulfide bond structures of the four subclasses of IgG antibodies were known in the 1960s (Milstein C. Biochem J 1966, 101:338 - 351; Pink JR, Milstein C. Nature 1967,
214:92-94; Frangione B, Milstein C. Nature 1967, 216:939 - 941; Pink JR, Milstein C. Nature 1967, 216:941 -942; Frangione B, et al. Biochem J 1968, 106,15 - 21; Frangione B, Milstein C. J Mol Biol 1968; 33:893 - 906; Edelman GM, et al. Proc Natl Acad Sci USA 1969; 63:78 -85; Frangione B, et al. Nature 196, 221: 145 -148, Spiegelberg, H. L. et al Biochemistry, 1975, 10, 2157-63 ). Disulfide bond structure is critical for the structure, stability, and biological functions of IgG molecules. Among the four subclasses of IgG antibodies, IgGi, IgG2, IgG3 and IgG4, each IgG contains a total of 12 intra-chain disulfide bonds; each disulfide bond is associated with an individual IgG domain. The two heavy chains are connected in the hinge region by a variable number of disulfide bonds: 2 for IgGi and IgG4, 4 for IgG2 and 11 for IgG3. The light chain of the IgGi is connected to the heavy chain by a disulfide bond between the last cysteine residue of the light chain and the fifth cysteine residue of the heavy chain. But, for IgG2, IgG3 and IgG4, the light chain is linked to the heavy chain by a disulfide bond between the last cysteine residue of the light chain and the third cysteine residue of the heavy chain (Liu, H. and May, K., 2012, mAbs
4, 17-23). On the ranks of the susceptibility of disulfide bonds in human IgGi antibodies by experimental reduction, differential alkylation, and LC-MS analysis (Liu, H, et al Anal. Chem., 2010, 82, 5219-5226), inter chain disulfide bonds are more susceptible to reduction than intra chain disulfide bonds, and the disulfide bonds between the light chain and heavy chain were more susceptible than disulfide bonds between the two heavy chains.
The upper disulfide bond of the two inter heavy chain disulfide bonds was more susceptible than the lower one. Furthermore, disulfide bonds in the CH2 domain were the most susceptible to reduction. Disulfide bonds in VL, CL, VH, and CHI domains had similar and moderate susceptibility, while disulfide bonds in the CH3 domain were the least susceptible to reduction (Liu, H, et al Anal. Chem., 2010, 82, 5219-5226).
Based on the more susceptibility of inter chain disulfide bonds in human IgGi antibodies, several institutions and companies adopted the chemically specific conjugation strategy through rebridging reduced interchain disulfide bonds of a native antibody, such as, using bromo or dibromo-maleimides, called next generation maleimides (NGMs) (Schumacher, F.F., et al 2014, Org. Biomol. Chem. 12, 7261-7269; UCL Cancer Institute), applying bis-alkylating reagents via a three-carbon bridge (Badescu, G., et al., 2014, Bioconjug. Chem. 25, 1124-1136., WO2013/ 190272, WO2014/064424 for PolyTherics Ltd), with di-substituted heteroaryl bridge (US Pat Appl. 2015/0105539 for Concortis Biosystem), or through di-maleimide as a bridge (WO2014/114207). We have also used bromo maleimide and dibromomaleimide linkers to conjugate both drugs and antibodies for a quite while (WO2014/009774, PCT/IB2012/053554). However, these above bridge linkers were designed in the way to conjugate only one cytotoxic agents to a pair of disulfide bonds, and therefore at most of time they only produced ADCs at DAR less than 2 (drugs per antibody), due to limited numbers (about two pairs) of reduced disulfide bonds are more accessible for conjugation.
As one of the major issues for ADCs is the limited numbers or amount of cytotoxic compound that ultimately reaches the tumor, and thus the favorable DAR over 3 is much important factor for improvement of ADC therapeutical index (Epenetos, A. A. et al, Cancer Res., 1986, 46, 3183-3191; Chari, R. V. Acc. Chem. Res., 2008, 41, 98-107, Zhao, R. Y. et al, 2011, J. Med. Chem. 54, 3606-3623), we therefore disclose novel disulfur bridge linkers of this invention that not only are able to conjugate two or more drugs per linker for achieving higher DARs (>4), but also can selectively rebridge pairs of reduced inter chain disulfide bonds on surface of antibody, which are generated by overloaded TCEP or DTT reduction agents. And the over reduced pairs of thiol groups that are inaccessibly reached by the bridge linkers can be recoupled (regenerated) by an oxide, e.g. dehydroascorbic acid (DHAA) or Cu(II), to form back disulfide bonds at the end of conjugation. In principal, this rebridging back of reduced disulfide bonds results in more stable or longer half-life of ADCs in comparison with traditional thiol linked ADCs. Furthermore, as the "ring-opened" succinimide ring linker bearing mono-thioether bond has improved in vitro stability, improved PK exposure, and improved efficacy as compared to the mono- thiol-maleimide-conjugated ADCs (Tumey, L. N, et al, 2014, Bioconjug. Chem. 25, 1871- 80; Lyon, R. P, et al. 2014, Nat. Biotechnol. 32, 1059-62), due to the latter is prone to payload loss via a retro-Michael type reaction of the maleimide conjugation (Shen, B. Q, et al, 2012, Nat Biotechnol. 30, 184-9; Tumey, L. N, et al, 2014 Bioconjug Chem. 25, 1871-
80), the bridge linkers of this invention containing a 2,3-disubstituted succinic group, or 2- monosubstituted, or 2,3-disubstituted fumaric or maleic (trans (E)~ or cis (Zj-butenedioic) group have less payload loss as compared to their nonhydrolyzed bromo or dibromo- maleimide linkers which were tested in our lab. In other words, the methods of the instant invention can be used to for the immunoconjugates that carry a combination of drugs, in particular different drugs, which can be delivered simultaneously and specifically to a particular target site, where the therapeutic molecules in the medicament are highly homogeneous, with lot-to-lot consistency. The major advantages of such immunoconjugates include: simultaneous targeted delivery of multiple drugs that act synergistically in target- ing malignant cells; combining drugs that act in different phases of the cell cycle to increase the number of target cells exposed to a particular pharmaceutical drug or effect; minimized exposure to non-target cells, tissues or organs; precise control over drug pay- loads and drug ratios leading to homogenous final products. In short, the bridge linkers of the invention can make homogeneous production of specific ADCs in a simple manner. SUMMARY OF THE INVENTION
The present invention provides linkers containing a 2,3-disubstituted succinic group, or 2-monosubstituted, or 2,3-disubstituted fumaric or maleic (trans (E)- or cis (Zj- butenedioic) group to link two drugs to a cell-binding agent (e.g., an antibody). The preferred formula of the cell-binding molecule-linker-drug conjugates can be represented as:
Figure imgf000007_0002
, wherein Cb is a cell-binding agent, L Cb is a cell-binding agent, L is a linker containing succinic, fumaric or maleic group; Drugl and Drug2 are a drug molecule; n is an integer from 1 to 30; and two S (sulfur) elements from Cb bridgely link to L, which covalently connects two or more drugs. The advantages in applying the linker in the cell molecule-drug conjugate are: a). Retaining the stability of the conjugates by covalently cross-linking (re-bridging) the pairs of reduced disulfur atoms of the cell- binding agents, particularly of antibodies; b). Enabling conjugation of the cytotoxic agents/drugs to specific sites of a cell-binding molecule, e.g. the inter chain disulfide bond sites of IgG antibodies, resulting in homogeneous production of ADC.
In one aspect of the present invention, the linker is represented by Formula (I)
Figure imgf000007_0001
Wherein
represents an optional single bond;
Figure imgf000007_0003
represents either a single bond or a double bond;
U and LP represent the same or different leaving group that can be substituted by a thiol. Such leaving groups are, but are not limited to, a halide (e.g., fluoride, chloride, bromide, and iodide), methanesulfonyl (mesyl), /?-toluenesulfonyl (tosyl),
trifluoromethylsulfonyl (triflate), trifluoromethylsulfonate, nitrophenol, N- hydroxysuccinimide (NHS), phenol; dinitrophenol; pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, 2-ethyl-5-phenylisoxazolium-3'-sulfonate, or an intermediate molecule generated with a condensation reagent for Mitsunobu reactions.
It provided that when
Figure imgf000007_0004
represents a single bond, both U and LP are not H; when represents a double bond, either U or LP can be H, but are not H at the same time.
Z1 and Z2 are the same or different a function group that enables to react with a cytotoxic drug, to form a disulfide, ether, ester, thioether, thioester, peptide, hydrazone, carbamate, carbonate, amine (secondary, tertiary, or quarter), imine, cycloheteroalkyane, heteroaromatic, alkyloxime or amide bond;
R1 and R2 are the same or different, and are absent, linear alkyl having from 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl or alkynyl, or 1-6 carbon atoms of esters, ether, amide, or polyethyleneoxy unit of formula (OCH2CH2)p, wherein p is an integer from 0 to about 1000, or combination thereof.
Additionally R1 and R2 are respectively a chain of atoms selected from C, N, O, S, Si, and P, preferably having 0-500 atoms, which covalently connects to X1 or X2 and Z1 or Z2. The atoms used in forming the R1 and R2 may be combined in all chemically relevant ways, such as forming alkylene, alkenylene, and alkynylene, ethers, polyoxyalkylene, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas,
semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, peptides, acyloxylamines, hydroxamic acids, or combination thereof.
X1 and X2 are independently selected from NH, N(R3), O, S or C H2; R3 is H, linear al- kyl having from 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl or alkynyl, or 1-6 carbon atoms of esters, ether, amide, or polyethyleneoxy unit of formula
Figure imgf000008_0001
H2 H2)p, wherein p is an integer from 0 to about 1000, or combination thereof.
In another aspect, this invention provides a cell-binding agent-drug conjugate of Formula (II), in which the cell-binding agent, Cb, and the drug, Drugl and Drug2, have reacted at the ends of the bridge linker:
Figure imgf000008_0002
(II)
Wherein:
Cb represents a cell-binding agent, preferred an antibody;
Inside the bracket (parentheses) are the linker-drug components that are conjugated to pairs of sulfur atoms of the cell-binding molecule. The sulfur atoms are preferred pairs of thiols reduced from the interchain disulfide bonds of the cell-binding agent by a reduction agent, such as DTT and/or TCEP;
Drug1 and Drug2 represent the same or different cytotoxic agents, which linked to the cell-binding agent via the bridge linker by a disulfide, thioether, thioester, peptide, hydra- zone, ether, ester, carbamate, carbonate, cycloheteroalkyane, heteroaromatic, alkoxime or amide bond;
Figure imgf000009_0003
are described the same previously in
Formula (I).
In a further aspect, the present invention provides a modified cell-binding agent of Formula (III), in which the cell-binding agent, Cb, through its pair of thiols generated with reduction of disulfide bonds, has reacted with the bridge linker, which has Z1 and Z2, the function groups capable of reacting with a drug:
Wherein Cb, Z1, Z2, n, R1, R2, X1, and X2 are defined the same as in
Figure imgf000009_0001
Formula (I) and (II).
In an even further aspect, the present invention provides a modified drug of Formula
(IV), in which the drug, Drug1 and Drug2, have reacted with the linker of Formula (I), which still has the 2,3-disubstituted succinic group, or 2-monosubstituted, or 2,3- disubstituted fumaric or maleic (trans (Eh or cis (Z)-butenedioic) group capable of reacting with a pair of sulfur atoms of the cell-binding agent:
Figure imgf000009_0002
Wherein
Figure imgf000009_0004
Drug1, Drug2, U, U', R1, R2, X1, and X2 are defined the same as in Formula (I) and (II).
The present invention further relates to a method of making a cell-binding molecule- drug conjugate of Formula (II), wherein the drugs, Drug1 and Drug2 are linked to a cell- binding agent via the bridge linker.
The present invention also relates to a method of making a modified cell-binding molecule of Formula (III), wherein the cell-binding molecule is reacted with the bridge linker of Formula (I).
The present invention also relates to a method of making a modified drug of formula (IV), wherein the drug is reacted with the bridge linker of Formula (I).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the synthesis of a bridge linker containing polyethylene glycols and the application of this linker in the conjugation of an antibody with drugs via amide bonds. Figure 2 shows the synthesis of a bridge linker containing polyethylene glycols and the application of this linker in the conjugation of drugs to an antibody via amide bonds.
Figure 3 shows the synthesis of a bridge linker containing polyethylene glycols and the application of this linker in the conjugation of drugs to an antibody via oxime linkage.
Figure 4 shows the synthesis of a bridge linker containing polyethylene glycols and the application of this linker in the conjugation of two drugs to an antibody via hydrazone linkage.
Figure 5 shows the synthesis of bridge linkers containing polyethylene glycols and the application in the conjugation of two different drugs per linker to an antibody via amide linkage.
Figure 6 shows the synthesis of bridge linkers and the application in the conjugation of two different drugs per linker to an antibody via hinder amide linkage.
Figure 7 shows the synthesis of bridge linkers containing peptides or polyethylene glycols and the application of these linkers in the conjugation of two (different) drugs to an antibody via hydrazone linkage..
Figure 8 shows the synthesis of the conjugatable analogs of MMAE, Tubulysin and PBD cytotoxic drugs.
Figure 9 shows the synthesis of the conjugatable analogs of PBD, MMAF, and Tubulysin D cytotoxic drugs.
Figure 10 shows the synthesis of the conjugates of cell-binding molecule-tubulysin analogs via the bridge-linker.
Figure 11 shows the synthesis of the conjugates of both PBD dimer analog and Tubulysin B analog per linker, or both MMAE and Tubulysin D analog per linker, to an antibody.
Figure 12 shows the synthesis of the conjugates of both PBD dimer analog and
MMAF analog per linker, or both PBD dimer and Tubulysin B analog per linker, to an antibody.
Figure 13 shows the synthesis of the conjugates of both Maytansinoid analog and Tubulysin B analog per linker, to an antibody.
Figure 14 shows the synthesis of the conjugates of both Maytansinoid analog and
PBD dimer analog per linker, or two Tubulysin B analogs per linker, to an antibody.
Figure 15 shows the synthesis of the conjugates of both two MMAF analogs per linker, or two Tubulysin B analogs per linker, containing polyethylene glycols, to an antibody Figure 16 shows the comparison of the anti-tumor effect of conjugate compounds 127, 129 and 142 with T-DM1 using human gastric tumor N87 cell model at dosing, 3 mg/kg, i.v., one injection. All the four conjugates did not cause the animal body weight loss (top figure). The animals at control group were sacrificed at day 37 due to the tumor volume larger than 1500 mm and they were too sick. The three compounds 127, 129 and 142 were better than T-DM1: All 6/6 animals at the groups of compound 127 and 129 had completely no tumor measurable at day 13 till day 60 (the end of experiment). All 6/6 animals at the group of Compound 142 group had no tumor measurable at day 21 and 2/6 animals had tumor growth (measurable) back at days 48, which still inhibited the tumor growth for over 55 days. In contrast T-DM1 at dose of 3 mg/Kg was not able to eradicate the tumors completely although it had inhibited the tumor growth for about 28 days.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
"Alkyl" refers to an aliphatic hydrocarbon group which may be straight or branched having 1 to 8 carbon atoms in the chain. "Branched" means that one or more lower C numbers of alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-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-methyl-hexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5- dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n- heptyl, isoheptyl, n-octyl, and isooctyl. A C1-C8 alkyl group can be unsubstituted or substituted with one or more groups including, but not limited to, -C1-C8 alkyl,-O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', - SR', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R') 2 and -CN; where each
R' is independently selected from -C1-C8 alkyl and aryl.
"Halogen" refers to fluorine, chlorine, bromine or iodine atom; preferably fluorine and chlorine atom.
"Heteroalkyl" refers to C2-C8 alkyl in which one to four carbon atoms are inde- pendently replaced with a heteroatom from the group consisting of O, S and N.
"Carbocycle" refers to a saturated or unsaturated ring having 3 to 8 carbon atoms as a monocycle or 7 to 13 carbon atoms as a bicycle. Monocyclic carbocycles have 3 to 6 ring atoms, more typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, arranged as a bicycle [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms arranged as a bicycle [5,6] or [6,6] system. Representative C3-C8 carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5- cycloheptatrienyl, -cyclooctyl, and -cyclooctadienyl.
A "C3-C8 carbocycle" refers to a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated nonaromatic carbocyclic ring. A C3-C8 carbocycle group can be unsubstituted or substituted with one or more groups including, but not limited to, -C1-C8 alkyl,-O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, - NHC(O)R', -SR', -S(O)R',-S(O)2R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R') 2 and -CN; where each R' is independently selected from -C1-C8 alkyl and aryl.
"Alkenyl" refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond which may be straight or branched having 2 to 8 carbon atoms in the chain. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, hexylenyl, heptenyl, octenyl.
"Alkynyl" refers to an aliphatic hydrocarbon group containing a carbon-carbon triple bond which may be straight or branched having 2 to 8 carbon atoms in the chain. Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5- pentynyl, n-pentynyl, hexylynyl, heptynyl, and octynyl.
"Alkylene" refers to a saturated, branched or straight chain or cyclic hydrocarbon rad- ical of 1-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to: methylene (-CH2-), 1,2- ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), and the like.
"Alkenylene" refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkene. Typical alkenylene radicals include, but are not limited to: 1,2-ethylene (- CH=CH-).
"Alkynylene" refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkyne. Typical alkynylene radicals include, but are not limited to: acetylene, propargyl and 4-pentynyl. "Aryl" or Ar refers to an aromatic or hetero aromatic group, composed of one or several rings, comprising three to fourteen carbon atoms, preferentially six to ten carbon atoms. The term of "hetero aromatic group" refers one or several carbon on aromatic group, preferentially one, two, three or four carbon atoms are replaced by O, N, Si, Se, P or S, preferentially by O, S, and N. The term aryl or Ar also refers to an aromatic group, wherein one or several H atoms are replaced independently by -R', -halogen, -OR', or - SR', -NR'R", -N=NR' -N=R' -NR'R",-NO2, -S(O)R' -S(O)2R', -S(O)2OR', - OS(O)2OR', -PR'R", -P(O)R'R", -P(OR')(OR"), -P(O)(OR')(OR") or - OP(O)(OR')(OR") wherein R', R" are independently H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, arylalkyl, carbonyl, or pharmaceutical salts.
"Heterocycle" refers to a ring system in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group of O, N, S, Se, B, Si and P. Preferable heteroatoms are O, N and S. Heterocycles are also described in The Handbook of Chemistry and Physics, 78th Edition, CRC Press, Inc., 1997-1998, p. 225 to 226, the disclosure of which is hereby incorporated by reference. Preferred nonaromatic heterocyclic include, but are not limited to epoxy, aziridinyl, thiiranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxolanyl, piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl,
dihydropyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl, as well as the fused systems resulting from the condensation with a phenyl group.
The term "heteroaryl" or aromatic heterocycles refers to a 5 to 14, preferably 5 to 10 membered aromatic hetero, mono-, bi- or multicyclic ring. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1,2,4-thiadiazolyl, isothiazolyl, triazoyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, pyridyl-N-oxide, as well as the fused systems resulting from the condensation with a phenyl group.
"Alkyl", "cycloalkyl", "alkenyl", "alkynyl", "aryl", "heteroaryl", "heterocyclic" and the like refer also to the corresponding "alkylene", "cycloalkylene", "alkenylene", "alkynylene", "arylene", "heteroarylene", "heterocyclene" and the likes which are formed by the removal of two hydrogen atoms.
"Arylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp carbon atom, is replaced with an aryl radical. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-l- yl, 2-phenylethen-l-yl, naphthylmethyl, 2-naphthylethan-l-yl, 2-naphthylethen-l-yl, naphthobenzyl, 2-naphthophenylethan-l-yl and the like.
"Heteroarylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen at- oms bonded to a carbon atom, typically a terminal or sp carbon atom, is replaced with a heteroaryl radical. Typical heteroarylalkyl groups include, but are not limited to, 2- benzimidazolylmethyl, 2-furylethyl and the like.
Examples of a "hydroxyl protecting group" include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, /?-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, i-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetate ester, substituted acetate esters, pivaloate, benzoate, methanesulfonate and /?-toluenesulfonate.
"Leaving group" refers to a functional group that can be substituted by another func- tional group. Such leaving groups are well known in the art, and examples include, but are not limited to, a halide (e.g., chloride, bromide, and iodide), methanesulfonyl (mesyl), /?- toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), and trifluoromethyl sulfonate.
The following abbreviations may be used herein and have the indicated definitions: Boc, ie/ -butoxy carbonyl; BroP, bromotrispyrrolidinophosphonium hexafluorophosphate; CDI, Ι,Γ-carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCE, dichloroethane;
DCM, dichloromethane; DIAD, diisopropylazodicarboxylate; DIBAL-H, diisobutyl- aluminium hydride; DIPEA, diisopropylethylamine; DEPC, diethyl phosphorocyanidate; DMA, Ν,Ν-dimethyl acetamide; DMAP, 4-(7V, N-dimethylamino)pyridine; DMF, N,N- dimethylformamide; DMSO, dimethylsulfoxide; DTT, dithiothreitol; EDC, l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; ESI-MS, electrospray mass spectrometry; HATU, O-(7-azabenzotriazol-l-yl)-N, N, N' , N'-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high pressure liquid chromatography; NHS, N-Hydroxysuccinimide; MMP, 4-methylmorpholine; PAB, /?- aminobenzyl; PBS, phosphate-buffered saline (pH 7.0-7.5); PEG, polyethylene glycol; SEC, size-exclusion chromatography; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Val, valine.
"Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. "Pharmaceutically acceptable solvate" or "solvate" refer to an association of one or more solvent molecules and a disclosed compound. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, etha- nol, methanol, DMSO, ethyl acetate, acetic acid and ethanolamine.
"Pharmaceutically acceptable excipient" includes any carriers, diluents, adjuvants, or vehicles, such as preserving or antioxidant agents, fillers, disintegrating agents, wetting agents, emulsifying agents, 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 for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions as suitable therapeutic combinations.
As used herein, "pharmaceutical salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, tartaric, citric, methanesulfonic, benzenesulfonic, glucuronic, glutamic, benzoic, salicylic, toluenesulfonic, oxalic, fumaric, maleic, lactic and the like. Further addition salts include ammonium salts such as tromethamine, meglumine, epolamine, etc., metal salts such as sodium, potassium, calcium, zinc or magnesium.
The pharmaceutical salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared via reaction the free acidic or basic forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418, the disclosure of which is hereby incorporated by reference.
"Administering" or "administration" refers to any mode of transferring, delivering, introducing or transporting a pharmaceutical drug or other agent to a subject. Such modes include oral administration, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous or intrathecal administration. Also contemplated by the present invention is utilization of a device or instrument in administering an agent. Such device may utilize active or passive transport and may be slow-release or fast-release delivery device.
The novel conjugates disclosed herein use the bridge linkers. Examples of some suitable linkers and their synthesis are shown in Figures 1 to 15.
THE BRIDGE LINKERS
The synthetic routes to produce bridge linkers as well as the preparation of the conjugates of drugs to a cell binding molecules of the present invention are shown in Figures 1- 15. The bridge linkers possess two elements: a) A Substituent that is a 2,3-disubstituted succinic group; or 2-monosubstituted, or 2,3-disubstituted fumaric group; or 2- monosubstituted, or 2,3-disubstituted maleic group; which can react to a pair of thiols to form covalent thioether bonds, and b) A group, such as but not limited to, a disulfide, maleimide, haloacetyl, aldehyde, ketone, azide, amine, alkoxyamine, hydrazide, ethenesulfonyl, acyl halide (acid halide), acryl (acryloyl), and/or acid anhydride group, capable of reaction with a drug. The bridge substituents of 2,3-disubstituted succinic group; or 2-monosubstituted, or 2,3-disubstituted fumaric group; or 2-monosubstituted, or 2,3- disubstituted maleic group; can be introduced by direct condensation of these 2,3- disubstituted succinic acid, or 2-monosubstituted or 2,3-disubstituted fumaric or maleic with an amine, an alcohol, or a thiol group to form amide, ester or thioester bonds. The synthesis of these bridge linkers is exampled in the Figures 1, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14 and 15.
Preferably, the bridge linkers are compounds of the Formula (I) below:
Figure imgf000016_0001
Wherein
represents an optional single bond;
Figure imgf000016_0002
represents either a single bond or a double bond;
It provided that when
Figure imgf000016_0003
represents a single bond, both U and LP are not H; when represents a double bond, either U or LP can be H, but are not H at the same time. Wherein the component:
Figure imgf000017_0001
, which can be 2,3-disubstituted succinic group, or 2-monosubstituted or 2,3-disubstituted fumaric group, or 2- monosubstituted or 2,3-disubstituted maleic group, is capable of reacting with a pair of sulfur atoms of the cell-binding agent; The sulfur atoms are preferred pairs of thiols re- duced from the interchain disulfide bonds of the cell-binding agent by a reducing agent, such as dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH) and tris (2- carboxyethyl) phosphine (TCEP), or/and beta mercaptoethanol (β-ΜΕ, 2-ME).
U and U' represent the same or different leaving group that can be substituted by a thiol. Such leaving groups are, but are not limited to, a halide (e.g., fluoride, chloride, bromide, and iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethyl- sulfonyl (triflate), trifluoromethylsulfonate, nitrophenol, N-hydroxysuccinimide (NHS), phenol; dinitrophenol; pentafluorophenol, tetrafluorophenol, difluorophenol,
monofluorophenol, pentachlorophenol, imidazole, dichlorophenol, tetrachlorophenol, 1- hydroxybenzotriazole, 2-ethyl-5-phenylisoxazolium-3 '-sulfonate, or an intermediate molecule generated with a condensation reagent for Mitsunobu reactions.
Z1 and Z2 are the same or different a function group that enables to react with a cytotoxic drug, to form a disulfide, thioether, thioester, peptide, hydrazone, ether, ester, carbamate, carbonate, amine (secondary, tertiary, or quarter), imine, cycloheteroalkyane, heteroaromatic, alkoxime or amide bond;
R1 and R2 are the same or different, and are absent, linear alkyl having from 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl or alkynyl, or 1-6 carbon atoms of esters, ether, amide, or polyethyleneoxy unit of formula (OCH2CH2)p, or polypropyleneoxy unit of formula (OCH2(CH3)CH2)p wherein p is an integer from 0 to about 1000, or combination thereof.
Additionally R1 and R2 are respectively a chain of atoms selected from C, N, O, S, Si, and P, preferably having 0-500 atoms, which covalently connects to X1 or X2 and Z1 or Z2. The atoms used in forming the R1 and R2 may be combined in all chemically relevant ways, such as forming alkylene, alkenylene, and alkynylene, ethers, polyoxyalkylene, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas,
semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, peptides, acyloxylamines, hydroxamic acids, or combination thereof. X1 and X2 are independently selected from NH, N(R3), O, S or CH2; Wherein R3 is H, linear alkyl having from 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl or alkynyl, or 1-6 carbon atoms of esters, ether, amide, or polyethyleneoxy unit of formula (OCH2CH2)p, wherein p is an integer from 0 to about 1000, or combination thereof.
In another embodiment, R1, R2, and R3, can be respectively a chain of atoms selected from C, N, O, S, Si, and P which covalently connects the cell-surface binding molecule and/or the conjugated drug. The atoms used in forming the bridge linker may be combined in all chemically relevant ways, such as forming alkylene, alkenylene, and alkynylene, ethers, polyoxyalkylene, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, acyloxylamines, hydroxamic acids, and many others. In addition, it is to be understood that the atoms forming the linker (L) may be either saturated or unsaturated, or may be radicals, or may be cyclized upon each other to form divalent cyclic structures, including cyclo alkanes, cyclic ethers, cyclic amines, arylenes, heteroarylenes, and the like in the linker.
Examples of the functional groups, Z1 and Z2, which enable linkage of a cytotoxic drug, include groups that enable linkage via a disulfide, thioether, thioester, peptide, hydrazone, ester, carbamate, carbonate, alkoxime or an amide bond. Such functional groups include, but are not limited to, thiol, disulfide, amino, carboxy, aldehydes, ketone, maleimido, haloacetyl, hydrazines, alkoxyamino, and/or hydroxy.
Examples of the functional groups, Z1 and Z2, that enable reaction with the terminal of amine of a drug/ cytotoxic agent can be, but not limited to, N-hydroxysuccinimide esters, p-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl esters, carboxylic acid chlorides or carboxylic acid anhydride; With the terminal of thiol can be, as but not limited to, pyridyldisulfides, nitropyridyldisulfides, maleimides, haloacetates,
methylsulfone phenyloxadiazole (ODA), carboxylic acid chlorides and carboxylic acid anhydride; With the terminal of ketone or aldehyde can be, as but not limited to, amines, alkoxyamines, hydrazines, acyloxylamine, or hydrazide; With the terminal of azide can be, as but not limited to, alkyne. Examples of these function groups are displayed below:
Figure imgf000018_0001
maleimide;
Figure imgf000019_0001
(dinitrophenoxy)acetyl;
Figure imgf000019_0002
2- (fluoro henoxy)- acetyl;
Figure imgf000019_0003
2-(dmuorophenoxy)-acetyl;
2-(pentafluorophenoxy)acetyl; O h l lf h l di l (ODA) id
Figure imgf000019_0004
Figure imgf000019_0005
Wherein X1 is F, CI, Br, I or Lv3; X2 is O, NH, N(R1), or CH2; R5 and R3 are H, R1, aromatic, heteroaromatic, or aromatic group wherein one or several H atoms are replaced independently by -R1, -halogen, -OR1, -SR1, -NR1R2, - NO2, -S(O)R1, - S(O)2R1, or -COOR1; Lv3 is a leaving group selected from nitrophenol; N-hydroxysuc- cinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol;
difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5- phenylisoxazolium-3 '-sulfonate, anhydrides formed its self, or formed with the other anhydride, e.g. acetyl anhydride, formyl anhydride; or an intermediate molecule generated with a condensation reagent for peptide coupling reactions, or for Mitsunobu reactions.
In preferred embodiments, R1, R2, and R3, are linear alkyl having from 1-6 carbon atoms, or polyethyleneoxy unit of formula (OCH2CH2)p p = 1~100.
The key step of synthesis of the bridge linker containing 2,3-disubstituted succinic group, or 2-monosubstituted or 2,3-disubstituted fumaric group, or 2-monosubstituted or 2,3-disubstituted maleic group is the condensation of the 2,3-disubstituted succinic acid, or 2-monosubstituted or 2,3-disubstituted fumaric acid, or 2-monosubstituted or 2,3- disubstituted maleic acid, or its acid derivatives, with the other components containing an amine (1° or 2° amines), alcohol, or thiol on their terminal, as shown in the following scheme (la):
Figure imgf000020_0001
Wherein X is X1 or X2 described in Formula (I) as NH, N(R3), O, or S; R is R1 and/or R2 that described in Formula (I); R3 is the same defined in Formula (I).
Lvi and Lv2 are the same or independently OH; F; CI; Br; I; nitrophenol; N-hydroxy- succinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol;
difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5- phenylisoxazolium-3 '-sulfonate, anhydrides formed its self, or formed with the other anhydride, e.g. acetyl anhydride, formyl anhydride; or an intermediate molecule generated with a condensation reagent for peptide coupling reactions, or for Mitsunobu reactions, e.g. condensation reagents are: EDC (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide), DCC (Dicyclohexyl-carbodiimide), Ν,Ν'-Diisopropylcarbodiimide (DIC), N-Cyclohexyl- N'-(2-morpholino-ethyl)carbodiimide metho-p-toluenesulfonate (CMC,or CME-CDI), 1,1 '- Carbonyldiimi-dazole (CDI),TBTU (O-(Benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), N,N,N',N'-Tetramethyl-O-(lH-benzotriazol-l-yl)uronium
hexafluorophosphate (HBTU), (Benzotriazol- l-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (Benzotriazol- l-yloxy)tripyrrolidinophosphonium
hexafluorophosphate (PyBOP), Diethyl cyanophosphonate (DEPC), Chloro-Ν,Ν,Ν',Ν'- tetramethylformamidinium hexafluorophosphate, l-[Bis(dimethylamino)methylene]-lH- l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), l-[(Dimethylami- no)(morpholino) methylene]- lH-[l,2,3]triazolo[4,5-b]pyridine-l-ium 3-oxide hexafluorophosphate (HDMA), 2-Chloro-l,3-dimethylimidazolidinium hexafluorophosphate (CIP), Chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), Fluoro-N,N,N',N'-bis(tetramethylene)formamidinium hexafluorophosphate (BTFFH), N,N,N',N'-Tetramethyl-S-(l-oxido-2-pyridyl)thiuronium hexafluorophosphate, O-(2-Oxo-l(2H)pyridyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TPTU), S-(l-
Oxido-2-pyridyl)-N,N,N',N'-tetramethylthiuronium tetrafluoroborate, O-[(Ethoxycarbonyl) cyano-methylenamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate (HOTU), (1- Cyano-2-ethoxy-2-oxoethylidenaminooxy) dimethylamino-morpholino-carbenium hexafluorophosphate(COMU), O-(Benzotriazol-l-yl)-N,N,N',N'-bis(tetramethylene) uronium hexafluorophosphate (HBPyU), N-Benzyl-N'-cyclohexylcarbodiimide (with, or without polymer-bound), Dipyrrolidino(N-succinimidyl-oxy)carbenium hexafluorophosphate (HSPyU), Chlorodipyrrolidinocarbenium hexafluorophosphate (PyClU), 2- Chloro-l,3-dimethylimidazolidinium tetrafluoroborate(CIB), (Benzotriazol-l-yloxy) dipiperidinocarbenium hexafluorophosphate (HBPipU), O-(6-Chlorobenzotriazol-l-yl)- Ν,Ν,Ν',Ν'-tetramethyluronium tetrafluoroborate (TCTU), Bromotris(dimethylamino)- phosphonium hexafluorophosphate (BroP), Propylphosphonic anhydride (PPACA, T3P®), 2-Morpholinoethyl isocyanide (MEI), N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate (HSTU), 2-Bromo-l-ethyl-pyridinium tetrafluoroborate (BEP), O- [(Ethoxycarbonyl)cyanomethylenamino]-N,N,N',N'-tetra-methyluronium tetrafluoroborate (TOTU), 4-(4,6-Dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium chloride (MMTM,
DMTMM), N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), O- (3,4-Dihydro-4-oxo-l,2,3-benzotriazin-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TDBTU), l, -(Azodicarbonyl)dipiperidine (ADD), Di-(4-chlorobenzyl) azodicar- boxylate (DCAD), Di-tert-butyl azodicarboxylate (DBAD), Diisopropyl azodicarboxylate (DIAD), Diethyl azodicarboxylate (DEAD).
The detail examples of the synthesis of the bridge linkers are shown in the figures
1- 10. Normally the bridge substituents of 2,3-disubstituted succinic group, or 2- monosubstituted or 2,3-disubstituted fumaric group, or 2-monosubstituted or 2,3- disubstituted maleic group can be condensated with linker components containing function groups capable to react to drugs of desired conjugation.
CELL-BINDING AGENT-DRUG CONJUGATES The conjugates of the present invention can be represented by the following formula,
Figure imgf000022_0001
wherein Cb is a cell-binding agent, L is linker containing succinic, fumaric or maleic group, Drug1 and Drug2 are a drug molecule, n is an integer from 1 to 30, and two S (sulfur) elements from Cb bridgely link to L, which covalently connects two or more drugs (per bridge linker L).
The bridge linker L may be composed of one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af"), p- aminobenzyloxycarbonyl ("PAB"), 4-thiopentanoate ("SPP"), 4-(N-maleimidomethyl)- cyclohexane- 1 carboxylate ("MCC"), (4-acetyl)aminobenzoate ("SIAB"), 4-thio-butyrate (SPDB), 4-thio-2-hydroxysulfonyl-butyrate (2-Sulfo-SPDB), ethyleneoxy -CH2CH2O- as one or more repeating units ("EO" or "PEO"). Additional linker components are known in the art and some are described herein.
Example structures of these components containing linkers are:
Figure imgf000022_0002
(ME, maleimidoethyl containing).
Figure imgf000023_0001
(valine-citrulline containing)
Figure imgf000023_0002
MCC, 4-(N-maleimidomethyl)cyclohexane-l carboxylate)
Figure imgf000023_0003
4-acetyl)aminobenzoate containing)
Figure imgf000023_0004
(4-thio-2-hydroxysulfonyl-butyrate, 2-sulfo-SPDB)
Preferably, the conjugates have the following Formula (Π):
Figure imgf000023_0005
(II)
wherein:
Cb represents a cell-binding agent, preferably an antibody, which conjugates to Drug1 and Drug2 via a pair of sulfur atoms (thiols). The conjugatable thiol groups can generally be generated from dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH) and tris (2-carboxyethyl) phosphine (TCEP), or/and beta mercaptoethanol (β-ΜΕ, 2-ME) reduction of pairs of disulfide bonds on the surface of cell-binding molecule.
Drug1 and Drug2 represent the same or different cytotoxic agents, linked to the cell- binding agent via the bridge linker through an alkyl, alkylene, alkenylene, alkynylene, ether, polyoxyalkylene, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, urethanes, amino acid, peptide, acyloxylamine, hydroxamic acid, disulfide, thioether, thioester, carbamate, carbonate, heterocyclic ring, heteroalkyl, heteroaromatic, or alkoxime bond, or combination thereof.
Figure imgf000024_0001
, R1 R2, X1 and X2 are described the same previously in
Formula (I).
As described in more detail below, Drug1 and Drug2 can be any of many small molecule drugs, including, but not limited to, tubulysins, calicheamicins, auristatins,
maytansinoids, CC-1065 analogs, morpholinos doxorubicins, taxanes, cryptophycins, epothilones, and benzodiazepine dimers (e.g., dimmers of pyrrolobenzodiazepine (PBD) or tomaymycin), indolinobenzodiazepines, imidazobenzothiadiazepines, or
oxazolidinobenzodiazepines).
To synthesize the conjugate, the cell-binding agent can be first modified with the bridge linkers of the present invention through reduction of disulfide bonds of the cell- binding molecule. The yielded a pair of free thiols can react to the bridge linker of Formula (I) at pH 5-9 aqueous media with or without addition of 0-30% of water mixable (miscible) organic solvents, such as DMA, DMF, ethanol, methanol, acetone, acetonitrile, THF, isopropanol, dioxane, propylene glycol, or ethylene diol, to introduce the reactive groups of Z1 and Z2 containing disulfide, maleimido, haloacetyl, azide, 1-yne, ketone, aldehyde, alkoxyamino, or hydrazide groups. Then a reactive group of a cytotoxic agent reacts to the modified cell-binding molecule accordingly. For example, synthesis of the cell-binding agent-drug conjugates linked via disulfide bonds is achieved by a disulfide exchange between the disulfide bond in the modified cell-binding agent and a drug containing a free thiol group. Synthesis of the cell-binding agent-drug conjugates linked via thioether is achieved by reaction of the maleimido or haloacetyl or ethylsulfonyl modified cell-binding agent and a drug containing a free thiol group. Synthesis of conjugates bearing an acid labile hydrazone can be achieved by reaction of a carbonyl group with the hydrazide moiety in the linker, by methods known in the art (see, for example, P. Hamann et al., Cancer Res. 53, 3336-334, 1993; B. Laguzza et al., J. Med. Chem., 32; 548-555, 1959; P. Trail et al., Cancer Res., 57; 100-105, 1997). Synthesis of conjugates bearing triazole linkage can be achieved by reaction of a 1-yne group of the drug with the azido moiety in the linker, through the click chemistry (Huisgen cycloaddition) (Lutz, J-F. et al, 2008, Adv.
Drug Del. Rev. 60, 958-970; Sletten, E. M. et al 2011, Acc Chem. Research 44, 666-676).
Alternatively, the drug can react with the bridge linkers of the present invention that have conjugated to a cell-binding molecule to give a modified cell-binding molecule linker of Formula (III) bearing functionalities. For example, a thiol-containing drug can be reached with the modified cell-binding molecule bridge linker of Formula (III) bearing a maleimdo, or a haloacetyl, or an ethylsulfonyl substituent at pH 5.5-9.0 in aqueous buffer to give a cell-binding molecule-drug conjugate via a thioether linkage. A thiol-containing drug can undergo disulfide exchange with a modified bridge linker of Formula (III) bear- ing a pyridyldithio moiety to give a conjugate a disulfide bond linkage. A drug bearing a hydroxyl group or a thiol group can be reacted with a modified bridge linker of Formula (III) bearing a halogen, particularly the alpha halide of carboxylates, in the presence of a mild base, e.g. pH 8.0-9.5, to give a modified drug bearing an ether or thiol ether link. A hydroxyl group containing drug can be condensed with a bridge cross linker of Formula (I) bearing a carboxyl group, in the presence of a dehydrating agent, such as EDC or DCC, to give ester linkage, then the subject drug modified bridge linker undergoes the conjugation with a cell-binding molecule. A drug containing an amino group can condensate with a carboxyl ester of NHS, imidazole, nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1- hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3 '-sulfonate on the cell-binding molecule-bridge linker of Formula (III) to give a conjugate via amide bond linkage.
The conjugate may be purified by standard biochemical means, such as gel filtration on a Sephadex G25 or Sephacryl S300 column, adsorption chromatography, and ion exchange or by dialysis. In some cases, a small molecule as a cell-binding agent (e.g. folic acid, melanocyte stimulating hormone, EGF etc) conjugated with a small molecular drugs can be purified by chromatography such as by HPLC, medium pressure column chromatography or ion exchange chromatography.
MODIFIED CELL-BINDING AGENTS/MOLECULES
The cell-binding agent modified by reaction with linkers of the present invention are preferably represented by the Formula (III)
Wherein Cb, Z1, Z2, n, R1, R2, Xi, and X2 are defined the same as in
Figure imgf000025_0001
Formula (I) and (II). In preferred embodiments, Z1 and Z2 are a disulfide substituent, maleimido, haloacetyl, alkoxyamine, azido, ketone, aldehyde, hydrazine, alkyne, an N-hydroxysuccinimide ester, or a carboxyl ester formed with phenol; dinitrophenol; pentafluorophenol; tetrafluoro- phenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole;
dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5- phenylisoxa-zolium-3 '-sulfonate. Z1 and Z2 can then react with a cytotoxic agent through disulfide, thioether, hydrazone, amide, alkoxime, carbamate, ester, ether bond or hetero- aromatic ring. The modified cell-binding agent can be prepared via a reaction of the cell- binding agent with the bridge linkers of Formula (I) as described in Formula (II) above.
In order to achieve a higher conjugation yield of the alkyne group on the bridge linkers with a pair of free thiols on the cell-binding molecule, preferably on an antibody, a small percentage of organic co- solvent may be required to add to the reaction mixture, as well in the solution after the reaction to maintain solubility of the Formula (III) in aqueous solution. To modify the cell-binding agents, the cross-linking reagent (bridge linker) of Formula (I) can be first dissolved in a polar organic solvent that is miscible with water, for example different alcohols, such as methanol, ethanol, and propanol, acetone, acetonitrile, tetrahydrofuran (THF), 1,4-dioxane, dimethyl formamide (DMF), dimethyl acetamide (DMA), or dimethylsulfoxide (DMSO) at a high concentration, for example 1-500 mM. Meanwhile, the cell-binding molecule, such as antibody dissolved in an aqueous buffer pH 5-9.5, preferably pH 6-8.5, at 1-35 mg/ml concentration was treated with 1-20 equivalent of TCEP or DTT for 20 min to 12 hour. After the reduction, DTT can be removed by SEC chromatographic purification. TCEP can be optionally removed by SEC chromatography too, or staying in the reaction mixture for the next step reaction without purification.
Furthermore, the reduction of antibodies or the other cell-binding agents with TCEP can be performed with a bridge linker of Formula (I), for which the cross-linking conjugation for the cell-binding molecules can be achieved simultaneously along with the TCEP reduction.
The aqueous solutions for the modification of cell-binding agents are buffered between pH 6 and 9, preferably between 6.5 and 7.5 and can contain any non-nucleophilic buffer salts useful for these pH ranges. Typical buffers include phosphate, triethanolamine HC1, HEPES, and MOPS buffers, which can contain additional components, such as cyclodextrins, sucrose and salts, for examples, NaCl and KC1. After the addition of the bridge linker of Formula (I) into the solution containing the reduced cell-binding molecules, the reaction mixture is incubated at a temperature of from 4 °C to 45 °C, preferably at ambient temperature. The progress of the reaction can be monitored by measuring the decrease in the absorption at 254 nm, or increase in the absorption at 280 nm, or the other appropriate wavelength. After the reaction is complete, isolation of the modified cell- binding agent can be performed in a routine way, using for example gel filtration chromatography, or adsorptive chromatography.
The extent of modification can be assessed by measuring the absorbance of the nitropyridine thione, dinitropyridine dithione, pyridine thione, carboxamidopyridine dithione and dicarboxamidopyridine dithione group released via UV spectra. For the conjugation without a chromophore group, the modification or conjugation reaction can be monitored by LC-MS, preferably by UPLC-QTOF mass spectrometry, or Capil- lary electrophoresis-mass spectrometry (CE-MS). The bridge cross-linkers described herein have diverse functional groups that can react with any drugs, preferably cytotoxic agents that possess a suitable substituent. For examples, the modified cell-binding molecules bearing an amino or hydroxyl substituent can react with drugs bearing an
N-hydroxysuccinimide (NHS) ester, the modified cell-binding molecules bearing a thiol substituent can react with drugs bearing a maleimido or haloacetyl group. Additionally, the modified cell-binding molecules bearing a carbonyl (ketone or aldehyde) substituent can react with drugs bearing a hydrazide or an alkoxyamine. One skilled in the art can readily determine which linker to use based on the known reactivity of the available functional group on the linkers. MODIFIED CYTOTOXIC DRUGS
The cytotoxic drugs modified by reaction with cross-linkers of the present invention are preferably represented by the Formula (IV):
Figure imgf000027_0001
Wherein
Figure imgf000027_0002
U, U', Drug1, Drug2, R1, R2, X1, and X2 are defined the same as in Formula (I) and (II).
The modified drugs can be prepared via reaction of the drug with the linkers of the Formula (I) to give a modified drug of Formula (IV) bearing functionality of an 2,3- disubstituted succinic group, or 2-monosubstituted or 2,3-disubstituted fumaric group, or 2-monosubstituted or 2,3-disubstituted maleic group. But for drugs containing a thiol, or the drugs undergoing to link a cell-binding molecule via the bridge linkers through thioether, thioester or disulfide bond, it is therefore preferred that the Drug1 or Drug2 may be synthesized to connect to R1, or R2 in a piece of components via the linkage of thioether, thioester or disulfide bond first. Then the synthesized R1-Drug1 or R2-Drug2 component is assembled to 2,3-disubstituted succinic acid, or 2-monosubstituted or 2,3-disubstituted fumaric acid, or 2-monosubstituted or 2,3-disubstituted maleic acid to form the bridge linker modified drugs of Formula (IV).
For examples of the synthesis, a thiol-containing drug can be reacted with the linker of components R1 or R2 bearing a maleimdo substituent at neutral pH in aqueous buffer to give a R1-Drug1 or R2-Drug2 compartment bearing thioether linkage, and following by condensation with either 2,3-disubstituted succinic acid, or 2-monosubstituted or 2,3- disubstituted fumaric acid, or 2-monosubstituted or 2,3-disubstituted maleic acid to give a modified drug of Formula (IV) bearing thioether linkage. A drug bearing a hydroxyl group can be reacted with a linker component R1 or R2 bearing a halogen, or a tosylate, or a mesylate, in the presence of a mild base, to give a R1-Drug1 or R2-Drug2 compartment bearing ether linkage, and following by condensation with 2,3-disubstituted succinic acid, or 2-monosubstituted or 2,3-disubstituted fumaric acid, or 2-monosubstituted or 2,3- disubstituted maleic acid to give a modified drug of Formula (IV) bearing thioether linkage. A hydroxyl group containing drug can be condensed with a linker of Formula (I) bearing a carboxyl group, in the presence of a dehydrating agent, such as EDC or dicyclohexylcarbodiimide (DCC), to give a modified drug of Formula (IV) via ester linkage. A drug bearing a thiol group can also react the linker of components R1 or R2 bearing a maleimido or a vinylsulfonyl, or a haloacetyl group, give a R1-Drug1 or R2-Drug2 compartment bearing thioether linkage, and following by condensation with a compartment of 2,3-disubstituted succinic acid, or 2-monosubstituted or 2,3-disubstituted fumaric acid, or 2-monosubstituted or 2,3-disubstituted maleic acid to give a modified drug of Formula (IV) bearing thioether linkage. An amino group containing drug can similarly undergo condensation with a carboxyl group on the bridge linker of Formula (I) to give a modified drug of Formula (IV) bearing amide bonds. The modified drug can be purified by standard methods such as column chromatography over silica gel or alumina, crystallization, preparatory thin layer chromatography, ion exchange chromatography, or HPLC.
CELL-BINDING AGENTS
The cell-binding molecule that comprises the conjugates and the modified cell-binding agents of the present invention may be of any kind presently known, or that become known, molecule that binds to, complexes with, or reacts with a moiety of a cell population sought to be therapeutically or otherwise biologically modified. The cell binding agents include, but are not limited to, large molecular weight proteins such as, for example, full-length antibodies (polyclonal antibodies, monoclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies); single chain antibodies; fragments of antibodies such as Fab, Fab', F(ab')2, Fv [Parham, J. Immunol. 131, 2895-2902 (1983)], fragments produced by a Fab expression library, anti-idiotypic
(anti-Id) antibodies, CDR's, diabody, triabody, tetrabody, miniantibody, small immune proteins (SIP), and epitope-binding fragments of any of the above which immuno- specifically bind to cancer cell antigens, viral antigens, microbial antigens or a protein generated by the immune system that is capable of recognizing, binding to a specific antigen or exhibiting the desired biological activity (Miller et al (2003) J. of Immunology
170:4854-4861); interferons (such as type I, II, III); peptides; lymphokines such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, GM-CSF, interferon-gamma (IFN-γ); hormones such as insulin, TRH (thyrotropin releasing hormones), MSH (melanocyte- stimulating hormone), steroid hormones, such as androgens and estrogens, melanocyte- stimulating hormone (MSH); growth factors and colony-stimulating factors such as epidermal growth factors
(EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factors (TGF), such as TGFa, TGFp, insulin and insulin like growth factors (IGF-I, IGF-II) G-CSF, M-CSF and GM-CSF [Burgess, Immunology Today, 5, 155-158 (1984)]; vaccinia growth factors (VGF); fibroblast growth factors (FGFs); smaller molecular weight proteins, poly-peptide, peptides and peptide hormones, such as bombesin, gastrin, gastrin- releasing peptide; platelet-derived growth factors; interleukin and cytokines, such as interleukin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory factors, granulocyte- macrophage colony- stimulating factor (GM-CSF); vitamins, such as folate; apoproteins and glycoproteins, such as transferrin [O'Keefe et al, 260 J. Biol. Chem. 932-937 (1985)]; sugar-binding proteins or lipoproteins, such as lectins; cell nutrient- transport molecules; and small molecular inhibitors, such as pro state- specific membrane antigen (PSMA) inhibitors and small molecular tyrosine kinase inhibitors (TKI), non-peptides or any other cell binding molecule or substance, such as bioactive polymers (Dhar, et al, Proc. Natl. Acad. Sci. 2008, 105, 17356-61); bioactive dendrimers (Lee, et al, Nat. Biotechnol. 2005, 23, 1517-26; Almutairi, et al; Proc. Natl. Acad. Sci. 2009, 106, 685-90); nanoparticles
(Liong, et al, ACS Nano, 2008, 19, 1309-12; 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); viral capsides (Flenniken, et al, Viruses Nanotechnol. 2009, 327, 71-93). In general, a monoclonal antibody is preferred as a cell-surface binding agent if an appropriate one is available. And the antibody may be murine, human, humanized, chimeric, or derived from other species.
Production of antibodies used in the present invention involves in vivo or in vitro pro- cedures or combinations thereof. Methods for producing polyclonal anti-receptor peptide antibodies are well-known in the art, such as in U.S. Pat. No. 4,493,795 (to Nestor et al). A monoclonal antibody is typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen (Kohler, G.; Milstein, C.
(1975). Nature 256: 495-497). The detailed procedures are described in "Antibodies— A Laboratory Manual", Harlow and Lane, eds., Cold Spring Harbor Laboratory Press, New
York (1988), which is incorporated herein by reference. Particularly monoclonal antibodies are produced by immunizing mice, rats, hamsters or any other mammal with the antigen of interest such as the intact target cell, antigens isolated from the target cell, whole virus, attenuated whole virus, and viral proteins. Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000. Fused hybrids are selected by their sensitivity to HAT (hypoxanthine-aminopterin-thymine). Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact specified receptors or inhibit receptor activity on target cells.
A monoclonal antibody used in the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well-known techniques, such as using protein-A affinity chromatography; anion, cation, hydrophobic, or size exclusive chromatographies (particularly by affinity for the specific antigen after protein A, and sizing column chromatography); centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
Media useful for the preparation of these compositions are both well-known in the art and commercially available and include synthetic culture media. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol. 8, 396 (1959)) supplemented with 4.5 gm/1 glucose, 0-20 mM glutamine, 0-20% fetal calf serum, several ppm amount of heavy metals, such as Cu, Mn, Fe, or Zn, etc, or/and the heavy metals added in their salt forms, and with an anti-foaming agent, such as polyoxyethylene- polyoxypropylene block copolymer.
In addition, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfec- tion with an oncovirus, such as Epstein-Barr virus (EBV, also called human herpesvirus 4
(HHV-4)) or Kaposi's sarcoma-associated herpesvirus (KSHV). See, U.S. Pat. Nos.
4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890. A monoclonal antibody may also be produced via an anti-receptor peptide or peptides containing the carboxyl terminal as described well-known in the art. See Niman et al., Proc. Natl. Acad. Sci. USA, 80: 4949-4953 (1983); Geysen et al., Proc. Natl. Acad. Sci.
USA, 82: 178-182 (1985); Lei et al. Biochemistry 34(20): 6675-6688, (1995). Typically, the anti-receptor peptide or a peptide analog is used either alone or conjugated to an immunogenic carrier, as the immunogen for producing anti-receptor peptide monoclonal antibodies.
There are also a number of other well-known techniques for making monoclonal antibodies as binding molecules in this invention. Particularly useful are methods of making fully human antibodies. One method is phage display technology which can be used to select a range of human antibodies binding specifically to the antigen using methods of affinity enrichment. Phage display has been thoroughly described in the literature and the construction and screening of phage display libraries are well known in the art, see, e.g.,
Dente et al, Gene. 148(1):7-13 (1994); Little et al, Biotechnol Adv. 12(3):539-55 (1994); Clackson et al., Nature 352:264-628 (1991); Huse et al., Science 246: 1275-1281 (1989).
Monoclonal antibodies derived by hybridoma technique from another species than human, such as mouse, can be humanized to avoid human anti-mouse antibodies when infused into humans. Among the more common methods of humanization of antibodies are complementarity-determining region grafting and resurfacing. These methods have been extensively described, see e.g. U.S. Pat. Nos. 5,859,205 and 6,797,492; Liu et al, Immunol Rev. 222:9-27 (2008); Almagro et al, Front Biosci. 13: 1619-33 (2008); Lazar et al, Mol Immunol. 44(8): 1986-98 (2007); Li et al, Proc. Natl. Acad. Sci. U S A. 103(10):3557-62 (2006) each incorporated herein by reference. Fully human antibodies can also be prepared by immunizing transgenic mice, rabbits, monkeys, or other mammals, carrying large portions of the human immunoglobulin heavy and light chains, with an immunogen.
Examples of such mice are: the Xenomouse. (Abgenix/Amgen), the HuMAb-Mouse (Medarex/BMS), the VelociMouse (Regeneron), see also U.S. Pat. No. 6,596,541 , 6,207,418, No. 6,150,584, No. 6,111,166, No. 6,075,181, No. 5,922,545, Nos. 5,661,016, 5,545,806, 5,436,149 and 5,569,825. In human therapy, murine variable regions and human constant regions can also be fused to construct called "chimeric antibodies" that are considerably less immunogenic in man than murine mAbs (Kipriyanov et al, Mol
Biotechnol. 26:39-60 (2004); Houdebine, Curr Opin Biotechnol. 13:625-9 (2002) each incorporated herein by reference). In addition, site-directed mutagenesis in the variable region of an antibody can result in an antibody with higher affinity and specificity for its antigen (Brannigan et al, Nat Rev Mol Cell Biol. 3:964-70, (2002)); Adams et al, J Immunol Methods. 231:249-60 (1999)) and exchanging constant regions of a mAb can improve its ability to mediate effector functions of binding and cytotoxicity.
Antibodies immuno specific for a malignant cell antigen can also be obtained commercially or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immuno specific for a malignant cell antigen can be obtained commercially, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
Apart from an antibody, a peptide or protein that bind/block/target or in some other way interact with the epitopes or corresponding receptors on a targeted cell can be used as a binding molecule. These peptides or proteins could be any random peptide or proteins that have an affinity for the epitopes or corresponding receptors and they don't necessarily have to be of the immunoglobulin family. These peptides can be isolated by similar techniques as for phage display antibodies (Szardenings, J Recept Signal Transduct Res. 2003; 23(4):307-49). The use of peptides from such random peptide libraries can be similar to antibodies and antibody fragments. The binding molecules of peptides or proteins may be conjugated on or linked to a large molecules or materials, such as, but is not limited, an albumin, a polymer, a liposome, a nano particle, a dendrimer, as long as such attachment permits the peptide or protein to retain its antigen binding specificity.
Examples of antibodies used for conjugation of drugs via the bridge linkers of this prevention for treating cancer, autoimmune disease, and/or infectious disease include, but are not limited to, 3F8 (anti-GD2), Abagovomab (anti CA-125), Abciximab (anti CD41
(integrin alpha-lib), Adalimumab (anti-TNF-a), Adecatumumab (anti-EpCAM, CD326), Afelimomab (anti-TNF-a); Afutuzumab (anti-CD20), Alacizumab pegol (anti-VEGFR2), ALD518 (anti-IL-6), Alemtuzumab (Campath, MabCampath, anti- CD52), Altumomab (anti-CEA), Anatumomab (anti-TAG-72), Anrukinzumab (IMA-638, anti-IL-13), Apolizumab (anti-HLA-DR), Arcitumomab (anti-CEA), Aselizumab (anti-L-selectin (CD62L), Atlizumab (tocilizumab, Actemra, RoActemra, anti-IL-6 receptor),
Atorolimumab (anti-Rhesus factor), Bapineuzumab (anti-beta amyloid), Basiliximab (Simulect, antiCD25 (a chain of IL-2 receptor), Bavituximab (anti-phosphatidylserine), Bectumomab (LymphoScan, anti-CD22), Belimumab (Benlysta, LymphoStat-B, anti-
BAFF), Benralizumab (anti-CD125), Bertilimumab (anti-CCLl l (eotaxin-1)),
Besilesomab (Scintimun, anti-CEA-related antigen), Bevacizumab (Avastin, anti-VEGF- A), Biciromab (FibriScint, anti-fibrin II beta chain), Bivatuzumab (anti-CD44 v6), Blinatumomab (BiTE, anti-CD19), Brentuximab (cACIO, anti-CD30 TNFRSF8),
Briakinumab (anti-IL-12, IL-23) Canakinumab (Ilaris, anti-IL-1), Cantuzumab (C242, anti-
CanAg), Capromab, Catumaxomab (Removab, anti-EpCAM, anti-CD3), CC49 (anti-TAG- 72), Cedelizumab (anti-CD4), Certolizumab pegol (Cimzia anti-TNF-a), Cetuximab (Er- bitux, IMC-C225, anti-EGFR), Citatuzumab bogatox (anti-EpCAM), Cixutumumab (anti- IGF-1), Clenoliximab (anti-CD4), Clivatuzumab (anti-MUCl), Conatumumab (anti- TRAIL-R2), CR6261 (anti-Influenza A hemagglutinin), Dacetuzumab (anti-CD40),
Daclizumab (Zenapax, anti-CD25 (a chain of IL-2 receptor)), Daratumumab (anti-CD38 (cyclic ADP ribose hydrolase), Denosumab (Prolia, anti-RANKL), Detumomab (anti-B- lymphoma cell), Dorlimomab, Dorlixizumab, Ecromeximab (anti-GD3 ganglioside), Eculizumab (Soliris, anti-C5), Edobacomab (anti-endotoxin), Edrecolomab (Panorex, MAbl7-lA, anti-EpCAM), Efalizumab (Raptiva, anti-LFA-1 (CD1 la), Efungumab
(Mycograb, anti-Hsp90), Elotuzumab (anti-SLAMF7), Elsilimomab (anti-IL-6),
Enlimomab pegol (anti-IC AM- 1 (CD54)), Epitumomab (anti-episialin), Epratuzumab (anti-CD22), Erlizumab (anti-ITGB2 (CD 18)), Ertumaxomab (Rexomun, anti-HER2/neu, CD3), Etaracizumab (Abegrin, anti-integrin ανβ3), Exbivirumab ( anti-hepatitis B surface antigen), Fanolesomab (NeutroSpec, anti-CD15), Faralimomab (anti-interferon receptor),
Farletuzumab (anti-folate receptor 1), Felvizumab (anti-respiratory syncytial virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF-1 receptor), Fontolizumab (anti-IFN-γ), Foravirumab (anti-rabies virus glycoprotein), Fresolimumab (anti-TGF-β), Galiximab (anti-CD80), Gantenerumab (anti- beta amyloid), Gavilimomab (anti-CD 147 (basigin)), Gemtuzumab (anti-CD33), Girentuximab (anti-carbonic anhydrase 9), Glembatumumab
(CR011, anti-GPNMB), Golimumab (Simponi, anti-TNF-a), Gomiliximab (anti-CD23 (IgE receptor)), Ibalizumab (anti-CD4), Ibritumomab (anti-CD20), Igovomab (Indimacis- 125, anti-CA-125), Imciromab (Myoscint, anti-cardiac myosin), Infliximab (Remicade, anti-TNF-a), Intetumumab (anti-CD51), Inolimomab (anti-CD25 (a chain of IL-2 recep- tor)), Inotuzumab (anti-CD22), Ipilimumab (anti-CD152), Iratumumab (anti- CD30 (TNFRSF8)), Keliximab (anti-CD4), Labetuzumab (CEA-Cide, anti-CEA), Lebrikizumab (anti- IL-13), Lemalesomab (anti-NCA-90 (granulocyte antigen)), Lerdelimumab (anti- TGF beta 2), Lexatumumab (anti-TRAIL-R2), Libivirumab (anti-hepatitis B surface antigen), Lintuzumab (anti-CD33), Lucatumumab (anti-CD40), Lumiliximab (anti- CD23
(IgE receptor), Mapatumumab (anti-TRAIL-Rl), Maslimomab (anti- T-cell receptor), Matuzumab (anti-EGFR), Mepolizumab (Bosatria, anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab (anti-CD74), Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3 ganglioside), Morolimumab (anti-Rhesus factor), Motavizumab (Numax, anti- respiratory syncytial virus), Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolomab
(anti-C242), Naptumomab (anti-5T4), Natalizumab (Tysabri, anti-integrin (¾), Nebacumab (anti-endotoxin), Necitumumab (anti-EGFR), Nerelimomab (anti-TNF-a), Nimotuzumab (Theracim, Theraloc, anti-EGFR), Nofetumomab, Ocrelizumab (anti-CD20), Odulimomab (Afolimomab, anti-LFA-1 (CDl la)), Ofatumumab (Arzerra, anti-CD20), Olaratumab (anti-PDGF-R a), Omalizumab (Xolair, anti-IgE Fc region), Oportuzumab (anti-EpCAM),
Oregovomab (OvaRex, anti-CA-125), Otelixizumab (anti-CD3), Pagibaximab (anti- lipoteichoic acid), Palivizumab (Synagis, Abbosynagis, anti-respiratory syncytial virus), Panitumumab (Vectibix, ABX-EGF, anti-EGFR), Panobacumab (anti- Pseudomonas aeruginosa), Pascolizumab (anti-IL-4), Pemtumomab (Theragyn, anti-MUCl), Pertuzumab (Omnitarg, 2C4, anti-HER2/neu), Pexelizumab (anti-C5), Pintumomab (anti- adenocarcinoma antigen), Priliximab (anti-CD4), Pritumumab (anti-vimentin), PRO 140 (anti-CCR5), Racotumomab (1E10, anti-(N-glycolylneuraminic acid (NeuGc, NGNA)- gangliosides GM3)), Rafivirumab (anti-rabies virus glycoprotein), Ramucirumab (anti- VEGFR2), Ranibizumab (Lucentis, anti-VEGF-A), Raxibacumab (anti-anthrax toxin, protective antigen), Regavirumab (anti-cytomegalovirus glycoprotein B), Reslizumab
(anti-IL-5), Rilotumumab (anti-HGF), Rituximab (MabThera, Rituxanmab, anti-CD20), Robatumumab (anti-IGF-1 receptor), Rontalizumab (anti-IFN-a), Rovelizumab
(LeukArrest, anti-CDl l, CD18), Ruplizumab (Antova, anti-CD154 (CD40L)), Satumomab (anti-TAG-72), Sevirumab (anti-cytomegalovirus), Sibrotuzumab (anti-FAP), Sifalimumab (anti-IFN-a), Siltuximab (anti-IL-6), Siplizumab (anti-CD2), (Smart) MI95 (anti-CD33),
Solanezumab (anti-beta amyloid), Sonepcizumab (anti-sphingosine-1 -phosphate),
Sontuzumab (anti-episialin), Stamulumab (anti-myo statin), Sulesomab (LeukoScan, (anti- NCA-90 (granulocyte antigen), Tacatuzumab (anti-alpha-fetoprotein), Tadocizumab (anti- integrin αι¾β3), Talizumab (anti-IgE), Tanezumab (anti-NGF), Taplitumomab (anti-CD 19), Tefibazumab (Aurexis, (anti-clumping factor A), Telimomab, Tenatumomab (anti-tenascin C), Teneliximab (anti-CD40), Teplizumab (anti-CD3), TGN1412 (anti-CD28),
Ticilimumab (Tremelimumab, (anti-CTLA-4), Tigatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13), Tocilizumab (Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor),
Toralizumab (anti-CD154 (CD40L)), Tositumomab (anti-CD20), Trastuzumab (Herceptin,
(anti-HER2/neu), Tremelimumab (anti-CTLA-4), Tucotuzumab celmoleukin (anti- EpCAM), Tuvirumab (anti-hepatitis B virus), Urtoxazumab (anti- Escherichia coli), Ustekinumab (Stelara, anti-IL-12, IL-23), Vapaliximab (anti-AOC3 (VAP-1)),
Vedolizumab, (anti-integrin α4β7), Veltuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1), Visilizumab (Nuvion, anti-CD3), Vitaxin (anti-vascular integrin avb3),
Volociximab (anti-integrin α5β1, Votumumab (HumaSPECT, anti-tumor antigen
CTAA16.88), Zalutumumab (HuMax-EGFr, (anti-EGFR), Zanolimumab (HuMax-CD4, anti-CD4), Ziralimumab (anti-CD 147 (basigin)), Zolimomab (anti-CD5), Etanercept (Enbrel®), Alefacept (Amevive®), Abatacept (Orencia®), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (Iron Regulatory Protein 2)], 14G2a (anti-GD2 ganglioside, from Nat. Cancer
Inst, for melanoma and solid tumors), J591 (anti-PSMA, Weill Cornell Medical School for prostate cancers), 225.28S [anti-HMW-MAA (High molecular weight-melanoma- associated antigen), Sorin Radiofarmaci S.R.L. (Milan, Italy) for melanoma], COL-1 (anti- CEACAM3, CGMl, from Nat. Cancer Inst. USA for colorectal and gastric cancers), CYT- 356 (Oncoltad®, for prostate cancers), HNK20 (OraVax Inc. for respiratory syncytial virus), ImmuRAIT (from Immunomedics for NHL), Lym-1 (anti-HLA-DRlO, Peregrine Pharm. for Cancers), MAK-195F [anti-TNF (tumor necrosis factor; TNFA, TNF-alpha; TNFSF2), from Abbott / Knoll for Sepsis toxic shock], MEDI-500 [T10B9, anti-CD3, TRaP (T cell receptor alpha/beta), complex, from Medlmmune Inc for Graft-versus-host disease], RING SCAN [ anti-TAG 72 (tumour associated glycoprotein 72), from Neoprobe
Corp. for Breast, Colon and Rectal cancers], Avicidin (anti-EPCAM (epithelial cell adhesion molecule), anti-TACSTDl (Tumor-associated calcium signal transducer 1), anti- GA733-2 (gastrointestinal tumor-associated protein 2), anti-EGP-2 (epithelial glycoprotein 2); anti-KSA; KS 1/4 antigen; M4S; tumor antigen 17-1A; CD326, from NeoRx Corp. for Colon, Ovarian, Prostate cancers and NHL]; LymphoCide (Immunomedics, NJ), Smart
ID 10 (Protein Design Labs), Oncolym (Techniclone Inc, CA), Allomune (BioTransplant, CA), anti-VEGF (Genentech, CA); CEAcide (Immunomedics, NJ), IMC-1C11 (ImClone Systems, NJ) and Cetuximab (ImClone, NJ) . Other antibodies as cell binding molecules/ligands include, but are not limited to, are antibodies against the following antigens: Aminopeptidase N (CD13), Annexin Al, B7-H3 (CD276, various cancers), CA125 (ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carci- nomas), CA242 (colorectal), placental alkaline phosphatase (carcinomas), prostate specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma), CD3 epsilon (T cell lymphoma, lung, breast, gastric, ovarian cancers, autoimmune diseases, malignant ascites), CD 19 (B cell malignancies), CD20 (non-Hodgkin's lymphoma), CD22 (leukemia, lym- phoma, multiple myeloma, SLE), CD30 (Hodgkin's lymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD51 (Metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lung cancers, ovarian cancer, Merkel cell carcinoma, and the liquid tumor, multiple myeloma), CD66e (cancers), CD70 (metastatic renal cell carcinoma and non-Hodgkin lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (cancers), mucin (carcinomas), CD221 (solid tumors), CD227 (breast, ovarian cancers), CD262 (NSCLC and other cancers), CD309 (ovarian cancers), CD326 (solid tumors), CEACAM3 (colorectal, gastric cancers), CEACAM5 (carcinoembryonic antigen; CEA, CD66e) (breast, colorectal and lung cancers), DLL4 (delta-like-4), EGFR (Epidermal Growth Factor Receptor, vari- ous cancers), CTLA4 (melanoma), CXCR4 (CD 184, Heme-oncology, solid tumors),
Endoglin (CD 105, solid tumors), EPCAM (epithelial cell adhesion molecule, bladder, head, neck, colon, NHL prostate, and ovarian cancers), ERBB2 (Epidermal Growth Factor Receptor 2; lung, breast, prostate cancers), FCGR1 (autoimmune diseases), FOLR (folate receptor, ovarian cancers), GD2 ganglioside (cancers), G-28 (a cell surface antigen glyvolipid, melanoma), GD3 idiotype (cancers), Heat shock proteins (cancers), HER1
(lung, stomach cancers), HER2 (breast, lung and ovarian cancers), HLA-DR10 (NHL), HLA-DRB (NHL, B cell leukemia), human chorionic gonadotropin (carcinoma), IGF1R (insulin-like growth factor 1 receptor, solid tumors, blood cancers), IL-2 receptor (interleu- kin 2 receptor, T-cell leukemia and lymphomas), IL-6R (interleukin 6 receptor, multiple myeloma, RA, Castleman's disease, IL6 dependent tumors), Integrins (ανβ3, α5β1, α6β4, α11β3, α5β5, ανβ5, for various cancers), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE 4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma), MS4A1 (membrane-spanning 4-domains subfamily A member 1, Non- Hodgkin's B cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast, ovarian, cervix, bronchus and gastrointestinal cancer), MUC16 (CA125) (Ovarian cancers), CEA (colorectal), gplOO (melanoma), MARTI (melanoma), MPG (melanoma), MS4A1 (membrane- spanning 4-domains subfamily A, small cell lung cancers, NHL), Nucleolin, Neu oncogene product (carcinomas), P21 (carcinomas), Paratope of anti-(N-glycolylneuraminic acid, Breast, Melanoma cancers), PLAP-like testicular alkaline phosphatase (ovarian, testicular cancers), PSMA (prostate tumors), PSA (prostate), ROBO4, TAG 72 (tumour associated glycoprotein 72, AML, gastric, colorectal, ovarian cancers), T cell transmembrane protein (cancers), Tie (CD202b), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, 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 (Tumor necrosis apoprosis Inducing ligand Receptor 1, lymphoma, NHL, colorectal, lung cancers), VCAM-1 (CD 106, Melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (various cancers). Some other tumor associated antigens recognized by antibodies have been reviewed (Gerber, et al, mAbs 1:3, 247-253 (2009); Novellino et al, Cancer Immunol Immunother. 54(3), 187-207 (2005). Franke, et al, Cancer
Biother Radiopharm. 2000, 15, 459-76).
The cell-binding agents, more preferred antibodies, can be any agents that are able to against tumor cells, virus infected cells, microorganism infected cells, parasite infected cells, autoimmune cells, activated cells, myeloid cells, activated T-cells, B cells, or mela- nocytes. More specifically the cell binding agents can be any agent/molecule that is able to against any one of the following antigens or receptors: CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CDl la, CDl lb, CDl lc, CD12w, CD14, CD15, CD16, CDwl7, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD51, CD52, CD53, CD54,
CD55, CD56, CD58, CD59, CD61, CD62E, CD62L, CD62P, CD63, CD66, CD68, CD69, CD70, CD72, CD74, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96, CD98, CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD125, CD126, CD127, CD133, CD134, CD135, CD138, CD141, CD142, CD143, CD144, CD147, CD151, CD147, CD152, CD154, CD156,
CD158, CD163, CD166, .CD168, CD174, CD180, CD184, CDwl86, CD194, CD195, CD200, CD200a, CD200b, CD209, CD221, CD227, CD235a, CD240, CD262, CD271, CD274, CD276 (B7-H3), CD303, CD304, CD309, CD326, 4-lBB, 5AC, 5T4 (Trophoblast glycoprotein, TPBG, 5T4, Wnt- Activated Inhibitory Factor 1 or WAIF 1), Adenocarcinoma antigen, AGS-5, AGS-22M6, Activin receptor-like kinase 1, AFP, AKAP-4, ALK, Alpha intergrin, Alpha v beta6, Amino-peptidase N, Amyloid beta, Androgen receptor,
Angiopoietin 2, Angiopoietin 3, Annexin Al, Anthrax toxin protective antigen, Anti- transferrin receptor, AOC3 (VAP-1), B7-H3, Bacillus anthracis anthrax, BAFF (B-cell activating factor), B-lymphoma cell, bcr-abl, Bombesin, BORIS, C5, C242 antigen, CA125
(carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, Canis lupus familiaris IL31, Carbonic anhydrase IX, Cardiac myosin, CCL11(C-C motif chemokine 11), CCR4 (C-C chemokine receptor type 4, CD194), CCR5, CD3E (epsilon), CEA (Carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (Factor D), Ch4D5, Cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18),
Clumping factor A, CRIPTO, FCSF1R (Colony stimulating factor 1 receptor, CD115), CSF2 (colony stimulating factor 2, Granulocyte-macrophage colony- stimulating factor (GM-CSF)), CTLA4 (cytotoxic T-lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4 (CD 184), C-X-C chemokine receptor type 4, cyclic ADP ribose hydrolase, Cyclin B l, CYP1B 1, Cytomegalovirus, Cytomegalovirus glycoprotein B, Dabigatran,
DLL4 (delta-like-ligand 4), DPP4 (Dipeptidyl-peptidase 4), DR5 (Death receptor 5), E. coli shiga toxin type-1, E. coli shiga toxin type-2, ED-B, EGFL7 (EGF-like domain- containing protein 7), EGFR, EGFRII, EGFRvIII, Endoglin (CD 105), Endothelin B receptor, Endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2, Episialin, ERBB2 (Epidermal Growth Factor Receptor 2), ERBB3, ERG (TMPRSS2 ETS fusion gene), Escherichia coli, ETV6-AML, FAP (Fibroblast activation protein alpha), FCGR1, alpha-Fetoprotein, Fibrin II, beta chain, Fibronectin extra domain-B, FOLR (folate receptor), Folate receptor alpha, Folate hydrolase, Fos-related antigen l.F protein of respiratory syncytial virus, Frizzled receptor, Fucosyl GM1, GD2 ganglioside, G-28 (a cell surface antigen glyvolipid), GD3 idiotype, GloboH, Glypican 3, N-glycolylneuraminic acid, GM3,
GMCSF receptor a-chain, Growth differentiation factor 8, GP100, GPNMB
(Transmembrane glycoprotein NMB), GUCY2C (Guanylate cyclase 2C, guanylyl cyclase C(GC-C), intestinal Guanylate cyclase, Guanylate cyclase-C receptor, Heat-stable entero- toxin receptor (hSTAR)), Heat shock proteins, Hemagglutinin, Hepatitis B surface antigen, Hepatitis B virus, HER1 (human epidermal growth factor receptor 1), HER2, HER2/neu,
HER3 (ERBB-3), IgG4, HGF/SF (Hepatocyte growth factor/scatter factor), HHGFR, HIV- 1, Histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB , HMWMAA, Human chorionic gonadotropin, HNGF, Human scatter factor receptor kinase, HPV E6/E7, Hsp90, hTERT, ICAM-1 (Intercellular Adhesion Molecule 1), Idiotype, IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-γ, Influeza hemagglutinin, IgE, IgE Fc region, IGHE, IL-1, IL-2 receptor (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), Integrins (α4, αιιbβ3, ανβ3, α4β7, α5β1, α6β4, α7β7, α11β3, α5β5, ανβ5), Interferon gamma-induced protein, ITGA2, ITGB2,
KIR2D, LCK, Le, Legumain, Lewis- Y antigen, LFA-1 (Lymphocyte function-associated antigen 1, CDl la), LHRH, LINGO-1, Lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT- 1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE Al, MAGE A3, MAGE 4, MARTI, MCP-1, MIF (Macrophage migration inhibitory factor, or glycosylation- inhibiting factor (GIF)), MS4A1 (membrane- spanning 4-domains subfamily A member 1),
MSLN (mesothelin), MUCl(Mucin 1, cell surface associated (MUC1) or polymorphic epithelial mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCPl(monocyte chemotactic protein 1), MelanA/MARTl, ML-IAP, MPG, MS4A1 (membrane-spanning 4-domains subfamily A), MYCN, Myelin- associated glycoprotein, Myostatin, NA17, NARP-1, NCA- 90 (granulocyte antigen), Nectin-4 (ASG-22ME), NGF, Neural apoptosis-regulated proteinase 1, NOGO-A, Notch receptor, Nucleolin, Neu oncogene product, NY-BR-1, NY- ESO-1, OX-40, OxLDL (Oxidized low-density lipoprotein), OY-TES 1, P21, p53 nonmutant, P97, Page4, PAP, Paratope of anti-(N-glycolylneuraminic acid), PAX3, PAX5, PCSK9, PDCD1 (PD-1, Programmed cell death protein 1,CD279), PDGF-Ra (Alpha-type platelet-derived growth factor receptor ), PDGFR-β, PDL- 1 , PLAC 1 , PLAP-like testicular alkaline phosphatase, Platelet-derived growth factor receptor beta, Phosphate- sodium co- transporter, PMEL 17, Polysialic acid, Proteinase3 (PR1), Prostatic carcinoma, PS
(Phosphatidylserine), Prostatic carcinoma cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, Rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD240), Rhesus factor, RANKL, RhoC, Ras mutant, RGS5, ROBO4, Respiratory syncytial virus, RON, Sarcoma translocation breakpoints, SART3, Sclerostin, SLAMF7 (SLAM family member
7), Selectin P, SDC1 (Syndecan 1), sLe(a), Somatomedin C, SIP (Sphingosine-1- phosphate), Somatostatin, Sperm protein 17, SSX2, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), STEAP2, STn, TAG-72 (tumor associated glycoprotein 72), Survivin, T-cell receptor, T cell transmembrane protein, TEMl (Tumor endothelial marker
1), TENB2, Tenascin C (TN-C), TGF-a, TGF-β (Transforming growth factor beta), TGF- βΐ, TGF^2 (Transforming growth factor-beta 2), Tie (CD202b), Tie2, TIM-1 (CDX-014), Tn, TNF, TNF-a, TNFRSF8, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblast glycoprotein), TRAIL-R1 (Tumor necrosis apoprosis Inducing ligand Receptor 1), TRAILR2 (Death receptor 5 (DR5)), tumor-associated calcium signal transducer 2, tumor specific glycosylation of MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, Tyrosinase, VCAM-1 (CD 106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, or vimentin, WT1, XAGE 1, or cells expressing any insulin growth factor receptors, or any epidermal growth factor receptors.
In another specific embodiment, the cell-binding ligand-drug conjugates via the bridge linkers of this invention are used for the targeted treatment of cancers. The targeted cancers include, but are not limited, Adrenocortical Carcinoma, Anal Cancer, Bladder Cancer, Brain Tumor (Adult, Brain Stem Glioma, Childhood, Cerebellar Astrocytoma, Cerebral
Astrocytoma, Ependymoma, Medulloblastoma, Supratentorial Primitive Neuroectodermal and Pineal Tumors, Visual Pathway and Hypothalamic Glioma), Breast Cancer, Carcinoid Tumor, Gastrointestinal, Carcinoma of Unknown Primary, Cervical Cancer, Colon Cancer, Endometrial Cancer, Esophageal Cancer, Extrahepatic Bile Duct Cancer, Ewings Family of Tumors (PNET), Extracranial Germ Cell Tumor, Eye Cancer, Intraocular Melanoma,
Gallbladder Cancer, Gastric Cancer (Stomach), Germ Cell Tumor, Extragonadal, Gestational Trophoblastic Tumor, Head and Neck Cancer, Hypopharyngeal Cancer, Islet Cell Carcinoma, Kidney Cancer (renal cell cancer), Laryngeal Cancer, Leukemia (Acute Lymphoblastic, Acute Myeloid, Chronic Lymphocytic, Chronic Myelogenous, Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer (Non-Small Cell, Small Cell, Lymphoma (AIDS -Related, Central Nervous System, Cutaneous T-Cell, Hodgkin's Disease, Non-Hodgkin's Disease, Malignant Mesothelioma, Melanoma, Merkel Cell Carcinoma, Metasatic Squamous Neck Cancer with Occult Primary, Multiple Myeloma, and Other Plasma Cell Neoplasms, Mycosis Fungoides, Myelodysplastic Syndrome, Myeloproli- ferative Disorders, Nasopharyngeal Cancer, Neuroblastoma, Oral Cancer, Oropharyngeal
Cancer, Osteosarcoma, Ovarian Cancer (Epithelial, Germ Cell Tumor, Low Malignant Potential Tumor), Pancreatic Cancer (Exocrine, Islet Cell Carcinoma), Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pheochromocytoma Cancer, Pituitary Cancer, Plasma Cell Neoplasm, Prostate Cancer Rhabdomyosarcoma, Rectal Cancer, Renal Cell Cancer (kidney cancer), Renal Pelvis and Ureter (Transitional Cell),
Salivary Gland Cancer, Sezary Syndrome, Skin Cancer, Skin Cancer (Cutaneous T-Cell Lymphoma, Kaposi's Sarcoma, Melanoma), Small Intestine Cancer, Soft Tissue Sarcoma, Stomach Cancer, Testicular Cancer, Thymoma (Malignant), Thyroid Cancer, Urethral Cancer, Uterine Cancer (Sarcoma), Unusual Cancer of Childhood, Vaginal Cancer, Vulvar Cancer, Wilms' Tumor.
In another specific embodiment, the cell-binding-drug conjugates via the bridge likers of this invention are used in accordance with the compositions and methods for the treat- ment or prevention of an autoimmune disease. The autoimmune diseases include, but are not limited, Achlorhydra Autoimmune Active Chronic Hepatitis, Acute Disseminated Encephalomyelitis, Acute hemorrhagic leukoencephalitis, Addison's Disease,
Agammaglobulinemia, Alopecia areata, Amyotrophic Lateral Sclerosis, Ankylosing Spondylitis, Anti-GBM/TBM Nephritis, Antiphospholipid syndrome, Antisynthetase 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 polyendocrine syndrome Types I, II, & III, Autoimmune progesterone dermatitis, Autoimmune thrombocytopenic purpura, Autoimmune uveitis, Balo disease/Balo concentric sclerosis, Bechets Syndrome, Berger's disease, Bickerstaff s encephalitis, Blau syndrome, Bullous Pemphigoid, Castleman's disease, Chagas disease, Chronic Fatigue Immune Dysfunction Syndrome, Chronic inflammatory demyelinating polyneuropathy, Chronic recurrent multifocal ostomyelitis, Chronic lyme disease, Chronic obstructive pulmonary disease, Churg-Strauss syndrome, Cicatricial Pemphigoid, Coeliac Disease, Cogan syndrome, Cold agglutinin disease, Complement component 2 deficiency, Cranial arteritis, CREST syndrome, Crohns Disease (a type of idiopathic inflammatory bowel diseases), Cushing's Syndrome, Cutaneous leukocytoclastic angiitis, Dego's disease, Dercum's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetes mellitus type 1, Diffuse cutaneous systemic sclerosis, Dress- ler's syndrome, Discoid lupus erythematosus, Eczema, Endometriosis, Enthesitis-related arthritis, Eosinophilic fasciitis, Epidermolysis bullosa acquisita, Erythema nodosum, Essential mixed cryoglobulinemia, Evan's syndrome, Fibrodysplasia ossificans
progressiva, Fibromyalgia, Fibromyositis, Fibrosing aveolitis, Gastritis, Gastrointestinal pemphigoid, Giant cell arteritis, Glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis,
Haemolytic anaemia, Henoch- Schonlein purpura, Herpes gestationis, Hidradenitis suppurativa, Hughes syndrome (See Antiphospholipid syndrome), Hypogammaglobulinemia, Idiopathic Inflammatory Demyelinating Diseases, Idiopathic pulmonary fibrosis, Idiopathic thrombocytopenic purpura (See Autoimmune thrombocytopenic purpura), IgA nephropathy (Also Berger's disease), Inclusion body myositis, Inflammatory demyelinating polyneuopathy, Interstitial cystitis, Irritable Bowel Syndrome , Juvenile idiopathic arthritis, Juvenile rheumatoid arthritis, Kawasaki's Disease, Lambert-Eaton myasthenic syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Linear IgA disease (LAD), Lou Gehrig's Disease (Also Amyotrophic lateral sclerosis), Lupoid hepatitis, Lupus erythematosus, Majeed syndrome, Meniere's disease, Microscopic polyangiitis, Miller-Fisher syndrome, Mixed Connective Tissue Disease, Morphea, Mucha- Habermann disease, Muckle-Wells syndrome, Multiple Myeloma, Multiple Sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic's Disease),
Neuromyotonia, Occular cicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord thyroiditis, Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria, Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis, Pemphigus, Pemphigus vulgaris, Pernicious anaemia, Perivenous encephalo- myelitis, POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica, Polymyositis,
Primary biliary cirrhosis, Primary sclerosing cholangitis, Progressive inflammatory neuropathy, Psoriasis, Psoriatic Arthritis, Pyoderma gangrenosum, Pure red cell aplasia, Rasmussen's encephalitis, Raynaud phenomenon, Relapsing polychondritis, Reiter's syndrome, Restless leg syndrome, Retroperitoneal fibrosis, Rheumatoid arthritis, Rheumatoid fever, Sarcoidosis, Schizophrenia, Schmidt syndrome, Schnitzler syndrome, Scleritis,
Scleroderma, Sjogren's syndrome, Spondyloarthropathy, Sticky blood syndrome, Still's Disease, Stiff person syndrome, Subacute bacterial endocarditis, Susac's syndrome, Sweet syndrome, Sydenham Chorea, Sympathetic ophthalmia, Takayasu's arteritis, Temporal arteritis (giant cell arteritis), Tolosa-Hunt syndrome, Transverse Myelitis, Ulcerative Colitis (a type of idiopathic inflammatory bowel diseases), Undifferentiated connective tissue disease, Undifferentiated spondyloarthropathy, Vasculitis, Vitiligo, Wegener's granulomatosis, Wilson's syndrome, Wiskott-Aldrich syndrome
In another specific embodiment, a binding molecule used for the conjugate via the bridge linkers of this invention for the treatment or prevention of an autoimmune disease can be, but are not limited to, anti-elastin antibody; Abys against epithelial cells antibody;
Anti-Basement Membrane Collagen Type IV Protein antibody; Anti-Nuclear Antibody; Anti ds DNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG; anti-celiac antibody; Anti Phospholipid Antibody IgK, IgG; Anti SM Antibody; Anti Mitochondrial Antibody; Thyroid Antibody; Microsomal Antibody, T-cells antibody; Thyroglobulin Antibody, Anti SCL-70; Anti- Jo; Anti-U.sub.lRNP; Anti-La/SSB; Anti SSA; Anti SSB; Anti Perital Cells Antibody; Anti Histones; Anti RNP; C-ANCA; P-ANCA; Anti centromere; Anti- Fibrillarin, and Anti GBM Antibody, Anti-ganglioside antibody; Anti-Desmogein 3 antibody; Anti-p62 antibody; Anti-s lOO antibody; Anti-Mitochondrial(M2) antibody; Rheu- matoid factor antibody; Anti-MCV antibody; Anti-topoisomerase antibody; Anti- neutrophil cytoplasmic(cANCA) antibody.
In certain preferred embodiments, the binding molecule for the conjugate in the present invention, can bind to both a receptor and a receptor complex expressed on an activated lymphocyte which is associated with an autoimmune disease. The receptor or receptor complex can comprise an immunoglobulin gene superfamily member (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 TNF receptor super- family member (e.g. CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, INF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-Rl, TRAIL-R2, TRAIL- R3, TRAIL-R4, and APO-3), an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin (C-type, S-type, or I-type), or a complement control protein.
In another specific embodiment, useful cell binding ligands that are immuno specific for a viral or a microbial antigen are humanized or human monoclonal antibodies. As used herein, the term "viral antigen" includes, but is not limited to, any viral peptide, polypeptide protein (e.g. HIV gpl20, HIV nef, RSV F glycoprotein, influenza virus neuramimi- dase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g. gB, gC, gD, and gE) and hepatitis B surface antigen) that is capable of eliciting an immune response. As used herein, the term "microbial antigen" includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., a bacteria, fungi, pathogenic protozoa, or yeast polypeptides including, e.g., LPS and capsular polysaccharide 5/8) that is capable of eliciting an immune response. Examples of antibodies available 1 for the viral or microbial infection include, but are not limited to, Palivizumab which is a humanized anti-respiratory syncytial virus monoclonal antibody for the treatment of RSV infection; PRO542 which is a CD4 fusion antibody for the treatment of HIV infection; Ostavir which is a human antibody for the treatment of hepatitis B virus; PROTVIR which is a humanized IgG.sub. l antibody for the treatment of cytomegalovirus; and anti-LPS antibodies. The cell binding molecules-drug conjugates via the bridge linkers of this invention can be used in the treatment of infectious diseases. These infectious diseases include, but are not limited to, Acinetobacter infections, Actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired immune deficiency syndrome), Amebiasis, Anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereus infection, Bacterial pneumonia, Bacterial vaginosis, Bacteroides infection, Balantidiasis,
Baylisascaris infection, BK virus infection, Black piedra, Blastocystis hominis infection, Blastomycosis, Bolivian hemorrhagic fever, Borrelia infection, Botulism (and Infant botulism), Brazilian hemorrhagic fever, Brucellosis, Burkholderia infection, Buruli ulcer,
Calicivirus infection (Norovirus and Sapovirus), Campylobacteriosis, Candidiasis
(Moniliasis; Thrush), Cat-scratch disease, Cellulitis, Chagas Disease (American trypanosomiasis), Chancroid, Chickenpox, Chlamydia, Chlamydophila pneumoniae infection, Cholera, Chromoblastomycosis, Clonorchiasis, Clostridium difficile infection, Coccidioidomycosis, Colorado tick fever, Common cold (Acute viral rhinopharyngitis;
Acute coryza), Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever, Cryptococcosis, Cryptosporidiosis, Cutaneous larva migrans, Cyclosporiasis, Cysticercosis, Cytomegalovirus infection, Dengue fever, Dientamoebiasis, Diphtheria, Diphyllobothriasis, Dracunculiasis, Ebola hemorrhagic fever, Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworm infection), Enterococcus infection, Enterovirus infection, Epidemic typhus,
Erythema infectiosum (Fifth disease), Exanthem subitum, Fasciolopsiasis, Fasciolosis, Fatal familial insomnia, Filariasis, Food poisoning by Clostridium perfringens, Free-living amebic infection, Fusobacterium infection, Gas gangrene (Clostridial myonecrosis), Geotrichosis, Gerstmann-Straussler-Scheinker syndrome, Giardiasis, Glanders, Gnathosto- miasis, Gonorrhea, Granuloma inguinale (Donovanosis), Group A streptococcal infection,
Group B streptococcal infection, Haemophilus influenzae infection, Hand, foot and mouth disease (HFMD), Hantavirus Pulmonary Syndrome, Helicobacter pylori infection, Hemolytic-uremic syndrome, Hemorrhagic fever with renal syndrome, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infec- tion, Human bocavirus infection, Human ewingii ehrlichiosis, Human granulocytic anaplasmosis, Human metapneumo virus infection, Human monocytic ehrlichiosis, Human papillomavirus infection, Human parainfluenza virus infection, Hymenolepiasis, Epstein- Barr Virus Infectious Mononucleosis (Mono), Influenza, Isosporiasis, Kawasaki disease, Keratitis, Kingella kingae infection, Kuru, Lassa fever, Legionellosis (Legionnaires' disease), Legionellosis (Pontiac fever), Leishmaniasis, Leprosy, Leptospirosis, Listeriosis, Lyme disease (Lyme borreliosis), Lymphatic filariasis (Elephantiasis), Lymphocytic choriomeningitis, Malaria, Marburg hemorrhagic fever, Measles, Melioidosis (Whitmore's disease), Meningitis, Meningococcal disease, Metagonimiasis, Microsporidiosis,
Molluscum contagiosum, Mumps, Murine typhus (Endemic typhus), Mycoplasma pneumonia, Mycetoma, Myiasis, Neonatal conjunctivitis (Ophthalmia neonatorum), (New) Variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardiosis, Onchocerciasis (River blindness), Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis, Pasteurellosis, Pediculosis capitis (Head lice), Pediculosis corporis (Body lice), Pediculosis pubis (Pubic lice, Crab lice), Pelvic inflammatory disease, Pertussis (Whooping cough),
Plague, Pneumococcal infection, Pneumocystis pneumonia, Pneumonia, Poliomyelitis, Prevotella infection, Primary amoebic meningoencephalitis, Progressive multifocal leukoencephalopathy, Psittacosis, Q fever, Rabies, Rat-bite fever, Respiratory syncytial virus infection, Rhinosporidiosis, Rhinovirus infection, Rickettsial infection, Rickettsial- pox, Rift Valley fever, Rocky mountain spotted fever, Rotavirus infection, Rubella, Salmonellosis, SARS (Severe Acute Respiratory Syndrome), Scabies, Schistosomiasis, Sepsis, Shigellosis (Bacillary dysentery), Shingles (Herpes zoster), Smallpox (Variola),
Sporotrichosis, Staphylococcal food poisoning, Staphylococcal infection, Strongyloidiasis, Syphilis, Taeniasis, Tetanus (Lockjaw), Tinea barbae (Barber's itch), Tinea capitis (Ring- worm of the Scalp), Tinea corporis (Ringworm of the Body), Tinea cruris (Jock itch),
Tinea manuum (Ringworm of the Hand), Tinea nigra, Tinea pedis (Athlete's foot), Tinea unguium (Onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxocariasis (Ocular Larva Migrans), Toxocariasis (Visceral Larva Migrans), Toxoplasmosis, Trichinellosis, Trichomoniasis, Trichuriasis (Whipworm infection), Tuberculosis, Tularemia, Ureaplasma urealyticum infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever,
Viral pneumonia, West Nile Fever, White piedra (Tinea blanca), Yersinia pseudotuberculosis infection, Yersiniosis, Yellow fever, Zygomycosis.
The cell binding molecule, which is more preferred to be an antibody described in this patent that are against pathogenic strains include, but are not limit, Acinetobacter baumannii, Actinomyces israelii, Actinomyces gerencseriae and Propionibacterium propionicus, Trypanosoma brucei, HIV (Human immunodeficiency virus), Entamoeba histolytica, Anaplasma genus, Bacillus anthracis, Arcanobacterium haemolyticum, Junin virus, Ascaris lumbricoides, Aspergillus genus, Astroviridae family, Babesia genus, Bacillus cereus, multiple bacteria, Bacteroides genus, Balantidium coli, Baylisascaris genus, BK virus, Piedraia hortae, Blastocystis hominis, Blastomyces dermatitides, Machupo virus, Borrelia genus, Clostridium botulinum, Sabia, Brucella genus, usually Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerans, Caliciviridae family, Campylobacter genus, usually Candida albicans and other Candida species, Bartonella henselae, Group A Streptococcus and Staphylococcus, Trypanosoma cruzi, Haemophilus ducreyi, Varicella zoster virus (VZV), Chlamydia trachomatis, Chlamydophila
pneumoniae, Vibrio cholerae, Fonsecaea pedrosoi, Clonorchis sinensis, Clostridium difficile, Coccidioides immitis and Coccidioides posadasii, Colorado tick fever virus, rhinoviruses, coronaviruses, CJD prion, Crimean-Congo hemorrhagic fever virus, Crypto- coccus neoformans, Cryptosporidium genus, Ancylostoma braziliense; multiple parasites,
Cyclospora cayetanensis, Taenia solium, Cytomegalovirus, Dengue viruses (DEN-1, DEN- 2, DEN-3 and DEN-4) - Flaviviruses, Dientamoeba fragilis, Corynebacterium diphtheriae, Diphyllobothrium, Dracunculus medinensis, Ebolavirus, Echinococcus genus, Ehrlichia genus, Enterobius vermicularis, Enterococcus genus, Enterovirus genus, Rickettsia prowazekii, Parvovirus B 19, Human herpesvirus 6 and Human herpesvirus 7, Fasciolopsis buski, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Clostridium perfringens, Fusobacterium genus, Clostridium perfringens; other Clostridium species, Geotrichum candidum, GSS prion, Giardia intestinalis, Burkholderia mallei, Gnathostoma spinigerum and Gnathostoma hispidum, Neisseria gonorrhoeae, Klebsiella granulomatis, Streptococcus pyogenes, Streptococcus agalactiae, Haemophilus influenzae,
Enteroviruses, mainly Coxsackie A virus and Enterovirus 71, Sin Nombre virus, Helicobacter pylori, Escherichia coli O157:H7, Bunyaviridae family, 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, Ancylostoma duodenale and Necator americanus, Hemophilus influenzae, Human bocavirus, Ehrlichia ewingii, Anaplasma phagocytophilum, Human metapneumovirus, Ehrlichia chaffeensis, Human papillomavirus, Human parainfluenza viruses, Hymenolepis nana and Hymenolepis diminuta, Ep- stein-Barr Virus, Orthomy-xoviridae family, Isospora belli, Kingella kingae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Kuru prion, Lassa virus, Legionella pneumophila, Legionella pneumophila, Leishmania genus, Mycobacterium leprae and Mycobacterium lepromatosis, Leptospira genus, Listeria monocytogenes, Borrelia burgdorferi and other Borrelia species, Wuchereria bancrofti and Brugia malayi, Lymphocytic choriomeningitis virus (LCMV), Plasmodium genus, Marburg virus, Measles virus, Burkholderia pseudomallei, Neisseria meningitides, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, numerous species of bacteria (Actinomycetoma) and fungi (Eumycetoma), parasitic dipterous fly larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion, Nocardia asteroides and other Nocardia species, Onchocerca volvulus, Paracoccidioides brasiliensis, Paragonimus westermani and other Paragonimus species, Pasteurella genus, Pediculus humanus capitis, Pediculus humanus corporis, Phthirus pubis, Bordetella pertussis, Yersinia pestis, Streptococcus pneumoniae, Pneumocystis jirovecii, Poliovirus, Prevotella genus, Naegleria fowleri, JC virus, Chlamydophila psittaci, Coxiella burnetii, Rabies virus, Streptobacillus moniliformis and Spirillum minus, Respiratory syncytial virus, Rhino sporidium seeberi, Rhinovirus, Rickettsia genus, Rickettsia akari, Rift Valley fever virus, Rickettsia rickettsii, Rotavirus, Rubella virus, Salmonella genus, SARS coronavirus, Sarcoptes scabiei, Schistosoma genus, Shigella genus, Varicella zoster virus, Variola major or Variola minor, Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus, Staphylococcus aureus, Streptococcus pyogenes, Strongyloides stercoralis, Treponema pallidum, Taenia genus, Clostridium tetani,
Trichophyton genus, Trichophyton tonsurans, Trichophyton genus, Epidermophyton floccosum, Trichophyton rubrum, and Trichophyton mentagrophytes, Trichophyton rubrum, Hortaea werneckii, Trichophyton genus, Malassezia genus, Toxocara canis or Toxocara cati, Toxoplasma gondii, Trichinella spiralis, Trichomonas vaginalis, Trichuris trichiura, Mycobacterium tuberculosis, Francisella tularensis, Ureaplasma urealyticum,
Venezuelan equine encephalitis virus, Vibrio colerae, Guanarito virus, West Nile virus, Trichosporon beigelii, Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fever virus, Mucorales order (Mucormycosis) and Entomophthorales order (Entomophthora- mycosis), Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Edwardsiella tarda, Yersinia pestis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei,
Salmonella typhimurium, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Pneumocystis carinii, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Clamydia spp.; pathogenic fungi (Aspergillus fumigatus, Candida albicans, Histoplasma capsulatum); protozoa (Entomoeba histolytica, Trichomonas tenas,
Trichomonas hominis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms). Other antibodies as cell binding ligands used in this invention for treatment of viral disease include, but are not limited to, antibodies against antigens of pathogenic viruses, including as examples and not by limitation: Poxyiridae, Herpesviridae, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, Oncovirus [such as, HBV (Hepatocellular carcinoma), HPV (Cervical cancer, Anal cancer), Kaposi's sarcoma-associated herpesvirus (Kaposi's sarcoma), Epstein-Barr virus (Nasopharyngeal carcinoma, Burkitt's lymphoma, Primary central nervous system lymphoma), MCPyV (Merkel cell cancer), SV40 (Simian virus 40), HCV (Hepatocellular carcinoma), HTLV-I (Adult T-cell leukemia/lymphoma)], Immune disorders caused virus: [such as Human Immunodeficiency Virus (AIDS)]; Central nervous system virus: [such as, JCV (Progressive multifocal leukoencephalopathy), MeV (Subacute sclerosing panencephalitis), LCV (Lymphocytic choriomeningitis), Arbovirus encephalitis, Orthomyxoviridae (probable) (Encephalitis lethargica), RV (Rabies), Chandipura virus, Herpesviral meningitis, Ramsay Hunt syndrome type II; Po- liovirus (Poliomyelitis, Post-polio syndrome), HTLV-I (Tropical spastic paraparesis)]; Cytomegalovirus (Cytomegalovirus retinitis, HSV (Herpetic keratitis)); Cardiovascular virus [such as CBV (Pericarditis, Myocarditis)]; Respiratory system/acute viral nasopharyngitis/viral pneumonia: [Epstein-Barr virus (EBV infection/Infectious mononucleosis), Cytomegalovirus; SARS coronavirus (Severe acute respiratory syndrome) Orthomyxoviridae: Influenzavirus A/B/C (Influenza/Avian influenza), Paramyxovirus: Human parainfluenza viruses (Parainfluenza), RSV (Human respiratory syncytial virus), hMPV]; Digestive system virus [MuV (Mumps), Cytomegalovirus (Cytomegalovirus esophagitis); Adenovirus (Adenovirus infection); Rotavirus, Norovirus, Astrovirus, Coronavirus; HBV (Hepatitis B virus), CBV, HAV (Hepatitis A virus), HCV (Hepatitis C virus), HDV (Hepatitis D virus), HEV (Hepatitis E virus), HGV (Hepatitis G virus)]; Urogenital virus [such as, BK virus, MuV (Mumps)].
According to a further object, the present invention also concerns pharmaceutical compositions comprising the conjugate via the bridge linkers of the invention together with a pharmaceutically acceptable carrier, diluent, or excipient for treatment of cancers, infections or autoimmune disorders. The method for treatment of cancers, infections and autoimmune disorders can be practiced in vitro, in vivo, or ex vivo. Examples of in vitro uses include treatments of cell cultures in order to kill all cells except for desired variants that do not express the target antigen; or to kill variants that express undesired antigen. Examples of ex vivo uses include treatments of hematopoietic stem cells (HSC) prior to the performance of the transplantation (HSCT) into the same patient in order to kill diseased or malignant cells. For instance, clinical ex vivo treatment to remove tumour cells or lym- phoid cells from bone marrow prior to autologous transplantation in cancer treatment or in treatment of autoimmune disease, or to remove T cells and other lymphoid cells from allogeneic bone marrow or tissue prior to transplant in order to prevent graft-versus-host disease, can be carried out as follows. Bone marrow is harvested from the patient or other individual and then incubated in medium containing serum to which is added the conjugate of the invention, concentrations range from about 1 pM to 0.1 mM, for about 30 minutes to about 48 hours at about 37 °C. The exact conditions of concentration and time of incubation (=dose) are readily determined by the skilled clinicians. After incubation, the bone marrow cells are washed with medium containing serum and returned to the patient by i.v. infusion according to known methods. In circumstances where the patient receives other treatment such as a course of ablative chemotherapy or total-body irradiation between the time of harvest of the marrow and reinfusion of the treated cells, the treated marrow cells are stored frozen in liquid nitrogen using standard medical equipment.
For clinical in vivo use, the conjugate via the linkers of the invention will be supplied as solutions or as a lyophilized solid that can be redissolved in sterile water for injection. Examples of suitable protocols of conjugate administration are as follows. Conjugates are given weekly for 8-20 weeks as an i.v. bolus. Bolus doses are given in 50 to 500 ml of normal saline to which human serum albumin (e.g. 0.5 to 1 mL of a concentrated solution of human serum albumin, 100 mg/mL) can be added. Dosages will be about 50 μg to 20 mg/kg of body weight per week, i.v. (range of 10 μg to 200 mg/kg per injection). 4-20 weeks after treatment, the patient may receive a second course of treatment. Specific clinical protocols with regard to route of administration, excipients, diluents, dosages, times, etc., can be determined by the skilled clinicians.
Examples of medical conditions that can be treated according to the in vivo or ex vivo methods of killing selected cell populations include malignancy of any types of cancer, autoimmune diseases, graft rejections, and infections (viral, bacterial or parasite).
The amount of a conjugate which is required to achieve the desired biological effect, will vary depending upon a number of factors, including the chemical characteristics, the potency, and the bioavailability of the conjugates, the type of disease, the species to which the patient belongs, the diseased state of the patient, the route of administration, all factors which dictate the required dose amounts, delivery and regimen to be administered.
In general terms, the conjugates via the linkers of this invention may be provided in an aqueous physiological buffer solution containing 0.1 to 10% w/v conjugates for parenteral administration. Typical dose ranges are from 1 g/kg to 0.1 g/kg of body weight per day; a preferred dose range is from 0.01 mg/kg to 20 mg/kg of body weight per day, or per week, or an equivalent dose in a human child. The preferred dosage of drug to be administered is likely to depend on such variables 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 compound selected, the formulation of the compound, the route of administration
(intravenous, intramuscular, or other), the pharmacokinetic properties of the conjugates by the chosen delivery route, and the speed (bolus or continuous infusion) and schedule of administrations (number of repetitions in a given period of time).
The conjugates via the linkers of the present invention are also capable of being ad- ministered in unit dose forms, wherein the term "unit dose" means a single dose which is capable of being administered to a patient, and which can be readily handled and packaged, remaining as a physically and chemically stable unit dose comprising either the active conjugate itself, or as a pharmaceutically acceptable composition, as described hereinafter. As such, typical total daily/weekly/biweekly/monthly dose ranges are from 0.01 to 100 mg/kg of body weight. By way of general guidance, unit doses for humans range from 1 mg to 3000 mg per day, or per week, per two week or per month. Preferably the unit dose range is from 1 to 500 mg administered one to four times a week, and even more preferably from 1 mg to 100 mg, once a week. Conjugates provided herein can be formulated into pharmaceutical compositions by admixture with one or more pharmaceutically acceptable excipients. Such unit dose compositions may be prepared for use by oral administration, particularly in the form of tablets, simple capsules or soft gel capsules; or intranasal, particularly in the form of powders, nasal drops, or aerosols; or dermally, for example, topically in ointments, creams, lotions, gels or sprays, or via trans-dermal patches.
DRUGS/CYTOTOXIC AGENTS
Drugs that can be conjugated to a cell-binding molecule in the present invention are small molecule drugs including cytotoxic agents, which can be linked to or after they are modified for linkage to the cell-binding agent. A "small molecule drug" is broadly used herein to refer to an organic, inorganic, or organometallic compound that may have a molecular weight of for example 100 to 1800, more suitably from 120 to 1400. Small molecule drugs are well characterized in the art, such as in WO05058367A2, and in U.S. Patent No. 4,956,303, among others and are incorporated in their entirety by reference. The drugs include known drugs and those that may become known drugs.
Drugs that are known include, but not limited to,
1). Chemotherapeutic agents: a). Alkylating agents: such as Nitrogen mustards:
chlorambucil, chlornaphazine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, mitobronitol, melphalan, mitolactol, pipobroman, novembichin, phenesterine, prednimustine, thiotepa, trofosfamide, uracil mustard; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); Duocarmycin (including the synthetic analogues, KW-2189 and CBI- TMI); Benzodiazepine dimers (e.g., dimmers of pyrrolobenzodiazepine (PBD) or tomaymycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidino- benzodiazepines); Nitrosoureas: (carmustine, lomustine, chlorozotocin, fotemustine, nimustine, ranimustine); Alkylsulphonates: (busulfan, treosulfan, improsulfan and piposulfan); Triazenes: (dacarbazine); Platinum containing compounds: (carboplatin, cisplatin, oxaliplatin); aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemel- amine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomel- amine]; b). Plant Alkaloids: such as Vinca alkaloids: (vincristine, vinblastine, vindesine, vinorelbine, navelbin); Taxoids: (paclitaxel, docetaxol) and their analogs, Maytansinoids (DM1, DM2, DM3, DM4, maytansine and ansamitocins) and their analogs, cryptophycins (particularly cryptophycin 1 and cryptophycin 8); epothilones, eleutherobin, discodermo- lide, bryostatins, dolostatins, auristatins, tubulysins, cephalostatins; pancratistatin; a sarcodictyin; spongistatin; c). DNA Topoisomerase Inhibitors: such as [Epipodophyllins:
(9-aminocamptothecin, camptothecin, crisnatol, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acids (retinols), teniposide, topotecan, 9-nitrocamptothecin (RFS 2000)); mitomycins: (mitomycin C)]; d). Anti-metabolites: such as { [Anti-folate: DHFR inhibitors: (methotrexate, trimetrexate, denopterin, pteropterin, aminopterin (4-aminopteroic acid) or the other folic acid analogues); IMP dehydrogenase
Inhibitors: (mycophenolic acid, tiazofurin, ribavirin, EICAR); Ribonucleotide reductase Inhibitors: (hydroxyurea, deferoxamine)]; [Pyrimidine analogs: Uracil analogs:
(ancitabine, azacitidine, 6-azauridine, capecitabine (Xeloda), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-Fluorouracil, floxuridine, ratitrexed (Tomudex)); Cytosine analogs: (cytarabine, cytosine arabinoside, fludarabine); Purine analogs: (azathioprine, fludarabine, mercaptopurine, thiamiprine, thioguanine)]; folic acid replenisher, such as frolinic acid}; e). Hormonal therapies: such as {Receptor antagonists: [Anti-estrogen: (megestrol, raloxifene, tamoxifen); LHRH agonists: (goscrclin, leuprolide acetate); Anti-androgens: (bicalutamide, flutamide, calusterone, dromostanolone propionate, epitiostanol, goserelin, leuprolide, mepitiostane, nilutamide, testolactone, trilostane and other androgens inhibitors)]; Retinoids/Deltoids: [Vitamin D3 analogs: (CB 1093, EB 1089 KH 1060, cholecalciferol, ergocalciferol); Photodynamic therapies: (verteporfin, phthalocyanine, photo sensitizer Pc4, demethoxy-hypocrellin A); Cytokines: (Interferon- alpha, Interferon-gamma, tumor necrosis factor (TNFs), human proteins containing a TNF domain)] }; f). Kinase inhibitors, such as BIBW 2992 (anti-EGFR/Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib. vandetanib, E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534), bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, Trastuzumab,
Ranibizumab, Panitumumab, ispinesib; g). antibiotics, such as the enediyne antibiotics (e.g. calicheamicins, especially calicheamicin γΐ, δΐ, α1 and β1, see, e.g., J. Med. Chem., 39 (11), 2103-2117 (1996), Angew Chem Intl. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A and deoxydynemicin; esperamicin, kedarcidin, C-1027, maduropeptin, as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin;
chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, nitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; f). Others: such as Polyketides (acetogenins), especially bullatacin and bullatacinone; gemcitabine, epoxomicins (e. g. carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allovectin-7, Xegeva, Provenge, Yervoy, Isoprenylation inhibitors (such as Lovastatin), Dopaminergic neurotoxins (such as l-methyl-4-phenylpyridinium ion), Cell cycle inhibitors (such as staurosporine), Actinomycins (such as Actinomycin D, dactinomycin), Bleomycins (such as bleomycin A2, bleomycin B2, peplomycin), Anthracyclines (such as daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, mtoxantrone, MDR inhibitors (such as verapamil), Ca2+ATPase inhibitors (such as thapsigargin), Histone deacetylase inhibitors (Vorinostat, Romidepsin, Panobinostat, Valproic acid, Mocetinostat (MGCD0103), Belinostat, PCI-24781, Entinostat, SB939, Resminostat, Givinostat, AR-42, CUDC-101, sulforaphane, Trichostatin A) ; Thapsigargin,
Celecoxib, glitazones, epigallocatechin gallate, Disulfiram, Salinosporamide A.; Anti- adrenals, such as aminoglutethimide, mitotane, trilostane; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; arabinoside, bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; eflornithine (DFMO), elfomithine; elliptinium acetate, etoglucid; gallium nitrate; gacytosine, hydroxyurea; ibandronate, lentinan;
lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucarin A, roridin A and anguidine); urethane, siRNA, antisense drugs, and a nucleolytic enzyme.
2) . An anti- autoimmune disease agent includes, but is not limited to, cyclosporine, cyclosporin A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g. amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, f uocortolone danazol, dexamethasone, Triamcinolone acetonide, beclometasone dipropionate), DHEA, enanercept,
hydroxychloroquine, infliximab, meloxicam, methotrexate, mofetil, mycophenylate, prednisone, sirolimus, tacrolimus.
3) . An anti-infectious disease agent includes, but is not limited to, a). Aminoglycosides: amikacin, astromicin, gentamicin (netilmicin, sisomicin, isepamicin), hygromycin B, kanamycin (amikacin, arbekacin, bekanamycin, dibekacin, tobramycin), neomycin
(framycetin, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, verdamicin; b). Amphenicols: azidamfenicol, chloramphenicol, f orfenicol, thiamphenicol; c). Ansamycins: geldanamycin, herbimycin; d). Carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem; e). Cephems:
carbacephem (loracarbef), cefacetrile, cefaclor, cefradine, cefadroxil, cefalonium, cefaloridine, cefalotin or cefalothin, cefalexin, cefaloglycin, cefamandole, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin, cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin, cefpimizole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime, ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cephamycin (cefoxitin, cefotetan, cefmetazole), oxacephem (flomoxef, latamoxef); f). Glycopeptides: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin; g). Glycylcyc lines: e. g. tigecycline; g). β- Lactamase inhibitors: penam (sulbactam, tazobactam), clavam (clavulanic acid); i).
Lincosamides: clindamycin, lincomycin; j). Lipopeptides: daptomycin, A54145, calcium- dependent antibiotics (CDA); k). Macrolides: azithromycin, cethromycin, clarithromycin, dirithromycin, erythromycin, flurithromycin, josamycin, ketolide (telithromycin, cethromycin), midecamycin, miocamycin, oleandomycin, rifamycins (rifampicin, rifampin, rifabutin, rifapentine), rokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandomycin, telithromycin; 1). Monobactams: aztreonam, tigemonam; m). Oxazolidinones: linezolid; n). Penicillins: amoxicillin, ampicillin (pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin), azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethyl-penicillin, clometocillin, procaine benzylpenicillin, carbenicillin (carindacillin), cloxacillin, dicloxacillin, epicillin, flucloxacillin, mecillinam (pivmecillinam), mezlocillin, meticillin, nafcillin, oxacillin, penamecillin, penicillin, pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin, sulbeniciUin, temocillin, ticarcillin; o). Polypeptides: bacitracin, colistin, polymyxin B; p).
Quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, kano trovafloxacin, levofloxacin, lomefloxacin, marbofloxacin,
moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q).
Streptogramins: pristinamycin, quinupristin/dalfopristin); r). Sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim- sulfamethoxazole (co-trimoxazole); s). Steroid antibacterials: e.g. fusidic acid; t). Tetracyclines: doxycycline, chlortetracycline, clomocycline,
demeclocycline, lymecycline, meclocycline, metacycline, minocycline, oxytetracycline, penimepicycline, rolitetracycline, tetracycline, glycylcyclines (e.g. tigecycline); u). Other types of antibiotics: annonacin, arsphenamine, bactoprenol inhibitors (Bacitracin),
DADAL/AR inhibitors (cycloserine), dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitors (e. g.
fosfomycin), nitrofurantoin, paclitaxel, platensimycin, pyrazinamide,
quinupristin/dalfopristin, rifampicin (rifampin), tazobactam tinidazole, uvaricin;
4) . Anti-viral drugs: a). Entry/fusion inhibitors: aplaviroc, maraviroc, vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab); b). Integrase inhibitors: raltegravir,
elvitegravir, globoidnan A; c). Maturation inhibitors: bevirimat, vivecon; d). Neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e). Nucleosides &_nucleotides: abacavir, aciclovir, adefovir, amdoxovir, apricitabine, brivudine, cidofovir, clevudine,
dexelvucitabine, didanosine (ddl), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU), 3 '-fluoro-substituted 2', 3 '-dideoxynucleoside analogues
(e.g. 3'-fluoro-2',3'-dideoxythymidine (FLT) and 3'-fluoro-2',3'-dideoxyguanosine (FLG), fomivirsen, ganciclovir, idoxuridine, lamivudine (3TC), 1-nucleosides (e.g. β-Ι- thymidine and ?-l-2'-deoxycytidine), penciclovir, racivir, ribavirin, stampidine, stavudine (d4T), taribavirin (viramidine), telbivudine, tenofovir, trifluridine valaciclovir,
valganciclovir, zalcitabine (ddC), zidovudine (AZT); f). Non-nucleosides: amantadine, ateviridine, capravirine, diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphonoformic acid), imiquimod, interferon alfa, loviride, lodenosine, methisazone, nevirapine, NOV-205, peginterferon alfa,
podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), tromantadine; g). Protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h). Other types of anti- virus drugs: abzyme, arbidol, calanolide a, ceragenin, cyanovirin-n, diarylpyrimidines, epigallocatechin gallate (EGCG), foscarnet, griffithsin, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, portmanteau inhibitors, ribavirin, seliciclib.
5) . The drugs used for conjugates via a bridge linker of the present invention also in- elude radioisotopes. Examples of radioisotopes (radionuclides) are
Figure imgf000055_0001
Figure imgf000055_0002
Rad Λi·oi ·sotope labeled antibodies are useful in receptor targeted imaging experiments or can be for targeted treatment such as with the antibody-drug conjugates of the invention (Wu et al
(2005) Nature Biotechnology 23(9): 1137-1146). The cell binding molecules, e.g. an antibody can be labeled with ligand reagents through the bridge linkers of the present patent that bind, chelate or otherwise complex a radioisotope metal, using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley- Interscience, New York, Pubs. (1991). Chelating ligands which may complex a metal ion include DOTA, DOTP, DOTMA, DTP A and TETA (Macrocyclics, Dallas, Tex. USA).
6). The pharmaceutically acceptable salts, acids or derivatives of any of the above drugs.
In another embodiment, the drug in the Formula (II) and (IV) can a chromophore molecule, for which the conjugate can be used for detection, monitoring, or study the interaction of the cell binding molecule with a target cell. Chromophore molecules are a compound that have the ability to absorb a kind of light, such as UV light, florescent light, IR light, near IR light, visual light; A chromatophore molecule includes a class or subclass of xanthophores, erythrophores, iridophores, leucophores, melanophores, and cyanophores; a class or subclass of fluorophore molecules which are fluorescent chemical compounds re- emitting light upon light; a class or subclass of visual phototransduction molecules; a class or subclass of photophore molecules; a class or subclass of luminescence molecules; and a class or subclass of luciferin compounds.
The chromophore molecule can be selected from, but not limited, Non-protein organic fluorophores, such as: Xanthene derivatives (fluorescein, rhodamine, Oregon green, eosin, and Texas red); Cyanine derivatives: (cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine); Squaraine derivatives and ring-substituted squaraines, including Seta, SeTau, and Square dyes; Naphthalene derivatives (dansyl and prodan derivatives); Coumarin derivatives; Oxadiazole derivatives (pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole); Anthracene derivatives (anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange); Pyrene derivatives (cascade blue, etc); Oxazine derivatives (Nile red, Nile blue, cresyl violet, oxazine 170 etc). Acridine derivatives (proflavin, acridine orange, acridine yellow etc). Arylmethine derivatives (auramine, crystal violet, malachite green). Tetrapyrrole derivatives (porphin, phthalocyanine, bilirubin).
Or a chromophore molecule can be selected from any analogs and derivatives of the following fluorophore compounds: CF dye (Biotium), DRAQ and CyTRAK probes (BioStatus), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLight Fluor (Thermo Scientific, Pierce), Atto and Tracy (Sigma Aldrich), FluoProbes (Interchim), Abberior
Dyes (Abberior), DY and MegaStokes Dyes (Dyomics), Sulfo Cy dyes (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTau and Square Dyes (SETA BioMedicals), Quasar and Cal Fluor dyes (Biosearch Technologies), SureLight Dyes (APC, RPEPerCP,
Phycobilisomes)(Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech); Examples of the widely used fluorophore compounds which are reactive or
conjugatable with the linkers of the invention are: Allophycocyanin (APC), Aminocou- marin, APC-Cy7 conjugates, BODIPY-FL, Cascade Blue, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, Fluorescein, FluorX, Hydroxycoumarin, IR-783, Lissamine Rhodamine B, Lucifer yellow, Methoxycoumarin, NBD, Pacific Blue, Pacific Orange, PE-Cy5 conjugates, PE-Cy7 conjugates, PerCP, R-Phycoerythrin(PE), Red 613, Seta-555-Azide, Seta- 555-DBCO, Seta-555-NHS, Seta-580-NHS, Seta-680-NHS, Seta-780-NHS, Seta-APC- 780, Seta-PerCP-680, Seta-R-PE-670, SeTau-380-NHS, SeTau-405-Maleimide, SeTau- 405-NHS, SeTau-425-NHS, SeTau-647-NHS, Texas Red, TRITC, TruRed, X-Rhodamine.
The fluorophore compounds that can be linked to the linkers of the invention for study of nucleic acids or proteins are selected from the following compounds or their derivatives: 7-AAD (7-aminoactinomycin D, CG-selective), Acridine Orange, Chromomycin A3, CyTRAK Orange (Biostatus, red excitation dark), DAPI, DRAQ5, DRAQ7, Ethidium Bromide, Hoechst33258, Hoechst33342, LDS 751, Mithramycin, Propidiumlodide (PI), SYTOX Blue, SYTOX Green, SYTOX Orange, Thiazole Orange, TO-PRO: Cyanine
Monomer, TOTO-1, TO-PRO- 1, TOTO-3, TO-PRO-3, YOSeta-1, YOYO-1. The fluorophore compounds that can be linked to the linkers of the invention for study cells are selected from the following compounds or their derivatives: DCFH (2'7'Dichorodihydro- fluorescein, oxidized form), DHR (Dihydrorhodamine 123, oxidized form, light catalyzes oxidation), Fluo-3 (AM ester. pH > 6), Fluo-4 (AM ester. pH 7.2), Indo-1 (AM ester, low/high calcium (Ca2+)), SNARF(pH 6/9). The preferred fluorophore compounds that can be linked to the linkers of the invention for study proteins/antibodies are selected from the following compounds or their derivatives: Allophycocyanin(APC), AmCyanl (tetram- er, Clontech), AsRed2 (tetramer, Clontech), Azami Green (monomer, MBL), Azurite, B- phycoerythrin(BPE), Cerulean, CyPet, DsRed monomer (Clontech), DsRed2 ("RFP",
Clontech), EBFP, EBFP2, ECFP, EGFP (weak dimer, Clontech), Emerald (weak dimer, Invitrogen), EYFP (weak dimer, Clontech), GFP (S65A mutation), GFP (S65C mutation), GFP (S65L mutation), GFP (S65T mutation), GFP (Y66F mutation), GFP (Y66H mutation), GFP (Y66W mutation), GFPuv, HcRedl, J-Red, Katusha, Kusabira Orange (mono- mer, MBL), mCFP, mCherry, mCitrine, Midoriishi Cyan (dimer, MBL), mKate
(TagFP635, monomer, Evrogen), mKeima-Red (monomer, MBL), mKO, mOrange, mPlum, mRaspberry, mRFPl (monomer, Tsien lab), mStrawberry, mTFPl, mTurquoise2, P3 (phycobilisome complex), Peridinin Chlorophyll (PerCP), R-phycoerythrin(RPE), T- Sapphire, TagCFP (dimer, Evrogen), TagGFP (dimer, Evrogen), TagRFP (dimer, Evrogen), TagYFP (dimer, Evrogen), tdTomato (tandem dimer), Topaz, TurboFP602 (dimer, Evrogen), TurboFP635 (dimer, Evrogen), TurboGFP (dimer, Evrogen), TurboRFP (dimer, Evrogen), Turbo YFP (dimer, Evrogen), Venus, Wild Type GFP, YPet, ZsGreenl (tetramer, Clontech), Zs Yellow 1 (tetramer, Clontech).
In another embodiment, the drug in the Formula (II) and (IV) can be a polyalkylene glycols that are used for extending the half-life of the cell-binding molecule when administered to a mammal. Polyalkylene glycols include, but are not limited to, poly(ethylene glycols) (PEGs), poly(propylene glycol) and copolymers of ethylene oxide and propylene oxide; particularly preferred are PEGs, and more particularly preferred are
monofunctionally activated hydroxyPEGs (e.g., hydroxyPEGs activated at a single terminus, including reactive esters of hydroxyPEG-monocarboxylic acids, hydroxyPEG- monoaldehydes, hydroxyPEG-monoamines, hydroxyPEG-monohydrazides, hydroxyPEG- monocarbazates, hydroxyPEG-monoiodoacetamides, hydroxyPEG-monomaleimides, hydroxyPEG-monoorthopyridyl disulfides, hydroxyPEG-monooximes, hydroxyPEG- monophenyl carbonates, hydroxyPEG-monophenyl glyoxals, hydroxyPEG- monothiazolidine-2-thiones, hydroxyPEG-monothioesters, hydroxyPEG-monothiols, hydroxyPEG-monotriazines and hydroxyPEG-monovinylsulfones).
In certain such embodiments, the polyalkylene glycol has a molecular weight of from about 10 Daltons to about 200 kDa, preferably about 88 Da to about 40 kDa; two branches each with a molecular weight of about 88 Da to about 40 kDa; and more preferably two branches, each of about 88 Da to about 20 kDa. In one particular embodiment, the polyalkylene glycol is poly(ethylene) glycol and has a molecular weight of about 10 kDa; about 20 kDa, or about 40 kDa. In specific embodiments, the PEG is a PEG 10 kDa (linear or branched), a PEG 20 kDa (linear or branched), or a PEG 40 kDa (linear or branched). A number of US patents have disclosed the preparation of linear or branched "non- antigenic" PEG polymers and derivatives or conjugates thereof, e.g., U.S. Pat. Nos. 5,428,128;
5,621,039; 5,622,986; 5,643,575; 5,728,560; 5,730,990; 5,738,846; 5,811,076; 5,824,701; 5,840,900; 5,880,131; 5,900,402; 5,902,588; 5,919,455; 5,951,974; 5,965,119; 5,965,566; 5,969,040; 5,981,709; 6,011,042; 6,042,822; 6,113,906; 6,127,355; 6,132,713; 6,177,087, and 6,180,095. The structure of the conjugates of the antibody-polyalkylene glycols via the bridge linker is as following PgOl:
Figure imgf000059_0001
Wherein mAb is an antibody; n is 1-30; R' and R" are independently H or CH3; m3 and m4 are independently 0-5000; " X1, X2, R1, R2, and R3 are the same defined
Figure imgf000059_0002
in Formula (I) and (II); R4 is OH, H, or R1, or R3 that is defined as in Formula (I).
In yet another embodiment, the preferred cytotoxic agents that conjugated to a cell- binding molecule via a bridge linker of this patent are tubulysins, maytansinoids, taxanoids (taxanes), CC-1065 analogs, daunorubicin and doxorubicin compounds, benzodiazepine dimers (e.g., dimers of pyrrolobenzodiazepine (PBD), tomaymycin, anthramycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidino-benzodiazepines), calicheamicins and the enediyne antibiotics, actinomycin, azaserines, bleomycins, epirubicin, tamoxifen, idarubicin, dolastatins, auristatins (e.g. monomethyl auristatin E, MMAE , MMAF, auristatin PYE, auristatin TP, Auristatins 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)), duocarmycins, thiotepa, vincristines, hemiasterlins, nazumamides, microginins, radiosumins, alterobactins, microsclerodermins, theonellamides,
esperamicins, PNU- 159682, and their analogues and derivatives above thereof.
Tubulysins that are preferred for conjugation in the present invention are well known in the art and can be isolated from natural sources according to known methods or prepared synthetically according to known methods (e. g. Balasubramanian, R.; et al. J. Med.
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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.; et al.
Mol. Pharmaceutics, 2012, 9, 168-175. Friestad, G. K.; et al. Org. Lett., 2009, 11, 1095- 1098. Kubicek, K.; et al., Angew Chem Int Ed Engl, 2010. 49: p. 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 applications: Zanda, M. ; et al, Can.
Pat. Appl. CA 2710693 (2011). Chai, Y.; et al. Eur. Pat. Appl. 2174947 (2010), PCT WO 2010034724. Leamon, C; et al, PCT WO 2010033733, WO 2009002993. Ellman, J.; et al, PCT WO 2009134279; PCT WO 2009012958, US appl. 20110263650, 20110021568, Matschiner, G.; et al, PCT WO 2009095447. Vlahov, I.; et al, PCT WO 2009055562, WO 2008112873. Low, P.; et al, PCT WO 2009026177. Richter, W., PCT WO 2008138561. Kjems, J.; et al, PCT WO 2008125116. Davis, M.; et al, PCT WO 2008076333. Diener, J.; et al, U.S. Pat. Appl. 20070041901, WO 2006096754. Matschiner, G.; et al, PCT WO 2006056464. Vaghefi, F.; et al, 5 PCT WO 2006033913. Doemling, A., Ger. Offen. DE 102004030227; PCT WO 2004005327; WO 2004005326; WO2004005269. Stanton, M.; et al, U.S. Pat. Appl. Publ. 20040249130. Hoefle, G.; et al, Ger. Offen. DE 10254439 ; DE 10241152; DE 10008089. Leung, D.; et al, WO 2002077036. Reichenbach, H.; et al, Ger.
Offen. DE 19638870; Wolfgang, R.; US 20120129779, Chen, H.,US appl. 20110027274. The preferred structure of tubulysins for conjugation of cell binding molecules are described in the patent application of PCT/IB2012/053554.
Examples of the structures of the conjugates of the antibody-tubulysin analogs via the bridge linker are T01, T02, T03, T04, T05, T06 and T07 as following:
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Wherein mAb is an antibody; Z3 and Z'3 are independently H, OP(O)(OMi)(OM2), OCH2OP(O)(OMi)(OM2), OSO3M1, R1, or O-glycoside (glucoside, galactoside, mannoside, glucuronoside, alloside, fructoside, etc), NH-glycoside, S-glycoside or CH2- glycoside; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, NR1R2R3; n is 1-30;
Figure imgf000062_0003
X1, X2, R1, R2 and R3 are the same defined in Formula (I) and (II).
Calicheamicins and their related enediyne antibiotics that are preferred for cell- binding molecule-drug conjugates of this patent are described in: Nicolaou, K. C. et al, Science 1992, 256, 1172-1178; Proc. Natl. Acad. Sci USA. 1993, 90, 5881-5888), U.S. Patent Nos. 4,970,198; 5,053,394; 5,108,912; 5,264,586; 5,384,412; 5,606,040; 5,712,374; 5,714,586; 5,739,116; 5,770,701; 5,770,710; 5,773,001; 5,877,296; 6,015,562; 6,124,310; 8,153,768. An Example of the structure of the conjugate of the antibody-Calicheamicin analog via the bridge linker is C01 as the following:
Figure imgf000062_0002
Wherein mAb is an antibody; n is 1-30;
Figure imgf000062_0004
X1, X2, R1, R2 and R3 are the same defined in Formula (I) and (II).
Maytansinoids that are preferred to be used in the present invention including maytansinol and its analogues are described in U.S. Patent Nos. 4,256,746, 4,361,650, 4,307,016, 4,294,757, 4,294,757, 4,371,533, 4,424,219, 4,331,598, 4,450,254, 4,364,866, 4,313,946, 4,315,929 4,362,663, 4,322,348, 4,371,533, 4,424,219, 5,208,020, 5,416,064, 5,208,020; 5,416,064; 6,333.410; 6,441,163; 6,716,821, 7,276,497, 7,301,019, 7,303,749, 7,368,565, 7,411,063, 7,851,432, and 8,163,888 . An example of the structure of the conjugate of the antibody- Maytansinoids via the bridge linker is as the following M01:
Figure imgf000063_0001
Wherein mAb is an antibody; n is 1~30; X1, X2, R1, R2 and R3 are the same
Figure imgf000063_0003
defined in Formula (I) and (II).
Taxanes, which includes Paclitaxel (Taxol), a cytotoxic natural product, and docetaxel (Taxotere), a semi-synthetic derivative, and their analogs which are preferred for conjugation via the bridge linkers of the present patent are exampled in:. K C. Nicolaou et al., J. Am. Chem. Soc. 117, 2409-2420, (1995); Ojima et al, J. Med. Chem. 39:3889-3896 (1996); 40:267-278 (1997); 45, 5620-5623 (2002); Ojima et al., Proc. Natl. Acad. Sci., 96:4256-4261 (1999; Kim et al., Bull. Korean Chem. Soc, 20, 1389-1390 (1999); Miller, et al. J. Med. Chem., 47, 4802-4805(2004); U.S. Patent No. 5,475,011 5,728,849,
5,811,452; 6,340,701; 6,372,738; 6,391,913, 6.436,931; 6,589,979; 6,596,757; 6,706,708; 7,008,942; 7,186,851; 7,217,819; 7,276,499; 7,598,290; and 7,667,054.
Examples of the structures of the conjugate of the antibody- taxanes via the bridge linker are as the following Tx01, Tx02 and Tx03.
Figure imgf000064_0001
Wherein mAb is an antibody; n is 1~30;
Figure imgf000064_0002
X1, X2, R1 and R2 are the same defined in Formula (I) and (II).
CC-1065 analogues and doucarmycin analogs are also preferred to be used for a conjugate with the bridge linkers of the present patent. The examples of the CC-1065 analogues and doucarmycin analogs as well as their synthesis are described in: e.g.
Warpehoski, et al, J. Med. Chem. 31:590-603 (1988), D. Boger et al., J. Org. Chem; 66; 6654-6661, 2001; U. S. Patent Nos: 4169888, 4391904, 4671958, 4816567, 4912227, 4923990, 4952394, 4975278, 4978757, 4994578, 5037993, 5070092, 5084468, 5101038, 5117006, 5137877, 5138059, 5147786, 5187186, 5223409, 5225539, 5288514, 5324483, 5332740, 5332837, 5334528, 5403484, 5427908, 5475092, 5495009, 5530101, 5545806, 5547667, 5569825, 5571698, 5573922, 5580717, 5585089, 5585499, 5587161, 5595499, 5606017, 5622929, 5625126, 5629430, 5633425, 5641780, 5660829, 5661016, 5686237, 5693762, 5703080, 5712374, 5714586, 5739116, 5739350, 5770429, 5773001, 5773435, 5786377 5786486, 5789650, 5814318, 5846545, 5874299, 5877296, 5877397, 5885793, 5939598, 5962216, 5969108, 5985908, 6060608, 6066742, 6075181, 6103236, 6114598, 6130237, 6132722, 6143901, 6150584, 6162963, 6172197, 6180370, 6194612, 6214345, 6262271, 6281354, 6310209, 6329497, 6342480, 6486326, 6512101, 6521404, 6534660, 6544731, 6548530, 6555313, 6555693, 6566336, 6,586,618, 6593081, 6630579,
6,756,397, 6759509, 6762179, 6884869, 6897034, 6946455, 7,049,316, 7087600, 7091186, 7115573, 7129261, 7214663, 7223837, 7304032, 7329507, 7,329,760,
7,388,026, 7,655,660, 7,655,661, 7,906,545, and 8,012,978. Examples of the structures of the conjugate of the antibody- CC-1065 analogs via the bridge linker are as the following CCOl, CC02, and CC03.
Figure imgf000065_0001
Wherein mAb is an antibody; n is 1-30; Z4 and Z'4 are independently H, ΡΟ(ΟΜι)(ΟΜ2), CH2PO(OM1)(OM2), SO3M1, CH3N(CH2CH2)2NC(O)-, O(CH2CH2)2NC(O)-, R1, or glycoside; X3 and X'3 are independently O, NH, NHC(O), OC(O), -C(O)O, R1, or absent;
Figure imgf000065_0002
, , X1, X2, R1 R2 M1, and M2 are the same defined in Formula (I) and (II). Daunorubicin/Doxorubicin Analogues are also preferred for conjugation via the bridge linkers of the present patent. The preferred structures and their synthesis are exam- pled in: Hurwitz, E., et al., Cancer Res. 35, 1175-1181 (1975). Yang, H. M., and Reisfeld, R. A., Proc. Natl. Acad. Sci. 85, 1189-1193 (1988); Pietersz, C. A., E., et al., E., et al.," Cancer Res. 48, 926-9311 (1988); Trouet, et al., 79, 626-629 (1982); Z. Brich et al., J. Controlled Release, 19, 245-258 (1992); Chen et al., Syn. Comm., 33, 2377-2390, 2003; King et al., Bioconj. Chem., 10, 279-288, 1999; King et al., J. Med. Chem., 45, 4336- 4343, 2002; Kratz et al., J Med Chem. 45, 5523-33. 2002; Kratz et al., Biol Pharm Bull. Jan. 21, 56-61 , 1998; Lau et al., Bioorg. Med. Chem. 3, 1305-1312, 1995; Scott et al., Bioorg. Med.l Chem. Lett. 6, 1491-1496; 1996; Watanabe et al., Tokai J. Experimental Clin. Med. 15, 327-334, 1990; Zhou et al., J. Am. Chem. Soc. 126, 15656-7, 2004; WO 01/38318; U.S. Patent No.). 5,106,951; 5,122,368; 5,146,064; 5,177,016; 5,208,323; 5,824,805; 6,146,658; 6,214,345; 7569358; 7,803,903; 8,084,586; 8,053,205. Examples of the structures of the conjugate of the antibody- CC-1065 analogs via the bridge linker are as the following DaOl, Da02, Da03 and Da04.
Figure imgf000066_0001
Da02
Figure imgf000067_0001
Wherein mAb is an antibody; n is 1~30; X3 and X'3 are independently H, O, NH, NHC(O),
NHC(O)NH, C(O), R1, or OC(O);
Figure imgf000067_0002
X1, X2, R1, and R2 are the same defined in Formula (I) and (II).
Auristatins and dolastatins are preferred in conjugation via the bridge linkers of this patent. The auristatins (e. g. auristain E (AE) auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), Monomethylauristatin (MMAF), Auristatin F phenylene diamine (AFP) and a phenylalanine variant of MMAE) which are synthetic analogs of dolastatins, are described in Int. J. Oncol. 15:367-72 (1999); Molecular Cancer
Therapeutics, vol. 3, No. 8, pp. 921-932 (2004); U.S. Application Nos. 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, 5635483, 5654399, 5663149, 5665860,
5708146, 5714586, 5741892, 5767236, 5767237, 5780588, 5821337, 5840699, 5965537,
6004934, 6033876, 6034065, 6048720, 6054297, 6054561, 6124431, 6143721, 6162930,
6214345, 6239104, 6323315, 6342219, 6342221, 6407213, 6569834, 6620911, 6639055,
6884869, 6913748, 7090843, 7091186, 7097840, 7098305, 7098308, 7498298, 7375078,
7462352, 7553816, 7659241, 7662387, 7745394, 7754681, 7829531, 7837980, 7837995, 7902338, 7964566, 7964567, 7851437, 7994135. Examples of the structures of the conjugate of the antibody-auristatins via the bridge linker are as the following AuOl, Au02, Au03, Au04, and Au05.
Figure imgf000068_0001
Wherein mAb is an antibody; n is 1~30; X3 and X'3 are independently CH2, O, NH, NHC(O), NHC(O)NH, C(O), OC(O) R1, or absent; X4 and X'4 are independently CH2, C(O), C(O)NH, C(O)N(R1), R1, NHR1, NR1, C(O)R1 or C(O)O; Z3 and Z'3 are independently H, R1, OP(O)(OMi)(OM2), NHR1, OCH2OP(O)(OM1)(OM2), OSO3M1, or O- glycoside (glucoside, galactoside, mannoside, glucuronoside, alloside, fructoside), NH- glycoside, S-glycoside, or CH2-glycoside; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, NR1R2R3; X1, X2, R1, R2 and R are the same defined in Formula
Figure imgf000069_0002
(I) and (II).
The benzodiazepine dimers (e. g. dimmers of pyrrolobenzodiazepine (PBD) or (tomaymycin), indolinobenzodiazepines, imidazobenzothiadiazepines, or
oxazolidinobenzodiazepines) which are preferred cytotoxic agents according to the present invention are exampled in the art: US Patent Nos . 8,163,736; 8,153,627; 8,034,808;
7,834,005; 7,741,319; 7,704,924; 7,691,848; 7,678,787; 7,612,062; 7,608,615; 7,557,099; 7,528,128; 7,528,126; 7,511,032; 7,429,658; 7,407,951; 7,326,700; 7,312,210; 7,265,105; 7,202,239; 7,189,710; 7,173,026; 7,109,193; 7,067,511; 7,064,120; 7,056,913; 7,049,311; 7,022,699; 7,015,215; 6,979,684; 6,951,853; 6,884,799; 6,800,622; 6,747,144; 6,660,856; 6,608,192; 6,562,806; 6,977,254; 6,951,853; 6,909,006; 6,344,451; 5,880,122; 4,935,362;
4,764,616; 4,761,412; 4,723,007; 4,723,003; 4,683,230; 4,663,453; 4,508,647; 4,464,467; 4,427,587; 4,000,304; US patent appl. 20100203007, 20100316656, 20030195196. Examples of the structures of the conjugate of the antibody- benzodiazepine dimers via the bridge linker are as the following PB01, PB02, PB03, PB04, PB05, PB06, PB07, PB08, PB PB lO and PB l l.
Figure imgf000069_0001
IB2015/056083
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Wherein mAb is an antibody; n is 1~30; X3 and X'3 are independently CH2, O, NH, NHC(O), NHC(O)NH, C(O), OC(O), OC(O)(NR3), R1, NHR1, NR1, C(O)R1 or absent; X4 and X'4 are independently CH2, C(O), C(O)NH, C(O)N(R1), R1, NHR1, NR1, C(O)R1 or C(O)O; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, NR1R2R ;
Figure imgf000072_0003
X1, X2, R1, R2 and R3 are the same defined in Formula (I) and (II). In addition, R1 and/or R2 can be absent.
In yet another embodiment, two or more different cytotoxic agents are preferred conjugated to a cell-binding molecule via a bridge linker of this patent. The two or more different cytotoxic agents can be selected from any combinations of tubulysins, maytansinoids, taxanoids (taxanes), CC-1065 analogs, daunorubicin and doxorubicin compounds, benzodiazepine dimers (e.g., dimers of pyrrolobenzodiazepine (PBD), tomaymycin, anthramycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidino-benzodiazepines), calicheamicins and the enediyne antibiotics, actinomycin, azaserines, bleomycins, epirubicin, tamoxifen, idarubicin, dolastatins, auristatins (e.g. monomethyl auristatin E, MMAE , MMAF, auristatin PYE, auristatin TP, Auristatins 2- AQ, 6-AQ, EB (AEB), and EFP (AEFP)), duocarmycins, thiotepa, vincristines, hemiasterlins, nazumamides, microginins, radiosumins, alterobactins, microsclerodermins, theonellamides, esperamicins, PNU- 159682, and their analogues and derivatives above thereof. Examples of the structures of the conjugates containing two or more different cytotoxic agents via the bridge linker are as the following Z01, Z02, Z02, Z04, Z05, Z06,
Figure imgf000072_0002
Figure imgf000073_0001
Figure imgf000074_0001
73
Figure imgf000075_0001
74
Figure imgf000076_0001
Wherein mAb is an antibody; n is 1-30; X3 and X'3 are independently CH2, O, NH, NHC(O), NHC(O)NH, C(O), OC(O), OC(O)(NR3), R1, NHR1, NR1, C(O)R1 or absent; X4 and X'4 are independently H, CH2, OH, O, C(O), C(O)NH, C(O)N(R1), R1, NHR1, NR1, C(O)R1 or C(O)O; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, NR1R2R3;
Figure imgf000076_0002
, X1, X2, R1 R2 and R3 are the same defined in Formula (I) and (II). In addition, R1 and/or R2 can be absent.
In yet another embodiment, cell-binding ligands or receptors can be conjugated to a cell-binding molecule via a bridge linker of this patent. These conjugated cell-binding ligands or receptors, in particular, antibody-receptor conjugates, can be not only to work as a targeting conductor/director to deliver the conjugate to malignant cells, but also be used to modulate or co-stimulate a desired immune response or altering signaling pathways. In the immunotherapy, the cell-binding ligands or receptors are preferred to conjugate to an antibody of TCR (T cell receptors) T cell, or of CARs (chimeric antigen receptors) T cells, or of B cell receptor (BCR), or the cytotoxic cells. The cell-binding ligands or receptors are selected, but not limited, from: Folate derivatives (binding to the folate receptor, a protein over-expressed in ovarian cancer and in other malignancies) (Low, P. S. et al 2008, Acc. Chem. Res. 41, 120-129); Glutamic acid urea derivatives (binding to the prostate specific membrane antigen, a surface marker of prostate cancer cells) (Hillier, S. M.et al, 2009, Cancer Res. 69, 6932-6940); Somatostatin (also known as growth hormone-inhibiting hormone (GHIH) or somatotropin release-inhibiting factor (SRIF)) or somatotropin re- lease-inhibiting hormone) and its analogues such as octreotide (Sandostatin) and lanreotide
(Somatuline) (particularly for neuroendocrine tumors, GH-producing pituitary adenoma, paraganglioma, nonfunctioning pituitary adenoma, pheochromocytomas) (Ginj, M., et al, 2006, Proc. Natl. Acad. Sci. U.S.A. 103, 16436-16441); certain Aromatic sulfonamides, specific to carbonic anhydrase IX (a marker of hypoxia and of renal cell carcinoma) (Neri, D., et al, Nat. Rev. Drug Discov. 2011, 10, 767-777); Pituitary adenylate cyclase activating peptide (PACAP) (PAC1) for pheochromocytomas and paragangliomas; Vasoactive intestinal peptide (VIP/PACAP) (VPAC1, VPAC2) for cancers of lung, stomach, colon, rectum, breast, prostate, pancreatic ducts, liver, and urinary bladder; Cholecystokinin (CCK) (CCK1 (formerly CCK-A) and CCK2 for small cell lung cancers, medullary thy- roid carcinomas, astrocytomas, and ovarian cancers; Bombesin(Pyr-Gln-Arg-Leu-Gly-
Asn-Gln-Trp-Ala-Val-Gly-H is-Leu-Met-NH2)/gastrin-releasing peptide (GRP) (BB 1, GRP receptor subtype (BB2), the BB3 and BB4) for renal cell, breast, lung, gastric and prostate carcinomas, and neuroblastoma (and neuroblastoma (Ohlsson, B., et al, 1999, Scand. J, Gastroenterology 34 (12): 1224-9; Weber, H. C, 2009, Cur. Opin. Endocri. Diab. Obesity 16(1): 66-71, Gonzalez N, et al, 2008, Cur. Opin. Endocri. Diab. Obesity
15(1), 58-64 ); Neurotensin (NTR1, NTR2, NTR3) for small cell lung cancer,
neuroblastoma, pancreatic and colonic cancer; Substance P (NK1 receptor) for Glial tumors; Neuropeptide Y (Y1-Y6) for breast carcinomas; Homing Peptides include RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), the dimeric and multimeric cyclic RGD peptides (e.g. cRGDfV) that recognize receptors (integrins) on tumor surfaces (Laakkonen P,
Vuorinen K. 2010, Integr Biol (Camb). 2(7-8): 326-337; Chen K, Chen X. 2011, Theranostics. 1: 189-200; Garanger E, et al, Anti-Cancer Agents Med Chem. 7 (5): 552- 558; Kerr, J. S. et al, Anticancer Research, 19(2A), 959-968; Thumshirn, G, et al, 2003 Chem. Eur. J. 9, 2717- 2725), and TAASGVRSMH and LTLRWVGLMS (chondroitin sulfate proteoglycan NG2 receptor) and F3 peptide (31 amino acid peptide that binds to cell surface-expressed nucleolin receptor) (Zitzmann, S., 2002 Cancer Res., 62, 18, pp. 5139-5143, Temminga, K., 2005, Drug Resistance Updates, 8, 381-402; P. Laakkonen and K. Vuorinen, 2010 Integrative Biol, 2(7-8), 326-337; M. A. Burg, 1999 Cancer Res., 59(12), 2869-2874; K. Porkka, et al 2002, Proc. Nat. Acad. Sci. USA 99(11), 7444-9); Cell Penetrating Peptides (CPPs) (Nakase I, et al, 2012, J. Control Release. 159(2),181- 188); Peptide Hormones, such as luteinizing hormone-releasing hormone (LHRH) agonists and antagonists, and gonadotropin-releasing hormone (GnRH) agonist, acts by targeting follicle stimulating hormone (FSH) and luteinising hormone (LH), as well as testosterone production, e.g. buserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-
NHEt), Gonadorelin (Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), Goserelin (Pyr- His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-AzGly-NH2), Histrelin (Pyr-His-Trp-Ser-Tyr- D-His(N-benzyl)-Leu-Arg-Pro-NHEt), leuprolide (Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg- Pro-NHEt), Nafarelin (Pyr-His-Trp-Ser-Tyr-2Nal-Leu-Arg-Pro-Gly-NH2), Triptorelin (Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2), Nafarelin, Deslorelin, Abarelix (Ac-
D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-isopropylLys-Pro- DAla-NH2), Cetrorelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-Cit- Leu-Arg-Pro-D-Ala-NH2), Degarelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser- 4-aminoPhe(L-hydroorotyl)-D-4-aminoPhe(carba-moyl)-Leu-isopropylLys-Pro-D-Ala- NH2), and Ganirelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-(N9, N10- diethyl)-homoArg-Leu-(N9, N10-diethyl)-homoArg-Pro-D-Ala-NH2) (Thundimadathil, J., J. Amino Acids, 2012, 967347, doi: 10.1155/2012/ 967347; Boccon-Gibod, L.; et al, 201 1, Therapeutic Advances in Urology 3 (3): 127-140; Debruyne, F., 2006, Future Oncology, 2(6), 677-696,); and Pattern Recognition Receptors (PRRs), such as Toll-like receptors (TLRs), C-type lectins and Nodlike Receptors (NLRs) (Fukata, M., et al, 2009, Semin.
Immunol. 21, 242-253; Maisonneuve, C, et al, 2014, Proc. Natl. Acad. Sci. U. S. A. Ill, 1-6; Botos, I., et al, 2011, Structure 19, 447-459; Means, T. K., et al, 2000, Life Sci. 68, 241-258) that range in size from small molecules (imiquimod, guanisine and adenosine analogs) to large and complex biomacromolecules such as lipopolysaccharide (LPS), nucleic acids (CpG DNA, polyLC) and lipopeptides (Pam3CSK4) (Kasturi, S. P., et al,
2011, Nature 470, 543-547; Lane, T., 2001, J. R. Soc. Med. 94, 316; Hotz, C, and Bourquin, C, 2012, Oncoimmunology 1, 227-228; Dudek, A. Z., et al, 2007, Clin. Cancer Res. 3, 7119-7125).
The cell-binding ligands or receptors can be Ig-based and non-Ig-based protein scaf- fold molecules. The Ig-Based scaffolds can be selected, but not limited, from Nanobody (a derivative of VHH (camelid Ig)) (Muyldermans S., 2013 Annu Rev Biochem. 82, 775- 797); Domain antibodies (dAb, a derivative of VH or VL domain) (Holt, L. J, et al, 2003, Trends Biotechnol. 21, 484-490); Bispecific T cell Engager (BiTE, a bispecific diabody) (Baeuerle, P. A, et al, 2009, Curr. Opin. Mol. Ther. 11, 22-30); Dual Affinity ReTargeting (DART, a bispecific diabody) (Moore P. A. P, et al. 2011, Blood 117(17), 4542-4551); Tetravalent tandem antibodies (TandAb, a dimerized bispecific diabody) (Cochlovius, B, et al. 2000, Cancer Res. 60(16):4336-4341). The Non-Ig scaffolds can be selected, but not limited, from Anticalin (a derivative of Lipocalins) (Skerra A. 2008, FEBS J., 275(11): 2677-2683; Beste G, et al, 1999 Proc. Nat. Acad. USA. 96(5): 1898-1903; Skerra, A. 2000
Biochim Biophys Acta, 1482(l-2):337-350; Skerra, A. 2007, Curr Opin Biotechnol.
18(4):295-304; Skerra, A. 2008, FEBS J. 275(11):2677-2683); Adnectins (10th FN3 (Fibronectin)) (Koide, A, et al, 1998 J. Mol. Biol., 284(4): 1141-1151; Batori V, 2002, Protein Eng. 15(12): 1015-1020; Tolcher, A. W, 2011, Clin. Cancer Res. 17(2):363-371; Hackel, B. J, 2010, Protein Eng. Des. Sel. 23(4):211-219); Designed Ankyrin Repeat
Proteins (DARPins) (a derivative of ankrin repeat (AR) proteins) (Boersma, Y.L, et al, 2011 Curr Opin Biotechnol. 22(6): 849-857), e.g. DARPin C9, DARPin Ec4 and DARPin E69_LZ3_E01 (Winkler J, et al, 2009 Mol Cancer Ther. 8(9), 2674-2683; Patricia M-K. M., et al, Clin Cancer Res. 2011; 17(1): 100-110; Boersma Y. L, et al, 2011 J. Biol. Chem. 286(48),41273-41285); Avimers (a domain A/low-density lipoprotein (LDL) receptor)
(Boersma Y. L, 2011 J. Biol. Chem. 286(48): 41273-41285; Silverman J, et al, 2005 Nat. Biotechnol., 23(12): 1556-1561).
Examples of the structures of the conjugate of the antibody-cell-binding ligands or receptors via the bridge linker are as the following: LB01 (PMSA ligand conjugate), LB02 (Folate receptor conjugate), LB03 (Somatostatin receptor conjugate), LB04 (Octreotide, a
Somatostatin analog receptor conjugate), LB05 (Lanreoiide, a Somatostatin analog receptor conjugate), LB06 (CAIX receptor conjugate), LB07(CAIX receptor conjugate), LB08 (luteinizing hormone-releasing hormone (LH-RH) ligand and GnRH conjugate), LB09 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand conjugate), LB 10 (GnRH antagonist, Abarelix conjugate), LB 11 (cobalamin, VB 12 analog conjugate), LB 12
(Gastrin releasing peptide receptor (GRPr), MBA conjugate), LB 13 (ανβ3 integrin receptor, cyclic RGD pentapeptide conjugate), LB 14 (hetero-bivalent peptide ligand for VEGF receptor conjugate), LB 15 (Neuromedin B conjugate), LB 16 (a G-protein coupled receptor, bombesin conjugate) and LB 17 (a Toll-like receptor, TLR2 conjugate).
Figure imgf000080_0001
mAb
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
LB 17
Wherein mAb is an antibody; n is 1~30; X3 and X'3 are independently CH2, O, NH, NHC(O), NHC(O)NH, C(O), OC(O), OC(O)(NR3), R1, NHR1, NR1, C(O)R1 or absent; X4 and X'4 are independently H, CH2, OH, O, C(O), C(O)NH, C(O)N(R1), R1, NHR1, NR1, C(O)R1 or C(O)O; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, NR1R2R3; m3 and m4 is 0-5000,
Figure imgf000084_0002
X1, X2, R1 R2 and R3 are the same defined in Formula (I) and (II). In addition, R1 and/or R2 can be absent.
The drugs/ cytotoxic agents used for conjugation via a bridge linker of the present patent can be any analogues and/or derivatives of drugs/molecules described in the present patent. One skilled in the art of drugs/cytotoxic agents will readily understand that each of the drugs/cytotoxic agents described herein can be modified in such a manner that the resulting compound still retains the specificity and/or activity of the starting compound. The skilled artisan will also understand that many of these compounds can be used in place of the drugs/cytotoxic agents described herein. Thus, the drugs/cytotoxic agents of the present invention include analogues and derivatives of the compounds described herein.
All references cited herein and in the examples that follow are expressly incorporated by reference in their entireties.
EXAMPLES
The invention is further described in the following examples, which are not intended to limit the scope of the invention. Cell lines described in the following examples were maintained in culture according to the conditions specified by the American Type Culture Collection (ATCC) or Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany (DMSZ), or The Shanghai Cell Culture Institute of Chinese Acadmy of Science, unless otherwise specified. Cell culture reagents were obtained from Invitrogen Corp., unless otherwise specified. All anhydrous solvents were commercially obtained and stored in Sure-seal bottles under nitrogen. All other reagents and solvents were purchased as the highest grade available and used without further purification. The preparative HPLC separations were performed with Varain PreStar HPLC. NMR spectra were recorded on Varian Mercury 400 MHz Instrument. Chemical shifts (.delta.) are reported in parts per million (ppm) referenced to tetramethylsilane at 0.00 and coupling constants (J) are reported in Hz. The mass spectral data were acquired on a Waters Xevo QTOF mass spect equipped with Waters Acquity UPLC separations module and Acquity TUV detector.
Example 1: tert-Butyl 3-(2-(2-hydroxyethoxy)ethoxy)propanoate (84)
Figure imgf000085_0001
To 350 mL of anhydrous THF was added 80 mg (0.0025 mol) of sodium metal and diethylene glycol 83 (150.1 g, 1.41 mol) with stirring. After the sodium had completely dissolved, tert-butyl acrylate (24 mL, 0.33 mol) was added. The solution was stirred for 20 h at room temperature and neutralized with 8 mL of 1.0 M HC1. The solvent was removed in vacuo and the residue was suspended in brine (250 mL) and extracted with ethyl acetate (3 x 125 mL). The combined organic layers were washed with brine (100 mL) then water
(100 mL), dried over sodium sulfate, and the solvent was removed. The resulting colorless oil was dried under vacuum to give 60.27 g (78% yields) of product 84. 1H NMR: 1.41 (s, 9H), 2.49 (t, 2H, J=6.4 Hz), 3.59-3.72 (m, 10H). ESI MS m z- CnH21O5 (M-H), cacld. 233.15, found 233.40.
Exam le 2. tert-Butyl 3- 2-(2-(tosylox )ethoxy)ethoxy)propanoate (85)
Figure imgf000085_0002
A solution of 84 (10.0 g, 42.70 mmol) in dichloromethane (50.0 mL) was treated with pyridine (20.0 mL). A solution of methanesulfonyl chloride (7.50 g, 65.81 mmol) in 50 mL dichloromethane was added dropwise via an addition funnel over 30 minutes. After 5 h TLC analysis revealed that the reaction was complete. The pyridine hydrochloride that had formed was filtered off and the solvent was removed. The residue was purified on silica gel by eluting from with 20% ethyl acetate in hexane to with neat ethyl acetate to give 10.39 g (76% yield) of compound 85. 1H NMR: 1.40 (s, 9H), 3.23 (s, 3H), 2.45 (t, 2H, J=6.4 Hz), 3.54-3.70 (m, 10H); ESI MS m/z+ C12H25O7S (M+H), cacld. 313.10, found 313.30. Example 3. tert-But l - 2- 2-azidoethox ethox ro anoate
Figure imgf000086_0001
To 50 niL of DMA was added tert-butyl 3-(2-(2-(Mesyloxy)ethoxy)ethoxy)ethoxy)- propanoate 85 (4.0 g, 12.81 mmol) and sodium azide (0.90 g, 13.84 mmol) with stirring. The reaction was heated to 80 °C. After 4 h TLC analysis revealed that the reaction was complete. The reaction was cooled to room temperature and quenched with water (25 mL). The aqueous layer was separated and extracted into ethyl acetate (3 x 35 mL). The combined organic layers were dried over anhydrous magnesium sulfate, filtered, concentrated in vacuo and purified on silica gel by eluting from with 15% ethyl acetate in hexane to with neat ethyl acetate to give 2.88 g (87% yield) of compound 86. 1H NMR (CDC13): 1.40 (s, 9H), 2.45 (t, 2H, J=6.4 Hz), 3.33 (t, 2H, J=5.2 Hz), 3.53-3.66 (m, 8H). ESI MS m/z+ C11H22N3O7 (M+H), cacld. 260.13, found 260.20.
Ex mple 4. 3-(2-(2-azidoethox ethoxy)propanoic acid (87).
Figure imgf000086_0002
The azide compound 86 (2.51 g, 9.68 mmol) dissolved in 1,4-dioxane (30 mL) and was added 10 ml of HC1 (conc). The mixture was stirred for 35 min, diluted with EtOH (30 ml) and toluene (30 ml) and evacuated under vacuum. The crude mixture was purified on silica gel using a mixture of methanol (from 5% to 10%) and 1% formic acid in methylene chloride as the eluant to give title compound 87 (1.63 g, 83% yield), ESI MS m/z- C7H12N3O4 (M-H), cacld. 202.06, found 202.30.
-dioxopyrrolidin-l-yl 3-(2-(2-azidoethoxy)ethoxy)propanoate (88).
Figure imgf000086_0003
To compound 87 (1.60 g, 7.87 mmol) in 30 mL of dichloromethane was NHS (1.08 g, 9.39 mmol) and EDC (3.60 g, 18.75 mmol) with stirring. After 8 h TLC analysis revealed that the reaction was complete, the reaction mixture was evaporated and purified on silica gel using a mixture of ethyl acetate (from 5% to 10%) in methylene chloride as the eluant to give title compound 88 (1.93 g, 82% yield). ESI MS m/z+ CnH17N4O6 (M+H), cacld. 301.11, found 301.20.
Example 6. (4R)-4-(2-((lR,3R)-l-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-l- methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)- 5-(3-(3-(2-(2-azidoethoxy)ethoxy)propanamido)-4-hydroxyphenyl)-2-methylpentanoic acid
Figure imgf000087_0001
To a solution of (4R)-4-(2-((lR,3R)-l-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-l- methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)- 5-(3-amino-4-hydroxyphenyl)-2-methylpentanoic acid, 93 (Huang Y. et al, Med Chem. #44, 249th ACS National Meeting, Denver, CO, Mar. 22-26, 2015; WO2014009774) (100 mg, 0.131 mmol) in the mixture of DMA (10 ml) and NaH2PO4 buffer (5 ml, 1.0 M, pH 7.5) was added compound 88 (80.0 mg, 0.266 mmol) in four portions in 2 h. The mixture was stirred overnight, concentrated and purified on C-18 preparative HPLC (3.0 x 25 cm, 25 ml/min) eluted with from 80% water/20% methanol to 10% water/90% methanol in 45 min to afford the title compound (101.5 mg, 82% yield). LC-MS (ESI) m/z calcd. forC45H70N9O11S [M+H]+: 944.48, found: 944.70.
Example 7. (4R)-4-(2-((lR,3R)-l-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-l-methyl- piperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(3-(3- (2-( -aminoethoxy)ethoxy)propanamido)-4-hydroxyphenyl)-2-methylpentanoic acid (95).
Figure imgf000087_0002
Compound 94 (100.0 mg, 0.106 mmol) in methanol (25 ml) containing 0.1% HC1 in a hydrogenation vessel was added Pd/C (25 mg, 10% Pd, 50% wet). After air was vacuumed out in the vessel, 35 psi H2 was conducted in. The mixture was shaken for 4 h, filtered through celite, concentrated, and purified on C-18 preparative HPLC (3.0 x 25 cm, 25 ml/min) eluted with from 85% water/15% methanol to 15% water/85% methanol in 45 min to afford the title compound (77.5 mg, 79% yield). LC-MS (ESI) m/z calcd. for C45H72N7OiiS [M+H]+: 918.49, found: 918.60.
Example 8. 4-(benzyloxy)-3-methoxybenzoic acid
Figure imgf000087_0003
4-Hydroxy-3-methoxybenzoic acid (50.0 g, 297.5 mmol) in the mixture of ethanol (350 ml) and NaOH solution (2.0 ML 350 ml) was added BnBr (140.0 g. 823.5 mmol ). The mixture was stirred at 65 °C for 8 h, concentrated, co-evaporated with water (2 x 400 ml) to -400 ml, acidified with 6 M HC1 to pH 3.0, filtered the solid, crystallized with EtOH, dried over the oven at 45 °C with vacuum to afford the title compound (63.6 g, 83% yield). ESI MS m/z+ 281.2 (M + Na).
Example 9. 4-(benz loxy)-5-m ethoxy-2-nitrobenzoic acid
Figure imgf000088_0001
4-(Benzyloxy)-3-methoxybenzoic acid (63.5 g, 246.0 mmol) in the mixture of CH2CI2 (400 ml) and HO Ac (100 ml) was added HNO3 (fuming, 25.0 ml, 528.5 mmol). The mixture was stirred for 6 h, concentrated, crystallized with EtOH. dried over the oven at 40 °C with vacuum to afford the title compound (63.3 g, 85% yield). ESI MS m/z+ 326.1 (M + Na).
Example 10. (2S,4R)-methyl 4-hydroxypyrrolidine-2-carboxylate, hydrochloric salt.
Figure imgf000088_0002
Tra n s -4- h yd ro x y - L-p ro l i ne ( 15.0 g. 1 14.3 mmol ) in dry methanol (250 niL) at 0 ~ 4 °C, was added dropwise thionyl chloride (17 m L 231 mmol). The resulting mixture was stirred for at RT overnight, concentrated, crystallized with EtOH/hexane to provide the title compound (18.0 g, 87% yield), ESI MS m/z+ 168.2 (M + Na).
Exa -l-tert-butyl 2-methyl 4-hydroxypyrrolidine-l,2-dicarboxylate
Figure imgf000088_0003
To a solution of trans-4-hydroxy-L-proline methyl ester (18.0 g. 107.0 mmol ) in the mixture of MeOH ( 150 ml ) and sodium bicarbonate solution (2.0 M. 350 ml ) was added (BOC)2O (30.0 g, 137.6 mmol) in three portions in 4 h. After stirring for an additional 4 h, the reaction was concentrated to -350 ml and extracted with EtOAc (4 x 80 m L). The combined organic layers were washed with brine (100 niL). dried (MgSO4), filtered, concentrated and purified by SiO2 chromatography (1 : 1 hexanes/EtOAc) to give the title compound (22.54 g. 86% yield ). ESI MS m/z+ 268.2 (M + Na). Example 12. (S)-l -tert-butyl 2-methyl 4-oxopyrrolidine- l .2-dicarboxylate
Figure imgf000089_0001
The title compound prepared through Dess-Martin oxidation was described in: Franco Man fre et al. J. Org. Chem. 1992, 57, 2060-2065. Alternatively Swern oxidation procedure is as following: A solution of (COCl)2 (13.0 ml. 74.38 mmol ) in CH2C12 (350 ml) cooled to -78 °C was added dry DM SO (26.0 mL). The solution was stirred at -78 °C for 15 min and then (2S,4R)-l-tert-butyl 2-methyl 4-hydroxypyrrolidine-l , 2-dicarboxylate (8.0 g, 32.63 mmol ) in CH2CI2 (100 ml ). After stirred at -78 °C for 2 h. triethylamine (50 ml. 180.3 mmol) was added dropwise, and the solution was warmed to room temperature. The mixture was diluted with NaH2PO4 (400 ml. 1 .0 M ) solution and separated. The acjucous layer was extracted with CH2Cl2 (2 x 60 ml). The organic layers were combined, dried over MgSO4, filtered, concentrated and purified by SiO2 chromatography (7:3 hexanes/ EtOAc) to give the title compound (6.73 g, 85% yield). ESI MS m/z+ 266.2 (M + Na).
-l -tert-butyl 2-methyl 4-methylenepyrrolidine- 1 .2-dicarboxylate
Figure imgf000089_0002
A solution of methyltriphenylphosphonium bromide (19.62 g, 55.1 1 mmol) in THE ( 150 mL) at 0°C was potassium- t-butoxide (6.20 g, 55.30 mmol ) in anhydrous THF (80 mL). After stirred at 0°C for 2 h, the resulting yellow ylide suspension was added the solution of (S)-l-tert-butyl 2-methyl 4-oxopyrrolidine- 1.2-dicarboxylate (6.70 g, 27.55 mmol) in THF (40 mL). After stirring at RT for 1 h, the reaction mixture was concentrated, diluted with EtOAc (200 mL), washed with H2O (150 mL), brine (150 mL), dried over MgSO4, concentrated purified on SiO2 flash chromatography (9: 1 hexanes/EtOAc) to yield the title compound (5.77 g, 87% yield). EIMS m/z+ 264 (M + Na).
-methyl 4-methylenepyrrolidine-2-carboxylate
Figure imgf000089_0003
(S)-l-tert-butyl 2-methyl 4-methylenepyrrolidine-l, 2-dicarboxylate (5.70 g, 23.63 mmol ) in EtOAc (40 ml ) at 4 °C was added HC1 (10 ml. 12 M ). The mixture was stirred for 1 h. diluted with toluene (50 ml), concentrated, and crystallized with EtOH/hexane to yield the title compound as HCl salt (3.85 g, 92% yield). EIMS m/z+ 142.2 (M +H).
Example 15. (S)-methyl l-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4- methylenepyrrolidine-2-carboxylate
Figure imgf000090_0001
A catalytic amount of DMF (30 μl) was added to a solution of 4-(benzyloxy)-5- methoxy-2-nitrobenzoic acid (2.70 g, 8.91 mmol) and oxalyl chloride (2.0 mL, 22.50 mmol) in anhydrous CH2CI2 (70 mL) and the resulting mixture was stirred at room temperature (RT) for 2 h. Excess CH2CI2 and oxalyl chloride was removed with rotavap. The actyl chloride was resuspended in fresh CH2CI2 (70 mL) and was added dropwise to a solution of 4-methylene-L-proline methyl ester HCl salt (1.58 g, 8.91 mmol), Et3N (6 mL) at 0°C under argon atmosphere. The reaction mixture was allowed to warm to RT and stirring was continued for 8 h. After removal of CH2CI2 and Et3N, the residue was partitioned between H2 O and EtOAc (70/70 mL). The aqueous layer was further extracted with EtOAc (2 x 60 mL). The combined organic layers were washed with brine (40 mL). dried
(MgSO4) and concentrated. Purification of the residue with flash chromatography (silica gel, 2:8 hexanes/EtOAc) yielded (S)-methyl l-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)- 4-methylencpyrrolidine-2-carboxylate (2.88 g, 76.1% yield); EIMS ml/, 449.1 ([M]++Na).
Example 16. (S)- l-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-methylenepyrro- lidine-2-carbaldehyde
Figure imgf000090_0002
To a vigorously stirred solution of (S)-methyl l-(4-(benzyloxy)-5-methoxy-2- nitrobenzoyl)-4-methylenepyrrolidme-2-carboxylate (2.80 g, 6.57 mmol) in anhydrous CH2CI2 (60 mL) was added dropwise solution of DIBAL-H (10 mL of a 1 M solution in CH2CI2) at -78 °C under argon atmosphere. After the mixture was stirred for an additional
90 min, excess reagent was decomposed by addition of 2 ml of methanol followed by 5% HCl (10 mL). The resulting mixture was allowed to warm to 0°C. Layers were separated and the aqueous layer was further extracted with CH2CI2 (3 x 50 mL). Combined organic layers were washed with brine, dried (MgSO4) and concentrated. Purification of the resi- due with flash chromatography (silica gel, 95:5 CHCl3/MeOH ) yielded (S)-l-(4- (ben/yloxy)-5-methoxy-2-nitroben/oyl )-4-methylenepyrrolidine-2-carbaldehyde (2.19 g, 84% yield). EIMS m/z 419.1 ([M]++Na).
Example 17. (S)-8-(benzyloxy)-7-methoxy-2-methylene-2,3-dihydro-lH-benzo[e]- pyrrolo 1 ,2-a] azepin-5( 1 1 aH)-one
Figure imgf000091_0001
(5)- l -(4-(ben/yloxy)-5-methoxy-2-nitrobenzo yl )-4-methylenepyrrolidine-2- carbaldehyde (2.18 g. 5.50 mmol ) and Na2S2O4 (8.0 g, 45.97 mmol) in the mixture of THF (60 ml ) and H2O (40 ml ) were stirred at RT for 20 h. Solvents were removed under high vacuum. The residue was re-suspended in MeOH (60 m L). and HC1 (6M ) was added dropwise until pH ~ 2. The resulting mixture was stirred at RT for 1 h. The reaction was work-up by removing most of MeOH, then diluted with EtOAc (100 m L). The EtOAc solution was washed with sat. aq. NaHCO3, brine, dried (MgSO4), and concentrated. Purification of the residue with flash chromatography (silica gel, 97:3 CHCl3/MeOH ) yielded (S)-8-(benzyloxy)-7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[l ,2- a]azepin-5(l laH)-one (1.52 g, 80%). EIMS m/z 372.1 ([M]++Na).
Example 18. (S)-8-hydroxy-7-methoxy-2-methylene-2,3-dihydro-lH-benzo[e]- pyrrolo l,2-a]azepin-5(l laH)-one
Figure imgf000091_0002
(S)-8-(benzyloxy)-7-methoxy-2-methylene-2,3-dihydro-lH-benzo[e]pyrrolo[l,2- a]azepin-5(l laH)-one (1.50 g, 4.32 mmol) in 70 ml of CH2C12 at 0°C was added 25 ml of CH2SO3H. The mixture was stirred at 0°C for 10 min then RT for 2 h, diluted with CH2C12 , neutralized with cold 1.0 M NaHCO3 to pH 4, filtered. The aqueous layer was extracted with CH2C12 (3x 60 ml ). The organic layers were combined. dried over Na2SO4, filtered, evaporated and purified on SiO2 chromatography eluted with CH3OH/CH2C12 (1: 15) to afford 81 1 mg (73% yield) of the title product. EIMS m/z 281 .1 < [ M]++Na).
Example 19. (1 laS,l la'S)-8,8'-(pentane-l,5-diylbis(oxy))bis(7-methoxy-2- methylene-2,3-dihydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-5(l laH)-one) (97)
Figure imgf000092_0001
To a stirred suspended solution of Cs2CO3 (0.761 g, 2.33 mmol ) in butanone (8 ml ) were added (S)-8-hydroxy-7-methoxy-2-methylene-2,3-dihydro-lH-benzo[e]pyrrolo[ 1,2- a][l,4]diazepin-5(l laH)-one (401 mg, 1.55 mmol ) and 1.5-diiodopcntane (240 mg. 0.740 mmol). The mixture was stirred at RT overnight, concentrated, and purified on SiO;
chromatography eluted with EtOAc/CH2Cl2 (1 : 10) to afford 337 mg (78% yield) of the title product. EIMS m/z 607.2 ([M]++Na).
Example 20. (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo- 2.3.5.10.1 1.1 1 a-hexahydro- 1 H-benzo [e ] p yr rolo [ 1 ,2-a] [ 1 ,4] diazepin- 8-yl)oxy)pentyl)oxy)- -methylene-2,3-dihydro-lH-benzo[e]pyrrolo[ 1 ,2-a][1.4]dia/epin-5( 1 1 aH )-one (98)
Figure imgf000092_0002
( 1 1 aS.1 1 a'S )-8.8'-(pcntanc- 1.5-diylbis(oxy ))bis(7-mcthoxy-2-mcthylene-2.3-dihydro- 1 H- benzo[e]pyrrolo[l,2-a][l,4]diazepin-5(11aH)-one) ( 150 mg. 0.256 mmol ) in anhydrous dichloromethane (1 m L) and absolute ethanol ( 1 .5 mL) was added sodium borohydride in methoxyethyl ether (85μ1, 0.5 M, ().042mmol ) at 0 °C. The ice bath was removed after 5 minutes and the mixture was stirred at room temperature for 3 hours, then cooled to 0 °C, quenched with saturated ammonium chloride, diluted with dichloromethane, and separated. The organic layer was washed with brine, dried over anhydrous Na2S()4 and filtered through Celite and concentrated. The residue was purified by reverse phase HPLC (CI 8 column, acetonitrile/ water). The corresponding fractions were extracted with dichloromethane and concentrated to afford the title compound (98), (S)-7-methoxy-8-((5-(((S)-7- mcthoxy-2-methylene-5-oxo-2.3.5.10.1 1 .1 1 a-hexahydro- lH-benzo[e]pyrrolo[ 1,2- a] [ 1 ,4] diazepin- 8-yl)oxy)pentyl)oxy)-2-methylene-2,3 -dihydro- 1 H-benzo [e]pyrrolo [ 1.2- a][l,4]diazepin-5(l l aH)-one, (64.7 mg, 43%), MS m/z+ 609.2 (M + Na), 625.3 (M + K), 627.2 (M + Na+ H2O); the full reduced compound. ( 1 1 aS.1 1 a'S )-8.8'-(pentane- 1.5- diylbis(oxy))bis(7-methoxy-2-methylene-2,3, 11 , 1 1 a-tetrahydro- 1 H-ben/o[eJpyrrolo[ 1.2- a][l,4]diazepin-5(10H)-one), (16.5 mg, 11.1 %), MS m/z+ 61 1 .2 (M + Na), 627.2 (M + K ). 629.2 (M + Na+ H2O); and the unreacted starting material (10.2 mg, 6.8%), MS m/z+ 607.2 (M + Na), 625.2 ( M + Na+ H2O). Example 21. ( S )-8-(( 5-( ( ( S )- 10 -( 3-(2-( 2-azidoethoxy )ethoxy )propanoyl )-7-methoxy- 2-methylene-5-oxo-2,3 ,5 ,10,11,11 a-hexahydro- 1 H-benzo[e]pyrrolo[ 1 ,2-a] [ 1 ,4]diazepin-8- yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-2,3-dihydro- lH-benzo[e]pyrrolo[ 1.2- a][l,4]diazepin-5(l laH)-one, (99)
Figure imgf000093_0001
To the mixture of compound 98 (60.0 mg, 0.102 mmol) and compound 88 (40.5 mg, 0.134 mmol) in dichloromethane (5 ml) was added EDC (100.5 mg, 0.520 mmol). The mixture was stirred at RT overnight, concentrated, and purified on SiO2 chromatography cluted with EtOAc/CH2Cl2 (1:6) to afford 63.1 mg (81% yield) of the title product 99. ESI MS m/z+ Q0H50N7O9 (M+H), cacld. 772.36, found 772.30.
Example 22. (S)-8-((5-(((S)-10-(3-(2-(2-aminoethoxy)ethoxy)propanoyl)-7-methoxy- 2-methylene-5-oxo-2,3 ,5.10.1 1.1 1 a-hexahydro- 1 H-benzo[e]pyrrolo[ 1 ,2-a] [ 1 ,4]diazepin-8- yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-2,3-dihydro- 1 H-benzo[e]pyrrolo[ 1 ,2- a][l,4]diazepin-5(l laH)-one, (100).
Figure imgf000093_0002
To the compound 99 (60 mg, 0.078 mmol) in the mixture of THF (5 ml) and
NaH2PO4 buffer (50 mM, pH 5.0, 1 ml) was added PPh3 (70 mg, 0.267 mmol). The mixture was stirred at RT overnight, concentrated, and purified on C-18 chromatography eluted with water/CH3CN (from 90% water to 35% water in 35 min ) to afford 45.1 mg (79% yield) of the title product 100 after dried with high vacuum pump. ESI MS m/z+ C4oH52N5O9 (M+H), cacld. 746.37, found 746.50.
-tert-butyl 2-(hydroxymethyl)pyrrolidine-l-carboxylate
Figure imgf000093_0003
Boc-L-proline (10.0 g, 46.4 mmol) dissolved in 50 mL THF was cooled to 0 °C, to which BH3 in THF (1.0 M, 46.4 mL) was added carefully. The mixture was stirred at 0 °C for 1.5 h then poured onto ice water and extracted with ethyl acetate. The organic layer was washed with brine (50 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give the title compound (8.50 g, 91% yield) as a white solid. H NMR
(500 MHz, CDC13) δ 3.94 (dd, J = 4.9, 2.7 Hz, 2H), 3.60 (ddd, J = 18.7, 11.9, 9.3 Hz, 2H), 3.49 - 3.37 (m, 1H), 3.34 - 3.23 (m, 1H), 2.06 - 1.91 (m, 1H), 1.89 - 1.69 (m, 2H), 1.65 - 1.51 (m, 1H), 1.49 - 1.40 (m, 9H).
-tert-butyl 2-formylpyrrolidine-l-carboxylate
Figure imgf000094_0001
To a solution of (S)-tert-butyl 2-(hydroxymethyl)pyrrolidine-l-carboxylate (13.0 g, 64.6 mmol) in dimethyl sulfoxide (90 mL) was added triethylamine (40 mL) and the stirring was continued for 15 min. The mixture was cooled over ice bath and sulfur triox- ide-pyridine complex (35.98 g, 226 mmol) was added in portions over a 40 min period. The reaction was warmed to r.t. and stirred for 2.5 h. After addition of ice (250 g), the mixture was extracted with dichloromethane (150 mL x 3). The organic phase was washed with 50% citric acid solution (150 mL), water (150 mL), saturated sodium bicarbonate solution (150 mL), and brine (150 mL), dried over anhydrous Na2SO4. Removal of solvent in vacuo yielded the title compound (10.4 g, 81% yield) as dense oil which was used without further purification. 1H NMR (500 MHz, CDCI3) δ 9.45 (s, 1H), 4.04 (s, 1H), 3.53 (dd, J = 14.4, 8.0 Hz, 2H), 2.00 - 1.82 (m, 4H), 1.44 (d, J = 22.6 Hz, 9H).
-4-methyl-5-phenyl-3-propionyloxazolidin-2-one
Figure imgf000094_0002
n-Butyllithium in hexane (21.6 mL, 2.2 M, 47.43 mmol) was added dropwise at -78 °C to a stirred solution of 4-methyl-5-phenyloxazolidin-2-one (8.0 g, 45.17 mmol) in THF (100 mL) under N2. The solution was maintained at -78 °C for 1 h then propionyl chloride (4.4 mL, 50.59 mmol) was added slowly. The reaction mixture was warmed to -50 °C, stirred for 2 h then quenched by addition of a saturated solution of ammonium chloride (100 mL). The organic solvent was removed in vacuo and the resultant solution was extracted with ethyl acetate (3 x 100 mL). The organic layer was washed with saturated sodium bicarbonate solution (100 mL) and brine (100 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (20% ethyl acetate/hexanes) to afford the title compound as dense oil (10.5 g, 98% yield). 1H NMR (500 MHz, CDC13) δ 7.45 - 7.34 (m, 3H), 7.30 (d, 7 = 7.0 Hz, 2H), 5.67 (d, 7 = 7.3 Hz, 1H), 4.82 - 4.70 (m, 1H), 2.97 (dd, 7 = 19.0, 7.4 Hz, 2H), 1.19 (t, 7 = 7.4 Hz, 3H), 0.90 (d, 7 = 6.6 Hz, 3H).
Example 26. (S)-tert-butyl 2-((lR,2R)-l-hydroxy-2-methyl-3-((4R,5S)-4-methyl-2- oxo- -phenyloxazolidin-3-yl)-3-oxopropyl)pyrrolidine-l-carboxylate.
Figure imgf000095_0001
To a solution of (4R,5S)-4-methyl-5-phenyl-3-propionyloxazolidin-2-one (9.40 g, 40.4 mmol) in dichloromethane (60 mL) was added Et3N (6.45 mL, 46.64 mmol) at 0 °C, followed by 1M dibutylboron triflate in dichloromethane (42 mL, 42 mmol). The mixture was stirred at 0 °C for 45 min, cooled to -70 °C, (S)-tert-butyl 2-formylpyrrolidine-l- carboxylate (4.58 g, 22.97 mmol) in dichloromethane (40 mL) was then added slowly over a 30 min period. The reaction was stirred at -70 °C for 2 h, 0 °C 1 h, and r.t. 15 min, and then quenched with phosphate buffer solution (pH 7, 38 mL). After the addition of MeOH- 30% H2O2 (2: 1, 100 mL) at below 10 °C and stirring for 20 min, water (100 mL) was added and the mixture was concentrated in vacuo. More water (200 mL) was added to the residue and the mixture was extracted with ethyl acetate (3 x 100 mL). The organic layer was washed with IN KHSO4 (100 mL), sodium bicarbonate solution (100 mL) and brine (100 mL), dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by flash column chromatography (10% - 50% ethyl acetate/hexanes) to afford the title compound as a white solid (7.10g, 7 ] % yield). 1H NMR (500 MHz, CDC13) δ 7.39 (dt, 7 = 23.4, 7.1 Hz, 3H), 7.30 (d, 7 = 7.5 Hz, 2H), 5.67 (d, 7 = 7.1 Hz, 1H), 4.84 - 4.67 (m, 1H), 4.08 - 3.93 (m, 3H), 3.92 - 3.84 (m, 1H), 3.50 (d, 7 = 9.0 Hz, 1H), 3.24 (d, 7 = 6.7 Hz, 1H), 2.15 (s, 1H), 1.89 (dd, 7 = 22.4, 14.8 Hz, 3H), 1.48 (d, 7 = 21.5 Hz, 9H), 1.33 (d, 7 = 6.9 Hz, 3H), 0.88 (d, 7 = 6.4 Hz, 3H).
Example 27. (S)-tert-butyl 2-((lR,2R)-l-methoxy-2-methyl-3-((4R,5S)-4-methyl-2- oxo-5-phenyloxazolidin-3-yl)-3-oxopropyl)pyrrolidine-l-carboxylate
Figure imgf000095_0002
To a mixture of (S)-tert-butyl 2-((lR,2R)-l-hydroxy-2-methyl-3-((4R,5S)-4-methyl- 2-oxo-5-phenyloxazolidin-3-yl)-3-oxopropyl)pyrrolidine-l-carboxylate. (5.1 g 11.9 mmol) and molecular sieves (4 A, 5 g) was added anhydrous dichloroethane (30 mL) under Nj. The mixture was stirred at room temperature for 20 min and cooled to 0 °C. Proton sponge (6.62 g, 30.9 mmol) was added, followed by trimethyloxonium tetrafluoroborate (4.40 g, 29.7 mmol). Stirring was continued for 2 h at 0 °C and 48 h at r.t. The reaction mixture was filtrated and the filtrate was concentrated and purified by column chromatography (20-70% ethyl acetate/hexanes) to afford the title compound as a colorless solid ( 1.80 g,
35% yield). 1H NMR (500 MHz, CDC13) δ 7.46 - 7.27 (m, 5H), 5.65 (s, 1H), 4.69 (s, 1H), 3.92 (s, 1H), 3.83 (s, 1H), 3.48 (s, 3H), 3.17 (s, 2H), 2.02 - 1.68 (m, 5H), 1.48 (d, J = 22.3 Hz, 9H), 1.32 (t, J = 6.0 Hz, 3H), 0.91 - 0.84 (m, 3H).
Example 28. (2R,3R)-3-((S)- l-(tert-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2- methylpropanoic acid
Figure imgf000096_0001
To a solution of (S)-tert-butyl 2-((lR,2R)-l-methoxy-2-methyl-3-((4R,5S)-4-methyl- 2-oxo-5-phenyloxazolidin-3-yl)-3-oxopropyl)pyrrolidine-l-carboxylate (1.80 g, 4.03 mmol) in THF' (30 mL) and ¾() (7.5 mL), 30% H2O 2 (1 .44 mL, 14.4 mmol) was added over a 5 min period at 0 °C , followed by a solution of LiOH (0.27 g, 6.45 mmol) in water (5 mL). After stirring at 0 °C for 3 h, 1 N sodium sulfite (1.5.7 mL) was added and the mixture was allowed to warm to r.t. and stirred overnight. THF was removed in vacuo and the aqueous phase was wash with dichloromethane (3 x 50 mL) to remove the
oxazoiidinone auxiliary. The aqueous phase was acidified to pH 3 with 1N HCl and extracted with ethyl acetate (3 x 50 mL). The organic layer was washed with brine (50 mL), dried over Na2SO4, filtered and concentrated in vacuo to afford the title compound as colorless oil (1.15 g. 98% yield). 1H NMR (500 MHz, CDC13) δ 3.99 - 3.74 (m, 2H), 3.44 (d, 7 = 2.6 Hz, 3H), 3.23 (s, 1H), 2.60 - 2.45 (m, 1H), 1.92 (tt, J = 56.0, 31.5 Hz, 3H), 1.79 - 1.69 (m, 1H), 1.58 - 1.39 (m, 9H), 1.30 - 1.24 (m, 3H).
Example 29. (4S,5S)-ethyl 4-((tert-butoxycarbonyl)amino)-5-methyl-3- oxoheptanoate
Figure imgf000096_0002
To an ice-cooled solution of (2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methyl- pentanoic acid (4.55 g, 19.67 mmol) in THF (20 mL) was added Ι,Γ-carbonyldiimidazole (3.51 g, 21.63 mmol). After evolution of gas ceased, the resultant mixture was stirred at r.t. for 3.5 h. A solution of freshly prepared isopropylmagnesium bromide in THF (123 mmol, 30mL) was added dropwise to a pre-cooled (0 °C) solution of ethyl hydrogen malonate (6.50 g, 49.2 mmol) at such a rate to keep the internal temperature below 5 °C. The mixture was stirred at r.t. for 1.5 h. This solution of the magnesium enolate was then cooled over an ice-water bath, followed by the gradual addition of the imidazolide solution over a 1 h period via a double-ended needle at 0 °C. The resultant mixture was stirred at 0 °C for 30 min then r.t. 64 h. The reaction mixture was quenched by addition of 10% aqueous citric acid (5 mL), and acidified to pH 3 with an additional 10% aqueous citric acid (110 mL). The mixture was extracted with ethyl acetate (150 mL x 3). The organic extracts were washed with water (50 mL), saturated aqueous sodium hydrogen carbonate (50 mL), and saturated aqueous sodium chloride (50 mL), dried over Na2SO4, and concen- trated in vacuo. The residue was purified by column chromatography on silica gel using ethyl acetate/hexane (1:4) as an eluent to give title compound (5.50 g, 93% yield). 1H NMR (500 MHz, CDC13) δ 5.04 (d, J = 7.8 Hz, 1H), 4.20 (p, J = 7.0 Hz, 3H), 3.52 (t, J = 10.7 Hz, 2H), 1.96 (d, J = 3.7 Hz, 1H), 1.69 (s, 2H), 1.44 (s, 9H), 1.28 (dd, J = 7.1, 2.9 Hz, 3H), 0.98 (t, J = 6.9 Hz, 3H), 0.92 - 0.86 (m, 3H).
Example 30. (3R,4S,5S)-ethyl 4-((tert-butoxycarbonyl)amino)-3-hydroxy-5-methyl- he tanoate
Figure imgf000097_0001
To a solution of (4S,5S)-ethyl 4-((tert-butoxycarbonyl)amino)-5-methyl-3- oxoheptanoate (5.90 g, 19.83 mmol) in ethanol (6 mL) at -60 °C was added sodium borohydride (3.77 g, 99.2 mmol) in one portion. The reaction mixture was stirred for 5.5 h below -55 °C then quenched with 10% aqueous citric acid (100 mL). The resultant solution was acidified to pH 2 with an additional 10% aqueous citric acid, followed by extraction with ethyl acetate (100 mL x 3). The organic extracts were washed with saturated aqueous sodium chloride (100 mL), dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (10-50% ethyl acetate/hexane) to give pure diastereomer (3R,4S,5S)-ethyl 4-((tert-butoxycarbonyl)amino)-3-hydroxy-5-methyl- heptanoate (2.20 g, 37% yield) and a mixture of (3R,4S,5S)-ethyl 4-((tert-butoxycarbonyl) amino)-3-hydroxy-5-methyl-heptanoate and (3S,4S,5S)-ethyl 4-((tert-butoxycarbonyl)- amino)-3-hydroxy-5-methylheptanoate (2.0g, 34% yield, about 9: 1 ratio). 1H NMR (500 MHz, CDC13) δ 4.41 (d, J = 9.3 Hz, 1H), 4.17 (tt, J = 7.1, 3.6 Hz, 2H), 4.00 (t, J = 6.9 Hz, 1H), 3.55 (dd, J= 11.7, 9.3 Hz, 1H), 2.56 - 2.51 (m, 2H), 2.44 (dd, J= 16.4, 9.0 Hz, 1H), 1.79 (d, J= 3.8 Hz, 1H), 1.60 - 1.53 (m, 1H), 1.43 (s, 9H), 1.27 (dd, J = 9.3, 5.0 Hz, 3H), 1.03 - 0.91 (m, 7H).
Example 31. (3R,4S,5S)-4-((tert-butoxycarbonyl)amino)-3-hydroxy-5-methyl- heptanoic acid
Figure imgf000098_0001
To a solution of compound (3R,4S,5S)-ethyl 4-((tert-butoxycarbonyl)amino)-3- hydroxy-5-methyl-heptanoate (2.20 g, 7.20 mmol) in ethanol (22 mL) was added 1 N aqueous sodium hydroxide (7.57 mL, 7.57 mmol). The mixture was stirred at 0 °C for 30 min then r.t. 2 h. The resultant solution was acidified to pH 4 by addition of 1 N aqueous hydrochloric acid, which was then extracted with ethyl acetate (50 mL x 3). The organic extracts were washed with 1 N aqueous potassium hydrogen sulfate (50 mL), and saturated aqueous sodium chloride (50 mL), dried over Na2SO4, and concentrated in vacuo to give the title compound (1.90 g, 95% yield). 1H NMR (500 MHz, CDC13) δ 4.50 (d, J = 8.7 Hz, 1H), 4.07 (d, J = 5.5 Hz, 1H), 3.59 (d, J = 8.3 Hz, 1H), 2.56 - 2.45 (m, 2H), 1.76 - 1.65 (m, 1H), 1.56 (d, J = 7.1 Hz, 1H), 1.45 (s, 9H), 1.26 (t, J = 7.1 Hz, 3H), 0.93 (dd, J = 14.4, 7.1 Hz, 6H).
Example 32. (3R,4S,5S)-4-((tert-butoxycarbonyl)(methyl)amino)-3-methoxy-5- meth lheptanoic acid
Figure imgf000098_0002
To a solution of (3R,4S,5S)-4-((tert-butoxycarbonyl)amino)-3-hydroxy-5-methyl- heptanoic acid (1.90 g, 6.9 mmol) in THF (40 mL) was added sodium hydride (60% oil suspension, 1.93 g, 48.3 mmol) at 0 °C. After stirring for lh, methyl iodide (6.6 mL, 103.5 mmol) was added. The stirring was continued at 0 °C for 40 h before saturated aqueous sodium hydrogen carbonate (50 mL) was added, followed by water (100 mL). The mixture was washed with diethyl ether (50 mL x 2) and the aqueous layer was acidified to pH 3 by 1 N aqueous potassium hydrogen sulfate, then extracted with ethyl acetate (50 mL x 3). The combined organic extracts were washed with 5% aqueous sodium thiosulfate (50 mL) and saturated aqueous sodium chloride (50 mL), dried over Na2SO4, and concentrated in vacuo to give the title compound (1.00 g, 48% yield). 1H NMR (500 MHz, CDC13) δ 3.95 (d, J = 75.4 Hz, 2H), 3.42 (d, J = 4.4 Hz, 3H), 2.71 (s, 3H), 2.62 (s, 1H), 2.56 - 2.47 (m, 2H), 1.79 (s, 1H), 1.47 (s, 1H), 1.45 (d, 7 = 3.3 Hz, 9H), 1.13 - 1.05 (m, 1H), 0.96 (d, J = 6.7 Hz, 3H), 0.89 (td, J = 7.2, 2.5 Hz, 3H).
Example 33. General procedure for the removal of the Boc function with
trifluoroacetic acid.
To a solution of the N-Boc amino acid (1.0 mmol) in methylene chloride (2.5 mL) was added trifluoroacetic acid (1.0 mL). After being stirred at room temperature for 1-3 h, the reaction mixture was concentrated in vacuo. Co-evaporation with toluene gave the deprotected product, which was used without any further purification.
Example 34. (S)-tert-butyl 2-((lR,2R)-l-methoxy-3-(((S)-l-methoxy-l-oxo-3- phenylpropan-2-yl)amino)-2-methyl-3-oxopropyl)pyrrolidine-l-carboxylate
Figure imgf000099_0001
To a solution of (2R,3R)-3-((S)-l-(tert-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2- methylpropanoic acid (100 mg, 0.347 mmol) and L-Phenylalanine methyl ester hydrochloride (107.8 mg, 0.500 mmol) in DMF (5 mL) at 0 °C was added diethyl cyanophosphonate (75.6 μL, 0.451 mmol), followed by Et3N (131 μL, 0.94 mmol). The reaction mixture was stirred at 0 °C for 2 h, then warmed to r.t. and stirred overnight. The reaction mixture was then diluted with ethyl acetate (80 mL), washed with 1 N aqueous potassium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium hydrogen carbonate (40 mL), and saturated aqueous sodium chloride (40 mL), dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (15-75% ethyl ace- tate/hexanes) to afford the title compound (130 mg, 83% yield) as a white solid. 1H NMR (500 MHz, CDC13) δ 7.28 (dd, J = 7.9, 6.5 Hz, 2H), 7.23 (t, J = 7.3 Hz, 1H), 7.16 (s, 2H), 4.81 (s, 1H), 3.98 - 3.56 (m, 5H), 3.50 (s, 1H), 3.37 (d, J = 2.9 Hz, 3H), 3.17 (dd, J = 13.9, 5.4 Hz, 2H), 3.04 (dd, 7 = 14.0, 7.7 Hz, 1H), 2.34 (s, 1H), 1.81 - 1.69 (m, 2H), 1.65 (s, 3H), 1.51 - 1.40 (m, 9H), 1.16 (d, J = 7.0 Hz, 3H).
Example 35. (S)-methyl 2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((tert-butoxycarbonyl)- (methyl)amino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2- methylpropanamido)-3-phenylpropanoate
Figure imgf000100_0002
To a solution of the deprotected product from (S)-tert-butyl 2-((lR,2R)-l-methoxy-3- (((S)-l-methoxy-l-oxo-3-phenylpropan-2-yl)amino)-2-methyl-3-oxopropyl)pyrrolidine-l- carboxylate (0.29 mmol) and (3R,4S,5S)-4-((tert-butoxycarbonyl)(methyl)amino)-3- methoxy-5-methylheptanoic acid (96.6 mg, 0.318 mmol) in DMF (5 mL) at 0 °C was added diethyl cyanophosphonate (58 μΐ,. 0.347 mmol), followed by Et3N (109 μL,. 0.78 mmol). The reaction mixture was stirred at 0 °C for 2 h, then warmed to r.t. and stirred overnight. The reaction mixture was diluted with ethyl acetate (80 mL), washed with 1 N aqueous potassium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium hydrogen carbonate (40 mL), and saturated aqueous sodium chloride (40 mL), dried over
Na.2SO4 and concentrated in vacuo. The residue was purified by column chromatography (15-75% ethyl acetate/hexanes) to afford the title compound (150 mg, 81% yield) as a white solid. LC-MS (ESI) m/z calcd. for C34H55N3O8 [M+H]+: 634.40, found: 634.40.
Example 36. (S)-methyl 2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-2-((tert- butoxycarbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)- pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate
Figure imgf000100_0003
To a solution of the deprotected product from (S)-methyl 2-((2R,3R)-3-((S)-l- ((3R,4S,5S)-4-((tert-butoxycarbonyl)-(methyl)amino)-3-methoxy-5-methylheptanoyl)- pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate (0.118 mmol) and
Boc-Val-OH (51.8 mg, 0.236 mmol) in DCM (5 mL) at 0 °C was added
bromotris(dimethylamino)- phosphonium hexafluorophosphate (BroP, 70.1 mg, 0.184 mmol), followed by diisopropylethylamine (70
Figure imgf000100_0001
0.425 mmol). The mixture was shielded from light and stirred at 0 °C for 30 min then at r.t. for 2 days. The reaction mixture was diluted with ethyl acetate (80 mL), washed with 1 N aqueous potassium hydrogen sulfate
(40 mL), water (40 mL), saturated aqueous sodium hydrogen carbonate (40 mL), and saturated aqueous sodium chloride (40 mL), dried over Na2SO4 and concentrated in vacuo.
The residue was purified by column chromatography (20-100% ethyl acetate/hexanes) to afford the title compound (67 mg, 77% yield) as a white solid. LC-MS (ESI) m/z calcd. for C39H64N4O9 [M+H]+: 733.47, found: 733.46. Example 37. Preparation of compound Boc-N-Me-Val-OH
Figure imgf000101_0001
To a solution of Boc-L-Val-OH (2.00 g, 9.2 mmol) and methyl iodide (5.74 mL, 92 mmol) in anhydrous THF (40 mL) was added sodium hydride (3.68 g, 92 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 1.5 h, then warmed to r.t. and stirred for 24 h.
The reaction was quenched by ice water (50 mL). After addition of water (100 mL), the reaction mixture was washed with ethyl acetate (50 mL x 3) and the aqueous solution was acidified to pH 3 then extracted with ethyl acetate (50 mL x 3). The combined organic phase was dried over Na2SO4 and concentrated to afford Boc-N-Me-Val-OH (2.00 g, 94% yield) as a white solid. 1H NMR (500 MHz, CDC13) δ 4.10 (d, J = 10.0 Hz, 1H), 2.87 (s,
3H), 2.37 - 2.13 (m, 1H), 1.44 (d, J = 26.7 Hz, 9H), 1.02 (d, J = 6.5 Hz, 3H), 0.90 (t, J = 8.6 Hz, 3H).
Example 38. (S)-methyl 2-((2R,3R)-3-((S)-l-((6S,9S,12S,13R)-12-((S)-sec-butyl)- 6,9-diisopropyl-13-methoxy-2,2,5,l l-tetramethyl-4,7,10-trioxo-3-oxa-5,8,l l-triazapenta- decan-15-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate
Figure imgf000101_0002
To a solution of the deprotected product from (S)-methyl 2-((2R,3R)-3-((S)-l-
((3R,4S,5S)-4-((S)-2-((tert-butoxycarbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-
5-methylheptanoyl)-pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3- phenylpropanoate (0.091 mmol) and Boc-N-Me-Val-OH (127 mg, 0.548 mmol) in DMF
(5 mL) at 0 °C was added diethyl cyanophosphonate (18.2 μL, 0.114 mmol), followed by 4-methylmorpholine (59 μL, 0.548 mmol). The reaction mixture was stirred at 0 °C for 2 h, then warmed to r.t. and stirred overnight. The reaction mixture was diluted with ethyl acetate (80 mL), washed with 1 N aqueous potassium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium hydrogen carbonate (40 mL), and saturated aqueous sodium chloride (40 mL), dried over sodium sulfate, and concentrated in vacuo. The residue was purified by column chromatography (20-100% ethyl acetate/hexanes) to afford the title compound (30 mg, 39% yield) as a white solid. LC-MS (ESI) m/z calcd. for C5H75N5O10 [M+H]+: 846.55, found: 846.56. Example 39. (S)-2-((2R,3R)-3-((S)-l-((6S,9S,12S,13R)-12-((S)-sec-butyl)-6,9- diisopropyl- 13-methoxy-2,2,5, 1 l-tetramethyl-4,7, 10-trioxo-3-oxa-5,8,l 1-triazapenta- deca -15-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanarnido)-3-phenylpropanoic acid
Figure imgf000102_0001
(S)-methyl 2-((2R,3R)-3-((S)-l-((6S,9S,12S,13R)-12-((S)-sec-butyl)-6,9-diisopropyl- 13-methoxy-2,2,5,l l-tetramethyl-4,7, 10-trioxo-3-oxa-5, 8,11-triazapenta-decan- 15- oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate (30 mg, 0.035 mmol) in THF (1.0 ml) was added LiOH in water (1.0M, 0.8 ml). The mixture was stirred at RT for 35 min, neutralized with 0.5M H3PO4 to pH 6, concentrated and purified on SiO2 column eluted with CH3OH/CH2Cl2/HOAc (1: 10:0.01) to afford the title compound (25.0 mg, 85% yield). LC-MS (ESI) m/z calcd. for C44H74N5O10 [M+H]+: 832.54, found:
832.60.
Example 40. (S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3- methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)- p henylpropanoic acid (101)
Figure imgf000102_0002
(S)-2-((2R,3R)-3-((S)-l-((6S,9S,12S,13R)-12-((S)-sec-butyl)-6,9-diisopropyl-13- methoxy-2,2,5,11 -tetramethyl-4,7,10-trioxo-3-oxa-5, 8,11-triazapenta-decan- 15- oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (25 mg, 0.030 mmol) in the mixture of HC1 (cone. 0.3 ml) and 1,4-dioxane (0.9 ml) was stirred at
RT for 35 min. The mixture was diluted with EtOH (1.0 ml) and toluene (1.0 ml), concentrated and re-evaporated with EtOH/toluene (2: 1) to afford the title compound as a white solid (22 mg, -100% yield) for the next step without further purification. LC-MS (ESI) m/z calcd. for C39H66N5O8 [M+H]+: 732.48, found: 732.60.
Example 41. (2S)-2-((2R,3R)-3-((2S)-l-((l lS,14S,17S)-l-azido-17-((R)-sec-butyl)-
11 , 14-diisopropyl- 18-methoxy- 10,16-dimethyl-9, 12,15 -trioxo-3 ,6-dioxa- 10,13,16-triazai- cosa -20-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid
Figure imgf000102_0003
To the crude compound 101 (22 mg, 0.030 mmol) in the mixture of DMA (0.8 ml) and NaH2P04 buffer (0.7 ml, 1.0 M, pH 7.5) was added compound 88 (18.0 mg, 0.060 mmol) in four portions in 2 h. The mixture was stirred overnight, concentrated and purified on Si02 column eluted with CH3OH/CH2Cl2/HOAc (1:8:0.01) to afford the title compound (22.5 mg, 82% yield). LC-MS (ESI) m/z calcd. for C46H77N8O11 [M+H]+: 917.56, found: 917.60.
Example 42. (2S)-2-((2R,3R)-3-((2S)-l-((l lS,14S,17S)-l-amino-17-((R)-sec-butyl)- 11 , 14-diisopropyl- 18-methoxy- 10, 16-dimethyl-9, 12, 15-trioxo-3 ,6-dioxa- 10, 13,16- triazaicosan-20-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (103).
Figure imgf000103_0001
Compound 102 (22.0 mg, 0.024 mmol) in methanol (5 ml) in a hydrogenation vessel was added Pd/C (5 mg, 10% Pd, 50% wet). After air was vacuumed out in the vessel, 25 psi H2 was conducted in. The mixture was shaken for 4 h, filtered through celite, concentrated to afford the crude title product (~20 mg, ~92% yield). for the next step without further purification. ESI MS m/z+ C46H79N60ii (M+H), cacld. 891.57, found 891.60.
Example 43. 2,3-dibromosuccinic anhydride (70)
Figure imgf000103_0002
2,3-dibromosuccinic acid (10.00 g, 36.51 mmol) in dry CH2C12 (100 ml) at 0°C was added phosphorus pentoxide (12.21 g, 85.84 mmol). The mixture was stirred at 0°C for 2 h and then RT for 5 h, filtered through short Si02 column, and rinsed the column with EtOAc/CH2Cl2 (1:6). The filtered solutions were combined, evaporated and solidified with EtOAc/Hexane to afford the title compound (6.63 g, 71% yield). ESI MS m/z+ C4H2Br203 (M+H), cacld. 256.85, found 256.70.
Example 44. 2,3-dibromo-4-((2-(2-(3-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2- methylene-5-oxo-2,3,5,l la-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-8- yl)oxy)pentyl)oxy)-2-methylene-5-oxo-2,3 ,11,11 a-tetrahydro- lH-benzo[e]pyrrolo[ 1 ,2- a][l,4]diazepin-10(5H)-yl)-3-oxopropoxy)ethoxy)ethyl)amino)-4-oxobutanoic acid (124)
Figure imgf000104_0001
To the compound 100 (40.0 mg, 0.068 mmol) in the mixture of DCM (4 ml) and DIPEA (12 uL, 0.069 mmol) was added 2,3-dibromosuccinic anhydride (38.0 mg, 0.148 mmol) at 0°C. The mixture was stirred at 0°C for 2 h and then RT 5h. The mixture was concentrated and purified on SiO2 column eluted with CH3OH/CH2Cl2/HOAc (1:6:0.01) to afford the title compound (56.5 mg, 83% yield). LC-MS (ESI) m/z calcd. for
C44H53Br2N5O12 [M+H]+: 1002.21, found: 1002.40, 1004.40 (M+2+H).
Example 45. 2,5-dioxopyrrolidin-l-yl 2,3-dibromo-4-((2-(2-(3-((S)-7-methoxy-8-((5- (((S)-7-methoxy-2-methylene-5-oxo-2,3,5,l la-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]- diazepin-8-yl)oxy)pentyl)oxy)-2-methylene-5-oxo-2,3,l l,l la-tetrahydro-lH-benzo[e]pyr- rolo[l,2-a][l,4]diazepin-10(5H)-yl)-3-oxopropoxy)ethoxy)ethyl)amino)-4-oxobutanoate (125
Figure imgf000104_0002
Compound 125 (55.0 mg, 0.054 mmol) in CH2C12 (3 ml) was added NHS (10.0 mg, 0.086 mmol) and EDC (30.5 mg, 0.158 mmol). The mixture was stirred at RT overnight, concentrated and purified on SiO2 column eluted with EtOAc/CH2Cl2 (1:5) to afford the title compound (50.5 mg, 85% yield). LC-MS (ESI) m/z calcd. for
Figure imgf000104_0003
[M+H]+: 1099.22, found: 1099.40, 1101.40 (M+2+H), 1119.50 (M+2+H+H2O).
Example 46. (2S)-2-((2R,3R)-3-((2S)-l-((13S,26S,29S,32S)-12,13-dibromo-32-((R)- sec-butyl)-26,29-diisopropyl-33-methoxy-l-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2- methylene-5-oxo-2,3,5,l la-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-8- yl)oxy)pentyl)oxy)-2-methylene-5-oxo-2,3 ,11,11 a-tetrahydro- lH-benzo[e]pyrrolo[ 1 ,2- a][l,4]diazepin-10(5H)-yl)-25,31-dimethyl-l,l l,14,24,27,30-hexaoxo-4,7,18,21-tetraoxa- 10,15,25, 28, 31 -pentaazapentatriacontan-35-oyl)pyrrolidin-2-yl)-3 -methoxy-2- methylpropanamido)-3-phenylpropanoic acid (126)
Figure imgf000105_0001
Compound 103 (~20 mg, 0.022 mmol) in the mixture of DMA (1 ml) and NaH2PO4 buffer (0.6 ml, 0.15 M, pH 7.5) was added compound 125 (30.0 mg, 0.027 mmol). The mixture was stirred for 7 h, concentrated, and purified on C-18 chromatography ( Φ 2.0 cm x 25 cm ) eluted with water/CH 3CN (from 90% water to 15% water in 50 min at 10 ml/min) to afford the title product 126 (26.1 mg, 63% yield) after dried with high vacuum pump. ESI MS m/z+ C9oH13oBr2N11O22 (M+H), cacld. 1874.77, found 1874.50.
Example 47. Conjugated compound 126 to an antibody for 127.
Figure imgf000105_0002
To a mixture of 2.0 mL of 10 mg/ml Herceptin in pH 6.0-8.0, were added of 0.70 -
2.0 mL PBS buffer of 100 mM NaH2PO4, pH 6.5-7.5 buffers, TCEP (28 μ L, 20 mM in water) and the compound 126 (14 μL, 20 mM in DMA). The mixture was incubated at RT for 4-16 h, then DHAA (135 μL, 50 mM) was added in. After continuous incubation at RT overnight, the mixture was purified on G-25 column eluted with 100 mM NaH2PO4, 50 mM NaCl pH 6.0-7.5 buffer to afford 16.5-17.7 mg of the conjugate compound 127
(82%~88% yield) in 13.1-15.0 ml buffer. The drug/antibody ratio (DAR) (the combination of PBD dimer and MMAF per antibody) was 3.85, which was determined via UPLC- Qtof mass spectrum. It was 96-99% monomer analyzed by SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID x 30 cm, 0.5 ml/min, 100 min) and a single band measured by SDS-PAGE gel.
Example 48. (4R)-4-(2-((lR,3R)-l-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-l- methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)- 5-(3-(12,13-dibromo-24-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo- 2,3,5,1 la-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-8-yl)oxy)pentyl)oxy)-2- methylene-5-oxo-2,3, 11 , 1 la-tetrahydro- lH-benzo[e]pyrrolo[ 1 ,2-a] [ 1 ,4]diazepin- 10(5H)- yl)-l l,14,24-trioxo-4,7,18,21-tetraoxa-10,15-diazatetracosanamido)-4-hydroxyphenyl)-2- methylpentanoic acid (128)
Figure imgf000106_0001
Compound 95 (20 mg, 0.021 mmol) in the mixture of DMA (1 ml) and NaH2PO4 buffer (0.6 ml, 0.15 M, pH 7.5) was added compound 125 (30.0 mg, 0.027 mmol). The mixture was stirred for 8 h, concentrated, and purified on C- 18 chromatography (Φ 2.0 cm x 25 cm ) eluted with water/CH 3CN (from 90% water to 20% water in 50 min at 10 ml/min ) to afford the title product 128 (26.6 mg, 64% yield) after dried with high vacuum pump. ESI MS m/z+ C89H123Br2N12O22S (M+H), cacld. 1901.69, found 1901.90.
Example 49. Conjugated compound 128 to an antibody for 129.
Figure imgf000106_0002
To a mixture of 2.0 mL of 10 mg/ml Herceptin in pH 6.0-8.0, were added of 0.70 ~ 2.0 mL PBS buffer of 100 mM NaH2PO4, pH 6.5-7.5 buffers, TCEP (28 μΐ,, 20 mM in water) and the compound 128 (14 μL, 20 mM in DMA). The mixture was incubated at RT for 4-16 h, then DHAA (135 μL, 50 mM) was added in. After continuous incubation at RT overnight, the mixture was purified on G-25 column eluted with 100 mM NaH2PO4, 50 mM NaCl pH 6.0-7.5 buffer to afford 16.4-17.6 mg of the conjugate compound 129 (82%~88% yield) in 13.2-15.1 ml buffer. The drug/antibody ratio (DAR) (the combination of PBD dimer and Tubulysin analog per antibody) was 3.9, which was determined via UPLC-Qtof mass spectrum. It was 96-99% monomer analyzed by SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID x 30 cm, 0.5 ml/min, 100 min) and a single band measured by SDS-PAGE gel. -dioxopyrrolidin-l-yl) 2,3-dibromosuccinate (9)
Figure imgf000107_0001
The solution of 2,3-dibromosuccinic acid (5.0 g, 18.25 mmol), N-hydroxysuccinimide (NHS) (5.01 g, 43.56 mmol) and EDC (12.02 g, 62.60 mmol) in dichloromethane (100 ml) was stirred at RT overnight, concentrated and purified on SiO2 column eluted with
EtOAc/CH2Cl2 (1:6) to afford the title compound (6.74 g, 79% yield). LC-MS (ESI) m/z calcd. for Ci2HnBr2N2O8 [M+H]+: 468.88, [M+H+2]+:470.88, found: 468.70, 470.70.
Example 51. (R,R,S,S,R,4R,4'R)-5,5'-(((12,13-dibromo-11,14-dioxo-4,7,18,21- tetraoxa-10,15-diazatetracosane-l,24-dioyl)bis(azanediyl))bis(4-hydroxy-3,l-phenylene))- bis(4-(2-((lR,3R)-l-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-l-methylpiperidine-2- carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-2-methylpentanoic acid
Figure imgf000107_0002
Compound 95 (40 mg, 0.042 mmol) in the mixture of DMA (1 ml) and NaH2PO4 buffer (0.6 ml, 0.15 M, pH 7.5) was added Bis(2,5-dioxopyrrolidin-l-yl) 2,3- dibromosuccinate (9) (18.0 mg, 0.038 mmol). The mixture was stirred for 8 h, concentrated, and purified on C - 18 chromatography (Φ 2.0 cm x 25 cm ) eluted with water/CH3CN (from 90% water/10% CH3CN to 20% water/90% CH3CN in 50 min at 10 ml/min ) to afford the title product 141 (38.5 mg. 49% yield) after dried with high vacuum pump. ESI MS m/z+ C94H143Br2Ni4O24S2 (M+H), cacld. 2073.81, found 2073.60.
Example 52. Conjugated compound 141 to an antibody for 142.
Figure imgf000108_0001
To a mixture of 2.0 mL of 10 mg/ml Herceptin in pH 6.0-8.0, were added of 0.70 ~ 2.0 mL PBS buffer of 100 mM NaH2PO4, pH 6.5-7.5 buffers, TCEP (28 μ L, 20 mM in water) and the compound 141 (14 μL, 20 mM in DMA). The mixture was incubated at RT for 4-16 h, then DHAA (135 μL, 50 mM) was added in. After continuous incubation at
RT overnight, the mixture was purified on G-25 column eluted with 100 mM NaH2PO4, 50 mM NaCl pH 6.0-7.5 buffer to afford 16.4-17.6 mg of the conjugate compound 92 (82%~88% yield) in 13.1-15.2 ml buffer. The drug/antibody ratio (DAR) was 3.9, which was determined via UPLC-Qtof mass spectrum. It was 96-99% monomer analyzed by SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID x 30 cm, 0.5 ml/min, 100 min) and a single band measured by SDS-PAGE gel.
Example 53. In vitro cytotoxicity evaluation of conjugates 127, 129 and 142 in comparison with T-DM1:
The cell lines used in the cytotoxicity assays were HL-60, a human promyelocytic leukemia cell line; NCI-N87, a human gastric carcinoma cell line; BT-474, a human invasive ductal carcinoma cell line; and SKOV3, a human ovarian carcinoma cell line. For HL-60, NCI-N87, and BT-474 cells, the cells were grown in RPMI-1640 with 10% FBS. For SKOV3 cells, the cells were grown in McCoy's 5 A Medium with 10% FBS. To run the assay, the cells (180 μΐ, 6000 cells) were added to each well in a 96-well plate and incubated for 24 hours at 37°C with 5% CO2. Next, the cells were treated with test compounds (20 μΐ) at various concentrations in appropriate cell culture medium (total volume, 0.2 mL). The control wells contain cells and the medium but lack the test compounds. The plates were incubated for 120 hours at 37°C with 5% CO2. MTT (5 mg/ml) was then added to the wells (20 μΐ) and the plates were incubated for 1.5hr at 37°C. The medium was carefully removed and DMSO (180 μΐ) was added afterward. After it was shaken for
15min, the absorbance was measured at 490 nm and 570 nm with a reference filter of 620nm. The inhibition% was calculated according to the following equation: inhibition% = [1-(assay-blank)/(control-blank)] x 100.
The cytotoxicity results: ICso (nM) N87 cell (Ag+) SK-OV-3 cell (Ag+ HL60 cell (Ag-
Conjugate 127 0.009 nM 0.010 nM >8 nM
Conjugate 129 0.012 nM 0.015 nM >8 nM
Conjugate 142 0.097 nM 0.083 nM >15 nM
T-DM1 0.263 nM 0.187 nM >15 nM
Specificity of conjugate 127 for N87 cell was over 889 (IC50> 8/ IC50= 0.009), and for SK-OV-3 cell was over 800; Specificity of conjugate 129 for N87 cell was over 666 (ICso> 8/ IC50= 0.012), and for SK-OV-3 cell was over 533; Specificity of conjugate 142 for N87 cell was over 155 (IC50> 15/ IC50= 0.097), and for SK-OV-3 cell was over 180; Specificity of conjugate T-DM1 for N87 cell was over 57 (IC50> 15/ IC50= 0.263), and for SK-OV-3 cell was over 80.
The three new conjugates 127, 129 and 142 were extremely more potent than the commercial conjugate T-DM1.
Example 54. Antitumor Activity In vivo.
The in vivo efficacy of conjugates 127, 129 and 142 along with T-DM1 were evaluated in a human gastric carcinoma N-87 cell line tumor xenograft models. Five- week-old female BALB/c Nude mice (30 animals) were inoculated subcutaneously in the area under the right shoulder with N-87 carcinoma cells (5 x 106 cells/mouse) in 0.1 mL of serum- free medium. The tumors were grown for 8 days to an average size of 133 mm3. The animals were then randomly divided into 5 groups (6 animals per group). The first group of mice served as the control group and was treated with the phosphate-buffered saline vehicle. The remaining three groups were treated with conjugates 127, 129, 142 and T- DM1 respectively at dose of 3 mg/Kg administered intravenously. Three dimensions of the tumor were measured every 4 days and the tumor volumes were calculated using the formula tumor volume =1/2 (length x width x height). The weight of the animals was also measured at the same time. A mouse was sacrificed when any one of the following criteria was met: (1) loss of body weight of more than 20% from pretreatment weight, (2) tumor volume larger than 1500 mm , (3) too sick to reach food and water, or (4) skin necrosis. A mouse was considered to be tumor-free if no tumor was palpable. The results were plotted in Figure 16. All the four conjugates did not cause the animal body weight loss. And the animals at control group were sacrificed at day 37 due to the tumor volume larger than 1500 mm and they were too sick. All 6/6 animals at the groups of compound 127 and 129 had completely no tumor measurable at day 13 till day 60 (the end of experiment). All 6/6 animals at the group of Compound 142 group had no tumor measurable at day 21 and 2/6 animals had tumor growth (measurable) back at days 48. In contrast T-DMl at dose of 3 mg/Kg was not able to eradicate the tumors and it only inhibited the tumor growth for 28 days.

Claims

CLAIMS What is claimed is:
1. A bridge linker compound of the Formula (I)
Figure imgf000111_0001
Wherein:
represents an optional single bond;
Figure imgf000111_0003
represents either a single bond or a double bond;
It provided that when
Figure imgf000111_0004
represents a single bond, both U and LP are not H; when
Figure imgf000111_0005
represents a double bond, either U or LP can be H, but are not H at the same time.
Figure imgf000111_0002
, which can be 2,3-disubstituted suc- cinic group, or 2-monosubstituted or 2,3-disubstituted fumaric group, or 2-monosubstituted or 2,3-disubstituted maleic group, is capable of reacting with a pair of sulfur atoms of the cell- binding agent; The sulfur atoms are preferred pairs of thiols reduced from the interchain disulfide bonds of the cell-binding agent by a reducing agent, such as dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH) and tris (2-carboxyethyl) phosphine (TCEP), or/and beta mercaptoethanol (β-ΜΕ, 2-ME).
U and LP represent the same or different leaving group that can be substituted by a thiol. Such leaving groups are, but are not limited to, a halide (e.g., fluoride, chloride, bromide, and iodide), methanesulfonyl (mesyl), /?-toluenesulfonyl (tosyl), trifluoromethyl-sulfonyl (triflate), trifluoromethylsulfonate, nitrophenol, N-hydroxysuccinimide (NHS), phenol; dinitrophenol; pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, 2-ethyl-5- phenylisoxazolium-3 '-sulfonate, or an intermediate molecule generated with a condensation reagent for Mitsunobu reactions.
R1 and R2 are the same or different, and are absent, linear alkyl having from 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl or alkynyl, or 1-6 carbon atoms of esters, ether, amide, or polyethyleneoxy unit of formula (OCH2CH2)p, or polypropyleneoxy unit of formula (OCH2(CH3)CH2)p wherein p is an integer from 0 to about 1000, or combination thereof.
Additionally R1 and R2 are respectively a chain of atoms selected from C, N, O, S, Si, and P, preferably having 0-500 atoms, which covalently connects to X1 or X2 and Z1 or Z2. The atoms used in forming the R1 and R2 may be combined in all chemically relevant ways, such as forming alkylene, alkenylene, and alkynylene, ethers, polyoxyalkylene, esters, amines, imines, poly- amines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, peptides, acyloxylamines, hydroxamic acids, or combination thereof.
X1 and X2 are independently selected from NH, N(R3), O, S or CH2; Wherein R3 is H, linear alkyl having from 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl or alkynyl, or 1-6 carbon atoms of esters, ether, amide, or polyethyleneoxy unit of formula (OCH2CH2)p, wherein p is an integer from 0 to about 1000, or combination thereof.
Z1 and Z2 are the same or different a function group that enables to react with a cytotoxic drug, to form a disulfide, thioether, thioester, peptide, hydrazone, ether, ester, carbamate, carbonate, amine (secondary, tertiary, or quarter), imine, cycloheteroalkyane, heteroaromatic, alkoxime or amide bond; The function groups of Z1 and Z2 are displayed below:
Figure imgf000112_0001
l halide (acid halide),
2-
Figure imgf000112_0002
- nitrophenoxy)acetyl;
Figure imgf000112_0003
-(dinitrophenoxy)acetyl;
Figure imgf000112_0004
2-(fluorophenoxy)-acetyl; 2-
O
Tf—0
(difluorophenoxy) -acetyl; 2 2-(((trifluoromethyl)-sulfonyl)oxy)acetyl;
Figure imgf000112_0005
2-(pentafluorophenoxy)acetyl;
Figure imgf000113_0001
N(R1), or CH2; R5 and R3 are H, R1, aromatic, heteroaromatic, or aromatic group wherein one or several H atoms are replaced independently by -R1, -halogen, -OR1, -SR1, -NR1R2, - NO2, - S(O)R1, -S(O)2R1, or -COOR1; Lv3 is a leaving group selected from nitrophenol; N-hydroxysuc- cinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1- hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3 '-sulfonate, anhydrides formed its self, or formed with the other anhydride, e.g. acetyl anhydride, formyl anhydride; or an intermediate molecule generated with a condensation reagent for peptide coupling reactions, or for Mitsunobu reactions.
2. A cell-binding agent-drug conjugate compound of Formula (II)
Figure imgf000113_0002
Wherein:
Cb represents a cell-binding agent, preferably an antibody, which conjugates to Drug1 and Drug2 via a pair of sulfur atoms (thiols). The conjugatable thiol groups can generally be generated from dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH) and tris (2-carboxyethyl) phosphine (TCEP), or/and beta mercaptoethanol (β-ΜΕ, 2-ME) reduction of pairs of disulfide bonds on the surface of cell-binding molecule.
Drug1 and Drug2 represent the same or different cytotoxic agents, linked to the cell-binding agent via the bridge linker through an alkyl, alkylene, alkenylene, alkynylene, ether,
polyoxyalkylene, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea,
semicarbazide, carbazide, alkoxyamine, urethanes, amino acid, peptide, acyloxylamine, hydroxamic acid, disulfide, thioether, thioester, carbamate, carbonate, heterocyclic ring, heteroalkyl, heteroaromatic, or alkoxime bond, or combination thereof.
n is 1 ~ 30; R1, R2, X1 and X2 are described the same previously in Claim 1.
Figure imgf000113_0003
3. A compound of Formula (III):
Figure imgf000114_0001
Wherein:
Cb, Z1, Z2, n, R1, R2, X1, and X2 are defined the same as in Claims 1 and 2.
Figure imgf000114_0003
4. A compound of Formula (IV):
Figure imgf000114_0004
Wherein:
Figure imgf000114_0005
U, U', Drug1, Drug2, R1, R2, X1, and X2 are defined the same as in Claims 1 and 2.
5. The bridge linker compound of Formula (I) of claims 1, wherein the 2,3-disubstituted succinic group, or 2-monosubstituted or 2,3-disubstituted fumaric group, or 2-monosubstituted or 2,3-disubstituted maleic group is synthesized through the condensation of the 2,3- disubstituted succinic acid, or 2-monosubstituted or 2,3-disubstituted fumaric acid, or 2- monosubstituted or 2,3-disubstituted maleic acid, or its acid derivatives, with the other components containing an amine (1° or 2° amines), alcohol, or thiol on their terminal as shown in the following scheme (la):
Figure imgf000114_0002
Wherein X is X1 or X2 as described in Claim 1 as NH, N(R3), O, or S; R is R1 and/or R2 R1, R2 and R3 are the same described in Claim 1;
Lvi and Lv2 are the same or independently OH; F; CI; Br; I; nitrophenol; N-hydroxy- succinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluoro- phenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol;
tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium- 3 '-sulfonate, anhydrides formed its self, or formed with the other anhydride, e.g. acetyl anhydride, formyl anhydride; or a intermediate molecule generated with a condensation reagent for peptide coupling reactions, or for Mitsunobu reactions, e.g. condensation reagents are: EDC (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide), DCC (Dicyclohexyl-carbodiimide), Ν,Ν'- Diisopropylcarbodiimide (DIC), N-Cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p- toluenesulfonate (CMC,or CME-CDI), Ι,Ι '-Carbonyldiimidazole (CDI),TBTU (O-(Benzotria- zol- 1 -yl)-N,N,N ',Ν '-tetramethyluronium tetrafluoroborate) , Ν,Ν,Ν ',Ν '-Tetramethyl-O-( 1 H- benzotriazol-l-yl)uronium hexafluorophosphate (HBTU), (Benzotriazol-l-yloxy)tris(dimethyl- amino)phosphonium hexafluorophosphate (BOP), (Benzotriazol-l-yloxy)tripyrrolidinophos- phonium hexafluorophosphate (PyBOP), Diethyl cyanophosphonate (DEPC), Chloro- Ν,Ν,Ν',Ν'-tetramethylformamidinium hexafluorophosphate, l-[Bis(dimethylamino)- methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), 1- [(Dimethylamino)(morpholino) methylene]- lH-[l,2,3]triazolo[4,5-b]pyridine-l-ium 3-oxide hexafluorophosphate (HDMA), 2-Chloro-l,3-dimethylimidazolidinium hexafluorophosphate (CIP), Chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), Fluoro-Ν,Ν,Ν',Ν'- bis(tetramethylene)formamidinium hexafluorophosphate (BTFFH), N,N,N',N'-Tetramethyl-S- (l-oxido-2-pyridyl)thiuronium hexafluorophosphate, O-(2-Oxo-l(2H)pyridyl)-N,N,N',N'- tetramethyluronium tetrafluoroborate (TPTU), S-(l-Oxido-2-pyridyl)-N,N,N',N'-tetramethyl- thiuronium tetrafluoroborate, O-[(Ethoxycarbonyl) cyano-methylenamino]-N,N,N',N'- tetramethyluronium hexafluorophosphate (HOTU), (l-Cyano-2-ethoxy-2-oxoethylidenamino- oxy)dimethylamino-morpholino-carbenium hexafluorophosphate(COMU), O-(Benzotriazol-l- yl)-N,N,N',N'-bis(tetramethylene) uronium hexafluorophosphate (HBPyU), N-Benzyl-N'- cyclohexylcarbodiimide (with, or without polymer-bound), Dipyrrolidino(N-succinimidyloxy)- carbenium hexafluoro -phosphate (HSPyU), Chlorodipyrrolidinocarbenium hexafluorophosphate (PyClU), 2-Chloro-l,3-dimethylimidazolidinium tetrafluoroborate(CIB), (Benzotriazol- 1-yloxy) dipiperidinocarbenium hexafluorophosphate (HBPipU), O-(6-Chlorobenzotriazol-l- yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TCTU), Bromotris(dimethylamino)- phosphonium hexafluorophosphate (BroP), Propylphosphonic anhydride (PPACA, T3P®), 2- Morpholinoethyl isocyanide (MEI), N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate (HSTU), 2-Bromo-l-ethyl-pyridinium tetrafluoroborate (BEP), O- [(Ethoxycarbonyl)cyanomethylenamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate (TOTU), 4-(4,6-Dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium chloride (MMTM, DMTMM), N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), O- (3,4-Dihydro-4-oxo-l,2,3-benzotriazin-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoro-borate (TDBTU), l,l '-(Azodicarbonyl)dipiperidine (ADD), Di-(4-chlorobenzyl) azodicar-boxylate (DCAD), Di-tert-butyl azodicarboxylate (DBAD), Diisopropyl azodicarboxylate (DIAD), Diethyl azodicarboxylate (DEAD).
6. The Formula (II) and (IV) of claims 2, and 4 wherein the Drug1 and Drug2 are the same or independently selected from: 1). Chemotherapeutic agents: a). Alkylating agents: such as Nitrogen mustards:
chlorambucil, chlornaphazine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, mitobronitol, melphalan, mitolactol, pipobroman, novembichin, phenesterine, prednimustine, thiotepa, trofosfamide, uracil mustard; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); benzodiazepine dimers (e.g., dimmers of pyrrolobenzodiazepine (PBD) or tomaymycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinobenzodiazepines);
Nitrosoureas: (carmustine, lomustine, chlorozotocin, fotemustine, nimustine, ranimustine); Alkylsulphonates: (busulfan, treosulfan, improsulfan and piposulfan); Triazenes: (dacarbazine); Platinum containing compounds: (carboplatin, cisplatin, oxaliplatin); aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaor- amide and trimethylolomelamine] ; b). Plant Alkaloids: such as Vinca alkaloids: (vincristine, vinblastine, vindesine, vinorelbine, navelbin); Taxoids: (paclitaxel, docetaxol) and their analogs, Maytansinoids (DM1, DM2, DM3, DM4, DM5, DM6, DM7, maytansine and ansamitocins) and their analogs, cryptophycins (particularly cryptophycin 1 and cryptophycin 8); epothilones, eleutherobin, discodermolide, bryostatins, dolostatins, auristatins, tubulysins, cephalostatins; pancratistatin; a sarcodictyin; spongistatin; c). DNA Topoisomerase Inhibitors: such as
[Epipodophyllins: (9-aminocamptothecin, camptothecin, crisnatol, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acids (retinols), teniposide, topotecan, 9-nitrocamptothecin (RFS 2000)); mitomycins: (mitomycin C)]; d). Antimetabolites: such as { [Anti-folate: DHFR inhibitors: (methotrexate, trimetrexate, denopterin, pteropterin, aminopterin (4-aminopteroic acid) or the other folic acid analogues); IMP dehydrogenase Inhibitors: (mycophenolic acid, tiazofurin, ribavirin, EICAR); Ribonucleotide reductase Inhibitors: (hydroxyurea, deferoxamine)]; [Pyrimidine analogs: Uracil analogs: (ancitabine, azacitidine, 6- azauridine, capecitabine (Xeloda), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-Fluorouracil, floxuridine, ratitrexed (Tomudex)); Cytosine analogs: (cytarabine, cytosine arabinoside, fludarabine); Purine analogs: (azathioprine, fludarabine, mercaptopurine, thiamiprine, thioguanine)]; folic acid replenisher, such as frolinic acid}; e). Hormonal therapies: such as {Receptor antagonists: [Anti-estrogen: (megestrol, raloxifene, tamoxifen); LHRH agonists: (goscrclin, leuprolide acetate); Anti-androgens: (bicalutamide, flutamide, calusterone, dromostanolone propionate, epitiostanol, goserelin, leuprolide, mepitiostane, nilutamide, testolactone, trilostane and other androgens inhibitors)]; Retinoids/Deltoids: [Vitamin D3 ana- logs: (CB 1093, EB 1089 KH 1060, cholecalciferol, ergocalciferol); Photodynamic therapies: (verteporfin, phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A); Cytokines: (Inter- feron-alpha, Interferon-gamma, tumor necrosis factor (TNFs), human proteins containing a TNF domain)] }; f). Kinase inhibitors, such as BIBW 2992 (anti-EGFR/Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib. vandetanib, E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534), bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, Trastuzumab, Ranibizumab, Panitumumab, ispinesib; g). antibiotics, such as the enediyne antibiotics (e.g. calicheamicins, especially calicheamicin .γΐ, δΐ, al and βΐ; dynemicin, including dynemicin A and deoxydynemicin; esperamicin, kedarcidin, C-1027, maduropeptin, as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin; chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, nitomycins, mycophe- nolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; f). Others: such as Polyketides (acetogenins), especially bullatacin and bullatacinone; gemcitabine, epoxomicins (e. g. carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allovectin-7, Xegeva, Provenge, Yervoy, Isoprenylation inhibitors (such as Lovastatin), Dopaminergic neurotoxins (such as l-methyl-4- phenylpyridinium ion), Cell cycle inhibitors (such as staurosporine), Actinomycins (such as Actinomycin D, dactinomycin), Bleomycins (such as bleomycin A2, bleomycin B2,
peplomycin), Anthracyclines (such as daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, mtoxantrone, MDR inhibitors (such as verapamil),
Ca2+ATPase inhibitors (such as thapsigargin), Histone deacetylase inhibitors (Vorinostat, Romidepsin, Panobinostat, Valproic acid, Mocetinostat (MGCD0103), Belinostat, PCI-24781, Entinostat, SB939, Resminostat, Givinostat, AR-42, CUDC-101, sulforaphane, Trichostatin A) ; Thapsigargin, Celecoxib, glitazones, epigallocatechin gallate, Disulfiram, Salinosporamide A.; Anti-adrenals, such as aminoglutethimide, mitotane, trilostane; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; arabinoside, bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; eflornithine (DFMO), elfomithine; elliptinium acetate, etoglucid; gallium nitrate; gacytosine, hydroxyurea; ibandronate, lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;
podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran;
spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucarin A, roridin A and anguidine); urethane, siRNA, antisense drugs;
2) . Anti-autoimmune disease agents: cyclosporine, cyclosporine A, aminocaproic acid, aza- thioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g. amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, fluocortolone danazol, dexamethasone, Triamcinolone acetonide, beclometasone dipropionate), DHEA, enanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mofetil, mycophenylate, prednisone, sirolimus, tacrolimus.
3) . Anti-infectious disease agents, a). Aminoglycosides: amikacin, astromicin, gentamicin (netilmicin, sisomicin, isepamicin), hygromycin B, kanamycin (amikacin, arbekacin, bekanamycin, dibekacin, tobramycin), neomycin (framycetin, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, verdamicin; b). Amphenicols:
azidamfenicol, chloramphenicol, florfenicol, thiamphenicol; c). Ansamycins: geldanamycin, herbimycin; d). Carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem; e). Cephems: carbacephem (loracarbef), cefacetrile, cefaclor, cefradine, cefadroxil, cefalonium, cefaloridine, cefalotin or cefalothin, cefalexin, cefaloglycin, cefamandole, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin, cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin, cefpimizole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime, ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cephamycin (cefoxitin, cefotetan, cefmetazole), oxacephem (flomoxef, latamoxef); f). Glyco- peptides: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin; g). Glycylcyclines: e. g. tigecycline; g). β-Lactamase inhibitors: penam (sulbactam, tazobactam), clavam (clavulanic acid); i). Lincosamides: clindamycin, lincomycin; j).
Lipopeptides: daptomycin, A54145, calcium-dependent antibiotics (CDA); k). Macrolides: azithromycin, cethromycin, clarithromycin, dirithromycin, erythromycin, flurithromycin, josamycin, ketolide (telithromycin, cethromycin), midecamycin, miocamycin, oleandomycin, rifamycins (rifampicin, rifampin, rifabutin, rifapentine), rokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandomycin, telithromycin; 1). Monobac- tams: aztreonam, tigemonam; m). Oxazolidinones: linezolid; n). Penicillins: amoxicillin, ampi- cillin (pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin), azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethylpenicillin, clometocillin, procaine benzylpenicillin, carbenicillin (carindacillin), cloxacillin, dicloxacillin, epicillin, flucloxaciUin, mecillinam (pivmecillinam), mezlocillin, meticillin, nafcillin, oxacillin, penamecillin, penicillin, pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin, sulbenicillin, temocillin, ticarcillin; o). Polypeptides: bacitracin, colistin, polymyxin B; p). Quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, kano trovafloxacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q). Streptogramins: pristinamycin, quinupristin/dalfopristin); r). Sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanamide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole (co- trimoxazole); s). Steroid antibacterials: e.g. fusidic acid; t). Tetracyclines: doxycycline, chlortet- racycline, clomocycline, demeclocycline, lymecycline, meclocycline, metacycline, minocycline, oxytetracycline, penimepicycline, rolitetracycline, tetracycline, glycylcyclines (e.g. tigecycline); u). Other types of antibiotics: annonacin, arsphenamine, bactoprenol inhibitors (Bacitracin), DADAL/AR inhibitors (cycloserine), dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitors (e. g. fosfomycin), nitrofurantoin, paclitaxel, platensimycin, pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampin), tazobactam tinidazole, uvaricin;
4). Anti-viral drugs: a). Entry/fusion inhibitors: aplaviroc, maraviroc, vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab); b). Integrase inhibitors: raltegravir, elvitegravir, globoidnan A; c). Maturation inhibitors: bevirimat, vivecon; d). Neuraminidase inhibitors:
oseltamivir, zanamivir, peramivir; e). Nucleosides &_nucleotides: abacavir, aciclovir, adefovir, amdoxovir, apricitabine, brivudine, cidofovir, clevudine, dexelvucitabine, didanosine (ddl), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU), 3'-fluoro- substituted 2', 3'-dideoxynucleoside analogues (e.g. 3'-fluoro-2',3'-dideoxythymidine (FLT) and 3'-fluoro-2',3'-dideoxyguanosine (FLG), fomivirsen, ganciclovir, idoxuridine, lamivudine (3TC), 1-nucleosides (e.g. -1-th ymidine and β-1-2 '-deoxycytidine), penciclovir, racivir, ribavirin, stampidine, stavudine (d4T), taribavirin (viramidine), telbivudine, tenofovir, trifluridine valaciclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f). Non-nucleosides: amantadine, ateviridine, capravirine, diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphonoformic acid), imiquimod, interferon alfa, loviride, lodenosine, methisazone, nevirapine, NOV-205, peginterferon alfa, podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), tromantadine; g). Protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, fos amprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h). Other types of anti-virus drugs:
abzyme, arbidol, calanolide a, ceragenin, cyanovirin-n, diary lpyrimidines, epigallocatechin gallate (EGCG), foscarnet, griffithsin, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, portmanteau inhibitors, ribavirin, seliciclib.
5) . A radioisotope that can be selected from (radionuclides) 3H, 11C, 14C, 18F, 32P, 35S, 64Cu,
68 Ga, 86 Y, 99 Tc, 111 In, 123 I, 124I, 125I, 131T, 133 Xe, 177 Lu, 21 1 At, or 213 Bi.
6) . A chromophore molecule, which can be one that has the ability to absorb a kind of light, such as UV light, florescent light, IR light, near IR light, visual light; A class or subclass of xanthophores, erythrophores, iridophores, leucophores, melanophores, cyanophores, fluorophore molecules which are fluorescent chemical compounds re-emitting light upon light, visual phototransduction molecules, photophore molecules, luminescence molecules, luciferin compounds; Non-protein organic fluorophores, such as: Xanthene derivatives (fluorescein, rhodamine, Oregon green, eosin, and Texas red); Cyanine derivatives: (cyanine,
indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine); Squaraine derivatives and ring-substituted squaraines, including Seta, SeTau, and Square dyes; Naphthalene derivatives (dansyl and prodan derivatives); Coumarin derivatives; Oxadiazole derivatives
(pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole); Anthracene derivatives
(anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange); Pyrene derivatives (cascade blue, etc); Oxazine derivatives (Nile red, Nile blue, cresyl violet, oxazine 170 etc).
Acridine derivatives (proflavin, acridine orange, acridine yellow etc). Arylmethine derivatives (auramine, crystal violet, malachite green). Tetrapyrrole derivatives (porphin, phthalocyanine, bilirubin); Any analogs and derivatives of the following fluorophore compounds: CF dye (Biotium), DRAQ and CyTRAK probes (BioStatus), BODIPY (Invitrogen), Alexa Fluor (Invi- trogen), DyLight Fluor (Thermo Scientific, Pierce), Atto and Tracy (Sigma Aldrich),
FluoProbes (Interchim), Abberior Dyes (Abberior), DY and MegaStokes Dyes (Dyomics), Sulfo Cy dyes (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTau and Square Dyes (SETA BioMedicals), Quasar and Cal Fluor dyes (Biosearch Technologies), SureLight Dyes (APC, RPEPerCP, Phycobilisomes)(Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco- Biotech), Allophycocyanin (APC), Aminocoumarin, APC-Cy7 conjugates, BODIPY-FL, Cascade Blue, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, Fluorescein, FluorX, Hydroxycoumarin, Lissamine Rhodamine B, Lucifer yellow, Methoxycoumarin, NBD, Pacific Blue, Pacific Orange, PE-Cy5 conjugates, PE-Cy7 conjugates, PerCP, R-Phycoerythrin(PE), Red 613, Seta-555- Azide, Seta-555-DBCO, Seta-555-NHS, Seta-580-NHS, Seta-680-NHS, Seta-780-NHS, Seta- APC-780, Seta-PerCP-680, Seta-R-PE-670, SeTau-380-NHS, SeTau-405-Maleimide, SeTau- 405-NHS, SeTau-425-NHS, SeTau-647-NHS, Texas Red, TRITC, TruRed, X-Rhodamine, 7- AAD (7-aminoactinomycin D, CG-selective), Acridine Orange, Chromomycin A3, CyTRAK Orange (Biostatus, red excitation dark), DAPI, DRAQ5, DRAQ7, Ethidium Bromide,
Hoechst33258, Hoechst33342, LDS 751, Mithramycin, Propidiumlodide (PI), SYTOX Blue, SYTOX Green, SYTOX Orange, Thiazole Orange, TO-PRO: Cyanine Monomer, TOTO-1, TO- PRO- 1, TOTO-3, TO-PRO-3, YOSeta-1, YOYO-1. The fluorophore compounds that can be linked to the linkers of the invention for study cells are selected from the following compounds or their derivatives: DCFH (2'7'Dichorodihydro-fluorescein, oxidized form), DHR
(Dihydrorhodamine 123, oxidized form, light catalyzes oxidation), Fluo-3 (AM ester. pH > 6), Fluo-4 (AM ester. pH 7.2), Indo-1 (AM ester, low/high calcium (Ca2+)), SNARF(pH 6/9), Allophycocyanin(APC), AmCyanl (tetramer, Clontech), AsRed2 (tetramer, Clontech), Azami Green (monomer, MBL), Azurite, B-phycoerythrin(BPE), Cerulean, CyPet, DsRed monomer (Clontech), DsRed2 ("RFP", Clontech), EBFP, EBFP2, ECFP, EGFP (weak dimer, Clontech), Emerald (weak dimer, Invitrogen), EYFP (weak dimer, Clontech), GFP (S65A mutation), GFP (S65C mutation), GFP (S65L mutation), GFP (S65T mutation), GFP (Y66F mutation), GFP (Y66H mutation), GFP (Y66W mutation), GFPuv, HcRedl, J-Red, Katusha, Kusabira Orange (monomer, MBL), mCFP, mCherry, mCitrine, Midoriishi Cyan (dimer, MBL), mKate
(TagFP635, monomer, Evrogen), mKeima-Red (monomer, MBL), mKO, mOrange, mPlum, mRaspberry, mRFPl (monomer, Tsien lab), mStrawberry, mTFPl, mTurquoise2, P3
(phycobilisome complex), Peridinin Chlorophyll (PerCP), R-phycoerythrin(RPE), T-Sapphire, TagCFP (dimer, Evrogen), TagGFP (dimer, Evrogen), TagRFP (dimer, Evrogen), TagYFP (dimer, Evrogen), tdTomato (tandem dimer), Topaz, TurboFP602 (dimer, Evrogen), TurboFP635 (dimer, Evrogen), TurboGFP (dimer, Evrogen), TurboRFP (dimer, Evrogen), Turbo YFP (dimer, Evrogen), Venus, Wild Type GFP, YPet, ZsGreenl (tetramer, Clontech), ZsYellowl (tetramer, Clontech).
7). The pharmaceutically acceptable salts, acids or derivatives of any of the above drugs.
7. The compound of Formula (II) and (IV) of claims 2 and 4, wherein "Drug1" and "Drug2" are a chromophore molecule, the conjugate compounds of Formula (II) and (IV) of claims 2, and 4, can be used for detecting, monitoring, or studying the interactions and/or functions of the cell binding molecule, and/or of the conjugate with a target, particularly, a targeted cell.
8. The compound of Formula (II) and (IV) of claims 2 and 4, wherein "Drug1" and "Drug2" can be a polyalkylene glycols or a polyalkylene glycol analogs, which are used for extending the half-life of the cell-binding molecule when administered to a mammal. Polyalkylene glycols are poly(ethylene glycols) (PEGs), poly(propylene glycol), copolymers of ethylene oxides or propylene oxides that has a molecular weight of from about 10 Daltons to about 200 kDa
9. The compound of Formula (II) and (IV) of claims 2 and 4, wherein "Drug1" and "Drug2" can be a cell-binding ligands or receptors, or receptor analogs, so that the conjugates can work as a targeting conductor/director to deliver the conjugate to malignant cells, and also can modulate or co-stimulate a desired immune response or altering signaling pathways.
10. The conjugate compound of claims 2 and 4, wherein "Drug1" and "Drug2" are preferred selected from tubulysins, calicheamicins, auristatins, maytansinoids, CC-1065 analogs, daunorubicin and doxorubicin compounds, taxanoids (taxanes), cryptophycins, epothilones, benzodiazepine dimers (e.g., dimmers of pyrrolobenzodiazepine (PBD), tomaymycin, anthramycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinobenzodiaze- pines), calicheamicins and the enediyne antibiotics, actinomycin, azaserines, bleomycins, epirubicin, tamoxifen, idarubicin, dolastatins/auristatins (e.g. monomethyl auristatin E, MMAE , MMAF, auristatin PYE, auristatin TP, Auristatins 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)), duocarmycins, thiotepa, vincristine, hemiasterlins, nazumamides, microginins, radiosumins, alterobactins, microsclerodermins, theonellamides, esperamicins, siRNA, nucleolytic enzymes, and/or pharmaceutically acceptable salts, acids, or/and their analogues derivatives of any of the above molecules.
11. The conjugate compounds of claims 2 and 3 wherein the cell binding agent/molecule is selected from an antibody, a protein, a vitamin (e.g. folate), peptides, a polymeric micelle, a liposome, a lipoprotein-based drug carrier, a nano-particle drug carrier, a dendrimer, and a molecule above coating with cell-binding ligands, or a combination of above thereof.
12. The cell-binding molecule/agent according to claims 2, 3, and 11 is preferred an antibody, full-length antibodies (polyclonal antibodies, monoclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies); a single chain antibody, an antibody fragment that binds to the target cell, a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment that binds the target cell, a chimeric antibody, a chimeric antibody fragment that binds to the target cell, a domain antibody, a domain antibody fragment that binds to the target cell, a resurfaced antibody, a resurfaced single chain antibody, or a resurfaced antibody fragment that binds to the target cell, a humanized antibody or a resurfaced antibody, a humanized single chain antibody, or a humanized antibody fragment that binds to the target cell, anti-idiotypic (anti-Id) antibodies, CDR's, diabody, triabody, tetrabody, miniantibody, small immune proteins (SIP), a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient-transport molecule, or large molecular weight proteins.
13. The cell-binding molecule/agent according to claims 2, 3, and 11 can be any agent that is able to target against a tumor cell, a virus infected cell, a microorganism infected cell, a parasite infected cell, an autoimmune disease cell, an activated tumor cells, a myeloid cell, an activated T-cell, an affecting B cell, or a melanocyte.
14. The cell-binding molecule/agent according to claims 2, 3, and 11 can be any
agent/molecule that is able to against any one of the following antigens or receptors: CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CDl la, CDl lb, CDl lc, CD12w, CD14, CD15, CD16, CDwl7, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD51, CD52, CD53, CD54, CD55, CD56, CD58, CD59, CD61, CD62E, CD62L, CD62P, CD63, CD66, CD68, CD69, CD70, CD72, CD74, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96, CD98, CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD125, CD126, CD127, CD133, CD134, CD135, CD137, CD138, CD141, CD142, CD143, CD144, CD147, CD151, CD147, CD152, CD154, CD156, CD158, CD163, CD166, .CD168, CD174, CD180, CD184, CDwl86, CD194, CD195, CD200, CD200a, CD200b, CD209, CD221, CD227, CD235a, CD240, CD262, CD271, CD274, CD276 (B7-H3), CD303, CD304, CD309, CD326, 4-1BB, 5 AC, 5T4 (Trophoblast glycoprotein, TPBG, 5T4, Wnt-Activated Inhibitory Factor 1 or WAIF1), Adenocarcinoma antigen, AGS-5, AGS-22M6, Activin receptorlike kinase 1, AFP, AKAP-4, ALK, Alpha intergrin, Alpha v beta6, Amino-peptidase N, Amyloid beta, Androgen receptor, Angiopoietin 2, Angiopoietin 3, Annexin Al, Anthrax toxin protective antigen, Anti-transferrin receptor, AOC3 (VAP-1), B7-H3, Bacillus anthracis anthrax, BAFF (B-cell activating factor), B-lymphoma cell, bcr-abl, Bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, Canis lupus familiaris IL31, Carbonic anhydrase IX, Cardiac myosin,
CCL11(C-C motif chemokine 11), CCR4 (C-C chemokine receptor type 4, CD194), CCR5, CD3E (epsilon), CEA (Carcinoembryonic antigen), CEACAM3, CEACAM5
(carcinoembryonic antigen), CFD (Factor D), Ch4D5, Cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18), Clumping factor A, CRIPTO, FCSF1R (Colony stimulating factor 1 receptor, CD 115), CSF2 (colony stimulating factor 2, Granulocyte-macrophage colony- stimulating factor (GM-CSF)), CTLA4 (cytotoxic T-lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4 (CD 184), C-X-C chemokine receptor type 4, cyclic ADP ribose hydrolase, Cyclin B l, CYP1B 1, Cytomegalovirus, Cytomegalovirus glycoprotein B, Dabigatran, DLL4 (delta-like - ligand 4), DPP4 (Dipeptidyl-peptidase 4), DR5 (Death receptor 5), E. coli shiga toxin type-1, E. coli shiga toxin type-2, ED-B, EGFL7 (EGF-like domain-containing protein 7), EGFR, EGFRII, EGFRvIII, Endoglin (CD 105), Endothelin B receptor, Endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2, Episialin, ERBB2 (Epidermal Growth Factor Receptor 2), ERBB3, ERG (TMPRSS2 ETS fusion gene), Escherichia coli, ETV6-AML, FAP (Fibroblast activation protein alpha), FCGR1, alpha-Fetoprotein, Fibrin II, beta chain, Fibronectin extra domain-B, FOLR (folate receptor), Folate receptor alpha, Folate hydrolase, Fos-related antigen l.F protein of respiratory syncytial virus, Frizzled receptor, Fucosyl GM1, GD2 ganglioside, G-28 (a cell surface antigen glyvolipid), GD3 idiotype, GloboH, Glypican 3, N-glycolylneuraminic acid, GM3, GMCSF receptor a-chain, Growth differentiation factor 8, GP100, GPNMB
(Transmembrane glycoprotein NMB), GUCY2C (Guanylate cyclase 2C, guanylyl 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/SF (Hepatocyte growth factor/scatter factor), HHGFR, HIV-1, Histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB , HMWMAA, Human chorionic gonadotropin, HNGF, Human scatter factor receptor kinase, HPV E6/E7, Hsp90, hTERT, ICAM-1 (Intercellular Adhesion Molecule 1), Idiotype, IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-γ, Influenza hemagglutinin, IgE, IgE Fc region, IGHE, IL-1, IL-2 receptor (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), Integrins (α4, αιιβ3, ανβ3, α4β7, α5β1, α6β4, α7β7, α11β3, α5β5, ανβ5), Interferon gamma- induced protein, ITGA2, ITGB2, KIR2D, LCK, Le, Legumain, Lewis-Y antigen, LFA- l(Lymphocyte function-associated antigen 1, CDl la), LHRH, LINGO-1, Lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE Al, MAGE A3, MAGE 4, MARTI, MCP-1, MIF (Macrophage migration inhibitory factor, or gly- cosylation-inhibiting factor (GIF)), MS4A1 (membrane- spanning 4-domains subfamily A member 1), MSLN (mesothelin), MUCl(Mucin 1, cell surface associated (MUC1) or polymorphic epithelial mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCPl(monocyte chemotactic protein 1), MelanA/MARTl, ML-IAP, MPG, MS4A1 (membrane-spanning 4-domains subfamily A), MYCN, Myelin-associated glycoprotein, Myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), Nectin-4 (ASG-22ME), NGF, Neural apoptosis-regulated proteinase 1, NOGO-A, Notch receptor, Nucleolin, Neu oncogene product, NY-BR-1, NY-ESO-1, OX-40, OxLDL (Oxidized low-density lipoprotein), OY-TES 1, P21, p53 nonmutant, P97, Page4, PAP, Paratope of anti-(N-glycolylneuraminic acid), PAX3, PAX5, PCSK9, PDCD1 (PD-1, Programmed cell death protein 1,CD279), PDGF-Ra (Alpha-type platelet-derived growth factor receptor ), PDGFR-β, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, Platelet- derived growth factor receptor beta, Phosphate-sodium co-transporter, PMEL 17, Polysialic acid, Proteinase3 (PR1), Prostatic carcinoma, PS (Phosphatidylserine), Prostatic carcinoma cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, Rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD240), Rhesus factor, RANKL, RhoC, Ras mutant, RGS5, ROBO4, Respiratory syncytial virus, RON, Sarcoma translocation breakpoints, SART3, Sclerostin, SLAMF7 (SLAM family member 7), Selectin P, SDC1 (Syndecan 1), sLe(a), Somatomedin C, SIP (Sphingosine- 1-phosphate), Somatostatin, Sperm protein 17, SSX2, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), STEAP2, STn, TAG-72 (tumor associated glycoprotein 72), Survivin, T-cell receptor, T cell transmembrane protein, TEM1 (Tumor endothelial marker 1), TENB2, Tenascin C (TN-C), TGF-a, TGF-β (Transforming growth factor beta), TGF-βΙ, TGF-β2 (Transforming growth factor-beta 2), Tie (CD202b), Tie2, TIM-1 (CDX-014), Tn, TNF, TNF-a, TNFRSF8, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B),
TNFRSF13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblast glycoprotein), TRAIL-R1 (Tumor necrosis apoprosis Inducing ligand Receptor 1), TRAILR2 (Death receptor 5 (DR5)), tumor-associated calcium signal transducer 2, tumor specific glycosylaton of MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, Tyrosinase, VCAM-1 (CD 106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, or vimentin, WT1, XAGE 1, or cells expressing any insulin growth factor receptors, or any epidermal growth factor receptors.
15. The tumor cell according to claim 13 is selected from lymphoma cells, myeloma cells, renal cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cancer cells, small-cell lung cancer cells, none small-cell lung cancer cells, testicular cancer cells, malignant cells, or any cells that grow and divide at an unregulated, quickened pace to cause cancers.
16. The linkage components R1 and/or R2 according to claims 1, 2, 3, and/or 4, can be composed of one or more linker components of: 6-maleimidocaproyl (MC), maleimido propanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), lysine-phenylalanine (lys-phe), p-aminobenzyloxycarbonyl (PAB), 4-thio-pentanoate (SPP), 4-(N-maleimidomethyl)cyclo- hexane-l-carboxylate (MCC), 4-thio-butyrate (SPDB), maleimidoethyl (ME), 4-thio-2- hydroxysulfonyl-butyrate (2-Sulfo-SPDB), pyridinyl-dithiol (PySS), alkoxy amino (AOA), ethyleneoxy (EO), 4-methyl-4-dithio-pentanoic (MPDP), azido (N3), alkynyl, dithio, peptides, and/or (4-acetyl)aminobenzoate (SIAB).
17. The conjugate compound of claim 2, wherein "Drug1" and "Drug2" are a Tubulysin analog, the conjugate compound of Formula (II) is preferred structures of T01, T02, T03, T04, T05, T06 and T07 as following:
Figure imgf000126_0001
Figure imgf000127_0001
Wherein mAb is an antibody; Z3 and Z'3 are independently H, OP(O)(OM1)(OM2),
OCH2OP(O)(OM1)(OM2), OSO3M1, R1, or O-glycoside (glucoside, galactoside, mannoside, glucurono side, alloside, fructoside, etc), NH-glycoside, S-glycoside or CH2-glycoside; M1 and
M2 are independently H, Na, K, Ca, Mg, NH4, NR1R2R3; n is 1-30 X1, X2, R1
Figure imgf000127_0002
R2 and R3 are the same defined in Claims 1 and 2.
18. The conjugate compound of claim 2, wherein "Drug1" and "Drug2" are a Calicheamicin analog, the conjugate compound of Formula (II) is preferred structures of C01 as following:
Figure imgf000128_0001
Wherein mAb is an antibody; n is 1~30;
Figure imgf000128_0004
, X1, X2, R1, R2 and R3 are the same defined in Claims 1 and 2.
19. The conjugate compound of claim 2, wherein "Drug1" and "Drug2" are a Maytansinoid analog, the conjugate compound of Formula (II) is preferred structures of M01 as following:
W
Figure imgf000128_0002
herein mAb is an antibody; n is 1~30; , X1, X2, R1, R2 and R3 are the same defined in Claims 1 and 2.
20. The conjugate compound of claim 2, wherein "Drug1" and "Drug2" are a Taxane analog, the conjugate compound of Formula (II) is preferred structures of TxOl, Tx02 and Tx03 as following:
Figure imgf000128_0003
Figure imgf000129_0001
Wherein mAb is an antibody; n is 1~30;
Figure imgf000129_0003
X1, X2, R1 and R2 are the same defined in Claims 1 and 2.
21. The conjugate compound of claim 2, wherein "Drug1" and "Drug2" are a CC-1065 analogue and/or doucarmycin analog, the conjugate compound of Formula (II) is preferred structures of CCOl, CC02, and CC03 as following:
Figure imgf000129_0002
Figure imgf000130_0001
Wherein mAb is an antibody; n is 1-30; Z4 and Z'4 are independently H, PO(OM1)(OM2), CH2PO(OM1)(OM2), SO3M1, CH3N(CH2CH2)2NC(O)-, O(CH2CH2)2NC(O)-, R1, or glycoside;
X3 and X'3 are independently O, NH, NHC(O), OC(O), -C(O)O, R1, or absent;
Figure imgf000130_0003
X 1, X2, R1, R2, M1, and M2 are the same defined in Claims 1 and 2.
22. The conjugate compound of claim 2, wherein "Drug1" and "Drug2" are a Daunorubicin or Doxorubicin analogue, the conjugate compound of Formula (II) is preferred structures of Da01, Da02, Da03 and Da04 as following:
Figure imgf000130_0002
Figure imgf000131_0001
Wherein mAb is an antibody; n is 1-30; X3 and X'3 are independently H, O, NH, NHC(O),
NHC(O)NH, C(O), R1, or OC(O); X1 X2, R1, and R2 are the same defined in
Figure imgf000131_0003
Claims 1 and 2.
23. The conjugate compound of claim 2, wherein "Drug1" and "Drug2" are an Auristatin and dolastatin analogue, the conjugate compound of Formula (II) is preferred structures of Au01, Au02, Au03, Au04, and Au05 as following:
Figure imgf000131_0002
Figure imgf000132_0001
Wherein mAb is an antibody; n is 1~30; X3 and X'3 are independently CH2, O, NH, NHC(O), NHC(O)NH, C(O), OC(O) R1, or absent; X4 and X'4 are independently CH2, C(O), C(O)NH, C(O)N(R1), R1, NHR1, NR1, C(O)R1 or C(O)O; Z3 and Z'3 are independently H, R1,
OP(O)(OMi)(OM2), NHR1, OCH2OP(O)(OMi)(OM2), OSO3M1, or O-glycoside (glucoside, galactoside, mannoside, glucuronoside, alloside, fructoside), NH-glycoside, S-glycoside, or
CH2-glycoside; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, NR1R2R3;
Figure imgf000132_0003
X1, X2, R1, R2 and R3 are the same defined in Claims 1 and 2.
24. The conjugate compound of claim 2, wherein "Drug1" and "Drug2" are a benzodiazepine dimer analogues, the conjugate compound of Formula (II) is preferred structures of PB01, PB02,
Figure imgf000132_0002
Figure imgf000134_0001
Figure imgf000135_0001
Wherein mAb is an antibody; n is 1-30; X3 and X'3 are independently CH2, O, NH, NHC(O), NHC(O)NH, C(O), OC(O), OC(O)(NR3), R1, NHR1, NR1, C(O)R1 or absent; X4 and X'4 are independently CH2, C(O), C(O)NH, C(O)N(R1), R1, NHR1, NR1, C(O)R1 or C(O)O; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, NR1R2R3;
Figure imgf000135_0002
X1, X2, R1, R2 and R3 are the same defined in Claims 1 and 2. In addition, R1 and/or R2 can be absent.
25. The conjugate compound of claim 2, wherein "Drug1" and "Drug2" are preferred two different cytotoxic agents selected from any combinations of tubulysins, maytansinoids, taxanoids (taxanes), CC-1065 analogs, daunorubicin and doxorubicin compounds, benzodiazepine dimers (e.g., dimers of pyrrolobenzodiazepine (PBD), tomaymycin, anthramycin, indolinobenzodia- zepines, imidazobenzothiadiazepines, or oxazolidino-benzodiazepines), calicheamicins and the enediyne antibiotics, actinomycin, azaserines, bleomycins, epirubicin, tamoxifen, idarubicin, dolastatins, auristatins (e.g. monomethyl auristatin E, MMAE , MMAF, auristatin PYE, auristatin TP, Auristatins 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)), duocarmycins, thiotepa, vincristines, hemiasterlins, nazumamides, microginins, radiosumins, alterobactins,
microsclerodermins, theonellamides, esperamicins, PNU-159682, and their analogues and derivatives. The more preferred structures of the conjugates containing two or more different cytotoxic agents via the bridge linker are as the following Z01, Z02, Z02, Z04, Z05, Z06, Z07, Z08, Z09, Z10, Z12, Z13, Z14, Z15, Z16, Z17 and Z18:
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Wherein mAb is an antibody; n is 1~30; X3 and X'3 are independently CH2, O, NH, NHC(O), NHC(O)NH, C(O), OC(O), OC(O)(NR3), R1, NHR1, NR1, C(O)R1 or absent; X4 and X'4 are independently H, CH2, OH, O, C(O), C(O)NH, C(O)N(R1), R1, NHR1, NR1, C(O)R1 or C(O)O; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, NR1R2R3;
Figure imgf000139_0003
X1, X2, R1, R2 and R3 are the same defined in Claims 1 and 2. In addition, R1 and/or R2 can be absent.
26. The conjugate compound of claim 2, wherein "Drug1" and "Drug2" are a polyalkylene glycol analog, the conjugate compound of Formula (II) is preferred structures of PgOl:
Figure imgf000139_0002
Wherein mAb is an antibody; n is 1-30; R' and R" are independently H or CH3; m3 and m4 are independently 1-5000;
Figure imgf000140_0002
X1, X2, R1, R2 and R3 are the same defined in Claims 1 and 2, R4 is OH, H, or R1, or R3 that is defined as in Claim 1.
27. The conjugate compound of claim 2, wherein "Drug1" and "Drug2" are a cell-binding ligand or receptor analog, the conjugate compound of Formula (II) is preferred structures: LB01 (PMSA ligand conjugate), LB02 (Folate receptor conjugate), LB03 (Somatostatin receptor conjugate), LB04 (Octreotide, a Somatostatin analog receptor conjugate), LB05 (Lanreotide, a Somatostatin analog receptor conjugate), LB06 (CAIX receptor conjugate), LB07(CAIX receptor conjugate), LB08 (luteinizing hormone-releasing hormone (LH-RH) ligand and GnRH conjugate), LB09 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand conjugate), LB 10 (GnRH antagonist, Abarelix conjugate), LB 11 (cobalamin, VB 12 analog conjugate), LB 12 (Gastrin releasing peptide receptor (GRPr), MBA conjugate), LB 13 (ανβ3 integrin receptor, cyclic RGD pentapeptide conjugate), LB 14 (hetero-bivalent peptide ligand for VEGF receptor conjugate), LB 15
(Neuromedin B conjugate), LB 16 (a G-protein coupled receptor, bombesin conjugate) and LB 17 a Toll-like receptor, TLR2 conjugate).
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Wherein mAb is an antibody; n is 1-30; X3 and X'3 are independently CH2, O, NH, NHC(O), NHC(O)NH, C(O), OC(O), OC(O)(NR3), R1, NHR1, NR1, C(O)R1 or absent; X4 and X'4 are independently H, CH2, OH, O, C(O), C(O)NH, C(O)N(R1), R1, NHR1, NR1, C(O)R1 or C(O)O; Mi and M2 are independently H, Na, K, Ca, Mg, NH4, NR1R2R3; m3 and m4 is 0-5000,
Figure imgf000145_0001
X1,
X2, R1, R2 and R3 are the same defined in Claims 1 and 2. In addition, R1 and/or R2 can be absent.
28. A pharmaceutical composition comprising a therapeutically effective amount of the conjugate compounds of claims 2, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and/or 27, and a pharmaceutically acceptable salt, carrier, diluent, or excipient therefore, or a combination thereof, for the treatment or prevention of a cancer, or an autoimmune disease, or an infectious disease.
29. The conjugate of claim 2, 17, 18, 19, 20, 21, 22, 23 24, 25, 26, and/or 27, having in vitro, in vivo or ex vivo cell killing activity.
30. The conjugate compound of claim 2, 17, 18, 19, 20, 21, 22, 23 24, 25, 26, and/or 27, can comprise either a peptides of 1-20 units of natural or unnatural amino acids, or a p-aminobenzyl unit, or a 6-maleimidocaproyl unit, or a disulfide unit, or a thioether unit, or a hydrozone unit, a triazole unit, or an alkoxime unit, among the linkage components R1 and/or R2.
31. The conjugate of claim 2, 17, 18, 19, 20, 21, 22 23, 24, 25, 26, and/or 27, wherein the linkage components R1 and/or R2 can be cleavable by a protease.
32. A pharmaceutical composition comprising a therapeutically effective amount of the conjugate of claim 2, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and/or 28, administered concurrently with the other therapeutic agents such as the chemotherapeutic agent, the radiation therapy, immunotherapy agents, autoimmune disorder agents, anti-infectious agents or the other conjugates for synergistically effective treatment or prevention of a cancer, or an autoimmune disease, or an infectious disease.
33. The synergistic agents according to claim 32 are preferably selected from one or several of the following drugs: Abatacept (Orencia), Abiraterone acetate (Zytiga®), Acetaminophen /hydrocodone, Adalimumab, afatinib dimaleate (Gilotrif®), alemtuzumab (Campath®),
Alitretinoin (Panretin®), ado-trastuzumab emtansine (Kadcyla™), Amphetamine mixed salts (Amphetamine/dextroamphetamine, or Adderall XR), anastrozole (Arimidex®), Aripiprazole, Atazanavir, Atezolizumab (MPDL3280A), Atorvastatin, axitinib (Inlyta®), belinostat
(Beleodaq™), Bevacizumab (Avastin®), Cabazitaxel (Jevtana®), Cabozantinib
(Cometriq™), bexarotene (Targretin®), blinatumomab (Blincyto™), Bortezomib
(Velcade®), bosutinib (Bosulif®), brentuximab vedotin (Adcetris®), Budesonide,
Budesonide/formoterol, Buprenorphine, Capecitabine, carfilzomib (Kyprolis®), Celecoxib, ceritinib (LDK378/Zykadia), Cetuximab (Erbitux®), Ciclosporin, Cinacalcet, crizotinib
(Xalkori®), Dabigatran, dabrafenib (Tafinlar®), Darbepoetin alfa, Darunavir, imatinib mesylate (Gleevec®), dasatinib (Sprycel®), denileukin diftitox (Ontak®), Denosumab (Xgeva®), Depakote, Dexlansoprazole, Dexmethylphenidate, Dinutuximab (Unituxin™), Doxycycline, Du- loxetine, Emtricitabine/Rilpivirine/Tenofovir disoproxil fumarate,
Emtricitabine/tenofovir/efavirenz, Enoxaparin, Enzalutamide (Xtandi®), Epoetin alfa, erlotinib (Tarceva®), Esomeprazole, Eszopiclone, Etanercept, Everolimus (Afinitor®), exemestane (Aromasin®), everolimus (Afinitor®), Ezetimibe, Ezetimibe/simvastatin, Fenofibrate,
Filgrastim, fingolimod, Fluticasone propionate, Fluticasone/salmeterol, fulvestrant (Faslodex®), gefitinib (Iressa®), Glatiramer, Goserelin acetate (Zoladex), Imatinib (Gleevec), Ibritumomab tiuxetan (Zevalin®), ibrutinib (Imbruvica™), idelalisib (Zydelig®), Infliximab, Insulin aspart, Insulin detemir, Insulin glargine, Insulin lispro, Interferon beta la, Interferon beta lb, lapatinib (Tykerb®), Ipilimumab (Yervoy®), Ipratropium bromide/salbutamol, Lanreotide acetate (Somatuline® Depot), lenaliomide (Revlimid®), lenvatinib mesylate (Lenvima™), letrozole (Femara®), Levothyroxine, Levothyroxine, Lidocaine, Linezolid, Liraglutide,
Lisdexamfetamine, MEDI4736 (AstraZeneca, Celgene), Memantine, Methylphenidate,
Metoprolol, Modafinil, Mometasone, Nilotinib (Tasigna®), Nivolumab (Opdivo®),
ofatumumab (Arzerra®), obinutuzumab (Gazyva™), olaparib (Lynparza™), Olmesartan, Olmesartan/hydrochlorothiazide, Omalizumab, Omega-3 fatty acid ethyl esters, Oseltamivir, Oxycodone, palbociclib (Ibrance®), Palivizumab, panitumumab (Vectibix®), panobinostat (Farydak®), pazopanib (Votrient®), pembrolizumab (Keytruda®), Pemetrexed (Alimta), pertuzumab (Perjeta™), Pneumococcal conjugate vaccine, pomalidomide
(Pomalyst®), Pregabalin, Quetiapine, Rabeprazole, radium 223 chloride (Xofigo®),
Raloxifene, Raltegravir, ramucirumab (Cyramza®), Ranibizumab, regorafenib
(Stivarga®), Rituximab (Rituxan®), Rivaroxaban, romidepsin (Istodax®), Rosuvastatin, ruxolitinib phosphate (Jakafi™), Salbutamol, Sevelamer, Sildenafil, siltuximab
(Sylvant™), Sitagliptin, Sitagliptin/metformin, Solifenacin, Sorafenib (Nexavar®), Sunitinib (Sutent®),Tadalafil, tamoxifen, Telaprevir, temsirolimus (Torisel®), Tenofovir/emtricitabine, Testosterone gel, Thalidomide (Immunoprin, Talidex), Tiotropium bromide, toremifene
(Fareston®), trametinib (Mekinist®), Trastuzumab, Tretinoin (Vesanoid®), Ustekinumab, Valsartan, vandetanib (Caprelsa®), vemurafenib (Zelboraf®), vorinostat (Zolinza®), ziv- aflibercept (Zaltrap®), Zostavax., and their analogs, derivatives, pharmaceutically acceptable salts, carriers, diluents, or excipients therefore, or a combination above thereof.
PCT/IB2015/056083 2015-08-10 2015-08-10 Novel linkers and their uses in specific conjugation of drugs to a biological molecule Ceased WO2015155753A2 (en)

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