Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C233/00—Carboxylic acid amides
  • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
  • C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
  • C07C233/17—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
  • C07C233/20—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a carbon atom of an acyclic unsaturated carbon skeleton
• • 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
• • 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
  • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
• • 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
  • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
• • 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
  • A61K47/6835—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
  • A61K47/6869—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
• • 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
  • 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
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
  • C07C59/40—Unsaturated compounds
  • C07C59/76—Unsaturated compounds containing keto groups
• • 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
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

• the present invention relates to the preparation of novel linkers used for the specific conjugation of a compound, in particular, a cytotoxic agent to a biological molecule.
• 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.
• Proteins, specifically antibodies have been extensively used in therapeutic applications, in vitro assays as research reagents and in vivo as diagnostic tools or as therapeutic drags (Gad, S. C. Drug discovery handbook, published by Wiley-Interscience, 2005).
• the protein needs to be modified with an interesting group, such as a cytotoxic drug, a radio label element or a chromatographic molecule for use in therapy or a detection agent when used in diagnostics (Teicher, B. A. et al. Clin. Cancer Res. 2011, 17, 6389-97; Elsadek, B. et al., J. Control Release, 2012, 157, 4-28).
• ADCs antibody-drug conjugates
• 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
• ADCs obtained by conjugation to cysteine side chains often display limited stability in circulation, leading to premature disconnection of the cytotoxic payload before the tumor site is reached (Junutula, I. R., et al 2008, Nat. Biotechnol. 26, 925-32).
• Disulfide bond structure is critical for the structure, stability, and biological functions of IgG molecules.
• IgGi, IgG 2 , IgG 3 and IgG 4 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. But, for IgG 2 , IgG 3 and IgG 4 , 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
• 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
• acetylenedicarboxyl group particularly when two cytotoxic agents are linked at both ends of the stretch-out bridge linker, forming a quite large size (>20 A) of molecule which in turn hardly accesses to the other disulfide bond sites, such as intra chain disulfide bonds beneath the complex antibodies.
• the disulfide bridge linkers of this invention therefore can be used for selective bridging the pairs of free thiols on the inter chain of antibody, which are generated by overloaded TCEP or DTT, and for producing an ADC having DAR (drugs per antibody) over four.
• bridge linkers can be recoupled (regenerated) by an oxide, e.g. dehydroascorbic acid (DHAA) or Cu(II), at the end of conjugation.
• DHAA dehydroascorbic acid
• Cu(II) Cu(II)
• the present invention provides linkers containing an acetylenedicarboxylic 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:
• Cb is a cell-binding agent
• L is a linker
• Drugl and Drug2 are a drug molecule
• n is an integer from 1 to 20
• S (sulfur) elements from Cb bridgely link to L which covalently connects two or more drugs (per bridge linker L).
• 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 bonds 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 sites of IgG antibodies, resulting in homogeneous production of ADC.
• the linker is represented by Formula (I)
• acetylenedicarboxyl group on the linker is capable of reacting with a pair of sulfur atoms of the cell-binding agent
• Zi and Z 2 are the same or different a function group that enables to react with a cytotoxic drug.
• the functional group Zi or Z 2 can link to a cytotoxic drug via a disulfide, ether, ester, thioether, thioester, peptide, hydrazone, carbamate, carbonate, amine (secondary, tertiary, or quartary), imine, cycloheteroalkyane, heteroaromatic, alkoxime or amide bond;
• Ri and R 2 are the same or different, and are absent, linear alkyl having from 1-6 car- bon 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.
• Ri 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 Xi or X 2 and Zi or Z 2 .
• the atoms used in forming the Ri and R 2 may be combined in all chemically relevant ways, such as forming alkylate, 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.
• Xi and X 2 are independently selected from N(R 3 ), O, S or CH 2 ;
• 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.
• 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
• Drugi 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;
• n 1 ⁇ 20; Ri, R 2 , Xi and X 2 are described the same previously in Formula (I).
• the present invention provides a modified cell-binding agent of Formula (III), in which the cell-binding agent, Cb, has reacted with the bridge linker, which has Zi and Z 2 , the function groups capable of reacting with a drug:
• the present invention provides a modified drug of Formula (IV), in which the drug, Drugi and Drug 2 , have reacted with the linker of Formula (I), which still has the acetylenedicarboxyl group capable of reacting with a pair of sulfur atoms of the cell-binding agent:
• Drugi Drug 2 , Ri, R 2 , Xi, and X 2 are defined the same as in Formula (I) and
• the present invention further relates to a method of making a cell-binding molecule- drug conjugate of Formula (II), wherein the drugs, Drugi 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
• Figure 1 shows the synthesis of a bridge linker containing polyethylene glycol groups and the application of this linker in the conjugation of an antibody with drugs.
• Figure 2 shows the synthesis of a bridge linker and the application of this linker in the conjugation of drugs to an antibody via oxime linkage.
• Figure 3 shows the synthesis of a bridge linker containing a peptide and the application of this linker in the conjugation of drugs to an antibody via amide linkage.
• Figure 4 shows the synthesis of a bridge linker containing peptides, polyethylene glycol.
• Figure 5 shows the synthesis of a bridge linker containing peptides and polyethylene glycols, and the application in the conjugation of two or four drugs per linker to an antibody via amide linkage.
• Figure 6 shows the synthesis of tubulysin analogs which are modified with the bridge- linker containing peptides and polyethylene glycols.
• Figure 7 shows the synthesis of the conjugates of cell-binding molecule-tubulysin analogs via the bridge-linker containing polyethylene glycols.
• Figure 8 shows the synthesis of the conjugates of cell-binding molecule- maytansinoids via the bridge-linker.
• Figure 9 shows the synthesis of the conjugates of cell-binding molecule-MMAF analogs via the bridge-linker.
• Figure 10 shows the synthesis of the conjugates of cell-binding molecule-tubulysin analogs via the bridge-linker.
• 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, z ' -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, w-octyl, and iso
• Ci-Cg alkyl group can be unsubstituted or substituted with one or more groups including, but not limited to, -Ci-Cg alkyl,-0-(Ci-Cg alkyl), -aryl, -C(0)R', -OC(0)R', -C(0)OR', -C(0)NH 2 , -C(0)NHR', -C(0)N(R') 2 , -NHC(0)R', - SR', -S(0) 2 R', -S(0)R', -OH, -halogen, -N 3 , -NH 2 , -NH(R'), -N(R') 2 and -CN; where each R' is independently selected from -Ci-Cg alkyl and aryl.
• "Halogen” refers to fluorine, chlorine, bromine or iodine atom; preferably fluorine and chlorine atom.
• Heteroalkyl refers to C 2 -Cs 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.
• 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 "C 3 -Cg carbocycle” refers to a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated nonaromatic carbocyclic ring.
• a C 3 -Cs carbocycle group can be unsubstituted or substituted with one or more groups including, but not limited to, -Q-Cs alkyl,-0-(Ci-C8 alkyl), -aryl, -C(0)R', -OC(0)R', -C(0)OR', -C(0)NH 2 , -C(0)NHR', -C(0)N(R) 2 , - NHC(0)R', -SR', -S(0)R',-S(0) 2 R', -OH, -halogen, -N 3 , -NH 2 , -NH(R'), -N(R') 2 and -CN; where each R' is independently selected from -Ci-Cs 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.
• 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, rc-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 3 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 p-toluenesulfonate.
• leaving group refers to a functional group that can be substituted by another functional 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 trifluoromethylsulfonate.
• a halide e.g., chloride, bromide, and iodide
• methanesulfonyl meyl
• /?- toluenesulfonyl tosyl
• triflate trifluoromethylsulfonate
• DMSO dimethylsulf oxide
• DTT dithiothreitol
• EDC l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride
• ESI-MS electrospray mass spectrometry
• HATU 0-(7- azabenzotriazol-l-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate
• HOBt 1- hydroxybenzotriazole
• HPLC high pressure liquid chromatography
• NHS N- Hydroxysuccinimide
• MMP 4-methylmorpholine
• ⁇ ⁇ -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
• “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.
• 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.
• novel conjugates disclosed herein use the bridge linkers. Examples of some suitable linkers and their synthesis are shown in Figures 1 to 10.
• 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- 9.
• the bridge linkers possess two elements: a) A Substituent that is acetylenedicarboxyl group that 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 and hydrazide, capable of reaction with a drug.
• the bridge substitu- ents of acetylenedicarboxyl can be introduced by direct condensation of
• acetylenedicarboxyl can be introduced by condensation of acetylene with acid halides or acid anhydrides to form carbon-carbon bonds acetylenedicarboxyl sites.
• the synthesis of these bridge linkers is exampled in the Figure 2 and 10.
• the bridge linkers are compounds of the Formula (I) below:
• acetylenedicarboxyl group on the linker is capable of reacting with a pair of sulfur atoms of the cell-binding agent
• Zi and Z 2 are the same or different a function group that enables to react with a cytotoxic drug.
• the functional group Zi or Z 2 can link to a cytotoxic drug via a disulfide, thioether, thioester, peptide, hydrazone, ether, ester, carbamate, carbonate, amine (second- ary, tertiary, or quarter), imine, cycloheteroalkyane, heteroaromatic, alkoxime or amide bond;
• Ri and R 2 are the same or different, and are 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 , or polypropyleneoxy unit of formula (OCH 2 (CH 3 )CH 2 ) p wherein p is an integer from 0 to about 1000, or combination thereof.
• Ri 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 Xi or X 2 and Zi or Z 2 .
• the atoms used in forming the Ri and R 2 may be combined in all chemically relevant ways, such as forming alkylate, 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.
• Xi and X 2 are independently selected from N(R 3 ), O, S or CH 2 ;
• 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.
• Ri, R 2 , and R 3 can be respectively a chain of atoms selected from C, N, O, S, Si, and P that covalently connects the cell-surface binding molecule and the conjugated drug.
• the atoms used in forming the bridge linker may be combined in all chemically relevant ways, such as forming alkylate, 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 group, Zi 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 group, Zi 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; with the terminal of thiol can be, as but not limited to, pyridyldisulfides, nitropyridyldisulfides, maleimides, haloacetates and carboxylic acid chlorides; with the terminal of ketone or aldehyde can be, as but not limited to, amines, alkoxyamines, hydrazines, acyloxylamine; with the terminal of azide can be, as but not limited to, alkyne.
• the key step of synthesis of the bridge linker containing acetylenedicarboxyl groups is the condensation of the acetylenedicarboxylic 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 Xi or X 2 described in Formula (I) as N(R 3 ), O, or S ;
• R is Ri and/or R 2 that described in Formula (I);
• R3 is the same in Formula (I).
• Lvi and Lv 2 are the same or independently OH; F; CI; Br; I; 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, anhydrides formed its self, or formed with the other anhydride, e.g.
• condensation reagents are: EDC (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide), DCC (Dicyclohexyl-carbodiimide), ⁇ , ⁇ '-Diisopropylcarbodiimide (DIC), N-Cyclohexyl- N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC,or CME-CDI), 1, 1 '- Carbonyldiimidazole (CDI),TBTU (0-(Benzotriazol- l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), N,N,N',N
• hexafluorophosphate HBTU
• B OP Benzotriazol- l-yloxy
• PyBOP Benzotriazol- 1 -yloxy
• DEPC Diethyl cyanophosphonate
• HATU Chloro- ⁇ , ⁇ , ⁇ ', ⁇ '- tetramethylformamidinium hexafluorophosphate, l-[Bis(dimethylamino)methylene]-lH- l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
• HDMA 2-Ch
• CIP Chlorotripyrrolidinophosphonium hexafluorophosphate
• PyCloP Chlorotripyrrolidinophosphonium hexafluorophosphate
• BFFH Fluoro-N,N,N',N'-bis(tetramethylene)formamidinium hexafluorophosphate
• BTFFH Fluoro-N,N,N',N'-bis(tetramethylene)formamidinium hexafluorophosphate
• BTFFH Tripyrrolidinophosphonium hexafluorophosphate
• BTFFH Tripyrrolidinophosphonium hexafluorophosphate
• BTFFH Fluoro-N,N,N',N'-bis(tetramethylene)formamidinium hexafluorophosphate
• TPTU 0-(2-Oxo-l(2H)pyridyl)-N,N,N',
• 2-Morpholinoethyl isocyanide MEI
• HSTU N,N,N',N'-Tetramethyl-0-(N-succinimidyl)uronium hexafluorophosphate
• BEP 2-Bromo-l-ethyl-pyridinium tetrafluoroborate
• TOTU O- [(Ethoxycarbonyl)cyanomethylenamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate
• TOTU 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-0-(N-succinimid
• TDBTU 0-(3,4-Dihydro-4-oxo-l,2,3-benzotriazin-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate
• LD -(Azodicarbonyl)dipiperidine
• DCAD Di-(4-chlorobenzyl) azodicar- boxylate
• DBAD Di-tert-butyl azodicarboxylate
• DID Diisopropyl azodicarboxylate
• DEAD Diethyl azodicarboxylate
• M is Na, K, Li, Cu, CuLi, Sn, Ti, Ca, Mg or Zn.
• bridge linkers The detail examples of the synthesis of the bridge linkers are shown in the figures 1-10. Normally the bridge substituents of acetylenedicarboxyl can be condensated with linker components containing function groups capable to react to drugs of desired conjugation.
• the conjugates of the present invention can be represented by the following formula, s a cell-binding agent, L is a linker, Drugl and
• Drug2 are a drug molecule, n is an integer from 1 to 20, 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 -CH 2 CH 2 0- one or more repeating units (“EO” or “PEO”). Additional linker components are known the art and some are described herein.
• Cb represents a cell-binding agent, preferably an antibody
• Drugi 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.
• Drugi 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 Zi and Z 2, whose reactive groups can be a disulfide, maleimido, haloacetyl, azide, 1- yne, ketone, aldehyde, alkoxyamino, or hydrazide.
• the 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., Hinman, L. M., 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
• 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) bearing 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)
• Zi and Z 2 are a disulfide substituent, maleimido, haloacetyl, alkoxyamine, azido, ketone, aldehyde, hydrazine group, an N-hydroxysuccinimide ester, or a carboxyl ester formed with phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxa- zolium-3 '-sulfonate.
• Zi and Z 2 can then react with a cytotoxic agent through thioether, hydrazone, amide, alkoxime, carbamate, ester, ether or disulfide bond.
• 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 KCl.
• the progress of the reaction can be monitored by measuring the decrease in the absorption at 254 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 Capillary electrophoresis-mass spectrometry (CEMS).
• 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):
• Drugi, Drug 2 , Z 1; Z 2 , n, R 1; R 2 , Xi, and X 2 are defined the same as in Formu- la (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 acetylenedi-carboxyl group capable of reacting with a pair of thiol groups of a cell-binding agent.
• the acetylenedicarboxyl group is synthesized through condensation with acetylene via the methods described in reaction equation (la), (lb), (Ic), (Id), (Ie), (If), (Ig) and (Hi).
• the Drugi or Drug 2 may be synthesized to connect to Ri, or R 2 in a piece of components via the linkage of thioether, thioester or disulfide bond first. Then the synthesized Ri- Drugi or R 2 -Drug 2 component is assembled to an acetylenedicarboxyl group to form the bridge linker modified drugs of Formula (IV).
• a thiol-containing drug can be reacted with the linker of components Ri or R 2 bearing a maleimdo substituent at neutral pH in aqueous buffer to give a Ri -Drugi or R 2 -Drug 2 compartment bearing thioether linkage, and following by condensation with a compartment of acetylenedicarboxyl group to give a modified drug of
• Formula (IV) bearing thioether linkage A drug bearing a hydroxyl group can be reacted with a linker component Ri or R 2 bearing a halogen, or a tosylate, or a mesylate, in the presence of a mild base, to give a Ri-Drugi or R 2 -Drug 2 compartment bearing ether linkage, and following by condensation with a compartment of acetylenedicarboxyl group to give a modified drug of Formula (IV) bearing thioether linkage.
• Ri or R 2 bearing a halogen or a tosylate, or a mesylate
• 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 Ri or R 2 bearing a maleimido or a vinylsulfonyl, or a haloacetyl group, give a Ri-Drugi or R 2 -Drug 2 compartment bearing thioether linkage, and following by condensation with a compartment of acetylenedicarboxyl group 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 alumi- na, 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.
• fragments produced by a Fab expression library fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR's, diabody, triabody, 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
• interferons such as type I, II, III
• 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 (melano- cyte-stimulating hormone), steroid hormones, such as androgens and estrogens, melano- cyte-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
• 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
• PSMA pro state- specific membrane antigen
• TKI tumor necrosis factor-like protein
• 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.
• 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.
• DMEM Dulbecco's minimal essential medium
• 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).
• EBV Epstein-Barr virus
• HHV-4 human herpesvirus 4
• 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. 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.
• 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);
• 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.
• 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.
• 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).
• 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, I Immunol Methods.
• Antibodies immunospecific 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 immunospecific 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 Aselizumab (anti-L-selectin
• 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 1 (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-CD 19), 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
• TRAIL-R2 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 gerlimomab
• Dorlixizumab Ecromeximab
• Eculizumab Soliris, anti-C5
• Edobacomab anti-endotoxin
• Edrecolomab Panorex,
• MAM7-1A anti-EpCAM
• Efalizumab Raptiva, anti-LFA-1 (CDl la)
• Efungumab Mycograb, anti-Hsp90
• Elotuzumab anti-SLAMF7
• Elsilimomab anti-IL-6
• Enlimomab pegol (anti-ICAM- 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), Fre
• 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 receptor)), Inotuzumab (anti-CD22), Ipilimumab (anti-CD 152), Iratumumab (anti- CD30 (TNFRSF8)),
• 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 04), Nebacumab (anti-endo toxin), Necitumumab (anti-EGFR), Nere
• VEGFR2 VEGFR2
• Ranibizumab Luciferis, 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
• 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 ⁇ 4 ⁇ ? ), Veltuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1), Visilizumab (Nuvion, anti-CD3), Vitaxin (anti-vascular integrin avb3),
• Volociximab anti-integrin asPi
• Votumumab HuaSPECT
• 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.
• 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 TNFSF2
• MEDI-500 T10B9
• anti-CD3, TR P 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 adhe- sion molecule)
• anti-TACSTDl Tumor-associated calcium signal transducer 1
• GA733-2 gastrointestinal tumor-associated protein 2
• anti-EGP-2 epidermal glycoprotein 2
• anti-KSA epidermal glycoprotein 2
• KS1/4 antigen antigen 17-1 A
• LymphoCide Immunomedics, NI), Smart ID 10 (Protein Design Labs), Oncolym (Techniclone Inc, CA), Allomune (BioTransplant, CA), anti-VEGF (Genentech, CA); CEAcide (Immunomedics, NJ), IMC- 1C11 (ImClone
• antibodies as cell binding molecules/ligands include, but are not limited to, are antibodies against the following antigens: Aminopeptidase N (CD 13), 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 (carcinomas), 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), CD19 (B cell malignancies), CD20 (non-Hodgkin's lympho
• Receptor 2 lung, breast, prostate cancers
• FCGR1 autoimmune diseases
• FOLR farnesol 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 breast2
• HLA-DR10 HLA-DRB
• IGF1R IGF1R
• 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 anti-transferrin receptor
• 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), ROB04, TAG 72 (t
• 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 melanocytes. 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,
• 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),
• 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, CYP1B1, 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,
• 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- ⁇ , PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, Platelet-derived growth factor receptor beta, Phosphate- sodium co- transporter, PMEL 17, Polysialic acid, Proteinase3 (PR1), Pro
• Phosphatidylserine Prostatic carcinoma cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, Rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD240), Rhesus factor,
• T-cell receptor T cell transmembrane protein
• TEMl Tumor endothelial marker 1
• TENB2 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 (tumor necrosis factor receptor superfamily member 10B), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B)
• TPBG Tropoblast 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 (CD106), 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.
• 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, E tragonadal, 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 Car
• Plasma Cell Neoplasms Mycosis Fungoides, Myelodysplastic Syndrome, Myeloproliferative 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,
• the cell-binding-drug conjugates via the bridge likers of this invention are used in accordance with the compositions and methods for the treatment 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,
• 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,
• 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, Vit
• 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;
• 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
• 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 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 e.g. CD27, CD40, CD95/Fa
• 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, polypep- tide 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.
• 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; PR0542 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.
• 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 metapneumovirus 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), Plague, Pneumococcal infection, Pneumocystis pneumonia, Pneumonia, Poliomyelitis, Prevotella infection, Primary amoebic meningoencephalitis, Progressive
• 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
• 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
• 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 0157: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
• Varicella zoster virus Variola major or Variola minor, Sporofhrix 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 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 (
• 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 (Ra- bies), 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 nas
• 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, Coro- navirus; 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)] .
• 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 auto- immune 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.
• 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/niL) can be added. Dosages will be about 50 ⁇ g to 20 nig/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).
• human serum albumin
• 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 (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 administered 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).
• Vinca alkaloids (vincristine, vinblastine, vindesine, vinorelbine, navelbin)
• Taxoids (paclitaxel, docetaxol) and their analogs
• Maytansinoids DM1,
• DNA Topoisomerase Inhibitors such as [Epipodophyllins: (9-aminocamptothecin, camptothecin, crisnatol, daunomycin, etoposide, etoposide phosphate, mnotecan, 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
• 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 propio- nate, 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- al
• 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,
• calicheamicins especially calicheamicin ⁇ , ⁇ , al and ⁇ , 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, Salinospor amide 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, fluocortolone danazol, dexamethasone, Triamcinolone acetonide, beclometasone dipropionate), DHEA, enanercept,
• corticosteroids e.g. amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, fluocortolone danazol, dexamethasone, Triamcinolone acetonide, beclometasone dipropionate
• 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
• Glycopep tides 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, 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,
• Sulfonamides mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanamide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim- sulfamethoxazole (co-trimoxazole); s).
• Steroid antibacterials e.g. fusidic acid; t).
• Tetracyclines doxycycline, chlortetracycline, clomocycline,
• antibiotics include 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,
• Bacitracin Bactoprenol inhibitors
• DADAL/AR inhibitors cycloserine
• dictyostatin discodermolide
• eleutherobin epothilone
• ethambutol etoposide
• faropenem fusidic acid
• furazolidone
• 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; c). Maturation inhibitors: bevirimat, becon; d). Neuramini- dase 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.
• 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, diary lpyrimidines, 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 include radioisotopes.
• radioisotopes are 3 H, n C, 14 C, 18 F, 32 P,
• 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, N.Y., Pubs. (1991).
• Chelating ligands which may complex a metal ion include DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, Tex.).
• 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.
• chromophore molecule can be selected from any analogs and derivatives of the following fluorophore compounds: CF dye (Biotium), DRAQ and CyTRAK probes
• 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, 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-Maleimi
• 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),
• 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.
• 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 (monomer, MBL), mCFP, mCherry, mCitrine, Midoriishi Cyan (dimer, MBL), mKate
• 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
• 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.
• tubulysins for conjugation of cell binding molecules are described in the patent application of PCT/IB2012/053554.
• mAb is an antibody
• 3 ⁇ 4 is H, OP(0)(OMi)(OM 2 ), OCH 2 OP(0)(OM 1 )(OM 2 ), OSO 3 M 1 , or 0-, NH-, S- or CH 2 - glycoside (glucoside, galactoside, mannoside, glucuronoside, alloside, fructoside, etc);
• Mi and M 2 are independently H, Na, K, Ca, Mg, NR 1 R 2 3 ; n is 1-20;
• Xi, X 2 , Ri, R 2 and R3 are the same defined in Formula (I).
• mAb is an antibody; n is 1-20; Xi, X 2 , Ri, R 2 and R 3 are the same defined in Formula (I).
• 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 1-20; Xi, X 2 , Ri, R 2 and R 3 are the same defined ' Formula (I).
• 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.,
• Examples of the structures of the conjugate of the antibody- taxanes via the bridge linker are as the following TxOl, Tx02 and Tx03.
• mAb is an antibody; n is 1-20; Xi, X 2 , Ri and R 2 are the same defined in Formula (I).
• CC-1065 analogues and doucarmycin analogs are also preferred to be used for a con- jugate 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:
• mAb is an antibody; n is 1-20; Z 4 is H, PO(OMi)(OM 2 ), SO 3 M 1 ,
• 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,
• mAb is an antibody; n is 1-20; X 3 is H, O, NH, NHC(O), NHC(0)NH, C(O), or OC(O); X 1; X 2 , Ri , and R 2 are the same defined in Formula (I).
• 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
• mAb is an antibody; n is 1-20; X 3 is CH 2 , O, NH, NHC(O), NHC(0)NH, C(O), OC(O) or absent; X 4 is CH 2 , C(O), C(0)NH, C(0)N(Ri), or C(0)0; X X 2 , Ri , R 2 and R 3 are the same defined in Formula (I).
• 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;
• 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 and PB08.
• mAb is an antibody; n is 1-20; X 3 is CH 2 , O, NH, NHC(O), NHC(0)NH, C(0), OC(O) or absent; X 4 is CH 2 , C(O), C(0)NH, C(0)N(Ri), or C(0)0; X t , X 2 , Ri, R 2 and R 3 are the same defined in Claim 1. In addition, Ri and/or R 2 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.
• 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 drug s/cy to toxic agents described herein.
• the drugs/cytotoxic agents of the present invention include analogues and derivatives of the compounds described herein.
• Example 4 13-Amino-4,7,10-trioxadodecanoic acid tert-butyl ester, 37; 13-Amino- bis(4,7,10-trioxadodecanoic acid tert-Butyl Ester), 38.
• the crude azide material 36 (5.0 g, -14.84 mmol) was dissolved in ethanol (80 mL) and 300 mg of 10% Pd/C was added. The system was evacuated under vacuum and placed under 2 atm of hydrogen gas via hydrogenation reactor with vigorous stirring. The reaction was then stirred overnight at room temperature and TLC showed that the starting materials disappeared. The crude reaction was passed through a short pad of celite rinsing with ethanol.
• Example 10 17-dioxo-4,7, 10,21 ,24,27 -hexaoxa- 13,18-diazatriacont- 15-yne- 1 ,30- dioic acid 86.
• Example 14 (R,R,S,S,R,4R,4'R)-5,5'-(((14,17-dioxo-4,7,10,21,24,27-hexaoxa- 13 , 18-diazatriacont- 15-yne- 1 ,30-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-carboxami- do)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-2-methylpentanoic acid), 90
• Example 15 Conjugated compound 90 to an antibody for 91.
• the mixture was purified on G-25 column eluted with 100 mM NaH 2 P0 4 , 50 mM NaCl pH 6.0-7.5 buffer to afford 16.8-17.9 mg of the conjugate compound 91 (-87% yield) in 13.1-14.9 ml buffer.
• the drug/antibody ratio (DAR) was 4.0, 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 16 Compound 92 (containing 4 tubulysin analogs per bridge linker).
• Example 17 Conjugated compound 92 to an antibody for 93
• 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 5A 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% C0 2 .
• MTT (5mg/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 15min, the absorbance was measured at 490nm and 570nm with a reference filter of 620nm.
• conjugate 91 and conjugate 93 were extremely more potent than the commercial conjugate T-DM1.

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