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  • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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Definitions

• a method consistent with the present invention relates to targeting specific cell populations using cell-binding agent maytansinoid conjugates linked via a non-cleavable linker. Another method consistent with the present invention is a method of making the conjugate.
• a composition consistent with the present invention relates to novel cell-binding agent maytansinoid conjugates where the maytansinoid is linked via a non-cleavable linker to the cell- binding agent.
• Another composition consistent with the present invention relates to novel maytansinoid esters.
• Maytansinoids are highly cytotoxic drugs. Maytansine was first isolated by Kupchan et al. from the east African shrub Maytenus serrata and shown to be 100- to 1000-fold more cytotoxic than conventional cancer chemotherapeutic agents like methotrexate, daunorubicin, and vincristine (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that some microbes also produce maytansinoids, such as maytansinol and C-3 esters of maytansinol (U.S. Pat. No.
• esters of group (b) were found to be much more cytotoxic than esters of group (a).
• Maytansine is a mitotic inhibitor. Treatment of L1210 cells in vivo with maytansine has been reported to result in 67% of the cells accumulating in mitosis. Untreated control cells were reported to demonstrate a mitotic index ranging from between 3.2 to 5.8% (Sieber et al., 43 Bibl. Haematol. 495-500 (1976)).
• Maytansine has also been shown to be active in vivo. Tumor growth in the P388 lymphocytic leukemia system was shown to be inhibited over a 50- to 100-fold dosage range, which suggested a high therapeutic index; also significant inhibitory activity could be demonstrated with the L1210 mouse leukemia system, the human Lewis lung carcinoma system and the human B-16 melanocarcinoma system (Kupchan, 33 Ped. Proc 2288-2295 (1974)). [09] Because the maytansinoids are highly cytotoxic, they were expected to be of use in the treatment of many diseases such as cancer. This expectation has yet to be realized. Clinical trials with maytansine were not favorable due to a number of side effects (Issel et al., 5 Cancer Treat.
• a conjugate of maytansinoids linked to the anti-Her2 breast cancer antibody TA.1 via the non-cleavable linker SMCC was shown to be 200- fold less potent than a conjugate of maytansinoids linked to TA.l via a linker having a cleavable disulfide bond (Chari et al., 52 Cancer Res. 127-133 (1992)).
• One aspect of the present invention is a method for targeting a maytansinoid to a selected cell population comprising contacting a cell population or tissue suspected of containing cells from said selected cell population with a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids is linked to the cell-binding agent via a non-cleavable linker.
• Another aspect of the present invention is a method for treatment of tumors, autoimmune diseases, graft rejections, graft versus host disease, viral infections, parasite infections, and other diseases that can be treated by targeted therapy wherein the targeting agent is a cell-binding agent, said method comprising administering to a subject in need of treatment an effective amount of a cell-binding agent maytansinoid conjugate wherein one or more maytansinoids is linked to the cell-binding agent, or a pharmaceutically acceptable formulation or solvate of said conjugate.
• Another aspect of the present invention is a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids is linked to a cell-binding agent via a non-cleavable linker.
• Another aspect of the present invention is a composition comprising the above-described conjugate.
• Another aspect of the present invention is a method of making the above-described conjugate.
• Another aspect of the present invention is novel maytansinoid esters.
• FIG. 1 shows the structure of SMCC.
• FIG. 2 shows the structure of DM 1.
• FIG. 3 shows graphically results of a FACS binding assay comparing huC242 antibody to the antibody-maytansinoid conjugate huC242-SMCC-DMl.
• FIG. 4 shows graphically the cytotoxicity of huC242-SMCC-DM 1.
• FIG. 5 shows size exclusion chromatography for huC242-SMCC-DMl .
• FIGS. 6A-C and FIG. 7 show graphically the cytotoxicity of huC242-SMCC-DMl compared to conjugates prepared with disulfide-containing linkers.
• FIGS. 8A-D show graphically the cytotoxicity of SMCC-DM1 conjugates linked to various cell-binding agents.
• FIG. 9 shows graphically the cytotoxicity of antibody-maytansinoid conjugate huC242-
• FIG. 10A shows graphically the antitumor activity of huC242-SMCC-DMl against COLO205 human colon cancer xenografts in SCID mice.
• FIG. 1 OB shows graphically the antitumor activity of huC242-SMCC-DM 1 against SNU16 human gastric tumor xenografts in SCID mice.
• FIG. IOC shows graphically the anti-tumor efficacy of trastuzumab-SMCC-DMl against human MCF7 tumor xenografts in SCID mice.
• FIG. 11 shows graphically plasma clearance rates of huC242-SMCC-DMl compared to conjugates prepared with disulfide-containing linkers.
• FIGS . 12 A-C show graphically results of acute toxicity studies of huC242-SMCC-DM 1 compared to conjugates prepared with disulfide-containing linkers.
• FIG. 13 shows the durability of cell-cycle arrest and cell destroying activity demonstrated by huC242-SMCC-DMl compared to conjugates prepared with disulfide-containing linkers.
• FIGS. 14A-D show the minimal bystander effect activity of huC242-SMCC-DMl compared to conjugates prepared with disulfide-containing linkers.
• FIG. 15 shows representative structures of maleimido-based cross-linking agents.
• FIG. 16 shows representative structures of haloacetyl-based cross-linking agents.
• FIG. 17 shows the structure of antibody-SMCC-DMl conjugates.
• FIG. 18 shows the structure of antibody-SIAB-DMl conjugates.
• FIG. 19 shows the structure of antibody-SMCC-DM4 conjugates.
• FIG. 20 shows the structure of antibody-SIAB-DM4 conjugates.
• FIG. 21 shows the synthesis of a maytansinoid cell-binding agent conjugate linked via a non-S-containing non-cleavable linker.
• FIG. 22 shows graphically cytotoxicity of huC242 -non-S-containing non-cleavable
• FIG. 23 shows graphically results of a FACS binding assay of huC242 -non-S-containing non-cleavable linker-DMl.
• FIG. 24 shows graphically results of a HER2 ECD plate-binding assay comparing trastuzumab antibody to the antibody-maytansinoid conjugate trastuzumab-SMCC-DMl.
• FIG. 25 shows graphically the cytotoxicity and specificity of trastuzumab-SMCC-DMl.
• FIG. 26 shows size exclusion chromatography for trastuzumab-SMCC-DMl .
• FIG. 27 shows graphically results of a HER2 ECD plate-binding assay comparing trastuzumab antibody to the antibody-maytansinoid conjugate trastuzumab-SIAB-DMl.
• FIG. 28 shows graphically the cytotoxicity and specificity of trastuzumab-SIAB-DMl.
• FIG. 29 shows size exclusion chromatography for trastuzumab-SIAB-DMl .
• 5,208,020 demonstrate that potent cytotoxic agents can be created by linking maytansinoids to appropriate cell-binding agents via cleavable linkers, especially cleavable linkers containing disulfide groups.
• Cell-binding agent maytansinoid conjugates permit the full measure of the cytotoxic action of the maytansinoids to be applied in a targeted fashion against unwanted cells only, thereby avoiding side effects due to damage to non-targeted, healthy cells.
• the present inventors have unexpectedly discovered that maytansinoids linked to cell- binding agents via non-cleavable linkers are superior in several important respects to maytansinoids linked via cleavable linkers. In particular, when compared to conjugates containing cleavable linkers, conjugates with non-cleavable linkers show equivalent antitumor activity both in vitro and in vivo, but demonstrate a marked decrease in plasma clearance rate and in toxicity.
• this invention provides an improved method for targeting cells, especially cells that are to be destroyed, such as tumor cells (particularly solid tumor cells), virus infected cells, microorganism infected cells, parasite infected cells, autoimmune cells (cells that produce autoantibodies), activated cells (those involved in graft rejection or graft vs. host disease), or any other type of diseased or abnormal cells, while exhibiting a minimum of side effects.
• tumor cells particularly solid tumor cells
• virus infected cells virus infected cells
• microorganism infected cells include parasite infected cells
• autoimmune cells cells that produce autoantibodies
• activated cells such as graft rejection or graft vs. host disease
• any other type of diseased or abnormal cells while exhibiting a minimum of side effects.
• the conjugate used in the inventive method has one or more maytansinoids linked to a cell-binding agent via a non-cleavable linker.
• a cell- binding agent for example an antibody
• a cross-linking reagent such as SMCC
• a reactive maytansinoid having a thiol group such as DM1
• the maytansinoid can be modified with a cross-linking reagent before being reacted with a cell- binding agent. See, for example, U.S. patent no. 6,441,163 Bl. Suitable Maytansinoids
• Maytansinoids suitable for use in the present invention are well known in the art, and can be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al., 99 PNAS 7968-7973 (2002)), or prepared synthetically according to known methods.
• Examples of suitable maytansinoids include maytansinol and maytansinol analogues.
• Suitable maytansinol analogues include those having a modified aromatic ring and those having modifications at other positions.
• Suitable maytansinol analogues having a modified aromatic ring include: (1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by LAH reduction of ansamytocin
• Suitable maytansinol analogues having modifications of other positions include: (1) C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the reaction of maytansinol with H 2 S or P 2 S 5 ); (2) C-14-alkoxymethyl(demethoxy/CH 2 OR)(U.S. Pat. No. 4,331,598); (3) C-14-hydroxymethyl or acyloxymethyl (CH 2 OH or CH 2 OAc) (U.S. Pat. No. 4,450,254) (prepared from Nocardia); (4) C-15-hydroxy/acyloxy (U.S. Pat. No.
• Particularly preferred maytansinoids comprising a free thiol group include N-methyl-alanine- containing esters and N-methyl-cysteine-containing esters of maytansinol are C-3 esters of maytansinol and its analogs.
• Preferred esters include N-methyl-alanine-containing esters and N- methyl-cysteine-containing esters of maytansinol. Synthesis of esters of maytansinol having a free thiol group has been previously described, for example in U.S. Patent No. 5,208,020, Chari et al., 52 Cancer Res., 127-131 (1992), and Liu et al., 93 Proc Natl. Acad.
• the maytansinoid contains a sterically hindered thiol group and is represented by formula (II'-L), (II'-D), or (II'-D,L):
• A, B, and D each independently is cyclic alkyl or cyclic alkenyl having 3 to 10 carbon atoms, simple or substituted aryl, or heterocyclic aromatic or heterocycloalkyl radical.
• Ri to R ⁇ are each independently linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical, and in addition, R 2 to Rj 2 can be H.
• 1, m, n, o, p, q, r, s, t, and u are each independently 0 or an integer of from 1 to 5, provided that at least two of 1, m, n, o, p, q, r, s, t and u are not both zero. May represents a maytansinoid that bears a side chain at C-3 hydroxyl, C-14 hydroxymethyl, C-15 hydroxyl or C-20 desmethyl.
• Another maytansinoid useful in the invention is represented by formula (II-L), (II-D), or (II-D,L): D D,L
• Ri to R 8 are each independently linear alkyl or alkenyl having l to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, heterocyclic aromatic or heterocycloalkyl radical, and in addition R 2 to R 8 can be H.
• 1, m and n are each independently an integer of from 1 to 5, and in addition n can be 0.
• Ri and R 2 are methyl.
• linear alkyls or alkenyls having 1 to 10 carbon atoms include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, propenyl, butenyl and hexenyl.
• Examples of branched alkyls or alkenyls having 3 to 10 carbon atoms include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 1 -ethyl-propyl, isobutenyl and isopentenyl.
• Examples of cyclic alkyls or alkenyls having from 3 to 10 carbon atoms include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl, and cyclohexenyl.
• Simple aryls include aryls having 6 to 10 carbon atoms, and substituted aryls include aryls having 6 to 10 carbon atoms bearing at least one alkyl substituent containing from 1 to 4 carbon atoms, or alkoxy substituent such as methoxy, ethoxy, or a halogen substituent or a nitro substituent.
• Examples of simple aryl that contain 6 to 10 carbon atoms include, but are not limited to, phenyl and naphthyl.
• substituted aryl examples include, but are not limited to, nitrophenyl, dinitrophenyl.
• Heterocyclic aromatic radicals include groups that have a 3 to 10-membered ring containing one or two heteroatoms selected from N, O or S.
• heterocyclic aromatic radicals include, but are not limited to, pyridyl, nitro- pyridyl, pyrollyl, oxazolyl, thienyl, thiazolyl, and furyl.
• Heterocycloalkyl radicals include cyclic compounds, comprising 3 to 10-membered ring systems, containing one or two heteroatoms, selected form N, O or S.
• heterocycloalkyl radicals include, but are not limited to, dihydrofuryl, tetrahydrofuryl, pyrrolidinyl, piperidinyl, piperazinyl,and morpholino.
• Particularly preferred maytansinoids comprising a side chain that contains a sterically hindered thiol bond are maytansinoids N -deacetyl-N- (4-mercapto-l-oxopentyl)-maytansine (termed DM3) and N -deacetyl-N " (4-methyl-4-mercapto-l-oxopentyl)-maytansine (termed DM4).
• DM3 and DM4 are represented by the following structural formulae:
• Cell-binding agents may be of any kind presently known, or that become known and include peptides and non-peptides. Generally, these can be antibodies (especially monoclonal antibodies), lymphokines, hormones, growth factors, vitamins, nutrient-transport molecules (such as transferrin), or any other cell-binding molecule or substance that specifically binds a target.
• cell-binding agents that can be used include: polyclonal and monoclonal antibodies, including fully human antibodies; single chain antibodies (polyclonal and monoclonal); fragments of antibodies (polyclonal and monoclonal) such as Fab, Fab', F(ab') 2 , and Fv
• lymphokines such as IL-2, IL-3, IL-4, IL-6; hormones such as insulin, TRH (thyrotropin releasing hormone), MSH (melanocyte- stimulating hormone), steroid hormones, such as andr ⁇ gens and estrogens; growth factors and colony-stimulating factors such as EGF, TGF-alpha, FGF, VEGF, G- CSF, M-CSF and GM-CSF (Burgess, 5 Immunology Today 155-158 (1984)); transferrin (O'Keefe et al., 260 J. Biol. Chem. 932-937 (1985)); and vitamins, such as folate.
• lymphokines such as IL-2, IL-3, IL-4, IL-6
• hormones such as insulin, TRH (thyrotropin releasing hormone), MSH (melanocyte- stimulating hormone), steroid hormones, such as andr ⁇ gens and estrogens
• growth factors and colony-stimulating factors such as EGF
• Monoclonal antibody techniques allow for the production of extremely specific cell- binding agents in the form of specific monoclonal antibodies.
• Particularly well known in the art are techniques for creating monoclonal antibodies 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 such as viral coat proteins.
• Sensitized human cells can also be used.
• Another method of creating monoclonal antibodies is the use of phage libraries of scFv (single chain variable region), specifically human scFv (see e.g., Griffiths et al., U.S. Patent Nos.
• the monoclonal antibody J5 is a murine IgG2a antibody that is specific for
• CALLA Common Acute Lymphoblastic Leukemia Antigen
• the monoclonal antibody MY9 is a murine IgGi antibody that binds specifically to the
• CD33 antigen J.D. Griffin et al 8 Leukemia Res., 521 (1984)
• AML acute myelogenous leukemia
• the monoclonal antibody anti-B4 interchangeably also called B4, is a murine
• IgG that binds to the CD19 antigen on B cells (Nadler et al, 131 J. Immunol. 244-250 (1983)) and can be used if the target cells are B cells or diseased cells that express this antigen such as in non-Hodgkin's lymphoma or chronic lymphoblastic leukemia.
• HuC242 is a humanized form of the monoclonal antibody C242 that is described in U.S. patent No. 5,552,293 and for which the hybridoma is deposited with the EC ACC identification Number 90012601.
• a humanized form can be prepared by either applying the CDR-grafting methodology (US. Patents Nos. 5,585,089; 5,693,761; and
• HuC242 can also be used to treat CanAg expressing tumors, such as colorectal, pancreatic, non-small cell lung, and gastric cancers.
• the antibody trastuzumab can be used to treat breast and other cancers, such as prostate and ovarian cancers that express the Her2 antigen.
• Anti-IGF-IR antibodies that bind to insulin growth factor receptor are also useful.
• Ovarian cancer and prostate cancer can be successfully targeted with, for example, an anti-MUCl antibody, such as anti-HMFG-2 (Taylor-Papadimitriou et al., 28. Int. J. Cancer 17- 21, 1981) or hCTMOl (56 Cancer Res. 5179-5185, 1996) and an anti-PSMA (prostate-specific membrane antigen), such as J591 (Liu et al. 57 Cancer Res. 3629-3634, 1997) respectively.
• Non-antibody molecules can also be used to target specific cell populations.
• GM-CSF which binds to myeloid cells
• IL-2 which binds to activated T- cells
• MSH which binds to melanocytes
• Folic acid can be used to target the folate receptor expressed on ovarian and other tumors.
• Epidermal growth factor (EGF) can be used to target squamous cancers such as lung and head and neck.
• Somatostatin can be used to target neuroblastomas and other tumor types. Cancers of the breast and testes can be successfully targeted with estrogen (or estrogen analogues) or androgen (or androgen analogues) respectively as cell-binding agents.
• Cross-Linking Reagents [98] The maytansinoid is linked to the cell-binding agent by means of a cross-linking reagent that, when reacted, forms a non-cleavable linker between the maytansinoid and the cell-binding agent.
• a "linker” is any chemical moiety that links a cell-binding agent covalently to a maytansinoid.
• part of the linker is provided by the maytansinoid.
• DM1 a thiol-containing maytansinoid (Fig. 2), is a derivative of the natural maytansinoid, maytansine, and provides part of the linker.
• the side chain at the C-3 hydroxyl group of maytansine ends in -CO-CH 3
• the side chain of DM1 ends in -CO-CH 2 -CH 2 - SH. Therefore the final linker is assembled from two pieces, the cross-linking reagent introduced into the cell-binding agent and the side chain from the DM1.
• Cleavable linkers are linkers that can be cleaved under mild conditions, i.e. conditions under which the activity of the maytansinoid drug is not affected. Many known linkers fall in this category and are described below.
• Disulfide containing linkers are linkers cleavable through disulfide exchange, which can occur under physiological conditions.
• Acid-labile linkers are linkers cleavable at acid pH.
• certain intracellular compartments such as endosomes and lysosomes, have an acidic pH (pH 4-5), and provide conditions suitable to cleave acid-labile linkers.
• Linkers that are photo-labile are useful at the body surface and in many body cavities that are accessible to light. Furthermore, infrared light can penetrate tissue. [104] Some linkers can be cleaved by peptidases. Only certain peptides are readily cleaved inside or outside cells, see e.g. Trouet et al., 79 Proc. Natl. Acad. Sci. USA, 626-629 (1982) and Umemoto et al. 43 Int. J. Cancer, 677-684 (1989). Furthermore, peptides are composed of ⁇ - amino acids and peptidic bonds, which chemically are amide bonds between the carboxylate of one amino acid and the ⁇ -amino group of a second amino acid. Other amide bonds, such as the
• Some linkers can be cleaved by esterases. Again only certain esters can be cleaved by esterases present inside or outside cells. Esters are formed by the condensation of a carboxylic acid and an alcohol. Simple esters are esters produced with simple alcohols, such as aliphatic alcohols, and small cyclic and small aromatic alcohols. For example, the present inventors found no esterase that cleaved the ester at C-3 of maytansine, since the alcohol component of the ester, maytansinol, is very large and complex.
• a non-cleavable linker is any chemical moiety that is capable of linking a maytansinoid to a cell-binding agent in a stable, covalent manner and does not fall under the categories listed above as cleavable linkers.
• non-cleavable linkers are substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage.
• substantially resistant to cleavage means that the chemical bond in the linker or adjoining the linker in at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% of the cell-binding agent maytansinoid conjugate population remains non-cleavable by an acid, a photolabile-cleaving agent, a peptidase, an esterase, or a chemical or a physiological compound that cleaves the chemical bond (such as a disulfide bond) in a cleavable linker, for within a few hours to several days of treatment with any of the agents described above.
• non-cleavable refers to the ability of the chemical bond in the linker or adjoining to the linker to withstand cleavage induced by an acid, a photolabile-cleaving agent, a peptidase, an esterase, or a chemical or a physiological compound that cleaves a disulfide bond, at conditions under which the maytansinoid or the cell binding agent does not lose its activity.
• a person of ordinary skill in the art would readily distinguish non-cleavable from cleavable linkers.
• An example of an appropriate control for testing whether a linker is substantially resistant to cleavage is a linker with a chemical bond, such as a disulfide bond, that is susceptible to cleavage by any of the agents described above.
• a linker is substantially resistant to cleavage by measuring the stability of the conjugates by ELISA, HPLC, or other suitable means, over a period of time extending from between a few hours to several days, typically 4 hours to 5 days.
• ELISA assays can be used to measure the level of stable conjugate in the plasma concentration.
• Non-cleavable linkers are also characterized in that the in vivo half-life of conjugates comprising non-cleavable linkers is generally about 20% higher than that of conjugates comprising cleavable linkers. In mice, the in vivo half-life of IgG-maytansinoid conjugates linked via non-cleavable linkers is at least 4 days.
• Suitable cross-linking reagents that form non-cleavable linkers between the maytansinoid and the cell-binding agent are well known in the art, and can form non-cleavable linkers that comprise a sulfur atom (such as SMCC) or that are without a sulfur atom.
• Preferred cross-linking reagents that form non-cleavable linkers between the maytansinoid and the cell-binding agent comprise a maleimido- or haloacetyl-based moiety.
• non-cleavable linkers are said to be derived from maleimido- or haloacetyl-based moiety.
• Cross-linking reagents comprising a maleimido-based moiety include N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N- succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-carboxy-(6-amidocaproate), which is a
• SMPB N-(p-maleimidophenyl)isocyanate
• PMPI N-(p-maleimidophenyl)isocyanate
• Cross-linking reagents comprising a haloacetyl-based moiety include N-succinimidyl-4-
• active esters described in Figs. 15 and 16 are comprised of ⁇ -succinimidyl and sulfosuccinimidyl esters
• other active esters such as ⁇ -hydroxy phthalimidyl esters, ⁇ -hydroxy sulfophthalimidyl esters ortho-nitrophenyl esters, para-nitrophenyl esters, 2,4-dinitrophenyl esters, 3-sulfonyl-4-nitrophenyl esters, 3-carboxy-4-nitrophenyl esters, pentaflurophenyl esters, and sulfonyl tetrafluorophenyl esters can also be used.
• cross-linking reagents form non-cleavable linkers that do not contain a sulfur atom.
• Fig. 21 shows a maytansinoid molecule derivatized with a cross-linking reagent that is derived from an ⁇ , ⁇ -dicarboxylic acid (an alkane or alkene dioic acid wherein the alkane or alkene has 3-24 carbon atoms).
• the cross- linking reagent When reacted with the cell-binding agent, the cross- linking reagent will form a non-sulfur containing non-cleavable linker (non-S-containing non- cleavable linker).
• the maytansinoid molecule of Fig. 21 is made as follows. First a monoester of adipic acid (also known as hexanedioic acid or 1,6-hexanedicarboxylic acid) is prepared by treatment with one equivalent of 2-trimethysilyl ethanol in the presence of dicyclohexylcarbodiimide. Activation of the remaining carboxylic acid group with isobutyl chloroformate, followed by reaction with N-methyl-L-alanine, provides the acylated N-methyl-L-alanine.
• adipic acid also known as hexanedioic acid or 1,6-hexanedicarboxylic acid
• Non-cleavable linkers that do not contain a sulfur atom can also be derived from other dicarboxylic acid based moieties using the method described above.
• Other suitable dicarboxylic acid based moieties include but are not limited to ⁇ , ⁇ -dicarboxylic acids of general formula (IV): HOOC-X,-Y n -Z m -COOH (IV)
• X is a linear or branched alkyl, alkenyl or alkynyl group having 2 to 20 carbon atoms
• Y is a cycloalkyl or cycloalkenyl group bearing 3 to 10 carbon atoms
• Z is a substituted or unsubstituted aromatic group bearing 6 to 10 carbon atoms or a substituted or unsubstituted heterocyclic group wherein the hetero atom is selected from N, O or S, and wherein 1 ,m and n are each 0 or 1, provided that they are all not 0 at the same
• X, Y, Z, 1, m and n are all defined as for formula (IV) above, and further wherein E together with the carbonyl group forms an active ester such as N-hydroxy succinimidyl and sulfosuccinimidyl esters, N-hydroxy phthalimidyl ester, N-hydroxy sulfophthalimidyl ester ortho-nitrophenyl ester, para-nitrophenyl ester, 2,4-dinitrophenyl ester, 3-sulfonyl-4-nitrophenyl ester, 3-carboxy-4-nitrophenyl ester, pentaflurophenyl ester, and sulfonyl tetrafluorophenyl ester.
• an active ester such as N-hydroxy succinimidyl and sulfosuccinimidyl esters, N-hydroxy phthalimidyl ester, N-hydroxy sulfophthalimidyl ester ortho-nitrophenyl ester,
• n represents an integer from 3 to 24, and E has the same definition as for the maytansinoid of formula 5.
• a more preferred embodiment is the derivatized maytansinoid represented by formula 7:
• linear alkyl, alkenyl, or alkynyl groups having 2 to 20 carbon atoms include, but are not limited to, ethyl, propyl, butyl, pentyl, hexyl, propenyl, butenyl, and hexenyl.
• Examples of branched alkyl, alkenyl, or alkynyl groups having 2 to 20 carbon atoms include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 1-ethyl- propyl, isobutenyl, isopentenyl, ethynyl, propynyl (propargyl), 1 -butynyl, 2-butynyl, and 1- hexynyl.
• Examples of cycloalkyl or cycloalkenyl groups having from 3 to 10 carbon atoms include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl, cyclohexenyl, and cycloheptadienyl.
• aromatic groups that contain 6 to 10 carbon atoms include, but are not limited to, phenyl and naphthyl.
• substituted aromatic groups include, but are not limited to, nitrophenyl and dinitrophenyl.
• Heterocyclic aromatic groups include, but are not limited to, groups that have a 3 to 10- membered ring containing one or two heteroatoms selected from N, O or S.
• Examples of substituted and unsubstituted heterocyclic aromatic groups include, but are not limited to, pyridyl, nitro-pyridyl, pyrollyl, oxazolyl, thienyl, thiazolyl, and furyl.
• Heterocycloalkyl radicals include, but are not limited to, cyclic compounds, comprising 3 to 10-membered ring systems, containing one or two heteroatoms, selected from N, O or S.
• heterocycloalkyl radicals include, but are not limited to, dihydro furyl, tetrahydrofuryl, tetrahydropyrollyl, piperidinyl, piperazinyl, and morpholino.
• Examples of ⁇ , ⁇ -dicarboxylic acids of the general formula HOOC-X ⁇ -Y n -Z m -COOH include, but are not limited to, adipic acid, glutaric acid, pimelic acid, hexene-l,6-dioc acid, pentene-l,5-dioc acid, cyclohexane-dioic acid, and cyclohexene-dioic acid Synthesis of Cytotoxic Conjugates [134] Conjugates of cell-binding agents and maytansinoids can be formed using any techniques presently known or later developed.
• Methods of conjugation of cell-binding agents with maytansinoids generally involve two reaction steps.
• a cell-binding agent such as an antibody
• a cross-linking reagent to introduce one or more, usually 1- 10, reactive groups.
• the modified cell-binding agent is then reacted with one or more thiol- containing maytansinoids to produce a conjugate.
• a thiol-containing maytansinoid can first be modified with a cross-linking reagent, followed by reaction of the modified maytansinoid with a cell-binding agent.
• the thiol-containing maytansinoid can be reacted with the maleimido compounds described in Fig. 15 or with the haloacetyl compounds described in Fig. 16, to give a maytansinoid thioether bearing an active succinimidyl or sulfosuccinimidyl ester.
• Reaction of these maytansinoids containing an activated linker moiety with a cell-binding agent provides another method of producing a non-cleavable cell- binding agent maytansinoid conjugate.
• a maytansinoid that does not contain a sulfur atom can first be derivatized by a dicarboxylic acid based cross-linking reagent, followed by reaction with the cell-binding agent, to form a conjugate in which the maytansinoid is linked to the cell-binding agent via a non-S-containing non-cleavable linker.
• a conjugate in which the maytansinoid is linked to the cell-binding agent via a non-S-containing non-cleavable linker.
• an average of 1-10 maytansinoids per antibody are linked.
• the conjugate can be purified through a Sephadex G-25 column.
• Representational conjugates of the invention are antibody-maytansinoid derivatives, antibody fragment-maytansinoid derivatives, growth factor-maytansinoid conjugates, such as epidermal growth factor (EGF)-maytansinoid derivatives, hormone-maytansinoid conjugates, such as melanocyte stimulating hormone (MSH)-maytansinoid derivatives, thyroid stimulating hormone (TSH)-maytansinoid derivatives, estrogen-maytansinoid derivatives, estrogen analogue-maytansinoid derivatives, androgen-maytansinoid derivatives, androgen analogue- maytansinoid derivatives, androgen analogue- maytansinoid derivatives, and vitamin-maytansinoid conjugates, such as folate maytansinoid.
• EGF epidermal growth factor
• hormone-maytansinoid conjugates such as melanocyte stimulating hormone (MSH)-maytansinoid derivatives, thyroid stimulating hormone (
• Maytansinoid conjugates of antibodies, antibody fragments, protein hormones, protein growth factors and other proteins are made in the same way.
• peptides and antibodies can be modified with the non-cleavable cross-linking reagents mentioned above.
• a solution of an antibody in aqueous buffer may be incubated with a molar excess of an antibody- modifying cross-linking reagent such as succinimidyl 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (SMCC), sulfo-SMCC, -maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS, succinimidyl-iodoacetate, or N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-carboxy-(6-amidocaproate), which is a molar excess of an antibody- modifying cross-linking reagent such as succinimidyl 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (SM
• succinimidyl ester (KMUA), sulfo-KMUA, ⁇ -maleimidobutyric acid N-succinimidyl ester
• GMBS methyl methacrylate
• EMCS ⁇ -maleimidcaproic acid N-hydroxysuccinimide ester
• the modified antibody is then treated with the thiol-containing maytansinoid (1.25 molar equivalent/maleimido or iodoacetyl group) to produce a conjugate.
• the mixtures are incubated overnight at about 4°C.
• the antibody-maytansinoid conjugates are purified by gel filtration through a Sephadex G-25 column.
• the number of maytansinoid molecules bound per antibody molecule can be determined by measuring spectrophotometrically the ratio of the absorbance at 252 nm and 280 nm. Typically, an average of 1-10 maytansinoids per antibody are linked.
• a preferred method is to modify antibodies with succinimidyl 4-( ⁇ -maleimidomethyl)- cyclohexane-1 -carboxylate (SMCC) to introduce maleimido groups followed by reaction of the modified antibody with a thiol-containing maytansinoid to give a thioether-linked conjugate.
• SMCC succinimidyl 4-( ⁇ -maleimidomethyl)- cyclohexane-1 -carboxylate
• conjugates with 1 to 10 drug molecules per antibody molecule result. Examples of antibody-maytansinoid conjugates are shown in Figs. 17-20.
• estrogen and androgen cell-binding agents such as estradiol and androstenediol can be esterified at the C-17 hydroxy group by reaction with an appropriately protected thiol group-containing carboxylic acid chloride such as 3-S-acetylpropanoyl chloride.
• an appropriately protected thiol group-containing carboxylic acid chloride such as 3-S-acetylpropanoyl chloride.
• Other methods of esterif ⁇ cation can also be employed as described in the literature (Haslam, 36 Tetrahedron 2400-2433 (1980)).
• the protected or free thiol-containing androgen or estrogen can then be reacted with a thiol-containing maytansinoid to produce conjugates.
• the conjugates can be purified by column chromatography on silica gel or by HPLC.
• a particularly preferred method is to modify maytansinol with a cross-linking reagent that results in a linkage that does not contain any sulfur atoms, followed by reaction of the modified maytansinoid with an antibody to produce conjugates.
• Therapeutic efficacy of the cytotoxic conjugates of the invention is to modify maytansinol with a cross-linking reagent that results in a linkage that does not contain any sulfur atoms, followed by reaction of the modified maytansinoid with an antibody to produce conjugates.
• Cell-binding agent maytansinoid conjugates of the invention can be evaluated for their ability to suppress proliferation of various cell lines in vitro.
• cell lines such as the human colon carcinoma line COLO205, the human melanoma cell line A375, the human myeloid leukemia cell line HL60, the human breast carcinoma line SKBR3, or the human epidermoid carcinoma cell line KB can be used for the assessment of cytotoxicity of these conjugates.
• Cells to be evaluated can be exposed to the compounds for 24 hours and the surviving fractions of cells measured in direct assays by known methods. (See, e.g. Goldmacher et al., 135 J. Immunol. 3648-3651 (1985), and Goldmacher et al., 102 J.
• IC 0 values can then be calculated from the results of the assays.
• High cytotoxicity can be defined as exhibiting a toxicity having an IC 50 (the inhibiting concentration of a toxic substance that leaves a surviving fraction of 0.5) of about 10 "8 M or less when measured in vitro with SKBR3 cells upon a 24 hour exposure time to the drug.
• IC 50 the inhibiting concentration of a toxic substance that leaves a surviving fraction of 0.5
• Conjugates of huC242 with DM1 using the cross-linking reagent SMCC are highly potent in destroying antigen positive SKBR3 cells, with an IC 50 value of 3.5 x 10 "12 M.
• antigen negative A375 cells are about 800-fold less sensitive demonstrating that maytansinoid conjugates of the present invention are highly potent and specific.
• conjugates with non-cleavable linkers that show specific activity in vitro are not restricted to the SMCC linker.
• a huC242 conjugate of DM1 synthesized with the non- cleavable linker SIAB showed potent and antigen-specific cytotoxicity in clonogenic assays in vitro (Fig. 9).
• a trastuzumab conjugate of DM1 synthesized with SIAB was also cytotoxic in clonogenic assays (Fig. 28).
• a huC242 -non-S-containing non-cleavable linker-DMl conjugate also demonstrated potent and antigen-specific cytotoxicity in clonogenic assays in vitro (Fig. 22).
• Antibody conjugates with DM1 using the SMCC linker show anti-tumor efficacy against human tumor xenografts in mice (Fig. 10A-C). Marked inhibition of tumor growth was observed upon treatment ofSNUl ⁇ gastric tumor xenografts with huC242-SMCC-DMl (Fig. 10A). This anti-tumor activity is observed at conjugate doses that have no effect on mouse body weight, a measure of drug toxicity. Treatment of mice bearing COLO205 colon carcinoma tumor xenografts with the huC242-SMCC-DMl conjugate resulted in complete regression of tumors, with some mice remaining free of detectable tumors for over 2 months post-treatment (Fig. 10B).
• a trastuzumab-SMCC-DMl conjugate also showed significant tumor regression, in a mouse tumor xenograft model with the MCF-7 breast carcinoma cell line (Fig. IOC).
• Plasma clearance of antibody-maytansinoid conjugate synthesized with the non-cleavable linker SMCC is very slow and comparable to the clearance of antibody alone. This is in sharp contrast to plasma clearance of conjugates prepared with relatively labile disulfide bonds such as huC242-SPP-DMl .
• the half-life for clearance of the SMCC conjugate is approximately 320 hours, while the half-life for the SPP conjugate is in the range of 40-50 hours (Fig. 11).
• the clearance of the antibody component for each type of conjugate is identical, suggesting that the difference in measured conjugate clearance rate is due to the loss of maytansinoid from the antibody conjugate in the case of the SPP-DM1 conjugate.
• the non- cleavable SMCC linkage is therefore much more resistant to maytansinoid-Iinker cleavage activities present in vivo than the SPP-DMl conjugate.
• Maytansinoid conjugates prepared with non-cleavable linkers such as SMCC show an unexpected increased tolerability in mice compared with conjugates prepared with cleavable disulfide linkers. An acute toxicity test with a single intravenous dose was carried out in female CD-I mice.
• Maytansinoid conjugates are thought to impart their cell destroying activity through the inhibition of microtubule polymerization. This inhibition of microtubule polymerization leads to an arrest of the cell cycle principally at G2/M. The antigen-dependent arrest of cells at G2/M by antibody-maytansinoid conjugates can be monitored by flow cytometry analysis (Fig. 13). Treatment of COLO205 cells with huC242-SPP-DMl or huC242-SMCC-DMl conjugate results in a complete G2/M arrest by 6-10 hours.
• the above-described conjugates can be used in a method for targeting maytansinoids to a selected cell population, the method comprising contacting a cell population or tissue suspected of containing the selected cell population with a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids is covalently linked to the cell-binding agent via a non- cleavable linker and the cell-binding agent binds to cells of the selected cell population.
• the above-described conjugates can also be used in a method of destroying cells, the method comprising contacting the cells with a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids is covalently linked to the cell-binding agent via a non- cleavable linker and the cell-binding agent binds to the cells.
• the above-described conjugates can also be used in a method of treatment of afflictions including but not limited to malignant tumors, autoimmune diseases, graft rejections, graft versus host disease, viral infections, microorganism infections, and parasite infections, the method comprising administering to a subject in need of treatment an effective amount of a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids is covalently linked to the cell-binding agent via a non-cleavable linker and the cell-binding agent binds diseased or infected cells of the affliction.
• Examples of medical conditions that can be treated according to the methods of the present invention include but are not limited to malignancy of any type including, for example, cancer of the lung, breast, colon, prostate, kidney, pancreas, ovary, and lymphatic organs; autoimmune diseases, such as systemic lupus, rheumatoid arthritis, and multiple sclerosis; graft rejections, such as renal transplant rejection, liver transplant rejection, lung transplant rejection, cardiac transplant rejection, and bone marrow transplant rejection; graft versus host disease; viral infections, such as CMV infection, HIV infection, AIDS, etc.; and parasite infections, such as giardiasis, amoebiasis, schistosomiasis, and others as determined by one of ordinary skill in the art.
• the methods can be practiced in vitro or in vivo.
• the above-described conjugates can be used in a method of in vitro use to treat, for example, autologous bone marrow cells prior to their transplant into the same patient in order to destroy diseased or malignant cells; bone marrow cells or other tissue prior to their transplantation in order to destroy T cells and other lymphoid cells and prevent graft-versus-host- disease (GVHD); cell cultures in order to destroy all cells except for desired variants that do not express the target antigen; or cell cultures in order to destroy variant cells that express undesired antigen; the method comprising treating the cells with an effective amount of a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids is covalently linked to the cell-binding agent via a non-cleavable linker and the cell-binding agent binds the cells that are to be destroyed.
• a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids is covalently linked to the cell-binding agent via
• the treated marrow cells can be stored frozen in liquid nitrogen using standard medical equipment.
• the cytotoxic agent can be supplied as a solution or a lyophilized powder that is tested for sterility and for endotoxin levels. Examples of suitable protocols of conjugate administration are as follows. Conjugates can be given weekly for 4 weeks as an intravenous bolus each week. Bolus doses can be given in 50 to 500 ml of normal saline to which 5 to 10 ml of human serum albumin can be added.
• a cell-binding agent maytansinoid conjugate having at least one maytansinoid linked to a cell-binding agent via a non-cleavable linker, provided that the linker does not comprise a group derived from a cross-linking agent selected from the group consisting of: succinimidyl 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (SMCC), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS, and succinimidlyl-iodoacetate when the cell-binding agent is an antibody.
• SMCC succinimidyl 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate
• MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester
• succinimidlyl-iodoacetate when the cell-binding
• the carrier may be a pharmaceutically acceptable carrier, diluent or excipient.
• Suitable carriers, diluents and/or excipients include: (1) Dulbecco's phosphate buffered saline, pH about 7.4, containing or not containing about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose; and may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20.
• One of the processes of making the cell-binding agent maytansinoid conjugate comprises: (a) providing the cell-binding agent (b) modifying the cell-binding agent with a cross-linking agent, and (c) conjugating the modified cell-binding agent with a maytansinoid or a thiol-containing maytansinoid thereby providing the non-cleavable linker between the cell-binding agent and the maytansinoid or thioLcontaining maytansinoid to produce the conjugate.
• Another process of making the cell-binding agent maytansinoid conjugate comprises: (a) providing the maytansinoid or a thiol-containing maytansinoid, (b) modifying the maytansinoid or thiol-containing maytansinoid with a cross-linking agent to thereby form a non-cleavable linker, and (c) conjugating the modified maytansinoid or thiol-containing maytansinoid with the cell- binding agent, thereby providing the non-cleavable linker between the cell-binding agent and the maytansinoid or thiol-containing maytansinoid to produce the conjugate.
• An additional process of making the cell-binding agent maytansinoid conjugate comprises: (a) providing the maytansinoid, (b) modifying the maytansinoid to provide a non-sulfur-containing maytansinol having an active ester, and
• Solvents used in the following experiments were dimethylsulfoxide (DMSO), dimethylacetamide (DMA), ethanol (EtOH), and lOOmM Ellman's Reagent (DTNB, available from Cayman Chemical) in DMSO.
• DMSO dimethylsulfoxide
• DMA dimethylacetamide
• EtOH ethanol
• DTNB lOOmM Ellman's Reagent
• the antibody was split into two samples; one was modified using a 7.5-fold molar excess of SMCC cross-linker, the other with a 8.5-fold molar excess of SMCC cross-linker. Samples were reacted at 8 mg/mL antibody. The reactions were carried out in Buffer A (95% v/v) with
• Trastuzumab antibody was obtained from Genentech for conjugation to DM1 using the non-cleavable heterobifunctional cross-linking reagent SMCC.
• the antibody was buffer- exchanged from 50 mM potassium phosphate/2 mM EDTA, pH 6.0 into 50 mM potassium phosphate/50 mM sodium chloride/2 mM EDTA, pH 6.5 (Buffer A).
• the antibody was then reacted with 7.5-fold molar excess SMCC linker and purified by Sephadex G25 resin before it was conjugated with DM1.
• the final conjugate was again purified by Sephadex G25 resin.
• the resulting conjugate contained 3.1 moles of DM1 per mole of antibody.
• a 10 mM solution of DM1 (free thiol form) was prepared in DMA (7.37 mg/mL) (Fig, 2).
• the absorbance of dilutions of the stock solution in EtOH was measured at 280 nm.
• the concentration of stock DM1 was calculated by using a molar extinction coefficient of 5700 M " 'cm "1 at 280 nm.
• the concentration of free -SH in the stock DM1 preparation was measured using Ellman's reagent (DTNB). Dilutions of the stock solution were prepared in Assay buffer made to 3% (v/v) DMA, and then 100 mM DTNB in DMSO (1/100 th volume) was added.
• the trastuzumab-SMCC reaction mixture was gel-filtered through a 1.5 x 4.9 cm prepacked column of Sephadex G25 resin equilibrated in Buffer A. The load and elution volumes were according to manufacturer's instructions (Amersham Biosciences). The concentration of the modified antibody solution was assayed spectrophotometrically using the extinction coefficient described above. The yield of modified antibody was 88 % based on protein concentration.
• Conjugation of Trastuzumab-SMCC with DM1 [202] The modified antibody was reacted with a 1.7-fold excess of DM1 over linker (assuming 5 linkers per antibody).
• the DMl/antibody ratio was found to be 3.13 and the conjugation step yield was 95.7%.
• the overall yield of conjugated trastuzumab was 84% based on the starting antibody.
• the resulting conjugate was analyzed for binding, cytotoxicity, specificity, extent of aggregation and free drug content.
• the antibody was modified using a 7.0-fold molar excess of SIAB at 20 mg/mL antibody.
• the reaction was carried out in Buffer A (95% v/v) with DMSO (5% v/v) for 2 hours at room temperature with stirring in the dark. e. G25 Chromatography to remove excess SIAB
• trastuzumab antibody and trastuzumab-SIAB-DMl were compared using the HER2 ECD plate binding assay provided by Genentech. The results are shown in Fig. 27. Naked trastuzumab and trastuzumab-SIAB-DMl had similar binding affinities (1.2x 10 "10 M for the antibody and 1.9x 10 "10 M apparent K D for the conjugate). b.
• DM1 is 4.7 (in ETOH) while ⁇ 52 / ⁇ 280 for this cross-linking reagent solution (in pH 7.5 buffer)

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