NO347360B1 - Cell-binding agent maytansinoid conjugate of formula trastuzumab-SMCC-DM1 or trastuzumab-SIABDM1, method for producing these and an in vitro method for directing maytansinoids to a selected cell population or to eliminate cells, as well as application. - Google Patents

Description

Field of the invention

The present invention relates to cell binding agent-maytansinoid conjugates of the formula trastuzumab-N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate (SMCC)-N<2>'-deacetyl-N<2>'-(3-mercapto-1-oxopropyl)-maytansine (DM1) or trastuzumab-N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB)-DM1, compositions comprising these and its use in the treatment of tumors.

The invention also relates to methods for producing the cell binding agent maytansinoid conjugates.

The invention further relates to in vitro methods for administering maytansinoids to a selected cell population or for eliminating cells.

Background for the invention

Maytansinoids are highly cytotoxic drugs. Maytansin was first isolated by Kupchan et al., from the East African shrub Maytenus serrata and was shown to be 100 to 1000 times more cytotoxic than conventional cancer chemotherapeutic agents such as methotrexate, daunorubicin and vincristine (US Patent 3,896,111). After this, it was discovered that some microbes also produce maytansinoids, such as maytansinol and C-3 esters of maytansinol (US patent 4,151,042). Synthetic C-3 esters of maytansinol and analogs of maytansinol have also been reported [Kupchan et al., 21 J. Med. Chem. 31-37 (1978); Higashide et al. 270 Nature 721-722 (1977); Kawai et al., 32 Chem. Pharm. Bull. 3441-3451 (1984)]. Examples of analogues of maytansinol from which C-3 esters have been prepared include maytansinol with modifications on the aromatic ring (e.g. dechloro) or at C-9, C-14 (e.g. hydroxylated methyl group), C-15, C- 18, C-20 and C-4,5.

The naturally occurring and synthetic C-3 esters can be classified into two groups:

(a) C-3 esters with single carboxylic acids (US Patents 4,248,870, 4,265,814,

4 308 268, 4 308 269, 4 309 428, 4 322 348 and 4 331 598), and

(b) C-3 esters with derivatives of N-methyl-L-alanine (US Patent 4,137,230 and

4,260,608; and Kawai et al., 32 Chem. Pharm. Bull. 3441-3451 (1984)).

Esters in group (b) were found to be much more cytotoxic than esters in group (a).

Maytansine is a mitotic inhibitor. Treatment of L1210 cells in vivo with maytansine has been reported to cause 67% of these cells to accumulate in mitosis. Untreated control cells were reported to show a mitotic index ranging from between 3.2 to 5.8% (Sieber et al., 43 Bibl.Haematol. 495-500 (1976)). Experiments with eggs from sea urchins and eggs from clams suggest that maytansine inhibits mitosis by interfering with the formation of microtubules via the inhibition of the polymerization of the microtubule protein tubulin (Remilliard et al., 189 Science 1002-1005(1975)).

In vitro, murine P388, L1210 and LY5178 leukemia suspensions have been found to be inhibited by maytansine at doses of 10<-3 > to 10<-1 >µg/ml, with the P388 line being most sensitive. Maytansine has also been shown to be an active inhibitor of in vitro growth of human nasopharyngeal carcinoma cells, and the human acute lymphoblastic leukemia line C.E.M. was reported to be inhibited by concentrations as low as 10<-7 >µg/ml (Wolpert-DeFillippes et al., 24 Biochem. Pharmacol. 1735-1738 (1975)).

Maytansin has also been shown to be active in vivo. Tumor growth in the P388 lymphocyte leukemia system was shown to be inhibited over a 50- to 100-fold dose range, indicating a high therapeutic index, and significant inhibitory activity could also be demonstrated with the L1210 mouse leukemia system, the human Lewis lung carcinoma system, and the human The B-16 melanocarcinoma system (Kupchan, 33 Ped. Proc 2288-2295 (1974)).

Because the maytansinoids are highly cytotoxic, it is expected that they can be used in the treatment of many diseases, such as cancer. This expectation has still not been realized. Clinical trials with maytansine were not beneficial due to a number of side effects (Issel et al., 5 Cancer Treat. Rev. 199-207 (1978)).

Adverse effects on the central nervous system and gastrointestinal symptoms were responsible for some patients resisting further therapy (Issel at 204), and maytansine appeared to be associated with peripheral neuropathy that could be cumulative (Issel at 207).

Accordingly, targeting techniques to selectively deliver drugs to the target cell were used. Both cleavable and non-cleavable linkers have been investigated for several drugs, but in most cases, including the case of the maytansinoid, in vitro cytotoxicity tests have revealed that antibody-drug conjugates rarely achieve the same cytotoxic potential as the free unconjugated drugs. Thus, it has generally been accepted that for targeted delivery of maytansinoids to be effective, the bond between maytansinoid and the cell binding agent must be cleavable.

In the field of immunotoxins, conjugates containing linkers with disulfide bridges between monoclonal antibodies and catalytically active protein toxins were shown to be more cytotoxic than conjugates containing other linkers. See Lambert et al., J. Biol. Chem. 12035-12041 (1985); Lambert et al., in Immunotoxins 175-209 (A.

Frankel, ed. 1988) and Ghetie et al., 48 Cancer Res. 2610-2517 (1988). This was attributed to the high intracellular concentration of glutathione which contributes to the efficient cleavage of the disulfide bond between an antibody molecule and a toxin. Recently, 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.1 via a linker that had a cleavable disulfide bond (Chari et al., 52 Cancer Res. 127-133 (1992)).

Thus, cytotoxic conjugates bound via disulfide-containing cleavable linkers have been searched for. Shen et al. describes the conversion of methotrexate to mercaptoethyl amide derivative followed by conjugation with poly-D-lysine via disulfide bond (260 J. Biol. Chem. 10905-10908 (1985)). Preparation of a conjugate of the trisulfide-containing toxic drug calicheamycin with an antibody was also described (Menendez et al., Fourth International Conference on Monoclonal Antibody Immunoconjugates for Cancer, San Diego, Abstract 81 (1989)).

US patents 5,208,020 and 5,416,064 disclose cytotoxic conjugates comprising cell binding agents bound to specific maytansinoids via cleavable linkers, such as linkers containing disulfide groups, linkers containing acid-labile groups, linkers containing photolabile groups, linkers containing peptidaselabile groups and linkers containing esteraselabile groups.

US Patent 6,333,410 B1 discloses a process for the preparation and purification of thioline-containing maytansinoids for binding to cell binding agents, and US Patent 6,441,163 B1 discloses a one-step process for the preparation of cytotoxic conjugates of maytansinoids and cell binding agents, wherein the linker is a disulfide-containing cleavable linker.

Furthermore, US patent document 5,208,020 describes antibody-maytansinoid conjugates with non-cleavable linkers, where the linker comprises a maleimido group. Nevertheless, the reference contains no experimental data showing that such conjugates are effective in treating disease.

The patent documents EP 1354896 A1, WO 0124763 A2, WO 02098883 A1 and the publication Chari Ravi V.J. et al.; "Immunoconjugates containing novel maytansinoids: promising anticancer drugs"; Cancer Research, vol. 52, No. 1, 1992, pp. 127-131, ISSN 0008-5472, describes cell binding agent-maytansinoid conjugates, but does not describe the antibody trastazumab.

It has now unexpectedly been found that cytotoxic conjugates of maytansinoids and cell binding agents linked via non-cleavable linkers are extremely potent and in many cases have unexpected advantages over conjugates of maytansinoids and cell binding agents of cleavable linkers.

Summary of the invention

The present invention relates to a cell binding agent-maytansinoid conjugate, characterized by the following formulas:

trastuzumab-N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate (SMCC)-N<2>'-deacetyl-N<2>'-(3-mercapto-1-oxopropyl)-maytansine (DM1) or trastuzumab-N-succinimidyl -4-(iodoacetyl)-aminobenzoate (SIAB)-DM1.

The invention also relates to a composition comprising the cell binding agent maytansinoid conjugate according to the invention and a carrier.

The invention also relates to a method for producing the cell binding agent-maytansinoid conjugate according to the invention, where the method 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 the thiol-containing maytansinoid to produce the conjugate.

The invention also relates to a method for producing the cell binding agent-maytansinoid conjugate according to the invention, where the method 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 to thereby provide the non-cleavable linker between the cell-binding agent and the maytansinoid or the thiol-containing maytansinoid to produce the conjugate.

The invention also relates to an in vitro method for administering maytansinoids to a selected cell population, characterized in that the method comprises contacting a cell population or a tissue suspected of containing the selected cell population with a cell binding agent-maytansinoid conjugate, where the cell binding agent maytansinoid-conjugated has following formula: trastuzumab-SMCC-DM1 or trastuzumab-SIAB.

The invention also relates to an in vitro method for eliminating cells, characterized in that the method comprises contacting the cells with a cell binding agent-maytansinoid conjugate where the cell binding agent-maytansinoid conjugate has the following formula: trastuzumab-SMCC-DM1 or trastuzumab-SIAB.

The invention also relates to the cell binding agent-maytansinoid conjugates with the formula trastuzumab-SMCC-DM1 or trastuzumab-SIAB-DM1, where the cells are diseased or infected cells from tumors, for use in a method for treating said tumor, where the method comprises administering to a individual in need of treatment an effective amount of said conjugate.

The present invention and embodiments thereof are specified in the subsequent patent claims.

Brief description of the figures

Figure 1 shows the structure of the SMCC.

Figure 2 shows the structure of DM1.

Figure 3 shows graphical results of a FACS binding assay comparing huC242 antibody with the antibody-maytansinoid conjugate huC252-SMCC-DM1.

Figure 4 graphically shows the cytotoxicity of huC252-SMCC-DM1.

Figure 5 shows size exclusion chromatography for huC252-SMCC-DM1.

Figures 6A-C and Figure 7 graphically show the cytotoxicity of huC252-SMCC-DM1 compared to conjugates prepared with disulfide-containing linkers.

Figures 8A-D graphically show the cytotoxicity of SMCC-DM1 conjugates bound to various cell binding agents.

Figure 9 graphically shows the cytotoxicity of antibody-maytansinoid conjugate huC252-SMCC-DM1.

Figure 10A graphically shows the antitumor activity of huC252-SMCC-DM1 against human COLO205 colon cancer xenografts in SCID mice.

Figure 10B graphically shows the antitumor activity of huC252-SMCC-DM1 against human SNU16 gastric tumor xenografts in SCID mice.

Figure 10C graphically shows the antitumor activity of transtuzumab-SMCC-DM1 against human MCF7 tumor xenografts in SCID mice.

Figure 11 graphically shows plasma depletion rates for huC252-SMCC-DM1 compared to conjugates prepared with disulfide-containing linkers.

Figures 12A-C graphically show the results of acute toxicity studies for huC252-SMCC-DM1 compared to conjugates prepared with disulfide-containing linkers.

Figure 13 shows the duration of cell cycle arrest and cell killing activity exhibited by huC252-SMCC-DM1 compared to conjugates prepared with disulfide-containing linkers.

Figures 14A-C show the minimal bystander activity for huC242-SMCC-DM1 compared to conjugates prepared with disulfide-containing linkers.

Figure 15 shows representative structures for maleimido-based cross-linking agents.

Figure 16 shows representative structures for haloacetyl-based cross-linking agents.

Figure 17 shows the structure of antibody-SMCC-DM1 conjugates.

Figure 18 shows the structure of antibody-SIAB-DM1 conjugates.

Figure 19 shows the structure of antibody-SMCC-DM4 conjugates.

Figure 20 shows the structure of antibody-SIAB-DM4 conjugates.

Figure 21 shows the synthesis of maytansinoid cell binding agent conjugate bound via a non-S-containing non-cleavable linker.

Figure 22 graphically shows cytotoxicity for huC242-non-S-containing non-cleavable linker-DM1.

Figure 23 graphically shows the result for a FACs assay with huC242-non-S-containing non-cleavable linker-DM1.

Figure 24 graphically shows the results of a HER2 ECD plate binding assay comparing trastuzumab antibody to the antibody-maytansinoid conjugate trastuzumab-SMCC-DM1.

Figure 25 graphically shows the cytotoxicity and specificity of trastuzumab-SMCC-DM1.

Figure 26 shows size exclusion chromatography for trastuzumab-SMCC-DM1. Figure 27 graphically shows the results for the HER2 ECD plate binding assay comparing trastuzumab antibody with the antibody-maytansinoid conjugate trastuzumab-SIAB-DM1.

Figure 28 graphically shows the cytotoxicity and specificity of trastuzumab-SIAB-DM1. Figure 29 shows size exclusion chromatography for trastuzumab-SIAB-DM1.

Detailed description

The field reveals that it is extremely difficult to modify existing drugs without reducing their cytotoxic ability. Nevertheless, US patent documents show

6,441,163 B1, 6,333,410 B1, 5,416,064 and 5,208,020 that potent cytotoxic agents can be formed by binding maytansinoids to appropriate cell binding agents via cleavable linkers, particularly cleavable linkers containing disulfide groups.

Cell binding agent maytansinoid conjugates allow the full cytotoxic effect of the maytansinoids to be used in a targeted manner only against unwanted cells, thereby avoiding side effects due to damage to the non-targeted healthy cells.

The present invention has unexpectedly discovered that maytansinoids bound to cell binding agents via non-cleavable linkers are superior in several ways to maytansinoids bound via cleavable linkers. When compared to conjugates containing cleavable linkers, conjugates with non-cleavable linkers particularly show equivalent antitumor activity both in vitro and in vivo, but show a marked reduction in plasma clearance rate and in toxicity.

The conjugates according to the invention have one or more maytansinoids bound to a cell binding agent via a non-cleavable linker. In one method of preparing the conjugate, a cell-binding agent, for example an antibody, is first modified with a cross-linking reagent such as SMCC. In a second step, a reactive maytansinoid having a thiol group, such as DM1, is reacted with the modified antibody to produce antibody-maytansinoid conjugates. Alternatively, the maytansinoid may be modified with a cross-linking reagent before being reacted with a cell binding agent. See, for example, US patent 6,441,163 B1.

Appropriate maytansinoids

Maytansinoids are well known in the art and can be isolated from natural sources according to known methods, prepared 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 analogs. Examples of suitable maytansinol analogs include those having a modified aromatic ring and those having modifications at other positions.

Specific examples of suitable maytansinol analogs having a modified aromatic ring include:

(1) C-19-dechlor (US Patent 4,256,746) (produced by LAH reduction of ansamytocin-2),

(2) C-20-hydroxy (or C-20-demethyl)+/-C-19-dechlor (US Pat.

4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH) and (3) C-20-demethyloxy, C-20-acyloxy (-OCOR), /-dechlor (US patent document 4 294 757) (produced by acylation using acyl chlorides). Specific examples of suitable maytansinol analogs having modifications at other positions include;

(1) C-9-SH (US Patent 4,424,219) (prepared by reaction of maytansinol with H2S or P2S5,

(2) C-14-Alkoxymethyl(demethoxy/CH2OR) (US Patent 4,331,598),

(3) C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (US Patent 4,450,254) (made from Nocardia),

(4) C-15-hydroxy/acyloxy (US Patent 4,364,866) (produced by the conversion of maytansinol by Streptomyces),

(5) C-15-Methoxy (US Patents 4,313,946 and 4,315,929) (isolated from Trewia nudlflora),

(6) C-18-N-demethyl (US Patents 4,362,663 and 4,322,348) (produced by the demethylation of maytansinol by Streptomyces) and

(7) 4,5-deoxy (US Patent 4,371,533) (prepared by titanium trichloride/LAH reduction of maytansinol).

Many positions on maytansinol are known to be useful as the binding site, depending on the type of binding. For the formation of an ester bond, for example, the C-3 position which has a hydroxy group, the C-14 position which is modified with hydroxymethyl, the C-15 position which is modified with a hydroxyl group and the C-20 position which has a hydroxyl group all appropriate. Nevertheless, the C-3 position is preferred and the C-3 position of maytansinol is particularly preferred.

A preferred maytansinoid has a free thiol group. Particularly preferred maytansinoids comprising a free thiol group include N-methylalanine-containing esters and N-methylcysteine-containing esters of maytansinol and are C-3 esters of maytansinol and its analogs. Preferred esters include N-methylalanine-containing esters and N-methylcysteine-containing esters of maytansinol. Synthesis of esters of maytansinol having a free thiol group has previously been described, for example in US Patent 5,208,020, Chari et al., 52 Cancer Res., 127-131 (1992), and Liu et al., 93 Proc Natl . Acad. Sci., 8618-8623 (1996). Furthermore, US Patent 6,333,410 B1 provides an improved method for the preparation and purification of thioline-containing maytansinoids suitable for binding to the cell binding agent.

The conjugates according to the present invention use the thioline-containing maytansinoid DM1, formally designated N<2'>-deacetyl-N<2'>-(3-mercapto-1-oxopropyl)-maytansine. DM1 is represented by the following structural formula:

The synthesis of the thioline-containing maytansinoid DM1 has previously been described (US patent 5,208,020).

US Patent Application 10/849,136 describes sterically hindered thioline-containing maytansinoids bearing one or two alkyl substituents on the α-carbon bearing the thiol functionality. In addition, the acyl group on the acylated amino acid side chain of the maytansinoid bearing the sulfhydryl group contains a linear chain length of at least three carbon atoms between the carbonyl group on the amide and the sulfur atom. These new maytansinoids are suitable for use as described.

The synthesis of the maytansinoids having a sterically hindered thiol group can be described with reference to US patent application 10/849,136, especially in Figure 3.

Other maytansinoids that comprise a side chain containing a sterically hindered thiol bond are the maytansinoids N<2'>-deacetyl-N<2'>-(4-mercapto-1-oxopentyl)-maytansine (designated DM3) and N<2'>- deacetyl-N<2'>-(4-methyl-4-mercapto-1-oxopentyl)-maytansine (designated DM4). DM3 and DM4 are given by the following structural formulas:

Cell binding agents

The effectiveness of the described conjugates as therapeutic agents depends on the judicious selection of an appropriate cell binding agent. Cell binding agents can be of a type that per are currently known or will become known and include peptides and non-peptides. In general, 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.

More specific examples of cell binding agents that may 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 (Parham, 131 J. Immunol. 2895-2902 (1983), Spring et al., 113 J.

Immunol. 470-478 (1974), Nisonoff et al., 89 Arch. Biochem. Biophys. 230-244 (1960)), chimeric antibodies and antigen-binding fragments thereof, domain antibodies (dAbs) and antigen-binding fragments thereof, including camelid antibodies (Desmyter et al., 3 Nature Struct. Biol. 752, 1996),

shark antibodies called novel antigen receptors (IgNAR) (Greenberg et al., 374 Nature, 168, 1995, Standfield et al. 305 Science 1770-1773, 2004),

interferons (eg alpha, beta, gamma),

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 androgens and estrogens, growth factor 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)) and vitamins such as folate.

Monoclonal antibody techniques allow the production of extremely specific cell binding agents in the form of specific monoclonal antibodies. Particularly well known in the art are techniques for preparing monoclonal antibodies which 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 viruses, truncated whole viruses and viral proteins such as viral envelope proteins. Sensitized human cells can also be used. Another method of preparing monoclonal antibodies is the use of phage libraries of scFv (single chain variable region), especially human scFv (see, for example, Griffiths et al., US Patents 5,885,793 and 5,969,108, McCafferty et al., WO 92/01047 , Liming et al., WO99/06587). In addition, surface-treated antibodies disclosed in US patent 5,639,641 can also be used, and so can humanized antibodies.

Selection of the appropriate cell binding agent is a choice that depends on the particular cell population to be targeted, but generally human monoclonal antibodies are preferred if one is conveniently available.

The monoclonal antibody J5 is a murine IgG2a antibody specific for common acute lymphoblastic leukemia antigen (CALLA) (Ritz et al, 283 Nature 583-585 (1980)) and can for example be used if the targeted cells express CALLA as in diseases with acute lymphoblastic leukemia.

The monoclonal antibody MY9 is a murine IgG1 antibody that binds specifically to the CD33 antigen (J.D. Griffin et al 8 Leukemia Res., 521 (1984)) and can be used if the target cells express CD33 as in acute myelogenous leukemia (AML) diseases .

Similarly, the monoclonal antibody anti-B4, which is also called B4 and is a murine IgG1 that binds to the CD19 antigen on G cells (Nadler et al, 131 J.

Immunol. 244-250 (1983)) 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.

In addition, the monoclonal antibody C242 that binds to the CanAg antigen (US Patent 5,552,293) can be used to treat CanAg-expressing tumor, such as colorectal cancer, pancreatic cancer, non-small cell lung cancer and gastric cancer. HuC242 is a humanized form of the monoclonal antibody C242 which is described in US patent document 5,552,293 and for which the hybridoma has been deposited at ECACC with identification number 90012601. A humanized form can be produced by either using CDR transplantation methodology (US patent document 5,585,089 , 5,693,761 and 5,693,762) or the surface modification methodology (US Patent 5,639,641). HuC242 can also be used to treat CanAg-expressing tumours, such as colorectal cancer, pancreatic cancer, non-small cell lung cancer and stomach cancer.

Furthermore, the antibody trastuzumab can be used to treat breast cancer or other forms of cancer, such as prostate cancer and ovarian cancer that express the Her2 antigen.

Anti-IFG-IR antibodies that bind to the insulin growth factor receptor are also useful.

Ovarian and prostate cancer can be successfully targeted with, for example, an anti-MUC1 antibody, such as anti- HMFG-2 (Taylor-Papadimitriou et al., 28. Int. J. Cancer 17-21. 1981) or hCTM01 (56 Cancer Res. 5179-5185, 1996) and an anti-PSMA (prostate-specific membrane antigen), such as J591 (Liu et al. 57 Cancer Res. 36329-3634, 1997).

Non-antibody molecules can also be used to target specific cell populations. For example, GM-CSF that binds to myeloid cells can be used as a cell-binding agent to target disease-affected cells from acute myelogenous leukemia. In addition, IL-2 that binds to activated T cells can be used for the prevention of transplant rejection, for the therapy and prevention of graft versus host disease, and for the treatment of acute T-cell leukemia. MSH that binds to melanocytes can be used in the treatment of melanoma. Folic acid can be used to target the folate receptor that was expressed on ovarian tumors and other tumors. Epidermal growth factor (EGF) can be used to target squamous cancers such as lung cancer and head and neck cancer.

Somatostatin can be used to target neuroblastomas and other tumor types.

Cancer forms in the breast and in the testicles can be successfully targeted with estrogen (or estrogen analogues) or androgen (or androgen analogues) as cell binding agents.

Cross-linking reagents

The maytansinoid is bound to the cell-binding agent by means of a cross-linking reagent which, when reacted, forms a non-cleavable linker between the maytansinoid and the cell-binding agent.

As used herein, a "linker" is any chemical entity that covalently binds a cell binding agent together with a maytansinoid. In some cases, part of the linker is provided by the maytansinoid. For example, DM1, which is a thioline-containing maytansinoid (Figure 2), is a derivative of the natural maytansinoid maytansine and provides part of the linker. The side chain of the C-3 hydroxyl group of maytansine ends in -CO-CH3, the side chain of DM1 ends in -CO-CH2-CH2-SH. Therefore, the final link is composed of two parts, the cross-linking reagent introduced into the cell binding agent and the side chain from DM1.

Cleavable linkers are linkers that can be cleaved under mild conditions, i.e. conditions where the activity of the maytansinoid drug is not affected. Many well-known linkers fall into this category and are described below.

Disulfide-containing linkers are linkers that are cleavable via disulfide exchange, which can take place under physiological conditions.

Acid-labile linkers are linkers that are cleavable at acidic pH. Certain intracellular compartments, such as endosomes and lysosomes, for example, have an acidic pH (pH 4-5), providing conditions suitable for cleaving acid-labile linkers.

Linkers that are photolabile are useful on the body surface and in many body cavities that are exposed to light. Furthermore, infrared light can penetrate tissue.

Some linkers can be cleaved with peptidase. Only certain peptides are readily cleaved inside or outside cells, see, for example, Touet et al., 79 Proc.Natl.Axad.Sci.USA, 626-627 (1982) and Umemoto et al. 43 Int. J. Cancer, 677-684 (1989). Furthermore, the peptide is composed of α-amino acids and peptide bonds, which are chemically amide bonds between the carboxylate of one amino acid and the α-amino group of another amino acid. Other amide bonds, such as the bond between a carboxylate and the ε-amino group on lysine, are understood not to be peptide bonds and are considered to be non-cleavable.

Certain linkers can be cleaved by esterases. Again, only certain esters can be cleaved by esterases present on the inside or outside of cells. Esters are formed by the condensation of a carboxylic acid and an alcohol. Simple esters are esters made with simple alcohols, such as aliphatic alcohols and small cyclic or small aromatic alcohols. For example, the inventor of the present invention found no esterase that cleaved the ester of C-3 maytansine, since the alcohol component of the ester, maytansinol, is very large and complex.

A non-cleavable linker is any chemical entity capable of binding a maytansinoid to a cell binding agent in a stable covalent manner and does not fall under the categories presented above as cleavable linkers. Thus, 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 that binds together 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 does not remain -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 within a few hours to several days of treatment with each of the means described above.

Furthermore, "non-cleavable" refers to the ability of the chemical bond in the linker or that is attached to the linker to receive 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 with conditions in which the maytansinoid or cell binding agent does not lose its activity.

A person skilled in the art will easily be able to distinguish between non-cleavable and cleavable links.

An example of a suitable control for testing whether a linker is substantially resistant to cleavage is a linker with a chemical bond, such as a disulfide bond, which is susceptible to cleavage by any of the agents described above. One can test whether a linker is substantially resistant to cleavage by measuring the stability of the conjugates by means of ELISA, HPLC or other appropriate means, over a period of time ranging from between a few hours to several days, typically 4 hours to 5 days . ELISA assay can be used to measure the level of stable conjugate in the plasma concentration.

Non-cleavable are also characterized in that the in vivo half-life of conjugates comprising non-cleavable linkers is generally approximately 20% higher than that of conjugates comprising cleavable linkers. In mice, the in vivo half-life of the IgG-maytansinoid conjugates linked via non-cleavable linkers is at least 4 days.

Suitable cross-linking agents that form non-cleavable linkers between the maytansinoid and the cell binding agent are well known in the art and may form non-cleavable linkers that comprise a sulfur atom (such as DMCC) or that are without a sulfur atom.

Cross-linking reagents that form non-cleavable linkers between the maytansinoid and the cell binding agent include a maleimido- or haloacetyl-based unit. according to the present invention, such non-cleavable linkers are said to be derived from maleimido- or haloacetyl-based units. Cross-linking reagents comprising a maleimido-based unit include N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate), which is a "long chain" analog of SMCC (LC-SMCC) κ-maleimido-undecanoic acid-N-succinimidonedecanoic acid-N-succinimidyl ester (KMUA), γ-maleimido-butyric acid-N-succinimidyl ester (GMBS), εmaleimidocaproic acid-N-hydroxysuccinimide ester (EMCS), N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl 6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl-4-(pmaleimidophenyl)-butyrate ( SMPB), and N-(p-maleimidophenyl)isocyanate (PMPI) (see Figure 15 for representative structures of maleimido-based crosslinkers). These cross-linking reagents form non-cleavable linkers that are derived from maleimido-based units.

Cross-linking reagents comprising a haloacetyl-based unit include N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA) and N-succinimidyl-3-(bromoacetamido)propionate (SBAP) ( see Figure 16 for representative structures of haloacetyl-based cross-linking agents). These cross-linking reagents form non-cleavable linkers that are derived from haloacetyl-based units.

While the active esters described in Figures 15 and 16 are composed of N-succinimidyl and sulfosuccinimidyl esters, other active esters such as N-hydroxyphthalimidyl esters,

N-hydroxysulfophthalimidyl esters, orthonitrophenyl esters, paranitrophenyl esters, 2,4-dinitrophenyl esters, 3-sulfonyl-4-nitrophenyl esters, 3-carboxy-4-nitrophenyl esters, pentafluorophenyl esters and sulfonyl tetrafluorophenyl esters can also be used.

Particularly preferred cross-linking reagents form non-cleavable linkers that do not contain a sulfur atom. Figure 21 shows a maytansinoid molecule that is derivatized with a cross-linking reagent that is derived from an α,ω-dicarboxylic acid (an alkane or alkenedic acid where the alkane or alkene has 3-24 carbon atoms). 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 in Figure 21 was prepared 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-trimethylsilylethanol in the presence of decyclohexylcarbodiimide. 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. Reaction with maytansinol in the presence of dicyclohexylcarbodiimide and zinc chloride followed by the protecting trimethylsilyl group with tetrabutylammonium fluoride provides the maytansinoide ester bearing a free carboxy group. Esterification of the carboxyl group by reaction with sulfo-N-hydroxysuccinimide in the presence of decyclohexylcarbodiimide provides the active ester of maytansinol which can react with a cell binding agent to give a non-cleavable conjugate that does not contain a sulfur atom.

Synthesis of cytotoxic conjugates

Conjugates of cell binding agents and maytansinoids can be formed using any technique currently known or that will later be developed.

Methods for conjugating cell binding agents with maytansinoids generally involve two reaction steps. In a method described in US Patent 5,208,020, a cell-binding agent such as an antibody can be modified with a cross-linking reagent to introduce one or more, usually 1-10 reactive groups. The modified cell binding agents were then reacted with one or more thioline-containing maytansinoids to produce a conjugate.

Alternatively, as disclosed in US Patent 6,441,163 B1, a thioline-containing maytansinoid may first be modified with a cross-linking reagent, followed by reaction of the modified maytansinoid with a cell binding agent. For example, the thioline-containing maytansinoid can be reacted with the maleimido compounds described in Figure 15 or with the haloacetyl compounds described in Figure 16 to give a maytansinoid thioether bearing an active succinimidyl or sulfosuccinimidyl ester. Reaction of these maytansinoids containing an activated linker unit with a cell binding agent provides another method for preparing a non-cleavable cell binding agent maytansinoid conjugate.

In another example, as indicated above, a maytansinoid that does not contain a sulfur atom may first be derivatized with a dicarboxylic acid-based cross-linking reagent, followed by reaction with the cell-binding agent to form a conjugate in which the maytansinoid is bound to the cell-binding agent via a non-S-containing non- cleavable linker.

Typically, the average is 1-10 maytansinoids per antibody bound. The conjugate can be purified through a Sephadex G-25 column.

US Patent Nos. 5,208,020 and 6,441,163 B1 are hereby incorporated by reference. Examples of conjugates 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 analog maytansinoid derivatives, androgen-maytansinoid derivatives, androgen analog-maytansinoid derivatives and vitamin-maytansinoid conjugates such as folate maytansinoid.

Maytansinoid conjugates of antibodies, antibody fragments, protein hormones, protein growth factors and other proteins were similarly prepared. For example, peptides and antibodies can be modified with the non-cleavable cross-linking reagents mentioned above. A solution of an antibody in aqueous buffer can be incubated with a molar excess of an antibody-modifying cross-linking reagent such as succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-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-1-carboxy-(6-amidocaproate)) which is a "long chain" analog of SMCC (LC-SMCC), sulfo-LC-SMCC, κ-maleimidodecanoic acid-N-succinimidyl ester (KMUA), sulfo-KMUA, γ-maleimido-butyric acid-N-succinimidyl ester (GMBS), sulfo-GMBS . , N-succinimidyl-4-(p-maleimidophenyl)-butyrate (SMPB), sulfo-SMPH, N-(p-maleimidophenyl)-isocyanate (PMPI), N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N- succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA) and N-succiminidyl-3-(bromoacetamido)-propionate (SBAP), as described in the literature. See Yoshitake et al., 101 Eur. J. Biochem. 395-399 (1979), Hashida et al., J. Applied Biochem. 56-63 (1984), and Liu et al., 18 690-697 (1979), Uto et al., 138 J. Immunol. Meth. 87-94 (1991), Rich et al., 18 J. Med. Chem. 1004-1010 (1975), Kitagawa and Aikawa, 79 J. Biochem. (Tohyo) 233-236 (1976), Tanimori et al., 62 J. Immunol. Meth. 123-128 (1983), Hashida et al., 6 J. Appl. Biochem. 56-63 (1984), Thorpe et al., 140 Eur. J. Biochem. 63-71 (1984), Chrisey et al., 24 Nucl. Acid Res. 3031-3039 (1996), Annunziato et al., 4 Bioconjugate Chem. 212-218 (1993), Rector et al., 24 J. Immunol. Meth. 321-336 (1978), and Inman et al., 2 Bioconjugate Chem. 458-463 (1991).

The modified antibody was then treated with the thioline-containing maytansinoid (1.26 molar equivalent/maleimido or iodoacetyl group) to prepare a conjugate. The mixtures are incubated overnight at approximately 4 ºC. 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 spectrophotometrically measuring the ratio between the absorbances at 252 nm and 280 nm. Typically, an average of 1-10 maytansinoids per antibody bound.

A preferred method is to modify the antibody with succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) to introduce maleimido groups followed by reaction of the modified antibody with a thioline-containing maytansine iodide to give a thioether-linked conjugate. Again, this conjugate leads to 1-10 drug molecules per antibody molecule. Examples of antibody-maytansinoid conjugates are shown in figures 17-20.

Likewise, for example, estrogen and androgen cell binding agents such as estradiol and androstenediol can be esterified on the C-17 hydroxy group by reaction with an appropriately protected thiol group containing carboxyl chloride such as 3-S-acetylpropanoyl chloride. Other methods for esterification can also be used as described in the literature (Haslam, 36 Tetrahedron 2400-2433 (1980)). The protected or free thioline-containing androgen or estrogen can then be reacted with a thioline-containing maytansinoid to provide conjugates. The conjugates can be purified by means of column chromatography on silica gel or by means of HPLC.

A particularly preferred method is to modify maytansinol with a cross-linking reagent which leads to a bond containing no sulfur atoms, followed by reaction of the modified maytansinoid with an antibody to produce the conjugate.

Therapeutic Efficacy of Cytotoxic Conjugates Cell binding agent maytansinoid conjugates can be evaluated for their ability to suppress proliferation for various cell lines in vitro. For example, cell lines such as the human colon carcinoma cell line COLO205, the human melanoma cell line A375, the human myeloid leukemia cell line HL60, the human breast carcinoma cell line SKBR3 or the human epidermoid carcinoma cell line KB can be used for the investigation of cytotoxicity of these conjugates. Cells to be evaluated can be exposed to the compounds for 24 hours and the surviving fraction of cells can be measured in direct analysis using known methods. (See, for example, Goldmacher et al., 135 J. Immunol. 3648-3651 (1985), and Goldmacher et al., 102 J. Cell Biol. 1312-1319 (1986).) IC50 values can then be calculated from the results of the analysis.

High cytotoxicity can be defined as the demonstration of a toxicity that has an IC50 (the inhibitory concentration of a toxic substance that gives a survival fraction of 0.5) of about 10<-8 >M or less when measured in vitro with SKBR3- cells during a 24-hour exposure to the drug.

The in vitro potency and target specificity of the antibody-maytansinoid conjugates are shown in Figure 4. Conjugates of huC242 with DM1 using the cross-linking reagent SMCC are highly potent in killing antigen-positive SKBR3 cells with an IC50 value of 3.5 x 10< -12 >M. In contrast, antigen-negative A375 cells are approximately 800 times less sensitive, demonstrating that maytansinoid conjugates of the present invention are highly potent and specific.

The huC242-SMCC-DM1 conjugate was equally potent or more potent compared to conjugates prepared with disulfide-containing linkers in clonogenic (Figure 6A-C) and indirect cytotoxicity assays (Figure 7). These results were unexpected based on previously published data showing that an anti-Her2 antibody conjugated to maytansinoids via SMCC showed no specific activity (Chari et al., 52 Cancer Res. 127-133 (1992).

Activity of conjugates prepared with SMCC-non-cleavable linker is not limited to huC242 conjugates. Specific activity in vitro was also observed with SMCC-DM1 conjugates of trastuzumab, which is an anti-Her2 antibody, My9-6 which is an anti-CD33 antibody, KS77 which is an anti-EGFR antibody and N901 which is an anti-CD56 antibody (Figures 8A-D and 25).

In addition, conjugates with non-cleavable linkers that show specific activity in vitro are not limited to the SMCC linker. A huC242 conjugate of DM1 synthesized with the non-cleavable linker SIAB showed strong and antigen-specific cytotoxicity in clonogenic assays in vitro (Figure 9). Furthermore, a trastuzumab conjugate of DM1 synthesized with SIAB was also cytotoxic in clonogenic assays (Figure 28). Furthermore, a huC242-non-S-containing non-cleavable linker-DM1 conjugate also showed strong and antigen-specific cytotoxicity in clonogenic assays in vitro (Figure 22).

Antibody conjugates with DM1 utilizing the SMCC linker show antitumor efficacy against human tumor xenografts in mice (Figure 10A-C). As shown in Figure 10A, marked inhibition of tumor growth was first observed upon treatment of COLO205 colon tumor xenografts with huC242-SMCC-DM1. In this experiment, a group of five animals bearing established subcutaneous tumors were treated with huC242-SMCC-DM1 at a dosage of 150 μg/kg of conjugated DM1. Tumor sizes were measured periodically and used in a graph against time after tumor inoculation. All five treated animals had complete remission, although three animals subsequently relapsed at various time points, while two animals remained tumor-free until the end of the experiment (Figure 10 A). Additionally, as shown in Figure 10B, this antitumor activity was observed at conjugate doses that have no effect on mouse body weight, which is a measure of drug toxicity. In this experiment, three groups of five animals each bearing established subcutaneous SNU tumors were treated with huC242-SMCC-DM1 at doses of 15 μg/kg, 30 μg/kg, and 60 μg/kg of conjugated DM1, respectively. Tumor sizes were measured periodically and used in a graph against time after tumor inoculation. HuC242-SMCC-DM1 showed a dose-dependent antitumor effect. The results show that treatment of mice bearing COLO205 colon carcinoma tumor xenografts with huC242-SMCC-DM1 conjugate led to complete regression of tumors, with some mice remaining without detectable tumors for over two months after treatment (Figure 10A). Again, this activity was obtained at a conjugate concentration that showed no effect on mouse body weight. A trastuzumab-SMCC-DM1 conjugate also showed significant tumor regression in a mouse tumor xenograft model with the MCF-7 breast carcinoma cell line (Figure 10C).

Plasma clearance of the antibody-maytansinoid conjugate synthesized with the non-cleavable linker SMCC is very late and comparable to the clearance of antibody alone. This is in sharp contrast to the plasma depletion of conjugates prepared with relatively labile disulfide bonds such as huC242-SPP-DM1. For example, the depletion half-life of the SMCC conjugate is approximately 320 hours, while the half-life of the SPP conjugate is in the range of 40-50 hours (Figure 11). Nevertheless, the depletion of the antibody component for each type of conjugate is identical, suggesting that the difference in measured conjugate depletion is due to the loss of maytansinoid from the antibody conjugate in the case of the SPP-DM1 conjugate. The non-cleavable SMCC bond is therefore much more resistant to maytansinoid linker cleavage activities present in vivo than the SPP-DM1 conjugate. Furthermore, the decreased depletion rate of the SMCC-bound conjugates compared to the SPP-DM1 conjugates leads to an almost 5-fold increase in total maytansinoid exposure to the animal as measured by the area under the curve (AUC). This increased exposure can have a significant impact on drug effectiveness in some cases.

Maytansinoid conjugates prepared with non-cleavable linkers such as SMCC show an unexpectedly increased tolerability in mice compared to conjugates prepared with cleavable disulfide linkers. An acute toxicity test with a single intravenous dose was performed in CD-1 female mice. A comparison of the tolerability of a huC242-SMCC-DM1 conjugate (non-cleavable) with huC242 conjugates prepared with linkers containing cleavable disulfide bonds was performed by monitoring mouse death (Figure 12A and B) and signs of toxicity (Figure 12C and D) over a series of 4 escalating doses of each conjugate. The maximum tolerated dose (MTD) of the SMCC-DM1 conjugate was higher than the highest dose tested (150 mg/kg) while the MTD of the disulfide-linked conjugate SPP-DM1 was in the range of 45-90 mg/kg. At 150 mg/kg, all mice in the SMCC-DM1-induced group survived while lethal toxicity was observed for all mice in the SPP-DM1-treated group at 96 hours post-treatment.

Maytansinoid conjugates are thought to exert their cytotoxic activity via 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 (Figure 13). Treatment of COLO205 cells with huC242-SPP-DM1 or huC242-SMCC-DM1 conjugates leads to a complete G2/M arrest at 6-10 hours. At 30 hours after treatment, however, some of the cells arrested by treatment with the disulfide-bonded huC242-SPP-DM1 conjugate escape from cell cycle arrest and begin cell division again. Surprisingly, cells treated with the non-cleavable conjugate do not escape the cell cycle arrest at this later time point. The difference in the duration of activity of these two conjugates is also reflected in the percentage of dead cells at the 30 hour time point, as assessed by a dye exclusion assay using trypan blue. These results demonstrate an unexpected duration of the molecular events induced by treatment with the non-cleavable SMCC linker conjugates.

A further aspect of conjugates prepared with non-cleavable linkers compared to conjugates having cleavable disulfide linkers is the absence of activity against antigen-negative cells when in proximity to antigen-positive cells, referred to herein as the bystander effect. This means that the conjugates prepared with non-cleavable linkers have minimal bystander activity. Both huC242-SPP-DM1 (cleavable) and huC242-SMCC (non-cleavable) conjugates show strong cytotoxic activity against the antigen-positive COLO205 cell line and have no activity against the antigen-negative Namalwa cell line when grown separately (Figure 14A-C). . Nevertheless, treatment of cocultures of COLO205 and namalwa cells with huC242-SPP-DM1 reveals dramatic cytotoxic activity of the conjugate against even the antigen-negative namalwa cells. In contrast, the huC242-SMCC-DM1 conjugate shows no such bystander activity under these conditions. No cytotoxic activity against namalwa cells is observed with the huC242-SMCC-DM1 conjugate even when co-cultured with the antigen-positive COLO205 cells. This minimal bystander activity for the non-cleavable conjugate, as measured in this in vitro assay, may contribute to the increased tolerability of conjugates with non-cleavable linkers observed in acute toxicity studies.

Results from the above experiments show that the maytansinoid conjugates with non-cleavable linkers according to the present invention have greatly improved antitumor activity compared to previously described cell binding agent maytansinoid conjugates.

Applications

The conjugates described above may be used in an in vitro method for administering maytansinoids to a selected cell population, wherein the method comprises contacting a cell population or a tissue suspected of containing the selected cell population with a cell binding agent maytansinoid conjugate, wherein one or more maytansinoids is bound to the cell binding agent via a non-cleavable linker and the cell binding agent binds to cells in the selected cell population.

The above-described conjugates can also be used in an in vitro method for destroying cells, where the method comprises contacting the cells with a cell-binding agent maytansinoid conjugate, where one or more maytansinoids are covalently bound to the cell-binding agent via a non-cleavable linker and the cell-binding agent binds to the cells.

Examples of medical conditions that may be treated include, but are not limited to, malignant cancers of any type including, for example, cancers of the lung, breast, colon, prostate, kidney, pancreas, ovaries and lymphatic organs, autoimmune diseases such as systemic lupus, rheumatoid arthritis and multiple sclerosis, transplant rejections such as kidney transplant rejection, liver transplant rejection, lung transplant rejection, heart transplant rejection and bone marrow transplant rejection, graft versus host disease, viral infections such as CMV infection, HIV infection, AIDS, etc., and parasitic infections such as giardiasis, amoebiasis, schistosomiasis, and others as determined by a professional in the field.

The methods may be practiced in vitro or in vivo.

The above-described conjugates can be used in a method using in vitro to treat, for example, autologous bone marrow cells before their transplantation into the same patient to destroy diseased or malignant cells, bone marrow cells or other tissues before their transplantation to destroy T cells and other lymphoid cells and prevent graft versus host disease (GVHD), cell culture to destroy all cells except desired variants that do not express the target antigen, or cell cultures to destroy variant cells that express the unwanted antigen, the method comprising treating the cells with an effective amount of a cell binding agent-maytansinoid conjugate, where one or more maytansinoids are covalently bound to the cell binding agent via a non-cleavable linker and the cell binding agent binds to the cells to be destroyed.

The conditions for clinical and non-clinical in vitro use can really be determined by a person skilled in the art.

For example, processing can be carried out as follows. Bone marrow may be harvested from the patient or other individual and then incubated in medium containing serum to which has been added the cytotoxic agent of the invention, concentration range from about 10 pM to 1 nM for about 30 minutes to about 48 hours at about 37 ºC. The exact conditions for concentration and time for incubation, i.e. the dose, can easily be determined by a person skilled in the field. After incubation, the bone marrow cell can be washed with a medium containing serum and returned to the patient intravenously according to known methods. In cases where the patient receives other treatment, such as in a round of ablative chemotherapy or total body radiation between the time of marrow harvesting and reinfection of treated cells, the treated marrow cells can be stored frozen in liquid nitrogen using standard medical equipment.

For in vivo clinical use, the cytotoxic agent can be administered as a solution or a lyophilized powder that has been tested for sterility and for endotoxin levels.

Examples of suitable protocols for conjugate administration are as follows. Conjugates can be given weekly for four weeks as an intravenous bolus every week. Bolus doses may be given in 50 to 500 ml of normal physiological saline to which 5 to 10 ml of human serum albumin may be added. Dosages will be 10 mg to 2,000 per administration intravenously (in the range of 100 ng to 20 mg/kg per day). After four weeks of treatment, the patient can continue to receive treatment on a weekly basis.

Specific in vivo clinical protocols with respect to route of administration, excipients, diluents, dosages, times, etc. may be determined by one skilled in the art as the clinical situation requires.

If desired, other active agents such as other antitumor agents may be administered together with the conjugate.

New conjugates, preparations and methods for preparing the conjugates

While some conjugates are antibodies of maytansinoids linked with a non-cleavable linker are known, others are new. Therefore, there is provided a cell-binding agent-maytansinoid conjugate having at least one maytansinoid bound 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-1-carboxylate (SMCC), sulfo-SMCC, mmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS and succinimidyl iodoacetate when the cell binding agent is an antibody.

The new conjugates can be prepared and used as described above.

The preparation comprises the cell binding agent maytansinoid conjugate and a carrier.

The carrier may be a pharmaceutically acceptable carrier, diluent or excipient.

Suitable pharmaceutically acceptable carriers, diluents and excipients are well known and can be determined by those skilled in the art as the clinical situation requires.

Examples of suitable carriers, diluents and/or excipients: (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% physiological 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.

For these new conjugates, synthetic procedures are also provided.

One of the methods for producing the cell binding agent maytansinoid conjugate includes:

(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 thioline-containing maytansinoid thereby providing the non-cleavable linker between the cell-binding agent and the maytansinoid or the thioline-containing maytansinoid to produce the conjugate.

Another method for preparing the cell binding agent maytansinoid conjugate comprises:

(a) providing the maytansinoid or a thioline-containing maytansinoid;

(b) modifying the maytansinoid or the thioline-containing maytansinoid with a cross-linking agent to thereby form a non-cleavable linker and

(c) conjugating the maytansinoid or the thioline-containing maytansinoid with the cell-binding agent to thereby provide the non-cleavable linker between the cell-binding agent and the maytansinoid or the thioline-containing maytansinoid to produce the conjugate.

A further process for 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

(c) conjugating the modified maytansinoid with the cell binding agent thereby providing a non-S-containing non-cleavable linker between the cell binding agent and maytansinol to produce the conjugate.

These methods are described in detail above and in the US patents referenced herein.

EXAMPLES

The invention will now be illustrated with reference to examples. Unless otherwise stated, all percentages, ratios, parts, etc. are by weight. Examples 2 and 3 illustrate the invention, the other examples are reference examples.

The buffers used in the following experiments were: 50 mM potassium phosphate (KPi)/50 mM sodium chloride (NaCl)/2 mM ethylenediaminetetraacetic acid (EDTA), pH 6.5 (buffer A), 1x phosphate-buffered saline (PBS), pH 6 .5 (buffer B), and 0.1 M KPi buffer/2 mM EDTA at pH 7.5 (assay buffer).

SMCC (Product No. 22360, M.W. 334.33 g/mole) and SIAB (Product No. 22329, M.W. 412.15 g/mole) were purchased from Pierce. The huC242 antibody is a humanized form of the monoclonal antibody C242, described in US patent document 5,552,293, where the hybridoma has been deposited with ECACC identification number 90012601). Tastuzumab antibody was obtained from Genentech. DM1 (free thiol form, M.W. 737.5 g/mole) was prepared as previously described in US Patents 5,208,020 and 6,333,410 B1.

Chromatography was performed using chromatography columns purchased from Amersham Biosciences (Sephadex G25 NAP-25 prepacked columns (Amersham 17-0852-02), HiPrep 26/10 desalting columns, Sephadex G25 fine resin, 3 connected in series (Amersham 17-5087 -01)). TSK-GEL G3000SWXL chromatography columns (TOSOH Bioscience, 08541) were also used with TSK Column Guard SWxl (TOSOH Bioscience 08543).

Solvents used in the following experiments were dimethylsulfoxide (DMSO), dimethylacetamide (DMA), ethanol (EtOH) and 100 mM Ellman's reagent (DTNB, available from Cayman Chemical) in DMSO.

EXAMPLE 1A - REFERENCE EXAMPLE

Preparation of huC242-SMCC-DM1 conjugate

a. Preparation and measurement of huC242 antibody

The concentration of antibody was measured using an extinction coefficient of 1.48 (mg/ml)<-1 >at 280 nm and a molecular weight of 147,000 g/mole.

b. Preparation and measurement of SMCC stick solution

A 20 mM solution of SMCC (6.69 mg/ml) was prepared in dimethyl sulfoxide (DMSO). The solution was diluted 1/40 in analysis buffer and the absorbance of the samples was measured at 302 nm. The concentration of the stock solution was calculated using an extinction coefficient of 602 M<-1>cm<-1>.

c. Preparation and measurement of DM1 stick solution

A 10 nM solution of DM1 (free thiol form) was prepared in dimethylacetamide (DMA) (7.37 mg/ml) (Figure 2). The absorbance in the dilutions of the stock solution in ethanol (EtOH) was measured at 280 nm. The concentration in cane DM1 was calculated using an extinction coefficient of 5,700 M<-1 >at 280 nm. The concentration of free sulfhydryl or thiol groups (-SH) in the stick DM1 preparation was determined using Ellman's reagent (DTNB). Dilutions of the stock solution were made in assay buffer to 3% (vol/vol) DMA and then 100 nM DTNB in DMSO (1/100 vol) was added.

The increase in absorbance at 412 nm was measured against a blank and the concentration was calculated using an extinction coefficient of 14,150 M<-1>cm<-1>. The concentration of –SH that emerged from the Ellman analysis was used to represent the DM1 stick concentration in calculations for conjugation conditions.

d. Modification of huC242 with SMCC crosslinker

The antibody was split into two samples, one modified using a 7.5-fold molar excess of SMCC cross-linker, the other with an 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 DMSO (5% v/v) for 2 h at room temperature with stirring.

e. G25 chromatography to remove excess SMCC

The huC242-SMCC reaction mixtures were gel filtered through 1.5 x 4.9 cm prepacked columns with Sephadex G25 resin equilibrated in buffer A. The loading and elution volumes were according to the manufacturer's instructions. The eluted modified antibody was analyzed to determine the concentration of the antibody using the extinction coefficient described above. The yield of modified antibody was 74.6% for the reaction with a 7.5-fold molar excess of SMCC and 81.6% for the reaction with an 8.5-fold molar excess of SMCC.

f. Conjugation of huC242-SMCC with DM1

The modified antibody samples were reacted with a 1.7-fold excess of DM1 relative to linker (assuming 5 linkers per antibody). The reaction was performed at an antibody concentration of 2.5 mg/ml in buffer A (97% v/v) with DMA (3% v/v). After addition of DM1, the reaction was incubated at room temperature for about 20 hours with stirring.

g. Conjugation purification using G25 comatograph The conjugation reaction mixtures were gel filtered via 1.5 x 4.9 cm pre-packed columns with Sephadex G25 resin equilibrated in buffer B. The loading and elution volumes were according to the manufacturer's instructions. The number of DM1 molecules that are bound per moles of huC242 were determined by measuring absorbance in the eluted material at both 252 nm and 280 nm. The DM1/antibody ratio for the 7.5-fold molar excess SMCC sample was found to be 3.54 and the ratio for the 8.5-fold molar excess SMCC sample was found to be 3.63.

The conjugation step yields were 83.7% and 75.4%, respectively. Both conjugates were pooled, sterile filtered and reanalyzed for drug and antibody concentrations. The pooled sample was assigned to Lot # 1713-146C and analyzed for binding, cytotoxicity, specificity, extent of aggregation and free drug content.

Table 1. Characteristics of huC242-SMCC-DM1

EXAMPLE 1B - REFERENCE EXAMPLE

In vitro testing of huC242-SMCC-DM1

a. Binding

The binding affinities for huC242 antibody and huC242-SMCC-DM1 were compared using an indirect method on COLO205 cells, where 5 x 10<3 >cells per well were used, with a primary incubation on ice for three hours. The results are shown in Figure 3. The naked antibody bound with a KD of 5.1 x 10<-10 >M and the conjugated version bound with a KD of 5.52 x 10<-10 >M. Thus, conjugation with DM1 does not appear to change the binding affinity for hu242.

b. Cytotoxicity and specificity

The in vitro cytotoxicity and specificity of huC242-SMCC-DM1 conjugate was investigated using a continuous exposure clonogenic assay. The results are shown in Figure 4. huC242-SMCC-DM1 was effective in destroying the antigen-positive SKBR3 cells (IC50 = 3.5 x 10<-12 >M). Specificity was shown by comparing the IC 50 value for target SKBR3 cells with that of the antigen-negative cell line, A375, where the IC 50 of the conjugate was greater than 3.0 x 10<-9 >M.

c. Size exclusion chromatography analysis

The conjugate was analyzed using a TSK3000 size exclusion column (Figure 5). Peak 4 represents the monomer fraction of the conjugate while earlier peaks represent multimer and later peaks represent fragment. The area under each curve divided by total peak areas represents the peak's contribution to the sample. The conjugate sample was found to be 96.0% monomeric.

d. Free drug

The percentage of free drug was measured by ELISA and was found to be 4.4%.

EXAMPLE 2A

Preparation of trastuzumat-SMCC-DM1 conjugate

Trastuzumab antibody was obtained from Genentech for conjugation to DM1 using the non-cleavable, heterobifunctional, cross-linking reagent SMCC. Buffer was changed on the antibody from 50 mM potassium phosphate/2 mM EDTA, pH 6.0 to 50 mM potassium phosphate/50 mM sodium chloride/2 mM EDTA, pH 6.5 (buffer A). The antibody was then reacted with a 7.5-fold molar excess of SMCC linker and purified using Sephadex-G25 resin before being conjugated with DM1. The final conjugate was again purified with Sephadex-G25 resin. The resulting conjugate contained 3.1 M of DM1 per moles of antibody.

a. Production and measurement of trastuzumab antibody

Trastuzumab antibody in 50 mM potassium phosphate/2 mM EDTA, pH 6.0 buffer was passed over a Sephadex-G25 column equilibrated with buffer A and eluted into buffer A. All buffers used in this experiment were tested to be certain that they were free of endotoxin using a chromogenic Lymulus amoebocyte lysate (LAL) method (Cambrex). The concentration of antibody was measured using an extinction coefficient of 1.45 ml mg<-1 >cm<-1 >at 280 nm and a molecular weight of 145,423 g.

b. Preparation and measurement of SMCC stick solution

A 20 mM solution of SMCC (6.69 mg/ml) was prepared in DMSO. The solution was diluted 1/40 in analysis buffer and the absorbance of the sample was measured at 203 nm.

The concentration of the stock solution was calculated using a molar extinction coefficient of 602 M<-1>cm<-1>.

c. Preparation and measurement of DM1 stick solution

A 10 mM solution of DM1 (free thiol form) was prepared in DMA (7.37 mg/ml) (Figure 2). The absorbance of dilutions of the stock solution in EtOH was measured at 280 nm.

The concentration of DM1 stock solution was calculated using a molar extinction coefficient of 5700 M<-1>cm<-1 >at 280 nm. The concentration of free SH in the DM1 stick solution was measured using Ellman's reagent (DTNB). Dilutions of the stock solution were prepared in assay buffer and made to 3% (vol/vol) DMA and then 100 nM DTNB in DMSO (1/100 vol) was added. The increase in absorbance at 412 nm was measured against a blank and the concentration was calculated using an extinction coefficient of 14,159 M<-1>cm<-1>. The concentration of -SH obtained from the Ellman assay was used to represent DM1 to the stock solution concentration in calculations for conjugation conditions.

d. Modification of trastuzumab with SMCC crosslinker

The antibody was modified using a 7.5-fold molar excess of SMCC at 20 mg/ml antibody. The reaction was carried out in buffer A (95% v/v) with DMSO (5% v/v) for 2 h at room temperature with stirring.

e. G25 chromatography to remove excess SMCC

The trastuzumab-SMCC reaction mixture was gel filtered through a 1.5 x 4.9 cm prepacked column with Sephadex-G25 resin equilibrated to buffer A. The loading and elution volumes were according to the manufacturer's instructions (Amersham Biosciences). The concentration of the modified antibody solution was analyzed spectrophotometrically using the extinction coefficient described above. The yield of modified antibody was 88% based on protein concentration.

f. Conjugation of trastuzumab-SMCC with DM1

The modified antibody was reacted with a 1.7-fold excess of DM1 relative to linker (assuming 5 linkers per antibody). The reaction was performed at 10 mg/ml antibody concentration in buffer A (94% v/v) with DMA (6% v/v). After addition of DM1, the reaction was incubated at room temperature for 16.5 hours with stirring.

g. Conjugation Purification by G25 Chromatography The conjugation reaction mixture was gel filtered through a 1.5 x 4.9 cm prepacked column with Sephadex-G25 resin equilibrated in buffer B. The loading and elution volumes were according to the manufacturer's instructions (Amersham Biosciences). The number of DM1 molecules bound per moles of trastuzumab was determined by measuring absorbance at both 252 nm and 280 nm of the eluted material. The DM1/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.

Table II. Characteristics of trastuzumab-SMCC-DM1

EXAMPLE 2B

In vitro testing of trastuzumat-SMCC-DM1

Binding studies showed that the conjugation of antibody to DM1 did not affect the apparent KD, both naked trastuzumab antibody and trastuzumab-SMCC-DM1 conjugate had the same binding affinity to ECD plates (5.5 x 10<-11 >M). Evaluation of the in vitro cytotoxicity of the sample showed that the trastuzumab-SMCC-DM1 conjugate is both highly toxic (IC<50 >3.6 x 10<-12 >M on antigen-positive cell line) and specific (IC<50 >greater than 3 .0 x 10<-9 >M on antigen-negative cell line).

a. Binding

The binding affinity of trastuzumab antibody and trastuzumab-SMCC-DM1 was compared using the HER2-ECD plate binding assay provided by Genetech. The results are shown in Figure 24. Both the naked antibody and the conjugated version start with an apparent KD of 5.5 x 10<-11 >M. Thus, conjugation with DM1 does not change the binding affinity of trastuzumab.

b. Cytotoxicity and specificity

The in vitro cytotoxicity and specificity of the trastuzumab-SMCC-DM1 conjugate were evaluated using a continuous exposure clonogenic assay.

The results are shown in Figure 25. Trastuzumab-SMCC-DM1 was effective in destroying the antigen-positive SKBR3 cells (IC<50>1.6 x 10<-12 >M). Specificity was shown when the IC50 was compared for the target SKBR3 cells and the antigen-negative cell line A375, where the IC50 of the conjugate was greater than 3.0 x 10<-9 >M.

c. Size exclusion chromatography analysis

The conjugate was analyzed using a TSK3000 size exclusion column (Figure 26). Peak 1 represents multimer, peak 2 represents dimer and peak 3 represents monomer. The area under each curve divided by total peak areas represents the peak's contribution to the sample. The conjugate sample was found to be 95.3% monomeric (Figure 26).

d. Analysis of free drug

The percentage of free drug was measured by ELISA and found to be 3.4%.

e. Endotoxin level

The conjugate was tested using a chromatographic LAL test and found to contain 0.03 EU/mg.

EXAMPLE 3A

Preparation of trastuzumat-SIAB-DM1 conjugate

Trastuzumab antibody was obtained from Genetech for conjugation to DM1 using the non-cleavable heterobifunctional cross-linker SIAB. The antibody was reacted with a 7.0-fold molar excess of SIAB linker at pH 6.5 and purified using Sephadex-G25F resin. Antibody-containing fractions were pooled and reacted with DM1 overnight at standard conjugation conditions of pH 6.5 and room temperature, but in the dark. A volume was removed from the reaction tube and analyzed to determine incorporation of DM1. The volume was measured after a NAP-5 filtration to have only 1.4 drugs/Ab. An additional 8-fold excess of SIAB was added to the reaction for 2 h and then the pH was raised to 8 immediately prior to the addition of an additional 1.5-fold excess of DM1/SIAB. The reaction was allowed to proceed and was purified using Sephadex-G25F resin. The resulting conjugate contained 3.42 moles of DMI per moles of antibody.

a. Measurement of trastuzumab antibody

The concentration of antibody was measured using an extinction coefficient of 1.45 ml mg<-1 >in cm<-1 >at 280 nm and a molecular weight of 145,423 g.

b. Production and measurement of SIAB cane solution

An 18 mM solution of SIAB (7.2 mg/ml) was prepared in DMSO. A wavelength scan of the solution diluted in pH 4 buffer was recorded for information purposes only.

c. Preparation and measurement of DM1 stick solution

A solution of DM1 (free thiol form) of approximately 30 mM was prepared in DMA.

The concentration of free -SH in the DM1 stock solution was measured using Ellman's reagent (DTMB). Dilutions of the stock solution were prepared in the assay buffer and made to 3% (vol/vol) DMA, and then 100 mM DTNV in DMSO (1/100 vol) was added.

The increase in absorbance at 412 nm was measured against a blank and the concentration was calculated using a molar extinction coefficient of 14,150 M<-1>cm<-1>.

The concentration of -SH resulting from the Ellman analysis was used to represent DM1 stick concentration in calculations for conjugation conditions.

d. Modification of trastuzumab with SIAB crosslinker

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 h at room temperature with stirring in the dark.

e. G25 chromatography to remove excess SIAB

The trastuzumab-SIAB reaction mixture was gel filtered through HiPrep 26/10 desalting columns equilibrated in buffer A. There appeared to be an interference at 280 nm from the SIAB reagent so the yield of modified antibody was assumed to be 100% and a modification of 5 linkers/antibody was assumed to determine the amount of DM1 in the conjugation reaction.

f. Conjugation of trastuzumab-SIAB with DM1

The modified antibody was reacted with a 1.7-fold excess of DM1 relative to linker assuming 100% yield and 5 crosslinkers/antibody as mentioned above. The concentration of antibody in the reaction was calculated to be 12.5 mg/ml and the reaction was performed in buffer A (97% v/v) with DMA (3% v/v). After addition of DM1, the reaction was incubated at room temperature in the dark for 16.5 hours with stirring.

g. Conjugation reaction analysis

A volume of 0.25 ml from the reaction mixture was removed and gel filtered through a pre-packed G25-Sephadex column equilibrated in buffer B. The number of DM1 molecules bound per moles of trastuzumab was determined by measuring absorbance at both 252 nm and 280 nm in the eluted material. The ratio DM1/antibody was only 1.4.

h. Additional modification/conjugation reaction

An additional 8-fold molar excess of SIAB was added and allowed to incubate for 2 hours at room temperature. A 1.5 times molar excess of DM1 relative to SIAB was added and the pH of the reaction was increased to 8 with the addition of 1 N NaOH.

The reaction was incubated at room temperature in the dark and gel filtered through a column of G25F resin equilibrated in buffer B.

i. Aggregation and characterization of conjugate

Protein-containing fractions were pooled, filtered and measured by absorbance at 252 and 280 nm. Samples of the conjugate were tested for endotoxin level, binding, specific and non-specific cytotoxicity, percent monomer and free drug level.

Table III. Characteristics of trastuzumab-SIAB-DM1

EXAMPLE 3B

In vitro testing of trastuzumab-SIAB-DM1

Binding studies showed that conjugation of antibody to DM1 did not affect the apparent KD, both naked trastuzumab and trastuzumab-SIAB-DM1 had similar binding affinities (1.2 x 10<-10 >M Ab and 1.9 x 10<-10 >M apparent KD conjugate). Evaluation of the in vitro cytotoxicity of the sample showed that the trastuzumab-SIAB-DM1 conjugate is both highly toxic (IC505 x 10<-12 >M on antigen-positive cell line SKBR3) and specific (IC50 greater than 3.0 x 10<-9 > M on antigen-negative cell line A375).

a. Binding

The binding affinity of trastuzumab antibody and trastuzumab-SIAAB-DM1 was compared using the HER2-ECD plate binding assay provided by Genentech. The results are shown in figure 27. Naked trastuzumab and trastuzumab-SIAB-DM1 had similar binding affinities (1.2 x 10<-10 >M for the antibody and 1.9 x 10<-10 >M apparent KD for the conjugate).

b. Cytotoxicity and specificity

Evaluation of the in vitro cytotoxicity of the sample showed that the trastuzumab-SIAB-DM1 conjugate is both highly toxic (IC50=5 x 10<-12 >M on antigen-positive cell line SKBR3) and specific (IC50 greater than 3.0 x 10<- 9 >M on antigen-negative cell line A375). See Figure 28.

c. Size exclusion chromatography analysis

The conjugate was analyzed using a TSK3000 size exclusion column (Figure 29). Peak 1 represents dimer and peak 2 represents monomer. The area under each curve divided by the total peak area represents the peak's contribution to the sample.

The conjugate sample was found to be 96.4% monomeric (Figure 29).

d. Free drug

The percentage of free drug was measured by ELISA and was found to be 0.35%.

e. Endotoxin level

The conjugate was tested using a chromatographic LAL test and found to contain >0.04 EU/mg.

EXAMPLE 4 - REFERENCE EXAMPLE

Conjugation of huC242 with a cross-linker that forms a non-S-containing non-cleavable linker

a. Synthesis

A stock solution of the crosslinking reagent (see Figure 21 for structure) was prepared in DMA, insoluble precipitate was spun off and the concentration in the remaining solution was determined using an extinction coefficient of ε<280>= 5,700 M<-1 >cm<- 1 >which is the extinction for DM1 at this wavelength. Since the real extinction coefficient for this material has not been measured, this is only an estimate of the concentration. It should be pointed out that the ratio ε<252>/ ε<280 > for DM1 is 4.7 (in EtOH) while ε<252>/ ε<280 > for the crosslinking reagent solution (in pH 7.5 buffer) was measured to be 1, 42, which does not indicate either various extinctions or impurities.

The conjugation reaction was performed on a 2 mg scale using 2.8 mg/ml huC242 antibody in 16% DMA in buffer E, pH 7.5 (buffer E = 50 mM sodium phosphate, 150 mM NaCl, 10 mM EDTA). Based on the estimated cross-linking reagent concentration in the stock solution, 30 equivalents of cross-linker/antibody were used (a previous experiment using 10 eq of cross-linker/antibody produced a conjugate of only 0.9 DM1/antibody). The reaction was allowed to proceed for 3 hours and then the conjugate was purified by passage through a Nap-10 (G25) column. After filtration (Millex GV filter, 0.2 µm pore size), the conjugate had 2.56 DM1/antibody (Lot # 1749-119A, antibody recovery = 78%). A volume of the conjugate was examined by HPLC (HiPrep column) for free DM1 and a DM1 peak was observed at 12.09'. The sample was therefore dialysed in buffer B to get rid of this peak and then reanalyzed. The final conjugate sample (Lot # 1749-124A) had no free DM1 by HPLC and had 1.84 DM1/antibody. SEC-HPLC was performed on the conjugate to show that it was 97% monomeric antibody.

b. Cytotoxicity and binding

The inventors performed binding and cytotoxicity studies on the huC242-non-S-containing non-cleavable linker-DM1 conjugate. First, the binding affinities of huC242 antibody, huC242-SMNP-DM3, and huC242-non-S-containing, non-cleavable linker-DM1 were compared using an indirect method on COLO205 cells.

5 x 10<3 >cells per well were used, with a primary incubation on ice for three hours.

The results are shown in Figure 23, which shows that the huC242-non-S-containing non-cleavable linker-DM1 conjugate had an approximately two-fold higher apparent dissociation constant relative to free antibody (see Figure 23). In addition, the huC242 non-S-containing non-cleavable linker-DM1 conjugate had an in vitro cytotoxicity comparable to huC242-SMNP-DM3 (IC50 of the non-S-containing non-cleavable linker conjugate = 7.0 x 10<- 12 >M) (see figure 22).

Claims (13)

PATENT CLAIMS 1. Cell binding agent-maytansinoid conjugate having the following formula: trastuzumab-N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate (SMCC)-N<2>'-deacetyl-N<2>'-(3-mercapto-1-oxopropyl )-maytansine (DM1) or trastuzumab-N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB)-DM1. 2. Cell binding agent-maytansinoid conjugate according to claim 1, wherein the cell binding agent binds to cells selected from the group consisting of breast cancer cells, ovarian cancer cells, non-small cell lung cancer cells and pancreatic cancer cells. 3. Cell binding agent-maytansinoid conjugate according to claim 1, where the cell binding agent binds to breast cancer cells. 4. Cell binding agent-maytansinoid conjugate according to claim 1, where the cell binding agent binds to tumor cells. 5. A composition comprising the cell binding agent-maytansinoid conjugate according to any one of claims 1 to 4 and a carrier. 6. Method for producing the cell binding agent-maytansinoid conjugate according to claim 1, wherein the method 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 the thiol-containing maytansinoid to produce the conjugate. 7. Method for producing the cell binding agent-maytansinoid conjugate according to claim 1, where the method comprises: (a) providing the maytansinoid or a thiol-containing maytansinoid, (b) modifying the maytansinoid or the 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 to thereby provide the non-cleavable linker between the cell-binding agent and the maytansinoid or the thiol-containing maytansinoid to produce the conjugate. 8. In vitro method for administering maytansinoids to a selected cell population, characterized in that the method comprises contacting a cell population or a tissue suspected of containing the selected cell population with a cell binding agent-maytansinoid conjugate, where the cell binding agent-maytansinoid conjugated has the following formula: trastuzumab-SMCC-DM1 or trastuzumab-SIAB. 9. In vitro method for the elimination of cells, characterized in that the method comprises contacting the cells with a cell binding agent-maytansinoid conjugate where the cell binding agent-maytansinoid conjugate has the following formula: trastuzumab-SMCC-DM1 or trastuzumab-SIAB. 10. Method according to claim 8 or 9, where the cell binding agent binds to tumor cells or cells expressing the Her-2 antigen. 11. Method according to claim 8 or 9, wherein the cell binding agent binds to cells selected from the group consisting of breast cancer cells, non-small cell lung cancer cells and pancreatic cancer cells. 12. Cell binding agent-maytansinoid conjugate having the following formula: trastuzumab-SMCC-DM1 or trastuzumab-SIAB-DM1, where the cells are diseased or infected cells from tumors, for use in a method for treating said tumors, where the method comprises administering to an individual in need of treatment an effective amount of said conjugate. 13. Conjugate according to claim 12, wherein the tumors are selected from the group consisting of breast cancer, non-small cell lung cancer, pancreatic cancer cells and ovarian cancer.