The specificity of cytotoxic agents can be greatly improved by targeted delivery through linkage of the cytotoxic agents to cell-binding agents.
Many reports have appeared on the attempted specific targeting of tumor cells with monoclonal antibody-drug conjugates (Sela et al, in Immunoconjugates 189-216 (C. Vogel, ed. 1987); Ghose et al, in Targeted Drugs 1-22 (E. Goldberg, ed. 1983); Diener et al, in Antibody mediated delivery systems 1-23 (J. Rodwell, ed. 1988); Pietersz et al, in Antibody mediated delivery systems 25-53 (J. Rodwell, ed. 1988); Bumol et al, in Antibody mediated delivery systems 55-79 (J. Rodwell, ed. 1988). All references and patents cited herein are incorporated by reference.
Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, and chlorambucil have been conjugated to a variety of murine monoclonal antibodies. In some cases, the drug molecules were linked to the antibody molecules through an intermediary carrier molecule such as serum albumin (Garnett et al, Cancer Res. 46: 2412 (1986); Ohkawa et al, Cancer Immunol. Immunother. 23: 86 (1986); Endo et al, Cancer Res. 47: 1076-1080 (1980)), dextran (Hurwitz et al, Appl. Biochem. 2: 25-35 (1980); Manabi et al, Biochem. Pharmacol. 34: 289-291 (1985); Dillman et al, Cancer Res. 46: 4886-4891 (1986); Shoval et al, Proc. Natl. Acad. Sci. 85: 8276-8280 (1988)), or polyglutamic acid (Tsukada et al, J. Natl. Canc. Inst. 73: 721-729 (1984); Kato et al, J. Med. Chem. 27: 1602-1607 (1984); Tsukada et al, Br. J. Cancer 52: 111-116 (1985)).
A wide array of linker technologies has been employed for the preparation of such immunoconjugates and both cleavable and non-cleavable linkers have been investigated. In most cases, the full cytotoxic potential of the drugs could only be observed, however, if the drug molecules could be released from the conjugates in unmodified form at the target site.
One of the cleavable linkers that has been employed for the preparation of antibody-drug conjugates is an acid-labile linker based on cis-aconitic acid that takes advantage of the acidic environment of different intracellular compartments such as the endosomes encountered during receptor mediated endocytosis and the lysosomes. Shen and Ryser introduced this method for the preparation of conjugates of daunorubicin with macromolecular carriers (Biochem. Biophys. Res. Commun. 102: 1048-1054 (1981)). Yang and Reisfeld used the same technique to conjugate daunorubicin to an anti-melanoma antibody (J. Natl. Canc. Inst. 80: 1154-1159 (1988)). Dillman et al. also used an acid-labile linker in a similar fashion to prepare conjugates of daunorubicin with an anti-T cell antibody (Cancer Res. 48: 6097-6102 (1988)).
An alternative approach, explored by Trouet et al, involved linking daunorubicin to an antibody via a peptide spacer arm (Proc. Natl. Acad. Sci. 79: 626-629 (1982)). This was done under the premise that free drug could be released from such a conjugate by the action of lysosomal peptidases. In vitro cytotoxicity tests, however, have revealed that antibody-drug conjugates rarely achieved the same cytotoxic potency as the free unconjugated drugs. This suggested that mechanisms by which drug molecules are released from the antibodies are very inefficient.
In the area of immunotoxins, conjugates formed via 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. 260: 12035-12041 (1985); Lambert et al, in Immunotoxins 175-209 (A. Frankel, ed. 1988); Ghetie et al, Cancer Res. 48: 2610-2617 (1988). This was attributed to the high intracellular concentration of glutathione contributing to the efficient cleavage of the disulfide bond between an antibody molecule and a toxin. Despite this, there are only a few reported examples of the use of disulfide bridges for the preparation of conjugates between drugs and macromolecules. (Shen et al, J. Biol. Chem. 260: 10905-10908 (1985)) described the conversion of methotrexate into a mercaptoethylamide derivative followed by conjugation with poly-D-lysine via a disulfide bond. Another report described the preparation of a conjugate of the trisulfide containing toxic drug calicheamycin with an antibody (Hinman et al, Cancer Res. 53: 3336-3342 (1993)).
One reason for the lack of disulfide linked antibody-drug conjugates is the unavailability of cytotoxic drugs possessing a sulfur atom containing moiety that can be used readily to link the drug to an antibody via a disulfide bridge. Furthermore, chemical modification of existing drugs is difficult without diminishing their cytotoxic potential.
Another major drawback with existing antibody-drug conjugates is their inability to deliver a sufficient concentration of drug to the target site because of the limited number of targeted antigens and the relatively moderate cytotoxicity of cancerostatic drugs like methotrexate, daunorubicin, and vincristine. In order to achieve significant cytotoxicity, linkage of a large number of drug molecules, either directly to the antibody or through a polymeric carrier molecule, becomes necessary. However, such heavily modified antibodies often display impaired binding to the target antigen and fast in vivo clearance from the blood stream.
In spite of the above-described difficulties, useful cytotoxic agents comprising cell-binding moieties and the group of cytotoxic drugs known as maytansinoids have been reported (U.S. Pat. No. 5,208,020, U.S. Pat. No. 5,416,064, and R. V. J. Chari, Advanced Drug Delivery Reviews 31: 89-104 (1998)). Similarly, useful cytotoxic agents comprising cell-binding moieties and analogues and derivatives of the potent antitumor antibiotic CC-1065 have also been reported (U.S. Pat. No. 5,475,092 and U.S. Pat. No. 5,585,499).
It has also been shown that the linkage of highly cytotoxic drugs to antibodies using a cleavable link, such as a disulfide bond, ensures the release of fully active drug inside cells, and such conjugates are cytotoxic in an antigen specific manner (R. V. J. Chari et al, Cancer Res. 52: 127-131 (1992); U.S. Pat. No. 5,475,092; and U.S. Pat. No. 5,416,064).
Taxanes are a family of compounds that includes paclitaxel (Taxol), a cytotoxic natural product, and docetaxel (Taxotere), a semi-synthetic derivative (see FIGS. 1 and 4), two compounds that are widely used in the treatment of cancer, E. Baloglu and D. G. I. Kingston, J. Nat. Prod. 62: 1448-1472 (1999). Taxanes are mitotic spindle poisons that inhibit the depolymerization of tubulin, resulting in cell death. While docetaxel and paclitaxel are useful agents in the treatment of cancer, their antitumor activity is limited because of their non-specific toxicity towards normal cells. Further, compounds like paclitaxel and docetaxel themselves are not sufficiently potent to be used in conjugates of cell-binding agents.
Recently, a few new docetaxel analogs with greater potency than either docetaxel or paclitaxel have been described (I. Ojima et al, J. Med. Chem., 39: 3889-3896 (1996)). However, these compounds lack a suitable functionality that allows linkage via a cleavable bond to cell-binding agents (FIG. 1).
The synthesis of novel taxanes that retain high cytotoxicity and that can be effectively linked to cell-binding agents has been described recently (U.S. Pat. No. 6,340,701, U.S. Pat. No. 6,372,738 and U.S. Pat. No. 6,436,931, and FIGS. 2 and 4). In these disclosures, taxanes were modified with chemical moieties, ones containing thiol or disulfide groups in particular, to which appropriate cell-binding agents could be linked. As a result, these novel taxanes preserved, and in some cases even enhanced, the cytotoxic potency of known taxanes.
In the taxanes described in the aforementioned patents, the linking group was introduced at the C-10, C-7 or the C-2′ position of the taxane.
In the cases where the linking group was at C-7, the C-10 position did not have a free hydroxyl substituent but was rather an ester, ether or carbamate substituent. It has been previously shown (I. Ojima et al, J. Med. Chem., 39: 3889-3896 (1996)) that the presence of an ester or carbamate substituent at C-10 produced taxoids of high potency. However, there have been no studies on the potency of taxanes bearing a free hydroxyl group at C-10 and a linking group at C-7.
As described herein, the potency of taxanes bearing a free hydroxyl group at C-10 and a linking group at C-7 was found to meet or exceed the potency of taxanes bearing an ester, ether or carbamate substituent at C-10 and a linking group at C-7. Thus, in a first aspect, the present invention provides these novel taxanes bearing a free hydroxyl group at C-10 and a linking group at C-7 and having potent cytotoxic activity.
Further, in the taxanes described in the aforementioned patents, the linking group was introduced at the C-10, C-7 or the C-2′ position of the taxane. In all taxanes, the substituents at C-3′N and C-3′, were named —NHCOR4 and R3, respectively. Furthermore, the substituent at C-3′N, —NHCOR4, was either a benzamido group (R4=phenyl), like in paclitaxel, or a tert-butyloxycarbonylamino moiety (—NH-t-BOC, R4=t-butoxy), like in docetaxel. Based on published data, it was assumed that altering these substituents would cause a loss in potency. The substituent at C-3′ (R3) was either aryl or a linear, branched or cyclic alkyl group having 1 to 10 carbon atoms. Since, based on published data, it was thought that the substituents at C-3′N and C-3′ could not be altered without a loss in drug activity, the linking group was always introduced at a different position of the taxane, namely at C-7, C-10 or C-2′. Also, the inability to change the substituents at C-3′ or C-3′N greatly limited the variety of disulfide-containing taxanes that could be synthesized.
In a second aspect, the present invention is also based on the unexpected finding that the substituents at both C-3′ and C-3′N do not have to be limited to that present in the known taxanes. As described herein, the potency of taxanes bearing a variety of different substituents at C-3′N meets or exceeds the potency of taxanes bearing a benzamido or —NH-t-BOC substituent at this position. The new substituent at C-3′ or C-3′N can also contain a linking group that allows for linkage to cell-binding agents. The present invention discloses these novel highly potent taxanes bearing a variety of different substituents at C-3′ and C-3′N. The linking group can now be incorporated at any one of the five positions: C-3′, C-3′N, C-10, C-7 or C-2′.