Organic compounds -- part of the class 532-570 series – Organic compounds – Unsubstituted hydrocarbyl chain between the ring and the -c-...
Reexamination Certificate
2001-05-31
2002-08-27
Kifle, Bruck (Department: 1624)
Organic compounds -- part of the class 532-570 series
Organic compounds
Unsubstituted hydrocarbyl chain between the ring and the -c-...
C540S462000
Reexamination Certificate
active
06441163
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an improved method for preparing cytotoxic conjugates comprising maytansinoids and cell binding agents. These conjugates have therapeutic use as they are delivered to a specific cell population in a targeted fashion. The present invention also relates to a method for preparing maytansinoids having a disulfide moiety that bears a reactive group which may be used in the preparation of cytotoxic conjugates. The present invention further relates to novel maytansinoids.
BACKGROUND OF THE INVENTION
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). 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.
46:2407-2412 (1986); Ohkawa et al.
Cancer Immumol. Immunother.
23:81-86 (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)). Recently, 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. described the conversion of methotrexate into a mercaptoethylamide derivative followed by conjugation with poly-D-lysine via a disulfide bond (
J. Biol. Chem.
260:10905-10908 (1985)). In addition, a report described the preparation of a conjugate of the trisulfide-containing toxic drug calicheamycin with an antibody (Menendez et al. Fourth International Conference on Monoclonal Antibody Immunoconjugates for Cancer, San Diego, Abstract 81 (1989)). Another report described the preparation of a conjugate of the trisulfide-containing toxic drug calicheamycin with an antibody (Hinman et al, 53
Cancer Res.
3336-3342 (1993)).
One reason for the lack of disulfide linked antibody-drug conjugates is the unavailability of cytotoxic drugs that bear a sulfur atom containing moiety that can be readily used 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.
Maytansinoids are highly cytotoxic drugs. Maytansine was first isolated by Kupchan et al. from the east African shrub
Maytenus serrata
and shown to be 100 to 1000 fold more cytotoxic than conventional cancer chemotherapeutic agents like methotrexate, daunorubicin, and vincristine (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that some microbes also produce maytansinoids, such as maytansinol and C-3 esters of maytansinol (U.S. Pat. No. 4,151,042). Synthetic C-3 esters of maytansinol and analogues of maytansinol have also been reported (Kupchan et al.
J. Med. Chem.
21:31-37 (1978); Higashide et al.
Nature
270:721-722 (1977); Kawai et al. Chem. Pharm. Bull. 32:3441-3451 (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 the 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 simple carboxylic acids (U.S. Pat. Nos. 4,248,870; 4,265,814; 4,308,268; 4,308,269; 4,309,428; 4,317,821; 4,322,348; and 4,331,598), and
(b) C-3 esters with derivatives of N-methyl-L-alanine (U.S. Pat. Nos. 4,137,230; 4,260,608; 5,208,020; and
Chem. Pharm. Bull.
12:3441 (1984)).
Esters of group (b) were found to be much more cytotoxic than esters of group (a).
Maytansine is a mitotic inhibitor. Treatment of L1210 cells in vivo with maytansine has been reported to result in 67% of the cells accumulating in mitosis. Untreated control cells were reported to demonstrate a mitotic index ranging from between 3.2 to 5.8% (Sieber et al. 43
Comparative Leukemia Research
1975, Bibl. Haemat. 495-500 (1976)). Experiments with sea urchin eggs and clam eggs have suggested that maytansine inhibits mitosis by interfering with the formation of microtubules through the inhibition of the polymerization of the microtubule protein, tubulin (Remillard et al.
Science
189:1002-1005 (1975)).
In vitro, P388, L1210, and LY5178 murine leukemic cell suspen
Chari Ravi V. J.
Widdison Wayne C.
Immunogen Inc.
Kifle Bruck
Sughrue & Mion, PLLC
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