`
`
`
`
`
`
`PHIGENIX
`PHIGENIX
`Exhibit 1024
`Exhibit 1024
`
`

`

`United States Patent
`
`[19]
`
`. [11] Patent Number:
`
`5,208,020
`
`Chari et a1.
`
`[45] Date of Patent:
`
`May 4, 1993
`
`USOOSZO802OA
`
`[54]
`
`[75]
`
`[73]
`
`[21]
`
`[22]
`
`[63]
`
`[51]
`
`[521
`
`[58]
`
`[56]
`
`CYTOTOXIC AGENTS COMPRISING
`MAYTANSINOIDS AND THEIR
`THERAPEUTIC USE
`
`Inventors: Ravi J. Chari, Boston; Victor S.
`Goldmacher, Newton Center; John
`M. Lambert, Cambridge; Walter A.
`Blattler, Brookline, all of Mass.
`
`Assignee:
`
`ImmunoGen Inc, Cambridge, Mass.
`
`Appl. No.: 911,380
`
`Filed:
`
`Jul. 13, 1992
`
`Related US. Application Data
`Continuation of Ser. No. 426,247, Oct. 25, 1989, aban-
`doned.
`
`Int. c1.s ................... 11611; 39/00, A61K 31/535;
`A61K 49/00; AOIN 57/00
`US. Cl. .............................. 424/8591; 514/279.5;
`540/462; 530/388.8; 530/388.15; 530/391.7;
`530/3889
`Field of Search .................. 424/8591; 514/229.5;
`540/462; 530/390
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`
`
`7/1975 Kupchan ............................. 540/462
`3,896,111
`4,137,230 1/ 1979 Hashimoto
`....... 540/462
`4,248,870
`2/1981 Miyashita .....
`514/2295
`4,256,246
`3/1981 Miyashita .....
`514/2295
`4,260,608
`4/1981 Miyashita .....
`514/2295
`4,263,294
`4/1981 Miyashita .....
`514/2295
`4,264,596 4/1981 Miyashita .
`514/2295
`4,294,757 10/1981 Asai ................. 540/462
`4,307,016 12/1981 Asai ................. 540/462
`4,308,268 12/1981 Miyashita .....
`514/2295
`4,308,269 12/1981 Miyashita .....
`514/2295
`4,309,428
`1/1982 Miyashita .....
`514/2295
`4,313,946 2/1982 Powell ......
`514/2295
`4,362,663 12/1982 Kida ............ 540/462
`4,364,866 12/1982 Asai ............. 540/462
`4,418,064 11/1983 Powell .............................. 514/2295
`
`
`
`4,424,219
`
`1/1984 Hashimoto ....................... 514/2295
`
`FOREIGN PATENT DOCUMENTS
`
`.
`0205326 12/1986 European Pat. Off.
`.
`0376176 7/1990 European Pat. Off.
`OTHER PUBLICATIONS
`
`Ravi V. J. Chari et a1, “Immunoconjugates Containing
`Novel Maytensinoids: Promising Anticancer Drugs”
`(Cancer Research), Cambridge, Mass, pp. 127-131,
`(Jan. 1, 1992).
`Greenfield et al Cancer Research, 50: 6600~6607 Oct.
`15, 1990.
`,
`Mueller et a1, Bioconjugate Chem. 1: 325—330 (1990).
`Endo et al, Cancer Research 47; 1076—1080 (Feb. 15,
`1987).
`
`Primary Examiner—Frederick E. Waddell
`Assistant Examiner—Gregory Hook
`Attorney, Agent, or Firm—Sughrue, Mion, Zinn,
`Macpeak 8: Seas
`
`[57]
`
`ABSTRACT
`
`A cytotoxic agent comprising one or more maytan-
`sinoids linked to a cell binding agent. A therapeutic
`agent for killing selected cell populations comprising:
`(a) a cytotoxic amount of one or more maytansinoids
`linked to a cell binding agent, and (b) a pharmaceuti-
`cally acceptable carrier diluent or excipient. A method
`for killing selected cell populations comprising contact-
`ing a cell population or tissue suspected of containing
`cells from said selected cell-population with a cytotoxic
`amount of a cytotoxic agent comprising one or more
`maytansinoids linked to a cell binding agent. An N-
`methyl-alanine-containing ester of maytansinol or an
`analogue of maytansinol, said N-methyl-alanine-con-
`taining ester comprising a linking group capable of
`linking an N-methyl-alanine-containing maytansinoid
`ester to a chemical moiety. N-methyl-cystcine-contain-
`ing ester of maytansinol or an analogue of maytansinol.
`
`18 Claims, 4 Drawing Sheets
`
`PHIGENIX
`
`Exhibit 1024-01
`
`

`

`US. Patent
`
`May 4, 1993
`
`Sheet 1 of 4
`
`5,208,020
`
`FIG.
`
`I
`
`PRIOR ART
`
`
`
`13
`
`H
`
`FIG. 2
`
`CH3SSOZCH3
`HS(CH2)n COOH ——-—> H3CSS(CH2)n COOH
`
`Z
`
`n=1
`(1
`bn=2
`c n=3
`00:4
`
`1 0 n=1
`bn=2
`c n=3
`d
`[1:4
`
`{W}
`2! ——————-———> x-Q—SSKHflzCOOH
`
`PHIGENIX
`
`Exhibit 1024-02
`
`02
`
`H N
`
`0
`
`b
`
`X
`
`x
`
`g
`
`nsstcnyncomcugcntcnycoon
`n
`R
`CH3
`CH3
`CH3
`
`l. (cuyzcncuzocoa/Egu
`————-—>
`2.
`cmnucntcuycoon
`
`RSS CH ) COOH
`
`(
`
`2 "
`
`

`

`US. Patent
`
`May 4, 1993
`
`Sheet 2 of 4
`
`5,208,020
`
`'
`MAYTANSINOL
`
`...____..._
`DCC/ZNClz
`
`5
`
`CH3
`
`ion SR
`0%;
`l
`2’" s
`
`
`
`é a
`b
`C
`6
`I
`f
`
`DTT
`
`R
`
`"
`
`(L)
`1
`CH3
`(L)
`2
`CH3
`CH3 3“.)
`CH3
`4 (D)
`CSHS 5“)
`C5H5 6 (0)
`
`(L)
`
`(L)
`
`2 (L)
`3_
`
`n
`
`1
`
`I a
`
`b
`c
`
`PHIGENIX
`
`Exhibit 1024-03
`
`

`

`US. Patent
`
`May 4,1993
`
`Sheet 3 of 4
`
`5,208,020
`
`FIG. 4a
`
`3'”
`°
`1““
`:2
`HOG—(IZH—N—H & HOG-(IZH-N-H
`
`n
`
`n=1-4
`
`g
`
`R=CH3, HIGHER ALKYL,ARYL
`
`n=1= u-usmncvsrems
`n=2: N-METHYLHOIOCYSTEINE
`
`3o
`0
`ll
`I
`u
`(CH312CHCH20COCl/E'3N
`1.
`9
`HOC(CH2)XCH3 28————— Hoc—CH-N—mcnz)x CH3
`'-
`I
`: 0-,0
`(cuzlnssn
`
`x
`
`CH
`
`2
`
`MAYTANSINOL
`DCC/ZnClz
`
`2 x = 0-10
`n = 1-4
`
`R = CH3, HIGHER ALKYL, ARYL
`
`FIG. 4b
`
`o
`
`CL
`
`S/S—R
`2’n
`0
`
`I
`
`(
`
`i
`
`H
`
`I
`
`
`
`PHIGENIX
`
`Exhibit 1024-04
`
`

`

`US. Patent
`
`May 4, 1993
`
`Sheet 4 of 4
`
`5,208,020
`
`FIG. 5
`
`FIG. 60
`
`CYTOTOXICITY .OF AT-NAYTANSINOID
`CONJUGATE (72 HR. EXPOSURE)
`
`CONPETINC BINDING ASSAY:
`HT-29 CELLS
`
`
`
`
`. HT-29 CELLs
`(AT-POSITIVE)
`0' KIBIITCENLIELgATIVEI
`
`
`
`
`o
`
`2
`
`I
`8
`5
`4
`CONCENTRATION x10 0N
`
`I2
`
`CONP TING IN IN As AY:
`
`55mg chfs 5
`
`
`
`
`0A7
`
`
`0 A? x SHAY
`
`A A? x 6 MAY
`
`I ANTI-84
`
`
`
`m
`:1
`8
`3
`
`E
`‘9
`5
`a
`;
`a
`
`FITC-LABELEDATBOUNDI%CONTROL)
`
`0.01
`
`‘00
`
`80
`
`60
`
`40
`
`20
`
`O
`
`CONCENTRATION OF COMPETING
`ANTIBODY 0R CONJUGATE InNI
`
`100
`
`‘3 BO
`8
`.—
`g
`‘5 60
`
`
`
`
`
`
`
`
`
`o A? x 3 MAY
`A A? x 6 MAY
`-
`I ANTI-B4
`
`
`
`
`I
`.
`IO
`CONCENTRATION OF CONPETING
`ANTIBODY OR CONJUGATE (nMI
`
`TOO
`
`BLOOD CLEWNCES 0F
`
`1961 AND IgGI-MAYTANSINOID
`
`0 (125-1) IgGI
`o IIzs-I) IgGI NAYTANSINOID
`
`'
`
`§ 40
`<
`a
`—J
`g
`:4 20
`2—3
`
`O
`
`mo
`
`80
`
`
`
`II25)-IINBLOOD"/0OF2MINUTEPOINT
`
`0
`
`0
`
`10
`
`.
`4O
`30
`20
`HOURS POST INJECTION
`
`50
`
`FIG. 6b
`
`F "3. 7
`
`PHIGENIX
`
`Exhibit 1024-05
`
`

`

`1
`
`5,208,020
`
`CYTOTOXIC AGENTS COMPRISING
`MAYTANSINOIDS AND THEIR THERAPEUTIC
`USE
`
`This is a continuation of application Ser. No.
`07/426,247 filed Oct. 25, 1989, now abandoned.
`
`FIELD OF THE INVENTION
`
`invention relates to novel cytotoxic
`The present
`agents and their therapeutic use. More specifically the
`invention relates to novel cytotoxic agents comprising
`maytansinoids and their therapeutic use. These novel
`cytotoxic agents have therapeutic use as a result of
`delivering the maytansinoids to a specific cell popula-
`tion in a targeted fashion by chemically linking the
`maytansinoid to a cell binding agent.
`BACKGROUND OF THE INVENTION
`
`In recent years, a myriad of reports have appeared on
`the attempted specific targeting of tumor cells with
`monoclonal antibody-drug conjugates (Sela et al.
`in
`Immunoconjagates 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
`(I. Rodwell, ed. 1988); Pietersz et al, in Antibody medi-
`ated delivery systems 25—53 (J. Rodwell, ed. 1988);
`Bumol et al, in Antibody mediated delivery system 55-79
`(J. Rodwell, ed. 1988). Cytotoxic drugs such as metho-
`trexate, daunorubicin, doxorubicin, vincristine, vinblas—
`tine, 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 intermedi-
`ary carrier molecule such as serum albumin (Gamett et
`a1. 46 Cancer Res 2407—2412 (1986); Ohkawa et a1 23
`Cancer Immumol. Immunother. 81—86 (1986); Endo et
`a1, 47 Cancer Res 1076—1080 (1980)), dextran (Hurwitz
`et a1, 2 Appl. Biochem. 25—35 (1980); Manabi et a1, 34
`Biochem. Pharmacol. 289—291 (1985); Dillman et al, 46
`Cancer Res. 4886—4891 (1986); Shoval et a1, 85 Proc.
`Natl. Acad. Sci. 8276—8280 (1988)), or polyglutamic acid
`(Tsukada et al, 73 J. Natl. Canc. Inst 721—729 (1984);
`Kato et a1 27 J. Med. Chem. 1602—1607 (1984); Tsukada
`et a1. 52 Br. J. Cancer 111—116 (1985)).
`A wide array of linker technologies have been em-
`ployed 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 intra-
`cellular compartments such as the endosomes encoun-
`tered during receptor mediated endocytosis and the
`lysosomes. Shen and Ryser introduced this method for
`the preparation of conjugates of daunorubicin with
`macromolecular carriers (102 Biochem. Biophys Res.
`Commun. 1048—1054 (1981)). Yang and Reisfeld used
`the same technique to conjugate daunorubicin to an
`anti-melanoma antibody (80 J. Natl. Canc.
`Inst.
`1154—1159 (1988)). Recently, Dillman et al also used an
`acid-labile linker in a similar fashion to prepare conju-
`gates of daunorubicin with an anti-T cell antibody (48
`Cancer Res 6097—6102 (1988)).
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`55
`
`65
`
`2
`An alternative approach, explored by Trouet et a1,
`involved linking daunorubicin to an antibody via a pep-
`tide spacer arm (79 Proc. Natl. Acad. Sci. 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 disul-
`fide bridges between monoclonal antibodies and cata-
`lytically active protein toxins were shown to be more
`cytotoxic than conjugates containing other linkers. See,
`Lambert et al, 260 J. Biol. Chem. 12035—12041 (1985);
`Lambert et al, in Immunotoxins 175—209 (A. Frankel,
`ed. 1988) Ghetie et al 48 Cancer Res. 2610—2617 (1988).
`This was attributed to the high intracellular concentra-
`tion 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 prepara-
`tion of conjugates between drugs and macromolecules.
`Shen et al described the conversion of methotrexate
`into a mercaptoethylamide derivative followed by con-
`jugation with poly—D-lysine via a disulfide bond (260 J.
`Biol Chem. 10905—10908 (1985)). A recent report de-
`scribed 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)).
`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
`readily used to link the drug to an antibody via a disul-
`fide bridge. Furthermore, chemical modification of
`existing drugs is difficult without diminishing their cy-
`totoxic 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 meth-
`otrexate, daunorubicin and vincristine. In order to
`achieve significant cytotoxicity, linkage of a large num-
`ber of drug molecules either directly to the antibody or
`through a polymeric carrier molecule becomes neces-
`sary. 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. Maytan-
`sine 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, daunorubi-
`cin, and vincristine (U.S. Pat. No. 3,896,111). Subse-
`quently it was discovered that some microbes also pro-
`duce 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, 21 J. Med.
`Chem 31—37 (1978); Higashide et al. 270' Nature
`721—722 (1977); Kawai et a1, 32 Chem. Pharm. Bull
`3441—3451 (1984)). Examples of analogues of maytan-
`sinol from which 03 esters have been prepared include
`maytansinol with modifications on the aromatic ring
`
`'
`
`PHIGENIX
`
`Exhibit 1024-06
`
`

`

`3
`(e.g. dechloro) or at the 09, C-14 (e.g. hydroxylated
`methyl group), 015, C-l8, 020 and 04,5.
`The naturally occurring and synthetic C-3 esters can
`be classified into two groups:
`.
`(a) G3 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) 03 esters with derivatives of N-methyl-L-alanine
`(U.S. Pat. Nos. 4,137,230; 4,260,608; and 12 Chem.
`Pharm. Bull 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, Bib]. Haemat
`495—500 (1976)). Experiments with sea urchin eggs and
`clam eggs have suggested that maytansine inhibits mito-
`sis by interfering with the formation of microtubules
`through the inhibition of the polymerization of the mi-
`crotubule protein, tubulin (Remillard et al, 189 Science
`1002—1005 (1975)).
`_
`In vitro P388, L1210, and LY5178 murine leukemic
`cell suspensions have been found to be inhibited by
`maytansine at doses of 10—3 to 10—1 microgram/ml
`with the P388 line being the 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 inhibited by concentrations as low as
`10—7 microgram/ml (Wolpert-DeFillippes et al, 24 Bio-
`chem. Pharmacol. 1735-1738 (1975)).
`In vivo, maytansine has also been shown to be active.
`Tumor growth in the P388 lymphocytic leukemia sys-
`tem was shown to be inhibited over a 50- to lOO-fold
`dosage range which suggested a high therapeutic index;
`also significant
`inhibitory activity could be demon-
`strated with the L1210 mouse leukemia system, the
`human Lewis lung carcinoma system and the human
`B-16 melanocarcinoma system (Kupchan, 33 Fed. Proc
`2288-2295 (1974)).
`Because the maytansinoids are highly cytotoxic, they
`were expected to be of use in the treatment of many
`diseases such as cancer. This expectation has yet to be
`realized. Clinical trials with maytansine were not favor-
`able due to a number of side effects (Issel et al, 5 Can.
`Trtmnt. Rev. 199—207 (1978)). Adverse effects to the
`central nervous system and gastrointestinal symptoms
`were responsible for some patients refusing further ther-
`apy (Issel at 204), and it appeared that maytansine was
`associated with peripheral neuropathy that might be
`cumulative (Issel at 207).
`Accordingly, a method of treating diseases with may-
`tansinoids wherein their side effects are reduced with-
`out compromising their cytotoxicity is greatly needed.
`SUMMARY OF THE INVENTION
`
`10
`
`15
`
`20
`
`30
`
`35
`
`45
`
`55
`
`Thus, one object of the present invention is to pro-
`vide maytansinoids in a form that are highly cytotoxic
`and that can still be effectively used in the treatment of
`many diseases. Another object of the present invention
`is to provide novel maytansinoid esters.
`These and other objects have been achieved by pro-
`viding a cytotoxic agent comprising one or more may-
`tansinoids linked to a cell binding agent.
`
`65
`
`5,208,020
`
`4
`In a second embodiment, the present invention pro-
`vides a therapeutic agent for killing selected cell popu-
`lations comprising:
`(a) a cytotoxic amount of one or more maytansinoids
`linked to a cell binding agent, and
`(b) a pharmaceutically acceptable carrier, diluent or
`excipient.
`In a third embodiment, the present invention pro-
`vides a method for killing selected cell populations
`comprising contacting a cell population or tissue sus-
`pected of containing cells from said selected cell popu-
`lation with a cytotoxic amount of a cytotoxic agent
`comprising one or more maytansinoids linked to a cell
`binding agent.
`In a fourth embodiment, the present invention pro-
`vides an N-methyl-alanine-containing ester of maytan-
`sinol or an analogue of maytansinol, said N-methyl-ala-
`nine-containing ester comprising a linking group capa-
`ble of linking an Ncmethyl-alanine-containing maytan-
`sinoid ester to a chemical moiety.
`In a fifth embodiment, the present invention provides
`an N-methyl-cysteine-containing ester of maytansinol
`or an analogue of maytansinol.
`
`25
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 shows maytansine (1a) and maytansinol (1b).
`FIG. 2 shows the synthesis of disulfidecontaining
`derivatives of N-methyl-L-alanine.
`FIG. 3 shows the synthesis of disulfide- and thiol-
`containing maytansinoids which can be linked to cell
`binding agents via a disulfide or any other sulfur-con-
`taining link such as thioether or thioester links. The
`synthesis starts with the intermediates of FIG. 2.
`FIG. 4(A) shows the synthesis of disulfide- and thiol-
`containing derivatives of N-methyl-L-cysteine.
`FIG. 4(B) shows the synthesis of disulfide- and thiol-
`containing maytansinoids from the intermediates of
`FIG. 4(A) that can be conjugated to cell binding agents
`via a disulfide or any other sulfur-containing link such
`as thioether or thioester links.
`FIG. 5 shows graphically the cytotoxicity of anti-
`body-maytansinoid conjugates.
`FIGS. 6(A) and 6(B) show graphically results of
`competitive binding assays of antibody-maytansinoid
`conjugates.
`FIG. 7 shows graphically the blood clearance of an
`antibody-maytansinoid conjugate in mice.
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`This invention is based on the synthesis of novel may-
`tansinoid derivatives that retain high cytotoxicity and
`that can be effectively linked to cell binding agents. The
`art reveals that it is extremely difficult to modify exist—
`ing drugs without diminishing their cytotoxic potential.
`The disclosed invention overcomes this problem by
`modifying the disclosed maytansinoid derivatives with
`chemical moieties, especially ones containing thiol or
`disulfide groups,
`to which appropriate cell binding
`agents can be linked. As a result, the disclosed novel
`maytansinoid derivatives preserve and in some cases
`even enhance the cytotoxic potency of the naturally
`occurring maytansinoids. The cell binding agent-
`maytansinoid derivative conjugates permit the full mea-
`sure of the cytotoxic action of the maytansinoid deriva-
`tives to be applied-in a targeted fashion against un-
`wanted cells only, therefore, avoiding side effects due
`to damage to non-targeted healthy cells. This invention
`
`PHIGENIX
`
`Exhibit 1024-07
`
`

`

`5,208,020
`
`5
`permits the maytansinoid derivatives to live up to their
`potential, something their undirected cytotoxic effects
`had previously made impossible. Thus the invention
`provides useful agents for the elimination of diseased or
`abnormal cells that are to be killed or lysed such as
`tumor cells (particularly solid tumor cells), virus in-
`fected cells, microorganism infected cells, parasite in-
`fected cells, autoimmune cells (cells that produce auto-
`antibodies), activated cells (those involvedin graft re-
`jection or graft vs. host disease), or any other type of
`diseased or abnormal cells, while exhibiting a minimum
`of side effects.
`
`Thus, this invention teaches the synthesis of maytan-
`sinoid derivatives that can be chemically linked to a cell
`binding agent while keeping a high cytotoxicity either
`in bound form or in released form or in both states. High
`cytotoxicity is defined as exhibiting a toxicity having an
`ICso-—~the inhibiting concentration of a toxic substance
`that leaves a surviving fraction of 0.5——of about 10—3M
`or less when measured in vitro with KB cells upon a 24
`hour exposure time to the drug.
`
`Cytotoxic Agent
`
`The cytotoxic agent according to the present inven-
`tion comprises one or more maytansinoids linked to a
`cell binding agent.
`In order to link the maytansinoid to a cell binding
`agent, the maytansinoid must first be modified
`Maytansinoids that can be usedin the present inven-
`tion to produce the modified maytansinoid capable of3
`being linked to a cell binding agent are well knownin
`the art and can be isolated from natural sources accord-
`ing to known methods or prepared synthetically ac-
`cording to known methods.
`Examples of suitable maytansinoids include maytan-
`sinol and maytansinol analogues. Examples of suitable
`maytansinol analogues include those having a modified
`aromatic ring and those having modifications at other
`positions.
`Specific examples of suitable analogues of maytan-
`sinol having a modified aromatic ring include:
`(1) C-19-dechloro (US. Pat. No. 4,256,746) (prepared
`by LAH reduction of ansamytocin P2);
`(2) C-20-hydroxy (or C—20—demethyl) +/-C-l9-
`dechloro (U.8. Pat. Nos. 4,361,650 and 4,307,016) (pre-
`pared by demethylation using Streptomyces or Actino-
`myces or dechlorination using LAH); and
`(—OCOR),
`(3) C-20—demethoxy, C-20-acyloxy
`+/—-‘dechloro (U.S. Pat. No. 4,294,757) (prepared by
`acylation using acyl chlorides).
`Specific examples of suitable analogues of maytan-
`sinol having modifications of other positions include:
`(1) C-9-Sl-I (US. Pat. No. 4,424,219) (prepared by the
`reaction of maytansinol with H28 or P255);
`(2)
`C-14-alkoxymethyl(demethoxy/CHzOR)(U.S.
`Pat. No. 4,331,598);
`(3) C-l4—hydroxymethyl or acyloxymethyl (CH20H
`or CHzoAc) (US. Pat. No. 4,450,254) (prepared from
`Nocardia);
`(4) C-15-hydroxy/acyloxy (US Pat. No. 4,364,866)
`(prepared by the conversion of maytansinol by Strepto-
`myceS);
`(5) C-lS-methoxy (U.8. Pat. Nos. 4,313,946 and
`4,315,929) (isolated from Trewia nudlflora);
`(6) C-lS-N-demethyl (US. Pat. Nos. 4,362,663 and
`4,322,348) (prepared by the demethylation of maytan-
`sinol by Streptomyces); and
`
`45
`
`55
`
`65
`
`10
`
`15
`
`25
`
`35
`
`6
`(7) 4,5—deoxy (US. Pat. No. 4,371,533) (prepared by
`the titanium trichloride/LAH reduction of maytan-
`sinol).
`In order to link the maytansinoid to the cell binding
`agent, a linking group is used.
`Suitable linking groups are well known in the art and
`include disulfide groups, thioether groups, acid labile
`groups, photolabile groups, peptidase labile groups and
`esterase labile groups. Preferred are disulfide groups
`and thioether groups.
`According to the present invention the linking group
`is part of a chemical moiety that is covalently bound to
`the maytansinoid through conventional methods. In a
`preferred embodiment,
`the chemical moiety can be
`covalently bound to the maytansinoid via. an ester link-
`age.
`Many positions on maytansinoids are expected to be
`useful as the linkage position, depending upon the type
`of link. For example, for forming an ester linkage, the
`03 position having a hydroxyl group, the C-14 position
`modified with hydroxymethyl, the C-15 position modi-
`fied with hydroxy and the C-20 position having a hy-
`droxy group are all expected to be useful. However the
`C-3 position is preferred and the 03 position of maytan-
`sinol is especially preferred.
`Also preferred is an N-methyl-alanine-containing C—3
`ester and an N-methyl-cysteine-containing C-3 ester of
`maytansinol or its analogues.
`
`Synthesis of Esters of Maytansinol Having a Linking
`Group
`
`While the synthesis of esters of maytansinol having a
`linking group is described belowin terms of thiol and
`disulfide linking groups, one of skillin the art will un-
`derstand that other linking groups specific representa-
`tive examples of which are set forth in Example 4, can
`also be used with the present invention as can other
`maytansinoids.
`The synthesis of maytansinoid derivatives can be
`described by reference to FIGS. 1, 2, 3, 4(A) and 4(B),
`where disulfide-containing maytansinoid esters are pre-
`pared by condensing maytansinol 1b with freshly pre-
`pared N-methyl-L-alanine or N-methyl-L-cysteine de-
`rivatives containing a disulfide group.
`w-Mercapto-carboxylic
`acids of varying chain
`lengths are converted into their respective methyl-
`dithio, e.g. 3a to 3d (where a: 1—10, including branched
`and cyclic aliphatics), or aryl-dithio, e.g. 4a to 4b, deriv-
`atives by reacting them with methyl methanethiolsul-
`fonate or aryldisulfides, such as diphenyldisulfide and
`ring substituted diphenyldisulfides and heterocyclic
`disulfides such as 2,2—dithiopyridine. The carboxylic
`acids are activated and then reacted with N-methyl-L-
`alanine to form the desired carboxylic acid compounds,
`e.g. 5a to 5f for condensation with maytansinol 1b.
`Esterification of maytansinol lb or an analogue with
`the carboxylic acids 53. to 5f gives the disulfide-contain-
`ing maytansinoids 6a to 6f. Cleavage of the disulfide
`group in 6a to 6f with dithiothreitol gives the thiol-con-
`taining maytansinoids 7a to 7c, which are readily linked
`via disulfide or thioether links to cell binding agents.
`N-methyl-Lflanine can be prepared as described in
`the literature (See, Fu, S. J. & Bimbauin, S. M., 75 J.
`Amer. Chem. Soc. 1953); or is obtainable commercially
`(Sigma Chemical Company».
`In another embodiment, N-methyl—cysteine or N-
`methylhomocysteine can be converted to the respective
`disulfide derivatives 8 (n=1 and 2, respectively) which
`
`PHIGENIX
`
`Exhibit 1024-08
`
`

`

`7
`are then acylated to yield the desired carboxylic acids 9
`(n = 1 and 2, respectively). Maytansinol is then esterified
`with 9 (n: 1) to give disulfide-containing ester 10. Re-
`duction of 10a with dithiothreitol as described for 7b
`
`5,208,020
`
`produces the thiol-containing maytansinoid 11 which
`can be conjugated to cell binding agents.
`N-methyl-cysteine can be prepared as described in
`Undheim and Eidem, 23 Acta Chem. Scand. 3129—3133
`(1970).
`More specifically, maytansinol 1b is derived from
`maytansine 1a or other esters of maytansinol by reduc-
`tion such as with lithium aluminum hydride. (Kupchan,
`S. M. et al 21 J. Med. Chem. 31—37 (1978); U.S. Pat. No.
`4,360,462). It is also possible to isolate maytansinol from
`the microorganism Nocardia, see, Higashide et al, U.S.
`Pat. No. 4,151,042 (1979). Maytansinol
`is then con-
`verted to the different ester derivatives, 6a to 6f and 10,
`using a suitable agent such as dicyclohexylcarbodiimide
`(DCC) and catalytic amounts of zinc chloride (See, U.S.
`Pat. No. 4,137,230; Kawai et a1, 32 Chem. Pharm. Bull
`3441-3951 (1984); U.S. Pat. No. 4,260,609). The two
`diastereomeric products containing the D and L-
`aminoacyl side chains result. The diastereomeric may-
`tansinoid esters are readily separated by preparative
`TLC on silica gel. For example, using Analtech GF
`plates (1000 microns) and developing with 6% metha-
`nol in chloroform yields distinct banding: the desired
`bands are scraped off the plate and the products ex-
`tracted with ethyl acetate. See, Kupchan, S. M., 21 J.
`Med. Chem 31—37 (1978); and Higashide et a1, U.S. Pat.
`No. 4,360,462 (1982).
`Reduction of the disulfide—containing maytansinoids
`to the corresponding mercapto-maytansinoids 7a, 7b, 7c
`and 11,
`is achieved by treatment with dithiothreitol
`(DTT) and purification by HPLC using a Waters radial-
`pak 018 column and eluting with a linear gradient of
`55% to 80% acetonitrile in H20 over 10 min. at a flow
`rate of 1.5 ml/min.
`
`When analogues of maytansinol are used as the start-
`ing material to give analogous disulfide-containing may-
`tansinoid esters, the analogues are prepared before re-
`acting them with the N-methyl-L-alanine or N—methyl-
`L-cysteine derivatives.
`Specific examples of N-methyl-alanine-containing
`maytansinoid derivatives useful in the present invention
`are represented by the formulae (I), (II), (III) and (IV).
`
`CH3 0
`II
`/\
`
`0
`
`Y3}?
`
`9
`may
`
`CH3
`
`(CH2)ISZo
`
`8
`
`CH
`
`‘fz
`T1
`u
`3
`ofi/E‘IAcn—CH—(CHMSZI ‘
`
`9
`
`CH3
`
`1)
`
`a
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`55
`
`miy
`
`wherein:
`
`R1 and R2, which may be the same or different, repre-
`sents H, CH3 or CH2CH3;
`Z] represents H or SR3, wherein R3 represents
`methyl, linear alkyl, branched alkyl, cyclic alkyl, simple
`or substituted aryl, or heterocyclic:
`m represents 0, l, 2 or 3; and may represents a may-
`tansinoid.
`
`(III)
`
`ofi/VN "M6}
`111:3:
`CH3
`
`wherein:
`
`Z2 represents H or SR4, wherein R4 represents
`methyl, linear alkyl, branched alkyl cyclic alkyl, simple
`or substituted aryl, or heterocyclic;
`n represents an integer of 3 to 8; and may represents
`a maytansinoid.
`
`(IV)
`
`
`
`wherein:
`
`Zorepresents H or SR, wherein R represents methyl,
`linear alkyl, branched alkyl, cyclic alkyl, simple or sub-
`stituted aryl or heterocyclic:
`1 represents 1, 2 or 3;
`yo represents C1 or H; and
`X3 represents H or CH3.
`Specific examples of N-methyl-cysteine—containing
`maytansinoid derivatives useful in the present invention
`are represented by the formulae (V) and (VI).
`
`wherein:
`
`Zorepresents H or SR, wherein R represents methyl,
`linear alkyl, branched alkyl, cyclic alkyl, simple or sub-
`stituted aryl or heterocyclic;
`1 represents an integer of l to 10; and may represents
`a maytansinoid.
`
`65
`
`wherein:
`
`5.7-3
`(Cl-12);, 0
`II
`0%},I A(CH2)PCH3
`
`CH3
`
`0
`may
`
`PHIGENIX
`
`Exhibit 1024-09
`
`

`

`9
`23 represents H or SR5, wherein R5 represents
`methyl, linear alkyl, branched alkyl, cyclic alkyl, simple
`or substituted aryl, or heterocyclic;
`0 represents 1, 2 or 3;
`p represents 0 or an integer of 1 to 10; and may repre-
`sents a maytansinoid.
`
`(VI)
`
`/\
`
`H
`(CH2)qC 3
`
`H If
`
`
`
`wherein:
`
`Z3 represents H or SR5, wherein R5 represents
`methyl, linear alkyl, branched alkyl, cyclic alkyl, simple
`or substituted aryl or heterocyclic:
`0 represents 1, 2, or 3;
`q represents 0 or an integer of 1 to 10;
`Yo represents C1 or H; and
`X3 represents H or CH3.
`Examples of linear alkyls include methyl, ethyl, pro-
`pyl, butyl, pentyl and hexyl.
`Examples of branched alkyls include isopropyl, iso-
`butyl, sec-butyl, tert.-butyl, isopentyl and l-ethyl-pro-
`pyl-
`Examples of cyclic alkyls include cyclopropyl, cyclo-
`butyl, cyclopentyl and cyclohexyl.
`Examples of simple aryls include phenyl and naph-
`thyl.
`Examples of substituted aryls include aryls such as
`those described above substituted with alkyl groups,
`with halogens, such as Cl, Br, F, nitro groups, amino
`groups, sulfonic acid groups, carboxylic acid groups
`hydroxy groups and alkoxy groups.
`Examples of heterocyclics are compounds wherein
`the heteroatoms are selected from O, N and S, and
`include pyrrollyl, pyridyl, fury] and thiophene.
`Disulfide-containing and mercapto-containing may-
`tansinoid drugs of the invention can be evaluated for
`their ability to suppress proliferation of various un-
`wanted cell lines In vitro. For example, cell lines such as
`the human epidermoid carcinoma line KB, the human
`breast tumor line SKBR3 and the Burkitt’s lymphoma
`line Namalwa can easily be used for the assessment of
`cytotoxicity of these compounds. Cells to be evaluated
`can be exposed to the compounds for 24 hours and the
`surviving fractions of cells measured in direct assays by
`known methods. IC50 values can then be calculated
`from the results of the assays.
`
`Preparation of Cell Binding Agents
`
`The effectiveness of the compounds of the invention
`as therapeutic agents depends on the careful selection of
`an appropriate cell binding agent. Cell binding agents
`may be of any kind presently known, or that become
`known and include peptides and non-peptides. Gener-
`ally,
`these can be antibodies (especially monoclonal
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`5,208,020
`
`10
`lymphokines, hormones, growth factors,
`antibodies),
`nutrient-transport molecules (such as transferrin), or
`any other cell binding molecule or substance.
`More specific examples of cell binding agents that can
`be used include:
`monoclonal antibodies:
`fragments of antibodies such as Fab, Fab’, and
`F(ab’)2 (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));
`interferons (e.g. a, B, 7); ‘
`lymphokines such as 1L2, 1L3, IL-4, IL—6;
`hormones such as insulin, TRH (thyrotropin releas-
`ing hormone), MSH (melanocyte-stimulating hor-
`mone), steroid hormones, such as androgens and estro-
`gens;
`growth factors and colony-stimulating factors such as
`EGF, TGF-a, G-CSF, M-CSF and GM-CSF (Burgess,
`5 Immunology Today 155—158 (1984)); and
`transferrin (O'Keefe et al, 260 J. Biol Chem. 932—937
`(1985)).
`Monoclonal antibody techniques allow for the pro-
`duction of extremely specific cell binding agents in the
`form of specific monoclonal antibodies. Particularly
`well known in the art are techniques for creating mono-
`clonal antibodies produced by immunizing mice, rats,
`hamsters or any other mammal with the antigen of
`interest such as the intact target cell, antigens isolated
`from the target cell, whole virus, attenuated whole
`virus, and viral proteins such as viral coat proteins.
`Sensitized human cells can also be used.
`Selection of the