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`European Patent Office
`Office européen des brevets
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`(11)
`
`EP 0 745 390 A2
`
`(12)
`
`EUROPEAN PATENT APPLICATION
`
`(43) Date of publication:
`04.12.1996 Bulletin 1996/49
`
`(51) Int. 01.6: A61 K 47/48
`
`(21) Application number: 96108570.1
`
`(22) Date of filing: 30.05.1996
`
`(84) Designated Contracting States:
`AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC
`NL PT SE
`
`(30) Priority: 31.05.1995 us 460152
`
`(71) Applicant: BRISTOL-MYERS SQUIBB COMPANY
`Princeton, NJ 08543-4000 (US)
`
`
`
`(72) Inventor: Senter, Peter D.
`Seattle, WA 98115 (US)
`
`(74) Representative: Kinzebach, Werner, Dr. et al
`Patentanwalte
`
`Reitstétter, Kinzebach und Partner
`Postfach 86 06 49
`
`81633 Milnchen (DE)
`
`(54)
`
`Polymeric prodrugs for beta-lactamase and uses thereof
`
`The instant invention relates to a method for
`(57)
`the delivery of antitumor drugs to tumor cells by the
`administration of a tumor-selective antibody- fi-lacta-
`mase conjugate that binds to tumor cells, and the addi-
`tional administration of a novel polymeric cephalosporin
`prodrug that is converted at the tumor site, in the pres-
`ence of the antibody-[i-lactamase, to an active cytotoxic
`drug. According to a preferred embodiment of this
`invention, the polymeric cephalosporin prodrug con-
`tains a polyethylene glycol or a branched polyethylene
`glycol moiety. The methods, antibody-enzyme conju-
`gate, prodrugs, pharmaceutical compositions, and com-
`binations of
`this
`invention provide for enhanced
`selective killing of tumor cells and are thus useful in the
`treatment of cancers and other tumors.
`
`EP0745390A2
`
`Primed by Rank Xerox (UK) Business Services
`2.13.8/3.4
`
`

`

`Description
`
`EP 0 745 390 A2
`
`The present invention relates generally to novel prodrugs and a method for delivering these prodrugs to a tumor
`cell site where they are converted to active cytotoxic agents. More particularly, the invention relates to polymeric cepha-
`losporin prodrugs, which when administered with a tumor-secific-antibody-B-lactamase conjugate, are converted at the
`tumor site to active cytotoxic drugs.
`Targeted drug delivery systems provide a mechanism for delivering cytotoxic agents directly to cancerous cells.
`The selective delivery of cytotoxic agents to tumor cells is desirable because systemic administration of these agents
`oflen kills normal cells within the body as well as the tumor cells sought to be eliminated. Antitumor drug delivery sys-
`tems currently in use typically utilize a cytotoxic agent conjugated to a tumor-specific antibody to form an immunocon-
`jugate. This immunoconjugate binds to tumor cells and thereby "delivers" the cytotoxic agent to the site of the tumor.
`The immunoconjugates utilized in these targeting systems include antibody-drug conjugates (see, e.g., Baldwin et al.,
`Lancet, pp. 603-605, March 15, 1986) and antibody-toxin conjugates (see, e.g., Thorpe, in Monoclonal Antibodies '84:
`'
`'
`lini
`IA li
`i n , A. Oinchera et al., eds., pp 475-506, 1985).
`Both polyclonal antibodies and monoclonal antibodies have been utilized in these immunoconjugates (see, e.g.,
`Ohkawa et al., Cancer Immunol. Immunother. 23: 81, 1986; Rowland et al., Cancer Immunol. Immunother., 21: 183,
`
`1986). Drugs used in these immunoconjugates include daunomycin (see, e.g., Gallego et al., Int. J. Cancer 33: 737,
`1984; Arnon et al., Immunological Rev., 62: 5, 1982; mexotrexate (Endo etal., Cancer Research, 47: 1076, 1987), mito-
`mycin C (Ohkawa et al., supra), and vindesine (Rowland et al., supra). Toxins used in the antibody-toxin conjugates
`include bacterial toxins such as ricin (see e.g., Moolten et al., Immunol. Rev., 62: 47, 1982).
`Despite the amount of research directed towards the use of immunoconjugates for therapeutic purposes, several
`limitations involved in these delivery approaches have become apparent (see, e.g., Embleton, Biochem. Society Trans-
`actions, 14: 393, 615th Meeting, Belfast, 1986). For example, the large amount of drug required to be delivered to the
`target tumor cell to effect killing of the cell is often unattainable because of limitations imposed by the number of tumor-
`associated antigens on the surface of the cells and the number of drug molecules that can be attached to any given
`antibody molecule. This limitation has led to the use of more potent cytotoxic agents such as plant toxins in these con-
`jugates and to the development of polymer-bound antibody-drug conjugates having very high drug multiplicity ratios
`(see, e.g., Thorpe, supra, pp. 475-506, and Baldwin et al., in Monoclonal Antibodies and Cancer Therapy pp. 215-231,
`Alan R. Liss, Inc., 1985). However, even with the large drug loading ratios or with the use of potent toxins, many immu-
`noconjugates still display suboptimal cytotoxic activity and are unable to effect complete killing at doses where all avail-
`able antigenic sites are saturated.
`It has also been recognized that the cytotoxic activity of an immunoconjugate is often dependent on its uptake,
`mediated by the antibody component of the conjugate into the tumor cell (see, e.g., J.M. Lambert et al., J. Biol. Chem.,
`260: 12035, 1985). This internalization is crucial when using an antibody-drug conjugate in which the drug has an intra-
`cellular site of action or when using antibody-toxin conjugates. However, the vast majority of tumor-associated antigens
`and thus the antibody-drug or antibody-toxin conjugates bound to those antigens, are not internalized. Those conju-
`gates that are internalized are often transported to the lysosome of the cell where the drug or toxin is degraded (see
`Vitetta et al., Science, 238: 1098, 1987). Accordingly, although an antibody-drug or antibody toxin conjugate may have
`excellent tumor-binding characteristics, the conjugate may nonetheless have a limited cytotoxic utility due to an inability
`to reach its site of action within the cell.
`
`In addition, it is well established that tumor cell populations are often heterogeneous with respect to antigen expres-
`sion (see, e.g., Albino et al., J. Exp. Med., 154: 1764, 1981). Furthermore, it has been demonstrated that antigen-pos-
`itive tumor cells may give rise to antigen-negative progeny (see, e.g., Yeh et al., J. Immunol, 126: 1312, 1981). Thus, in
`any population of tumor cells, there will be a certain number of cells that do not possess the antigen for which a partic-
`ular immunoconjugate is specific. The immunoconjugate will therefore not be able to bind to these cells and mediate
`their killing.
`Due to these drawbacks, the currently utilized antitumor drug or toxin delivery systems have had a limited amount
`of success, especially when used for in vivo treatment.
`In addition to the immunoconjugates discussed above, antibody-enzyme conjugates have been studied in vitro in
`combination with a second untargeted enzyme for the conversion of iodide or arsphenamine to their toxic forms in order
`to amplify antibody-mediated cytotoxicity (see, e.g., Parker et al., Proc. Natl. Acad. Sci. USA, 72: 338, 1975: Philpott et
`al., Cancer Research, 34: 2159, 1974).
`According to these in vitro studies, the enzyme, glucose oxidase, is attached to an antibody and used in combina-
`tion with an untargeted peroxidase enzyme to convert iodide or arsphenamine to cytotoxic iodine or arsenical, respec-
`tively. This approach, therefore, requires not only the targeting of glucose oxidase to tumor cells with antibody, but also
`the presence at the tumor site of two other untargeted events. The likelihood that all three of these agents will be
`present in vivo at the tumor site at the same time is small.
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`EP 0 745 390 A2
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`Canadian Patent No. 1,216,791, discloses the conjugation to an antibody of an enzyme capable of liberating
`ammonium ions from substrates. The ammonium ions are then said to potentiate the cytotoxic action of certain immu-
`notoxins targeted to the tumor site.
`European Patent Application No. 84302218.7 discloses a method for treating a diseased cell population such as a
`tumor wherein an antibody is used to target a non-metabolizable antigen to tumor cells. The antigen accumulates within
`at least a percentage of the tumor cells, which are then lysed to release the antigen into a ubiquitous fibronectin cap-
`turing matrix formed at the tumor site. An iodine-containing ligand which is specific for and will bind to the antigen
`affixed to the matrix is administered. The cytotoxic iodine acts to kill the tumor cells at that site. Also suggested is the
`use of an antibody-conjugate to target enzyme to a tumor site and the addition of a non-lethal substrate which the
`enzyme can convert to a cytotoxic material (see European Application No. 843022187, pp. 34-35). However, nowhere
`in the application is there any disclosure of how one is perform this embodiment. Similarly, Hellstrom et al., in Controlled
`Drug Delivery (2d ed), Robinson and Lee (eds) p. 639, 1987, suggest that "drugs which would be nontoxic until acti-
`vated by an agent (e.g., an enzyme) localized to a tumor may be another approach..."
`US. Patent No. 4,975,278, hereby incorporated by reference in its entirety, provides a method for delivering cyto-
`toxic agents to tumor cells by the combined use of antibody-enzyme conjugates and prodrugs. According to this inven-
`tion, an enzyme that is capable of converting a poorly or non-cytotoxic prodrug into an active cytotoxic drug is
`conjugated to a tumor-specific antibody. This antibody-enzyme conjugate is administered to a tumor-bearing mamma-
`lian host and binds, due to the antibody specificity, to the surface of those tumor cells which possess the tumor antigen
`for which the antibody is specific. The prodrug is then administered to the host and is converted at the tumor site by the
`action of the antibody-bound enzyme into a more active cytotoxic drug.
`Nitrogen mustards have long been recognized as cytotoxic agents (See, e.g., Stock, in Drug Design, E. J., Ariens,
`ed., Vol.
`II, pp. 532-571, Academic Press, New York, 1971 .) Benn. et al.. J. Chem. Soc.. 2365 (1961) prepared a variety
`of amides, including urethanes and ureas, from N,N-di-2'-chloroethyl-para-phenylenediamine that are useful for reac-
`tions with various functional groups that are of potential value for the attachment of nitrogen mustards to a wide variety
`of other units. The attachment of the electron-attracting urethane group deactivates the highly toxic nitrogen mustard.
`Reactivation of the nitrogen mustard at the tumor site may occur if the urethane is decomposed by fission of the ester
`or peptide linkage.
`Mobashery, et al. (J. Am. Chem. Soc., 108:1685, 1986) teaches the use of fi-lactamases resident in bacteria resist-
`ant to the fi-lactam antibiotics, to hydrolyze cephalosporin-toxophore derivatives to effect the release of the toxophore
`within the bacterium.
`
`Mobashery et al., (J. Biol. Chem., 261: 7879, 1986) synthesized an antibacterial agent consisting of the antibiotic
`peptide fiCl-LAla-fiCl-LAla linked through a C10 ester to the cephem nucleus of cephalosporin. The hydrolytic cleavage
`of the fi-lactam ring by fi-lactamase resident in the bacterium releases the heteroatom-linked C10 substituent.
`A general discussion of the chemistry of the cephalosporins is provided by Abraham, MyreVieViIs-—Chemical
`Society, 21 :231, 1967, and Abraham et al., inW E.H. Flynn, ed.,
`Academic Press, N.Y., 1972, pp 1-26.
`US. Patent No. 3,484,437 teaches derivatives of cephalosporanic acid formed by the reaction of a deacylated
`cephalosporin salt with isocyanates to form carbamates.
`US. Patent No. 3,355,452 teaches the 0-desacetyl-O-carbamoyl-7-acylamino-cephalosporanic acid derivatives of
`7-amino-cephalosporanic acid, where the 7-N-acyl group is a carboxylic acid radical and the CO group is bonded to a
`carbon atom.
`
`There has been a great deal of investigation concerning the use of polymers as carriers of anticancer drugs
`(Maeda, H., et al. (1992), Bioconiugate Chem. 3, 351-362; Duncan, R. (1992),W 3, 175-210). The
`molecular weight carriers can lead to reductions in systemic toxicity, longer retention time in the body, alterations in bio-
`logical distribution, and in improvements in therapeutic efficacy. Polyethylene glycol (PEG1, Zalipsky, S,. et al., (1983)
`Eur. Polym. J. E, 1177-1183; Ouchi, T., et al., (1992), Drug Design Discovery 9, 93-105); Caliceti, R, et al., (1993), u
`Farmaco fl, 919-932; Panarin, E. F., et al., (1989), J. Controlled Rel. E. 119-129), PEG copolymers (Poiani, G. J., et
`al., (1994), Bioconiugate Chem. 5, 621-630), dextran (Munechika, K., et al., (1994), Biol. Pharm. Bull. 17, 1193-1198),
`hydroxypropylmethacrylamide (Seymore, L. W., et al., (1994), Br. J. Cancer m, 636-641), and poly(styrene-co-maleic
`acid) (Maeda, H, (1992), J. Controlled Rel., E, 315-324) are but a few examples of polymers that have been used to
`deliver anticancer drugs and other biologically active molecules to target issues.
`In most cases, release of active anticancer drugs from the polymer support is mediated by simple aqueous hydrol-
`ysis or by proteolytic or esterase enzymes (Maeda, H., et al. (1992), Bioconiugate Chem. 3, 351-362; Duncan, R.
`(1992), Anticancer Drugs 3, 175-210). Since the conditions for these reactions are not preferentially confined to tumor
`tissues, some nonspecific drug release is inevitable. Consequently, there may be advantages in developing strategies
`for drug release that exploit some of the physiological and biochemical differences between neoplastic and normal tis-
`sues. Such differences may either be inherent, or established by targeting enzymes to tumor cell surfaces in the form
`of mAb-enzyme conjugates that recognize tumor associated antigens. This targeting strategy, which has been success-
`fully applied to the activation of a number of low molecular weight anticancer prodrugs (Bagshawe, K. D. (1994), m
`
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`

`EP 0 745 390 A2
`
`P—harmacokinet. 2_7, 368-376; Deonarain, M. P, et al (1994), Br. J. Cancer 70 786-794; Senter, P. D., etaL, (1993) B_io-
`coniugate Chem 4, 3-9-), should also be applicable to the release of active drugs that are covalently bound to polymer
`supports.
`Many of the enzymes utilized for prodrug activation carry out reactions not normally occurring in mammalian sys-
`tems. For example, cephalosporin containing prodrugs undergo drug elimination when hydrolyzed by B-lactams (Vrud-
`hula, V. M., et al., (1993), Bioconiugate Chem. 4, 334-340; Meyer, D. L., et al., (1993) Cancer Res. fl, 3956-3963).
`Heretofore, polymeric cephalosporin prodrugs have been unknown.
`The present invention is based on the discovery of novel polymeric cephalosporin-related prodrugs, capable of
`conversion to antitumor agents at the tumor site using a fi-lactamase-antibody conjugate. The antibody is directed
`against a tumor antigen present on the surface of the specific tumor type targeted.
`The present invention provides polymeric cephalosporin prodrugs of the general formula (I)
`
`QNH
`
`/S
`
`/ G-Fl
`
`O
`
`COZH
`
`(I)
`
`wherein Q is a pharmaceutically acceptable polymeric moiety; G is -NH- or is of the formula
`
`0
`
`—L—(O);u\
`
`L is a direct bond or -S-(CH2)n-; R is an agent capable of exerting a cytotoxic effect on tumor cells when released from
`said cephalosporin-prodrug; n is 2, 3, or 4; and m is 0 or 1 with the proviso that when L is a direct bond, m is 1; or a
`pharmaceutically acceptable salt thereof.
`It is preferred that the polymeric moiety, ii, "Q" is the residue of a polyethylene glycol or a branched polyethylene
`glycol.
`The cytotoxic compound is one having at least one functional group amenable to chemical modification to provide
`the cephalosporin prodrug. Generally, such functional groups are selected from amino, carboxyl, and hydroxyl groups
`such that the linkage between the cytotoxic agent and the cephalosporin component is of the carbamate, amide, ester,
`and carbonate types.
`In one aspect, the present invention provides as one subclass of compounds of formula (I) cephalosporin prodrugs
`of the general formula (II) in which the cytotoxic agent is linked to the cephalosporin nucleus via carbamate or amide
`group
`
`QNH
`
`S
`
`o
`
`/
`
`/
`COzl-_l
`
`’1OL
`\(0)... NR3
`
`L
`
`(ll)
`
`wherein Q, L, and m are as defined under formula (I); and NFla is a nitrogen containing cytotoxic drug; or a pharmaceu-
`tically acceptable salt thereof.
`In another aspect the present invention provides a cephalosporin-cytotoxic agent prodrug linked via an amine
`group such as a cephalosporin-mitomycin prodrug having the formula (Ila)
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`

`EP 0 745 390 A2
`
`QNH
`
`S
`
`’
`
`o
`
`0
`
`
`/ NH\
`OC(O)NH2
`OCH3
`
`
`COZH
`
`(Ila)
`
`wherein Q is as defined above under formula (I) and Rd is hydrogen or C1_3 alkyl.
`Another embodiment of the subject invention is directed to a method for delivering a cytotoxic agent to tumor cells
`by administering a pharmaceutically effective amount of at least one antibody-fi-Iactamase conjugate comprising an
`antibody reactive with an antigen on the surface of the tumor cells. A pharmaceutically effective amount of a polymeric
`cephalosporin prodrug is also administered, where the polymeric cephalosporin prodrug comprises a polymeric moiety
`linked in the cephalosporin which is in turn linked to the cytotoxic agent.
`In an alternative embodiment, the present invention is directed to a method of delivering a cytotoxic agent to tumor
`cells wherein the antigen binding region of an antibody reactive with a tumor-associated antigen is linked to at least a
`functionally active part of [i-Iactamase, and is administered with a pharmaceutically effective amount of a polymeric
`cephalosporin prodrug.
`In another embodiment, the subject invention is directed to a method of treating mammalian tumors which includes
`the step of administering to a mammal a pharmaceutically effective amount of at least one antibody-fi-lactamase con-
`jugate and a pharmaceutically effective amount of at least one polymeric cephalosporin prodrug.
`These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view
`of the disclosure herein.
`
`Figure 1. Mechanism of drug release by fi-Iactamase.
`Figure 2. HPLC analyses of PEG-AC-Dox samples. A: M-PEG-AC-Dox. B: B-PEG-AC-Dox. C: M-PEG-AC-Dox +
`bL (solid line), Dox (dotted line). D: B-PEG-AC-Dox + bL (solid line), doxorubicin (dotted line). See the example section
`for the meaning of the abbreviations.
`Figure 3. Cytotoxic effects of PEG-AC-Dox samples imAb-bL conjugates on H2981 lung adenocarcinoma cells as
`determined by the incorporation of [3H] thymidine into DNA compared to untreated control cells. A: Cells were exposed
`to L6-bL, washed and then treated with M-PEG-AC-Dox for 1 hour. The cytotoxic effects were determined aproximately
`24 hours later. B: Cells were exposed to conjugates, washed, and then treated with B-PEG-AC-Dox for 2 hours.
`Figure 4 Pharmacokinetics of PEG-AC-Dox samples and doxorubicin in nude mice bearing subcutaneous 3677
`human tumor xenografts. Tumor and blood samples were extracted, and the amount of prodrug and drug was quantified
`by HPLC. A: Blood levels of M-PEG-AC-Dox and B-PEG-AC-Dox after intravenous prodrug injection (52 umol/kg). The
`results are compared with doxorubicin levels in animals receiving 15.5 umol/kg doxorubicin. B: Tumor levels of M-PEG-
`AC-Dox, B-PEG-AC-Dox, and doxorubicin after intravenous injection (52 umol polymer/kg, 15.5 umol free Dox/kg).
`The practice of the present invention will employ, unless otherwise indicated, conventional techniques of synthetic
`organic chemistry, protein chemistry, molecular biology, microbiology, and recombinant DNA technology, which are
`within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Scopes, R.K., Protein Purifica-
`tion Principles and Practices, 2d ed. (Springer-Verlag, 1987), Methods in Enzymology (S. Colowick and N. Kaplan, eds.,
`Academic Press, Inc.), Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Press,
`Cold Spring Harbor, NY, 1989, Handbook of Experimental Immunology, Vols. l-IV (D.M. Weir and CC. Blackwell, eds,
`1986, Blackwell Scientific Publications); House,W, 2nd ed., Benjamin /Cummings, Menlo
`Park, Cal., 1972.
`All patents, patent applications, and publications mentioned herein, whether s_um or m, are hereby incorporated
`by reference in their entirety.
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`A. Definitions
`
`In defining the present invention, the following terms will be employed, and are intended to be defined as indicated
`below.
`
`

`

`EP 0 745 390 A2
`
`The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active
`substance that is less cytotoxic to cells compared to the parent drug and is capable of being enzymatically activated or
`converted into the more active parent form. See, e.g, Wilman, Biochem. Society Transactions, 14:375 (615th Meeting,
`Belfast, 1986); Stella et al., Directed Drug Delivery B. Borchardt et al., ed., 247-267 (Humana Press, 1985). The terms
`"parent drug", "drug" and "cytotoxic agent" are used interchangeably herein.
`The term "cephalosporin prodrug" as used herein refers to a prodrug generated by the linkage of a parent com-
`pound as described above to a cephalosporin as defined below.
`The term "fi-lactamase" as used herein refers to any enzyme capable of hydrolyzing the CO - N bond of a fi-lactam
`ring. The fi-Iactamases are reviewed in Bush, Antimicrobial. Agents Chemother, 33:259, 1989.
`The term "nitrogen mustard" as used herein refers to a compound of the general structure RN(CHZCHZCI)2, where
`B may be an alkyl, aryl, or aralkyl group substituted with a functional group amenable to further chemical modification,
`for example, an amino or a carboxyl group. Nitrogen mustards having more than one nitrogen atom are also included,
`such that both chloroethyl groups need not be attached to the same nitrogen atom. In some nitrogen mustards, the chlo-
`rine atoms may be replaced with other halogen atoms, especially bromine, or mesylates. See, eg., Stock,
`in m
`Design, E. J., Ariens, ed., Vol.
`II, pp. 532-571, Academic Press, New York, 1971.
`The term "cephalosporin" as used herein refers to derivatives of 7-aminocephalosporanic acid having the charac-
`teristic fi-lactam dihydrothiazine ring of cephalosporin C, occurring either naturally or synthetically. Examples of these
`derivatives and a review of the chemistry of the cephalosporins is given in Abraham, Quarterly reviews - Chemical Soci-
`e_ty, 21: 231, 1967. The term "cephem" is sometimes used herein to refer to a cephalosporin. The structure of cepha-
`losporin C is shown below:
`
`H
`
`-—
`_
`_
`020 CH (CH2)3 CONH
`
`/3
`
`MHz
`
`/
`
`OCCH3
`
`COZH
`
`The term "cephalosporin mustard" as used herein refers to a cephalosporin as described above, wherein the
`cephalosporin has been derivatized with a nitrogen mustard as described above.
`The term "cytotoxic" as used herein refers to the property of causing cell growth retardation or cell death, particu-
`larly as measured by a colony inhibition assay or 3H-thymidine uptake assay (see, eg., Hellstrom et al., in In Vitro Meth-
`ods in Cell-Mediated Immunity Bloom and Glade, eds., 1971, and the examples herein).
`The term "pharmaceutically acceptable polymeric moiety" refers to the polymeric residues of a polymer that is
`found upon reaction of a polymer with a cephalosporin or cephalosporin prodrug, wherein the polymeric moiety does
`not result in any substantial side effects and does not substantially interfere with the efficacy of the cytotoxic agents.
`The term "polymeric residue" refers to the portion of the polymer that becomes covalently bonded to the 7-nitrogen
`of the cephalosporin ring.
`
`B. General Methods
`
`The present invention relates to a novel method for the delivery of cytotoxic agents to tumor cells and provides for
`enhanced selective killing of tumor cells in the treatment of cancers, such as carcinomas and melanomas, as well as
`other tumors.
`
`According to the method of the invention, an antibody-enzyme conjugate is administered to a tumor-bearing mam-
`malian host. This antibody-enzyme conjugate consists of a tumor-selective antibody linked to a fi-lactamase that is
`capable of converting a prodrug that is less cytotoxic to cells than the parent drug into the more active parent drug.
`When introduced into the host, the antibody component of the conjugate, which is reactive with an antigen found on the
`tumor cells, directs the conjugate to the site of the tumor and binds to the tumor cells. The antibody thus delivers the
`enzyme to the site of the tumor. A prodrug that is a substrate for the fi-lactamase is also introduced into the host and is
`converted, at the tumor site, by the enzyme into an active cytotoxic drug. The drug is thus activated extracellularly and
`can diffuse into all of the tumor cells at that site, i.e., those cells bearing the particular tumor antigen to which the anti-
`body of the conjugate is specific and to which the antibody has bound as well as those cells that are negative for that
`antigen but are nonetheless present at the site of the tumor. The method of this invention therefore overcomes the cur-
`rent problems of tumor antigen heterogeneity and the requirement of antigen/conjugate internalization associated with
`conventional immunoconjugate drug delivery techniques.
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`EP 0 745 390 A2
`
`Furthermore, because the present method does not require the drug to be bound directly to the antibody and
`thereby limit the amount of drug that can be delivered, the common-place problem of drug potency at the tumor site
`does not arise.
`In fact, the present method amplifies the number of active drug molecules present at the tumor site
`because the antibody-bound enzyme of the conjugate can undergo numerous substrate turnovers, repeatedly convert-
`ing prodrug into active drug. Moreover, the present method is capable of releasing the active drug specifically at the
`tumor site as opposed to release to other tissues. This is so because the concentration of the enzyme at the tumor site
`is higher than its concentration at other tissues due to the coating of the tumor cells with the antibody-enzyme conju-
`gate.
`The antibody of the immunoconjugate of the invention includes any antibody which binds specifically to a tumor-
`associated antigen. Examples of such antibodies include, but are not limited to, those which bind specifically to antigens
`found on carcinomas, melanomas, lymphomas, and bone and soft tissue sarcomas as well as other tumors. Antibodies
`that remain bound to the cell surface for extended periods or that are internalized very slowly are preferred. These anti-
`bodies may be polyclonal or preferably, monoclonal, may be intact antibody molecules or fragments containing the
`active binding region of the antibody, e.g., Fab or F(ab')2, and can be produced using techniques well established in the
`art. See, e.g., FLA. DeWeger et al., Immunological Rev.. 62: 29-45. 1982 (tumor-specific polyclonal antibodies produced
`and used in conjugates): Yeh et al., Proc. Natl. Acad. Sci. USA, 76:2927, 1979; Brown et al., J. lmmun., 127:539, 1981
`(tumor-specific monoclonal antibodies produced); and Mach et al., in Monoclonal Antibodies for Cancer Detection and
`Therapy, R.W Baldwin et al., eds., pp 53-64, Academic Press, 1985 (antibody fragments produced and used to localize
`tumor cells). In addition, if monoclonal antibodies are used, the antibodies may be of mouse or human origin or chimeric
`antibodies (see, e.g., Oi, Biotechnigues, 4:214, 1986).
`Examples of antibodies which may be used to deliver the fi-lactamase to the tumor site include, but are not limited
`to. L6. an lgG2a monoclonal antibody (hybridoma deposit no. ATCC HB8677) that binds to a glycoprotein antigen on
`human lung carcinoma cells (Hellstrom, et al., Proc. Natl. Acad. Sci. USA, 83:7059, 1986); 96.5, an lgG2a monoclonal
`antibody that is specific for p97, a melanoma-associated antigen (Brown, et al., J. Immunol. 127:539, 1981); 1F5, an
`lgG2a monoclonal antibody (hybridoma deposit no. ATCC HB9645) that is specific for the CD-20 antigen on normal and
`neoplastic B cells (Clark et al., Proc. Natl. Acad. Sci. USA, 82:1766, 1985).
`An alternative strategy is to use antibodies that internalize, providing that the prodrug can also internalize, or that
`a sufficient amount of antibody also remains on the surface of the cell. An example of such antibodies may be found in
`Cancer Research 56:2183 (1990).
`The enzyme component of the immunoconjugate of the invention includes any enzyme capable of hydrolyzing the
`CO - N bond of a fi-lactam. Some of these enzymes are available commercially, such as E. M or E. cereus fi-lactama-
`ses. These and other fi-lactamases may be cloned and expressed using recombinant DNA techniques well known in
`the art.
`
`The fi-lactamases of this invention can be covalently bound to antibodies by techniques well known in the art such
`as the use of the heterobifunctional crosslinking reagents SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate) or
`SMCC (succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (see, e.g., Thorpe et al., Immunol. Rev., 62:
`119, 1982; Lambert et al., supra, at p. 12038; Rowland et al., supra, at pp 183-184; Gallego et al., supra, at pp. 737-
`7138). Alternatively, fusion proteins comprising at least the antigen binding region of an antibody linked to at least a
`functionally active portion of a fi-lactamase can be constructed using recombinant DNA techniques well known in the
`art (see, e.g., Neuberger et al., Nature, 312:604, 1984). These fusion proteins act in essentially the same manner as
`the antibody-enzyme conjugates described herein.
`The prodrugs of the invention contain an antitumor agent linked to a polymeric cephalosporin or polymeric cepha-
`losporin derivative. The antitumor agent is activated or otherwise converted into a more active form upon cleavage of
`the prodrug with fi-lactamase.
`In the preferred embodiment, the antitumor agent is a nitrogen mustard, as defined
`above. A representative nitrogen mustard is shown below:
`
`H N_©_N/\/°'
`
`\/\CI
`
`2
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`

`

`Other preferred antitumor agents include doxorubicin, which has the general formula:
`
`EP 0 745 390 A2
`
`CH30
`
`OH
`
`0
`
`H3
`
`HO
`
`0
`
`NH2
`
`and mitomycin C, which has the general formula:
`
`HZN
`
`H3C
`
`0
`
`O
`
`OCONHZ
`
`,ocr-i3
`
`NH
`
`N
`
`The prodrugs of this invention are not limited to these compounds, and may include other antitumor agents that can
`be derivatized into a prodrug form for use in a cephalosporin conjugate. Such antitumor agents include etoposide, ten-
`iposide, daunomycin, carminomycin, aminopterin, dactinomycin, cis-platinum and cis-platinum analogues, bleomycins,
`esperamicins (see US. Patent 4,675,187), and 5-fluorouracil.
`In one preferred embodiment of this invention, an anthracycline-cephalosporin prodrug is synthesized by reaction
`of an anthracycline with a carboxyl protected 3-[(carbonyloxy)methyl]cephem such as the diphenylmethyl esters of 3-
`[[(p-nitrophenoxy)
`carbonyloxy]methyl]cephem and 3-(1,2,2,2-tetrachloroethoxy)carbonyloxy]methyl]cephem. The
`resulting prodrug contains an anthracycline linked to the cephalosporin by the amino group of the former through a car-
`bamate bond.
`
`In another preferred embodiment of this invention, a cephalosporin mustard is synthesized by reaction of a 3-
`hydroxymethyl cephalosporin salt with an isocyanate, as described in US. Patent Nos. 3,355,452, and 3,484,437, and
`Belgian Patent No. 741,381, herein incorporated by reference in their entirety. Such a reaction is also described in detail
`in the examples.
`More generally, the present invention provides cephalosporin prodrugs of the general formula (I)
`
`QNH
`
`3
`
`0
`
`/
`
`/
`
`COZH
`
`i
`We)...
`
`L
`
`R
`
`wherein Q is a pharmaceutically acceptable polymeric moiety; L is a direct bond or -S-(CH2)n-; R is an agent capable
`of exerting a cytotoxic effect on tumor cells when released from said cephalosporin-prodrug; n is 2, 3, or 4; and m is 0
`or 1 with the proviso that when L is a direct bond, m is 1; or a pharmaceutically acceptable salt thereof.
`The pharmaceutically acceptable polymeric moiety can be any of the polymeric moieties known in the art to be
`capable of being linked to the cephalosporin. Examples include polyethylene glycol, a branched polyethylene glycol, a
`polyethylene glycol copolymer, dextran, hydroxypropylmethacrylamide, poly(styrene-co-maleic acid), polygalacturonic
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`

`

`EP 0 745 390 A2
`
`acid, carboxymethylated fi-D-glucan, N-(2-hydroxypropyl)methacrylamide copolymer, N-(2-hydroxyethyl)methacrylate-
`vinyl-pyrrolidone copolymer, poly(hydroxyethyl-L-glutamine), poly(oc-maleic acid), a polyamino acid, and a