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`PHIGENIX
`PHIGENIX
`Exhibit 1014
`Exhibit 1 0 14
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`Update
`
`Oncologic, Endocrine & Metabolic
`
`The development of antibody delivery systems to target
`cancer With highly potent maytansinoids
`
`Changnian Liu & Ravi VJ Chari
`
`Improving the tumour selectivity of cytotoxic drugs through
`conjugation to tumour-reactive mono clonal antibodies may lead
`to novel, more potent agents for cancer therapy. The maytansi—
`noid drugs are 100— to lOOO—fold more cytotoxic in Vitro than
`current clinical anticancer drugs. We recently demonstrated that
`conjugation of maytansinoid drugs to monoclonal antibodies
`renders them highly efficacious against cancers of breast and
`colon in both in Vitro and in in V1'V0 tumour models. Antibody—
`maytansinoids represent a new generation of immunoconju—
`gates that may yet fulfil the promise of effective cancer therapy
`through antibody targeting of cytotoxic agents.
`
`Keywords: antibody, cancer therapy, immunoconjugates, maytansinoids
`
`Exp. Opin. Invest. Drugs (1997) 6(2):] 69-] 72
`
`
`
`
`
`Forpersonaluseonly.
`
`1. Antibody-drug conjugates and cancer
`therapy
`
`Cancer chemotherapy could be greatly improved by
`utilising agents with enhanced potency and cancer
`specificity. The development of conjugates between
`potent cytotoxic agents and monoclonal antibodies
`with tumour reactivity has now advanced to a stage
`that antibody—drug conjugates look very promising as
`novel anticancer agents with the above characteristics.
`
`treatments were commenced before the tumours were
`
`well—established [4] or when extremely large doses
`were used [5]. For example,
`it has been recently
`reported that an immunoconjugate, prepared with the
`monoclonal antibody BRQG and cloxorubicin, cured
`athymic mice bearing human tumour xenografts [5].
`However, this effect was only achieved at the maxi—
`mum tolerated dose (MTD) of the immunoconjugate
`(a doxorubicin dose of 20 mg/kg/d x 3).
`
`A few antibody—drug conjugates have been further
`evaluated in humans [6—8]. In general, no significant
`anticancer effects have been observed with these
`
`The early development of monoclonal antibody—drug
`conjugates focused on the use of well—established
`agents in clinical trials. Indeed, the peak circulating
`clinical anticancer agents, such as doxorubicin,
`serum concentrations of conjugate were typically in
`methotrexate, vinblastine, mitomycin C, and melpha—
`the range of their in Vitro ICSO values (inhibitory
`lan, in conjugated form [1—3]. However, evaluation of
`concentration resulting in 50% cell death) values and,
`in Vitro cytotoxicity revealed that most of these conju—
`thus, capable of eliminating at best only about 50% of
`gates were not potent enough to be clinically useful.
`Drug levels achieved inside target cells were too low,
`tumour cells. Lack of clinical success with these early
`antibody—drug conjugates suggests that
`it was not
`and only marginal antigen—specific killing of cultured
`possible to achieve the intratumoural and intracellular
`tumour cells was observed. Not surprisingly, therefore,
`in tumour xenograft animal models, therapeutic effects
`concentrations of drugs sufficient to kill large numbers
`of cancer cells.
`with these conjugates were observed only when the
`
`169
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`
`
`1997 © Ashley Hlbliaations Ltd. 189‘! 1354-3784
`
`NW“ Hum. minim». wmm v.i\\.\\\t\tr«t
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`
`
`PHIGENIX
`
`Exhibit 1014-01
`
`

`

`
`
`
`170 The development of antibody delivery systems to target cancer — Liu & Chan‘
`
`Figure 1: Structural representation ofmaytansine (1), DMl (2), and antibody—DMI (3).
`
`t
`
`
`
`1 Maytan sine
`
`Possible reasons for these outcomes may be:
`
`0
`
`0
`
`lack of cytotoxic potency — the majority of com—
`monly used anticancer drugs are only moderately
`cytotoxic at clinically achievable concentrations in
`antibody— drug conjugate form (large numbers of
`drug molecules have to be intemalised to cause cell
`death);
`
`tumour cells only express limited numbers of target
`antigens, which restricts the amount of drug deliv—
`ered;
`
`0 poor penetration of irnmunoconjugates into tu—
`mours, and inefficient intemalisation of antigen—
`antibody complexes;
`
`-
`
`inefficient release of the active drug from the anti—
`body inside target cells.
`
`We reasoned that immunoconjugates must be com—
`posed of drugs possessing much higher potency than
`the currently used anticancer agents if therapeutic
`levels of conjugates at
`the tumour sites are to be
`achieved in patients. We have recently reported anti—
`body conjugates with the maytansinoid drug DMl
`[9,10], a sulfydryl—containing derivative of maytansine
`(Takeda, Osaka) (Figure l). Maytansinoids effect cell
`killing by interfering with the formation of micro—
`tubules and depolymerisation of already formed rni—
`crotubules [11]. They are 100— to 1000—fold more
`cytotoxic than chemotherapeutic drugs such as dox—
`orubicin, methotrexate, and Vinca alkaloids. DMl
`is
`linked to the antibody Via a novel disulfide linker
`which allows for rapid release of the fully active drug
`inside the target cells. Disulfide linkers are superior to
`other linkers in that they are more stable during storage
`and in serum, but are still able to release the active
`
`
`
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`drugs from the conjugates inside target cells efficiently
`[12,13].
`
`2. In vitro cytotoxicity and specificity of
`
`antibody-maytansinoid conjugates
`
`Antibody—maytansinoid conjugates were assessed for
`in Vitro cytotoxicity against human cancer cell
`lines
`using a clonogenic assay [14]. The disulfide—linked
`maytansinoid immunoconjigates exhibited high anti—
`gen—specific cytotoxicity [10,11]. The C242—maytansi—
`noid conjugate (C242—DM1) prepared with the
`monoclonal antibody C242 (Pharmacia Oncology,
`Lund, Sweden), which recognises the CanAg antigen
`[15] expressed on all human colorectal cancers, killed
`antigen—positive COLO 205 cells with an IC5o value of
`3.2 x 10‘11 M (3.5 pg/ml), with > 99.999% of the cells
`killed at a conjugate concentration of 4.5 x 10'9 M (3.3
`ng/ml)
`(all concentrations refer to DMl; one micro—
`gram of DM1 corresponds to 54 ug of C242—DM1
`conjugate). In contrast, the conjugate was MOO—fold
`less cytotoxic towards antigen—negative A—375 mela—
`noma cells (IC50 = 3.6 x 10'8 M, 26.5 ng/ml), demon-
`strating that the cytotoxicity effect of C242—DM1 is
`antigen—specific. Both cell
`lines were found to be
`equally sensitive to unconjugated maytansinoid (IC50
`= 4 x 10'11 M). Similar results were observed with
`TAl—DMl conjugate prepared with the monoclonal
`antibody TA.1, which binds to the HER—Z/neu onco-
`gene protein expressed on the surface of human breast
`
`cancer cells [16]. The TA. 1—DM1 conjugate was highly
`cytotoxic to artigen-positive SK—BR—3 breast cancer
`cells (IC50 = 1.6 x 10'11 M). The conjugate was at least
`lOOO—fold less cytotoxic to antigen—negative human
`oral epidermoid carcinoma KB cells (leo > 2 x 10‘8 M)
`[10].
`
`
`© Ashley Publications Ltd. All rights reserved.
`
`Exp. Opin. Invest. Drugs (1997) 6(2)
`msumrsmmm}
`
`
`
`PHIGENIX
`
`Exhibit 1014-02
`
`

`

`
`
`
`Oncologic, Endocrine & Metabolic — Update 171
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`Figure 2: Anti-tumour6activity of C242-DM1 against large COLO 205 tumours (mean tumour size= 260 mm3.) Each mouse was
`inoculated with 5 x 106 COLO 205 cells and treatments were started on day 7 after tumour inoculation. Each group contained 8 ani-
`mals.
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`Days after tumour inoculation
`
`3. C242-DM1 in treatment of human colon
`
`cancer xenografts in severe combined
`immunodeficient mice
`
`Animals bearing COLO 205 colon tumours (homoge—
`neous antigen expression) were treated either with
`
`five daily injections of C242—DM1 at a dose of 300
`ug/kg/d, or with an equivalent dose of the isotype—
`matched non—binding conjugate N901-DM1, or with a
`mixture consisting of corresponding amounts of C242
`antibody and unconjugated DMl [10]. Treatment with
`C242—DM1 completely eliminated all
`tumours within
`two weeks of the initiation of therapy, and all eight
`animals were tumour—free for 200 days (duration of the
`experiment). In contrast, very little antitumour activity
`was observed in animals treated with non—targeted
`conjugate or with the mixture of antibody and free
`DMl. In a dose—response study, C242-DM1 eliminated
`COLO 205 tumours in all 8 animals at a dose as low
`as 225 ug/kg/d x 5, which is 59% of the maximum
`tolerated dose (MTD = 380 pg/kg/d X 5) [10].
`
`These results encouraged us to evaluate the therapeu—
`tic efficacy of C2342—DMl in mice bearing larger (aver—
`age size 260 mm3) subcutaneous COLO 205 xenografts
`(Figure 2). Animals received two courses of 5— day
`treatment with C242—DM1 (300 ug/kg/d) or, for com—
`parison,
`treatment with 5—fluorouracil
`(5—FU),
`the
`standard chemotherapeutic drug used for the treat—
`ment of colorectal cancer. C242-DM1 again cured all
`animals rendering them tumour—free for greater than
`200 days (duration of the experiment) without intoler—
`
`able toxicities. This therapeutic effect on large tumours
`is especially remarkable in View of the finding that
`5—FU at its MTD (15 mg/kg/d x 5) only slightly (by
`about 5 days) delayed tumour growth [10].
`
`C242—DM1 was then evaluated against established
`colon tumour xenografts from die LoVo and HT—29
`colon cancer cell
`lines which express the CanAg
`antigen heterogeneously on only 20 — 30 % of their
`cells [10]. Animals bearing LoVo tumour xenografts
`were treated with either one or two courses of C242—
`
`DMl (300 ug/kg/d x 5). Remarkably, complete tumour
`regressions lasting 5 weeks were observed in all
`animals treated with one course of C242—DM1. The
`
`period of complete regression could be prolonged to
`9 weeks by a second course of treatment with C242—
`DMl initiated 21 days after the start of the first course,
`suggesting that using multiple cycles of this immuno—
`conjugate for treatment of colorectal cancer may be a
`feasible clinical regimen. Similar effects were obtained
`in the PIT—29 colon tumour model [10].
`
`4. Conclusion
`
`The use of antibodyedrug conjugates for the treatment
`of cancers, i.e., the selective delivery of cytotoxic drugs
`to tumour cells, seems to be a prOmising approach [17].
`However, no such agent has yet demonstrated signifi—
`cant antitumour activity in the clinical setting. Anti—
`body—maytansinoid conjugates represent a new
`generation of irnmunoconjugates that may fulfil the
`promise of effective cancer therapy through antibody
`targeting of cytotoxic drugs. From the preclinical data,
`
`’51
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`© Ashley Publications Ltd. All rights reserved.
`
`Exp. Opin. Invest. Drugs (1997) 6(2)
`flinmrammaa} I‘
`
`
`PHIGENIX
`
`Exhibit 1014-03
`
`

`

`
`
`
`172 The development of antibody delivery systems to target cancer - Liu & Chari
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`C242—DM1 stands out as a promising new candidate
`for clinical evaluation against colorectal cancer. The
`conjugates made using humanised antibodies [18] will
`allow patients to be treated with several courses of
`targeted chemotherapy, potentially increasing clinical
`benefit substantially.
`‘
`
`Acknowledgements
`
`This review is supported in part by a Phase 1 grant
`from the NIH—SBIR program. We are grateful to Drs
`John M Lambert and Walter A Blattler for reading the
`manuscript and for making suggestions.
`
`Bibliography
`1.
`
`SELA M, HURWITZ E: Conjugates of antibodies with
`cytotoxic drugs. In: Immunoconjugates. Vogel CW (Ed),
`Oxford University Press, New York (1987):189—216.
`
`RODWELL JD: Antibody-Mediated Delivery System. Mar—
`cel Dekker, New York (1988).
`
`Pl'ETERSZ GA: The linkage of cytotoxic drugs to mono-
`clonal antibodies for the treatment of cancer. BioCon—
`jugate Chem, (1990) 1:89—95.
`STARLING J], MACLAK RS, LAW KL er al.: In vivo antitu—
`mour activity ofa monoclonal antibody— Vinca alkaloid
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`pression and nonintemalization of antibody—antigen
`complexes. Cancer Res. (1991) 51:2965-2972.
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`TRAIL PA, WILLN'ER D, LASCH SJ eta]: Cure ofxenografted
`human carcinoma by BR96-doxorubicin immunocon-
`jugates. Science (1993) 261:121-215.
`ELIAS DJ, HlRSHOWlTZ L, KLINE LE eta1.: Phase I clinical
`comparative study of monoclonal antibody KSl/4 and
`KS 1/4-methotrexate immunoconjugate in patients with
`non-small cell lung cancer. Cancer Res. (1990) 50:4154—
`4159.
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`ELIAS DJ, KLINE LE, ROBBINS EA et a1.: Monoclonal
`antibody KSl/4-methotrexate immunoconjugate in
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`Med. (1994) 150:1114-1122.
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`SCHNECK D, BUTLER F. DUGAN W era]; Disposition of
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`'c01. flier. (1990) 47:36~41.
`
`CHARI RVJ, MAR"E;L BA, GROSS JL er 2].: Immunoconju—
`gates containing novel maytansinoids: promising anti—
`cancer drugs. CancerRes. (1992) 52:127—131.
`LlU C, TADAYOlKl BM, BOURRET IA er a1: Eradication of
`large colon tumour xenografts by targeted delivery of
`maytansinoids. Proc. Natl. Acad. Sci. USA (1996) 93: 8618—
`8623.
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`REMELLARD S, REBHUN LI, HOWE GA, KU'PCHAN SM:
`Anfimitotic activity of the potent tumour inhibitor
`maytansine. Science (1977) 189:1002—1005.
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`LAMBERT IM, BLATTLER WA, MCLATYRE GD et aI.: Immu—
`notoxins containing single chain ribosome-inacfivat—
`ing proteins. In: Immunotoxins. Frankel AE (Ed), Kluwer
`Academic Publishers, Norwell, MA (1988):175—209.
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`SHEN WC, RYSER HP, LAMANNA L: Disulphide spacer
`between methotrexate and poly(D-lysine). ]. Biol. Chem.
`(1985) 260:10905—10908.
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`GO LDMACHER VS, TINNEL NL, NELSON BC: Evidence that
`pinocytosis in lymphoid cells has a low capacity. J. Cell.
`Bio]. (1986) 102:1312—1319.
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`
`
`BAECKSTROM D, HANSSON GC, N SSON 0 er a1.: Purifi—
`cation and characterization of a mernbrane—bound and
`secreted mucin—type glycoprotein carrying the carci—
`noma—associated sialyl—Lea epitope ondistinct core pro—
`teins. ]. Bio]. Chem. (1991) 266:21537—21547.
`
`
`
`
`MCKEN * SJ, MARKS P], LAM T at 2.1.: Generation and
`characterization of monoclonal antibodies specific for
`the human neu oncogene product p 185. Oncogene (1989)
`42543—548.
`
`BLATTLER WA, CHARI RV], LAMBERT JM: Immunoconju—
`gates In: The Cancer Jherapeun'cs Handbook. Telcher B
`(Ed), Humana Press, Inc, Totowa, NJ (l996):369—392.
`ROGUSKA MA, PEDERSON JT, KEDDY CA et a]; Humani—
`zation of murine monoclonal antibodies through vari—
`able domain resurfacing. Proc. Natl. Acad. Sci. USA (1994)
`91:969—973.
`
`
`
`Changnian Liu & Raw‘ V] Chari
`ImmunoGen, Inc, 148 Sidney Street, Cambridge, MA 02139—4239,
`USA.
`
`© Ashley Publications Ltd. All rights reserved.
`
`
`
`Exp. 0pm Invest. Drugs (1997) 6(2)
`minwratiinm}
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`
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`1W”WWWW%W§%§%
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`
`
`PHIGENIX
`
`Exhibit 1014-04
`
`