1996
`Peptides
`Robert Ramage and Roger Epton (Eds.)
`© 1998 The European Peptide Society
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`409
`Immunotherapy of a Human Small Cell Lung Carcinoma
`(SCLC) Xenograft Model by the Bispecific Molecule
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`(BsMol) mAb22xLys3-Bombesin (M22xL-BN)
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`Jie-Hua Zhou1, Jian Chen1, Michael Mokotoff2, Rui-Kun Zhong1,
`Leonard D. Shultz3 and Edward D. Ball1
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`'Division of Hematology I BMT & 2Department of Pharmaceutical Sciences,
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`University of Pittsburgh, Pittsburgh, PA 15261, USA
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`1 Jackson Laboratory, Bar Harbor, ME 04609, USA
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`Introduction
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`Among the approaches being studied in cancer therapy is the application of bispecific
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`monoclonal antibodies (BsAb), which are molecules that link two mAbs, each having
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`different antigenic specificities. These BsAb are able to bridge autologous immune
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`effector cells to tumor cells, thereby enhancing the specific lysis of the target tumor cells
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`[I]. Also being studied for cancer immunotherapy are peptide growth factors linked to
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`mAbs and these are known as bispecific molecules (BsMol) [2].
`We have constructed a BsMol by conjugating a bombesin/gastrin releasing peptide
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`(BN/GRP) analog, Lys3-BN (L-BN), to the FcyRl-specific mAb22 (M22), and call this
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`M22xL-BN [3]. We chose the BN/GRP receptor as our target because: I) the majority
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`of small cell lung carcinoma (SCLC) cell lines and biopsy specimens from SCLC
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`patients express BN/GRP receptors [4], 2) BN, and its mammalian equivalent, GRP, can
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`act as an autocrine growth factor in the proliferation of SCLC cells [5], and 3) synthetic
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`BN/GRP antagonists and a mAb against GRP have been shown to inhibit SCLC tumor
`growth in vitro as well as in vivo [6-8]. On the other hand, FcyRI (CD64) is a potent
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`immune trigger molecule that is expressed on a number of immune effector cells, such as
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`monocytes (Mo), is up-regulated by a variety of cytokines, and binds to M22 without the
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`interference of circulating immunoglobulins
`[9].
`Our earlier studies have shown that M22xL-BN can effectively enhance the in vitro
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`cytotoxicity of SCLC cells by cytokine-activated human Mo [3]. In order to further
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`study the effectiveness of this immunotherapeutic approach, a SCLC xenograft model
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`was established by innoculation of human SCLC (DMS273) cells into SCIO/NOD mice.
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`Immunotherapy was tested by injection of cytokine-activated Mo and M22xL-BN into
`these mice after the SCLC cells had been growing for 3 days.
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`Results and Discussion
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`The construction of the BsMol, M22xL-BN, was begun by reacting L-BN with SATA
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`(Pierce) and the L-BN-SATA conjugate was purified by C18-HPLC. Deacetylation with
`NH20H gave the free sulfhydryl,
`L-BN-SH, which was purified by C18-HPLC. M22
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`BI Exhibit 1018
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`Jie-Hua Zhou et al.
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`Table 1. Values of two experiments
`on the left represent tumor weight (mg± SD), while the two
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`on the right represent the number of peritoneal tumor cells (1 xUf ±SD).
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`Group A
`Group B
`Group C
`Group D
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`490±130
`710±400
`320±170
`540±200
`630±250
`820±360
`90±60
`25±25
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`3.1±2.8 1.9±0.8
`2.3±0.2 1.6±1.4
`4.4±2.0 1.3±0.7
`0.5±0.1 0.2±0.2
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`(Medarex, Inc.) was reacted with Sulfo-SMCC (Pierce) to produce a maleimide­
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`containing Ab. The final conjugation of L-BN-SH with the maJeimide-containing Ab was
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`effected by mixing equimolar amounts overnight at RT [3].
`SCID/NOD mice 6-8 weeks old (Jackson Lab) were maintained in pathogen-free
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`and irradiated with 300 rad immediately before the ip injection of lxl06
`facilities
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`DMS273 cells. All mice were checked periodically for evidence of tumor growth and
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`all received were sacrificed at day 28. Mice (12-16) were divided into four groups and
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`DMS273 cells on day l . Cytokine-activated Mo (Ix I 07) were mixed with either M22
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`(Group C) or M22xL-BN (Group D) prior to injecting the cells on day 3 and day 6.
`Control mice (Group A) received medium only and Group B received cytokine-activated
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`Mo only on days 3 and 6. After the mice were sacrificed the peritoneal cavity was
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`washed with 10 mL of normal saline and peritoneal exudate cells counted, stained and
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`for human CD15 as positive analyzed by flow cytometry. DMS273 cells were identified
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`(mAb PM81) and negative for mouse CD45. The peritoneal cavity was also examined
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`for gross tumor nodules, which were dissected and weighed. The results from two
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`separate experiments are shown in Table 1.
`studies in model for immunotherapy W e have established a human SCLC xenograft
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`SCID/NOD mice and found that irnmunotherapy with the BsMol M22xL-BN is effective
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`in this xenograft model and potentially applicable in a clinical setting, after optimal
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`conditions are determined.
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`References
`
`I. Fanger, M.W., Morganelli, P.M. and Guyre, P.M., Crilical
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`IOI.
`Rev. lmmuno/., 12 (1992)
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`2. Mokotoff, M., Chen, J., Zhou, J-H. and Ball, E.D., Curr. Med. Chem., 3 (1996) 87.
`3. Chen, J., Mokotoff, M., Zhou, J-H., Fanger, M.W. and Ball, E.D., C/in. Cancer Res.,
`(1995) 425.
`4 . Moody, T.W. and Cuttitta,
`F.,LifeSci., 52 (1993) 1161.
`
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`5. Camey, D.N., Cuttitta, F., Moody, T.W. and Minna, J.D., Cancer Res., 47 (1987) 821.
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`6. Avis, I.L., Kovacs, T.O.G., Kasprzyk, P.G., Treston, A.M., Bartholomew, R., Walsh J.H.,
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`Cuttitta, F. and Mulshine, J.L., J. Natl. Cancer Inst.,
`83 (1991) 1470.
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`7. Heimbrook, D.C., Saari, W.S., Balishin, N.L., Fisher, T.W., Friedman, A., Kiefer, D.M.,
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`Rotberg, N.S., Wallen, J.W. and Oliff, A., J. Med. Chem., 34 (1991) 2102.
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`8. Staley, J., Coy, D., Taylor, J.E., Kjm, S. and Moody, T.W., Peptides,
`12 (1991) 145.
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`9. Guyre, P.M., Graziano, R.F., Vance, B.A., Morganelli, P.M. and Fanger, M.W., J. lmmuno/.,
`143 (1989) 1650.
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