`Robert RatTUJge and Roger Epton (Eds.)
`© 1998 The European Peptide Society
`
`409
`Immunotherapy of a Human Small Cell Lung Carcinoma
`(SCLC) Xenograft Model by the Bispecifie Molecule
`(BsMol) mAb22xLys3-Bombesin (M22xL-BN)
`
`Jie-Hua Zhou1, Jian Chen1, Michael Mokotoff2
`, Rui-Kun Zhong1,
`Leonard D. Shultz3 and Edward D. Ball1
`
`1 Division of Hematology I BMT & 2Department of Pharmaceutical Sciences,
`University of Pittsburgh, Pittsburgh, PA 15261, USA
`3 Jackson Laboratory, Bar Harbor, ME 04609, USA
`
`Introduction
`
`Among the approaches being studied in cancer therapy is the application of bispecific
`monoclonal antibodies (BsAb), which are molecules that link two mAbs, each having
`different antigenic specificities. These BsAb are able to bridge autologous immune
`effector cells to tumor cells, thereby enhancing the specific lysis of the target tumor cells
`[1]. Also being studied for cancer immunotherapy are peptide growth factors linked to
`mAbs and these are known as bispecific molecules (BsMol) [2].
`We have constructed a BsMol by conjugating a bombesin/gastrin releasing peptide
`(BN/GRP) analog, Lys3-BN (L-BN), to the FcyRI-specific mAb22 (M22), and call this
`M22xL-BN [3]. We chose the BN/GRP receptor as our target because: 1) the majority
`of small cell lung carcinoma (SCLC) cell lines and biopsy specimens from SCLC
`patients express BN/GRP receptors [4], 2) BN, and its mammalian equivalent, GRP, can
`act as an autocrine growth factor in the proliferation of SCLC cells [5], and 3) synthetic
`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
`immune trigger molecule that is expressed on a number of immune effector cells, such as
`monocytes (Mo), is up-regulated by a variety of cytokines, and binds to M22 without the
`interference of circulating immunoglobulins [9].
`Our earlier studies have shown that M22xL-BN can effectively enhance the in vitro
`cytotoxicity of SCLC cells by cytokine-activated human Mo [3].
`In order to further
`study the effectiveness of this immunotherapeutic approach, a SCLC xenograft model
`was established by innoculation of human SCLC (DMS273) cells into SCID/NOD mice.
`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.
`
`Results and Discussion
`
`The construction of the BsMol, M22xL-BN, was begun by reacting L-BN with SATA
`(Pierce) and the L-BN-SATA conjugate was purified by C 18-HPLC. Deacetylation with
`NH20H gave the free sulfhydryl, L-BN-SH, which was purified by C 18-HPLC. M22
`
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`PETITIONER'S EXHIBITS
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`Exhibit 1018 Page 1 of 2
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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
`on the right represent the number of peritoneal tumor cells ( 1 xi a5 ± SD).
`
`Group A
`Group B
`Group C
`Group D
`
`490±130
`320±170
`820±360
`25±25
`
`710±400
`540±200
`630±250
`90±60
`
`3.1±2.8
`2.3±0.2
`4.4±2.0
`0.5±0.1
`
`1.9±0.8
`1.6±1.4
`1.3±0.7
`0.2±0.2
`
`(Medarex, Inc.) was reacted with Sulfo-SMCC (Pierce) to produce a maleimide(cid:173)
`containing Ab. The final conjugation of L-BN-SH with the maleimide-containing Ab was
`effected by mixing equimolar amounts overnight at RT [3].
`SCID/NOD mice 6-8 weeks old (Jackson Lab) were maintained in pathogen-free
`facilities and irradiated with 300 rad immediately before the ip injection of lxl06
`DMS273 cells. All mice were checked periodically for evidence of tumor growth and
`were sacrificed at day 28. Mice (12-16) were divided into four groups and all received
`DMS273 cells on day 1. Cytokine-activated Mo (lxl07
`) were mixed with either M22
`(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
`Mo only on days 3 and 6. After the mice were sacrificed the peritoneal cavity was
`washed with 10 mL of normal saline and peritoneal exudate cells counted, stained and
`analyzed by flow cytometry. DMS273 cells were identified as positive for human CD15
`(mAb PM81) and negative for mouse CD45. The peritoneal cavity was also examined
`for gross tumor nodules, which were dissected and weighed. The results from two
`separate experiments are shown in Table 1.
`We have established a human SCLC xenograft model for immunotherapy studies in
`SCID/NOD mice and found that immunotherapy with the BsMol M22xL-BN is effective
`in this xenograft model and potentially applicable in a clinical setting, after optimal
`conditions are determined.
`
`References
`
`I. Fanger, M.W., Morganelli, P.M. and Guyre, P.M., Critical Rev. lmmunol., 12 (1992) 101.
`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., Clin. Cancer Res.,
`(1995) 425.
`4. Moody, T.W. and Cuttitta, F., Life Sci., 52 (1993) 1161.
`5. Camey, D.N., Cuttitta, F., Moody, T.W. and Minna, J.D., Cancer Res., 47 (1987) 821.
`6. Avis, LL., Kovacs, T.O.G., Kasprzyk, P.G., Treston, A.M., Bartholomew, R., Walsh J.H.,
`Cuttitta, F. and Mulshine, J.L., J. Natl. Cancer Inst., 83 (1991) 1470.
`7. Heimbrook, D.C., Saari, W.S., Balishin, N.L., Fisher, T.W., Friedman, A., Kiefer, D.M.,
`Rotberg, N.S., Wallen, J.W. and Oliff, A., J. Med. Chem., 34 (1991) 2102.
`8. Staley, J., Coy, D., Taylor, J.E., Kim, S. and Moody, T.W., Peptides, 12 (1991) 145.
`9. Guyre, P.M., Graziano, R.F., Vance, B.A., Morganelli, P.M. and Fanger, M.W., J. lmmunol.,
`143 (1989) 1650.
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