Monocyte-and neutrophil-mediated lysis of SCCL by a
`
`
`molecule comprised of Lys3-BN and mAb22
`bispecific
`
`J. Chen•, M. Mokotoffb, J.-H. Zhoua, M.W. Fangerc and E.D. Balla
`
`
`
`
`"Hematology/Bone Marrow Tra11splcmtation, U11iversity of Pi{lsburgh Medical Center and
`
`Pi11sb11rgh Cancer lnstit111e, Pi{lsburgh. PA 15261. U.S.A.
`
`
`
`
`b Department of Plwrmace111ical Sciences. School of Pharmacy, U11ii•ersity of Pi11sb11rgh.
`
`Pi11sb11rgh. PA 15261. U.S.A.
`
`
`
`'"Department of Microbiology. Dartmouth Medical School. Leba11011. NH 03756, U.S.A.
`
`r otroduction
`
`Small cell carcinoma of the lung (SCCL) grows rapidly, metastasizes early and
`
`
`
`
`eventually causes the death of about 30,000 people per year in the U.S.A. Various
`
`
`
`combinations of chemotherapy have been used with liule improvement in the long-term
`
`
`survival rate. For these reasons recent efforts have focused on developing other
`
`
`
`
`therapeutic strategics for the treatment of SCCL, such as immunological and hormonal
`
`
`therapy [I], and intensive chemotherapy with autologous bone marrow or peripheral
`
`stem cell support (2]. Most human SCCL cell lines produce gastrin releasing peptide
`(GRP), which is similar in action and sequence to bombesin (BN), a peptide from frog
`
`skin. These SCCL cell lines produce GRP and express a single class of high-affinity
`
`receptors for BN/GRP (3].
`As a result of BN/GRP receptors having limited distribution in the body, these
`
`
`
`
`
`
`receptors could serve as targets for directing specific immune reactions. We have
`
`
`developed a novel approach of immunotherapy using the BN/GRP receptors expressed
`
`
`
`IC) molecule (immunoconjugatc, on SCCL cells as targets. We have made a bispccific
`
`between Lys3-BN and a monoclonal antibody (mAb22) [4] against the human high­
`
`
`
`affinity Fe gamma receptor (FcyRI, CD64), which is expressed on the surface of human
`
`
`
`monocytes (Mo) and IFNy-activated polymorphonuclcar neutrophils (PMN). We
`
`
`
`hypothesized that this IC should be able to redirect these immune effector cells towards
`
`SCCL cells and, thus, elicit specific antibody-dependent cell-mediated cytotoxicity
`(ADCC) of the carcinoma cells.
`
`Results and Discussion
`
`The IC, mAb22-Lys-BN, was obtained by the reaction of Lys3-BN with N-suecinimidyl
`
`
`
`
`S-acetylthioacetate (SAT A). The Lys3-BN-SA TA conjugate was purified by reversed
`
`
`
`
`phase HPLC. Deacetylation to the free sulfhydryl, Lys3-BN-SH, was accomplished with
`
`
`hydroxylamine (NH20H) and purification was performed by HPLC. The antibody
`
`mAb22, or its F(ab'h fragment (Medarcx, Inc.), was reacted with sulfosuccinimidyl
`
`
`4-(N-maleimidomcthyl)-eyclohexanc-l-carboxylatc (Sulfo-SMCC, Pierce) lo produce a
`malcimide-containing
`
`Ab. The final conjugation of Lys3-BN-SH with the maleimidc­
`
`
`
`containing Ab was effected by mixing equimolar amounts overnight at RT.
`The ability of two immunoconjugatcs, mAb22-Lys-BN and F(ab'h-Lys-BN, LO bind
`
`
`
`
`
`to four SCCL cell lines (NCI-H69, H345. SHP-77, and DMS273) was determined by
`
`
`
`now cytometric analysis, using the indirect staining method. The binding was directly
`proportional
`
`to the amount of IC used to stain the cells. This was manifested both by
`
`
`
`an increase in the absolute percentage of cells stained positively and by an augmentation
`
`819
`
`1 of 2
`
`BI Exhibit 1017
`
`

`

`of the mean nuorescence intensity (MR) of the entire cell population. In general. the IC
`
`
`
`
`
`prepared between the whole antibody. mAb22, and Lys '-BN had a higher MR than that
`
`prepared between the F(ab'h fragment and Lys'-BN.
`AOCC assays used target (T) SCCL cells that were incubated with 51Cr. The
`
`
`effector (E) cells, Mo or PMN. were obtained from different donors and activated by
`
`
`
`prior incubation with rlFNy. The cytotoxic potencies varied among donors. In all four
`
`SCCL cell lines. more than 80% of the cells were lysed by Mo from different donors.
`Tumor cell lysis was primarily dependent on the EfT ratio used for each donor. The
`
`achieved at an EfT ratio of
`
`greatest lysis, with either Mo or PMN, was consistently
`
`
`donors, 100:1. About 80% of SHP-77 cells were lysed by PMN from two different
`while with the H69 cells the lysis was about 45%. Since PMN do not express FCyRI
`
`
`on their cell surface without rfFNy stimulation, non-stimulated PMN had much less
`
`cytotoxicity on target cells.
`cytotoxicity was studied under different assay conditions in each of the
`
`
`
`Mo-mediated
`
`
`
`four SCCL cell lines. When the target cells were incubated with activated effector (Mo)
`
`
`cells in the absence of IC, about 15-60% tumor cell lysis was observed, depending upon
`
`donor. The addition of mAb22 did not cause any further cell lysis. However, the addition
`
`
`of IC (mAb22-Lys-BN) resulted in an incrense in tumor cell lysis to about 50-95%. The
`
`IC-induced SCCL cell lysis could be significantly (p < 0.05) blocked by adding an
`
`excess amount of either mAb22 or Lys3-BN. Similarly, with two SCCL lines, PMN
`
`
`tumor cell Iris could be significantly IC-induced blocked by adding an excess amount
`of either mAb22 or Lys -BN.
`
`Conclusions
`
`We have produced a novel IC between mAb22 and Lys3-BN. which binds to BN/GRP
`
`receptors on the cell surface of four SCCL cell lines and to FcyRJ on both human Mo
`
`
`cells and PMN. As hypothesized, IC mAb22-Lys-BN can direct immune effector
`
`
`towards target tumor cells and elicit a specific AOCC, which leads to the lysis of target
`
`
`SCCL cells. This cytotoxicity is dependent on Elf-cell ratios. can be induced in a wide
`
`range of IC concentrations, and can be blocked by the addition of an excess amount of
`
`either parental molecule, mAb22 or Lys3-BN.
`
`References
`
`Avis. 1.L .• Kovacs. T.0.G .. Kasprzyk. PG. Trc,ion. A.M. Banholorncw. R .• Wobh. J.H .. Cuuiua. F.
`
`and Mulshinc. J.L .. J. N:ul. Cancer ln,L. 83(1991)1470
`2 Vredenburgh. JJ. nnd Ball. E.D. Cancer Res .. 50( 1990)7216
`3 M ood y. T.W. and Cuni11a,
`F .. Life Sci. 52( 1993)1161
`4 Guyrc. P.M .. Graziano. R.F .• Vance. B.A .. Morgancllt.
`P.M and Fangcr. M.W. J lmmunol.,
`143( 1989) 1650.
`
`820
`
`2 of 2
`
`BI Exhibit 1017
`
`