identified T-celJ costimulatory ligands (eg B7-2 etc.) are induced is not known. However, y-IFN is known to 
induce costimulatory CD28 ligand expression on some antigen producing cells (Nadler L, personal 
communication). ylFN transduction appeared to augment an allogeneic mixed lymphocyte-tumor cell 
reaction (MLTR) using transduced and nontransduced SK-N-RA cells, suggesting augmentation of antigen- 
presentation following transduction (Table VII). Whether the observed enhancement of MLTR is due to direct 
cytc^ne effects, phenotypic effects on tumor cells, or both, is under investigation. 
In contrast to MHC-I/II, expression of most “tumor associated” antigens (NAAg), as determined by flow 
cytometry using monoclonal antibodies, is stable or actually enhanced. One such neuroblastoma antigenic 
determinant, HSAN1.2, was expressed at a higher level following transduction of SK-N-AS cells (Figure 17). 
Other neuroblastoma associated antigens (eg Gd 2. 459, 390, and HNK-1) remained unchanged in expression. 
Whether antigen processing was altered in transduced cells is not known. However, continued expression of 
5/5 NAAg suggests that y-EFN will not substantially change tumor surface characteristics, and hopefully will 
augment presentation of NAAg, in the context of MHC-I/Il. Whether any of the NAAg may serve as 
anitgenic targets is not known. 
3.4 CONCLUDING REMARKS 
The biological effects of y-IFN transduced cells in vivo have been investigated using 4 distinct murine tumor models 
(a fibrosarcoma, a melanoma, a colorectal carcinoma, and a lung carcinoma). In all tumor models tested, we have 
shown that y-IFN-transduced cells secrete biologically active y-EFN, increase the level of surface-associated Class I 
MHC, and are non-tumorigenic due, at least in part, due to T cell responses, presumably CTL (see Table IV and Figs 
5,6,7). CTL induction was stimulated with y-lFN transduced tumor cells in all 3 tumor models tested 
(representative data is shown for CT26 in Fig.8). The CTLs thus generated were CD8+ and MHC -restricted 
(Figs. 10). CTL generation due to injection of y-IFN tranduced tumor cells resulted in systemic immunity to 
subsequent challenge with unmodified tumor in the B16 tumor model (Fig. 11). Many of these observations have 
been substantiated by other investigators including Watanabe et. al. (28), Gansbacher et. al. (30), and Porgador et. al. 
(82). The latter investigators demonstrated that tumor-bearing animals could be cured of metastatic disease by 
subsequent injection of y-IFN transduced tumor cells, but not by injection of unmodified tumor cells (82). Crowley 
and Seigler have also shown that injection of human anti melanoma CTL resulted in cleaning of human tumor 
xenografts in nude mice (80,81). Reproduction of these key references may be found in Appendix K. 
In addition to these murine sUidies, we have also demonstrated the following: (1) human neuroblastoma cell cultures 
can be readily established and transduced with retroviral vectors expressing y-IFN ; (2) the transduced neuroblastoma 
cells secreted y-EFN and increased the expression of Class I and Class II MHC; (3) y-IFN vector transduction of 
neuroblastoma cells resulted in potential improvement in antigen presentation as determined by in vitro stimulation 
of lymphocyte responses against allogeneic antigens. In addition, Seigler and coworkers (RAC protocol #9306-043) 
have shown that in vitro stimulation of patient PBMC with tumor cells transduced with the y-IFN gene result in 
increased CTL activity against the unmodified tumor cell line (Fig. 18). 
The use of y-IFN transduced tumor cells has already been tqjproved by the RAC for the treatment of melanoma 
patients (Seigler et al., RAC protocol #9306-043). The parallel results observed in this proposal between in vivo 
murine studies and in vitro human neuroblastoma studies support the potential viability of the approach in 
neuroblastoma and warrant testing in high risk or relapsed neuroblastoma patients for which satisfactory ^temative 
therapy is not currently available. 
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