These data indicate that for the rejection of LLC-I12 both an effective T-cell response and an NK j 
cell response is required; a B-cell response is not essential since SCID mice, lacking both T and B cells, 
have the same survival times as Nude mice, lacking only T cells. 
I 
c. LLC-I12 delays growth of untransfected LLC 
Several studies have already established that the rejection of 112 producing tumors establishes a 
protective immunity to a subsequent tumor challenge with normal tumors. We wished to determine 
whether the immunization with LLC-I12 was able to reject or delay the growth of already established 
LLC at a different site. This question approaches the situation in human tumors, attempting to influence 
tumor growth of already established tumors by immunization. Injection of 5xl0 6 LLC-I12 on days 1, 
3, 5, 7 after transplantation of LLC into the contralateral side resulted in prolongation of survival from 
33.6±6.7 to 47.8+4.7 days (p < 0.01) see manuscript, Appendix E. This is a significant therapeutic 
effect, that we hope to improve by further modification of the injection dose, schedule and combination 
of lymphokines. It will be possible for instance to increase the dose of injected LLC-I12 by first 
irradiating the cells so as to prevent their outgrowth as tumor. 
d. Generation of a CD4 cytotoxic response by LLC-D2 
Immunization of mice with 5xl0 6 LLC-I12 on day 1, 3, 5, 7 and 49, followed one week later by 
the analysis of tumor specific cytotoxicity in unfractionated spleen cells, generated 20 % tumor lysis at 
a 100: 1 spleen cell to tumor cell ratio in 4h Cr-release assays. Both LLC and LLC-I12 could serve as 
targets. Spleen cells from control animals had no cytotoxic activity towards either LLC or LLC-I12. The 
cytolytic activity of spleen cells from immunized mice was completely blocked by anti CD4 antibodies 
but not by anti CD 8 antibodies added during the assay (Fig. 1 , see Appendix C). In accord with this 
finding we observed a expression of MHC class II antigen on LLC, which did not change upon 
transfection with pBMG-Neo-112 (Fig. 2, see Appendix C). The summary of cytokine transfection on 
the rejection rate of LLC, see Table 5, Appendix C). 
As far as combinations are concerned the combined use of 112 and 114, based on our model studies, i 
appears promising. In addition it is believed that 112 and 114 are necessary for the generation of a 
memory CTL response and it has been reported that the presence of 114 together with 112 suppresses a 
promiscuous CTL response and generates instead an antigen restricted response. This hypothesis will 
be further tested in the laboratory. 
We have not yet conducted studies with y-Ifn in our LLC model. However this lymphokine appears 
attractive in combination with 112, since it is known to upregulate MHC expression, required for T-cell 
recognition, and to increase cytotoxic activity. We are therefore in the process of including 7 -Ifn in our 
studies. 
1.4 Human Small Cell Lung Cancer 
Lung cancer is one of the most common (150,000 new cases per year) and most lethal (130,000 
deaths per year) cancers in the U.S.A. (46). The two major classes of lung cancer are non-small cell 
carcinoma (NSCLC) and small cell carcinoma (SCLC); the latter accounts for 1/4 of all lung cancers. 
Combination chemotherapy ±_ radiation therapy is the treatment of choice for SCLC, which is a rapidly 
progressive disease that disseminates widely and early. Unlike complex staging classifications of most 
other cancers, the stages of SCLC consist only of limited disease (LD) and extensive disease (ED). LD 
refers to SCLC which is confined largely to one hemithorax so that the identifiable areas of disease can 
be encompassed in a single radiation therapy treatment field. ED includes any disease outside the 
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