The tumorigenic properties of L33 ylFN-expressing tumor cell lines were also tested in 
immunocompromised athymic nude mice. The L33 tumors grew almost as well as the unmodified tumor 
(Fig 7), indicating that the decreased tumorigenicity observed in normal animals was at least partly due to an 
immune response involving T cells, most likely CTL. 
3.2.4. GENERATION OF TUMOR-SPECIFIC CTL. The immunological mechanisms for the decreased 
tumorigenicity of tumor cells expressing y-IFN were of interest. There were at least three possible 
explanations for immune protection observed for y-IFN-expressing, but not unmodified, tumor cells. Either 
1) the unmodified cells were not capable of eliciting cytotoxic lymphocyte responses, or 2) tumor-specific 
cytotoxic lymphocytes exist but the cells were resistant to lysis, or 3) both. The exceptionally low levels of 
Class I MHC on most tumor cells and the need for Class 1 MHC for antigen presentation (3, 4) suggested 
that immune responses against the y-IFN -expressing tumor cells might not have been effective against the 
unmodified tumor. 
To lest these possibilities, the CTL induction characteristics of unmodified and y-IFN-expressing tumor cells were 
compared for B16, Lewis lung tumor, and CT26, using the unmodified tumor cell as a target Cytolytic 
activity was substantially increased for y-IFN transduced B16 and Lewis lung tumor (data not shown). 
Representative data in Fig 8 indicate that injection of unmodified CT26 irradiated tumor cells resulted in a 
relatively poor CTL response. Injection with y-IFN -expressing CT26, however, resulted in improved CTL 
responses. Control experiments using unrelated cell lines, BCIOME and B16F10 as CTL targets, indicated 
that the anti-CT26 immune response was specific (Fig 9). Antibody depletion studies indicate that the CTL 
are CD8 -f T cells (Fig. 10). 
These data show that the lack of measurable response against the unmodified tumor is due to insufficient CTL 
induction , not due to the inability of the cell to be lysed by CTL. These results have been confirmed by other 
investigators with another tumor model (30, 82). 
3.2.5. MURINE TUMOR VA(XINATION STUDIES. To determine whether the observed immune responses 
could protect mice from challenge with unmodified tumor cells, vaccination studies were performed by 
priming mice with either unmodified, or y-IFN-transduced, irradiated B16F10 tumor cells. The animals were 
then challenged with the unmodified parental tumor cell. A single pre- injection with as little as 4x10^ y- 
IFN-expressing tumor cells, but not unmodified tumor cells, resulted in a measurable (~ 2.5-fold) protection 
against tumor challenge. A single injection with larger numbers of cells resulted a 5-fold decrease in the 
number of lung metastasis (Fig.ll). y-IFN-transduced tumor cells may therefore be capable of generating 
systemic, protective, anti-tumor immunity. 
3.3. Human Neuroblastoma Data 
3.3.1. MHC-I EXPRESSION IN NEUROBLASTOMA CELLS: In prep^tion for the current trial, we have 
characterized over 46 untreated neuroblastoma cell lines for overall exjM^ssion of class I MHC using the W6/32 
antibody. Only 5 of 46 tumors expressed class I antigens as determined by flow cytometry with W6/32 and 
most of these did so weakly (Reynolds CP, Seeger R, unpublished data; (64). Virtually all neuroblastoma 
subsets do not express MHC-I. Effective gene therapy strategy therefore would likely require induction of 
MHC-I. 
Over 140 neuroblastoma cell lines have been established in the laboratories of Drs. Seeger and Reynolds, from 
tumor, bone marrow and/or blood of patients with neuroblastoma. As many as 70 of these lines have been 
recently established from patients who are still alive. Hence, a large variety of autologous and/or HLA 
partially matched allogeneic cell lines are available for the study. Our preliminary experience suggests that 
approximately two thirds of the lines will have adequate transduction growth characteristics, following y-IFN 
transduction and will be available for use in the trial. However, a proportion of patients’ cell lines do not grow 
adequately following y-IFN transduction or may be difficult to transduce. The large neuroblastoma cell bank 
insures that for many of these patients, particularly those with common HLA haplotypes (eg Al, A2, etc.), 
suitable allogeneic cell lines will be available. We aim to develop a panel of transduced allogeneic cell lines 
for this purpose. 
[Recombinant DNA Research, Volume 19 
[95] 
