TABLE OF CONTENTS 
1.0 OBJECTIVES Page 1 
2 . 0 INTRODUCTION AND RATIONALE 1 
2.1 CANCER IMMUNOLOGY 1 
2.2 RETROVIRAL VECTOR PACKAGING CELL LINES 3 
2.3 CLINICAL APPROACH 4 
3.0 PRE-CLINICAL STUDIES 5 
3.1 SUMMARY 5 
3.2 METHODS 6 
3.2.1. Construction of vector producing cell lines 6 
3.2. 1 . 1 . Construction of Human y- Interferon retroviral Provector 
plasmid pHuy-IFN 6 
3.2. 1.2. Construction of Murine y-IFN retroviral provector, 
pMuy-IFN 6 
3 . 2 . 1 . 3 . Generation of Retroviral Packaging Cell Lines 7 
3.2. 1.4. Generation of Vector Producing Clones 7 
3.2.2. Vector Characterization 8 
3.2.2. 1. G418 resistance titer assay 8 
3. 2. 2. 2. Quality control (QC) testing 8 
3. 2. 2. 3. Assay for Replication-Competent Retrovirus (RCRV) 
in vector-containing samples 8 
3. 2.2. 4. Assay for RCRV in the vector-producing cell line, VCLHuy. . . .9 
3.2.3. Protein Expression Assays 9 
3 . 2 . 3 . 1 . Murine Class I MHC Expression Assays 9 
3. 2. 3. 2. Biological Activity ofy-EFN 9 
3.3 RESULTS AND DISCUSSION 9 
3.3.1. Retroviral Vector Producing Cell Lines 9 
3.3. 1.1. Packaging Cell Lines 9 
3.3. 1 .2. Characterization of the human y-IFN vector 
producing cell line, VCLHuy 10 
3.3.2. Murine Tumor Model Data 11 
3.3.2. 1. Murine y-IFN vector expression and MHC 11 
3. 3. 2. 2. Tumorigenicity of murine y-IFN-expressing cells 11 
3 . 3 . 2 . 3 . Generation of tumor-specific CTL 12 
3. 3. 2. 4. Murine tumor vaccination studies 13 
3.3.3. Human Melanoma Data 13 
3.3.3. 1. Transducibility of human melanoma cells in vitro 13 
Recombinant DNA Research, Volume 17 
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