The Clinical Trials Team supports the clinical program of the Vaccine Branch under the direction of Dr. Jay Berzofsky. Our current trials of in vitro matured dendritic cells to present mutated ras and p53 peptide antigens have been closed. These studies conducted in collaboration with the Department of Transfusion Medicine demonstrated our ability to reproducibly generate mature dendritic cells that can safely be administered to patients with cancer. These mature dendritic cells are pulsed with patient-specific peptides that encode the sequence of the mutated ras or p53 gene specific for that patient's colon or lung tumor respectively. Administration of peptide-pulsed dendritic cells has been well tolerated with no acute infusional reactions in any patient. Only limited evidence of immunization has been documented; one of 21 patients tested showed reactivity to the mutated oncogene. No tumor responses were observed. These trials have demonstrated the difficulty of accruing patients with mutated oncogenes as targets. The screening process requires that the patients undergo HLA testing and express HLA-A*0201 thereby eliminating 55% of subjects screened. A second screen for expression of the mutated ras oncogene eliminated two-thirds of the HLA-A*0201 positive subjects. In addition the 4-6 week period required for analysis of these parameters resulted in loss of eligibility for treatment on the trial for about half of these patients due to disease progression . An additional problem with this approach is the lack of immungenicity observed thus far. The ras and p53 peptides have not been epitope enhanced to increase their immunogenicity which appears to be an important step for producing an immune response to most peptides. As a result of these difficulties new targets have been identified that are almost universally expressed in a given tumor type and that have been epitope enhanced to improve their immunogenicity. A novel protein expressed in patients with prostate and breast cancer has recently been described. This 58 amino acid protein, T-cell receptor ? alternate reading frame protein (TARP), was identified with the expressed sequence database. The mRNA is initiated in the J? 1 exon of the TCR ? and the protein expressed is initiated in an alterntive reading frame than the TCR ? coding sequence. The protein is expressed both by normal and malignant prostate cancer tissue with over 90% of prostate cancer specimens positive for its expression. Two HLA A2 epitopes that produce cytolytic T cell responses were determined. These sequences map to amino acids 27-35 and 29-37. TARP27-35 was found to bind with an affinity that was 10 times greater than that of TARP29-37. These peptides were demonstrated to be immunogenic by immunizing A2Kb transgenic mice (expressing human HLA0201) with dendritic cells pulsed with these peptides or with DNA encoding the peptide. Dendritic cell immunization produced a higher level of immunity than DNA immunzation and as expected due to its higher binding affinity, TARP27-35 produced a higher level of CD8+ T cell response than TARP29-37. Epitope enhancement of the TARP peptides was performed to increase the level of immunity that could be generated with these peptides. Amino acid substitutions in the TARP27-35 peptide did not increase binding affintity but two amino acid substitutions in TARP29-37 did produce higher binding affinity peptides. For TARP29-37, Arg at position 3 and Leu at position 9 were substituted with Ala (TARP29-37-3A) and Val (TARP29-37-9V), respectively. Substitution at position 3 with Ala in TARP29-37 resulted in the greatest increase in the binding affinity of the peptide. Although TARP29-37-9V showed a lower binding affinity to HLA-A2 than TARP29-37-3A did, substitution of Leu at position 9 with Val did enhance the binding affinity compared with the wild-type peptide, TARP29-37.
- National Institutes of Health
Amino Acid Substitution
Tumor Suppressor Protein p53