The Clinical Trials Team supports the clinical program of the Vaccine Branch under the direction of Dr. Jay Berzofsky. We are using peptide vaccines directed at mutated oncogenes as targets for immune recognition. The beauty of this approach is that if these pathways are required for maintenance of the malignant phenotype then immune recognition of these mutated peptides will require the cells to eliminate their expression to survive. Initial clinical trials carried out in the Branch with mutated ras and p53 peptides administered with adjuvant showed that these peptides were immunogenic and were associated with clinical benefit in a subset of patients. Our current trials have attempted to improve on this result through the use of in vitro matured dendritic cells to present peptide antigens. In collaboration with the Department of Transfusion Medicine we have generated mature dendritic cells that are pulsed with mutated ras or p53 peptides for clinical trials. Elutriated monocytes are incubated for 96 hours with GM-CSF and IL4 followed by an overnight exposure to CD40Ligand. This results in a population of cells that expresses high levels of the T cell costimulatory molecules B7.1 and B7.2 and the MHC class I and II molecules. For the purpose of this trial we have defined cells that are positive for expression of CD83 as mature dendritic cells. The addition of CD40 Ligand reproducibly upregulates expression of CD83 on GM-CSF and IL4 treated monocytes which are negative or have low level CD83 expression, consistent with immature dendritic cells. We are able to reproducibly generate mature dendritic cells in vitro from 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. Administration of peptide-pulsed dendritic cells has been well tolerated with no acute infusional reactions in any patient. The goal of the trial is to induce the development of T cells specific for the mutated peptides and in a preliminary fashion to evaluate the antitumor activity of this approach. Immune monitoring includes both cytokine production in primary stimulations and cytotoxicity assays with repetitively stimulated cells. At this time one of 15 patients tested has shown evidence of immune reactivity to the mutated oncogene. No tumor responses have been observed yet. 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-A0201 thereby eliminating 55% of subjects screened. A second screen for expression of the mutated ras oncogene eliminated two-thirds of the HLA-A0201 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. When the immunogenicity of these peptides was evaluated in A2Kb transgenic mice both of the epitope enhanced peptides produced a higher percentage of CD8+ T cells specific than the wild type sequence. It was also shown that T cells generated with the epitope enhanced TARP29-37 sequences reacted with targets pulsed with the wild type TARP29-37 peptide in the mouse. Althugh immunogenicity of these peptides was demonstrated in the mouse it is important to confirm their immungenicity and cross reactivity in humans. Studies of these peptides in human cells showed that TARP29-37, TARP29-37-3A, and TARP29-37-9V were immunogenic in human T cells. TARP29-37-9V specific T cells recognize targets pulsed with all three peptides equally well whereas TARP29-37-3A specific T cells recognized only targets pulsed with TARP29-37-3A, and that TARP29-37 specific T cells recognized targets pulsed with the epitope enhanced peptides less well. This would suggest that the TARP29-37-3A peptide would not be appropriate for immunization in humans whereas the TARP29-37-9V would be more likely to generate T cells that recognize the wild type sequence. Human T cells specific for TARP27-35 recognized targets pulsed with that sequence as anticipated. In addition to their ability to kill targets pulsed with TARP peptides, CD8+ T cells specific for TARP peptides were able to kill tumor targets that were HLA-A2 positive and that expressed TARP sequences. The availability of tetramers that react with CD8+ T cells specific for TARP provides a simple means of evaluating the ability to stimulate immunity to the TARP peptides. In a limited survey tetramer positive cells ranged from 0.66% to 3.9% of the CD8+ T cells in prostate and breast cancer patients compared with .01-.6% in normal controls. A clinical trial has been written to examine TARP as a target for immune recognition and will use peptide pulsed dendritic cells administered on an every other week basis. Although peptide immunization with epitope-enhanced peptides reproducibly generates T cell responses in patients, tumor regression is infrequent. In addition immune therapies in general produce long-lasting tumor regressions in a small minority of patients. These observations suggest that there are immune mechanisms that prevent the induced immune response from producing tumor regression. A number of checkpoint controls in immune regulation can be targeted including TGF-?, CTLA-4 and the CD4+, CD25+ T cell regulatory population.
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