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Diagnostic Approaches to Maximize Therapeutic Effect 195
same samples simultaneously (multiparameter flow cytometry) enables a new
biomarker-based approach for monitoring multiple markers of immune
responses, which hopefully will be capable of predicting or correlating to clinical
effect.
In multiparameter flow cytometry, the antigen-specific T cells are first
identified by tetramer or intracellular staining and characterized further by
functional and phenotypic markers. The markers of interest include those associated
with differentiation and activation status. It has been reported that central
memory T cells with the phenotype of CD45RA – CCR7 þ CD62L high CD27 þ
CD28 þ confer superior protective and therapeutic immunity (46–48). CD107a,
perforin, and granzyme B expression correlates directly with cytotolytic activity
of T cells (49). The proliferation capacity can be assessed by CFSE dilutions by
flow cytometry (50). In addition to intracellular staining of IFN-g, accumulation
of other cytokines including TNF-a, IL-2, and IL-5, among others can be
detected using the same principle (51). Regulatory T cells hallmarked by CD25
and Fox-P3 expression can be identified from a polyclonal population (52).
Antigen-specific T cells have to infiltrate the tumor site to exert their antitumor
function. Chemokine receptor and adhesion molecule expression on the T-cell
surface will predict the possibility of T-cell migration to tumor sites. In a study
analyzing chemokine receptor profile of melanoma-specific T cells in patients,
the presence of CXCR3 expressing tumor antigen–specific T cells was associated
with increased survival (53). The detailed phenotypic and functional analysis of
tumor-specific cells and the correlation with clinical response certainly will
improve our current understanding of antitumor response and guide development
of future immunotherapy strategies.
The multiparameter flow cytometry has been successfully applied in our
cancer vaccine preclinical development (54). In the current MKC1106-PP
clinical trial, pre- and post-vaccination samples from patients will be analyzed
and their phenotype and functionality will be compared.
Use Tetramer and ELISPOT Assay to Monitor Immune
Response in Clinical Trial of MKC1106-PP
MKC1106-PP utilizes plasmid prime, peptide boost strategy. Each treatment
cycle includes four administrations of plasmid followed by two administrations
of peptides. Patients will receive two cycles of vaccination initially. If there is
no progression of disease, the patient may receive up to an additional four
cycles for a total of six cycles of treatment. To evaluate the efficacy of
MKC1106-PP, the immune responses induced by MKC1106-PP will be
monitoredbybothtetramerandELISPOTassay.Theassayswillbeperformed
on samples before the treatment, after plasmid priming but before peptide
administration, and after peptide boost in each cycle. A substantial increase in
the result of tetramer and ELISPOT assays after dosing would indicate that
an immune response has been induced in a patient. These two assays have been