Immune Response Monitoring as Potential Tool for Therapy Guidance

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MKC's cancer vaccine is an active immunotherapy aimed at inducing or augmenting tumor-specific T cells in vivo that leads to tumor regression and survival benefit. With the initiation of various vaccination trials, accurate and reliable assays for testing T-cell function is crucial for the evaluation, comparison, and further development of these approaches. The cellular immune responses have been evaluated using methods measuring cytotoxicity, proliferation, or release of cytokines in a bulk culture. However, these assays often require in vitro stimulation prior to performing them. A selection bias is automatically introduced with culturing of the effector cells, and the results subsequently obtained from these assays may not reflect in vivo T-cell function.

The emergence of ex vivo assays represented by tetramer analysis, ELI-SPOT assay, and intracellular cytokine staining has significantly improved our ability to measure T-cell response to vaccine attributing to their capability of detecting antigen-specific cell at the single cell level and therefore providing quantitative information. The evolved multiparameter flow cytometry allows us to characterize T-cell subpopulations and provide a better understanding of antitumor immunity.

The immune function varies among individuals, and the variation is amplified among cancer patients. It is common that patients respond to cancer immunotherapy heterogeneously. Therefore, it is important to monitor each individual's immune response to vaccine treatment and adjust the treatment strategy accordingly to achieve clinical benefit. It is logical to measure the increase of tumor-reactive T cells, in vivo if any, by tetramer and ELISPOT assays, after vaccine administration. However, recent findings indicate that generation of a large in vivo population of tumor-reactive CD8 T cells alone is insufficient to achieve clinically significant tumor regression. Studies applying multiparameter analysis of T-cell phenotypes and functions demonstrate that it is the effective memory response that has a superior antitumor activity (35-37). No doubt, the multiparameter flow cytometry is a valuable addition to tetramer and ELISPOT assay for monitoring immune responses to vaccines.

Tetramer Analysis

The use of MHCl/peptide tetrameric technology to directly visualize and quantify antigen-specific CTLs was first described by Altman et al. in 1996 (38) in which soluble, fluorescently labeled, multimeric MHC/peptide complex bind stably, specifically, and avidly to antigen-specific T cells. This assay is easy to perform; generally 30 minutes staining of tetramer at room temperature is sufficient. Both fresh and cryopreserved PBMC samples have been successfully analyzed and have achieved comparable results (39). The tetramer is able to identify all the T cells specifically recognizing the MHCl/peptide complex composing the tetramer regardless of their functional status. Since the tetramer analysis is a flow cytometry-based assay, it can be used together with other cell surface staining to obtain further characterization of tetramer-positive cells. Alternatively, the tetramer-labeled population can be sorted for additional assays to study its functionality. However, compared with functional assays ELISPOT and intracellular cytokine staining, the sensitivity of the assay is relatively low, and sequences of the antigen epitope peptides have to be available for forming respective tetramers. Some low-affinity clinically important peptide epitopes may not be able to form tetramer efficiently (40). In most cases of immune monitoring, tetramer analysis is accompanied with other functional assays to address functional status of the cells. The immune monitoring workshop in 2002 sponsored by the Society for Biological Therapy recommended that tetramer assay be used in conjunction with ELISPOT or cytokine flow cytometry for evaluating immune responses induced by cancer vaccine (41).

ELISPOT Assay

The ELISPOT assay was originally established to enumerate antibody secreting B cells at the single cell level (42) and later adapted to quantitatively measure the frequency of IFN-g-producing cells (43). The ELISPOT assay is based on the principle of the ELISA. A 96-well microtiter plate with nitrocellulose or PVDF membrane is coated with a monoclonal antibody against the cytokine of interest. Unseparated PBMCs or isolated CD8+ or CD4+ T cells are incubated with an appropriate antigen for 6-48 hours. In response to recognition of the antigen, cytokine is released by T cells and captured by membrane-bound antibody in the local environment of the cytokine-secreting cells. The cells are washed off and a biotinylated secondary antibody specific to a second epitope of the cytokine is added. To make the antibody-cytokine-antibody sandwich visible, an avidin-enzyme complex and an insoluble enzyme-specific substrate are added. The end result is an area with colored spots, each spot representing a single cell that secretes cytokine.

Similar to ELISA, ELISPOT assay is simple, easy to perform, and amenable to high throughput. This assay is highly sensitive with the reported limit of detection of 1/100,000 (0.001%) compared to 1/10,000 (0.01%) for tetramer analysis (44,45). ELISPOT assay can be performed with either fresh or cry-opreserved PBMC samples with similar results (39). However ELISPOT assay is unable to distinguish reactive cell types in polyclonal populations such as PBMCs. Another pitfall of this assay is that each sample can only provide limited information due to the difficulty of multiplexing this assay. How to minimize the operator-dependent variability is another challenge of the assay.

Multiparameter Flow Cytometry

The immune response against the tumor is far more complicated than it was thought before. Not only the magnitude but also the quality of the immune response elicited by the cancer vaccine determines the clinical outcome. The capability of current flow cytometry to measure multiple components of the 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 CD45RACCR7+CD62LhighCD27+ CD28+ 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 monitored by both tetramer and ELISPOT assay. The assays will be performed 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 developed and validated using antigen-specific T cells generated by in vitro immunizations.

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