Immune Monitoring Assays of Cancer Vaccines

The discipline of immune monitoring for cancer vaccine and immunotherapy trials is experiencing an era of unparalleled growth because of an increased understanding of the fundamental basis of human cancer and host–tumor interactions. Developing standardized methods and validating individual assays is a major priority for tumor immunologists.

A set of robust assays needs to be established that are standardized and validated in both academic and industry laboratories. Creative Biolabs has experts to help you select and develop particular assays. A variety of tests are available for immune monitoring of cancer vaccine and immunotherapy trials.

While ELISA has become the standard for detecting antibody responses, there are many assays for monitoring T cell responses. Three assays—ELISpot, tetramer staining, and CFC—have emerged as first-line methods for monitoring T cell responses in most clinical trials. These assays are relatively reliable and sensitive and allow ex vivo T cell analysis at the single-cell level. In addition to these standard assays, other approaches, such as flow cytometry, proliferation, and cytotoxicity assays may provide additional functional information. Other more complex methods, such as quantitative real-time PCR, microarray analysis, and proteomics have been useful for gene and protein profiling of the tumor microenvironment and as complements to first-line monitoring assays.

Enzyme-Linked Immunospot Assay (ELISpot)

The interferon-γ (INFγ) ELISpot assay is a commonly used procedure and has been extensively validated for the detection and quantification of functional T cell responses in patients treated with cancer vaccines. ELISpot is an attractive and powerful assay that allows tracking of antigen-specific T cells in the peripheral blood of patients who receive vaccines provided enough T cells are available for pre- and postimmunization sampling. ELISpot also permits quantitative measurement of cytokine secreting cells at the single-cell level directly ex vivo or after in vitro stimulation, which can be used to expand low-frequency T cells. The function of cells can be inferred from the types of cytokines secreted by cells in response to antigenic stimuli. ELISpot assay is often the first choice in vaccine evaluation since high-throughput screening using a microtiter plate format is efficient and can accommodate large numbers of samples and multiple antigens. In general, the ELISpot assay has the lowest limit of detection and can often detect as few as 1 in 100,000 T cells.

Tetramer Analyses

Tetramer analysis is a flow-cytometry-based technique that replaces monoclonal antibodies with folded peptide–MHC complexes attached to a color substrate. The assay can be used to quantitate the number of antigen-specific T cells in samples with as few as 10,000 cells. Thus, the availability of sufficient cells for the assay is rarely a problem for tetramer analysis.

Flow Cytometry

Multiparametric characterization of cytokine production on a single-cell basis with high throughput of samples is an attractive feature of flow cytometric analysis. The technique allows simultaneous use of four or more antibodies and is thus particularly useful for the small subsets of lymphocytes modulated by immunotherapy.

Quantitative Real-Time RT-PCR

The polymerase chain reaction (PCR) revolutionized the field of molecular biology and allowed for accurate detection of gene transcripts from minute volumes of genetic material. The assay has been most useful in evaluating the tumor microenvironment, where there are quantitative or qualitative differences in immune modulating molecules and cells, for example, the balance between effector and regulatory T cell accumulation in tumors. The qRT-PCR assay may also be more reliable since a few studies have suggested that cellular immune assays may not be reliable for cells located within established tumors.

Microarray Analysis

Microarray technology is based on the availability of an expressed sequences tag (EST) database of the human genome and allows collection of extensive biological data. These data can be used to enhance our understanding of physiological processes and disease states by profiling tissue and single-cell populations at the RNA and protein levels. The technique is particularly useful for comparative analysis of genome wide mRNA expression patterns. In cancer research, high-throughput evaluation of genetic or protein profiles in specific tissues can be used to identify unique signatures in tumor compared to normal tissue, or in primary compared to metastatic tissue. Microarray analysis has generated significant information on tumor-associated and tumor-specific biomarkers for cancer diagnosis and as targets for therapeutic drug development.

Proteomics Analysis

While PCR and microarray data provide information on gene and gene expression status, it is clear that gene expression does not always correlate with actual protein expression. The direct analysis of proteins within cells or tissue samples is referred to as proteomics analysis. This technique can provide data on all proteins in a particular tissue sample, and has been used for a variety of diagnostic and monitoring purposes.

T Cell Proliferation Assay

Although more sophisticated techniques, such as ELISpot and flow cytometry, are typically used for monitoring immune responses, standard T cell proliferation and cytotoxicity assays can often be used to help validate the more complex assays. The T cell proliferation assay is based on the ability of T cells to undergo proliferation on antigen exposure (usually in the presence of APC), and the proliferation can be measured by standard 3H- thymidine incorporation during DNA synthesis. The assay is relatively easy to perform but can be a lengthy procedure that requires radioactive material.

T Cell Cytotoxicity Assay

The potential cytotoxic activity of CD8⁺ T cells can be directly measured by lysis of target cells expressing the cognate antigen recognized by the T cells. MHC-expressing cells pulsed with specific peptides or intact MHC-restricted tumor cells have been used as targets in T cell cytotoxicity assays.

Each assay has unique advantages and disadvantages, and the selection of appropriate immune assays for monitoring clinical trials is not a trivial issue. The evaluation of cancer vaccines and immunotherapy regimens will require standardization of the selected assays and statistical analysis plans in place before an assay can be used for immune monitoring. Creative Biolabs is a leader in the field of vaccine development and has focused on the cancer vaccines for years. We have experienced experts and advanced platforms that are able to provide excellent services. If you are interested in our services, please contact us for more details.

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