Evaluation of Dendritic Cell Products Generated for Human Therapy and Post-Treatment Immune Monitoring - - BioPharm International

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Evaluation of Dendritic Cell Products Generated for Human Therapy and Post-Treatment Immune Monitoring


BioPharm International
Volume 21, Issue 3

Population-based Assays

Several population-based assays measuring either proliferation of autologous T cells co-incubated with antigen-loaded DCs or target-cell cytotoxicity, in which CTLs generated in co-cultures (IVS) are tested for the ability to lyse the relevant target, are available for DC product characterization. These population-based assays are generally less informative than single-cell assays. These assays can be used not only for assessments of the DC-based products' potential to ex vivo stimulate T-cell responses, but also for immune monitoring of PBMC obtained from cancer patients treated with DC products.

In the absence of a defined antigen test, the capability of DCs to present antigens to autologous T cells can be assessed using recall antigens, such as CMV or influenza virus antigens, that re-stimulate memory T-cell responses. Pre-existing immunity against one or both of these antigens is widespread. Therefore, DCs obtained from individuals immunized with these antigens could be loaded with the antigens, allowing for the re-stimulation of the corresponding autologous T cells in vitro and confirmation of DC function.37 However, these tests do not confirm the ability of the DCs to invoke primary immune responses, which may be a critical requirement for DCs to achieve high immune potency.

SELECTING ASSAYS TO MONITOR DENDRITIC CELL–BASED CANCER VACCINES

Immune monitoring of vaccination protocols requires assays that can accurately measure vaccine-induced changes in the frequency and function of antitumor effector T cells. Assays selected for monitoring of immune cells following therapy must be adaptable to serial testing with a minimal loss of accuracy. It is important to select assays that can accurately discriminate between therapy-induced changes in an immune response relative to that measured at baseline.


Table 5. Assays currently available for immune monitoring of vaccine-based protocols
Many new types of immune assays are available for immune monitoring of DC-based clinical trials. It is possible to prioritize phenotypic versus functional, specific versus non-specific, direct versus indirect, and single cell versus bulk assays (Table 5).36 The choice of assays for immune monitoring often depends on available resources. Current emphasis has been on targeted assays that can evaluate specific activation pathways or even individual signaling molecules. Assays that identify and measure the frequency of subsets of cells or individual cells engaged in a response to a specific stimulus are of special interest. In general, tumor antigen-specific assays now can be performed reliably and precisely are replacing older and less informative nonspecific tests. Multiplex assays that define an immunologic profile (e.g., Th1 versus Th2 cytokine profile), are more desirable than assays able to measure only one analyte. A better understanding of immune mechanisms and hypothesis-driven monitoring are being combined in establishing immune measures as biomarkers of tumor progression or patient survival. The objective is to reliably use immune measures as surrogate endpoints of clinical responses to biotherapy. It is, however, unrealistic to expect that a single assay will recapitulate in vivo responses to the DC-based vaccine because several mechanisms contribute to vaccine-related upregulation of immune responses following DC transfers. Some of these mechanisms might be orchestrated by the tumor, for example, and they could suppress anti-tumor immune activity.38 Thus, the knowledge of mechanisms operating in the tumor microenvironment is an important component of immune-monitoring strategies.

Once an assay that accurately reflects vaccine-induced changes in the phenotype or function of immune cells is selected, it may require adaptation to serial monitoring may be to evaluate cryopreserved "batched" specimens. Specimen batching is required to test all serial samples collected from a patient in the course of therapy in the same assay. This practice avoids inter-assay variability and increases reliability. This also requires the laboratory to compare the assay performance on both fresh and cryopreserved/thawed cells before its acceptance for monitoring. Certain assays, notably those that measure cytotoxicity, cannot be performed reliably with cryopreserved/thawed mono-nuclear cells.39 Assays that must be performed on freshly harvested specimens require documentation of inter-assay variability so that vaccine-induced changes in an immune measure can be distinguished from assay-related daily variability. Reliable assays performed with cryopreserved/thawed specimens are the best candidates for serial monitoring of DC-based cancer vaccines.


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