The use of a wide variety of different immunoassays to assess immunological endpoints in cancer immunotherapy clinical trials has provoked recommendations that standardization and rigorous validation of these immunoassays is needed [1, 11]. In response to these recommendations, we put three immunoassays, the tetramer, ELISPOT, and real time RT-PCR assays through a rigorous validation process in preparation for our cancer vaccine clinical trials. These assays met key validation criteria necessary for generating reliable clinical data. The assays were determined to be specific for each antigen, gp100 or MART-1. Assay precision for cell based functional assays met the criteria with % CV < 20% (intra day) and < 25% (inter day).
Assays were found to be sensitive with the real time RT-PCR being the most sensitive at 1 in 50,000 PBMCs. The tetramer flow cytometric method sensitivity was determined to be 1/4545–6667 (Tetramer Assay collecting 1 million events) and the ELISPOT sensitivity was at 1/10,000–20,000 (using high PBMC assay), similar to data reported by others . For ELISPOT, assessment of assay sensitivity depends on number of TIL cells spiked into the number of PBMCs as the negative cell population. Due to the limited number of PBMC that could be obtained from melanoma patients, we also validated the ELISPOT assay using a low PBMC number and assay sensitivity was poor (1/2000); this is due to a mathematical calculation where responder TIL cells were spiked into a smaller PBMC population and this smaller number served as the denominator. Higher TIL cell numbers resulted in a larger number of secreting cells (100–200 cells/well), which were difficult to count due to poor resolution. We performed a TIL cell titration study and demonstrated that 10–50 cells/well provided significant resolution to achieve a reliable assessment of cell numbers.
Similarly, a larger number of total events collected for the tetramer assay will improve assay sensitivity. With limited patient PBMC samples and the need for assay throughput and cell quality (viability) during sample acquisition, we validated the tetramer assay with ~500,000 total events collected. When one million PBMC was collected, assay sensitivity was improved but samples acquired at a later time showed poor cell viability. We also evaluated the use of fixed cells after staining, and found the MFI to be much lower suggesting tetramer binding to fixed TCR was poor.
Similar differences in sensitivity between different immunoassays have been previously observed . Assay sensitivity is also influenced by the T cell line (TIL cells) used to validate an immunoassay, and few groups use the same T cell lines. For example, only 33% of the cells in the TIL1520 cell line were responsive to peptide stimulation . Comparisons between laboratories will likely be in closer agreement when the same cell lines are used to validate an immunoassay and same TIL cells number/PBMC number is used. As an example, the sensitivity of our ELISPOT assay was in close agreement with a previously published report where the TIL1520 were used to determine ELISPOT sensitivity . A set of standard cell lines would enable a comparison of assay performance between laboratories.
While effector T cell responses can reliably be measured by each of these immunoassays, an important challenge is in determining the value that constitutes a positive response. A strong positive immunologic response measured by the MART-1 tetramer assay, such as the example shown in Figure 7, is often indisputable. Such a response profile showed a clear defined MART-1 tetramer positive CD8+ T cell population that was well separated from the tetramer negative CD8+ T cell population. This clearly suggests that immunization successfully enhanced the immune response. Low percentages of tetramer positive cells were seen in pre-treatment baseline sample. The binding resembles the tetramer positive cells specific for foreign antigens (Flu) in Figure 1, demonstrating breaking of tolerance to self antigen (MART-1).
On the other hand, positive responses are more likely to be detected at low percentages in the blood making it much more difficult to define a positive immunological response to a cancer vaccine. Therefore, guidelines need to be implemented on data analysis and interpretation based on assay performance characteristics such as precision and LOD. Use of proper negative controls such as the negative control tetramer, will help distinguish a positive response by setting the correct quadrant for data analysis to reduce subjectivity, especially when tetramer positive cells are not well separated from the negative population. Fold increase (>2 fold) of post-treatment response over the baseline value has been used, however, baseline values near zero value could result in an artificially high fold increase. Subtraction of the post-treatment value from the baseline value and subtraction of data from the negative control have also been used; analysis and interpretation of negative values remain challenging.
Interestingly, MART-1 tetramer positive CD8+ T cells were detected among both healthy volunteers and in melanoma patients who received cancer vaccines. Among healthy volunteers, MART-1 positive cells showed low MFI (median) probably reflecting low affinity/avidity (MFI is not shown). In patients, however, MART-1 positive cells had high MFI (Figure 7). Function of these MART-1 positive CD8+ T cells were reported that the cells in healthy volunteers may be of naïve phenotype, which lacks effector function (presence of CTL precursors); however, in cancer patients, these cells have the memory phenotype [15–17]. Their effector function was demonstrated in vitro upon MART-1 peptide stimulation (in the presence of APC such as dendritic cells) using methods such as cytokine production (IL-2, GM-CSF, IFNγ) and CTL activity.
Correlation of MART-1 specific CD8+ T cells in peripheral blood with the presence of CTL cells at the tumor site and clinical response in vivo is still not fully established [15, 17–20]. A majority of the peptide reactive CD8+ cells may not be tumor reactive due to various mechanisms such as down modulation of HLA class I on tumor cell surface and presence of regulatory T cells and TGFβ, etc. Sorting of the tetramer positive cells for generation of CTL in vitro has been used as adoptive transfer (cell based therapy) in melanoma patients [21, 22]. Correlation of immunologic response to clinical response (tumor regression) still needs to be established [15, 17, 20]. MART-1 specific CD8+ T cell response in one patient, as an example, was detected by all three validated clinical assays, HLA-A2 MART-1 tetramer assay, IFNγ real time RT-PCR and ELISPOT (Figure 7 and Table 10), demonstrating the assay utility in monitoring patient T cell response. Correlation to clinical response, however, was not demonstrated. A better understanding of the immune response seen in peripheral blood vs. the response at the tumor site will help us more fully understand the mechanisms of cancer vaccine and its potency.
The use of validated methods for clinical patient monitoring is important. When following patient longitudinal responses over time, our understanding of assay performance will assist us in implementing procedures that reduce assay variability and the use of QC samples will allow us to monitor assay long term performance; making data generated from these validated methods more meaningful. While the validation of these three T cell assays was challenging, the experience we obtained during validation studies and conducting patient screening will assist us and others in the field to validate similar assays for assessment of patient T cell responses to not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analysis.