- Standardized collection of high quality PBMC patient samples in this multi-center trial by a proven European laboratory network. These samples will be essential for WP4 as well as WP2 and WP5.
- Determination of immunological efficacy (proof of concept) of F16-IL2 by detecting and characterizing T cellactivation
- Validation of the relevance of novel MCV epitopes identified in WP3 will establish these as parameter formonitoring or even therapeutic applications in future MCC trials
- Identification of new cellular markers and potential surrogates for immunological efficacy of F16-IL2 to discover patient populations with a better chance to respond clinically.
Standardized PBL collection
Immatics has established and validated standardized PBMC collection and immunomonitoring. The feasibility of these SOPs have been confirmed in several international multicentric trials (188 patients respective1352 patient isolations in total, 103 sites, 56 contracted and trained PBMC laboratories) demonstrating high success rates (>95%). Briefly, standardized PBMC collection will be performed by assigned PBMC laboratories from collaboration partners situated in close distance from the clinical sites that allow venipuncture, shipment and PBMC isolations within a total time of 8 h. PBMC operators trained and qualified by immatics will prepare the PBMCs (lymphocytes and monocytes), count, and cryopreserve these according to immatics SOPs. All critical consumables will be provided by immatics, including PBMC Kits containing components for isolation and freezing and cryoproof labels allowing unambiguous identification of each cryovial. Isolation and cryoconservation will be documented in PBMC process slips that will be faxed to immatics. PBMC samples will be stored at -80°C prior to shipment and will then sent to immatics on dry ice by a trained courier service within seven days. Alternatively, PBMCs can be stored for longer than seven days at the PBMC lab if a standardized liquid nitrogen cryostorage is installed. After arrival at immatics, frozen PBMC samples will be transferred into acentral gas phase liquid nitrogen cryostorage system for storage before assays are performed.
Standardized centralized analysis of specific and functional T cells will be performed to monitor the patient immune competence. F16-IL2 administration is expected to lead to activation of MCV- and MCC-specificcytotoxic T cells (CTLs) directed towards naturally occurring epitopes inducing their proliferation and acquisition of effector functions. Thus, T cell response to peptide antigens identified in WP3 will be monitored. This serves two purposes: (i) MCC-specific immunological response profiles before and after application of F16-IL2 will be assessed in detail on basis of molecularly defined antigens to scrutinize the immunological effects after application of F16-IL2; and (ii) Validation of the relevance of novel MCV epitopes identified in WP3 will establish these as parameter for monitoring or even therapeutic applications in other MCC trials.
Cytotoxic T cells
Induced CTL effector functions can vary, including the ability to secrete cytokines and chemokines such as IFN-γ, TNF-α and MIP1-β, respectively or lysis of target cells; the latter is marked by the translocation of CD107a to the cell surface. To address the different features of induced T-cell responses the following complementary assays will be used:
- MHC multimer flow cytometric (FCM) assays allow the precise and very sensitive detection of CD8+ T cells that are specific for a given peptide/MHC complex. Albeit processing of large numbers of samples by FCM is time and resource consuming, this assay has been established as gold standard due to its high sensitivity and the fact that valuable additional information can be obtained on a single-cell level.
- Intracellular cytokine staining (ICS) assays address the quality of CTL activation: Several functional markers of T-cell activation can be assessed in parallel on a single cell basis by FCM including the synthesis of cytokines and chemokines as well markers for lytic capacity or recently performed cell lysis.
With respect to the fact that on the one hand the available PBMC cell numbers per patient will be limited and on the other hand many immune-dominant T cell epitopes from the MCV and MCC tumor antigens may be identified in WP3, immunomonitoring assays will be adapted to include cell saving technologies:
- immatics will implement and validate the highly cell saving Multimer Multiplexing technology (also used in WP3) and compare its sensitivity to immatics’ routine Multimer technology
- immatics will adapt new cell saving technologies to minimize losses in routine ICS protocols
Regulatory T cells and myeloid-derived suppressor cell
Other relevant immune cell populations i.e. Foxp3+ regulatory T cells (Tregs) and six different myeloid-derived suppressor cell (MDSCs) populations will be analyzed to extensively characterize changes in immunological parameters upon F16-IL2 therapy. For quantification and characterization of both populations from patient’s PBMC samples, a highly standardized proprietary FCM panel including validated markers for Tregs and MDSC has been established for phenotypic and even functional characterization of these. The statistical analysis will be performed with respect to the possible identification of cellular markers and potential surrogates for immunological efficacy of F16-IL2 in WP2.