Alignment of practices for data harmonization across multi-center cell therapy trials: a report from the Consortium for Pediatric Cellular Immunotherapy

Abstract

Immune effector cell (IEC) therapies have revolutionized our approach to relapsed B-cell malignancies, and interest in the investigational use of IECs is rapidly expanding into other diseases. Current challenges in the analysis of IEC therapies include small sample sizes, limited access to clinical trials and a paucity of predictive biomarkers of efficacy and toxicity associated with IEC therapies. Retrospective and prospective multi-center cell therapy trials can assist in overcoming these barriers through harmonization of clinical endpoints and correlative assays for immune monitoring, allowing additional cross-trial analysis to identify biomarkers of failure and success. The Consortium for Pediatric Cellular Immunotherapy (CPCI) offers a unique platform to address the aforementioned challenges by delivering cutting-edge cell and gene therapies for children through multi-center clinical trials. Here the authors discuss some of the important pre-analytic variables, such as biospecimen collection and initial processing procedures, that affect biomarker assays commonly used in IEC trials across participating CPCI sites. The authors review the recent literature and provide data to support recommendations for alignment and standardization of practices that can affect flow cytometry assays measuring immune effector function as well as interpretation of cytokine/chemokine data. The authors also identify critical gaps that often make parallel comparisons between trials difficult or impossible.

Keywords: CAR T; biomarkers; cellular therapy; correlative studies; harmonization; immunotherapy.

Figure 1:. Pre-analytic, analytic, and post-analytic variables that impact correlative data harmonization efforts across sites participating in multi-center cell therapy trials.

Pre-analytic (patient and sample related), analytic (assay related), and post-analytic (data related) factors can impact consistency and reproducibility of assays commonly used to evaluate IEC trial-related samples. An in-depth discussion of the variables highlighted in bold are included within the scope of this work, including pre-analytic variables related to biospecimen collection and processing, in addition to providing recommendations for analytes to be measured using cytokine and flow-based assays.

Figure 2:. Recovery of mononuclear cells, CD34⁺ and CD3⁺ cells in freshly isolated versus cryopreserved samples, and CD3 and CAR marker detection in fresh versus cryopreserved matched pairs.

(A) Comparison of percent recovery of cryopreserved and fresh participant-matched PBMC samples; fresh samples normalized to 100%. Mononuclear cell (MNC) recovery was calculated by comparing MNC cell counts prior to freezing with the live cell counts of the thawed cells for each sample (n=60). Percentages of each antigen expression are derived from Lymphocytes of CD45⁺ events. Similarly, CD3⁺ (n=20) and CD34⁺ (n=46) recovery was calculated by comparing flow cytometry expression between the fresh and thawed samples. Cell viability data (n=62) was calculated by examining 7-AAD expression of the thawed cells. Significance was determined using paired t test. (B) Target population recovery/stability was evaluated for CAR T cells by examining the frequency of lymphocytes expressing markers of interest in participant-matched fresh and cryopreserved cells. Fresh samples (n=26) were obtained from peripheral blood specimens treated with RBC Lysis prior to staining, while frozen samples (n=26) underwent mononuclear cell isolation by ficoll density gradient separation prior to cryopreservation. Gating strategy included viable singlet non-myeloid cell isolation prior to selection of the lymphocyte population of interest. Significance was determined using the Wilcoxon matched-pairs signed rank test.

Figure 3:. Time course of CAR T cell transduction marker detection upon co-culture with target cells.

Healthy donor T cells underwent transduction with two CAR constructs expressing either the EGFRt or Her2tG reporter molecule. Following the post-transduction expansion period, an aliquot was removed for flow cytometry staining, and the remaining culture was cryopreserved. Cells were later thawed for stimulation and culture expansion via CAR T Rapid Expansion Protocol (REP), with periodic sampling for flow cytometric analysis of the CAR reporter molecules. While detection of EGFRt and Her2tG CAR markers dramatically decreased immediately post-thaw, Her2tG expression and/or detection recovered to pre-freeze levels during the course of the culture.

Figure 4:. CAR T cell transduction marker detection with or without rest in media with or without IL-2.

Enhanced CAR transduction-marker recovery in cryopreserved cells was attempted by the addition of a rest period post-thaw, with or without addition of IL-2. Transduced CAR T cells were examined pre- and post-cryopreservation, as described in Figure 3. Following thaw, cells were resuspended in either R10 media or R10 media supplemented with 50U/mL of rhIL-2 cytokine. Cells were incubated at 37°C and evaluated by flow cytometry staining on day 1 (D1) and day 2 (D2) post-thaw. The addition of IL-2 showed enhanced CAR T cell transduction-marker staining compared to day 0 (D0; immediately post-thaw) and the unsupplemented culture; extending the rest period beyond one day did not result in any further increase in reporter molecule detection in either culture.