Rapid Manufacturing of Highly Cytotoxic Clinical-Grade SARS-CoV-2-specific T Cell Products Covering SARS-CoV-2 and Its Variants for Adoptive T Cell Therapy

Abstract

Objectives: Evaluation of the feasibility of SARS-CoV-2-specific T cell manufacturing for adoptive T cell transfer in COVID-19 patients at risk to develop severe disease. Methods: Antiviral SARS-CoV-2-specific T cells were detected in blood of convalescent COVID-19 patients following stimulation with PepTivator SARS-CoV-2 Select using Interferon-gamma Enzyme-Linked Immunospot (IFN-γ ELISpot), SARS-CoV-2 T Cell Analysis Kit (Whole Blood) and Cytokine Secretion Assay (CSA) and were characterized with respect to memory phenotype, activation state and cytotoxic potential by multicolor flow cytometry, quantitative real-time PCR and multiplex analyses. Clinical-grade SARS-CoV-2-specific T cell products were generated by stimulation with MACS GMP PepTivator SARS-CoV-2 Select using CliniMACS Prodigy and CliniMACS Cytokine Capture System (IFN-gamma) (CCS). Functionality of enriched T cells was investigated in cytotoxicity assays and by multiplex analysis of secreted cytotoxic molecules upon target recognition. Results: Donor screening via IFN-γ ELISpot allows for pre-selection of potential donors for generation of SARS-CoV-2-specific T cells. Antiviral T cells reactive against PepTivator SARS-CoV-2 Select could be magnetically enriched from peripheral blood of convalescent COVID-19 patients by small-scale CSA resembling the clinical-grade CCS manufacturing process and showed an activated and cytotoxic T cell phenotype. Four clinical-grade SARS-CoV-2-specific T cell products were successfully generated with sufficient cell numbers and purities comparable to those observed in donor pretesting via CSA. The T cells in the generated products were shown to be capable to replicate, specifically recognize and kill target cells in vitro and secrete cytotoxic molecules upon target recognition. Cell viability, total CD3⁺ cell number, proliferative capacity and cytotoxic potential remained stable throughout storage of up to 72 h after end of leukapheresis. Conclusion: Clinical-grade SARS-CoV-2-specific T cells are functional, have proliferative capacity and target-specific cytotoxic potential. Their function and phenotype remain stable for several days after enrichment. The adoptive transfer of partially matched, viable human SARS-CoV-2-specific T lymphocytes collected from convalescent individuals may provide the opportunity to support the immune system of COVID-19 patients at risk for severe disease.

Keywords: COVID-19; SARS-CoV-2; adoptive T cell therapy; antiviral T cells; immunotherapy.

FIGURE 1

Donor screening and pretesting. (A) Summarized results of donor screening via Interferon-gamma (IFN-γ) Enzyme-Linked ImmunoSpot (ELISpot) assay, n = 198 (all) and n = 104 (responders only) convalescent COVID-19 patients. unstim.: unstimulated control; spw: spots per well. Data are shown as violin plots, each symbol represents data obtained from one donor. ****p < 0.0001; Wilcoxon matched-pairs signed rank test. (B) Representative FACS plots and summarized results from intracellular cytokine staining in whole blood after stimulation with PepTivator SARS-CoV-2 Select using the SARS-CoV-2 T Cell Analysis Kit (Whole Blood) (Miltenyi Biotec). unstim. control: unstimulated control; TNF-α: Tumor Necrosis Factor-alpha. Data are shown as mean of data obtained from n = 12 convalescent COVID-19 patients. (C) Summarized frequencies of IFN-γ-secreting T cell subsets detected and magnetically enriched using Cytokine Secretion Assay (CSA); left: pre-enrichment (corresponding to pre drug substance („preDS“) in clinical manufacturing), values obtained from unstimulated control were subtracted; right: post-enrichment (corresponding to „DS“/drug product („DP”) in clinical manufacturing). Data are shown as mean + SD of data obtained from n = 22 convalescent COVID-19 patients. Each symbol represents data obtained from one donor. (D) Correlation of data obtained from ELISpot Assay (spw/2.5 × 10⁵ PBMCs) and CSA (%CD3⁺/IFN-γ⁺) pre-enrichment (preDS) and post-enrichment (DS). Each dot corresponds to data obtained from one donor, n = 22 convalescent COVID-19 patients. Linear regression analysis was performed to calculate statistical significance. (E) Activation marker (CD69, CD137, and CD154) expression on indicated samples obtained via CSA, compared to unstimulated control (unstim. control). Data are shown as mean + SD of data obtained from n = 3 convalescent COVID-19 patients. (F) mRNA levels of IFN-γ, granzyme B, and perforin of indicated samples obtained via CSA. Data are shown as mean + SD of data obtained from n = 3 convalescent COVID-19 patients.

FIGURE 2

Clinical manufacturing of SARS-CoV-2-specific T cells (n = 4). SARS-CoV-2-specific T cells were magnetically enriched under GMP-compliant conditions using Cytokine Capture System (CCS) and CliniMACS Prodigy and analyzed via flow cytometry. (A) Enrichment of SARS-CoV-2-specific Interferon-gamma (IFN-γ)-secreting T cells using CCS and CliniMACS Prodigy. Data are shown as mean of data obtained from n = 4 manufacturing processes. (B) Representative FACS plots depicting IFN-γ production in indicated T cell subsets of in-process control (pre-enrichment; preDS) and the magnetically enriched T cell product (drug substance, DS). (C) Summarized results of clinical-grade manufacturing in comparison to corresponding donor pretesting Cytokine Secretion Assay (CSA) and process-accompanying CSA. Bars represent mean, each symbol represents data obtained from one donor (same colors indicate matched data), n = 4 convalescent COVID-19 patients. Statistical analysis was performed using Friedman Test, followed by Dunn’s multiple comparison; ns not significant.

FIGURE 3

Characterization of clinical-grade SARS-CoV-2-specific T cells (n = 4). SARS-CoV-2-specific T cells were magnetically enriched under GMP-compliant conditions using Cytokine Capture System (CCS) and CliniMACS Prodigy and analyzed via flow cytometry. (A) Impurities of SARS-CoV-2-specific T cell product. Shown is the fold-reduction of indicated immune cell subsets in the T cell product (drug substance, DS) compared to preDS (pre-enrichment). Bars and lines represent mean and SD, n = 4 manufacturing runs. IFN-γ: Interferon-gamma; NK cells: Natural Killer cells, NKT cells: Natural Killer T cells. (B) Memory phenotype composition of T cell product (DS) compared to preDS. Numbers above bars indicate fold change of naïve T cells (T_N) between DS and preDS within indicated T cell subsets. T_CM: T central memory; T_EM: T effector memory; T_EMRA: T effector memory re-expressing CD45RA. (C) Stability of SARS-CoV-2-specific T cell product during shelf-life in terms of viability (left; Frequency of 7-AAD^- cells among CD45⁺ leukocytes) and total viable CD3⁺ T cell numbers (right) as determined via flow cytometry. 24 h/72 h: time after end of leukapheresis. Each symbol represents data obtained from one manufacturing run (same colors indicate matched data), horizontal lines represent the mean.

FIGURE 4

Cytotoxic potential of clinical-grade SARS-CoV-2-specific T cells (n = 4). SARS-CoV-2-specific T cells obtained by Cytokine Capture System (CCS) and CliniMACS Prodigy were expanded for 7–13 days and subjected to cytotoxicity assays using unloaded and SARS-CoV-2 Select-loaded autologous PBMCs as target cells. (A) Frequencies of cells with active caspase-3/7 or 7-AAD⁺ cells among target cells (CellTrace Proliferation dye positive). Light bars: unloaded target cells. Dark bars: loaded target cells. Results are displayed as individual results and as means. (B) Cell culture supernatants from cytotoxicity assays from day 12/13 were analyzed with respect to presence of cytotoxic effector molecules by LEGENDPlex Assay. Results are displayed as individual results and as means, n = 4 manufacturing processes. Statistical significance was calculated using Two-way ANOVA and Sidak’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.