Single-Cell Multiplex Proteomics Reveals Synergistic Impact of Antigen and Rimiducid-Dependent Stimulatory Signals on Promoting Polyfunctional GoCAR-T Cells Targeting Prostate Stem Cell Antigen (PSCA)

Background: The GoCAR-T cell products targeting tumor antigens and incorporated with a rimiducid-controlled switch for T cell full activation have emerged as next generation CAR-T cell therapy in solid tumor. To fully characterize the cytokine/chemokine profile of pre-infusion GoCAR-T cell products, we employed a single-cell detection and bioinformatic platform to deeply assess functional attributes of GoCAR-T cells by simultaneous detection of the 32 proteins secreted from single GoCAR-T cells upon various stimulations.

Methods: GoCAR-T cells were manufactured from human peripheral blood mononuclear cells (PBMCs) of healthy donors transduced with a rimiducid-inducible costimulatory unit, MyD88/CD40 (iMC), and a first-generation CAR targeting PSCA. CD4+ and CD8+ GoCAR-T cells were enriched by anti-CD4 or anti-CD8 microbeads and stimulated with PSCA-expressing human pancreatic adenocarcinoma cell line (HPAC) in the presence or absence of rRimiducid at 37°C, 5% CO₂. After 24-hour coculture, CD4+ and CD8+ GoCAR-T cells were collected and loaded into a single-cell barcode chip (SCBC) containing ~12,000 microchambers. Each chamber (~1.2 nl) was pre-patterned with a complete copy of a 32-plex antibody array. Cells on the SCBC were imaged and incubated for 16 hrs at 37°C, 5% CO₂; single-cell cytokine signals were captured with a microarray scanner. The polyfunctional profile (2+ cytokines per cell) of single GoCAR-T cells was evaluated across 5 functional groups: Effector: Granzyme B, TNF-α, IFN-γ, MIP-1α, Perforin, TNF-β; Stimulatory: GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, IL-21; Chemoattractive: CCL11, IP-10, MIP-1β, RNATES; Regulatory: IL-4, IL-10, IL-13, IL-22, sCD137, sCD40L, TGF-β1; Inflammatory: IL-6, IL-17A, IL-17F, MCP-1, MCP-4, IL-1β.

Results: Single-cell multiplex proteomic analysis demonstrates the synergistic effects of PSCA-specific and rimiducid-induced stimulation on polyfunctional upregulation in both CD4+ and CD8+ GoCAR-T cells across donors. Polyfunctional strength index (PSI) reveals the PSCA-induced polyfunctional increase in GoCAR-T cells was predominated by antitumor-associated proteomics including Granzyme B, IFN-γ, TNF-α, Perforin, MIP-1α, IL-2, IL-8, IP-10, MIP-1β, sCD137 and sCD40L, and further elevated in GM-CSF by the costimulatory signals induced by rimiducid. In addition, the polyfunctional heatmap uncovers the upregulated polyfunctional cell subsets of single GoCAR-T cells that co-secreted combinatorial proteins of Granzyme B, IFN-γ, TNF-α, Perforin, MIP-1α, IL-2, IL-8, IP-10, MIP-1β, sCD137 and sCD40L in response to PSCA and/or rimiducid.

Conclusions: Single-cell multiplexed precision profiling reveals antitumor polyfunctional upregulation and heterogeneity of GoCAR-T cell products with the stimulation of PSCA+ tumor cells and/or iMC activation by rimiducid. The presented polyfunctional metrics may provide insights into quality check of pre-infusion GoCAR-T cell products and potential biomarker discovery to predict efficacy, persistence and safety of GoCAR-T cell therapy in solid tumor.