Abstract 898: Single-cell multiplex proteomics reveals synergistic activity of antigen and MyD88/CD40 stimulatory signals on promoting polyfunctional chimeric antigen receptor T cells
Authors: Foster Aaron , Shaw Joanne , Collinson-Pautz Matthew , Mahendravada Aruna , Gagliardi Christine , Khalil Mariam , Paczkowski Patrick , Mackay Sean , Zhou Jing 
Published: September 2020

Abstract
Introduction: GoCAR is a chimeric antigen receptor (CAR) platform that utilizes a rimiducid-dependent activation switch (inducible MyD88/CD40; iMC) to enhance CAR-T cell proliferation and to stimulate the host immune system. To understand how these unique CAR signaling molecules compare to conventional costimulatory domains (e.g., CD28 and 4-1BB), we employed a detection and bioinformatics platform to assess the functional attributes of CD19-specific CAR-T cells by simultaneous detection of 32 proteins secreted from single CAR-T cells upon various stimulation conditions.
Methods: T cells were transduced with retrovirus encoding CD19-specific CARs with conventional costimulatory molecules (CD19.ζ, CD19.28.ζ or CD19.BB.ζ) or with GoCAR, a vector encoding iMC and a CD19-specific CAR (iMC-CD19.ζ). Control or modified T cells were enriched by anti-CD4 or anti-CD8 microbeads and stimulated with CD19 or ΔNGFR-expressing K562 cells with or without 10 nM rimiducid. After 24-hour, CD4 and CD8 T cells were collected and loaded into an IsoCode chip containing ~12,000 microchambers. Each chamber (~1.2 nL) was pre-patterned with a complete copy of a 32-plex antibody array. Cells on-chip were imaged, and single-cell cytokine signals were captured with a microarray scanner. The T cell polyfunctional profile (2+ cytokines per cell) of single T cells was evaluated across 5 functional groups: Effector: Granzyme B, TNFα, IFNγ, MIP1α, Perforin, TNFβ; 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, RANTES; Regulatory: IL-4, IL-10, IL-13, IL-22, sCD137, sCD40L, TGFβ1; Inflammatory: IL-6, IL-17A, IL-17F, MCP-1, MCP-4, IL-1β.
Results: Single-cell multiplex proteomic analysis demonstrated the synergistic effects of CD19-specific and rimiducid-induced stimulation on polyfunctional upregulation in both CD4 and CD8 GoCAR-T cells across donors. In the “off” state (i.e., no rimiducid), CD19-specific GoCAR-T showed a reduced polyfunctional strength index (PSI) compared to CD28 and 4-1BB CAR-T cells, which was predominated by antitumor-associated proteins. In contrast, activation of iMC dramatically increased GoCAR-T PSI, which was characterized by elevated “stimulatory” (GM-CSF, IL-2, IL-5, IL-8, IL-9), “chemotactic” (MIP-1b) and “regulatory” (IL-4, sCD137) molecules compared to conventional CAR constructs. Polyfunctional heatmaps revealed that GoCAR-T cells co-secreted combinatorial proteins in response to CD19-expressing target cells in combination with iMC-based costimulation. Further, both CD4+ and CD8+ GoCAR-T cells acquired the ability to produce IL-2 as part of their polyfunctional profile, which was only observed with iMC costimulation.
Conclusions: Single-cell multiplexed proteomic profiling reveals that polyfunctionality can be regulated by small molecule activation of an inducible costimulatory switch, producing distinct “on/off” states. iMC signaling also enhances production of important stimulatory and chemoattractive molecules which may improve CAR-T cell activity and provoke a host anti-tumor immune response. Thus, the GoCAR-T platform may potentially be used to optimize CAR-T activity through rimiducid administration to maximize efficacy while mitigating CAR-T-related toxicities.
Citation Format: Aaron Foster, Joanne Shaw, Matthew Collinson-Pautz, Aruna Mahendravada, Christine Gagliardi, Mariam Khalil, Patrick Paczkowski, Sean Mackay, Jing Zhou. Single-cell multiplex proteomics reveals synergistic activity of antigen and MyD88/CD40 stimulatory signals on promoting polyfunctional chimeric antigen receptor T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 898.