Constitutively active MyD88/CD40 costimulation enhances expansion and efficacy of chimeric antigen receptor T cells targeting hematological malignancies

Abstract

Successful adoptive chimeric antigen receptor (CAR) T-cell therapies against hematological malignancies require CAR-T expansion and durable persistence following infusion. Balancing increased CAR-T potency with safety, including severe cytokine-release syndrome (sCRS) and neurotoxicity, warrants inclusion of safety mechanisms to control in vivo CAR-T activity. Here, we describe a novel CAR-T cell platform that utilizes expression of the toll-like receptor (TLR) adaptor molecule, MyD88, and tumor-necrosis factor family member, CD40 (MC), tethered to the CAR molecule through an intentionally inefficient 2A linker system, providing a constitutive signal that drives CAR-T survival, proliferation, and antitumor activity against CD19+ and CD123+ hematological cancers. Robust activity of MC-enhanced CAR-T cells was associated with cachexia in animal models that corresponded with high levels of human cytokine production. However, toxicity could be successfully resolved by using the inducible caspase-9 (iC9) safety switch to reduce serum cytokines, by administration of a neutralizing antibody against TNF-α, or by selecting “low” cytokine-producing CD8+ T cells, without loss of antitumor activity. Interestingly, high basal activity was essential for in vivo CAR-T expansion. This study shows that co-opting novel signaling elements (i.e., MyD88 and CD40) and development of a unique CAR-T architecture can drive T-cell proliferation in vivo to enhance CAR-T therapies.

MyD88/CD40 signaling retains CAR T cells in a less differentiated state

“…To evaluate if the benefit of MC costimulation can be extended to other CARs, we designed an MC-CAR with an HER2-specific scFv. T cells expressing this CAR had decreased effector function (Supplemental Figure 15), which is similar to findings by other investigators, who evaluated a CD19-specific MC-CAR (14). To overcome this obstacle, we generated T cells expressing an HER2-CAR and an EphA2-CAR that only contained an MC costimulatory endodomain.…”

“…We and others have previously shown that expression of an inducible costimulatory molecule that consists of 2 FKBP12 v36 binding domains, MyD88, and CD40 (iMC) significantly enhances the effector function of T cells expressing a CAR with a CD3 ζ endodomain (ζ-CAR) (12,13). More recently, investigators showed that constitutive expression of MyD88 and CD40 (cMC) improved the effector function of T cells expressing CARs with a ζ-signaling domain, while incorporating MyD88 and CD40 signaling domain into the signaling domain of a CD19-specific CAR reduced its antitumor activity (14). MyD88, the canonical adaptor molecule for TLR and IL-1 receptor family signaling (15), and CD40, a known T cell costimulatory molecule with a role in memory formation (16,17), are both well-studied molecules.…”

“…MyD88, the canonical adaptor molecule for TLR and IL-1 receptor family signaling (15), and CD40, a known T cell costimulatory molecule with a role in memory formation (16,17), are both well-studied molecules. However, mechanistic studies with iMC and cMC ζ-CAR T cells have been limited, showing decreased PD-1 expression after stimulation of iMC and cMC CAR T cells; activation of NF-κB, TNF, and antiapoptotic pathway signaling after iMC activation; and baseline cytokine expression of cMC CAR T cells (12)(13)(14).…”

CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?

“…37,38 Since then, signaling domains from a broad range of molecules have been explored, including OX40, CD27, and ICOS. 37,[45][46][47][48][49] CD28 and 41BB co-stimulation in the context of CAR T cells has been extensively studied, including detailed phosphoproteomic and single-cell RNA sequencing (RNA-seq) analyses. [50][51][52] They activate different pathways within T cells, with CD28 signaling promoting glycolytic metabolism and an effector memory phenotype, in contrast to 41BB signaling, which induces oxidative metabolism and a central memory phenotype.…”

4-1BB Signaling Boosts the Anti-Tumor Activity of CD28-Incorporated 2nd Generation Chimeric Antigen Receptor-Modified T Cells

“…Current clinically applied CARs consist of an extracellular antigen-binding domain, a hinge and transmembrane domain, co-stimulatory domain, and a CD3z activation domain (9). Optimization of the CAR design to improve its persistence and cytotoxicity is a focus of current CAR-T studies (10)(11)(12). Various approaches have been reported to achieve these goals.…”