Double Strike Approach for Tumor Attack: Engineering T Cells Using a CD40L:CD28 Chimeric Co-Stimulatory Switch Protein for Enhanced Tumor Targeting in Adoptive Cell Therapy

Abstract

Activation of co-stimulatory pathways in cytotoxic T lymphocytes expressing chimeric antigen receptors (CARs) have proven to boost effector activity, tumor rejection and long-term T cell persistence. When using antigen-specific T cell receptors (TCR) instead of CARs, the lack of co-stimulatory signals hampers robust antitumoral response, hence limiting clinical efficacy. In solid tumors, tumor stroma poses an additional hurdle through hindrance of infiltration and active inhibition. Our project aimed at generating chimeric co-stimulatory switch proteins (CSP) consisting of intracellular co-stimulatory domains (ICD) fused to extracellular protein domains (ECD) for which ligands are expressed in solid tumors. The ECD of CD40L was selected for combination with the ICD from the CD28 protein. With this approach, it was expected to not only provide co-stimulation and strengthen the TCR signaling, but also, through the CD40L ECD, facilitate the activation of tumor-resident antigen-presenting cells (APCs), modulate activation of tumor endothelium and induce TCR-MHC independent apoptotic effect on tumor cells. Since CD28 and CD40L belong to different classes of transmembrane proteins (type I and type II, respectively), creating a chimeric protein presented a structural and functional challenge. We present solutions to this challenge describing different CSP formats that were successfully expressed in human T cells along with an antigen-specific TCR. The level of surface expression of the CSPs depended on their distinct design and the state of T cell activation. In particular, CSPs were upregulated by TCR stimulation and downregulated following interaction with CD40 on target cells. Ligation of the CSP in the context of TCR-stimulation modulated intracellular signaling cascades and led to improved TCR-induced cytokine secretion and cytotoxicity. Moreover, the CD40L ECD exhibited activity as evidenced by effective maturation and activation of B cells and DCs. CD40L:CD28 CSPs are a new type of switch proteins designed to exert dual beneficial antitumor effect by acting directly on the gene-modified T cells and simultaneously on tumor cells and tumor-supporting cells of the TME. The observed effects suggest that they constitute a promising tool to be included in the engineering process of T cells to endow them with complementary features for improved performance in the tumor milieu.

High-resolution profiling of neoantigen-specific T cell receptor activation signatures links moderate stimulation patterns to resilience and sustained tumor control

“…In a synopsis, experimental outcomes like these and ours, have implications for T cell engineering and vaccination strategies (61,62) currently focusing mainly on enhancing co-stimulatory receptor interactions (63,64), reducing inhibitory signals (65) or TCR affinity maturation (66,67). We show that individual TCR-intrinsic factors play a major role in determining T cell activation and sustained functionality in addition to p-HLA-complex density, antigen expression, co-signaling interactions and immunosuppressive factors in the TME.…”

Coengineering specificity, safety, and function into T cells for cancer immunotherapy

“…Phillip Darcy and Paul Beavis demonstrated that the combination of TCR‐ or CAR T cells engineered to secrete Fms‐like tyrosine kinase 3 ligand (FLT3L), together with immune agonists poly (I:C) and anti‐4‐1BB, expanded intratumoral conventional type 1 DCs, induced epitope spread, and enabled enhanced tumor control upon ACT 302 . The group of Prof. Renier Brentjens has coengineered CAR T cells to express CD40L 358 (normally only transiently expressed after TCR stimulation), and others have developed a CD40L‐CD28 switch receptor 395 . Notably, CD40L coengineering of anti‐CD19‐CAR T cells was associated with higher expression of HLA, adhesion and costimulatory molecules, and superior tumor control 358 .…”

Evolution by innovation as a driving force to improve TCR-T therapies

“…At the same time, TCR-T cells can be engineered to enhance their capacities to proliferate, function and survive in hostile tumor settings. Here costimulatory switch proteins (CSP) that turn negative signals into positive signals are at the forefront of development of next generation TCR-T therapies ( 141 , 142 ). Two CSP are developed in the E2E platform to armor and/or enhance the functional capacities of TCR-T cells to directly attack tumor cells and to reshape the TME.…”