CAR-T cell efficacy can also be significantly limited by the hostile tumor microenvironment (TME), comprised of anti-inflammatory cytokines and immune cells that directly suppress T cell function. Armored CAR-T cells (alternatively referred to as fourth generation CAR-T cells, Fig. 1) are designed to dampen the suppression signal, transform it into an activation signal, or recruit bystander immune effectors to promote tumor cell targeting. For example, armored CAR-T cells expressing dominant negative (dn) cytokine receptors, such as dnTGFβRII, can effectively deplete locally secreted cytokines, such as TGFβ. Alternatively, CARs engineered with a switch receptor, such as the TGFRβR linked to a second-generation CAR intracellular signaling domain, converts an inhibitory stimulus to an activating signal. Switch receptors can also transduce homeostatic signals by expressing a CAR with the IL-4 receptor ectodomain linked to an IL-7, IL-2, or IL-15 receptor intracellular signaling domain.
Fig1. Armored CAR-T cells. Chimeric antigen receptor (CAR)-T cell in vivo persistence and anti-tumor efficacy can be inhibited by anti-inflammatory cells and cytokines in the tumor micro environment (TME). Armored CAR-T cells are designed to combat these barriers by attenuating suppressive signals or by promoting endogenous immune effectors and inflammatory cytokines. For example, (a) CAR-T cells can circumvent inhibitory immune checkpoint signaling via targeting of the programmed cell death 1 (PD-1) pathway. This can be accomplished via knockdown of PD-1, engineered dominant-negative PD-1 or PD-1 switch receptors, or direct secretion of anti- PD- 1 antibodies. (b) CAR-T cells can be engineered to express peptides that recruit endogenous effector T cells and foster a supportive microenvironment such as CD40 ligand (CD40L) or 4-1BB ligand (4-1BBL). (c) Finally, CAR-T cells may promote an inflammatory milieu by direct expression and secretion of cytokines. (APC, antigen-presenting cell; CSR, chimeric switch receptor; DNR, dominant negative receptor; scFv, single-chain variable fragment; shRNA, short hairpin RNA
T cell function can be suppressed by direct binding to inhibitory ligands, such as PD-L1, expressed on tumor cells and tumor-associated myeloid cells. Binding of PD-L1 to its receptor (PD-1) on T cells promotes T cell exhaustion diminishes CAR-T cell persistence. Several armored CARs have been designed to com bat this pathway, including expressing a dnPD-1 receptor, using a PD-1 switch receptor with a CD28 intracellular signaling domain, engineering the CAR to secrete an anti-PD-1 antibody or a blocking scFv, and silencing PD-1 expression using short hairpin RNAs or CRISPR-Cas9 (Fig. 1a). CAR-T cells that secrete anti-PD-1 antibodies or scFvs exhibit autocrine and paracrine effects, thereby bolstering the endogenous and CAR-T cell-mediated anti-tumor response.
More recently, CAR-T cells have been engineered to express co-stimulatory ligands, such as the proinflammatory marker CD40 ligand (CD40L) and 4-1BB ligand (4-1BBL) to foster a more supportive TME (Fig. 1b). CD40L is expressed on activated T cells and binds to CD40 receptor on antigen presenting cells (APCs). Interaction of CD40L with its cognate receptor results in APC activation and down stream secretion of proinflammatory cytokines. CAR-T cells expressing either membrane bound or soluble CD40L activate APCs in the TME and promote endogenous (non-CAR)-T cells to recognize and eliminate tumor cells. CAR-T cells co-expressing membrane-bound 4-1BBL exhibited enhanced persistence and decreased expression of exhaustion markers in a murine lymphoma model. These were later used in a phase I clinical trial in patients with relapsed or refractory B cell NHL, and demonstrated a complete response rate of 59% with durable remission achieved in 29% of patients.
CAR-T cells can be programmed to secrete their own cytokines, which promotes proliferation, survival, and antitumor activity of T cells while also altering the immune milieu of the TME. (Fig. 1c). Cytokines such as IL-12, IL-15, and IL-18, can promote T cell persistence and expansion. IL-12 secreting CAR-T cells exhibit enhanced expansion and persistence, have greater cytotoxicity, and are more resistant to apoptosis and suppression by PD-1. A clinical trial of these armored CAR-T cells is currently underway in patients with ovarian cancer (NCT02498912). IL-15 similarly promotes expansion, antitumor activity, and persistence of CAR-T cells when secreted, or tethered to the cell mem brane, but has demonstrated little effect on the surrounding TME. IL-18 also enhances functionality of CAR-T cells in humanized mouse models, while altering the TME by increasing the abundance of proinflammatory macrophages, depleting anti-inflammatory macrophages and T regulatory cells, and recruiting endogenous T cells. Cytokine-secreting CAR-T cells can, however, promote CRS in animal models despite the fact that secretion predominantly occurs locally. So far, pre-clinical models have confirmed this strategy to be safe with minimal systemic toxicity at lower doses.
