CAR T Cells for Solid Tumors: New Strategies for Finding, Infiltrating, and Surviving in the Tumor Microenvironment

Abstract

Chimeric antigen receptor (CAR) T cells, T cells that have been genetically engineered to express a receptor that recognizes a specific antigen, have given rise to breakthroughs in treating hematological malignancies. However, their success in treating solid tumors has been limited. The unique challenges posed to CAR T cell therapy by solid tumors can be described in three steps: finding, entering, and surviving in the tumor. The use of dual CAR designs that recognize multiple antigens at once and local administration of CAR T cells are both strategies that have been used to overcome the hurdle of localization to the tumor. Additionally, the immunosuppressive tumor microenvironment has implications for T cell function in terms of differentiation and exhaustion, and combining CARs with checkpoint blockade or depletion of other suppressive factors in the microenvironment has shown very promising results to mitigate the phenomenon of T cell exhaustion. Finally, identifying and overcoming mechanisms associated with dysfunction in CAR T cells is of vital importance to generating CAR T cells that can proliferate and successfully eliminate tumor cells. The structure and costimulatory domains chosen for the CAR may play an important role in the overall function of CAR T cells in the TME, and "armored" CARs that secrete cytokines and third- and fourth-generation CARs with multiple costimulatory domains offer ways to enhance CAR T cell function.

Keywords: T cell; adoptive T cell immunotherapy; chimeric antigen receptor; engineered T cells; solid tumors.

Figure 1

A representative figure of an armored 3rd generation CAR in a T cell and a schematic of the transgene, which includes the extracellular scFv, two intracellular costimulatory domains (4-1BB and CD28), the ζ chain, a 2A linker, and the gene of interest to be coexpressed (61, 62). Examples of “armor” added to the CAR T cell are the CCR2 receptor (63), which has been shown to increase T cell migration and homing to the tumor site (64, 65) or constitutive secretion of the cytokine IL-7 and chemokine CCL19, which are important to memory differentiation and T cell migration, respectively (66). CARs that constitutively secrete IL-12 have also been used in several studies to boost survival and cytotoxicity (67). Also depicted is an example of an inducible suicide gene, tEGFR, which consists of the truncated transmembrane and extracellular portion of the EGFR protein. When targeted by the antibody Cetuximab, the receptor triggers apoptosis in the cell, providing a safety switch to protect against potential toxicity (68). Inducible caspase 9 (iC9) and HSV-TK are other common suicide genes that have been coexpressed with CARs.

Figure 2

T cell extravasation into the TME and subsequent exhaustion mediated by inhibitory ligands on tumor and tumor-associated cells. Endothelial cells experiencing inflammation express adhesion molecules including selectins, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule (ICAM-1). P- and E-Selectins (the latter shown in the figure) bind cutaneous lymphocyte antigen (CLA), a specially glycosylated form of P-selectin glycoprotein ligand 1 (PSGL-1) that is expressed on activated T cells (114). VCAM-1 binds very late antigen-1 (VLA-4) and ICAM-1 binds lymphocyte function-associated antigen-1 (LFA-1) (115). Upon binding endothelial cell ligands, T cells undergo tethering and rolling before adhering to the endothelium and transmigrating through it as shown. Once in the tumor microenvironment, T cells are in an environment full of tumor-associated, immunosuppressive cells including tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), myeloid-derived suppressor cells (MDSCs), T-regulatory cells (Tregs), and cancer-associated fibroblasts (CAFs) (95). These cells express inhibitory molecules, including CD80/CD86, which bind the inhibitory receptor CTLA-4 (pictured), and secrete soluble factors that suppress or cause apoptosis in T cells. CAFs also serve as a physical barrier between T cell and tumor cell. Additionally, tumor cells themselves express ligands such as Gal9 and PDL-1, which bind to the T cell inhibitory receptors TIM-3 and PD-1, respectively. All these factors serve to promote an “exhausted” phenotype in the T cell, characterized by upregulation of inhibitory receptors such as PD-1, TIM-3, TIGIT, and LAG-3, loss of CCR7, CD62L, and CD45R0, loss of cytotoxicity, and apoptosis (111).

Figure 3

Some inhibitory soluble factors and molecules secreted by tumor cells and tumor-associated cells such as MDSCs, TAMs, TANs, CAFs, and Tregs. High levels of lactate and an acidic environment are generated because of the tumor cells' preferential use of glycolysis, which impairs T cell function (111). The hypoxic environment also limits oxidative phosphorylation, a metabolic requirement for central memory T cells. High levels of reactive oxygen species (ROS) are generated by tumor cells and by induced mitochondrial dysfunction in T cells, which can be toxic to the cell. The soluble factors VEGF, TGFβ, indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), and adenosine are secreted by tumor and tumor-associated cells and can have damaging effects on T cells (109, 117). Adenosine enters the T cell through the receptor A2AR and stimulates production of cyclic AMP, which inhibits T cell proliferation, trafficking and cytotoxicity (118). PGE2 enters through the receptor EP4 and inhibits phosphorylation of STAT3, dampening proliferation, development of favorable memory phenotype, and cytotoxic function in T cells (119). Competition with glycolytic tumor cells for glucose results in downregulation of the glucose receptor GLUT1 because of decreased AKT/mTOR signaling and consequently, the T cell's metabolic capacities are further diminished (120).