DIALing-up the preclinical characterization of gene-modified adoptive cellular immunotherapies

Abstract

The preclinical characterization of gene modified adoptive cellular immunotherapy candidates for clinical development often requires the use of mouse models. Gene-modified lymphocytes (GML) incorporating chimeric antigen receptors (CAR) and T-cell receptors (TCR) into immune effector cells require in vivo characterization of biological activity, mechanism of action, and preclinical safety. Typically, this characterization involves the assessment of dose-dependent, on-target, on-tumor activity in severely immunocompromised mice. While suitable for the purpose of evaluating T cell-expressed transgene function in a living host, this approach falls short in translating cellular therapy efficacy, safety, and persistence from preclinical models to humans. To comprehensively characterize cell therapy products in mice, we have developed a framework called "DIAL". This framework aims to enable an end-to-end understanding of genetically engineered cellular immunotherapies in vivo, from infusion to tumor clearance and long-term immunosurveillance. The acronym DIAL stands for Distribution, Infiltration, Accumulation, and Longevity, compartmentalizing the systemic attributes of gene-modified cellular therapy and providing a platform for optimization with the ultimate goal of improving therapeutic efficacy. This review will discuss both existent and emerging examples of DIAL characterization in mouse models, as well as opportunities for future development and optimization.

Keywords: CAR-T cells; adoptive cell therapy; animal models; immunotherapy; tumor microenvironment.

Figure 1

The DIAL framework. Distribution, infiltration, accumulation, and longevity are classified as key attributes and areas to focus on for enhancing cell therapy products. Examples of interventions are provided for each attribute and discussed in the text.

Figure 2

Distribution. (A) T cells isolated from patient’s peripheral blood or mouse lymphoid organs are engineered to express a CAR. CAR-T cells are expanded ex vivo and infused back into the patient or in tumor-bearing mice. Upon infusion, CAR-T cells distribute through the body, migrate to the tumor, and kill target-expressing tumor cells. (B) delivery of mRNA-encoding CAR ligands using lipid nanoparticles (LNPs) to lymph nodes results in expression of the target by APC. Target-expressing APC prime and activate CAR-T cells, allowing for expansion and migration to the tumor.

Figure 3

Infiltration. (A) Chemokine receptors can be engineered into CAR-T cells, enhancing the sensitivity to chemokine gradients and ultimately migration and infiltration to the tumor site. (B) Extracellular matrix-(ECM) rich tumor types physically restrict lymphocytic infiltration, creating an immune-excluded TME. Use of combination agents that directly or indirectly target tumor fibrosis may facilitate GML infiltration into the TME.

Figure 4

Accumulation. (A) The TME is rich of immunosuppressive factors that can inhibit CAR-T activation, proliferation, and accumulation even in presence of target antigen. (B) CAR-T can be engineered to alleviate distinct challenges of the TME. Armoring strategies can shield CAR-T cell, decreasing their sensitivity to inhibiting factors (TGFβ, PD-L1, adenosine), or can endow them with the ability to target immunosuppressive cells (anti-MDSCs).

Figure 5

Longevity. (A) Cytokines employed for CAR-T ex vivo expansion can affect their differentiation and ability to self-renew and persist in the host upon infusion. Longevity is measured by quantification of CAR-T in peripheral blood, and results in durable remission. Persistence of human CAR-T in immunocompromised mice is complicated by potential GvHD and lack of homeostatic cytokines. (B) Engineering of cytokines enhancing CAR-T cell stemness and fitness directly or indirectly improves endogenous T cell recruitment. CRISPR KO of intracellular checkpoint inhibitors or combination with CPI blocking antibodies can further improve CAR-T persistence.