Chimeric antigen receptor (CAR)-engineered lymphocytes for cancer therapy

Abstract

Introduction: Chimeric antigen receptors (CARs) usually combine the antigen binding site of a monoclonal antibody with the signal activating machinery of a T cell, freeing antigen recognition from MHC restriction and thus breaking one of the barriers to more widespread application of cellular therapy. Similar to treatment strategies employing monoclonal antibodies, T cells expressing CARs are highly targeted, but additionally offer the potential benefits of active trafficking to tumor sites, in vivo expansion and long-term persistence. Furthermore, gene transfer allows the introduction of countermeasures to tumor immune evasion and of safety mechanisms.

Areas covered: The basic structure of so-called first and later generation CARs and their potential advantages over other immune therapy systems. How these molecules can be grafted into immune cells (including retroviral and non-retroviral transduction methods) and strategies to improve the in vivo persistence and function of immune cells expressing CARs. Examples of tumor-associated antigens that have been targeted in preclinical models and clinical experience with these modified cells. Safety issues surrounding CAR gene transfer into T cells and potential solutions to them.

Expert opinion: Because of recent advances in immunology, genetics and cell processing, CAR-modified T cells will likely play an increasing role in the cellular therapy of cancer, chronic infections and autoimmune disorders.

Figure 1. The basic structure of a monoclonal antibody (mAb)-derived chimeric antigen receptor (CAR)

The most common CARs combine the extracellular antigen recognition site of a mAb and the intracellular domains of a T cell receptor complex (TCR) molecule, such as the ζ-chain. Clustering of CARs induced by antigen binding is thought to be responsible for initiating signal transduction that leads to T-cell activation and killing of the cells expressing the target antigen.

Figure 2. First versus second generation CARs

First generation CARs include a single stimulatory domain. Because most tumors do not express costimulatory molecules (“signal 2”), T cells are incompletely activated even when the CAR is engaged by the target antigen. Second generation CARs contain one additional costimulatory endodomain, which is thought to improve T cell activation and proliferation, and thus promote better killing of the target tumor cells.

Figure 3. Retroviral transduction of T cells and expansion of CAR-expressing T cells

T cells are activated from the patient’s peripheral blood mononuclear cells (PBMC) by stimulation with anti-CD3 antibody (OKT3) and these activated T cells are expanded with interleukin 2 (IL-2) and transduced with a replication incompetent retrovirus encoding the CAR. Further expansion in IL-2-containing medium is done until sufficient numbers for clinical application are reached.

Figure 4. Epstein-Barr Virus (EBV) and tumor bispecific cytotoxic T lymphocytes (CTLs)

Unlike most tumor cells (including malignant lymphocytes), EBV-transformed cells not only express specific antigens but also high levels of many different costimulatory molecules, including CD40L, CD80 and CD86. Because they express both class I and class II human leukocyte antigen (HLA) molecules, they can present viral epitopes that will stimulate both CD8 and CD4 virus-specific cells, favoring the cognate interactions between T cell subsets that are critical for optimal and sustained immune responses. CTLs with native receptor (αβ-TCR) specificity directed to EBV receive physiological costimulation in vivo during their encounters with persistent viral antigens on professional antigen presenting cells. If these EBV-specific CTLs are modified to co-express a CAR directed to a tumor associated antigen, they will obtain physiologic costimulatory signals following engagement of their native receptors, which in turn will enhance their survival and their CAR-mediated anti-tumor activity.