Calibrated CAR Activation Potential Directs Alternative T Cell Fates and Therapeutic Potency

Immunotherapy with second-generation chimeric antigen receptor (CAR) T cells has achieved great clinical success against hematological malignancies. CD19-specific CARs incorporating CD28 and CD3z signaling domains have demonstrated remarkable potency resulting in the frequent induction of complete remissions and were recently approved by the US FDA for use in some refractory B cell malignancies. We previously demonstrated that CD28-based CARs program strong effector functions, but impart a relatively limited T cell lifespan. Increasing functional T cell persistence is therefore likely to further enhance the therapeutic success of 1928z CAR T cells.

We hypothesized that excessive signal strength arising from redundancy of combined CD3z and CD28 signals might foster terminal T cell differentiation and exhaustion. We therefore proceeded to titrate the activation potential of CD28-based CARs and assess the impact thereof on the function, longevity and therapeutic potency of CAR T cells.

We analyzed the contribution of individual immunoreceptor tyrosine-based activation motifs (ITAMs) to the phenotype and function of 1928z CAR T cells. ITAM-mutated CAR T cells demonstrated similar expression levels in retrovirally transduced primary T cells and directed comparable short-term cytotoxicity and proliferation capacity in vitro. However, remarkable differences in their therapeutic potency emerged when T cells expressing different mutant CARs were tested in the pre-B acute lymphoblastic leukemia NALM6 mouse model. The CAR "stress test" model revealed that a CAR containing a single functional ITAM - depending on its position - outperformed wild-type 1928z CARs, achieving rapid and durable tumor eradication even at low T cell doses, by delaying T cell differentiation and exhaustion. CAR T cells retrieved from the bone marrow of treated mice demonstrated that inactivation of two ITAM domains augmented CAR persistence at the tumor sites with a higher percentage of central memory cells and a decreased proportion of terminally differentiated effector cells. Deletion mutants further revealed the importance of ITAM location within second-generation CAR T cells.

These findings were rigorously tested by directing the mutant CAR cDNAs to the T-cell receptor α constant (TRAC) locus using CRISPR/Cas9 technology, thereby ruling out potentially confounding effects arising from different CAR expression levels. TRAC-1928z mutants demonstrated superior antitumor efficacy compared to conventional TRAC-1928z CARs and prevented terminal T cell differentiation and exhaustion.

Genome-wide transcriptional profiles of TRAC-edited naïve peripheral blood T cells further demonstrated that CARs encoding different ITAMs direct T cells to different fates. While TRAC-1928z CARs demonstrated similarity to transcriptional profiles of effector cells, reducing the number of ITAMs to one ITAM preserved a less-differentiated T cell state and promoted greater T cell persistence. We identified one 1928z mutant CAR, which improved therapeutic potency and induced a transcriptional profile similar to that of stem cell memory T cells (T_SCM). Another 1928z mutant CAR with further reduction of the activation potential resulted in a naïve-like phenotype with great proliferation potential and persistence, but greatly diminished anti-tumor efficacy.

In conclusion, we demonstrate that the number and position of ITAMs in 1928z CAR T cells influence functional, phenotypic and transcriptional programs resulting in profound effects on therapeutic potency. Balancing T cell differentiation and acquisition of effector functions is essential to optimize therapeutic potency of CAR T cells and can be intrinsically regulated by defined mutations in the CD3z chain of 1928z CAR T cells.

Improved therapeutic potency of CAR T cells can thus be achieved by calibrating activation strength, thus retaining memory functions and preventing exhaustion, without compromising effector functions. Importantly, we were able to identify a novel CAR design which programs a favorable balance of effector and memory signatures, inducing increased persistence of highly functional CARs with the replicative capacity of long-lived memory cells and potent effector functions. Clinical studies evaluating the new CAR design are in preparation.