Session: 614. Acute Lymphoblastic Leukemia: Therapy, excluding Transplantation: Poster I
Hematology Disease Topics & Pathways:
Biological, Therapies, CAR-Ts, Biological Processes, Technology and Procedures, gene editing, Clinically relevant, immune mechanism
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 (TSCM). 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.
Disclosures: Sadelain: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Fate Therapeutics Inc.: Research Funding.
See more of: Oral and Poster Abstracts