All Chimeric Antigen Receptors (CARs) are not made equal, especially when it comes to their antigen sensitivity. That was the message imparted by Dr. Michel Sadelain in his talk “Emerging principles to overcome tumor antigen escape” at the recent AACR 2022 meeting in New Orleans. Stating that CAR T cells have different sensitivities for their targeted antigens, for example, with CD8 CARs being usually more sensitive than 4-1BB CARs, Dr. Sadelain explained how low antigen density and inadequate CAR sensitivity are associated with CAR antigen-escape and tumor relapse.
Engineered T cell receptors with improved sensitivity. Michel Sadelain, Memorial Sloan Kettering Cancer Center, NY, at AACR Annual Meeting 2022, Adoptive Cellular Therapy: Treatment for the Masses.
CAR T cell therapy has been highly efficacious for refractory blood malignancies, such as acute lymphoblastic leukemia (ALL), often providing long-lasting responses. Nevertheless, albeit the high efficacy of CAR T cells, such as those targeting CD19 in B cell malignancies which may result in 70 to 90% durable responses, relapses do occur (Sterner and Sterner, 2021).
One process known to underscore disease recurrence is the reduced or complete loss of the therapeutic target in cancer cells following treatment with single-antigen targeting CAR T cells. For example, CAR antigen-escape occurs following treatments targeting CD19 or BCMA. A similar phenomenon occurs in solid tumors following CAR T cell therapy targeting IL13Ra2 (Sterner and Sterner, 2021).
CAR antigen-escape may occur through various mechanisms, including antigen mutation or alternative splicing, epitope masking, low antigen density, and linage switch. As shared by Sadelain, several approaches may be leveraged to cope with reduced antigen density within a tumor, including the use of dual specific CARs or multiple CARs with different signaling properties. Else, co-expression of molecules that assist CARs, such as chimeric co-stimulatory receptors, may help improve T cell low-antigen recognition.
“Resistance mechanisms associated with antigen loss following CD19 CAR T-cell therapy. (A) CAR T-cells bind with tumor-associated antigens and represent antitumor activity. (B) Due to CD19 CAR mutations or alternative splicing, CAR T-cells cannot bind the antigens and therefore, cancer cells may be resistant to therapy. (C) Due to CD19 CAR binding to CD19 antigen and subsequent masking of the CD19 epitope, CAR T-cells cannot attack the cancer cells. (D) Down-regulated antigen density prevents cancer cells against CD19 CAR T-cell therapy. (E) The surface antigen is changed from lymphoid to myeloid lineage, which prevents CD19 CAR T-cells from binding to the cancer cells.” Retrieved without modifications from Song et al. 2019. https://creativecommons.org/licenses/by/4.0/
Dr. Sadelain’s team has compared antigen sensitivity thresholds for T cells engineered using CRISPR/Cas9-based tools and AAV vector gene delivery. Specifically, that of T cells modified by targeting the T-cell receptor α constant chain (TRAC) locus, resulting in TRAC-CARs and the newly developed HLA Independent T cell receptor (TRAC-HIT) or HIT T cells (Eyquem et al. 20217, Mansilla-Soto et al. 2022). Both TRAC-CAR (CD3-independent) and TRAC-HIT (CD3-dependent) receptors re-direct T cell receptor antigen specificity and support HLA independent antigen binding.
The team found that among T cells engineered to detect CD19, through either TRAC-HIT or TRAC-CAR T receptors, HIT T cells were more sensitive, effectively detecting and targeting cells in vitro expressing lower levels of CD19, even as low as ~20 CD19 molecules. In contrast,TRAC-CAR T cells had a higher threshold for detecting and inducing cell lysis, not being able to target effectively cells expressing below ~200 CD19 molecules.
In association with HIT T cells’ increased sensitivity and lysis activity under conditions of low antigen expression, Sadelain’s group also found a greater cytokine response in these cells than in TRAC-CAR T cells. Together, these findings suggested that HIT T receptors may support more robust signaling upon activation by low antigen levels, which they demonstrated by increased phosphorylation of CD3ζ and various downstream targets. Significantly, in vivo responses on tumor burden mirrored the in vitro findings, with TRAC-HIT T cells outperforming CAR T cells, including those with CD28-based costimulatory domain, known to be more sensitive.
Overall, these findings may provide new options for solid tumor therapies. Solid tumors are notoriously difficult to treat with CAR T cells due to several challenges, such as their immunosuppressive microenvironment, limited tumor-specific target options with many being intracellular proteins, tumor antigen heterogeneity, and reduced antigen density (Titov et al. 2020). T cells engineered with more sensitive receptors would provide an advantage against solid tumors with limited target densities.
Advancements in CRISPR/Cas9 tools have made it possible to modify T cells with outstanding specificity. Additionally, new approaches to editing T cells following fully non-viral CRISPR/Cas-based methods continue to evolve, providing opportunities for more efficient gene insertions and safer T cell medicines.
