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Restoration of LAT activity improves CAR T-cell sensitivity and persistence in response to antigen-low acute lymphoblastic leukemia

ABSTRACT: Chimeric antigen receptor (CAR) T-cells induce responses in patients with relapsed/refractory leukemia; however, long-term efficacy is frequently limited by post-CAR relapses. The inability to target antigen-low cells is an intrinsic vulnerability of second-generation CAR T-cells and underlies the majority of relapses following CD22BBz CAR T-cell therapy. We interrogated CD22BBz CAR signaling in response to low antigen and found inefficient phosphorylation of LAT, limiting downstream signaling. To overcome this, we designed the Adjunctive LAT-Activating CAR T-cell (ALA-CART) platform, pairing a second-generation CAR with a LAT-CAR incorporating the intracellular domain of LAT. ALA-CART cells demonstrated reduced differentiation during manufacturing and increased LAT phosphorylation, MAPK signaling and AP-1 activity. Consequently, ALA-CART cells showed improved cytotoxicity, proliferation, persistence and efficacy against antigen-low leukemias that were refractory to clinically-active CD22BBz CAR T-cells. These data suggest restoration of LAT signaling through the ALA-CART platform represents a promising strategy for overcoming multiple mechanisms of CAR T-cell failure.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell, Jurkat Cell

DISEASE(S): Acute Lymphocytic Leukemia

SUBMITTER: Christopher Ebmeier

LAB HEAD: Mark Eric Kohler MD PhD

PROVIDER: PXD058699 | Pride | 2025-05-02

REPOSITORIES: Pride

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			Action	DRS
	CCE20211221_Cathy_Danis_EricKoehlerLab_Phospho1_f1_hfx1.raw	Raw
	CCE20211221_Cathy_Danis_EricKoehlerLab_Phospho1_f1_hfx2.raw	Raw
	CCE20211221_Cathy_Danis_EricKoehlerLab_Phospho1_f2_hfx1.raw	Raw
	CCE20211221_Cathy_Danis_EricKoehlerLab_Phospho1_f2_hfx2.raw	Raw
	CCE20211221_Cathy_Danis_EricKoehlerLab_Phospho1_f3_hfx1.raw	Raw

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Project description:Chimeric antigen receptor (CAR) T cell therapy have demonstrated remarkable success in treating B-cell malignancies that are refractory to standard therapies; however, preclinical and clinical studies have demonstrated that CAR T cell efficacy is greatly reduced against antigen-low tumors. This was exemplified in a clinical trial of CD22-directed CAR T cells where post-CAR relapses were driven by a decrease in the surface expression of the CD22 antigen. To address the poor response of CAR T cells to antigen-low tumor cells, we performed global phosphoproteomics using a SILAC/mass spectrometry approach to examine signal transduction events within CAR T cells in response to high- and low-levels of CD22 antigen. Stimulation with low levels of CD22 antigen resulted in decreased activation of several canonical T cell signaling pathways in CAR T cells, suggesting poor utilization of LAT. To overcome this inefficiency of LAT activation, we designed a bicistronic construct consisting of a clinically active 2nd generation CD22-BBz CAR along with a novel Adjunctive LAT Activating CAR incorporating the intracellular signaling domain of LAT (ALA-CART). ALA-CART cells demonstrated enhanced phosphorylation of LAT and restored downstream signaling in response to low levels of CD22. In xenograft models, ALA-CART cells completely eradicated CD22-low leukemia, significantly extending survival of mice in comparison to the clinically-active, standard CD22-BBz CAR T cells. Compared to the standard CD22-BBz CAR T cells, ALA-CART cells exhibited transcriptional differences in the resting state reflecting a less differentiated state, which corresponded to an enrichment of T stem cell memory cells and enhanced in vivo persistence. Thus, through the identification of CAR signaling deficits, we rationally designed the ALA-CART platform which improves the sensitivity and persistence of CAR T cells to target antigen-low cells, overcoming a mechanism of resistance to standard CAR therapies.

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Restoration of LAT activity improves CAR T-cell sensitivity and persistence in response to antigen-low acute lymphoblastic leukemia

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