Single residue in CD28-costimulated CAR-T cells limits long-term persistence and antitumor durability
Sonia Guedan, … , Carl H. June, Avery D. Posey Jr.
Sonia Guedan, … , Carl H. June, Avery D. Posey Jr.
Published February 18, 2020
Citation Information: J Clin Invest. 2020;130(6):3087-3097. https://doi.org/10.1172/JCI133215.
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Research Article Immunology

Single residue in CD28-costimulated CAR-T cells limits long-term persistence and antitumor durability

Abstract

Chimeric antigen receptor–T (CAR-T) cell therapies can eliminate relapsed and refractory tumors, but the durability of antitumor activity requires in vivo persistence. Differential signaling through the CAR costimulatory domain can alter the T cell metabolism, memory differentiation, and influence long-term persistence. CAR-T cells costimulated with 4-1BB or ICOS persist in xenograft models but those constructed with CD28 exhibit rapid clearance. Here, we show that a single amino acid residue in CD28 drove T cell exhaustion and hindered the persistence of CD28-based CAR-T cells and changing this asparagine to phenylalanine (CD28-YMFM) promoted durable antitumor control. In addition, CD28-YMFM CAR-T cells exhibited reduced T cell differentiation and exhaustion as well as increased skewing toward Th17 cells. Reciprocal modification of ICOS-containing CAR-T cells abolished in vivo persistence and antitumor activity. This finding suggests modifications to the costimulatory domains of CAR-T cells can enable longer persistence and thereby improve antitumor response.

Authors

Sonia Guedan, Aviv Madar, Victoria Casado-Medrano, Carolyn Shaw, Anna Wing, Fang Liu, Regina M. Young, Carl H. June, Avery D. Posey Jr.

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Figure 1

Short-term signaling through 28z-YMFM CAR closely resembles CD28 signaling.

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Short-term signaling through 28z-YMFM CAR closely resembles CD28 signali...
(A) Upper panel: Schematic representation of a panel of chimeric receptors that contain the SS1 scFv and differ in the intracellular domain. The CD28(YMFM)z mutant contains the CD28 transmembrane and intracellular domain with a point mutation in the YMNM motif (the asparagine [N] is mutated to phenylalanine [F]). The ICOS(YMNM)z mutant contains the ICOS transmembrane and intracellular domains with a point mutation in the YMFM motif [the phenylalanine [F] is mutated to an asparagine [N]). Lower panel: The full amino acid sequences for CD28 and ICOS intracellular domains and their corresponding mutants are shown. (B) Characterization of tonic signaling in CAR-T cells during primary expansion. Representative histograms showing the expression of the CAR protein and markers of activation, inhibition, and differentiation in CAR-T cells at 14 days after stimulation with anti-CD3/CD28 beads. (C) CAR-T cells were cocultured with mesothelin+ Capan-2 cells. Supernatants were obtained 24 hours later, and cytokine production was analyzed by Luminex. Representative of 2 donors. (D) A real-time cytotoxicity assay (xCELLigence) was used to evaluate the lysis of Capan-2 tumor cells when treated with CAR-T cells (E/T = 3:1) over a 120-hour period. (E) CAR-T cells from 2 different healthy donors were stimulated with immobilized recombinant mesothelin. Transcriptome analysis by RNA-sequencing was performed 6 days following antigen recognition. Scatter plots show the gene expression levels in 28z-YMFM or ICOSz CAR-T cells versus 28z CAR-T cells. The number of upregulated (orange) and downregulated (blue) genes is indicated.