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 3

Signaling through a CD28-based CAR containing the ICOS YMFM motif shows enhanced AKT phosphorylation with reduced p-PLCγ, p-VAV, and calcium flux.

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Signaling through a CD28-based CAR containing the ICOS YMFM motif shows ...
(A–E) CAR-T cells were stimulated with magnetic beads coated with recombinant mesothelin. Cell lysates were obtained at 5 and 10 minutes following antigen encounter and phosphorylation levels for VAV, PLCγ, ERK, and AKT were analyzed by Western blot (A and D) and densitometry (B and E). Basal phosphorylation was evaluated without stimulation (minute 0). (C) T cells from 2 to 5 different healthy donors were stimulated as in A–E, and AKT, VAV, and PLCγ phosphorylation was analyzed by densitometry. The mean ± SD from 4 independent experiments is shown. *P < 0.05; **P < 0.01 by 1-way ANOVA with Tukey’s post hoc test. (F) Calcium influx was measured in CAR-T cells at baseline for 30 seconds, and then after stimulation with mesothelin-coated magnetic beads for 6 minutes, followed by stimulation with biotinylated OKT3 and avidin for 6 minutes, and treated with ionomycin. The mean Indo-1 ratio of violet/blue fluorescence emission is displayed on the y axis and the time of sample collection in seconds is displayed on the x axis. Representative of 3 different experiments using 3 different normal donors.