CD28-CAR-T cell activation through FYN kinase signaling rather than LCK enhances therapeutic performance

Abstract

Signal transduction induced by chimeric antigen receptors (CARs) is generally believed to rely on the activity of the SRC family kinase (SFK) LCK, as is the case with T cell receptor (TCR) signaling. Here, we show that CAR signaling occurs in the absence of LCK. This LCK-independent signaling requires the related SFK FYN and a CD28 intracellular domain within the CAR. LCK-deficient CAR-T cells are strongly signaled through CAR and have better in vivo efficacy with reduced exhaustion phenotype and enhanced induction of memory and proliferation. These distinctions can be attributed to the fact that FYN signaling tends to promote proliferation and survival, whereas LCK signaling promotes strong signaling that tends to lead to exhaustion. This non-canonical signaling of CAR-T cells provides insight into the initiation of both TCR and CAR signaling and has important clinical implications for improvement of CAR function.

Keywords: CAR; CAR-T; CD28; FYN; LCK independent signaling; T cell receptor; allogeneic CAR-T; chimeric antigen receptor T cell; co-stimulation; signal transduction; specificity.

Graphical abstract

Figure 1

CAR-T cells can transduce T cell signaling without LCK (A) Top: western blot detection of LCK or FYN expression in CAR or TCR-Jcam1.6 cell. Bottom: CAR or TCR responsiveness in LCK-deficient Jcam1.6 cells. CHO-L2 is the artificial APC presenting the LMP2A_426-434 (CLGGLLTMV) (L2) target peptide on HLA-A2. CHO-GAG presents the irrelevant GAG (SLYNTVATL) peptide. (B) Phosphorylation of TCR signaling pathway molecules PLCγ1, ERK, and CD3ζ at different time points by LCK-deficient CD28CAR-Jcam cells. LCK-sufficient TCR-Jurkat cells were used as positive control. Phosphorylation of PLCγ1, ERK1/2, or CD3ζ was calculated relative to intensity of total PLCγ1, ERK, or ERK, respectively. (C) Schematic of LCK locus-targeted homologous directed repair (HDR) by CRISPR-Cas9 editing. The anti-CD19-CD28CAR construct has either a Myc or His tag. (D) CAR-His and CAR-Myc expression after LCK locus-targeted HDR. (E) Western blot detection of LCK expression after sorting of CAR-His⁺, CAR-Myc⁺, and CAR-DP (Myc⁺/His⁺) CD8⁺-T cells. Control represents mock-edited CD8⁺-T cells. (F) Cytotoxicity of CAR-His⁺, CAR-Myc⁺, and CAR-DP CD8⁺-T cells. Control group was the target cells with mock-edited CD8⁺-T cells. (G) Cytokine secretion of CAR-His⁺, CAR-Myc⁺, and CAR-DP CD8⁺-T cells upon activation. CAR-T cells were incubated with CD19-expressing Daudi cells at a 1:1 ratio for 18 h, then stained for intracellular TNF or IFNγ. The left panel is the representative fluorescence-activated cell sorting (FACS) data, and the right panel is the statistical summary and analysis. All data are representative of 3 independent experiments, plotted as mean ± SD of technical triplicates. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; not significant (NS) p > 0.05. (A)–(G) were analyzed by Student’s t test. See Figure S1 for additional data.

Figure 2

LCK-independent CAR signaling relies on CD28 co-stimulatory domain (A) The effects of recognition domain in disLCK-CAR-T cell signaling. Anti-CD19-CD28CAR construct has been replaced with anti-HER2 scFv. CHO-CD19- and HER2-expressing SKBR3 cells were used as target cells and mixed with its respective disLCK-CAR-T cells at a 1:1 ratio for 18 h. The top panel is the representative FACS data, and the bottom panel is the schematic of the constructs with different recognition domains and an analytical summary. (B) The effects of intracellular domain in disLCK-CAR-T cell signaling. The top panel is the representative FACS data, and the bottom panel is the schematic of the constructs with different intracellular domains and an analytical summary. (C) Cytokine secretion of disLCK-CAR-T cells with different CD28-CAR mutations. The top panel is the representative FACS data, and the bottom panel is the schematic of CD28-CAR mutations and an analytical summary. (D) Cytokine secretion of disLCK-CD137-CAR-T with or without CD80/CD86 co-stimulation on endogenous CD28. The top panel is the representative FACS data, and the bottom panel is the analytical summary. Data are representative of 3 independent experiments, plotted as mean ± SD of technical triplicates. p values denoted as in the Figure 1 legend by Student’s t test. See Figure S2 for additional data.

Figure 3

FYN is suggested to mediate CD28-CAR-T cell activation in the absence of LCK (A) The cytotoxicity of primary CAR-T and TCR-T cells with or without PP2 inhibitor (10 μM). CAR-T targets CD19, and TCR-T targets HLA/A2-L2. Daudi cells, which express CD19 and were transduced with HLA/A2-L2, were used as target cells. The CAR-T cells were mixed with target cells at an E:T ratio of 4:1. (B) Phosphorylation (pY420) of FYN in LCK-deficient CAR- or TCR-Jcam cells at different time points. APC stands for artificial antigen-presenting cell CHO-L2, to which the CAR and TCR responded. The number shown below indicates band intensity of pY420 relative to total FYN (FYN pY420 was stained by anti-pSRC family pY416). (C) LCK and FYN expression in LCK or FYN-KO Jurkat clone after CRISPR-Cas9 editing. C-CBL was used as loading control. (D) IL-2 production of TCR and CAR in LCK or FYN-KO Jurkat upon activation by CHO-L2. (E) Association of FYN with CD28CAR variants. Jcam cells with different CD28CAR variants were activated by CHO-L2 for 5 min, and anti-Myc antibody was used to immunoprecipitate CAR. The association strength (intensity ratio) was calculated by the relative intensity of FYN to that of immunoprecipitated CAR. (F) Correlation of FYN association strength with IL-2 production by Jcam cells with different CD28-CAR variants. The intensity ratio in abscissa is from (E), and the statistical significance is calculated by compare IL-2 production by CD28CAR variants with that of wild type (WT). The CAR and TCR mentioned above (B–E) both specifically target HLA/A2-L2, and CD28CAR was used. Data are representative of 3 independent experiments (except 2 for E), plotted as mean ± SD of technical triplicates. p values denoted as in the Figure 1 legend by Student’s t test. See Figure S3 for additional data.

Figure 4

disLCK-CAR-T cells selectively activate CAR signaling (A) TCR and CAR responsiveness selectivity in LCK-sufficient or -deficient Jurkat T cells. The top panel shows the schematics of E183 peptide-specific TCR (E183-TCR) or LMP2 peptide-specific CAR (L2-CAR) expressed in LCK-sufficient (1–3) or -deficient Jurkat T cells (4). The bottom panel shows the responsiveness of each group against CHO-E183 or CHO-L2, respectively. CHO-E183 is a mono-peptide CHO APC presenting only the E183 peptide. Similarly, CHO-L2 presents only the LMP2A peptide. (B) Schematics of activation of endogenous TCR in disLCK-CAR-T or conventional CAR-T cells. Anti-CD3 Ab was used to activate endogenous TCR. (C) Cytokine secretion of CAR-T cells upon anti-CD3 activation. The left panel is the representative data, and the right panel is the statistical summary and analysis. (D) CAR expression after anti-CD3 antibody activation. The left panel is the representative data, and the right panel is the statistical summary and analysis. Data are representative of 3 independent experiments, plotted as mean ± SD of technical triplicates. p values denoted as in the Figure 1 legend by Student’s t test. See Figure S4 for additional data.

Figure 5

LCK-disrupted CAR-T cells enhance in vivo therapeutic efficacy (A) Cytokine secretion of disLCK-CAR-T and conventional CAR-T (generated by lentivirus transduction) upon activation by target cells. The left panel is the representative FACS data, and the right panel is the analytical summary. (B) Comparison of cytotoxicity of disLCK-CAR-T and conventional CAR-T to CD19 expressing Daudi cells at a serial E:T ratio. (C) Real-time killing curve of disLCK-CAR and conventional CAR-T to CHO cells overexpressing CD19. Cell index represents the cell growth. Arrow points show when the CAR-T cells were added. The respective E:T ratio is indicated in parentheses. (D) Schematic of in vivo mouse model of liquid cancer or solid tumor. (E) Kaplan-Meier analysis of survival in standard leukemia cancer model (Nalm-6 cells and CAR-T cells were administered at 0.5 × 10⁶ and 3 × 10⁶ cells, respectively, log rank Mantel-Cox test). The CAR-T cells in left graph were generated as described in (A) (n = 3–4 mice per group) and were generated by lentivirus transduction. The right graph shows treatment with cells containing both shRNA (either non-targeting control [shNTC] or LCK targeting [shLCK]) and CAR expression cassette (n = 7 mice per CAR-T group, n = 4 for control group treated by PBS). (F) In vivo efficacy of anti-solid tumor mouse model. 2 × 10⁶ breast cancer cell line MCF7-HER2 cells were administered subcutaneously (s.c.) at day 0, and 8 × 10⁶ anti-HER2CAR-T cells were administered on day 5. Tumor size was measured and monitored over time (n = 5 mice per group). Data in (A)–(C) are representative of 3 independent experiments, plotted as mean ± SD of technical triplicates. p values denoted as in the Figure 1 legend using Student’s t test for (A) and two-way ANOVA for (B), (C), and (F). See Figure S5 for additional data.

Figure 6

disLCK-CAR-T cell activation by FYN leads to a more proliferative, memory-retaining, and exhaustion-reduced state (A) Signal pathway analysis of FYN and LCK group molecules. The 330 molecules linked to either FYN or LCK can be divided into FYN unique (blue, 58 molecules); FYN more-strongly linked (light blue, 11 molecules); shared linked (dark gray, 149 molecules); LCK more-strongly linked (orange, 10 molecules); and LCK unique (red, 102 molecules). FYN or LCK unique and strongly linked molecules are grouped in the FYN or LCK group, respectively, and analyzed in the KEGG signal pathway database. The pathways are ranked by the number of genes in the respective pathways. (B) CAR-T cell proliferation upon target cell activation. The proliferation event was detected by FACS after 5-day incubation with Nalm-6 cells. The left panel shows the representative graph, and the right panel is the statistical summary. (C) CAR, exhaustion, and memory marker expression in resting T cells (5 days after sorting and restimulation by feeder cells). (D) Radar chart summary of exhaustion, and memory marker expression in T cells after encountering target cells at different E:T ratios. Axes show the percentage of expression of each marker. (E) Memory subtypes of CAR-T cells after stimulation at different E:T ratios. T cells were stained with anti-CD45RA and anti-CD62L after 18-h incubation with Daudi cells at the corresponding E:T ratio. Data are representative of 3 (B) or 2 (C–E) independent experiments, plotted as mean ± SD of technical triplicates. p values denoted as in the Figure 1 legend calculated by Student’s t test. See Figures S6 and S7 for additional data.

Figure 7

disLCK-CAR-T cells have an enhanced therapeutic profile in vivo (A) Schematic of the ex vivo analysis of CAR-T cells after administration in vivo. (B) Bone marrow CAR-T cells were analyzed at day 11 or 18 after cancer cells injected, and percentage of memory T cells (CD45RO⁺) was detected by FACS. The left panel shows the representative FACS data, and the right panel shows the statistical summary. n = 4 mice per group, each dot = one mouse, CAR-T = blue square, disLCK-CAR-T = red round dot. (C) Exhaustion marker expression on the CAR-T cells from bone marrow analyzed on day 10. The left panel shows the representative FACS data, and the right panel shows the statistical summary. n = 4 mice per group, each dot = one mouse, CAR-T = blue square, disLCK-CAR-T = red round dot. Cells with 3, 2, or 1 exhaustion marker expression (TIM-3, LAG-3, and PD-1) are designated as exhaustion (+), and cells without exhaustion marker expression are designated as exhaustion (−). (D) Cell number of the CAR-T cells from bone marrow and spleen at days 10 and 16 after cancer cells were injected. 1.8 × 10⁶ CAR-T cells were administered in each mouse at day 4 after 1.5 × 10⁶ Nalm-6 cells were injected at day 0. n = 4 mice per group. (E) Principal-component analysis (PCA) of conventional CAR-T and disLCK-CAR-T cell in vivo performance. Each dot = one mouse, disLCK-CAR-T = red round dots, conventional CAR-T = blue square dots. (F) Heatmap of the in vivo performances of CAR-T cells after PCA. The columns are biological repeats. Each row represents PCA values of ex vivo data of each factor, memory is the CD45RO⁺ group, and exhaustion (−) and (+) are defined the same as in (C). Data in (B)–(D) are means ± SD. Boxes in (B) (right panels) represent 95% confidence intervals. p values denoted as in the Figure 1 legend using Student’s t test. See Figure S7F for additional data.