Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation

Abstract

Successful tumor eradication by chimeric antigen receptor-expressing (CAR-expressing) T lymphocytes depends on CAR T cell persistence and effector function. We hypothesized that CD4+ and CD8+ T cells may exhibit distinct persistence and effector phenotypes, depending on the identity of specific intracellular signaling domains (ICDs) used to generate the CAR. First, we demonstrate that the ICOS ICD dramatically enhanced the in vivo persistence of CAR-expressing CD4+ T cells that, in turn, increased the persistence of CD8+ T cells expressing either CD28- or 4-1BB-based CARs. These data indicate that persistence of CD8+ T cells was highly dependent on a helper effect provided by the ICD used to redirect CD4+ T cells. Second, we discovered that combining ICOS and 4-1BB ICDs in a third-generation CAR displayed superior antitumor effects and increased persistence in vivo. Interestingly, we found that the membrane-proximal ICD displayed a dominant effect over the distal domain in third-generation CARs. The optimal antitumor and persistence benefits observed in third-generation ICOSBBz CAR T cells required the ICOS ICD to be positioned proximal to the cell membrane and linked to the ICOS transmembrane domain. Thus, CARs with ICOS and 4-1BB ICD demonstrate increased efficacy in solid tumor models over our current 4-1BB-based CAR and are promising therapeutics for clinical testing.

Keywords: Immunology; Immunotherapy.

Figure 1. In vitro characterization of CD4⁺ and CD8⁺ T cells redirected with SS1-CARs.

(A) Schematic representation of chimeric receptors that contain the SS1 single chain fragment that binds to mesothelin and differ in the transmembrane and intracellular domains. (B) Surface expression of the SS1-CARs on human CD4⁺ T cells at the end of the primary expansion. Representative of 3 donors. (C) A real-time, impedance-based cytotoxicity assay (xCelligence) was used to evaluate the lysis of non–small cell lung L55 tumor cells when treated with CAR T cells at 1:1 E:T ratio over a 20-hour period. Representative of 2 donors. (D) CD4⁺ CAR T cells were cocultured with pancreatic cancer cells (Capan-2) that express mesothelin. Supernatants were obtained 24 hours after coculture, and cytokine production was analyzed by Luminex. Representative of 3 different experiments performed under similar conditions. (E) Ratios of cytokine expression for 28z and ICOSz were calculated using results from C. (F) CD4⁺ CAR T cells from different donors (n = 3–6) were cocultured with APC cells modified to express mesothelin. Supernatants were obtained 24 hours after coculture, and cytokine production was analyzed by ELISA. Ratios of cytokine expression for 28z and ICOSz are shown. Box plots show median (line) and 25th to 75th percentile (box). The end of the whiskers represents the minimum and the maximum of all of the data. *P < 0.05 by t-test. (G) CD4⁺ CAR T cells were cocultured with Capan-2 or immobilized mesothelin. Supernatants were obtained 24 hours after coculture, and IL-6 production was analyzed by Luminex. Representative of 3 donors (H) CD4⁺ CAR T cells were stimulated with magnetic beads coated with recombinant mesothelin. Cell lysates were obtained at different time points (5, 10, 30 and 60 min) and phosphorylation levels for AKT and ERK were analyzed by Western Blot and densitometry. Basal phosphorylation was evaluated without stimulation (minute 0). Representative of 3 donors.

Figure 2. Helper effect of CD4⁺ CAR T cells expressing an ICOS-based CAR significantly increased the persistence of coinjected CD8⁺ CAR T cells expressing 4-1BB– or CD28-based CARs.

(A and B) NSG mice bearing s.c. L55 tumor cells were treated 30 days after tumor implantation with 2 doses of CD4⁺ and CD8⁺ T cells redirected with SS1-CARs. The concentration of (A) CD4⁺ T cells and (B) CD8⁺ T cells was determined in the blood of treated animals 22 days after T cell injection. Error bars represent ± SEM (n = 7–10). *P < 0.05; **P < 0.01, and ***P < 0.001 by 1-way ANOVA with Tukey post hoc test. (C and D) NSG mice bearing s.c. ovarian tumors (SKOV3) were treated 30 days after tumor implantation with 2 doses of CD4⁺ and CD8⁺ T cells redirected with anti-folate receptor α Mov19-CARs. CD8⁺ T cells were modified to express BBz, and CD4⁺ T cells were left untransduced or redirected with Z, 28z, BBz, or ICOSz signaling domains. (C) The concentration of CD4⁺ and CD8⁺ T cells were determined in the blood of treated animals 27 days after T cell injection. Error bars represent ± SEM (n = 6–8). T cell count data was transformed to reduce variance, and significance was analyzed by 1-way ANOVA with Tukey post hoc test. (D) Tumor volume was analyzed at indicated time points; legend indicates the signaling domain in the CD4⁺ and CD8⁺ T cells. Results are expressed as a mean tumor volume (± SEM) with n = 6–8 mice per group.

Figure 3. Third-generation CARs containing ICOS and 4-1BB intracellular domains mediate enhanced antitumor effects and increased T cell persistence.

(A) NSG mice bearing s.c. pancreatic tumors (Capan-2) were treated 15 days after tumor implantation with 2 doses of T cells redirected with SS1-CARs. A group containing a combination of CD4⁺ T cells redirected with ICOSz and CD8⁺ T cells redirected with BBz (ICOSz⁴-BBz⁸) was also included. Tumor volume was analyzed at indicated time points. Results are expressed as a mean tumor volume (± SEM) with n = 6–8 mice per group. *P < 0.05 by 2-way ANOVA with Tukey’s multiple comparison test. (B) Waterfall plots of the change in tumor volume on day 34 versus baseline for individual animals. (C) The concentration of CD4⁺ and CD8⁺ T cells were determined in the blood of treated animals 21 days after T cell injection. Error bars represent ± SEM. *P < 0.05 by 1-way ANOVA with Tukey post hoc test. ***P < 0.001. For CD8⁺ T cell counts, data was transformed to reduce variance, and significance was analyzed by 1-way ANOVA with Tukey post hoc test. (D) Correlation of numbers of CD4⁺ and CD8⁺ T cells per microliter of blood was plotted versus the tumor volume on day 21 for the animals treated with ICOSz⁴-BBz⁸. (E and F) NSG mice were sacrificed on day 34 after treatment, and human T cells were isolated from mouse spleens. (E) CAR expression was analyzed by flow cytometry. Representative of 3–5 animals. (F) CAR T cells recovered from spleen were cocultured with K562 that express mesothelin, and cytokine release was analyzed by ELISA 24 hours after coculture. Box plots show median (line) and 25th to 75th percentile (box). The end of the whiskers represents the minimum and the maximum of all of the data. *P < 0.05 by 1-way ANOVA with Tukey post hoc test.

Figure 4. Levels of surface CAR expression influence antitumor effects.

(A and B) T cells were transduced with indicated MOI with lentiviral vectors encoding CAR proteins. CAR expression (A) and mean cell volume (B) was analyzed on day 13 after T cell transduction by flow cytometry. (C) Surface expression of the CAR proteins on human T cells at the time of functional evaluation. Transduction efficiencies are indicated with MFI of the transduced populations in parentheses. (D) Fold-change CAR expression (MFI) in pGK300-BBz and EF-1α-BBz relative to EF-1α-ICOSBBz was analyzed in 3 donors. **P < 0.01 by 1-way ANOVA with Tukey post hoc test. (E) T cell volume during ex vivo expansion in the absence of cognate antigen. Results are expressed as the mean T cell volume (± SD) with n = 2 donors. (F) Representative histograms showing the expression of activation, differentiation, and exhaustion markers in CAR T cell 14 days after stimulation with anti-CD3/CD28 beads. Representative of 2 donors. (G) NSG mice bearing s.c. Capan-2 pancreatic tumors were treated 20 days after tumor implantation with 2 doses of UTD or CAR T cells with the indicated promoter and signaling domain. Tumor volume was analyzed at indicated time points. Results are expressed as the mean tumor volume (± SEM) with n = 7–10 mice per group. Statistical significance: *P < 0.05 by 2-way ANOVA with Tukey’s multiple comparison test.

Figure 5. ICOSBBz enhanced in vivo functions are only observed when ICOS is proximal to the cell membrane.

(A and B) CAR T cells obtained from 3 different healthy donors were cocultured with APC cells transduced with mesothelin (K562meso). (A) Supernatants were obtained 24 hours later, and cytokine production was analyzed by Luminex. Floating bars show mean (line) and the minimum and maximum values (box). *P < 0.05, **P < 0.01, and ***P < 0.001 by 1-way ANOVA with Tukey post hoc test. (B) Proliferation was assessed by determining the total number of live CAR T cells within the coculture flow cytometry using counting beads 8 days after coculture. Total cell count at the end of the experiment was normalized to the total cell count at day 0. (C and D) NSG mice bearing s.c. pancreatic tumors (Capan-2) were treated 20 days after tumor implantation with 2 doses of UTD or CART cells (n = 7–10 mice per group). (C) Kaplan–Meier survival curves are plotted. The predetermined end point was established when tumor volume exceeded 1,000 mm³. **P < 0.01 by a log rank (Mantel-Cox) test. (D) The ratio of CD4⁺ and CD8⁺ cells within the human CD45⁺ T cell population in the peripheral blood of treated animals was analyzed on day 44 by flow cytometry (n = 7–10 mice per group). ***P < 0.001 by 1-way ANOVA with Tukey post hoc test.

Figure 6. ICOS transmembrane domain is necessary for improved antitumor effect and increased persistence.

(A) Surface expression of the CAR proteins on human T cells at the time of functional evaluation. ICOSBBz has an ICOS transmembrane region and TM CD8–ICOSBBz and BBICOSz have a CD8α transmembrane domain. Transduction efficiencies are indicated with MFI of the transduced populations in parentheses. (B) Fold-change CAR expression (MFI) in CD8-ICOSBBz and BBICOSz relative to ICOSBBz was analyzed in 3 normal donors. *P < 0.05 by 1-way ANOVA with Tukey post hoc test. (C) T cell volume during ex vivo expansion in the absence of cognate antigen. Representative of 2–4 donors (D) Representative histograms showing the expression of activation, differentiation, and exhaustion markers in CAR T cells 13 days after stimulation with anti-CD3/CD28 beads. Representative of 2 donors. (E) CAR T cells were cocultured with APC cells transduced with mesothelin (K562meso). Supernatants were obtained 24 hours later, and cytokine production was analyzed by Luminex. (F and G) NSG mice bearing s.c. Capan-2 pancreatic tumors were treated 36 days after tumor implantation with 2 doses of UTD or CAR T cells. (F) Tumor volume was analyzed at indicated time points. Results are expressed as a mean tumor volume (± SEM) with n = 5–7 mice per group. *P < 0.05, **P < 0.01 by 2-way repeated-measure ANOVA. (G) The concentration of CD4⁺ T cells and CD8⁺ T cells was determined in the blood of treated animals 21 days after T cell injection. Error bars represent ± SEM (n = 5–7). To analyze differences among groups, T cell count data was transformed to reduce variance, and significance was analyzed by 1-way ANOVA with Tukey post hoc test; *P < 0.05.