Targeting and cytotoxicity of chimeric antigen receptor T cells grafted with PD1 extramembrane domain

Abstract

Background: Immunosuppression induced by programmed cell death protein 1 (PD1) presents a significant constraint on the effectiveness of chimeric antigen receptor (CAR)-T therapy. The potential of combining PD1/PDL1 (Programmed cell death 1 ligand 1) axis blockade with CAR-T cell therapy is promising. However, developing a highly efficient and minimally toxic approach requires further exploration. Our attempt to devise a novel CAR structure capable of recognizing both tumor antigens and PDL1 encountered challenges since direct targeting of PDL1 resulted in systemic adverse effects.

Methods: In this research, we innovatively engineered novel CARs by grafting the PD1 domain into a conventional second-generation (2G) CAR specifically targeting CD19. These CARs exist in two distinct forms: one with PD1 extramembrane domain (EMD) directly linked to a transmembrane domain (TMD), referred to as PE CAR, and the other with PD1 EMD connected to a TMD via a CD8 hinge domain (HD), known as PE8HT CAR. To evaluate their efficacy, we conducted comprehensive assessments of their cytotoxicity, cytokine release, and potential off-target effects both in vitro and in vivo using tumor models that overexpress CD19/PDL1.

Results: The findings of our study indicate that PE CAR demonstrates enhanced cytotoxicity and reduced cytokine release specifically towards CD19 + PDL1 + tumor cells, without off-target effects to CD19-PDL1 + tumor cells, in contrast to 2G CAR-T cells. Additionally, PE CAR showed ameliorative differentiation, exhaustion, and apoptosis phenotypes as assessed by flow cytometry, RNA-sequencing, and metabolic parameter analysis, after encountering CD19 + PDL1 + tumor cells.

Conclusion: Our results revealed that CAR grafted with PD1 exhibits enhanced antitumor activity with lower cytokine release and no PD1-related off-target toxicity in tumor models that overexpress CD19 and PDL1. These findings suggest that our CAR design holds the potential for effectively addressing the PD1 signal.

Keywords: Chimeric antigen receptor T cell; Hinge domain; Off-target toxicity; PD1; Tumor microenvironment.

Fig. 1

CAR grafted with PD1 with or without CD8 hinge exhibited different function to PDL1. A. Schematic representation of four CAR variants with different domains: 2G, PD1-S28, PE8HT, and PEPT CAR, showing variations in the hinge, extramembrane, and transmembrane domains. B. Cytotoxic percentages of targeted cells by mock-T and 2G, PD1-S28, PE8HT, and PEPT CAR-T cells were measured after 12–16 h of coculture in vitro. The E:T ratios (2.5:1 and 5:1) represent the ratios of the absolute number of CAR-T cells to target cells. The following cell types were used: CD19-PDL1- cells (K562), CD19 + PDL1- cells (K562-CD19 and NALM-6), CD19 + PDL1 + cells (K562-CD19-PDL1 and NALM-6-PDL1), and CD19-PDL1 + cells (K562-PDL1 and 786o). The number of mock-T cells was the same as in the 2G group. The results shown are representative of at least three independent experiments using T cells from different healthy donors. C. Proliferation of different effector cells was assessed after stimulation with CD19-PDL1+ (786o) and CD19 + PDL1+ (786o-CD19) cell lines. All effector cells were stained with CFSE. Proliferation was analyzed by flow cytometry by measuring CFSE dilution. The red, green, orange, and blue peaks represent cells before stimulation, cells without any additional stimulation after 48 h, cells incubated with 786o cells (at a 1:1 ratio of 10⁵:10⁵) after 48 h, and cells incubated with 786o-CD19 cells (at a 1:1 ratio of 10⁵:10⁵) after 48 h. CAR: chimeric antigen receptor; CFSE: carboxyfluorescein diacetate succinimidyl ester

Fig. 2

The proinflammatory cytokine release specific to the antigen was lower in PEPT CAR cells. A–C. IFN-γ, TNF-α, and IL-2 production by mock, 2G CAR, PD1-S28 CAR, PEPT CAR, and PE8HT CAR T cells. Cytokine concentrations in the media were measured after 24 h coincubation with K562, K562-CD19, NALM-6, 786o, K562-PDL1, K562-CD19-PDL1(KL19), 786o-CD19, and NALM-6-PDL1 cells at E:T of 1:1. Results are mean values ± SD of triplicate from one of two representative experiments. *P < 0.05; **P < 0.01; ***P < 0.005

Fig. 3

The PEPT CAR-T cells enhanced anti-tumor activity without off-target toxicity to PDL1 in vivo. A. Schematic representation of the experimental procedure for tumor challenge, T cell adoptive transfer, cytokine detection, and in vivo imaging. B. Representative bioluminescent images of tumor growth over time. C. Total body flux (photons/s) for each mouse was quantified and averaged per group. Error bars represent mean ± SEM.D. Kaplan-Meier survival analysis of NALM-6-PDL1-GFP-luc challenged mice. Overall survival curves were plotted using the Kaplan-Meier method and compared using the log-rank (Mantel-Cox) test (n = 4 or 5; * P < 0.05). E. Murine PDL1/2 expression in different tissues of NSG mice stained with human PDL1/2 antibodies by flow cytometry. F. On day 4, blood was collected from the tail vein. The samples from all mice of each group were pooled, and plasma was isolated. The concentration of IFN-γ was detected using an ELISA-kit in duplicate

Fig. 4

PE8T cells improved antitumor activity to CD19 + PDL1 + cells with reduced cytokine release and affinity for CD19 protein. A. Schematic of PE8T CAR containing variations in the hinge, extramembrane, and transmembrane domains. B. Cytotoxic percentages of K562, K562-PDL1, NALM-6, and NALM-6-PDL1 cells cocultured with mock-T, 2G, and PE8T CAR-T cells at E:T of 5:1 after 6–8 h in vitro. The results are representative of at least three independent experiments with T cells from different healthy donors (left); the proliferation of different effector cells after CFSE-staining, incubation with different target cells (K562, NALM-6, NALM-6-PDL1) in RPMI (10% FBS) for 48 h. The proliferation rate was analyzed by flow cytometry. (right) Results are mean values ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005. C. IFN-γ, IL-21, TNF-α, IL-10, IL-2, and IL-6 production by mock-T, 2G, and PE8T cells. The concentration of cytokines in the media was measured after 24 h coincubation with N(NALM-6) and NL(NALM-6-PDL1) at E:T of 1:1 (mean ± SD, n = 2). Results are mean values ± SD of triplicate from one of two representative independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005. D. Heat map of selected cytokines enriched in genes significantly upregulated or downregulated in 2G CAR-T vs. PE8T CAR-T cells after stimulation with NALM-6-PDL1 cells at E:T of 1:1 for 48 h. For each pathway, a single sample enrichment score was calculated, and the mean was taken per response group. A color gradient ranging from dark blue to dark red indicates the mean normalized enrichment score (ranging from − 2 to + 2) of pathways enriched in induced (red) or repressed (blue) genes. E. The affinity of CD19 protein to different CAR-T cells: 2G > PE8T. EC50 of CAR-T cells binding to CD19 protein was determined by flow cytometry. The results of D and E are mean values ± SD of triplicate from a single experiment. EC50, 50% maximal effective concentration

Fig. 5

PE8T CAR-T cells harnessed tumor and prolonged survival in vivo compared with 2G CAR-T cells. A. Schematic representation of the experimental procedure for tumor challenge, T cell adoptive transfer, cytokines detection, and in vivo imaging. B. Representative bioluminescent images of tumor over time. C. The logarithm of total body flux (photons/s) for each mouse was quantified and averaged per group (mean ± SEM, n = 6). D. Kaplan–Meier survival analysis of NALM-6-PDL1-GFP-luc challenged mice. Overall survival curves were plotted using the Kaplan–Meier method and compared utilizing the log-rank (Mantel–Cox) test (n = 6). *P < 0.05; **P < 0.01; ***P < 0.005. E. On day 5, 9, 16, 20, blood was collected from one group mixed to detect the concentration of IFN-γ and IL-2 using an ELISA-kit (mean ± SD, n = 2)

Fig. 6

The PE8T cells reduced exhaustion and increased oxygen consumption rate inculcated with CD19 + PDL1 + tumor cells. A. Proliferation curves of different CAR-T cells from day 3 to day 11 after isolation (mean ± SD, n = 3). Results are representative of triplicate with T cells from one healthy donor. B. The stage of T cell differentiation and development is defined as: naïve T (T_N): CD45RA + CD45RO−, stem cell memory T (T_SCM): CD45RA + CD45RO+, central memory T (T_CM): CD45RA + CCR7+, and effect memory T (T_EM): CD45RA − CCR7−. Phenotypic detection was performed on day 8 after isolation (mean ± SD, n = 3). Results are representative of triplicate with T cells from one healthy donor. 2G CAR-T vs.PE8T CAR-T.C. Representative GSEA results from running the 2G CAR-T vs. PE8T CAR-T cells rank list about differentiation gene ontology sets (mean ± SD, n = 3). Results are representative of triplicate with T cells from one healthy donor. CAR-T cells were collected after stimulation with NALM-6-PDL1 cells at E:T of 1:1 for 48 h. D. The OCR, basal OCR and maximum OCR of 2G CAR-T and PE8T CAR-T cells in culture under basal metabolic conditions and in response to mitochondrial inhibitors, as specified in the experimental procedures. E. ECAR, basal ECAR and maximum ECAR of 2G CAR-T and PE8T CAR-T cells in culture under basal metabolic conditions and in response to mitochondrial inhibitors, as specified in the experimental procedures. Data represent at least triplicate performed with cells from one healthy human donor plotted as mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.005.). One of three independent experiments is presented (n = 3). Data are represented as mean ± SD.F. Representative GSEA results from running the 2G CAR-T vs. PE8T CAR-T cells rank list about metabolism gene ontology sets (mean ± SD, n = 3). Results are representative of triplicate with T cells from one healthy donor. CAR-T cells were collected after stimulation with NALM-6-PDL1 cells at E:T of 1:1 for 48 h. One of three independent experiments is presented (n = 3). Data are represented as mean ± SD. ECAR, extracellular acidification rate; GSEA, gene set enrichment analysis; OCR, oxygen consumption rate