PD-1 blockade does not improve efficacy of EpCAM-directed CAR T-cell in lung cancer brain metastasis

Abstract

Background: Lung cancer brain metastasis has a devastating prognosis, necessitating innovative treatment strategies. While chimeric antigen receptor (CAR) T-cell show promise in hematologic malignancies, their efficacy in solid tumors, including brain metastasis, is limited by the immunosuppressive tumor environment. The PD-L1/PD-1 pathway inhibits CAR T-cell activity in the tumor microenvironment, presenting a potential target to enhance therapeutic efficacy. This study aims to evaluate the impact of anti-PD-1 antibodies on CAR T-cell in treating lung cancer brain metastasis.

Methods: We utilized a murine immunocompetent, syngeneic orthotopic cerebral metastasis model for repetitive intracerebral two-photon laser scanning microscopy, enabling in vivo characterization of red fluorescent tumor cells and CAR T-cell at a single-cell level over time. Red fluorescent EpCAM-transduced Lewis lung carcinoma cells (^EpCAM/tdtLL/2 cells) were implanted intracranially. Following the formation of brain metastasis, EpCAM-directed CAR T-cell were injected into adjacent brain tissue, and animals received either anti-PD-1 or an isotype control.

Results: Compared to controls receiving T-cell lacking a CAR, mice receiving EpCAM-directed CAR T-cell showed higher intratumoral CAR T-cell densities in the beginning after intraparenchymal injection. This finding was accompanied with reduced tumor growth and translated into a survival benefit. Additional anti-PD-1 treatment, however, did not affect intratumoral CAR T-cell persistence nor tumor growth and thereby did not provide an additional therapeutic effect.

Conclusion: CAR T-cell therapy for brain malignancies appears promising. However, additional anti-PD-1 treatment did not enhance intratumoral CAR T-cell persistence or effector function, highlighting the need for novel strategies to improve CAR T-cell therapy in solid tumors.

Keywords: Brain metastasis; CAR T cell; Lung cancer; PD-1-blockade.

Fig. 1

Experimental protocol. a Schematic illustrating the experimental setup, including chronology of tumor cell injection, CAR T-cell injection, ICB therapy, and imaging methodologies. b Illustration of the injection sites of tumor cells and (CAR) T-cell, respectively. c Intracerebral tumor growth following stereotactic implantation of ^EpCAM/tdTLL/2 tumor cells (red). Blood vessels are highlighted after i.v. injection of FITC-dextran (green). Images represent mosaics of multiple maximum intensity projections with 400 µm depth from the brain surface (Scale bar 200 µm left pictures, 1000 µm right picture). Note that the day count refers to the day after tumor cell injection. d Hematoxylin and eosin staining as well as immunofluorescence staining of EpCAM on brain tumor tissue. Scale bar 100 µm. e Flow cytometry of ^EpCAM/tdTLL/2 tumor cells after 48 h of co-culturing with ^EpCAM/GFPCAR T-cell and ^GFPT-cell, respectively

Fig. 2

TPLSM of ^EpCAM/GFPCAR T-cell and ^GFPT-cell after intraparenchymal injection 7 days after tumor cell inoculation. a, b Intratumoral (CAR) T-cell density (cells/mm³) on d4, d7, and d10 after local injection of ^EpCAM/GFPCAR T-cell (+ IgG isotype (light green; n = 8) or anti-PD-1 antibodies (dark green, n = 8)) and ^GFPT-cell (+ IgG isotype (n = 7)), respectively, as determined by two-photon laser scanning microscopy. c Comparison of (CAR) T-cell densities (cells/mm³) on day 4 after injection within the tumor and the contralateral, non-tumor-bearing hemisphere as assessed by TPLSM in maximum intensity projection (MIP). All results are displayed as mean ± SEM. d, e Representative images of EpCAM-directed CAR T- (n = 8) and undirected T-cell (n = 7) with isotype control IgG (D) or in combination with intraperitoneal anti-PD-1-treatment e on d4, d7, and d10 after intraparenchymal administration using in vivo TPLSM in MIP with 400 µm depth from the brain surface. Scale bars 100 µm. Mean ± SEM

Fig. 3

Tumor growth and survival after intraparenchymal injection of (CAR) T-cell with and without concomitant aPD-1 treatment. a, b, c Summarized tumor areas (mm²) of n = 8 animals receiving locally injected ^EpCAM/GFPCAR T-cell + IgG (A, B, light green) / + aPD-1 (B, dark green) and n = 8 animals receiving ^GFPT-cell + IgG (A, C, light gray) / + aPD-1 (C, dark gray), respectively. Growth behavior was determined by in vivo microscopy using epifluorescence. Mean ± SEM. ***p ≤ 0.0005. d, e, f Brain tumor growth illustrated by one representative animal on day -2, 4, 7, and 10 after local (CAR) T-cell administration and intraperitoneal anti-PD-1/IgG isotype injection measured by TPLSM using epifluorescence. Tumor cells are visualized by their red fluorescent signal. Scale bars 400 µm. g, h Individual tumor areas (mm²) of n = 8 ^EpCAM/GFPCAR T-cell + IgG (G) and n = 8 ^GFPT-cell + IgG (H), respectively, measured by in vivo TPLSM using epifluorescence on days − 2, 4, 7 and 10 after local injection. i, j Kaplan–Meier survival estimates for tumor-bearing mice (injected seven days prior to local (CAR) T-cell administration) treated with either ^EpCAM/GFPCAR T-cell + aPD-1 (dark green; n = 8), ^EpCAM/GFPCAR T-cell + IgG isotype antibody (light green; n = 8) or ^GFPT-cell + IgG isotype antibody (light gray, n = 8). Log-rank test, ****p < 0.0001

Fig. 4

Characterization of intratumoral CAR T-cell dynamics below visible depths using immunofluorescence. After termination of the experiment brains get excised and stained for (CAR) T-cell density. a Histological sections of brains after tumor cell and ^EpCAM/GFPCAR T-cell injection and intraperitoneal aPD-1 and IgG administration, respectively. Sections were stained with an antibody against GFP to identify CAR T-cell (green), against CD3 to visualize T-cell (pink) and DAPI for cell nuclei (blue). CAR T-cell density within the tumor and at the infiltration zone was analyzed. Please note: Tumor size does not differ significantly between both groups. Scale bars 1400 µm (top images) and 150 µm (small images). b Intratumoral CAR T-cell density (cells/mm³) at the end of the experiment with (n = 4) and without (n = 4) concomitant aPD-1 treatment. c Tumor volume (mm³) of ^EpCAM/GFPCAR T-cell treated animals with and without concomitant anti-PD-1 treatment. Mean ± SEM. d, e Percentage distribution of CAR T-cell within the tumor and at the infiltration zone determined by immunofluorescence. f Immunofluorescence staining of tumor sections. EpCAM expression by tumor cells was confirmed. Expression levels of the immune checkpoint molecules PD-1, PD-L1, TIM-3, and LAG-3 are illustrated in pink. Scale bars 100 µm. g Quantification of PD-1- and TIM-3-positive cells. ***p < 0.001