Validation of a PD-1/CD28 chimeric switch receptor to augment CAR-T function in dogs with spontaneous B cell lymphoma

Abstract

Chimeric antigen receptor (CAR) T cell therapy has achieved unprecedented clinical outcomes in patients with relapsed/refractory B cell leukemias; however, response rates in patients with large B cell lymphoma (LBCL) are less impressive. Expression of PD-1 on activated T cells and PD-L1 on malignant, stromal, and immune cells within the tumor microenvironment (TME) contribute to CAR-T exhaustion, hypofunction, and treatment failures. Here, a comparative approach is taken to develop a chimeric switch receptor (CSR) with potential to augment CAR-T persistence, function, and clinical efficacy in immune competent, pet dogs with spontaneous B cell lymphoma (BCL). We show that similar to human CAR-T cells, expression of a PD-1/CD28 CSR in canine CAR-T cells results in enhanced function against PD-L1⁺ targets and preserves central memory phenotype. We also demonstrate that these effects depend upon active CSR signaling. This work paves the way for in vivo studies in canine BCL patients to inform human trial design.

Keywords: Cancer; Therapeutics; Veterinary medicine.

Graphical abstract

Figure 1

Canine CD20-BB-ζ CAR-T cells express cPD-1 after CAR engagement (A) Schematic representation of cCD20-BB-ζ CAR construct. ICD, intracellular domain; TM, transmembrane domain. (B) Surface expression of cCD20-BB-ζ CAR on CD4⁺ and CD8⁺ T cells evaluated by flow cytometry 4 days post transduction. Plots are gated on lymphocytes>single>live>CD5⁺>CD4⁺ or CD8⁺ cells. (C) Surface expression of cCD20 and cPD-L1 on engineered K562 cells. Plots are gated on live single cells. (D) cPD-1 expression on CAR-T cells after co-culture with indicated target cells at different E:T ratios. CAR-T cells were generated as described and 6 days after transduction (8 days post activation) cells were co-cultured with either K562-WT or K562-cCD20 cells with or without cPD-L1, and cPD-1 expression was determined by flow cytometry after 72 h. Plots are gated on lymphocyte>single>live>CD5⁺>CAR⁺ cells.

Figure 2

cPD-L1 expressing targets suppress canine CAR-T cell effector functions (A) Proliferation index of canine CD4⁺ and CD8⁺ CD20-BB-ζ CAR T cells from 4 healthy donors, 72 h after stimulation with the indicated K562 target cells at an E:T ratio of 1:1. (B) cCD20-BB-ζ CAR-T cells were co-cultured with the indicated K562 target cells at different E:T ratios and cytotoxicity was measured after 24 h. Data are mean ± SD (n = 4 biological replicates). (C) IFN-γ, IL-2, and TNF-α production by cCD20-BB-ζ CAR T cells cultured with K562 (wild-type) or K562-cCD20 cells with or without PD-L1 at an E:T ratio of 1:1. Data are mean ± SD (n = 4 biological replicates). (D and E) Effects of target cPD-L1 expression on CAR-T cell phenotype. cCD20-BB-ζ CAR T cells were stimulated three times with K562-cCD20 or K562-cCD20-cPD-L1 cells and immunophenotyped 6 days after the third stimulation. (D) Representative two-dimensional dot plot of CD45RA and CD62L expression on CD8⁺ CAR T cells. (E) Data for each of the four dogs are shown. ∗p < 0.05; ∗∗p ≤ 0.01; ns: not significant. Naive-like (CD45RA⁺CD62L⁺), TCM-like (CD45RA⁻CD62L⁺), TEM-like (CD45RA⁻CD62L⁻), TEMRA-like (CD45RA⁺CD62L⁻).

Figure 3

An active PD-1/CD28 CSR augments cCD20-BB-ζ CAR T cell function against cPD-L1 expressing target cells (A) Design of CSR_WT and CSR_MUT CAR constructs. ECD, extracellular domain; TM, transmembrane; ICD, intracellular domain. (B) Cartoon showing the CD28 mutations that prevent Grb2 and Lck binding in the CSR_MUT construct. (C) Canine CD4⁺ and CD8⁺ T cells successfully co-express the cCD20-BB-ζ CAR and the CSR_WT and CSR_MUT constructs. Representative data from one of four CAR-T cell products generated from 4 healthy donor dogs 6 days post-transduction. Plots are gated on lymphocyte>single>live>CD5⁺>CD4⁺ or CD8⁺ cells. (D) CAR expression on CD4⁺ and CD8⁺ T cells from 4 healthy donor dogs transduced with CAR alone, CAR+CSR_WT or CAR+CSR_MUT. Data are mean ± SD (n = 4 biological replicates). ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ns: not significant.

Figure 4

An active PD-1/CD28 CSR augments CAR-T function against PD-L1 engineered target cells (A) IFN-γ, TNF-α, and IL-2 production by CAR+CSR_WT or CAR+CSR_MUT after 24 h co-culture with K562-cCD20 or K562-cCD20-cPD-L1 target cells at an E:T ratio of 1:1. Data are mean ± SD (n = 4 biological replicates). (B) Cytotoxicity of CAR, CAR+CSR_WT, or CAR+CSR_MUT against K562-cCD20 and K562-cCD20-cPD-L1 target cells at E:T ratio of 8:1. Data are mean ± SD (n = 4 biological replicates). (C) Memory phenotype of CAR, CAR+CSR_WT, or CAR+CSR_MUT T cells determined 6 days after the third stimulation with K562-cCD20 and K562-cCD20-cPD-L1 target cells at an E:T ratio of 1:1. Data for each of the four dogs are shown. ∗p < 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001, ns: not significant.

Figure 5

An active PD-1/CD28 CSR augments CAR-T function against canine B cell lymphoma (A) Schematic of experimental timeline of repeat stimulation of CAR-T cells with WT or CLBL-1ΔcPD-L1 target cells. (B) Confirmation of lack of cPD-L1 expression by CLBL-1ΔcPD-L1 cells by flow cytometry. Plots are gated on 7AAD⁻ cells. (C) Cell surface expression of CAR on CD4⁺ and CD8⁺ CAR-T and CAR+CSR_WT T cells from healthy donor dogs following cell sorting as determined by flow cytometry. Data from three individual dogs are shown. (D) IFN-γ and TNF-α production by CAR-T cells stimulated with CLBL-1 cells or CLBL-1ΔcPD-L1 cells after 5 rounds of stimulation at an E:T of 1:1. IFN-γ and TNF-αproduction was assessed in supernatants 24 h after the fifth stimulation. Data are mean ± SD of 3 technical replicates for each dog. (E) CAR-T and CSR_WTCAR-T cells from healthy dogs were co-cultured with CLBL-1 target cells at an E:T ratio of 1:1. Supernatants were harvested at 24 h after the 5^th stimulation and assayed for IFN-γ and TNF-α production. (F and G) CAR-T and CSR_WT CAR-T cells from 2 healthy dogs were stimulated 3 times every 3 days with K562-cCD20-cPD-L1 target cells and then co-cultured with (F) CLBL-1 cells or (G) CLBL-1 cells pre-treated with IFN-γ for 48 h, at increasing E:T ratios. Cytotoxicity was determined by luciferase assay at 24 h. Data are mean ± SD of 3 technical replicates for each dog are shown. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns: not significant.