Nonsignaling extracellular spacer regulates tumor antigen selectivity of CAR T cells - PubMed (original) (raw)

. 2024 Mar 2;32(2):200789.

doi: 10.1016/j.omton.2024.200789. eCollection 2024 Jun 20.

Yukiko Yamaguchi 1, Jackson Gibson 1, Ethan A Gerdts 1, Brook Jeang 1, Dileshni Tilakawardane 1, John P Murad 1, Wen-Chung Chang 1, Sarah L Wright 1, Michalina S Thiel 1, Stephen J Forman 1 2, Lawrence A Stern 3, Saul J Priceman 1 2

Affiliations

Nonsignaling extracellular spacer regulates tumor antigen selectivity of CAR T cells

Kelly T Kennewick et al. Mol Ther Oncol. 2024.

Abstract

Advancing chimeric antigen receptor (CAR)-engineered T cells for the treatment of solid tumors is a major focus in the field of cellular immunotherapy. Several hurdles have hindered similar CAR T cell clinical responses in solid tumors as seen in hematological malignancies. These challenges include on-target off-tumor toxicities, which have inspired efforts to optimize CARs for improved tumor antigen selectivity and overall safety. We recently developed a CAR T cell therapy targeting prostate stem cell antigen (PSCA) for prostate and pancreatic cancers, showing improved preclinical antitumor activity and T cell persistence by optimizing the intracellular co-stimulatory domain. Similar studies were undertaken to optimize HER2-directed CAR T cells with modifications to the intracellular co-stimulatory domain for selective targeting of breast cancer brain metastasis. In the present study, we evaluate various nonsignaling extracellular spacers in these CARs to further improve tumor antigen selectivity. Our findings suggest that length and structure of the extracellular spacer can dictate the ability of CARs to selectively target tumor cells with high antigen density, while sparing cells with low antigen density. This study contributes to CAR construct design considerations and expands our knowledge of tuning solid tumor CAR T cell therapies for improved safety and efficacy.

Keywords: CAR; HER2; MT: Regular Issue; PSCA; T cell immunotherapy; breast cancer; chimeric antigen receptor; nonsignaling extracellular spacer domain; prostate cancer.

© 2024 The Authors.

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Conflict of interest statement

S.J.P. is a scientific advisor, consultant, and/or receives royalties from Imugene, Mustang Bio, Bayer, Celularity, and Adicet Bio. S.J.F. is a scientific advisor to and receives royalties from Mustang Bio.

Figures

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Graphical abstract

Figure 1

Figure 1

PSCA-CAR T cells containing varying nonsignaling extracellular spacers (A) Diagram of the lentiviral expression cassette with PSCA-CARs containing the humanized scFv (A11 clone) targeting PSCA, a CD4 transmembrane domain, a cytoplasmic 4-1BB co-stimulatory domain, and a cytolytic CD3ζ domain. The nonsignaling extracellular spacer domain was altered to be either a 229-amino acid-modified human IgG4 Fc spacer with a double mutation (L235E; N297Q) within the CH2 region (EQ), a 129-amino acid-modified human IgG4 Fc spacer (void of the CH2 domain, ΔCH2), a 22-amino acid IgG4 HL, or a 10-amino acid synthetic linker (L). A nonsignaling CD19t, separated from the CAR with a T2A ribosomal skip sequence, was expressed as a surrogate marker of transduction. (B) Molecular weight of CARs with varying spacer length detected by western blotting CD3ζ protein. Endogenous T cell receptor was also detected (17–24 kDa). (C) UTD and PSCA-CAR T cells containing either EQ, ΔCH2, HL, or L spacers were evaluated by flow cytometry for CD19t expression to detect lentiviral transduction of CARs. (D) Protein L binding was evaluated by flow cytometry to detect cell surface expression of the scFv of the CAR. (E) Ex vivo expansion kinetics for UTD and PSCA-CAR T cells over 16 days in culture. All of the data are representative of at least 2 independent experiments with at least 2 donors.

Figure 2

Figure 2

Nonsignaling extracellular spacer regulates antigen sensitivity and functionality of PSCA-CAR T cells in vitro (A) Flow cytometry analysis of PSCA expression in lentivirally transduced human prostate cancer cell lines PC3 and DU145. (B) Fluorescence microscopy analysis of PSCA expression in PC3, PC3-PSCAlo, and PC3-PSCAhi tumor cells. (C and D) Percentage (C) and MFI (D) of CD137 expression in UTD and PSCA-CAR T cells containing either EQ, ΔCH2, HL, or L spacers. CD137 expression was evaluated by flow cytometry following 24-h coculture with the indicated tumor targets at a 1:2 E:T ratio. (E) IFN-γ production quantified by ELISA in supernatants from UTD or indicated PSCA-CAR T cells cultured overnight with tumor targets at a 1:1 E:T ratio. (F) IFN-γ production quantified by ELISA in supernatants from UTD or indicated PSCA-CAR T cells cultured overnight on plate-bound recombinant human PSCA at varying protein concentrations. All of the data are representative of at least 2 independent experiments performed with duplicates with at least 2 donors. Mean ± SEM is presented with p value (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 3

Figure 3

Short extracellular spacers improve selectivity of PSCA-CAR T cells to prostate tumor cells with higher antigen density UTD and PSCA-CAR T cells containing either EQ, ΔCH2, HL, or L spacers were cocultured with PC3, PC3-PSCAlo, and PC3-PSCA tumor cells at a 1:20 E:T ratio, and flow cytometry analysis was performed after 9 days. (A) Representative flow cytometry plots comparing the spacer variants. Gating strategy to distinguish tumor cells and T cells (left). (B–D) Quantification of tumor cell killing (B), T cell expansion (C), and PD-1 expression (D). Tumor cell killing was normalized to respective conditions containing control UTD T cells. PD-1 expression was assessed in CAR T cells detected by CD19t expression. All of the data are representative of at least 2 independent experiments performed with duplicates with at least 2 donors. Mean ± SEM is presented with p value (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 4

Figure 4

Nonsignaling extracellular spacer controls antigen sensitivity and functionality of HER2-CAR T cells in vitro (A) Diagram of the lentiviral expression cassette with HER2-CARs containing the humanized scFv (4D5 clone) targeting HER2 with a CD8 transmembrane domain, a cytoplasmic 4-1BB co-stimulatory domain, and a cytolytic CD3ζ domain. The nonsignaling extracellular spacer domain was altered to be either a 229-amino acid-modified human IgG4 Fc spacer with a double mutation (L235E; N297Q) within the CH2 region (EQ), a 129-amino acid-modified human IgG4 Fc spacer (void of the CH2 domain, ΔCH2), a 22-amino acid IgG4 HL, or a 10-amino acid synthetic linker (L). A nonsignaling CD19t, separated from the CAR with a T2A ribosomal skip sequence, was expressed as a surrogate marker of transduction. (B) UTD and HER2-CAR T cells containing either EQ, ΔCH2, HL, or L spacers were evaluated by flow cytometry for CD19t expression to detect lentiviral transduction of CARs. (C) Protein L binding was evaluated by flow cytometry to detect cell surface expression of the scFv of the CAR. (D) Ex vivo expansion kinetics for UTD and PSCA-CAR T cells over 18 days in culture. All of the data are representative of at least 2 independent experiments. (E) Flow cytometric analysis of HER2 expression in human breast cancer cell lines. HER2-MDA-MB-468 (468), MDA-MB-361 (361), MDA-MB-231BR (231BR), and MDA-MB-231BR cells engineered to overexpress HER2 (231BR-HER2), and BBM1 tumor cells have varying HER2 expression. (F) IFN-γ production quantified by ELISA in supernatants from UTD or indicated HER2-CAR T cells cocultured overnight with tumor targets at a 1:1 E:T ratio. (G) IFN-γ production quantified by ELISA in supernatants from UTD or indicated HER2-CAR T cells cultured overnight on plate-bound recombinant human HER2-Fc at varying protein concentrations. (H) Tumor cell killing by HER2-CAR T cells. HER2-CAR T cells containing either EQ, ΔCH2, HL, or L spacers were cocultured with indicated tumor cells at a 1:20 E:T ratio, and flow cytometry analysis was performed after 8 days. Tumor cell killing was calculated by normalizing to respective conditions containing control UTD T cells. All of the data are representative of at least 2 independent experiments performed with duplicates or triplicates with at least 2 donors. Statistical analysis was performed to compare EQ, HL, and L spacers. Mean ± SEM is presented with p value (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 5

Figure 5

Short extracellular spacers in PSCA-CAR T cells promote selective killing of prostate cancer cells with high antigen density in vivo (A) Illustration of in vivo model. Male NSG mice bearing subcutaneous PC3-PSCAhi and PC3-PSCAlo tumor cells (2.5 × 106 each site) on either flank with treated with 0.5 M Mock or indicated PSCA-CAR T cells by i.v. injection on day 22. (B–E) Average and individual tumor volumes (mm3) of PC3-PSCAhi (B and D) and PC3-PSCAlo (C and E) tumors. All of the data are representative of at least 2 independent experiments with at least 2 donors. Each group had N ≥ 5. Statistical significance shown indicates results of ANOVA performed on 56 days posttumor injection. Mean ± SEM is presented with p value (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 6

Figure 6

Nonsignaling extracellular spacer regulates antitumor activity of PSCA-CAR T cells against pancreatic ductal adenocarcinoma in vivo (A) PSCA expression in HPAC cells evaluated by flow cytometry. (B) Quantification of tumor cell killing by PSCA-CAR T cells containing either ΔCH2, HL, or L spacers following a 3-day coculture with PSCA+ HPAC tumor cells at a 1:1 E:T ratio. (C and D) Average (C) and individual (D) tumor volumes (mm3) in NSG mice bearing subcutaneous HPAC (2.5 × 106) tumors on day 0, and treated with 5 M UTD or indicated PSCA-CAR T cells by i.v. injection on day 16. Statistical significance shown indicates results of ANOVA performed on 51 days posttumor injection. (E) Immunohistochemistry staining of human CD3 in HPAC tumors from mice treated with UTD or PSCA-CAR T cells containing either ΔCH2, HL, or L spacers. (F) Quantification of CD3+ human T cells per unit area of HPAC tumors stained by immunohistochemistry. (G) Frequency of CAR T cells relative to the total cells in HPAC tumors measured by flow cytometry. (H) Concentration of circulating CAR T cells in peripheral blood measured by flow cytometry. All of the data are representative of at least 2 independent experiments with at least 2 donors. In vitro tumor killing assays were performed with duplicates (B). In vivo studies were performed with N = 10 per group. Circulating CAR T cells were quantified in all of the mice (H). Tumor was harvested from 2 mice (E–G), and tumor volume is shown with N = 8 (A and B). Mean ± SEM is presented with p value (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

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