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126 Early-phenotype Lewis Y CAR-T cells persist better in vivo and induce solid tumor regression in combination with anti-PD1
  1. Deborah Meyran1,
  2. Joe Zhu1,
  3. Jeanne Butler1,
  4. Sean Macdonald1,
  5. Daniela Tantalo1,
  6. Niko Thio1,
  7. Kevin Sek1,
  8. Paul Ekert2,
  9. Michael Kershaw1,
  10. Joe Trapani1,
  11. Phillip Darcy1 and
  12. Paul Neeson1
  1. 1Peter MacCallum Cancer Centre, Melbourne, Australia
  2. 2Children’s Cancer Institute, Sydney, Australia


Background Chimeric antigen receptor (CAR-T) cells are a promising new therapy for patients with cancer. However, in contrast to their success in B cell malignancies, CAR-T cells targeting solid cancers have had limited success so far due to their poor proliferation and poor long-term persistence in vivo. To address this issue, we used naïve T cells to generate second-generation CAR-T cells recognizing the tumor antigen Lewis Y (LeY), termed ‘early’ CAR-T cells.

Methods Purified naïve T cells were activated by CD3/CD28 soluble tetrameric antibody complex, retrovirally transduced (LeY scFv-CD3z-CD28 CAR) and expanded in IL-7/IL-15. The early LeY CAR-T cell function was tested in vitro for cytotoxicity (Cr-release and degranulation), proliferation, and cytokine secretion by CBA, either de novo or following chronic stimulation for 1 month. Finally, early CAR-T cell persistence and anti-tumor efficacy was assessed in the OVCAR3-NSG model, in the presence or absence of anti-PD-1.

Results The early-CAR-T cells comprised stem cell memory-like (CD95+, CD62L+, CD45RA+) and central memory phenotype (CD95+, CD62L+, CD45RA-) T cells with increased expression of ICOS, Ki67, TCF7 and CD27 (Figure 1). The early-CAR-T cells retained potent antigen-specific cytotoxicity, and secreted significantly higher levels of cytokines (IFN-?, TNF-a and IL-2) and increased proliferation compared to conventional CAR-T cells. Importantly, early-CAR-T cells had a significantly higher proliferative capacity after long-term chronic stimulation compared to conventional CAR-T cells (figure 2), and CD4+ CAR-T cells were critical for effective early CD8+ CAR-T cell proliferation capacity in vitro (figure 3). Early CAR-T cells had significantly better in vivo tumor control compared to conventional CAR-T cells (Figure 4), this was associated with increased CAR-T cell persistence. Because chronically stimulated early-LeY-CAR-T cells expressed PD-1 (figure 2), and OVCAR-3 cells expressed PD-L1 when co-cultured with LeY-CAR-T cells (figure 5), we combined early LeY-CAR-T cells with anti-PD-1 therapy and observed complete tumour regression in these mice. Interestingly, early LeY-CAR-T cell plus anti-PD-1 treatment also enhanced the percentage of circulating stem-cell memory like CAR-T cells in vivo (figure 5).

Abstract 126 Figure 1

Early-CAR-T protocol, including Naïve-T cells purification and expansion in IL-7 and IL-15 promotes the maintenance of a TSCM and TCM phenotype. A) Scheme of the 7-day production protocol for Early-CAR-T cells. B) Phenotype by FACs of the conventional CAR-T cells and the Early-CAR-T cells. Pooled data in triplicate for 6 donors. C) Phenotype by Mass cytometry comparing the Conventional-CAR-T cells vs Early-CAR-T cells vs Early-CD8-CAR-T cells. Data for one donor representative of 3 different donors

Abstract 126 Figure 2

Early-CAR-T cells are comparable in vitro to conventional CAR-T cells in terms of killing but have a better proliferation capacity that persists after chronic stimulation. The long-term stimulated early- CAR-T cells maintain their memory phenotype and upregulated PD-1. A) Chromium release assay against the LeY+ cell line (OVCAR3), data for one donor representative of 3 other donors. B) Cytokine secretion evaluated by CBA after coculture with the LeY+ cell line (OVCAR3) or with the LeY- cell line (MDA-MB435). C) Division index of CAR-T cells quantified with CTV. D) Evaluation of the differentiation, proliferation and cytotoxicity of the CAR-T cells after chronic stimulation

Abstract 126 Figure 3

Early-CD4+- CAR-T cells are critical for the proliferation capacity of the Early-CD8+-CAR-T cells. A) Scheme of the CD4-depletion protocol to compare Early-CD8-CAR-T proliferation with or without CD4-T cells. B) Division index of CD4-depleted Early-CAR-T cells, CD8-T cells from bulk Early-CAR-T cells, and from CD4+ T cells from bulk Early-CAR-T-cells quantified with CTV

Abstract 126 Figure 4

Early-CAR-T cells show in vivo a better persistence and a better proliferation capacity associated with a better tumoral control. A) Design of the in vivo experiment (n=7 mice per group) B) T-cell persistence in peripheral blood was measured by FACS. C) Speakman correlation (Day 13) between Tumor size and% CAR-T- cells. D) Tumor kinetic and Kaplan-Meier analysis of survival of OVCAR-bearing NSG mice treated with Conventional CAR-T cells, or Early-CAR-T cells or Low-dose of Early-CAR-T cells

Abstract 126 Figure 5

Anti-PD1 treatment enhance the efficacy of the Early-CAR-T cells. A) Upregulation of PD-L1 on OVCAR3 when expanded in the supernatant from co-culture of OVCAR3 with LeY-CAR-T cells. B) Design of the in vivo experiment (n=7 mice per group). C) T-cell persistence, phenotype and anti-human IgG4 in peripheral blood were measured by FACS. D) Tumor kinetic of OVCAR-bearing NSG mice treated with Early-CAR-T cells or Early-CAR-T cells + Nivolumab

Conclusions Our early CAR-T cells have better cytokine secretion and proliferation than conventional CAR-T cells. Early CAR-T cells also have superior anti-tumor efficacy in vivo, they have better persistence and maintain the circulating T cell memory pool. Importantly, low dose early-LeY-CAR-T cells combined with anti-PD1-treatment leads to complete clearance of LeY+ solid tumors in vivo. The early CAR-T cell production protocol is directly translatable for improving CAR-T cell efficacy in clinical trials for patients with solid tumors.

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