D2HGDH-mediated D2HG catabolism enhances the anti-tumor activities of CAR-T cells in an immunosuppressive microenvironment

Abstract BACKGROUND We found that CD70, as an immune modulator, in GBM plays a critical role in immunosuppression and tumor progression. Although CD70+ tumors recruit more CD3+ T cells than CD70— tumors do in patients with GBM, CD70 on GBM is also found to be involved in promoting CD8+ T cell death. The experiments by overexpressing or silencing CD70 in a primary tumor demonstrate that it can alter cell growth, survival, migration, and morphology of GBM cells. CD70 is negatively correlated with survival in patients with gliomas. These results suggest that CD70 is involved in immunosuppression in GBM. OBJECTIVE To determine if abrogating CD70 in tumor using CD70CAR-T cells could reverse the immunosuppressive microenvironment and enhance overall endogenous tumor immunity against both CD70+ and CD70— tumors, which might help to overcome a key obstacle— tumor-heterogeneity using single-targeted CAR-T cell therapy. METHODS CD70 was overexpressed (~75% positivity) in KR158 GBM line. Murine CD70CAR T cells were used to eliminate CD70+ tumors in an immunocompetent orthotopic tumor mouse model. Tumor-bearing mice were administered the CD70CAR T and vector-tranduced T cells, followed by IVIS imaging for tumor growth. The presentation and phenotype of CAR T cells and endogenous immune cell populations in tumors and spleens were measured. RESULTS Five weeks post treatment, CD4+ T cells were found to be the dominant T cell population in tumors for both CAR-T and endogenous T cells. While the CAR-T cells shrank the tumors, fewer PD-1 expressing endogenous T cells, as well as granulocytic MDSC, but not monocytic MDSC were observed in the tumor (not in spleen) for the CAR-T group, compared to the vector group. No significant changes were seen for NK cells and Tregs between the groups. CONCLUSION This study suggests that eliminating CD70+ tumor cells may reverse the immunosuppressive landscape of the tumor microenvironment.

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143 Mesothelin (MSLN) targeting allogeneic CAR T cells engineered to overcome tumor immunosuppressive microenvironment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.143 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A151-A151

Author(s):

Cecile Schiffer-Mannioui ◽

Sophie Leduc ◽

Isabelle Chion-Sotinel ◽

Diane le Clerre ◽

Valérie Guyot ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Solid Tumors ◽

Tgfβ Signaling ◽

Knock Out ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Immunosuppressive Microenvironment

BackgroundChimeric Antigen Receptor (CAR) T cell therapy is emerging as a potential treatment for solid tumors, even if only limited activity has been observed for CAR T therapies to date. Cellular therapies face indeed many hurdles in solid tumors, such as the immunosuppressive microenvironment. TGFβ is an important growth factor of the tumor microenvironment and has been shown to suppress anti-tumor immunity. Gene editing represents a powerful way to enhance properties of CAR T cells and can be used to circumvent the effect of TGFβ signaling. The tumor associated antigen mesothelin (MSLN) is an attractive target for cellular therapy; being expressed at high levels in several tumor types (e.g., pleural mesothelioma and pancreatic cancer) while only modestly expressed in healthy tissues.MethodsUCARTMeso, an allogeneic CAR T cell product targeting MSLN expressing cells is being developed by Cellectis. UCARTMeso bears an anti-MSLN CAR and a triple gene knock-out (KO) for TRAC, CD52 and TGFBR2 genes, all generated using TALEN® gene-editing technology. TRAC KO limits the risk of GvHD, while CD52 KO allows the use of alemtuzumab in the preconditioning regimen. The additional KO of TGFBR2 confers resistance to the immunomodulatory effects of TGFβ within the solid tumor microenvironment.ResultsPreclinical studies showed high specificity of the anti-MSLN CAR, as well as potent anti-tumor activity in vitro against different cell lines expressing MSLN. In addition, this activity was confirmed in mouse studies against pancreatic and pleural mesothelioma tumor models, with comparable activities observed in the latest model upon i.v. or intra-pleural administration of UCARTMeso. Also, we observed that TGFBR2 edited anti-MSLN CAR T cells displayed a blockade in the TGFβ signaling pathway, being able to respond to antigen stimulation in the presence of TGFβ (figure 1).Abstract 143 Figure 1Left panel: TGFβ-induced SMAD2/3 phosphorylation in anti-MSLN CAR T cells. UCARTMeso cells were stained with mesothelin recombinant protein for CAR detection and anti-pSMAD2/3 one hour post exposure to TGFβ. The lack of SMAD2/3 phosphorylation in TGFBR2 KO cells indicates that they are unable to trigger TGFβ signaling. Right panel: Antigen-induced anti-MSLN CAR T cell activation in the presence (blue histogram) or absence (red histogram) of TGFβ. CAR T cells were stained with anti-CD25 antibody and analyzed by flow cytometry 5 days post exposure to antigen ± TGFβ. The data shows that cells not edited at the TGFBR2 locus are unable to be activated upon target exposure in the presence of TGFβ, while edited cells were activated in the presence of TGFβ, triggering CD25 expression at similar levels as those of cells activated in the absence of TGFβ.ConclusionsAltogether, we have demonstrated potent antitumor activity in vitro and in vivo, and that addition of the third knock-out of TGFBR2 gene provide valuable additional properties to UCARTMeso cells, representing a very attractive strategy for their use in the treatment of solid tumors.

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ICG/LY@HES-PCL Nanomedicine Improving the Efficacy of CAR-T Therapy in Lymphoma

Blood ◽

10.1182/blood-2021-154349 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4806-4806

Author(s):

Yuxiang Tang ◽

Heng Mei ◽

ZhaoZhao Chen ◽

Weiqi Yao ◽

Chenggong Li ◽

...

Keyword(s):

T Cells ◽

Tumor Tissue ◽

Imaging System ◽

Conflicts Of Interest ◽

Photothermal Effect ◽

Car T Cells ◽

Car T ◽

Immunosuppressive Microenvironment

Abstract Background : The presence of tumor immunosuppressive microenvironment, abnormal vascular microenvironment, and the presence of a cross-linked matrix barrier may hinder the recruitment, proliferation, and activation of CAR-T to lymphoma tissue infiltration, which limit the efficacy of CAR-T in the treatment of lymphoma. The enhanced infiltration effect of ICG photothermal effect on CAR-T and the effect of LY on improving tumor immunosuppressive microenvironment have been confirmed. In this work, we have constructed LY/ICG@HES-PCL nanomedicine to improve the accumulation, proliferation and activation of CAR-T in lymphoma tissue, further improving the therapy efficacy of CAR-T in lymphoma. Methods: The effect of LY/ICG@HES-PCL on the proliferation and activation of CAR-T was studied by Transwell experiment. LY/ICG@HES-PCL was studied in vivo to investigate its improving the efficacy of CAR-T therapy in lymphoma. Biefly, 1×10 7 Raji human lymphoma cells were subcutaneously injected into both sides of each mouse. After ≈20 days, when the tumor volume reached ≈100 mm 3, mice were randomly assigned to 5 groups (n=6) and were treated by PBS, ICG@HES-PCL, ICG@HES-PCL+NIR, LY/ICG@HES-PCL, LY/ICG@HES-PCL+NIR, respectively. (4.0 mg/kg as ICG, 4.0 mg/kg as ICG). NIR groups were irradiated with an 808 nm laser at the power density of 0.3 W cm −2 for 10 mins. Two hours later, 1 × 10 7 CAR-T cells were intravenously injected into the mice. The tumor sizes were recorded by a digital caliper every 3 days. LY/ICG@HES-PCL in vivo was monitored using an in vivo bioluminescence imaging system. Lymphoma microenvironment was analyzed by immunofluorescence and FCM. Results: LY/ICG@HES-PCL increased CAR-T quantity by 3.7 times, IL-2 and IFN-λ release by 2.5 and 2.1 times in vitro, increased CAR-T accumulation in tumor tissue by 4.45 times within 3 days in vivo, achieving CAR-T activation, proliferation, and tumor accumulation enhancement. LY/ICG@HES-PCL improved CAR T efficacy in lymphoma by 3.4 times and CAR T longevity within 15 days by 1.6 times. Discussion: LY/ICG@HES-PCL co-delivered the photosensitifier ICG and TGF-β inhibitor LY to the lymphoma tissue. The photothermal effect of ICG decreased the density of lymphoma matrix and activated CD4+ T cells, CD8+ T cells and NK cells in tumor tissue. LY was used to inhibit PD-1 and CLTA-4 of CAR-T, so as to promotes CAR-T proliferation, improve CAR-T activity and longevity in vivo, and ultimately improve the anti-lymphoma efficacy of CAR-T cells. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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