Impact of Epichaperome Inhibitor PU-H71 on Anti-Tumor T Cell Responses and Its Implications for Immune and Cellular Therapy

The epichaperome is a tightly integrated network of proteins supporting critical roles in the tumor-associated microenvironment and cancer cell survival. The epichaperome inhibitor PU-H71 is under development in clinical trials. A patient with an unclassified myeloproliferative neoplasm harboring a novel PML-SYK fusion, who progressed to acute myeloid leukemia despite allogeneic stem cell transplantation was successfully treated using PU-H71 (Sugita et al. 2021, NPJ Precis Oncol). We reported changes in donor immune cells after PU-H71 treatment, suggesting that epichaperome inhibition was modulating antitumor immune responses and improving the efficacy of cellular immunotherapy. To investigate this hypothesis, we analyzed the effects of PU-H71 on antitumor immune responses, GvHD and CAR-T cells.

We tested the effects of PU-H71 on C57BL/6 mice immune responses to ovalbumine (OVA) immunization. OVA-specific T- and B-cell responses were evaluated by ELISPOT and ELISA, respectively. When administered at the time of priming, PU-H71 significantly increased the number of IFNγ-secreting T cells as compared to controls (median 126 vs 42 per million of cells, p=0.0002; n=8), without an impact on T cell phenotype or anti-OVA antibody levels. In contrast, PU-H71 had no effect when administered once the OVA-specific T cell response was ongoing. Then, we tested the effect of PU-H71 on antitumor immune responses in OVA-primed mice challenged with E.G7 (OVA⁺) or parental EL4 lymphoma cell lines (OVA^neg). PU-H71 improved in vivo anti-tumor immune responses in OVA-primed mice challenged with E.G7 but not EL4 cells (Mean survival 17.5 vs 14 days, p=0.0003, n=8 per group).

Since PU-H71 augmented early but not established T cell responses, we next evaluated whether it would have an effect on a GvHD murine model. Lethally-irradiated B6C3F1 mice received bone marrow and splenocytes from C57BL/6 mice and were treated with PU-H71 or vehicle at the first signs of GvHD (D11). GvHD clinical scores and histological analysis (colon, small intestine, liver and skin) were assessed. PU-H71 did not aggravate GvHD at both clinical and histological levels. Therefore, PU-H71 augments antigen-specific mouse T cell responses by acting during priming.

To better understand its effect on human T cells and its potential adjuvant effect during cellular therapy, we next evaluated in vitro and in vivo CAR-T cells phenotype and function in the presence of PU-H71. T cells were purified from healthy donor PBMCs and were transduced with a CD19.4-1Bb.3z lentiviral vector to obtain CART19 cells. First, we found that epichaperome indeed forms in CART19 cells upon activation with anti-CD3/CD28 beads vs control. In vitro cytotoxicity of CAR-T cells toward NALM6 B-ALL cells was significantly improved (55.39 vs 74.66%, p=0.0356) by PU-H71. Finally, the therapeutic effect of the combination of PU-H71 and CART19 was evaluated in a B-ALL NSG mouse model using NALM6-BLIV cells, with bioluminescence imaging and flow cytometry (CART19 and leukemic cells) monitoring. Results suggest that PU-H71 administration increases CART19 numbers in the bone marrow and improves their in vivo antitumor efficacy against NALM6.

Together, our results show that PU-H71 augments antigen-specific T cell responses during priming including CAR-T cell cytotoxicity, with little effect on ongoing immune responses including GvHD. Our data suggest that PU-H71 improves the in vivo efficiency of CAR-T cells in a B-ALL mouse model. In addition to its previously demonstrated direct anti-tumor effect, PU-H71 also had an adjuvant effect on anti-tumor T cell responses while not aggravating GvHD, which supports its clinical evaluation in the post-transplant setting or in combination with immune therapies like CAR-T cells.

Martinet:Grifols: Research Funding. Chiosis:Samus Therapeutics Inc: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Roboz:Clovis Oncology: Other: Travel and accommodation expenses; Sandoz: Consultancy, Other: Travel and accommodation expenses; Genentech/Roche: Consultancy, Other: Travel and accommodation expenses; Daiichi Sankyo: Consultancy; Amgen: Consultancy; Takeda: Consultancy; MedImmune: Consultancy, Research Funding; Celltrion: Consultancy, Other: Travel and accommodation expenses; Jazz: Consultancy, Other: travel; Janssen: Consultancy, Other: travel and accommodation expenses, Research Funding; Actinium: Consultancy; Astellas: Consultancy; Jasper Therapeutics: Consultancy; Sunesis Pharmaceuticals: Other: Travel and accommodation expenses, Research Funding; Otsuka: Consultancy; Bristol Myers Squibb: Consultancy; Roche: Consultancy; Helsinn Therapeutics: Consultancy; Mesoblast: Consultancy; Bristol Myers Squibb: Consultancy; Array BioPharma: Other: Travel and accommodation expenses; Novartis: Consultancy, Other: Travel and accommodation expenses, Research Funding; Pfizer: Consultancy, Honoraria, Other: Travel and accommodation expenses; Astex Pharmaceuticals: Consultancy, Other: Travel and Accommodation expenses, Research Funding; Amphivena Therapeutics: Other: Travel and accommodation expenses, Research Funding; Eisai: Other: Travel and accommodation expenses; Agios: Other: travel, Research Funding; Amgen: Consultancy, Other: travel; Karyopharm Therapeutics: Research Funding; CTI: Research Funding; Mofitt Cancer Center: Research Funding; GlaxoSmithKline: Consultancy; Bayer: Consultancy, Other: Travel and accommodation expenses; MEI Pharma: Consultancy, Research Funding; Celgene: Consultancy, Other: travel and accommodation expenses, Research Funding; Agios: Consultancy, Research Funding; AbbVie: Consultancy, Other: travel and accommodations, Research Funding; Onconova Therapeutics: Research Funding; Tensha Therapeutics: Research Funding. Boyer:Argenx: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; UCB: Consultancy, Honoraria, Patents & Royalties, Research Funding; OGD2: Consultancy, Honoraria; Egle Tx: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Guzman:Samus Therapeutics: Other: Inventor on licensed IP; Seq RX: Current holder of stock options in a privately-held company; BridgeMedicines: Research Funding.