Axicabtagene ciloleucel (axi-cel) is a chimeric antigen receptor (CAR) T cell therapy for relapsed or refractory large B cell lymphoma (LBCL). Here, we evaluated whether immune dysregulation, present prior to CAR-T cell therapy, associated with treatment failure. Tumor expression of interferon (IFN) signaling, high blood levels of monocytic myeloid-derived suppressor cells (M-MDSCs), and high blood IL-6 and ferritin each associated with a lack of durable response. Similar to other cancers, we found that in LBCL tumor IFN signaling is associated with the expression of multiple checkpoint ligands including PD-L1, and these were higher in patients who lacked durable responses to CAR-T therapy. Moreover, tumor IFN signaling and blood M-MDSCs associated with decreased axi-cel expansion. Finally, patients with high tumor burden had higher immune dysregulation with increased serum inflammatory markers and tumor IFN signaling. These data support that immune dysregulation in LBCL promotes axi-cel resistance via multiple mechanistic programs: insufficient axi-cel expansion associated with both circulating M-MDSC and tumor IFN signaling, that also gives rise to expression of immune checkpoint ligands.
The use of electric fields in vivo to deliver DNA, called electroporation, has the potential to broadly impact vaccination and disease treatment. The evidence for this has emerged from a large number of recently completed and ongoing clinical trials. The methods for applying electric fields to tissues traditionally involve contact between metal electrodes and the tissue. In this study, we investigated the use of helium plasma as a noncontact method for electrically treating tissue in a manner that results in the uptake and expression of foreign DNA in murine skin. More specifically, our goal was to demonstrate that DNA encoding a model-secreted protein could be delivered, detected in the blood, and remain functional to produce its known biological effect. Murine erythropoietin (EPO) was the model-secreted protein. Results clearly demonstrated that an intradermal DNA injection followed by plasma treatment for 2 min resulted in elevated levels of EPO in the blood and corresponding hemoglobin increases that were statistically significant relative to DNA injection alone.
Introduction: Approximately 60% of Large B cell Lymphoma (LBCL) patients that receive CD19 CAR T cell therapy with axicabtagene ciloleucel (axi-cel) experience lymphoma progression (Locke et al. Lancet Oncol. 2019) and the likelihood of response to subsequent therapy is low (Spiegel, Dahiya et al. ASCO 2019). Target loss of CD19 is observed in less than a third of patients experiencing relapse. Alternative mechanisms of resistance to axi-cel are poorly understood. Lymphoma patients with elevated serum markers of systemic inflammation, such as ferritin and IL-6, have worse outcomes following axi-cel (Locke, Neelapu et al. Mol.Ther.2017; Faramand et al. ASH 2018). We hypothesized that suppressive monocytic myeloid derived suppressor cells (M-MDSCs), which are associated with worse chemotherapy outcomes in LBCL (Azzaoui et al. Blood 2016), and tumor driven inflammation may be present and responsible for decreased efficacy of axi-cel in LBCL. Methods: LBCL patients undergoing axi-cel treatment were enrolled onto prospective sample collection protocols. Patients were stratified for analysis into ongoing responders (complete response or partial response) or relapsed (progressive disease) after a minimum of 3 months follow-up (range 3 - 15 months). M-MDSCs, defined as a Lin-, CD11b+, CD33+, CD15-, CD14+, HLA-DRlow population, were sorted from leftover apheresis material after collection for axi-cel manufacture. M-MDSC ability to suppress proliferation of autologous T cells stimulated with CD3/CD28 coated beads was measured by 3H thymidine incorporation. Circulating peripheral blood M-MDSCs, quantified by % of live cells by flow cytometry, were measured at the time of apheresis and serially after axi-cel infusion until day 30. In vitro mouse experiments utilized a CD19-CD28 CAR and cytokine-induced bone marrow MDSCs (Thevenot et al. Immunity 2014). Cytokines were measured by ELISA and cytotoxicity against CD19 bearing cell lines used xCELLigence real-time cell analysis, as we have done previously (Li et al. JCI Insight 2018).Tumor biopsies were taken within 1 month prior to infusion of axi-cel. Limited gene expression profiling of tumor microenvironment (TME) genes used the Nanostring IO360 panel (770 genes). Analysis used nSolver to identify cell types, GSEA and differential gene expression between groups. Results: First, we demonstrated that M-MDSCs sorted from patient apheresis material suppressed the proliferation of autologous T cells (n=6). We next enumerated M-MDSCs in the peripheral blood (n = 32). M-MDSC numbers initially decreased after lymphodepleting chemotherapy but recovered to baseline levels by day +10. The level of M-MDSCs following CAR T cell therapy strongly correlated with pre-CAR T baseline levels (R = 0.871, p <0.0001), suggesting that the number of M-MDSCs present during CAR T cell expansion is dependent on factors already present before therapy began. M-MDSC levels were significantly higher in patients who subsequently relapsed, both at baseline (p= 0.01) and after axi-cel (p=0.04), as compared to patients with durable response. Mouse MDSCs were able to suppress CAR T cell IFN-gamma excretion (p<0.0001) and cytotoxicity (p<0.0001) in vitro. To evaluate the role of the TME we interrogated limited set gene expression profiling on patient (n=27) pre-axi-cel tumor biopsies. By cell type scoring, the macrophage gene score was significantly higher in patients who relapsed after CAR T therapy (p <0.001). By differential gene expression and gene set enrichment, patients who relapsed had a significantly higher expression (p <0.01) of multiple genes indicative of chronic interferon (IFN) signaling including higher levels of OAS2, OAS3, IFI6 and IFIT1, as well as the IFN-stimulated macrophage gene SIGLEC-1/CD169. Conclusions: Systemic inflammatory myeloid cytokines, circulating M-MDSCs in the blood and chronic IFN in the TME all associate with LBCL relapse after axi-cel CAR T cell therapy. Our observations support that CAR T cells can be suppressed by baseline patient and tumor-related factors and strategies to overcome these factors should be targeted to improve patient outcomes. MDJ and HZ contributed equally. Disclosures Jain: Kite/Gilead: Consultancy. Bachmeier:Kite/Gilead: Speakers Bureau. Chavez:Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; Kite Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals, Inc.: Speakers Bureau. Shah:Jazz Pharmaceuticals: Research Funding; Incyte: Research Funding; Kite/Gilead: Honoraria; Celgene/Juno: Honoraria; Pharmacyclics: Honoraria; Adaptive Biotechnologies: Honoraria; Spectrum/Astrotech: Honoraria; Novartis: Honoraria; AstraZeneca: Honoraria. Mullinax:Iovance: Research Funding. Davila:Celgene: Research Funding; GlaxoSmithKline: Consultancy; Precision Biosciences: Consultancy; Novartis: Research Funding; Atara: Research Funding; Bellicum: Consultancy; Adaptive: Consultancy; Anixa: Consultancy. Locke:Kite: Other: Scientific Advisor; Novartis: Other: Scientific Advisor; Cellular BioMedicine Group Inc.: Consultancy.
Background: Remarkable durable responses are seen with chimeric antigen receptor (CAR) T cell therapy in B cell lymphoma, however the majority of patients relapse (Locke et al. Lancet Oncol. 2019). Improvements enabling CAR T cells (CAR-T) to circumvent mechanisms of resistance may increase efficacy. Hypoxia, nutrient deprivation and acidosis, all common in the tumor microenvironment (TME), impair metabolic function necessary for CAR-T to kill tumor (Chang et al. Cell 2015). The metabolic response gene peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) co-activates genes that upregulate mitochondrial and glycolytic machinery for ATP synthesis from myriad carbon sources. Post translational modifications (PTM) fine tune PGC-1α activity to meet energy demands (Luo et al. IJC 2019). We hypothesized that CAR-T co-expressing full-length PGC-1α or the truncated (ie. short) NT-PGC-1α isoform, with mutations that prevent suppressive PTMs, would confer metabolic flexibility to improve function under TME conditions. Methods: We constructed four PGC-1α encoded retroviral vectors with an IRES and DsRed fluorescent protein: full-length wild type (WT); full-length mutant (GA); wild type short isoform (NT); and mutant short isoform (mNT). GA contained T295A and S571A mutations to abrogate GSK3β and Akt mediated PTMs. mNT sequence contained K to A mutations at K78/K145/K184/K254 to prevent acetylation by GCN5, and L to A mutations of the nuclear export sequence corresponding to L29/L33/L36/L38. Human CD8 T cells were activated with αCD3/αCD28 beads + 100 IU IL-2/mL, and transduced at 48 hr. to express FMC63-CD28/CD3z CAR and non-functional truncated CD34. Cells were co-transduced with WT, or in the case of metabolically flexible CAR T cells (mfCAR-T) with a mutant and/or short isoform PGC-1α vector. After 7 days of expansion CD34+DsRed+ cells were isolated by FACS. In vitro experiments were performed within 2 weeks to characterize mitochondrial dynamics/oxidative stress (flow cytometry), cytokine secretion (ELISA), and real-time cytotoxicity (xCelligence). The effect of glucose restriction was evaluated in normal (10 mM) and low glucose (0.01 mM) medium. A Mitochondrial stress test (Seahorse) was performed 30 days after FACS. CAR-T (WT and control w/o co-transduction) and mfCAR-T were stimulated with CD19+ K562 or 3T3 cells. Results: Representative PGC-1α metabolic fitness target genes (ERRα, TFAM, and NRF2) were increased in mfCAR T cells (p≤0.001). mfCAR-T exhibited decreased mitochondrial biomass (p≤0.01) and mitochondrial membrane potential (MMP) (p≤0.01) in both glucose conditions. However, MMP:mitochondrial biomass and autophagy were greater (p≤0.01, p≤0.001), suggesting accelerated mitochondrial quality control (MQC). Oxidative stress was generally decreased (p≤0.01) in mfCAR-T, accompanied by reduced apoptosis. All mfCAR and control CAR T cells cytolysed 100% of targets at a 1:1 ratio but differed in cytolytic rate. Relative to CAR only, WT CAR-T and GA mfCAR-T killed 1.6 and 1.9 times faster, while shorter isoforms required 1.9 times longer to lyse all targets. IFNγ and IL-2 secretion by GA-mfCAR-T was increased above control CAR-T and other mfCAR T cells (p≤0.01), while others were similar. At 30 days both WT-CAR-T and all 3 mfCAR-T had increased spare respiratory capacity (SRC) compared to control CAR-T (p≤0.05); however ATP production and OCR/ECAR was increased (p≤0.001, p≤0.0.05) in mfCAR-T above control CAR-T and WT-CAR-T. Conclusion: Enforced expression of mutant or truncated PGC-1α in CAR-T enhanced mitochondrial quality control with commensurate function. mfCAR-T cells exhibited equivalent cytotoxicity in vitro, improved survival, and a metabolism less reliant on glucose. Stark differences in SRC, OCR/ECAR, and mitochondrial ATP production between WT and mfCAR-T suggest signaling pathways in CAR T cells may target PTM mediated suppression of PGC-1α and lead to metabolic exhaustion in the TME. mfCAR-T are a promising new strategy to improve the function of CAR-T cells in the TME. Further in vitro and in vivo experiments are needed to validate the approach. Disclosures Locke: Kite, a Gilead Company: Consultancy, Research Funding; Celgene/Bristol-Myers Squibb: Consultancy; Cellular Biomedicine Group: Other: Consultancy with grant options; Wugen: Consultancy; GammaDelta Therapeutics: Consultancy; Calibr: Consultancy; Allogene: Consultancy; Novartis: Consultancy.
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