Improved Anti-Tumor Response of Chimeric Antigen Receptor T Cell (CART) Therapy after GM-CSF Inhibition Is Mechanistically Supported By a Novel Direct Interaction of GM-CSF with Activated Carts

Abstract

Despite the remarkable activity of CD19 directed chimeric antigen receptor T cell (CART19) therapy in the treatment of B cell malignancies, the therapy is limited by the development of severe life-threatening complications such as neurotoxicity (NT) and cytokine release syndrome (CRS). Additionally, durable efficacy following CART19 therapy is not optimal. Emerging literature suggests that inhibitory myeloid cells and their cytokines play an important role in inducing CAR-T cell toxicities and also contribute to the inhibition of their effector functions. Specifically, GM-CSF was identified as a critical cytokine in the development of NT and CRS after CART19 therapy. Neutralization of GM-CSF in preclinical models has been shown to prevent CRS and enhance CART anti-tumor activity through modulation of myeloid cell behavior, resulting in reduction of tumor associated macrophages. In addition to the predominant effect of GM-CSF on myeloid cells, there appears to be a direct effect on CART19 cells. In this study, we aimed to evaluate the direct effect of GM-CSF on CART cells. Our initial finding of enhanced anti-tumor activity of CART19 cells after GM-CSF inhibition suggested a direct effect of GM-CSF on CART cells (Sterner et al. 2019, Blood). In these experiments, a guide RNA (gRNA) targeting exon 3 of GM-CSF in a CRISPR-Cas9 lentiviral vector was used to knock out GM-CSF during CART cell manufacturing. This resulted in a disruption efficiency of approximately 70% of the GM-CSF gene. Using a high tumor burden xenograft model for relapsed acute lymphoblastic leukemia established through the engraftment of the CD19+ luciferase+ NALM6 cell line (1x106 cells intravenously) in immunocompromised NOD-SCID-γ-/- mice, treatment with low doses of GM-CSFk/o CART19 resulted in improved anti-tumor activity and overall survival compared to GM-CSFwt CART19. The lack of myeloid cells in this model pointed to an intrinsic effect of GM-CSF on CAR-T cells. To ensure that this was not related to an off-target effect of the gRNA, whole exome sequencing (WES) of the modified cells was performed. There was no difference in the single nucleotide variants or indel counts between GM-CSFk/o CART19 and GM-CSFwt CART19 (Figure 1A). WES was significant for only two alterations in the exon 3 targeted by the gRNA (Figure 1B). The high efficiency and accuracy of targeting exon 3 of GM-CSF indicated that the improvement in CART function is unlikely related to an off-target effect of the gRNA and suggested the possibility of a direct interaction between GM-CSF and CART cells as a potential mechanism behind the improved anti-tumor activity. To investigate this interaction, we first assessed the expression of GM-CSF receptors on CART cells. While resting CART cells do not express any GM-CSF receptors, our analysis robustly indicates that activated CART cells significantly upregulate both α and β subunits of the GM-CSF receptor. This finding was significant both when CART cells are activated through their T cell receptor with CD3/CD28 beads (Figure 1C) or through the CAR with irradiated NALM6 cells (Figure 1D). Additionally, activated GM-CSFk/o CART19 cells also upregulated GM-CSF receptors, indicating this upregulation is induced by T cell stimulation. These results suggest a direct interaction between GM-CSF and upregulated GM-CSFR on activated CART cells. Having demonstrated that 1) GM-CSF depletion enhances CART19 efficacy in xenograft models in the absence of monocytes and 2) T cell activation increases GM-CSF receptor expression, we sought to uncover the downstream changes resulting from this effect. Transcriptome interrogation of GM-CSFk/o CART19 revealed a distinct signature including a significant inhibition of the Fas death pathway, a known critical pathway in inducing CART cell apoptosis. This suggests a potential mechanism for enhanced CART19 activity following GM-CSF depletion (Figure 1E). In summary, our results strongly indicate that CART cells increase expression of GM-CSF receptor subunits when activated, resulting in modulation of CART cell functions. Furthermore, GM-CSFk/o CART19 revealed a distinct transcriptome signature compared to GM-CSFwt CART19. These results illuminate a novel mechanism for a direct modulatory effect of GM-CSF on activated CART cells. Disclosures Cox: Humanigen: Patents & Royalties. Sterner:Humanigen: Patents & Royalties. Sakemura:Humanigen: Patents & Royalties. Ahmed:Humanigen: Employment. Chappell:Humanigen: Employment. Durrant:Humanigen: Employment. Parikh:Acerta Pharma: Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Genentech: Honoraria; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Ascentage Pharma: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Lentigen: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Kite/Gilead: Research Funding; Morphosys: Research Funding.

First Clinical Use of Lenzilumab to Neutralize GM-CSF in Patients with Severe and Critical COVID-19 Pneumonia

“…We have recently shown that GM-CSF depletion results in modulation of apoptosis pathways in T cells. 22 It is unclear at this time if the increase in lymphocyte count is secondary to clearance of SARS-CoV-2 virus, or a direct effect of GM-CSF on T cells; this question will be answered in the planned phase III trial. Figure 3 Five patients received other pharmacotherapies targeting COVID-19 besides lenzilumab.…”

“…21 GM-CSF depletion results in modulation of myeloid cell behavior, a specific decrease in their inflammatory cytokines, and a reduction in tissue trafficking, 21 while enhancing T-cell apoptosis machinery. 22 These biological effects prevented both CRS and neuro-inflammation after CART therapy in preclinical models and are being tested in a phase Ib/II clinical trial (NCT 04314843).…”

Akt-Fas to Quell Aberrant T Cell Differentiation and Apoptosis in Covid-19

“…Sorbera et al claim SARS-Cov-induced apoptosis via a caspase-dependent mechanism may suggest inhibition of the Fas/FasL interaction may have efficacy in Sars-Cov-2, but the rationale disregards differentiation ( 45 ). Finally, in a CRISPR-Cas9 lentiviral vector knock out of GM-CSF in CAR T cells, Fas expression was significantly inhibited, which the authors argue would reduce CAR-T apoptosis ( 46 ), and GM-CSF inhibition trials are undergoing. Considering 1) the role the immune system may have in Covid-19 pathogenesis, 2) the observation that FasL is upregulated ( 21 ), and 3) that Fas signaling induces Akt-mediated differentiation, investigation is warranted.…”

Advances in CAR T Therapy for Hematologic Malignancies

“…Granulocyte-macrophage colony stimulating factor (GM-CSF) is upregulated and released by activated CAR T which may promote macrophage activation. 73 The critical role of GM-CSF is further evidenced in animal models by reductions in CRS severity observed both with GM-CSF blockade following CAR T 74 or with GM-CSF gene knockout CAR T. 75 Release of IL-6 is further stimulated by release of IL-1 from APCs. 76 CAR T and other activated T cells appear to be the primary source of interferon (IFN)-ɣ and IL-2 during CRS.…”