763 Intratumoral delivery of high potency STING agonists modulates the immunosuppressive myeloid compartment and induces curative responses in checkpoint-refractory glioblastoma models

BackgroundGlioblastoma is an aggressive primary brain malignancy that is characterized by a highly suppressive tumor microenvironment, including myeloid-derived suppressor cells, tumor-associated macrophages, and brain-resident microglia, but lacking significant T cell infiltration.1 2 This phenotype is reflected in the recently developed QKi-/- Pten-/- P53-/- (QPP) tumor model,3 which we show is resistant to PD1 or CTLA-4 blockade, but sensitive to agonists of the innate immune sensor Stimulator of Interferon Genes (STING). We have previously shown that agonists of the innate dsDNA-sensing cGAS-STING pathway are capable of proinflammatory repolarization in in vitro models of suppressive myeloid cells, although their function in the context of the Glioblastoma myeloid compartment in vivo remains poorly understood.4MethodsWe utilized the synthetic cyclic di-nucleotide STING agonists IACS-8803 (8803) and ML-RR-S2-CDA (MLRR) to assess survival and tumor immune infiltrate functional reprogramming in two orthotopic transplantable human and murine Glioblastoma tumor models, U87 and the recently developed QPP8 (Qki-/- Pten-/- P53-/-). Using in vitro models of M2-polarized microglia, we investigated the ability of natural (2'3'-cGAMP) and synthetic (MLRR and 8803) STING agonists to reverse immunosuppressive microglial polarization.ResultsWe found that intratumoral delivery of STING agonists significantly prolonged survival in the murine QPP8 orthotopic Glioblastoma tumor model, in contrast to checkpoint blockade which had no benefit on survival. In huNOG-EXL mice engrafted with human hematopoietic stem cells implanted with orthotopic U87 Glioblastoma, intratumoral delivery of STING agonists significantly prolonged survival and reduced expression of CD163 and CD206 on human tumor-infiltrating myeloid populations. Preliminary data suggests that in vitro suppressively-polarized microglia reduce expression of M2 functional markers, and increase expression of iNOS, PD-L1, CD80, and CD86 in a STING agonist potency-dependent manner.ConclusionsWe found that STING agonists can induce curative responses in checkpoint-refractory murine Glioblastoma models and mediate significant extension of survival in a humanized mouse U87 xenograft setting. This prolonged survival is associated with a decrease in immunosuppressive M2 functional markers in human tumor infiltrating myeloid populations. Additionally, M2-polarized microglia demonstrated a reduction in M2 functional markers and upregulation of proinflammatory M1 markers following treatment with STING agonists. Together these results indicate that delivery of STING agonists can induce proinflammatory repolarization of the Glioblastoma myeloid stroma, including both infiltrating myeloid populations and brain-resident microglia, to drive prolonged survival in refractory models of Glioblastoma.ReferencesGabrusiewicz K, Rodriguez B, Wei J, et al. Glioblastoma-infiltrated innate immune cells resemble M0 macrophage phenotype. JCI Insight 2016;1(2).Quail DF, Joyce JA. The microenvironmental landscape of brain tumors. Cancer Cell 2017;31(3):326–41.Shingu T, Ho AL, Yuan L, et al. Qki deficiency maintains stemness of glioma stem cells in suboptimal environment by downregulating endolysosomal degradation. Nat Genet 2017;49(1):75–86.Ager C, Boda A, Rajapakshe K, et al. (2021) “High potency STING agonists engage unique myeloid pathways to reverse pancreatic cancer immune privilege. JITC (in press)Ethics ApprovalAll experiments were conducted according to protocols approved by the University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee.

Intratumoral delivery of engineered recombinant modified vaccinia virus Ankara express-ing Flt3L and OX40L generates potent antitumor immunity through activating the cGAS/STING pathway and depleting tumor-infiltrating regulatory T cells

Intratumoral (IT) delivery of immune-activating viruses can serve as an important strategy to turn cold tumors into hot tumors, resulting in overcoming resistance to immune checkpoint block-ade (ICB). Modified vaccinia virus Ankara (MVA) is a highly attenuated, non-replicative vaccinia virus that has a long history of human use. Here we report that IT recombinant MVA (rMVA), lacking E5R encoding an inhibitor of the DNA sensor cyclic GMP-AMP synthase (cGAS), ex-pressing a dendritic cell growth factor, Fms-like tyrosine kinase 3 ligand (Flt3L), and a T cell co-stimulator, OX40L, generates strong antitumor immunity, which is dependent on CD8+ T cells, the cGAS/STING-mediated cytosolic DNA-sensing pathway, and STAT1/STAT2-mediated type I IFN signaling. Remarkably, IT rMVA depletes OX40hi regulatory T cells via OX40L/OX40 inter-action and IFNAR signaling. Taken together, our study provides a proof-of-concept for improving MVA-based cancer immunotherapy, through modulation of both innate and adaptive immunity.

Efficient Secretion of Murine IL-2 From an Attenuated Strain of Clostridium sporogenes, a Novel Delivery Vehicle for Cancer Immunotherapy

Despite a history dating back to the 1800s, using Clostridium bacteria to treat cancer has not advanced beyond the observation that they can colonise and partially destroy solid tumours. Progress has been hampered by their inability to eradicate the viable portion of tumours, and an instinctive anxiety around injecting patients with a bacterium whose close relatives cause tetanus and botulism. However, recent advances in techniques to genetically engineer Clostridium species gives cause to revisit this concept. This paper illustrates these developments through the attenuation of C. sporogenes to enhance its clinical safety, and through the expression and secretion of an immunotherapeutic. An 8.6 kb sequence, corresponding to a haemolysin operon, was deleted from the genome and replaced with a short non-coding sequence. The resultant phenotype of this strain, named C. sporogenes-NT, showed a reduction of haemolysis to levels similar to the probiotic strain, C. butyricum M588. Comparison to the parental strain showed no change in growth or sporulation. Following injection of tumour-bearing mice with purified spores of the attenuated strain, high levels of germination were detected in all tumours. Very low levels of spores and vegetative cells were detected in the spleen and lymph nodes. The new strain was transformed with four different murine IL-2-expressing plasmids, differentiated by promoter and signal peptide sequences. Biologically active mIL-2, recovered from the extracellular fraction of bacterial cultures, was shown to stimulate proliferation of T cells. With this investigation we propose a new, safer candidate for intratumoral delivery of cancer immunotherapeutics.

Image-Guided Intratumoral Delivery of Immunotherapeutics in Gastrointestinal Malignancies

Intratumoral (IT) administration of immunotherapy is a promising treatment strategy under clinical development for gastrointestinal malignancies. Due to its targeted nature, IT immunotherapies can generate regional proinflammatory microenvironments that result in the focal recruitment of tumor-specific immune cells. Precision targeting of tumors via IT immunotherapy injection theoretically produces a more robust immune response to the treated tumor itself and to distant metastatic tumors that share tumor-specific antigens with those of the treated tumor, while also minimizing the priming of the adaptive immune system to nonspecific antigens. Diverse arrays of IT immunotherapeutic agents including but not limited to lyophilized bacteria, viral vectors, cellular-based agents, molecules, and peptides, both as monotherapies and in combination with systemic immunotherapies, are in various stages of preclinical and clinical development. In this review, we summarize the current state of the art for IT immunotherapy and highlight potential future directions and their relevance to image-guided interventionalists.