EXTH-81. RE-EXPRESSION OF CXCL9 FOLLOWING ADENO-ASSOCIATED VIRUS (AAV) TRANSGENE DELIVERY TRANSFORMS THE IMMUNOLOGICAL LANDSCAPE OF GLIOBLASTOMA, SENSITIZING TUMORS TO PD-1 IMMUNE CHECKPOINT BLOCKADE (ICB)

Abstract

Abstract BACKGROUND To date, few glioblastoma (GBM) patients have demonstrated clinical benefit from treatment with anti-PD-1 ICB. GBM is notoriously devoid of lymphocytes, signifying that a possible obstacle for effective ICB response may be the limited ability of cytotoxic T-lymphocytes (CTLs) to home to these tumors. CTLs are recruited via long-range signaling mediated by the diffusion of chemokines present in inflammatory environments. Following a comprehensive chemokine screen of clinical tumor specimens, we identified that CXC motif ligand 9 (CXCL9) is notably absent in GBM. To test if GBM reconstitution with CXCL9 could transform these intractable tumors into ICB-responsive, we leveraged AAV gene therapy to generate durable production of this potent lymphotactic signal. HYPOTHESIS AAV mediated re-expression of CXCL9 will improve lymphotaxis in GBM, sensitizing these tumors to PD-1 ICB. METHODS 3-dimensional whole brain fluorescent imaging was performed to characterize the distribution, tropism, and durability of AAV transgene expression in preclinical GBM models. Single-cell RNA sequencing (scRNAseq) and multiparameter flow cytometry was utilized to immunologically profile tumors in response to single and combination treatment. Survival studies with and without lymphocyte depletion were performed to evaluate immunogenic responsiveness of therapy. RESULTS Intra-tumor delivery of AAV6 yields stable transduction of tumor-reactive astrocytes, producing a localized, peri-tumoral pattern of transgene signal distribution. Combination therapy increases tumor infiltration by lymphocytes. scRNAseq analyses reveal widespread innate and adaptive immune activation in response to each CXCL9 and PD-1 ICB treatment. Infiltrating CD8 T-lymphocytes demonstrate inflammatory signatures associated with increased effector function, including antigen presentation, cytotoxicity, and TCR signaling. Combination treatment considerably improved overall survival in two distinct GBM models, with durable responses dependent on CD8 T-lymphocyte infiltration. CONCLUSIONS This work suggests direct conditioning of the tumor microenvironment by AAV-CXCL9 could potentially overcome a key resistance factor in GBM, sensitizing tumors to immune mediated therapies such as PD-1 ICB.