Abstract Background: TIL cell therapy can induce durable disease regression in solid tumors. TIL engineered with regulatable mbIL15 (cytoTIL15TM cells) demonstrate interleukin 2-independent expansion and function (Burga SITC 2023) and can lead to durable clinical responses (Amaria ASCO 2024). Furthermore, TIL engineered with co-regulated expression of mbIL15 and LIGHT show enhanced efficacy in fibroblast-containing, immunologically cold tumors (Koscso AACR 2024). Chondrosarcomas are often of mesenchymal stem-cell origin and show similar transcriptional signature as fibrotic tumors, including high expression of LTBR and HVEM (TNFRSF14; LIGHT co-receptor). Herein, we sought to determine if tumor-reactive TIL engineered with mbIL15 and LIGHT could be produced from immune-excluded tumor types, such as chondrosarcoma. Methods: This retrospective study used tumors from patients with chondrosarcoma treated at Northwestern University to characterize the tumor microenvironment using immunohistochemistry, TCR sequencing, and spatial transcriptomics. Punches from intratumoral and peritumoral areas of selected tumor sections were compared. We also prospectively collected chondrosarcoma tumors to expand TIL using a conventional (non-engineered) process or the Obsidian process. TIL were characterized via flow cytometry, TCR sequencing, and single-cell sequencing. TIL function was evaluated with LIGHT functional assays as well as co-cultures with autologous tumor spheroids, with cytotoxicity marked by caspase 3/7 staining. Results: Chondrosarcoma tumors had significantly higher peritumoral levels of cells expressing nearly every immunologic marker tested, including CD45, CD68, CD4, CD8, CD20, CD56, CD14, CD206, and MECA-79. Gene expression analysis showed that while large sections of chondrosarcomas have a more inflammatory gene expression profile compared with other similar sarcomas, these genes are confined to the tumor's periphery. TCR sequences detected in the intratumoral area appeared to be from semi-invariant alpha-beta TCR-expressing, innate-like MAIT cells, whereas a more polyclonal T-cell fraction was present in the peritumoral area (2–959 unique complementarity-determining region 3 [uCDR3] in peritumoral region, vs 460–18989 uCDR3 in peritumoral region immune clusters, n=5). Additionally, chondrosarcoma showed 2.58-fold higher expression of HVEM compared with other sarcoma types, providing a rationale for LIGHT-engineered TIL. Co-culture experiments with TIL engineered with co-regulated mbIL15 and LIGHT manufactured from chondrosarcomas demonstrated enhanced cytotoxicity against autologous spheroids compared with non-engineered TIL (ANOVA, p<0.005). Conclusions: While chondrosarcoma is usually classified as an immunologically cold tumor, we observe peritumoral immune cells in chondrosarcoma, and using the Obsidian TIL expansion process coupled with mbIL15 and LIGHT engineering generates potent tumor-specific lymphocytes. These findings support further efforts to develop TIL cell therapy for immunologically cold tumors such as chondrosarcoma. Citation Format: Zheng Ao, Rusul Al-Marayaty, Bulent Arman Aksoy, Carmela Passaro, Farres Obeidin, Rachel Burga, Nishita Roy, Samer Attar, Terrance Peabody, Juliana Ng, Weiqing Jing, Himaly Shinglot, Ali Zhang, Alonso Villasmil Ocando, Andres Alvarado, Dexue Sun, Dhruv Sethi, Jan ter Meulen, Michelle Ols, Seth M Pollack. Effective generation of potent tumor-infiltrating lymphocytes (TIL) expressing regulatable membrane-bound IL15 (mbIL15) and LIGHT (TNFSF14) from immune-excluded chondrosarcoma [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B001.

Abstract Introduction: Adoptive cellular therapies (ACT) have encountered challenges in solid tumors due in part to the immunosuppressive tumor microenvironment (TME). We have developed OBX-115, TIL engineered to express mbIL15 regulatable using the cytoDRiVE® platform, which allows for TIL expansion, persistence, and anti-tumor efficacy under control of the FDA-approved small-molecule ligand, acetazolamide (ACZ), eliminating the need for co-administration of IL2 (NCT05470283). LIGHT, a tumor necrosis factor family member, interacts with lymphotoxin beta receptor (LTbR) and herpes virus entry mediator (HVEM) found on various TME cell types, including stromal cells such as cancer associated fibroblasts (CAF). In preclinical studies, LIGHT expression within a tumor has been linked to the formation of tertiary lymphoid structures and vascular normalization (Ramachandran Cancer Cell 2023), both associated with better clinical outcomes (Sautès-Fridman Nat Rev Cancer 2019). We hypothesized that engineering TIL with regulatable mbIL15 and LIGHT expression could enhance their efficacy by modifying the TME. Methods: TIL from colorectal (CRC) and head and neck squamous cell carcinoma (HNSCC) were transduced with retroviral vectors to express regulatable mbIL15 and LIGHT. ACZ-induced surface expression of mbIL15 and LIGHT in expanded TIL was examined using flow cytometry. Functional signaling of LIGHT was assessed through co-culture with Jurkat-HVEM-NF-kappaB reporter cells and LTbR+ HUVEC cells. In vitro, engineered TIL were tested in stromal-rich tumor models (CRC and HNSCC) by co-culturing with autologous patient-derived tumor/CAF hybrid spheroids. In vivo, antigen-independent TIL persistence was assessed in NSG mice without exogenous IL2. Syngeneic studies were performed to assess the efficacy of adoptively transferred mbIL15 and LIGHT-engineered Pmel cells (CD8+ T cells transgenic for a gp100-specific T cell receptor) in a subcutaneous cold tumor model (B16-F10). Results: Engineered TIL were successfully expanded without exogenous IL2. ACZ-dependent mbIL15 and LIGHT expression were confirmed, validating co-regulation and functionality in vitro. TIL engineered with mbIL15 and LIGHT displayed significantly increased cytotoxicity against autologous tumor/CAF spheroids compared with TIL expressing mbIL15 alone (p<0.005) in CRC and HNSCC tumor/CAF hybrid models. TIL with mbIL15 and LIGHT expanded in vivo and persisted for ≥42 days without exogenous IL2 support. Moreover, Pmel cells engineered with mbIL15 and LIGHT demonstrated durable anti-tumor efficacy in B16-F10 tumor-bearing mice, which was greater than Pmel cells engineered with mbIL15 alone (p<0.01). Conclusions: These preclinical results suggest that TIL engineered with regulatable mbIL15 and LIGHT using the cytoDRiVE platform have the potential to address the high unmet clinical need in cold tumors with suppressive TME, which are currently not amenable to ACT. Citation Format: Balazs Koscso, Zheng Ao, Carmela Passaro, Nirzari Shah, Ngoc Ly, Patricia Timpug, Bulent A. Aksoy, Dexue Sun, Dan Jun Li, Kerri-Lynn Sheahan, Violet Young, Theresa Ross, Benjamin Primack, Meghan Langley, Jeremy Tchaicha, Dhruv K. Sethi, Jan ter Meulen, Michelle Ols. Tumor-infiltrating lymphocytes (TIL) engineered with regulatable membrane-bound IL15 (mbIL15) and LIGHT (TNFSF14) show enhanced efficacy in fibroblast-containing cold tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB065.

Supplementary Table 1. Dose-response effects of human glioblastoma cells after treatment with NG34 or rQNestin34.5. Supplementary Figure S1. A, The dose-response curves represent cell viability assay for human astrocytes. Intracellular ATP was measured as an index of cell viability, five days after infection of human astrocyte cells with NG34 or rQNestin34.5. Data were normalized to maximum and minimum values before plotting the averaged values of six replicates with S.D. error bars and non-linear dose-response curves. Ranges of 50% effective doses (ED50) at 95% confidence intervals are 0.00094-0.0034 pfu (NG34) and 0.0011-0.0024 puf (rQNestin34.5), and values of R2 are 0.9423 (NG34) and 0.9748 (rQNestin34.5), respectively. B, viral replication in normal tissues was measured by titrating viral yields collected from cultured human astrocytes after HSV1 F strain (wild-type), rHSVQ, NG34 or rQNestin34.5 at 0.1 of their MOIs. Error bars represent S.D (n = 4). Supplementary Figure S2. The dose-response curves represent are obtained from the cell viability assay used in Table. 1. Supplementary Figure S3. NG34-gCRliFluc and rHSVQ-gCRliFluc oHSV were generated as described in the Materials and Methods section . Supplementary Figure S4. Bioluminescent imaging of xenografted human glioma indicates tumor regression upon oHSV treatment. Supplementary Figure S5. Reproducibility of therapeutic efficacy of NG34 in different models in vivo. Supplementary Figure S6. Gene expression of cytokines in mouse brains with NG34 inoculation. Supplementary Figure S7. Immunohistochemistry of the brains of athymic mice after the HSV-1 inoculation.

Purification of the scFvPD-1 from HEK293 cells

Engineering of NG34 to insert the sequence of the scFv against PD-1

In vivo comparison of scFvPD-1 production and viral gene expression in mouse and human GBM cells

Tumor infiltrating lymphocytes (TIL) have shown promising efficacy in immunologically “hot” solid tumors that have a high level of T cell infiltration, such as melanoma. However, current treatment regimens require high dose IL-2 administration to support TIL survival, which limits their clinical applications due to IL-2 related toxicity. Obsidian Therapeutics is engineering TIL with membrane bound IL-15 (mbIL15) to eliminate the dependence of TIL on exogenous IL-2, potentially enhancing the tolerability of TIL therapies. Because there is a high unmet medical need in patients with tumors colder than melanoma, we evaluated mbIL15-engineered TIL expansion and functionality from colorectal cancer (CRC) and sarcoma biopsies. Using an IL2-independent, proprietary rapid expansion process (REP) we successfully expanded mbIL15-engineered TIL from both CRC and sarcoma, despite significantly lower T-cell numbers in the tumor tissues (average of 12-15% CD45+ TIL versus 64% in melanoma). mbIL15-engineered TIL showed an enrichment for CD8+ T cells throughout the REP and high T cell receptor variable beta chain (TCR Vbeta) diversity. mbIL15-engineered TIL also exhibited lower immune checkpoint expression (LAG3/PD-1) and higher activation marker expression (CD25/CD27/CD28) when compared with unengineered TIL expanded from the same tumors using a conventional REP with IL-2. mbIL15-engineered TIL were polyfunctional, as defined by expression of more than one effector molecules (CD107a, perforin, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and granzyme b) in response to CD3/CD28 stimulation. To examine the cytotoxic function of mbIL15-engineered TIL, we developed autologous cell lines from the same tumors that were used to generate the TIL. Using whole exome and RNA sequencing we found that the autologous tumor cell lines maintained expression of conserved tumor antigens and HLA-expression when compared with the primary tumor. When co-cultured with the autologous tumor cell lines, mbIL15-engineered TIL secreted higher levels of IFN-γ and induced higher cytotoxicity as compared to unengineered TIL cultured with IL-2. Taken together, these data demonstrate that mbIL15-engineered TIL can successfully be expanded from comparatively “cold” tumors with low T-cell infiltration, such as CRC and sarcoma, while maintaining high TCR diversity and polyfunctionality and demonstrating higher cytokine production and cytotoxic activity against autologous tumor lines, compared to conventional TIL with IL2. Citation Format: Zheng Ao, Carmela Passaro, Bulent A. Aksoy, Balazs Koscso, Rachel Burga, Kyle Pedro, Natasha Ly, Nirzari Shah, Alonso V. Ocando, Gauri Kulkarni, Trisha Timpug, Seth M. Pollack, Jan ter Meulen, Michelle L. Ols. Engineering tumor infiltrating lymphocytes from sarcoma and colorectal tumors with membrane bound IL-15 for IL-2 independent expansion and enhanced cytotoxicity against autologous tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB093.

Background The clinical impact of tumor infiltrating lymphocytes (TIL) cell products is currently limited by suboptimal persistence and potency, as well as the need for high-dose adjuvant IL-2 treatment, which is associated with severe toxicities. Thus, we engineered an IL-2-independent TIL product, based on regulated expression of interleukin 15 (cytoTIL15TM cells), which has shown anti-tumor efficacy and persistence in human melanoma PDX models. Since the immuno-suppressive tumor microenvironment (TME) hinders cell therapies, we hypothesized that combining pleotropic cytokines of the interferon (IFN), IL-1, or TNF families with IL-15 would further enhance antitumor activity and that our cytoDRiVE® platform would allow pharmacologic control of these potent immune mediators. We tested constitutive and regulated combinations of a representative member of these cytokines with IL-15 in human TIL for in vitro polyfunctionality and in vivo antigen-independent persistence. We also engineered mouse pmel-TCR cells with cytokine combinations for evaluation in the syngeneic B16 melanoma model. Methods Human TIL were expanded and engineered with lentiviral vectors to express IL-15 with IFN-alpha, IL-18 (IL-1 family member) or undisclosed TNFSF-X (TNF superfamily member). Expanded TIL were immunophenotyped and assessed for polyfunctionality by flow cytometry after CD3/CD28 stimulation. Engineered TIL were transferred into NSG mice to assess antigen-independent TIL persistence in the absence of exogenous IL-2. Cytokines modified with our carbonic anhydrase 2 (CA2)-based cytoDRiVE® drug responsive domain (DRD) were evaluated for control of protein levels with the CA2 ligand, acetazolamide (ACZ). Cytokine expression was evaluated in flow cytometry and Meso Scale Discovery assays. To assess anti-tumor and TME remodeling capabilities, we used a syngeneic model with transduced pmel-TCR cells adoptively transferred into mice bearing B16 melanomas. Results Engineered TIL expressing both IL-15 and either IFN-alpha, IL-18 or TNFSF-X showed similar fold expansion, immunophenotype and polyfunctionality in vitro as TIL expressing only IL-15. Combination cytokine-expressing TIL showed similar in vivo antigen-independent persistence in the absence of IL-2 as TIL engineered with only IL-15. As compared to control pmel cells, sub-optimal cell doses of pmel T cells expressing both IL-15 and either IFN-alpha or, IL-18, showed improved efficacy and TME remodeling, while combining IL-15 with TNFSF-X resulted in significant tumor growth arrest of B16 melanoma tumors without escape. Conclusions While IL-15 drives expansion and persistence of cytoTIL15TM cells without IL-2, adding pleotropic and highly immune-stimulatory members of the IFN, IL-1 or TNF families may provide enhanced efficacy for patients with solid tumors marked by an immunosuppressive TME. Ethics Approval All animal studies were IACUC approved

Oncolytic virus (OV) therapy, which is being tested in clinical trials for glioblastoma, targets cancer cells, while triggering immune cells. Yet OV sensitivity varies from patient to patient. As OV therapy is regarded as an anti-tumor vaccine, by making OV-infected cancer cells secrete immunogenic proteins, linking these proteins to transcriptome would provide a measuring tool to predict their sensitivity. A set of six patient-derived glioblastoma cells treated ex-vivo with herpes simplex virus type 1 (HSV1) modeled a clinical setting of OV infection. The cellular transcriptome and secreted proteome (separated into extracellular vesicles (EV) and EV-depleted fractions) were analyzed by gene microarray and mass-spectroscopy, respectively. Data validation and in silico analysis measured and correlated the secretome content with the response to infection and patient survival. Glioblastoma cells reacted to the OV infection in a seemingly dissimilar fashion, but their transcriptomes changed in the same direction. Therefore, the upregulation of transcripts encoding for secreted proteins implies a common thread in the response of cancer cells to infection. Indeed, the OV-driven secretome is linked to the immune response. While these proteins have distinct membership in either EV or EV-depleted fractions, it is their co-secretion that augments the immune response and associates with favorable patient outcomes.

CAR T cells are highly effective at inducing remissions in patients with refractory B cell malignancies. The ex vivo process used to manufacture these therapies limits patient access and exposes T cells to non-physiologic conditions in culture. Viral vectors are efficient at genetically modifying T cells and are used in the ex vivo manufacture of most CAR T cell products but lack target-cell specificity. Here we describe how viral envelope glycoproteins, termed fusogens, engineered to target the CD8 co-receptor can efficiently and specifically deliver a chimeric antigen receptor (CAR) to human T cells in vivo that support target cell killing and tumor eradication. Modification of viral envelope proteins to include antibody-based binding domains (CD8 binders) can produce fusogen-viral vector compositions that deliver genetic payloads to CD8 T cells with therapeutic effect (1, 2). Screening CD8-targeted binders identified fusogens with a range of on-target cell transduction efficiencies and off-target cell line specificities. CD8-targeted fusogens with the highest efficiency were comparable to VSVg-pseudotyped lentiviruses at transducing CD8 T cells, but unlike VSVg showed CD8 T cell specific transduction. Interestingly, CD8-targeted fusogens were superior to VSVg-pseudotyped lentivirus at transducing non-activated human T cells. In vivo fusogen-mediated delivery showed efficient and specific CD8 T cell transduction and expression of a GFP reporter gene. Second-generation CARs containing a CD19 binding domain and a 41BB costimulatory domain delivered by targeted fusogen demonstrated CD8 specific expression and functional activity against non-malignant B cells and CD19+ Nalm6 leukemia cells. To demonstrate targeted fusogen-mediated in vivo CAR delivery and activity, we established Nalm6 xenografts in mice into which unmodified activated human PBMCs were infused. Activated human PBMCs alone were unable to control tumor growth. A single intravenous delivery of a CD8 fusogen containing a second-generation CD19 CAR transgene across a range of functional titer doses resulted in the generation of CD8 CAR Ts that eradicated the CD19+ tumor xenografts. A high percentage of T cells demonstrated CAR expression after fusogen delivery with clear specificity for the CD8+ cells. Importantly, the fusogen was able to generate a functional CAR response regardless of prior activation status of the T cells. Targeted in vivo delivery of CAR into T cell subsets may represent a novel therapeutic approach for human B cell malignancies. The ability to specifically deliver a CAR gene to a T cell in vivo would be transformative in terms of patient access and is likely to generate a qualitatively superior therapeutic T cell. References: 1) Bender, R. R. et al. PLOS Pathogens 12, e1005641 (2016). 2) Agarwal, S. et al. OncoImmunology 8, 1-8 (2019). Citation Format: Akinola Emmanuel, Patty Cruite, Hanane Ennajdaoui, Carmela Passaro, Paige Baldwin, Victoria Duback, Anna Liang, Jess Elman, Samantha Crocker, Shirisha Amatya, Caspian Harding, Allyse Mazarelli, Sergey Lyubinetsky, Vidur Patel, Avani Parikh, Kelan Hlavaty, Jason Rodriguez, Lauren Pepper, Albert Ruzo, Salvatore Iovino, Kutlu Elpek, Michael Laska, Trevor McGill, Donna Dambach, Terry Fry, Jagesh Shah. In vivo CAR T therapy: targeted in vivo gene delivery of a CAR using a CD8-specific fusogen results in tumor eradication [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB105.

Introduction: Ex vivo manufactured chimeric antigen receptor (CAR) T cell therapies are highly effective for treating B cell malignancies. However, the complexity, cost and time required to manufacture CAR T cells limits access. To overcome conventional ex vivo CAR T limitations, a novel gene therapy platform has been developed that can deliver CAR transgenes directly to T cells through systemic administration of a fusosome, an engineered, target-directed novel paramyxovirus-based integrating vector that binds specific cell surface receptors for gene delivery through membrane fusion. Here, we demonstrate that systemic administration of a CD8a-targeted, integrating vector envelope (i.e., fusogen) encoding an anti-CD20 CAR into Southern pig-tail macaques (M. nemestrina), which is a species permissive to the integrating vector-mediated transduction, results in T cell transduction and B cell depletion with no treatment-related toxicities.

Tumor interferon (IFN) signaling promotes PD-L1 expression to suppress T cell-mediated immunosurveillance. We identify the IFN-stimulated non-coding RNA 1 (INCR1) as a long noncoding RNA (lncRNA) transcribed from the PD-L1 locus and show that INCR1 controls IFNγ signaling in multiple tumor types. Silencing INCR1 decreases the expression of PD-L1, JAK2, and several other IFNγ-stimulated genes. INCR1 knockdown sensitizes tumor cells to cytotoxic T cell-mediated killing, improving CAR T cell therapy. We discover that PD-L1 and JAK2 transcripts are negatively regulated by binding to HNRNPH1, a nuclear ribonucleoprotein. The primary transcript of INCR1 binds HNRNPH1 to block its inhibitory effects on the neighboring genes PD-L1 and JAK2, enabling their expression. These findings introduce a mechanism of tumor IFNγ signaling regulation mediated by the lncRNA INCR1 and suggest a therapeutic target for cancer immunotherapy.

The mode of action for oncolytic viruses (OVs) in cancer treatment is thought to depend on a direct initial cytotoxic effect against infected tumor cells and subsequent activation of immune cell responses directed against the neoplasm. To study both of these effects in a mouse model of glioblastoma (GBM), we employed murine GBM cells engineered to constitutively express the type I Herpes Simplex Virus (HSV1) HSV-1 receptor, nectin-1, to allow for more efficient infection and replication by oncolytic HSV (oHSV). These cells were further engineered with a surrogate tumor antigen to facilitate assays of T cell activity. We utilized MRI-based volumetrics to measure GBM responses after injection with the oHSV and bioluminescent imaging (BLI) to determine oHSV replicative kinetics in the injected tumor mass. We found increased infiltration of both surrogate tumor antigen- and oHSV antigen-specific CD8+ T cells within 7 days after oHSV injection. There was no increase in tumor infiltrating CD8+ T cells expressing “exhaustion” markers, yet oHSV infection led to a reduction in PD-1+ CD8+ T cells in injected GBMs and an increase in IFNγ+ CD8+ T cells. There was a significant direct correlation between oHSV-mediated reduction in GBM volume and increased infiltration of both viral and tumor antigen-specific CD8+ T cells, as well as oHSV intratumoral gene activity. These findings imply that CD8+ T cell cytotoxicity against both tumor and viral antigens as well as intratumoral oHSV gene expression are important in oHSV-mediated GBM therapy.

Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM); however, a mechanistic connection in vivo has not been established. The purpose of this study is to characterize the effects of murine CMV (MCMV) on GBM growth in murine models. Syngeneic GBM models were established in mice perinatally infected with MCMV. We found that tumor growth was markedly enhanced in MCMV+ mice, with a significant reduction in overall survival compared with that of controls (P < 0.001). We observed increased angiogenesis and tumor blood flow in MCMV+ mice. MCMV reactivation was observed in intratumoral perivascular pericytes and tumor cells in mouse and human GBM specimens, and pericyte coverage of tumor vasculature was strikingly augmented in MCMV+ mice. We identified PDGF-D as a CMV-induced factor essential for pericyte recruitment, angiogenesis, and tumor growth. The antiviral drug cidofovir improved survival in MCMV+ mice, inhibiting MCMV reactivation, PDGF-D expression, pericyte recruitment, and tumor angiogenesis. These data show that MCMV potentiates GBM growth in vivo by increased pericyte recruitment and angiogenesis due to alterations in the secretome of CMV-infected cells. Our model provides evidence for a role of CMV in GBM growth and supports the application of antiviral approaches for GBM therapy.

Abstract Purpose: Glioblastoma (GBM) is resistant to standard of care. Immune checkpoints inhibitors (such as anti-PD-1 mAbs) efficiently restore antitumor T-cell activity. We engineered a new oncolytic herpes simplex virus (oHSV) expressing a single-chain antibody against PD-1 (scFvPD-1) to evaluate its efficacy in mouse models of GBM. Experimental Design: NG34scFvPD-1 expresses the human GADD34 gene transcriptionally controlled by the Nestin promoter to allow replication in GBM cells and a scFvPD-1 cDNA transcriptionally controlled by the CMV promoter. ELISA assays were performed to detect binding of scFvPD-1 to mouse and human PD-1. In vitro cytotoxicity and replication assays were performed to measure NG34scFvPD-1 oncolysis, and scFvPD-1 expression and secretion were determined. In vivo survival studies using orthotopic mouse GBM models were performed to evaluate the therapeutic potency of NG34scFvPD-1. Results: NG34scFvPD-1–infected GBM cells express and secrete scFvPD-1 that binds mouse PD-1. The introduction of the scFvPD-1 sequence in the viral backbone does not alter the oncolytic properties of NG34scFvPD-1. In situ NG34scFvPD-1 treatment improved the survival with a tail of durable survivorship in 2 syngeneic immunocompetent mouse models of GBM. Mice that survived the first GBM challenge rejected the second challenge of GBM when implanted in the contralateral hemisphere. However, this was not true when athymic mice were employed as the recipients of the second challenge, consistent with the need for an intact immune system to obtain a memory response. Conclusions: NG34scFvPD-1 treatment induces a durable antitumor response in 2 preclinical mouse models of GBM with evidence for antitumor memory.