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Oncolytic virotherapy aims to activate host antitumor immunity, but the effectiveness of current intratumoral herpes-simplex virus (HSV) based monotherapy is limited. Here, to boost the potency of cancer cell targeting α-herpesviruses derived from HSV or Varicella-Zoster Virus (VZV), we coinjected them with myeloid cell-tropic β-herpesviruses derived from cytomegalovirus (CMV) which can powerfully modulate immune responses. Co-administered CMV was shown to promote intratumoral replication of HSV, increase local release of pro-inflammatory cytokines, inhibit adaptive antiviral immune responses, enhance systemic antitumor responses, and lead to superior control of both injected and contralateral tumors. CMV-encoded myeloid-tropic genes, NK evasins (i.e. the vMHC-I homolog), and the m02-m16 region (involved in downregulating classical MHC-I) were implicated in enhancing antitumor activities. CMV is a promising adjuvant to reshape and enhance the antitumor immune responses following α-herpesvirus therapy.

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Although chimeric antigen receptor T (CART)–cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor in vivo expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)–directed CART cells to express NIS (NIS+CART19 and NIS+BCMA-CART, respectively) and tested the sensitivity of 18F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NIS+CART19 and NIS+BCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive 125I uptake in vitro. 18F-TFB-PET detected NIS+CART cells in vivo to a sensitivity level of 40,000 cells. 18F-TFB-PET confirmed NIS+BCMA-CART-cell trafficking to the tumor sites in localized and systemic tumor models. In a xenograft model for CART-cell toxicity, 18F-TFB-PET revealed significant systemic uptake, correlating with CART-cell in vivo expansion, cytokine production, and development of CRS-associated clinical symptoms. NIS provides a sensitive, clinically applicable platform for CART-cell imaging with PET scan. 18F-TFB-PET detected CART-cell trafficking to tumor sites and in vivo expansion, correlating with the development of clinical and laboratory markers of CRS. These studies demonstrate a noninvasive, clinically relevant method to assess CART-cell functions in vivo.

Supplementary Table S1

Eligibility criteria to ensure pet dogs meet physical and laboratory candidacy for entry into this clinical trial

Summary of viral shedding in a variety of biological samples

A summary of canine patient disease types and methods to assess disease burden

Summary of viral transgene expression as assessed by measurement of human IFN-beta

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Representative histological sections of tumor removed from patient UTK01

Purpose: Attenuated measles viruses are promising experimental anticancer agents currently being evaluated in a phase I dose escalation trial for ovarian cancer patients. Virus attachment, entry, and subsequent intercellular fusion between infected and uninfected neighboring cells are mediated via the two measles receptors (CD46 and SLAM). To minimize potential toxicity due to measles virus–associated immunosuppression and infection of nontarget tissues, we sought to develop an ovarian cancer exclusive fully retargeted measles virus.Experimental Design and Results: Interactions of measles virus with its natural receptors were ablated, and a single-chain antibody (scFv) specific for α-folate receptor (FRα), a target overexpressed on 90% of nonmucinous ovarian cancer, was genetically engineered on the viral attachment protein (MV-αFR). Specificity of virus tropism was tested on tumor and normal cells. Biodistribution of measles virus infection was evaluated in measles-susceptible CD46 transgenic mice, whereas antitumor activity was monitored noninvasively by bioluminescence imaging in xenograft models. Tropism and fusogenic activity of MV-αFR was redirected exclusively to FRα without compromise to virus infectivity. In contrast to the parental virus, MV-αFR has no background infectivity on normal human cells. The antitumor activity of MV-αFR, as assessed by tumor volume reduction and overall survival increase, was equal to the parental virus in two models of human ovarian cancer (s.c. and i.p.).Conclusions: A FR-exclusive ovarian cancer targeted oncolytic virus was generated and shown to be therapeutically effective, thus introducing a new modality for FR targeting and a candidate measles virus for clinical testing.

Supplementary Figure 1 from Retargeted Oncolytic Measles Strains Entering via the EGFRvIII Receptor Maintain Significant Antitumor Activity against Gliomas with Increased Tumor Specificity

Among the best-characterized genetic alterations in gliomas is the amplification of the epidermal growth factor receptor (EGFR) gene, present in ∼40% of glioblastoma multiforme, and frequently associated with the EGFRvIII gene rearrangement. We have previously shown that attenuated vaccine strains of measles virus have potent antitumor activity against gliomas, and identified H protein mutations, which ablate recognition of the natural measles virus receptors CD46 and SLAM. Retargeted recombinant viruses were generated from the measles Edmonston-NSe vaccine strain displaying a single-chain antibody against EGFRvIII at the COOH terminus of H and containing the marker green fluorescent protein (GFP) gene in position 1. Two different H mutants were employed: HSNS (V451S, Y481N, and A527S)-CD46 blind, and HAA (Y481A and R533A)-CD46 and SLAM blind. MV-GFP virus was used as a positive control. Both EGFRvIII-retargeted viruses had significant antitumor activity against EGFRvIII-expressing glioblastoma multiforme but no cytopathic effect against normal cells. In an orthotopic model of EGFRvIII-expressing GBM39 xenografts, there was comparable therapeutic efficacy between retargeted strains and unmodified MV-GFP and statistically significant prolongation of survival in treated animals compared with the control group (P = 0.001). Formation of syncytia was observed in tumors treated with retargeted viruses, with a surrounding infiltrate consisting of macrophages and natural killer cells. In summary, EGFRvIII-retargeted oncolytic measles virus strains have comparable therapeutic efficacy with the unmodified MV-GFP strain against EGFRvIII-expressing glioma lines and xenografts with improved therapeutic index, a finding with potential translational implications in glioma virotherapy. (Cancer Res 2006; 66(24): 11840-50)

Supplementary Figure Legend from Retargeted Oncolytic Measles Strains Entering via the EGFRvIII Receptor Maintain Significant Antitumor Activity against Gliomas with Increased Tumor Specificity

Supplementary Figure 1 from Phase I Trial of Intraperitoneal Administration of an Oncolytic Measles Virus Strain Engineered to Express Carcinoembryonic Antigen for Recurrent Ovarian Cancer

Supplementary Figure 4 from Phase I Trial of Intraperitoneal Administration of an Oncolytic Measles Virus Strain Engineered to Express Carcinoembryonic Antigen for Recurrent Ovarian Cancer

Supplementary Figure Legends 1-5 from Phase I Trial of Intraperitoneal Administration of an Oncolytic Measles Virus Strain Engineered to Express Carcinoembryonic Antigen for Recurrent Ovarian Cancer

Supplementary Figure 5 from Phase I Trial of Intraperitoneal Administration of an Oncolytic Measles Virus Strain Engineered to Express Carcinoembryonic Antigen for Recurrent Ovarian Cancer

Supplementary Figure 2 from Phase I Trial of Intraperitoneal Administration of an Oncolytic Measles Virus Strain Engineered to Express Carcinoembryonic Antigen for Recurrent Ovarian Cancer

Supplementary Figure 3 from Phase I Trial of Intraperitoneal Administration of an Oncolytic Measles Virus Strain Engineered to Express Carcinoembryonic Antigen for Recurrent Ovarian Cancer

Supplementary Table 1 from Phase I Trial of Intraperitoneal Administration of an Oncolytic Measles Virus Strain Engineered to Express Carcinoembryonic Antigen for Recurrent Ovarian Cancer

Virus neutralizing antibody (vnAb) titers are the strongest laboratory correlate of protection from SARS-CoV-2. Providing individuals with real-time measures of their vnAb titers is predicted to improve their ability to make personal wellness decisions. Yet, widespread commercial testing of SARS-CoV-2 vnAbs does not currently occur. Here, we examined whether knowing their vnAb titer impacted wellness decision-making among individuals. To this end, starting on January 1, 2021, we offered all employees from two companies free IMMUNO-COV™ testing and conducted a survey to assess their behaviors and decisions regarding booster vaccination. IMMUNO-COV is a clinically validated, surrogate virus assay that quantitates serum titers of SARS-CoV-2 vnAbs. To help participants gauge their level of protection based on their vnAb titer, we calibrated IMMUNO-COV titers to the World Health Organization (WHO) International Standard (IU/mL), making them comparable to published reports of correlates of protection, and we fit historical IMMUNO-COV vnAb titer values into predictive models of immune protection from COVID-19. As expected, data for the 56 program participants showed variability in vnAb titers post vaccination, rates vnAb decay, and fold-increases in vnAb titers after booster vaccination. Based on the participant survey, the majority (66%) of participants indicated that knowing their vnAb titer impacted their social behaviors and/or their decision on the timing of a booster vaccination. Several participants indicated that knowing their vnAb titer contributed to their peace of mind regarding their high level of protection from COVID-19. Together, these data demonstrate that regular determination of SARS-CoV-2 neutralizing antibody titers can significantly impact decisions regarding social interactions and timing of booster vaccinations.

Oncologic viruses (OV) can induce tumor death through both direct oncolysis and immune-mediated destruction. Our study investigates the use of a novel OV with a Vesicular Stomatitis Virus (VSV) vector modified to express interferon-beta (IFN-b) and Tyrosinase Related Protein 1 (TYRP1) (VSV-IFNb-TYRP1). VSV is an attractive vector since humans are not naturally infected with VSV; thus, pre-existing immunity is minimal. IFN-b leads to increased T-cell responses and tumor cell apoptosis, while TYRP1, a melanocyte differentiation antigen, increases immunogenicity of the therapy. We conducted a Phase 1 clinical trial with a 3+3 design in patients with metastatic uveal melanoma. VSV-IFNb-TYRP1 was injected into a liver metastasis under image guidance, then administered on the same day as a single intravenous (IV) infusion. IV doses started at dose level (DL) 1 1 × 1010 TCID50, then escalated to DL2 3 × 10 10, DL3 1 × 1011, and DL4 3 × 1011. The dose delivered intratumorally (IT) varied based on the IV dose. The primary endpoints were safety and maximum tolerated dose (MTD). Efficacy was a secondary endpoint. Correlative studies focused on understanding viral pharmacokinetics (PK) and immunological responses induced by VSV-IFNb-TYRP1 therapy. Twelve patients with previously treated metastatic uveal melanoma were enrolled. Median follow up was 19.1 months. Four DLs were evaluated with one patient at DL4 experiencing dose limiting toxicities (DLTs), including decreased platelet count (grade 3), increased aspartate aminotransferase (AST), and cytokine release syndrome (CRS). Non-DLTs at DL4 were fatigue, fever, CRS, hematological toxicities (decreased platelets and lymphocytes), and AST elevation. Four patients had stable disease (SD) and 8 had progressive disease (PD) as their best response. ELIspot data show that three of the four patients with response to TYRP1 also had a response to gp100, suggesting possible epitope spreading. These four patients received immune checkpoint inhibitor treatment as the next line of therapy, which led to clinical benefit for two patients. Our study evaluated VSV-IFNb-TYRP1 administered via IT and IV routes in a previously treated population of metastatic uveal melanoma patients and found no major safety events. Although there were no clear objective responses to VSV-IFNb-TYRP1, dose-dependent immunogenicity to melanoma antigens was seen. While OV response alone is not sufficient for clinical benefit, the evidence of epitope spreading is encouraging. Future studies of VSV-IFNb-TYRP1 will evaluate combinations with other therapies. Citation Format: Katherine E. Smith, Adam Weisbrod, Carrie Strand, Jacob Allerd, Jose S. Pulido, Alysha Newsom, Lianwen Zhang, Nandakumar Packiriswamy, Heather Montane, Lisa Kottschade, Anastasios Dimou, Yiyi Yan, Markovic Svetomir, Kah Whye Peng, Richard G. Vile, Matthew S. Block, Roxana S. Dronca. Phase I trial of vesicular stomatitis virus expressing human interferon beta and tyrosinase related protein 1 (VSV-IFNb-TYRP1) in metastatic ocular melanoma [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 CT207.

TPS657 Background: Poorly differentiated neuroendocrine carcinoma (NEC) is an aggressive malignancy comprising both pulmonary and extrapulmonary primary sites. NEC includes both small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC), as well as other neuroendocrine carcinomas arising from any primary organ. A substantial portion (~40%) of NEC arises from gastrointestinal primary sites. The optimal systemic therapy beyond first line platinum and etoposide is not established. There is a critical need to improve upon the median survival in the second line, as most patients do not survive more than 6 months. The efficacy of single agent immune checkpoint inhibitors (ICIs) in NEC has been disappointing. One possible explanation for this is that the tumor microenvironment in NEC is non-inflamed. VSV-IFNβ-NIS is a vesicular stomatitis virus (VSV)-based oncolytic virus being tested in multiple early phase clinical trials. Preliminary studies of immune responses in patients receiving VSV-IFNβ-NIS therapy suggest some patients develop T cell responses to viral antigens and known tumor antigens. We hypothesize that VSV-IFNβ-NIS therapy may convert a non-inflamed or immune-excluded phenotype in NEC to a highly inflamed phenotype that sensitizes the tumor to ICIs. Methods: This is a phase 1-2 safety run-in study designed to determine the safety of VSV-IFNβ-NIS in combination with a single agent ICI, pembrolizumab, followed by dose expansion in patients with refractory non-small cell lung cancer (NSCLC) or NEC. The safety run-in portion of this study has been completed, and we are presently testing the recommended phase 2 dose (RP2D) of VSV-IFNβ-NIS in an expansion cohort of patients with SCLC or NEC of any primary site. Patients must have previously progressed on at least one line of systemic therapy. Prior treatment with checkpoint inhibitors is permitted. Patients are treated one time with the RP2D of 1.0x10^11 TCID50 VSV-IFNβ-NIS on day 1, followed by pembrolizumab on day 8 and then pembrolizumab every 21 days until progression, up to 2 years. The primary objective is to estimate the response rate by RECIST 1.1. Secondary objectives include estimation of disease-control rate, duration or response, progression-free survival, overall survival, and safety signals. The NEC expansion cohort will seek to enroll 10 patients. If at least one objective response is observed, and safety is confirmed, the regimen will be considered for future study. Clinical trial information: NCT03647163 .

TPS9595 Background: There is a need for novel immunotherapies to address the patient population that never or no longer responds to immune checkpoint inhibitors (CPI). VV1 is an oncolytic vesicular stomatitis virus engineered to express human interferon beta (IFNβ) to enhance cellular anti-tumor immune responses and tumor selectivity. Phase 1 studies demonstrated VV1 anti-tumor activity in several malignancies with or without a CPI. We are exploring ways to optimize VV1 efficacy in combination with cemiplimab, an anti-PD1 antibody approved for lung, basal and squamous cell skin cancers. Recent clinical data support a 5-fold higher dose of VV1 than was previously explored, and pre-clinical data show synergy between the oncolytic and an anti-CTLA4 antibody, in addition to cemiplimab, supporting exploration of a triplet. What was originally a five-arm study of intravenous (IV) VV1 in combination with IV cemiplimab has been amended to focus on 2 means of optimizing efficacy: use of a higher dose of VV1 and triplet combination in proof-of-concept populations. Methods: We are now enrolling pts with advanced melanoma (after progression on anti-PD1) and plan to include 1st-line NSCLC pts with PD-L1 expression ≥50%. The study is first exploring the preliminary anti-tumor activity, safety, and immunogenic activity of the combination of IV VV1 at a dose of 1.0 x 1011 TCID50 once on D1 followed by IV cemiplimab Q3W starting on D8, or the same regimen with an additional intratumoral injection of VV1 1.0 x 109 TCID50 once on D1 for pts with accessible lesions. Pts receive IV cemiplimab Q3W until confirmed disease progression or intolerable toxicity. Once at least 6 pts have been treated with acceptable safety across the 2 melanoma doublet cohorts using this higher dose of VV1, a 3rd melanoma cohort will open to add a single dose of ipilimumab 50 mg on D1 (all IV triplet). Once 6 melanoma pts have received the triplet safely, the 1st-line NSCLC cohort will open. All cohort decisions are guided by a Data Review Committee. Cohorts will be expanded based on a Simon 2-stage design using a type I error rate of 0.05 and power of 85%. Null ORR is 10% with a target of 35% for 2nd line melanoma and null ORR is 40% in 1st line NSCLC with a target of 70%. Each melanoma cohort will require a response in ≥2 of 10 pts in the 1st stage to add 11 more in the 2nd stage, while NSCLC will first need 5/9 evaluable pts to respond, then an additional 13 to complete the design. Response is assessed at week 7 then Q9W per RECIST v1.1. The study includes serial biopsies in ≥3/10 pts in Stage 1 of each of the IV melanoma cohorts (doublet and triplet therapy), all pts in Stage 2 of these IV melanoma cohorts, and all pts in both Stage 1 and Stage 2 of the IV/IT melanoma cohort, to permit a thorough investigation of the impact of the 3 immunotherapies under investigation. The study is currently ongoing in the USA. Clinical trial information: NCT04291105.

509 Background: Bladder cancer is a leading cause of cancer death in the United States. The histology in > 90% of cases is urothelial carcinoma (UC). Tumors may present either as non-muscle-invasive (NMIBC) or muscle-invasive disease (MIBC). Current standard of care for patients with high risk NMIBC includes transurethral resection of bladder tumor (TURBT) followed by intravesical immunotherapy with Bacillus Calmette-Guerin (BCG). Meanwhile, patients with BCG unresponsive NMIBC or MIBC are recommended to undergo radical cystectomy (RC), which adversely impacts quality of life and is associated with significant morbidity. MV-NIS is an investigational oncolytic measles virus with an excellent clinical safety profile. This ongoing phase I clinical study is designed to test the safety, efficacy and identify the recommended phase 2 dose (RP2D) of intravesical MV-NIS in patients with NMIBC or MIBC who are scheduled for RC and not eligible for neoadjuvant chemotherapy. Methods: Bladder UC patients were evaluated for eligibility and provided informed consent prior to enrolling. To date 8 patients have been enrolled: 4 to the single dose safety cohort, and 4 to the multi-dose expansion cohort. Patients were administered intravesical ̃1x109 TCID50 MV-NIS once at least 1 week prior to RC (safety cohort), or twice at 4 and 2 weeks prior to RC (expansion cohort). Patients were closely monitored during the 2-hour instillation period. Tumor specimens from the pre-treatment TURBT and post-treatment RC were analyzed to determine pre- and post-treatment pathological stage and evaluate tumor killing and immune cell infiltrate. Results: Intravesical MV-NIS treatment was well tolerated in all patients. Only a single Adverse Event (AE) attributable to MV-NIS treatment (Grade 1 hematuria). AEs Grade > 2 were related to post-surgical complications. Tumor downstaging was observed in 4 of 8 patients. Among 4 patients in the expansion cohort, 2 had no residual disease (pT0). Central assessment of RC tissues showed significant inflammatory infiltrate in all treated bladder specimens. Detailed analyses are ongoing to characterize MV infection and immune infiltrate in bladder tissue. Conclusions: The higher-than-expected rate of tumor downstaging and pT0 pathology, paired with the significant immune infiltrate observed in post-treatment bladder tissue, provide compelling evidence that intravesical MV-NIS has clinical activity against UC. These results support the use of two doses of ̃1x109 TCID50 as the RP2D in future clinical studies for BCG unresponsive NMIBC or MIBC patients. MV-NIS induced inflammation may act synergistically with checkpoint blockade therapies. Clinical trial information: NCT03171493.