Zhang, Lianwen

Abstract VSV-IFNβ-NIS (VV1) is a Vesicular Stomatitis Virus (VSV) that encodes the interferon beta (IFNβ) and the iodide symporter (NIS) gene. The IFNβ gene confers tumor selectivity and enhances host antitumor immunity while the NIS gene acts as an imaging biomarker, allowing noninvasive imaging of viral infection sites using PET/SPECT. Multiple myeloma (MM) is a cancer of plasma cells and poses a challenge for treatment due to its biological heterogeneity. As MM usually relapses and is refractory, current therapies face the issue of drug resistance and patients may have to turn to novel therapies. Therefore, we set out to understand the activity of VSV which could be a new approach for patients with relapsed refractory MM (RRMM). Our study utilized two unique bortezomib resistant Vk*MYC transplantable models, Vk12598 and Vk12653, with distinct genomic features. Upon re-transplantation of splenocytes from these mice, flow cytometry revealed that Vk12598 and Vk12653 mice had high MM populations in the spleen and bone marrow. To assess VV1 efficacy in tumor bearing mice, we performed a VV1 dose escalation study, showing efficacy in delaying MM development and prolonging survival in both mice models, with Vk12598 being the more responsive model. Later studies also showed that VV1 induces tumor remission in the Vk12598 model when treatment was initiated at low tumor burden (gamma/albumin ratio ∼0.2). IFNβ levels increased significantly post 24-72h of VV1 treatment, indicating targeted infection and replication of the virus in MM cells. However, high doses of VV1 led to toxicity, as observed from early mortality and weight loss. To determine the sites of MM metastasis and VV1 infection, timepoint harvests were carried out. Hematoxylin and eosin and immunostainings showed the onset of MM of Vk*MYC in the bone marrow and spleen first, then later in the liver. Immunofluorescent staining confirmed that the higher the tumor burden observed, the more VV1 was present in the tissues. To enhance VV1 efficacy, we performed in vivo combination therapies of VV1 with anti-PD-1, an inhibitor of PD-1 which is overexpressed on MM patient T cells; and bortezomib, which inhibits NF-κB which in turn prevents IFN pathway protein transcription, potentially enhancing the replication of VV1 virus. In Vk12598 mice, VV1 combined with both drugs induced tumor regression and doubled the median survival time compared to treatment with VV1 only or VV1 coupled with either of the drugs individually. Addition of VV1 to either anti-PD-1 or bortezomib alone also increased survival time significantly. Studies are still ongoing to understand the mechanisms of VSV and its combination treatments in Vk*MYC models. This translational study will allow us to gain the knowledge needed to refine VSV treatments and provide novel therapy combinations to improve MM patient survival. Citation Format: Yi Ying Ma, Lianwen Zhang, Rebecca A. Nace, Marta Chesi, Stephen J. Russell, Kah Whye Peng. Harnessing oncolytic virus VSV and combination therapy to advance treatments for relapsed refractory multiple myeloma using Vk*MYC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 940.

Background The varicella-zoster virus (VZV), belonging to the group of human α-herpesviruses, has yet to be developed as a platform for oncolytic virotherapy, despite indications from clinical case reports suggesting a potential association between VZV infection and cancer remission. Methods Here, we constructed oncolytic VZV candidates based on the vaccine strain vOka and the laboratory strain Ellen. These newly engineered viruses were subsequently assessed for their oncolytic properties in the human MeWo melanoma xenograft model and the mouse B16-F10-nectin1 melanoma syngeneic model. Results In the MeWo xenograft model, both vOka and Ellen exhibited potent antitumor efficacy. However, it was observed that introducing a hyperfusogenic mutation into glycoprotein B led to a reduction in VZV’s effectiveness. Notably, the deletion of ORF8 (encodes viral deoxyuridine triphosphatase) attenuated the replication of VZV both in vitro and in vivo, but it did not compromise VZV’s oncolytic potency. We further armed the VZV Ellen-ΔORF8 vector with a tet-off controlled mouse single-chain IL12 (scIL12) gene cassette. This augmented virus was validated for its oncolytic activity and triggered systemic antitumor immune responses in the immunocompetent B16-F10-nectin1 model. Conclusions These findings highlight the potential of using Ellen-ΔORF8-tet-off-scIL12 as a novel VZV-based oncolytic virotherapy.

Although the live-attenuated measles virus (MeV) is one of the safest and most efficacious human vaccines, a measles-vectored COVID-19 vaccine candidate expressing the SARS-CoV-2 spike failed to elicit neutralizing antibody (nAb) responses in a phase-1 clinical trial, especially in measles-immune individuals. Here, we constructed a comprehensive panel of MeV-based COVID-19 vaccine candidates using a MeV with extensive modifications on the envelope glycoproteins (MeV-MR). We show that artificial trimerization of the spike is critical for the induction of nAbs and that their magnitude can be significantly augmented when the spike protein is synchronously fused to a dodecahedral scaffold. Furthermore, preexisting measles immunity did not abolish heterologous immunity elicited by our vector. Our results highlight the importance of antigen optimization in the development of spike-based COVID-19 vaccines and therapies.

Background: Oncolytic virotherapy, an innovative immunomodulatory therapeutic approach, is currently being investigated for its potential in treating relapsed refractory (R/R) hematologic malignancies. This study focuses on the use of an engineered Vesicular Stomatitis Virus called VSV-IFNβ-NIS, a bullet-shaped rhabdovirus which encodes both interferon beta (IFNβ) and sodium iodine symporter (NIS) with particular tumor cell tropism. Viral IFNβ serves as an indicator of viral proliferation and boosts the host's anti-tumor immune response. This is a phase 1 clinical trial with the identifier NCT03017820, wherein patients (pts) with R/R multiple myeloma (MM), and non-Hodgkin lymphoma (NHL) are treated with a single systemic administration of VSV-IFNβ-NIS. Study Design and Methods: Patient Eligibility Criteria: The Mayo Clinic Institutional Review Board approved the protocol. The study includes adult patients with relapsed or refractory multiple myeloma (MM), or T-cell lymphoma (TCL). For MM or plasmacytoma, patients should have <15% plasma cells and a largest diameter of <5 cm. TCL patients should have a mass <5 cm. Study Design: This is a single-center phase 1 trial aiming to determine the maximum tolerated dose of VSV-IFNβ-NIS in patients with R/R MM, and TCL. Secondary objectives included assessing the safety profile and estimating the clinical response rate of VSV-IFNβ-NIS. Correlative objectives involved monitoring viral replication through plasma IFN-β transgene levels and using SPECT/CT imaging with the NIS gene. Viral genomes were measured through quantitative reverse transcription (qRT) polymerase chain reaction (PCR) of VSV RNA, and any virus shedding was examined in body fluids. The tested doses were 5 × 109 TCID50, 1.7 × 1010, 5 × 1010, and 1.7 × 1011 TCID50. The virus was administered via IV infusion over 30 minutes, and patients received 650 mg acetaminophen and 50 mg diphenhydramine hydrochloride before infusion and every 6 hours for 24 hours to manage fever and hypotension. Two expansion cohorts at DL4 have been added (20 additional PTCL, 10 B-cell lymphoma and histiocytic neoplasms) to obtain additional data on tolerability and efficacy and biomarkers of response in these histologies. Safety and Response Assessments: All patients undergo toxicity evaluation, and adverse events (AEs) assessment using the Common Terminology Criteria for Adverse Events (CTCAE) version 4 criteria, except for cytokine release syndrome (CRS) grading, which follows the 2014 Lee criteria. Dose escalation and tolerability are based on the standard 3+3 phase 1 trial design. Ruxolitinib is added from days 2-5 or day -1 to day 9 depending on the arm to mitigate IFNβ toxicity. Response assessment followed the International Myeloma Working Group criteria for MM and the Lugano Classification for NHL and histiocytic neoplasms. Pharmacodynamic and Pharmacokinetic Analyses: For viremia and viral shedding assessment, RNA is isolated from whole blood, mouth rinse, buccal swabs, and urine samples. Quantitative RT-PCR determined VSV-nucleocapsid (N) RNA copy number, and virus shedding is assessed through coculturing samples on Vero cells. Anti-VSV antibodies are evaluated using the standard plaque reduction neutralization assay. Plasma cytokine levels are measured with a 30-plex human cytokine kit, and human IFN-β levels is quantified using an enzyme-linked immunosorbent assay (ELISA) kit. Statistical Analysis: Incidence of dose-limiting toxicities and AEs are tabulated by dose level (DL). AEs are summarized by type, maximum grade, and duration across patients in each DL. Spearman rank correlation tests are used evaluated correlations between laboratory-based measures, and Wilcoxon rank sum tests compared continuous measures between response groups. Response rates will be summarized by DL and disease group, with estimated response rates and corresponding 95% binomial confidence intervals calculated.

e14594 Background: Voyager-V1 (VV1) is an oncolytic vesicular stomatitis virus engineered to express interferon beta (IFNβ) to boost cellular antitumor immunity and sodium iodine symporter (NIS). A phase 1 trial investigating a single infusion of VV1 in patients with relapsed refractory multiple myeloma and T-cell lymphoma (TCL) showed promising and durable clinical activity, primarily in patients with TCL. VV1 infects tumor cells and macrophages in the lymph nodes and spleen, resulting in release of tumor antigens and IFNβ that activates and boost cellular antitumor immunity. We present the preliminary pharmacokinetic analysis in patients given one intravenous (IV) dose of VV1. Methods: Patients received 5e9 to 1.7e11 TCID50 (50% tissue culture infective dose) of VV1 given over 30 minutes. We also explored the impact of infusion duration (0.25, 0.5,1 and 3 hours) in 17 patients with solid tumors. The objectives were to determine the impact of viral dose on pharmacokinetics (PK) of VSV and IFNβ. For VSV and IFNβ PK, whole blood was collected in 30 patients on days 1, 2, 3, and 8. Dose levels were 1.7E10, 5E10, and 1.7E11 TCID50. The plasma concentration-time data were estimated with non-compartmental analysis using WinNonlin 8.3. Results: The half-life of VV1 is comparable (10-12h) between dose levels of 1.7e10, 5e10 and 1.7e11. Ideal infusion rate was found to be 30 minutes and provides the highest Cmax and AUC. Most peak concentrations for viremia and IFNβ were achieved between ‘end of infusion’ and 2.5 hours, 4.5 and 24 hours, respectively. Following infusion, a second concentration peak is found in over half the patients leading to highly variable terminal elimination half-life, specifically where the second peak is at 24 hours. There was no second peak for IFNβ levels. Peak plasma concentration and systemic exposure increased in proportion to increased dose, consistent with linear clearance. Conclusions: The 30 minute infusion rate was considered the best as its PK provides the highest Cmax and AUC with no significant CRS differences among other groups. IL-6 and IFNα markers at hour 4 indicate a likely CRS and hour 24 provides a possible prediction of the grade. Cmax and AUC were also highest at 1.7e11 TCID50. This is the recommended dose that will be used in the expansion arm in patients with TCL. Clinical trial information: NCT03017820 , NCT02923466 . [Table: see text]

7530 Background: Oncolytic virotherapy is a novel immunomodulatory therapeutic approach currently under study for relapsed refractory (R/R) hematologic malignancies. The engineered Vesicular Stomatitis Virus VSV-IFNβ-NIS used in this study encodes interferon beta (IFNβ) and sodium iodine symporter (NIS). Virally-encoded IFNβ serves as an index of viral proliferation and enhances host anti-tumor immunity. Herein, we report the updated results of the phase 1 clinical trial NCT03017820 of a one-time administration of systemic VSV-IFNβ-NIS among patients (pts) with R/R multiple myeloma (MM), T cell Lymphoma (TCL) and Acute myeloid Leukemia (AML) with particular focus on the TCL cohort treated at the highest dose level (DL4) without Ruxolitinib. Methods: VSV-IFNβ-NIS was administered as a single IV dose following a classical 3+3 phase I trial, starting at 5e9 TCID50 DL1 through 1.7e11 TCID50 DL4. A dose expansion cohort of 8 additional R/R TCL pts was added at DL4 in view of an initial signal of activity. The primary objective was to determine the maximum tolerated dose of VSV-IFNβ-NIS as a single agent. Secondary objectives were determination of safety profile and preliminary efficacy of VSV-IFNβ-NIS. Adverse events (AEs) are reported based on CTCAE V4; cytokine release syndrome (CRS) grading was based on Lee (Blood 2014) criteria. Results: To date, a total of 43 pts received VSV-IFNβ-NIS: MM (N=21), TCL (N=21) and AML (N=1). In the TCL cohort: at DL4 Group A (no Ruxolitinib), 12 pts were registered, but only 11 pts were treated (mycosis fungoides (MF) N=3; ALK- ALCL N=1; PTCL-NOS N=2; AITL N=4; Nodal PTCL, TFH N=2; ages 33-79). These pts received a median of 3 lines of therapy (range: 1-5). No dose limiting toxicities were observed. Grades 3 and higher AEs were hematologic with transient lymphopenia (63.6%), neutropenia (36.4%) being the most common. The non-hematologic AEs observed included unique toxicities such as CRS grades 1-2 (81.8%); the transient transaminitis grade 1-2 (36.4%) and grade 3 (27.3%) along with thrombocytopenia grade 1-2 (63.6%) and grade 3 (18.2%) were a reflection IFNβ toxicity. All the AEs observed were short lived. Responses were seen in pts with PTCL. At DL4, in pts with systemic PTCL (N=9), there were 5 responses including 3 complete responses (CR) with two long-lasting at 24 months, an ongoing 15 month CR, and a 5 month CR, and 2 partial responses (PR) lasting 5 months in one pts and 6 months in the other. Conclusions: These updated results of single agent VSV-IFNβ-NIS which include pts with PTCL treated at the highest dose level and a dose expansion cohort demonstrates not only the safety of VSV-IFNβ-NIS but also a strong efficacy signal in pts with PTCL with impressive durable responses. Two expansion cohorts at DL4 have been added (20 additional PTCL, 10 B-cell lymphoma pts) to obtain additional data on tolerability and efficacy and biomarkers of response in these pts. Clinical trial information: NCT03017820 .

The natriuretic peptide system (NPS) and renin-angiotensin-aldosterone system (RAAS) function oppositely at multiple levels. While it has long been suspected that angiotensin II (ANGII) may directly suppress NPS activity, no clear evidence to date supports this notion. This study was designed to systematically investigate ANGII–NPS interaction in humans, in vivo, and in vitro. Circulating atrial, b-type, and c-type natriuretic peptides (ANP, BNP, CNP), cyclic guanosine monophosphate (cGMP), and ANGII were simultaneously investigated in 128 human subjects. Prompted hypothesis was validated in vivo to determine the influence of ANGII on ANP actions. The underlying mechanisms were further explored via in vitro approaches. In humans, ANGII demonstrated an inverse relationship with ANP, BNP, and cGMP. In regression models predicting cGMP, adding ANGII levels and the interaction term between ANGII and natriuretic peptides increased the predictive accuracy of the base models constructed with either ANP or BNP, but not CNP. Importantly, stratified correlation analysis further revealed a positive association between cGMP and ANP or BNP only in subjects with low, but not high, ANGII levels. In rats, co-infusion of ANGII even at a physiological dose attenuated cGMP generation mediated by ANP infusion. In vitro, we found the suppressive effect of ANGII on ANP-stimulated cGMP requires the presence of ANGII type-1 (AT1) receptor and mechanistically involves protein kinase C (PKC), as this suppression can be substantially rescued by either valsartan (AT1 blocker) or Go6983 (PKC inhibitor). Using surface plasmon resonance (SPR), we showed ANGII has low binding affinity to the guanylyl cyclase A (GC-A) receptor compared to ANP or BNP. Our study reveals ANGII is a natural suppressor for the cGMP-generating action of GC-A via AT1/PKC dependent manner and highlights the importance of dual-targeting RAAS and NPS in maximizing beneficial properties of natriuretic peptides in cardiovascular protection.

Abstract 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.

Abstract 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.

Clinical success with intravenous (IV) oncolytic virotherapy (OV) has to date been anecdotal. Here, we conducted a phase 1 clinical trial of systemic OV therapy and investigated the mechanisms of action in responding T-cell lymphoma (TCL) patients. A single IV dose of VSV-IFNβ-NIS was administered to patients with relapsed refractory hematologic malignancies to determine safety and efficacy across 4 dose levels (DL). Correlative studies were undertaken to evaluate viremia, virus shedding, virus replication and immune responses. Fifteen patients received VSV-IFNβ-NIS (7 multiple myeloma, 7 TCL, 1 acute myeloid leukemia); 3 patients were treated at each DL1 through DL3 (0.05, 0.17, and 0.5 x 1011 TCID50), and 6 at DL4 (1.7 x 1011 TCID50). There were no dose limiting toxicities. 3 of 7 patients with TCL had RECIST responses: a 3-month PR at DL2, a 6-month PR and a durable CR ongoing at 20 months at DL4. Viremia peaked at the end of infusion and no infectious virus shedding was detected. Plasma interferon-β levels, a biomarker of VSV-IFNβ-NIS replication, peaked between 4h and 48h post infusion. The patient with CR had robust viral replication with increased ell free DNA in her plasma, a very high peak IFNβ level of 18,213 pg/ml, a strong anti-VSV neutralizing antibody response, and increased numbers of tumor reactive T-cells. VSV-IFNβ-NIS as a single agent was effective in patients with TCL resulting in durable disease remissions in heavily pretreated patients. Correlative analyses suggest that responses may be due to a combination of direct oncolytic tumor destruction and immune-mediated tumor control. Further clinical testing is warranted. (This trial is registered at www.clinicaltrials.gov as NCT03017820).