Gayathri Vijayakumar, PhD

While single-cell sequencing technologies have revealed tissue heterogeneity, resolving mixed cellular libraries into cellular clones is essential for many pooled screens and clonal lineage tracing. Fluorescent proteins are limited in number, while DNA barcodes can only be read after cell lysis. To overcome these limitations, we used influenza virus hemagglutinins to engineer a genetically encoded cell-surface protein barcoding system. Using antibodies paired to hemagglutinins carrying… Show more

While single-cell sequencing technologies have revealed tissue heterogeneity, resolving mixed cellular libraries into cellular clones is essential for many pooled screens and clonal lineage tracing. Fluorescent proteins are limited in number, while DNA barcodes can only be read after cell lysis. To overcome these limitations, we used influenza virus hemagglutinins to engineer a genetically encoded cell-surface protein barcoding system. Using antibodies paired to hemagglutinins carrying combinations of escape mutations, we developed an exponential protein barcoding system which can label 128 clones using seven antibodies. This study provides a proof of principle for a strategy to create protein-level cell barcodes that can be used in vivo in mice to track clonal populations. Show less

Newcastle disease virus (NDV) is an attractive candidate for oncolytic immunotherapy due to its ability to replicate in tumor cells and potentially to overcome the inherently immunosuppressive nature of the tumor microenvironment. The advent of checkpoint blockade immunotherapy over the past few years represents a paradigm shift in cancer therapy. However, the prevalence of severe immune-related adverse events with CTLA4 and PD1 pathway blockade in clinical studies, especially in combination… Show more

Newcastle disease virus (NDV) is an attractive candidate for oncolytic immunotherapy due to its ability to replicate in tumor cells and potentially to overcome the inherently immunosuppressive nature of the tumor microenvironment. The advent of checkpoint blockade immunotherapy over the past few years represents a paradigm shift in cancer therapy. However, the prevalence of severe immune-related adverse events with CTLA4 and PD1 pathway blockade in clinical studies, especially in combination therapy groups, is a cause for concern. Immunotherapies with cytokines have also been extensively explored, but they have been associated with adverse events in clinical trials. Oncolytic vectors engineered to express checkpoint blockade antibodies and cytokines could provide an avenue for reducing the clinical toxicity associated with systemic therapy by concentrating the immunomodulatory payload at the site of disease. In this study, we engineered six different recombinant viruses: NDVs expressing checkpoint inhibitors (rNDV-anti-PD1 and rNDV-anti-PDL1); superagonists (rNDV-anti-CD28); and immunocytokines, where the antibodies are fused to an immunostimulatory cytokine, such as interleukin 12 (IL-12) (rNDV-anti-CD28-murine IL-12 [mIL-12], rNDV-anti-PD1-mIL-12, and rNDV-anti-PDL1-mIL-12). These six engineered viruses induced tumor control and survival benefits in both highly aggressive unilateral and bilateral B16-F10 murine melanoma models, indicative of an abscopal effect. The data represent a strong proof of concept on which further clinical evaluation could build. Show less

Newcastle disease virus (NDV) is an avian paramyxovirus that has been extensively studied as an oncolytic agent, in addition to being an economically important pathogen in the poultry industry. The establishment of a reverse genetics system for this virus has enabled the development of genetically modified recombinant NDV viruses with improved oncolytic and immunotherapeutic properties. In this chapter, we describe the materials and methods involved in the in vitro cloning and rescue of NDV… Show more

Newcastle disease virus (NDV) is an avian paramyxovirus that has been extensively studied as an oncolytic agent, in addition to being an economically important pathogen in the poultry industry. The establishment of a reverse genetics system for this virus has enabled the development of genetically modified recombinant NDV viruses with improved oncolytic and immunotherapeutic properties. In this chapter, we describe the materials and methods involved in the in vitro cloning and rescue of NDV expressing murine 4-1BBL as well as the in vivo evaluation of NDV expressing 4-1BBL in a B16-F10 murine melanoma model. Show less

Monoclonal antibodies (mAbs) targeting negative regulators, or checkpoint molecules (e.g. PD1/PD-L1 & CTLA4), of anti-tumoural T cells have demonstrated clinical efficacy in treating several neoplastic diseases. While many patients enjoy remarkable responses to checkpoint inhibitors, a majority show adverse effects. Understanding how checkpoint inhibitors may augment established chemotherapy or radiotherapy regimens or other immunotherapies like oncolytic viruses may lead to better clinical… Show more

Monoclonal antibodies (mAbs) targeting negative regulators, or checkpoint molecules (e.g. PD1/PD-L1 & CTLA4), of anti-tumoural T cells have demonstrated clinical efficacy in treating several neoplastic diseases. While many patients enjoy remarkable responses to checkpoint inhibitors, a majority show adverse effects. Understanding how checkpoint inhibitors may augment established chemotherapy or radiotherapy regimens or other immunotherapies like oncolytic viruses may lead to better clinical outcomes measured by improved efficacy with reduced toxicity. Here, we assess how Newcastle disease virus (NDV), an oncolytic virus in clinical testing, may interact with radiotherapy to enhance checkpoint inhibitor blockade. Show less

Influenza A virus (IAV) has shown promise as an oncolytic agent. To improve IAV as an oncolytic virus, we sought to design a transgenic virus expressing an immune checkpoint-inhibiting antibody during the viral life cycle. To test whether it was possible to express an antibody during infection, an influenza virus was constructed encoding the heavy chain of an antibody on the PB1 segment and the light chain of an antibody on the PA segment. This antibody-expressing IAV grows to high titers, and… Show more

Influenza A virus (IAV) has shown promise as an oncolytic agent. To improve IAV as an oncolytic virus, we sought to design a transgenic virus expressing an immune checkpoint-inhibiting antibody during the viral life cycle. To test whether it was possible to express an antibody during infection, an influenza virus was constructed encoding the heavy chain of an antibody on the PB1 segment and the light chain of an antibody on the PA segment. This antibody-expressing IAV grows to high titers, and the antibodies secreted from infected cells exhibit comparable functionality with hybridoma-produced antibodies. To enhance the anti-cancer activity of IAV, an influenza virus was engineered to express a single-chain antibody antagonizing the immune checkpoint CTLA4 (IAV-CTLA4). In mice implanted with the aggressive B16-F10 melanoma, intratumoral injection with IAV-CTLA4 delayed the growth of treated tumors, mediated an abscopal effect, and increased overall survival. Show less