Vaccinia viral (VV) vectors are increasingly used in oncolytic virus therapy and vaccine development for cancer and infectious diseases. However, their effectiveness is hindered by the strong anti-viral immune response induced by the viral vector. In this review, we discuss the strategies to deimmunize vaccinia viral vector. One approach is to mask the virus from the neutralization antibody responses by mapping and eliminating of B-cell epitopes on the viral membrane proteins. The recombinant VVs contain one or more viral glycoproteins with mutations in the neutralizing antibody epitopes, resulting in viral escape from neutralization. In addition, a regulator of complement activation (e.g., CD55) can be expressed on the surface of the virus particle, leading to increased resistance to complement-mediated neutralization.

Chimeric antigen receptor (CAR) - engineered natural killer (NK) cell therapy has emerged as a promising platform for adoptive immunotherapy for cancer. However, CAR-NK or CAR-T therapy so far has achieved limited efficacy in solid tumors compared with hematologic malignancies. One of the challenges is the lack of prevalent tumor-specific surface antigen target in solid tumors. Hence, we are developing a novel oncolytic vaccinia virus (VV) to deliver a dual functional glycol-immune checkpoint inhibitor and universal tumor cell marker combined with CAR-NK cell therapy to increase anti-tumor efficacy. Using the vaccinia virus deleted in both viral TK and VGF genes (vvDD) to drive the expression of a membrane-bound sialidase derived from actinomyces viscosus (avSial) under the viral late promoter, we observed efficient desialylation of both VV-infected and non-infected tumor cells, which would alleviate the suppression of sialic acid on NK and other immune cells within TME. Further, the surface bound avSial on VV-infected tumor cells can also serve as a universal target for avSial-CAR NK cells, with less concern for cross-reactivity to normal human tissues and antigen loss. We developed anti-avSial antibodies through mice immunization study. The selected single chain variable fragments (scFV) were constructed in CAR format containing CD28 CD3 ζ for co-stimulation and human IL-15 gene for better NK persistence and function. The binding affinity of anti-avSial scFv to avSialidase were further screened in CAR constructs-transfected 293T cells. The selected anti-avSial scFv CAR were packaged into gamma retroviral vectors to transduce activated and expanded NK cells derived from healthy donor peripheral blood. For proof-of-concept, target tumor cell lines A375, A549 and HT-29 expressing transmembrane sialidase and GFP were also generated. Compared with CD19 CAR NK and none transduced (NT) NK cells, avSial CAR NK had markedly increased cytotoxicity against avSial-expressing tumor cells in co-culture assays at low E:T ratios <= 1:2. Upon tumor cell rechallenge, avSial CAR NK also controlled the tumor growth significantly better than CD19 CAR NK or NT NK cells. Furthermore, avSial CAR NK completely eliminated A375 tumor spheroids expressing transmembrane sialidase whereas CD19 CAR NK or NT NK cells only transiently controlled the tumor spheroids growth. Taken together, we have developed an effective avSial targeting CAR IL15 NK that will be used to demonstrate enhanced efficacy and versatility in combination with avSial-armed VV for treatment of solid tumors. Citation Format: Xiaomei Wang, Guowei Wei, Keshav B. Karki, Winnie Chan, Mariya Viskovska, Andrew Williams, Nancy Chang, Haiyan Jiang. Developing a novel combination therapy using engineered chimeric antigen receptor natural killer cells targeting avsialidase with avsialidase-armed oncolytic vaccinia virus in solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6225.

Oncolytic virotherapy represents a promising therapeutic strategy for cancers that do not respond well to immune checkpoint inhibitors. Oncolytic viruses (OV) preferentially replicate in and lyse tumor cells, leading to systemic immune stimulation. Studies have shown that hypersialylation promotes tumor growth and metastasis, as well as the suppression of immune cells. Cancer-associated fibroblasts (CAFs) are known to facilitate tumor invasion and angiogenesis and maintain immunosuppressive microenvironment in solid tumors. We are developing vvDD-Sial-FAP/CD3, an oncolytic vaccinia virus expressing a membrane-bound sialidase, to remove sialic acids from the cell surface glycans, and fibroblast activation protein (FAP)-targeted T cell engager, to eliminate glycoimmune checkpoint and tumor stroma, respectively. vvDD-Sial-FAP/CD3 is an engineered vaccinia virus of Western Reserve (WR) strain with: (1) an insertional disruption of the viral thymidine kinase (TK) gene with the sialidase and FAP/CD3 transgenes. TK is an essential enzyme for the pyrimidine synthesis pathway; viral TK gene deletion thus results in selective replication of virus in rapidly dividing cancerous cells with high intracellular nucleotide pools, and (2) a deletion of the vaccinia growth factor (VGF) genes for greater dependence on the cell cycling status of the cancer cells. Our results showed that sialidase expressed from vvDD-Sial-FAP/CD3 efficiently cleaves the sialic acids from the cell surface and the Fc fused to the sialidase induced antibody-dependent cell-mediated cytotoxicity (ADCC) using an ADCC reporter assay. In vitro efficacy studies were conducted using HCT-116 human colon cancer cells mixed with FAP-expressing normal human dermal fibroblasts or FAP-positive HCC1143 human breast cancer cells in the presence of human peripheral blood mononuclear cells. In both tumor models, vvDD-Sial-FAP/CD3 induced activation of both CD4+ and CD8+ T cells, as measured by the upregulation of CD69 and CD25 markers and increased granzyme B release, which resulted in enhanced cell killing. Using A549 co-cultured with CAFs microtissues, we showed that vvDD-Sial-FAP/CD3 spread efficiently within the tumor spheroids. In addition, vvDD-Sial-FAP/CD3 increased tumor-infiltrated lymphocytes, leading to enhanced cell killing. Taken together, our results have demonstrated potent anti-tumor and -stroma effects induced by vvDD-Sial-FAP/CD3 in multiple cancer models. In vivo efficacy of vvDD-Sial-FAP/CD3, either alone or in combination with checkpoint inhibitor or NK cell therapy, is being evaluated. Citation Format: Winnie M. Chan, Mariya Viskovska, Micaela McArthur, Xue Mary Yu, Wei Liang, Zane Norman, Xiaotong Song, Nancy T. Chang, Haiyan Jiang. Glycoimmune checkpoint and tumor stroma-targeted oncolytic vaccinia virus for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3300.