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.