Engineering Neutralization-Resistant Oncolytic Vaccinia Virus for Effective Treatment of Multiple Myeloma

The goal of this study is to develop an effective oncolytic vaccinia virus (VV) therapy for multiple myeloma (MM). Despite significant advancements in the clinical management of MM over the past two decades, it remains incurable, necessitating innovative therapeutic strategies. Recent clinical studies have demonstrated significant activity of BCMA-targeted therapies, such as BCMA-CAR T cells and BCMA-ADC, in patients with relapsed and refractory MM (RRMM). However, these therapies may have side effects due to BCMA expression on normal plasma cells. Oncolytic VV, with its excellent safety profile from historical use in smallpox eradication and recent promising clinical trials against solid tumors, emerges as a promising new class of agents for MM treatment. However, current VV-based approaches face challenges, such as suboptimal infection of MM and systemic intravenous delivery being compromised by neutralizing antibodies against the virus.

To address these challenges, we developed a novel oncolytic vaccinia virus variant, designated as BCMA T-cell engager armed neutralization escape variant (BCMA-TEA-VVNEV). This innovative virus incorporates two critical modifications: genetically altered surface glycoproteins to evade neutralizing antibodies for repeated systemic administration and a BCMA-targeted bispecific antibody (BiTE) comprising two single-chain variable fragments (scFvs)—one specific for BCMA and the other for CD3, a T-cell engager. The BCMA-CD3 BiTE recruits and activates T cells to kill MM cells not directly infected by the virus, enhancing the oncolytic effect.

Genetic modification of VV glycoproteins to generate the neutralization escape variant (VVNEV) of the virus was performed by altering the major neutralizing antibody (NAb) antigens, including A27L, H3L, D8L, and L1R. We identified crucial H3L residues involved in antibody recognition through peptide arrays and epitope mapping, which were then mutated to generate the new H3L. The published A27L, D8L, and L1R residues involved in antibody recognition were utilized. Mammalian plasmids encoding alanine substitutions of these glycoproteins were transfected into CV-1 cells infected with VV, facilitating homologous recombination to replace wild-type genes with the modified ones. A GFP expression cassette was used to screen for successful recombinants, followed by removal using LoxP sites. In vitro analysis using the plaque reduction neutralization test (PRNT) confirmed that VVNEV could effectively evade neutralization by a panel of anti-VV polyclonal antibodies, demonstrating its enhanced ability to escape immune detection. Mouse studies revealed that VVNEV is less immunogenic, escaping pre-existing anti-VV and anti-NEV antibodies, and effectively facilitating T cell activation and enhancing tumor killing in B16 tumor models.

BCMA-TEA-VVNEV was successfully generated with an average yield of 10e9 PFU/ml. This modified virus effectively infected multiple MM cell lines, including RPMI8226 (BCMA-high), U266 (BCMA-high), ARP-1 (BCMA-low), and ARK (BCMA-low), at a multiplicity of infection (MOI) of 0.1, where unmodified VV failed. Additionally, BCMA-TEA-VVNEV demonstrated enhanced VV’s tumor lytic ability by over 2-fold.

In conclusion, we successfully generated BCMA-TEA-VVNEV, which can escape anti-VV neutralization antibodies and effectively activate human T cells. These results suggest that BCMA-TEA-VVNEV represents a promising candidate for further investigation in MM treatment. Our preliminary data indicate that BCMA-TEA-VVNEV can overcome the limitations of conventional oncolytic VV therapies by enhancing systemic delivery and immune-mediated targeting of MM cells. This dual approach improves direct oncolytic activity and leverages the immune system's capability to eradicate residual tumor cells, presenting a promising therapeutic strategy for multiple myeloma.