HSV1716 Infects Multiple Myeloma Cells and Leads to CD138 Downregulation

Multiple myeloma (MM) is a plasma cell malignancy that accounts for approximately 10% of all hematological cancers. MM is classified as incurable due to a high relapse rate after the initial therapy and failure to achieve long-term minimal residual disease (MRD). Therefore, new therapies and combinations are needed. Oncolytic viruses (OVs) are genetically modified viruses that preferentially infect and lyse malignantly transformed cells. HSV1716 is a replication-restricted herpes-simplex virus type I (HSV-1)-based OV (HSV-OV) which has been evaluated in several clinical trials. HSV-OVs have been shown to infect and kill MM cell lines and primary malignant plasma cells. Furthermore, HSV-OV mediated target cell killing was demonstrated to be enhanced by natural killer (NK) cells, an immune cell subset that naturally recognizes malignantly transformed or virally infected cells. The underlying mechanisms behind HSV-OV-enhanced NK-tumor cell killing, however, have not yet been elucidated. Here, we aim to monitor phenotypic changes of MM cells upon HSV1716 infection and thus explore the basis for NK cell recognition of HSV-OV infected target MM cells.

The MM cell lines RPMI8226 and MM1S were infected with HSV1716 at various multiplicities of infection (MOIs) over a three-day timecourse. Target cell death and expression of viral glycoproteins were measured at different timepoints by flow cytometry. HSV1716 caused oncolysis of 60% of RPMI8226, and 70% of MM1S cells 48h post-infection (p.i.) at an MOI of 0.5. Under these conditions, the expression of viral glycoprotein E on live cells reached 74% on RPMI8226 cells and 37% on MM1S cells. Primary NK (pNK) cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) and co-cultured with infected and uninfected MM cells for 4 hours. CD107a as a marker for NK cell degranulation, as well as the release of IFNγ and TNF were measured on CD3^-CD56⁺ pNK cells by flow cytometry. CD107a expression increased from 15.6% (+/- 0.9%) to 56.2% (+/- 2.4%) following infection of RPMI8226 cells and from 15.6% (+/- 3.4%) to 59.2% (+/- 4.5%) following infection of MM1S cells. Similarly, the expression of IFNγ and TNF increased by four- and three-fold, respectively, as compared to uninfected target cells.

To monitor changes in the expression of NK cell ligands on infected MM cells, a 16-color flow cytometry panel was designed. RPMI8226 and MM1S cells were infected at different MOIs, and ligand expression was analyzed at 24h, 48h, and 72h p.i.. Most significantly, an early and progressive downregulation of CD138 expression from 96% on uninfected cells to 79 % (24h MOI 0.5), 70% (48h MOI 0.5), and 22% (72h MOI 0.5) on live RPMI8226 cells and from 99% on uninfected cells to 82% (24h MOI 0.5), 65% (48h MOI 0.5) and 23% (72h MOI 0.5) on live MM1S cells was observed. Of note, the expression of CD38 remained stable at each of these timepoints. While downregulation of HLA class I molecules was observed, the expression of other NK cells ligands on RPMI8226 or MM1S cells did not notably differ between infected and uninfected cells.

Together, our data confirm that HSV-OV enhances NK cell-mediated killing of MM cells. While expression of ligands for activating NK cell receptors was not altered on infected versus uninfected RPMI8226 and MM1S cells, a strong downregulation of CD138 with maintained CD38 expression was observed upon HSV1716 infection. The implementation of oncolytic virotherapy as an immunotherapeutic platform for cancer treatment adds a new and promising therapeutic tool for combination therapies to the treatment options of MM.

Meinke:XNK Therapeutics AB: Consultancy. Conner:Sorrento Therapeutics: Current Employment. Powers:Sorrento Therapeutics: Current Employment. Allen:Sorrento Therapeutics: Current Employment. Wagner:VyGenBio: Consultancy, Current equity holder in publicly-traded company. Alici:Vycellix: Consultancy, Current equity holder in publicly-traded company.