Oncolytic Viruses in Lymphoma

Lymphoma is a heterogenous disease accounting for 4% of all UK cancer diagnoses. Its overall response to chemotherapy is good. However, in patients with relapsed or refractory disease, less than half will obtain durable remissions. Anti CD20 targeting by monoclonal antibodies such as rituximab have made great improvements in the treatment responses of B cell Lymphoma. However, resistance can occur, and relapse following treatment is commonly seen in indolent Lymphoma subtypes. Oncolytic virus (OV) therapies are an emerging treatment option for many malignancies and the first OV has recently been approved by the FDA for the treatment of melanoma. The aim of OV therapy is to effectively replicate within the host, specifically target and lyse tumour cells, and induce robust, long lasting tumour-specific immunity, without having any detrimental effects on healthy cells.

We therefore hypothesise that an oncolytic adenovirus that places transcriptional control of the adenoviral replication essential component E1A under a CD20 promoter will specifically infect and lyse B cell Lymphoma cells. The aim is that in combination with conventional chemotherapy this will not only lead to reduced tumour burden and remission, but long-term immunity allowing for longer periods of remission and a potential cure for those indolent lymphomas currently considered incurable.

HT and SC-1 cell lines were used in cell viability assays to assess the optimal plating density (2x104 cells per well) to allow further investigation, we then went on to determine chemotherapy (Vincristine and Doxorubicin) dosing to allow for subsequent virus and drug combination studies. HT and SC-1 cell lines tested in vitro showed expected dose dependent decreases in viability with approved lymphoma chemotherapies (Vincristine and Doxorubicin), but only up to 50%.

CD20 expression in B cell lymphoma is well documented in the literature, initially HT and SC-1 cell lines were checked for CD20 expression by flow cytometry and found to be positive (MFI 77 and 172 respectively).

Importantly, for the replication of the virus the CD20 promoter MS4A1 was assessed by qPCR in both HT and SC1 cell lines and high levels of expression were shown (relative expression 290 and 144.4 fold respectively compared to housekeeping gene). Lymphoma cell lines (HT and SC-1) were then used to assess viral infection and replication, by both immunohistochemistry and by flow cytometry using a reporter adenovirus (AdGFP). This confirmed that these cells were able to be infected by, and allow for replication of, the adenovirus.

As lymphoma cells lack the CAR receptor which is the recognised mechanism for adenoviral infection, other potential receptors including CD51/61 were assessed in order to explain infection.

MS4A1 was inserted upstream of E1a via the AdEasy system. This plasmid underwent homologous recombination with the Adenoviral backbone and transfection into HEK293 cells to produce the MS4A1Ad virus.

Ethical approval was gained for collection of patient samples to enable testing of the CD20 specific adenovirus in primary cell culture. Primary samples were obtained from patients being investigated for Lymphoma who consented to the study. Samples were also collected to be used to create patient derived xenograft models on which the OV could be tested prior to early phase clinical trials, given the potential for additional substantial benefit for patients with lymphoma. Twenty one chronic lymphocytic leukaemia (CLL) patient-derived cell samples were investigated for CD20 expression by flow cytometry and showed high levels of CD20 expression (50-98% of viable cells) and MS4A1 expression (relative expression to housekeeping gene was 177110). Due to numerous issues with virus construction, the effectiveness of the virus was unable to be assessed on Lymphoma cell lines or patient-derived cells; and due to time constraints xenograft models were not derived.

We conclude this MS4A1Ad virus has potential as a treatment for CD20 positive lymphomas, but its efficacy on lymphoma cells and in xenograft models need to be investigated further.