Gene delivery in malignant B cells using the combination of lentiviruses conjugated to anti‐transferrin receptor antibodies and an immunoglobulin promoter

Abstract

Background We previously developed an antibody-avidin fusion protein (ch128.1Av) specific for the human transferrin receptor 1 (TfR1; CD71) to be used as a delivery vector for cancer therapy and showed that ch128.1Av delivers the biotinylated plant toxin saporin-6 into malignant B cells. However, due to widespread expression of TfR1, delivery of the toxin to normal cells is a concern. Therefore, we explored the potential of dual targeted lentiviral-mediated gene therapy approaches to restrict gene expression to malignant B cells. Targeting occurs through the use of ch128.1Av or its parental antibody without avidin (ch128.1) and through transcriptional regulation using an immunoglobulin promoter. Methods Flow cytometry was used to detect the expression of enhanced green fluorescent protein (EGFP) in a panel of cell lines. Cell viability after specific delivery of the therapeutic gene FCU1, a chimeric enzyme consisting of cytosine deaminase genetically fused to uracil phosphoribosyltransferse that converts the 5-fluorocytosine (5-FC) prodrug into toxic metabolites, was monitored by an MTS assay. Results We found that EGFP was specifically expressed in a panel of human malignant B cells, but not in human T cell lines. EGFP expression was observed in all cell lines when a ubiquitous promoter was used. Furthermore, we show the decrease of cell viability in malignant plasma cells in the presence of 5-FC. Conclusions These studies demonstrate that gene expression can be restricted to malignant B cells and suggest that this dual targeted gene therapy strategy may help to circumvent the potential side effects of certain TfR1-targeted protein delivery approaches.

Insights into the effector functions of human IgG3 in the context of an antibody targeting transferrin receptor 1

“…NS0/1 murine myeloma cells were transfected with the human γ3 heavy and human κ light chain expression vectors to express the ch128.1 mutants as previously reported for wild type ch128.1 (Ng et al, 2006). Cells expressing the ch128.1 mutants and wild type ch128.1 were grown in roller bottles and antibodies were purified from cell culture supernatants using affinity chromatography as previously described (Helguera and Penichet, 2005; Leoh et al, 2014; Ng et al, 2006). SDS-PAGE analysis under reducing and non-reducing conditions was performed to confirm the molecular weight and assembly of the mutant antibodies.…”

“…Immulon-H2B plates (Thermo Fisher Scientific, Inc., Waltham, MA) were coated with 1 μg/mL human soluble TfR1 (sCD71) in 50 mM carbonate/bicarbonate buffer, pH 9.3. sCD71, the recombinant extracellular domain of TfR1, was expressed in BHK cells (kind gift of Dr. Anne Mason, University of Vermont, Burlington, VT) and purified as previously described (Leoh et al, 2014). Plates were washed with PBS then blocked with 3% BSA in PBS.…”

“…However, the levels of sensitivity to ch128.1 and ch128.1Av vary among cell lines (Daniels et al, 2007, 2011; Ng et al, 2006; Ortiz-Sanchez et al, 2009). As originally designed, ch128.1Av has been successfully used to deliver biotinylated toxin saporin, a ribosome inactivating protein (Daniels et al, 2007; Daniels-Wells et al, 2013), and lentivirus for gene therapy (Leoh et al, 2014; Morizono et al, 2009). Importantly, significant anti-tumor protection against xenograft models of the human B-cell malignancy multiple myeloma in SCID-Beige mice was observed using ch128.1 or ch128.1Av alone (Daniels et al, 2011).…”

Neurotoxin from Naja naja atra venom inhibits skin allograft rejection in rats

“…The MTS method was used to determine cell proliferation [26,32]. Briefly, 100 μl of spleen cell suspension (5 × 10 6 cells/ml) from each rat was added into individual wells of 96-well plates.…”

Anaplastic Thyroid Carcinoma: Current Treatments and Potential New Therapeutic Options with Emphasis on TfR1/CD71

“…The combinations of such antibodies against TfR on human tumor cells have been demonstrated to have antiproliferative effects both in vitro and in vivo [106, 109–114]. …”