We have made an anti-mesothelin immunotoxin in which the major human B cell epitopes have been silenced or removed. Roche has taken a license to this invention and manufactured a new immunotoxin named RG7787 that has an anti-mesothelin Fab fused to a mutated form of PE. RG7787 has good anti-tumor activity in animal models and when combined with a Taxane produces complete remissions in these models. We have also initiated a program to identify and remove human specific T cell epitopes. We have identified all the major T cell epitopes in PE38 and using alanine-scanning mutagenesis identified amino acids essential for T cell activation. Using this information we have constructed LMB-T20, which contains six mutations in domain III (R427A, F443A, L477H, R494A, R505A and L552E) and a deletion of all the epitopes in domain II. LMB-T20 has low immunogenicity, is very active on mesothelin-expressing cancer cells and has good anti-tumor activity in mice. It is a good candidate for clinical development We are attempting to make a new immunotoxin with mutations that will eliminate both B and T cell epitopes. In collaboration with D. FitzGerald we have begun to study how protein synthesis arrest leads to apoptosis and have identified BAK and Mcl-1 as critical players in this process. We have also begun to study how protein phosphorylation regulates sensitivity of cells to immunotoxins by doing knock down studies of tyrosine and serine/threonine kinases and measuring the effect of knock down on immunotoxin mediated cell death. In collaboration with Alan Wayne POB, we have analyzed the response of cells directly isolated from patients with ALL to HA22 killing and are trying to set up an assay that may help predict if patients will respond to treatment. We have also isolated HA22 resistant cell lines and are using these to understand the basis of clinical drug resistance. One of the mechanisms is the loss of ability to synthesize DPH4 due to methylation of the the promoter of that gene. A significant advance is described in Liu, Onda et al in PNAS this year. This paper shows the identification and removal of human B cell epitopes from PE based immunotoxins producing a new immunotoxin predicted to have very low immunogenicity in humans.
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