Abstract 4334: Identification and elimination of T-cell epitopes in recombinant immunotoxins used to treat cancer.
Authors: Mazor Ronit , Vassall Aaron N. , Eberle Jaime A. , Beers Richard , Hu Xiaobo , Benhar Itai , Pastan Ira 
Published: August 2014

Abstract
Moxetumomab pasudotox and SS1P are immunotoxins that have been developed in our lab to treat cancer. Moxetumomab pasudotox, which can be given for many treatment cycles, targets CD22 and has produced many complete remissions in drug-resistant hairy cell leukemia. However, SS1P, which targets mesothelin, could only be given for one cycle before ADA (anti drug antibodies) developed and neutralized the protein. We hypothesize that SS1P could be more effective if it were less immunogenic and more treatment cycles could be given.
To de-immunize the immunotoxin we sought to identify and remove T cell epitopes. To identify the epitopes we stimulated PBMCs from 50 normal donors with whole immunotoxin for 14 days to expand the T cells and then stimulated the T cells with peptides (15 mers) derived from the toxin, followed by ELISpot to identify the peptides that stimulated the T cells. We found that all 50 donors gave a positive response, which correlates with the frequency of ADAs in patients with normal immune systems. We used alanine-scanning mutagenesis to determine which amino acids in each peptide are important for T cell stimulation.
SS1P contains domains II and III of PE. In 46% of the donors we found a single highly immunodominant and promiscuous epitope that is located in domain II. We previously showed we could eliminate almost all of domain II while retaining excellent cytotoxic activity. These data indicate that immunogenicity should be eliminated in 34% of normal individuals donors and diminished in another 42% by deletion of domain II. In domain III we identified 5 peptides that stimulated T cell responses in different donors and also identified alanine mutations within each peptide that eliminated or greatly lowered T cell stimulation in most donors. We are using this information to make immunotoxins with mutations in domain III that should have a further decrease in immunogenicity.
We have shown that among many individuals with different HLA alleles there is a dominant T cell epitope in domain II of PE38 and 5 subdominant epitopes in domain III. We plan to use this information to make new immunotoxins in which the large majority of T cell epitopes are silenced resulting in diminished immunogenicity of the immunotoxin. We plan to combine these T cell mutations with B cell mutations previously described to make immunotoxins with very low immunogenicity allowing more treatment cycles to be given and better anti-tumor activity achieved.
Citation Format: Ronit Mazor, Aaron N. Vassall, Jaime A. Eberle, Richard Beers, Xiaobo Hu, Itai Benhar, Ira Pastan. Identification and elimination of T-cell epitopes in recombinant immunotoxins used to treat cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4334. doi:10.1158/1538-7445.AM2013-4334