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Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes.

ABSTRACT: Recombinant immunotoxins (RITs) are hybrid proteins used to treat cancer. These proteins are composed of an Fv that reacts with cancer cells joined to a portion of Pseudomonas exotoxin A, which kills the cell. Because the toxin is a foreign protein, it can induce neutralizing antibodies and thereby limit the number of doses a patient can receive. We previously identified seven major mouse B-cell epitopes in the toxin, and subsequently silenced them using point mutations that converted large hydrophilic amino acids to alanine, yet retained full antitumor activity. Here we present results in which we identify and silence human B-cell epitopes in the RIT HA22. We obtained B cells from patients with antibodies to RITs, isolated the corresponding variable fragments (Fvs), and constructed a phage-display library containing Fvs that bind to the RITs. We then used alanine scanning mutagenesis to locate the epitopes. We found that human and mouse epitopes frequently overlap but are not identical. Most mutations that remove mouse epitopes did not remove human epitopes. Using the epitope information, we constructed a variant immunotoxin, HA22-LR-LO10, which has low reactivity with human antisera, yet has high cytotoxic and antitumor activity and can be given to mice at high doses without excess toxicity. The toxin portion of this RIT (LR-LO10) can be used with Fvs targeting other cancer antigens and is suitable for clinical development.

SUBMITTER: Liu W

PROVIDER: S-EPMC3406843 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes.

Liu Wenhai W Onda Masanori M Lee Byungkook B Kreitman Robert J RJ Hassan Raffit R Xiang Laiman L Pastan Ira I

Proceedings of the National Academy of Sciences of the United States of America 20120702 29

Recombinant immunotoxins (RITs) are hybrid proteins used to treat cancer. These proteins are composed of an Fv that reacts with cancer cells joined to a portion of Pseudomonas exotoxin A, which kills the cell. Because the toxin is a foreign protein, it can induce neutralizing antibodies and thereby limit the number of doses a patient can receive. We previously identified seven major mouse B-cell epitopes in the toxin, and subsequently silenced them using point mutations that converted large hydr ...[more]

PMID: 22753489

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Project description:The surge of SARS-CoV-2 has created a wave of pandemic around the globe due to its high transmission rate. To contain this virus, researchers are working around the clock for a solution in the form of vaccine. Due to the impact of this pandemic, the economy and healthcare have immensely suffered around the globe. Thus, an efficient vaccine design is the need of the hour. Moreover, to have a generalised vaccine for heterogeneous human population, the virus genomes from different countries should be considered. Thus, in this work, we have performed genome-wide analysis of 10,664 SARS-CoV-2 genomes of 73 countries around the globe in order to identify the potential conserved regions for the development of peptide based synthetic vaccine viz. epitopes with high immunogenic and antigenic scores. In this regard, multiple sequence alignment technique viz. Clustal Omega is used to align the 10,664 SARS-CoV-2 virus genomes. Thereafter, entropy is computed for each genomic coordinate of the aligned genomes. The entropy values are then used to find the conserved regions. These conserved regions are refined based on the criteria that their lengths should be greater than or equal to 60 nt and their corresponding protein sequences are without any stop codons. Furthermore, Nucleotide BLAST is used to verify the specificity of the conserved regions. As a result, we have obtained 17 conserved regions that belong to NSP3, NSP4, NSP6, NSP8, RdRp, Helicase, endoRNAse, 2'-O-RMT, Spike glycoprotein, ORF3a protein, Membrane glycoprotein and Nucleocapsid protein. Finally, these conserved regions are used to identify the T-cell and B-cell epitopes with their corresponding immunogenic and antigenic scores. Based on these scores, the most immunogenic and antigenic epitopes are then selected for each of these 17 conserved regions. Hence, we have obtained 30 MHC-I and 24 MHC-II restricted T-cell epitopes with 14 and 13 unique HLA alleles and 21 B-cell epitopes for the 17 conserved regions. Moreover, for validating the relevance of these epitopes, the binding conformation of the MHC-I and MHC-II restricted T-cell epitopes are shown with respect to HLA alleles. Also, the physico-chemical properties of the epitopes are reported along with Ramchandran plots and Z-Scores and the population coverage is shown as well. Overall, the analysis shows that the identified epitopes can be considered as potential candidates for vaccine design.

Dataset Information

Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes.

Publications

Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes.

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