Improvement of a Recombinant Anti-Monkey Anti-CD3 Diphtheria Toxin Based Immunotoxin by Yeast Display Affinity Maturation of the scFv

Abstract

Recently, a bivalent recombinant anti-human CD3 diphtheria toxin (DT) based immunotoxin derived from the scFv of UCHT1 antibody has been made that shows enhanced bioactivity and is free from the side effects of Fc receptor interaction. In this case, the diminution of CD3 binding due to the placement of the scFv domain at the C-terminus of the truncated DT in single scFv immunotoxins was compensated by adding an additional scFv domain. However, this strategy was less successful for constructing an anti-rhesus recombinant immunotoxin derived from the scFv of FN18 antibody due to poor binding of the anti-rhesus bivalent immunotoxin. We report here that, by increasing the FN18 scFv affinity through random mutagenesis and selection with a dye-labeled monkey CD3epsilongamma recombinant heterodimer, we greatly improved the bioactivity of FN18 derived immunotoxin. The best mutant, C207, contained nine mutations, two of which were located in CDRs that changed the charge from negative to positive. Binding affinity of the C207 scFv to the monkey T cell line HSC-F increased 9.8-fold. The potency of the C207 bivalent immunotoxin assayed by inhibition of protein synthesis increased by 238-fold.

Development of a Diphtheria Toxin Based Antiporcine CD3 Recombinant Immunotoxin

“…It is also possible that increasing the affinity of the scFv (2-6-15) similar to the affinity maturation approach used for the anti-monkey CD3 recombinant immunotoxin may improve depletion function. Affinity maturation of the scFv (2-6-15) by yeast display 20 is currently in progress to isolate a higher affinity scFv (2-6-15) for improved porcine T cell depletion in vivo .…”

Recombinant anti-monkey CD3 immunotoxin depletes peripheral lymph node T lymphocytes more effectively than rabbit anti-thymocyte globulin in naïve baboons

“…A recombinant version with better production yields and bioactivity was produced [15] and in one rhesus macaque demonstrated to have LN depleting function [16]. …”

“…Transplantation studies using this reagent in monkeys showed improved graft survival of renal grafts [11–14]. Due to low production yields of the above agent, a recombinant version with higher T cell affinity and greater bioactivity in a DT based immunotoxin compared to FN18 was developed [15]. In a fold-back diabody format, affinity matured FN18 scFv (C207) has a 7-fold increase in T cell binding and showed a profound decrease in lymph node T cells from 66% to 18.7% in one treated rhesus macaque [16].…”

An Anti‐PSMA Bivalent Immunotoxin Exhibits Specificity and Efficacy for Prostate Cancer Imaging and Therapy

“…[14,15] The DT-based anti-CD3 bivalent immunotoxin, A-dmDT390-bisFv (UCHT1), has been shown to bind to its target and depletes T cells effectively [16] This immunotoxin has undergone preclinical studies [17,18] and is currently in clinical trials for treating cutaneous T-cell lymphoma (clinical trial identifier NCT00611208). In general, bivalent immunotoxins containing two scFv units have higher affinity and efficacy towards the targeted cells than those containing one scFv.…”