A fold-back single-chain diabody format enhances the bioactivity of an anti-monkey CD3 recombinant diphtheria toxin-based immunotoxin

Abstract

T-cell depleting anti-CD3 immunotoxins have utility in non-human primate models of transplantation tolerance and autoimmune disease therapy. We recently reported that an affinity matured single-chain (scFv) anti-monkey CD3 antibody, C207, had increased binding to T-cells and increased bioactivity in a diphtheria toxin (DT)-based biscFv immunotoxin compared with the parental antibody, FN18. However, FN18 scFvs and their mutant derivatives such as C207 did not exhibit robust bivalent character in the biscFv format. We now report that C207 in a diabody format exhibits a 7-fold increase in binding to T-cells over scFv (C207) indicating considerable divalent character for the diabody. This construct was formed by reducing the V(L)/V(H) linker to five residues and was secreted from Pichia pastoris as the non-covalent dimer. An immunotoxin based on this diabody format was secreted as a non-covalent dimer but was devoid of bioactivity and failed to bind T-cells, suggesting steric hindrance from the two large closely positioned truncated DT moieties. We constructed a single-chain diabody immunotoxin by fusing to the truncated DT C-terminus L1-VL-L1-VH-L2-VL-L1-VH where L1 is a five-residue linker and L2 is the longer (G4S)3 linker permitting interactions between the distal and proximal VL/VH domains. This 'fold-back' immunotoxin was secreted predominantly as the monomer and exhibited a 5- to 7-fold increase in bioactivity over DT390biscFv(C207) and depleted monkey T-cells in vivo.

Impact of irradiation and immunosuppressive agents on immune system homeostasis in rhesus macaques

“…Three female and one male RMs 2-3 years old received a single dose of 4 Gy TBI. Anti-monkey CD3 recombinant immunotoxin (CD3-IT) at 50 lg/kg was administered intravenously by slow bolus injection (NHP resource, material produced by the Massachusetts General Hospital-Dana Farber-Harvard Cancer Center Recombinant Protein Expression and Purification Core Facility) [19,23,24]. JAK inhibitor (tofacitinib citrate, CP-690550 citrate; Selleckchem, Houston, TX, USA) at 15 mg/kg diluted in 0Á5% methylcellulose was given for 5 consecutive days by oral gavage [29].…”

“…CD3-IT is a recombinant fusion protein consisting of a truncated diphtheria toxin fused to affinity matured anti-CD3 antibody FN18, which is able to deplete T cells [19]. Anti-CD3 immunotoxins have been evaluated in clinical settings for T cell lymphoma [20,21] and in numerous transplant models [22][23][24][25].…”

A bivalent recombinant immunotoxin with high potency against tumors with EGFR and EGFRvIII expression

“…[27][28][29][30] The full linear sequence of DT390-BiscFv806 was DT390-V L -(G 4 S) 3 -V H -(G 4 S) 3 -V L -(G 4 S) 3 -V H , which was confirmed by DNA sequencing. The cartoon structure of DT390-BiscFv806 was shown in Figure 1A, presenting a bivalent tandem scFv format.…”

“…Importantly, P. pastoris is capable of folding multi-domain RITs properly by forming disulfide bonds. [27][28][29][30] The expression and purification parameters of this system have been further optimized for production scale up of RITs according to the U.S. FDA GMP guidelines. An example is the anti-CD3 RIT, Resimmune (A-dmDT390-bisFv(UCHT1)), which is under Phase II clinical trials and has been produced at a raw yield of »207 mg/L and a purified yield of »144.2 mg/L after a 163-hour induction period in a single batch of 120 L bioreactor culture.…”

“…Our previous studies on different formats of RITs have confirmed the higher binding affinity of bivalent RITs than that of monovalent RITs. 28,29,40 The studies with RITs targeting PSMA (A-dmDT390-scfbDb(PSMA)) or CD3 (A-dmDT390-bisFv(UCHT1)) have shown that the binding affinity and cytoxicity of the bivalent format are >2.5-fold and >10-fold higher, respectively, than that of a monovalent format. 28,29,40 We expected that the bivalent DT390-BiscFv806 would have a high binding affinity and cytotoxicity to the targeted cells.…”