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

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.

Improvement of a recombinant anti-monkey anti-CD3 diphtheria toxin based immunotoxin by yeast display affinity maturation of the scFv.

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.

Polymorphisms of CD3epsilon in cynomolgus and rhesus monkeys and their relevance to anti-CD3 antibodies and immunotoxins.

The monoclonal antibody FN18 has been used as a marker for monkey T cells and as a T-cell-depleting reagent when conjugated to diphtheria toxin that was mutated to prevent binding to non-targeted cells. The antibody recognizes a conformational epitope on the ectodomain of monkey CD3epsilon and displays a range of binding activity to the T cells from different rhesus and cynomolgus monkeys. Our quantitative fluorescence-activated cell sorting analysis of the FN18 reactivity to T cells from different rhesus and cynomolgus monkeys showed that there are at least three levels of FN18 reactivity in the monkeys tested: high, moderate and low. On the basis of available DNA sequence information, we determined the gene structure of rhesus CD3epsilon chain and designed primers that can be used to amplify and quickly sequence the ectodomain of monkey CD3epsilon. Our sequence analysis revealed that the extent of nucleotide sequence variation in this area is greater than that previously reported. In addition to the amino acids at positions 45 and 50, we demonstrated that position 35 of CD3epsilon was also important and substitution of amino acid A for V at this position greatly reduced T-cell reactivity to FN18. We found that T cells from monkeys with high FN18 reactivity all had V, E and R at positions 35, 45 and 50 in CD3epsilon, respectively; those having low FN18 reactivity were homozygous in CD3epsilon with at least one of the changes: V35 to A, E45 to G and R to 50Q, whereas members in the moderate group are heterozygous, having both V and A, E and G, R and Q at these locations. A cytotoxicity assay revealed that T cells from a heterozygous rhesus monkey with moderate FN18 reactivity were much (about 40 times) less sensitive to a FN18-derived immunotoxin than those from a homozygous rhesus monkey having high FN18 reactivity.

Increasing secretion of a bivalent anti-T-cell immunotoxin by Pichia pastoris.

The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G(4)S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation. This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut(+)) under control of the AOX1 promoter. There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P. pastoris and the limited capacity of P. pastoris to secrete the immunotoxin. The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction. Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction. The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature. A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth. The effects of adding glycerol and yeast extract to the methanol feed were synergistic. Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production. The synergy was further enhanced by decreasing the induction temperature from 23 to 15 degrees C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system. This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive P. pastoris.

Gene optimization is necessary to express a bivalent anti-human anti-T cell immunotoxin in Pichia pastoris.

The bivalent anti-human anti-T cell immunotoxin A-dmDT390-bisFv(G(4)S) was developed for treatment of T cell leukemia, autoimmune diseases, and tolerance induction for transplantation. The multi-domain structure of the bivalent immunotoxin hinders efficient production in Escherichia coli and most eukaryotes are sensitive to the toxin. However, Pichia pastoris has a tolerance to levels of DT (diphtheria toxin) that were previously observed to intoxicate wild type eukaryotic cells, including Saccharomyces cerevisiae. This tolerance has permitted the optimization of the secreted expression of A-dmDT390-bisFv(G(4)S) in P. pastoris under the control of AOX1 (alcohol oxidase 1) promoter. The original DNA sequence of A-dmDT390-bisFv(G(4)S) was not expressed in P. pastoris because of several AT-rich regions, which induce an early termination of transcription. After DNA rebuilding for abolishing AT-rich regions and codon optimization, the immunotoxin could be expressed up to 10mg/L in the shake flask culture. No differences in the expression levels of immunotoxin were observed by using different secretional signal sequences, Mut(s) (methanol utilization slow phenotype) or Mut(+) (methanol utilization plus phenotype) phenotypes. Buffered complex medium (pH 7.0) having 1% casamino acids provided the highest expression in shake flask culture and PMSF (phenylmethylsulfonyl fluoride) in the range of 1 to 3mM further improved the expression level presumably by inhibiting protein degradation. The immunotoxin was purified by DEAE (diethylaminoethyl) Sepharose ion exchange chromatography and Protein L affinity chromatography. The immunotoxin purified from P. pastoris culture was as fully functional as that expressed in a toxin resistant mutant CHO (Chinese hamster ovary) cell line. Our results demonstrate that P. pastoris is an ideal system for expression of toxin-based fusion proteins.