Polymorphisms of CD3ε in cynomolgus and rhesus monkeys and their relevance to anti-CD3 antibodies and immunotoxins

Abstract

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 CD3ε 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 CD3ε chain and designed primers that can be used to amplify and quickly sequence the ectodomain of monkey CD3ε. 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 CD3ε 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 CD3ε, respectively; those having low FN18 reactivity were homozygous in CD3ε 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. © 2007 Australasian Society for Immunology Inc. All rights reserved.