title: The improvement of an anti-CD22 immunotoxin: conversion to single-chain and disulfide stabilized form and affinity maturation by alanine scan.
abstract: HA22-LR is a recombinant immunotoxin for the treatment of B-cell malignancies that contains the Fv portion of an anti-CD22 antibody fused to a functional portion of Pseudomonas exotoxin A. In the present study, we attempted to improve this molecule. First, we produced a single-chain version of HA22-LR (scdsFv-HA22-LR) in which a peptide linker was introduced between the disulfide-linked light and heavy chains to enable production via single fermentation. No difference in cytotoxic activity was observed between scdsFv-HA22-LR and prototype HA22-LR. Next, we attempted to increase the affinity of scdsFv-HA22-LR by using alanine scanning mutagenesis of complementarity determining regions (CDRs) to assess the specific contribution of each CDR residue to the antigen binding. We found that mutation of asparagine 34 in VLCDR1, which is located at the VL/VH interface, to alanine (N34A) caused a substantial increase in affinity and activity. Estimated KD values measured by fluorescence-activated cell sorting were lowered by 10-fold: 0.056 nM in the N34A mutant compared to 0.58 nM in wild type (WT). Cell viability assays of CD22-positive B-cell lymphoma and leukemia cell lines showed that the N34A mutant had increased cytotoxicity ranging from ~2 (HAL-1, IC 50(WT): 2.37 ± 0.62 ng/ml, IC 50(N34A): 1.32 ± 0.41 ng/ml) to 10 (SUDHL-6, IC 50(WT): 0.47 ± 0.090 ng/ml, IC 50(N34A): 0.048 ± 0.018 ng/ml)-fold compared to WT immunotoxin. The present study suggests that the N34A mutant of scdsFv-HA22-LR could have important consequences in a clinical setting.
authors: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors: Seiji
authors: S
authors: Kawa
authors: Masanori
authors: M
authors: Onda
authors: Mitchell
authors: M
authors: Ho
authors: Robert J
authors: RJ
authors: Kreitman
authors: Tapan K
authors: TK
authors: Bera
authors: Ira
authors: I
authors: Pastan
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000409:Alanine; D000595:Amino Acid Sequence; D000915:Antibody Affinity; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000633:toxicity*; D002460:Cell Line; D004220:Disulfides; D005098:Exotoxins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000633:toxicity*; D006801:Humans; D016393:Lymphoma, B-Cell / Q000276:immunology / Q000628:therapy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051918:Sialic Acid Binding Ig-like Lectin 2 / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000633:toxicity*; D037521:Virulence Factors / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000409:Alanine; D001427:Bacterial Toxins; D004220:Disulfides; D005098:Exotoxins; D011994:Recombinant Proteins; D051918:Sialic Acid Binding Ig-like Lectin 2; D057127:Single-Chain Antibodies; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C497867:CD22 protein, human; C541974:immunotoxin HA22