title: Improved cytotoxic activity toward cell lines and fresh leukemia cells of a mutant anti-CD22 immunotoxin obtained by antibody phage display.
abstract: Recombinant immunotoxins are fusion proteins composed of the Fv domains of antibodies fused to bacterial or plant toxins that are being developed for the targeted therapy of cancer. RFB4 (Fv)-Pseudomonas exotoxin 38 (PE38) is an immunotoxin that targets CD22 expressed on B cells and B-cell malignancies. A disulfide-stabilized form of RFB4 (Fv)-PE38 is being evaluated in a Phase I clinical trial. The aim of the present study was to improve the activity of RFB4 (Fv)-PE38 to more effectively treat patients with leukemias and lymphomas. To increase the affinity of RFB4 (Fv), we used the techniques of phage display and hot spot mutagenesis. We identified mutational hot spot sequences in heavy chain complementary determining region 3 (V(H) CDR3) and randomized these in a phage display library. Mutant phages were panned on CD22-positive Daudi cells. A variety of mutant Fvs were obtained, and the corresponding immunotoxins were prepared. Several mutant immunotoxins with increased binding affinity and cytotoxic activity were obtained. The most active immunotoxin contained amino acid residues Thr-His-Trp (THW) in place of Ser-Ser-Tyr (SSY) at positions 100, 100A, and 100B of the Fv and had an affinity improved from 85 nM to 6 nM. The THW mutant had a 5- to 10-fold increase in activity on various CD22-positive cell lines and was up to 50 times more cytotoxic to cells from patients with chronic lymphocytic leukemia and hairy-cell leukemia.
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-4264, USA.
authors.forename: Giuliana
authors.initials: G
authors.lastname: Salvatore
authors.forename: Richard
authors.initials: R
authors.lastname: Beers
authors.forename: Inger
authors.initials: I
authors.lastname: Margulies
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Kreitman
authors.forename: Ira
authors.initials: I
authors.lastname: Pastan
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000494:pharmacology*; D000915:Antibody Affinity; D015703:Antigens, CD / Q000276:immunology*; D000944:Antigens, Differentiation, B-Lymphocyte / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D015815:Cell Adhesion Molecules; D002460:Cell Line; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000494:pharmacology; D000922:Immunotoxins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D037102:Lectins; D007938:Leukemia / Q000188:drug therapy* / Q000473:pathology; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D011506:Proteins / Q000187:drug effects; D051918:Sialic Acid Binding Ig-like Lectin 2; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; C497867:CD22 protein, human; D015815:Cell Adhesion Molecules; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D037102:Lectins; D011506:Proteins; D051918:Sialic Acid Binding Ig-like Lectin 2; C055966:immunoglobulin Fv