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 (VH 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.
Footnotes
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 To whom requests for reprints should be addressed, at Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Building 37, Room 5106, NIH, 37 Convent Drive, MSC 4264, Bethesda, MD 20892-4264. Phone: (301) 496-4797; fax: (301) 402-1344; E-mail: pasta{at}helix.nih.gov
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↵2 The abbreviations used are: PE38, Pseudomonas exotoxin 38; HCL, hairy-cell leukemia; dsFv, disulfide stabilized Fv; DPBS, Dulbecco’s phosphate buffered saline; scFv, single-chain Fv; CLL, chronic lymphocytic leukemia; RIT, recombinant immunotoxin; CDR3, complementary determining region 3; WT, wild type.
- Accepted December 17, 2001.
- Received August 6, 2001.
- Revision received December 10, 2001.
Volume 8, Issue 4
