Improvement of a Recombinant Anti-Monkey Anti-CD3 Diphtheria Toxin Based Immunotoxin by Yeast Display Affinity Maturation of the scFv Zhirui Wang, Geun-Bae Kim, ² Jung-Hee Woo, ‡ Yuan Yi Liu, Askale Mathias, Scott Stavrou, and David M. Neville, Jr.* Section on Biophysical Chemistry, Laboratory of Molecular Biology, National Institute of Mental Health, Building 10 Rm 3D46, 10 Center Drive, Bethesda, Maryland 20892-1216. Received November 2, 2006; Revised Manuscript Received December 28, 2006 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 CD3ǫγ 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. INTRODUCTION Anti-CD3 immunotoxins induce profound but transient T cell depletion in vivo by inhibiting eukaryotic protein synthesis, and they have utility in nonhuman primate models of transplantation tolerance and autoimmune disease therapy. The chemically conjugated anti-human immunotoxin, UCHT1-CRM9, depleted 3 logs of xenografted human T leukemic cells in nude mice resulting in long-term regressions of 86% of the tumors in an animal model of T cell leukemia/lymphoma (1). The chemically conjugated anti-monkey immunotoxin, FN18-CRM9, depleted 1.5-2.0 logs of T cells from the lymph node compartment and markedly prolonged the survival of kidney allografts (2, 3). In combination with a short-term course of deoxyspergualin, FN18-CRM9 produced long-term tolerance to pancreatic islet allografts in 3 out of 7 recipients (functioning graft survival over 5 years) (4). An anti-porcine anti-T cell conjugate was effective in maintaining long-term hematopoietic stem cell transplants in the absence of graft versus host disease (5). These conjugated immunotoxins were formed by chemically cross- linking the antibody or antibody fragment to a diphtheria toxin binding site mutant, CRM9. In this way, the binding site of the immunotoxin was dictated by the antibody moiety (6). Analysis of these 1:1 conjugates by SDS 1 reducing gels revealed that all of the possible linkages between the toxin A and B chains and the antibody heavy and light chains were formed, even though the average link per molecule was maintained at one. Although immunoglobulin receptor interactions could be eliminated by conjugation with F(ab′) 2 2 antibody fragments, purified yields were low (2% of toxin input) and linkage heterogeneity remained. In order to overcome the disadvantages of chemically conjugated immunotoxins, recombinant anti-human anti-T cell immunotoxins were developed. These immunotoxins, utilizing diphtheria toxin truncated at amino acid residue 390, were restricted to having the antibody moiety placed C-terminal to the truncated toxin, because antibody domains fused to the N-terminal of the toxin interfered with translocation of biologi- cally active A chain (7, 8). However, it was found that when the scFv of UCHT1 was fused to the C-terminus of DT390 its binding activity toward T cells was reduced by a factor of 0.033 compared to the free scFv. Since the monovalent scFv was reduced in binding by a factor of 0.3 compared to the parental divalent antibody, the overall binding reduction of this fusion immunotoxin was 0.01 compared to the parental antibody. By adding a second scFv moiety separated through a (G 4 S) 3 linker, a 10-fold increase in binding activity was achieved compared * Corresponding author. National Institute of Mental Health, Bldg 10, Rm 3D46, 10 Center Drive, Bethesda, Maryland 20892-1216, Tel: (301) 496 6807, Fax: (301) 480 0446, E-mail: davidn@mail.nih.gov. ² Current address: Department of Animal Science and Technology, Chung-Ang University, Gyeonggi-Do 456-756 Korea. ‡ Current address: Cancer Research Institute of Scott and White Memorial Hospital, 5701 South Airport Road, Temple, Texas 76502. 1 Abbreviations: SDS, sodium dodecyl sulfate; dH2O, deionized H2O; FACS, fluorescent activated cell sorting; TE buffer, a buffer containing tris[hydroxymethyl]aminomethane and EDTA; TEG buffer, a similar buffer with the addition of 5% glycerol; 7-AAD, amino- actinomycin D; FITC, fluorescein; MCF, mean channel fluorescence; MCF′, background corrected mean channel fluorescence; PE, phyco- erythrin. 2 Abbreviations of antibody fragments and recombinant antibodies and immunotoxins: F(ab′)2, pepsin generated NH2 terminal antibody fragment containing the both variable light chains, VL, and both the variable heavy chains, VH, the constant light and heavy chains and the interchain disulfide hinge; FR, framework region of variable light or heavy chains; CDR, complimentary determining region of variable light or heavy chains; scFv, recombinant single chain variable region VL-L-VH where in this work L represents a glycine serine (G4S)3 linker; biscFv, a recombinant tandem repeat of scFv, VL1-L-VH1-L- VL2-L-VH2, numerals indicate the order of the chains; DT390-biscFv, a recombinant immunotoxin consisting of the catalytic and translocation domain of diphtheria toxin truncated at residue 390 and preceded by an alanine residue and containing a double mutation, dm, removing glycosylation sites fused to a biscFv following residue 390. Writing in parenthesis following Fv indicates the lineage of the Fv. 947 Bioconjugate Chem. 2007, 18, 947-955 10.1021/bc0603438 Not subject to U.S. Copyright. Published 2007 by American Chemical Society Published on Web 03/13/2007