Improvement of a Recombinant Anti-Monkey Anti-CD3 Diphtheria

Mar 13, 2007 - Recently, a bivalent recombinant anti-human CD3 diphtheria toxin (DT) based immunotoxin derived from the. scFv of UCHT1 antibody has ...
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Bioconjugate Chem. 2007, 18, 947−955

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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,† 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 CD3γ 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 crosslinking 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 SDS1 reducing gels revealed that all * 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: [email protected]. † 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; dH O, deionized 2 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, aminoactinomycin D; FITC, fluorescein; MCF, mean channel fluorescence; MCF′, background corrected mean channel fluorescence; PE, phycoerythrin.

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′)22 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 biologically 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 (G4S)3 linker, a 10-fold increase in binding activity was achieved compared 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-LVL2-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.

10.1021/bc0603438 Not subject to U.S. Copyright. Published 2007 by American Chemical Society Published on Web 03/13/2007

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to monovalent fusion immunotoxin. This fusion immunotoxin, A-dmDT390biscFv(UCHT1), displayed an increase in potency of 10- to 30-fold compared to the chemically conjugated immunotoxin and depleted 2.4 logs of T cells in the lymph node compartment of transgenic mice expressing human CD3 (9). The increase in potency of the fusion immunotoxins over the chemical conjugate normalized for equal cell binding was ascribed to an increase in the efficiency of postbinding processes within the intoxication pathway (10). The in vivo potency of the divalent fusion immunotoxin, A-dmDT390biscFv(UCHT1), was considered acceptable for clinical development. The expression and purification of this immunotoxin from P. pastoris under GMP compliant conditions has been described (11, 12). Currently, gram quantities have been produced and are undergoing testing for an IND (investigational new drug) submission for the treatment of refractory human T cell leukemia (36). This paper describes the steps used to develop a monkey analogue of the recombinant anti-T cell immunotoxin being readied for clinical trials. A monkey analogue is needed to explore preclinical immune tolerance protocols using a fusion immunotoxin that has similar potency, similar serum half-lives, and biodistribution as the clinical reagent. Preliminary evidence indicated that the scFv of the anti-monkey T cell antibody FN18 was even more sensitive than UCHT1 to positional affects of a neighboring DT390 domain, resulting in a much greater loss in affinity (13). We reasoned that increasing the affinity of FN18scFv would compensate for the loss in affinity of recombinant immunotoxins and consequently increase their bioactivity. Therefore, we endeavored to affinity mature the scFv of FN18 through random mutagenesis and selection by sorting flow cytometry using dye-labeled CD3γ ectodomain heterodimers, and techniques developed by Boder and Wittrup (14). Here, we report the selection of scFv(FN18) mutants with increased binding affinity and the bioactivities of recombinant immunotoxins made with the highest affinity mutants. In additionally, we compare binding data of scFv(UCHT1) and biscFv(UCHT1) to those of the mutant scFv(FN18) and mutant biscFv(FN18) to illustrate differences in the divalent character of biscFvs made from FN18 versus UCHT1.

EXPERIMENTAL PROCEDURES Plasmids, Bacterial and Yeast Strains, Antibodies, Cell Lines. Pichia pastoris strain GS115, plasmids pPICZR and pPIC9K, Saccharomyces cereVisiae strain EBY100, and antiV5 monoclonal antibody were purchased from Invitrogen. Plasmids pET17b and pET27b and E. coli BL21 (DE3) pLysS were purchased from Novagen. Goat anti-mouse IgG2a antibody conjugated with PE /FITC was purchased from Caltag. Streptavidin Alexa Fluor-488 conjugate and streptavidin-FITC was from Molecular Probes. Goat anti-mouse IgG-HRP was purchased from Santa Cruz Biotechnology and used to detect the expression of FN18 scFv in E. coli BL21 (DE3) pLysS. The hybridoma secreting FN18 was kindly provided by Dr. Margreet Jonker, Biomedical Primate Research Center Rijswijk, and was produced and purified by the National Cell Culture Center, Minneapolis, MN. Chemically conjugated anti-monkey anti-CD3 immunotoxin, FN18-CRM9, was prepared as previously described (15). Biocytin-labeled monkey cyno/rhesus CD3γ ectodomain heterodimer ligand with alanine or valine at amino acid residue 35 of CD3 was prepared as described by Wang and Neville (16). The Herpes Saimiri virus transformed cynomolgous cell T cell line HSC-F (17) was supplied by the Centralized Facility for AIDS Reagents supported by EU Programme EVA and the U.K. Medical Research Council. This cell line exhibits the moderate binding FN18 phenotype (37). The Herpes Saimiri transformed rhesus cell T cell line Mm357-2 was supplied by the NIH Nonhuman Primate Reagent Resource,

Wang et al.

Beth Israel Deaconess Medical Center, Boston MA. This cell line exhibits the high binding FN18 phenotype (37). Codon Optimization of scFv(FN18) and Construction of biscFv(FN18). Footnote 2 details the protein domain structure of the recombinant monovalent and bivalent single chain antibody fragments scFv, and biscFv and the diphtheria toxin based immunotoxins derived from these single chain antibodies. Our lab reported that codon optimization is necessary to express DT390-based immunotoxins in Pichia pastoris (18). We used the optimized DT390 nucleotide sequence described by Woo et al. (18) for the DT390 domain of the new anti-monkey immunotoxins. The initial anti-monkey immunotoxin A-dmDT390biscFv(FN18) was constructed from the scFv of FN18 following the strategy used to construct A-dmDT390biscFv(UCHT1) in P. pastoris (18). A-dmDT390biscFv(FN18) was cloned into pnPICZR (XhoI-EcoRI), subcloned into pPIC9K (SacI-EcoRI), which was used to transform P. pastoris strain GS115. Low yields (see results) prompted us to rebuild the new scFv(FN18) DNA using the P. pastoris preferred codons (19). Eight primers for VL and 10 primers for VH of scFv(FN18) were designed to cover the full length of scFv(FN18). The primer length was kept under 70 bases. There was about a 21 base overlap between any of the neighboring primers. Ten picomoles of the first and the last primers and 2 pmol of the rest of the primers were used. The PCR program was, after heating at 95 °C for 4 min, 25 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and then extension for another 10 min. The PCR products were checked with 1% agarose gel electrophoresis. The band with the correct size was cut out and extracted with the QIAquick Gel Extraction Kit. The VL PCR product was digested with BamHI and the VH PCR product with BglII. Then, the VL part and VH part were ligated together with T4 ligase for 6 h. PCR amplification was done with the first primer (VL1a) of the VL carrying NcoI site at the 5′ end and the last primer of the VH (VH10b) carrying the EcoRI site at the 3′ end to get the full length of scFv(FN18). The PCR product was checked again with 1% agarose gel electrophoresis. The band with the correct size (VL + VH) was cut out and extracted with the QIAquick Gel Extraction Kit. The PCR product was digested with both NcoI and EcoRI and cloned into pET17b-DT390 at NcoI-EcoRI for sequencing analysis and E. coli BL21 expression analysis. To construct the rebuilt biscFv(FN18), we amplified the first scFv(FN18) with primers VL1a carrying NcoI site and VH10a carrying BamHI site. We amplified the second scFv(FN18) with VL1b carrying BglII site and VH10b carrying stop codon and EcoRI site. Then, we cloned the first scFv(FN18) into pET27b and the second scFv(FN18) into pET17b for sequencing analysis. Then, the first and second scFv(FN18) were subcloned together into pET17b-DT390 (NcoI-BamHI/BglII-stop codon-EcoRI) and confirmed by E. coli BL21 expression analysis. The DT390-biscFv(FN18) was then subcloned into pnPICZR (XhoI-EcoRI) (18) and then subcloned into pPIC9K (SacI-EcoRI). The rebuilt DT390-biscFv(FN18) in pPIC9K was transformed into Pichia pastoris GS115. Random Mutagenesis. The optimized scFv(FN18) sequence insert described in the previous section was randomly mutated with error-prone PCR using nucleotide analogues (20) as described by Colby et al. (21). Briefly, 6 of 50 µL-PCR reactions were set up with different concentration (200, 20, and 2 µM; two reactions for each of the concentrations) of the two nucleotide analogues: 8-oxo-dGTP and dPTP (8-oxo-2′-deoxyguanosine-5′-triphosphate and 2′-deoxy-p-nucleoside-5′-triphosphate, respectively, both from TriLink Biotech). Other components of the PCR mix were as follows: 10× PCR buffer without MgCl2, 2 mM of MgCl2, 0.5 µM of forward primer PYD5 For (5′ CAG TTA CTT CGC TGT TTT TCA ATA TTT TCT GTT ATT GCT AGC 3′, NheI site underlined), 0.5 µM

Immunotoxin Affinity Maturation

of reverse primer PYD5 Rev (5′ ATC GAG ACC GAG GAG AGG GTT AGG GAT AGG CTT ACC GAA TTC 3′, EcoRI site underlined), 200 µM of dNTP, 0.1-1 ng of pYD5 DNA containing the codon optimized FN18-scFv DNA, 2.5 units of Taq polymerase, adding dH2O to the final volume of 50 µL. The PCR program used was 94 °C for 3 min; then cycles of 94 °C for 45 s, 55 °C for 30 s, 72 °C for 1 min, and finally an extension for another 10 min. PCR cycles were different for varying concentration of the nucleotide analogues (200 µM for 5 cycles, 200 µM for 10 cycles, 20 µM for 10 cycles, 20 µM for 20 cycles, 2 µM for 10 cycles, 2 µM for 20 cycles). The mutagenic PCR products were purified from SYBR Gold (Molecular Probes) stained agarose gel (1%) with QIAquick Gel Extraction Kit. In order to clear the DNA of incorporated nucleotide analogues and to generate sufficient mutated DNA for the transformations, a second round of PCR reactions was performed in the absence of the nucleotide analogues. Six 50 µL reactions were set up for each PCR product from the previous mutagenic reactions. This time, the PCR mix contained 2 µL of the gel-purified mutagenized DNA, 10× PCR-MgCl2 buffer, 1.5 mM of MgCl2, 0.2 µM of forward primer (same as above), 0.2 µM of reverse primer (same as above), 200 µM of dNTP, and 5 units of platinum Taq polymerase, with dH2O added to the final volume. The PCR program was 95 °C for 5 min, 30 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and extension for another 10 min at 72 °C. The PCR products were again gel-purified. This protocol produces 40-100 µg of PCR products. Preparation of the FN18-scFv Yeast Display Library. pYD5 (13) was digested with both NheI and EcoRI and purified with the QIAquick Gel Extraction Kit as the linear vector. The inserts produced by PCR and the linear vector were cotransformed into yeast Saccharomyces cereVisiae EBY100 using the standard transformation procedure in the Gietz Lab Transformation kit (Genomics One International, Inc., Buffalo, NY) as described by the instructions. The inserts were flanked by ∼42 nucleotides homologous to the 5′ and 3′ ends of the open vector, thus permitting homologous combination between the open vector and the insert within the yeast (upstream from NheI in the aga2 signal peptide region and downstream from EcoRI in the V5 epitope region). Transformations utilizing homologous recombination are highly efficient in S. cereVisiae (22). Library size (number of transformants) was determined by plating 0.1 µL, 1 µL, and 10 µL of the 1 mL of the resuspended transformed Saccharomyces cereVisiae EBY100 cells onto a 100 mm screening plate. Using 1 µg of the linear pYD5 vector DNA (NheI-EcoRI) and 10 µg of the mutagenized library insert DNA (PCR product) cotransformed into 1 × 108 yeast Saccharomyces cereVisiae EBY100 cells, we obtained about 1 × 106 transformants. Normally, ten reactions were done and pooled, yielding about 1 × 107 transformants. Growth and Induction. The growth and induction phases were modified as described by Wang et al. (13). The yeast display medium contained 0.67% YNB (Q.Biogene, with ammonium sulfate, without amino acids), 2% glucose (or galactose), 0.5% casamino acids (DIFCO), adjusting pH to 6.0 with pH 6.0 sodium phosphate buffer, 20 µg/mL of ampicillin, 20 µg/mL of kanamycin, 100 units/mL penicillin, and 100 µg/mL streptomycin. For plates, 1.5% agar and 1 M sorbitol were added to the medium. In the growth phase, glucose was used as the carbon source at 30 °C for 24 h, and in the induction phase, galactose was used as the carbon source at 20 °C for 24 h. FACS Analysis, Sorting, and Sequencing. FACS analysis of yeast displayed scFv and KD determination were conducted using biocytin-labeled monkey cyno/rhesus CD3γ ectodomain heterodimer ligand as described by Wang et al. (13). The sorting for higher-affinity FN18 sFv displaying yeast cells was per-

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formed on an Epics Elite ESP cell sorter (Beckman Coulter). The number of yeast cells screened was set as a factor of 10 greater than the library size to ensure that no unique clones were overlooked. Yeast cells were stained with anti-V5 antibody (Invitrogen) and CD3γ biocytin labeled ligand at 4 °C for 1.5 to 2 h. The amount of V5 antibody used was 1 µL/106 cells, while the molar amount of CD3γ biocytin to be used was predetermined by analytical FACS analysis and average KD measurement on the mixed clone population (usually 0.1-0.05 × KD). Yeast samples were then washed twice at 5000 g for 5 min at 4 °C. Staining with secondary reagents was done for 1 h at 4 °C with goat anti-mouse IgG2a-PE (Caltag) 5 µL and streptavidin Alexa Fluor 488 (Molecular Probes) 2 µL/106 cells, respectively. Samples were then washed twice at 5000 g for 5 min at 4 °C. Screening was done with an equilibrium screen (21) utilizing bivariate plots of the stained V5 and CD3γ ligand. Cells were initially gated on the characteristic of their forward and side light scatter to avoid cellular clumps and debris. If in the sort sample a double-positive diagonal was observed, the sample was sorted with a diagonal edge to normalize for expression of the scFv. In cases where no double-positive diagonal was present, almost the entire dual positive quadrant was collected. The first rounds of sorting were given a generous sort gate anywhere from 1% to 4% utilizing the gated logic mode. The purity mode was used for the third and fourth rounds as gate stringency was increased, reaching 0.1%. Sorted events were collected in yeast growth medium (see Growth and Induction section). After each round of mutagenesis, we did four rounds of sorting. Then, random clones picked up from selection plates were propagated, induced, and analyzed by FACS for their binding affinities as previously described (13). The yeast mutant plasmid DNA was extracted with a Zymoprep Yeast Plasmid Minipreparation kit (Zymo Research, Orange, CA) from those clones with the highest affinity and then transformed into E. coli. After the amplification in E. coli, the plasmid DNA was sequenced to determine the mutation(s) in the scFv sequence or used as template DNA for the next round of mutagenesis. We repeated the process until we failed to generate any new higher-affinity mutants. In this study, we did three rounds of the mutagenesis. Site-directed mutagenesis was applied to combine some the higher-affinity mutations into a single plasmid clone using a QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Expression and Purification of Mutant Immunotoxins in P. pastoris. To make DT390-biscFv(FN18-M20) bivalent immunotoxin, the first scFv(FN18-M20) and second scFv(FN18-M20) genes were amplified by PCR (template DNA, M20 yeast plasmid; primers for the first scFv, WP-051 and WP053; primers for the second scFv, WP-054 and WP-052. All of the WP and GP primers are listed in Table 1). The first scFv PCR product was digested with NcoI and BamHI, and the second scFv PCR product was digested with BamHI and EcoRI. Then, the two fragments were inserted into NcoI and EcoRI sites of pET17b-DT390 (pJW1019). The DT390-biscFv(FN18-M20) gene was subcloned into XhoI and EcoRI sites of pnPICZR. The resulting plasmid (pJW1020) was transformed into Pichia pastoris JW107, and the transformants were selected on YPD plates containing zeocin (100 µg/mL). Six colonies were randomly selected and cultivated in test tubes containing 5 mL YPD at 28 °C at 250 rpm for 2 days. The cultures were induced with methanol for 1 day, and then the culture supernatants were analyzed on SDS-PAGE under nonreducing condition. One clone (JW132) was selected and cultivated in shake-flasks to purify DT390-biscFv(FN18-M20) by phenyl hydrophobic interaction chromatography and then Super Q anion exchange chromatography. Solid sodium sulfate was added to 595 mL of culture supernatant to a final concentration of 500 mM. The

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Table 1. Primer Sequence for the Construction of scFvs and Immunotoxinsa

a

primers

sequence (5′f3′)

WP-051 WP-052 WP-053 WP-054 WP-061 WP-073 WP-075 GP-080 GP-089 GP-090 GP-102

TTCTT GCCAT GGGAC GTTGT TATGT CTCAA TCT CGTGA ATTCT TAAGA GGAGA CGGTG ACAGA GGT ATGGA ATTCA TGGGA TCCAC CGCCT CCGCT GCCTC CGCCT CCAGA GGAGA CGGTG ACAGA GGT GGTGG ATCCG GAGGG GGAGG CTCGG ACGTT GTTAT GTCTC AATCT TTCTT GCTCG AGAAA AGAGA CGTTG TTATG TCTCA ATCT TACAT ATTTT TGTTT TTGCT GTCAT TCGTT CAAGG CGACG TTGTT ATGTC TCAAT CT ATGTT CGAAA CGATG AATAT ATTTT ACATA TTTTT GTTTT TGCTG TCA TACAT ATTTT TGTTT TTGCT GTCAT TCGTT CAAGG CGACA TTGTT ATGTC TCAAT CT TTCTT GCCAT GGGAC TTTGT TATGT CTCAA TCT GGTGG ATCCG GAGGG GGAGG CTCGG ACTTT GTTAT GTCTC AATCT TACAT ATTTT TGTTT TTGCT GTCAT TCGTT CAAGG CGACT TTGTT ATGTC TCAAT CT

Restriction sites are underlined. Boldface indicates the DNA sequences used to anneal two primers.

sample was loaded onto 50 mL phenyl 650M (Tosoh) in a 5 cm × 30 cm × K50 column (Amersham Biosciences). The bound DT390-biscFv(FN18-M20) was eluted with TEG buffer containing 5% glycerol, 20 mM Tris-HCl, and 1 mM EDTA (pH 8.0). Pooled fractions containing DT390-biscFv(FN18M20) were dialyzed 3 times against TE buffer. The dialyzed sample was loaded to 3.5 mL of Super Q 650M (Tosoh) in a 1.5 cm × 10 cm Flex column (Kontes Glass Company). The bound DT390-biscFv(FN18-M20) was eluted with 50 mM NaCl in TEG buffer. To make DT390-biscFv(FN18-C207) bivalent immunotoxin, the first scFv(FN18-C207) and second scFv(FN18-C207) genes were amplified by PCR (template DNA, C207 yeast display plasmid; primers for the first scFv, GP-089 and WP053; primers for the second scFv, GP-090 and WP-052). The first scFv PCR product was digested with NcoI and BamHI, and the second scFv PCR product was digested with BamHI and EcoRI. Then, the two scFv parts in the vector pJW1020 (DT390-biscFv(FN18-M20) in pnPICZR) were sequentially replaced with C207 counterparts using NcoI and BamHI for the first scFv and BamHI and EcoRI for the second scFv. Purification of DT390-biscFv(FN18-C207) was performed as described for DT390-biscFv(FN18-M20) except that the loading of the anion exchange column was performed at pH 9.0 instead of pH 8.0 and the protein was eluted with 50 mM sodium borate at pH 8.5. In addition, the DT390-biscFv(FN18-C207) immunotoxin was fractionated on Superdex-200 10/300 GL to remove lower molecular weight breakdown products. Purity of both immunotoxins was judged by SDS gel electrophoresis and HPLC size exclusion chromatography on Superdex-200 10/300 GL (Amersham Biosciences). Expression and Purification of Wild-Type, Mutant scFvs and biscFvs. For the production of the following scFvs and biscFvs, the original R-factor signal sequence in the vector system was replaced with killer toxin (kt) signal peptide due to the incomplete cleavage in the Kex2 cleavage site between the R-factor signal sequence and the inserted protein sequences of interest. This cleavage can be variable, since it is influenced by the first two amino acid residues of the post-Kex2 insert (13). To obtain scFv(FN18-WT), the scFv(FN18-WT) gene was PCR-amplified from the scFv(FN18-WT) yeast display plasmid using the primers (GP-080 and WP-052). After the PCR product was purified by gel elution, it was used for the second PCR using the primers WP-075 and WP-052. Primer WP-075 contained the 5′-sequence of the signal sequence of killer toxin from KluyVeromyces lactis. After the second PCR, the product was digested with SfuI and EcoRI and purified by gel elution, and the treated PCR fragment was cloned into pwPICZR, which now contained the killer toxin signal peptide. The scFv(FN18M20) and scFv(FN18-C207) genes were obtained by the same procedure using different primer sets for the first PCR (WP073 and WP-052 for M20; GP-102 and WP-052 for C207). To make biscFv(FN18-C207), the first scFv(FN18-C207) and

second scFv(FN18-C207) genes were PCR-amplified from the C207 yeast display plasmid. The first scFv was obtained by the two-step PCR as described in the previous section (primers GP-102 and WP-053 for the first PCR; primers WP-075 and WP-053 for the second PCR) and the second scFv was PCRamplified using the primers (GP-090 and WP-052). The first scFv PCR product was digested with SfuI and EcoRI, purified by gel elution, and cloned into pwPICZR. This constructed vector was digested with BamHI and EcoRI and ligated with the scFv PCR product treated with the same enzymes. Transformation, growth, and induction conditions were similar to those used for the immunotoxins with the exception of biscFv(FN18C207) where a modified version of strain mutEF2JC307 was used (23, 36). Protein L affinity chromatography was used to purify the scFv and biscFv constructs; 1.8 mL of 5 M NaCl was added to 58 mL of culture supernatant and then loaded to 4 mL of Protein L agarose (Pierce) in a 1.5 cm × 10 cm Flex column (Kontes Glass Company) that was equilibrated with 100 mM potassium phosphate buffer, 150 mM NaCl (pH 7.0). The bound scFv(FN18-M20) was eluted with 100 mM glycine buffer (pH 2.5). 100 µL of 1 M Tris-HCl buffer (pH 8.0) was added to the eluted fractions. The eluted fractions were pooled and concentrated using an Amicon Ultra-4 (10 000 MWCO, Millipore). Purity was judged by SDS gel electrophoresis and HPLC Superdex 200 10/300 GL gel filtration (Amersham Biosciences). Purity was above 95% in all cases. Concentration was determined from the calculated absorption coefficients based on the amino acid sequences located at http://researchlink. labvelocity.com/tools/conversionTools/proteinExtinction. jhtml. Protein Synthesis Inhibition Assay. This was performed by measuring the incorporation of 3H-labeled leucine into HSC-F cells after a 20 h exposure to immunotoxins at different concentrations. Means and standard deviations of six replicates were calculated and divided by the means of non-immunotoxin treated controls × 100 to give % control protein synthesis as previously described (9). T Cell Binding Assay of Recombinant scFv Antibodies And Immunotoxins. FN18-FITC conjugate was purchased from Biosource and added to 250 000 HSC-F cells at 5 × 10-9 M in the presence of varying concentrations of scFv and immunotoxin binding competitors at 4 °C for 30 min. The displacement of FN18-FITC was measured by FACS on a Beckman-Coulter Cytomics FC 500 instrument after washing the cells two times at 400 g at 4 °C and resuspending in 0.5 mL of 1 × PBS containing 2.5 µg of propidium iodide to exclude dead cells from further analysis. Mean channel fluorescent values, MCI, were corrected (MCF′) by subtracting an appropriate FITC-labeled isotope control from both the FN18FITC tracer and the FN18-FITC tracer plus competitor MCF values. The % inhibition was determined as [(MCF′FN18-FITC - MCF′FN18-FITC+competitor)/MCF′FN18-FITC] × 100. The relative binding affinity between any two competitors was determined

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Immunotoxin Affinity Maturation

by dividing their respective concentrations at equal % inhibition obtained from plots of % inhibition versus concentration as previously described (9). Immunotoxin-Induced Loss of CD3+CD4+ Resting Monkey T Cells. T cells from normal monkeys were isolated from blood by two centrifugations through Lymphocyte Separation Medium from BioWhittaker following the manufacturer’s directions. The cells were washed and incubated in RPMI 1640 medium containing 25 mM HEPES with 10% fetal calf serum + 2 g/L NaHCO3, adjusted to pH 7.4, plus 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, and 50 µg/L of gentamicin sulfate with and without varying concentrations of immunotoxins for 72 h at 5% CO2, 37 °C. Cells were washed and then stained for 30 min at 4 °C with anti-CD3 (SP34-2PE BD Pharmingen), anti-CD4-FITC (BD Pharmingen), CD20PE Cy7 (BD Biosciences), and the vital dye 7-ADD (BD Pharmingen) and analyzed on a Beckman-Coulter Cytomics FC 500 instrument counting 104 events. Cells were analyzed for uptake of the vital dye, the percent of cells within the lymphocyte forward scatter/side scatter gate, and the percent of cells within the lymphocyte gate within the quadrant displaying the CD3+ and CD4+ epitopes by two-color FACS. The % of cells within the lymphocyte gate at each immunotoxin concentration was divided by % of cells within the lymphocyte gate for the 72 h no-treatment control (G1). The % of cells within the CD3+CD4+ quadrant at each immunotoxin concentration was divided by % of cells within the CD3+CD4+ quadrant for the 72 h no-treatment control (Q1). The % loss of CD3+CD4+ cells was calculated as G1 × Q1 × 100.

RESULTS Higher Immunotoxin Expression with Codon-Optimized scFv(FN18). Before codon optimization of the biscFv(FN18) domain of DT390biscFv(FN18), expression in P. pastoris strain GS115 yielded shake-flask culture supernatants with a barely visible band at 96.3 kDa corresponding to the DT390biscFv(FN18) by Coomassie staining of SDS gels. Five clones screened from 91 clones by dot blots for higher expression gave supernatant concentrations of 1.3 to 2.7 ng/µL by comparison with a known concentration standard of DT390biscFv(UCHT1) in SDS gels. After rebuilding, 5 out of 12 transformants in GS115 had supernatant concentrations equal to 11 ng/µL after 24 h of induction judged by SDS gels (gel data not shown). A four-step purification procedure utilized absorption to Thiophilic Resin (Clonetech) followed by elution according to manufacturer’s directions, dialysis versus 20 M Tris-HCl, pH 7.0, application to DEAE Sepharose (Amersham) followed by a step elution with 0.5 M NaCl and finally application to a Zorbax GF-250 column (DuPont). The major peak ran as a single band when analyzed on HPLC with a Zorbax GF-250 column. Purity was estimated as 95% by HPLC size exclusion and SDS gels. Higher-Binding Mutants Selected with CD3Eγ Ectodomain Heterodimer. When this work was begun (16), we were using the monkey CD3 sequence reported by Uda et al. (24) for the selecting ligand in the CD3γ ectodomain heterodimer used in flow cytometry. We encountered very low binding for the wildtype (WT) displayed scFvs and the initial mutant scFvs (13). Subsequently, through consulting the rhesus genome data base and by sequencing the CD3 ectodomain of 24 rhesus and cynomolgous monkeys, we determined that high FN18-binding monkeys have a valine at amino acid residue 35 of CD3. The presence of alanine at this position is a frequently occurring polymorphism that reduces FN18 binding even when present in the heterozygote state (37). In our recent work, mutant selections utilized valine at position 35 of CD3 of CD3γ ectodomain heterodimer. However, the change from A35 to V35 failed to select any new mutations.

Table 2. Representative scFv(FN18) Mutants Obtained from Yeast Display Selectiona scFv clones

Wt

KD (nM)

mutations and locations

L1

M20

C207

13.9 ( 1.0 5.5 ( 0.3 4.4 ( 1.1 2.9 ( 0.3

b

I2F I2V K 18 R S 32 F Q 35 R K 166 R R 168 K E 184 G A 207 V S 225 A

VL-FR1 VL-FR1 VL-FR1 VL-CDR1 VL-CDR1 VH-FR2 VH-FR2 VH-CDR2 VH-FR3 VH-FR3

+ + +

+ + +

+ +

+ + +

+ + + + + + + +

a

Clone name indicates the mutation and residue within the scFv. Residue numbering proceeds from the NH2 terminus of VL, the (G4S)3 linker and VH. b Mean equilibrium dissociation constants, KD, determined as in Figure 1 from two titrations and curve fittings with CD3γ ectodomain heterodimer35V batch L5-5. Errors are (1 SD.

Random mutagenesis of yeast displayed FN18 scFv libraries selected by dye-labeled ectodomain CD3γ heterodimers for increased affinity yielded 43 mutations. Seven occurred in the first round in 3 clones, 31 in the second round in 20 clones, and 27 in the third round in 14 clones. Of these 43 mutations, only 9 showed significant increases in affinity when combined in single clones. These 10 mutations and their locations are summarized in Table 2. Three of these mutations occur in the complementary determining regions (CDR), two in the VL and one in the VH chains. The highest-affinity mutant, C207, contains a net increase of two positive charges over the wildtype. C207 was first made by combining several mutations by site-directed mutagenesis after the second round of mutagenesis and selection. However, C207 appeared spontaneously after three rounds of mutagenesis and selection. After the second round of mutagenesis and selection, no new affinity-enhancing mutants were found, although new favorable combinations were found in the third round of mutagenesis and selection. The affinity of the yeast displayed scFv(FN18-C207) was increased 4.8-fold over the wild-type (WT) scFv. The titration curve and curve fitting for C207 and the WT scFv are shown in Figure 1. The binding curves appear saturable by inspection, and this is supported by the fact that the R values are >0.99 using a fitting equation that contains only a saturable term (see inserts in Figure 1 for the binding fitting equation and fit parameters). Nonspecific binding is by definition nonsaturable (25). If it were present here, it would appear as a linear increase in MFI as a function of ligand concentration superimposed on the saturable curve, and it would lower the R values (unless a nonsaturable term was added to the fitting equation). These considerations demonstrate that the measured increase in affinity caused by the C207 mutations is due to an increase in specific binding rather than nonspecific binding. The maximum mean fluorescent intensity at saturation increases by 2.4-fold in the C207 mutant compared to the WT, suggesting that the mutant has an increased protein folding stability within the yeast secretory pathway (26). Purity of P. pastoris Mutant Immunotoxins. Following the three-step purification procedure, DT390-biscFv(FN18-M20) and DT390-biscFv(FN18-C207) displayed a major band on Coomassie-stained SDS nonreducing gels (Figure 2) migrating near the 97.3 kDa marker. DT390-biscFv(FN18-C207) showed several faint bands migrating near the 66.3 and 55.4 kDa markers. Western blots identified these as breakdown products. On Superdex-200 chromatography, these appeared as a small trailing shoulder accounting for 5.4% of the major band peak height. These bands were removed by subsequent fractionation on Superdex-200 as shown in the Figure 3 SDS nonreducing gel.

952 Bioconjugate Chem., Vol. 18, No. 3, 2007

Figure 1. A comparison of titrations of CD3γ ectodomains labeled with streptavidin-488 on yeast displaying the wild-type scFv(FN18WT), (0); and the highest-affinity selected mutant, scFv(FN18-C207), (O), is shown. The mean fluorescence intensity, MFI, is plotted vs the CD3γ concentration on the x-axis. The data points are fitted using nonlinear least-squares by KaleidaGraph software (Synergy Software, Reading, PA) using the hyperbolic equation y ) m1 + m2 × M0/(m3 + M0), where y ) MFI at the given ligand concentration M0, m1 ) MFI at 0 ligand concentration, m2 ) MFI at saturation minus m2, and m3 ) KD, the equilibrium dissociation constant in nM units. The top inserted box contains the C207 mutant fit data with a KD of 3 nM. The CD3 preparation contains V at residue 35.

Wang et al.

Figure 3. SDS 4-12% gradient tris-glycine nonreducing gel of DT390biscFv(FN18-C207) after an additional purification step of fractionation on Superdex-G200 to remove lower molecular weight contaminants. From left to right, the lanes are (1) molecular weight markers, (2,3) 22 µg of DT390biscFv(FN18-C207), (4,5) 44 µg of DT390biscFv(FN18-C207), and (6,7) standard DT390biscFv(UCHT1).

Figure 4. The % inhibition of tracer FITC-labeled FN18 binding to HSC-F monkey T cells is plotted vs the concentration of binding competitors consisting of mutant and wild-type scFvs, biscFvs, and immunotoxins derived from these scFvs. The relative binding affinity for any two competitors can be estimated from the ratio of their concentrations at equal % inhibition values. Estimates are considered to be reliable when comparing parallel curves or parallel curve regions. Means from replicate experiments comparing binding affinities relative to scFv(FN18-WT) are shown in Table 3.

Figure 2. SDS 12% acrylamide bis-tris nonreducing gel of immunotoxins DT390biscFv(FN18-M20) and DT390biscFv(FN18-C207) are shown after the three-step purification procedure. From left to right, the lanes are (1) molecular weight markers, (2-4) immunotoxin standard DT390biscFv(UCHT1), (5-7) DT390biscFv(FN18-M20), and (8-10) DT390biscFv(FN18-C207). Numbers at the top indicate the load in µg applied. Marker MWs are given at the left.

Binding of Mutant scFvs and Their Derived Immunotoxins to T Cells. The binding activity of the P. pastoris expressed mutants and their derivative immunotoxins to the monkey TCR was explored in a binding competition assay using the continuous cultured monkey T cell line HSC-F. A tracer amount of FITC-labeled FN18 antibody was added to HSC-F cells along

with varying concentrations of competitors, and the % inhibition of the FN18-FITC binding was determined by FACS (Figure 4). The binding activity of the scFv(FN18-WT) is reduced over the parental divalent FN18 by a factor of 0.29. Likewise, the binding activity of the immunotoxin derived from the scFv(FN18-WT) is reduced by a factor of 0.01 compared to the parental antibody and by 0.04 compared to scFv(FN18-WT) as judged by the ratio of competitor concentrations at equal % inhibition values shown in Figure 4. The loss of binding activity of scFvs placed C-terminal to DT390 and other large proteins has been previously reported (9). However, the extent of the binding loss is 10-fold greater in the FN18 constructs compared to the anti-human CD3 UCHT1 constructs. This observation prompted us to undertake the affinity maturation of FN18 scFv in order to regain binding affinity within the immunotoxin

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Immunotoxin Affinity Maturation Table 3. Relative Affinity Changes of Mutant scFvs(FN18)a mutant scFv affinity increase by yeast display relative to scFv-WT WT M20 C207 C207

scFv 1.0 ( 0.07 2.5 ( 0.14 4.8 ( 0.4 biscFv

P. pastoris expressed mutant scFv affinity increase by T cell binding relative to scFv-WTb 1.0 ( 0.014 3.0 ( 0.14 9.8 ( 0.2.4 16.7 ( 0.3

Results are means + 1 SD for 2 or 3 determinations. b T cell binding affinity was measured displacement of FN18-FITC tracer from HSF cells by scFv competitors and ratioing equal displacement concentrations. a

format. The scFv(FN18-M20) and scFv(FN18-C207) do exhibit increased binding in this assay, and the binding of biscFv(FN18-C207) is further enhanced. These results are summarized in Table 3, right-hand column, where the mean increases in binding affinity of the mutant scFvs relative to scFv(FN18-WT) are listed from Figure 4 data and replicate experiments. The relative affinity changes determined from the yeast display KD values are also listed for comparison. In Figure 4, the increase in the scFv(FN18-M20) is seen to be effective in increasing the affinity of the M20 derived immunotoxin, DT390biscFv(FN18-M20). However, the increase is only about 4-fold over the WT immunotoxin, DT390-biscFv(FN18-WT). The DT390biscFv(FN18-C207) immunotoxin binding was not significantly different from the DT390-biscFv(FN18-M20) immunotoxin over the concentration range covered, although the steeper curve suggests that a difference might be observed at higher concentrations. Increased Cytotoxicity of FN18-scFv Mutant-Derived Immunotoxin. In spite of the relatively small increase in binding affinity provided by the mutant M20 and C207 immunotoxins, the bioactivity of these immunotoxins exhibited large increases over the WT immunotoxins. Dose-response curves of a protein synthesis inhibition assay in HSC-F cells are shown in Figure 5 after a 20 h exposure to immunotoxins. In comparison to the DT390-biscFv(FN18-WT) immunotoxin, the M20 mutant immunotoxin is increased 68-fold, while the C207 mutant immunotoxin is increased 228-fold. Relative to the chemically conjugated immunotoxin, FN18-CRM9, these two mutant immunotoxins are increased 17- and 57-fold respectively. The receptor specificity of the assay is shown by the ability of a 10-fold excess of the FN18 antibody to compete with the cytotoxicity of each mutant immunotoxin at its highest concentration (filled symbols), and the lack of toxicity of the noncross-reacting anti-human immunotoxin DT390biscFv(UCHT1). In another continuously cultured rhesus cell line, Mm357-2, M20 and C207 immunotoxins were increased 7-fold and 35fold, respectively, over the chemically conjugated immunotoxin (data not shown). In order to more closely replicate the in vivo status of T cells, we assayed the effects of these immunotoxins on freshly isolated T cells from peripheral monkey blood. Because the majority of these T cells are resting, the level of protein synthesis is too low to measure by the incorporation of labeled amino acids. However, resting T cells exposed to immunotoxins do incorporate the vital dye 7-AAD that is normally excluded from viable cells. We have found that these T cells also disappear from the normal lymphocyte gate. Those T cells that remain in the gate lose surface CD3 and CD4 epitopes in an immunotoxin dose- and time-dependent fashion. In Figure 6a, the DT390biscFv(FN18-C207) mutant is seen to be 7.7-fold more potent than the chemical conjugate, 5.4-fold ( 3.3 for two replicate assays, while in Figure 6b, the DT390biscFv(FN18-M20) mutant is

Figure 5. The % control inhibition of protein synthesis for four immunotoxins on HSC-F monkey T cells is plotted vs the immunotoxin concentrations on the x-axis after a 24 h exposure. Each point is the mean of six replicates and the positive sides of 1 SD single-sided error bars are shown. The mutant recombinant immunotoxin DT390biscFv(FN18-C207) is the most potent immunotoxin, exceeding the wild-type recombinant immunotoxin by 238-fold. The receptor specificity of the assay is shown by the ability of a 10-fold excess of the FN18 antibody to compete the cytotoxicity of each immunotoxin at its highest concentration (filled symbols) and the lack of toxicity of the non-crossreacting anti-human immunotoxin DT390biscFv(UCHT1).

seen to be 0.4-fold less potent than the chemical conjugate. Therefor, enhanced potency for the C207 mutant relative to the M20 mutant is 13.5-fold in resting T cells. Similar results were found when the assay measures the loss of the CD3+CD4- cell population (data not shown). The possible causes for differences in relative potency between resting T cells and continuously cultured T cell lines are considered in the next section.

DISCUSSION We increased the affinity of the anti-CD3 antibody FN18 by random mutagenesis combined with sorting flow cytometry selection using dye-labeled monkey CD3γ heterodimeric ectodomains. Although the affinity increase was modest in the best mutant, C207 (4.8-fold by yeast display), the increase was greater when judged by binding assays to T cells (9.8-fold) and further increased in the biscFv format used in diphtheria toxin based immunotoxins (16.7-fold). This resulted in an increase in bioactivity of 228-fold when measured on a continuously cultured monkey T cell line referenced to immunotoxin made with the wild-type scFv. Bioactivity toward monkey resting T cells isolated from peripheral blood increased 5.4-fold over the chemically conjugated immunotoxin, FN18-CRM9, that depletes resting T cells in monkey lymph nodes by 1.5-2 logs (2). Although the wild-type and the two mutant immunotoxins show the same rank order with regard to bioactivity between resting and transformed T cells, the large numerical differences imply differing rate-limiting steps within the intoxication pathway in resting versus transformed T cells. The rate-limiting step in diphtheria toxin intoxication of cells actively synthesizing protein has been ascribed to the translocation step of the toxin A chain into the cytosol compartment (27). However, resting T cells have very low rates of protein synthesis unless activated (28). In this situation, the toxin substrate, elongation factor-2, is continuously bound to the ribosome (as opposed to cycling on and off) and is protected against enzymatic inactivation by the toxin A chain (29). The rate of protein synthesis would then

954 Bioconjugate Chem., Vol. 18, No. 3, 2007

Figure 6. The loss of monkey peripheral blood CD3+CD4+ T cells from the FACS lymphocyte gate is shown after a 72 h exposure to immunotoxins as a function of immunotoxin concentration. In part a, the DT390biscFv(FN18-C207) mutant is seen to be 7.7-fold more potent than the chemical conjugate, 5.4-fold + 3.3 for two replicate assays; while in part b, the DT390biscFv(FN18-M20) mutant is seen to be 0.4-fold less potent than the chemical conjugate. The enhanced potency for the C207 mutant relative to the M20 mutant is 13.5-fold in resting T cells.

become rate-limiting for the intoxication process. It has been shown that the antibody moiety of an immunotoxin interacting with CD3 provides an activating signal (30). The strength of this interaction may be determined by subtle features of the interaction and thus could enhance the intoxication rate. These considerations may explain why recombinant immunotoxins made with mutant C207 are active in depleting blood and lymph node T cells in monkeys in vivo, providing a homogeneous reagent for preclinical testing of tolerance induction in organ transplantation protocols (38). The current study exploited the advantages of using Pichia pastoris for recombinant protein expression. All of the FN18 derivatives, mutant scFv, biscFv, and recombinant immunotoxins were expressed in P. pastoris using the secretory pathway ensuring optimal folding of each. This gave us confidence that the comparative numbers in binding and bioactivity assays reflected the advantages of the mutations rather than differences in protein folding that are often encountered when refolding proteins from insoluble granules in E. coli. Varying the signal peptide was found to be a valuable tool in optimizing the expression of these different constructs. Analysis of the mutations in mutant C207 revealed that there was a net gain of two positive charges over the WT scFv within

Wang et al.

two CDR regions. Two recent crystallographic structures of antihuman CD3 antibodies OKT3 and UCHT1 bound to the human CD3γ (31) and human CD3δ ectodomains (32) showed that the antibodies bind to an electronegative area on the exposed surface of CD3. It is possible that these new positive charges in the mutant are aiding the antibody CD3 interaction. The new mutations present in the C207 immunotoxin might cause some increase in immunogenicity and possibly compromise this methodology for human use. However, the major immunogen in this case is the toxin moiety. Preclinical animal studies have shown that T cell depletion sufficient for tolerating transplant protocols can be achieved with only 4 days of consecutive dosing, prior to the initiation of either a primary or secondary antibody response (9). Our binding studies show that the scFv of FN18 loses affinity by a factor of 0.29 compared to the parental divalent antibody. This is nearly identical to the case of UCHT1 (0.3-fold) (9). Both scFvs lose affinity when placed C-terminal to DT390 (33). The strategy of using the biscFv format in DT-based immunotoxins was to compensate for this positional loss of affinity by introducing a bivalent interaction. However, the binding data presented here show that the biscFv format works less well for DT390-biscFv(FN18) than for DT390-biscFv(UCHT1), and the difference is 10-fold. The increase in affinity on going from scFv(UCHT1) to biscFv(UCHT1) is 5-fold (9). The increase in affinity on going from C207-scFv to C207-biscFv is only 16.7/ 9.8- or 1.7-fold (Table 3). This suggests that the biscFv of FN18 mutants has less divalent character than the UCHT1 biscFv. This could be the result of pairing preferences. In the biscFv structure, VL1-L-VH1-L-VL2-L-VH2, optimal divalent character will be achieved by binding of VL1 with VH1 and VL2 with VH2. However, other options for pairing are possible that will limit the divalent character of the biscFv. In the future, it will be necessary to explore other antibody formats for the biscFv domain of the anti-monkey immunotoxins derived from FN18. Diabody constructs with shortened linkers may be an option to force optimal pairing to achieve better divalent character (34, 35).

ACKNOWLEDGMENT This research was supported by the Intramural Research Program of NIMH, NIH. We thank Jim Nagle of the NINDS/ NIH DNA Sequencing Facility for their generous support. Supporting Information Available: The eight primers for VL and ten primers for VH to rebuild the scFv(FN18) for P. pastoris optimized codons. This material is available free of charge via the Internet as http://pubs.acs.org/BC.

LITERATURE CITED (1) Neville, D. M., Jr., Scharff, J., and Srinivasachar, K. (1992) In vivo T cell ablation by a holo-immunotoxin directed at human CD3. Proc. Natl. Acad. Sci. U.S.A. 89, 2585-2589. (2) Hu, H., Stavrou, S., Baker, B. C., Tornatore, C., Scharff, J., Okunieff, P., and Neville, D. M., Jr. (1997) Depletion of T lymphocytes with immunotoxin retards the progress of experimental allergic encephalomyelitis in rhesus monkeys. Cell. Immunol. 177, 26-34. (3) Fechner, J. H., Jr., Vargo, D. J., Geissler, E. K., Graeb. C., Wang, J., Hanaway, M. J., Watkins, D. J., Piekarczyk, M., Neville, D. M., Jr., and Knechtle, S. J. (1997) Split tolerance induced by immunotoxin in a rhesus kidney allograft model. Transplantation 63, 13391345. (4) Contreras, J. L., Jenkins, S., Eckhoff, D. E., Hubbard, W. J., Lobashevsky, A., Bilbao, G., Thomas, F. T., Neville, D. M., Jr., and Thomas, J. M. (2003) Stable R- and β-islet cell function after tolerance induction to pancreatic islet allografts in diabetic primates. Am. J. Transplant. 3, 128-138.

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