Diphtheria toxin (DT) is often used in the construction of immunotoxins. One potential problem using DTbased immunotoxins is the pre-existing anti-DT antibodies present in human blood due to vaccination. The present study examined the effect of human serum with pre-existing anti-DT antibodies on the toxicity of UCHT1-CRM9, an immunotoxin directed against CD3 molecules on T-lymphocytes. Sera with detectable anti-DT antibodies at 1:100 or greater dilutions inhibited the immunotoxin toxicity.

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... iments with radiolabeled UCHT1-CRM9 indicate that anti-DT antibodies partially block its binding to the cell surface as well as inhibit the translocation from the endosome to the cytosol. The inhibitory effect could be adsorbed using a full-length DT mutant or B-subfragment. A C-terminal truncation mutant could not adsorb the inhibitory effect, suggesting that the last 150 amino acids contain the epitope(s) recognized by the inhibitory antibodies. Therefore, an anti-CD3 single-chain immunotoxin, sFv-DT390, was made with a truncated DT. The IC 50 of sFv-DT390 was 4.8 ؋ 10 ؊11 M, 1/16 the potency of the divalent UCHT1-CRM9. More importantly, sFv-DT390 toxicity was only slightly affected by the anti-DT antibodies in human sera. Mutated full-length and truncated diphtheria toxin (DT) 1 molecules are used for making immunotoxins. These immunotoxins show strong cytotoxic effects to their target cells, and some of them have already been used in clinical trials (1-7). Previously, our laboratory constructed an immunotoxin directed against the CD3⑀ molecule of the T-cell receptor complex, a pan T-cell marker. This construct is made with a monoclonal antibody of mouse-origin, UCHT1, and a binding site mutant of DT, CRM9 (8). The immunotoxin, UCHT1-CRM9, is capable of regressing established xenografted human T-cell (Jurkat) tumors in nude mice (9). A rhesus monkey analog of UCHT1-CRM9, FN18-CRM9, was capable of not only depleting circulating T-cells but also depleting resident T-cells in the lymph nodes. 2 This immunotoxin also delayed skin allograft rejection as compared to antibody treatment and non-treatment controls. FN18-CRM9 has also been used as an adjunct in inducing tolerance to mismatched kidney transplants (24). In contrast with ricin and Pseudomonas exotoxin based immunotoxins, there is a potential problem using UCHT1-CRM9, or other DT-based immunotoxins, in the treatment of human diseases. Most people have been immunized against DT. Therefore these people have a pre-existing anti-DT antibody titer which could potentially inhibit or alter the efficacy of these immunotoxins. This limitation also occurred in our rhesus monkey studies. FN18-CRM9 could deplete T cells in the blood, but to a much lesser extent in animals with anti-DT antibodies, and the T cells repopulated several days earlier compared to those monkeys without anti-DT titers. 2 In order to overcome this antibody mediated inhibition, we undertook the first examination of the effect and the mechanism of human sera containing anti-DT antibodies on UCHT1-CRM9 toxicity. A DT point mutant, a truncation mutant, and DT subfragments were used in an attempt to neutralize the anti-DT effect in human sera. Based on the neutralization data, a single-chain immunotoxin was constructed with a C-terminal deletion mutant of DT which could potentially bypass the inhibitory effect of the pre-existing anti-DT antibodies. MATERIALS AND METHODS Cells-Jurkat cells (ATCC) were maintained in RPMI 1640 supplemented with 10% fetal calf serum, 25 mM sodium bicarbonate, and 50 g/ml gentamycin sulfate. . Purification of the DT B-subfragment has been described (11). Immunotoxins-UCHT1-CRM9 synthesis has been described (12). The recombinant immunotoxin, sFv-DT390, was generated in two phases. First the coding sequences for the variable light (V L ) and variable heavy (V H ) chain regions of the UCHT1 antibody were amplified by a two-step protocol of reverse transcriptase-polymerase chain reaction using primers based on the published sequence (13). The 5Ј V L primer added a unique NcoI restriction enzyme site while the 3Ј V H primer added a termination codon at the J to constant region junction and an EcoRI site. The V L region was joined to the V H region by single-stranded overlap extension and the two regions are separated by a (Gly 3 Ser) 4 linker that should allow for proper folding of the individual variable domains to form a function antibody binding site (14). Second, genomic DNA was isolated from a strain of Corynebacterium diphtheriae producing the DT mutant CRM9 (C7[␤ htox-201tox-9h Ј]) as described (15). This DNA was used for polymerase chain reaction. The 5Ј primer was specific for the toxin gene beginning at the signal sequence and added a unique NdeI restriction site. The 3Ј primer was specific for the DT sequence terminating at amino acid 390 and added an NcoI site in-frame with the coding sequence. The polymerase chain reaction products were digested with the appropriate restriction enzymes and cloned into the Escherichia coli expression plasmid pET-17b (Novagen, * 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. ‡ Contributed equally to this work. § To whom reprint requests should be sent. 1 The abbreviations used are: DT, diphtheria toxin; ELISA, enzymelinked immunosorbent assay; MES, 4-morpholineethanesulfonic acid.