1 Fenske Laboratory, University Park, PA 16802, 2 Section on Biophysical Chemistry, Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, MD 28092-4034, 3 Novartis Pharmaceuticals, Summit, NJ 07901 and 5 Division of Transplantation Immunology, University of Alabama at Birmingham, AL 35294, USA
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Abstract |
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Keywords: CD3/depletion/divalent/immunotoxin/T cell/transplantation
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Introduction |
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The T cell receptor protein CD3 efficiently mediates the entry of diphtheria toxin-based immunotoxins (Neville et al., 1989
). In monkeys, reduction of 23 logs of lymph node T cells is achieved using a chemical conjugate of anti-rhesus CD3 antibody and CRM9, a diphtheria toxin binding-site mutant (Neville et al., 1996
; Hu et al., 1997
). Long-term tolerance to xenograft pancreatic islet transplants and renal allograft transplants have been described using this material (Contreras et al., 2000
; Thomas et al., 2000
). We have described a monovalent recombinant form of this conjugate and its anti-human counterpart, DT390-sFv (UCHT1), that has reduced sensitivity to anti-DT neutralizing antibodies (Thompson et al., 1995
; Ma et al., 1997
). The sFv moiety in this type of immunotoxin is restricted to the toxin C-terminus because the N-terminal toxin A chain can accept only a limited number of foreign residues before its enzymatic properties are compromised (Madshus et al., 1992
).
This paper reports an optimized derivative DT390-sFv (UCHT1) immunotoxin. Our focus has been on improving the binding affinity of this recombinant immunotoxin. It appeared to us that this could most easily be accomplished by increasing the valency of the immunotoxin to reflect the divalency of the parental antibody. Our work has been patterned on previous efforts to form multivalent recombinant antibody derivatives (Holliger et al., 1993; Shu et al., 1993
; Whitlow et al., 1993
; Kipriyanov et al., 1994
; Hu et al., 1996
), either through single-chain tandem sFv groups or through heavy chain disulfide bound dimeric structures, the so-called minibodies. We therefore made a systematic investigation of the binding of anti-CD3
antibody fragments and recombinant derivatives to CD3
expressing cells and the immunotoxins so derived. Linker size and placement were also investigated. In order to judge the in vivo potency of our optimized immunotoxin, we used a transgenic mouse that expresses human CD3
(Wang et al., 1998
), thereby acquiring pre-clinical data but avoiding the use of large non-human primates. Our data suggest that this newly optimized molecule has valuable in vivo potential for the treatment of T cell tumors as well as induction of tolerance for transplantation.
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Materials and methods |
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The construction of DT390-sFv has been described (Thompson et al., 1995). This construct contains the first 390 amino acids of native DT, including the secretory signal peptide, upstream of the UCHT1 sFv [in the variable light (VL) to variable heavy (VH) configuration]. The VLVHlinker is (G4S)3. A variant monovalent construct, M-DT389-sFv, was also produced (Hexham et al.). This construct was expressed in Escherichia coli without a signal peptide and was refolded and purified from cytoplasmic inclusion bodies. This construct encoded an N-terminal methionine residue. In addition to having one less DT residue, this construct also contained a six-residue flexible linker (ASAGGS) between the DT moiety and the sFv moiety and the sequence of the VLVHlinker was changed to (G3S)4, see Fig. 1
.
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The generation of the bisFv construct was completed in two separate steps. In the first step, the 5' end of the UCHT1-sFv (VL) was modified by polymerase chain reaction (PCR) to contain part of the linker sequence (the 3' end of the linker was added to the 5' end of the sFv). Additionally, the 3' end of the UCHT1-sFv (VH) was modified to incorporate the 5'end of the linker sequence. In the second step, the two sFv DNA sequences (VLVHlinker and linkerVLVH) were verified to encode the appropriate amino acid sequences and subcloned together (VLVHlinkerVLVH) using the unique BamHI restriction enzyme site. The bisFv sequence was then subcloned downstream of the DT390 sequence in pET15b to generate DT390-bisFv (G4S).
A similar scheme was used to create a bisFv construct using the CHB1 linker (Mallender and Voss, 1994), between the two sFv domains. For the CHB1 linker, the two sFv DNA sequences (VLVHlinker and linkerVLVH) were verified to encode the appropriate amino acid sequences and subcloned together (VLVHlinkerVLVH) using the unique XhoI restriction enzyme site. The bisFv sequence was then subcloned downstream of the DT390 sequence in pET-15b to generate DT390-bisFv (CHB1).
Construction, expression and quantification of UCHT1 derivatives
The Fab fragment of UCHT1 was prepared by papain digestion using cross-linked agarosepapain (Pierce) following the manufacturer's directions. Contaminating Fc fragments and undigested UCHT1 were removed by absorption on to protein A Sepharose (Pierce). Fab was quantified by size-exclusion HPLC (GF-250 Zorbax) and UV absorption and integration using a UCHT1 standard and a correction for MW reduction.
The sFv and bisFv of UCHT1 were amplified by PCR. The template was DT390-bisFv (G4S) (see above) and the primers were chosen from the 5' end of VL and the 3' end of VH. The PCR products were checked by gel electrophoresis and the bands of sFv and bisFv of UCHT1 were cut out and extracted with a QIAquik Gel Extraction Kit (Qiagen, Chatsworth, CA). The extracted DNA was digested with NdeI and EcoRI and cloned into expression vector pET17b (Novagen) at NdeI and EcoRI sites. These were transformed into E.coli strain: BL21 (DE3)pLysS competent cells (Novagen). A single colony was cultured overnight. The overnight culture was re-cultured for 34 h with fresh medium to reach an OD600 of 0.6, after which IPTG, 1 mM, was added to induce expression. The bacterial pellet was harvested by centrifugation. Inclusion bodies were prepared by the method of Buchner et al. (Buchner et al., 1992) and solubilization of inclusion bodies and refolding of the protein were performed according to Vallera et al. (Vallera et al., 1996
).
Minibody construction and expression
The [sFv(UCHT1)-H-CH3-h]2 minibody was constructed by PCR overlap extension. This construct was patterned on the flex minibody described by Hu et al. (Hu et al., 1996
). The sFv (UCHT1) template was DT390-sFv (see above). In the minibody, the sFv region is followed by the hinge region of human IgG1 (residue 216 to residue 229). These residues were amplified from a plasmid containing human IgG1 heavy chain (supplied by Dr Syed Kashmiri, National Cancer Institute). In the hinge, residue C220 was changed to P (Shu et al., 1993
) leaving C226 and C229 residues to form the interchain disulfide dimer. Between the hinge and
CH3, a flexible spacer, (G3S2)2, was inserted following the construction of Hu et al (Hu et al., 1996
). A histidine (h) tag of six residues was added at the C-terminus of
CH3 and a murine kappa signal peptide, MSVPTQVLGLLLLWLTDARC, was placed 5' to the sFv (Xiang, 1992
). This gene was cloned into pBacPAK8 (Clontech, Palo Alto, CA) and expressed in S9 cells at a level of 5 µg/ml as quantified by Coomassie Brilliant Blue staining using UCHT1 as a standard. Although the sFv of UCHT1 does not bind to Protein L Plus (Pierce) at pH 8.0 in PBS, it does bind in the presence of high salt. One volume of culture medium was mixed with 1.5 volumes of 2.5 M glycine5 M NaCl, pH 9.0, and applied to a 1 ml column volume of Protein-L plus equilibrated with 1.5 M glycine3 M NaCl, pH 9.0. The column was washed with two column volumes of application buffer and eluted with 4 ml of 0.25 M glycineHCl, pH 2.5. The divalent construct was quantified by Coomassie Brilliant Blue staining of SDS gels using a Fab (UCHT1) standard.
The [sFv(UCHT1)-µCH2-h]2 minibody construct was made by using PCR amplification, from previously cloned single-chain human IgM antibody construct (Ma et al., 1996). The sFv(UCHT1)-µCH2-h construct was amplified by using 5' sFv and 3' CH2 primers. A six histidine residue tag was introduced to the 3' end of the CH2 domain. Following purification of the amplified sFv(UCHT1)-µCH2-h fragment by gel elution and digestion with EcoRI and NotI, it was inserted between the EcoRI and NotI sites of pET17b vector (Novagen). E.coli XL-1 Blue strain was used for all plasmid constructions. For expression in Pichia pastoris, pPICZ
(Invitrogen, Carlsbad, CA) was used as the Pichia expression vector. The DNA sequence was confirmed by sequencing. KM71 was used as the host strain (Invitrogen). Maximum secretion of divalent anti-CD3 minibody could be obtained at 4 days after methanol induction in 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% yeast nitrogen base, 4x10-5% biotin, 1% methanol plus 1% casamino acids to retard proteolysis. Western blots from non-reducing and reducing gels probed with polyclonal anti human IgM (Life Technologies, Bethesda, MD) confirmed the disulfide dimer, which accounted for 75% of the secreted material. Partial purification was achieved by absorbing contaminating proteins on DEAE Sepharose at pH 8.5 and applying the flow-through to Protein L agarose (Pierce) in the presence of 1.5 M glycine and 3 M NaCl, pH 8.9, and eluting with PBS diluted 1:3 in water. The divalent construct was quantified by Coomassie Brilliant Blue staining of SDS gels using a Fab (UCHT1) standard.
Construction, expression and quantification of HSA-sFv(UCHT1)
The gene for the HSA-sFv(UCHT1) fusion construct codes from 5' to 3' the 609 residues of human serum albumin precursor variant A followed by a six-residue flexible linker (ASAGGS) and then the sFv moiety of sFv (UCHT1) used in DT389-sFv (see above). The fusion was performed by PCR overlap extension. This gene was cloned into the pHILD2 vector (Invitrogen) under the AOX1 promoter and expressed as a secreted form in Pichia pastoris GS115. HSA-sFv(UCHT1) was purified from the supernatant by ammonium sulfate precipitation, followed by cation-exchange chromatography on a Bio-Rad S2 column at pH 6.0, where the protein was in the unbound fraction. HSA-sFv(UCHT1) was quantified by Lowry protein assay using an HSA standard.
Expression of other immunotoxins
The production of recombinant immunotoxins such as dmDT390-sFv(His6) from stably transfected DT resistant CHO cell lines by means of the pSR-neo vector has been described (Liu et al., 2000
). The notation dm refers to the double mutation removing the potential N-glycosylation sites at positions 1618 in the DT A chain and positions 235237 in the DT B chain. The signal peptide used in CHO expression contained an additional terminal alanine to optimize the cleavage process at an AlaAla junction and therefore added an Ala residue to the DT N-terminus. This new construct is now called A-dmDT390-sFv (after genetic removal of the C terminal His 6 tag). For CHO cell expression of the bivalent single-chain construct, DT390 was removed at the NcoI site from the DT390sFv plasmid for E.coli expression and replaced by sp-dmDT390 yielding on expression A-dmDT390-bisFv (G4S). The production of the disulfide-linked immunotoxins (A-DT390-sFv-H-
CH3-h)2 and (A-DT390-sFv-µCH2-h)2 was performed in CHO cells as described above, except that the glycosylation sites were not removed by mutation. These constructs were treated with N-glycosidase prior to toxicity assays under conditions that removed all detectable glycosylated forms (Liu et al., 2000
). M-DT389-sFv was expressed in E.coli and purified from cytoplasmic inclusion bodies as indicated above. Routine quantification was by Lowry protein assay calibrated by mass spectrometry.
Protein synthesis inhibition assay
Inhibition assays were performed as described previously (Neville et al., 1989). Briefly, various concentrations of immunotoxin were incubated with Jurkat cells (1x104 cells/well of a 96-well plate) for 20 h. A 1 h pulse of [3H]leucine (4.5 µC/ml) was given before cells were collected on filters with a Skatron harvester (Skatron, Sterling, VA). Samples were counted in a scintillation counter. Each experiment was performed in quadruplicate. Results were calculated into a mean value and recorded as a percentage of control cells.
Recombinant antibody and immunotoxin affinity estimation by FACS analysis
The anti-human anti-CD3 antibody, UCHT1, kindly provided by Dr P.Beverley, Imperial Cancer Research Fund, was derivatized with FITC at 5 mol/mol Ab. This was used as a tracer at 5x10-9 M and competed with various concentrations of UCHT1 and test materials in parallel binding to Jurkat cells or in some cases human PBMC at 4°C for 30 min before washing by centrifugation and being subjected to FACS gating on live cells with propidium iodide. Mean channel fluorescent values (MCF') were corrected by subtracting an appropriate FITC non-binding isotype control. The binding affinity of a ligand relative to UCHT1 was calculated by dividing the concentration of UCHT1 by the concentration of ligand that gave equal MCF' values. When the observed MCF' were not equal, the UCHT1 concentration required to match a ligand MCF' value was interpolated from plots of MCF' versus log concentration of ligand and UCHT1. These plots were roughly linear between tracer displacement values between 10% and 90%; however, the curves were not uniformly parallel. Interpolations were performed at low, medium and high displacement values when possible and the means and SDs were calculated with values of n ranging between 2 and 6. In several cases the relative affinity of the chemical conjugate UCHT1CRM9 was compared with that of a recombinant immunotoxin by observing the MCF' values of polyclonal FITC-labeled anti-DT over a range of concentrations. The second antibody method gave comparable results to the competition method when the difference in affinity of UCHT1 and UCHT1CRM9 was included in the calculation.
T cell depletion in tg600+/-mice
tg600 homozygous mice were obtained from Dr Cox Terhorst (Beth Israel Hospital, Harvard Medical School, Boston, MA). In this work the heterozygote strain of the mouse, tg
600+/-, was used (F1 of tg
600xC57BL/6J). These mice contain three copies of the complete human CD3
gene under the transcriptional regulation of their endogenous promoters and enhancers (Wang et al., 1998
). A twice per day experimental dosing regimen was chosen because the monovalent immunotoxin was observed to have a short half-life in mice (<6 h). Accordingly, tg
600+/- were treated with immunotoxin twice daily for 4 days by tail vein administration using 0.1% murine serum albumin as a vehicle to prevent adsorption to plastic surfaces. Approximately 16 h after the final treatment, the lymph nodes and spleen were removed and single cell suspensions were prepared from individual mice. Total cells were counted by chamber counts. The percentage of CD3 positive cells was assessed by two-color FACS analysis using a FITC-conjugated anti-human CD3 antibody (UCHT1FITC) to measure human CD3 expression, phycoerythrin (PE)-conjugated anti-mouse CD3 antibody (500A2PE) to measure expression of mouse CD3 and the appropriately stained isotype controls. Viaprobe (Pharmingen, San Diego, CA) was used to gate on the live cells. Gates were set to count the fraction of double positive (DP) events (huCD3+muCD3+) for the isotype controls in non-treated animals and the fraction of DP events in treated animals. The fraction of DP isotype controls was subtracted from the fraction of DP events in treated animals and the result, DP', multiplied by the total cell count. This was subtracted from the DP' cells in control animals. The result was divided by the DP' cells in control animals to give the fractional depletion in DP cells at each immunotoxin concentration. (In non-treated animals 9899% of the lymph node and spleen T cells were DP cells, the remaining being single positive muCD3+.) Mean fractional lymph node and spleen T cell depletion values were calculated for each immunotoxin concentration. The data were fitted by probit analysis using a log transformation of the concentration scale using SPSS software (SPSS, Chicago, IL). In probit transformation instead of regressing the actual proportion responding to the values of the stimuli, each of the observed proportions is replaced with the value of the standard normal curve below which the observed proportion of the area is found (Finney, 1978
). Data points for monovalent and divalent immunotoxin were fitted alone or together to yield parallel curves with one regression coefficient.
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Results |
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One potential way to increase the toxicity of the immunotoxin is to increase the binding of the molecule to the target, i.e. increase the affinity. To increase the binding characteristics of the immunotoxin, we prepared a variety of recombinant bivalent anti-CD3 constructs and estimated their relative affinity compared to the parental antibody, UCHT1, and the monovalent Fab fragment of UCHT1. These studies were done to help us optimize the binding moiety of recombinant anti-CD3 immunotoxin. The mean relative binding values of the UCHT1 single-chain derivatives, lacking the DT moiety of the immunotoxin, were tested on Jurkat cells using the FACS-based assay (Table I
). Because plots of corrected mean channel fluorescence versus log concentration of competing UCHT1 or test ligands were not always parallel, comparisons were made at high, medium and low values of UCHT1FITC displacement and the means and SDs were calculated. High SD values reflect relatively non-parallel curves.
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To determine whether the observed increase in affinity of the bivalent constructs does increase the toxicity of immunotoxins, the DT390 moiety was added to each bivalent construct. The bivalent immunotoxins were tested for their toxicity on Jurkat cells in a 20 h protein synthesis inhibition assay (Table II). The disulfide-linked dimer immunotoxins were relatively non-toxic. The monovalent immunotoxins had similar activity to UCHT1CRM9 and an
3-fold increase in toxicity compared with the monovalent Fab-CRM9. There was no significant difference between the M-DT389-sFv that was produced in E.coli and refolded from inclusion bodies and the A-dmDT390-sFv construct that was secreted from CHO cells. However, it should be noted that the bacterial construct exhibited <3% nicking at the furin-sensitive bond between the A and B toxin chains, while the CHO material exhibited variable degrees of nicking from various preparations that ranged from 20 to 50%. Higher fractions of nicked material increased toxicity in Jurkat assays by a factor of 1.5 (data not shown). A bivalent construct utilizing a different linker sequence between the two sFv domains (Mallender and Voss, 1994
), DT390-bisFv (CHB1), demonstrated toxicity equivalent to DT390-sFv (data not shown). On the other hand, the DT390-bisFv (G4S) construct showed increased toxicity compared with DT390-sFv. The in vitro translated DT390-sFv is
1.5 logs less toxic than UCHT1CRM9 whereas purified DT390-sFv has similar toxicity to the chemical conjugate (Liu et al., 2000
). The relative toxicity between single-chain immunotoxins from the in vitro coupled transcription and translation has always predicted the relative toxicity of purified protein (data not shown).
Bivalent immunotoxin depletes human CD3 positive T-cells in tg600+/-mice
To demonstrate the in vivo significance of the increased activity of the bivalent immunotoxin, we compared the ability of the bivalent and monovalent immunotoxins to deplete human CD3 positive T-cells in tg600+/- mice. Increasing concentrations of immunotoxin were administered twice daily for 4 days. Spleens (Figure 2A
) and lymph nodes (Figure 2B
) from 39 animals were used to generate the means for each concentration. In both spleen and lymph node the mean depletion values from divalent immunotoxin are shifted to the left, indicating higher potency. The curves were fitted by probit analysis. The regression model is transformed Pi = A+ BlogXi, where Pi is the observed proportion responding at dose logXi and B is the regression coefficient. The regression coefficient is related to the fractional depletion F by the empirical equation F = XB/XB + (IC50)B. The divalent fitted curves are the solid lines. For the spleen fit, 54 cases were available for monovalent and 39 cases for divalent immunotoxin and the fits in Figure 2A
were performed individually. The regression coefficients shown in Table III
are nearly identical and the curves are nearly parallel. When both cases are fitted together (93 cases), the changes are minimal. The divalent immunotoxin is nine times more potent than the monovalent. The significance is high (Table III
). In the lymph node fit shown in Figure 2B
, both monovalent and divalent were fitted together (89 cases). The curves are more shallow compared with the spleen curves. This result is influenced by the lowest concentration mean value of the divalent immunotoxin, which has a mean value of 0.4 compared with 0 in the spleen. In the lymph node the divalent immunotoxin appears 34-fold more potent than the monovalent immunotoxin based on the probit model.
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Discussion |
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Our first attempts to increase the affinity involved the generation of divalent constructs including minibodies. Minibodies, first described by Hu et al. (Hu et al., 1996), use a single interactive heavy chain domain to stabilize a disulfide dimerized sFv construct such as (sFv-H-
CH3-h)2. We also did this to a previously undescribed minibody utilizing the µCH2 domain to provide the stabilizing interaction and the interchain disulfide bond. These divalent constructs had identical affinity when compared with the native antibody, UCHT1. However, addition of DT390 moeities to the N-termini of the divalent minibodies drastically reduced their binding and failed to show an increase in binding over DT390sFv. In addition, their potency was almost non-existent. Because the addition of a single toxin moiety to the UCHT1 antibody decreased its affinity 3-fold, we suspected that the toxin moiety was providing steric inhibition to the Fv binding domain. If this was the case, positional effects might be important. In fact, addition of the human serum albumin sequence at the N-terminus of the sFv lowered the relative binding 35-fold. This suggested that the N-terminus of the sFv was sensitive to the presence of large foreign protein domains resulting in a reduction of sFv binding affinity. The mechanism could be steric inhibition or, alternatively, the additional moiety could alter overall protein folding, thereby affecting bioactivity (Martsev et al., 2000
).
A single-chain construct consisting of two tandem sFv domains provided a possible way to achieve bivalent binding and to minimize steric interactions at the C-terminal sFv. The divalent sFv, bisFv, had binding values slightly better than the native antibody. Addition of the DT390 moiety to the bisFv, A-dmDT390-bisFv (G4S), showed increased relative binding by 67-fold over the A-dmDT390-sFv. Moreover, the divalent construct had a 13-fold increase in in vitro toxicity compared with the monovalent immunotoxin and the chemical conjugate, UCHT1CRM9. A bivalent construct utilizing an inter-sFv linker based on Mallender and Vos (Mallender and Voss, 1994) was only as toxic as the monovalent DT390-sFv (data not shown). This implies that the folding of the bivalent construct is an important variable in immunotoxin design. The DT390-bisFv (G4S) single-chain divalent immunotoxin represents a very significant increase in potency over previously described anti-CD3 immunotoxins and this increase in potency is due to an increase in binding to the CD3
epitope.
The 10-fold enhanced potency of the divalent recombinant immunotoxin relative to the monovalent immunotoxin observed in Jurkat cells was also seen in spleen T cell depletion in the tg600+/- mice that express human CD3
. At the highest dose tested, 1026 pmol/kg, the fractional T cell depletion was 0.995 by the probit model. This dose was associated with significant immunosuppressive effects in allograft survival studies (Weetall et al., personal communication). The enhanced potency of the divalent construct was 34-fold greater in the comparison of lymph nodes. This enhancement was within the range of the individual in vitro assays. Differences between lymph node and spleen data may reflect systematic experimental errors rather than real compartmental differences. However, one could expect a greater potency enhancement in vivo compared with in vitro owing to decreased renal clearance of the higher molecular weight divalent immunotoxin. Vallera et al. reported that a similar recombinant anti-murine anti-CD3 monovalent immunotoxin utilizing DT390 induces renal toxicity (Vallera et al., 2000
), presumably by renal filtration and renal tubular reabsorption of this relatively low-MW immunotoxin. Renal toxicity was reduced by adding a C-terminal cysteine that generated a disulfide dimer immunotoxin and thus permitted higher in vivo dosing. However, this dimer did not exhibit any increase in in vitro potency. We suggest that this result was due to steric hindrance generated by the two DT390 moieties. The configuration of one DT390 moiety followed by a bisFv (G4S) moiety appears to attenuate the steric effect of a single N-terminal DT390 moiety on sFv binding and offers increased in vivo and in vitro potency over all pre-existing anti-CD3 immunotoxin constructs.
In summary, this report delineates the optimization of an anti-CD3 immunotoxin from the parental chemically conjugated immunotoxin through a monovalent recombinant immunotoxin to a divalent single-chain immunotoxin, A-dmDT390-bisFv (G4S). The parental chemically conjugated anti-human CD3 immunotoxin has a demonstrated in vivo activity against leukemic T cells, reducing these cells 3 logs at a tolerable dose in a xenograft model (Neville et al., 1992). The chemically conjugated anti-rhesus analog can transiently deplete 2 logs of resting lymph node T cells in monkeys, a process that can induce long-term allograft transplantation tolerance (Huang et al., 2000
; Thomas et al., 2000
). The availability of the optimized construct with a 13-fold increase in potency suggests that this reagent will be efficacious in vivo against human CD3+ T cell leukemia/lymphoma. The fractional resting T cell depletion of 0.995 in the tg
600+/- mouse at 1026 pmol/kg is noteworthy because this dose of DT389-IL-2, another truncated DT immunotoxin, is well tolerated clinically (Saleh et al., 1998
). This suggests that the optimization achieved with A-dmDT390-bisFv (G4S) may have resulted in a therapeutic window that will permit the use of T cell depletion tolerance protocols in human transplantation.
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Notes |
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6 Present address: Eli Lilly & Company, Lilly Corporate Center, Indianapolis, IN 46286, USA
7 To whom correspondence should be addressed. E-mail: davidn{at}helix.nih.gov
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Acknowledgments |
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Received May 13, 2001; revised July 26, 2001; accepted September 10, 2001.