Improvement of a recombinant anti-monkey anti-CD3 diphtheria toxin based immunotoxin by yeast display affinity maturation of the scFv

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 CD3epsilongamma 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.     

Expression, purification, and characterization of an immunotoxin containing a humanized anti-CD25 single-chain fragment variable antibody fused to a modified truncated Pseudomonas exotoxin A

Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin therapy. In our previous study, a modified PE38KDEL, denoted PE38KDELKQK, was engineered to eliminate VLS. The PE38KDELKQK-based immunotoxin has been proved to retain potent anti-tumor activity but with a remarkable attenuation in VLS. In the present study, we have constructed and expressed a recombinant immunotoxin CD25-PE38KDELKQK containing humanized anti-CD25 single-chain antibody (scFv) genetically fused to PE38KDELKQK in Escherichia coli. After washing with buffer containing 2 M urea, the purity of inclusion body was about 82%. The denatured inclusion bodies were refolded on-column in Tris buffer (pH 8.0) containing 4mM of GSH and 1 mM of GSSG using a gradient of decreasing urea. We found that the presence of GSH/GSSG (4:1) in the on-column refolding buffer was important for efficient refolding. In addition, slow flow rate was another important factor could increase refolding. Under these conditions, the activity of the refolded protein could reach about 90% of that of the native protein. The refolded proteins were purified to homogeneity ( approximately 95% purity) by a combination of His-Ni(2+) metal affinity chromatography and gel filtration chromatography. The in vitro cytotoxicity assay indicated the purified immunotoxin CD25-PE38KDELKQK had specific cytotoxicity to CD25-positive leukemic cells comparable to wild-type CD25-PE38KDEL (wt). In contrast, CD25-PE38KDELKQK was shown to be much weaker in inducing VLS in mice than wt. The protein expression, purification, and refolding system established in this paper is important for further study on immunotoxin CD25-PE38KDELKQK.     

Development of a diphtheria toxin based antiporcine CD3 recombinant immunotoxin

Anti-CD3 immunotoxins, which induce profound but transient T-cell depletion in vivo by inhibiting eukaryotic protein synthesis in CD3+ cells, are effective reagents in large animal models of transplantation tolerance and autoimmune disease therapy. A diphtheria toxin based antiporcine CD3 recombinant immunotoxin was constructed by fusing the truncated diphtheria toxin DT390 with two identical tandem single chain variable fragments (scFv) derived from the antiporcine CD3 monoclonal antibody 898H2-6-15. The recombinant immunotoxin was expressed in a diphtheria-toxin resistant yeast Pichia pastoris strain under the control of the alcohol oxidase promoter. The secreted recombinant immunotoxin was purified sequentially with hydrophobic interaction chromatography (Butyl 650 M) followed by strong anion exchange (Poros 50 HQ). The purified antiporcine CD3 immunotoxin was tested in vivo in four animals; peripheral blood CD3+ T-cell numbers were reduced by 80% and lymph node T-cells decreased from 74% CD3+ cells pretreatment to 24% CD3+ cells remaining in the lymph node following 4 days of immunotoxin treatment. No clinical toxicity was observed in any of the experimental swine. We anticipate that this conjugate will provide an important tool for in vivo depletion of T-cells in swine transplantation models.     

Methylamine facilitates demonstration of specific uptake of diphtheria toxin by CHO cell and toxin-resistant CHO cell mutants

We have measured the specific uptake of ¹²⁵I-labelled diphtheria toxin in the presence of methylamine by a number of cell lines with different sensitivities to diphtheria toxin. The results show a strong correlation between the toxin sensitivities of the cell lines and the amount of specific uptake. The specific association of labelled toxin with cells was clearly demonstrated even with CHO cells, a cell line with relatively low sensitivity. Thus, CHO cell mutants that are resistant to diphtheria toxin could be classified as toxin-binding or non-binding cells by this method.     

Antibody-mediated routing of diphtheria toxin in murine cells results in a highly efficacious immunotoxin

The chemical coupling of diphtheria toxin to an antimurine Thy1 antibody resulted in the most efficacious immunotoxin to date. At 1 micrograms/ml the immunotoxin inhibited protein synthesis of a Thy+ AKR murine cell at a rate of 1.4 logs/h, within the order of magnitude of the efficacy of native toxins. This is unusual since murine cells are highly resistant to diphtheria toxin. The conjugate is highly specific; Thy- AKR cells display no intoxication at 1 microgram/ml even after 18 h. The effects of ammonia, acid pulsing of external media, and low temperature reveal some similarities and some differences between intoxication of sensitive cells by toxin and of murine cells by the antibody-toxin conjugate. The differences that result in the high efficacy of the antibody-toxin conjugate appear to result from the antibody-mediated routing. These results imply that murine cells possess an acidic compartment which can mediate toxin cytosolic entry. Unlike the Thy antigen, the toxin receptor on murine cells is unable to route the toxin to this cellular site.     

Characterization of a full-length, active-site mutant of diphtheria toxin.

A full-length recombinant mutant of diphtheria toxin containing serine in place of a crucial active-site glutamate has been purified and characterized. The serine substitution caused a minor structural alteration in the toxin as measured by trypsinolysis. ADP-ribosyltransferase activity and cytotoxicity of the mutant were both decreased by approximately 500-fold. A similar reduction in cytotoxicity was found when the enzymic fragments of both the wild-type and mutant toxins were introduced into the cytosol of fibroblasts by osmotically lysing pinosomes. The mutation did not alter the binding of the toxin to cell surface receptors and had no apparent effect on membrane translocation. The results suggest that the decreased cytotoxicity of the mutant is solely due to the reduced ADP-ribosyltransferase activity.     

Formation of a hybrid toxin from ricin agglutinin and a non-toxic mutant protein of diphtheria toxin

CRM45, a non-toxic mutant protein of diphtheria toxin, is treated with glutaraldehyde and conjugated to ricin agglutinin. The hybrid protein thus obtained is purified by gel filtration and affinity chromatography. The toxicity of the purified hybrid toxin is about 8–10 times grater than that of ricin agglutinin when tested in mice and cultured L cells.     

T-cell activation and B-cell depletion in chimpanzees treated with a bispecific anti-CD19/anti-CD3 single-chain antibody construct

BscCD19xCD3 is a bispecific single-chain antibody construct with exceptional cytotoxic potency in vitro and in vivo. Here, we have investigated the biological activity of bscCD19xCD3 in chimpanzee, the only animal species identified in which bscCD19xCD3 showed bispecific binding, redirected B-cell lysis and cytokine production comparable to human cells. Pharmacokinetic analysis following 2-h intravenous infusion of 0.06, 0.1 or 0.12 μg/kg of bscCD19xCD3 as part of a dose escalation study in a single female chimpanzee revealed a half-life of approximately 2 h and elimination of the bispecific antibody from circulation within approximately 8 h after the end of infusion. This short exposure to bscCD19xCD3 elicited a transient increase in serum levels of IFNγ, IL-6, IL-2, soluble CD25, and transiently upregulated expression of CD69 and MHC class II on CD8-positive cells. Cytokine release and upregulation of T-cell activation markers were not observed with vehicle controls. A multiple-dose study using 5 weekly doses of 0.1 μg/kg in two animals also showed transient cytokine release and an activation of peripheral T cells with a first-dose effect, accompanied by a transient lymphopenia. While oscillations of T-cell counts were relatively even during repeated treatments, the amplitudes of peripheral B cells declined with every infusion, which was not observed in a vehicle control animal. Our data show that bscCD19xCD3 can be safely administered to chimpanzees at dose levels that cause fully reversible T-cell activation and, despite a very short exposure time, cumulative loss of peripheral B lymphocytes. A clinical trial testing prolonged administration of bscCD19xCD3 (MT103) for improving efficacy is currently ongoing.     

The improvement of an anti-CD22 immunotoxin: Conversion to single-chain and disulfide stabilized form and affinity maturation by alanine scan

HA22-LR is a recombinant immunotoxin for the treatment of B-cell malignancies that contains the Fv portion of an anti-CD22 antibody fused to a functional portion of Pseudomonas exotoxin A. In the present study, we attempted to improve this molecule. First, we produced a single-chain version of HA22-LR (scdsFv-HA22-LR) in which a peptide linker was introduced between the disulfide-linked light and heavy chains to enable production via single fermentation. No difference in cytotoxic activity was observed between scdsFv-HA22-LR and prototype HA22-LR. Next, we attempted to increase the affinity of scdsFv-HA22-LR by using alanine scanning mutagenesis of complementarity determining regions (CDRs) to assess the specific contribution of each CDR residue to the antigen binding. We found that mutation of asparagine 34 in V_LCDR1, which is located at the V_L/V_H interface, to alanine (N34A) caused a substantial increase in affinity and activity. Estimated K_D values measured by fluorescence-activated cell sorting were lowered by 10-fold: 0.056 nM in the N34A mutant compared to 0.58 nM in wild type (WT). Cell viability assays of CD22-positive B-cell lymphoma and leukemia cell lines showed that the N34A mutant had increased cytotoxicity ranging from ∼2 (HAL-1, IC₅₀(WT): 2.37 ± 0.62 ng/ml, IC₅₀(N34A): 1.32 ± 0.41 ng/ml) to 10 (SUDHL-6, IC₅₀(WT): 0.47 ± 0.090 ng/ml, IC₅₀(N34A): 0.048 ± 0.018 ng/ml)-fold compared to WT immunotoxin. The present study suggests that the N34A mutant of scdsFv-HA22-LR could have important consequences in a clinical setting.Key words: immunotoxin, HA22, affinity-maturation, alanine scan, V_H/V_L interface     

Expression of a biologically active diphtheria toxin fragment B in Escherichia coli

The toxB gene of Corynebacterium diphtheriae bacteriophage β encoding the B fragment of diphtheria toxin was cloned into an inducible expression vector. When expressed In Escherichia coli, fragment B was not proteolysed and was indistinguishable, by immunological criteria, from wild-type C. diphthsriae derived fragment B. Soluble fragment B was partially purified from the cytoplasm by saline precipitation steps and was shown to compete with the wild-type diphtheria toxin for binding to receptors of sensitive eukaryotic cells. A complete diphtheria toxin was reconstituted by formation of the disulphide bridge between purified fragment A and recombinant fragment B, which migrates at the expected Mr on Western blots and which was able to block protein synthesis by ADP-ribosylation of elongation factor–2, thereby indicating that the recombinant fragment B had retained its biological activity.     

Genotoxicity of goniothalamin in CHO cell line

Goniothalamin (GTN) is a styrylpyrrone derivative from Goniothalamus umbrosus and other Annonaceae species. It has been shown to have anti-cancer and apoptosis-inducing properties against various human tumour and animal cell lines. The compound has also been shown to be active in vivo against DMBA-induced rat mammary tumours and was reported as an anti-fertility agent in rats. The aim of our study was to assess the genotoxicity of GTN in CHO cells using the UKEMS guidelines. A metabolic activation fraction (S9) was prepared according to standard methods. The methylthiazoletetrazolium (MTT) screening assay was then carried out to determine the cytotoxicity index (IC50) of GTN. The average IC50 value was 12.45 (+/- 3.63)microM. The mitotic index (MI) assay was then performed to determine the clastogenicity indices (MI(C25), MI(C50) and MI(C100)) of GTN. The chromosome aberration (CA) induction assay using air-dried metaphase spread was then performed to investigate the clastogenic effects of goniothalamin. Benzo[a]pyrene (BaP) and ethylmethanesulphonate (EMS) were used as positive controls in the presence and absence of S9 metabolic activation, respectively. The anti-genotoxicity effect of GTN was also assessed using a combination of GTN and EMS, and GTN and BaP. Dose-responses of CA frequencies were determined for both, the genotoxicity and anti-genotoxicity effects. GTN on its own and when combined with positive controls, was found to induce and enhance CA, respectively. Chromatid and whole chromosome breaks/gaps, as well as interchanges, endoreduplications and ring chromosomes were the main types of aberration induced by GTN. The overall clastogenic effect of GTN was statistically significant. In conclusion, GTN is potentially a genotoxic or clastogenic substance without any anti-genotoxic properties.     

Peroxisome division is impaired in a CHO cell mutant with an inactivating point-mutation in dynamin-like protein 1 gene

We earlier isolated a Chinese hamster ovary cell line ZP121 showing morphologically abnormal, tubular peroxisomes, and apparent dysmorphogenesis of mitochondria. Here, we identified an inactivating point-mutation in dynamin-like protein 1 gene, DLP1, responsible for the phenotype of ZP121. One allele of DLP1 possessed a point missense mutation resulting in G363D in the middle region of 699-amino-acid long DLP1, termed DLP1G363D, while the other allele was normal. DLP1G363D was apparently expressed at a higher level than DLP1. Abnormal morphogenesis of peroxisomes as well as mitochondria was restored when wild-type DLP1 was transfected. The GTPase activity of DLP1G363D was barely detectable, indicating that the G363D mutation severely affected the GTPase activity. Moreover, a higher level of DLP1G363D expression in CHO-K1 cells reproduced the ZP121-type phenotype, hence indicating its dominant-negative activity to the wild-type DLP1, most likely by forming a heteromeric tetramer. The G363D mutation also gave rise to a temperature-sensitive phenotype showing normal morphogenesis of peroxisomes and mitochondria at 40 degrees C. Microtubule organization was most likely involved in the elongation of peroxisomes. Furthermore, ZP121 was lowered in the level of phospholipids, plasmalogens, and phosphatidylethanolamine and was less sensitive to oxidative stresses. Thus, ZP121 is the first dlp1 mutant in mammalian cells.     

Expression and immunogenicity of a recombinant diphtheria toxin fragment A in Streptococcus gordonii

A nontoxic mutant diphtheria toxin fragment A (DTA) was genetically fused in single, double, or triple copy to the major surface protein antigen P1 (SpaP) and surface expressed in Streptococcus gordonii DL-1. The expression was verified by Western immunoblotting. Mouse antisera raised against the recombinant S. gordonii recognized the native diphtheria toxinm suggesting the recombinant DTA was immunogenic. When given intranasally to mice with cholera toxin subunit B as the adjuvant, the recombinant S. gordonii expressing double copies of DTA (SpaP-DTA(2)) induced a mucosal immunoglobulin A response and a weak systemic immunoglobulin G response. S. gordonii SpaP-DTA(2) was able to orally colonize BALB/c mice for a 15-week period and elicited a mucosal response, but a serum immunoglobulin G response was not apparent. The antisera failed to neutralize diphtheria toxin cytotoxicity in a Vero cell assay.     

Glycosylation of the hepatitis C virus envelope protein E1 occurs posttranslationally in a mannosylphosphoryldolichol-deficient CHO mutant cell line

The addition of N-linked glycans to a protein is catalyzed by oligosaccharyltransferase, an enzyme closely associated with the translocon. N-glycans are believed to be transferred as the protein is being synthesized and cotranslationally translocated in the lumen of the endoplasmic reticulum. We used a mannosylphosphoryldolichol-deficient Chinese hamster ovary mutant cell line (B3F7 cells) to study the temporal regulation of N-linked core glycosylation of hepatitis C virus envelope protein E1. In this cell line, truncated Glc(3)Man(5)GlcNAc(2) oligosaccharides are transferred onto nascent proteins. Pulse-chase analyses of E1 expressed in B3F7 cells show that the N-glycosylation sites of E1 are slowly occupied until up to 1 h after protein translation is completed. This posttranslational glycosylation of E1 indicates that the oligosaccharyltransferase has access to this protein in the lumen of the endoplasmic reticulum for at least 1 h after translation is completed. Comparisons with the N-glycosylation of other proteins expressed in B3F7 cells indicate that the posttranslational glycosylation of E1 is likely due to specific folding features of this acceptor protein.     

Increased cellular internalization of amphiphiles in a multidrug-resistant CHO cell line

The uptake of labeled palmitoyl carnitine and palmitoyl lysophosphatidylcholine by CHO cells was studied by measuring the extractability of these amphiphiles by bovine serum albumin. A multidrug-resistant cell line, CHRC5, showed a more rapid uptake, compared with the parental line, of these amphiphiles into a pool that was no longer susceptible to extraction with bovine serum albumin. The more rapid uptake by the drug-resistant cell line was reversed back to the rates observed with the parental cell line in the presence of verapamil, quinacrine or cyclosporin A. These latter three drugs also reverse the multidrug-resistant phenotype. These results demonstrate a relationship between the rate of amphiphile uptake and multidrug resistance.     

Expression cloning of a diphtheria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor.

A monkey cDNA (pDTS) encoding a diphtheria toxin (DT) sensitivity determinant was isolated by expression cloning in mouse L-M cells. Mouse cells are naturally resistant to DT, because they lack functional cell surface receptors for the toxin. Unlike wild-type L-M cells, pDTS-transfected mouse cells are extremely toxin sensitive and specifically bind radioiodinated DT. Intoxication of the transfected cells requires receptor-mediated endocytosis of the bound toxin. The cDNA is predicted to encode an integral membrane protein that is identical to the precursor of a heparin-binding EGF-like growth factor. The DT sensitivity protein is thus a growth factor precursor that DT exploits as a receptor.     

Virioimmunoassay for studying the interaction of diphtheria toxin with cell membranes]

A model for studying the interaction of diphtheria toxin with cell membranes includes immobilization of purified cell membranes on Sephadex G-25, adsorbtion of toxin on the membranes in the presence of protective colloid, and subsequent detection of adsorbed toxin by means of virioimmunoassay. Diphtheria toxin adsorbed rapidly on membranes of both sensitive (HeLa, macrophages) and resistent to tis action cells, but not on stromaof human erythrocytes. The rate of interaction depends on the concentration of toxin and the temperature of incubation. Adsorbed toxin may be eluted by acidic buffer, 8 M area and 4 M guanidin. HCl, but not by triton X-100, tween 20 and 80, sodium dodecylaulfate and basic buffer.