Monolith affinity chromatography for the rapid quantification of a single-chain variable fragment immunotoxin.

We developed a novel analytical method for concentration determination of tandem single-chain antibody diphtheria toxin (immunotoxin). The method is based on polymethacrylate monoliths with Protein L ligands as the binding moiety. Different buffers were tested for elution of the Protein L-bound immunotoxin and 4.5 M guanidinium hydrochloride performed best. We optimized the elution conditions and the method sequence resulting in a fast and robust method with a runtime <10 min. Fast determination of immunotoxin is critical if any process decisions rely on this data. We determined method performance and a lower limit of detection of 27 µg/mL and a lower limit of quantification of 90 µg/mL was achieved. The validity of the method in terms of residual analysis, precision, and repeatability was proven in a range from 100 to 375 µg/mL. The short runtime and ease of use of a high-performance liquid chromatography method is especially useful for a process analytical tool approach. Bioprocesses related to immunotoxin where fermentation or other process parameters can be adjusted in accordance to the immunotoxin levels will be benefited from this method to achieve the highest possible purity and productivity.

Bioanalysis of the Bicycle® toxin conjugate BT5528 and released monomethyl auristatin E via liquid chromatography-tandem mass spectrometry.

Background: The Bicycle® toxin conjugate BT5528 is a novel peptide therapeutic conjugated to the cytotoxic agent monomethyl auristatin E (MMAE). A bioanalytical assay was developed to quantify BT5528 and unconjugated MMAE in human plasma. Methodology: BT5528 quantitation used a protein precipitation procedure followed by LC-MS/MS detection. Quantitation of MMAE required a selective offline and online solid-phase extraction with detection via LC-MS/MS. Results: BT5528 was quantified over the assay range of 5-2500 ng/ml and free MMAE was quantified over the assay range of 0.05-50 ng/ml. Conclusion: Bioanalytical methods were used in the bioanalysis of intact BT5528 and released MMAE, in a phase I/IIa clinical trial; to date, over 2000 human patient samples have been analyzed.

Cytotoxin antibody-based colourimetric sensor for field-level differential detection of elapid among big four snake venom.

Development of a rapid, on-site detection tool for snakebite is highly sought after, owing to its clinically and forensically relevant medicolegal significance. Polyvalent antivenom therapy in the management of such envenomation cases is finite due to its poor venom neutralization capabilities as well as diagnostic ramifications manifested as untoward immunological reactions. For precise molecular diagnosis of elapid venoms of the big four snakes, we have developed a lateral flow kit using a monoclonal antibody (AB1; IgG1 - κ chain; Kd: 31 nM) generated against recombinant cytotoxin-7 (rCTX-7; 7.7 kDa) protein of the elapid venom. The monoclonal antibody specifically detected the venoms of Naja naja (p < 0.0001) and Bungarus caeruleus (p<0.0001), without showing any immunoreactivity against the viperidae snakes in big four venomous snakes. The kit developed attained the limit of quantitation of 170 pg/µL and 2.1 ng/µL in spiked buffer samples and 28.7 ng/µL and 110 ng/µL in spiked serum samples for detection of N. naja and B. caeruleus venoms, respectively. This kit holds enormous potential in identification of elapid venom of the big four snakes for effective prognosis of an envenomation; as per the existing medical guidelines.

Intracellular route and mechanism of action of ERB-hRNase, a human anti-ErbB2 anticancer immunoagent.

Human anti-ErbB2 immunoRNase with Erbicin fused to HP-RNase (ERB-hRNase) is a fully human immunoRNase made up of human pancreatic RNase fused to a human anti-ErbB2 scFv. It binds selectively with high affinity to ErbB2-positive cells, and specifically inhibits their proliferation, in vitro and in vivo. An investigation of its mechanism of action and its intracellular destination has revealed that ERB-hRNase depends on its RNase activity for cytotoxic action; it reaches the cytosol directly from the endosomal compartment; it is inhibited by the cytosolic RNase inhibitor (cRI), but the levels that ERB-hRNase reaches in the cytosol neutralize cRI, thus inducing cell death by apoptosis.

Chemical de-conjugation for investigating the stability of small molecule drugs in antibody-drug conjugates.

Antibody-drug conjugates (ADCs) offer new therapeutic options for advanced cancer patients through precision killing with fewer side effects. The stability and efficacy of ADCs are closely related, emphasizing the urgency and importance of gaining a comprehensive understanding of ADC stability. In this work, a chemical de-conjugation approach was developed to investigate the in-situ stability of the small molecule drug while it is conjugated to the antibody. This method involves chemical-mediated release of the small molecule drug from the ADC and subsequent characterization of the released small molecule drug by HPLC. The feasibility of this technique was demonstrated utilizing a model ADC containing a disulfide linker that is sensitive to the reducing environment within cancer cells. Five reducing agents were screened for use in de-conjugation; tris(2-carboxyethyl) phosphine (TCEP) was selected for further optimization due to its high efficiency and clean impurity profile. The optimized de-conjugation assay was shown to have excellent specificity and precision. More importantly, it was shown to be stability indicating, enabling the identification and quantification of the small molecule drug and its degradation products under different formulation pHs and storage temperatures. In summary, the chemical de-conjugation strategy demonstrated here offers a powerful tool to assess the in-situ stability of small molecule drugs on ADCs and the resulting information will shed light on ADC formulation/process development and storage condition selection.

Physical and Chemical Stability of Antibody Drug Conjugates: Current Status.

Antibody drug conjugates (ADCs) are an emerging class of chemotherapeutic cancer treatment agents that combine the targeting specificity of antibodies with the efficient cell-killing potential of cytotoxic drugs. Unlike their protein and small-molecule therapeutic counterparts, the stability and degradation properties of ADCs are relatively unknown. Theoretically, ADC stability could be governed by properties and processes stemming from both the antibody and the linker-toxin chemistry. Recently, systematic studies of intrinsic ADC molecule stability have been presented in the primary literature. As there are burgeoning industrial and academic efforts aimed at developing optimized conjugation chemistries and antibody engineering approaches for next-generation ADCs, it is important to capture the current state of understanding of ADC stability. In this minireview, we discuss aspects of physical and chemical stability of ADCs gathered from a survey of the literature and illustrate how investigations into stability enable the development of more effective ADC molecules for the future.

Linking the occurrence of cutaneous opportunistic fungal invaders with elemental concentrations in false killer whale (Pseudorca crassidens) skin.

Cetaceans, occupying the top levels in marine food chains, are vulnerable to elevated levels of potentially toxic trace elements, such as aluminium (Al), mercury (Hg) and nickel (Ni). Negative effects associated with these toxic metals include infection by opportunistic microbial invaders. To corroborate the link between the presence of cutaneous fungal invaders and trace element levels, skin samples from 40 stranded false killer whales (FKWs) were analysed using culture techniques and inductively coupled plasma-mass spectroscopy. Twenty-two skin samples yielded 18 clinically relevant fungal species. While evidence for bioaccumulation of Hg in the skin of the FKWs was observed, a strong link was found to exist between the occurrence of opportunistic fungal invaders and higher Al : Se and Al : Zn ratios. This study provides indications that elevated levels of some toxic metals, such as Al, contribute to immunotoxicity rendering FKWs susceptible to colonization by cutaneous opportunistic fungal invaders.

The effects of aromatic and aliphatic maleimide crosslinkers on anti-CD5 ricin immunotoxins.

The aromatic maleimide crosslinkers m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfosuccinimidyl 4-(p-maleimidophenyl) butyrate (S-SMPB) and m-maleimidobenzoylsulfosuccinimide ester (S-MBS) and the aliphatic crosslinker N-gamma-maleimidobutyryloxysuccinimide ester (GMBS) were used to make anti-CD5 intact ricin immunotoxins (IT). IT made with the various crosslinkers were compared under standard conjugation conditions for differences in yield, toxicity of the toxin moiety, binding of the antibody moiety, IT activity, and IT specificity. Our findings showed that IT yield was dramatically improved using crosslinkers with an aromatic, rather than an aliphatic configuration. Gel analysis showed that all IT were of similar, but not identical composition. Conjugation resulted in several IT species including antibody linked to one or two molecules of ricin. For MBS IT and S-SMPB IT, differences in amounts of IT in final fractions and IT in fractions after removal of IT species containing galactose binding sites showed that differences in yield may be attributable to the formation of IT species with obstructed galactose binding sites. All IT bound selectively by FACS analysis and blocking studies. The aliphatic GMBS crosslinker yielded the most toxic IT in cell-free translation assays as well as in shorter-term protein synthesis inhibition and mitogen assays. However, evaluation in the longer-term, more sensitive clonogenic assay showed that at 1000 ng/ml there were no differences in potency between any of the IT. We conclude that the yield of intact ricin IT can be improved using aromatic maleimide crosslinkers without sacrificing IT potency.

New biosensors for environmental analysis.

Environmental analysis requires fast and reliable measurement results. Biosensors, which facilitate integral monitoring as well as single substance analysis, achieve high sensitivities in a minimum of measuring time. Four new on-line biosensors, which cover a wide range of environmentally relevant substances, are introduced: A water-quality monitoring bacteria electrode, whose gradual development is described as an example, a heavy-metal screening urease inhibition sensor, a genotoxic potential as well as a immunotoxic potential indicating sensor. Future prospects are given.

Comparison of various in vitro assays for efficacy screening of immunotoxins.

Before using immunotoxins in vivo, their efficacy is evaluated in in vitro assays. In this study we compare six different assays for the evaluation of immunotoxins: protein and DNA synthesis inhibition assay, chromium release assay, cell line colony assay, limiting dilution assay and clonogenic assay. All assays except the chromium release assay show specificity of the immunotoxins in appropriate concentrations. The protein and DNA synthesis inhibition assays are easy to perform and, therefore, suitable for initial screening, while the clonogenic assay seems to be the best one for immunotoxin efficacy determination.

In vivo labeling of rabbit cholinergic basal forebrain neurons with fluorochromated antibodies.

Cholinergic basal forebrain neurons (CBFN) expressing the low-affinity neurotrophin receptor p75 (p75(NTR)) were previously selectively labeled in vivo with carbocyanine 3 (Cy3)-tagged anti-p75(NTR), but the applied 192IgG-conjugates recognized p75(NTR) only in rat. The antibody ME 20.4 raised against human p75(NTR) had been shown to cross-react with the receptor in monkey, raccoon, sheep, cat, dog, pig and rabbit. Hence, for in vivo labeling of rabbit CBFN in the present study, ME 20.4 was fluorochromated with Cy3-N-hydroxysuccinimide ester and purified Cy3-ME 20.4 was injected intracerebroventricularly. Two days post-injection, clusters of Cy3-ME 20.4 were found in CBFN displaying choline acetyltrans-ferase-immunoreactivity. Following photoconversion, electron microscopy revealed fluorochromated antibodies in secondary lysosomes. In conclusion, Cy3-ME 20.4 might become an appropriate marker for CBFN in live and fixed tissues of various mammalian species.

The role of the septo-hippocampal cholinergic projection in T-maze rewarded alternation.

Administration of 192IgG-saporin, a cholinergic neurotoxin, to the medial septum destroys the cell bodies from which the septo-hippocampal cholinergic projection originates, leading to reductions in both hippocampal acetylcholinesterase (AChE) and choline acetyltransferase (ChAT). Despite reports that 192IgG-saporin-induced cholinergic loss leads to post-operative impairments in acquisition and performance of spatial memory tasks, a number of other reports have described intact spatial memory performance following these lesions. Factors that might account for these different outcomes include variations in toxin injection sites or volumes, and post-operative testing at times that might permit regeneration of damaged neuronal processes. We, therefore, assessed the effects of intraseptal microinjection of 192IgG-saporin, in rats, on the post-operative retention of pre-operatively acquired discrete-trial rewarded alternation in the T-maze. This design allowed us to assess the effects of the lesion 7 days post-surgery, at which point, at best, incomplete neuronal regeneration would have been expected to have occurred. The lesion led to a profound loss of hippocampal AChE staining, and a clear inflammatory response, as assessed by proliferation of OX42-stained macrophages in the medial septum and diagonal band nuclei, but there was no impairment in spatial working memory.

Immunolesioning: selective destruction of neurons using immunotoxin to rat NGF receptor.

192 IgG, a monoclonal antibody to the rat nerve growth factor (NGF) receptor, was disulfide-coupled to saporin, a ribosome-inactivating protein. Systemic injection of 192 IgG-saporin destroyed sympathetic postganglionic neurons and some sensory neurons. Injection of 192 IgG-saporin into the lateral ventricle destroyed cholinergic neurons of the basal forebrain. These results show that antineuronal immunotoxins are a powerful approach that may prove useful in a variety of neurobiological applications.

Production of antibodies and development of a radioimmunoassay for okadaic acid.

An okadaic acid immunogen, prepared by conjugation of okadaic acid to bovine albumin with carbodiimide, was used to immunize two rabbits. The rabbits responded by producing antibodies that neutralized okadaic acid's stimulation of arachidonic acid metabolism and this neutralization increased during the course of immunization. The immune sera bound 3H-okadaic acid and this binding also increased with repeated immunization. After absorption of the rabbit IgG with a goat anti-rabbit IgG, binding was reduced greater than 99%. The binding of okadaic acid to the antibodies in one antiserum was inhibited by as little as 0.2 pmoles of unlabelled okadaic acid. The apparent association constant for binding with this antiserum was 4.17 x 10(9) M-1 (35 degrees C). Maitotoxin, teleocidin, 12-O-tetradecanoylphorbol-13-acetate, aplysiatoxin, palytoxin and brevetoxin B when tested at 29, 228, 168, 169, 3.7 and 112 pmole levels, respectively, did not inhibit binding. The serologic and biological activities of okadaic acid after incubation for 60 min in 0.01 N HCl at 35 degrees C or at 100 degrees C at pH 7.2 were unaffected.

Safety evaluation of biological and biotechnology-derived medicines.

Evaluating the 'safety' of drugs produced by biotechnology resembles the assessment of conventional 'new chemical entities,' but with certain major differences. The 'quality' of the product requires careful control because of concern about the carry-over of DNA, immunogenic proteins, endotoxin and process chemicals. Equally the potency and purity of the product must also be considered, as well as its identity. The toxicity testing of rDNA-derived proteins, monoclonal antibodies and vaccines, although increasingly being swept under the umbrella of conventional studies, should be empirically devised according to the nature and physiological effects of the substance, taking account of the responsiveness of suitable species for non-clinical testing, the potential immunogenicity of heterologous proteins and any effect the drug may have on physiological mechanisms and the immune status of the test animals. Conventional types of single and multidose and reproduction toxicity experiments can then be adapted to detect and investigate any hazard of the novel drug. Kinetics, metabolism and drug interactions should be explored and the regulatory demand for genotoxicity data satisfied. If appropriate, immunological actions, including auto-immunity, can be sought. 'Safety-in-use' should then be predictable with some confidence, because of the extent of the toxicological investigations and because activities that cannot be examined will have been delineated, e.g. lack of a responsive species or of a suitable laboratory procedure.

Immunocytochemical localization of an immunoconjugate (antibody IgG against prostatic acid phosphatase conjugated to 5-fluoro-2'-deoxyuridine) in human prostate tumors.

Current chemotherapeutic and/or endocrine treatments for adenocarcinoma of the prostate (CaP) do not selectively target neoplastic prostate cells. Therefore, new approaches are needed to improve treatment for prostate tumors. We hypothesized that because of the specific binding of antibody immunoglobulin G (IgG) against human prostatic acid phosphatase (PAcP), PAcP-IgG could function as a carrier protein for the conjugated chemotherapeutic drugs and that the immunoconjugate would then selectively localize (bind) to epithelial cells of human prostate tumors, but not to epithelial cells of other solid organs. Our objective was to test this hypothesis using human prostate, colon, and kidney tissue samples and human prostate pieces incubated in short-term organ culture. We used derivatives of 5-fluorouracil labeled with fluorescein isothiocyanate (FITC) and rabbit anti-PAcP-IgG tagged with CY3/rhodamine alone or as an immunoconjugate. Localization of PAcP-IgG alone and the immunoconjugate in prostate produced similar and specific immunostaining in prostate epithelial cells and their tumors, but not in epithelia of colon and kidney tissue sections or in prostate sections-treated with normal rabbit serum. Confocal microscopy showed co-localization of CY3 and FITC of the immunoconjugate in the same group of prostate epithelial cells and their tumors. Organ culture studies showed that human prostate tissue samples incubated with normal rabbit serum did not show any fluorescence whereas those cultured with PAcP-IgG immunoconjugate showed fluorescence in glandular epithelial cells. The later study also showed that in organ culture the immunoconjugate had penetrated and labeled prostate glands internal to the cut surfaces. Drug labeled with FITC did not localize specifically in the prostatic epithelium. Analysis of our data has shown that PAcP-IgG was needed for specific localization of the immunoconjugate in prostate glands. We conclude that PAcP-IgG was essential for delivery and binding of the drug in human prostate. This is the first report to show that PAcP-IgG-5-Fu-2'-d-based immunoconjugate was selective and specific to epithelial cells of human prostate and its tumors, as revealed by organ culture, immunocytochemical, and confocal microscopic techniques.

Assessment of potential immunotoxic effects caused by cypermethrin, fluoxetine, and thiabendazole using heat shock protein 70 and interleukin-1ß mRNA expression in the anuran Xenopus laevis.

The current study describes the effect of cypermethrin, fluoxetine, and thiabendazole, at environmentally relevant concentrations, on the expression of heat shock protein 70 (HSP70) and interleukin 1ß (IL-1ß), using Xenopus laevis larvae as animal model. Cytokines and interleukins are considered good predictors of the immunotoxic potential of xenobiotics. Tadpoles at stage 47 (normal tables of X. laevis) were exposed under static conditions to: 0.3 and 30 µg/L fluoxetine, 0.7 µg/L thiabendazole, and 0.24 µg/L cypermethrin. The effects were evaluated at 7, 24, and 72 h, and 6 and 9 d. Randomly chosen tadpoles were used as genetic material for detection of hsp70 and IL-1ß mRNA induction through reverse transcription PCR. Tadpoles exposed to 30 µg/L fluoxetine showed mRNA expression of both genes at all exposure times, whereas at 0.3 µg/L a peak response for hsp70 was observed after 24 h, and the increase in IL-1ß mRNA was statistically significant with respect to the control 72 h after exposure. Thiabendazole induced a high expression of mRNA for both hsp70 and IL-1ß at all exposure times. Cypermethrin increased the hsp70 mRNA levels, with a peak at 24 h, and provoked high expression of IL-1ß mRNA at all exposure times. Considering the relationship between HSP70 and IL-1ß and their involvement (mainly of IL-1ß) in immune responses, certain changes observed in their expression could be considered warning indicators of potential immunotoxic effects of these substances on Xenopus.