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.

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.

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.

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.

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.

Preliminary study to characterize differences in potential immunomodulatory effects of cyclosporine A using BALB/c and ICR mouse splenocytes / Estudo preliminar para caracterizar as diferenças nos efeitos imunomodulatórias de ciclosporina A esplenócitos usando BALB / c e ICR do mouse

Cyclosporine A (CsA) is widely used as an immunosuppressant for the treatment of autoimmune diseases and immune regulation in transplant patients. Due to its wide applicability, studies of unwanted side effects of CsA are imperative. It has been found that not all patients treated with CsA display the same types/patterns of adverse effects. To ascertain the bases for these differential responses, potential differing effects of CsA on B-lymphocytes were analyzed. This entailed an assessment of changes in CsA viability and mitotic activity within splenocyte populations from BALB/c and ICR mice. These particular strains were examined because: (1) in each of them, previously have been shown that differed in the respond to biological response modifiers, such as bacterial agents, and/or immunogens; (2) our own earlier studies showing strain-associated differences in ex vivo splenocyte/lymphocyte responses to other drug; and, (3) a potential immunomodulatory effect of any agent should be studied in at least two different strains during a broad toxicological evaluation. Splenocytes from each strain were treated with 200 μg/mL CsA, and CD4+, CD8+, and CD19+ cell viabilities were monitored at various time points during the exposure period. In general, there appeared to be a trend toward greater decreases in viability among BALB/c B-lymphocytes than their ICR counterparts as incubation progressed. Differences related with T-lymphocyte sensitivity to drug associated to strains was not observed, because it was uniformly lethal throughout. With regard to mitotic activity, cells from ICR mice were more susceptible to inhibition of spontaneous cell division at low concentrations of CsA (relative to the rates of blastogenesis by BALB/c counterparts). At higher concentrations of the drug however, there were no differences in the sensitivity of each strain. This work provides new insight into the mechanism of action of CsA and illustrates the need for at least two different strains of mice/rodents for the evaluation of the overall toxicological potential of any test agent.

Ciclosporina A (CsA) é amplamente usada como imunossupressor para o tratamento de doenças autoimunes e regulação imune nos pacientes transplantados. Devido à alta aplicabilidade, são imperativos os estudos sobre seus efeitos colaterais indesejáveis. Descobriu-se que nem todos os pacientes tratados com CsA apresentam os mesmos tipos/padrões de efeitos adversos. Para averiguar as bases dessas respostas diferentes, analisaram-se efeitos potenciais diferentes da CsA nos linfócitos B. Isto envolveu a avaliação de alterações na viabilidade da CsA e da atividade mitótica dentro das populações de esplenócitos de camundongos BALB/c e ICR. Essas espécies, em particular, foram examinadas porque: (1) cada uma delas mostrou, previamente, respostas diferentes a modificadores de respostas biológicas, tais como agentes bacterianos e/ou imunogênicos; (2) nossos estudos anteriores mostraram diferenças associadas às espécies em respostas ex vivo de esplenócitos/linfócitos a outro fármaco e (3) qualquer efeito imunomodulatório potencial de um agente em teste deveria ser estudado, no mínimo, em duas espécies diferentes durante a avaliação toxicológica ampla. Esplenócitos de cada espécie foram tratados com 200 μg/mL de CSA e a viabilidade das células CD4+, CD8+ e CD19+ foi monitorada em vários tempos durante o período de exposição. No geral, parece haver uma tendência em relação a aumentos maiores na viabilidade entre os linfócitos B de BALB/c do que no de ICR, à medida que a incubação progride. Não se observou diferenças na sensibilidade do linfócito T, uma vez que o fármaco foi uniformemente letal. Com relação à atividade mitótica, as células de camundongos ICR se mostraram mais suscetíveis à inibição da divisão celular espontânea em baixas concentrações de CsA (relativamente às taxas de blastogênese de BALB/c). Em concentrações maiores do fármaco, entretanto, não houve diferenças na sensibilidade em cada uma das espécies. Este trabalho propicia nova visão do mecanismo de ação de CsA e ilustra a necessidade de, pelo menos, duas espécies diferentes de camundongos/roedores para a avaliação da toxicidade potencial de qualquer agente em teste.

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.

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.

In contrast to specific B cells, human basophils are unaffected by the toxic activity of an allergen toxin due to lack of internalization of immunoglobulin E-bound allergen.

BACKGROUND: Specific immunotherapy is the only curative therapy for type I allergies and the alarming increase in allergy prevalence emphasizes the need for additional/alternative strategies for curative treatment. Allergen toxins (AT), fusion products of an allergen with an apoptosis inducing cytotoxin, are a new kind of immunotoxin. OBJECTIVE: AT should allow allergen-specific targeting and elimination of allergy-relevant cells, with B cells being the primary target. An important question is the fate of the effector cells, e.g. mast cells and basophils, which carry allergen-specific IgE: the immunotoxin might even prove to be harmful. METHODS: We established a reliable in vitro B cell model (using two mouse hybridoma cell lines) for testing specificity and toxicity of P5-ETA', a fusion protein of the major timothy grass pollen allergen Phl p 5b and truncated Pseudomonas Exotoxin A. In a second step, we investigated the impact of the AT on human basophils. RESULTS: P5-ETA' reliably eliminated Phl p 5-specific cells in the in vitro B cell model, leaving unspecific B cells unharmed. Human basophils of grass pollen allergic donors specifically bound P5-ETA', released IL-4 and up-regulated the activation marker CD203c, but were not subject to the toxic effect because of lack of internalization of IgE-bound allergen. CONCLUSION: According to our data, basophils are pure effector cells in the context of IgE-bound allergen and not involved in classical antigen presentation.

Expression of human endogenous retrovirus HERV-K18 superantigen is elevated in juvenile rheumatoid arthritis.

OBJECTIVE: To investigate the presence of a host-encoded superantigen as possible etiologic factor in pediatric rheumatic disease. We measured the expression and the ability of interferon-alpha (IFN-alpha) to induce the human endogenous retrovirus HERV-K18 superantigen in juvenile rheumatoid arthritis (JRA) and pediatric systemic lupus erythematosus (SLE). METHODS: Expression levels of HERV-K18 were measured in peripheral blood or synovial fluid mononuclear cells (SFMC) from 13 patients with JRA, 11 pediatric SLE patients, and 24 healthy controls, by semiquantitative reverse transcription-polymerase chain reaction, comparing 18S ribosomal transcripts as endogenous standard. IFN-alpha induction was tested by pretreatment of samples with 2000 U/ml. RESULTS: HERV-K18 expression was significantly elevated in peripheral blood from patients with JRA (mean ratio of HERV-K18 to 18S ribosomal transcripts 2.456, SD 2.122; p = 0.014), but not patients with SLE (mean 0.997, SD 0.579; p = 0.258), compared to controls (mean 0.749, SD 0.598). HERV-K18 transcripts were detected in SFMC of 7/7 JRA patients. IFN-alpha induced HERV-K18 strongly in JRA (mean fold induction = 8.934, SD 15.556) and controls (mean 8.270, SD 6.609), but weakly in SLE (mean 2.432, SD 2.219; p = 0.009). HERV-K18 levels were found to be independent of previously determined modifiers of expression, including Epstein-Barr virus infection, IFN-alpha levels, or the percentage of B cells in peripheral blood. CONCLUSION: HERV-K18 superantigen levels were elevated in JRA patients, but not pediatric patients with SLE, suggesting a possible mechanism for autoimmunity in the former group by superantigen stimulation of autoreactive T cells.

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.

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.

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.

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.

Preclinical studies with the anti-CD19-saporin immunotoxin BU12-SAPORIN for the treatment of human-B-cell tumours.

The immunotoxin BU12-SAPORIN was constructed by covalently coupling the single-chain ribosome-inactivating protein saporin to the anti-CD19 monoclonal antibody BU12 via a disulphide linker using the heterobifunctional reagent SPDP. The immunoreactivity and specificity of BU12-SAPORIN was identical to that of unmodified native BU12 antibody. BU12-SAPORIN was selectively cytotoxic in vitro in a dose-dependent manner for the CD19+ human common acute lymphoblastic leukaemia (cALL) cell line NALM-6 but exhibited no toxicity for the CD19- T-cell acute lymphoblastic leukaemia (T-ALL) cell line HSB-2. The survival of severe combined immunodeficient (SCID) mice with disseminated NALM-6 leukaemia was significantly prolonged compared with sham-treated control animals by a course of therapy with BU12-SAPORIN but not with the irrelevant anti-CD7 immunotoxin HB2-SAPORIN. BU12-SAPORIN had no therapeutic effect in SCID mice with disseminated CD19- HSB-2 leukaemia. These preclinical studies have clearly demonstrated the selective cytotoxicity of BU12-SAPORIN for CD19+ target cells both in vitro and in vivo. This, taken together with the lack of expression of the CD19 molecule by any normal life-sustaining tissue and its ubiquitous and homogeneous expression by the majority of cALL and B-NHL cells, provides the rationale for undertaking a phase I trial of systemic therapy with BU12-SAPORIN.