Antibodies, Volume 2, Issue 1 (March 2013) – 10 articles , Pages 1-167

To obtain thermostable immunoreagents specific for the spore form of Bacillus anthracis two llamas were immunized with a combination of six different recombinant proteins. These proteins BclA, gerQ, SODA1, SOD15, BxpB and the protein p5303 have all been shown as components of the B. anthracis spore and could potentially serve as targets for the detection of spores in multiplexed biosensors. Peripheral blood lymphocytes were used to construct a phage display library from which single domain antibodies (sdAbs) targeting each of the proteins were isolated. Unique sdAbs exhibiting nanomolar or better affinities for the recombinant proteins were obtained and most of the isolated sdAbs retained their ability to bind antigen after cycles of heating as determined by enzyme linked immunosorbent assay (ELISA). SdAbs targeting the BclA and gerQ proteins were able to successfully detect bacterial spores, whether broken or intact, using a direct ELISA; the sdAbs were specific, showing binding only to B. anthracis spores and not to other Bacillus species. Additionally, SODA1 and p5303 binding sdAbs detected spores in sandwich assays serving as both captures and tracers. Used in combination, sdAbs targeting B. anthracis proteins could be integrated into emerging biosensors to improve specificity in multiplex assays. Full article

The potential utility of immunotoxins for cancer therapy has convincingly been demonstrated in clinical studies. Nevertheless, the high immunogenicity of their bacterial toxin domain represents a critical limitation, and has prompted the evaluation of cell-death inducing proteins of human origin as a basis for less immunogenic immunotoxin-like molecules. In this review, we focus on the current status and future prospects of targeted fusion proteins for cancer therapy that employ granzyme B (GrB) from cytotoxic lymphocytes as a cytotoxic moiety. Naturally, this serine protease plays a critical role in the immune defense by inducing apoptotic target cell death upon cleavage of intracellular substrates. Advances in understanding of the structure and function of GrB enabled the generation of chimeric fusion proteins that carry a heterologous cell binding domain for recognition of tumor-associated cell surface antigens. These hybrid molecules display high selectivity for cancer cells, with cell killing activities similar to that of corresponding recombinant toxins. Recent findings have helped to understand and circumvent intrinsic cell binding of GrB and susceptibility of the enzyme to inhibition by serpins. This now allows the rational design of optimized GrB derivatives that avoid sequestration by binding to non-target tissues, limit off-target effects, and overcome resistance mechanisms in tumor cells. Full article

Monoclonal antibody (MAb) based therapies have achieved considerable success in oncology, primarily when used in combination with cytotoxic drugs. Antibody drug conjugates (ADCs) are a class of therapeutics that harness the antigen-selectivity of MAbs to deliver highly potent cytotoxic drugs to antigen-expressing tumor cells. The use of MAb directed delivery can confer a therapeutic index to highly potent cytotoxic drugs, increasing both the efficacy and safety of therapy. Although simple in concept, to achieve the design goal of improved therapeutic efficacy and reduced toxicity, each of the components of an ADC; the MAb, linker and drug need to considered in the context of the targeted antigen, the selectivity of antigen expression and the biology of the tumor type on which the target antigen is expressed. The characteristics of targets, MAbs, linkers and drugs being used in ADC design are discussed. Full article

An immune phage library derived from mice, hyperimmunized with morphine-conjugated BSA, was used to isolate a single-chain Fv (scFv) clone, M86, with binding activity to morphine-conjugated thyroglobulin (morphine-C-Tg) but not to codeine-, cocaine-, or ketamine-conjugated Tg. Surface plasmon resonance analysis using a morphine-C-Tg-coupled CM5 sensor chip showed that the K_d value was 1.26 × 10⁻⁸ M. To analyze its binding activity to free morphine and related compounds, we performed a competitive ELISA with M86 and morphine-C-Tg in the absence or presence of varying doses of free morphine and related compounds. IC₅₀ values for opium, morphine, codeine, and heroin were 257 ng/mL, 36.4, 7.3, and 7.4 nM, respectively. Ketamine and cocaine exhibited no competitive binding activity to M86. Thus, we established a phage library-derived scFv, M86, which recognized not only free morphine and codeine as opium components but also heroin. This characteristic of M86 may be useful for developing therapeutic reagents for opiate addiction and as a free morphine-specific antibody probe. Full article

Targeting and killing specific cells discriminately has been the goal of targeted therapy dating back to the era of Paul Ehrlich. The discovery of cancer stem cells has caused a paradigm shift within the cancer field and provided an opportunity to use targeted therapies such as targeted toxins to bind and kill these cells selectively. A number of targeted toxins have been developed against recently identified cancer stem cell markers. In this review we discuss the development and current status of these exciting novel drugs and their potential use to combat drug-refractory relapse. Full article

The single variable new antigen receptor domain antibody fragments (V_NARs) derived from shark immunoglobulin new antigen receptor antibodies (IgNARs) represent some of the smallest known immunoglobulin-based protein scaffolds. As single domains, they demonstrate favorable size and cryptic epitope recognition properties, making them attractive in diagnosis and therapy of numerous disease states. Here, we examine the stability of V_NAR domains with a focus on a family of V_NARs specific for apical membrane antigen 1 (AMA-1) from Plasmodium falciparum. The V_NARs are compared to traditional monoclonal antibodies (mAbs) in liquid, lyophilized and immobilized nitrocellulose formats. When maintained in various formats at 45 °C, V_NARs have improved stability compared to mAbs for periods of up to four weeks. Using circular dichroism spectroscopy we demonstrate that V_NAR domains are able to refold following heating to 80 °C. We also demonstrate that V_NAR domains are stable during incubation under potential in vivo conditions such as stomach acid, but not to the protease rich environment of murine stomach scrapings. Taken together, our results demonstrate the suitability of shark V_NAR domains for various diagnostic platforms and related applications. Full article

Elucidating the intracellular fate(s) of targeted toxins is of fundamental importance for their optimal use as anticancer drugs, since the biochemical targets of their enzymatic activity reside in the cell cytoplasm, as in the case of the plant ribosome inactivating proteins (RIP) saporin, ricin and of bacterial toxins. In this paper, we compared the cell surface binding and cytotoxic properties of the model RIP ricin to an immunotoxin constructed with a monoclonal antibody directed against the human T-cell marker CD7 covalently linked to saporin (CD7-SAP). Our results indicate that, despite the fact that internalization takes place via an apparently common entry route leading to the Golgi complex, surprisingly, the addition of an endoplasmic reticulum retrieval C-terminal signal (KDEL) to CD7-SAP does not potentiate its cytotoxicity. In addition, while ricin toxicity is clearly reduced by Brefeldin A under conditions where this fungal metabolite causes Golgi stack disruption, we paradoxically observed a potentiating effect by Brefeldin A on CD7-SAP cytotoxicity suggesting that this inhibitor interferes with retrograde route(s) other than the well established Trans-Golgi Network-ER retrograde route. Full article

Conventional cancer treatments lack specificity and often cause severe side effects. Targeted therapeutic approaches are therefore preferred, including the use of immunotoxins (ITs) that comprise cell-binding and cell death-inducing components to allow the direct and specific delivery of pro-apoptotic agents into malignant cells. The first generation of ITs consisted of toxins derived from bacteria or plants, making them immunogenic in humans. The recent development of human cytolytic fusion proteins (hCFP) consisting of human effector enzymes offers the prospect of highly-effective targeted therapies with minimal side effects. One of the most promising candidates is granzyme B (GrB) and this enzyme has already demonstrated its potential for targeted cancer therapy. However, the clinical application of GrB may be limited because it is inactivated by the overexpression in tumors of its specific inhibitor serpin B9 (PI-9). It is also highly charged, which means it can bind non-specifically to the surface of non-target cells. Furthermore, human enzymes generally lack an endogenous translocation domain, thus the endosomal release of GrB following receptor-mediated endocytosis can be inefficient. In this review we provide a detailed overview of these challenges and introduce promising solutions to increase the cytotoxic potency of GrB for clinical applications. Full article

Human cytolytic fusion proteins (hCFPs) are comprised of a specific cell-surface-binding moiety and an effector molecule of human origin. In contrast to common immunotoxins, including bacterial or plant toxins, they are considered not to be immunogenic. Two examples for human pro-apoptotic effector proteins are the serine protease Granzyme B and the RNase Angiogenin. Pre-clinical testing of functionality in in vitro and in vivo studies is essential for therapeutics. Establishing relevant animal models that have predictive value for therapeutic success is a great challenge in biomedical research. In this study, we investigated the species-dependent cytotoxic activity of two hCFPs prior to their application in a murine inflammation model. We found that in vitro and ex vivo either hCFP was able to kill human cells only, leaving murine cells unaffected. In contrast, no species-dependency was found for the bacterial Pseudomonas exotoxin A based immunotoxin H22(scFv)-ETA’. This species-dependent functioning has to be carefully considered when performing pre-clinical studies in animal models. Full article

TATA-box binding protein-associated factor 1 (TAF1), the largest subunit of the transcription factor IID complex, plays an important role in the RNA polymerase II-mediated gene transcription pathway regulating the transcription of a large number of genes related to cell division. The neuron-specific isoform of the TAF1 gene (N-TAF1) may have an essential role in neurons through transcriptional regulation of many neuron-specific genes. The present study reports the preparation and properties of a monoclonal antibody directed against N-TAF1. The monoclonal antibody, 3A-11F, specifically recognized N-TAF1 protein with no reactivity to TAF1 protein, as evidenced by immunocytochemistry and immunoprecipitation using cultured cells expressing recombinant N-TAF1 or TAF1 protein. Immunohistochemistry using 3A-11F showed that N-TAF1-imunoreactivity was detected in the nuclear region of neurons in the rat brain. The 3A-11F monoclonal antibody promises to be a useful tool for determining the expression pattern and biological function of N-TAF1 in the brain. Full article