Tuning the properties of maleimide reagents holds significant promise in expanding the toolbox of available methods for bioconjugation. Herein we describe aryloxymaleimides which represent 'next ...generation maleimides' of attenuated reactivity, and demonstrate their ability to enable new methods for protein modification at disulfide bonds.
Surgery is the cornerstone of oncologic therapy with curative intent. However, identification of tumor cells in the resection margins is difficult, resulting in nonradical resections, increased ...cancer recurrence and subsequent decreased patient survival. Novel imaging techniques that aid in demarcating tumor margins during surgery are needed. Overexpression of carcinoembryonic antigen (CEA) is found in the majority of gastrointestinal carcinomas, including colorectal and pancreas. We developed ssSM3E/800CW, a novel CEA‐targeted near‐infrared fluorescent (NIRF) tracer, based on a disulfide‐stabilized single‐chain antibody fragment (ssScFv), to visualize colorectal and pancreatic tumors in a clinically translatable setting. The applicability of the tracer was tested for cell and tissue binding characteristics and dosing using immunohistochemistry, flow cytometry, cell‐based plate assays and orthotopic colorectal (HT‐29, well differentiated) and pancreatic (BXPC‐3, poorly differentiated) xenogeneic human–mouse models. NIRF signals were visualized using the clinically compatible FLARE™ imaging system. Calculated clinically relevant doses of ssSM3E/800CW selectively accumulated in colorectal and pancreatic tumors/cells, with highest tumor‐to‐background ratios of 5.1 ± 0.6 at 72 hr postinjection, which proved suitable for intraoperative detection and delineation of tumor boarders and small (residual) tumor nodules in mice, between 8 and 96 hr postinjection. Ex vivo fluorescence imaging and pathologic examination confirmed tumor specificity and the distribution of the tracer. Our results indicate that ssSM3E/800CW shows promise as a diagnostic tool to recognize colorectal and pancreatic cancers for fluorescent‐guided surgery applications. If successfully translated clinically, this tracer could help improve the completeness of surgery and thus survival.
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The use of fluorescent tracers to identify cancerous cells at tumor margins could improve outcomes for surgery with curative intent. This study describes a newly developed near‐infrared fluorescent tracer targeted to carcinoembryonic antigen (CEA), a glycoprotein overexpressed in gastrointestinal cancers. In mice, the fluorescent agent, ssSM3E/800CW, accumulated specifically in tumor cells, enabling the detection and delineation of tumor borders on a clinically relevant timescale. If successfully translated into the clinic, ssSM3E/800CW could facilitate the visualization of cancerous tissue and improve the completeness of surgical resection.
A major obstacle to the efficient production of antibody conjugates for therapy and diagnosis is the non-ideal performance of commonly used chemical methods for the attachment of effector-molecules ...to the antibody of interest. Here we demonstrate that this limitation can be simply addressed using 3,4-substituted maleimides to bridge and thus functionalize disulfide bonds to generate homogeneous antibody conjugates. This one-step conjugation reaction is fast, site-specific, quantitative and generates products with full binding activity, good plasma stability and the desired functional properties. Furthermore, the rigid nature of this modification by disulfide bridging enables the successful detection of antigen with a spin labeled antibody fragment by continuous-wave electron paramagnetic resonance (cw-EPR), which we report here for the first time. Antigen detection is concentration dependent, observable in human blood and allows the discrimination of fragments with different binding affinity. We envisage broad potential for antibody based in-solution diagnostic methods by EPR or 'spinostics'.
This protocol is applicable to recombinant protein expression by small-scale fermentation using the Pichia pastoris expression system. P. pastoris has the capacity to produce large quantities of ...protein with eukaryotic processing. Expression is controlled by a methanol-inducible promoter, which allows a biomass-generation phase before protein production is initiated. The target protein is secreted directly into a protein-free mineral salt medium, and is relatively easy to purify. The protocol is readily interfaced with expanded bed adsorption for immediate capture and purification of recombinant protein. The setting up of the bioreactor plus the fermentation itself takes 1 wk. Making the master and user seed lots takes approximately 2 wk for each individual clone.
Abstract Superparamagnetic iron oxide nanoparticles (SPIONs) can substantially improve the sensitivity of magnetic resonance imaging (MRI). We propose that SPIONs could be used to target and image ...cancer cells if functionalised with recombinant single chain Fv antibody fragments (scFv). We tested our hypothesis by generating antibody-functionalised (ab f ) SPIONs using a scFv specific for carcinoembryonic antigen (CEA), an oncofoetal cell surface protein. SPIONs of different hydrodynamic diameter and surface chemistry were investigated and targeting was confirmed by ELISA, cellular iron uptake, confocal laser scanning microscopy (CLSM) and MRI. Results demonstrated that ab f -SPIONs bound specifically to CEA-expressing human tumour cells, generating selective image contrast on MRI. In addition, we observed that the cellular interaction of the ab f -SPIONs was influenced by hydrodynamic size and surface coating. The results indicate that ab f -SPIONs have potential for cancer-specific MRI.
Human peripheral blood lymphocytes can be transduced to express antigen-dependent CD3zeta chimeric immune receptors (CIRs), which function independently of the T-cell receptor (TCR). Although the ...exact function of these domains is unclear, previous studies imply that an extracellular spacer region is required for optimal CIR activity. In this study, four scFvs (in the context of CIRs with or without extracellular spacer regions) were used to target the human tumor-associated antigens carcinoembryonic antigen (CEA), neural cell adhesion molecule (NCAM), the oncofetal antigen 5T4, and the B-cell antigen CD19. In all cases human T-cell populations expressing the CIRs were functionally active against their respective targets, but the anti-5T4 and anti-NCAM CIRs showed enhanced specific cytokine release and cytotoxicity only when possessing an extracellular spacer region. In contrast, the anti-CEA and anti-CD19 CIRs displayed optimal cytokine release activity only in the absence of an extracellular spacer. Interestingly, mapping of the scFv epitopes has revealed that the anti-CEA scFv binds close to the amino-terminal of CEA, which is easily accessible to the CIR. In contrast, CIRs enhanced by a spacer domain appear to bind to epitopes residing closer to the cell membrane, suggesting that a more flexible extracellular domain may be required to permit the efficient binding of such epitopes. These results show that a spacer is not necessary for optimal activity of CIRs but that the optimal design varies.
The potential for protein-engineered biotherapeutics is enormous, but pharmacokinetic modulation is a major challenge. Manipulating pharmacokinetics, biodistribution, and bioavailability of small ...peptide/protein units such as antibody fragments is a major pharmaceutical ambition, illustrated by the many chemical conjugation and recombinant fusion approaches being developed. We describe a recombinant approach that leads to successful incorporation of polysialic acid, PSA for the first time, onto a therapeutically valuable protein. This was achieved by protein engineering of the PSA carrier domain of NCAM onto single-chain Fv antibody fragments (one directed against noninternalizing carcinoembryonic antigen-CEA and one against internalizing human epidermal growth factor receptor-2-HER2). This created novel polysialylated antibody fragments with desired pharmacokinetics. Production was achieved in human embryonic kidney cells engineered to express human polysialyltransferase, and the recombinant, glycosylated product was successfully fractionated by ion-exchange chromatography. Polysialylation was verified by glycosidase digestion and mass spectrometry, which showed the correct glycan structures and PSA chain length similar to that of native NCAM. Binding was demonstrated by ELISA and surface plasmon resonance and on live cells by flow cytometry and confocal immunofluorescence. Unexpectedly, polysialylation inhibited receptor-mediated endocytosis of the anti-HER2 scFv. Recombinant polysialylation led to an estimated 3-fold increase in hydrodynamic radius, comparable to PEGylation, leading to an almost 30-fold increase in blood half-life and a similar increase in blood exposure. This increase in bioavailability led to a 12-fold increase in tumor uptake by 24 h. In summary, recombinant polysialylation of antibody fragments in our system is a novel and feasible approach applicable for pharmacokinetic modulation, and may have wider applications.
Advances in biomolecular technology have allowed the development of genetically fused antibody-enzymes. Antibody-enzyme fusion proteins have been used to target tumors for cancer therapy in two ways. ...In one system, an antibody-enzyme is pretargeted to the tumor followed by administration of an inactive prodrug that is converted to its active form by the pretargeted enzyme. This system has been described as antibody-directed enzyme prodrug therapy. The other system uses antibody-enzyme fusion proteins as direct therapeutics, where the enzyme is toxic in its own right. The key feature in this approach is that the antibody is used to internalize the toxic enzyme into the tumor cell, which activates cell-death processes. This antibody-enzyme system has been largely applied to deliver ribonucleases. This article addresses these two antibody-enzyme targeting strategies for cancer therapy from concept to (pre)clinical trials.
An important step in drug development is the assignment of an International Nonproprietary Name (INN) by the World Health Organization (WHO) that provides healthcare professionals with a unique and ...universally available designated name to identify each pharmaceutical substance. Monoclonal antibody INNs comprise a -mab suffix preceded by a substem indicating the antibody type, e.g., chimeric (-xi-), humanized (-zu-), or human (-u-). The WHO publishes INN definitions that specify how new monoclonal antibody therapeutics are categorized and adapts the definitions to new technologies. However, rapid progress in antibody technologies has blurred the boundaries between existing antibody categories and created a burgeoning array of new antibody formats. Thus, revising the INN system for antibodies is akin to aiming for a rapidly moving target. The WHO recently revised INN definitions for antibodies now to be based on amino acid sequence identity. These new definitions, however, are critically flawed as they are ambiguous and go against decades of scientific literature. A key concern is the imposition of an arbitrary threshold for identity against human germline antibody variable region sequences. This leads to inconsistent classification of somatically mutated human antibodies, humanized antibodies as well as antibodies derived from semi-synthetic/synthetic libraries and transgenic animals. Such sequence-based classification implies clear functional distinction between categories (e.g., immunogenicity). However, there is no scientific evidence to support this. Dialog between the WHO INN Expert Group and key stakeholders is needed to develop a new INN system for antibodies and to avoid confusion and miscommunication between researchers and clinicians prescribing antibodies.
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