Botulism, caused by exposure to one or more of the eight serotypes of botulinum neurotoxins (BoNTs) (BoNT/A through H), is often fatal without rapid treatment. [...].

Many therapeutic targets are cell surface receptors, which can be challenging antigens for antibody generation. For many therapeutic applications, one needs antibodies that not only bind the cell surface receptor but also are internalized into the cell. This allows use of the antibody to deliver various payloads into the cell to achieve a therapeutic effect. Phage antibody technology has proven a powerful tool for the generation and optimization of human antibodies to any antigen. While applied to the generation of antibodies to purified proteins, it is possible to directly select cell binding and internalizing antibodies on cells. Potential advantages of this approach include: cell surface receptors are in native conformation on intact cells while this might not be so for recombinant proteins; antibodies can be selected for both cell binding and internalization properties; the antibodies can be used to identify their tumor associated antigens; and such antibodies can be used for human treatment directly since they are human in sequence. This review will discuss the factors that impact the successful selection of cell binding and internalizing antibodies. These factors include the cell types used for selection, the impact of different phage antibody library formats, and the specific selection protocols used.

Antibody-based therapeutics currently enjoy unprecedented success, growth in research and revenues, and recognition of their potential. It appears that the promise of the "magic bullet" has largely been realized. There are currently 22 monoclonal antibodies (mAbs) approved by the United States Food and Drug Administration (FDA) for clinical use and hundreds are in clinical trials for treatment of various diseases including cancers, immune disorders, and infections. The revenues from the top five therapeutic antibodies (Rituxan, Remicade, Herceptin, Humira, and Avastin) nearly doubled from $6.4 billion in 2004 to $11.7 billion in 2006. During the last several years major pharmaceutical companies raced to acquire antibody companies, with a recent example of MedImmune being purchased for $15.6 billion by AstraZeneca. These therapeutic and business successes reflect the major advances in antibody engineering which have resulted in the generation of safe, specific, high-affinity, and non-immunogenic antibodies during the last three decades. Currently, second and third generations of antibodies are under development, mostly to improve already existing antibody specificities. However, although the refinement of already known methodologies is certainly of great importance for potential clinical use, there are no conceptually new developments in the last decade comparable, for example, to the development of antibody libraries, phage display, domain antibodies (dAbs), and antibody humanization to name a few. A fundamental question is then whether there will be another change in the paradigm of research as happened 1-2 decades ago or the current trend of gradual improvement of already developed methodologies and therapeutic antibodies will continue. Although any prediction could prove incorrect, it appears that conceptually new methodologies are needed to overcome the fundamental problems of drug (antibody) resistance due to genetic or/and epigenetic alterations in cancer and chronic infections, as well as problems related to access to targets and complexity of biological systems. If new methodologies are not developed, it is likely that gradual saturation will occur in the pipeline of conceptually new antibody therapeutics. In this scenario we will witness an increase in combination of targets and antibodies, and further attempts to personalize targeted treatments by using appropriate biomarkers as well as to develop novel scaffolds with properties that are superior to those of the antibodies now in clinical use.

Background

Only Clostridium botulinum strain IBCA10-7060 produces the recently described novel botulinum neurotoxin type H (BoNT/H). BoNT/H (N-terminal two-thirds most homologous to BoNT/F and C-terminal one-third most homologous to BoNT/A) requires antitoxin to toxin ratios ≥1190:1 for neutralization by existing antitoxins. Hence, more potent and safer antitoxins against BoNT/H are needed.

Methods

We therefore evaluated our existing monoclonal antibodies (mAbs) to BoNT/A and BoNT/F for BoNT/H binding, created yeast-displayed mutants to select for higher-affinity-binding mAbs by using flow cytometry, and evaluated the mAbs' ability to neutralize BoNT/H in the standard mouse bioassay.

Results

Anti-BoNT/A HCC-binding mAbs RAZ1 and CR2 bound BoNT/H with high affinity. However, only 1 of 6 BoNT/F mAbs (4E17.2A) bound BoNT/H but with an affinity >800-fold lower (equilibrium dissociation binding constant [KD] = 7.56 × 10(-8)M) than its BoNT/F affinity (KD= 9.1 × 10(-11)M), indicating that the N-terminal two-thirds of BoNT/H is immunologically unique. The affinity of 4E17.2A for BoNT/H was increased >500-fold to KD= 1.48 × 10(-10)M (mAb 4E17.2D). A combination of mAbs RAZ1, CR2, and 4E17.2D completely protected mice challenged with 280 mouse median lethal doses of BoNT/H at a mAb dose as low as 5 µg of total antibody.

Conclusions

This 3-mAb combination potently neutralized BoNT/H and represents a potential human antitoxin that could be developed for the prevention and treatment of type H botulism.

A surface plasmon resonance based RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin is reported. Using detoxified recombinant type A botulinum neurotoxin as the surrogate, the aptasensor detects active toxin within 90 min. The detection limit of the aptasensor in phosphate buffered saline, carrot juice, and fat free milk is 5.8 ng/ml, 20.3 ng/ml and 23.4 ng/ml, respectively, while that in 5-fold diluted human serum is 22.5 ng/ml. Recovery of toxin from disparate sample matrices are within 91-116%. Most significant is the ability of this aptasensor to effectively differentiate the natively folded toxin from denatured, inactive toxin, which is important for homeland security surveillance and threat assessment. The aptasensor is stable for more than 30 days and over 400 injections/regeneration cycles. Such an aptasensor holds great promise for rapid detection of active botulinum neurotoxin for field surveillance due to its robustness, stability and reusability.

The blood-brain barrier (BBB) represents an obstacle in targeting and delivering therapeutics to the central nervous system. In order to discover new BBB-targeting molecules, we panned a phage-displayed nonimmune human single-chain antibody fragment (scFv) library against a representative BBB model comprised of hydrocortisone-treated primary rat brain endothelial cells. Parallel screens were performed with or without pre-subtraction against primary rat heart and lung endothelial cells in an effort to identify antibodies that may have binding selectivity toward brain endothelial cells. After three rounds of screening, three unique scFvs, scFv15, scFv38, and scFv29, were identified that maintained binding to primary rat brain endothelial cells, both in phage and soluble scFv format. While scFv29 and to a lesser extent, scFv15, exhibited some brain endothelial cell specificity in tissue culture, scFv29 did not appear to bind a BBB antigen in vivo. In contrast, both scFv15 and scFv38 were capable of immunolabeling rat brain vessels in vivo and displayed brain vascular selectivity with respect to all peripheral organs tested other than heart. Taken together, scFv15 and scFv38 represent two new antibodies that are capable of binding antigens that are expressed at the BBB in vivo.

The identification of tumor-specific cell surface antigens is a critical step toward the development of targeted therapeutics for cancer. The epitope space at the tumor cell surface is highly complex, composed of proteins, carbohydrates, and other membrane-associated determinants including post-translational modification products, which are difficult to probe by approaches based on gene expression. This epitope space can be efficiently mapped by complementary monoclonal antibodies. By selecting human antibody gene diversity libraries directly on the surface of prostate cancer cells, we have taken a functional approach to identifying fully human, tumor-specific monoclonal antibodies without prior knowledge of their target antigens. Selection conditions have been optimized to favor tumor-specific antibody binding and internalization. To date, we have discovered >90 monoclonal antibodies that specifically bind and enter prostate cancer cells, with little or no binding to control cells. These antibodies are able to efficiently deliver intracellular payloads when attached to nanoparticles such as liposomes. In addition, a subset of the antibodies displayed intrinsic antiproliferative activity. These tumor-specific internalizing antibodies are likely to be useful for targeted therapeutics either alone or in combination with effector molecules. The antigens they bind constitute a tumor-specific internalizing epitope space that is likely to play a significant role in cancer cell homeostasis. Targeting components of this epitope space may facilitate development of immunotherapeutic and small molecule-based strategies as well as the use of other therapeutic agents that rely upon delivery to the interior of the tumor cell.

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A-G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the current study, we have developed an enzyme-linked immunosorbent assay (ELISA)-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotypes A, B, C, D, E, and F. With engineered high-affinity antibodies, the BoNT assays have sensitivities in buffer ranging from 1.3fM (0.2pg/ml) to 14.7fM (2.2pg/ml). Using clinical and food matrices (serum and milk), the microarray is capable of detecting BoNT serotypes A to F to similar levels as in standard buffer. Cross-reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical, food, and environmental samples.

Background

The use of printed or electronic checklists and other cognitive aids has gained increasing interest from anesthesia providers and professional societies. While these aids are not currently considered standard of care, the perceptions of the clinician might have an impact on their adoption.

Objectives

We conducted a comprehensive survey to study the current opinions of anesthesia provider on the use of checklists and other cognitive aids.

Patients and methods

A questionnaire was developed by a departmental checklist focus group, which aimed to identify the perception of health care checklists in general as well as specific checklists for routine and crisis situations in anesthesia. Furthermore participants were asked regarding their perception of performing routine anesthesia and managing crisis situations without any cognitive aids. Using a web-based system, the survey was administered to all anesthesia providers at a single large United States academic medical center (University of California San Francisco). Demographic information included professional status (faculty, anesthesia resident, or nurse anesthetists [certified registered nurse anesthetists; CRNA]) and years of clinical experience.

Results

69% of 312 providers responded. 98% of the survey takers consider the procedural time-out (the widely used pre-incision operating room checklist) as important or very important. We found that many anesthesia providers acknowledged limitations in their ability to perform clinical tasks without any lapses, and a majority would use checklists and other cognitive aids if available. Their acceptances are especially high for crisis situations (87 - 97%, depending on years of experience) and routine care that providers do not perform often (76 - 91%). Printed or electronic aids for patient-care transition and shift hand-offs were also valued (61% and 58%). To prepare for and perform routine anesthesia care, 40% of providers claimed interest in using checklists, however, the interest differed significantly with clinical experience: While both the least and most experienced providers valued aids for routine anesthesia (54% and 50%), only 29% of providers with 2 - 10 years of anesthesia experience claimed interest in using them. Distraction from patient care and decreased efficiency were concerns expressed for the use of routine checklist (27% and 31%, respectively). The main factors found to support the successful implementation of checklists into clinical care are ease of use and thoughtful integration into the anesthesia workflow.

Conclusions

Providers at our large academic institution generally embrace the concept of checklists and other cognitive aids. This was true for all providers for checklists for procedural time outs, anesthesia crisis situations and those for routine procedures that providers rarely perform. Only very experienced and very junior providers appreciated the use of checklists for routine care. There remains a discrepancy between these claims and provider's perception on their clinical competency based on memory alone.