Antibody engineering to improve manufacturability.

Expression variation among antibodies produced by stably transfected Chinese Hamster Ovary (CHO) cells is well established. While developing CHO-K1 cell lines, we encountered a human monoclonal antibody, mAb B-c, with severe manufacturability issues, including very poor expression and high levels of heavy chain (HC) dimer and high molecular weight aggregates. Using transient expression in CHO-K1 cells, we identified light chain (LC) as the source of the manufacturability issues for this antibody. While other antibodies achieved optimal expression at 1:1 or 2:1 LC to HC ratios, mAb B-c required up to a 6:1 LC:HC for maximal expression, which was still significantly lower than that for other tested antibodies. To overcome the manufacturability issues, LC shuffling was performed with the original HC to select antibodies with unique LCs which retained acceptable binding affinities. Transient CHO-K1 expression evaluation of the new LCs co-expressed with the original HC identified antibodies with high expression at a 1:1 or 2:1 LC:HC; the expression levels of these new antibodies were comparable to those of other well-expressed antibodies. Expression of these new antibodies in stably transfected CHO-K1 cells confirmed these results. In addition, antibodies containing the new LCs had very low levels of high molecular weight aggregates and HC dimer. These results demonstrate that certain antibody manufacturability issues can be attributed to LC and that engineering antibodies by pairing HCs with alternate LCs can improve antibody expression and product quality while maintaining or improving affinity.

Disinfection of needleless connectors for catheters in one second using a hand-held UV device.

BACKGROUND: The standard of care for disinfecting needleless connectors (NCs) of central venous catheters includes alcohol-containing caps or up to a 15-second scrub with alcohol or chlorhexidine. Due to the clinical impact and high cost of treating Central line-associated bloodstream infections (CLABSI), reducing the incidence of CLABSI is a priority for public health and of the Centers for Disease Control. Alcohol-containing caps have been demonstrated to disinfect external NC surfaces, but not the internal surface. Ultraviolet light (UV-C) is a strategy for disinfection of NC internal and external surfaces. METHODS: Four clinically relevant bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa) and Candida albicans were inoculated on NCs. Disinfection efficacy was measured after exposure to one second of 285 nm UV-C light at 48 mW/cm2 in a proprietary handheld device and UV-C transparent NC or standard of care. Disinfection of internal and external surfaces of NC inoculated with S aureus using alcohol caps, and UV-C was also compared. RESULTS: A 4-log reduction in colony forming units (CFUs) on the interior and exterior surfaces of the UV-transparent NC of clinically relevant pathogens was observed with UV-C light at this power for 1 second. DISCUSSION: We demonstrated the efficacy of UV-C for the disinfection of NCs in one second using the UV-C device in benchtop studies. CONCLUSIONS: This device holds promise for reducing CLABSI, and clinical studies are planned.

A Three-Monoclonal Antibody Combination Potently Neutralizes BoNT/G Toxin in Mice.

Equine-derived antitoxin (BAT®) is the only treatment for botulism from botulinum neurotoxin serotype G (BoNT/G). BAT® is a foreign protein with potentially severe adverse effects and is not renewable. To develop a safe, more potent, and renewable antitoxin, humanized monoclonal antibodies (mAbs) were generated. Yeast displayed single chain Fv (scFv) libraries were prepared from mice immunized with BoNT/G and BoNT/G domains and screened with BoNT/G using fluorescence-activated cell sorting (FACS). Fourteen scFv-binding BoNT/G were isolated with KD values ranging from 3.86 nM to 103 nM (median KD 20.9 nM). Five mAb-binding non-overlapping epitopes were humanized and affinity matured to create antibodies hu6G6.2, hu6G7.2, hu6G9.1, hu6G10, and hu6G11.2, with IgG KD values ranging from 51 pM to 8 pM. Three IgG combinations completely protected mice challenged with 10,000 LD50s of BoNT/G at a total mAb dose of 6.25 µg per mouse. The mAb combinations have the potential for use in the diagnosis and treatment of botulism due to serotype G and, along with antibody combinations to BoNT/A, B, C, D, E, and F, provide the basis for a fully recombinant heptavalent botulinum antitoxin to replace the legacy equine product.

Multicolor fluorescence activated cell sorting to generate humanized monoclonal antibody binding seven subtypes of BoNT/F.

Generating specific monoclonal antibodies (mAbs) that neutralize multiple antigen variants is challenging. Here, we present a strategy to generate mAbs that bind seven subtypes of botulinum neurotoxin serotype F (BoNT/F) that differ from each other in amino acid sequence by up to 36%. Previously, we identified 28H4, a mouse mAb with poor cross-reactivity to BoNT/F1, F3, F4, and F6 and with no detectable binding to BoNT/F2, F5, or F7. Using multicolor labeling of the different BoNT/F subtypes and fluorescence-activated cell sorting (FACS) of yeast displayed single-chain Fv (scFv) mutant libraries, 28H4 was evolved to a humanized mAb hu6F15.4 that bound each of seven BoNT/F subtypes with high affinity (KD 5.81 pM to 659.78 pM). In contrast, using single antigen FACS sorting, affinity was increased to the subtype used for sorting but with a decrease in affinity for other subtypes. None of the mAb variants showed any binding to other BoNT serotypes or to HEK293 or CHO cell lysates by flow cytometry, thus demonstrating stringent BoNT/F specificity. Multicolor FACS-mediated antibody library screening is thus proposed as a general method to generate multi-specific antibodies to protein subtypes such as toxins or species variants.

A Monoclonal Antibody Combination against both Serotypes A and B Botulinum Toxin Prevents Inhalational Botulism in a Guinea Pig Model.

Botulinum neurotoxins (BoNT) are extremely potent and can induce respiratory failure, requiring long-term intensive care to prevent death. Recombinant monoclonal antibodies (mAbs) hold considerable promise as BoNT therapeutics and prophylactics. In contrast, equine antitoxin cannot be used prophylactically and has a short half-life. Two three-mAb combinations are in development that specifically neutralize BoNT serotype A (BoNT/A) and B (BoNT/B). The three-mAb combinations addressing a single serotype provided pre-exposure prophylaxis in the guinea pig inhalation model. A lyophilized co-formulation of six mAbs, designated G03-52-01, that addresses both A and B serotypes is in development. Here, we investigated the efficacy of G03-52-01 to protect guinea pigs against an aerosol exposure challenge of BoNT/A1 or BoNT/B1. Previously, it was found that each antibody demonstrated a dose-dependent exposure and reached maximum circulating concentrations within 48 h after intramuscular (IM) or intravenous (IV) injection. Here we show that G03-52-01, in a single IM injection of G03-52-01 administered 48 h pre-exposure, protected guinea pigs against an aerosol challenge of up to 238 LD50s of BoNT/A1 and 191 LD50s of BoNT/B1. These data suggest that a single IM administration of G03-52-01 provides pre-exposure prophylaxis against botulism from an aerosol exposure of BoNT/A1 or BoNT/B1.

Neutralizing Concentrations of Anti-Botulinum Toxin Antibodies Positively Correlate with Mouse Neutralization Assay Results in a Guinea Pig Model.

Botulinum neurotoxins (BoNT) are some of the most toxic proteins known and can induce respiratory failure requiring long-term intensive care. Treatment of botulism includes the administration of antitoxins. Monoclonal antibodies (mAbs) hold considerable promise as BoNT therapeutics and prophylactics, due to their potency and safety. A three-mAb combination has been developed that specifically neutralizes BoNT serotype A (BoNT/A), and a separate three mAb combination has been developed that specifically neutralizes BoNT serotype B (BoNT/B). A six mAb cocktail, designated G03-52-01, has been developed that combines the anti-BoNT/A and anti-BoNT/B mAbs. The pharmacokinetics and neutralizing antibody concentration (NAC) of G03-52-01 has been determined in guinea pigs, and these parameters were correlated with protection against an inhalation challenge of BoNT/A1 or BoNT/B1. Previously, it was shown that each antibody demonstrated a dose-dependent mAb serum concentration and reached maximum circulating concentrations within 48 h after intramuscular (IM) or intraperitoneal (IP) injection and that a single IM injection of G03-52-01 administered 48 h pre-exposure protected guinea pigs against an inhalation challenge of up to 93 LD50s of BoNT/A1 and 116 LD50s of BoNT/B1. The data presented here advance our understanding of the relationship of the neutralizing NAC to the measured circulating antibody concentration and provide additional support that a single IM or intravenous (IV) administration of G03-52-01 will provide pre-exposure prophylaxis against botulism from an aerosol exposure of BoNT/A and BoNT/B.

Identification of recombinant Fabs for structural and functional characterization of HIV-host factor complexes.

Viral infection and pathogenesis is mediated by host protein-viral protein complexes that are important targets for therapeutic intervention as they are potentially less prone to development of drug resistance. We have identified human, recombinant antibodies (Fabs) from a phage display library that bind to three HIV-host complexes. We used these Fabs to 1) stabilize the complexes for structural studies; and 2) facilitate characterization of the function of these complexes. Specifically, we generated recombinant Fabs to Vif-CBF-ß-ELOB-ELOC (VCBC); ESCRT-I complex and AP2-complex. For each complex we measured binding affinities with KD values of Fabs ranging from 12-419 nM and performed negative stain electron microscopy (nsEM) to obtain low-resolution structures of the HIV-Fab complexes. Select Fabs were converted to scFvs to allow them to fold intracellularly and perturb HIV-host protein complex assembly without affecting other pathways. To identify these recombinant Fabs, we developed a rapid screening pipeline that uses quantitative ELISAs and nsEM to establish whether the Fabs have overlapping or independent epitopes. This pipeline approach is generally applicable to other particularly challenging antigens that are refractory to immunization strategies for antibody generation including multi-protein complexes providing specific, reproducible, and renewable antibody reagents for research and clinical applications. The curated antibodies described here are available to the scientific community for further structural and functional studies on these critical HIV host-factor proteins.

A Four-Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotypes C and D.

Human botulism can be caused by botulinum neurotoxin (BoNT) serotypes A to G. Here, we present an antibody-based antitoxin composed of four human monoclonal antibodies (mAbs) against BoNT/C, BoNT/D, and their mosaic toxins. This work built on our success in generating protective mAbs to BoNT /A, B and E serotypes. We generated mAbs from human immune single-chain Fv (scFv) yeast-display libraries and isolated scFvs with high affinity for BoNT/C, BoNT/CD, BoNT/DC and BoNT/D serotypes. We identified four mAbs that bound non-overlapping epitopes on multiple serotypes and mosaic BoNTs. Three of the mAbs underwent molecular evolution to increase affinity. A four-mAb combination provided high-affinity binding and BoNT neutralization of both serotypes and their mosaic toxins. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing and neutralizing BoNT/C and BoNT/D serotypes and their mosaic toxins. A derivative of the four-antibody combination (NTM-1634) completed a Phase 1 clinical trial (Snow et al., Antimicrobial Agents and Chemotherapy, 2019) with no drug-related serious adverse events.

Pharmacokinetics of Human Recombinant Anti-Botulinum Toxin Antibodies in Rats.

Botulinum neurotoxins (BoNT) are potential biothreat agents due to their high lethality, potency, and ease of distribution, thus the development of antitoxins is a high priority to the US government. This study examined pre-clinical pharmacokinetic studies in rats of four oligoclonal anti-BoNT mAb-based therapeutics (NTM-1631, NTM-1632, NTM-1633, NTM-1634) for five BoNT serotypes (A, B, E, C, and D). NTM-1631, NTM-1632, and NTM-1633 each consist of three IgG1 mAbs, each with a distinct human or humanized variable region which bind to distinct epitopes on BoNT serotype A, B, or E respectively. NTM-1634 consists of four human immunoglobulin G1 (IgG1) mAbs binding BoNT C/D mosaic toxins. The mechanism of these antitoxins requires that three antibodies simultaneously bind toxin to achieve rapid clearance. Rats (total 378) displayed no adverse clinical signs attributed to antibody treatment from any of the antitoxins. Pharmacokinetic evaluation demonstrated that the individual mAbs are slowly eliminated, exhibiting dose-dependent exposure and long elimination half-lives ranging from 6.5 days to 10 days. There were no consistent differences observed between males and females or among the individual antibodies in each formulation in half-life. Anti-drug antibodies (ADA) were observed, as expected for human antibodies administered to rats. The results presented were used to support the clinical investigation of antibody-based botulism antitoxins.

Monoclonal Antibody Combinations Prevent Serotype A and Serotype B Inhalational Botulism in a Guinea Pig Model.

Botulinum neurotoxins (BoNT) are some of the most toxic proteins known, with a human LD50 of ~1 ng/kg. Equine antitoxin has a half-life in circulation of less than 1 day and is limited to a treatment rather than a prevention indication. The development of monoclonal antibodies (mAbs) may represent an alternative therapeutic option that can be produced at high quantities and of high quality and with half-lives of >10 days. Two different three mAb combinations are being developed that specifically neutralize BoNT serotypes A (BoNT/A) and B (BoNT/B). We investigated the pharmacokinetics of the anti-BoNT/A and anti-BoNT/B antibodies in guinea pigs (Cavia porcellus) and their ability to protect guinea pigs against an aerosol challenge of BoNT/A1 or BoNT/B1. Each antibody exhibited dose-dependent exposure and reached maximum circulating concentrations within 48 h post intraperitoneal or intramuscular injection. A single intramuscular dose of the three mAb combination protected guinea pigs against an aerosol challenge dose of 93 LD50 of BoNT/A1 and 116 LD50 of BoNT/B1 at 48 h post antibody administration. These mAbs are effective in preventing botulism after an aerosol challenge of BoNT/A1 and BoNT/B1 and may represent an alternative to vaccination to prevent type A or B botulism in those at risk of BoNT exposure.

Antibody-Drug Conjugates Targeting the Urokinase Receptor (uPAR) as a Possible Treatment of Aggressive Breast Cancer.

A promising molecular target for aggressive cancers is the urokinase receptor (uPAR). A fully human, recombinant antibody that binds uPAR to form a stable complex that blocks uPA-uPAR interactions (2G10) and is internalized primarily through endocytosis showed efficacy in a mouse xenograft model of highly aggressive, triple negative breast cancer (TNBC). Antibody-drug conjugates (ADCs) of 2G10 were designed and produced bearing tubulin inhibitor payloads ligated through seven different linkers. Aldehyde tag technology was employed for linking, and either one or two tags were inserted into the antibody heavy chain, to produce site-specifically conjugated ADCs with drug-to-antibody ratios of either two or four. Both cleavable and non-cleavable linkers were combined with two different antimitotic toxins-MMAE (monomethylauristatin E) and maytansine. Nine different 2G10 ADCs were produced and tested for their ability to target uPAR in cell-based assays and a mouse model. The anti-uPAR ADC that resulted in tumor regression comprised an MMAE payload with a cathepsin B cleavable linker, 2G10-RED-244-MMAE. This work demonstrates in vitro activity of the 2G10-RED-244-MMAE in TNBC cell lines and validates uPAR as a therapeutic target for TNBC.

An ambient temperature-stable antitoxin of nine co-formulated antibodies for botulism caused by serotypes A, B and E.

Safe and effective antitoxins to treat and prevent botulism are needed for biodefense. We have developed recombinant antibody-based therapeutics for botulinum neurotoxin (BoNT) serotypes A, B, and E. The mechanism of action of this antitoxin requires that three mAbs bind one toxin molecule to achieve clearance. Here we present a co-formulation of an antitoxin to the three most important serotypes. Combining these antibodies obviates the need to identify the serotype causing intoxication prior to drug administration, which would facilitate administration. The lyophilized powder formulation contains nine mAbs, three mAbs for each of the three serotypes (A, B, E). The formulation was stored as a liquid and lyophilized powder for up to one year, and characterized by binding affinity and multiple physicochemical methods. No significant increase in soluble higher order aggregates, cleavage products, or change in charge isoforms was measured after storage as a lyophilized powder at 50°C for one year. Furthermore, toxin-domain binding ELISA data indicated that each of the individual antibodies in the lyophilized drug product showed essentially full binding capability to their respective toxin domains after being stored at 50°C for one year. Physicochemical characterization of the formulation demonstrated the nine individual mAbs were remarkably stable. This work demonstrates feasibility of lyophilized, oligoclonal antibody therapies for biodefense with ambient temperature stability, that would facilitate stockpiling, distribution, and administration.

Discovery of internalizing antibodies to basal breast cancer cells.

We present a strategy to discover recombinant monoclonal antibodies (mAbs) to specific cancers and demonstrate this approach using basal subtype breast cancers. A phage antibody library was depleted of antibodies to common cell surface molecules by incubation with luminal breast cancer cell lines, and then selected on a single basal-like breast cancer cell line (MDA-MB-231) for binding associated receptor-mediated endocytosis. Additional profiling against two luminal and four basal-like cell lines revealed 61 unique basal-specific mAbs from a pool of 1440 phage antibodies. The unique mAbs were further screened on nine basal and seven luminal cell lines to identify those with the greatest affinity, specificity, and internalizing capability for basal-like breast cancer cells. Among the internalizing basal-specific mAbs were those recognizing four transmembrane receptors (EphA2, CD44, CD73 and EGFR), identified by immunoprecipitation-mass spectrometry and yeast-displayed antigen screening. Basal-like breast cancer expression of these four receptors was confirmed using a bioinformatic approach, and expression microarray data on 683 intrinsically subtyped primary breast tumors. This overall approach, which sequentially employs phage display antibody library selection, antigen identification and bioinformatic confirmation of antigen expression by cancer subtypes, offers efficient production of high-affinity mAbs with diagnostic and therapeutic utility against specific cancer subtypes.

A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes.

Human botulism is most commonly caused by botulinum neurotoxin (BoNT) serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb)-based antitoxin capable of binding and neutralizing multiple subtypes of BoNT/E. Libraries of yeast-displayed single chain Fv (scFv) antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent-toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting (FACS). A total of 10 scFv were isolated that bound one or more BoNT/E subtypes with nanomolar-level equilibrium dissociation constants (KD). By diversifying the V-regions of the lead mAbs and selecting for cross-reactivity, we generated three scFv that bound all four BoNT/E subtypes tested at three non-overlapping epitopes. The scFvs were converted to IgG that had KD values for the different BoNT/E subtypes ranging from 9.7 nM to 2.28 pM. An equimolar combination of the three mAbs was able to potently neutralize BoNT/E1, BoNT/E3, and BoNT/E4 in a mouse neutralization assay. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing multiple BoNT/E subtypes. A derivative of the three-antibody combination (NTM-1633) is in pre-clinical development with an investigational new drug (IND) application filing expected in 2018.

A Single Tri-Epitopic Antibody Virtually Recapitulates the Potency of a Combination of Three Monoclonal Antibodies in Neutralization of Botulinum Neurotoxin Serotype A.

The standard of treatment for botulism, equine antitoxin, is a foreign protein with associated safety issues and a short serum half-life which excludes its use as a prophylactic antitoxin and makes it a less-than-optimal therapeutic. Due to these limitations, a recombinant monoclonal antibody (mAb) product is preferable. It has been shown that combining three mAbs that bind non-overlapping epitopes leads to highly potent botulinum neurotoxin (BoNT) neutralization. Recently, a triple human antibody combination for BoNT/A has demonstrated potent toxin neutralization in mouse models with no serious adverse events when tested in a Phase I clinical trial. However, a triple antibody therapeutic poses unique development and manufacturing challenges. Thus, potentially to streamline development of BoNT antitoxins, we sought to achieve the potency of multiple mAb combinations in a single IgG-based molecule that has a long serum half-life. The design, production, and testing of a single tri-epitopic IgG1-based mAb (TeAb) containing the binding sites of each of the three parental BoNT/A mAbs yielded an antibody of nearly equal potency to the combination. The approach taken here could be applied to the design and creation of other multivalent antibodies that could be used for a variety of applications, including toxin elimination.

High-throughput screening: update on practices and success.

High-throughput screening (HTS) has become an important part of drug discovery at most pharmaceutical and many biotechnology companies worldwide, and use of HTS technologies is expanding into new areas. Target validation, assay development, secondary screening, ADME/Tox, and lead optimization are among the areas in which there is an increasing use of HTS technologies. It is becoming fully integrated within drug discovery, both upstream and downstream, which includes increasing use of cell-based assays and high-content screening (HCS) technologies to achieve more physiologically relevant results and to find higher quality leads. In addition, HTS laboratories are continually evaluating new technologies as they struggle to increase their success rate for finding drug candidates. The material in this article is based on a 900-page HTS industry report involving 54 HTS directors representing 58 HTS laboratories and 34 suppliers.