Stable nebulization and muco-trapping properties of regdanvimab/IN-006 support its development as a potent, dose-saving inhaled therapy for COVID-19.

The respiratory tract represents the key target for antiviral delivery in early interventions to prevent severe COVID-19. While neutralizing monoclonal antibodies (mAb) possess considerable efficacy, their current reliance on parenteral dosing necessitates very large doses and places a substantial burden on the healthcare system. In contrast, direct inhaled delivery of mAb therapeutics offers the convenience of self-dosing at home, as well as much more efficient mAb delivery to the respiratory tract. Here, building on our previous discovery of Fc-mucin interactions crosslinking viruses to mucins, we showed that regdanvimab, a potent neutralizing mAb already approved for COVID-19 in several countries, can effectively trap SARS-CoV-2 virus-like particles in fresh human airway mucus. IN-006, a reformulation of regdanvimab, was stably nebulized across a wide range of concentrations, with no loss of activity and no formation of aggregates. Finally, nebulized delivery of IN-006 resulted in 100-fold greater mAb levels in the lungs of rats compared to serum, in marked contrast to intravenously dosed mAbs. These results not only support our current efforts to evaluate the safety and efficacy of IN-006 in clinical trials, but more broadly substantiate nebulized delivery of human antiviral mAbs as a new paradigm in treating SARS-CoV-2 and other respiratory pathologies.

Phosphatidylserine Sensing by TAM Receptors Regulates AKT-Dependent Chemoresistance and PD-L1 Expression.

Tyro3, Axl, and Mertk (collectively TAM receptors) are three homologous receptor tyrosine kinases that bind vitamin K-dependent endogenous ligands, Protein S (ProS), and growth arrest-specific factor 6 (Gas6), and act as bridging molecules to promote phosphatidylserine (PS)-mediated clearance of apoptotic cells (efferocytosis). TAM receptors are overexpressed in a vast array of tumor types, whereby the level of expression correlates with the tumor grade and the emergence of chemo- and radioresistance to targeted therapeutics, but also have been implicated as inhibitory receptors on infiltrating myeloid-derived cells in the tumor microenvironment that can suppress host antitumor immunity. In the present study, we utilized TAM-IFNγR1 reporter lines and expressed TAM receptors in a variety of epithelial cell model systems to show that each TAM receptor has a unique pattern of activation by Gas6 or ProS, as well as unique dependency for PS on apoptotic cells and PS liposomes for activity. In addition, we leveraged this system to engineer epithelial cells that express wild-type TAM receptors and show that although each receptor can promote PS-mediated efferocytosis, AKT-mediated chemoresistance, as well as upregulate the immune checkpoint molecule PD-L1 on tumor cells, Mertk is most dominant in the aforementioned pathways. Functionally, TAM receptor-mediated efferocytosis could be partially blocked by PS-targeting antibody 11.31 and Annexin V, demonstrating the existence of a PS/PS receptor (i.e., TAM receptor)/PD-L1 axis that operates in epithelial cells to foster immune escape. These data provide a rationale that PS-targeting, anti-TAM receptor, and anti-PD-L1-based therapeutics will have merit as combinatorial checkpoint inhibitors.Implications: Many tumor cells are known to upregulate the immune checkpoint inhibitor PD-L1. This study demonstrates a role for PS and TAM receptors in the regulation of PD-L1 on cancer cells. Mol Cancer Res; 15(6); 753-64. ©2017 AACR.

Phosphatidylserine-targeting antibodies augment the anti-tumorigenic activity of anti-PD-1 therapy by enhancing immune activation and downregulating pro-oncogenic factors induced by T-cell checkpoint inhibition in murine triple-negative breast cancers.

BACKGROUND: The purpose of this study was to investigate the potential of antibody-directed immunotherapy targeting the aminophospholipid phosphatidylserine, which promotes immunosuppression when exposed in the tumor microenvironment, alone and in combination with antibody treatment towards the T-cell checkpoint inhibitor PD-1 in breast carcinomas, including triple-negative breast cancers. METHODS: Immune-competent mice bearing syngeneic EMT-6 or E0771 tumors were subjected to treatments comprising of a phosphatidylserine-targeting and an anti-PD-1 antibody either as single or combinational treatments. Anti-tumor effects were determined by tumor growth inhibition and changes in overall survival accompanying each treatment. The generation of a tumor-specific immune response in animals undergoing complete tumor regression was assessed by secondary tumor cell challenge and splenocyte-produced IFNγ in the presence or absence of irradiated tumor cells. Changes in the presence of tumor-infiltrating lymphocytes were assessed by flow cytometry, while mRNA-based immune profiling was determined using NanoString PanCancer Immune Profiling Panel analysis. RESULTS: Treatment by a phosphatidylserine-targeting antibody inhibits in-vivo growth and significantly enhances the anti-tumor activity of antibody-mediated PD-1 therapy, including providing a distinct survival advantage over treatment by either single agent. Animals in which complete tumor regression occurred with combination treatments were resistant to secondary tumor challenge and presented heightened expression levels of splenocyte-produced IFNγ. Combinational treatment by a phosphatidylserine-targeting antibody with anti-PD-1 therapy increased the number of tumor-infiltrating lymphocytes more than that observed with single-arm therapies. Finally, immunoprofiling analysis revealed that the combination of anti-phosphatidylserine targeting antibody and anti-PD-1 therapy enhanced tumor-infiltrating lymphocytes, and increased expression of pro-immunosurveillance-associated cytokines while significantly decreasing expression of pro-tumorigenic cytokines that were induced by single anti-PD-1 therapy. CONCLUSIONS: Our data suggest that antibody therapy targeting phosphatidylserine-associated immunosuppression, which has activity as a single agent, can significantly enhance immunotherapies targeting the PD-1 pathway in murine breast neoplasms, including triple-negative breast cancers.

Antibody-Mediated Phosphatidylserine Blockade Enhances the Antitumor Responses to CTLA-4 and PD-1 Antibodies in Melanoma.

In tumor-bearing animals, the membrane phospholipid phosphatidylserine (PS) suppresses immune responses, suggesting that PS signaling could counteract the antitumor effect of antibody-driven immune checkpoint blockade. Here, we show that treating melanoma-bearing mice with a PS-targeting antibody enhances the antitumor activity of downstream checkpoint inhibition. Combining PS-targeting antibodies with CTLA-4 or PD-1 blockade resulted in significantly greater inhibition of tumor growth than did single-agent therapy. Moreover, combination therapy enhanced CD4(+) and CD8(+) tumor-infiltrating lymphocyte numbers; elevated the fraction of cells expressing the proinflammatory cytokines IL2, IFNγ, and TNFα; and increased the ratio of CD8 T cells to myeloid-derived suppressor cells and regulatory T cells in tumors. Similar changes in immune cell profiles were observed in splenocytes. Taken together, these data show that antibody-mediated PS blockade enhances the antitumor efficacy of immune checkpoint inhibition. Cancer Immunol Res; 4(6); 531-40. ©2016 AACR.

Synthesis and evaluation against hepatitis C virus of 7-deaza analogues of 2'-C-methyl-6-O-methyl guanosine nucleoside and L-Alanine ester phosphoramidates.

7-Deazapurines are known to possess broad antiviral activity, however the 2'-C-methylguanosine analogue displays poor cell permeation and limited phosphorylation, thus is not an efficient inhibitor of hepatitis C virus (HCV) replication. We previously reported the 6-O-methyl entity as a prodrug moiety to increase liphophilicity of guanine nucleosides and the ProTide approach applied to 2'-C-methyl-6-O-methylguanosine has lead to potent HCV inhibitors now in clinical trials. In this Letter, we report the synthesis and biological evaluation of 2'-C-methyl-6-O-methyl-7-deaza guanosine and ProTide derivatives. In contrast to prior studies, removal of the N-7 of the nucleobase entirely negates anti-HCV activity compared to the 2'-C-methyl-6-O-methylguanosine analogues. To understand better this significant loss of activity, enzymatic assays and molecular modeling were carried out and suggested 2'-C-methyl-6-O-methyl-7-deaza guanosine and related ProTides do not act as efficient prodrugs of the free nucleotide, in marked contrast to the case of the parent guanine analogue.

Phosphorodiamidates as a promising new phosphate prodrug motif for antiviral drug discovery: application to anti-HCV agents.

We herein report phosphorodiamidates as a significant new phosphate prodrug motif. Sixty-seven phosphorodiamidates are reported of two 6-O-alkyl 2'-C-methyl guanosines, with significant variation in the diamidate structure. Both symmetrical and asymmetric phosphorodiamidates are reported, derived from various esterified amino acids, both d and l, and also from various simple amines. All of the compounds were evaluated versus hepatitis C virus in replicon assay, and nanomolar activity levels were observed. Many compounds were noncytotoxic at 100 µM, leading to high antiviral selectivities. The agents are stable in acidic, neutral, and moderately basic media and in selected biological media but show efficient processing by carboxypeptidases and efficiently yield the free nucleoside monophosphate in cells. On the basis of in vitro data, eight leads were selected for additional in vivo evaluation, with the intent of selecting one candidate for progression toward clinical studies. This phosphorodiamidate prodrug method may have broad application outside of HCV and antivirals as it offers many of the advantages of phosphoramidate ProTides but without the chirality issues present in most cases.

Dual pro-drugs of 2'-C-methyl guanosine monophosphate as potent and selective inhibitors of hepatitis C virus.

We have previously reported the power of combining a 5'-phosphoramidate ProTide, phosphate pro-drug, motif with a 6-methoxy purine pro-drug entity to generate highly potent anti-HCV agents, leading to agents in clinical trial. We herein extend this work with the disclosure that a variety of alternative 6-substituents are tolerated. Several compounds exceed the potency of the prior 6-methoxy leads, and in almost every case the ProTide is several orders of magnitude more potent than the parent nucleoside. We also demonstrate that these agents act as pro-drugs of 2'-C-methyl guanosine monophosphate. We have also reported the novel use of hepatocyte cell lysate as an ex vivo model for ProTide metabolism.

INX-08189, a phosphoramidate prodrug of 6-O-methyl-2'-C-methyl guanosine, is a potent inhibitor of hepatitis C virus replication with excellent pharmacokinetic and pharmacodynamic properties.

INX-08189 is an aryl-phosphoramidate of 6-O-methyl-2'-C-methyl guanosine. INX-08189 was highly potent in replicon assays, with a 50% effective concentration of 10±6 nM against hepatitis C genotype 1b at 72 h. The inhibitory effect on viral replication was rapid, with a 50% effective concentration (EC50) of 35±8 nM at 24 h. An intracellular 2'-C-methyl guanosine triphosphate (2'-C-MeGTP) concentration of 2.43±0.42 pmol/10(6) cells was sufficient to achieve 90% inhibition of viral replication. In vitro resistance studies confirmed that the S282T mutation in the NS5b gene conferred an approximately 10-fold reduction in sensitivity to INX-08189. However, the complete inhibition of S282T mutant replicons still could be achieved with an EC90 of 344±170 nM. Drug combination studies of INX-08189 and ribavirin indicated significant synergy in antiviral potency both in wild-type and S282T-expressing replicons. Genotype 1b replicons could be cleared after 14 days of culture when exposed to as little as 20 nM INX-08189. No evidence of mitochondrial toxicity was observed after 14 days of INX-08189 exposure in both HepG2 and CEM human cell lines. In vivo studies of rats and cynomolgus monkeys demonstrated that 2'-C-MeGTP concentrations in liver equivalent to the EC90 could be attained after a single oral dose of INX-08189. Rat liver 2'-C-MeGTP concentrations were proportional to dose, sustained for greater than 24 h, and correlated with plasma concentrations of the nucleoside metabolite 2'-C-methyl guanosine. The characteristics displayed by INX-08189 support its continued development as a clinical candidate for the treatment of chronic HCV infection.

Pharmacokinetics and safety of FV-100, a novel oral anti-herpes zoster nucleoside analogue, administered in single and multiple doses to healthy young adult and elderly adult volunteers.

FV-100 is the prodrug of the highly potent anti-varicella zoster virus bicyclic nucleoside analogue CF-1743. To characterize the pharmacokinetics and safety of oral FV-100, 3 randomized, double-blind, placebo-controlled clinical trials were conducted: (i) a single-ascending-dose study in 32 healthy subjects aged 18 to 55 years (100-, 200-, 400-, and 800-mg doses) with an evaluation of the food effect in the 400-mg group; (ii) a multiple-ascending-dose study in 48 subjects aged 18 to 55 years (100 mg once daily [QD], 200 mg QD, 400 mg QD, 400 mg twice a day, and 800 mg QD for 7 days); and (iii) a 2-part study in subjects aged 65 years and older with a single 400-mg dose in 15 subjects and a 400-mg QD dosing regimen for 7 days in 12 subjects. FV-100 was rapidly and extensively converted to CF-1743, the concentration of which remained above that required to reduce viral activity by 50% for the 24-hour dosing period. Renal excretion of CF-1743 was very low. A high-fat meal reduced exposure to CF-1743; a low-fat meal did not. Pharmacokinetic parameters for the elderly subjects were comparable to those for the younger subjects. FV-100 was well tolerated by all subjects. The pharmacokinetic and safety profiles of FV-100 support its continued investigation for the treatment of herpes zoster and prevention of postherpetic neuralgia with once-daily dosing and without dose modifications for elderly or renally impaired patients.

Design, synthesis and evaluation of a novel double pro-drug: INX-08189. A new clinical candidate for hepatitis C virus.

We herein report a novel double pro-drug approach applied to the anti-HCV agent 2'-beta-C-methyl guanosine. A phosphoramidate ProTide motif and a 6-O-methoxy base pro-drug moiety are combined to generate lipophilic prodrugs of the monophosphate of the guanine nucleoside. Modification of the ester and amino acid moieties lead to a compound INX-08189 that exhibits 10nM potency in the HCV genotype 1b subgenomic replicon, thus being 500 times more potent than the parent nucleoside. The potency of the lead compound INX-08189 was shown to be consistent with intracellular 2'-C-methyl guanosine triphosphate levels in primary human hepatocytes. The separated diastereomers of INX-08189 were shown to have similar activity in the replicon assay and were also shown to be similar substrates for enzyme processing. INX-08189 has completed investigational new drug enabling studies and has been progressed into human clinical trials for the treatment of chronic HCV infection.

Phosphoramidate ProTides of 2'-C-methylguanosine as highly potent inhibitors of hepatitis C virus. Study of their in vitro and in vivo properties.

Hepatitis C virus infection constitutes a serious health problem in need of more effective therapies. Nucleoside analogues with improved exposure, efficacy, and selectivity are recognized as likely key components of future HCV therapy. 2'-C-Methylguanosine triphosphate has been known as a potent inhibitor of HCV RNA polymerase for some time, but the parent nucleoside is only moderately active due to poor intracellular phosphorylation. We herein report the application of phosphoramidate ProTide technology to bypass the rate-limiting initial phosphorylation of this nucleoside. Over 30 novel ProTides are reported, with variations in the aryl, ester, and amino acid regions. l-Alanine compounds are recognized as potent and selective inhibitors of HCV in replicon assay but lack rodent plasma stability despite considerable ester variation. Amino acid variation retaining the lead benzyl ester moiety gives an increase in rodent stability but at the cost of potency. Finally l-valine esters with ester variation lead to potent, stable compounds. Pharmacokinetic studies on these agents in the mouse reveal liver exposure to the bioactive triphosphate species following single oral dosing. Systemic exposure of the ProTide and parent nucleoside are low, indicating possible low toxicity in vivo, while liver concentrations of the active species may be predictive of efficacy in the clinic. This represents one of the most thorough cross-species studies of ProTides to date.

Monoclonal antibodies specific for Candida albicans Als3 that immunolabel fungal cells in vitro and in vivo and block adhesion to host surfaces.

Two monoclonal antibodies (MAbs) were raised against the Candida albicans cell-surface glycoprotein Als3 using the N-terminal domain of the protein as the immunogen. ELISA was used to demonstrate the specificity of the MAbs for the Als3 fragment, but not for the corresponding N-terminal domain fragments from other proteins in the Als family. The anti-Als3 MAbs immunolabeled the surface of germ tubes from a diverse collection of wild-type C. albicans isolates, but did not label yeast cells, an als3Delta/als3Delta deletion mutant strain, nor isolates of other Candida species associated with human disease. Als3 was visualized readily in fresh and formalin-fixed, paraffin-embedded kidney tissue from a murine model of candidiasis. The anti-Als3 MAbs were also useful for immunogold electron microscopy and Western blotting. Both MAbs blocked C. albicans adhesion to vascular endothelial cells and buccal epithelial cells. These versatile MAbs are a valuable addition to the reagents available to study C. albicans cell surface dynamics and interaction of the fungus with host cells.

Effects of the cFMS kinase inhibitor 5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580) in normal and arthritic rats.

The cFMS (cellular homolog of the V-FMS oncogene product of the Susan McDonough strain of feline sarcoma virus) (Proc Natl Acad Sci U S A 83:3331-3335, 1986) kinase inhibitor 5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580) inhibits colony-stimulating factor (CSF)-1-induced monocyte growth and bone degradation in vitro and inhibits CSF-1 signaling through cFMS kinase in 4-day models in mice (Proc Natl Acad Sci U S A 102:16078, 2005). In the present study, the kinase selectivity of GW2580 was further characterized, and the effects of chronic treatment were evaluated in normal and arthritic rats. GW2580 selectively inhibited cFMS kinase compared with 186 other kinases in vitro and completely inhibited CSF-1-induced growth of rat monocytes, with an IC(50) value of 0.2 microM. GW2580 dosed orally at 25 and 75 mg/kg 1 and 5 h before the injection of lipopolysaccharide inhibited tumor necrosis factor-alpha production by 60 to 85%, indicating a duration of action of at least 5 h. In a 21-day adjuvant arthritis model, GW2580 dosed twice a day (b.i.d.) from days 0 to 21, 7 to 21, or 14 to 21 inhibited joint connective tissue and bone destruction as assessed by radiology, histology and bone mineral content measurements. In contrast, GW2580 did not affect ankle swelling in the adjuvant model nor did it affect ankle swelling in a model where local arthritis is reactivated by peptidoglycan polysaccharide polymers. GW2580 administered to normal rats for 21 days showed no effects on tissue histology and only modest changes in serum clinical chemistry and blood hematology. In conclusion, GW2580 was effective in preserving joint integrity in the adjuvant arthritis model while showing minimal effects in normal rats.

Automated sample preparation facilitated by PhyNexus MEA purification system for oligosaccharide mapping of glycoproteins.

A reproducible high-throughput sample cleanup method for fluorescent oligosaccharide mapping of glycoproteins is described. Oligosaccharides are released from glycoproteins using PNGase F and labeled with 2-aminobenzoic acid (anthranilic acid, AA). A PhyNexus MEA system was adapted for automated isolation of the fluorescently labeled oligosaccharides from the reaction mixture prior to mapping by HPLC. The oligosaccharide purification uses a normal-phase polyamide resin (DPA-6S) in custom-made pipette tips. The resin volume, wash, and elution steps involved were optimized to obtain high recovery of oligosaccharides with the least amount of contaminating free fluorescent dye in the shortest amount of time. The automated protocol for sample cleanup eliminated all manual manipulations with a recycle time of 23 min. We have reduced the amount of excess AA by 150-fold, allowing quantitative oligosaccharide mapping from as little as 500 ng digested recombinant immunoglobulin G (rIgG). This low sample requirement allows early selection of a cell line with desired characteristics (e.g., oligosaccharide profile and high specific productivity) for the production of glycoprotein drugs. In addition, the use of Tecan or another robotic platform in conjunction with this method should allow the cleanup of 96 samples in 23 min, a significant decrease in the amount of time currently required to process such a large number of samples.

Monoclonal antibodies recognizing the Enterococcus faecalis collagen-binding MSCRAMM Ace: conditional expression and binding analysis.

Enterococci are opportunistic pathogens known to cause numerous clinical infections and complications in humans. Adhesin-mediated binding to extracellular matrix (ECM) proteins of the host is thought to be a crucial step in the pathogenesis of these bacterial infections. Adhesin of collagen from Enterococcus faecalis (Ace) is a cell-wall anchored protein of E. faecalis that has been shown to be important for bacterial binding to the ECM. In this report, we characterize the conditions for Ace expression and demonstrate Ace binding to mammalian epithelial and endothelial cells as well as to collagens found in the ECM. To further characterize Ace expression and function, we report the generation of a panel of monoclonal antibodies (mAbs) directed against this important E. faecalis virulence factor. Through the use of multiple in vitro assays, surface plasmon resonance and flow cytometry, we have characterized this panel of mAbs which may prove to be not only beneficial in studies that address the precise biological role of adhesion of E. faecalis, but may also serve as beneficial therapeutic agents against E. faecalis infections.

A panel of monoclonal antibodies recognizing the Staphylococcus epidermidis fibrinogen-binding MSCRAMM SdrG.

Staphylococcus epidermidis is an important opportunistic human pathogen that has recently emerged as a major cause of foreign-body infections. The most important stage contributing to the pathogenesis of this bacteria is the initial adherence to host tissue. SdrG is a cell-wall-anchored fibrinogen-binding adhesin of S. epidermidis that has been shown to be necessary for bacterial binding to fibrinogen-coated foreign bodies, such as catheters. Here we report the generation and characterization of a panel of monoclonal antibodies (MAbs) directed against this S. epidermidis virulence factor. Through the use of multiple in vitro assays, surface plasmon resonance, and flow cytometry, we have characterized a diverse array of MAbs that may prove to be beneficial in studies that address the precise biologic role of SdrG.

Inhibition of colony-stimulating-factor-1 signaling in vivo with the orally bioavailable cFMS kinase inhibitor GW2580.

Colony-stimulating-factor-1 (CSF-1) signaling through cFMS receptor kinase is increased in several diseases. To help investigate the role of cFMS kinase in disease, we identified GW2580, an orally bioavailable inhibitor of cFMS kinase. GW2580 completely inhibited human cFMS kinase in vitro at 0.06 microM and was inactive against 26 other kinases. GW2580 at 1 microM completely inhibited CSF-1-induced growth of mouse M-NFS-60 myeloid cells and human monocytes and completely inhibited bone degradation in cultures of human osteoclasts, rat calvaria, and rat fetal long bone. In contrast, GW2580 did not affect the growth of mouse NS0 lymphoblastoid cells, human endothelial cells, human fibroblasts, or five human tumor cell lines. GW2580 also did not affect lipopolysaccharide (LPS)-induced TNF, IL-6, and prostaglandin E2 production in freshly isolated human monocytes and mouse macrophages. After oral administration, GW2580 blocked the ability of exogenous CSF-1 to increase LPS-induced IL-6 production in mice, inhibited the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity, and diminished the accumulation of macrophages in the peritoneal cavity after thioglycolate injection. Unexpectedly, GW2580 inhibited LPS-induced TNF production in mice, in contrast to effects on monocytes and macrophages in vitro. In conclusion, GW2580's selective inhibition of monocyte growth and bone degradation is consistent with cFMS kinase inhibition. The ability of GW2580 to chronically inhibit CSF-1 signaling through cFMS kinase in normal and tumor cells in vivo makes GW2580 a useful tool in assessing the role of cFMS kinase in normal and disease processes.

Characterization of a humanized monoclonal antibody recognizing clumping factor A expressed by Staphylococcus aureus.

We report the humanization and characterization of monoclonal antibody (MAb) T1-2 or tefibazumab, a monoclonal antibody that recognizes clumping factor A expressed on the surface of Staphylococcus aureus. We demonstrate that the binding kinetics of MAb T1-2 is indistinguishable compared to that of its murine parent. Furthermore, MAb T1-2 is shown to enhance the opsonophagocytic uptake of ClfA-coated latex beads, protect against an intravenous challenge in a prophylactic model of rabbit infective endocarditis, and enhance the efficacy of vancomycin therapy in a therapeutic model of established infective endocarditis.

Characterization of a protective monoclonal antibody recognizing Staphylococcus aureus MSCRAMM protein clumping factor A.

The Staphylococcus aureus MSCRAMM (microbial surface components recognizing adhesive matrix molecules) protein clumping factor A (ClfA) has been shown to be a critical virulence factor in several experimental models of infection. This report describes the generation, characterization, and in vivo evaluation of a murine monoclonal antibody (MAb) against ClfA. Flow cytometric analysis revealed that MAb 12-9 recognized ClfA protein expressed by all of the clinical S. aureus strains obtained from a variety of sources. In assays measuring whole-cell S. aureus binding to human fibrinogen, MAb 12-9 inhibited S. aureus binding by over 90% and displaced up to 35% of the previously adherent S. aureus bacteria. Furthermore, a single infusion of MAb 12-9 was protective against an intravenous challenge with a methicillin-resistant strain of S. aureus in a murine sepsis model (P < 0.0001). These data suggest that anti-ClfA MAb 12-9 should be further investigated as a novel immunotherapy for the treatment and prevention of life-threatening S. aureus infections.

In vivo expression of a GST-fusion protein mediates the rapid generation of affinity matured monoclonal antibodies using DNA-based immunizations.

Previously we demonstrated the rapid generation of affinity matured monoclonal antibody (MAb) producing cell lines following gene gun delivery of DNA using a mammalian expression vector (pAlpha/hFc), which enables the expression of human Fc-chimera proteins in vivo. Here we compare the pAlpha/hFc vector to modified vectors that replace human IgG(1) with either a Glutathione-S-Transferase (GST) fusion protein or a mouse IgG(2c) (mFc) fusion protein. We report that in vivo expression of a GST-chimera results in the rapid generation of affinity matured MAbs, comparable with antibodies raised using the pAlpha/hFc vector, that were reactive with annexin V. The mFc vector failed to induce early antigen-specific B-cell responses suitable for MAb development.