RESEARCH ARTICLE ANTIMICROBIAL THERAPEUTICS
A multifunctional bispecific antibody protects against Pseudomonas aeruginosa
Widespread drug resistance due to empiric use of broad-spectrum antibiotics has stimulated development of bacteria-specific strategies for prophylaxis and therapy based on modern monoclonal antibody (mAb) technologies. However, single-mechanism mAb approaches have not provided adequate protective activity in the clinic. We constructed multifunctional bispecific antibodies, each conferring three mechanisms of action against the bacterial pathogen Pseudomonas aeruginosa by targeting the serotype-independent type III secretion system (injectisome) virulence factor PcrV and persistence factor Psl exopolysaccharide. A new bispecific antibody platform, BiS4, exhibited superior synergistic protection against P. aeruginosa–induced murine pneumonia compared to parent mAb combinations or other available bispecific antibody structures. BiS4aPa was protective in several mouse infection models against disparate P. aeruginosa strains and unexpectedly further synergized with multiple antibiotic classes even against drug-resistant clinical isolates. In addition to resulting in a multimechanistic clinical candidate (MEDI3902) for the prevention or treatment of P. aeruginosa infections, these antibody studies suggest that multifunctional antibody approaches may be a promising platform for targeting other antibiotic-resistant bacterial pathogens.
INTRODUCTION Antibody therapy with animal serum targeting bacterial toxins or capsular polysaccharides predates the discovery and development of smallmolecule antibiotics (1, 2). Broad-spectrum antibiotic chemotherapy eventually displaced serum therapy and became the cornerstone of modern medicine given its relative safety in comparison to serum derived from nonhuman sources and because it enabled more convenient empiric therapy. However, increasing drug resistance to virtually all antibiotic classes, particularly within the designated ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pathogens (3), threatens modern medicine as we know it (4, 5). This severe situation and paucity of new antibiotic classes in development coupled with the belief that empiric broad-spectrum antibiotic therapy has led to bacterial cross-resistance are stimulating renewed interest in pathogen-specific strategies including monoclonal antibody (mAb) technology for the most problematic microorganisms. Although mAbs offer considerable potential, the principal challenge for singletarget mAb-based approaches is obtaining adequate protective activity against a broad range of strains and disease states. P. aeruginosa is one of the most recalcitrant ESKAPE pathogens and a leading cause of acute pneumonia in the hospital environment and of chronic lung infections in cystic fibrosis patients. Its intrinsic drug resistance, owing to its comparatively large genome and regulatory capacity, makes P. aeruginosa a challenging target for a single mAb approach. We previously reported the identification of protective mAbs that mediate serotype-independent opsonophagocytic killing of P. aeruginosa and inhibit adherence to cultured epithelial cells (6). The most protective mAb in multiple infection models was elucidated to target a determinant associated with exopolysaccharide Psl, an abundantly expressed MedImmune, LLC, One MedImmune Way, Gaithersburg, MD 20878, USA. *Corresponding author. E-mail: [email protected]
extracellular sugar polymer implicated in immune evasion and biofilm formation (6–8). In addition, we recently identified a new highly active mAb against the clinically validated target PcrV, which strongly inhibits P. aeruginosa type III secretion (T3S) transport of multiple virulence factors (9–11). Given the important role of Psl and T3S expression in the establishment of acute and persistent P. aeruginosa infections (12–15), we reasoned that a combination of the anti-Psl and anti-PcrV mAb mechanisms of action (MOAs) could enhance strain and disease coverage against P. aeruginosa. Although it is possible to co-administer antibody combinations, it is more practicable to develop a single-molecule clinical candidate. Bispecific antibody technology was first described with the derivation of hybrid hybridomas (16); however, this technology often resulted in mispaired byproducts and poor overall product yield. With the increased implementation of molecular biological methods and the development of single-chain variable fragments (scFvs) (17), construction of bispecific antibodies by fusion of scFv domains to free termini of mAb heavy chain or light chain sequences became feasible (18). Further developments in bispecific antibody technology were forged by engineering heterodimerization motifs into constant sequences, resulting in more than 50 bispecific platforms described to date (19). Whereas bispecific antibodies against viral targets have been reported (20), bispecific antibodies targeting bacterial antigens are limited (21, 22). Herein, we describe enhanced anti–P. aeruginosa activity afforded by a multimechanistic bivalent, bispecific antibody configuration targeting Psl and PcrV designated BiS4aPa. The potent serotype-independent activity of BiS4aPa observed against diverse strain types, including multidrug-resistant (MDR) strains, in multiple animal infection models in both prophylactic and therapeutic regimens, and the surprisingly potent in vivo synergy in adjunctive therapy with multiple antibiotic classes, supports this molecule as a promising clinical candidate (designated MEDI3902) for the prophylaxis or adjunctive treatment of P. aeruginosa infections. The successful application of this approach suggests a more broadly
www.ScienceTranslationalMedicine.org
12 November 2014
Vol 6 Issue 262 262ra155
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Antonio DiGiandomenico, Ashley E. Keller, Cuihua Gao, Godfrey J. Rainey, Paul Warrener, Mareia M. Camara, Jessica Bonnell, Ryan Fleming, Binyam Bezabeh, Nazzareno Dimasi, Bret R. Sellman, Jamese Hilliard, Caitlin M. Guenther, Vivekananda Datta, Wei Zhao, Changshou Gao, Xiang-Qing Yu, JoAnn A. Suzich, C. Kendall Stover*
RESEARCH ARTICLE
RESULTS Anti-pseudomonal MOAs are functional in bispecific mAb formats In efforts to identify serotype-independent protective mAbs against P. aeruginosa, we had previously identified unique progenitor mAbs targeting the Psl exopolysaccharide and the PcrV component of the T3S transport system (6, 11). Given the different roles of Psl and T3S in P. aeruginosa infection (12–15), and the protective activities afforded by the individual anti-Psl and anti-PcrV parent mAbs in multiple P. aeruginosa infection models (6, 11), we reasoned that combining both specificities and activities could significantly enhance protection and broaden P. aeruginosa strain coverage. A broad geographical survey of 269 recent P. aeruginosa clinical isolates showed that the vast majority of strains are capable of expressing Psl (89.8 to 91.2%) and PcrV (87.7 to 90.2%), whereas 97.3 to 100% of isolates expressed either or both targets (table S1). The relative prevalence of cytotoxic exoU and invasive exoS strain types was also consistent with previous reports (table S1) (23–25). Because the length of the surface-expressed T3S system needle is thought to extend 80 to 120 nm from the surface of the bacterium (26–28), it appeared plausible that a combination of anti-Psl and anti-PcrV binding units separated by suitable interparatopic distances on a single molecule, coupled with adequate flexibility and geometry, could allow simultaneous binding to both Psl and PcrV surface targets. We therefore constructed bispecific antibodies possessing antiPcrV and anti-Psl specificities with varying intramolecular distances between binding units using the anti-PcrV mAb, V2L2-MD [human immunoglobulin G1 (IgG1)] (11), as the bispecific antibody scaffold (Fig. 1A). Two previously described bispecific antibody formats (18, 29) were initially selected for this study, in which the anti-Psl scFv is genetically linked to the heavy chain N terminus (BiS2aPa) or the heavy chain C terminus (BiS3aPa), resulting in proximal and distal interparatopic distances, respectively (Fig. 1, B and C). In addition, we devised and constructed a unique bispecific configuration with an intermediate interparatopic distance between antigen binding sites, designated BiS4aPa, by genetically inserting the anti-Psl scFv in the upper hinge region of the anti-PcrV mAb coding scaffold (Fig. 1D). All bispecific constructs were assessed for their in vitro potency compared to each respective parental mAb. Opsonophagocytic killing activity (Fig. 2A), cell attachment inhibition (Fig. 2B), and inhibition of cytotoxicity (Fig. 2C) were measured for anti-Psl and anti-PcrV mAbs. Whereas all bispecific constructs exhibited strong anti-Psl opsonophagocytic killing activity, a modest reduction in opsonophagocytic killing activity was observed for the BiS2aPa and BiS4aPa bispecific antibodies, while the BiS3aPa construct exhibited the greatest reduction in opsonophagocytic killing activity relative to the parent anti-Psl mAb or mixture of anti-Psl and anti-PcrV mAbs (Fig. 2A). In contrast, BiS3aPa and BiS4aPa exhibited enhancement of anti-Psl–mediated inhibition of P. aeruginosa attachment to cultured epithelial cells in comparison to BiS2aPa, the parent anti-Psl mAb, or mAb mixture (Fig. 2B). The anti-PcrV component of the bispecific constructs was next evaluated for inhibition of P. aeruginosa T3S-mediated acute cytotoxicity, which is mediated by strains that express the ExoU phospholipase (30, 31). The constructs with the greatest interparatopic
spacing, BiS3aPa and BiS4aPa, displayed significantly enhanced anticytotoxic activity at lower antibody concentrations (
A multifunctional bispecific antibody protects against Pseudomonas aeruginosa
Widespread drug resistance due to empiric use of broad-spectrum antibiotics has stimulated development of bacteria-specific strategies for prophylaxis and therapy based on modern monoclonal antibody (mAb) technologies. However, single-mechanism mAb approaches have not provided adequate protective activity in the clinic. We constructed multifunctional bispecific antibodies, each conferring three mechanisms of action against the bacterial pathogen Pseudomonas aeruginosa by targeting the serotype-independent type III secretion system (injectisome) virulence factor PcrV and persistence factor Psl exopolysaccharide. A new bispecific antibody platform, BiS4, exhibited superior synergistic protection against P. aeruginosa–induced murine pneumonia compared to parent mAb combinations or other available bispecific antibody structures. BiS4aPa was protective in several mouse infection models against disparate P. aeruginosa strains and unexpectedly further synergized with multiple antibiotic classes even against drug-resistant clinical isolates. In addition to resulting in a multimechanistic clinical candidate (MEDI3902) for the prevention or treatment of P. aeruginosa infections, these antibody studies suggest that multifunctional antibody approaches may be a promising platform for targeting other antibiotic-resistant bacterial pathogens.
INTRODUCTION Antibody therapy with animal serum targeting bacterial toxins or capsular polysaccharides predates the discovery and development of smallmolecule antibiotics (1, 2). Broad-spectrum antibiotic chemotherapy eventually displaced serum therapy and became the cornerstone of modern medicine given its relative safety in comparison to serum derived from nonhuman sources and because it enabled more convenient empiric therapy. However, increasing drug resistance to virtually all antibiotic classes, particularly within the designated ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pathogens (3), threatens modern medicine as we know it (4, 5). This severe situation and paucity of new antibiotic classes in development coupled with the belief that empiric broad-spectrum antibiotic therapy has led to bacterial cross-resistance are stimulating renewed interest in pathogen-specific strategies including monoclonal antibody (mAb) technology for the most problematic microorganisms. Although mAbs offer considerable potential, the principal challenge for singletarget mAb-based approaches is obtaining adequate protective activity against a broad range of strains and disease states. P. aeruginosa is one of the most recalcitrant ESKAPE pathogens and a leading cause of acute pneumonia in the hospital environment and of chronic lung infections in cystic fibrosis patients. Its intrinsic drug resistance, owing to its comparatively large genome and regulatory capacity, makes P. aeruginosa a challenging target for a single mAb approach. We previously reported the identification of protective mAbs that mediate serotype-independent opsonophagocytic killing of P. aeruginosa and inhibit adherence to cultured epithelial cells (6). The most protective mAb in multiple infection models was elucidated to target a determinant associated with exopolysaccharide Psl, an abundantly expressed MedImmune, LLC, One MedImmune Way, Gaithersburg, MD 20878, USA. *Corresponding author. E-mail: [email protected]
extracellular sugar polymer implicated in immune evasion and biofilm formation (6–8). In addition, we recently identified a new highly active mAb against the clinically validated target PcrV, which strongly inhibits P. aeruginosa type III secretion (T3S) transport of multiple virulence factors (9–11). Given the important role of Psl and T3S expression in the establishment of acute and persistent P. aeruginosa infections (12–15), we reasoned that a combination of the anti-Psl and anti-PcrV mAb mechanisms of action (MOAs) could enhance strain and disease coverage against P. aeruginosa. Although it is possible to co-administer antibody combinations, it is more practicable to develop a single-molecule clinical candidate. Bispecific antibody technology was first described with the derivation of hybrid hybridomas (16); however, this technology often resulted in mispaired byproducts and poor overall product yield. With the increased implementation of molecular biological methods and the development of single-chain variable fragments (scFvs) (17), construction of bispecific antibodies by fusion of scFv domains to free termini of mAb heavy chain or light chain sequences became feasible (18). Further developments in bispecific antibody technology were forged by engineering heterodimerization motifs into constant sequences, resulting in more than 50 bispecific platforms described to date (19). Whereas bispecific antibodies against viral targets have been reported (20), bispecific antibodies targeting bacterial antigens are limited (21, 22). Herein, we describe enhanced anti–P. aeruginosa activity afforded by a multimechanistic bivalent, bispecific antibody configuration targeting Psl and PcrV designated BiS4aPa. The potent serotype-independent activity of BiS4aPa observed against diverse strain types, including multidrug-resistant (MDR) strains, in multiple animal infection models in both prophylactic and therapeutic regimens, and the surprisingly potent in vivo synergy in adjunctive therapy with multiple antibiotic classes, supports this molecule as a promising clinical candidate (designated MEDI3902) for the prophylaxis or adjunctive treatment of P. aeruginosa infections. The successful application of this approach suggests a more broadly
www.ScienceTranslationalMedicine.org
12 November 2014
Vol 6 Issue 262 262ra155
1
Downloaded from stm.sciencemag.org on March 14, 2015
Antonio DiGiandomenico, Ashley E. Keller, Cuihua Gao, Godfrey J. Rainey, Paul Warrener, Mareia M. Camara, Jessica Bonnell, Ryan Fleming, Binyam Bezabeh, Nazzareno Dimasi, Bret R. Sellman, Jamese Hilliard, Caitlin M. Guenther, Vivekananda Datta, Wei Zhao, Changshou Gao, Xiang-Qing Yu, JoAnn A. Suzich, C. Kendall Stover*
RESEARCH ARTICLE
RESULTS Anti-pseudomonal MOAs are functional in bispecific mAb formats In efforts to identify serotype-independent protective mAbs against P. aeruginosa, we had previously identified unique progenitor mAbs targeting the Psl exopolysaccharide and the PcrV component of the T3S transport system (6, 11). Given the different roles of Psl and T3S in P. aeruginosa infection (12–15), and the protective activities afforded by the individual anti-Psl and anti-PcrV parent mAbs in multiple P. aeruginosa infection models (6, 11), we reasoned that combining both specificities and activities could significantly enhance protection and broaden P. aeruginosa strain coverage. A broad geographical survey of 269 recent P. aeruginosa clinical isolates showed that the vast majority of strains are capable of expressing Psl (89.8 to 91.2%) and PcrV (87.7 to 90.2%), whereas 97.3 to 100% of isolates expressed either or both targets (table S1). The relative prevalence of cytotoxic exoU and invasive exoS strain types was also consistent with previous reports (table S1) (23–25). Because the length of the surface-expressed T3S system needle is thought to extend 80 to 120 nm from the surface of the bacterium (26–28), it appeared plausible that a combination of anti-Psl and anti-PcrV binding units separated by suitable interparatopic distances on a single molecule, coupled with adequate flexibility and geometry, could allow simultaneous binding to both Psl and PcrV surface targets. We therefore constructed bispecific antibodies possessing antiPcrV and anti-Psl specificities with varying intramolecular distances between binding units using the anti-PcrV mAb, V2L2-MD [human immunoglobulin G1 (IgG1)] (11), as the bispecific antibody scaffold (Fig. 1A). Two previously described bispecific antibody formats (18, 29) were initially selected for this study, in which the anti-Psl scFv is genetically linked to the heavy chain N terminus (BiS2aPa) or the heavy chain C terminus (BiS3aPa), resulting in proximal and distal interparatopic distances, respectively (Fig. 1, B and C). In addition, we devised and constructed a unique bispecific configuration with an intermediate interparatopic distance between antigen binding sites, designated BiS4aPa, by genetically inserting the anti-Psl scFv in the upper hinge region of the anti-PcrV mAb coding scaffold (Fig. 1D). All bispecific constructs were assessed for their in vitro potency compared to each respective parental mAb. Opsonophagocytic killing activity (Fig. 2A), cell attachment inhibition (Fig. 2B), and inhibition of cytotoxicity (Fig. 2C) were measured for anti-Psl and anti-PcrV mAbs. Whereas all bispecific constructs exhibited strong anti-Psl opsonophagocytic killing activity, a modest reduction in opsonophagocytic killing activity was observed for the BiS2aPa and BiS4aPa bispecific antibodies, while the BiS3aPa construct exhibited the greatest reduction in opsonophagocytic killing activity relative to the parent anti-Psl mAb or mixture of anti-Psl and anti-PcrV mAbs (Fig. 2A). In contrast, BiS3aPa and BiS4aPa exhibited enhancement of anti-Psl–mediated inhibition of P. aeruginosa attachment to cultured epithelial cells in comparison to BiS2aPa, the parent anti-Psl mAb, or mAb mixture (Fig. 2B). The anti-PcrV component of the bispecific constructs was next evaluated for inhibition of P. aeruginosa T3S-mediated acute cytotoxicity, which is mediated by strains that express the ExoU phospholipase (30, 31). The constructs with the greatest interparatopic
spacing, BiS3aPa and BiS4aPa, displayed significantly enhanced anticytotoxic activity at lower antibody concentrations (
