A multifunctional bispecific antibody against Pseudomonas aeruginosa as a potential therapeutic strategy

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative bacteria that is responsible for significant morbidity and mortality in human populations (1). It is a common cause of hospital-acquired and ventilator-associated pneumonia, opportunistic infections in immunosuppressed and burns patients, and most notably a frequent and troublesome bacteria in the airway of people with cystic fibrosis (1,2).

P. aeruginosa possesses several features that render it a formidable pathogen and a particularly challenging target to treat in patients (3). P. aeruginosa have a number of virulence factors and strategies to evade host-defense systems. Compounds such as exotoxin A, pyocyanin, phospholipase C, proteases and hydrogen cyanide are all released that cause host damage and subvert the host immune response (2). The bacteria are capable of entering different states including the formation of mucoid biofilm-forming colonies where they produce large quantities of alginate and are protected from both host-defense mechanisms and antibiotics (4). P. aeruginosa are also capable of quorum-sensing via a homoserine-L-lactone system (5).

In the lungs of people with cystic fibrosis it is known that genetically and phenotypically diverse populations of P. aeruginosa exist in chronic infection (6). In this situation a number of mutations leading to antimicrobial resistance appear, such as efflux pumps and hypermutability. The cystic fibrosis airway is particularly vulnerable to chronic infection with P. aeruginosa due to reduced mucociliary clearance, impaired innate immunity and abundance of extracellular DNA from necrotic neutrophils in mucus, which provides a support for the biofilm matrix and a hypoxic niche (2). P. aeruginosa infection is associated with significantly worse clinical outcomes in people with cystic fibrosis (7). Considerable efforts are therefore taken in clinical cystic fibrosis management to firstly prevent infection by avoiding contact with other patients, secondly in attempting to eradicate P. aeruginosa at the first isolation with aggressive antimicrobial regimens and thirdly in reducing associated morbidity in those that are chronically infected or colonised with the organism (8-10).

The use of antibiotics targeted against P. aeruginosa is a mainstay of cystic fibrosis treatment in the form of oral, intravenous or nebulised therapy. However, given that it is a life-long condition, problems with multiple drug resistance are often significant and eradication of P. aeruginosa infection becomes effectively impossible once it is chronically established in the majority of individual patients (11,12). Ultimately issues with pan-resistant P. aeruginosa are especially relevant in consideration of suitability of patients with advanced disease for lung transplantation where it is essential that an antimicrobial cocktail is available that will kill the bacteria in the immediate post-transplant phase when high levels of immunosuppression are required (13). Furthermore allergies to antibiotics are not uncommon in people with cystic fibrosis and may limit which antimicrobials can be prescribed (14).

These issues coupled with the relative dearth in the development of new antibiotics in general at present mean that alternative approaches to tackle bacterial infections are urgently required (15). The use of antibodies targeted against bacteria, so-called “passive immunisation”, represent one such option. The general concept is not new and indeed dates back to the pre-antibiotic era when hyper-immune serum was used to treat infections such as diphtheria and tetanus (16). In both of these examples serum was an effective treatment due to its ability to neutralise the toxins that are a key part of disease pathogenesis. Serum treatment was less effective against other bacteria such as pneumococcus or Staphylococcus aureus, reflecting more diverse associated pathophysiology and heterogeneity amongst the organisms themselves, and the subsequent advent of antibiotics made such approaches effectively redundant (16). By way of an example, the use of passive immunisation with palivizumab to protect against respiratory syncytial virus in high-risk infants during winter months is widely accepted (17).

In a paper by DiGiandomenico et al. published in Science Translational Medicine in November 2014, the authors report work performed by MedImmune to develop a multifunctional bispecific antibody against P. aeruginosa as a potential therapeutic and/or preventative strategy (18). Data is presented showing a positive protective effect of the antibody against P. aeruginosa infection in the lungs of mice (18).

The authors have previously developed monoclonal antibodies directed at epitopes of P. aeruginosa Psl, an exosaccharide required for biofilm formation that also reduces host phagocytic function, and the PcrV protein, which plays a key role in enhancing the type III secretion system and subsequent cytotoxicity by bacterial toxin injection into host cells (19). Other investigators have performed an early phase clinical study of an antibody, KB001, targeted against the PcrV protein and type III secretion system in P. aeruginosa in people with cystic fibrosis. The study was primarily designed around safety and pharmacodynamics but results suggested a trend towards reduced airway inflammation at 28 days but generated no statistically significant differences in P. aeruginosa density or clinically relevant outcomes (20).

In the 2014 paper DiGiandomenico et al. hypothesised that combining mechanisms of action against both PsI and PcrV in one monoclonal antibody would be more practical than co-administration of two antibodies and potentially more efficacious. The authors therefore constructed bispecific antibodies targeting both Psl and PcrV with varying intermolecular distances between antigen binding sites. In vitro assessment of opsonophagocytosis and inhibition of cytotoxicity and attachment of P. aeruginosa to epithelial cells, showed one particular construct, BiS4αPa, to provide better protection than other constructs with differing interparatopic distances (BiS2αPa and BiS3αPa), individual mAbs for Psl and PcrV and the combination of these mAbs. This was attributed to the optimal interparatopic distance provided by BiS4αPa to allow simultaneous binding at both anti-Psl and anti-PcrV sites (18).

In vivo assessments of prophylactic protection conferred by BiS4αPa against P. aeruginosa strains were made using a murine lethal acute pneumonia model. Significantly improved protection for acute pneumonia was found with lower concentrations of BiS4αPa administered 24 h prior to infection with the highly pathogenic 6206 and the multi-drug resistant 6077 P. aeruginosa strains in comparison to either monoclonal antibodies to Psl or PcrV alone. Therapeutic effects were also evident when BiS4αPa was administered an hour post infection. Using this model, administration of BiS4αPa resulted in reduced bacterial dissemination and histological evidence of lung injury following infection with each of these strains (18).

Opsonophagocytic activity and anti-cytotoxic effects against clinical isolates of P. aeruginosa following protection with BiS4αPa in this model were also significantly better than those seen with an immunoglobulin G control. However, information regarding how this effect compared with individual or the combination of mAbs against Psl and PcrV is not included. Furthermore prophylactic and therapeutic administration of BiS4αPa against P. aeruginosa strains with multi-drug resistance (with the exception of resistance to colistin) in this acute pneumonia model prevented lethality. BiS4αPa was also found to show dose dependent protective and therapeutic effects in comparison to an immunoglobulin G control in multiple other models including immunocompromised pneumonia, thermal injury and bacteraemia (18).

To ascertain the mechanism of superior anti-cytotoxic BiS4αPa action, the bispecific antibody construct was modified further by firstly replacing the anti-Psl binding unit with negative control sequence, secondly replacing the anti-PcrV binding unit with a negative control sequence, and thirdly the inclusion of a point mutation to reduce opsonophagocytic killing function without affecting PcrV and Psl binding. In vitro assessment of these modified constructs together with investigation in the acute pneumonia model suggested that potentiated effects of BiS4αPa were due to the anti-cytotoxic activity enhanced by its action against Psl, in addition to its opsonophagocytic killing and inhibition of cell attachment (18).

To determine the benefits of BiS4αPa use in conjunction with existing antibiotic therapy sub-therapeutic doses of ciprofloxacin or meropenem were administered with sub-prophylactic doses of BiS4αPa 24 h pre-infection in the murine acute pneumonia model. All mice were still alive 120 h post-infection following combination therapy, whereas those who received either BiS4αPa or either antibiotic in isolation died from infection. Combination therapy of BiS4αPa with tobramycin against the tobramycin-resistant P. aeruginosa strain 6077 showed improved survival and a significantly lower bacterial burden in the murine pneumonia model. These findings suggest that in this model, there are advantages of complementing antibiotic therapy with this bispecific construct, particularly in the context of drug resistant strains of P. aeruginosa (18).

This work has generated a multi-mechanistic clinical candidate known as MEDI3902 for potential use in the treatment or prevention of P. aeruginosa infections. The authors also comment that multifunctional antibodies may be a promising platform for targeting other antibiotic-resistant pathogens (18).

Such approaches are complex however and are not without associated problems and possible limitations. The first and most obvious issue is that the antibody remains at an early-stage of development and assessment of its efficacy. The work described in the paper was performed in a murine acute model of pneumonia that arguably does not replicate the situation in humans during P. aeruginosa pulmonary infection. Careful and cautious evaluation of the antibody in man is required. There are several examples in the literature of antibacterial antibodies that have shown promising efficacy in pre-clinical animal models only to be proven ineffective in human studies (16,21-23). In particular the airway in people with cystic fibrosis, where P. aeruginosa is such a clinical problem, is notably more complex, with a combination of defective host defense, challenging physiology and adaptation of P. aeruginosa to facilitate chronic infection (2).

Other important factors include safety concerns about the administration of biological therapies and risk of potentially significant allergic reactions, administration involving injections and high financial cost. With such specific treatment clearly accurate and rapid microbiological diagnosis is an essential requirement that is often challenging in current clinical practice. In addition most antibiotics provide some spectrum of coverage of multiple bacteria that is not likely to be provided by a targeted antibody.

Despite these concerns the development of multifunctional antibacterial antibodies represents an exciting new therapeutic approach that may potentially address an area of great clinical need. In future years as knowledge of bacterial pathogenesis advances along with refinements in monoclonal antibody design and manufacturing plus development of rapid molecular diagnostics in clinical microbiology the relevance and importance of antibody-based approaches is only likely to increase (16).

Acknowledgements

M Brodlie is supported by a Medical Research Council Clinician Scientist Fellowship.

Footnote

Provenance: This is a Guest Editorial commissioned by the Section Editor Hongcheng Zhu, MD, PhD (Department of Radiation Oncology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China).

Conflicts of Interest: The authors have no conflicts of interest to declare.

References

1. Sousa AM, Pereira MO. Pseudomonas aeruginosa Diversification during Infection Development in Cystic Fibrosis Lungs-A Review. Pathogens 2014;3:680-703. [PubMed]
2. Williams HD, Davies JC. Basic science for the chest physician: Pseudomonas aeruginosa and the cystic fibrosis airway. Thorax 2012;67:465-7. [PubMed]
3. Folkesson A, Jelsbak L, Yang L, et al. Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective. Nat Rev Microbiol 2012;10:841-51. [PubMed]
4. Rasamiravaka T, Labtani Q, Duez P, et al. The formation of biofilms by Pseudomonas aeruginosa: a review of the natural and synthetic compounds interfering with control mechanisms. Biomed Res Int 2015;2015:759348.
5. Jimenez PN, Koch G, Thompson JA, et al. The multiple signaling systems regulating virulence in Pseudomonas aeruginosa. Microbiol Mol Biol Rev 2012;76:46-65. [PubMed]
6. Rodríguez-Rojas A, Oliver A, Blázquez J. Intrinsic and environmental mutagenesis drive diversification and persistence of Pseudomonas aeruginosa in chronic lung infections. J Infect Dis 2012;205:121-7. [PubMed]
7. Henry RL, Mellis CM, Petrovic L. Mucoid Pseudomonas aeruginosa is a marker of poor survival in cystic fibrosis. Pediatr Pulmonol 1992;12:158-61. [PubMed]
8. Davidson AG, Chilvers MA, Lillquist YP. Effects of a Pseudomonas aeruginosa eradication policy in a cystic fibrosis clinic. Curr Opin Pulm Med 2012;18:615-21. [PubMed]
9. Ratjen F, Munck A, Kho P, et al. Treatment of early Pseudomonas aeruginosa infection in patients with cystic fibrosis: the ELITE trial. Thorax 2010;65:286-91. [PubMed]
10. Langton Hewer SC, Smyth AR. Antibiotic strategies for eradicating Pseudomonas aeruginosa in people with cystic fibrosis. Cochrane Database Syst Rev 2014;11:CD004197. [PubMed]
11. Ciofu O, Tolker-Nielsen T, Jensen PØ, et al. Antimicrobial resistance, respiratory tract infections and role of biofilms in lung infections in cystic fibrosis patients. Adv Drug Deliv Rev 2015;85:7-23. [PubMed]
12. Potron A, Poirel L, Nordmann P. Emerging broad-spectrum resistance in Pseudomonas aeruginosa and Acinetobacter baumannii: Mechanisms and epidemiology. Int J Antimicrob Agents 2015;45:568-85. [PubMed]
13. Haja Mydin H, Corris PA, Nicholson A, et al. Targeted Antibiotic Prophylaxis for Lung Transplantation in Cystic Fibrosis Patients Colonised with Pseudomonas aeruginosa Using Multiple Combination Bactericidal Testing. J Transplant 2012;2012:135738.
14. Parmar JS, Nasser S. Antibiotic allergy in cystic fibrosis. Thorax 2005;60:517-20. [PubMed]
15. Sherrard LJ, Tunney MM, Elborn JS. Antimicrobial resistance in the respiratory microbiota of people with cystic fibrosis. Lancet 2014;384:703-13. [PubMed]
16. Oleksiewicz MB, Nagy G, Nagy E. Anti-bacterial monoclonal antibodies: back to the future? Arch Biochem Biophys 2012;526:124-31. [PubMed]
17. Harkensee C, Brodlie M, Embleton ND, et al. Passive immunisation of preterm infants with palivizumab against RSV infection. J Infect 2006;52:2-8. [PubMed]
18. DiGiandomenico A, Keller AE, Gao C, et al. A multifunctional bispecific antibody protects against Pseudomonas aeruginosa. Sci Transl Med 2014;6:262ra155.
19. DiGiandomenico A, Warrener P, Hamilton M, et al. Identification of broadly protective human antibodies to Pseudomonas aeruginosa exopolysaccharide Psl by phenotypic screening. J Exp Med 2012;209:1273-87. [PubMed]
20. Milla CE, Chmiel JF, Accurso FJ, et al. Anti-PcrV antibody in cystic fibrosis: a novel approach targeting Pseudomonas aeruginosa airway infection. Pediatr Pulmonol 2014;49:650-8. [PubMed]
21. Domanski PJ, Patel PR, Bayer AS, et al. Characterization of a humanized monoclonal antibody recognizing clumping factor A expressed by Staphylococcus aureus. Infect Immun 2005;73:5229-32. [PubMed]
22. Teng NN, Kaplan HS, Hebert JM, et al. Protection against gram-negative bacteremia and endotoxemia with human monoclonal IgM antibodies. Proc Natl Acad Sci U S A 1985;82:1790-4. [PubMed]
23. Bebbington C, Yarranton G. Antibodies for the treatment of bacterial infections: current experience and future prospects. Curr Opin Biotechnol 2008;19:613-9. [PubMed]