antimicrobial therapeutics a multifunctional bispecific … · 2014. 11. 10. · geting the psl...

R E S EARCH ART I C L E

ANT IM ICROB IAL THERAPEUT I CS

A multifunctional bispecific antibody protects againstPseudomonas aeruginosaAntonio 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*

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Widespread drug resistance due to empiric use of broad-spectrum antibiotics has stimulated development ofbacteria-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. Weconstructedmultifunctional bispecific antibodies, each conferring threemechanisms of action against the bacterialpathogen 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, exhib-ited superior synergistic protection against P. aeruginosa–induced murine pneumonia compared to parent mAbcombinations or other available bispecific antibody structures. BiS4aPa was protective in several mouse infectionmodels against disparate P. aeruginosa strains and unexpectedly further synergized with multiple antibiotic classesevenagainst drug-resistant clinical isolates. In addition to resulting in amultimechanistic clinical candidate (MEDI3902)for thepreventionor treatment ofP. aeruginosa infections, these antibody studies suggest thatmultifunctional antibodyapproaches may be a promising platform for targeting other antibiotic-resistant bacterial pathogens.

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INTRODUCTION

Antibody therapy with animal serum targeting bacterial toxins or cap-sular polysaccharides predates the discovery and development of small-molecule antibiotics (1, 2). Broad-spectrum antibiotic chemotherapyeventually displaced serum therapy and became the cornerstone ofmodern medicine given its relative safety in comparison to serumderived from nonhuman sources and because it enabled more conve-nient empiric therapy. However, increasing drug resistance to virtuallyall antibiotic classes, particularly within the designated ESKAPE(Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacterspp.) pathogens (3), threatens modern medicine as we know it (4, 5).This severe situation and paucity of new antibiotic classes in develop-ment coupled with the belief that empiric broad-spectrum antibiotictherapy has led to bacterial cross-resistance are stimulating renewedinterest in pathogen-specific strategies including monoclonal antibody(mAb) technology for the most problematic microorganisms. AlthoughmAbs offer considerable potential, the principal challenge for single-target mAb-based approaches is obtaining adequate protective activityagainst a broad range of strains and disease states. P. aeruginosa is oneof the most recalcitrant ESKAPE pathogens and a leading cause ofacute pneumonia in the hospital environment and of chronic lung in-fections in cystic fibrosis patients. Its intrinsic drug resistance, owingto its comparatively large genome and regulatory capacity, makesP. aeruginosa a challenging target for a single mAb approach. We pre-viously reported the identification of protective mAbs that mediateserotype-independent opsonophagocytic killing of P. aeruginosa andinhibit adherence to cultured epithelial cells (6). The most protectivemAb in multiple infection models was elucidated to target a determi-nant associated with exopolysaccharide Psl, an abundantly expressed

MedImmune, LLC, One MedImmune Way, Gaithersburg, MD 20878, USA.*Corresponding author. E-mail: [email protected]

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extracellular sugar polymer implicated in immune evasion and biofilmformation (6–8). In addition, we recently identified a new highly activemAb against the clinically validated target PcrV, which stronglyinhibits P. aeruginosa type III secretion (T3S) transport of multiplevirulence factors (9–11). Given the important role of Psl and T3S ex-pression in the establishment of acute and persistent P. aeruginosa in-fections (12–15), we reasoned that a combination of the anti-Psl andanti-PcrV mAb mechanisms of action (MOAs) could enhance strainand disease coverage against P. aeruginosa. Although it is possible toco-administer antibody combinations, it is more practicable to developa single-molecule clinical candidate. Bispecific antibody technologywas first described with the derivation of hybrid hybridomas (16); how-ever, this technology often resulted in mispaired byproducts and pooroverall product yield. With the increased implementation of molecularbiological methods and the development of single-chain variable frag-ments (scFvs) (17), construction of bispecific antibodies by fusion ofscFv domains to free termini of mAb heavy chain or light chain se-quences became feasible (18). Further developments in bispecific an-tibody technology were forged by engineering heterodimerizationmotifs into constant sequences, resulting in more than 50 bispecificplatforms described to date (19). Whereas bispecific antibodies againstviral targets have been reported (20), bispecific antibodies targetingbacterial antigens are limited (21, 22). Herein, we describe enhancedanti–P. aeruginosa activity afforded by a multimechanistic bivalent,bispecific antibody configuration targeting Psl and PcrV designatedBiS4aPa. The potent serotype-independent activity of BiS4aPa ob-served against diverse strain types, including multidrug-resistant (MDR)strains, in multiple animal infection models in both prophylactic andtherapeutic regimens, and the surprisingly potent in vivo synergy inadjunctive therapy with multiple antibiotic classes, supports this mol-ecule as a promising clinical candidate (designated MEDI3902) for theprophylaxis or adjunctive treatment of P. aeruginosa infections.The successful application of this approach suggests a more broadly

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applicable strategy to combat other serious antibiotic-resistant bac-terial pathogens with multifunctional antibodies.

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RESULTS

Anti-pseudomonal MOAs are functional in bispecificmAb formatsIn efforts to identify serotype-independent protective mAbs againstP. aeruginosa, we had previously identified unique progenitor mAbs tar-geting the Psl exopolysaccharide and the PcrV component of the T3Stransport system (6, 11). Given the different roles of Psl and T3S inP. aeruginosa infection (12–15), and the protective activities affordedby the individual anti-Psl and anti-PcrV parent mAbs in multipleP. aeruginosa infection models (6, 11), we reasoned that combiningboth specificities and activities could significantly enhance protectionand broaden P. aeruginosa strain coverage. A broad geographical sur-vey of 269 recent P. aeruginosa clinical isolates showed that the vastmajority of strains are capable of expressing Psl (89.8 to 91.2%) andPcrV (87.7 to 90.2%), whereas 97.3 to 100% of isolates expressed eitheror both targets (table S1). The relative prevalence of cytotoxic exoU andinvasive exoS strain types was also consistent with previous reports(table S1) (23–25). Because the length of the surface-expressed T3S sys-tem needle is thought to extend 80 to 120 nm from the surface of thebacterium (26–28), it appeared plausible that a combination of anti-Psland anti-PcrV binding units separated by suitable interparatopic dis-tances on a single molecule, coupled with adequate flexibility and ge-ometry, could allow simultaneous binding to both Psl and PcrV surfacetargets. We therefore constructed bispecific antibodies possessing anti-PcrV and anti-Psl specificities with varying intramolecular distancesbetween binding units using the anti-PcrV mAb, V2L2-MD [humanimmunoglobulin 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 genet-ically linked to the heavy chain N terminus (BiS2aPa) or the heavy chainC terminus (BiS3aPa), resulting in proximal and distal interparatopicdistances, respectively (Fig. 1, B and C). In addition, we devised andconstructed a unique bispecific configuration with an intermediateinterparatopic distance between antigen binding sites, designatedBiS4aPa, by genetically inserting the anti-Psl scFv in the upper hingeregion of the anti-PcrV mAb coding scaffold (Fig. 1D).

All bispecific constructs were assessed for their in vitro potency com-pared to each respective parental mAb. Opsonophagocytic killing ac-tivity (Fig. 2A), cell attachment inhibition (Fig. 2B), and inhibition ofcytotoxicity (Fig. 2C) were measured for anti-Psl and anti-PcrV mAbs.Whereas all bispecific constructs exhibited strong anti-Psl opsonopha-gocytic killing activity, a modest reduction in opsonophagocytic killingactivity was observed for the BiS2aPa and BiS4aPa bispecific anti-bodies, while the BiS3aPa construct exhibited the greatest reduction inopsonophagocytic killing activity relative to the parent anti-Psl mAb ormixture of anti-Psl and anti-PcrV mAbs (Fig. 2A). In contrast, BiS3aPaand BiS4aPa exhibited enhancement of anti-Psl–mediated inhibition ofP. aeruginosa attachment to cultured epithelial cells in comparison toBiS2aPa, the parent anti-Psl mAb, or mAb mixture (Fig. 2B).

The anti-PcrV component of the bispecific constructs was nextevaluated for inhibition of P. aeruginosa T3S-mediated acute cyto-toxicity, which is mediated by strains that express the ExoU phos-pholipase (30, 31). The constructs with the greatest interparatopic

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spacing, BiS3aPa and BiS4aPa, displayed significantly enhanced anti-cytotoxic activity at lower antibody concentrations (<1 nM) com-pared to the parent anti-PcrV mAb (P = 0.0001 for both BiS3aPa andBiS4aPa), whereas no difference in activity was observed between theanti-PcrVmAb, themAbmixture, and BiS2aPa in the in vitro cytotoxicinhibition assay (Fig. 2C). This finding suggests that the BiS2 platformcontaining the shortest interparatopic distance between anti-Psl andanti-PcrV binding units may not be adequately spaced to confer thebenefit of anti-Psl binding avidity or simultaneous engagement withboth targets. Together, these in vitro data for the three distinct MOAssuggested a collective superiority for BiS4aPa in comparison to theother bispecific constructs, parent mAbs, or mAb combination.

BiS4aPa exhibits enhanced protective activity in vivoWe next evaluated each bispecific antibody in comparison to individualparental anti-Psl and anti-PcrVmAbs and the mAbmixture for pro-tection against lethality in a murine lung infection model caused bycytotoxic (ExoU+) strain 6206, which is representative of themost high-ly pathogenic P. aeruginosa challenge strains we have tested (6, 11)(Table 1). Prophylactic administration of the single-parent anti-PslmAb provided no protection against strain 6206, whereas anti-PcrV,BiS2aPa, and BiS3aPa mAbs protected 40 to 80% of the challengedanimals at 5 mg of antibody per kilogram of body weight (mpk) and

Fig. 1. P. aeruginosa bispecific antibodies compared to IgG1. (A) Paren-tal IgG1. (B to D) Bispecific constructs using anti-PcrV mAb V2L2-MD (11) as a
scaffold. scFvs of anti-Psl mAb Psl0096 (derived from Cam-003) (6) werelinked via a 10–amino acid linker (GGGGSx2) to the V2L2-MD heavy chainN terminus (B), heavy chain C terminus (C), or between C220 and D221 ofthe upper hinge region flanked by 10–amino acid linkers (GGGGSGGGGS)(D). Fab light chain is light blue, and heavy chain is dark blue; CDR1-3H,red; CDR1-3L, salmon; scFv variable light (VL) chain is light orange and variableheavy (VH) chain is dark orange; hinge and Fc are green. Linkers between VHand VL in scFv and between scFv and IgG sequences are gray. Carbohydratesat the N297 Fc glycosylation site are rendered in the stick conformation. Illus-trations depict the complementarity determining regions (CDRs) of the scFvspointing outward toward antigen binding sites. Subunit images were ren-dered in PyMOL (Delano Scientific) and assembled in PowerPoint.



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provided minimal protection (10 to 20%) at the 1 mpk dose (Table 1).In contrast, we consistently observed enhanced activity in mice dosedprophylactically with both themAbmixture (1mpk for eachmAb) andBiS4aPa (1 mpk total) (78 and 88%, respectively). Whereas isobolo-gram analysis was not possible given the lack of activity for the anti-Psl mAb at all tested doses for strain 6206, the enhanced activity forthe mAb combination and BiS4aPa was synergistic given the markedincrease in potency over the anti-PcrV parent mAb in the absence ofobservable anti-Psl activity. Further titration of BiS4aPa (0.5 mpk)

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and the mAb mixture (0.5 mpk for each mAb) indicated enhanced ef-ficacy for BiS4aPa (P < 0.0001), even though the larger–molecularweight BiS4aPa construct (200.3 kD versus 150 kD for an IgG1, respec-tively) and its anti-Psl and anti-PcrV binding units are at a molar andweight disadvantage in these studies. The enhanced BiS4aPa activitycompared with the mAb mixture was also confirmed in studies withanother P. aeruginosa cytotoxic (ExoU+) clinical isolate, MDR strain6077 (Table 1), which is more representative of a broader range ofclinical isolates used as challenge strains in the murine pneumonia

Fig. 2. Bispecific antibody anti-Psl and anti-PcrV activity againstP. aeruginosa. (A) Opsonophagocytic killing activity of antibody constructsusing the P. aeruginosa luminescent reporter strain PAO1.lux. Samples wererun in duplicate and are representative of three independent experiments.(B) Anti–cell attachment activity of antibody constructs using PAO1.lux re-porter strain. Error bars indicate the SEM of four samples and are represent-ative of four independent experiments. (C) P. aeruginosa epithelial cellcytotoxicity assay using cytotoxic P. aeruginosa strain 6077 (ExoU+). Sampleswere run in duplicate and are representative of four independent experi-ments. Statistical comparisons were conducted by calculating the area underthe curve (AUC) comparing bispecific constructs to anti-Psl mAb (A and B)and anti-PcrV mAb (C).

Table 1. Protection against lethal P. aeruginosa pneumonia. Summaryof lethal pneumonia survival studies completed for the indicated mAbsagainst P. aeruginosa strains 6206 and 6077. Mice were treated prophylacti-cally with antibody 24 hours before infection. No survival was observedin mice treated with the isotype control IgG against either strain. Differencesin survival for BiS4aPa versus the anti-Psl/anti-PcrV mAb mixture at 0.5 mpk

for 6206 and 0.02 mpk for 6077 were evaluated by the log-rank test (*P <0.0001). 6206: Results were compiled from 15 independent experiments; n isindicated in parentheses. 6077: Results were compiled from 11 independentexperiments; n is indicated in parentheses. For the anti-Psl and anti-PcrVmAb mixture, the indicated mpk is for each mAb. Parentheses indicate totalnumber of animals analyzed at each antibody dosage. nd, not determined.

Antibody percent protection

Strain 6206
Dose (mpk) Anti-Psl Anti-PcrV BiS2aPa
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5
0 (10) 80 (10) 70 (10) 40 (20) 100 (60) 100 (10)
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0 (10) 20 (10) 10 (10) 10 (20) 88 (60) 78 (40)
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nd 10 (10) 0 (10) 0 (20) *60 (60) 15 (40)
Strain 6077
Dose (mpk) Anti-Psl Anti-PcrV BiS4aPa Anti-Psl + anti-PcrV
0.5
64 (24) 100 (16) 100 (10) 100 (10)
0.2
60 (24) 88 (24) 96 (26) 100 (18)
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6.3 (16) 83 (24) 92 (26) 69 (18)
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model. Activity against strain 6077 was also confirmed to be synergisticin comparison to the individual parental mAbs by isobologram analysis[isobole (I) = 0.43] (fig. S1A). In addition to prophylactic activity, ther-apeutic administration (1 hour after infection) of BiS4aPa also resultedin potent protective activity against challenge strains 6206 and 6077 inthe murine lethal pneumonia model (fig. S2, A and B).

Given the consistently enhanced in vivo activity of BiS4aPa, we fo-cused our remaining analyses on this molecule as a potential candidatefor clinical development. We first evaluated the impact of BiS4aPa onbacterial burden in lung and distal organ tissues using the lethalpneumonia mouse model. All doses of BiS4aPa significantly reducedP. aeruginosa lung colony-forming units (CFUs) in comparison tomice dosed prophylactically with control IgG (P < 0.05) (fig. S3, Aand B). BiS4aPa also significantly reduced bacterial dissemination tothe spleen and kidneys in comparison to control IgG (P < 0.05) (fig.S3, A and B). Furthermore, histopathological evaluation of lungs frominfected mice dosed prophylactically with BiS4aPa alone 24 hoursbefore infection or treated 4 hours after infection revealed marked re-ductions in pathological markers of pneumonia and cellular damagein comparison to mice treated with control IgG (Fig. 3, A and B).

The protective activity of BiS4aPa was also evaluated against a col-lection of recent clinical isolates of P. aeruginosa. BiS4aPa exhibitedhigh-level in vitro anti-Psl–mediated opsonophagocytic killing activity(fig. S4, A to D) and anti-PcrV–mediated anti-cytotoxic activity (fig.S5, A to I) against all strains tested. Furthermore, prophylactic or ther-

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apeutic administration of BiS4aPa prevented lethality from acute pneu-monia (Fig. 4, A to D) against isolates known to be resistant to allanti-pseudomonal standard-of-care antibiotics with the exception of colis-tin [minimal inhibitory concentrations (MICs) presented in table S2].

Multiple BiS4aPa MOAs contribute to synergistic protectionGiven the superior in vivo activity of BiS4aPa in comparison to theparent mAb mixture, we next investigated the potential mechanism bywhich BiS4aPa mediates enhanced anti-cytotoxic activity in compar-ison to the anti-PcrV mAb or parent mAb mixture. We hypothesizedthat the high-avidity lower-affinity binding of the BiS4aPa anti-Pslmodule (6) to the abundant surface Psl exopolysaccharide effectivelyincreases the local antibody concentration and residence time aroundthe bacterium without impeding the higher-affinity engagement of theanti-PcrV component (11) of the bispecific antibody to its target,thereby allowing cooperative target engagement while increasing anti-PcrV activity around the bacterium. To test this hypothesis, we con-structed several BiS4aPa antibody variants that lacked one or morefunctional activities contained within parental BiS4aPa. First, BiS4aPcrVwas constructed with a negative control scFv in place of anti-Psl bindingunit. The in vitro anti-cytotoxic activity of BiS4aPcrV was significantlyreduced in comparison to BiS4aPa (P < 0.001) but was not differentwhen compared to the parental anti-PcrV mAb (P = 0.434) (Fig. 5A).A significant difference was also observed in a direct comparison ofBiS4aPa versus anti-PcrV (P < 0.001). The increased anti-cytotoxicactivity of BiS4aPa in comparison to mAbs lacking anti-Psl specificitybecame apparent at mAb concentrations lower than 1 nM in the in vitrodose-response curve. Consistent with these results, no difference inactivity was observed between the anti-cytotoxic activities of BiS4aPa,BiS4aPcrV, and the parental anti-PcrV mAb when evaluated against aPsl-deficient strain of P. aeruginosa (6206DpslA) (Fig. 5B). These in vitrodata suggested that the enhanced anti-cytotoxic activity observed withBiS4aPa is associated with Psl engagement by BiS4aPa. These data werefurther confirmed in the acute pneumonia mouse model induced bystrain 6206DpslA, in which no difference in activity was observed inmice prophylactically treated with either BiS4aPa or the parent anti-PcrV mAb at molar equivalent doses (Fig. 5C).

In addition to enhancing the anti-cytotoxic activity mediated byanti-PcrV, the anti-Psl component of BiS4aPamediates opsonophagocytickilling in the presence of effector cells (Fig. 2A) and inhibits P. aeruginosabinding to epithelial cells (Fig. 2B). We next evaluated the relative con-tribution of anti-Psl opsonophagocytic killing and inhibition of cellattachment to the overall activity of BiS4aPa by constructing variantsthat lacked anti-cytotoxic activity as well as a clone deficient for effec-tor function necessary for opsonophagocytic killing activity. BiS4aPsl,lacking anti-cytotoxic activity, was constructed by replacing the anti-PcrV binding unit sequence of BiS4aPa with an irrelevant negative con-trol antibody sequence. In addition, we constructed BiS4aPa Fc domainvariant, with an N297Q point mutation previously shown to diminishFcgR engagement and C1q binding (32), thereby diminishing effectorcell–mediated opsonophagocytic killing activity without affecting Psland PcrV target binding. No difference in the opsonophagocytic kill-ing (Fig. 5D) and adherence inhibitory activity (Fig. 5E) was observedbetween BiS4aPsl and BiS4aPa. In contrast, BiS4aPa-N297Q exhib-ited significantly impaired anti-Psl opsonophagocytic killing activityin comparison to BiS4aPa (P < 0.001) (Fig. 5D), without affectingthe enhanced in vitro inhibition of Psl-mediated adherence (Fig. 5E)or the anti-PcrV–mediated anti-cytotoxic activity (Fig. 5A). The activity

Fig. 3. BiS4aPa-mediated reduction in pneumonia lung pathology inmice. (A and B) Mice received IgG control or BiS4aPa prophylactically (n = 3
for each, respectively) (A) or as a single-agent treatment at 4 hours afterinfection with P. aeruginosa strain 6077 (n = 3 for each, respectively) (B).Mice receiving control IgG presented with hemorrhagic interstitial pneu-monia involving most of the examined lung section, along with markedbronchial epithelial damage and severe edema surrounding the bronch-ioles and blood vessels. In addition, control IgG–treated animals alsoshowed the presence of bacterial colonies (inset) in three of three micein the treatment group and in one of three mice in the prophylaxis group.In contrast, mice receiving BiS4aPa alone in prophylaxis or treatment ex-hibited minimal inflammatory infiltrate in the alveolar spaces with no de-tectable bacteria, primarily showing bronchial pneumonia with little or noalveolar hemorrhage and edematous changes surrounding the largerblood vessels. The pathologist was blinded to the treatment groups.



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of BiS4aPa, associated variants, and parent mAbs was then compared inthe pneumonia model induced by wild-type P. aeruginosa strain 6206. Asignificant reduction in activity was observed with all variant constructsin comparison to BiS4aPa (Fig. 5F). No difference in activity was ob-served between BiS4aPcrV and the anti-PcrV mAb. Although BiS4aPa-N297Q exhibited intermediate activity, reduced in comparison to thecomplete BiS4aPa (P = 0.01) construct, its activity was significantlygreater than that of the BiS4aPcrV (P < 0.0001) or parental anti-PcrVmAbs (P < 0.0001) (Fig. 5F). Together, these results indicate that Psltargeting potentiates BiS4aPa anti-cytotoxic activity and that anti-Pslopsonophagocytic killing and possibly anti–cell attachment activity al-so contribute to efficacy in the murine pneumonia model. In addition,these data further confirm that targeting both Psl and PcrV with BiS4aParesults in significantly enhanced efficacy in comparison to the single-parent mAbs with the potential of broadening coverage againstP. aeruginosa strains that might not express one of the two targets.

BiS4aPa is protective in multiple infection modelsGiven that P. aeruginosa infections are particularly problematic in im-munosuppressed patients and because BiS4aPa functions at least in partby effector cell–mediated opsonophagocytic killing, we further evaluatedBiS4aPa activity in a cyclophosphamide-induced immunocompromisedpneumonia model in which total white blood cell and neutrophil countsare markedly reduced (33). BiS4aPa provided concentration-dependent

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protection in both the prophylactic and post-infection treatment modelsin these immunosuppressed mice (fig. S6, A and B).

P. aeruginosa also remains a significant cause of bacteremia andmortality in the critically ill and in serious infections of burn wounds(34, 35). We therefore evaluated BiS4aPa in murine thermal injuryand bacteremia infection models. In the thermal injury model, attach-ment and colonization of injured murine tissues with P. aeruginosaresult in the formation of a biofilm (36) followed by systemic bacterialdissemination as a consequence of immune cell anergy (37). Prophy-laxis or treatment with BiS4aPa provided significant protection at allantibody concentrations evaluated for both cytotoxic (ExoU+) (fig. S6,C and D) and invasive (ExoS+) (fig. S6, E and F) clinical isolates; theExoS+ strain is highly lethal in the thermal injury model as indicatedby the low LD100 (absolute lethal dose) challenge dose. BiS4aPa admin-istered prophylactically or therapeutically also demonstrated potentactivity against bacteremia induced by intravenous challenge withmultiple strains of P. aeruginosa (fig. S6, G to J).

BiS4aPa promotes synergistic protection withantibiotic therapyGiven the steady increase in global MDR infection rates, we testedwhether BiS4aPa antibody–mediated therapy could complement theantibacterial activity of multiple antibiotics used to treat P. aeruginosa.We first selected marginal subtherapeutic antibiotic doses of ciprofloxacin

Fig. 4. BiS4aPa protective activity against MDR P. aeruginosa lethalpneumonia. (A to D) BiS4aPa was screened against MDR isolates

calculated by the log-rank test by comparing each concentration ofBiS4aPa to the control IgG. (A to D) Data are representative of three

ARC3928 (A and B) and ARC3502 (C and D) under both prophylactic (Aand C) (T = 24 hours before infection) and therapeutic regimens [T = 1 hourafter infection (B) and T = 0.15 hour after infection (D)]. Results are repre-sented as Kaplan-Meier survival curves; significant differences were

independent experiments. (A) Control IgG, n = 8; BiS4aPa at 1.0, 0.3, and0.1 mpk, n = 8; BiS4aPa at 0.03 mpk, n = 7. (B) Control IgG, n = 8; BiS4aPa,n = 10 for all groups. (C) Control IgG, n = 8; BiS4aPa, n = 8 for all groups.(D) Control IgG, n = 8; BiS4aPa, n = 8 for all groups.




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(CIP) and meropenem (MEM) (two antibiotics with disparate MOAs)to simulate the insufficient drug exposure for antibiotic-resistant strainsin the lethal pneumonia model. These subtherapeutic antibiotic doseswere then used to treat mice in which subprotective prophylactic dosesof BiS4aPa had been administered 24 hours before infection to evaluatethe potential for synergistic activity. Animals receiving control IgG orsingle-agent treatments all succumbed to infection, whereas mice receiv-ing the subtherapeutic combinations of antibiotic plus BiS4aPa survivedchallenge (Fig. 6, A and B). Isobologram analyses confirmed synergisticactivity when combining BiS4aPa with either CIP (I = 0.21) or MEM(I = 0.45) (fig. S1, B and C). We next evaluated if this activity could be

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recapitulated in a post-infection treatment setting. Subtherapeuticdoses of individual drugs and the BiS4aPa/antibiotic combinationswere adjunctively delivered to mice 4 hours after infection. Enhancedprotection was observed in mice receiving the BiS4aPa/antibiotic com-binations in comparison to little or no protection in animals treatedwith control IgG or individual antibiotics (Fig. 6, C and D). We furtherevaluated the impact of BiS4aPa on P. aeruginosa bacterial burden andlung pathology in mice receiving CIP with BiS4aPa treatment at 4 hoursafter infection. CIP-treated groups demonstrated significant reduc-tions in bacterial burden in comparison to non-CIP–treated groups inall tissues evaluated with or without co-administration of the control

Fig. 5. Contributions of BiS4aPa MOAs to enhanced activity. (A)P. aeruginosa epithelial cell cytotoxicity assay with wild-type P. aeruginosa.

Error bars represent the SEM for three samples and are representative ofthree independent experiments. (E) Anti–cell attachment activity with lu-

Error bars represent the SEM for six samples and are representative of sixindependent experiments. (B) Cytotoxicity assay with Psl-deficient P. aeruginosa.Samples were run in duplicate and are representative of three independentexperiments. (C) Acute pneumonia mouse model. Mice were treated withmolar equivalent doses of BiS4aPa and anti-PcrV mAb (V2L2-MD) 24 hoursbefore infection with 6206DpslA. No statistical difference was observed bythe log-rank test between treatment groups (n = 10). Representative datafrom three independent experiments. (D) Opsonophagocytic killing assay.

minescent P. aeruginosa. Error bars represent the SEM for three samplesand are representative of two independent experiments. (F) Acute pneu-monia model. Mice were treated with molar equivalent doses of the indi-cated antibodies followed by infection with wild-type strain 6206.Results compiled from six independent experiments. Control IgG, n = 48;anti-Psl, n = 16; BiS4aPsl, n = 24; anti-PcrV, n = 16; BiS4aPcrV, n = 16; BiS4aPa-N297Q, n = 16; BiS4aPa, n = 48. (A, B, D, and E) Statistical comparisons wereconducted by calculating the AUC.




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mAb or subtherapeutic dose of BiS4aPa at 4 hours after infection (Fig.6E). The combination of CIP and BiS4aPa provided only marginal ad-ditional CFU reduction over CIP treatment alone in the lung. However,only the combination of BiS4aPa and CIP prevented death in thismodel, illustrating that P. aeruginosa lethality is not solely dependenton bacterial burden and that antibody functional activities can furtherenhance antibiotic therapy by disarming pathogenic mechanisms andreducing pathology while at the same time promoting bacterial clearance(Fig. 6E). This protection was evident in further histopathological evalua-tion of lungs from infected animals. Control IgG– and CIP-treated animalspresented with severe bronchial epithelial damage, edema, and severe

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hemorrhagic interstitial pneumonia, whereas these pathological char-acteristics were absent in mice treated with BiS4aPa and CIP (Fig. 6F).

The synergy observed with subtherapeutic doses of BiS4aPa andmultiple antibiotic classes strongly suggested the potential for enhancingmarginally active antibiotics against drug-resistant strains of P. aeruginosa.We further confirmed this potential by testing BiS4aPa combinedwith a dose regimen of the aminoglycoside tobramycin (TOB) approx-imating human drug exposure versus both a TOB-susceptible andTOB-resistant strain of P. aeruginosa in the murine acute pneumoniamodel. As expected, the human equivalent multidose regimen expo-sure of TOB protected against susceptible P. aeruginosa strain 6206

Fig. 6. Synergistic activity of BiS4aPa and antibiotics against P. aeruginosapneumonia. (A and B) Previously determined subprotective doses of

differences in survival were calculated by the log-rank test for the BiS4aPaand antibiotic combination versus antibiotic treatment alone. (A to D) n = 6

BiS4aPa were administered to mice 24 hours before intranasal infectionwith P. aeruginosa strain 6206. Subtherapeutic doses of CIP (A) or MEM(B) were administered 1 hour after infection. (C and D) Mice were treatedwith subprotective doses of both BiS4aPa and CIP (C) or MEM (D) 4 hoursafter intranasal infection with P. aeruginosa strain 6206. (F) Histologicalevaluation of lungs from mice treated with IgG control (n = 3) or BiS4aPa(n = 3) in combination with subtherapeutic doses of CIP 4 hours after in-fection. (A to D) Results are represented as Kaplan-Meier survival curves;

for all antibody treatment groups. Representative data from two (A and B),five (C), and four (D) independent experiments. (E) Organ burden analysisof animals treated with BiS4aPa and CIP 4 hours after infection. Statisticalcomparisons were performed by one-way analysis of variance (ANOVA)with Bonferroni’s multiple comparison test. Results are presented as a scat-ter dot plot and are marked by the SEM and mean. Control IgG, Control IgG+ CIP, CIP alone and BiS4aPa + CIP, n = 10; BiS4aPa alone, n = 9. Data arerepresentative of three independent experiments.




(MIC: 0.5 mg/ml) (Fig. 7A) but failed to protect against TOB-resistantP. aeruginosa strain 6077, which has an MIC eightfold above the Clin-ical and Laboratory Standards Institute (CLSI)–established MIC breakpoint (1 mg/ml) for human TOB therapy (Fig. 7B). In contrast, even ata low single dose of 0.1 mg/kg, which is subtherapeutic under theconditions of this experiment, BiS4aPa was synergistically protectivein adjunctive therapy with TOB against this resistant strain (Fig. 7C).We next evaluated the impact of BiS4aPa on strain 6077 bacterial bur-den in mice receiving control mAb, TOB, or BiS4aPa individually andBiS4aPa and TOB in combination.Whereas control IgG– and TOB-treatedmice yielded similar bacterial burdens, mice receiving the subtherapeuticdose of BiS4aPa in combination with TOB exhibited significantly lowerbacterial burden in evaluated tissues (P = 0.0006) (Fig. 7D).

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DISCUSSION

In addition to the treatment of primary community or trauma-associatedinfections, much of modern medical practice is only possible with theuse of antibiotics to prevent or treat opportunistic infections associatedwith high-risk surgeries. However, empiric use of broad-spectrum anti-biotics has led to the progressive selection of cross-resistance to mostantibiotic classes. With insufficient potential for new antibiotic classeson the horizon, the antibiotic resistance crisis has brought more focus

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on strategies for preserving the valuable antibiotics we have left or willhave in the future. This dire situation has also stimulated work onpathogen-specific biological strategies using modern antibody-basedtechnologies as one possible approach to preserve and augment anti-biotic options. mAbs offer considerable potential for preventing ormanaging bacterial infections including enhanced specificity and safe-ty, lack of drug-drug interactions, long half-lives, and complementaryMOAs that can enhance antibiotic activities without driving antibioticresistance or disruption of the beneficial microbiota. However, inmany circumstances, mAbs aimed at single targets may offer onlymarginal effectiveness against diverse strain types across different dis-ease manifestations. Among the drug-resistant bacterial ESKAPE path-ogens, P. aeruginosa may present the greatest challenge because of itslarge genome coding capacity and complex regulatory networks,allowing the bacterium to phenotypically adapt to environmental pres-sures and to form persistent biofilm communities. Multiple P. aeruginosaproteins and carbohydrates have been explored individually as antibodytargets in the laboratory, few of which are currently under clinical eval-uation (9, 38).

For challenging drug-resistant bacterial infections, multidrug thera-pies withmultipleMOAs are increasingly required to reduce the poten-tial for increased drug resistance. Here, we reasoned that a combinationof complementary mAbs with different MOAs against the TS3 systemandPsl exopolysaccharide, targets that are expressed bymostP. aeruginosa

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Fig. 7. Synergistic activity of BiS4aPa and TOB against resistantP. aeruginosa. (A to C) Efficacy of TOB against (A) TOBS strain 6206 and

ment. (A to C) Data are representative of three independent experiments.(A) n = 6, (B) n = 5, and (C) n = 8 for all treatment groups. (D) Organ

(B) TOBR strain 6077. (C) Mice were treated with subprotective doses ofboth BiS4aPa (T = 24 hours before infection) and TOB (T = 1 hour) afterintranasal infection with P. aeruginosa strain 6077. (A to C) Results arerepresented as Kaplan-Meier survival curves. Significant differences werecalculated by the log-rank test comparing (A and B) TOB-treated mice tothe diluent control, and (C) TOB treatment alone to BiS4aPa + TOB treat-

burden analysis in 6077-infected animals treated with BiS4aPa + TOB.Results are presented as a scatter dot plot and are marked by the SEMand mean. Differences in CFU were determined by the Mann-Whitney U testfor pairwise comparison of TOB versus BiS4aPa + TOB–treated groups.Control IgG, n = 7; TOB, BiS4aPa, and BiS4aPa + TOB, n = 8. Data are repre-sentative of two independent experiments.




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primary clinical isolates (table S1), could provide enhanced activity againstacute infection and prevent the establishment of persistent infections.Indeed, the anti-cytotoxic activity provided by anti-PcrV to bluntthe pathogenesis and invasive progression of infection coupled withanti-Psl–mediatedopsonophagocytic killing, clearance, and anti-adherenceactivities augmented activity againstP. aeruginosa–induced lethal pneu-monia in mice. In addition, we hypothesized that in a bispecific mAbformat, the high-avidity binding activity against the abundant Psl targetcould enhance activity against the low-abundance PcrV target, therebyresulting in a higher local mAb concentration “cloud” around the bac-terium, and thus effectively increasing bacterial cell surface–associatedanti-PcrV activity, which would not be observed with an anti-PcrV mAbalone. This hypothesis was supported by the enhanced anti-cytotoxicactivity for the two bispecific constructs in vitro, particularly at lowconcentrations where the control PcrV mAb alone was ineffective. Ofthe three bispecific mAb configurations tested, the BiS4 configurationwas the only construct exhibiting enhanced cytotoxic activity withouta substantial deficiency in the anti-Psl–mediated opsonophagocytickilling activity. The novelty of the BiS4aPa bispecific format lies inthe placement of the anti-Psl scFv, which is inserted into the upper hingeregion rather than appended to the N or C termini of the antibodyheavy chain as previously described in other bispecific configurations.This configuration offers an intermediate distance between paratopesand appears to be most optimal for dual target engagement in theapplications examined herein. Although the BiS4 format worked bestfor this target combination, these studies also demonstrate that havinga sufficient range ofmultispecific formatsmay be necessary for successin other antibacterial applications. These studies may also shed lighton what types of antibacterial mAb target combinations might resultin synergistic outcomes.

Because antibodies generally do not directly kill bacteria, it may seemmost reasonable to consider these agents for preventing diseasebefore bacterial burden is high and the infection is established. Itwould also seem that antibody prophylaxis would be most practicalin severely ill patients at highest risk for infections causing high mor-bidity and mortality, and for infections that are extremely expensiveto treat. P. aeruginosa infections in intensive care settings certainlyfall into this class (39). However, the data provided herein for BiS4aPaalso suggest a potential beyond prophylaxis, particularly for the treat-ment of acute drug-resistant P. aeruginosa infection for which fewdesirable antibiotic options remain. Although BiS4aPa showed con-siderable potential in monotherapy with multiple clinical strain typesin multiple animal models, also striking was the unexpected synergyobserved in combination with multiple antibiotic classes, even with strainsthat are highly resistant to the partner antibiotic. These data indicatethat BiS4aPa has the potential to complement the bactericidal or bac-teriostatic activities of antibiotics, perhaps by limiting the tissue dam-age caused by the cytotoxic pathogenic mechanisms of the bacteria whilealso promoting bacterial clearance, thereby minimizing the potential fora concomitant destructive hyper-inflammatory innate host response.These complementary and synergistic protective activities coupled withthe long antibody half-life and unlikely drug-drug interactions betweenantibody and antibiotics offer the attractive option of combinationtherapy with antibiotics. The data provided herein demonstrate consid-erable potential for the prevention and treatment of acute P. aeruginosalung infections. However, we have also demonstrated protective post-infection treatment potential in the thermal injury mouse model, acomparatively less acute infection model in which biofilms are reported

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to form (36). Given that the Psl exopolysaccharide target has beenimplicated in biofilm formation and maintenance, it seems reason-able to consider that antibodies targeting Psl with attendant antibody-dependent effector cell engagement may have the potential to aid incontaining established biofilm communities. However, additional studieswould be required to assess anti-biofilm activities in an in vivo setting ofinfection (40, 41).

If the full potential for antibody-based strategies to augment ourantibacterial options is to be realized, multifunctional approaches mustbe pursued. However, mAb combinations and recombinant polyclonalapproaches present formidable and arduous clinical development chal-lenges including the potential requirement to develop multiple biopro-cesses and to produce, purify, and clinically assess each individual mAb.Notwithstanding the potential advantages for single-molecule multi-functional bispecific mAbs versus antibody mixtures, the potential forundesirable biophysical properties or more immunogenic structuresmay be greater for non-germline, engineered multispecific antibodystructures. Although not addressed in the study here, biophysical for-mulation stability must also be monitored in the decision schema forselection of a multispecific clinical candidate. However, immunogenic-ity liabilities that could affect mAb candidate pharmacokinetics or ac-tivities in humans cannot be adequately assessed in preclinical studiesand may only be finally assessed in human clinical trials. Our derivationof BiS4aPa portends one possible multifunctional bispecific strategy toameliorate some of the challenges associated with mAb combinations.Additionally, these findings support a significant benefit of synergistical-ly augmenting antibiotic bactericidal activities through inhibiting cy-totoxicity, bacterial adherence, and promoting effector cell clearancemediated by advanced biological therapeutics. While providing sup-porting data for BiS4aPa as a human clinical candidate (designated here-after as MEDI3902) to prevent or possibly treat acute P. aeruginosainfections, these results also suggest a promising new bispecific anti-body platform, enabling multimechanistic strategies to augment andpreserve our dwindling antibiotic options for serious drug-resistantbacterial infections.

MATERIALS AND METHODS

Study designWe constructed multimechanistic bispecific antibodies with specifici-ties against P. aeruginosa Psl exopolysaccharide and PcrV, a proteinrequired for type III secretion injectisome activity, to evaluate thepotential of enhanced strain and disease coverage against P. aeruginosa.All antibody constructs were first tested in in vitro functional ac-tivity screens followed by validation and comparison of efficacy in aP. aeruginosa acute pneumonia mouse model. One new multime-chanistic bispecific antibody, BiS4aPa, exhibiting synergistic protec-tion against lethal challenge in the pneumonia model, was selectedas the lead clinical candidate and further evaluated against lethal in-fection in P. aeruginosa mouse models of thermal injury and bactere-mia. In addition, we evaluated BiS4aPa activity when combinedadjunctively with antibiotics. Experts in statistics and experimental de-sign were consulted to validate the design of all in vivo experimentsbefore execution. All in vivo studies were performed in accordancewith federal, state, and institutional guidelines and were approved bythe MedImmune Institutional Animal Care and Use Committee inan Association for Assessment and Accreditation of Laboratory Animal




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Care International–accredited facility. Animals were monitored closelyfor survival up to 120 hours after challenge. Guidelines for humaneendpoints were strictly followed for all in vivo experiments; moribundanimals were immediately euthanized by CO2 asphyxiation and re-corded as a nonsurvivor. Sample sizes in all animal studies for eachmodel were estimated using log-rank test with 5% type I error rate and80% power. The hypothesized effect size for each comparison was de-rived from historical data or pilot study data. Sample sizes were cal-culated using nQuery Advisor software. All animals were randomlyassigned to treatment groups using a randomization tool implementedin MS Excel. Animal allocations were not blinded to study scientistsfor containment and personnel considerations. Pathologists that eval-uated tissue sections were blinded to study groups.

Synergistic effects between different antibodies and antibodies in com-bination with antibiotics were evaluated using a typical isobologramapproach as described (42, 43). All samples were included in allexperiments. All in vitro and in vivo experiments were repeated atleast three times unless otherwise stated within the figure legends.

Bispecific antibody constructionAntibody sequences were cloned into cytomegalovirus promoter-drivenexpression vectors. Anti-PcrV and control specificities in the Fab po-sition of the molecules were transferred from existing expression plas-mids using restriction digestion and ligation. scFv sequences weregenerated by gene synthesis (Eurofins MWG Operon), amplified bypolymerase chain reaction using primers that generated a 15-nucleotideoverlap to the target insertion site, and inserted using InFusion (BDClontech). Anti-Psl scFv sequences were generated in a VH-VL orien-tation with a 20–amino acid linker (GGGGSx4). Negative control scFvsequences were generated similarly but in a VL-VH orientation. AllscFv sequences were stabilized by engineering a VH-VL interdomaindisulfide bond generated by mutating VH44 and VL100 (Kabatnumbering) to Cys. BiS2 was constructed by fusing scFv sequencesto the N terminus of the heavy chain, separated by a 10–amino acidlinker (GGGGSx2). Similarly, BiS3 was constructed by appending thelinker-scFv to the C terminus of the heavy chain. BiS4 constructs weregenerated by flanking the scFv with 10–amino acid linkers (GGGGSx2)and inserting into the upper hinge region between C220 and D221(EU numbering). All proteins were expressed by transient transfectionin 293 cells, purified by protein A affinity chromatography, and pol-ished using size exclusion chromatography. The integrity of the moleculeswas verified using mass spectrometry, both intact mass and peptidemapping, to ensure proper formation of engineered and endogenousdisulfide bonds.

Opsonophagocytosis killing assayAssays were performed as described (6, 44). Briefly, assays were per-formed in 96-well plates using 0.025 ml of each component, lumines-cent P. aeruginosa strains, diluted baby rabbit serum, differentiatedHL-60 cells, and mAb. Data were acquired using an Envision Multi-label plate reader (Perkin Elmer) and plotted as percent killing com-pared to a control lacking antibody.

Cell attachment assayAssays were performed as described (6). Briefly, antibodies were addedto confluent A549 cells grown in opaque 96-well plates (Nunc Nun-clon Delta). Log-phase luminescent PAO1 was added at a multiplicityof infection (MOI) of 10. After incubation at 37°C for 1 hour, cells

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were washed followed by addition of LB + 0.5% glucose. Bacteria werequantified after a brief incubation at 37°C.

Cytotoxicity assayAssays were performed as described (11). Briefly, antibodies wereadded to A549 cells seeded in white 96-well plates (Nunc NunclonDelta) in Dulbecco’s modified Eagle’s medium plus 10% fetal bovineserum. Log-phase P. aeruginosa clinical isolates capable of expressingExoU were added at an MOI of 10 and incubated for 2 hours at 37°/5%CO2 followed by measurement of lactate dehydrogenase released fromlysed cells.

Calculation of MICsMICs were performed using the materials, standards, and methods setforth by the CLSI (45).

P. aeruginosa acute pneumonia mouse modelThe P. aeruginosa acute pneumonia was performed as described (6, 44).Antibodies or phosphate-buffered saline was intraperitoneally admin-istered 24 hours before or 1 to 4 hours after infection. Antibiotics wereadministered subcutaneously 1 or 4 hours after infection. For acutepneumonia organ burden experiments, mice were infected followedby harvesting of lungs, spleens, and kidneys 24 hours after infectionfor determination of CFU.

P. aeruginosa immunocompromised pneumonia mouse modelMice were rendered immunocompromised by intraperitoneal deliveryof cyclophosphamide monohydrate (CyM) (150 and 100 mg/kg) sus-pended in 0.9% saline on days −4 and −1, respectively, followed byP aeruginosa intranasal challenge on day 0. Depletion of total whiteblood cells was confirmed by counting blood cells at day 0 from CyM-treated mice compared to nontreated controls using the SysmexXT-2000i Automated Hematology Analyzer.

P. aeruginosa thermal injury and bacteremia mouse modelsThe thermal injury and bacteremia models were performed as de-scribed with modifications (6, 44). For thermal injury, 11-week-oldnon-Swiss albino–1 mice were shaved dorsally and anesthetized withisoflurane before exposure to a custom-made aluminum platform.This platform was designed to induce a 12 to 15% total body surfacearea thermal injury as determined using the Meeh equation (46).Animals were exposed to a constant temperature of 92°C for 5 s fol-lowed by intraperitoneal injection of 0.5 ml of saline for hydration.P. aeruginosa was then injected subcutaneously under the thermalwound. Buprenorphine hydrochloride was administered to mice twicedaily for the duration of the experiment to mitigate pain and distressfrom the thermal injury. Animals were monitored closely for 5 daysafter infection. In the bacteremia model, 7-week-old BALB/c micewere treated with control IgG or BiS4aPa by intraperitoneal adminis-tration 24 hours before intravenous challenge with P. aeruginosathrough the tail vein.

HistopathologyInfected mice were euthanized; lungs were removed, inflated, andfixed in 10% neutral buffered formalin (VWR). All lung samplesunderwent routine histological processing and paraffin embedding;4-mm histological sections were stained with Gills hematoxylin(Mercedes Medical) and eosin (Surgipath). All stained sections

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were analyzed using a Nikon 80i microscope with 10× and 40× objec-tives and reviewed by a pathologist who was blinded to the treatmentgroups.

Statistical analysesThe log-rank test was used to compare Kaplan-Meier survival curvesbetween different treatment groups generated in GraphPad Prism ver-sion 5. ANOVA analysis with Bonferroni correction was used forcomparison of antibody-treated groups in lung burden studies. Thelung burden CFU in each group was normally distributed after logtransformation using D’Agostino-Pearson normality test. The samplevariances were also similar in each group. GraphPad Prism version 5.0software was used for construction of figures and for ANOVA. TheAUC for each antibody activity-response curve was calculated usingthe linear trapezoidal rule on the means at different concentrationsin the log scale. AUC calculation and statistical comparisons betweendifferent antibodies were performed using PK package in R software.

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SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/6/262/262ra155/DC1Table S1. P. aeruginosa strain survey for Psl, PcrV, exoU, and exoS.Table S2. Determination of MICs.Fig. S1. Isobologram analysis for synergistic activity.Fig. S2. BiS4aPa treatment protects mice from lethal pneumonia.Fig. S3. BiS4aPa reduces P. aeruginosa organ burden.Fig. S4. BiS4aPa opsonophagocytic killing activity against P. aeruginosa clinical isolates.Fig. S5. BiS4aPa anti-cytotoxic activity against P. aeruginosa clinical isolates.Fig. S6. BiS4aPa protection in P. aeruginosa lethal challenge models.

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Acknowledgments: We thank J. Lin and L. Shirinian for expressing and purifying antibodyconstructs. We thank M. Jones, S. Oldham, A. Alfaro, and G. Wilson for excellent technicalassistance with animal experiments. In addition, we thank J. Goldberg (Emory University)and V. Fowler (Duke University) for P. aeruginosa clinical isolates. Funding: Research wassupported by MedImmune, LLC. Author contributions: A.D., C.K.S., G.J.R., and B.R.S.contributed to experimental design; A.D., P.W., and C.M.G. conducted key in vitro assaysand clinical isolate strain surveys for antibody activities; A.D., A.E.K., M.M.C., J.B., and J.H. con-ducted key multiple in vivo infection model work and independent in vivo replicateexperiments; N.D., B.B., Cuihua Gao, R.F., Changshou Gao, and G.J.R. contributed to the design,construction, and production of bispecific antibodies; W.Z. and X.-Q.Y. performed statisticaland isobologram analyses; V.D. performed blinded histological analyses; A.D., J.A.S., G.J.R., andC.K.S. prepared the manuscript. Competing interests: This work was funded by MedImmune,LLC, a wholly owned subsidiary of AstraZeneca Pharmaceuticals. All authors were employed byMedImmune, LLC when work was executed and may currently hold AstraZeneca stock or stockoptions. Patents describing the activity of the lead antibody (BiS4aPa or MEDI3902) in this workhave been filed by MedImmune: PCT/US2012/041538, PCT/US2012/063639, PCT/US2012/063722,PCT/US2013/068609, PCT/US2014/037839, and national applications thereof.

Submitted 29 May 2014Accepted 24 October 2014Published 12 November 201410.1126/scitranslmed.3009655

Citation: A. DiGiandomenico, A. E. Keller, C. Gao, G. J. Rainey, P. Warrener, M. M. Camara,J. Bonnell, R. Fleming, B. Bezabeh, N. Dimasi, B. R. Sellman, J. Hilliard, C. M. Guenther,V. Datta, W. Zhao, C. Gao, X.-Q. Yu, J. A. Suzich, C. K. Stover, A multifunctional bispecificantibody protects against Pseudomonas aeruginosa. Sci. Transl. Med. 6, 262ra155 (2014).

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Pseudomonas aeruginosaA multifunctional bispecific antibody protects against

Vivekananda Datta, Wei Zhao, Changshou Gao, Xiang-Qing Yu, JoAnn A. Suzich and C. Kendall StoverBonnell, Ryan Fleming, Binyam Bezabeh, Nazzareno Dimasi, Bret R. Sellman, Jamese Hilliard, Caitlin M. Guenther, Antonio DiGiandomenico, Ashley E. Keller, Cuihua Gao, Godfrey J. Rainey, Paul Warrener, Mareia M. Camara, Jessica

DOI: 10.1126/scitranslmed.3009655, 262ra155262ra155.6Sci Transl Med

bacterial pathogens.that multifunctional bispecific antibodies may be a promising platform for targeting other antibiotic-resistant immunosuppression and synergistically enhanced treatment with multiple antibiotic classes. This study suggestsdesignated clinical candidate MEDI3902, was also protective in mouse models of thermal injury, bacteremia, and

Pa construct, nowαmodel of lung infection compared to the parent monoclonal antibody combination. This BiS4 in a mouseP. aeruginosaPa exhibited targeted synergistic protection against αantibody platform called BiS4

.). A new multimechanistic bispecificet alPcrV and the persistence factor Psl exopolysaccharide (DiGiandomenico by targeting both the type III secretion injectisome virulence factorPseudomonas aeruginosabacterial pathogen

Multifunctional bispecific antibodies were constructed conferring three mechanisms of action against thePseudomonasBispecific Antibodies Protect Against

ARTICLE TOOLS http://stm.sciencemag.org/content/6/262/262ra155

MATERIALSSUPPLEMENTARY http://stm.sciencemag.org/content/suppl/2014/11/10/6.262.262ra155.DC1

CONTENTRELATED

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REFERENCES

http://stm.sciencemag.org/content/6/262/262ra155#BIBLThis article cites 45 articles, 14 of which you can access for free

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