8. A. Coros, B. Callahan, E. Battaglioli, K. M. Derbyshire,Mol. Microbiol. 69, 794–808 (2008).

9. J. L. Flint, J. C. Kowalski, P. K. Karnati, K. M. Derbyshire,Proc. Natl. Acad. Sci. U.S.A. 101, 12598–12603(2004).

10. L. M. Parsons, C. S. Jankowski, K. M. Derbyshire,Mol. Microbiol. 28, 571–582 (1998).

11. T. A. Gray, J. A. Krywy, J. Harold, M. J. Palumbo,K. M. Derbyshire, PLOS Biol. 11, e1001602 (2013).

12. P. A. DiGiuseppe Champion, M. M. Champion, P. Manzanillo,J. S. Cox, Mol. Microbiol. 73, 950–962 (2009).

13. E. N. Houben, K. V. Korotkov, W. Bitter, Biochim. Biophys. Acta1843, 1707–1716 (2014).

14. S. E. Converse, J. S. Cox, J. Bacteriol. 187, 1238–1245(2005).

15. G. Koraimann, M. A. Wagner, Front. Cell. Infect. Microbiol. 4, 54(2014).

16. P. K. Singh, W. J. Meijer, FEMS Microbiol. Lett. 358, 119–128(2014).

17. G. M. Dunny, R. P. Berntsson, J. Bacteriol. 198, 1556–1562(2016).

18. N. Agarwal, S. C. Woolwine, S. Tyagi, W. R. Bishai, Infect.Immun. 75, 452–461 (2007).

19. S. Raman et al., J. Bacteriol. 188, 8460–8468 (2006).

ACKNOWLEDGMENTS

We thank J. Wade for valuable discussions and technical assistanceand the Wadsworth Center Core Facilities for Media andBioinformatics. Primary data can be found in the supplementarymaterials, and RNA-seq data sets have been deposited at theNational Center for Biotechnology Information with accessionnumber SRP080861. This work was supported by NationalInstitute of Allergy and Infectious Diseases grants R01AI097191and R21AI07258 (to K.M.D. and T.A.G.). R.R.C. generated strains,

performed conjugation, and carried out RT-PCR experiments;N.B. identified the initial transposon mutant, constructed mutants,and performed conjugation assays; C.S. prepared RNA-seqlibraries; P.L. analyzed RNA-seq data; K.M.D. contributed studydesign and data analyses; T.A.G. contributed study design,targeted mutations, and data analyses; and K.M.D. and T.A.G.wrote the manuscript.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/354/6310/347/suppl/DC1Material and MethodsFigs. S1 to S4Tables S1 and S2References (20–22)

6 May 2016; accepted 31 August 201610.1126/science.aag0828

EBOLAVIRUSES

A“Trojan horse” bispecific-antibodystrategy for broad protectionagainst ebolavirusesAnna Z. Wec,1* Elisabeth K. Nyakatura,2* Andrew S. Herbert,3* Katie A. Howell,4

Frederick W. Holtsberg,4 Russell R. Bakken,3 Eva Mittler,1 John R. Christin,5

Sergey Shulenin,4 Rohit K. Jangra,1 Sushma Bharrhan,1 Ana I. Kuehne,3

Zachary A. Bornholdt,6 Andrew I. Flyak,7 Erica Ollmann Saphire,6,8

James E. Crowe Jr.,7,9,10† M. Javad Aman,4† John M. Dye,3†Jonathan R. Lai,2† Kartik Chandran1†

There is an urgent need for monoclonal antibody (mAb) therapies that broadly protectagainst Ebola virus and other filoviruses. The conserved, essential interaction between thefilovirus glycoprotein, GP, and its entry receptor Niemann-Pick C1 (NPC1) provides anattractive target for such mAbs but is shielded by multiple mechanisms, including physicalsequestration in late endosomes. Here, we describe a bispecific-antibody strategy totarget this interaction, in which mAbs specific for NPC1 or the GP receptor–binding siteare coupled to a mAb against a conserved, surface-exposed GP epitope. Bispecificantibodies, but not parent mAbs, neutralized all known ebolaviruses by coopting viralparticles themselves for endosomal delivery and conferred postexposure protection againstmultiple ebolaviruses in mice. Such “Trojan horse” bispecific antibodies have potential asbroad antifilovirus immunotherapeutics.

The development of therapeutics targetingEbola virus (EBOV) and other filovirusesis a global health priority. The success ofZMapp—a cocktail of three monoclonalantibodies (mAbs) targeting the EBOV

surface glycoprotein GP—in reversing Ebolavirus disease in nonhuman primates (NHPs) hasunderscored the promise of antiviral immuno-therapy (1). However, most available mAbs havea narrow antiviral spectrum, because they recog-nize variable surface-exposed GP epitopes (2).ZMapp protects against EBOV but not againstother filoviruses with known epidemic poten-tial, including the ebolaviruses Bundibugyo virus(BDBV) and Sudan virus (SUDV) and the moredivergent marburgviruses. Given the scientificand logistical challenges inherent in develop-ing a separate mAb cocktail for each filovirus,as well as the need for preparedness againstnewly emerging or engineered viral variants,

broadly protective antifilovirus immunotherapiesare highly desirable. A few mAbs have showncross-neutralization and protection in rodents,indicating that cross-species protection by asingle molecule is possible; however, such anti-bodies are rare (3–8).An unusual feature of cell entry by filoviruses

is the proteolytic cleavage of GP in endosomesto reveal “cryptic” epitopes (9, 10), includingthe receptor-binding site (RBS) that engages thecritical intracellular receptor, Niemann-Pick C1(NPC1) (fig. S1) (11–18). Engagement of NPC1’ssecond luminal domain, NPC1-C, by this highly con-served RBS in cleaved GP (GPCL) is required forcell entry and infection by all filoviruses (11, 19–21).Consistent with this, MR72, an RBS-specific mAbisolated from a Marburg virus (MARV) diseasesurvivor, blocked GPCL-NPC1 interaction in vitroand broadly neutralized viruses bearing in vitrocleaved GPCL (15, 22, 23). However, MR72 failed

to neutralize infection by uncleaved ebolaviruses,likely because it could not gain access to lateendosomes, where the GPCL RBS becomes un-masked (15). Therefore, the development of broadlyprotective immunotherapies targeting the GPCL-NPC1 interaction is challenged by the endosomalsequestration of this virus-receptor complex.We envisioned a bispecific antibody (bsAb)–

engineering strategy to block intracellular GPCL-NPC1 interaction by a “Trojan horse” mechanism.We reasoned that, by coupling receptor or RBS–targeting mAbs to a delivery mAb directed againsta broadly conserved epitope in uncleaved GP,virions themselves could be coopted to transportbsAbs to the appropriate endosomal compart-ments (Fig. 1, A and B). To block the filovirus-receptor interaction, we chose mAbs targetingboth its viral and host facets: MR72, a humanmAbthat recognizes the GPCL RBS (above), and mAb-548, a novel murine mAb that engages humanNPC1-C. mAb-548 bound with picomolar affinityto an NPC1-C epitope that overlaps the GPCL-binding interface and blocked GPCL–NPC1-Cassociation in vitro at pH 5.5, the presumptivepH of late endosomes (figs. S1 and S2). mAb-548resembled MR72 in its lack of neutralizing ac-tivity against uncleaved viruses (Fig. 2, A and B,and fig. S6), likely because NPC1 is absent fromthe cell surface (11, 24). To deliver mAb-548 andMR72 to endosomes, we selected the macaquemAb FVM09, which recognizes a conserved linear

350 21 OCTOBER 2016 • VOL 354 ISSUE 6310 sciencemag.org SCIENCE

1Department of Microbiology and Immunology, AlbertEinstein College of Medicine, Bronx, NY 10461, USA.2Department of Biochemistry, Albert Einstein College ofMedicine, Bronx, NY 10461, USA. 3U.S. Army MedicalResearch Institute of Infectious Diseases, Fort Detrick, MD21702, USA. 4Integrated Biotherapeutics Inc., Gaithersburg,MD 20878, USA. 5Department of Cell Biology, Albert EinsteinCollege of Medicine, Bronx, NY 10461, USA. 6Department ofImmunology and Microbial Science, The Scripps ResearchInstitute, La Jolla, CA 10550, USA. 7Department of Pathology,Microbiology and Immunology, Vanderbilt University,Nashville, TN 37235, USA. 8The Skaggs Institute forChemical Biology, The Scripps Research Institute, La Jolla,CA 10550, USA. 9Department of Pediatrics, VanderbiltUniversity, Nashville, TN 37232, USA. 10Vanderbilt VaccineCenter, Vanderbilt University, Nashville, TN 37232, USA.*These authors contributed equally to this work. †Correspondingauthor. Email: kartik.chandran@einstein.yu.edu (K.C.); jon.lai@einstein.yu.edu (J.R.L.); john.m.dye1.civ@mail.mil (J.M.D.);javad@integratedbiotherapeutics.com (M.J.A.); james.crowe@vanderbilt.edu (J.E.C.)

RESEARCH | REPORTSon M

arch 25, 2021

http://science.sciencemag.org/

Dow

nloaded from

epitope in the GP glycan cap of all known ebola-viruses (Fig. 1A and fig. S4) (8). FVM09 does notneutralize infection and confers limited in vivoprotection against EBOV (8).The heavy- and light-chain variable domains

(VH and VL, respectively) of FVM09 were fusedto mAb-548 and MR72 by using the dual–variabledomain immunoglobulin (DVD-Ig) design strategy(25). The DVD-Ig format was chosen as a test casebecause it allows bivalent binding of both com-bining sites but does not use long polypeptidelinkers that may be susceptible to proteolysisor immunogenic presentation. The FVM09~548and FVM09~MR72 DVD-Igs could be readily iso-lated from transiently transfected human em-bryonic kidney 293 (HEK293) cells (fig. S3A).Size-exclusion chromatography–multiangle lightscattering indicated a monodisperse popula-tion of monomers, with some higher aggregatepresent (fig. S3, B and C). Each DVD-Ig couldbind to EBOV GP via the FVM09 “outer” variabledomains, with no loss of affinity relative to theparent FVM09 immunoglobulin G (IgG), as de-termined by biolayer interferometry (BLI) (Fig.1C and table S1). FVM09~548 could recognizehuman NPC1-C, by means of its “inner” variabledomains, with a subpicomolar equilibrium disso-ciation constant (KD). TheMR72 variable domainsalso retained subnanomolar affinity toward GPCLin the DVD-Ig format. Two-phase binding studies,

in which each DVD-Ig was first exposed to EBOVGP and then to NPC1-C or GPCL (Fig. 1D), indi-cated that there were no steric restrictions toengagement of both combining sites.We tested the DVD-Igs for their capacity to

neutralize infection in human cells by recombi-nant vesicular stomatitis viruses bearing EBOVGP (rVSV–EBOV GP) or control nonfilovirus glyco-proteins derived from VSV and Andes hantavirus(Fig. 2, A and B, and fig. S5) (26). Both FVM09~548and FVM09~MR72 specifically and potently neu-tralized rVSV–EBOV GP, whereas the parentalmAbs FVM09, mAb-548, and MR72 had little orno neutralizing activity. Equimolar mixtures ofthe “delivery” IgG, FVM09, with each receptor-RBS–targeting IgG (mAb-548 or MR72) also didnot neutralize infection (Fig. 2, A and B); this in-dicated that DVD-Ig antiviral activity requires thephysical linkage of delivery and receptor-RBS–binding specificities. Overall, the DVD-Ig half-maximal inhibitory concentration (IC50) valueswere in the nanomolar range, similar to the mea-sured KD of the FVM09-GP complex but higherthan those of the mAb-548–NPC1-C and MR72-GPCL complexes.The GPCL-NPC1 interaction is conserved among

filoviruses (11, 15, 21, 27, 28), and thus, we pos-tulated that the DVD-Igs would exhibit broadneutralizing activity. rVSVs bearing GP proteinsfrom the four other ebolaviruses were sensitive

to neutralization by both DVD-Igs, whereas rVSV–MARV GP was resistant (Fig. 2, C, D, and G), con-sistent with the known specificity of FVM09toward ebolaviruses (8). We next tested the DVD-Igsagainst authentic EBOV, BDBV, and SUDV (Fig. 2,E and F). Each ebolavirus was neutralized by bothreceptor- and RBS-targeting DVD-Igs but not bythe individual parent IgGs (Fig. 2G and fig. S6).The success of antibody therapeutics has fueled

the development of a panoply of optimized bsAbarchitectures, several of which (including theDVD-Ig) are in clinical trials (29). To explore thegenerality of our strategy to other formats, wegenerated an FVM09*MR72 “asymmetric IgG”using the DuoBody platform (figs. S7 and S8)(30). FVM09*MR72 broadly neutralized rVSVsbearing ebolavirus GPs, albeit with reduced po-tency against SUDV GP, possibly because of its lossof bivalent recognition of GP and/or GPCL. None-theless, these results illustrate that endosomaltargeting of the ebolavirus-receptor interaction isamenable to other bispecific-antibody formats.Our observation that bsAbs combining two

nonneutralizing antibodies could confer potentneutralization implied critical roles for both bind-ing specificities. This hypothesis is supported bythree pieces of evidence. First, the activity of theDVD-Igs against rVSV–EBOV GP particles con-taining two point mutations in the FVM09 epi-tope was greatly reduced (Fig. 3A and fig. S9).

SCIENCE sciencemag.org 21 OCTOBER 2016 • VOL 354 ISSUE 6310 351

Fig. 1. Dual–variable domain Ig (DVD-Ig) moleculescombining extracellular delivery and endosomalreceptor–RBS–binding specificities can recognizeboth of their respective antigens. (A) Schematicof mAb-548 and MR72 endosomal receptor or RBS–specific mAbs and FVM09 GP–specific delivery mAb(top row), as well as DVD-Igs engineered to combinethem (bottom row). (B) A hypothetical mechanismfor delivery of DVD-Igs (bottom), but not parent IgGs(top), to the endosomal sites of GPCL-NPC1 interac-tion. LE, late endosomes. (C) Kinetic binding curvesfor DVD-Ig–antigen interactions were determinedby BLI. FVM09~548 (left) and FVM09~MR72 (right)were loaded onto probes, which were then dipped inanalyte solutions (FVM09~548: EBOV GP and humanNPC1-C; FVM09~MR72: EBOVGP and GPCL). Gray linesshow curve fits to a 1:1 binding model. See table S1 forkinetic binding constants. (D) Two-phase binding experi-ments for the DVD-Igs by BLI. Each DVD-Ig–bearingprobe was sequentially dipped in analyte solutionscontaining EBOV GP and then NPC1-C (FVM09~548),or EBOV GP and then GPCL (FVM09~MR72). (C) and(D) Representative curves from two independent ex-periments are shown.

RESEARCH | REPORTSon M

arch 25, 2021

http://science.sciencemag.org/

Dow

nloaded from

352 21 OCTOBER 2016 • VOL 354 ISSUE 6310 sciencemag.org SCIENCE

Fig. 2. DVD-Igs, butnot parent IgGs ortheir mixtures, havebroad neutralizingactivity againstebolaviruses. (A to D)Neutralization of rVSVsencoding enhancedgreen fluorescentprotein (eGFP) andbearing filovirusGP proteins in humanU2OS osteosarcomacells. Virions werepreincubated withincreasing concentra-tions of each parentIgG, DVD-Ig, or equi-molar mixtures of par-ent IgGs (e.g., FVM09 +mAb-548) (1:1 mixture)and then exposed to cells for 12 to 14 hours at 37°C. Infection wasmeasured by automated counting of eGFP+ cells and normalizedto infection obtained in the absence of Ab.TAFV,Taï forest virus;RESTV, Reston virus; MARV, Marburg virus. (E to F) Neutraliza-tion of authentic filoviruses in human U2OS osteosarcomacells, measured in microneutralization assays. Infected cells were immunostained for viral antigen at 48 hours postinfection and enumerated by automatedfluorescence microscopy. (A) to (F) Averages ± SD for four to six technical replicates pooled from two or three independent experiments. (G) Data in (A)to (F) were subjected to nonlinear regression analysis to derive Ab concentrations at half-maximal neutralization (IC50 ± 95% confidence intervals fornonlinear curve fit). *IC50 values derived from curves that did not reach 90% neutralization at the highest concentration tested in the experiments are shown.

Fig. 3. Roles of deliv-ery and endosomalreceptor–RBS–targeting specificitiesin ebolavirus neutrali-zation by DVD-Igs.(A) Neutralizing activityof DVD-Igs againstrVSVs bearing WT GPor a GP(E288D/W292R) mutant, inwhich Asp replacesGlu288 and Arg replacesTrp292 (see fig. S9).(B) Neutralizing activityof mutant DVD-Igsbearing mAb-548 orMR72 combiningsites with mutationsin the third VH

complementaritydetermining region(FVM09~548Mut andFVM09~MR72Mut,respectively). (C)Neutralizing activity of DVD-Igs against rVSV–EBOV GP in U2OS cells bearingendogenous levels of NPC1 (NPC1WT) or ectopically overexpressing NPC1(NPC1High). (A) to (C) Averages ± SD for six technical replicates pooled fromtwo independent experiments. (D) Internalization of labeled Abs into cells inthe absence or presence of viral particles. A schematic of the experiment isshown at the left. Parent IgGs and DVD-Igs covalently labeled with the acid-dependent fluorophore pHrodo Red were incubated with rVSV–EBOV GPparticles and exposed to cells. Virus– Ab+ and virus+ Ab+ populations were mea-sured by flow cytometry. Averages ± SD for four technical replicates pooledfrom two independent experiments are shown. Group means for the percentageof Ab+ cells were compared by two-factor analysis of variance (ANOVA) (see

fig. S11). Šídák’s post hoc test was used to compare the capacity of eachAb to internalize into virus– versus virus+ cell populations (••••P < 0.0001;ns, not significant). Dunnett’s post hoc test was used to compare the inter-nalization of each Ab to that of the “no Ab” control in virus+ cell populations(****P < 0.0001; all other Ab versus no Ab comparisons were not signif-icant). (E) Delivery of Abs to NPC1+ endosomes. FVM09~548 was incu-bated with rVSV–EBOV GP particles and exposed to cells expressing anNPC1–enhanced blue fluorescent protein–2 fusion protein. Viral particles,Ab, and NPC1 were visualized by fluorescence microscopy (also see figs.S12 and S13). Representative images from two independent experimentsare shown. Scale bar, 20 mm.

RESEARCH | REPORTSon M

arch 25, 2021

http://science.sciencemag.org/

Dow

nloaded from

Second, DVD-Igs bearing mAb-548 and MR72variable domains with mutations that abolishbinding (FVM09~548Mut and FVM09~MR72Mut)lacked neutralizing activity (Fig. 3B). Third,FVM09~548 could not neutralize rVSV–EBOV GPinfection in a cell line bearing supraphysiologicallevels of NPC1, likely because NPC1 overexpres-sion saturates available mAb-548 combining sites(Fig. 3C). Viral neutralization by FVM09~MR72was unaffected in NPC1-overexpressing cells, con-sistent with the higher affinity of the GPCL-MR72complex (55 pM) (table S1), relative to the GPCL–NPC1-C complex (150 mM) (16).We postulated that the bsAbs harness extra-

cellular virions for their delivery to endosomalsites of filovirus-receptor interaction in the con-text of natural infection. Accordingly, we eval-uated the internalization of DVD-Igs and theirparent IgGs into cellular endosomes (Fig. 3Dand figs. S10 and S11). Each Ab was covalentlylabeled with the acid-dependent fluorescent probepHrodo Red and exposed to cells, either aloneor following preincubation with fluorescent rVSV–EBOV GP particles (19, 26). Cells were measuredfor both virus- and Ab-associated fluorescenceby flow cytometry. Virus-negative cells displayedlittle Ab signal; this indicated that neither theDVD-Igs nor their parent IgGs could internal-ize into cells without virions. By contrast, virus-positive cells were strongly positive for theDVD-Igs but not for the parent mAb-548 andMR72 IgGs. Concordantly, only FVM09 and theDVD-Igs could efficiently colocalize with virions(Fig. 3E and fig. S12) or Ebola virus–like par-ticles (VLPs) (fig. S13) in NPC1+ late endosomes,where viral membrane fusion takes place (19, 31).These results, together with the capacity of FVM09to bind EBOV GP with high affinity between pH5.5 and 7.5 (fig. S14 and table S1), suggest thatvirion-bsAb complexes remain associated in earlyendosomes and traffic together to late endosomes,where proteolytic removal of the GP glycan capdislodges FVM09, and where mAb-548 and MR72

can engage their respective cellular and viraltargets. Collectively, our findings support a two-step “deliver-and-block” mechanism for bsAbneutralization.Finally, we evaluated the protective efficacy

of the DVD-Igs in two murine models of lethalebolavirus challenge. Because our prior exper-iments were conducted in human cells, we firsttested DVD-Ig neutralization activity in murineNIH/3T3 cells (fig. S15). Whereas FVM09~MR72retained full activity, FVM09~548 exhibited poorneutralization in murine cells (fig. S15, A and B).This could be readily explained by FVM09~548’sreduced binding affinity for the murine NPC1ortholog (fig. S15C) and consequent reduced ca-pacity to block the GPCL-NPC1 interaction (fig. S15E).The discrepancy between binding of mAb-548to human and mouse NPC1 likely arises fromspecies-dependent amino acid sequence differ-ences in the mAb-548–binding region of NPC1-C(fig. S16). By contrast, this region in human NPC1-Cis identical to those of rhesus macaques and crab-eating (cynomolgus) macaques (fig. S16), whichprovide the two NHP models of filovirus chal-lenge currently in use. mAb-548 bound stronglyto, and inhibited GPCL interaction with, an NHPNPC1 ortholog derived from the mantled guereza,which also shares an identical mAb-548–bindingregion (fig. S15, D and F). Therefore, althoughhost species–specific differences in NPC1 bindingmay affect FVM09~548’s efficacy in rodents, theyare unlikely to do so in NHPs and humans.Both DVD-Igs were tested for their capac-

ity to protect BALB/c mice when administered2 days after a lethal challenge with EBOV-MA(Fig. 4A) (32). FVM09~MR72 afforded a high lev-el of protection (70%) relative to the untreatedgroup, whereas no significant survival was re-corded for FVM09~548 and parent IgG mixtures.We also evaluated the DVD-Igs for postexposureprotection against a lethal human SUDV isolatein the immunocompromised, type 1 interferona/b receptor–deficient (IFNa/b R−/−) mouse

model (Fig. 4B) (7, 33). FVM09~MR72 was fullyprotective, and FVM09~548 provided partial pro-tection, relative to the untreated group. The lim-ited in vivo efficacy of FVM09~548 was consistentwith its reduced capacity to inhibit the GPCL–murine NPC1 interaction (fig. S15). These find-ings provide evidence that a bsAb targeting thecritical intracellular virus-receptor interactioncan confer broad protection against lethal ebola-virus challenge, even under stringent conditionsof postexposure treatment.Recent antibody discovery efforts have dem-

onstrated the existence of conserved GP surfaceepitopes that can elicit broadly reactive mAbs withcross-protective potential (3–7, 34). Herein, wedescribe a complementary strategy to generatebroadly protective Abs that target highly con-served epitopes at the intracellular filovirus-receptor interface, which are normally shieldedfrom GP-specific mAbs. Because the cryptic epitope–targeting components of the bsAbs engineeredin this study block endosomal receptor bindingby all known filoviruses (15) (this study), next-generation molecules combining them withappropriate delivery mAbs of viral or cellularorigin may afford coverage against all filoviruses,including newly emerging and engineered var-iants. This Trojan horse bispecific-antibody ap-proach may also find utility against other viralpathogens known to use intracellular receptors[e.g., Lassa virus (35)], or more generally, totarget entry-related virus structural rearrange-ments that occur only in the endolysosomalpathway.

REFERENCES AND NOTES

1. X. Qiu et al., Nature 514, 47–53 (2014).2. C. D. Murin et al., Proc. Natl. Acad. Sci. U.S.A. 111, 17182–17187

(2014).3. K. A. Howell et al., Cell Rep. 15, 1514–1526 (2016).4. A. I. Flyak et al., Cell 164, 392–405 (2016).5. J. C. Frei et al., Sci Rep. 6, 19193 (2016).6. W. Furuyama et al., Sci Rep. 6, 20514 (2016).7. F. W. Holtsberg et al., J. Virol. 90, 266–278 (2016).8. Z. Y. Keck et al., J. Virol. 90, 279–291 (2016).9. K. Chandran, N. J. Sullivan, U. Felbor, S. P. Whelan,

J. M. Cunningham, Science 308, 1643–1645 (2005).10. K. Schornberg et al., J. Virol. 80, 4174–4178 (2006).11. E. H. Miller et al., EMBO J. 31, 1947–1960 (2012).12. J. E. Carette et al., Nature 477, 340–343 (2011).13. M. Côté et al., Nature 477, 344–348 (2011).14. A. S. Herbert et al., MBio 6, e00565-e15 (2015).15. Z. A. Bornholdt et al., MBio 7, e02154-e15 (2016).16. H. Wang et al., Cell 164, 258–268 (2016).17. M. A. Brindley et al., J. Virol. 81, 7702–7709

(2007).18. B. Manicassamy, J. Wang, H. Jiang, L. Rong, J. Virol. 79,

4793–4805 (2005).19. J. S. Spence, T. B. Krause, E. Mittler, R. K. Jangra, K. Chandran,

MBio 7, e01857-e15 (2016).20. M. J. Aman, MBio 7, e00346-e16 (2016).21. M. Ng et al., Virology 468-470, 637–646 (2014).22. T. Hashiguchi et al., Cell 160, 904–912 (2015).23. A. I. Flyak et al., Cell 160, 893–903 (2015).24. J. P. Davies, Y. A. Ioannou, J. Biol. Chem. 275, 24367–24374

(2000).25. C. Wu et al., Nat. Biotechnol. 25, 1290–1297 (2007).26. L. M. Kleinfelter et al., MBio 6, e00801-15 (2015).27. M. Ng et al., Elife 4, e11785 (2015).28. E. Ndungo et al., mSphere 1, e00007-16 (2016).29. C. Spiess, Q. Zhai, P. J. Carter, Mol. Immunol. 67 (2 Pt A),

95–106 (2015).30. A. F. Labrijn et al., Proc. Natl. Acad. Sci. U.S.A. 110, 5145–5150

(2013).

SCIENCE sciencemag.org 21 OCTOBER 2016 • VOL 354 ISSUE 6310 353

Fig. 4. FVM09~MR72 affords broadpostexposure protection from lethalebolavirus challenge. (A) BALB/cmice were challenged with mouse-adapted EBOV (EBOV-MA), and thentreated with single doses of parentIgG mixtures [300 mg, ~15 mg perkilogram body weight (mg/kg)] orDVD-Igs (400 mg, ~20 mg/kg, adjustedfor molecular weight), or vehicle(phosphate-buffered saline, PBS) at2 days postchallenge. (B) Type1 IFNa/b R−/− mice were challengedwith WT SUDV and then treated withtwo doses of parent IgG mixtures(300 mg total per dose, ~15 mg/kg);DVD-Igs (400 mg per dose, ~20 mg/kg);or vehicle (PBS) 1 and 3 days post-challenge. “n” indicates the numberof animals per group. Data fromsingle cohorts are shown. Survival ineach group was compared with that in the PBS group by log-rank (Mantel-Cox) test (*P < 0.05; ***P <0.001); all other comparisons to the untreated group were not significant.

RESEARCH | REPORTSon M

arch 25, 2021

http://science.sciencemag.org/

Dow

nloaded from

31. J. A. Simmons et al., J. Virol. 90, 605–610 (2016).32. M. Bray, K. Davis, T. Geisbert, C. Schmaljohn, J. Huggins,

J. Infect. Dis. 178, 651–661 (1998).33. J. M. Brannan et al., J. Infect. Dis. 212 (suppl. 2), S282–S294

(2015).34. Z. A. Bornholdt et al., Science 351, 1078–1083

(2016).35. L. T. Jae et al., Science 344, 1506–1510 (2014).

ACKNOWLEDGMENTS

The data presented in this manuscript are tabulated in the mainpaper and in the supplementary materials. We thank C. Haroldand T. Alkutkar for technical support. We acknowledge M. D. Scharff,S. Buhl, and the Einstein Macromolecular Therapeutics DevelopmentFacility for assistance with generation of mAb-548; E. Ndungofor assistance with hybridoma screening and for provision ofpurified human-Russell’s viper NPC1 chimeras; E. L. Snapp andL. Costantini for generating an NPC1-eBFP2 fusion construct;and A. F. Labrijn, P. W. H. I. Parren, and Genmab for the gift of

purified b12*MR72 DuoBody. This work is supported by NIHgrants U19 AI109762 (Centers for Excellence in TranslationalResearch) to K.C., J.R.L., J.M.D., and E.O.S.; R01 AI088027 to K.C.;and 1R41 AI122403 to J.R.L. and M.J.A.; Joint Science and TechnologyOffice–Defense Threat Reduction Agency (JSTO-DTRA) projectCB04088 to J.M.D.; and a DTRA contract (HDTRA1-13-C-0015) toM.J.A. E.K.N. was also supported by a DAAD (DeutscherAkademischer Austauschdienst, German Academic ExchangeService) fellowship. M.J.A. is president of and owns stocks, andF.W.H. and S.S. own stock options, in Integrated Biotherapeutics.M.J.A., F.W.H., K.A.H., and S.S. are named coinventors on a patentapplication (WO 2015/200522 A2) submitted by IntegratedBiotherapeutics, that covers the endosomal targeting of filovirusbispecific antibodies. A.Z.W., E.K.N., J.R.L., and K.C. are namedcoinventors on a patent application (PCT/US/16/30652)submitted by Albert Einstein College of Medicine that coversthe use of mAb-548 and bispecific antibodies incorporatingmAb-548. A.I.F. and J.E.C. are named coinventors on a patentapplication (PCT/US2016/019644) submitted by Vanderbilt

University Medical Center that covers the use of the MR72antibody and bispecific antibodies incorporating MR72.mAb-548 and bispecific antibodies incorporating it are availablefrom K.C. under a material transfer agreement with AlbertEinstein College of Medicine. MR72 antibody and bispecificantibodies incorporating it are available from J.E.C. undera material transfer agreement with Vanderbilt UniversityMedical Center.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/354/6310/350/suppl/DC1Materials and MethodsFigs. S1 to S16Table S1References (36–48)

13 June 2016; accepted 25 August 2016Published online 8 September 201610.1126/science.aag3267

ANTIGEN PRESENTATION

A large fraction of HLA class Iligands are proteasome-generatedspliced peptidesJuliane Liepe,1* Fabio Marino,2,3 John Sidney,4 Anita Jeko,2,3 Daniel E. Bunting,1

Alessandro Sette,4 Peter M. Kloetzel,5,6 Michael P. H. Stumpf,1

Albert J. R. Heck,2,3 Michele Mishto5,6*

The proteasome generates the epitopes presented on human leukocyte antigen (HLA)class I molecules that elicit CD8+ T cell responses. Reports of proteasome-generatedspliced epitopes exist, but they have been regarded as rare events. Here, however,we show that the proteasome-generated spliced peptide pool accounts for one-thirdof the entire HLA class I immunopeptidome in terms of diversity and one-fourth interms of abundance. This pool also represents a unique set of antigens, possessingparticular and distinguishing features. We validated this observation using a rangeof complementary experimental and bioinformatics approaches, as well as multiplecell types. The widespread appearance and abundance of proteasome-catalyzed peptidesplicing events has implications for immunobiology and autoimmunity theories andmay provide a previously untapped source of epitopes for use in vaccines andcancer immunotherapy.

The presentation of epitopes on the cell sur-face is a key mechanism by which organismsidentify the presence of pathogens, meta-bolic malfunctioning, or tumors. The HLAclass I (HLA-I) immunopeptidome—the set of

epitopes allocated onto the HLA-I molecules—impinges on the CD8+ T cell repertoire and thecell-mediated immune response (1). HLA-I immuno-peptidomes are usually investigated by sequence

identification of peptides eluted from HLA-I mol-ecules by means of tandem liquid chromatography–mass spectrometry (LC-MS/MS) (fig. S1). The keystep for the transformation of a protein into HLA-I–restricted epitopes is usually processing by theproteasome (1), which cuts proteins into peptides;alternatively, the proteasome can also cut andpaste peptide sequences, thereby releasing peptideantigens that do not correspond to the originalprotein sequence (2) (fig. S2). This proteasome-catalyzed peptide splicing (PCPS) has long beenconsidered to occur only rarely; partly this has beenbecause the screening of the HLA-I immunopep-tidome for proteasome-generated spliced pep-tides was impeded by methodological challenges.To overcome these problems, we developed

an analytical strategy that accounts for recentdiscoveries underpinning the PCPS mechanismand can handle the vast proteome-wide humanspliced peptide database (fig. S3). With this strat-egy, we initially analyzed the HLA-I–eluted im-

munopeptidome of the GR lymphoblastoid cellline (GR-LCL); for a deeper coverage of the im-munopeptidome, we adopted a two-dimensional(2D) peptide prefractionation strategy followed bya hybrid peptide fragmentation method [electron-transfer higher-energy collision dissociation(EThcD)] for peptide identification (3, 4) (fig. S1),supplemented by an adapted target-decoy ap-proach (fig. S4). Our analysis led to the identi-fication of 6592 nonspliced and 3417 splicedpeptides 9 to 12 amino acid residues in length(9- to 12-mer peptides) (table S1). The latter num-ber represents 34% of the total of identified anti-genic peptides (Fig. 1A), thereby increasing thenumber of identified HLA-I ligands by some 50%.We confirmed the authenticity of the identifiedspliced antigenic peptides by comparing the LC-MS/MS spectra of 98 exemplary spliced peptideswith their corresponding synthetic peptides andcomputing their correlation score (table S2 andfig. S5). In addition, we verified the proteasome-dependent generation of the spliced antigenicpeptides in vitro for three examples by digestionsof synthetic polypeptides harboring the correspond-ing antigenic peptides by purified 20S proteasome(fig. S6).We queried the HLA-I immunopeptidome mass

spectrometry data of GR-LCL against the stan-dard Swissprot human proteome database, whichdoes not account for spliced peptides; this re-vealed that 655 peptides (i.e., 9% of the totalnonspliced antigenic peptides) would be erro-neously matched against this incomplete data-base as nonspliced peptides, because they havemuch better hits as spliced peptides in our searchagainst the combined spliced and nonspliced pep-tide database (Fig. 1A). The correct sequencesfor a set of these antigenic spliced peptides wereverified by comparing the LC-MS/MS spectra ofthe synthetic spliced and nonspliced candidateswith the corresponding LC-MS/MS of the GR-LCLHLA-I immunopeptidome (fig. S7, A to I). Ouridentifications are further supported by the ionscore distributions (see fig. S4) of the nonsplicedpeptides and of those spliced peptides that werewrongly assigned as nonspliced peptides usingthe standard Swissprot human proteome data-base, which differ only slightly in their medianbut not in the overall shape (fig. S7J).

354 21 OCTOBER 2016 • VOL 354 ISSUE 6310 sciencemag.org SCIENCE

1Centre for Integrative Systems Biology and Bioinformatics,Department of Life Sciences, Imperial College London,London SW7 2AZ, UK. 2Biomolecular Mass Spectrometry andProteomics, Bijvoet Center for Biomolecular Research andUtrecht Institute for Pharmaceutical Sciences, UtrechtUniversity, 3584 CH Utrecht, Netherlands. 3NetherlandsProteomics Centre, CH Utrecht, Netherlands. 4Division ofVaccine Discovery, La Jolla Institute for Allergy andImmunology, La Jolla, CA 92037, USA. 5Institut fürBiochemie, Charité–Universitätsmedizin Berlin, 10117 Berlin,Germany. 6Berlin Institute of Health, 10117 Berlin, Germany.*Corresponding author. Email: michele.mishto@charite.de(M.M.); j.liepe@imperial.ac.uk (J.L.)

RESEARCH | REPORTSon M

arch 25, 2021

http://science.sciencemag.org/

Dow

nloaded from

A ''Trojan horse'' bispecific-antibody strategy for broad protection against ebolaviruses

Flyak, Erica Ollmann Saphire, James E. Crowe Jr., M. Javad Aman, John M. Dye, Jonathan R. Lai and Kartik ChandranMittler, John R. Christin, Sergey Shulenin, Rohit K. Jangra, Sushma Bharrhan, Ana I. Kuehne, Zachary A. Bornholdt, Andrew I. Anna Z. Wec, Elisabeth K. Nyakatura, Andrew S. Herbert, Katie A. Howell, Frederick W. Holtsberg, Russell R. Bakken, Eva

originally published online September 8, 2016DOI: 10.1126/science.aag3267 (6310), 350-354.354Science 

, this issue p. 350; see also p. 284Scienceotherwise lethal ebolavirus infection.either NPC1 or the NPC1 binding site on GP. Treating mice therapeutically with these antibodies allowed them to survive Parren). They coupled an antibody specific for a conserved, surface-exposed epitope of GP to antibodies that recognizeantibody strategy targeting all known ebolaviruses that overcomes this problem (see the Perspective by Labrijn and

now report a bispecificet al.cleave the Ebola virus surface glycoprotein (GP) to reveal its receptor binding site. Wec receptor, NPC1, inside the cell within endosomes rather than on the cell surface. Furthermore, enzymes in endosomesand other filoviruses. Neutralizing ebolaviruses with antibodies is a challenge because the viruses bind their entry

The recent major Ebola virus outbreak in West Africa high-lighted the need for effective therapeutics against thisTreating Ebola with a Trojan horse

ARTICLE TOOLS http://science.sciencemag.org/content/354/6310/350

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2016/09/07/science.aag3267.DC1

CONTENTRELATED

http://stm.sciencemag.org/content/scitransmed/9/384/eaai8711.fullhttp://stm.sciencemag.org/content/scitransmed/7/317/317ps24.fullhttp://stm.sciencemag.org/content/scitransmed/7/290/290ra89.fullhttp://stm.sciencemag.org/content/scitransmed/7/307/307rv5.fullhttp://stm.sciencemag.org/content/scitransmed/8/329/329ra33.fullhttp://science.sciencemag.org/content/sci/354/6310/284.full

REFERENCES

http://science.sciencemag.org/content/354/6310/350#BIBLThis article cites 48 articles, 20 of which you can access for free

PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions

Terms of ServiceUse of this article is subject to the

is a registered trademark of AAAS.ScienceScience, 1200 New York Avenue NW, Washington, DC 20005. The title (print ISSN 0036-8075; online ISSN 1095-9203) is published by the American Association for the Advancement ofScience

Copyright © 2016, American Association for the Advancement of Science

on March 25, 2021

http://science.sciencem

ag.org/D

ownloaded from