HIV THERAPY

Trispecific broadly neutralizing HIVantibodies mediate potent SHIVprotection in macaquesLing Xu,1* Amarendra Pegu,2* Ercole Rao,1 Nicole Doria-Rose,2 Jochen Beninga,1

Krisha McKee,2 Dana M. Lord,1 Ronnie R. Wei,1 Gejing Deng,1 Mark Louder,2

Stephen D. Schmidt,2 Zachary Mankoff,2 LanWu,1 Mangaiarkarasi Asokan,2

Christian Beil,1 Christian Lange,1 Wulf Dirk Leuschner,1 Jochen Kruip,1 Rebecca Sendak,1

Young Do Kwon,2 Tongqing Zhou,2 Xuejun Chen,2 Robert T. Bailer,2 KeyunWang,2

Misook Choe,2 Lawrence J. Tartaglia,3,4 Dan H. Barouch,3,4 Sijy O’Dell,2 John-Paul Todd,2

Dennis R. Burton,4,5 Mario Roederer,2 Mark Connors,6 Richard A. Koup,2 Peter D. Kwong,2

Zhi-yong Yang,1 John R. Mascola,2† Gary J. Nabel1†

The development of an effective AIDS vaccine has been challenging because of viralgenetic diversity and the difficulty of generating broadly neutralizing antibodies (bnAbs).We engineered trispecific antibodies (Abs) that allow a single molecule to interact withthree independent HIV-1 envelope determinants: the CD4 binding site, the membrane-proximal external region (MPER), and the V1V2 glycan site. Trispecific Abs exhibited higherpotency and breadth than any previously described single bnAb, showed pharmacokineticssimilar to those of human bnAbs, and conferred complete immunity against a mixture ofsimian-human immunodeficiency viruses (SHIVs) in nonhuman primates, in contrast tosingle bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutictargets through a single protein, and they may be applicable for treatment of diversediseases, including infections, cancer, and autoimmunity.

Avariety of broadly neutralizing antibodies(bnAbs) have been isolated from HIV-1–infected individuals (1–3), but their poten-tial to treat or prevent infection in humansmay be limited by the potency or breadth

of viruses neutralized (4, 5). The targets of theseantibodies have been defined according to anunderstanding of the HIV-1 envelope structure(6–9). Although bnAbs occur in selected HIV-1–infected individuals (usually after several yearsof infection), it remains a challenge to elicit themby vaccination because broad and potent HIV-1 neutralization often requires unusual antibodycharacteristics, such as long hypervariable loops,interaction with glycans, and a substantial levelof somaticmutation. Strategies have thus shiftedfrom active to passive immunization, both to pro-tect against infection and to target latent virus(10–14).We and others have begun to explore com-

binations of bnAbs that optimize potency and

breadth of protection, thus reducing the likeli-hood of resistance and viral escape (15–17). Anti-bodies directed to the CD4 binding site (CD4bs),membrane-proximal external region (MPER),and variable-region glycans are among the com-binations that so far provide optimal neutraliza-tion (18). In addition, alternative combinationshave also been investigated for the immunotherapyof AIDS, specifically by directing T lymphocytesto activate latent viral gene expression and en-hance lysis of virally infected cells (19, 20). Be-causemultiple antibodiesmay help to reduce theviral replication that sustains chronic HIV-1 in-fection, we report here the generation of multi-specific antibodies designed to increase thepotential efficacy of HIV-1 antibodies for preven-tion or therapy.

Design of bispecific antibodies andevaluation of neutralization breadth

Although individual anti–HIV-1 bnAbs can neu-tralize naturally occurring viral isolates with highpotency, the percentage of strains inhibited bythese monoclonal Abs (mAbs) varies (21, 22). Inaddition, resistant viruses can be found in thesame patients from whom bnAbs were isolated,which suggests that immune pressure against asingle epitope may not optimally treat HIV-1infection or protect against it. We hypothesizedthat the breadth and potency of HIV-1 neutral-ization by a single antibody could be increasedby combining the specificities against differentepitopes into a single molecule. This strategywould be expected not only to improve efficacybut also to simplify treatment regimens, as well

as the regulatory issues required for clinicaldevelopment.To test this concept, we initially incorporated

prototype bnAbs to the CD4bs and MPER sitesinto a modified bispecific Ab. When two var-iable regions are linked in tandem, the distalsite typically retains its ability to bind antigenwhile the proximal binding is markedly dimin-ished. We therefore used an alternative configu-ration, termed CODV-Ig, that introduced linkersand inverted the order of the antibody bind-ing site in light and heavy chains to alter theorientation of the variable regions, allowing eachregion to interact with its target (23). Severalknown bnAbs, including VRC01, 10E8, PGT121,and PGT128 [reviewed in (1)], were evaluated fortheir ability to neutralize a select panel of viruseswith known resistance or sensitivity to these anti-bodies (fig. S1). Initially, we determined whetherthe position of the variable regions from VRC01and 10E8 in the proximal or distal positions (Fig.1A) could affect neutralization breadth and po-tency. Inclusion of both variable regions in eitherorientation in the bispecific antibody reducedthe number of resistant strains relative to theparental antibodies alone (Fig. 1B). Better po-tency was observed when VRC01 was proximaland 10E8 distal, although neither bispecific anti-body was as potent as a mixture of the two anti-bodies alone.To explorewhether other bnAbs could perform

better in the bispecific format, we evaluated twodifferent combinations: VRC01 plus PGT121, orVRC01 plus PGT128. For PGT121, expressionwasobserved only with VRC01 in the distal position.When this antibodywas compared to the paren-tal antibodies alone, it providedmarginally betterneutralization (Fig. 2A). In contrast, VRC01 couldbe expressedwith PGT128 in both positions, withgreater breadth observedwhen VRC01 was distal(Fig. 2B). Together, these data indicate that im-provements in breadth could be achieved with abispecific format; however, in this case, the po-tency was not consistently improved relative toeach Ab alone. We therefore sought an alterna-tive format to improve the potency and breadthof neutralization.

Generation and comparison of broadand potent trispecific antibodies

To achieve our goal, we used a previously un-described trispecific Ab format. Three speci-ficities were combined by using knob-in-holeheterodimerization (24) to pair a single arm de-rived from a normal immunoglobulin (IgG) witha double arm generated in the CODV-Ig. A panelof bnAbs was evaluated, including those directedagainst the CD4bs that included VRC01 and N6,as well as PGT121, PGDM1400, and 10E8 (fig. S1).A modified version of the latter, termed 10E8v4,was used because of its greater solubility (25).We first determined which bispecific arms showedthe best potency, breadth, and yield. This screen-ing analysis revealed that combinations contain-ing PGDM1400, CD4bs, and 10E8v4 showed thehighest level of production and greatest potencyof neutralization (fig. S2).

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1Sanofi, 640 Memorial Drive, Cambridge, MA 02139, USA.2Vaccine Research Center, National Institute of Allergy andInfectious Diseases, Bethesda, MD 20892, USA. 3Center forVirology and Vaccine Research, Beth Israel DeaconessMedical Center, Harvard Medical School, Boston, MA 02215,USA. 4Ragon Institute of MGH, MIT, and Harvard, Cambridge,MA 02139, USA. 5Department of Immunology andMicrobiology, International AIDS Vaccine Initiative (IAVI)Neutralizing Antibody Center, Center for HIV/AIDS VaccineImmunology and Immunogen Discovery, The ScrippsResearch Institute, La Jolla, CA 92037, USA. 6NationalInstitute of Allergy and Infectious Diseases, Bethesda, MD20892, USA.*These authors contributed equally to this work.†Corresponding author. Email: gary.nabel@sanofi.com (G.J.N.);jmascola@mail.nih.gov (J.R.M.)

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We then evaluated different combinations ofsingle-arm and double-arm specificities fromPGDM1400, CD4bs, and 10E8v4 Abs for theirexpression levels and activity against a smallpanel of viruses (fig. S3), leading ultimately tothe identification of trispecific antibodies VRC01/PGDM1400-10E8v4 and N6/PGDM1400-10E8v4as lead candidates. When analyzed against apanel of 208 viruses (18) and compared to theparental antibodies alone, the highest neutrali-zation potency and breadth were observed withN6/PGDM1400-10E8v4, with only 1 of the 208viruses showing neutralization resistance and amedian inhibitory concentration (IC50) of less than0.02 mg/ml (Fig. 3A). VRC01/PGDM1400-10E8v4also displayed high potency and breadth, andonly four resistant viruses were found. Someparental mAbs displayed either high breadth(e.g., 10E8, N6) or high potency (PGDM1400),but none displayed a combination of breadthand potency as optimal as the trispecific Abs(Fig. 3B). For example, the most potent and broadparental mAb, N6, was around 20% as potent asthe N6/PGDM1400-10E8v4 trispecific Ab andtargeted only a single epitope, which could in-crease the chance of viral escape mutations. As asingle recombinant protein, the trispecific Abs dem-onstrated potency and breadth superior to anysingle antibody yet defined (Fig. 3 and fig. S4).We also determined the binding affinity of

each component of the trispecific Ab and com-pared each to its parental Fab. The equilibriumbinding constant Kd of each binding site in thetrispecific Ab, determined by surface plasmonresonance, was comparable to the affinity of theparental Fab, with PGDM1400 showing a slightdecrease in affinity (factor of ~3) and VRC01 and10E8v4 exhibiting approximately a logarithmicincrease (fig. S5). In addition, the trispecific Abwas able to bind sequentially to each of the threeantigens (Fig. 3C), indicating that there is inde-pendent binding of each epitope.The N6 trispecific Ab also showed greater

potency and breadth relative to three relatedbispecific Abs when tested against a panel of20 viruses that were selected for resistance tobnAbs (table S1). This finding is consistent withprevious studies comparing the efficacy of mix-tures of two versus three bnAbs (18) and pro-vides additional support for the multitargetingconcept. In addition to their greater efficacy,the trispecific Abs also yielded higher proteinlevels and greater solubility than the bispecificmodel (compare fig. S2A and fig. S3), whichwould facilitate large-scale production and clin-ical translation.

Fc modification to extend half-life andcrystal structure

To identify the optimal candidate for furtherdevelopment, we determined the half-life of thetrispecific Abs in nonhuman primates (NHPs).We previously showed that in the context of theVRC01 mAb, mutations that increased bindingto the neonatal Fc receptor (FcRn), which recyclesIgG in intestinal epithelial cells and increaseslevels in the serum, extended half-life, enhanced

Xu et al., Science 358, 85–90 (2017) 6 October 2017 2 of 5

Fig. 1. CODV-Ig bispecific antibody design and neutralization titers of the VRC01/10E8bispecific antibodies. (A) CODV-Ig bispecific antibody design with two different orientations of 10E8and VRC01. (B) Neutralization titers (IC50), in mg/ml, of VRC01/10E8 bispecific Abs and parental Absagainst a select panel of 19 circulating HIV-1 strains; values highlighted in red, orange, and yellowindicate highest, medium, and lowest potency, respectively.

A B

Fig. 2. Neutralization titers of VRC01/PGT121- and VRC01/PGT128-based bispecific antibodies.(A and B) Neutralization titers (IC50), in mg/ml, of the VRC01/PGT121 (A) and VRC01/PGT128 (B)bispecific Abs against a select panel of 20 circulating HIV-1 strains, with colors as in Fig. 1.

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mucosal localization, and conferred more efficientprotection against lentivirus infection relative tothe wild-type antibody (26). One such mutationwas incorporated into the trispecific Abs as wellas the parental VRC01 and N6 Abs. Abs werethen infused into rhesus macaques, and serumlevels were analyzed over a 14-day time frame.Ab VRC01 displayed a longer half-life than the

more broad and potent N6, which was also di-rected to the CD4bs (Fig. 4, VRC01 versus N6).Similarly, the trispecific Ab containing VRC01showed greater persistence and a longer half-life (7.43 days, based on day 1–day 14 serum con-centrations) than the N6 trispecific (4.79 days) invivo (Fig. 4, VRC01/PGDM1400-10E8v4 and N6/PGDM1400-10E8v4). For this reason, and because

the N6 trispecific Ab yielded less product withdecreased solubility, we studied the VRC01/PGDM1400-10E8v4 trispecific Ab further.Further characterization was performed by

solving the crystal structure of the bispecificarm of the trispecific Ab, PGDM1400-10E8v4CODV Fab, at 3.55 Å resolution (Fig. 5, A and B).The light chain was well resolved in the electrondensity (with the exception of the two mostC-terminal residues), whereas the heavy chainshowed some regions of dynamic disorder.The most notable region consisted of part ofPGDM1400 complementary-determining regionheavy chain 3 (CDRH3) and the linker betweenPGDM1400 Fv and the heavy chain constantdomain (residues 280 to 305). Similar to theanti–interleukin-4 (IL-4)/IL-13 CODV Fab crystalstructures (23), the PGDM1400 and 10E8v4 Fvsopposed one another, with the CDRs well ex-posed to the solvent. The distance between theCDRH3s of PGDM1400 and 10E8v4 exceeded100 Å. The PGDM1400 and 10E8v4 Fvs super-posed very well with their respective parentalFv structures [RMSD (Ca) ≈ 1 Å] (fig. S6) (25, 27),confirming that their antigen-binding propertieshave been well preserved in the CODV format.Most important, the orientations of the CDRsin two Fvs were 180° from each other, whichsuggests that each antibody-combining site canindependently engage its antigen without ob-structing the other Fv structure. A model for thetrispecific Ab was constructed by combining thePGDM1400-10E8v4 CODV Fab with VRC01 (6)and the intact b12 (28) IgG crystal structures(Fig. 5C). Similar to a natural IgG, the distancebetween the monovalent fragment of antigenbinding (Fab) and CODV Fab was about 150 Å.Two of three antigens (gp120 core and gp41MPER) were also included in the model, althoughwe do not have direct evidence that all three HIVepitopes can be engaged simultaneously by asingle trispecific Ab.

Enhanced cross-protection anddecreased viral escape in vivo

To evaluate the VRC01/PGDM1400-10E8v4 tri-specific Ab for its ability to protect against in-fection, we used a mixture of two SHIVs thateach differed in neutralization sensitivity tothe parental bnAbs. In vitro assessment of thereplication-competent SHIV challenge stocksshowed that SHIV BaLP4 was sensitive to VRC01and the trispecific antibody but was resistantto PGDM1400 (Fig. 6A). In contrast, SHIV 325Cvirus was sensitive to PGDM1400 and the tri-specific Ab but was resistant to VRC01 (Fig. 6A).In a neutralization assay with an equal mixtureof SHIV BaLP4 and SHIV 325c, we observedthat the trispecific Ab could achieve completeneutralization of the viral mixture, whereasVRC01 or PGDM1400 could not (fig. S7). Whennaïve rhesus macaques were infused with thehalf-life–extended VRC01, PGDM1400, or VRC01/PGDM1400-10E8v4 (5mg/kg), respectively, serumconcentrations were maintained at levels of≥1 mg/ml formore than 14days for all Abs (Fig. 6B).A decrease in serum levels at later time points for

Xu et al., Science 358, 85–90 (2017) 6 October 2017 3 of 5

Fig. 3. Neutralization titers of trispecific antibodies and broadly neutralizing antibodies, andsequential binding of alternative Env epitopes. (A) Neutralization titers (IC50) of different bnAbsand trispecific Abs against a genetically diversepanel of 208 circulatingHIV-1 strains.The solid line denotesthe median IC50 neutralization titer of sensitive viruses; the dashed line indicates median titers of all208 viral strains. Percentages of resistant viruses are shown in the top line. (B) Breadth and potency ofthe trispecific Abs relative to other bnAbs. Colors associated with each Ab designate first-generationAbs of lower breadth (orange); second-generation bnAbs or an engineered Ab-like molecule, eCD4-IgG(brown); structurally enhanced bnAbs (green); or trispecifics (red). (C) Sequential binding of threeantigens to the trispecific Ab,VRC01/PGDM1400-10E8v4, in the indicated order.The RSC3 (45) antigenrepresents monomeric gp120 optimized for the CD4 binding site Ab VRC01. MPER peptide interactswith 10E8 (7); gp140 trimer for PGDM1400 was derived from the gp140DN6 (BG505) protein.

Fig. 4. Serum antibody levels in rhe-sus macaques infused with parentaland trispecific antibodies. Concen-trations of VRC01 (green), N6 (black),and the two indicated trispecific Abs(brown, blue) containing a Fc mutationto extend half-life were measured inserum over the course of 14 days afterintravenous administration of a single10 mg/kg dose of each antibody. Eachdata point represents the mean ± SEMof the values from two to six animalsper group (VRC01, n = 6; N6, n = 4;each trispecific Ab, n = 2) anddetermined in replicates from twoindependent experiments.

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the trispecific Ab correlatedwith the developmentof monkey anti-human Abs but arose almost2 weeks after the SHIV challenge.To ensure an adequate challenge dose, we

first challenged naïve animals with each virusindependently. For SHIV 325c, four naïve rhesusmacaques were inoculated one time intrarectallywith 1 ml of undiluted viral stock. All four ani-mals were infected and showed persistent vire-mia for up to 90 days (fig. S8). For SHIV BaLP4,the same stock and dose of virus were used asdescribed in several of our prior publications(26, 29, 30). In total, 30 control animals werepreviously challenged with a single 1-ml intra-rectal inoculation of SHIV BaLP4, and all becameinfected.To assess in vivo protection, we challenged

NHPs mucosally with a mixture of these dif-ferentially sensitive SHIVs, 5 days after Ab in-fusion in two separate experiments, with fouranimals in each group. In total, six of eightmacaques (75%) infused with VRC01 alone, andfive of eight animals (62%) treated solely withPGDM1400, became infected. In contrast, noneof the eight animals in the trispecific-treatedgroup were infected (Fig. 6C; P = 0.0058, two-tailed Fisher exact test). These data confirm thatthe improved breadth and potency of the tri-specific Ab conferred protection against virusesthat otherwise show resistance to single bnAbsalone.

DiscussionNext-generation HIV-1 bnAbs

A hallmark of HIV-1 infection is the remarkablegenetic diversity of the virus. Since 2010, sub-stantial progress has been made in the identi-fication of bnAbs that show exceptional breadthand potency [reviewed in (1)]. Several of theseantibodies have progressed into clinical trialsfor prevention or treatment, and there is renewedinterest in exploring their potential in the clinicalmanagement of HIV-1 infection (5, 12, 14). Here,we explored the potential of different bnAbs tocombine into a single protein that confers pro-tection against diverse HIV-1 strains. Among theclasses of bnAbs, we found that trispecific Absderived from bnAbs with CD4bs, MPER, andV1V2 glycan specificities had broad specificity,were potent, and could be produced in sufficientquantities to allow evaluation in NHPs and even-tually in humans. When tested in NHPs with vi-ruses resistant to individual parental bnAbs, thetrispecific Ab demonstrated complete protectionagainst both viruses, whereas infection was es-tablished in most animals treated with individ-ual parental antibodies VRC01 and PDGM1400.In addition, the ability of this trispecific Ab totarget three independent epitopes may improvetreatment efficacy in humans.In HIV-1–infected patients, reductions in viral

load have been observed after one infusion of asingle bnAb, thus demonstrating the biologicalactivity of HIV-1 bnAbs (31–34). A modest exten-sion of viral rebound was also observed whenindividual bnAbs were infused after antiretroviraldrugs were discontinued in previously suppressed

HIV-1–infected subjects (32, 33). NHP and humanpassive transfer studies have also suggested thatsuch bnAbs can enhance antiviral immunity thatmay contribute to improved viral control (35, 36).In addition, NHP studies demonstrate the im-portance of mAb potency and prolonged anti-body half-life in mediating protection againstinfection (26, 29). The generation of trispecificAbs with improved potency and breadth mayfurther enhance the efficacy of either passive im-munity or passive-active immunization strategies.Although bnAbs show exceptional breadth and

potency, resistant viral strains have been de-tected in patients who make these Abs (6, 37)and among natural viral isolates (38–40), raisingthe concern that resistance and escape mutationsmay arise. Such escape mutations are producedfrequently with antiviral drug therapy (41), andcountermeasures to reduce the likelihood ofescape would increase the likelihood of develop-ing a globally relevant therapy. Such breadth ofcoverage might alternatively be generated byadministering multiple bnAbs, and protectiveefficacy in a NHP model has recently been dem-onstrated against a mixture of SHIV viruses usingan antibody cocktail (42), providing further sup-port for the multitargeting concept. CombinationmAb therapy increases the complexity, develop-ment pathway, cost, and regulatory burdens oftheir use for treatment or prevention, in contrastto a single biologic therapy. The potency of the

trispecific Abs described here also exceeds thatof a broad and potent recombinant form of CD4(43), termed eCD4-Ig (fig. S4), and this lattermolecule is also directed to a single, albeit highlyconserved, HIV-1 Env epitope. The availability of asingle protein that targets multiple independentepitopes on virus also reduces the potentialgeneration of escape mutations. This advantagecould be related, in part, to the presence of threeindependent binding specificities at all times, incontrast to mixtures of antibodies where selec-tive pressure by individual mAbs with shorterhalf-lives may wane.

Clinical translation

The trispecific Abs have not yet been evaluatedfor safety and efficacy in humans. Initial char-acterization of their half-life in NHPs suggeststhat they behave similarly to conventional anti-bodies, but it remains unknown whether theycould be immunogenic in vivo. The administra-tion of a bispecific antibody to the human cyto-kines IL-4 and IL-13, which uses a related formatand linkers (44), may provide guidance in thisregard. This bispecific antibody has been evalu-ated in humans, where single subcutaneous dosesof SAR156597, ranging from 10 to 300 mg/kg,were well tolerated in healthy subjects, with lowtiters of anti-drug antibody (ADA) in only 4 of36 subjects (44). This trial showed amean half-lifeof about 2 weeks (44), similar to natural mAbs.

Xu et al., Science 358, 85–90 (2017) 6 October 2017 4 of 5

Fig. 5. Crystal structure of the CODV Fab and a structure model of the trispecific antibody.(A) Configuration of the trispecific antibody, color-coded by parental antibody. Dark shades (purple orgreen) denote heavy chain peptides; light shades denote light chain peptides. (B) Crystalstructure of the PGDM1400-10E8v4 CODV Fab in side and top views. CDRH3s from the two Fvsare labeled to highlight the antigen-binding region gp41 MPER, which was modeled in bysuperposing PDB 5IQ9 onto the 10E8v4 Fv. (C) VRC01/gp120 structure (PDB 4LST) and theCODV Fab were modeled onto the b12 structure (PDB 1HZH) by overlaying the CH1-CL domains.Color codes are matched in (A), (B), and (C).

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Although further human trials are neededto assess the full potential of the trispecific Abplatform, the data from the NHP challenge studydescribed here, as well as the previous experiencein humans with bispecific Abs (44), suggest thatthe approach merits further clinical investiga-tion. Studies in HIV-infected subjects, alone or incombination with other immune interventions,will address the potential of trispecific Abs toprovide durable protective immunity against in-fection or sustained viral control in HIV-infectedsubjects during drug holidays or in the absenceof antiretroviral therapy. The recognition of in-dependent target sites with multispecific anti-bodies can also be applied to other infectiousdiseases, cancer, and autoimmunity. These anti-bodies can promote recognition and binding tocritical antigenic determinants on target cellswhile simultaneously allowing engagement ofimmune cells that can stimulate relevant effec-tor function without the complications and ex-pense of delivering multiple recombinant proteins.

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ACKNOWLEDGMENTS

We thank C. Lawendowski for excellent program management;A. E. Schroeer and B. DelGiudice for graphic arts support;K. Radošević, C. J. Wei, M. Hollis, S. Rao, and B. Zhang fororganizational support; S.-Y. Ko for pharmacodynamics analysis;H. Qiu and B. Brondyk for technical advice; and L. Hou andA. Park from Sanofi for expressing and purifying the10E8v4-PGDM1400 CODV Fab used in the crystallization. Thedata are tabulated in the main text and supplementary materials.The coordinates and crystal structure factors were deposited inthe Protein Data Bank (PDB) under code 5WHZ. L.X., Z.-y.Y.,G.J.N., R.R.W., J.B., J.K., E.R., W.D.L., C.B., C.L., M. Connors, J.R.M.,R.A.K., N.D-R., T.Z., P.D.K., Y.D.K., and A.P. are inventors on patentapplication WO 2017/074878 submitted by Sanofi U.S. and theNational Institutes of Health that discloses the use of anti-HIVantibodies. Supported by Sanofi Global R&D (L.X., L.W., E.R., J.B.,J.K., D.M.L., R.R.W., G.D., C.B., C.L., W.D.L., R.S., Z.-y.Y., andG.J.N.); the National Institute of Allergy and Infectious Diseases(NIAID) Division of Intramural Research and Vaccine ResearchCenter (A.P., N.D.-R., K.M., M.L., M.A., Y.D.K., T.Z., S.D.S., R.T.B., K.W.,M. Choe, Z.M., S.O., J.-P.T., X.C., M.R., M. Connors, P.D.K., R.A.K.,and J.R.M.); NIH grants AI096040, AI124377, and AI126603 (D.H.B.and L.J.T.); and NIAID grant UM1AI100663 and IAVI (D.R.B.). Authorcontributions: Z.-y.Y., M.R., P.D.K., R.A.K., J.R.M., D.R.B., and G.J.N.designed the research; Z.-y.Y., A.P., N.D.-R., E.R., J.B., J.K., R.S.,and R.B.T. carried out the research; Z.-y.Y., L.X., A.P., L.W., M.A.,D.M.L., R.R.W., G.D., K.M., M.L., C.B., C.L., W.D.L., S.D.S., R.T.B.,K.W., M. Choe, Z.M., S.O., J.-P.T., and X.C. performed the experiments;Y.D.K., T.Z., D.H.B., M.R., and M. Connors contributed newreagents/viral strains; Z.-y.Y., L.X., A.P., N.D.-R., R.R.W., G.D., P.D.K.,J.R.M., and G.J.N. analyzed the data; and Z.-y.Y., A.P., N.D.-R.,R.R.W., J.R.M., and G.J.N. wrote the paper. All authors reviewedthe paper. VRC01, VRC07, VRC07-523, and CAP256-VRC26.25antibodies are available from J.R.M. and 10E8 and N6 antibodies areavailable from M. Connors under a material transfer agreementwith NIH. All other requests for data and further information shouldbe directed to the corresponding authors.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/358/6359/85/suppl/DC1Materials and MethodsFigs. S1 to S8Tables S1 and S2References (46–48)

30 May 2017; accepted 28 August 2017Published online 20 September 201710.1126/science.aan8630

Xu et al., Science 358, 85–90 (2017) 6 October 2017 5 of 5

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Fig. 6. Trispecific and broad neutralizing antibody sensitivity of SHIVs, plasma antibodylevels, and viremia in rhesus macaques. (A) IC50 neutralizing titers, in mg/ml, of VRC01, PGDM1400,and VRC01/PGDM1400-10E8v4 against replication-competent SHIV BaLP4 or SHIV 325c, with colorsas in Fig. 1. (B) Plasma levels of VRC01, PGDM1400, and VRC01/PGDM1400-10E8v4 in rhesusmacaques (n = 8 on each arm, done in two separate experiments with four animals each). All animalswere administered 5 mg/kg of the indicated antibody intravenously. Each data point represents themean ± SEM of the values from all eight animals per group. (C) Plasma viral loads in rhesus macaques(n = 8 per group) challenged with a mixture of SHIV BaLP4 and SHIV 325c, 5 days after intravenousadministration of VRC01, PGDM1400, or VRC01/PGDM1400-10E8v4.

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Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques

Mario Roederer, Mark Connors, Richard A. Koup, Peter D. Kwong, Zhi-yong Yang, John R. Mascola and Gary J. NabelBailer, Keyun Wang, Misook Choe, Lawrence J. Tartaglia, Dan H. Barouch, Sijy O'Dell, John-Paul Todd, Dennis R. Burton, Lange, Wulf Dirk Leuschner, Jochen Kruip, Rebecca Sendak, Young Do Kwon, Tongqing Zhou, Xuejun Chen, Robert T.Gejing Deng, Mark Louder, Stephen D. Schmidt, Zachary Mankoff, Lan Wu, Mangaiarkarasi Asokan, Christian Beil, Christian Ling Xu, Amarendra Pegu, Ercole Rao, Nicole Doria-Rose, Jochen Beninga, Krisha McKee, Dana M. Lord, Ronnie R. Wei,

originally published online September 20, 2017DOI: 10.1126/science.aan8630 (6359), 85-90.358Science 

, this issue p. 85; see also p. 46Sciencesimplify HIV treatment regimens and improve therapy response.

99% of HIV viruses. This approach could potentially∼trispecific antibodies were highly potent and broadly neutralized site (CD4bs) recruits killer T lymphocytes that can eliminate the virus. When tested against >200 different HIV strains, Cohen and Corey). This ''trispecific'' antibody uses two sites (V1V2 and MPER) to bind HIV-infected cells, while the thirdsingle antibody molecule to recognize three highly conserved proteins needed for HIV infection (see the Perspective by

engineered aet al.broadly neutralizing antibodies that can recognize and conquer multiple viral strains or subtypes. Xu The HIV virus continually evolves tricks to evade elimination by the host. Prevention and a cure will likely rely on

A triple threat for HIV

ARTICLE TOOLS http://science.sciencemag.org/content/358/6359/85

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2017/09/19/science.aan8630.DC1

CONTENTRELATED

http://stm.sciencemag.org/content/scitransmed/9/405/eaam5441.fullhttp://stm.sciencemag.org/content/scitransmed/9/402/eaaf1483.fullhttp://stm.sciencemag.org/content/scitransmed/9/406/eaal1321.fullhttp://science.sciencemag.org/content/sci/358/6359/46.fullhttp://stm.sciencemag.org/content/scitransmed/9/408/eaao4235.full

REFERENCES

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