Proc. Natl. Acad. Sci. USAVol. 90, pp. 11683-11687, December 1993Immunology

Active immunity against the CD4 receptor by using an antibodyantigenized with residues 41-55 of the first extracellular domain

(antigenized antibody/human immunodeficency virus/anti-receptor immunity/vacdnation)

PAOLA LANZA, RoSARIo BILLETTA, SVETLANA ANTONENKO, AND MAURIZIO ZANETTI*Department of Medicine and Cancer Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0961

Communicated by J. Edwin Seegmiller, July 26, 1993

ABSTRACT Using the process of "antibody antigeniza-tion," we engineered two antibody molecules carrying in thethird complementarity-determining region of the heavy chainvariable domain a 7-mer or a 15-mer peptide epitope ofthe firstextraceliular domain (D1) of human CD4 receptor-namely,Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser (SFLTKGPS; positions 42through 49) and Gly-Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser-Lys-Leu-Asn-Asp-Arg-Ala (GSFLTKGPSKLNDRA; positions 41through 55). These amino acid sequences are contained in theconsensus binding site for the human immunodeficiency virus(HIV) on CD4 receptor. Both antigenized antibodies (AgAbs)bound recombinant gpl20 and were recognized by a prototypemonoclonal antibody to CD4 whose binding site is within aminoacid residues 41-55. AgAbs were then used as immunogens inrabbits and mice to elicit a humoral response against CD4.Only the AgAb carrying the sequence 41GSFLTKGPSKLN-DRA5s induced a response against CD4. The induced antibod-ies showed specificity for the amino acid sequence of CD4engineered in the AgAb molecule, were able to inhibit theformation of syncytia between human CD4+ T cells MOLT-3and 8E5 (T cells that are constitutively infected with HIV), andstained human CD4+ CEM T cells. Four murine monoclonalantibodies were used to analyze the relationship betweensyncytia inhibition and CD4 binding at the single antibodylevel, and indicated that recognition of native CD4 is not anabsolute requirement for inhibition of syncytia. This studydemonstrates that antigenized antibodies can be used as im-munogens to elicit site-specific and biologically active immunityto CD4. The importance of this approach as a general way toinduce anti-receptor immunity and as a possible new measureto immunointervention in HIV infection is discussed.

Strategies of immunotherapeutic intervention in human im-munodeficiency virus (HIV) infection have focused recentlyon the prevention or disruption of the interaction betweenHIV and CD4 by using engineered forms ofhuman CD4 (1-7).However, clinical trials based on the passive administrationof high doses of recombinant CD4 (rCD4) or chimeric CD4-conjugated to an immunoglobulin constant region (immu-noadhesin) have shown little efficacy (8-10). Among thereasons that may account for the apparent inefficacy are (i)induction by CD4 of gp120 variants with reduced affinity andincreased fusion capacity (11); (ii) higher resistance to neu-tralization by CD4 of fresh isolates from HIV-infected pa-tients than of laboratory strains of HIV (10-12); (iii) CD4-promoted release of gpl20 from virions (13, 14) and infectedcells (15), with local formation ofgpl20-CD4 complexes (16);and (iv) blockade of CD4 by immunoglobulins or antigen-antibody complexes via the immunoglobulin binding site ofCD4 (17, 18).

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Active immunity to CD4 has been indicated recently as anew therapeutic approach for HIV infection: monkeys ac-tively immunized with human rCD4 (19) or rCD4 in completeFreund's adjuvant (CFA) (20) produced autoantibodies toautologous CD4 and mounted a generalized "anti-viral"immunity. Although it is not known whether the effect ofactive therapy was due to blockade ofvirus binding at the cellsurface or inhibition of postbinding events, these experi-ments suggest that presentation of CD4 in an immunogenicform may be sufficient to induce a humoral response oftherapeutic value.Immunization against the whole CD4 receptor molecule

could either result in the induction of antibodies against aregion of CD4 involved in major histocompatibility complexbinding (21) or in poor immunogenicity because of self-tolerance. To circumvent these drawbacks, we sought toinduce active immunity against CD4 using antibodies engi-neered to carry selected oligopeptides within the molecularenvironment of their complementarity-determining regions(CDR)-"antigenization of antibody" (22). We conforma-tionally constrained within the molecular environment of theCDR3 of the heavy (H) chain variable (V) domain of anantibody molecule two oligopeptides of CD4 that map to theputative binding sites for the virus (23, 24). We demonstratethat immunization with an antibody "antigenized" withamino acid residues 41 through 55 ofCD4 (Gly-Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser-Lys-Leu-Asn-Asp-Arg-Ala; GSFLT-KGPSKLNDRA in single-letter code) yielded antibodies thatbound CD4 and inhibited the formation of syncytia in vitro,an event principally determined by the interaction betweengpl20 on the surface of infected cells and CD4 on uninfectedcells.

METHODSAnimals. Adult New Zealand White rabbits were pur-

chased from a local breeder. Eight-week-old C57BL/6 (H-2b)mice were purchased from The Jackson Laboratory.

Generation of Antigenized Antibodies (ASAb). A host VHgene containing a single Kpn I/Asp-718 site (25) was used toclone complementary oligonucleotides coding for humanCD4-(42-49) or CD4-(41-55) peptide fragments, yielding theVHCD4A or VHCD4B genes contained in a 2.3-kilobase (kb)EcoRI fragment. These were cloned upstream from a humanIgGl constant region gene contained in the 12.8-kb vectorpNyl by our method (26). Thirty micrograms of the finalDNA constructs pNylCD4A or pNylCD4B was electropo-rated in the murine J558L cell line (20 x 106 cells) at a field

Abbreviations: AgAb, antigenized antibody; H, heavy; L, light; V,variable; BSA, bovine serum albumin; CFA, complete Freund'sadjuvant; CDR, complementarity-determining region; FITC, fluo-rescein isothiocyanate; HIV, human immunodeficiency virus; HRP,horseradish peroxidase; rCD4, recombinant CD4; VH, variable re-gion ofan immunoglobulin heavy chain; mAb, monoclonal antibody.*To whom reprint requests should be addressed.

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Proc. Nati. Acad. Sci. USA 90 (1993)

strength of 750 V/cm. Transfected cells were incubatedwithout selection for 24 hr and then selected in the presenceof 1.2 mg of G418 (GIBCO) per ml. G418-resistant clonessecreting a high level of AgAb were identified by ELISA withhorseradish peroxidase (HRP)-conjugated goat antibody tohuman immunoglobulin (Sigma). Culture supernatants wereprecipitated by (NH4)2SO4, and the AgAb was purified byaffinity chromatography on a protein A-Sepharose column(Pharmacia-LKB) equilibrated with 3 M NaCl/1 M glycine,pH 8.9. Elution was performed with 0.1 M glycine hydro-chloride/0.5 M NaCl, pH 2.8. The eluted fractions wereneutralized with 1 M Tris HCl (pH 8.0) and dialyzed against0.15 M phosphate-buffered saline (PBS; pH 7.3). The purityof the antibodies was assessed by electrophoresis on aSDS/10% PAGE.

Preparation of Monoclonal Antibodies (mAbs). Six-week-old C57BL/6 mice were immunized with y1CD4B (100 ,ug perinjection) intraperitoneally at monthly intervals first in com-plete Freund's adjuvant (CFA) and subsequently in incom-plete Freund's adjuvant. A mouse with the highest titer ofsyncytia-inhibiting antibodies served as a spleen donor. Hy-bridomas were produced by fusing spleen cells withP3X63Ag8 nonsecreting cells according to conventional tech-niques. Antibodies in the culture supernatants were screenedfor their ability to inhibit syncytia formation in the assaydescribed below. Positive wells were subcloned twice bylimiting dilution. Antibodies were purified from culture su-pernatants by protein A-Sepharose chromatography. Theirpurity was checked by SDS/PAGE.Immunochemical Reagents. Soluble recombinant gpl20

(SF2 isolate) was kindly obtained from Chiron. Soluble rCD4(Receptin) was the gift of Biogen. Leu3a, a murine mAb(IgG2a,K) to CD4 specific for amino acid residues 35-47 (27),was a gift of D. Buck (Becton Dickinson). OKT4D, a murinemAb (IgGl,K) to CD4 specific for amino acid residues 44-52(27), was the gift of D. E. Woods (R. W. Johnson Pharma-ceutical Research Institute, Raritan, NJ). HRP-conjugatedgoat antibody to human immunoglobulin (-chain specific)and goat antibody to mouse immunoglobulin (y-chain spe-cific) were purchased from Sigma. Goat antibody to rabbitimmunoglobulin [H and light (L) chains] conjugated to fluo-rescein isothiocyanate (FITC) and FITC-conjugated goatantibody to mouse immunoglobulin (H and L chain-specific)were purchased from Biomeda (Foster City, CA).

Immunological Methods. ELISA. Direct binding to gpl20was performed on 96-well microtiter plates (Dynatech)coated (1 ,ug/ml) with soluble gpl20 (Chiron) in 0.9% NaCl byovernight incubation at 4°C. AgAbs in PBS containing 1%bovine serum albumin (BSA) and 0.05% Tween 20 (PBSA)

a I A-42 49

pr SF LTKGP S--1--VGSFLTKGPSKLNDRA]41 55

VH Cy,H KYPYSHGI CHl CH2

CDR3

J558L (Xl)

were incubated for 2 hr at 37°C. Bound AgAbs were revealedby using HRP-conjugated goat antibody to human immuno-globulin (<-chain specific) (Sigma). Bound peroxidase wasrevealed by adding o-phenylenediamine dihydrochloride andH202. Tests were done in duplicate. The wild-type AgAb(y1WT) and the serum of a HIV-infected patient (HIV+serum) (1:1000 dilution) served as the negative and positivecontrol, respectively. The inhibition ofthe binding ofOKT4Dto y1CD4B was done on 96-wel microtiter plates coated withOKT4D (Pharmaceutical Research Institute) (2.5 ug/ml) in0.9% NaCl by incubation overnight at 4°C. y1CD4B conju-gated with HRP was mixed 1:1 (vol:vol) with inhibitoryantibodies in PBSA for 2 hr at room temperature and incu-bated (50 u1 per well) in individual wells for 1 hr at roomtemperature. The bound y1CD4B was washed and revealed asdetailed above. Tests were done in triplicate.

Western blot. Western blot was performed as described byTowbin et al. (28). Briefly, 1 ug of purified ASAb waselectrophoresed on a SDS/10% polyacrylamide gel underreducing conditions (5% 2-mercaptoethanol). Proteins wereblotted onto a 0.45-pm polyvinylidene difluoride (PVDF)membrane (Millipore) and blocked by soaking the membranein 5% dry milk in PBS and incubating for 1 hr at roomtemperature with OKT4D (2.5 ;tg/ml). The bound antibodywas revealed by 1251-labeled goat antibody to mouse K lightchain. PVDF membrane was exposed to Kodak X-OMATAR fim for 3 days at -70°C. The wild-type AAb (y1WT)served as a control.

Syncytia Formation Assay. Syncytia-inhibiting antibodieswere detected by using a syncytia formation assay thatutilizes CD4+ human T cells, MOLT3, and the human T-cellline 8E5, which harbors. an integrated defective HIV-LAVretrovirus genome (29). Briefly, 8E5 cells (1.2 x 106 per ml)and MOLT3 cels (2 x 106 per ml) were resuspended incomplete RPMI 1640 medium and mixed 1:1 (vol:vol), and100 ;LI of the mixture was incubated in 96-well round-bottomplates (Costar) at 37°C in a humidified CO2 incubator withorbital shaking every 5 min. After 30 min the plates werecentrifuged for 5 min at 300 x g and incubated for 3 hr at 37°C.Next, the cells were transferred to a 96-well flat-bottom plate(Costar), and the formation of syncytia was determined bymicroscopic inspection on an inverted microscope. Syncytiawere defined as cells having a balloon degeneration. Thepercent inhibition was calculated as follows: [(no. of syncytiawith the immune serum - no. of syncytia with the preimmuneserum)/no. of syncytia with the preimmune serum] x 100. Inthe case of murine mAbs, an isotype-matched antibodyspecific for thyroglobulin served as a control. The percentinhibition values were corrected for the percent inhibition

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"A "PANFIG. 1. Structure and epitope expression of AgAb, carrying oligopeptides of human CD4 and reactivity with an anti-CD4 antibody. (a)

Schematic view of AgAbs carrying human CD4-(42-49) peptide (A) or CD4-(41-55) peptide (B) epitopes in the CDR3 region of the H chain.Peptides were inserted between residues Val-94 and Pro-95. CD4 peptides, SFLTKGPS (Upper) or GSFLTKGPSKLNDRA (Lower), wereflanked at each side by a Val-Pro doublet. The A1 L chain is provided by the murine myeloma J558L used for transfection. (b) Western blotrecognition of the two AgAbs by OKT4D, a mAb to CD4. A band corresponding to the H chain is visible on both ylCD4A and .y1CD4B but notwith y1WT, the wild-type AgAb control.

11684 Immunology: Lanza et al.

Proc. Natl. Acad. Sci. USA 90 (1993) 11685

0.8 HIVc 0.6 -1 Serum

co0.2

0.2 ~ 11 10 100

Antibody, /,g/ml

FIG. 2. y1CD4A and ylCD4B bind to recombinant gpl20 inELISA. Values represent the mean absorbance (A492) of two inde-pendent experiments. Tests were done in duplicate. The wild-typeAgAb (ylWT) and the serum of a HIV-infected patient (HIV+ serum)at 1:1000 dilution served as the negative and positive control,respectively. Values of normal (HIV-) human serum at the samedilution was <0.500 A492.

value with preimmune serum at the same dilution. Routinely,Leu3a served as a positive control.FACS Analysis. Binding of rabbit and mouse antibodies to

CD4 was studied by flow cytometry using the CD4+ CEMhuman T-cell line. Typically, CEM cells were harvested inexponential growth phase, washed twice in Dulbecco's mod-ified Eagle's medium containing 1% BSA, 0.01% NaN3, and10 mmol Hepes and was incubated (1.5 x 106 cells in 200 ,ul)with rabbit serum (1:500 dilution) or murine mAbs in 1.8-mlpolycarbonate Eppendorf tubes for 90 min at 4°C. The cellswere washed and then incubated with the FITC-conjugatedgoat antibody to rabbit Ig (H and L chains) or a FITC-conjugated goat antibody to mouse immunoglobulin(Biomeda) for 45 min at 4°C, washed twice, resuspended in1% paraformaldehyde, and analyzed in an Ortho Cytofluo-rograf II.

Immunizations. Adult New Zealand White rabbits wereimmunized with .y1CD4A and y1CD4B (100 ,g per injection)subcutaneously five times at monthly intervals first in CFAand subsequently in incomplete adjuvant. Sera were col-lected 10 days after each booster immunization and tested at1:40 dilution in complete RPMI 1640 medium.

RESULTSWe engineered two antibodies to carry amino acid residues42-49 and 41-55 of the first extracellular domain (D1) of

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human CD4. Several studies indicate that amino acid residues38-56 constitute the core of the virus-binding site (23, 24).The topology of the region corresponds to the CDR2 loop ofhuman immunoglobulin L chain (30). The structure andoverall anatomy of the two engineered immunoglobulins,denoted y1CD4A and y1CD4B, are illustrated in Fig. la.OKT4D, a mAb to human CD4 that binds an epitope betweenresidues 44 and 52 (31), bound the H chain of both proteins,hence demonstrating the expression of the grafted CD4oligopeptide in an immunologically accessible way (Fig. lb).Both AgAbs Zy1CD4A and y1CD4B but not control y1WTbound purified gp120 in a solid-phase assay (Fig. 2). Thebinding of a reference standard serum from a HIV+ patient isshown by comparison. These results indicate that the CDR3loop tailored to express CD4 sequences imparted the anti-genized antibody with the ability to bind viral gpl20, hencemimicking the receptor site of CD4.The in vivo immunogenicity of the two AgAbs was assessed

by testing their ability to induce antibodies capable of inhib-iting syncytia formation as direct evidence of their binding toand interfering with a biologically relevant site of the CD4receptor. Four of five rabbits immunized with .ylCD4B [CD4-(41-55)] mounted a humoral response able to inhibit syncytiaformation in vitro (Fig. 3A). Rabbits similarly immunizedwith y1CD4A [CD4-(42-49)] did not inhibit syncytia forma-tion. Antibodies in the serum of all four rabbits also boundnative CD4 as detected by flow cytometry on human CD4+CEM T cells. A representative experiment is shown in Fig.3B, where staining of CEM cells by the immune serum iscompared to that of Leu3a, a canonical mAb to CD4.To confirm this observation and, more importantly, to

analyze the relationship between syncytia inhibition and CD4binding at the single antibody level, hybridomas were pro-duced in mice. From a C57BL/6 mouse immunized with theAgAb y1CD4B with a high titer for antibodies inhibitingsyncytia formation (250% inhibition at 1:500 dilution), weidentified hybridomas whose supernatant inhibited the syn-cytia formation reproducibly. Four clones (all IgGl,K) wereretained for further study, purified, and characterized. Allfour mAbs inhibited syncytia formation in a dose-dependentmanner (range of inhibition, 25-50%o) (Fig. 4a). We used anELISA inhibition assay to demonstrate that they were spe-cific for the amino acid sequence grafted into the CDR3 loopof the AgAb. As shown in Fig. 4b the binding of HRP-conjugated y1CD4B to OKT4D immobilized on polyvinylmicrotiter wells could be inhibited by each ofthe four murine

10 100

Fluorescence Intensity

FIG. 3. Anti-CD4 response in the serum of rabbits immunized with y1CD4A and y1CD4B. (A) Detection of syncytia-inhibiting antibodies.Rabbits 1-5 were immunized with y1CD4A, and rabbits 6-10 were immunized with y1CD4B. Preimmune sera were used as negative controls.Anti-CD4 mAb Leu3a served as a positive control. The syncytia formation assay was done with 8E5 (HIV-infected human T-cell line) andMOLT3 (CD4+ human T-cell line) cells. (B) Flow cytometry staining of CD4+ CEM cells using the serum of a rabbit (no. 7) immunized withy1CD4B. -.- (shaded area), Immune rabbit serum (1:500 dilution); , mAb to CD4 Leu3a (0.1 ,Lg/ml); * * *, preimmune rabbit serum (1:500dilution); and , FITC-conjugated goat anti-rabbit antibody.

Immunology: Lanza et al.

Proc. Natl. Acad. Sci. USA 90 (1993)

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100 50 25 12.5

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FIG. 4. (a) Dose-response analysis of syncytia-inhibiting mAbs produced by immunization with Vy1CD4B. Inhibition by OKT4D, a prototypemAb to CD4 that binds amino acid residues 44-52 and an isotype matched mAb of unrelated specificity are shown for comparative purposes.(b) mAbs to V/1CD4B inhibit the interaction between y1CD4B and OKT4D. (c) Western blot binding to human rCD4 by murine mAbs. mAbOKT4D (positive control) and an isotype-matched antibody (negative control) are also shown. (d) Flow cytometry staining ofCD4+ CEM cellsby the four mAbs generated by immunization with y1CD4B. Binding of the mAbs to CEM cells (--- -) was revealed by using FITC-conjugatedgoat antibody to mouse immunoglobulin (H and L chain specific) for 1 hr at 4°C. -, Leu3a as a positive control; * * *, cells incubated withthe FITC-conjugated goat antibody only.

mAbs, hence proving that they were specific for the loopcontaining the sequence GSFLTKGPSKLNDRA engineeredin 'y1CD4B. By immunoblot (Western blot) on rCD4, all fourantibodies bound the same molecular mass (46-50 kDa) bandand were identified by OKT4D, a mAb to CD4 whose bindingsite includes residue 47 (Fig. 4c). Last, we used flow cytometryto determine whether the fourmAbs also bound native CD4 onCD4+ CEM cells. As shown in Fig. 4d only antibody PL1stained CD4+ CEM cells. Collectively, the analysis of mAbscan be summarized as follows: four of four mAbs inhibitedsyncytia formation and bound the immunizing AgAb and rCD4,but only one reacted with native CD4 on CD4+ CEM cells.

DISCUSSIONWe proved that the antibodies elicited by immunization withan AgAb carrying a 15-mer peptide from the sequence of theputative gpl20 binding site of CD4 were (i) specific for thesequence ofCD4 grafted into the H chain CDR3 of the AgAb,(ii) reactive with rCD4 in Western blot, and (iii) active ininhibiting cell fusion and syncytia formation between infectedand uninfected T cells. Sera displaying syncytia-inhibitingactivity stained CD4+ CEM cells, implying immunologicalrecognition of CD4 in its native configuration. However, a

comparison between biological activity and binding charac-teristics at the single antibody level suggested that recogni-tion ofCD4 onCEM cells was not a prerequisite for inhibitionof syncytia formation. Several explanations can account forthis apparent paradox. One is that binding to native CD4requires higher binding affinity than that yielded by ourimmunization procedure. Another possibility is that the con-

formation of the CD4 peptide expressed in the antibody foldonly partially resembles the corresponding loop in the nativereceptor. Finally, the epitope expressed in the antibody looppossesses a limited degree of flexibility and may mimic atransitional state of CD4 during cell contact. Indeed, amodification of the virion structure upon contact with CD4has already been hypothesized (32), and it is plausible toenvision similar conformational changes for CD4 as well. Itis worth mentioning that chimpanzees immunized with theintact extracellular portion ofhuman CD4 produce protectiveantibodies that do not recognize native CD4 (33).Adhesion between infected and noninfected cells likely

represents a key event in the pathogenesis of HIV infectionas no polymorphism is known for CD4, and the virus-bindingsite for CD4 is conserved in different isolates of HIV withsubstantially divergent env gene sequences (34). UninfectedCD4+ cells can fuse with infected T lymphocytes (35) andmonocytes/macrophages (36). Syncytia are found in thebrain of AIDS patients (37) and can form around dendriticcells during antigen presentation (38). In all likelihood, cellfusion can cause consumption of CD4+ cells. Althoughsuppression of syncytia formation may not necessarily reflectinhibition of HIV infection (39), recent epidemiological dataindicate that the syncytia-forming phenotype ofHIV may beassociated with an accelerated drop in CD4+ cell count andan overall poor progression of the disease (D. Richman,personal communication). Thus, active humoral immunitytargeted at preventing syncytia formation, alone or in com-bination with other immunotherapeutic or pharmacologicalapproaches, may be clinically beneficial.

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11686 Immunology: Lanza et al.

Proc. Natl. Acad. Sci. USA 90 (1993) 11687

The approach described in here to conformationally con-strain active sites of receptors within an antibody CDR loopis another way to generate immunogens for the induction ofanti-receptor immunity. Owing to the fact that animals re-spond to isologous and autologous idiotypic determinants(40, 41), immunization by antibodies antigenized with dis-crete stretches of somatic cell receptors is also a way tocircumvent unresponsiveness to self proteins (modified self)and induce anti-receptor immunity ofexquisite molecular andbiological specificity. The present demonstration validatesantibody mimicry as a viable concept and points to a differentdirection of study in the structure-function of antibodies,including practical applications for antigenized antibodies(42).Members ofthe immunoglobulin gene superfamily serve as

receptors not only for HIV but also for poliovirus (43, 44) andrhinovirus (45, 46). Therefore, the strategy indicated in thispaper may be exploited to induce active anti-receptor immu-nity against viruses other than HIV or against receptorsinvolved in the pathogenesis of autoimmune diseases.

We thank Ms. Kelly Cooper for technical assistance. We aregrateful to the following companies for their kind gifts: Biogen forsoluble rCD4 (Receptin), Chiron for soluble gpl20, Becton Dickinsonfor Leu3a, and R. W. Johnson Pharmaceutical Research Institute forOKT4D. This work was supported by National Institutes of HealthGrant HD25787.

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Immunology: Lanza et al.