Proc. Nati. Acad. Sci. USAVol. 89, pp. 3175-3179, April 1992Biochemistry

Human combinatorial antibody libraries to hepatitis Bsurface antigenSUZANNE L. ZEBEDEE*, CARLOS F. BARBAS I1t, YAO-LING HOM*, ROGER H. CAOTHIEN*, RICHARD GRAFF*,JULI DEGRAW*, JAYASHREE PYATI*, ROBERT LAPOLLA*, DENNIS R. BURTONt, RICHARD A. LERNERt,AND GEORGE B. THORNTON**The R. W. Johnson Pharmaceutical Research Institute, 3535 General Atomics Court, Suite 100, San Diego, CA 92121; and tThe Scripps Research Institute,Departments of Molecular Biology, Immunology and Chemistry, 10666 North Torrey Pines Road, La Jolla, CA 92037

Contributed by Richard A. Lerner, December 26, 1991

ABSTRACT Human antibody Fab fragments that bind tohepatitis B surface antigen (HBsAg) were generated by using arecombinant phage surface-display expression system. Char-acterization ofHBsAg-specific Fab fragments isolated from twovaccinated individuals reveals diversity in specificity of antigenbinding and in the sequences of the complementarity-determining region. The sequence results show examples ofhuman light-chain promiscuity that result in fine specificitychanges and a strong relationship to a human germ-line gene.This application illustrates further that this technique is apowerful tool to isolate distinct human antibodies againstimmunogenic viral targets.

The expression of diverse human monoclonal antibodiesthrough the use of combinatorial systems has potential for theidentification and development of these reagents as thera-peutics. Use of a modified bacteriophage A genome forbacterial expression ofboth human and mouse antibodies hasbeen described (1-6). The essence of this approach is that arepertoire of antibodies can be re-created from randompairings of cloned heavy- and light-chain genes.

Recently, improvements to the expression of combinato-rial antibody libraries were developed to allow for antigen-specific screening of larger libraries (7, 8). These improve-ments are based on reports demonstrating the expression ofpeptides and proteins on the surface of filamentous phage(9-18). The combinatorial antibody approach directs theexpression of recombinant antibody Fab fragments to thesurface of filamentous phage through the selection of aphagemid that coexpresses the combinations of heavy- andlight-chain genes. Heavy-chain expression is linked to thephage coat protein gene III membrane anchorage domain,and the resulting fusion protein becomes anchored into theperiplasmic space. Expression of the light chain from theplasmid DNA as a secreted protein permits Fab assemblywithin the periplasmic space. After helper phage infection,this antibody Fab then becomes incorporated into a phage byreplacement of the wild-type gene III protein, and the Fab isdisplayed on the phage surface.The main advantage of the phage surface technology is the

ability to sort large combinatorial libraries by a powerfulenrichment and selection process. The selection processmakes the combinatorial antibody technology attractive forthe isolation of specific high-affinity human antibodies andfor antibody isolation from individuals with low serum titersto the desired antigen.To examine the application of the phage surface-display

technology to a viral system, we chose to generate andcharacterize human antibodiest to hepatitis B surface antigen(HBsAg). The availability of vaccines, purified antigen, and

murine antibodies for epitope mapping makes the hepatitistarget an ideal model system to study the immune responseto a viral antigen.

MATERIALS AND METHODSImmunization and RNA Preparation. A human volunteer

was immunized with the hepatitis B virus recombinant vac-cine Recombivax (Merck). On day 7 following the secondbooster injection (individual BO), whole blood was collected.A second volunteer, who received Heptavax (Merck) injec-tions in 1986, followed by the Recombivax immunizationschedule in 1990 (individual JM), donated lymphocytes byleukophoresis on day 14 following the second booster injec-tion. The cells from both individuals were pelleted, andleukocytes were separated on Histopaque gradients (Sigma).RNA was prepared by sequential extractions with 6 M and7.5 M guanidinium chloride (19). Serum titers to HBsAg werechecked with the AUSAB test (Abbott).

Library Construction. RNA isolated from JM and BO wassubjected to reverse transcription (Perkin-Elmer) using the3'-specific heavy-chain (yl, Fd region) and K and A light-chainprimers previously described (4, 20). The antibody DNAswere then amplified in the presence of the corresponding5'-specific primers for heavy- and light-chain genes by poly-merase chain reaction (PCR) techniques. Heavy-chain PCRproducts were combined and digested with Xho I and Spe I,and K and A light-chain DNAs were separately combined anddigested with Xba I and Sac I to yield three populations ofdigested antibody DNAs (per individual) for gel purificationand cloning as described (21). These fragments were clonedinto the pComb3 vector (7, 21, 22). Following bacterialtransformation, the four combinatorial libraries were treatedas described by Barbas and Lerner (21) to prepare phage.

Solid-Phase Selection. Microtiter wells were coated with100 Ag ofHBsAg (adw and ayw subtypes; Boston Biomedica)overnight at 40C. The wells were blocked, incubated with oneof the four phage libraries (typically >101" colony-formingphage per well), washed, and eluted (7, 22). The selectedphage were then allowed to infect Escherichia coli XL1-Bluecultures (Stratagene), used to prepare new panned phagestocks, and reincubated with antigen in microtiter wells.From each of three incubations with HBsAg, the outputphage titer was quantitated as described (7, 21).

Nitrocellulose Filter Assay. Bacterial clones from eachround of antigen panning were streaked on agar plates,induced, transferred to nitrocellulose, and treated with chlo-roform (21, 23). The filters were blocked and incubated with

Abbreviations: HBsAg, hepatitis B surface antigen; CDR, comple-mentarity-determining region; FR, framework; HRP, horseradishperoxidase.MThe sequences reported in this paper have been deposited in theGenBank data base (accession nos. M88309-M88319).

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HBsAg (ad/ay) particles (0.5 Ag/ml) in phosphate-bufferedsaline containing 1% bovine serum albumin, washed in phos-phate-buffered saline containing 0.05% Tween-20, incubatedwith horseradish peroxidase (HRP)-conjugated mouse anti-HBsAg antibody (16D; Ortho Diagnostics), and developedwith 600 ,ug of 3,3'-diaminobenzidine (Sigma) per ml and0.03% hydrogen peroxide.

Conversion to Soluble Fab Fragments and Periplasmic Ex-tract Preparation. Purified phage DNA, isolated from thethird round of infected bacterial cells, was digested as de-scribed (21) to remove gene III sequences. Single clones werepicked from the resulting transformation, grown in 5 ml ofLBbroth with carbenicillin (50 ,ug/ml) to an OD6w of 1.0, andinduced with 2 mM isopropyl P-D-thiogalactopyranoside(IPTG) overnight at 370C. Culture supernatants were takenafter the bacteria were pelleted. Extracts were prepared,essentially as described (24), from the pellet after resuspen-sion in residual medium, incubation with 50 ttl of chloroformat 250C for 30 min, addition of400 1.d of 10 mM Tris, pH 8.0/1mM phenymethylsulfonyl fluoride, and centrifugation at 7000rpm in a Sorval SS34 rotor to remove bacterial debris.Supernatants and extracts were stored at 40C.Fab Concentration Determination and Indirect ELISAs. To

determine the concentration of human Fab in each periplas-mic extract, a sandwich ELISA was performed as follows:sheep anti-human Fab (Cappel Laboratories) was coated inCostar 96-well plates overnight at 4°C, periplasmic extractwas added in serial 3-fold dilutions for seven rows, followedby HRP-conjugated anti-human Fab and substrates for colordevelopment. Supernatant and extract Fab amounts wereplotted and quantitated according to known concentrations ofhuman IgG (Cappel) as a standard. For indirect ELISAs,plates were coated with either adw or ayw subtype HBsAg(0.5 ,ug/ml) and then incubated with a standardized amountof protein extract in serial 5-fold dilutions for eight rows,followed by HRP-conjugated anti-human Fab for detection.

Competition ELISAs. Mouse ascites fluids from six hybrid-oma lines (two d epitope-specific lines, two a lines, and twoy lines) were obtained from Dave Milich (The Scripps Re-search Institute). HBsAg (adw or ayw; Boston Biomedica)was coated onto microtiter plates at 0.5 ,g/ml overnight at4°C. Wells were blocked, incubated with human Fab at 4times maximal binding levels (OD490 of 2.5, determined byindirect ELISA) for 1 hr at 37°C, and then incubated withmouse antibody for 1 hr at 37°C at a concentration to yield anOD of 2.5. Wells were washed and developed with HRP-conjugated anti-mouse (Fab)2. Percent inhibition was calcu-lated as 100 - {[experimental OD/control OD (without initialcompetitor)] x 100}. For the reciprocal assay, mouse anti-body was incubated with HBsAg-coated plates at a 4-foldexcess, and human Fab to yield an OD of 2.5 was added anddetected with goat anti-human Fab, essentially as describedabove.

Nucleotide Sequence Analysis. Plasmid DNA was preparedas described (19) and nucleotide sequence was determined byusing Sequenase 2.0 (United States Biochemical). To obtainthe heavy-chain sequences, primers from the 5' vector se-quence (5'-GGCCGCAAATTCTATTTCAAGG-3') and theCH1 constant region (5'-CGCTGTGCCCCCAGAGGT-3')were used. To obtain the light-chain sequence, the 5' vectorsequence (5'-CTAAACTAGCTAGTCGCC-3') and CAprimer (5'-GAGACACACCAGTGTGGC-3'), or CK primer(5'-CACAACAGAGGCAGTTCC-3') were used. The se-quences were assembled by using MacVector analysis pro-grams (IBI).

Electron Microscopy. Phage that contained membrane-bound Fab fragments were isolated from individualJM clonesafter the initial (negative) or third (positive) panning round toHBsAg and confirmed for binding by using the filter assay asdescribed above. The phage were diluted to 104 colony-

forming units per jul and incubated with adw/ayw HBsAg(International Enzymes, Fallbrook, CA) (0.5 ,ug/ml) for 30min at 250C. This mixture was incubated with 300-mesh gridspreviously bound with rabbit anti-HBsAg for 10 min and thenwas fixed and treated with uranyl acetate for staining. Thefields were viewed with a Hitachi HU12a at x 30,000 andx60,000 magnification.Western Blot Analysis. Human Fab fragments from IPTG-

induced 5-ml culture supernatants were concentrated (Cen-tricon 10; Amicon) and separated in a nonreducing 10%oacrylamide gel (19). The proteins were transferred to nitro-cellulose (25), blocked, incubated with HRP-conjugated goatanti-human Fab, and developed.

RESULTSTo prepare human monoclonal antibodies that recognizehepatitis B virus, the antibody repertoires from two individ-uals vaccinated with recombinant HBsAg were cloned andexpressed by a phage surface-display technique (7). Eachindividual was immunized with recombinant HBsAg vaccine,and lymphocytes were collected on day 7 (individual BO) orday 14 (individual JM) following the second Recombivaxboost. Serum titers to HBsAg were examined and found to be1:200 (BO) and 1:10,000 (JM). RNA was extracted from eachof the cell samples and used to prepare PCR-amplifiedantibody-specific DNAs corresponding to heavy-chain (yl,Fd region) and K and A light-chain families for cloning into thepComb3 vector system. Four libraries were generated andexpressed on the surface of phage: BO heavy chains with BOK or A light chains, and JM heavy chains with JM K or A lightchains.To select the population ofhuman Fab fragments that bind

to HBsAg, the phage libraries were incubated with HBsAg(adw and ayw subtypes) in a microtiter well. The phage thatbound to the well were eluted, reamplified in bacteria, andreincubated (panned) with HBsAg for three rounds ofantigenselection. This enrichment process was monitored by titeringthe phage colony-forming units eluted from the antigen-coated well at each stage (Table 1). Two of the four pannedlibraries, BOA and JMK, were enriched for phage that bind toHBsAg as demonstrated by the increasing number of colony-forming units eluted as the panning proceeded. Two of thelibraries did not show any specific enrichment for HBsAg-positive phage (BOK, JMA).To confirm the enrichment numbers, a nitrocellulose filter

assay for the detection of HBsAg antibodies was devised.This assay involves incubating the membrane-bound Fabfragments with HBsAg particles and detection with a mousemonoclonal antibody to HBsAg (HRP-conjugated). The num-ber of clones that expressed HBsAg-specific Fab fragmentsfor the BOA and JMK libraries increased as the panning stagesprogressed (Fig. 1). This result parallels the enrichmentnumbers and shows that the third-pan phage stocks were95-100%o positive for anti-HBsAg. Interestingly, two positiveJMA clones were also detected by this assay on the third-panfilter, whereas the enrichment data did not identify thepresence of positive binders in this library.The expression of anti-HBsAg Fabs on the surface of

phage allows electron microscopic examination of the phage

Table 1. Enrichment of repertoire libraries to HBsAgColony-forming units

Library First pan Second pan Third pan

BOK 2.3 x 10 4.0 x 1O 3.79 X 104BOA 6.2 x 103 4.99 x 104 5.12 x 106JMK 1.6 x 10 9.5 x 105 3.6 x 106JMA 9.6 x 104 3.1 x 104 3.5 x 104

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First Pan Second Pan

/..* 4. ,... .w ~~~v

BO

jm!1.21

BJM x

Ame..'.1* I

.

JM x

FIG. 1. Nitrocellulose filter assay to detect HBsAg antibodies.Agar plates were streaked with the bacterial colonies from the initial(pre-pan), first, second, or third panning stage and transferred tonitrocellulose. After chloroform treatment, the filters were incubatedwith HBsAg (Boston Biomedica) (1 ug/ml), washed, and thenincubated with HRP-conjugated mouse anti-HBsAg (Ortho Diagnos-tics) prior to substrate development. Panning stages and libraries areas indicated.

interaction with the HBsAg particle. Freshly prepared phagefrom several JMK clones (chosen from pre-pan or third-panfilter) were incubated with adw/ayw subtype HBsAg parti-cles, secured to the microscopy grid by rabbit anti-HBsAg,and negatively stained (Fig. 2). Electron micrographs frompositive (third pan; Fig. 2B) phage show an association of thefilamentous phage with HBsAg particles on one end of thephage. Interactions of single phage with a particle, two phageper particle, and single phage with two particles bound wereobserved (data not shown).To characterize further the antibody Fab fragments, each

of the third-pan phage populations from the positive libraries(BOA, JMK) was converted from membrane-bound to solubleFab fragments. This manipulation requires removal of thegene III sequences from the pComb3 phagemid and subse-quent library transformation into bacteria for the productionof soluble Fab. To verify Fab expression in the periplasmicspace and in the medium, proteins from individual convertedclones were separated by gel electrophoresis and examinedfor Fab production by Western blot analysis using goatanti-human Fab secondary antibody. Under nonreducingconditions, a single positive band at 50 kDa (or 25 kDa forreducing conditions), equivalent to the human Fab controlwas seen for each of the clones examined (data not shown).To examine the population of converted soluble clones for

reactivity to HBsAg, Fab concentration was quantitated tonormalize for the Fab production of each clone. The extractFab concentrations ranged from 0.1 to 16.0 pug/ml (data notshown). The clones were tested for HBsAg binding by anindirect ELISA with equivalent amounts of Fab from 24clones from each of the two positive libraries (BOA, JMK).Thirty-two clones that bound to HBsAg in a concentration-dependent manner were identified (see Fig. 3 for examples).Sixteen clones that bound HBsAg were selected (8 from eachindividual) for further analysis and comparison.

FIG. 2. Electron microscopy ofHBsAg particles and filamentousphage. Fresh phage were prepared from the pre-pan stage (A) or thirdpan stage (B) of the JMK library and incubated with HBsAg particlesfor 30 min. These complexes were added to rabbit anti-HBsAgantibody attached to a grid and then were washed, fixed, andnegatively stained. The grids were viewed with a Hitachi electronmicroscope. (x 35,000.)

Competition ELISAs using mouse monoclonal antibodieswith defined specificities to the group (a) or subtype (d, w)epitopes of HBsAg were performed on selected Fab clonesfrom both libraries (Table 2). The assays were performedusing the human Fab fragments as the competitor and showedcompetition of mouse antibody binding. The results were

3-

2-

0 -

0

1-

10.02 0.05 0.16 0.47

Fab, ng/ml

l1.4 4.22

FIG. 3. Specificity of binding of human soluble Fab to HBsAg.Equivalent concentrations of clone periplasmic extracts were dilutedand incubated with bound HBsAg in a 96-well plate. After the platewas washed, anti-human Fab coupled to HRP was added, followedby reagents for color development. m, BO clone 08; o, BO clone 10;*, BO clone 12.

Initial

BO i.

Third PanX.e

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Table 2. Percent inhibition of mouse monoclonal antibody binding to HBsAg with human Fab fragmentsHuman antibody Fab Polyclonal

Mouse BO JM serumantibody 08 09 10 12 L2 01 10 15 HIV* BO JM5D 14.3 0 0 0 0 45.9 62.4 22.4 0 84.5 43.315D 33.4 24.3 27.2 44.5 0 26.0 18.3 0 0 66.0 70.010A 25.9 11.3 13.9 24.3 2.1 2.4 0 4.0 0 47.3 57.014A 0.5 0 0.7 0 12.6 7.6 27.2 4.2 0 70.0 29.22Y 0 2.0 0.1 0 0 0 0 3.0 0 12.4 25.27Y 0 0 14.5 0 12.2 0 0 8.0 0 29.0 31.0Assays were performed with adw subtype (5D, 15D, 10A, 14A) or ady subtype (2Y, 7Y).

*Human immunodeficiency virus human Fab clone 14 (ref. 22).

confirmed to be similar in a reciprocal assay with mousemonoclonal HBsAg antibody as the competitor (data notshown). The general binding trends of the human Fab frag-ments show differences between BO and JM clones, but allofthe selected Fabs from each individual appear to recognizea similar epitope (except the JMA clone). Clones isolatedfrom the BOA library inhibit binding of the 15D and 10Amouse antibodies, whereas the JMK clones inhibit the bindingof the 5D mouse line. Data obtained for the polyclonal serumfrom BO and JM (Table 2) demonstrate that these antibodyspecificities were present in the sera of both individuals.The nucleotide sequences encoding the variable domains

of heavy and light chains from each of the selected cloneswere determined. Fig. 4 compares the predicted amino acidsequences for the HBsAg binders. Of the eight selected BOAclones, five share identical heavy- and light-chain sequences(as shown for clone 08 light chain and BO heavy chain, Fig.4), and the others differ only in the light chain (clones 09, 10,12). A comparison of the four different A chain sequencesshows mostly identical FR structure (26) with amino acidsubstitutions in CDR1, CDR2, and CDR3 domains among theclones. The FR4 region of the light-chain J domain has alsocontributed to the diversity of these clones.The nucleotide sequence results for the JM clones exam-

ined predict four distinct light-chain proteins and two heavy-chain proteins (Fig. 4). The two positive A clones identifiedfrom the HBsAg filter assay (see Fig. 1) were sequenced andfound to be identical (Fig. 4, L2 and JMa). The same heavychain (JMa) that pairs with this A light chain was also foundto associate with two other K clones (Fig. 4, clones 01 and 15).The other sequenced K clones (five separate clones) from JM

were found to use a different pairing ofheavy chain (3Mb) andlight chain (Fig. 4, clone 10).The K sequences ofJM share a similar FR (26) but differ in

the length and composition of the CDR domains. The twoisolated JM heavy chains have similar FR sequences exceptfor FR2 length variability; the CDR sequences contain aminoacid substitutions, with the most variation observed in CDR3.

DISCUSSIONCombinatorial antibody libraries generated from human sam-ples have been used to isolate high-affinity human antibodyFab fragments to tetanus toxoid (4, 7), human immunodefi-ciency virus type 1 gpl20 (22), and HBsAg (this work). Wehave used the phage surface-display technique to isolateHBsAg-specific binders from the lymphocytes of vaccinatedindividuals with both high and low titers to HBsAg and haveshown that high-affinity antibodies with sequence variationcan be selected. The data further demonstrate the power andselection capabilities ofthe combinatorial antibody techniquefor the cloning of human antibodies.HBsAg is a well-characterized molecule that can readily be

used to study the gene III expression system and antigenimmunogenicity in humans. Our electron microscopy resultsconfirm both that antibody Fab fragments are expressed onthe phage surface (7) and that the Fab-gene III fusion isproperly located at the phage end (27, 28). The rare obser-vation ofa single Fab-expressing phage bound to two HBsAgparticles suggests that some of the phage may express morethan one Fab on their surface with this system. However,since the majority of the observed particle-phage associa-

LIGHT CHAIN SEQUENCE

FR1 CDR1 FR2 CDR208 AELTQPPSASGTPGQRVTISC SGSSSNIGTNTVN WYQQLPGRAPKLLIY SNNERPS

a 09 AELTQPPSASGTPGQRVTISC SGSSSNIGTNTVN WYQQLPGAAPKLLIY SNNERPS10 AELTQPPSASGTPGQRVTISC SGSTSNIGTNTVN WYQQLPGTAPKLLIY SNSERPS12 AELTQPPSASGTPGQRVTISC SGSSSNIGSNTVN WYQQLPGTAPKLLIY SNNERPS

FR3 CDR3 FR4 CLGVPDRFSGSKAGTSASLAISGLOSEDEADYYC EAWDDNLHGPV FGGGTRLTVLR QPGVPDRFSGSKSGTSASLAISGLOSEDEADYYC EAWDDSLQGPL FGGGTKLTVLG QPGVPDRFSGSKSGTSASLAISGLQSEDEAEYYC EAWDDSLQGPV FGGGTKVTVLG QPGVPDRFSASKSGTSASLAISGLQSEDEADYYC AAWDDSLHGPV FGGGTKLTVLR QP

JMX L2 AELTQPPSVSGAPGQRVTISC TGSSSNIGADSEVT WYTRLAGAAPKLLIF DNSFRPS GVPDRFSVSRSGTSASLAITGLQAEDEGDYYC QSFDKEPEESN FRRGTRVTVLR QP

01 AELTQSPGTLSLSPGERATLSC RASQSVFSNYLAJMK 10 AELTQSPATLSLSPGERATLSC RASQSVSSYLA

15 AELTQSPGTLSLSPGERATLSC RASQSVSSSYLA

WYQQKPGQAPRLLIY GASSRAT GIPDRFSGGGSGTDFTLTINRLEPEpFAVYYC QQYGSSPSTWYQQKPGQAPRLLIY DASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPPSWYQQKPGQAPRLLIY GASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPT

HEAVY CHAIN SEQUENCE

FRI CDR1 FR2 CDR FR3 CDR3 FR4 CHIBO AQVKLLESGAEVRKPGASVKVSCKASGYTFT GYYLH WVRQAPGQGLEWMG WIHPNNGGTFHPPKFRG RVTMTRDTSISTAYMELSSLRSDDTAVYYCAI EYGDTDCGGDCYSI WGQGTLVTVSS AS

JMa AQVKLLESGPGLVKPSETLSLTCTITGGSIS SSSYFWG WIRQPREGAEWIG SIYSSGTTYYNPSLES RVTMSVDTSKNQFSLNLDSVTAADTAVYYCAR PLRLNYNSRSYYPGIGPFDM WGQGTMVTVSS ASJMb AQVKLLESGPGQVKPSETLSLTCTVSGGPLS SSSNYWG WIRQTPGKRLEWIG SIYNSGTAYYNPSLET RVTISVDTSKNQVSLNLKSVTAADTAMYYCAR PLTHDDFLTAYYPGGGYFDL WGRGTLVTVSS AS

FIG. 4. Predicted amino acid sequences for the selected BO and JM anti-HBsAg clones. The nucleotide sequences of eight BOA, two JMA,and seven JMK clones were determined and translated into protein sequence' (single-letter code). The sequences are segregated intocomplementarity-determining regions (CDRs) and framework (FR) regions based on homology to known sequences (26). Only the clones withunique sequences are presented here for the light chain and the heavy chain. Sequences are as follows, from top to bottom: light chains BOA08,BOA09, BOA10, BOA12, JMA2, JMKO1, JMK10, and JMK15 and heavy chains BO (assembles with all BO light chains), JMa (assembles with JMA,JMKO1, and JMK15), and JMb (assembles with JMK10). Five clones of the BOA library and of the JMK library were sequenced and found to beidentical to the BOA08 and JMK10 sequences.

FGQGTKVELK RTFGGGTKVEIK RTFGGGTKVELK RT

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tions were one particle per phage, the monovalent Fabdisplay model for this technique is supported.As expected, the individual with low serum titer to HBsAg

(BO) showed a more limited immune response to the vaccinethan the high-titer individual (JM). Of the characterized BOanti-HBsAg binders, only limited light-chain sequence diver-sity was observed: the light chains were found in associationwith the same heavy chain, and the Fab fragments allrecognize a similar epitope of HBsAg. However, among theclones analyzed from the high-serum-titer (JM) library, sig-nificantly more diversity was observed. Two heavy-chainproteins were identified and several light chains showeddifferences in CDR length and composition, especially in theCDR3 and FR4 regions. The hypervariability of the CDR3domain has been noted previously (29, 30) and can becontributed by various mechanisms of D-region diversity.A comparison of the CDR3 heavy-chain sequences from the

human anti-HBsAg clones with the antibody data base did notidentify exact matches with other known antibody sequences.However, light-chain comparisons revealed that the K chainfrom JM clone 15 is identical in FR, CDR1, and CDR2 to thegerm-line HumKv325 gene (31) and homologous in all the CDRsto the autoantibody TayKv322 (32). Other antibody sequencesthat are related to the HumKv325 gene include antibodies torheumatoid factor, chronic lymphocytic leukemia, Sjogren syn-drome, and cytomegalovirus (32-35). The significance of thisK-chain similarity between the HBsAg antibodies and the au-toimmune antibodies is unclear; but some viruses may play arole in inflammation (34-36), or alternatively, the human germ-line light-chain antibody repertoire may be limited.Antibody characterization data for the HBsAg Fab frag-

ments suggest that much of the antigen recognition is depen-dent upon the human heavy chain, and the light chains showmore promiscuity. For example, the same BO heavy chainassociates with several light-chain sequences to recognize thesame epitopes on HBsAg. Also, the specificity of the germ-line-related JM15 clone for HBsAg is strictly dependent onthe heavy chain, as an identical light chain has been docu-mented to assemble with distinct heavy chains to binddifferent antigens (31-35). Human heavy-chain promiscuitywas also observed among the JM clones; one heavy chain(JMa) was able to assemble with both K and A light chains,although the two classes of antibodies recognize differentepitopes (see Table 2). These results demonstrate that chainpromiscuity can be utilized to affect specificity changes andthat chain shuffling experiments can be utilized to re-engineerantibodies via the combinatorial approach (37).

Eight different variable-region clones with sequences thatvary in heavy- and light-chain combinations, framework sub-groups, and CDR length and composition have been charac-terized. It cannot be determined from the combinatorial ap-proach used to generate the positive Fab fragments whetherthese exact heavy/light chain pairings occur in the antibodyrepertoire of the vaccinated individuals, but only that theseparate heavy and light chains exist. The importance of eachisolated heavy/light chain combination is the identification ofa functional Fab that has high affinity and specificity for thetarget antigen. Changes in the antibody sequences from eachof the original individual repertoires, such as potential muta-tions from PCR error and cloning construction (at the aminoterminus of each mature protein), have been tolerated by thebinding of the expressed antibody to HBsAg.The isolation of human antibody Fabs that bind to hepatitis

B virus will allow their examination as passive immunizationagents for the prevention and therapy of viral hepatitis B.Antibodies to HBsAg are known to protect against infection(38). We have described human antibodies that possess theprotective group (a) reactivity. These antibodies, generatedfrom HBsAg-selected combinatorial libraries, represent pro-totype passive immunotherapy candidates.

We thank Didier Leturcq and Ann Moriarity for help in assaydevelopment and Cheng-Ming Chang for electron microscopy work.We thank Dave Milich and Raju Koduri for discussions, T. T. Wu forhelp with sequence comparisons, and John L. Gerin, Jack Geltosky,and Raju Koduri for reviewing the manuscript.

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