Neutralizing Recombinant Human Antibodies to a Conformational V2- and CD4-Binding Site-Sensitive Epitope of HIV-1 gpl20 Isolated by Using an Epitope-Masking Procedure'

Henrik J. Ditzel,2*t James M. Binley,* John P. Moore,* Joseph Sodroski,§ Nancy Sullivan,' Lynette S. W. Sawyerjl R. Michael Hendryjl Wei-Ping Yang,' Carlos F. Barbas Ill,' and Dennis R. Burton2*'

*Departments of Immunology and ¶Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037; 'Department of Medical Microbiology, Odense University Medical School, Odense, Denmark; *Aaron Diamond AIDS Research Center, New York University School of Medicine, New York, NY 1001 6; 'Division of Human Retrovirology, Dana-Farber Cancer Institute, Boston, MA 021 15; and IlViral and Rickettsial Disease Laboratory, California Department of Health Services, Berkeley, CA 94704

As part of the goal of assembling a mixture of neutralizing human mAbs for possible prophylaxis and therapy of HIV-1 disease, we describe a strategy by which neutralizing human Abs to a weakly immunogenic epitope can be accessed. From a phage display library derived from an asymptomatic HIV-1 seropositive donor, a panel of recombinant Fabs against the CD4 binding site (CD4bs) of gpl20 was retrieved by affinity selection using recombinant gpl20 (strain LAI). Two Fabs corresponding to the dominant clones were used to mask the CD4bs epitope(s) before repeating the selection procedure. Four Fabs were then retrieved that had novel heavy chain sequences. Three recognized a novel epitope distinct from that recognized by conventional CD4bs Abs and were defined by the following criteria: 1) second V region (V2 region) dependence indicated by sensitivity to amino acid changes in the V2 loop and by competition with murine anti-V2 mAbs; 2) CD4bs dependence indicated by sensitivity to amino acid changes usually associated with CD4 binding and by inhibition of Fab binding to gpl20 by soluble CD4; this dependence seemed to arise via conformational changes rather than by direct binding, as CD4bs Abs enhanced binding of two of the novel Fabs and, in a reversal of the competition format, the novel Fabs did not inhibit soluble CD4 binding to gp120; and 3) equivalent binding to glycosylated and deglycosylated gpl20 and significant, although much reduced, binding to denatured gpl20 in con- trast with CD4bs Abs, which do not bind to deglycosylated or denatured gpl20. One of the novel Fabs efficiently neutralized the MN and LA1 strains of HIV-1. These results indicate the presence of a novel neutralizing conformational epitope on gpl20 sensitive to the V2 loop and the CD4bs and further highlight the conformational flexibility of gpl20. The strategy of masking highly immunogenic epitopes with Abs to rescue a broader range of specific Abs from com- binatorial libraries should be widely applicable. The journal of Immunology, 1995, 154: 893-906.

B oth the cellular and humoral immune systems are involved in the immune response against HIV-1, the etiologic agent of AIDS (1, 2). Abs against

epitopes on the HIV-1 surface glycoprotein gp120 and, to

Received for publication May 12, 1994. Accepted for publication October 20, 1994.

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

and by Johnson &Johnson. H.J.D. was supported by the Danish Research Council ' This work was supported by National Institutes of Health Grant A133292-02

and Odense University School of Medicine. ].P.M. was supported by the Aaron Diamond Foundation and New York University School of Medicine CFAR. J.S. was supported by National Institutes of Health Grants A124755 and A131 783, by Cancer Center (CA06516) and Center for AIDS Research (A128691) Grants to the

Charitable Foundation and from the late William McCarty-Cooper. C.F.B. is a Dana-Farber Cancer Institute, and by gifts from the G. Harold and Leila Y. Mathers

Scholar of the American Foundation for AIDS Research and a recipient of a Scholar Award from the Cancer Research Institute.

lesser extent, the transmembrane glycoprotein gp41 con- stitute most of the neutralizing humoral response to HIV-1 (3, 4). Such serum Abs are capable of inhibiting both HIV-1 cellfree infectivity and cell-to-cell spread.

gp120 is a heavily glycosylated protein with multiple internal disulfide bonds (5-7). Comparison of divergent HIV-1 isolates has revealed that the structure of gp120 can be separated into five relatively conserved (C1 through C5) and five variable regions (V1 through VS) (7, 8). Three regions on the gp120 molecule seem to be espe- cially important for Ab-mediated virus neutralization: the

'Address correspondence and reprint requests to Dr. Dennis Burton or Dr. Henrik Ditzel, Department of Immunology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037.

Copyright 0 1995 by The American Association of Immunologists 0022.1 767/95/$02.00

894 NEUTRALIZING HUMAN HIV-1 Abs FROM PHAGE LIBRARIES

V33 region and the discontinuous epitope forming the CD4 binding site (CD4bs) have long been recognized as epitopes for neutralizing Abs (9-13), and recent data in- dicate that rodent Abs recognizing the V2 region also me- diate virus neutralization (14-18). Whereas the CD4bs is responsible for the initial binding of the virus to the CD4 molecule on the surface of target cells (19, 20), both the V2 and the V3 regions are important for the subsequent virus-cell fusion process, although the exact mechanisms are unknown (21-27). It has often been considered that the CD4bs, V2 region, and V3 region comprise separate epitopes, although recent reports indicate some interde- pendence between distinct domains of the same protein

Given the strain heterogeneity of the HIV-1 virus (31), an Ab-based strategy for therapy or prophylaxis will likely re- quire Abs targeted to several epitopes. The preparation of combinatorial libraries from variable heavy and light chain Ab genes provides an efficient route for the selection of high affinity human mAbs (32). From such libraries, Abs of inter- est can be cloned, using Ag-binding as the selection system. The construction of such Ab libraries on the surface of m13 bacteriophage has been described (33-35), as has their ap- plication to the generation of a large range of human mAbs against viruses, including HIV-1, RSV, HSV-1, HSV-2, VZV, CMV, and hepatitis B (12, 36-40).

Obtaining Abs with different specificities against a given Ag from combinatorial libraries can be difficult be- cause certain epitopes may dominate. This would seem to be the case for the CD4bs of gp120, in that virtually all of the Abs we obtained from a library derived from an asymptomatic HIV-1 seropositive donor were directed to this site and were competitive with one another (12, 36, 37). Abs recognizing the V3 loop of HIV-1 MN were se- lected from the same library by using a constrained pep- tide corresponding to the tip of the loop (36, 37).

Here we report on the preparation of an additional li- brary from a long-term asymptomatic HIV-1 seropositive individual and describe a method by which Abs to minor epitopes can be obtained. Human monoclonal Fabs to a novel epitope sensitive to both the V2 region and the CD4bs of gp120 were generated along with Fabs directed against the CD4bs. Several of the Fabs to the CD4bs, and one to the novel epitope, showed potent neutralizing abil- ity for laboratory strains of HIV-1.

Materials and Methods Lymphocyte RNA preparation and library construction

(17, 28-30).

RNA was prepared from 5 ml of bone marrow lymphocytes aspirated from a long-term (>6 years) asymptomatic HIV-1 seropositive donor.

Abbreviations used in this paper: V3 region, third variable region; V2 region, second variable region; CD4bs, CD4 binding site; SB medium, superbroth medium; AP, alkaline phosphatase; SATA, N-succinimidyl-S-acetylthioac- etale; NPP, nitrophenol substrate.

After reverse-transcription, the yl-(Fd region) and K-chains were ampli- fied by the PCR, as described (32). To increase the efficiency of restric- tion enzyme cutting of PCR-amplified material and subsequent library construction, a number of extension primers were used, as described (40). These oligonucleotides contain a poly(GA) tail 5’ to the original primer sequences, increasing the number of bases between the cutting site and the end of the molecule. The subsequent construction of 1 g G l ~ Fab li- braries using the pComb3 m13 surface display system has also been described previously (33,34,36). In brief, the amplified light chain DNA was cut with the restriction enzymes Sac1 and XbaI and ligated with SucIiXbuI-linearized pComb3 vector for 3 h. The ligated material was purified and transformed by electroporation into 200 p,l Escherichia coli XL1-Blue cells. After transformation, the culture was grown overnight first in SOC medium and then superbroth (SB) medium containing car- benicillin and tetracycline. For cloning of the heavy chain, phagemid DNA was digested as described above, except that the restriction en- zymes SpeI and XhoI were used. The transformed final IgGlK Fab library was grown in SOC medium for 1 h at 37°C after addition of SB medium containing carbenicillin (50 pg/ml) and tetracycline (10 pg/rnl). After 3 h, helper phage VCS-m13 (10” plaque-forming units) was added, and the culture was shaken for an additional 2 h. Kanamycin (70 pg/ml) was then added, and the culture was incubated at 30°C overnight. The super- natant was cleared by centrifugation (4000 X g for 20 min) at 4°C. Phage were precipitated by addition of 5% polyethylene glycol and 0.15 M NaCl and incubation on ice for 30 min followed by another centrifuga- tion. Phage pellets were resuspended in PBS buffer containing l% BSA and microcentrifuged for 3 min to pellet debris.

Amplification of Ag binding phage through library panning

Panning of the combinatorial libraries without epitope masking was con- ducted as described previously (36). In brief, four microtiter wells were coated overnight with 0.5 &well recombinant gp120, W strain (gp120 BRU, American BioTechnologies, Cambridge, MA) (41) in 0.1 M bi- carbonate buffer, pH 8.6, at 4°C. Plates were washed three times with water and blocked with PBS containing 3% BSA for 1 h at 37°C. The BSA was discarded, and S O p1 phage resuspended in PBS were added to each well and incubated for 2 h. For Ab masking, the wells were previ- ously incubated with the purified Fab fragment L42 and b3 ((37) a Fab with an aminoacid sequence nearly identical to L28) in a concentration of 40 Fg/ml for 1 h at 37°C. One-half of the volume was then removed before adding 50 pl of phage. Unbound phage were removed by vigorous washing 10 times with PBS containing 0.05% Tween 20 (PBS-Tween). Bound phage, enriched for those bearing Ag-binding surface Fabs, were eluted with acid. The eluted phage were amplified by infection of E. coli and superinfection with M13 helper phage. The panning procedure was repeated 4 times, following which soluble Fab fragments were obtained by excision of the cpIII gene, achieved by NheI and SpeI digestion and subsequent ligation of the compatible cohesive ends of the vector.

ELISA analysis of soluble Fab fragments

Fabs were prepared as bacterial supernatants through a freeze-thawing procedure, as reported earlier (33, 34, 36). To assess specificity, super- natants were screened in an ELISA system (36). Coating of ELISA wells with gp120 at a concentration of 0.1 Fg/well and subsequent blocking were conducted as described above. To assess specificity, Fabs were tested against BSA, OVA, human placental DNA (all from Sigma Chem- ical Co., St. Louis, MO), and transferrin. Briefly, supernatants were in- cubated for 2 h at 37°C. Plates were washed 10 times with PBS-Tween, and bound Fabs were detected with an alkaline phosphatase (AP)-labeled goat anti-human IgG F(ab’), Ab (Pierce, Rockford, IL) diluted 1500 in PBS, visualized with nitrophenol substrate (NPP substrate) (Sigma Chemical Co.), and read at 405 nm. Donor sera from the donor was evaluated for the presence of specific Abs against the gp120 by the Same ELISA. To investigate whether the epitopes recognized by the Fab frag- ments were conformational, gp120 was denatured and reduced by boiling for 5 min in PBS containing 1% SDS and 50 mM DTT before 10-fold dilution into PBS containing 1% Nonidet P-40 to the concentration used (0.1 pg/well). Native or denatured gp120 was then captured on a solid phase via the carboxyl terminus by using sheep polyclonal Ab D7324 (Aalto Bioreagents, Dublin, Ireland). A murine Ab BAT-085 (17), which

The Journal of Immunology 895

has been shown to react almost as well with denatured gp120 as with the native molecule. was used as a control.

Purification of Fabs

gpl20-binding Fabs were purified as previously described (38) with mi- nor modifications. In brief, E. coli containing appropriate clones were inoculated separately into 1-liter cultures of SB containing carbenicillin (50 pg/ml), tetracycline (10 pg/ml), and MgCl, (20 mM), and grown at 37”C, with shaking, for 6 h. Protein expression was then induced with 2 mM isopropyl P-o-thiogalactopyranoside and grown at 30°C overnight. Soluble Fab was purified from bacterial supernatants by affinity chroma- tography, using a sheep anti-human matrk (Schleicher & Schuell, Keene, NH). The column was washed with a PBS solution and Ab-eluted in 0.2 M glycine-HC1 buffer of pH 2.2 and immediately brought to neutral pH with 1 M Tris-HC1, pH 9.0.

Nucleic acid sequencing

Nucleic acid sequencing was conducted on a 373A automated DNA se- quencer (ABI, Foster City, Ca) by using a Taq fluorescent dideoxy ter- minator cycle sequencing kit (ABI). Primers for the elucidation of heavy chain sequence were SEQGz (S’-GTCGTTGACCAGGCAGCCCAG-3’) hybridizing to the (+) strand and the T3 primer (S’-A’ITAACCCTCAC TAAAG-3‘) hybridizing to the (-) strand. For the light chain, SEQKb primer (5’-ATAGAAGTTG’ITCAGCAGGCA-3’) and KEF primer (5’- GAATI’CTAAACTAGCTAG’ITCG-3’) were used, binding to the (+) and (-) strands, respectively.

Alkaline-phosphatase labeling of Fabs

The Abs L39, L40, L78, L28, and L42 were labeled with AP by using the two-step maleimide method (42). In brief, the heterobifunctional cross- linker (sulfosuccinimidyl 4(N-maleimidomethyl) cyclohexane-l-carbox- ylate) (sulfo-SMCC) in approximately 50-fold molar excess was allowed to react with primary amines on calf intestinal AP for 30 min at room temperature to form a stable amide bond, and the maleimide-activated AP was subsequently separated by gel filtration. To form sulfhydryl groups on the Fab fragments, N-succinimidyl-S-acetylthioacetate (SATA) in approximately 50-fold molar excess was allowed to react for 30 min at room temperature with 1 ml(200 to 700 pg/ml) of Fab in 100 mM phosphate, 100 mM EDTA buffer, pH 7.1, and then transferred to a vial containing 5 mg hydroxylamine-hydrochloride. After incubation for 2 h at room temperature, the deacetylated Fab derivative was separated from hydroxylamine-HC1 and byproducts by gel filtration. Finally, 0.7 ml of SATA-derivatized Fab (ap- proximately 0.4 mg/ml) was incubated with 1 mg of malehide-activated AP for 2 h. Labeled Abs were tested for reactivity against gpl20, BSA, and OVA to assure that the binding specificity was not affected by the labeling procedure.

Fab competition ELISA

Cross-competition experiments were performed between the directly AP- labeled and unlabeled human Fabs for binding to immobilized gp120. In each case, a fixed concentration of labeled Fab, which, in earlier titration experiments, had been determined to give 75% of maximum binding, was incubated with gp120 and unlabeled Fab in twofold dilution steps (0.01 to 100 pg/ml) in PBS for 2 h. The wells were washed, and bound M - labeled Fab was detected with NPP substrate.

CD4 competition EL /SA

gp120 was coated onto ELISA wells and blocked with BSA as described above. Recombinant Fab in twofold dilution (from 0.3 to 100 pg/ml) were incubated 1 h with the immobilized gp120 before soluble recom- binant CD4 (AIDS Research and Reference Reagent Program, Division of AIDS, National Institutes of Health, Bethesda, MD) at a fixed con- centration of 1 p d m l was added and incubated for an additional 2 h at 37°C. After 10 washes with PBS-Tween, a mouse anti-CD4 mAb Q425 (42) (AIDS Research and Reference Reagent Program, Division of AIDS, National Institutes of Health) was added and incubated for 1 h. The wells were again washed with PBS-Tween, incubated with an AP- labeled goat anti-mouse IgG F(ab’), (Pierce), and visualized with NPP

substrate. The CD4-Ab competition experiment was also reversed in for- mat: soluble CD4 in 10-fold dilution (lo”, to M) was incubated for 1 h at 37°C with immobilized gp120 before Fab at a fixed concen- tration, which, in earlier titration experiments, gave 75% of maximum binding, was added and incubated for another 2 h. After washing with PBS-Tween, bound human Fab were detected with A P anti-human IgG F(ab‘), and visualized as described above.

Inhibition €LISA to estimate Fab binding affinities

Relative Fab binding affinities were estimated by competition ELISAs, as described previously (12). gp120 was coated onto ELISA wells and blocked with BSA, as described above. Fab fragments, which had been determined by titration experiments to give 75% of maximum binding, were incubated with free gp120 (10”’ to 10” M) for 2 h at 37°C. Bound human Fab were detected as described above.

Surface plasmon resonance to measure Fab binding affinities

The kinetics for Fab binding to recombinant gp120 (MN or W strain) were determined by surface plasmon resonance-based measurements us- ing the BIAcore instrument (Pharmacia, Piscataway, NJ). The sensor chip was activated for immobilization with N-hydroxysuccinimide and N-ethyl-N’-(3-diethyl aminopropyl) carbodiimide. gp120 was coupled to the surface by injection of 50 p l of a SO-pg/ml sample. Excess activated esters were quenched with 30 pI ethanolamine, 1 M, pH 8.5. Typically, 4000 resonance units were immobilized. Binding of Fab fragments to immobilized gp120 was studied by injection of Fab in a range of con- centrations (0.5 to 80 pglml) at a flow rate of 5 pl/rnin. The association was monitored as the increase in resonance units per unit time. Dissoci- ation measurements were acquired after the end of the association phase but with a flow rate of 50 pl/min. The binding surface was regenerated with HCI, 1 M NaCI, pH 3, and remained active for 20 to 40 measure- ments. The association and dissociation rate constants, k,, and k,,, were determined from a series of measurements, as described (43-45). Equi- librium association and dissociation constants were deduced from the rate constants.

Deglycosylation of gp 120

The sequential removal of sialic acid and galactose from gpl20 coated onto microtiter plates was conducted as previously described (46, 47). Briefly, gp120 microtiter wells were coated overnight, as described above. After washing, the wells were treated with 100 mU/ml sialidase (Behringwerke) (50 pl/well) at 37°C for 2 h and thereafter rinsed with 50 mM sodium acetate buffer, pH 4.5, before exposure to 25 mM sodium periodate in 50 mM sodium acetate buffer, pH 4.5, for 1 h at room temperature. Alternatively, the plates were treated with 10 U/well E. coli /3-galactosidase (Sigma Chemical Co., grade 1% in 0.1 M phosphate buff- er/l mM MgCl, for 24 h at 37”C, after sialidase digestion. After washing and blocking with 3% BSA in PBS for 1 h, the plates were incubated with Fabs to gp120. Bound Fabs were visualized as described above.

Competition with murine V2 and V3 Abs

Cross-competition experiment were performed between the human Fabs and two murine anti-V2 mAbs, SC258 and 684-238 (17) (kindly pro- vided by Dr. Gerry Robey, Abbott Laboratories, Irving, TX), and with two murine anti-V3 mAbs, RN3-50.1 and IIIB-V3-13 (48,49) (AIDS Research and Reference Reagent Program, National Institutes of Health). Coating of gp120 was conducted as described above. Competing Ab at a concentration 100 times that giving 75% maximum binding in previous titration experiments was incubated with the human Fab for 2 h after washing and detection of the human Fab, as described above. The assay was also reversed so that the human Fab was added at a concentration 10 times and 100 times that giving 75% maximum binding in previous ti- tration experiments. The murine Ab was detected with an ”labeled secondary Ab. In each case, controls with no competing Ab and irrele- vant Ab were included.

896 NEUTRALIZING HUMAN HIV-1 Abs FROM PHAGE LIBRARIES

Binding of Fabs to gp120 mutants

COS-1 cells were transfected with pSVIIlenw plasmid expressing either wild-type or mutant HIV-1 (HXBc2) envelope glycoproteins. After 48 h incubation, the gp120 in the culture supernatant (supplemented with 1 % Nonidet P-40 detergent) was captured onto a solid phase using a sheep anti-gpl20 Ab D7324, as described elsewhere (17, 30). A fixed concen- tration of recombinant Fab, determined by previous titration curves to give approximately 75% of the level of binding at saturating Fah con- centration, was added in TMTSS buffer (17, 30). After incubation and washing, bound Fabs were visualized by using AP-conjugated anti-Fab Ab, followed by the AMPAK amplification system (Dako Diagnostics) (50). Because the level of binding was nonsaturating for the detection system used (OD,,, values of 0.3 to 0.8 for most Fabs), this method measures both enhancing and inhibitory amino acid substitutions. As a reference, the binding of a pool of HIV seropositive serum to each mutant was determined. The binding of a Fab to each mutant was tested in triplicate and the mean value was expressed as a binding index, which is the ratio of OD,, for the Fab to that for the reference serum corrected for background (mock transfection supernatant). The average value of this ratio for the whole mutant panel was calculated. Ratios deviating <0.5 times or >1.5 times from the mean ratio were considered significant inhibitory or enhancing amino acid substitutions, respectively.

HIV-microplaque neutralization assay

The assay to determine neutralizing Ab titer was conducted as described, with minor modifications (51). In brief, Fabs were 3-fold serially diluted in 50% assay medium and 50% NHPP and preincubated in quadruplicate with equal volume (25 ~ 1 ) containing 20 plaque-forming units (pfu) of HIV-1 (MN or LA1 strain) per well for 18 h in 96-we11 microtiter plates in 5% C02/95% air atmosphere at 37°C. Thereafter, 90,000 MT-2 cells in a volume of 25 @I were added to each well and incubated an additional h at 37°C. Assay medium (75 @I) containing 1.6% SeaPlaque Agarose (FMC Bioproducts, Rockland, Maine) heated to 39.5"C was added to each well and the plates were immediately centrifuged (500 X 8 ) for 20 min at 20°C to form cell monolayers. Plates were incubated for 6 days at 37°C and then stained with 50 &ml propidium iodide in PBS. After 24 to 48 h, fluorescent plaques were counted on a transilluminator (304 nm). Each Fab was run at least twice against each strain, and the most potent neutralizers were titered. The neutralizing titer was defined as the con- centration of Fab required to give a 50% reduction in plaque numbers as compared with controls containing no Fab. This dilution was interpolated between data points, Within each run, the intrinsic statistical error of the interpolated titers averages 2 30%. A well-characterized neutralizing Fab was included in each test as an internal control.

Neutralization of HIV- 1 by using an envelope complementation assay

The ability of recombinant Fabs to neutralize the MN isolate and the HXBc2 molecular clone of the HTLV-IIIB (LAI) isolate were assessed in an envelope complementation assay (52). Briefly, COS-1 cells were co- transfected with a plasmid expressing envelope glycoproteins and a plas- mid containing an env-defective HIV-1 virus encoding the bacterial chloramphenicol acetyltransferase gene. Equal fractions of the cell su- pernatants containing recombinant virions were incubated at 37°C for 1 h with varying concentrations of Fab before incubation with Jurkat cells. Three days post-infection, Jurkat cells were lysed, and chloramphenicol acetyltransferase activity was measured.

Results Library construction and Ab selection

An IgGlK Fab library was prepared from the bone marrow of a long-term asymptomatic HIV-1 seropositive male do- nor. The library, designated L, consisted of 2 X lo6 mem- bers. Donor serum, taken concomitantly with bone mar- row samples, showed serologic activity with an Ab titer of 1:400 for gp120 LAI. The library was panned against re- combinant gp120 LA1 coated onto microtiter wells to en-

rich for specific Ag-binding clones, Four rounds of pan- ning resulted in a 50-fold amplification of eluted phage. Phagemid DNA was prepared from the second, third, and fourth rounds of panning and the gene 111 fragment was re- moved by treatment with the enzymes NheI and SpeI fol- lowed by religation. The reconstructed phagemid was used to transform XL1-Blue cells to produce clones secreting soluble Fab fragments.

From the fourth round of panning, 15 randomly picked clones were grown in 10 ml culture, and the supernatants containing the Fab fragments were screened for reactivity with recombinant gp120 LA1 in an ELISA system. All clones examined reacted with gp120, but not with BSA. From the second and third round of panning, 7 of 15 and 12 of 15 clones showed reactivity against gp120. Sequence analysis of the variable regions of the heavy chain re- vealed that 10 of the 34 positive clones were unique. As shown in Figure 1A, the sequences could be organized into four distinct heavy chain types. The sequence discrepan- cies within the groups beginning with L28 and L4l are very small and could arise from the PCR or the reverse transcription method. However, there is a marked differ- ence of 8 amino acids between the two clones in the group beginning with L42 that is more likely attributed to in vivo somatic mutation. The variable light chains (V,) of the gp120 binding clones were also sequenced and organized into the groups defined by Figure lA, as shown in Figure 1C. As also observed with the gpl20-binding Fabs from the M library (37), extensive chain promiscuity was seen; different V, were able to combine with the same, or a very similar, V, without affecting the Ag-binding ability in the gp120 ELISA, as exemplified in the group beginning with L28. We also observed the opposite result on one occa- sion: different V, binding (L28A, LA2) to the same V,. In the further characterization of the Ahs, we chose one as representative from each group, namely L28, L33, L42, and L52. L28 and L42 dominated the Ab repertoire ob- tained, constituting 21 of 34 gpl20-binding clones.

Library panning after epitope masking by Fabs

To examine the possibility of obtaining a larger panel of Abs reacting with different regions of gp120, Fabs corre- sponding to the two dominant clones from the initial pan- ning of the L library were purified and used to mask their respective epitopes on gp120, before repeating panning of the L library. After four rounds of panning, 15 of 20 clones were positive. Sequence analysis of the heavy chains re- vealed that all the clones obtained contained V, sequences different from those obtained without epitope masking (Fig. 1B). These new clones could be organized into 4 new groups of Abs according to their V, sequence. The V, sequences of the clones are shown in Fig. 1D. For further characterization of these Abs, we chose one representative from each group (L39, L40, LA1, and L78).

The Journal of Immunology 897

F R 1 C D R l F R 2 C D R 2 F R 3 CDR3 FR4

A L 2 8 L E E S G G G L V K ~ G S L R L S C A G S G F ~ ~ T NAWMT W R Q S P G K G L N A S I K S K F m G S S H Y h G P V E G RFTISRNDLEDKLFLEMSGLWEDTGVYYCAT KYPRYYDMMRGVRNHYYMDV WGKGl"JIVss ~ 2 8 ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ 2 8 ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . p . . . . . P . . . . . . . . . . p . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ 2 8 ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

~ 3 3 LEQSGAELKKPGESSKISCKSGY~P GHFIH WRQAPGQGLWK RINPNTGGTHYVEKFKD RVTMTRDSS~TAYMELNRLTSDDTAIYFCAR DFKLRNYAWLLDPYYMDV W~'X:?TvlVss

~ 4 2 LESGFGLVKPSATLSLSC?*JSGGSLN ~ y y w WIRQPPGKGLEWLG SLHYSGlTDYNPSLGS RISISMDASXNQFSLh?TSJSA~'DAARYYCAR SKGDYDFFRGYPRYYFDS WGQGiiLVAVSA ~ 4 2 ~ ... D . . . . . . . . . . . . . . . . . A . . . ... C . . . . . . . . . . . . . . . . D . . . . . . . . . . . . . . . . . E . . . . . . . . . . . . . R . . . . . . . . . . . ................. . T . ,

L 5 2 LEQSGPGLVEPSKTLSIHLCNPPGTJSLT NWAWN WIRQSPSRGLEWLG RTYYRSKWSNDYA>JSVKS RITINPDTSXNQFSLQLNSJTPEDTAVYYCAR GAPSYDFWSGYPAYFDY 1.IGQGTLVWSS

. . . .

B L 3 9 LESGPGLVKPSQTLSLSCWSRGSIGRP GYYWS WIRQHPGLEWIG YIYYMGSTYYNPSFES RVLIsVDTSQDQFSLKLSsVTAhDTAIYYCAR VPLSTJPGALAYYFDY WGKGTLVWSS

L 4 0 LESGPGLVKPSQTLSLTCWSGASVSS SYYWS WIRQPAGKGLEWIG HIYSSGSTNYNPSLKS RVTMSVDRSKNQFSLKLSSVTMDTAVYYCAR ERYDNVWGRLWFDP WGQGTLV?Z'SS

~ 4 1 LESGGGWQPGRSLRLSCAC~GFTFS DYGW KLrRQkPGKGLRrNA HVWDCGSYQNYRDS'YKG RFT1SRDNSKNTLYLQMNSLRAEDTA~'YYCAP. U T F G V L R L L K G W F D P HZQGTL'r'T:SS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

L 4 1 6 T R . . . . ~ 4 1 ~ , . D .

. . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

L 7 8 LEESGGGVVQPGRSLRLSCAVSGFIFD NSGMN WRQAPGKGLEWJA VISYDAQHQDYGDSVRG RFTISRDNSKNTLFLQMNRLRTDDTA'JYFCAK GAWGLGYSYMDV NGSGTAVWSS

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

CLONE FR1

C L 2 8 L 2 8 A L 2 8 6 L 2 8 C

L 3 3

L 4 2 L 4 2 A

L 5 2 ~

D L 3 9

L 4 0

L 4 1 L 4 1 A L 4 l 6

L 7 8

MAELTQSPGTLSLSPGARATLSC . . . . . . . . D . . . . . RGE . . . . . . . . . . . . L S . P V T . . E P . S I . . . . . . . . . . D . . . . . R . K . . . . . .

MAELTQSPSPPCLCLQGKEPTLSC

MAEL'IQSPLSLPVTPWRAGVSISC . . . . . A . . . . . H . . . . R P . . . Y

~ E L T Q S P S L P V C A S V G D R V T I ~

MAEL~SPPSLSr?F ' jGDRVTIAC

MAELTQSPSSLSASVGDRVTISC

MAELTPSPSSLSASVGDRVTFAC . . . V . . . . . . . . . . . . . . . . I . . . . . . . . . . T . . . . . . . . . . . I . .

MAELTQSPSSLSASVGDRLTITC

C D R l F R 2

RASQSVSSNLA WYQQKPGQTPRLLIY

WS . . . LLHSNGYNYLD . . L . . . . L S . Q . . V . A , . . . . .

. . . . . . . . . . . . . . . . . . . A , . . . .

. . . . . . . . . . . . . . . . . . .

RASQSVSSNLA WYQQKFGQAPRLLIY

WSSQSLLHSNGYNYLD WYLQKFGLSPQLLW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

QASQDI'JNRLN WYQLKPGRAPYLLIY

CDR2

DASTRAT G . . . . . . L G . N . .S G.. . . .

DTSNRAT

V . . . . . . LGSNRAS

DTSNLEE

FR 3

GIPDRFSGSGSGTDFTLTISRVEPEDFAVYYC . . A . . . . . . . . . E . . . . . . S L Q S . . . . . . . . .'I. . . . . . . . . Y . . . K . . . . . A . . VGT . . . . A . . . . . . . . E . . . . . . S L Q S . . . . . . .

GIPARFSGSGSGTDFTLTISSLEPEDSAVYYC

GVPDRFSGDTSGTYFTLKISRVEAEDVGVYYC .S . . . . . . SG . . . . . . . . . . . . . . . . . . . . . .

G'IPARFSGDTSGTEFTLTISSLQPEDVATYYC

C D R 3 F R 4

Q Q Y G S F P I T FGQGTRLDLKRWA . . . NNR.Y. . . . . W E 1 . . . . . M . h L Q T . Y . . . . . . K . E I . . . . . . .NNR . Y . . . . . .WJEI . . . . .

QQYGSRPGYT FGQGTKLEIKRTJA

MQhLQTPYT FGQGTRLEIKRTIA . . . . . . . . . . . . . . . . . . . . . ZQTSAFPLT FGQGTRLEIKRTIA

RASQPISNSLN WYQHKPGMPNLLIY TASRLRS i~lPSRFSGSGSGTDFTLTIISLQPEDFATYYC LQSYGPPPT FGQGTKVFI'KRTIA

PASQNIASRLA WYQQKPGMPNLLIY D A S N L W GVPSRFSGDTSGTEFTLTISSLQPEDVATYYC QQTSAFPLT FGQGTRLEIKRTJA

RASQSISNYLN WYQQKVGWFKLLIY AASTLQS G'JPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGYSTPEYT FGQGTKLEIKRWA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

RASQDISEYLA WYQQKPGFAPKLLIY AASTLQS G'IPARFSGSGSGTDFTLTISSLQPEDFATYFC QQFKIYPLT FGGGTKVEIRRTJA

FIGURE 1. Amino acid sequences of variable heavy ( A and €3) and light ( C and D) domains of anti-gpl20 Fabs retrieved without ( A and C) and with (Band D) epitope masking. The N-terminal sequence LEQSG arises from the VH1 a and LEESG from the VH3a primers (36). Identity with the first sequence in a group is indicated by dots.

To confirm that the new Abs selected were not related to some irreproducibility in the method, the L library was panned again against unmasked gp120 LAI. After four rounds of panning all of the 9 positive gpl20-binding clones had sequences identical with those obtained in the first panning experiment without epitope masking. The L library was also panned against gp120 of the SF2 strain. After four rounds of panning, five positive clones were sequenced. Two clones had V, sequences similar to L28, two had sequences similar to LA2 and the last sequence was identical with L33: all of these clones were obtained before epitope masking using gp120 of the LA1 strain. The results underline the reproducibility of the method and also confirm that the primarily selected Abs are strain cross-reactive.

Ab competition studies

To determine if the Abs obtained after epitope masking were directed against novel epitopes on gp120, the Fabs L39, LAO, L41, and L78 were labeled with AP by using the two-step maleimide method. Competition ELISA with la- beled Ab and masking Abs is depicted in Figure 2 A . The

data show that binding to gp120 of Fabs L39, L40, or L78 was not inhibited by pooled L28 and L42. Binding of L78, and especially of L40, was in fact enhanced by previous binding of L28 and LA2. In contrast, the binding of Fab L41 was inhibited by L28 and L42 (Table I).

The two Abs used for masking were also AP-labeled to insure that the previous competition results were not a re- sult of labeling artifacts. Competition ELISA with the la- beled masking Ab and Fabs L39, L40, L41, and L78 are depicted in Figure 2B. In this assay, LA1 blocked the bind- ing of L42. In contrast, Fabs L39, L40, and L78 did not inhibit L42. Similar results were obtained with L28 (data not shown). This implies that L39, L40, and L78 are di- rected to a different site on gp120 than L28 and L42, whereas L41 is directed to a closely related epitope.

Ab characterization and epitope mapping

To estimate the affinities of the Fab fragments for gp120 LAI, inhibition ELISAs were conducted by utilizing sol- uble recombinant gp120 to compete with microtiter well- bound gp120 for Fab binding. As shown in Figure 3, all the Abs had apparent affinities of 1 X lo7 to 5 X lo8 M-'.

A 0

0

!.a&€& CI L39 0 L40 E II L41 II II

0 L78 I I I I n I I m

,001 .01 . 1 1 10 100 1000

pg/ml competing Fabs (L28 and L42)

B

CI 0

II

Conmetitor

CI L39 L40

II L41 0 L78

.001 .01 . 1 1 1 0 100 1000

pg/ml Competing Fab

FIGURE 2. A ) competition of AP-labeled L39, L40, L41, or L78 with unlabeled L28 and L42. B ) competition of AP-la- beled L42 with unlabeled L39, L40, L41, or L78. In each case, a fixed concentration of the labeled Fab was used, which had been determined by titration experiments to give 75% of maximum binding. Bound labeled Fab was visual- ized with nitrophenol substrate.

For more detailed binding analysis, several Fabs were cho- sen for further study using real-time biospecific interaction analysis (BIAcore). Association constants (K,) and disso- ciation constants (Kd) for Fab binding to gp120 MN and LAI strains are presented in Table 11. Binding constants for LAI correlated to those obtained by inhibition ELISA, with K, in the range of 5 X lo7 to 5 X 10‘ M-‘; i.e., Kd in the range of 2 X lo-’ to 1 X M. Fabs L41 and L42, both directed to the CD4bs, showed equivalent bind- ing constants for MN and LAI, whereas Fab L28 showed

NEUTRALIZING HUMAN HIV-1 Abs FROM PHAGE LIBRARIES

tighter binding to LAI. All previously tested CD4bs Fabs have also shown tighter binding to LAI (data not shown). Of the Abs obtained with epitope-masking, LA1 had the tight- est binding of all the Fabs studied with a Kd for gp120 in the nanomolar range. Fabs L40 and L78 had slightly lower af- finities and bound gp120 LAI more strongly than MN.

To investigate the ability of the panel of Fabs to com- pete with soluble CD4 for gp120 binding, competition ELISAs with gp120 coated onto microtiter wells were conducted. Fab in increasing concentration was allowed to react with gp120 before a constant amount of soluble CD4 was added. All Fabs retrieved without epitope masking, and Fab L41 retrieved with epitope masking, inhibited binding of CD4 to gp120. In contrast, L39, LAO, and L78 did not inhibit CD4 binding. The competition experiment was also reversed by incubating increasing amounts of sol- uble CD4 with Fabs at a fixed concentration giving 75% of maximum binding. In this assay, all the Fabs in the panel were competed by soluble CD4 (Fig. 4). The most favored interpretation of these results is that Fabs L28, L33, L41, L42, and L52 are directed against the CD4bs, whereas Fabs L39, LAO, and L78 are directed against an epitope distinct from the CD4bs. However, the binding of soluble CD4 to gp120 induces a conformational change of gp120 which abrogates the binding of these Abs. The same con- formational change in gp120 does not seem to be induced by the anti-CD4bs Abs described above. Instead the bind- ing of anti-CD4bs Abs actually enhances binding of LAO and L78 to their epitopes (Table I).

The panel of Fabs was further assessed for reaction with gp120 LAI, either in native form or after denaturation by boiling in the presence of DTT and SDS. As shown in Figure 5 and Table I, the binding of all of the Fabs was reduced by denaturation of the gp120 LA1 molecule. How- ever, whereas denaturation totally abolished reactivity with the CD4bs Fabs, Fabs L39, L40, and L78 still exhib- ited significant binding.

To further characterize the epitopes recognized, recom- binant gp120 LAI was treated with neuraminidase/&ga- lactosidase or neuraminidase/periodate to remove (pl-4)- linked galactose from N-linked sugars. As shown in Figure 6 and Table I, binding of all the Fabs obtained before Ab masking and L41 obtained after masking, was absolutely dependent on this glycosylation. In contrast, Fab L40 binding was only partially affected by removal of periph- eral monosaccharides, and binding of Fabs L39 or L78 was unaffected by either sialidaselp-galactosidase or pe- riodate treatment.

A panel of gp120 mutants expressed in COS-1 cells was also used to characterize the epitopes recognized by the panel of combinatorial human Fabs. This panel of mutants, generated by mutations in the env gene of the HXBc2 HIV-1 molecular clone and cloned into the pSVIIIenv plasmid, have previously been used to characterize the CD4bs and panels of Abs to gp120. The OD value of Fab binding to a given mutant was compared with the binding

The Journal of Immunology 899

Table I. Binding pattern of the panel of human recombinant Fabs

Ahs on Fab Binding sCD4 Bindlng Fah Binding Denatured Deglycosylated Sensitivity to v 2 Effect of CD4bs Effect of Fah on Effect of sCD4 on Binding to Binding to

Fab Designation to gp120 to gpl20 to gpl20 gP120 gP120 Mutations

Retrieved without masking L28 inhibit inhibit inhibit no L33 inhibit inhibit inhibit no L42 inhibit inhibit inhibit no L52 inhibit inhibit inhibit no

- no

no no

- no -

-

Retrieved witt masking L39 no effect no effect inhibit weak + Yes L40 enhance no effect inhibit weak + Ye5 L41 inhibit inhibit inhibit no no L78 enhance no effect inhibit weak + Yes

-

120 , I

I DL28 - DL33 - DL42 7 DL52 "t DL39 - DL41 - DL40

1 DL78

- 1 3 - 1 2 - 1 1 - 1 0 - 9 - 8 -7 - 6 Log [competing gp120 IllB]

FIGURE 3. Relative affinities of recombinant human Fabs for gpl20 LA1 as measured by inhibition ELISA.

of the Fab to the wild-type gp120 and expressed as a bind- ing ratio. Fabs L28 and L42, were first studied (Table 111) and their binding to HXBc2 gp120 in the ELISA system was abolished or strongly impaired by amino acid substi- tutions previously shown to abrogate or greatly reduce CD4 and CD4bs Ab binding (10, 53), i.e., 368 D/R, 368 Dm, 370 E/R, and 257 T/R. In addition, L42 binding was moderately reduced by changes 266 A/E and 477 D/V, and L28 binding was also reduced by changes 370 EIQ, 475 M/S, 102 EL, and 463 NID. Binding of L28 was enhanced by changes at 45 W/S, 298 R/G, 381 E/P, 382 FIL, 420 I/R, 435 Y/H, and 435 Y/S and the removal of the whole V3 region, whereas LA2 binding was significantly en- hanced by changes at 381 E/P and 382 FIL.

The Fabs obtained after the epitope masking were then studied. Substitutions at 368 D/R, 370 EIR, and 257 T/R abolished or markedly reduced Fab L78 binding. However in addition, changes within the V2 loop also strongly im- paired binding of this Fab, i.e., 1521153 GE/SM, 1831184 PI/SG, 191/193, and YL/GS, and the excision of the whole

Vl/V2 loop structure also moderately impaired the bind- ing. A substitution at 262N/T in the C3 region abolished the binding of L78 and a substitution at 314 G/W at the tip of the V3 loop moderately inhibited L78 binding. In con- trast, substitutions in the C4 and C5 region (420 I/R, 435 YIS, 435 Y/H, 438 P/R, 475 MIS, and 495 G/K) enhanced L78 binding. As shown in Table 111, Fabs L39 and L40 showed similar binding patterns to L78, with only minor differences. Fab L41, retrieved after epitope masking but competitive with masking Abs, was also tested against the gp120 mutants. Although this Fab showed many similar- ities with L28 and L42, such as an inability to bind to mutants with substitutions at 257 T/R, 368 D/R, 368 D E , 370 E/Q, and 370 E/R, L41 binding was also completely abolished or significantly reduced by substitutions at 113 D/R, 256 S j Y , 384 Y E , and 421 WL. These substitutions either enhanced or had no effect on the binding of L28 and L42. Substitutions at 380 GJF and 381 EIP and the removal of the whole V3 region enhanced LA1 binding.

Additional evidence that Fabs L39, L40, and L78 are influenced by the V2 region in binding to gp120 was pro- vided by competition assays between these Fabs and two previously described murine anti-V2 mAbs (Fig. 7). As shown in Figure 7a, Abs SC258 and 684-238 both inhib- ited binding of Fabs L39, L40, and L78 to gp120; in the reverse format, these Fabs inhibited SC258 and 684-238 binding to gp120 (Fig. 7, b and c). In contrast, SC258 and 684-238 Abs did not inhibit LA1 binding to gp120, nor did Fab L41 block binding of these anti-V2 Abs. The panel of human Fabs were also tested against two murine anti-V3 loop mAbs, RN3-50.1 and IIIB-V3-13, and no competi- tion was observed (data not shown).

We have previously reported on a very potent neutral- izing Fab, b12, which, in mutant studies, has been mapped to the CD4bs (11, 54). However, b12 binding has also been found to be reduced by substitution at 1831184 PI/SG (binding ratio = 0.33) and excision of the VlfV2 loop (0.23) (54). Because of this V2 dependence, we investi- gated the similarities and differences between L78 and b12. In competition experiments, b12 did not compete

900 NEUTRALIZING H U M A N HIV-1 Abs FROM PHAGE LIBRARIES

Table II. Kinetic constants and affinity constants for the binding of selected Fabs to LA/ and MN gpl20 measured by surface plasmon resonance'

Fab Kon KO, K, ("'1 Kd (M) Kd ? SD (nM)

gpl20 LA1 L28 1.98 X 104 5.80 x 10-5 3.40 X lo8 2.90 X 10-9 L42 2.50 x 104 1.40 X 10-4 1.80 X 10' 5.6 x 10-9 L41 L40 1.80 X 104 1.14 X 10-4 L78

L28 L42 L4 1 L40 7.4 x 103 8.80 x 1 0 - ~ L78

2.9 (0.33) 5.6 (0.06) 1.7 (0.1 3)

1.90 x 104 I 20 x 10-4 6.1 (0.56)

1.10 x l o8 9.50 X 9.5 (0.05)

7.6 x 103 1.30 x 1 o - ~ 6.00 x 10' 1.60 x 104

1.70 X lo-@ 9.5 x 10-5 1.70 X lo8

4.90 x 104 6.00 x 10-9

1.15 x 4.30 X 10' 2.40 x 10-9 8.40 x l o 7

7.9 x 103 1.20 x 10-8

1.77 x 4.50 x l o 7 2.20 x 10-8 22 (2.2)

7.4 x 104 1.30 x 10-4 5.7 x l o 8 1.70 X 10-9 6.10 x 10-9 1.60 X lo8

gpl20 MN 17 (0.5) 6 (0.5)

2.4 10.2) 12 (0.1 4)

a The equilibrium association and dissociation constants were calculated from the experimentally determined kinetic constants where K, = k,Jk,, and = kJk,,.

with L78 for binding to gp120 (Fig. 7d), nor did b12 com- Pete with the two murine anti-V2 region Abs (SC258 and 684-238). A whole IgG construct of b12 was used for these experiments to allow detection of b12 by a labeled anti-human IgG Fc Ab. In CD4 competition experiments, preincubation of b12, in contrast with L78, inhibited CD4 binding to gp120. Finally, b12 binding, in contrast with L78, was abolished after gp120 denaturation as well as sialidaselp-galactosidase and sialidase/periodate deglyco- sylation of gp120 (data not shown).

Virus neutralization

Affinity-purified Fab from each of the eight Abs in the panel were examined for neutralizing ability in infectivity assays by using MN and LAI strains of HIV-1. Neutral- ization was determined as the ability of the Fab fragments to inhibit infection, as measured by a plaque reduction assay using MT-2 cells. As shown in Table IV, a signifi- cant difference in the neutralization ability of the different anti-HIV-1 Fabs were found. Three of the CD4bs Abs (L42, L41, and L28) showed 50% inhibition of the LAI strain of HIV-1 at concentrations below 2 pg/ml and 50% inhibition of the MN strain below 10 pg/ml. The CD4bs Fab LA2 was an especially efficient neutralizer, with 50% inhibition at 0.7 pg/ml and 0.9 pg/ml for the MN and LAI strain of HIV-1, respectively. On the basis of this limited initial study, only two of the CD4bs Fabs (L33 and L4l) seemed to be relatively strain-specific. Of the V2-depen- dent Fabs, only L78 showed good neutralization ability, with 50% inhibition at 2 pg/ml and 1.6 pg/ml for the MN and LA1 strains of HIV-1, respectively.

To further examine the neutralization ability of the Fabs, those showing potent neutralization in the plaque reduction assay were analyzed in an envelope complementation assay using the LAI and MN strain. The CD4bs Ab LA2 showed 50% neutralization at less than 1 pg/ml for the MN and at 1 p d m l for the LAI strain. LA1 showed 50% neutralization at 1 pg/ml for both MN and LAI strains. Finally, the V2-de- pendent Fab L78 showed 50% neutralization at 10 pdml for

the MN strain, but did not reach 50% neutralization of the LAI strain at this concentration.

Discussion The use of combinatorial libraries displayed on the surface of filamentous bacteriophage offers an efficient route to obtain a diverse set of human mAbs from an immune do- nor (55). However, certain epitope specificities may dom- inate the cloned Abs because, for example, the epitope is immunodominant or because Abs to that epitope have a selective advantage in the panning process arising from a higher affinity. In this report, we describe an epitope- masking strategy by which Abs to an extended set of epitopes can be retrieved. The strategy has been used to successfully clone a neutralizing human Ab directed to a previously undefined conformational epitope influenced by both the V2 region and the CD4bs of the gp120 mol- ecule on HIV-1 (Table I).

When using recombinant gp120 LAI as the Ag in af- finity selection our experience with several libraries con- structed from asymptomatic long term HIV-1 seropositive donors is that the great majority of the retrieved Fabs are directed against the CD4bs (11,36,37). Two factors prob- ably contribute to this observation. First, the immune re- sponse in these patients is strongly directed to this epitope (56), and second, the retrieved Abs are not selected by gp120 from the infecting strain, but rather by that of the divergent strain LAI. An important part of our overall strategy to retrieve broadly reactive Abs potentially useful for immunotherapy involves using a strain far from that generally causing infection in North America, effectively eliminating strain-specific Abs. This tends to focus Ab se- lection onto the CD4bs, but even in hypervariable regions such as the V2 and V3 loops some conserved features are found (57-60), and the challenge is to retrieve these minor strain cross-reactive Ab specificities from libraries. For the V3 loop, we have used V3 peptides (37). An alternative is to block the major CD4bs specificity with existing Abs before affinity selection from the library. This approach

The journal of Immunology 901

0 v)

* 1-

P 0 - L28

."c L42 - L30 -t L40 - L41

I

-P- L78

0 I I . 1 1 1 0 1 0 0

pglml competing Fab

120

I S I

' "0- L28 1 - L33 1 - L42 I - L 4 1 I

2

E VI 3 n 1 0

n " . . - . .

L28 L33 L42 L52 L39 L40 L41 L78 BAT-085

FIGURE 5. Binding of recombinant Fab fragments to native (a) and denatured m) gp120 LAI. The Fabs were used at a concentration giving 75% maximum binding to native gpl20 in titration experiments.

v) 0 d

n 1

0

0 L28 ~ 3 3 ~ 4 2 ~ 5 2 ~ 3 9 L ~ O L ~ I ~ 7 a

FIGURE 6. Binding of recombinant Fabs after sequential removal of carbohydrates from gpl20 LAI. Effect of combined sialidaselp-galactosidase (H) or sialidasdperiodate (0) treat- ment on the reactivity of the panel of Fab fragments as com- - L70 I pared with untreated gpl20 4).

0 , I I I I I

- 1 2 - 1 1 - 1 0 - 9 - 8 - 7 - 6 - 5

log [competing CD4] FIGURE 4. Competition between Fabs and sCD4 for bind-

ficity of the Ab repertoire in rodents immunized with re-

ing to recombinant gp120. A) in this set of competition ex- combinant HIV-1 proteins or peptides will be identical periments, Fabs in increasing concentration were allowed to with that in humans infected with the HIV-1 virus. The react with gp120 before a constant amount of soluble CD4 Prevalence of Vz-dePendent Abs in the human immune was added. The binding of CD4 was detected with a mono- response against HIV-1 is difficult to determine merely by - clonal anti-CD4 Ab. B) in a second set of competition ex- periments, soluble CD4 in increasing concentration was al- lowed to react with gpl20 before a constant amount of Fab fragment was added. The binding of human Fab was detected with a labeled anti-human IgG F(ab'), Ab.

has yielded high-affinity strain cross-reactive Abs that rec- ognize a complex epitope dependent on the V2 loop.

Several groups have recently described V2-dependent regions of gp120 containing neutralizing linear and con- formational epitopes using murine mAbs (14-18). It has been predicted that V2 Abs are also part of the human Ab response. However, it seems unlikely that the fine speci-

serology. Previous evaluations have used peptides, but, as indicated previously (17, 61) and by our experience, arti- factual binding can occur and information is limited to the fraction of the response not dependent on conformation. Another approach (17) has been to evaluate the ability of sera from HIV-infected individuals to inhibit binding of murine V2-dependent mAbs. Serum from a subset of HIV-1 seropositive individuals inhibited these murine Abs (17), but one problem with this approach is that HIV-1 sera contains Abs that enhance as well as those that inhibit gp120 binding of V2 Abs (17). An additional complication of serologic analysis of HIV-1 infection is that infected individuals may have high levels of Abs that recognize, with moderate affinity, a wide variety of Ags (62). To

902 NEUTRALIZING HUMAN HIV-1 Abs FROM PHAGE LIBRARIES

Table Ill. Relative binding of human recombinant Fabs to sCD4 binding to gp120, it might be expected that CD4 selected HXBc2 gp120 mutant9 binding would not inhibit Fab binding. However, in a sec-

ond set of competition experiments wherein sCD4 was meincubated with ~0120. inhibition of the V2-deoendent

L28 L39 L40 L41 L42 L78

45 WIS 102 EjL 106 VA 113 D/R 117 KhV 152/153

G E/S M 168 K/L 183/184

PVSC 19211 94

YSL/GSS AV1 N2 207 K h V 256 S r / 257 T/R 262 N/T 266 NE 281 A/V 298 WC 314 G/W A V3 368 D/R 368 D/r 370 EIR 370 E/Q 380 C/F 381 E/P 382 F/L 384 YIE 420 IIR 421 K/L 432 KIA 435 YIH 435 YIS 438 P/R 435 Y/S 438 PIR 457 D/A 463 NID 475 MIS 477 DN

1.60 0.35 0.65 1.03 1.03 0.68 0.46 0.65 0.42 0.66 0.88 0.59 0.91 0.41 0.23 1.1 1 1.05 0.59 1.40 0.53 1.13 0.30 1.28 0.83 1.01 0.71 1.32 1.20 1.24 1.24 0.70 0.18 0.48 0.80 0.93 0.46

0.78 0.47 0.19 0.88 1 .OO 0.83 0.82 0.18 0.13 0.53 0.63 0.19

0.82 0.29 0.32 0.94 0.93 0.29

1.07 0.53 0.52 1.16 0.95 0.54 1.15 2.47 0.94 1.14 1 .I4 1.37 0.64 0.47 0.42 0.00 0.29 0.66 0.04 0.41 0.06 0.02 0.01 0.15 1.06 0.47 0.00 0.34 0.72 0.00 1.03 0.88 0.58 0.97 0.55 0.76 0.69 0.18 0.65 0.75 0.64 0.93 1.60 0.76 1.39 1.27 1.04 1.49 0.59 0.24 0.52 0.47 0.62 0.24 1.88 0.88 1.26 1.64 1.45 1.17 0.18 0.24 0.00 0.00 0.01 0.10 0.13 0.06 0.00 0.00 0.05 0.05 0.00 0.00 0.00 0.00 0.00 0.00 0.18 0.47 0.10 0.00 0.97 0.29 1.54 1.06 1.29 1.52 1.39 0.93 2.18 1.29 1.23 1.44 1.67 1.22 1.72 ND ND 1.08 1.55 ND 1.21 0.47 0.45 0.00 1.40 0.59 1.70 1.47 1.81 1.28 1.38 1.73 0.82 0.76 1.19 0.00 0.83 1.37 1.59 1.76 2.00 1.02 1.16 1.41 1.73 2.06 2.32 1.17 1.21 1.73 1.93 2.59 2.58 1.39 1.18 1.83 1.20 1.47 2.22 1.11 1.09 1.56 1.93 2.59 2.58 1.39 1.18 1.83 1.20 1.47 2.22 1.1 1 1.09 1.56 0.62 1.06 0.58 0.78 0.84 0.68 0.48 1.88 1.42 0.58 0.63 1.22 0.13 3.41 2.06 0.89 1.03 1.54 0.50 1.47 0.23 0.39 0.46 0.46

491 IIF 0.68 1.94 1.52 1.05 0.89 1.46

produce a binding index 10 .5 or >1.5 for any of the Fabs tested: 36 VIL, 4 0 " The following mutants were also included in the test panel but did not

YID, 69 WIL, 76 P/v, 80 NIR, 88 NIP, 103 Q/F, 1 13 DIA, 120I121 VWLE, 125

33+1, 313 PIS, 356 N/i, 386 N/Q, 392 N/E 397 N/E, 395 WIS, 406 N/G, 420 LC, 17611 77 FY/AT, 179/180 LD/DL, 252 W, 257 TIA, 267 VL, 269 E/L, MT

470 P/L, 485 KjV, and 493 PIK. ND, not done. I/R, 427 WIS, 429 KJL, 430 VIS, 433 AJL, 450 TIN, 456 WK, 457 DIR, 470 PIG,

study the humoral response to such epitopes requires ac- cess to human mAbs. Compared with murine V2-depen- dent Abs, the human VZsensitive Fab fragments showed many similar characteristics, but also some important dif- ferences, as outlined below.

The epitope(s) recognized by the novel Fabs were probed by a series of competition experiments between Fabs and CD4 and CD4bs Abs. Competition experiments between Fabs and sCD4 showed that, whereas all the anti- CD4bs Fabs inhibited sCD4 from binding, Fabs L39, L40, and L78 had no effect, indicating a binding site distinct from the CD4bs. As Fab L39, LAO, and L78 did not inhibit

Fabs was observed. This surprising behavior has also been described independently from two studies on murine V2- dependent Abs (16, 17) and implies some elements of ir- reversibility in the interaction of CD4 and gp120. It also implies that CD4 binding decreases the availability of the V2-dependent epitope, adding to the body of evidence sug- gesting CD4-induced conformational changes in a 1 2 0 (63).

Competition experiments between the CD4bs Abs and the V2-dependent Abs indicated that preincubation of two CD4bs Abs, L28 and L42, enhanced binding of V2 Abs LAO, and L78, whereas binding of L41 (CD4bs Ab) was inhibited. Again, similar results were obtained in the mu- rine system, in that binding of VZdependent murine Abs to gp120 was enhanced by CD4bs Abs (17), suggesting that CD4bs Abs induce conformational changes in gp120 that increase the exposure of the V2 epitope. Indeed, this enhanced exposure may be a key factor in the retrieval of the V2-dependent Abs after CD4bs masking. It should be noted, however, that other anti-CD4bs Abs have been reported that do not enhance binding of V2-dependent Abs (17).

Using a panel of gp120 mutants expressed in COS-1 cells, the binding specificities of the three novel Fabs were mapped. All were sensitive to V2 region changes and, to a lesser extent, to excision of the V1W2 loop. These changes are not believed to produce global changes in gp120 conformation as they do not generally perturb Ab binding to the CD4bs (10,30,54) although they do perturb CD4 binding (21). Perhaps unexpectedly, binding of the novel Fabs was also sensitive to changes at residues 368 D/R, 368 DIT, and 370 E/R, generally considered essential for binding of CD4 and CD4bs Abs (10, 53). In addition, a substitution at 262 NIT in the C3 region abolished L78 binding. None of the previously described murine V2-de- pendent Abs were sensitive to these latter changes (17). Binding of the three V2-dependent human Fabs was en- hanced by substitutions within the C5 region, as previ- ously reported for V2-dependent murine Abs (17). Further evidence that the three novel Fabs were dependent on the V2 region for gp120 binding was provided by competition between the Fab fragments and the previously described murine VZdependent Abs, SC258 and 684-238 (17).

From the above, it is clear that the epitope we have described is distinct from that recognized by the V2-de- pendent murine Abs characterized to date. In this regard, it should again be emphasized that the murine Abs have been generated by immunization with recombinant pro- teins or peptides, whereas the human Abs arise from nat- ural infection.

Surprisingly, L41, the last of the four Abs retrieved after panning on masked gp120, competed with the two CD4bs masking Abs and, in mutant studies, LA1 was mapped to the CD4bs. The differences between L41, L28, and L42

-1 1 - ~ ~

The Journal of Immunology 903

1.2 -I i

1.6 1.0

E 1.2 e v) 0

0.6

* 0.8

0

* n 8 0.4

0.4 0.2

0.0 0.0 L 3 9 L 4 0 L 7 8 L 4 1 SC258 L 3 9 L 4 0 L 7 8 L 4 1

alone + competing recombinant Fabs

1.6

C

1.6 1.2

z E 1.2

0 v v) 0.8 v)

n n 0 * 0.8

0 0.4

0.4

0.0 0.0 6 8 4 - 2 3 8 L 3 9 L 4 0 L 7 8 L 4 1

alone + competing recombinant Fabs I g G l b l 2 L 7 8 b12 SC258 684-2

alone + competing antibodies 8

FIGURE 7. a) Binding of Fabs L39, L40, L78 and L41 (in a concentration giving 75% maximum binding) to gpl20 LA1 either alone (.) or competed with mouse anti-V2 Ab SC258 (El) or 684-238 (0) at a concentration 100 times that giving 75% maximum binding in previous titration experiments. b) Binding of mouse anti-V2 Ab SC258 (at a concentration giving 75% maximum binding) to gpl2O either alone or competed with Fabs L39, L40, L78 or L41 at a concentration 10 times (B) or 100 times (Ed) that giving 75% maximum binding in previous titration experiments. c) Binding of mouse anti-V2 Ab 684-238 (at a concentration giving 75% maximum binding) to gp120 either alone or competed with Fabs L39, L40, L78 or L41 at a con- centration 10 times @) or 100 times (.) that giving 75% maximum binding in previous titration experiments. d) Binding of whole human lgGl b12 (at a concentration giving 75% maximum binding) to gpl20 either alone or competed with Fabs L78 (60 pghnl), b12 (25 pghnl), SC258 or 684-238 at a concentration 100 times that giving 75% maximum binding in previous titration experiments.

are that the former is affected by several amino acid changes in addition to those affecting L28 and LA2 and has a somewhat higher affinity for gp120. However, the cur- rent data do not clearly explain why L41 was only re- trieved after masking.

To further explore the nature of the novel epitope, stud- ies on the effects of gp120 glycosylation and conformation on Fab binding were conducted. Glycans represent ap- proximately 50% of the total m.w. of gp120 and seem to play an important role in reducing the efficacy of the hu- moral immune response to viruses, e.g., by masking neu- tralizing sites (64-66). The epitopes of all the CD4bs Abs were greatly affected by changes in glycosylation, indicat- ing either that (pl-4)-linked galactose from N-linked sug- ars is included in the binding site or that the carbohydrates

Table IV. Neutralization of HIV-1 by recombinant human Fab fragments as measured by microplaque assay"

Neutralization Titer

M N 1a1 Fab (pdml) (pdml)

L28 L33 L42 L52 L39 L40 L4 1 L78 b12

4.2

0.7 >40

>50 >50

40.8 8.2 2 .o 0.1

4 (59%) 1.1

0.9 >50 >50 >SO

0.8 1.6 0.7

Fab required to inhibit plaque formation to 50%. a The Ab neutralization titer is expressed as the concentration (pLp/ml) of

904 NEUTRALIZING HUMAN HIV-1 Abs FROM PHAGE LIBRARIES

are responsible for keeping the tertiary structure of this region of the molecule intact. However, the Abs are not directed to carbohydrate alone, inasmuch as they did not react with reduced but fully glycosylated gp120. In con- trast with CD4bs Abs, binding of the three V2-dependent Abs was not, or only slightly, affected by the enzymatic treatment, indicating that these carbohydrates are not in- cluded in their binding site.

It has been reported that most broadly neutralizing hu- man Abs are directed against discontinuous epitopes on gp120 (67). All Abs obtained from this HIV-1 donor li- brary seem to recognize such discontinuous epitopes, as binding of all the Abs was affected by gp120 denaturation. There were, however, differences in the extent to which binding was affected. Whereas binding of all Abs to the CD4bs was completely abolished, the three V2-dependent Abs were still able to bind appreciably to denatured gp120, although apparently with much reduced affinity as com- pared with intact gp120.

Several of the V2-dependent murine mAbs described in the literature are fairly broadly neutralizing, suggesting the epitopes recognized are relatively conserved across strains (14, 15). One of the VZdependent Fab (L78) showed ef- ficient neutralization of both the MN and LA1 strains of HIV-1, as measured by a plaque reduction assay using MT-2 target cells. Against both strains, this Fab exhibited 50% neutralization at approximately 2 Fg/ml. Using an envelope complementation assay, L78 exhibited 50% neu- tralization at about 10 Fg/ml for the MN strain HIV-1. Of the retrieved anti-CD4bs Fabs, L42 in particular showed potent neutralization against the two strains, with 50% neutralization at less than 1 &ml in both assays. The ability of L78 and L42 to neutralize primary isolates of HIV-1 will be assessed after their conversion to whole Abs (68). The neutralizing efficacy of the Fab fragments may be enhanced by using an affinity maturation procedure in- volving random mutation in the CDRs and affinity selec- tion. Recently, with use of this procedure, we have in- creased the affinity of a potent neutralizing Fab directed to the CD4bs by 10-fold (69). This increase was paralleled by a corresponding increase in neutralization ability.

In this report, CD4bs human Fabs were incubated on gpl20-coated ELISA wells to mask their epitopes before panning the HIV-1 library. An alternative approach would be to initially coat the CD4bs Ab onto the ELISA wells followed by the Ag. By using this approach, epitope mask- ing is ensured by the nature of the coating, and, further- more, nonpurified Ag may be used. Indeed, we have used this strategy elsewhere (P. P. Sanna, R. A. Williamson, A. de Logue, R. Burioni, S. E. Bloom, and D. R. Burton, manuscript in preparation). One disadvantage of the cap- ture approach is that all the Ags will be oriented in the same manner, which may limit the epitopes exposed to Ab-phage particles.

It should also be noted that one of the clones, L28, has a sequence similar to an Ab retrieved from a different

HIV-1 donor library and reported previously as s7 (11, 37). Only three amino acids were different in the heavy chain variable region. The reason for this could be that these sequences are conserved or, more likely, that the L library was cross-contaminated during construction with phage from the other library. We have found that such cross-contamination can be a complicating factor in re- trieving Abs against a given epitope from libraries con- structed from different patients. However, the possible phage contamination, if any, does not interfere with the conclusions reported in this paper. Rather, the described masking method may be a useful strategy to avoid re- trieval of known contaminating phageIAb specificities from libraries.

In conclusion, the masking procedure described is shown to be a valuable approach to retrieve Abs reactive with more minor epitopes, as demonstrated by the suc- cessful isolation of human mAbs to a novel neutralizing conformational epitope influenced by the V2 and the CD4bs regions. Because Fab L78 is fairly broadly and po- tently neutralizing, it may be a useful component of a mixture of anti-HIV-1 human mAbs for passive immunotherapy.

Acknowledgments

We are particularly grateful to the donors for their cooperation, and to Clifford Lane and Robert Walker (NIAID, National Institutes of Health) for kindly providing clinical samples used in this study. We thank Glenn Pilkington, Roman Rozenshteyn, Anthony Williamson, Terri Jones, and Shu-Wing Poon for their contributions to this work. We express gratitude to Jim Chambers for sequencing. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health: mAb 1IIB-V3-13 from Dr. Jon Laman; mAb V3 (RN3-50.1) from Repligen Corporation; mAb Q425 from Drs. Peter Kwong and Dr. Quentin Sattentau; and soluble recombinant CD4 from Dr. Ray Sweet.

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