AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 11, 1992 Mary Ann Liebert, Inc., Publishers

Monoclonal Antibodies to the Extracellular Domain of HIV-1 IIIB gpl60 that Neutralize Infectivity, Block Binding to CD4, and React with Diverse Isolates MP> A I nVDM 4 1/IM 1AX1CC D DTIDTCD 2 GERALD R. NAKAMURA,11 DA RANDAL P. PORTER, BYRN,4 KIM DnCC\ITUA[_ ROSENTHAL,22 JAMES 1 MAURINE R. HOBBS,1 LAVON RIDDLE,3 DONNA J. EASTMAN DONALD DOWBENKO,1 TIM GREGORY,3 BRIAN M. FENDLY,2 and PHILLIP W. BERMAN1

ABSTRACT Ten monoclonal antibodies prepared against a soluble, recombinant form of gpl60, derived from the IIIB isolate of HIV-1, were characterized. Four of the antibodies neutralized HIV-1IIIB infectivity in vitro, three blocked the binding of recombinant gpl20 to CD4, three were reactive with gp41, and one preferentially reacted with an epitope on gp 120 within the gpl60 precursor. All three CD4 blocking antibodies bound to distinct epitopes, with one mapping to the Cl domain, one mapping to the C4 domain, and one reactive with a conformation-dependent, discontinuous epitope. Of these, the antibody reactive with the discontinuous epitope exhibited neutralizing activity against homologous and heterologous strains of HIV-1. The binding of these monoclonal antibodies to a panel of seven recombinant gpl20s prepared from diverse isolates of HIV-1 was measured, and monoclonal antibodies with broad cross reactivity were identified. The epitopes recognized by 7 of the 10 monoclonal antibodies studied were localized by their reactivity with synthetic peptides and with fragments of gpl20 expressed as fusion proteins in a \gt-ll gpl60 epitope library.

INTRODUCTION

UNDERSTANDI NG gpl20 gp41,

the relationship between the structure and the function of the HIV-1 envelope glycoproteins, and is central to the development of strategies for the prevention and treatment of acquired immunodeficiency syndrome (AIDS). One powerful method for the localization of functional domains of proteins is through the use of monoclonal antibodies (MAbs). In principle, MAbs to functional domains of gpl20 or gp41 should be able to inhibit HIV-1 infectivity by interfering with such processes as: ( 1 ) the attachment of the virus to target cells, (2) the transport of the virus into cells after virus has attached, (3) the uncoating of the virus once inside the cell, and (4) syncytia formation between uninfected CD4 bearing target cells and virus infected cells. Thus far MAbs have enabled the identification of a sequence within the CD4 domain of gp 120 (residues 397 and 439) important for binding to CD4,1 and a

region within the V3 domain (residues 301-324) that constitutes the principal type-specific neutralizing determinant (PND).2,3 In previous studies1,4 the properties of monoclonal antibodies to recombinant gpl20 from the IIIB strain of HIV-1 (IIIB-rgpl20) were described. Although immunization with this protein allowed the isolation of a MAb that defined a region on gpl20 critical for the binding of CD4,1 MAbs that neutralized HIV-1 infectivity were not obtained.4 One explanation for the inability of IIIB-gpl20 to elicit MAbs with neutralizing activity was that the conformation of gpl20 on the surface of virions or virus infected cells might significantly differ from that of monomeric gpl20. Because gpl20 occurs on the surface of the virus as a noncovalent oligomeric complex with gp41,5-7 we have considered the possibility that the immunogenicity of monomeric gpl20 differs from that of gp 120 as it occurs within gp 160 or the gp 120/gp41 complex.

Departments of 'Immunobiology, 2Hybridoma Development, and 'Recovery Process Sciences, Genentech, Inc., S. San Francisco, CA 94080. 4Department of Hematology and Oncology, New England Deaconess Hospital, Harvard Medical School, Boston, MA. 1875

NAKAMURA ET AL.

1876 In the present studies we describe the properties of 10 monoclonal antibodies elicited against a gpl60 variant8 of the IIIB strain of HIV-1, termed recombinant soluble gpl60 (IIIBrsgpl60), produced in Chinese hamster ovary (CHO) cells. Immunization with IIIB-rsgpl60 allowed for the production of four MAbs able to neutralize HIV-1 infectivity in vitro and several other MAbs with novel and unique properties. For example, the 6E10 M Ab bound to a conformation-dependent epitope distinct from the principal neutralizing determinant (PND) and able to neutralize the infectivity of homologous and heterologous isolates of HIV-1. Another MAb, 5B3, is notable in that it blocked the binding of IIIB-rgpl20 to CD4, yet bound to a site in the amino terminus of IIIB-rgp 120 that is distinct from ' the CD4 binding site previously described. Comparative bindderived from diverse isolates of HIV-1 studies ing using rgpl20 have enabled us to identify two MAbs with broad cross reactivity. Epitope mapping studies involving the use of proteolytic fragments of IIIB-rgp 120, synthetic peptides, and a bacteriophage Xgt-11 IIIB-gpl60 epitope library have enabled us to localize the binding sites of seven of the ten MAbs that were

produced. METHODS

Antigens gpl20 (IIIB-rgpl20) and gpl60 (IIIB-rsgpl60) purified by affinity chromatography from growth-condi-

Recombinant were

tioned cell culture medium of the D5319 and D683.DC.9 cell lines described previously.8 For ELISA assays a gp41 fusion protein (LE41) consisting of an amino terminal fragment of the Tip E gene fused to 100 amino acids from the amino terminus of gp41 was expressed in Escherichia coli and purified as described previously.10 Reduced and carboxymethylated IIIB-rgp 120 and

IIIB-rsgpl60 were prepared by dialysis of IIIB-rsgpl60 and IIIB-rgp 120 into 0.36 mM Tris/HCl buffer (pH 8.6) containing 8

M urea and 10 mM EDTA. Dithiothreitol (DTT) was added to a final concentration of 10 mM, and the proteins were mixed at room temperature for 4 hours. Iodoacetic acid was added to a concentration of 25 mM, and the samples were mixed in the dark for 30 min. DTT was added to a concentration of 100 mM, and the samples were dialyzed into ammonium bicarbonate buffer. These proteins were used to coat microtiter plates for ELISA

analysis. Monoclonal

antibody production

Balb/c mice were immunized with IIIB-rsgpl60 incorporated complete Freund's adjuvant. Each mouse received 20 u.g of IIIB-rsgpl60 per injection intraperitoneally (i.p.) and intravenously (i.v.), and was boosted three days prior to fusion. The mouse with the highest antisera titer was selected for fusion. Spleen cells were fused with NP3x63. Ag8.653 mouse myeloma cells in a 4:1 ratio using 50% polyethylene glycol (PEG)." Hybrids were selected for growth in media containing hypoxanthine and azaserine. Positive parental supernatants were identified by screening individual wells against IIIB-rsgpl60 in a solid-phase enzyme-linked immunosorbent assay (ELISA). Reactive wells were expanded, cloned by limiting dilution, and the in

cells injected into pristine-primed Balb/c mice. Ascites fluid was collected, pooled, and the immunoglobulin was purified by protein-A column chromatography using standard techniques. Fab fragments of MAbs to IIIB-rsgpl60 were prepared as described previously. '

hybridoma

Enzyme-linked immunoadsorbant assays (ELISA) The binding of MAbs to various recombinant proteins was measured by ELISA performed by methods similar to those described previously. Briefly, ELISA plates were prepared by treating the wells of microtiter plates (Maxisorb, Nunc) with HIV-1 proteins or synthetic peptides diluted to a final concentration of 2 p.g/ml in PBS or 0.05 M carbonate buffer, pH 9.6 (100 p-l/well). After overnight incubation at 4°C, the plates were washed with wash buffer (phosphate-buffered saline containing 0.05% Tween-20), and then treated with blocking buffer (0.5% bovine serum albumin (Sigma) in PBS). Following blocking, the wells were incubated with hybridoma supernatants, ascites fluid, or purified antibody at room temperature for 1-2 h. The wells were then washed four times with wash buffer and the labeled horseradish peroxidase-conjugated goat anti-mouse IgG (Cappel) was added to each well and incubated for 1 h. The plates were then washed three times with wash buffer, and the colorometric substrate o-phenylenediamine dihydrochloride was added. 2.5 M H2S04 was added to stop the colorometric reaction, and the absorbance of the solution in each well was measured at 492 nm using a microtiter plate reading spectrophotometer. Antibody isotype analysis was performed using an ID EIA kit (Zymed, So. San Francisco, CA) according to the manufacturer's instructions. For this analysis, plates were coated with IIIB-rsgpl60 at 1.0 p-g/ml.

Epitope cross-competition

ELISA

ELISA microtiter plates were coated with IIIB-rsgpl60 at 1.0 After blocking with blocking buffer, 50 p.1 of purified MAb at 100 p,g/ml was added and incubated at room temperature for 1 h. Without washing, 50 p,l of each antibody coupled to HRP was added for an additional hour. The plate was washed, substrate added, and read at 490 nm.

p.g/ml.

Assays to

the ability of monoclonal antibodies binding of IIIB-rgp 120 to CD4

to measure

block the

Two different assays were used to measure the ability of antibodies toblock the binding of IIIB-rgp 120 to CD4. The first assay used was essentially that described previously" where 125I-labeled IIIB-rgp 120 in solution was bound to CHO cells transfected with human CD4. Briefly, 1.5 x 104 CHO cells expressing CD4 were plated into 96-well microtiter plates, and cultured overnight at 37°C. The cells were then washed in assay medium consisting of Hepes (12 mM) buffered Dulbecco Modified Eagle's medium containing 10% fetal bovine serum and 0.1 % NaN3. In a separate 96-well microtiter plate, 125I-labeled IIIB-rgpl20 (2 nM) was mixed with the MAbs, serially diluted in assay medium, and then transferred onto the cells. The mixture was allowed to react with the cells for 2 h at room temperature, and then were washed four times with assay medium. The washed cells are then solubilized with 1 N NaOH (100 p.1) for 30 min and 90 p.1 was removed for counting in a

MAbs TO

IIIB-rsgplóO

1877

gamma counter (LKB). To determine specific binding of 125Ilabeled IIIB-rgpl 20 to cell associated CD4, duplicate binding reactions were performed in an excess (30 p.g) of noniodinated IIIB-rgp 120. The average cpm in these samples were subtracted from that of the experimental samples. Percentage binding was calculated by dividing the specific cpm obtained in the presence of the IIIB-rsgpl60-specific MAbs divided by cpm obtained when the binding reaction was carried out in the presence of an isotype-matched control MAb. For assay using recombinant soluble CD4 (rsCD4), ELISA microtiter plates were coated at 1.0 pg/ml with antibody to CD4 (L104.5) that does not interfere with IIIB-rgpl20 binding. In a separate rection, serially diluted MAbs were incubated with IIIB-rgp 120 overnight at 4°C, and then incubated with rsCD4 for 1 h at room temperature. The resulting mixture was then transferred to the L104.5-coated plate and washed. rsCD4 that was not bound to IIIB-rsgpl20 was detected with a horseradish peroxidase-conjugated MAb that binds to the gpl20 binding site on rsCD4 (Leu3a; Becton

Dickenson). Immunoblot assays Commercial HIV-1 immunoblot strips (Dupont) were used to the reactivity of the MAbs with authentic HIV-1 derived viral proteins. In these studies 10 p.1 of ascites were diluted to 3 ml in blocking buffer and incubated with the nitrocellulose strips for 1 hr at room temperature. The strips were then washed three times with 3 ml of blocking buffer and then incubated with a 1:1000 dilution of alkaline phosphatase conjugated, affinity purified, goat anti mouse immunoglobulin (Cappel) for 1 h. The strips were then washed three times and stained with a phosphatase developing kit obtained from Kirkegaard and Perry (Gaithersberg, MD). measure

Recombinant gp 120 from diverse strains Recombinant

gpl20s

from

seven

of HIV-1

diverse isolates of HIV-1

(IIIB, MN, JrCSF, Z6, Z321, NY-5, and B244) were produced

purified by methods similar to those described of the rgpl20s used in these studies were expressed as fusion proteins where 25 amino acids from the mature amino terminus of herpes simplex virus type 1 glycoprotein D were fused to amino acid residues corresponding to amino acids 11 or 12 (Kpnl site) at the amino terminus of HIV-1 gpl20. The recombinant envelope glycoproteins all terminate at the normal dibasic gpl20/gp41 processing site. A comparative listing of the amino acid sequences of all gpl20s used in these studies, with the exception of A244 is provided by Myers et al. The sequence of the gpl20 from the A244 isolate, the construction of the cell lines used to produce the recombinant materials, and the biological and immunologie properties of these proteins will be described elsewhere (P.W. Berman et al., manuscript in preparation). gpl20 from the MN strain of HIV-1 (MN-rgpl20) was prepared as described previously.12 We thank Dr. M. Martin (NIH) for providing the env gene from the NY-5 isolate,

dation) for providing a DNA fragment containing the from

a

env

gene

Northern Thailand HIV-1 isolate termed A244.

Neutralization assay Neutralization assays were performed as previously described.15 Briefly, dilutions of serum (50 pi), or mouse ascites, were preincubated with 100 TCID50 of virus (50 p.1) for 1 h at 4°C. H9targetcells,161850p.lof5 x 106 cells/ml, were added

and incubated for 1 h at 37°C. The cells were then suspended in 2 ml of complete culture medium (20% FCS, RPMI-1640, +P/S, L-glutamine, 2 pg/ml polybrene) and incubated at 37°C in a 24-well plate. The cultures were split 1/2 on day 4 with complete medium, and the supernatant harvested on day 7 for analysis in a reverse transcriptase (RT) assay.iy Virus stocks were prepared in H9 cells by the "shake" method of Vujcic, Kim, et al.20,21 TCID50 were determined in the same H9 cell, 7-day, RT-based assay described above, with 1 TCID50 being defined by the amount of virus that produced greater than 1 SD of RT activity over uninfected cell control levels. H9 target cells and HIV-1H1B were originally obtained from Dr. Robert Gallo, National Cancer Institute.16"18 HIV-1 AL and HIV-1R,4029 were isolated from Haitian donors and HIV-1906 was isolated from a European donor known to have had a sexual contact from Chad, as previously described.14 The HIV-2 strain LAV-2ROD was obtained from Dr. Chermann.22

Epitope mapping studies

in

\gt-l I

The location of amino acid residues defining MAb binding sites is specified with reference to the sequence of the IIIB isolate of HIV-1 described by Muesing et al.,23 where the initiator methionine residue, at the start of the signal sequence, corresponds to residue 1. Epitope mapping studies were carried out according to the method of Mehra et al.24 using a gpl60 epitope library described previously.4 Synthetic peptides used for epitope mapping were kindly provided by Dr. John Burnier

(Genentech).

in CHO cells and

previously.9,12 All

Drs. Schocetman and Srinivasan (Centers for Disease Control, Atlanta) for providing the env genes from the African isolates Z6 and Z321, Dr. A. Ashkenazi (Genentech) for providing the expression plasmid14 used to produce rgpl20 from the JrCSF strain, and Dr. Francine McCutchan (Henry M. Jackson Foun-

RESULTS Antibodies to

IIIB-rgpl20 or ¡IIB-rsgpI60

Previous studies8 described the structure, purification, and of IIIB-rsgpl60, a variant of the HIV-1 envelope glycoprotein precursor that contained the extracellular domains of gpl20 and gp41, but lacked the transmembrane domain, cytoplasmic tail, and the polyglycoprotein processing site of the wild-type envelope glycoprotein. Expression of this variant in CHO cells allowed for the production of a 140 kD protein that was folded into a functionally relevant conformation, glycosylated, and secreted into growth-conditioned cell culture medium. Unlike IIIB-rgp 120, this protein self-associated to form noncovalent dimers and higher order structures. Binding studies showed that IIIB-rsgp 160 bound to CD4 with an affinity comparable to IIIB-rgpl 20.8 In the present studies, mice were immunized with IIIB-rsgp 160 and ten stable cell lines producing monoclonal antibodies against IIIB-rsgp 160 were isolated as described above. Initial characterization (data not shown) revealed that 8 of the 10 antibodies were of the IgG,

immunogenicity8,25

NAKAMURA ET AL.

1878 Table 1. Summary

ELISA

Antibody 5B3 6E10 9E3 10C1 10D8 10F6 11G5 13H8 14F12 15G7

of

Monoclonal Antibodies

reactivity'

to

IIIB-rsgp160

Conformation

rsgplóO

rgpl20

p4I

0.91 0.77 0.67 0.49 0.55 0.57 0.47 0.67 0.38 0.47

1.05 1.04 0.07 0.02 1.11 1.12 1.07 0.92 0.05 0.04

0.03 0.04 0.98 0.03 0.03 0.03 0.04 0.02 0.07 0.04

dependent*

HlV-lWB

CD4 inhibition"

neutralization'1

+ +

ND 2.01 ND ND