Immunology 1990 71 10-15

Differences in affinity of anti-CD4 monoclonal antibodies predict their effects on syncytium induction by human immunodeficiency virus D. WILKS, L. WALKER, J. O'BRIEN, J. HABESHAW & A. DALGLEISH Retroviral Research Group, Division of Immunology, Clinical Research Centre, Harrow

Acceptedfor publication 6 June 1990

SUMMARY A panel of 20 anti-CD4 monoclonal antibodies (mAb) was ranked in terms of affinity, using an inhibition radioimmunoassay. The ability of these antibodies to inhibit the induction of syncytia by human immunodeficiency virus (HIV) and to prevent binding of the HIV envelope glycoprotein 120 (gp 120) to CD4 was also measured. Syncytium inhibition correlated strongly with affinity (P < 0-001) but only weakly with inhibition of gp120 binding (P= 0-038). Some antibodies partially blocked binding of gp 120 to CD4 but did not inhibit syncytia, and some antibodies inhibited syncytia but only weakly blocked binding of gp 120. These results suggest that the syncytium inhibition assay is highly affinity-dependent, and that epitopes on CD4 concerned with virus binding are distinct from those involved in syncytium formation.

INTRODUCTION The CD4 antigen binds to major histocompatability complex (MHC) class II antigens (Doyle & Strominger, 1987; Gay et al., 1987; Clayton et al., 1989; Lamarre et al., 1989) and functions as a co-receptor with the T-cell receptor/T3 complex (Janeway, 1989). Its expression on T lymphocytes identifies a subset ofcells that shows class II restriction, is predominantly associated with helper/inducer functions and is depleted in the acquired immune deficiency syndrome (AIDS) (Schroff et al., 1983). CD4 has also been shown to act as the major cellular receptor for the human immunodeficiency virus (HIV) (Dalgleish et al., 1984; Klatzmann et al., 1984; Maddon et al., 1986); viral tropism for CD4+ cells is mediated by the interaction of the HIV major envelope glycoprotein 120 (gp 120) and CD4, which occurs with high affinity in vitro (Smith et al., 1987). Monoclonal antibodies (mAb) to CD4 can prevent the induction of syncytia by HIV in lymphocyte cultures in vitro (Dalgleish et al., 1984; Klatzmann et al., 1984; Sattentau et al., 1986), and can block the attachment of whole HIV particles or recombinant gp120 (rgpl20) to CD4+ cells (McDougal et al., 1986; Lundin et al., 1987), presumably either by steric hindrance or by direct competition for the gp 120 binding site itself. A large number of anti-CD4 mAb has been raised, and since they vary in their ability to inhibit the formation of syncytia of MHC class II-dependent immune functions, it is of interest to identify which epitopes of CD4 are recognized by which mAb. Previous CD4 epitope mapping studies have used the inhibition of syncytium formation or gpl2O binding to suggest

which mAb bind to CD4 at or near the gp120 binding site (Sattentau et al., 1986, 1989). The ability to inhibit syncytium formation and the ability to block binding of gpl2O are not equivalent (Linsley et al., 1988; Camerini & Seed, 1990) and both of these functions may be dependent on antibody affinity. We have used a radioimmunoassay (RIA) modification of an inhibition ELISA (Rath, Stanley & Steward, 1988) to rank a panel of anti-CD4 mAb in order of relative functional affinity and we have examined the degree of correlation between affinity, the ability to inhibit syncytium formation, and the ability to block the binding of gpl2O to CD4.

Correspondence: Dr D. Wilks, Retroviral Research Group, Division of Immunology, Clinical Research Centre, Watford Road, Harrow HAI 3UJ, U.K.

Recombinant antigens Soluble CD4 (sCD4) was kindly donated by Dr R. Sweet (Smith, Kline and French, King of Prussia, PA) and Dr D. Capon

MATERIALS AND METHODS Monoclonal anti-CD4 antibodies The following anti-CD4 mAb were kindly supplied by BectonDickinson Immunocytometry Systems (San Jose, CA): antiLeu-3a, L201, L204, L215, L216, L217, L218. MT151 and MT31O were provided by Dr P. Rieber (University of Munich, FRG). The OKT4 series was kindly provided by Dr P. Rao (Ortho Pharmaceuticals, Raritan, NJ). 6.3G4, 7.2G4, 7.3Fl 1, 12.22F5 and 12.16A2 are anti-CD4 mAb recently raised at the Clinical Research Centre by the authors under the AIDSDirected Programme of the Medical Research Council. All the anti-CD4 mAb used were raised by immunization of mice with CD4+ cells, with the exception of 12.16A2 and 12.22F5, which were raised by preliminary immunization with CD4+ cells, followed by boosting with soluble CD4 (sCD4).

10

I1I

Affinity of anti-CD4 mAb (Genentech, San Francisco, CA). Both of these constructs consist of the four extracellular domains of CD4 but lack the transmembrane and cytoplasmic domains. Mammalian recombinant gpl20 (rgp 120) was obtained from Celltech, Slough, Berks, U.K., through the AIDS-Directed programme of the Medical Research Council (AIDS-Directed Programme repository numbers ADP 604; this preparation is greater than 95% pure and contains less than 15% 70,000/50,000 MW cleavage products).

Syncytium inhibition assay Supernatant from HIV-l infected H9 or CEM cells was serially diluted in RPMI with 10% foetal calf serum supplemented with penicillin, streptomycin and glutamine and added to CD4+ C8166 cells (6 x 103 cells/well). After incubation for I week the wells were examined for the presence of syncytia and the dilution of supernatant that caused syncytia in 50% of wells was determined. This dilution was regarded as the TCID50. Serial doubling dilutions of mAb in RPMI were prepared starting at a variety of known concentrations. Fifty-microlitre

Cell lines and virus strains HPB-ALL is a human T-cell line that expresses CD4. It was provided by Dr D. Buck (Becton-Dickinson Corp., San Jose, CA). NALM-6 is a CD4- human B-cell line, which was provided by Dr M. Larkin (Clinical Research Centre). H9 cells infected with HIV- 1 strains HTLV-IIIB and HTLVIIIRF were provided by Dr R. Gallo (National Cancer Institute, MD). CBL- I was isolated in London, and was grown in CEM cells.

volumes of these dilutions were incubated for 1 hr at 370 with C8166 cells (6 x I03 cells in 20 pi) and then 80 p1 of virus supernatant were added at a dilution sufficient to give 100 times the previously determined TCID50. The wells were scored for the presence of syncytia at 48 hr. The highest dilution of mAb that completely inhibited the formation of syncytia was recorded.

Inhibition RIA for the measurement of relativefunctional affinity Rabbit anti-mouse immunoglobulin (Ig) antibody (Dakopatts, Glostrup, Denmark) was radioiodinated using the lodogen method (Fracker & Speck, 1978) to a specific activity of approximately 35 pCi/pg. Functional affinity was measured using a RIA modification of the method of Rath et al. (1988). Stage 1. Flexible 96-well plates (Falcon 3912; BectonDickinson) were coated with 50 pi of sCD4 at 1 Mg/ml in 0 05 M carbonate buffer, pH 9-6, overnight at 4°. After washing twice in water, the plates were quenched with 0 3% gelatine in phosphate-buffered saline (PBS) at room temperature (RT) for 1 hr. mAb were diluted in PBS with 0-1% Tween 20, 1% bovine serum albumin (BSA) and 0 3% gelatine to a final concentration of 16 ,g/ml and this dilution was then used to prepare a series of doubling dilutions in the sCD4-coated plates. The plates were incubated for 5 hr at room temperature and then washed three times in PBS with 0-1% Tween 20 (PBS-T). Radiolabelled anti-mouse immunoglobulin antibody was added to each well at 2 x 105 counts per minute (c.p.m.) in 50 pi per well. After incubation for 1 hr at room temperature, the plates were washed and bound radioactivity counted on an LKB Clinigamma 1272 gamma counter (Pharmacia, Uppsala, Swe-

den).

RIA for inhibition of CD4-gp120 interaction Recombinant gpl 20 was radiolabelled by the Iodogen technique to a specific activity of 105 pCi/pg. Plastic 96-well plates (Falcon 3912) were quenched overnight at 4° with 0 3% gelatine and then washed with PBS. AntiCD4 mAb were serially diluted in the plates, from a starting concentration of 100 pg/ml in a 25 pi volume of RPMI medium supplemented with 0-1% gelatine, 0 1% BSA and 20 mm sodium azide. HPB-ALL cells were added at 106 cells/well in 25 p1 and the plates were incubated for 60 min at room temperature. Radiolabelled rgp 120 was added at a concentration giving approximately 106 c.p.m./well in 25 p,, and the plates were incubated for a further 150 min at room temperature. Controls were included on each plate as follows: no mAb, sCD4 (5 pg/ml), anti-Leu-3a (50 pg/ml). A further plate was prepared with labelled gp12O and CD4- NALM-6 cells (106 cells/well), and also wells with no cells at all, to control for non-specific binding to cell membranes and to the plastic wells,

respectively. Following incubation, the cell pellets were washed as above and cell-associated radioactivity determined as above. Inhibition of gpl 20 binding to CD4 was calculated according to the formula:

(B-T)

The concentration of each antibody that gave 60% of maximum binding (approximately 4000 c.p.m. well) was calculated for use in the second stage of the assay. The absolute number of c.p.m. bound by a concentration of mAb of 4 pg/ml was also used for comparison with binding of mAb to CD4+ cells. Stage 2. The assay was repeated using a fixed concentration of mAb, determined as above, in the presence of variable concentrations of sCD4 (0-5 pg/ml). The percentage inhibition (I) was calculated from the following formula, after correction for non-specific background binding: I = 100 x 1

-

c.p.m. bound at given sCD4 conc. c.p.m. bound in the absence of sCD4

and plotted against sCD4 concentration. The concentration of

sCD4 required to inhibit binding by 50% (C50) was derived, and antibodies were ranked in ascending order of C50, and thus in descending order of functional affinity.

10X(B -L)

where B=c.p.m. bound in the absence of mAb, T=c.p.m. bound in the presence of test mAb at any particular concentration and L=c.p.m. bound in the presence of anti-Leu-3a at 50,pg/ml. The concentration required for 50% inhibition of binding and the maximum inhibition observed in the presence of excess mAb were derived. In the absence of mAb approximately 2% of added radiolabelled gpl20 bound to the CD4+ cells in this assay. Backgound binding, either in empty wells or on CD4NALM-6 cells amounted to approximately 0-3% of added gpl20. Indirect RIA of anti-CD4 mAb binding to CD4+ cells Plastic 96-well plates were quenched and washed as above. 2 x 105 HPB-ALL cells were added per well, pelleted by centrifugation at 430 g for 2 min and resuspended in their residual volume. Anti-CD4 mAb were added at 4 pg/ml in 50 p1 of RPMI medium supplemented as for the gp l 20 binding assay.

D. Wilks et al.

12

Table 1. In vitro activity of anti-CD4 mAb

Antibody 7.2G4 6.3G4 L204 Leu-3a L218 12.22F5 7.3F1 l OKT4A MT310 MT151 L201 OKT4D 12.16A2 L216 L217 OKT4E L215 OKT4F OKT4C OKT4

Functional affinity (C50) 009 0 13 0 13 0-16 0.19 020 021 022 025 028 031 0 80 1-00 1 12 1 36 1 94 285 3 13 376

Inhibition of gpI2O/CD4 binding

Syncytium inhibition assay

(mg/i)

C50

0 16 0 13 0 10 0 17 4 1 25 075 083 070 40 1 88 240 > 320 223 100 320 500 > 320 320 >500

023 020 026 026 300 400 026 ND ND 046 026 008

Maximum % inhibition 100 100 100 100 50 100 100 ND ND 98 100 100 25 34 89 88 90 95 78 2

1-10 1 20 1 25 1 23 1 30

Indirect RIA sCD4 D4+ cells (total c.p.m. bound) 26,727 26,516 26,379 19,110 8484 23,988 21,450 ND ND 16,188 12,768 8307 20,397 6390 4737 5310 6009 3675 5928 2127

10,200 10,503 10,923 7044 6669 10,182 11,934 ND ND

10,308 7320 5079 5343 6363 6279 5592 4989 3771 8472 2235

L

z=0 73, P