Proc. Natl. Acad. Sci. USA Vol. 88, pp. 3238-3242, April 1991 Immunology
Production of site-selected neutralizing human monoclonal antibodies against the third variable domain of the human immunodeficiency virus type 1 envelope glycoprotein (acquired immunodeficiency syndrome/lymphoblastoid lnes/beterohybridoma)
MIROSLAW K. GORNY*, JIAN-YIN XU*, VASILIKI GIANAKAKOSt, SYLVIA KARWOWSKA*, CONSTANCE WILLIAMSt, HAYNES W. SHEPPARDt, CAkL V. HANSONI, AND SUSAN ZOLLA-PAZNER*t§ *Department of Pathology, New York University Medical Center, 550 First Avenue, New York, NY 10016; tVeterans Affairs Medical Center, 423 East 23rd Street, New York, NY 10010; and tViral and Rickettsial Disease Laboratory, California Department of Health Services, 2151 Berkeley Way, Berkeley, CA 94704
Communicated by Michael Heidelberger, December 21, 1990
provide an alternative means of prophylaxis and treatment. For example, Prince et al. (18) have shown that passively administered human immunoglobulin preparations with high anti-HIV neutralizing titers, injected prior to HIV challenge, prevent primate infection, and several studies of pregnant HIV-infected women have shown a correlation between maternal Abs to the V3 loop and protection of the fetus from in utero or perinatal infection (19-21). Therefore, for passive immunization, human mAbs to the V3 loop that are capable of HIV neutralization might be particularly useful. Methods for generating human mAbs to HIV have been developed resulting in the generation of several lines making mAbs specific for gp4l and p24 with various biological functions (22-25) but none possess neutralizing activity. With the description of the V3 loop of gpl20 as a principal neutralizing domain and the identification of MN as a representative of one of the most prevalent HIV strains in North America and Europe (17, 26, 27), the work described below was initiated to derive human mAbs site-selected for specificity and function directed against the V3 loop of HIVMN.
Cell lines secreting IgG1 human monoclonal ABSTRACT antibodies (mAbs) to the envelope glycoprotein, gpl2O, of human immunodeficiency virus (IRV) have been produced by transformation of peripheral blood cells front IIV-infected individuals and by fusion of transformed cells to a humanmouse heteromyeloma cell line (SHM-D33). Two human mAbs were site-selected by means of a 23-mer synthetic peptide spanning a portion of the third variable domain of gpI20 from the MN strain of HIV. The two heterohybridomas produce three times more IgG than do their parent lymphoblastoid cell lines. The specificities of these mAbs have been mapped to sequences near the tip of the disulfide loop of the gpl20 third variable domain, Lys-Arg-lle-His-Ile and His-Ie-Gly-Pro-GlyArg, respectively. The mAbs have dissociation constants of 3.7 x 10-6 M and 8.3 x 10-7 M, neutralize HIVMN in vitro at nanogram levels, and bear the characteristics of antibodies associated with protective immunity in vivo.
Neutralizing antibodies (Abs) are considered to be essential for protection against many viral infections, including those caused by retroviruses. Neutralizing Abs against human immunodeficiency virus type 1 (HIV-1) have also been described and their specificities have been mapped to several locations within the structural proteins of the virion such as the core protein p17 (1, 2), the envelope glycoprotein gpl20, and the transmembrane protein gp4l (3-7). Some of these epitopes encompass genetically conserved regions of the envelope (4-6, 8, 9), but they do not appear to be principal neutralizing domains since immunization against these epitopes elicits polyclonal antisera with low neutralizing titers (6) or gives rise to murine monoclonal antibodies (mAbs) that neutralize the virus only at relatively high concentrations (5, 10). The CD4 binding domain of gp120, a broadly conserved region, may give rise to group-specific neutralizing Abs that appear in infected humans several months after infection.O In various systems, this region of gpl20 has given rise to neutralizing Abs (5, 11) or to Abs that affect binding of gpl20 to CD4 (12, 13). A principal neutralizing domain has been identified (1416). It resides within the third variable domain (V3) of gpl20, located between Cys-301 and Cys-335, I which are bridged by a disulfide bond (56). The tip of this loop consists of four highly conserved amino acids (Gly-Pro-Gly-Arg) flanked by amino acids that differ between isolates (17, 56). Whereas active immunization against gpl20 and the V3 loop is being studied as a mode of prevention of HIV infections passive immunization with Abs to gp120 and the V3 loop might
MATERIALS AND METHODS Subjects. Cells from two asymptomatic homosexual men were used in these experiments. The volunteers had been seropositive for at least 3 years and 1 year, respectively, and had CD4' cell counts of 628 and 871 per mm3. Each gave informed consent to participate in these studies as specified
by the Institutional Review Boards. Synthetic Peptide Used for Screening of Reactive Cells by ELISA. A peptide that spans 23 amino acids of the gp120 V3 loop of the MN strain of HIV-1 (23-mer peptide) was synthesized by Peninsula Laboratories. The peptide, derived from the sequence published by Myers et al. (56k, has the following amino acid sequence: YNKRKRIHIGPGRAFYTTKNIIG. Establishment of Epstein-Barr Virus-Transformed Cell Lines and Heterohybridomas. The method for producing cell lines synthesizing human mAbs to HIV-1 was as described (22). Briefly, mononuclear cells were obtained from blood and incubated overnight with Epstein-Barr virus. Cyclosporin A (Sandoz, East Hanover, NJ) was incorporated into the Abbreviations: mAb, monoclonal antibody; Ab, antibody; HIV, human immunodeficiency virus; V3, third variable domain of gp120; RIP, radioimmunoprecipitation. §To whom reprint requests should be addressed. lBlattner, W., Nara, P., Shaw, G., Hahn, B., Kong, L., Matthews, T., Bolognesi, D., Waters, D. & Gallo, R., Fifth International Conference on AIDS, June 4-9, 1989, Montreal, page 510, abstr. MC07. "The numbering system used throughout for HIVMN gp120 was designated by Myers et al. (56).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3238
Immunology: Gorny et al. medium at 0.5 ,ug/ml for the first week after infection of cells with Epstein-Barr virus. Cells were cultured for 3-4 weeks in 96-well plates and then screened for Abs in the supernatant specific for the 23-mer peptide. Positive cultures were expanded, subcultured several times, and finally expanded into flasks. Lymphoblastoid cells were fused with the mouse x human heteromyeloma SHM-D33 (kindly provided by Nelson Teng, Stanford University, Stanford, CA). Briefly, the SHM-D33 cells and lymphoblastoid cells were mixed at a ratio of 1:3 and centrifuged to a pellet. Then, 1 ml of 50%o (vol/vol) polyethylene glycol 1300-1600 (Sigma) was added dropwise over 1 min, followed by slow dilution with Iscove's modified Dulbecco's medium. After resuspension in Iscove's medium supplemented with 15% (vol/vol) fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 ,ag/ml), the cells were plated in 96-well culture plates in 100 Al at 8 x 104 cells per well. The next day, 1 x 104 mouse peritoneal cells were added per well as feeder cells with 0.5 mM hypoxanthine/0.2 ,uM aminopterin/16,uM thymidine/1 juM ouabain-(Sigma). After 2-3 weeks, all culture wells were screened for Ab production against the aforementioned peptide and hybrids producing reactive Abs were expanded in 24-well plates. Hybrids that produced the highest level of Abs and IgG, measured by ELISA, were cloned repeatedly at 100 to 1 cells per well. Hybridomas are denoted by the suffix "D" in the cell line designation. Antibody Detection and Characterization. Culture supernatants were screened by ELISA as described (22) for reactivity to the 23-mer that was coated overnight at 40C at a concentration of 1 Ag/ml in 0.05 M sodium carbonate (pH 9.6). The specificity ofAb binding was assessed by radioimmunoprecipitation (RIP). RIP assays were carried out by the method of Pinter and Honnen (28) with 30 ,ug of strain HTLV-IIIB lysate (Organon) and/or strain MN lysate (Advanced Biotechnologies, Silver Spring, MD) labeled with 125I using the Bolton-Hunter reagent (New England Nuclear). Culture supernatants were incubated with labeled viral lysate and further processed for electrophoretic analysis as described (22, 28). Antibody subclasses were determined by ELISA and IgG quantitation was performed (22). The light chain type of mAbs was analyzed by ELISA using microtiter plates coated with rabbit anti-human K chain or rabbit anti-human A chain Abs (Dakoplatts, Glostrup, Copenhagen). The developing Abs were alkaline phosphatase-coupled goat anti-human K chain and goat anti-human A chain (Sigma), respectively. Epitope Mapping. The fine specificity of the mAbs was determined using the Epitope Mapping Kit (Cambridge Research Biochemicals, Valley Stream, NY). Briefly, 18 sequential overlapping hexapeptides that span the 23-mer were synthesized in situ on plastic pins. The peptides were deprotected, washed, and dried according to the manufacturer's instructions. Because the configuration of the pins fits into 96-well microtiter plates, the ELISA assays were carried out in standard microplates as recommended by the manufacturer. Thus, all pins were allowed to react with sera or culture supernatants from the various cell lines diluted 1:100 or 1:10, respectively, in phosphate-buffered saline containing 0.1% Tween-20, 1% ovalbumin, and 1% bovine serum albumin. Thereafter, the pins were washed and immersed in horseradish peroxidase-conjugated goat anti-human IgG. The color reaction was read in a Dynatech MR-700 at 405 nm. Determination of mAb Affinity. Determination of the dissociation constants (Kd) of human mAbs was performed by an ELISA methodology according to Friguet et al. (29). Briefly, the culture supernatants of 257-2D, 257-2, 268-liD, and 268-11 were tested at concentrations of 2.5, 1.1, 1.25, and 0.5 ,(g/ml, respectively. The 23-mer (Mr, 2638) was dissolved in 0.05 M acetic acid to a concentration of 1 mg/ml (3.8 x
Proc. Natl. Acad. Sci. USA 88 (1991)
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10' M), diluted in phosphate-buffered saline (pH 7.2), and used at concentrations ranging from 10-5 to 10-8 M. Supernatant and peptide were mixed in equal volumes and after 16 hr, the mixture was added to plates coated with the 23-mer (1 gg/ml) and the amount of unbound mAb was measured by ELISA. Data were plotted according to the Friguet modification of Klotz (29) to determine the Kd. Neutralization Assay. A plaque assay, by Hanson et al. (30), that measures the inhibition of HIV infection of MT-2 cells was used to detect the neutralizing activity of mAbs in the presence or absence of human complement. Thus, mAbs were serially diluted in 50%o (vol/vol) assay medium (30) plus 50% (vol/vol) of a normal human plasma pool. The plasma pool served as the source of human complement; for studies in the presence of complement, mAb and virus were incubated for 18 hr at 370C. For tests in the absence of complement, the plasma pool was heat-inactivated and the mAb and virus were incubated under these conditions for 1 hr at 370C. The dilution at which 50% of the input virus was neutralized on the basis of plaque counts was calculated by interpolation using a third-order regression analysis of the mean plaque count at each dilution. RESULTS Peripheral blood specimens from two subjects provided 17.2 and 92.0 x 106 mononuclear cells, which were cultured in microtiter wells at 60,000 and 85,000 cells per well, respectively. In both cases, initial screening of wells after 3-4 weeks of culture revealed two positive wells for Abs reactive with the 23-mer of the V3 domain of gpl20 of HIVMN. Cells in these wells were expanded and cloned (22) and one clone from each volunteer was stabilized. The cell lines were designated 257-2 and 268-11, respectively. Each line was cloned three times at 100 cells per well and found to produce an IgG1 A mAb to the 23-mer. Simultaneous with the original cloning, cells from expanded cultures that gave rise to cell lines 257-2 and 268-11 were fused to the SHM-D33 heteromyeloma. Two resulting heterohybridomas, 257-2D and 268liD, were cloned at least four times each, with the final two clonings performed at 1 cell per well. Although the lymphoblastoid cell lines 257-2 and 268-11 produced 6.4 and 3.8 jig of IgG per ml per 106 cells per 24 hr, respectively, the related heterohybridomas, 257-2D and 268-liD produced 20.5 and 11.3 ,ug of IgG per ml per 106 cells per 24 hr, respectively. The mAbs were shown to react in an ELISA with the 23-mer when the latter was bound to the wells of microtiter plates at concentrations as low as 1 ng/ml (data not shown). The specificity of these mAbs was further defined by RIP, the results of which are shown in Fig. 1. Both mAbs react 12 3 4 5 6 7 8
FIG. 1. RIP assay of human mAbs with HIV lysates. Lanes: 1 and 2, reactivity of a serum specimen from an HIV-infected individual; 3 and 4, reactivity of supernatant 257-2D; 5 and 6, reactivity of supernatant 268-11D; 7 and 8, reactivity of supernatant 280-2 (which is unreactive with HIV antigens); 1, 3, 5, and 7, reactivity of specimens to HIVMN lysate; 2, 4, 6, and 8, reactivity of specimens to
HTLV-111B lysate.
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Proc. Natl. Acad. Sci. USA 88 (1991)
with the env-encoded protein gp120 of HIVMN but not with the gp120 derived from strain HTLV-IIIB, revealing the type-specificity of these mAbs. Fine mapping of the epitopes of these mAbs was performed using the Epitope Mapping Kit, which utilizes the method developed by Geysen et al. (31) to synthesize hexapeptides on plastic pins. Each mAb was tested against a set of 18 hexapeptides that spans the region of the 23-mer, each hexapeptide overlapping its neighbor by five amino acids. The hexapeptides, bound to the plastic pins on which they were synthesized, were incubated with sera from seronegative or seropositive subjects and with supernatant fluids of heterohybridomas 257-2D and 268-liD. Seronegative sera did not react with any pins. A seropositive serum sample reacted above background levels with all pins, giving peak reactions with three pins attached to peptides spanning the region PGRAFYTT located at the tip and right side of the V3 loop (Fig. 2A). The mAb 257-2D bound strongly to two adjacent hexapeptides, RKRIHI and KRIHIG (Fig. 2B), indicating that the smallest reactive peptide (core of the epitope) that this mAb recognizes is KRIHI, located to the left of the conserved tip of the V3 loop. The flanking N- and C-terminal arginine and glycine residues may also contribute to the binding of this mAb. The mAb 268-liD bound to a single hexapeptide consisting of HIGPGR, which spans the tip of the loop and the two adjacent amino acids to the left. The Kd values of these mAbs were determined using the method of Friguet et al. (29) and mAbs 257-2D and 268-liD were found to have Kd values of 3.7 x 10-6 and 8.3 x 10-7 M, respectively (Fig. 3). These figures are in the range of those reported for IgG mAbs (29, 32). The Kd values of mAbs produced by the lymphoblastoid cell lines (257-2 and 268-11) and by the heterohybridomas (257-2D and 268-liD) were similar (data not shown). One should note that these Kd values are derived from the reaction between the mAbs and the 23-mer peptide. The Kd value of the mAbs for the native gp120 molecule may be lower by virtue of contributions of conformation of the whole molecule to the epitopes to which the mAbs react. Finally, these mAbs were tested for their ability to neutralize virus in a plaque reduction assay (30). When supernatant fluids from 257-2D and 268-liD were incubated with HIVMN for 1 hr in the absence of complement prior to addition to permissive MT-2 cells, 50% neutralization was achieved at dilutions of 1:4700 and 1:2000, corresponding to mAb concentrations of 3.0 and 23.0 ng/ml, respectively (Table 1). No neutralization was obtained when the mAbs were tested against HTLV-IIIB. When 257-2D and 268-liD were tested in a more sensitive assay, by incubation with virus for 18 hr in the
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FIG. 3. Determination of the Kd for two human mAbs to gpl20 performed according to the method of Friguet et al. (29). Klotz plots are shown in which the slopes of the lines are equivalent to the Kd of 257-2D (o) and 268-liD (o). v is the fraction of bound Ab and 1/v corresponds to Ao/(Ao - A), where Ao is the absorbance of Ab in the absence of peptide and A equals the absorbance of Ab after incubation with peptide. ao is the peptide concentration.
presence of human complement, neutralization was achieved at dilutions of 1:44,000 and 1:41,000, corresponding to mAb concentrations of 0.3 and 1.1 ng/ml, respectively. Again, no neutralization of HTLV-IIIB occurred under these conditions. A representative dose-response curve for the activity of mAb 257-2D against HIVMN, in the absence and presence of complement, is shown in Fig. 4. Human mAb 50-69 directed against the HIV transmembrane protein gp4l and mAb 71-31 directed to the core protein p24, described by Gorny et al. (22), were tested in parallel and displayed essentially no neutralizing activity for either strains of HIV.
DISCUSSION Despite the technical difficulties encountered over the past decade in producing human mAbs, several have been obtained to HIV (11, 22, 33-40). The success in generating human mAbs to HIV, as opposed to other antigens, appears to be due to the chronic antigenic stimulation of HIV-infected individuals that results in hyperimmunization and continual circulation of Ab-producing B cells. Consequently, Abproducing B cells can be obtained from the peripheral blood and the majority of these cells produce IgG antibodies. Of the human mAbs produced, most have been directed to gp4l or p24 as they have generally been identified and selected for reactivity with whole viral lysates that contain a preponderance of these structural proteins. The site-selected approach in the screening procedure for selecting the mAbs described
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tides homologous with the 23-mer of the HIVMN V3 loop. The reactivity of each hexapeptide with seronegative (solid bars) or seropositive (hatched bars) sera (A) or with supernatant from heterohybridoma 257-2D (open bars) or 268-liD (solid bars) (B) is shown on the ordinate and each hexapeptide is designated by the single-letter code of its N-terminal residue and the subsequent five amino acids. Thus, the sequence appearing on the abscissa is the sequence of the 23-mer.
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Proc. Natl. Acad. Sci. USA 88 (1991)
Table 1. Neutralizing activity of human mAbs against HIV HIVMN neutralizing Ab titers, dilution 1 hr/no C mAb Specificity 18 hr/+ C 257-2D gpl20 1:4700 (3.0) 1:44,000 (0.3) 268-liD gp120 1:2000 (23.0) 1:41,000 (1.1) 50-69 gp41 1:3 1:3 71-31 p24 Negative Negative C, complement. All data with strain HTLV-IIIB were negative. Numbers in parentheses are ng of mAb per ml.
herein was designed to pick cells producing Abs to a particular neutralizing domain rather than identifying mAbs to random HIV epitopes as was previously done (11, 22, 33-40). The mechanisms by which Abs to the V3 loop mediate neutralization have yet to be elucidated. Some have postulated a requirement for high-affinity Abs to effect neutralization (20); however, the data shown above demonstrate that mAbs with average dissociation constants are quite effective neutralizing Abs, at least under in vitro conditions. Stephens et al. (41) have suggested that proteolytic cleavage of the V3 loop is essential for infectivity and it is thus attractive to postulate that Abs to the V3 loop may interfere with this cleavage step. The data presented above suggest another mechanism, that, in addition to the physical inactivation or 120
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Antibody dilution (1/log10) FIG. 4. HIV neutralization by serial dilution of mAb 257-2D. Data normalized as a percentage of the mean plaque count in 12 or 24 replicate control wells (o) for experiments shown in A and B, respectively. In these examples, control plaque numbers equaled 9.6 (SEM = 1.4) and 9.1 (SEM = 1.1) for A and B, respectively. Error bars represent one SEM above and below the mean at each dilution. The starting concentration of 257-2D was 14.0 gtg/ml. (A) Neutralization of HIVMN incubated with 257-2D in the absence of complement for 1 hr. (B) Neutralization of HIVMN incubated with 257-2D in the presence of complement for 18 hr. are
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blocking conferred by the Ab moiety, components of the complement cascade may contribute to virus neutralization by means of virolysis, a phenomenon described with HIV by Spear et al. (42). Other mechanisms of virus neutralization (for review, see ref. 43) must also be considered, a task that will be facilitated by the availability of these and other neutralizing mAbs. Studies of the cross-reactivity of type-specific anti-HIV Abs have been based on serologic studies of reactivity with peptides from the V3 region (17, 20). Although these studies have revealed a high frequency of reactivity for certain viral sequences, little is known about the extent to which sera with these defined reactivities will neutralize viruses with the homologous sequences in vitro, and essentially nothing is known about the in vivo function of these Abs. Moreover, the hypervariability of the V3 loop and the ability of HIV and other retroviruses to mutate under the pressure of an effective immune response (44, 45) implies that passive immunization with a single mAb may be insufficient to protect or treat a large population of high-risk individuals or single individuals over a long period of time. These considerations may dictate the need for a mixture of mAbs. However, the feasibility of producing a variety of human mAbs to HIV is demonstrated by the data presented herein and elsewhere (11, 22, 33-40). The epitope mapping of the mAbs to the V3 loop point to the possibility of generating mAbs to variable regions of the virus with reactivity to more than one virus strain. Thus, the epitope of mAb 268-11 is shared by a number of members of the MN family (MN, SC, and JH3) and appears in more distantly related isolates that possess the same sequence near the tip of the V3 loop (HAN, WMJ1, and WMJ3) (20, 56). The epitope of mAb 257-2 occurs in a more variable portion of the loop and, therefore, might react with a more restricted group of viruses. Nonetheless, both mAbs neutralize SF-2 as well as MN despite disparities in the relevant epitopes at amino acids 311 and 313 (K. Steimer, N. Haigwood, M.K.G., S.K., J.-Y.X., C.V.H., H.W.S., and S.Z.-P., unpublished data). Studies of the sensitivity of field isolates may define further the degree to which these mAbs will be capable of neutralizing the most prevalent strains derived directly from patients, but these studies must await standardization of methods to test the sensitivity of field isolates to antibodydependent neutralization. The usefulness of these mAbs as reagents for passive immunization can only be determined by in vivo studies. Nonetheless, precedents exist for the prevention of retroviral-induced diseases in mice by passive immunization (for review, see ref. 46), and, as noted above, there is increasing evidence that passive immunization of nonhuman primates can also protect against challenge with HIV-1 (18, 47). Preliminary studies of passive immunization of HIV-infected patients with plasma (48-50) or immunoglobulin preparations from sera of asymptomatic HIV-infected donors** have also suggested the usefulness of pursuing passive immunotherapy as a modality of treatment and prophylaxis. It is probable that human mAbs would have several advantages over immunoglobulin preparations from sera of HIV-positive donors. (i) Immunoglobulin pools prepared from patients' sera will contain enhancing Abs (51-53) that may counteract the benefits of protective Abs; however, human mAbs can be selected with protective but no enhancing function. (ii) Protective Abs represent an extremely small proportion of polyclonal immunoglobulins, necessitating the administration of gram quantities of immunoglobulin by the **Rhame, F. S., Goodroad, B. K., Cummins, L. M., Fletch, C. V., Henry, K., Shah, N., Condie, R. M., Balfour, H. H., Jr., & Allain, J. P., Sixth International Conference on AIDS, June 20-23, 1990, San Francisco, vol. 3, page 211, abstr. SB500.
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intravenous route. Potent mAbs, on the other hand, may be administered in milligram amounts (54, 55), allowing frequent administration by the intramuscular route. (iii) Higher levels of specific protective Abs could be more readily achieved using mAb preparations than using polyclonal immunoglobulin. This rationale, then, provides the basis for the identification and development of human mAbs to HIV with known protective function. This work was supported by research funds from the National Institutes of Health (Grants Al 72658 and Al 82693) and from the Department of Veterans Affairs. 1. Papsidero, L. D., Sheu, M. & Ruscetti, F. W. (1989) J. Virol. 63, 267-272. 2. Sarin, P. S., Sun, D. K., Thornton, A. H., Naylor, P. H. & Goldstein, A. L. (1986) Science 232, 1135-1137. 3. Javaherian, K., Langlois, A. J., McDanal, C., Ross, K. L., Eckler, L. I., Jelfis, C. L., Profy, A. T., Rusche, J. R., Bolognesi, D. P., Putney, S. D. & Matthews, T. J. (1989) Proc. Nat!. Acad. Sci. USA 86, 6768-6772. 4. Ho, D. D., Kaplan, J. C., Rackauskas, I. E. & Gurney, M. E. (1988) Science 239, 1021-1023. 5. Sun, N., Ho, D. D., Sun, C. R. Y., Liou, R.-S., Gordon. W., Fung, M. S. C., Li, X.-L., Ting, R. C., Lee, T.-H., Chang, N. T. & Chang, T.-W. (1989) J. Virol. 63, 3579-3585. 6. Chanh, T. C., Dreesman, G. R., Kanda, P., Linette, G. P., Sparrow, J. T., Ho, D. D. & Kennedy, R. C. (1986) EMBO J. 5, 3065-3071. 7. Thomas, E. K., Weber, J. N., McClure, J., Clapham, P. R., Singhal, M. C., Shriver, M. K. & Weiss, R. A. (1988) AIDS 2, 25-29. 8. Ho, D. D., Sarngadharan, M. G., Hirsch, M. S., Schooley, R. T., Rota, T. R., Kennedy, R. C., Chanh, T. C. & Sato, V. L. (1987) J. Virol. 61, 2024-2028. 9. Modrow, S., Hahn, B. H., Shaw, G. M., Gallo, R. C., Wong-Staal, F. & Wolf, H. (1987) J. Virol. 61, 570-578. 10. Dalgleish, A. G., Chanh, T. C., Kennedy, R. C., Kanda, P., Clapham, P. R. & Weiss, R. A. (1988) Virology 165, 209-215. 11. Robinson, J. E., Holton, D., Pacheco-Morell, S., Liu, J. & McMurdo, H. (1990) AIDS Res. Human Retroviruses 6, 567-579. 12. Lasky, L. A., Nakamura, G., Smith, D. H., Fennie, C., Shimasaki, C., Patzer, E., Berman, P., Gregory, T. & Capon, D. J. (1987) Cell 50, 975-985. 13. Linsley, P. S., Ledbetter, J. A., Kinney-Thomas, E. & Hu, S. (1988) J. Virol. 62, 3695-3702. 14. Rusche, J. R., Javaherian, K., McDanal, C., Petro, J., Lynn, D. L., Grimaila, R., Langlois, A., Gallo, R. C., Arthur, L. O., Fischinger, P. J., Bolognesi, D. P., Putney, S. D. & Matthews, T. J. (1988) Proc. Nat!. Acad. Sci. USA 85, 3198-3202. 15. Palker, T. J., Clark, M. E., Langlois, A. L., Matthews, T. J., Weinhold, K. J., Randall, R. R., Bolognesi, D. P. & Haynes, G. (1988) Proc. Nat!. Acad. Sci. USA 85, 1758-1762. 16. Goudsmit, J., Boucher, C. A. B., Meloen, R. H., Epstein, L. G., Smit, L., Van Der Hoek, L. & Bakker, M. (1988) AIDS 2, 157-164. 17. LaRosa, G. J., Davide, J. P., Weinhold, K., Waterbury, J. A., Profy, A. T., Lewis, J. A., Langlois, A. J., Dreesman, G. R., Boswell,. R. N., Shadduck, P., Holley, L. H., Karplus, M., Bolognesi, D. P., Matthews, T. J., Emini, E. A. & Putney, S. D. (1990) Science 249, 932-935. 18. Prince, A. M., Horowitz, B., Shulman, R. W., Pascual, D., Hewlett, I., Epstein, J. & Eichberg, J. W. (1990) in Vaccines 90: Modern Approaches to New Vaccines Including Prevention of AIDS, ed. Brown, F. (Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY), pp. 347-351. 19. Rossi, P., Moschese, V., Broliden, P. A., Fundaro, C., Quinti, I., Plebani, A., Giaquinto, G., Tovo, P. A., Ljunggren, K., Rosen, J., Wigzell, H., Jondal, M. & Wahren, B. (1989) Proc. Natl. Acad. Sci. USA 86, 8055-8058. 20. Devash, Y., Calvelli, T. A., Wood, D. G., Reagan, K. J. & Rubinstein, A. (1990) Proc. Natl. Acad. Sci. USA 87, 3445-3449. 21. Goedert, J. J., Mendez, H., Drummond, J. E., Robert-Guroff, M., Minkoff, H. L., Holman, S., Stevens, R., Rubinstein, A., Blattner, W. A., Willoughby, A. & Landesman, S. H. (1989) Lancet H, 1351-1354. 22. Gorny, M. K., Gianakakos, V., Sharpe, S. & Zolla-Pazner, S. (1989) Proc. Nat!. Acad. Sci. USA 86, 1624-1628. 23. Till, M., Zolla-Pazner, S., Gorny, M. K., Patton, J. S., Uhr, J. & Vitetta, E. S. (1989) Proc. Nat!. Acad. Sci. USA 86, 1987-1991. 24. Robinson, W. E., Jr., Kawamura, T., Gorny, M. K., Montefiori, D. C., Mitchell, W. M., Lake, D., Xu, J., Matsumoto, Y., Sugano, T., Masuho, Y., Hersh, E. & Zolla-Pazner, S. (1990) Proc. Nat!. Acad. Sci. USA 87, 3185-3189.
Proc. Natl. Acad. Sci. USA 88 (1991) 25. Tyler, D. S., Stanley, S. D., Zolla-Pazner, S., Gorny, M. K., Shadduck, P. P., Langlois, A. J., Matthews, T. J., Bolognesi, D. P., Palker, T. J. & Weinhold, K. J. (1990) J. Immunol. 145, 3276-3282. 26. Devash, Y., Matthews, T. J., Drummond, J. E., Javaherian, K., Waters, D. J., Arthur, L. O., Blattner, W. A. & Rusche, J. R. (1990) AIDS Res. Hum. Retroviruses 6, 307-316. 27. Goudsmit, J., Zwart, G., de Jong, J., Wolfs, T., de Ronde, A. & Nara, P. L. (1990) in Vaccines 90: Modern Approaches to New Vaccines Including Prevention of AIDS, ed. Brown, F., Chanock, R. M., Ginsberg, H. S. & Lerner, R. A. (Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY), pp. 291-2%. 28. Pinter, A. & Honnen, W. J. (1988) J. Immunol. Methods 112, 235-241. 29. Friguet, B., Chaffotte, A. F., Djavadi-Ohaniance, L. & Goldberg, M. E. (1985) J. Immunol. Methods 77, 305-319. 30. Hanson, C. V., Crawford-Mikaya, L. & Sheppard, H. W. (1990)J. Clin. Microbiol. 28, 2030-2034. 31. Geysen, H. M., Meloen, R. H. & Barteling, S. J. (1984) Proc. Natl. Acad. Sci. USA 81, 3998-4002. 32. Larsson, A., Ghosh, R. & Hammarstrom, S. (1987) Mol. Immunol. 24, 569-576. 33. Banapour, B., Rosenthal, K., Rabin, L., Sharma, V., Young, L., Fernandez, J., Engleman, E., McGrath, M., Reyes, G. & Lifson, J. (1987) J. Immunol. 139, 4027-4033. 34. Grunow, R., Jahn, S., Porstmann, T., Kiessig, S. S., Steinkellner, H., Steindl, F., Mattanovich, D., Gurtler, L., Deinhardt, F., Katinger, H. & Baehr, R. V. (1988) J. Immunol. Methods 106, 257-265. 35. Desgranges, C. D., Boyer, V., Souche, S., Sprecher, S., Burny, A., Gallo, R., Bernard, J., Reveil, B. & Zagury, D. (1988) Lancet i, 935-936. 36. Evans, L. A., Homsy, J. M., Morrow, W., Gaston, I., Sooy, C. D. & Levy, J. A. (1988) J. Immunol. 140, 941-943. 37. Sugano, T., Musuho, Y., Matsumoto, Y., Lake, D., Gschwind, C., Petersen, E. A. & Hersh, E. M. (1988) Biochem. Biophys. Res. Commun. 155, 1105-1112. 38. Amadori, A., Climinale, V., Calabro, M. L., Tessarollo, L., Francavilla, E. & Chieco-Bianchi, L. (1989) AIDS Res. Hum. Retroviruses 5, 73-78. 39. Ohlin, M., Broliden, P. A., Danielson, L., Wahren, B., Rosen, J., Jondal, M. & Borrebaeck, C. A. (1989) Immunology 68, 325-331. 40. Scott, C. F., Jr., Silver, S., Profy, A. T., Putney, S. D., Langlois, A., Weinhold, K. & Robinson, J. E. (1990) Proc. Natl. Acad. Sci. USA 87, 8597-8601. 41. Stephens, P. E., Clements, G. & Yarranton, G. T. (1990) Nature (London) 343, 219. 42. Spear, G. T., Sullivan, B. L., Landay, A. L. & Lint, T. F. (1990) J. Virol. 64, 5869-5873. 43. Mandel, B. (1985) in Immunochemistry of Viruses: the Basisfor Serodiagnosis and Vaccines, ed. van Regenmortel, M. H. V. (Elsevier, New York), p. 53. 44. Reitz, M. S., Wilson, C., Naugle, C., Gallo, R. C. & Robert-Guroff, M. (1988) Cell 54, 57-63. 45. Hussain, K., Issel, C. J., Schnorr, K. L., Rwambo, P. M. & Monteflaro, R. C. (1987) J. Virol. 61, 2956-2%1. 46. Gorny, M., Pinter, A. & Zolla-Pazner, S. (1989) Progress in AIDS Pathology, eds. Rotterdam, H. & Sommers, S. C. (Field & Wood, New York), pp. 181-199. 47. Emini, E. A., Nara, P. L., Schleif, W. A., Lewis, J. A., Davide, J. P., Lee, D. R., Kessler, J., Conley, S., Matsushita, S., Putney, S. D., Gerety, R. & Eichberg, J. W. (1990) J. Virol. 64, 3674-3678. 48. Jackson, G. G., Rubenis, M., Knigge, M., Perkins, J. T., Paul, D. A., Despotes, J. C. & Spencer, P. (1988) Lancet il, 647-651. 49. Karpas, A., Hill, F., Youle, M., Cullen, V., Gray, J., Byron, N., Hayhoe, F., Tenant-Flowers, M., Howard, L., Gilgen, D., Oates, J. K., Hawkins, D. & Gazzard, B. (1988) Proc. Natl. Acad. Sci. USA 85, 9234-9237. 50. Karpas, A., Hewlett, I. K., Hill, F., Gray, J., Byron, N., Gilgen, D., Bally, V., Oates, J. K., Gazzard, B. & Epstein, J. E. (1990) Proc. Nat!. Acad. Sci. USA 87, 7613-7617. 51. Robinson, W. E., Jr., Montefiori, D. C. & Mitchell, W. M. (1988) Lancet i, 790-794. 52. Homsy, J., Meyer, M., Tateno, M., Clarkson, S. & Levy, J. A. (1989) Science 244, 1357-1360. 53. Matsuda, S., Gidlund, M., Chiodi, F., Cafaro, A., Nygren, A., Morein, B., Nilsson, K., Fenyo, E. M. & Wigzell, H. (1989) Scand. J. Immunol. 30, 425-434. 54. Khazaeli, M. B., Rogers, K. J. & LoBuglio, A. F. (1989) J. Immunol. Methods 117, 175-180. 55. Hale, G., Clark, M. R., Marcus, R., Winter, G., Dyer, M. J. S., Phillips, J. M., Riechmann, L. & Waldmann, H. (1988) Lancet H, 1394-1399. 56. Myers, G., Rabson, A., Bersofsky, J., Smith, T. & Wong-Staal, F. (1990) Human Retroviruses and Aids, Theoret. Biol. and Biophys. Publ., Los Alamos Nat]. Lab., Los Alamos, NM).
Production of site-selected neutralizing human monoclonal antibodies against the third variable domain of the human immunodeficiency virus type 1 envelope glycoprotein (acquired immunodeficiency syndrome/lymphoblastoid lnes/beterohybridoma)
MIROSLAW K. GORNY*, JIAN-YIN XU*, VASILIKI GIANAKAKOSt, SYLVIA KARWOWSKA*, CONSTANCE WILLIAMSt, HAYNES W. SHEPPARDt, CAkL V. HANSONI, AND SUSAN ZOLLA-PAZNER*t§ *Department of Pathology, New York University Medical Center, 550 First Avenue, New York, NY 10016; tVeterans Affairs Medical Center, 423 East 23rd Street, New York, NY 10010; and tViral and Rickettsial Disease Laboratory, California Department of Health Services, 2151 Berkeley Way, Berkeley, CA 94704
Communicated by Michael Heidelberger, December 21, 1990
provide an alternative means of prophylaxis and treatment. For example, Prince et al. (18) have shown that passively administered human immunoglobulin preparations with high anti-HIV neutralizing titers, injected prior to HIV challenge, prevent primate infection, and several studies of pregnant HIV-infected women have shown a correlation between maternal Abs to the V3 loop and protection of the fetus from in utero or perinatal infection (19-21). Therefore, for passive immunization, human mAbs to the V3 loop that are capable of HIV neutralization might be particularly useful. Methods for generating human mAbs to HIV have been developed resulting in the generation of several lines making mAbs specific for gp4l and p24 with various biological functions (22-25) but none possess neutralizing activity. With the description of the V3 loop of gpl20 as a principal neutralizing domain and the identification of MN as a representative of one of the most prevalent HIV strains in North America and Europe (17, 26, 27), the work described below was initiated to derive human mAbs site-selected for specificity and function directed against the V3 loop of HIVMN.
Cell lines secreting IgG1 human monoclonal ABSTRACT antibodies (mAbs) to the envelope glycoprotein, gpl2O, of human immunodeficiency virus (IRV) have been produced by transformation of peripheral blood cells front IIV-infected individuals and by fusion of transformed cells to a humanmouse heteromyeloma cell line (SHM-D33). Two human mAbs were site-selected by means of a 23-mer synthetic peptide spanning a portion of the third variable domain of gpI20 from the MN strain of HIV. The two heterohybridomas produce three times more IgG than do their parent lymphoblastoid cell lines. The specificities of these mAbs have been mapped to sequences near the tip of the disulfide loop of the gpl20 third variable domain, Lys-Arg-lle-His-Ile and His-Ie-Gly-Pro-GlyArg, respectively. The mAbs have dissociation constants of 3.7 x 10-6 M and 8.3 x 10-7 M, neutralize HIVMN in vitro at nanogram levels, and bear the characteristics of antibodies associated with protective immunity in vivo.
Neutralizing antibodies (Abs) are considered to be essential for protection against many viral infections, including those caused by retroviruses. Neutralizing Abs against human immunodeficiency virus type 1 (HIV-1) have also been described and their specificities have been mapped to several locations within the structural proteins of the virion such as the core protein p17 (1, 2), the envelope glycoprotein gpl20, and the transmembrane protein gp4l (3-7). Some of these epitopes encompass genetically conserved regions of the envelope (4-6, 8, 9), but they do not appear to be principal neutralizing domains since immunization against these epitopes elicits polyclonal antisera with low neutralizing titers (6) or gives rise to murine monoclonal antibodies (mAbs) that neutralize the virus only at relatively high concentrations (5, 10). The CD4 binding domain of gp120, a broadly conserved region, may give rise to group-specific neutralizing Abs that appear in infected humans several months after infection.O In various systems, this region of gpl20 has given rise to neutralizing Abs (5, 11) or to Abs that affect binding of gpl20 to CD4 (12, 13). A principal neutralizing domain has been identified (1416). It resides within the third variable domain (V3) of gpl20, located between Cys-301 and Cys-335, I which are bridged by a disulfide bond (56). The tip of this loop consists of four highly conserved amino acids (Gly-Pro-Gly-Arg) flanked by amino acids that differ between isolates (17, 56). Whereas active immunization against gpl20 and the V3 loop is being studied as a mode of prevention of HIV infections passive immunization with Abs to gp120 and the V3 loop might
MATERIALS AND METHODS Subjects. Cells from two asymptomatic homosexual men were used in these experiments. The volunteers had been seropositive for at least 3 years and 1 year, respectively, and had CD4' cell counts of 628 and 871 per mm3. Each gave informed consent to participate in these studies as specified
by the Institutional Review Boards. Synthetic Peptide Used for Screening of Reactive Cells by ELISA. A peptide that spans 23 amino acids of the gp120 V3 loop of the MN strain of HIV-1 (23-mer peptide) was synthesized by Peninsula Laboratories. The peptide, derived from the sequence published by Myers et al. (56k, has the following amino acid sequence: YNKRKRIHIGPGRAFYTTKNIIG. Establishment of Epstein-Barr Virus-Transformed Cell Lines and Heterohybridomas. The method for producing cell lines synthesizing human mAbs to HIV-1 was as described (22). Briefly, mononuclear cells were obtained from blood and incubated overnight with Epstein-Barr virus. Cyclosporin A (Sandoz, East Hanover, NJ) was incorporated into the Abbreviations: mAb, monoclonal antibody; Ab, antibody; HIV, human immunodeficiency virus; V3, third variable domain of gp120; RIP, radioimmunoprecipitation. §To whom reprint requests should be addressed. lBlattner, W., Nara, P., Shaw, G., Hahn, B., Kong, L., Matthews, T., Bolognesi, D., Waters, D. & Gallo, R., Fifth International Conference on AIDS, June 4-9, 1989, Montreal, page 510, abstr. MC07. "The numbering system used throughout for HIVMN gp120 was designated by Myers et al. (56).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3238
Immunology: Gorny et al. medium at 0.5 ,ug/ml for the first week after infection of cells with Epstein-Barr virus. Cells were cultured for 3-4 weeks in 96-well plates and then screened for Abs in the supernatant specific for the 23-mer peptide. Positive cultures were expanded, subcultured several times, and finally expanded into flasks. Lymphoblastoid cells were fused with the mouse x human heteromyeloma SHM-D33 (kindly provided by Nelson Teng, Stanford University, Stanford, CA). Briefly, the SHM-D33 cells and lymphoblastoid cells were mixed at a ratio of 1:3 and centrifuged to a pellet. Then, 1 ml of 50%o (vol/vol) polyethylene glycol 1300-1600 (Sigma) was added dropwise over 1 min, followed by slow dilution with Iscove's modified Dulbecco's medium. After resuspension in Iscove's medium supplemented with 15% (vol/vol) fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 ,ag/ml), the cells were plated in 96-well culture plates in 100 Al at 8 x 104 cells per well. The next day, 1 x 104 mouse peritoneal cells were added per well as feeder cells with 0.5 mM hypoxanthine/0.2 ,uM aminopterin/16,uM thymidine/1 juM ouabain-(Sigma). After 2-3 weeks, all culture wells were screened for Ab production against the aforementioned peptide and hybrids producing reactive Abs were expanded in 24-well plates. Hybrids that produced the highest level of Abs and IgG, measured by ELISA, were cloned repeatedly at 100 to 1 cells per well. Hybridomas are denoted by the suffix "D" in the cell line designation. Antibody Detection and Characterization. Culture supernatants were screened by ELISA as described (22) for reactivity to the 23-mer that was coated overnight at 40C at a concentration of 1 Ag/ml in 0.05 M sodium carbonate (pH 9.6). The specificity ofAb binding was assessed by radioimmunoprecipitation (RIP). RIP assays were carried out by the method of Pinter and Honnen (28) with 30 ,ug of strain HTLV-IIIB lysate (Organon) and/or strain MN lysate (Advanced Biotechnologies, Silver Spring, MD) labeled with 125I using the Bolton-Hunter reagent (New England Nuclear). Culture supernatants were incubated with labeled viral lysate and further processed for electrophoretic analysis as described (22, 28). Antibody subclasses were determined by ELISA and IgG quantitation was performed (22). The light chain type of mAbs was analyzed by ELISA using microtiter plates coated with rabbit anti-human K chain or rabbit anti-human A chain Abs (Dakoplatts, Glostrup, Copenhagen). The developing Abs were alkaline phosphatase-coupled goat anti-human K chain and goat anti-human A chain (Sigma), respectively. Epitope Mapping. The fine specificity of the mAbs was determined using the Epitope Mapping Kit (Cambridge Research Biochemicals, Valley Stream, NY). Briefly, 18 sequential overlapping hexapeptides that span the 23-mer were synthesized in situ on plastic pins. The peptides were deprotected, washed, and dried according to the manufacturer's instructions. Because the configuration of the pins fits into 96-well microtiter plates, the ELISA assays were carried out in standard microplates as recommended by the manufacturer. Thus, all pins were allowed to react with sera or culture supernatants from the various cell lines diluted 1:100 or 1:10, respectively, in phosphate-buffered saline containing 0.1% Tween-20, 1% ovalbumin, and 1% bovine serum albumin. Thereafter, the pins were washed and immersed in horseradish peroxidase-conjugated goat anti-human IgG. The color reaction was read in a Dynatech MR-700 at 405 nm. Determination of mAb Affinity. Determination of the dissociation constants (Kd) of human mAbs was performed by an ELISA methodology according to Friguet et al. (29). Briefly, the culture supernatants of 257-2D, 257-2, 268-liD, and 268-11 were tested at concentrations of 2.5, 1.1, 1.25, and 0.5 ,(g/ml, respectively. The 23-mer (Mr, 2638) was dissolved in 0.05 M acetic acid to a concentration of 1 mg/ml (3.8 x
Proc. Natl. Acad. Sci. USA 88 (1991)
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10' M), diluted in phosphate-buffered saline (pH 7.2), and used at concentrations ranging from 10-5 to 10-8 M. Supernatant and peptide were mixed in equal volumes and after 16 hr, the mixture was added to plates coated with the 23-mer (1 gg/ml) and the amount of unbound mAb was measured by ELISA. Data were plotted according to the Friguet modification of Klotz (29) to determine the Kd. Neutralization Assay. A plaque assay, by Hanson et al. (30), that measures the inhibition of HIV infection of MT-2 cells was used to detect the neutralizing activity of mAbs in the presence or absence of human complement. Thus, mAbs were serially diluted in 50%o (vol/vol) assay medium (30) plus 50% (vol/vol) of a normal human plasma pool. The plasma pool served as the source of human complement; for studies in the presence of complement, mAb and virus were incubated for 18 hr at 370C. For tests in the absence of complement, the plasma pool was heat-inactivated and the mAb and virus were incubated under these conditions for 1 hr at 370C. The dilution at which 50% of the input virus was neutralized on the basis of plaque counts was calculated by interpolation using a third-order regression analysis of the mean plaque count at each dilution. RESULTS Peripheral blood specimens from two subjects provided 17.2 and 92.0 x 106 mononuclear cells, which were cultured in microtiter wells at 60,000 and 85,000 cells per well, respectively. In both cases, initial screening of wells after 3-4 weeks of culture revealed two positive wells for Abs reactive with the 23-mer of the V3 domain of gpl20 of HIVMN. Cells in these wells were expanded and cloned (22) and one clone from each volunteer was stabilized. The cell lines were designated 257-2 and 268-11, respectively. Each line was cloned three times at 100 cells per well and found to produce an IgG1 A mAb to the 23-mer. Simultaneous with the original cloning, cells from expanded cultures that gave rise to cell lines 257-2 and 268-11 were fused to the SHM-D33 heteromyeloma. Two resulting heterohybridomas, 257-2D and 268liD, were cloned at least four times each, with the final two clonings performed at 1 cell per well. Although the lymphoblastoid cell lines 257-2 and 268-11 produced 6.4 and 3.8 jig of IgG per ml per 106 cells per 24 hr, respectively, the related heterohybridomas, 257-2D and 268-liD produced 20.5 and 11.3 ,ug of IgG per ml per 106 cells per 24 hr, respectively. The mAbs were shown to react in an ELISA with the 23-mer when the latter was bound to the wells of microtiter plates at concentrations as low as 1 ng/ml (data not shown). The specificity of these mAbs was further defined by RIP, the results of which are shown in Fig. 1. Both mAbs react 12 3 4 5 6 7 8
FIG. 1. RIP assay of human mAbs with HIV lysates. Lanes: 1 and 2, reactivity of a serum specimen from an HIV-infected individual; 3 and 4, reactivity of supernatant 257-2D; 5 and 6, reactivity of supernatant 268-11D; 7 and 8, reactivity of supernatant 280-2 (which is unreactive with HIV antigens); 1, 3, 5, and 7, reactivity of specimens to HIVMN lysate; 2, 4, 6, and 8, reactivity of specimens to
HTLV-111B lysate.
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Proc. Natl. Acad. Sci. USA 88 (1991)
with the env-encoded protein gp120 of HIVMN but not with the gp120 derived from strain HTLV-IIIB, revealing the type-specificity of these mAbs. Fine mapping of the epitopes of these mAbs was performed using the Epitope Mapping Kit, which utilizes the method developed by Geysen et al. (31) to synthesize hexapeptides on plastic pins. Each mAb was tested against a set of 18 hexapeptides that spans the region of the 23-mer, each hexapeptide overlapping its neighbor by five amino acids. The hexapeptides, bound to the plastic pins on which they were synthesized, were incubated with sera from seronegative or seropositive subjects and with supernatant fluids of heterohybridomas 257-2D and 268-liD. Seronegative sera did not react with any pins. A seropositive serum sample reacted above background levels with all pins, giving peak reactions with three pins attached to peptides spanning the region PGRAFYTT located at the tip and right side of the V3 loop (Fig. 2A). The mAb 257-2D bound strongly to two adjacent hexapeptides, RKRIHI and KRIHIG (Fig. 2B), indicating that the smallest reactive peptide (core of the epitope) that this mAb recognizes is KRIHI, located to the left of the conserved tip of the V3 loop. The flanking N- and C-terminal arginine and glycine residues may also contribute to the binding of this mAb. The mAb 268-liD bound to a single hexapeptide consisting of HIGPGR, which spans the tip of the loop and the two adjacent amino acids to the left. The Kd values of these mAbs were determined using the method of Friguet et al. (29) and mAbs 257-2D and 268-liD were found to have Kd values of 3.7 x 10-6 and 8.3 x 10-7 M, respectively (Fig. 3). These figures are in the range of those reported for IgG mAbs (29, 32). The Kd values of mAbs produced by the lymphoblastoid cell lines (257-2 and 268-11) and by the heterohybridomas (257-2D and 268-liD) were similar (data not shown). One should note that these Kd values are derived from the reaction between the mAbs and the 23-mer peptide. The Kd value of the mAbs for the native gp120 molecule may be lower by virtue of contributions of conformation of the whole molecule to the epitopes to which the mAbs react. Finally, these mAbs were tested for their ability to neutralize virus in a plaque reduction assay (30). When supernatant fluids from 257-2D and 268-liD were incubated with HIVMN for 1 hr in the absence of complement prior to addition to permissive MT-2 cells, 50% neutralization was achieved at dilutions of 1:4700 and 1:2000, corresponding to mAb concentrations of 3.0 and 23.0 ng/ml, respectively (Table 1). No neutralization was obtained when the mAbs were tested against HTLV-IIIB. When 257-2D and 268-liD were tested in a more sensitive assay, by incubation with virus for 18 hr in the
1.0 _
1
2
3 k
x
4
5
6
104M
FIG. 3. Determination of the Kd for two human mAbs to gpl20 performed according to the method of Friguet et al. (29). Klotz plots are shown in which the slopes of the lines are equivalent to the Kd of 257-2D (o) and 268-liD (o). v is the fraction of bound Ab and 1/v corresponds to Ao/(Ao - A), where Ao is the absorbance of Ab in the absence of peptide and A equals the absorbance of Ab after incubation with peptide. ao is the peptide concentration.
presence of human complement, neutralization was achieved at dilutions of 1:44,000 and 1:41,000, corresponding to mAb concentrations of 0.3 and 1.1 ng/ml, respectively. Again, no neutralization of HTLV-IIIB occurred under these conditions. A representative dose-response curve for the activity of mAb 257-2D against HIVMN, in the absence and presence of complement, is shown in Fig. 4. Human mAb 50-69 directed against the HIV transmembrane protein gp4l and mAb 71-31 directed to the core protein p24, described by Gorny et al. (22), were tested in parallel and displayed essentially no neutralizing activity for either strains of HIV.
DISCUSSION Despite the technical difficulties encountered over the past decade in producing human mAbs, several have been obtained to HIV (11, 22, 33-40). The success in generating human mAbs to HIV, as opposed to other antigens, appears to be due to the chronic antigenic stimulation of HIV-infected individuals that results in hyperimmunization and continual circulation of Ab-producing B cells. Consequently, Abproducing B cells can be obtained from the peripheral blood and the majority of these cells produce IgG antibodies. Of the human mAbs produced, most have been directed to gp4l or p24 as they have generally been identified and selected for reactivity with whole viral lysates that contain a preponderance of these structural proteins. The site-selected approach in the screening procedure for selecting the mAbs described
A
0.5 Y
N
K
R
K
R
H
I
I
G
P
G
R
A
F
Y
T
FIG. 2. Serum and mAb reactivities
T
by an ELISA with overlapping hexapep-
0 2.0
1.5 _
0.5
]
i
3
-
q Y
N
K
R
K
R
I
H
I
G
P G
R
A
F
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T
T K N
I
I
G
tides homologous with the 23-mer of the HIVMN V3 loop. The reactivity of each hexapeptide with seronegative (solid bars) or seropositive (hatched bars) sera (A) or with supernatant from heterohybridoma 257-2D (open bars) or 268-liD (solid bars) (B) is shown on the ordinate and each hexapeptide is designated by the single-letter code of its N-terminal residue and the subsequent five amino acids. Thus, the sequence appearing on the abscissa is the sequence of the 23-mer.
Immunology: Gorny et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
Table 1. Neutralizing activity of human mAbs against HIV HIVMN neutralizing Ab titers, dilution 1 hr/no C mAb Specificity 18 hr/+ C 257-2D gpl20 1:4700 (3.0) 1:44,000 (0.3) 268-liD gp120 1:2000 (23.0) 1:41,000 (1.1) 50-69 gp41 1:3 1:3 71-31 p24 Negative Negative C, complement. All data with strain HTLV-IIIB were negative. Numbers in parentheses are ng of mAb per ml.
herein was designed to pick cells producing Abs to a particular neutralizing domain rather than identifying mAbs to random HIV epitopes as was previously done (11, 22, 33-40). The mechanisms by which Abs to the V3 loop mediate neutralization have yet to be elucidated. Some have postulated a requirement for high-affinity Abs to effect neutralization (20); however, the data shown above demonstrate that mAbs with average dissociation constants are quite effective neutralizing Abs, at least under in vitro conditions. Stephens et al. (41) have suggested that proteolytic cleavage of the V3 loop is essential for infectivity and it is thus attractive to postulate that Abs to the V3 loop may interfere with this cleavage step. The data presented above suggest another mechanism, that, in addition to the physical inactivation or 120
A
1001 801 an I %vI
40
20L
2----------------------
--------
-
O
c
0
1
f
2
2.3 2.6
2.9
3.2 3.5
3.8
4.1
4.4
I
el
2.3
2.6
3.2
3.8
4.4
5.0
5.6
6.2
6.8
Antibody dilution (1/log10) FIG. 4. HIV neutralization by serial dilution of mAb 257-2D. Data normalized as a percentage of the mean plaque count in 12 or 24 replicate control wells (o) for experiments shown in A and B, respectively. In these examples, control plaque numbers equaled 9.6 (SEM = 1.4) and 9.1 (SEM = 1.1) for A and B, respectively. Error bars represent one SEM above and below the mean at each dilution. The starting concentration of 257-2D was 14.0 gtg/ml. (A) Neutralization of HIVMN incubated with 257-2D in the absence of complement for 1 hr. (B) Neutralization of HIVMN incubated with 257-2D in the presence of complement for 18 hr. are
3241
blocking conferred by the Ab moiety, components of the complement cascade may contribute to virus neutralization by means of virolysis, a phenomenon described with HIV by Spear et al. (42). Other mechanisms of virus neutralization (for review, see ref. 43) must also be considered, a task that will be facilitated by the availability of these and other neutralizing mAbs. Studies of the cross-reactivity of type-specific anti-HIV Abs have been based on serologic studies of reactivity with peptides from the V3 region (17, 20). Although these studies have revealed a high frequency of reactivity for certain viral sequences, little is known about the extent to which sera with these defined reactivities will neutralize viruses with the homologous sequences in vitro, and essentially nothing is known about the in vivo function of these Abs. Moreover, the hypervariability of the V3 loop and the ability of HIV and other retroviruses to mutate under the pressure of an effective immune response (44, 45) implies that passive immunization with a single mAb may be insufficient to protect or treat a large population of high-risk individuals or single individuals over a long period of time. These considerations may dictate the need for a mixture of mAbs. However, the feasibility of producing a variety of human mAbs to HIV is demonstrated by the data presented herein and elsewhere (11, 22, 33-40). The epitope mapping of the mAbs to the V3 loop point to the possibility of generating mAbs to variable regions of the virus with reactivity to more than one virus strain. Thus, the epitope of mAb 268-11 is shared by a number of members of the MN family (MN, SC, and JH3) and appears in more distantly related isolates that possess the same sequence near the tip of the V3 loop (HAN, WMJ1, and WMJ3) (20, 56). The epitope of mAb 257-2 occurs in a more variable portion of the loop and, therefore, might react with a more restricted group of viruses. Nonetheless, both mAbs neutralize SF-2 as well as MN despite disparities in the relevant epitopes at amino acids 311 and 313 (K. Steimer, N. Haigwood, M.K.G., S.K., J.-Y.X., C.V.H., H.W.S., and S.Z.-P., unpublished data). Studies of the sensitivity of field isolates may define further the degree to which these mAbs will be capable of neutralizing the most prevalent strains derived directly from patients, but these studies must await standardization of methods to test the sensitivity of field isolates to antibodydependent neutralization. The usefulness of these mAbs as reagents for passive immunization can only be determined by in vivo studies. Nonetheless, precedents exist for the prevention of retroviral-induced diseases in mice by passive immunization (for review, see ref. 46), and, as noted above, there is increasing evidence that passive immunization of nonhuman primates can also protect against challenge with HIV-1 (18, 47). Preliminary studies of passive immunization of HIV-infected patients with plasma (48-50) or immunoglobulin preparations from sera of asymptomatic HIV-infected donors** have also suggested the usefulness of pursuing passive immunotherapy as a modality of treatment and prophylaxis. It is probable that human mAbs would have several advantages over immunoglobulin preparations from sera of HIV-positive donors. (i) Immunoglobulin pools prepared from patients' sera will contain enhancing Abs (51-53) that may counteract the benefits of protective Abs; however, human mAbs can be selected with protective but no enhancing function. (ii) Protective Abs represent an extremely small proportion of polyclonal immunoglobulins, necessitating the administration of gram quantities of immunoglobulin by the **Rhame, F. S., Goodroad, B. K., Cummins, L. M., Fletch, C. V., Henry, K., Shah, N., Condie, R. M., Balfour, H. H., Jr., & Allain, J. P., Sixth International Conference on AIDS, June 20-23, 1990, San Francisco, vol. 3, page 211, abstr. SB500.
3242
Immunology: Gorny et al.
intravenous route. Potent mAbs, on the other hand, may be administered in milligram amounts (54, 55), allowing frequent administration by the intramuscular route. (iii) Higher levels of specific protective Abs could be more readily achieved using mAb preparations than using polyclonal immunoglobulin. This rationale, then, provides the basis for the identification and development of human mAbs to HIV with known protective function. This work was supported by research funds from the National Institutes of Health (Grants Al 72658 and Al 82693) and from the Department of Veterans Affairs. 1. Papsidero, L. D., Sheu, M. & Ruscetti, F. W. (1989) J. Virol. 63, 267-272. 2. Sarin, P. S., Sun, D. K., Thornton, A. H., Naylor, P. H. & Goldstein, A. L. (1986) Science 232, 1135-1137. 3. Javaherian, K., Langlois, A. J., McDanal, C., Ross, K. L., Eckler, L. I., Jelfis, C. L., Profy, A. T., Rusche, J. R., Bolognesi, D. P., Putney, S. D. & Matthews, T. J. (1989) Proc. Nat!. Acad. Sci. USA 86, 6768-6772. 4. Ho, D. D., Kaplan, J. C., Rackauskas, I. E. & Gurney, M. E. (1988) Science 239, 1021-1023. 5. Sun, N., Ho, D. D., Sun, C. R. Y., Liou, R.-S., Gordon. W., Fung, M. S. C., Li, X.-L., Ting, R. C., Lee, T.-H., Chang, N. T. & Chang, T.-W. (1989) J. Virol. 63, 3579-3585. 6. Chanh, T. C., Dreesman, G. R., Kanda, P., Linette, G. P., Sparrow, J. T., Ho, D. D. & Kennedy, R. C. (1986) EMBO J. 5, 3065-3071. 7. Thomas, E. K., Weber, J. N., McClure, J., Clapham, P. R., Singhal, M. C., Shriver, M. K. & Weiss, R. A. (1988) AIDS 2, 25-29. 8. Ho, D. D., Sarngadharan, M. G., Hirsch, M. S., Schooley, R. T., Rota, T. R., Kennedy, R. C., Chanh, T. C. & Sato, V. L. (1987) J. Virol. 61, 2024-2028. 9. Modrow, S., Hahn, B. H., Shaw, G. M., Gallo, R. C., Wong-Staal, F. & Wolf, H. (1987) J. Virol. 61, 570-578. 10. Dalgleish, A. G., Chanh, T. C., Kennedy, R. C., Kanda, P., Clapham, P. R. & Weiss, R. A. (1988) Virology 165, 209-215. 11. Robinson, J. E., Holton, D., Pacheco-Morell, S., Liu, J. & McMurdo, H. (1990) AIDS Res. Human Retroviruses 6, 567-579. 12. Lasky, L. A., Nakamura, G., Smith, D. H., Fennie, C., Shimasaki, C., Patzer, E., Berman, P., Gregory, T. & Capon, D. J. (1987) Cell 50, 975-985. 13. Linsley, P. S., Ledbetter, J. A., Kinney-Thomas, E. & Hu, S. (1988) J. Virol. 62, 3695-3702. 14. Rusche, J. R., Javaherian, K., McDanal, C., Petro, J., Lynn, D. L., Grimaila, R., Langlois, A., Gallo, R. C., Arthur, L. O., Fischinger, P. J., Bolognesi, D. P., Putney, S. D. & Matthews, T. J. (1988) Proc. Nat!. Acad. Sci. USA 85, 3198-3202. 15. Palker, T. J., Clark, M. E., Langlois, A. L., Matthews, T. J., Weinhold, K. J., Randall, R. R., Bolognesi, D. P. & Haynes, G. (1988) Proc. Nat!. Acad. Sci. USA 85, 1758-1762. 16. Goudsmit, J., Boucher, C. A. B., Meloen, R. H., Epstein, L. G., Smit, L., Van Der Hoek, L. & Bakker, M. (1988) AIDS 2, 157-164. 17. LaRosa, G. J., Davide, J. P., Weinhold, K., Waterbury, J. A., Profy, A. T., Lewis, J. A., Langlois, A. J., Dreesman, G. R., Boswell,. R. N., Shadduck, P., Holley, L. H., Karplus, M., Bolognesi, D. P., Matthews, T. J., Emini, E. A. & Putney, S. D. (1990) Science 249, 932-935. 18. Prince, A. M., Horowitz, B., Shulman, R. W., Pascual, D., Hewlett, I., Epstein, J. & Eichberg, J. W. (1990) in Vaccines 90: Modern Approaches to New Vaccines Including Prevention of AIDS, ed. Brown, F. (Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY), pp. 347-351. 19. Rossi, P., Moschese, V., Broliden, P. A., Fundaro, C., Quinti, I., Plebani, A., Giaquinto, G., Tovo, P. A., Ljunggren, K., Rosen, J., Wigzell, H., Jondal, M. & Wahren, B. (1989) Proc. Natl. Acad. Sci. USA 86, 8055-8058. 20. Devash, Y., Calvelli, T. A., Wood, D. G., Reagan, K. J. & Rubinstein, A. (1990) Proc. Natl. Acad. Sci. USA 87, 3445-3449. 21. Goedert, J. J., Mendez, H., Drummond, J. E., Robert-Guroff, M., Minkoff, H. L., Holman, S., Stevens, R., Rubinstein, A., Blattner, W. A., Willoughby, A. & Landesman, S. H. (1989) Lancet H, 1351-1354. 22. Gorny, M. K., Gianakakos, V., Sharpe, S. & Zolla-Pazner, S. (1989) Proc. Nat!. Acad. Sci. USA 86, 1624-1628. 23. Till, M., Zolla-Pazner, S., Gorny, M. K., Patton, J. S., Uhr, J. & Vitetta, E. S. (1989) Proc. Nat!. Acad. Sci. USA 86, 1987-1991. 24. Robinson, W. E., Jr., Kawamura, T., Gorny, M. K., Montefiori, D. C., Mitchell, W. M., Lake, D., Xu, J., Matsumoto, Y., Sugano, T., Masuho, Y., Hersh, E. & Zolla-Pazner, S. (1990) Proc. Nat!. Acad. Sci. USA 87, 3185-3189.
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