Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
  • C07K16/1036—Retroviridae, e.g. leukemia viruses
  • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
  • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• One aspect of this invention relates to methods an compositions for influencing the immunogenicity of human im munodeficiency virus (HIV) antigens and more specifically t methods and compositions for raising antibodies that inhibi the propagation of HIV infection.
• HIV human im munodeficiency virus
• Another aspect of this invention relates to antibodie that have the ability to inhibit such propagation.
• HIV human immunodeficienc virus
• T4 helper/inducer lymphocytes
• the viral envelope includes a population of glycoproteins (called gp 160) anchored in the viral cell membrane bilayer via their C-terminal region.
• gp 160 glycoproteins anchored in the viral cell membrane bilayer via their C-terminal region.
• Each glycoprotein contains two segments: the N-terminal segment, called gpl20, which protrudes from the membrane into the surrounding medium; and the C-terminal segment, gp 41, which spans the membrane.
• HIV infection is propagated by direct lymphocyte-lymphocyte fusion between virus-infected cells (which have been shown to express gpl20 and gp 41 on their surface) and uninfected CD4 + cells. This fusion takes place even in the absence of free HIV in the surrounding medium. Lifson, J.D. et al. Science, 232:1123-1127. 1986; Sodroski, J. et al. Nature, 322:470-474. 1986; and Lifson, J.D. et al, Nature, 313:725-728, 1986.
• gpl20 The properties and isolation of gpl20 from HIV par ⁇ ticles and its sequencing from different HIV isolates are well- known and have been extensively described e.g. in the foregoing references.
• the preparation of gpl20 via recombinant DNA techniques has been described in Lasky, L.A. et al. Science, 233:209, 1986 and in published European patent application of Genentech, Inc. published on August 24, 1988, Serial No. 279,688 (based on U.S.S.N. 155,336 filed 02/12/88) naming Berman, P. .; Gregory, T.J.; Lasky, L.A. ; Nakamura, G.R; et al.
• CD4 the cellular receptor of gpl20, has been isolate from lymphocytes. Synthetic (soluble) and recombinant CD4 hav been described in Smith, D.H. et al. Science, 2_38.:1704-1707, 1987. Other methods as well as properties of CD4 have bee described in Jameson, B.A. et al. Science, 240:1335-1339, 1988; Fisher, R.A. et al. N ture, 331:76-78, 1988; Hussey, R.E. et al. Nature. 3_31:78-81, 1988; Deen, K.C. et al. Nature. 331:82- 84, 1988; Traunecker, A. et al.. Nature, 3_3_l:84-86, 1988; and Lifson, J.D. et al. Science. 241:712-716. 1988.
• the first region has been placed between residues 307 and 330 of gpl20 and represents an immunodominant epitope since animals immunized with whole gp 160 or gpl20 (or with fragments of gpl20 containing the epitope) produce high titers of HIV- neutralizing antibodies (i.e., antibodies that inhibit virion- lymphocyte fusion) .
• This immunodominant epitope is situated in a highly variable segment of gpl20 that varies from isolate to isolate and, as a result, the antibodies are also isolate- specific. This limits their utility in immunological studies and in therapy against (or prevention of) HIV infection. Also, sera from HIV-infected humans do not contain high titers of these antibodies.
• HlV-neutra- lizing antibodies specifically antibodies that inhibit the binding of viral gpl20 to lymphocytic CD4 directed to other epitopes of gpl20 which have not been precisely identified (though it is thought to be proximal to the CD4-binding site of gpl20); Lasky, L.A. et al. Cell. 50.:975-985, 1987.
• the antibodies are group- and not isolate-specific, which indicates that this second epitope is located in a more conserved domain of gpl20.
• animals immunized with gpl20, gp 160 or various fragments of gpl20 have not produced HIV-neutralizing antibodies.
• one object of this invention is to provide significant amounts of antibodies that neutralize the infec- tivity of HIV virus (i.e., inhibit its ability to invade T4 lymphocytes) .
• Another object of this invention is to provide an ⁇ tibodies that prevent HIV-induced cell fusion between healthy T4 lymphocytes and lymphocytes infected with HIV.
• Yet another object of this invention is to use such antibodies to improve the understanding of the pathogenesis of HIV and to inhibit propagation of HIV infection in human T4 lymphocytes.
• Still another object is to provide compositions and methods for raising such antibodies.
• Further objects of this invention include use of such antibodies in research to elucidate the structure and function of HIV components and the mechanism of HIV infectivity and use of such antibodies in the passive or active immunization of humans for propylactic or therapeutic purposes.
• Figure 1 A-C is a series of plots comparing the magnitude and cell fusion-blocking ability of various antibody titers in successive bleedings of animals immunized with CD4, gpl20 and a combination of CD4 and gpl20.
• Figure 2 is a series of graphs illustrating the time course of an experiment with three groups of mice respectively injected with CD4 (A,D,G), CD4-gpl20 complex (B,E.H) and gpl20 (G,H,I).
• the weekly serum samples were assayed individually for gpl20-binding antibodies (A,B,C), CD4-binding antibodies (D,E,F), and syncytia-blocking capacity (G,H,I).
• mice immunized with the complex showed a somewhat higher anti-gpl20 response than those immunized with gpl20 alone (panels B versus C) ; a markedly lower titer of CD4 binding as compared to those receiving CD4 alone (panel E versus D); and a significantl higher syncytia-blocking response (panel H versus G) .
• Figure 3 is a graph showing titration of 11-week seru from 4 mice injected with CD4-gpl20 complex for III j - j and R syncytia-blocking capacity. The parallel behavior of in dividual sera in the two tests suggests that the antibodies ar directed at group-specific determinants.
• Figure 4 is a graph showing the effect of antibodies o rCD4-phosphatase binding to solid phase rgpl20.
• panel A th antibody is OKT4A; in panel B, 94; in panel C, OKT4, in pane D, 55.
• Abscissa antibody concentration, from left t right, 0, 0.3, 1, 3, 10 micrograms/ml.
• Figure 5 is a graph showing effect of rgpl20 on rCD4 phosphatase binding to various antibodies captured on soli phase goat anti-mouse Ig.
• the antibody is OKT4A; i panel B, 94; in panel C, 0KT4; in panel D, 55.
• Abscissa concentration of gpl20 (0, 0.4, 4, 40 micrograms/ml).
• Figure 6 is a graph showing the effect of rgpl20 o phosphatase-labeled antibody binding to solid phase rCD4.
• I panel A the antibody is 94; in panel B, 55.
• Abscis ⁇ sa antibody concentrations (0.1, 1, 3, 10 micrograms/ml).
• gpl20 shall mean not only gpl20 itself but also any other molecule that binds with CD4 in a similar manner and that when so bound has the same conformation.
• the term will include fragments of gpl20 that bind to CD4 as well as analogs and derivatives of gpl20 that possess the ability to bind CD4 and to generate antibodies with the cell-fusion blocking ability of the antibodies of the present invention.
• this definition of gpl20 shall include gpl20 from any HIV isolate since methods for sequencing this protein are known and are independent of the particular isolate of HIV from which the native protein is derived.
• CD4 shall mean CD4 and/or fragments, derivatives or analogs containing the gpl20-binding site of CD4, such as CD4 isolated from the lymphocytic surface and CD4 or CD4 deriva ⁇ tives (such as CD4-Immunoadhesin and analogs (e.g. soluble CD4) produced by synthetic (including but not limited to recombinant DNA) techniques.
• gpl20/CD4 complex shall mean a bimolecular (i.e., noncovalent) conjugate or complex between gpl20 and CD4 (or between gpl20 and antiidiotypic antibody bearing a CD4 internal image) .
• a simple mixture of gpl20 and CD4 contains this complex because of the high affinity between gpl20 and its cellular receptor. It is not necessary that the complex be made of isolated gpl20 and CD4.
• whole lymphocytes having gpl20 bound to the CD4 on their surface are envisioned as a possible form of an immunogen encompassing the gpl20/CD4 complex of the present invention.
• the native lymphocytes of an HIV-infected human would not act as such an immunogen in that human because of the ability of autologous CD4 + lymphocytes to act as antigen-presenting cells (APC), as reported by Lanzavec- chia, A. et al, Nature, 334:530-532. 1988. (Consequently, the gpl20/CD4 complex even if it exists on the surface of these human lymphocytes could not act as an immunogen. )
• antibodies of the type of the present invention could be induced in a human by immunization with the foregoing complex in one or more of its forms contemplated herein.
• Antibodies of the present invention shall mean (a) polyclonal, group-specific an- tibodies raised by immunization with the gpl20/CD4 complex and capable of inhibiting lymphocyte fusion; and/or (b) monoclonal antibodies raised against this immunogen and possessing the same fusion inhibiting property.
• Negative of HIV shall mean inhibition of the ability of HIV to bind to and invade a susceptible lymphocyte.
• Cell fusion inhibition shall mean inhibition of the ability of HIV-infected lymphocytes to form syncytia with healthy CD4 + lymphocytes (i.e. lymphocytes possessing surface CD4 ) in the absence of free HIV.
• CD4 + lymphocytes i.e. lymphocytes possessing surface CD4
• the titers of such antibodies are not very high either in absolute terms or by comparison to these individuals' titers of nonneutralizing antibodies.
• HIV-neutralizing antibodies directed against an epitope located within a more conserved area of gpl20, and distinct from the immunodominant epitope.
• Such antibodies are group-specific and constitute useful investigative tools in the pathogenesis of HIV and in research efforts to produce abate ⁇ ment or prevention of HIV infection.
• human group-specific antibodies neutralized HIV in vitro and inhibited lymphocyte fusion in vitro. they failed to inhibit lymphocyte invasion by free HIV introduced in the serum of primates passively immunized with the human antibodies.
• a third group of animals were immunized with a mixture of CD4 and gpl20.
• the immune response showed the presence of anti-CD4 antibodies (although the titer was significantly lower compared to the anti-CD4 elicited by immunization with CD4 alone) and anti-gpl20 antibodies (in amounts comparable to those elicited by immunization with gpl20 alone).
• the sera showed a very high titer of cell-fusion inhibiting antibodies.
• the antibodies of the present invention are not simply anti (CD4), since the cell-fusion inhibiting titer does not correlate with the titer obtained by the immunization with CD4 alone and since anti CD4 do not possess the cell-fusio inhibiting ability of the antibodies of the present invention
• the antibodies of the present inventio do not appear to be simply anti gpl20.
• th present antibodies are not the same as previously observe antibodies which inhibit the event of binding between CD4 an gpl20 because the titers of the present antibodies do no correlate with the titers of the previously observed binding inhibiting antibodies.
• the antibodies of the presen invention are not elicited except in the presence of th gpl20/CD4 complex, as will be illustrated below, and therefor constitute novel and distinct entities.
• the HIV-neutralizing ability of the antibodies of th present invention has been measured by a cell-fusion assa developed by Skinner, M.A. et al, J. Virol.. .62:4195, 1988.
• This assay exploits the ability of HIV-infected lymphocytes t form syncytia (fused cells) with healthy but HIV-susceptible
• CD4 + lymphocytes in the absence of free HIV
• the assay thus compares the ability of HIV-infected lymphocytes to form such syncytia under experimental conditions with the ability of the infected cells to form syncytia under control conditions, i.e. in the absence of a potential fusion- inhibitor.
• This assay is a stringent indicator of HIV-infection inhibition ability by a given inhibitor and in particular by an antibody.
• Another fusion assay is available that measures the ability of free HIV to invade lymphocytes (i.e., the fusion takes place between the virion and the lymphocytes).
• lymphocytes i.e., the fusion takes place between the virion and the lymphocytes.
• antibodies are available that can inhibit lymphocyte infection by free virus, very few antibodies also inhibit lymphocyte-lymphocyte fusion in the absence of free virus.
• most, if not all, antibodies that inhibit cell fusion also inhibit infection by free virus.
• the performance of the present antibodies in the lymphocyte-lymphocyte cell fusion assay constitutes good evidence of the HIV-inhibiting ability of such lymphocytes.
• the antibodies of the present invention may be thus used to inhibit both invasion of lymphocytes by HIV and spread of HIV infection via lymphocyte fusion and hence constitute good candidates for passive immunization (both prophylactic and therapeutic).
• passive immunization may be combined with cytotoxic agents or coadministered with other HIV inhibitors, such as ricin toxin A chain which when linked to recombinant CD4 has been shown to be selectively toxic to infected T- lymphocytes. Till, M.A., et al. Science. 242:1166-1168, 1988.
• immunization of susceptible mammals with a combination of gpl20/CD4 is expected to improve the effective ⁇ ness of the immune response of these mammals against HIV infec- tion both preventively and therapeutically.
• antibodies of the present invention include use in screening tests for the presence of the gpl20/CD4 complex; as research tools to identify new epitopes of gpl20 and specifically epitopes that are available only by changes in the conformation of gpl20 by CD4 binding and/or vice versa.
• Monoclonal antibodies in accordance with the invention and especially human monoclonal antibodies represent a preferred form of the present invention and can be used for passive immunization in humans.
• many other uses are contemplated as will be apparent to those of ordinary skill in the art.
• Amounts used for immunization in mammals can generally vary from about 10 to about 100 micrograms CD4/kg body weight and from about 13 to about 130 micrograms of gpl20/kg body weight.
• the foregoing amounts are based on the assumption that equivalent amounts of CD4 and gpl20 will be used, which is preferred but not necessary. It will be appreciated of course by those of ordinary skill in the field that an excess of one or the other constituent of the complex (i.e., an amount in addition to that sufficient to form a complex with the avail ⁇ able amount of the other constituent) is not fatal to the operability of the present invention but an equimolar mixture of CD4 and gpl20 is preferred.
• Well-known immunization protocols may be used with o without adjuvant.
• One preferred protocol involved use of th complex in complete Freund's adjuvant as set forth in Example (of course any other well-known immunization adjuvant can b used or adjuvant can be omitted altogether).
• a single immunization is sufficient, but immunizatio may be repeated 4 weeks after the first injection with an additional optional booster 4 weeks after the second injection in incomplete adjuvant (or without adjuvant) .
• the immunogenic ability of the complex of the present invention can be boosted by use of carriers such as tetanus toxoid, keyhole limpet hemocyanin, vaccinia virus, diphtheria toxoid, etc., as is well known in the art.
• Concentrations of the antibodies of the present invention effective in inhibiting lymphocyte-lymphocyte fusion should be at least sufficient to prevent successful carrying out of the sequence of events that lead to either invasion of the lymphocytes by free HIV or lymphocyte-lymphocyte fusion between any available gpl20 (whether on the viral envelope or on the surface of an infected lymphocyte) and CD4 + lymphocytes.
• the upper limit of the effective concentration is irrelevant in vitro. In vivo, the upper limit of the effective antibody concentration may be limited by factors outside the binding mechanism, such as on immune response of the host against the antibodies.
• the antibodies of the present invention may be purified by well-known techni ⁇ ques for purification of immunoglobulins, including but not limited to use of precipitation techniques (such as ammonium sulfate precipitation) and/or immunoaffinity chromatography methods (with an antigen as the adsorbent) wherein the desired antibody is preferentially bound to the column or excluded in the eluant; protein A sepharose chromatography; Affigel-blue chromatography; high performance liquid chromatography and combinations of these techniques.
• precipitation techniques such as ammonium sulfate precipitation
• immunoaffinity chromatography methods with an antigen as the adsorbent
• Monoclonal antibodies to the complex of the present invention may be raised according to well-known techniques, such as those described in Kohler and Milstein, Nature, 256:495, 1975 and in Goding, infra.
• human monoclonal antibodies may be raised from immortalized human lymphocytes sensitized against the complex of the present invention in vitro (as described in Reading, C.L., 1982, J_. Immunol. Meth. , S3:261; Hoffman, M.K. et al in Engleman, E.G. et al (Eds) Human Hybridomas and Monoclonal Antibodies, Plenum Press, New York, 1985, p. 466; Borrebaeck, C.A.K., Trends in Biotechnology, 4.:147, 1986) or in vivo (by immunizing humans with the complex as described herein and selecting B- ⁇ ells with the appropriate specificity) .
• Epstein-Barr virus can be obtained from the filtered supernatant of the marmoset cell line B95-8 (Miller, G., and Lipman, M., 1973, PNAS (USA) 7 :190) available from the ATCC under Accession No. CRL-1612. Infected lymphocytes are then washed and cultured in RPMI-1640 medium in the presence of fetal bovine serum, glutamine, penicillin and streptomycin in 96-well plates at 10 ⁇ cells per well. After screening for antibody production, positive cultures can be expanded and cultured further.
• Cultures with supernatants showing specific reactivity to the complex of the present invention can be subcultured on feeder layers of GK5 human lymphoblastoid cells (derived from GM1500 as described in Kearny, J., j_ Engl. J. Med.. 309:217. 1983) irradiated with 3000 rads of gamma- radiation. Stable clones can then be subcultured at low densities (10-100 cells) on feeder cells and the subcultures can be expanded. The specificity of the antibodies can be tested by the Skinner et al assay referenced and described herein.
• Antiidiotype antibodies can be obtained by immunizing syngenic mice with anti-CD4 monoclonal antibody.
• a most efficient protocol of immunization is to inject the monoclonal antibodies four times a day at weekly intervals. The total amount injected is 13 doses of 20 micrograms per mouse at each time.
• the antibody is injected coupled to KLH (keyhole limpet hemocyanin) in complete Freund's adjuvant; on day 6 the antibody is injected coupled to KLH in incomplete Freund's adjuvant (IFA); on day 13 the antibody is injected coupled to KLH in saline; on day 27 the antibody is injected alone in incomplete Freund's adjuvant.
• KLH keyhole limpet hemocyanin
• IFA incomplete Freund's adjuvant
• the animals are boosted after 4 to 12 weeks after the last injection by injecting again the monoclonal antibody in incomplete Freund's adjuvant in saline.
• Spleen fusion with an appropriate myeloma partner is performed 3 days after the boost. Fusion and hybrid growth and selection are then performed in accordance with well-known techniques.
• the antiidiotypi ⁇ antibodies bearing CD4 internal image can be identified by testing for binding to gpl20 (e.g., by radioim- muno assay, enzyme-linked immunosorbent assay, etc. ) and purified using well-known methods including immunoaffinity chromatography using gpl20 as the adsorbent.
• the complex of the present invention when antiidiotype antibodies ( "AA") are used can be formed by mixing gpl20 and AA preferably in equimolar amounts and waiting for the two constituents to complex with each other.
• the materials used in the present invention may be purified from natural sources or synthesized by well-known (recombinant and other) techniques as described above.
• recombinant CD4 and CD4-Immunoadhesin are available from Genentech, Inc. and so is recombinant gpl20. If not already obtained in purified form, these materials should preferably be purified prior to use in immunization.
• Techni ⁇ ques for purification are well-known; see, e.g., U.S. Patent No. 4,725,669 of Essex et al. issued February 16, 1988.
• the antibodies of the present invention must be present in the vicinity of infected T4 lymphocytes (or HIV- susceptible uninfected T4 lymphocytes) before the gpl20 ex- pressed on the surface of infected T4 lymphocytes (or the gpl20 on the surface of the virion) binds to its receptor (CD4) on the surface of an uninfected lymphocyte (or on the surface of a susceptible lymphocyte in the case of infection by free virus) .
• the antibodies of the present invention will be present in such vicinity prior to encounter between the gpl20- bearing infected lymphocyte (or virus) and the target CD4 + lymphocyte.
• mice were im ⁇ munized as follows:
• the first group was injected once intraperitoneally (i.p.) with 10 micrograms of CD4 in 0.2 ml of complete Freund's adjuvant.
• the second group was injected once i.p. with 12.5 micrograms of gpl20 in 0.2 ml of complete Freund's adjuvant.
• the third group was injected once i.p. with a mixture of 10 micrograms of CD4 and 12.5 micrograms of gpl20 (previously incubated together for 30 minutes) in 0.4 ml of complete Freund's adjuvant.
• mice were bled weekly over a three-month period and the sera were monitored for CD4- and gpl20-binding titers. In addition, the sera were monitored for their ability to block lymphocyte-lymphocyte fusion, according to an assay described in Example 2 below, and for their ability to inhibit binding between CD4 and gpl20, according to an assay described in Example 3.
• CD4 and gpl20 titers were determined as follows: Binding antibodies were assayed by ELISA. Microtiter plates were coated with the appropriate antigen (CD4 or gpl20, respectively) at a concentration of 3 micrograms/ml in car ⁇ bonate buffer 0.1 M, pH 9.6 overnight at 4°C, washed and blocked by incubating them with 1% bovine serum albumin (BSA) in phosphate buffer saline (PBS) for 45 min. at room tempera ⁇ ture. A 0.1 ml sample of test serum at various dilutions in PBS were then added and incubated for two hours at room temperature.
• BSA bovine serum albumin
• PBS phosphate buffer saline
• PNPP sodium p-nitrophenyl phosphate
• Figure 1A is a plot of the anti-gpl20 titer (i micrograms/ml serum) of sera elicited by immunization of mic with gpl20 (solid line), CD4 (broken line) and the gpl20/CD complex (- • -•-•-) for each weekly bleeding.
• immunization with gpl2 alone elicited anti-gpl20 antibodies
• immunization with CD4 alone elicited no anti-gpl20 antibodies
• immunization with th complex also elicited anti-gpl20 antibodies (i.e. the comple generated formation of antibodies against gpl20 alone).
• Figure IB is a plot of the anti-CD4 titer (i micrograms/ml serum) of sera elicited by immunization with CD4 alone (broken line), gpl20 alone (solid line) and complex (-*- • - • -).
• the assay to determine the immune serum capacity to block lymphocyte-lymphocyte fusion was described in Skinner, supra. Ten microliters of different dilutions (at least 1:10) of the test serum were distributed in Costar 96 A/2 (half-well) plates.
• 5xl0 3 or lOxlO 3 infected cell partners (from CB/IIIB or CEM/RF lymphocyte cell line publicly available from National Institute of Allergy and Infectious Disease, Reagent Program respectively infected with HTLV IIIB or HTLV RF isolate available from Dr. Gallo at the National Institute of Health) were added to each well contained in 40 microliters of culture medium. Then, 7x10"* uninfected CD4 + human lymphocytes (Molt4 available from American Type Culture Collection, Rockville, MD, Accession No. CRL-1582) in 40 microliters of culture medium containing fetal bovine serum were added to the wells and the plates were incubated at 37"C in a 5% C * 2 atmosphere for 20-24 hours.
• the plates were then read for the occurrence and the number of lymphocyte syncytia in an inverted microscope at a 40-fold enlargement.
• Giant cells having a size of at least five times the area of normal cells were scored as syncytia produced by cell fusion.
• the syncytia were 50-80/well (control).
• Fusion-blocking units were calculated by converting the percent decrease in syncytia scored relative to the control value and taking into account the serum dilution (1 FBU is defined as the amount of antibody that reduces the number of syncytia to 50% of the control value). The results, in FBU, are plotted in Figure IC.
• the solid line represents FBU achieved by immune sera of mice immunized with gpl20 alone.
• this value is essentially the same as the control.
• the broken line represents the fusion-blocking ability of immune sera elicited by immunization with CD4 alone.
• this value is positive but not very high. This is attributable to the fact that anti-CD4 will bind some of the CD4 on the uninfected lym ⁇ phocyte surfaces and thus prevent the gpl20 of the infected lymphocytes from binding to the CD4.
• the line -•-•-•- represents the fusion- blocking ability of immune sera elicited by immunization with gpl20/CD4 complex.
• the FBU of this sera starts at about that of the anti-CD4 sera in week 1 and extends to about 50 times that of the anti-CD4 sera.
• Example 3 Monoclonal Antibodies
• Monoclonal antibodies will be raised by immunizing mic with complex in accordance with the method of Example 1 (excep that two injections can be used spaced 4 weeks apart) optional ly with a booster using incomplete Freund's adjuvant 4 week after the second injection.
• spleen cells will be obtained, purified and fuse with myeloma cells.
• the spleen of the mouse with the highes fusion-blocking ability will be excised using well-know dissertion ' techniques.
• a single-cell suspension will be mad up by teasing the spleen as described in Goding, J.W. Monoclonal Antibodies: Principles and Practice, Academic Press Inc., New York 1983, pp. 50-97 and specifically on p.
• Th spleen cells will be harvested by centrifugation (e.g. 400x for 5 min. ) and washed. Erythrocytes will be removed b ammonium chloride lysis followed by centrifugation. The splee cells will be counted and approximately 10° cells will be use for fusion with commercially available mouse myeloma cell (e.g. SP 20 from American Type Culture Collection unde Accession No. CRL-1581). (2 - 3)xl0 7 myeloma cells will b mixed with the spleen cells in serum-free media and centrifuge at 400xg for 5 min. Any remaining medium will be removed b suction.
• mouse myeloma cell e.g. SP 20 from American Type Culture Collection unde Accession No. CRL-1581.
• the cell pellet will be suspended in 0.5-1 ml of war fusion medium containing 10 g of 50% w/v PEG ( .W. 1500) and 1 ml Dulbecco's modified Eagles' Minimum Essential Medium, p 7.6.
• the mixture will be stirred, centrifuged and resuspende in fetal bovine serum-containing medium using normal splee cells as feeders.
• the cells will then be exposed to HA selective media and grown in such media in an atmospher containing 5% CO2.
• the cultures will be pulled and fed. Hybrids will be growing and screenable at 10-15 days afte incubation begins. Positive clones (i.e.
• clones secretin fusion-blocking antibody will be identified and recloned until their secretion of the desirable immunoglobulin is steady an reliable. Monoclonals will thus be obtained.
• the Skinner et al assay can be used to determine specificity of the desired antibody. Difficulty in obtaining such monoclonals is not expected because the titers of fusion-blocking antibody are relatively high as demonstrated in previous Examples. If desired, spleen cells secreting anti-CD4 and/or anti-gpl20 will be separated first before fusion to maximize the probability of obtaining fusion-blocking monoclonals.
• CD4 + cells can be prevented by antibodies specific for the gpl20 binding site on CD4, defined as the VI domains of CD4 involved in the initial CD4-gpl20 binding event, i.e., the region homologous to CDR-2, amino and residues 41-52 (Peterson, A., et al.. Cell. 54:65, 1988; Landau, N.R. et al.. Nature. 334:159, 1988; Clayton, L.K., Nature. 335:363, 1988; Jameson, B.A. et al.. Science. 240:1355.
• the VI domains of CD4 involved in the initial CD4-gpl20 binding event i.e., the region homologous to CDR-2, amino and residues 41-52 (Peterson, A., et al.. Cell. 54:65, 1988; Landau, N.R. et al.. Nature. 334:159, 1988; Clayton, L.K., Nature. 335:363,
• mice were injected intraperitoneally (i.p with antigen emulsified in Complete Freund's Adjuvant (CF Difco, Detroit, MI).
• the antigens and doses were a) CD4, micrograms/mouse; b) gpl20, 12.5 micrograms/mouse, c) CD gpl20, 16 micrograms CD4 and 12.5 micrograms gpl20, thorough mixed and incubated for 20 min, and then emulsified in CF
• the mice were bled prior to immunization and every week afte for 13 weeks. Serum samples were stored at -20 C C. Enzymes and substrates.
• Alkaline phosphatase and glutara dehyde used to label antibodies for Enzyme Linked Immunoso bent Assay (ELISA) tests, and substrate paranitrophany phosphate (PNPP) were acquired from Sigma Chemicals (St. Loui MO) .
• Monoclonal antibodies Monoclonal antibodies.
• the monoclonal antibodies are F-91-36, F-91-55 and F-91-94, hereinaft called 36, 55 and 94 respectively. They were derived from fusion of mouse 91, immunized with CD4-gpl20 complex described above. All these mAbs are IgG ⁇ subclass. Antibody binding of gpl20 and CD4.
• ELISA tests were performe by coating plates with 10 micrograms/ml gpl20 or, respectively with 3 micrograms/ml rCD4 and incubating them with seria dilutions of the mouse sera starting at 1:100 dilution Phosphatase-labeled host anti-mouse IgG (obtained from Sigm Chemicals) was used to reveal bound antibodies. Readings were performed in a Titertek automated photometer.
• the cell fusion among CD4 + cell lines acutely infected by the virus requires gpl20-CD4 specific binding.
• the test was performed according to Matthews et al. (Proc. Natl. Acad. Sci USA £4.:5424, 1987). Briefly, GEM cells chronically infected with either HTLV3-III B or HTLV3-RF were used for each determination. Sera diluted 1:10 were distributed in 96-well A/2 plates (Costar). Five to 10 x 10 3 HIV-infected cells were added. 70 x 10 3 uninfected Molt 4 cells were admixed and the plates were incubated overnight at
• CD4-gpl20 responders is absorbed by CD4 and not by gp!20.
• CD4 binding (mi ⁇ rogram Blocking gpl20 binding Fusion blocking Ab/mli ( ⁇ 50 /ml)* U 50 / ⁇ tl)
• *0ne blocking U50 is the amount of antibody that reduces to 50% the amount of gpl20 bound to CD4-coated wells or, respectively, the number of syncytia in the fusion test.
• U5 Q /ml was calculated by multiplying 1
• Table 3 List of hybridomas obtained from fusion 91, hierarchically ordered according to their supernatant's capacity to bind CD4, and tested for binding gpl20 and blocking syncytia formation.
• Table 3 shows an early test of 170 wells with hybridoma clones, ordered according to their CD4 binding capacity.
• Table 4 shows a further characterization of the hybridomas when the inhibition of gpl20-CD4 binding test was performed.
• the anti-CD4 mAbs can be divided into three categories: a) those that do not inhibit gpl20 binding and do not block syncytia; b) those that do not inhibit gpl20 binding and block syncytia; and c) those that inhibit gpl20 binding and block syncytia.
• MAbs 55 and 94 were further studied, as representa- tives of the latter two categories, respectively. Both were of IgG ⁇ isotype. 6. Preliminary mapping experiments.
• the binding site of both mAbs 55 and 94 was localized within the first two domains (VI- V2) of CD4 by binding experiments using the CD4 IgG immunoad- hesin (Genentech) which contains the two external domains of CD4 spliced to an immunoglobulin constant region (data not shown) .
• the binding of these mAbs to solid phase CD4 was tested in the presence of a series of anti-CD4 monoclonal antibodies whose epitopes and binding characteris- tics are shown or partially known from the literature. The results are set forth in Table 5 below.
• mice receiving the complex Immunoadhesin-rgpl20 responded to gpl20 with antibody titers similar to those receiving only rgpl20 while these mice responded to CD4 with antibody titers similar to those receiving Immunoadhesin alone.
• mice which were immunized with Immunoadhesin-rgpl20 were the only group of mice which showed significant-titers of syncitia- blocking antibodies.
• CD4-Immunoadhesin Genentech Inc. South San Francisco, CA

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Virology (AREA)
• Genetics & Genomics (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Biophysics (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Hematology (AREA)
• Oncology (AREA)
• AIDS & HIV (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Peptides Or Proteins (AREA)
• Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Applications Claiming Priority (2)

Publications (2)

Family

ID=24359218

Family Applications (1)

Country Status (4)

Cited By (1)

Families Citing this family (12)

Citations (3)

• 1991
  • 1991-08-26 EP EP91916608A patent/EP0550516A1/de not_active Withdrawn
  • 1991-08-26 WO PCT/US1991/006079 patent/WO1992005799A1/en not_active Application Discontinuation
  • 1991-08-26 CA CA 2091253 patent/CA2091253A1/en not_active Abandoned
  • 1991-08-26 AU AU86317/91A patent/AU653366B2/en not_active Ceased

Patent Citations (3)

Non-Patent Citations (4)

Also Published As

Similar Documents

Legal Events