EP0512023A1 - Medications et procedes de traitement du sida et du complexe relatif au sida (arc) utilisant des anticorps anti-carbohydrates et des antigenes de carbohydrates - Google Patents

Medications et procedes de traitement du sida et du complexe relatif au sida (arc) utilisant des anticorps anti-carbohydrates et des antigenes de carbohydrates

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Publication number
EP0512023A1
EP0512023A1 EP91903499A EP91903499A EP0512023A1 EP 0512023 A1 EP0512023 A1 EP 0512023A1 EP 91903499 A EP91903499 A EP 91903499A EP 91903499 A EP91903499 A EP 91903499A EP 0512023 A1 EP0512023 A1 EP 0512023A1
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Prior art keywords
sialyl
antibodies
monoclonal antibody
medicament
hybridoma
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German (de)
English (en)
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EP0512023A4 (en
Inventor
John-Erik Stig Hansen
Jens Ole Nielsen
Bent Faber Vestergaard
Henrik Clausen
Sen-Itiroh Hakomori
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Biomembrane Institute
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Biomembrane Institute
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • C07K16/34—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to treatment of acquired immune deficiency syndrome (AIDS) and AIDS related complex (ARC), and in particular the present invention relates to medicaments and methods for treating AIDS and ARC employing the anti-carbohydrate antibodies Le y , A I and sialyl-Tn and employing the carbohydrate antigens Le y , A I and sialyl-Tn.
  • AIDS acquired immune deficiency syndrome
  • ARC AIDS related complex
  • inhibitory lectins may bind to glycans adjacent to the binding site and thereby sterically interfere with T4-gp120 binding as has been found for neutralizing antibodies (Bahraoui, E., et. al. (1988) AIDS 2:165-169; Linsley, P.S., et. al. (1988) J. Virol. 62:3695.. Glycans so far identified on gp120 by lectin studies are rather ubiquitous, and the therapeutic potential of lectin-based treatment therefore seems small.
  • the present invention is particularly directed to acquired immune deficiency syndrome (AIDS) which is recognized as a distinct new disease whose etiology has been identified as being associated with infection of a new class of lymphotrophic retrovirus termed human immunodeficiency virus (HIV) (previously called, in various terms, human T cell lymphotropic virus III (HTLV-III), human lymphadenopathy-associated virus (LAV), or AIDS-related virus (ARV)) (Gallo, R.C. et. al., (1986) Prog. Allergy. 37:1; Montagnier, L. (1986) Allergy. 37:46).
  • HAV human immunodeficiency virus
  • HTLV-III human T cell lymphotropic virus III
  • LAV human lymphadenopathy-associated virus
  • ARV AIDS-related virus
  • the disease is characterized by a disorder associated with an impaired cell- mediated immunity and absolute lymphopenia, particularly reduced helper T lymphocytes (T4 + or CD4). This is due to the fact that HIV preferentially infects the CD4 lymphocyte population. AIDS may be preceded by a presyndrome that is usually manifested by a complex of designated clinical features and helper T lymphopenia (Friedman-Kien, A.E., et. al., (1982) Ann, int. Med. 96:693; Gottling, M.S., et. al., (1981) N. Engl. J. Med. 305:1425; Masur, H., et. al., (1981) M. Bngl. J.
  • AIDS-related Complex The presyndrome is called AIDS-related Complex (ARC). Diagnosis of infection with HIV is usually made on the basis of detecting antibodies directed against HIV. The exact antibody profile may vary with the stage of the disease (Gallo, R.C. et. al., (1986) Prog. Allergy. 37:1). Despite significant progress in understanding the pathogenesis of HIV, no studies have been performed regarding carbohydrate antigens in T lymphoid cells infected with HIV, although carbohydrate changes associated with functional change of cellular phenotypes have been well documented (Hakomori, S. (1981) Annu. Rev. Biochem. 50:733).
  • Le y determinant is highly expressed at the surface of human T cell lines only after infection with HIV, as well as in T lymphocytes of peripheral blood of patients with AIDS and ARC but not in normal lymphocytes of healthy individuals (Adachi, M., et. al., (1988) J. Expt. Med. 167:323).
  • one object of the present invention is to find monoclonal antibodies that inhibit in vitro HIV infectively, either by binding to virus or to the target cells used.
  • Another object of the present invention is to use the monoclonal antibodies found to inhibit in vitro HIV infection to define carbohydrate structures expressed as viral glycoproteins and glycosphingolipids associated with the virus, thereby identifying glycans of the viral capsule as targets that are expected to be useful for immuno-therapy and/or vaccine development.
  • AIDS acquired immune deficiency syndrome
  • ARC AIDS related complex
  • the present invention also provides a method for inhibiting or slowing the progression of AIDS and ARC comprising administering to a subject having AIDS or ARC a medicament comprising:
  • (B) A pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention provides a vaccine against AIDS and ARC, comprising:
  • A An immunologically effective amount of one or more antigens selected from the group consisting of Le y , A I and sialyl-Tn, and
  • (B) A pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention further provides a method of actively immunizing against AIDS and ARC, comprising administering to a subject a medicament comprising:
  • A An immunologically effective amount of one or more antigens selected from the group consisting of Le y , A I and sialyl-Tn, and
  • (B) A pharmaceutically acceptable carrier, diluent or excipient.
  • the anti-Le y , anti-A I and anti-sialyl-Tn antibodies are monoclonal antibody BM1 produced by hybridoma BM1 having ATCC deposit no. HB 10312, monoclonal antibody AH21 produced by hybridoma AH21 having ATCC deposit No. HB 10226, and monoclonal antibody TKH2 produced by hybridoma TKH2 (BM4) having ATCC deposit No. HB 9654.
  • Fig. 1 is a bar graph showing the inhibitory effect of various monoclonal antibodies on HIV infection of cells.
  • the ordinate represents antigen production, in arbitrary units, and the abscissa represents control with untreated virus ( ⁇ AB), control without virus ( ⁇ HIV), or hybridoma supernatant containing the designated monoclonal antibodies.
  • Figs. 2A to 2D are bar graphs showing inhibition of HIV by various monoclonal antibodies.
  • Fig. 2A anti-A I monoclonal antibody (MAb) AH21;
  • Fig. 2B anti- Le y MAb BM1;
  • Fig. 2C anti-sialyl-Tn MAb TKH2;
  • Fig. 2D anti-sialyl-Tn MAb B72.3.
  • the ordinate represents HIV antigen production (% of control), and the abscissa represents the concentration of monoclonal antibody relative to virus inoculum ( ⁇ g/TCID 50 ).
  • Carbohydrate structures are often involved in initial adhesion of pathogens to target cells.
  • a panel of anti- carbohydrate monoclonal antibodies was tested for their ability to inhibit in vitro HIV infectivity, either by binding to the virus or the target cells used.
  • Monoclonal antibodies to three different carbohydrate antigens i.e., Le y , A I and sialyl-Tn, were able to block infection by cell virus as well as inhibit syncytium formation.
  • the inhibition of virus infectivity was independent of virus strain (HTLV III B and a patient isolate SSI-002), cell line used for virus propagation (H9 and MT4) or cell type used as infection target (MT4, PMC and T4 lymphocytes).
  • the present invention provides a medicament for inhibiting or slowing the progression of acquired immune deficiency syndrome (AIDS) and AIDs related complex (ARC) comprising:
  • (B) A pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention provides a method for inhibiting or slowing the progression of AIDS and ARC comprising administering to a subject having AIDS or ARC a medicament comprising:
  • any antibody can be used as long as it is specific for the carbohydrate antigen Le y having the following structure:
  • R represents ceramide or a carrier molecule.
  • Preferred antibodies specific to the carbohydrate antigen Le y have an affinity for Le y corresponding to a KD of about 5x10 -7 .
  • An especially preferred monoclonal antibody specific to the carbohydrate antigen Le y is BM1 produced by hybridoma BM1 having ATCC Deposit No. HB 10312, and any other monoclonal antibodies having the identifying characteristics of monoclonal antibody BM1 are preferred.
  • Monoclonal antibody BM1 has the following identifying characteristics:
  • (1) has a KD of about 10 -8 ;
  • monoclonal antibody BM1 is a murine monoclonal antibody.
  • Polyclonal antibodies to the carbohydrate antigen Le y can be prepared by known methods using the purified antigens described in Abe, K., et al (1983) Biol. Chem. 258:11793.
  • Monoclonal antibodies to Le y can also be prepared by methods known in the art, such as, for example, by the method described by Abe, K., et. al., (1983) J. Biol. Chem. 253:1179.
  • Monoclonal antibody BM1 can be prepared by methods known in the art using human gastric cancer cell line MKN 74 as the immunogen.
  • the antibody to carbohydrate antigen A I any antibody can be used as long as it is specific to the carbohydrate antigen A I having the following structure:
  • Examples of such preferred monoclonal antibodies specific to the carbohydratic antigen A i include monoclonal antibody AH21 produced by hybridoma AH21 having ATCC deposit No. HB 10226, monoclonal antibody
  • AH16 (Abe, K. et. al., (1984) J. Immunol . 132:1951) and any other monoclonal antibodies having the identifying characteristics of either of these monoclonal antibodies.
  • Monoclonal antibody AH21 is especially preferred.
  • Monoclonal antibody AH21 has the following identifying characteristics:
  • (1) has a KD of about 5x10 -7 ;
  • monoclonal antibody AH21 is a murine monoclonal antibody.
  • Monoclonal antibody AH16 has the following identifying characteristics:
  • (1) has a KD of about 10 -8 ;
  • Polyclonal antibodies to the carbohydrate antigen A I can be prepared by known methods, using the purified antigens described in Abe, K., et. al., (1983) J. Immunol. 132:1951.
  • Monoclonal antibodies to the carbohydrate antigen A I can be prepared by methods known in the art, such as, for example, by the method described in Abe,
  • Monoclonal antibody AH21 can be prepared by methods known in the art using purified type 1 chain A as the immunogen.
  • Monoclonal antibody AH16 can be prepared by methods known in the art using human gastric cancer cell lines as the immunogen.
  • any antibody can be used as long as it is specific for the sialyl-Tn carbohydrate antigen having the following structure:
  • the isotypes and affinities of suitable anti- sialyl-Tn antibodies for the antigen vary extensively from one antibody to the next, and thus the specificity to sialyl-Tn is the critical identifying characteristic of these antibodies.
  • Examples of preferred monoclonal antibodies include TKH2 produced by hybridoma TKH2, having ATCC Deposit No. HB 9654, TKH1 (Kjeldsen, T. et. al., (1988) Cancer Res. 48:2214-2220), B72.3 (Colcher, D. et. al., (1981) Proc. Natl. Acad. Sci. USA. 78:3199- 3203), and any other monoclonal antibodies having the identifying characteristics of these monoclonal antibodies.
  • Monoclonal antibody TKH2 is especially preferred.
  • B72.3 is their isotypes which are IgM, IgG I and IgG I , respectively.
  • monoclonal antibody TKH2 is a murine monoclonal antibody.
  • Polyclonal antibodies to the carbohydrate antigen sialyl-Tn can be prepared by known methods, Kjeldsen, T. et. al., (1988) Cancer Res. 48:2214.
  • Monoclonal antibodies to the carbohydrate antigen sialyl-Tn can be prepared by methods known in the art, such as those described in Kjeldsen, T., et. al., (1988) Cancer Res. 48:2214 and Nuti, M., et. al.,
  • Monoclonal antibodies to sialyl-Tn can also be prepared by the following novel method disclosed in co-pending application U.S. Serial No. 317,492, the entire disclosure of which is incorporated herein by reference.
  • the immunogen used to obtain the antibody to sialyl-Tn antigen is the sialyl-Tn antigen which is a core structure of many mucin-type glycoproteins.
  • Mucin-type glycoprotein as used herein, means a high molecular weight protein (Mr>10 6 ) with a high degree of O-linked glycosylation at serine or theronine residues. Mucin-type glycoproteins are further polymerized by S-S-dependent linkage and are the major components of epithelial secretions.
  • core structure of mucin-type glycoprotein means a basic carbohydrate structure without peripheral substitution and which is directly linked to the protein moiety of a mucin-type glycoprotein.
  • any sialyl-Tn core structure of a mucin-type glycoprotein can be used as an immunogen as long as the glycoprotein has a high molecular weight (relative molecular weight > 10 6 daltons) and is glycosylated to the same degree as mucin, i.e., more than 50% of total weight is glycosylated.
  • Animal mucins containing the sialyl- Tn antigen can also be used as an immunogen.
  • the sialyl-Tn antigen immunogen can be obtained by enzymatic or chemical modification of a mucin-type glycoprotein to expose the sialyl-Tn antigen core structure or by isolation of mucins having on them sialyl-Tn core structures. These mucins are present in some animal species. Examples of types of enzymatic modifications that can be used to expose the sialyl-Tn core structure of various mucin-type glycoproteins include the elimination of the terminally located ⁇ 2 ⁇ 3 sialyl residue by influenza virus sialidase or the total elimination of all sialic acid residues by Clostridium perfringense sialidase.
  • Enzymatic modification can also include treatment with ⁇ -galactosidase (preferably from Charonia lampas), ⁇ -fucosidase, and N-acetylhexosaminidase.
  • ⁇ -galactosidase preferably from Charonia lampas
  • ⁇ -fucosidase preferably from Charonia lampas
  • N-acetylhexosaminidase acetylhexosaminidase.
  • Examples of chemical reactions which can be used to expose the sialyl-Tn core structure of mucin-type glycoproteins include periodate oxidation followed by reduction with sodium borohydride and treatment with weak acid. The procedure is called Smith degradation (Sprio. G., Methods Enzymol. (1972) 28:3-43). This chemical treatment eliminates non-reducing terminals of carbohydrate residues except sialic acid, which can be eliminated by sialidase treatment, as described above.
  • mucins isolated from animals that can be used as immunters include ovine submaxillary mucin (OSM) in which 90% of the carbohydrate chains consist of the sialyl-Tn antigen and bovine submaxillary mucin (BSM) in which 50% of the carbohydrate chains consist of the sialyl-Tn antigen and 20% of the carbohydrate chains consist of T antigen and other unidentified residues.
  • OSM ovine submaxillary mucin
  • BSM bovine submaxillary mucin
  • the sialyl-Tn antigen immunogen is isolated and purified according to conventional methods.
  • mucin-type glycoproteins which will be enzymatically or chemically modified to produce a sialyl-Tn core structure can be isolated by gel filtration through Sepharose 4B or Sephacryl 200S.
  • the isolated glycoprotein is then enzymatically or chemically modified, by methods described above, to expose the sialyl-Tn core structure, and the sialyl- Tn core structure is purified for use as immunogen as follows.
  • the modified mucin can be separated by gel filtration through Sepharose 4B or Sephacryl 200. High pressure chromatography on a synthetic molecular filter column (fast liquid chromatography; Pharmacia) is also useful to separate enzymatically or chemically modified mucins.
  • the modified mucin does not need to be purified. The presence of a small quantity of unmodified mucin will not be harmful to use as an immunogen according to the present invention. Further, modification is usually quantitative, if proper routine precautions are taken.
  • Mucins which are derived from animal species and contain glycoproteins already in the form of a sialyl- Tn core structure are obtained by conventional methods. For example, by gel filtration through Sepharose 4B, Sephacryl 200, or FPLC, as described above. The thus derived animal mucins can be further purified for use as immunogens by the same methods, such as conventional gel filtration or FPLC as described above. However, the purity of the immunogen is not essential for use to produce a monoclonal antibody.
  • Human erythrocyte glycophorin can be obtained from human erythrocyte membranes by the method originally described by Marchesi, V.T. and Andrews, E.D. (Science (1971) 174:1247-1248).
  • Whether a glycoprotein has the sialyl-Tn core structure can be determined by affinity chromatography with a peanut lectin column (Carter, W.G., and Sharon, N., Arch. Biochem. Biophys., (1977) 180:570- 582) or by immunoblotting of glycoproteins with anti- Tn antibody (available from ChemBiomed, Edmonston, Alberta, Canada).
  • sialyl-Tn antigen The distribution of the sialyl-Tn antigen is rather limited.
  • one source of the sialyl- Tn antigen which is useful in the present invention is culture supernatants of squamous lung carcinoma cell lines QG 56 and LU-65 (Hirohashi S, et. al., (1985) Proc. Natl. Acad. Sci. USA, 82:7039-7043).
  • a second source is again ovine submaxillary mucin, which contains a high density of sialyl-Tn.
  • sialyl-Tn antigen in a form useful as the immunogen for the present invention from culture supernatant, the various cell lines described above are cultured according to known methods. The culture supernatants are treated to obtain sialyl-Tn antigen as follows.
  • the spent culture medium from cells cultured in suspension is lyophilized to reduce its volume to 1/50 of the original volume.
  • the concentrated spent medium is then dialyzed extensively against phosphate- buffered saline containing 0.01% sodium azide at 4oC.
  • the dialyzed material is placed on Sepharose 4B and gel filtrated.
  • the void volume is pooled, concentrated further, and re-chromatographed on Sephacryl 200.
  • the glycoprotein fraction in the void volume is used as immunogen. All procedures must be completed within a limited time and at low temperature (4°C), since the sialyl linkage is unstable.
  • the immunogen prepared as described above, is treated for immunization as follows.
  • the sialyl-Tn antigen e.g., 4.0 mg
  • distilled water e.g., 4 ml
  • an appropriate amount of acid-treated Salmonella minnesota e.g., 16 mg
  • the dried mixture is suspended in a suitable volume (e.g., 4.0 ml) of an appropriate carrier (e.g., 140 mM NaCl containing 20 mM phosphate buffer, pH 7.0), and aliquots of about 100 ⁇ g of mucin and 400 ⁇ g of bacteria are injected intravenously. Immunization can also be made with complete
  • the host used for immunization can be a mouse or rat of any strain or any other type of animal whose splenocytes are suitable for preparation of hybridomas, i.e., susceptible to cell fusion with HAT- sensitive myeloma cell lines to establish stable hybridomas.
  • the immunization schedule depends upon the host animal susceptibility to mucin immunization, but the protocol described above is suitable for mice. Alternative conditions can also be applied. Suitable immunization schedules can be determined by the skilled artisan.
  • a suitable immunization schedule for Balb/c mice is to inject the immunogen preparation intravenously through the caudal vein once a week for
  • the amount of immunogen preparation administered to the host depends upon the molecular weight of the mucin, the exposure of the carbohydrate epitope, and the novelty and density of the epitope associated with mucin-type glycoproteins.
  • the range of glycoprotein injected in mice is 3-5 ⁇ g coated on 30-50 ⁇ g of Salmonella minnesota suspended in 100 ⁇ l of saline, intravenously injected in each individual mouse, whose body weight range is 100-150 g.
  • complete Freund's adjuvant is used, about
  • HAT-sensitive myeloma cells can be NS-1, SP-1 or SP-2, for example, but any type of HAT-sensitive myeloma cells can be used. Occasionally, hybridomas can be established after fusion of host splencytes with myeloma cells even though no antibody was detectable in the host serum. Therefore, it is not essential to detect antibodies before cell fusion. Fusions are usually carried out in polyethylene glycol, as detailed by Young, W.W., et. al. (supra). A preferred myeloma cell line for use in the present invention is SP-2.
  • the fused cells are cultured in 96-well plates until miniclones are formed. It is important to use splenocytes 48-72 hours after the last booster injection and to fuse with well proliferating myeloma cells to obtain a number of surviving fused cells to grow. Moisture and CO 2 concentration in the incubator must be carefully controlled at the initial stage of culturing the fused cells.
  • Feeder cells are not necessary to grow the fused cells according to the process of the present invention.
  • hybridomas secreting antibodies that react with the sialyl-Tn antigen that was used to immunize are cloned and subcloned by limiting dilution, i.e., by diluting to a point where less than one cell per new culture will be expected, and then plating into the wells.
  • each well of a 96-well plate is coated with mucin glycoprotein containing the sialyl- Tn core structure used as immunogen by incubation of each well with glycoprotein solution in PBS, i.e., 5- 10 ⁇ g/50 ⁇ l/well is added and incubated overnight. Glycoprotein solution is removed, washed, and further incubated with bovine serum albumin (10 ⁇ g/50 ⁇ l/well) to block the plate before using it to screen antibodies. This method is described by Hirohashi et al. (supra).
  • the hybridomas that secrete the antibodies that react with the particular antigens are screened as follows. Antibody bound to an antigen-coated well is usually detected by secondary antibody (anti-mouse IgM and IgG goat or rabbit antibodies) followed by 125 I- labeled protein A as initially described by Young, W.W., et al. (supra). The method is still more sensitive than available ELISA assays, although an ELISA can also be used. ELISA's can be more conveniently processed by use of automated readers and ELISA kits available commercially.
  • Monoclonal antibodies to sialyl-Tn antigen secreted by hybridomas thus isolated can be produced in quantity by growing large batches of hybridoma cell cultures and purifying the antibody from the supernatant or by injecting mice with the hybridoma line to stimulate the production of ascites fluid. Both methods are well known in the art.
  • the hybridomas isolated according to the present invention can be grown in large batches in suspension culture, or more conveniently in a fiberglass container in which cells are packed and grown in high density, wherein antibodies can diffuse into the culture medium.
  • the monoclonal antibodies can be purified by known methods, for example, by affinity separation using protein A or high pressure liquid chromatography on reverse phase alkylated silica gel or with a synthetic polystyrene gel filtration column.
  • Suitable doses of the above-described medicament can readily be determined by the skilled artisan, depending upon the mode of administration.
  • a suitable dosage for intravenous administration of antibodies to suppress further AIDS infectivity at the initial stage of patients with AIDS should be determined by various conventional experiments, since TKH2 antibody can inhibit HIV infection at 2 ⁇ g/ml concentration in vitro. 15.6 mg of TKH2 is believed sufficient to prevent HIV infectivity when administered to a man of 60 kg weight who has approximately 7.8 1 of blood. Humanized antibodies are highly preferable to perform this in vivo inhibition.
  • Antibodies can be administered intravenously, however, other forms of administration are also possible.
  • Suitable pharmaceutically acceptable carriers, diluents and excipients are readily determined by the skilled artisan.
  • the antibodies can be administered when solubilized in a physiological buffer solution.
  • no carrier is considered especially preferable.
  • the present invention also provides a vaccine against AIDS and ARC, comprising:
  • the present invention provides a method of actively immunizing against AIDS and ARC, comprising administering to a subject a medicament comprising:
  • a pharmaceutically acceptable carrier, diluent or excipient A pharmaceutically acceptable carrier, diluent or excipient.
  • the antigen Le y whose structure is defined above, can be prepared either synthetically by known methods (Hindsgaul, O. et. al., (1982) Carbohydrate Res. 109:102-142 and Spohr, U. et. al., (1985) Canadian J. Cham. 63:2644-2652) or, alternatively, can be isolated from naturally occurring sources by known methods, such as described in Abe, K. et. al., (1983) J. Biol. Chem. 258:11793.
  • the antigen A I having the structure set forth above, can also be prepared synthetically by known methods (see Lemieux, R.U. Chemical Society Reviews (1978) Vol. 7 423-452; IUPAC Frontiers of Chemistry 1982 K.J. Laidler, ed. 3-24 for the synthesis of Type 1 chain A) or can be isolated from natural sources, also by known methods such as that described in Abe, K. et. al., 11980 J. Immunol. 132.1951.
  • the antigen sialyl-Tn whose structure is set forth above, can be isolated from natural sources by known methods, such as that described in Kjeldsen, T. et. al., (1988) Cancer Res. 48:2214, or by the method described above for preparing purified immunogen comprising the sialyl-Tn antigen.
  • Suitable immunologically effective doses and pharmaceutically acceptable carriers, diluents and excipients of the above-described vaccine can readily be determined by the skilled artisan.
  • an immunologically active dose of sialyl-Tn antigen as a vaccine will be approximately 55 ⁇ g per injection.
  • the carrier molecule is important.
  • BCG coated with sialyl-Tn antigen is considered to be a useful way to prevent AIDS infectivity.
  • One example is 50 ⁇ g of sialyl-Tn antigen coated on 500 ⁇ g of BCG injected intradermally or subcutaneously.
  • Suitable methods of administration of the above- described vaccine can also be readily determined by the skilled artisan.
  • Antibodies tested, their specificity, isotype, and reference for production, are listed in Table I.
  • the twenty different monoclonal antibodies shown in Table I define carbohydrate structures or glycosphingolipids found at the periphery of both N- linked and O-linked, or exclusively O-linked, chains of glycoproteins and also glycosphingolipids (for a review see Clausen, H., and S. Hakomori (1989) Vox Sang. 56:1).
  • FCS fetal calf serum
  • TKH2, B72.3, BM1 and AH21 were used after purification from culture supernatant (Example 2; Figs. 2A to 2D).
  • TKH2 (IgG 1 ) and B72.3 (IgG 1 ) were purified by protein A-Sepharose chromatography, and AH21 and BM1 by ion-exchange chromatography as described by the manufacturer (Pharmacoa, Upsala, Swedan).
  • T4-lymphocyte cell lines H9 Popovic, M., et. al. (1984) Science 224:497)
  • CEM Foley, G.E., et. al. (1965) cancer 18:522)
  • MT4 Harada, S., et. al. (1985) Science 229:563
  • FCS 5% for CEM cells
  • growth medium 100 IU/ml penicillin, 20 ⁇ g/ml gentamicin and 100 IU/ml streptomycin (growth medium).
  • PMC Peripheral blood mononuclear cells
  • T4-lymphocytes were selected by panning (Wysocki, L.J., et. al. (1978) Proc. Natl. Acad. Sci. USA 75:2844). Donor PMC and T4-lymphocytes were cultured as above in growth medium supplemented with 20 IU/ml Interleukin-2.
  • HIV-1 strain SSI-002 was isolated from an HIV-infe ⁇ ted patient (CDC II) as previously described (Nara, P.L. et. al., (1989) Proc. Natl. Acad. Sci. USA 86:7139- 43). The virus was then passed three times in MT4 cells, and stored as above. Before use, the TCID 50 of the virus preparations was determined in MT4 cells and PMC. Toxicity
  • Toxicity of the monoclonal antibody preparations was examined by incubating 0.5x10 6 MT4 cells in 24-well cell culture plates in growth medium containing from 20 to 0 ⁇ g/ml purified antibody, and supernatants were exchanged with fresh medium containing appropriate concentrations of antibody after 4 days of culture. Live cell counts were obtained on days 0, 4 and 7 using trypan blue exclusion.
  • the primers used were SK29/SK30 (Ou, C.-Y., et. al. (1988) Science 239:295).
  • the mixture was heated to 94oC for 5 minutes and then 2 units Taq- polymerase (Perkin-Elmer Cetus, Norwalk, USA) were added. Forty rounds of amplification were carried out in a DNA Thermal Cycler (Perkin-Elmer Cetus). Each cycle lasted 1 min at 94°C, 1 min at 65°C and 1 min at 72oC.
  • One ⁇ l of the amplified product was mixed with
  • Membranes were prehybridized for 2 hours at 42°C in hybridization solution (50% deionized formamide, 1% sodium dodecyl sulphate (SDS), 1 M NaCl, 50 mg/ml dextran sulphate and 0.1 mg/ml sheared salmon sperm DNA), whereafter 20 pmol of 32 P-labeled probe (SK31) was added and hybridization was performed overnight at 42°C. Membranes were washed 10 minutes in 2 X SSC, 0.1% SDS at room temperature, followed by 30 minutes at 2 X SSC, 0.1% SDS at 62oC. Finally, the membrane was rinsed 2x15 minutes in 0.2 X SSC, 0.1% SDS at room temperature and exposed to Kodak XAR films overnight.
  • hybridization solution 50% deionized formamide, 1% sodium dodecyl sulphate (SDS), 1 M NaCl, 50 mg/ml dextran sulphate and 0.1 mg/ml sheared salmon sper
  • Binding of the monoclonal antibodies to cells was determined by indirect immunofluorescence microscopy (Inanue, E.R., et. al. (1986) In:D. M. Weir (ed) , Immunochemistry 1, 23:1). Briefly, 10 6 cells resuspended in 50 ⁇ l suspension buffer (PBS containing 1% BSA, 2% human serum and 0.1% azide) were incubated with 50 ⁇ l hybridoma supernatant for 45 min at 4°C.
  • Ten TCID 50 HTLV iii B was mixed with 0.5 ml hybridoma supernatant and incubated for 1 hour at 37°C.
  • MT4 cells (1x10 6 ) were suspended in this mixture and incubated for 2 hours at 37°C. After extensive washing the cells were resuspended in growth medium (4ml) containing 10% v/v of the corresponding hybridoma supernatant; and duplicates (1.5 ml) were transferred to a 24 well cell-culture plate. The cells were cultured for 7 days at 37°C, 5% CO 2 , and cell-free supernatant (750 ⁇ l) was exchanged with fresh medium without hybridoma supernatant after 2, 4 and 7 days.
  • HIV antigen output was measured by ELISA as described above on the supernatants. Dialyzed hybridoma supernatants (200 ⁇ l) were preincubated with 10 TCID 50 HTLV III B or SSI-002 before inoculation and cultured as described above.
  • Purified monoclonal antibody was preincubated with 10 TCID 50 HTLV III B which was then used to inoculate 1 x 10 6 MT4 cells, 4 x 10 6 3-day PHA stimulated PMC or selected T4-lymphocytes. After incubation for 2 hours at 37°C, cells were washed and resuspended in 5 ml growth medium. Quadruplicates of 1 ml cell suspension were transferred to a 24-well cell culture plate. Supernatant (500 ⁇ l) was exchanged with fresh growth medium after 4 and 7 days of culture.
  • Fig. 1 The results are shown in Fig. 1.
  • the ordinate represents antigen production, in arbitrary units, and the abscissa represents control with untreated virus ( ⁇ AB), control without virus ( ⁇ HIV), or hybridoma supernatant containing the designated monoclonal antibodies.
  • TKH2, BM1, AH21 and AH16 having similar but broader specificity than AH21.
  • Cells from these cultures were analyzed for HIV-DNA by polymerase chain reaction after 4 days of culture. All cultures with HIV antigen negative supernatants (TKH2, BM1, AH21, AH16 and the uninfected control) were also negative for HIV-DNA, and cultures with antigen positive supernatants were also positive for HIV-DNA.
  • TKH2, BM1, AH21, AH16 and the uninfected control were also negative for HIV-DNA, and cultures with antigen positive supernatants were also positive for HIV-DNA.
  • TCID 50 HTLV III B was incubated in a total volume of 60 ⁇ l RPMI 1640 containing affinity-purified monoclonal antibodies in varying concentrations for 1 hr at 37°C.
  • Figs. 2A to 2D the ordinate represents HIV antigen production (% of control), and the abscissa represents the concentration of monoclonal antibody relative to virus inoculum ( ⁇ g/ICID 50 ).
  • Figs. 2A to 2D show that purified antibodies of hybridoma culture supernatant of AH21, BM1, TKH2, and as a control B72.3 (having the same specificity as
  • TKH2 showed a concentration dependent inhibition of infection.
  • the inhibitory antibody concentration was defined as the antibody concentration resulting in an 80% reduction of HIV antigen expression after 4 days of culture, as described for neutralizing monoclonal antibody (MAb) titers (Kinney Thomas, E., et. al.
  • the inhibitory concentrations were interpolated to be: B72.3:0.18 ⁇ g/TCID 50 ; AH21: 0.32 ⁇ g/TCID 50 ; BM1: 1.16 ⁇ g/TCID 50 ; TKH2: 1.6 ⁇ g/TCID 50 .
  • the monoclonal antibodies inhibited infection with both HIV-1 reference strain HTLV III B and isolate SSI-002 when the virus preparation was preincubated with MAb before inoculation onto MT4 cells. No reduction in HIV infection was found when the cells were preincubated with MAb and then inoculated with untreated virus.
  • a cell immunofluorescence assay was conducted as described above.
  • Table 2 summarizes staining results with the selected monoclonal antibodies.
  • AH21 and AH16 did not label cells.
  • TKH2 weakly labeled a small population of cells, whereas B72.3 did not stain cells.
  • BM1 did not stain MT4 cells, uninfected or infected, and only a small percentage of T4 lymphocytes purified from a normal donor by panning were positive.
  • Host cell glycolipids may be incorporated in virions (Klenk. H.-D., et. al. (1970) Proc. Natl. Acad. Sci. USA 66:57), which should thus show a glycosylation pattern similar to the host cell.
  • the glycosylation of viral proteins is also believed to be performed by the host cell gene-encoded glycosyltransferases. Therefore, it is generally expected that the host cell and proteins of viruses propagated therein show similar glycosylation patterns.
  • the final glycan structure of a glycoprotein is a result of both exoglycosidase and glycosyltransferase activity as well as primary structure of the core protein, which in the case of viral glycoproteins could result in novel glycosylation patterns.
  • virus infection could induce expression of abnormal glycosyltransferase genes in the host cell.
  • the present results showed no correlation between inhibition of infection and immunofluorescence staining of cells used as targets or as virus source. The virus inhibition of the MAbs was thus independent of cell type used for virus propagation.
  • Syncytial activity was defined as the minimal number of infected H9 cells where syncytium formation (giant-cells were more than 5 nuclei) was detectable in the cultures.
  • the identified antigen may be used to provoke an immune response in patients by active immunization.
  • a panel of murine monoclonal antibodies was employed defining peripheral carbohydrate structures (generally 2-5 residues) which may be found on poly-N-acetyllacetosamine structures of both N- and O-linked glycoprotein and glycospingolipid types, as well as structures restricted to O-linked glycoprotein types (Table I).
  • Monoclonal antibody BM1 directed to Le y and previously found to be expressed on HIV-infected lymphocytes (Adachi, M., et. al. (1988) J. Earn. Med. 167:323, PincUS, S.H., et. al. (1989) J. Immunol.
  • HIV-infected patients have also been produced in vitro using selected synthetic peptides from gp120 and gp41 (Weber J.N., et. al. (1989) Lancet i:119; Chanh, T.C, et. al. (1986) EMBO J. 5:3065; Lasky, L.A., et. al. (1986) Science 233:209). These do not seem to protect against disease progression and are type-specific. "Escape" mutants of HIV, containing mutations in the env gene, have been shown to arise in vitro, whereby the virus is able to escape the effect of formerly neutralizing antibodies (Weiss, R.A. (1988) J. Acq. Imm. Def. Syn. 1:536). The anti-HIV reactivity of antisera against
  • Equine infectious anemia virus is described against a carbohydrate part of the HIV envelope (Montelaro, R.C, et. al. (1988) J. Gen. Virol. 69:1711), but HIV-neutralization by carbohydrate-specific antibodies has not been described earlier.
  • Carbohydrate epitopes may not be expected to be influenced as readily by mutations in the genome coding for the peptide part of the HIV envelope.
  • the present results indicate that viral glycans can be considered targets for anti-viral immunotherapy and/or vaccine development.
  • Ovine submaxillary mucin was used as the source of the sialyl-Tn antigen. Approximately 90% of the carbohydrate chain on OSM consists of the sialyl- Tn antigen.
  • OSM was isolated from ovine submaxillary glands by conventional methods. (Tettamanti, G. and Pigman, W. (1968) Arch. Biochem. Biophys., 124:45- 50).
  • aqueous extract of submaxillary glands was precipitated at acidic pH (e.g., 3.5). This is called a mucin clot.
  • the mucin clot was centrifuged, dissolved in water, the pH adjusted to neutral, and fractional ethanol precipitation in sodium acetate was performed.
  • the thus isolated OSM was used as was for the immunogen composition.
  • the high molecular weight sialyl-Tn antigen isolated as described above was dissolved in distilled water in an amount of 4.0 mg protein/4 ml water and 16 mg acid-treated salmonella minnesota, obtained by conventional methods, was added. The mixture was thoroughly mixed for 1 hour at 57oC, and then lyophilized. The lyophilized sample was then suspended in 4.0 ml of PBS and aliquots of 100 ⁇ l (i.e., 100 ⁇ g of the glycoprotein and 400 ⁇ g of bacteria) were injected intravenously through the caudal vein into each of five Balb/c mice.
  • mice were boosted with 200 ⁇ l of the antigen suspension (i.e., 200 ⁇ g of glycoprotein and 800 ⁇ g of the bacteria).
  • the animals were sacrificed, the spleen cells were removed and splenocytes were fused with mouse myeloma SP-2 cells by conventional methods (Hirohashi, S. et. al., (1984) Proc. Natl. Acad. Sci. USA, 82:7039-7043 and Fukushi, Y. et. al. (1984) J. Biol. Chem., 259:4681- 4685).
  • Hybridomas which grew on selective media were screened by conventional methods for monoclonal antibody reactivity with the above-described OSM, desialyated OSM, bovine submaxillary mucin (BSM) (approximately 50% of the carbohydrate chains on BSM consist of sialyl-Tn antigen), and glycophorin A.
  • Bovine submaxillary mucin (BSM) and glycophorin A were purchased from Sigma Chemical Company, St. Louis, Missouri.
  • OSM was desialyated by treatment with 0.1 unit/ml of neurominodase from Clostridium Perfrinoens Type X
  • Hybridomas secreting monoclonal antibodies showing strong reactivity with OSM, weak reactivity with BSM, and no reactivity with glycophorin A or sialidase-treated OSM were found.
  • the deposits were made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. All restrictions to access will be irrevocably removed upon grant of a U.S. patent on the instant application.

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Abstract

Médication d'inhibition ou de ralentissement de la progression du syndrome immunodéficitaire acquis (SIDA) et du complexe relatif au SIDA (ARC) comprenant une quantité efficace sur le plan pharmaceutique d'un ou plusieurs anticorps sélectionnés à partir du groupe consistant en anti-LeY, anti-AI et anti-sialyle-Tn et une charge, un diluant ou un excipient acceptables sur le plan pharmaceutique. Procédé d'inhibition ou de ralentissement de la progression du SIDA et de l'ARC comprenant l'administration à un patient atteint du SIDA ou de l'ARC d'une médication incluant une quantité efficace sur le plan pharmaceutique d'un ou plusieurs anticorps sélectionnés à partir du groupe consistant en anti-LeY, anti-AI et anti-sialyle-Tn, et une charge, un diluant ou un excipient acceptables sur le plan pharmaceutique. Vaccin contre le SIDA et l'ARC comprenant une quantité efficace sur le plan immunologique d'un ou plusieurs antigènes sélectionnés à partir du groupe consistant en LeY, AI et sialyle-Tn et une charge, un diluant ou un excipient acceptables sur le plan pharmaceutique. Procédé d'immunisation active contre le SIDA et l'ARC comprenant l'administration à un patient d'une médication incluant une quantité efficace sur le plan immunologique d'un ou plusieurs antigènes sélectionnés à partir du groupe consistant en LeY, AI et sialyle-Tn et une charge, un diluant ou un excipient acceptables sur le plan pharmaceutique.
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US6800468B1 (en) 1998-11-13 2004-10-05 Henrik Clausen UDP-galactose: β-N-acetyl-glucosamine β1,3galactosyltransferases, β3Gal-T5
WO2005078443A1 (fr) * 2004-02-13 2005-08-25 Inverness Medical Switzerland Gmbh Determination d'infection par la reponse immunitaire a un groupe fonctionnel glucidique

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EP0304663A1 (fr) * 1987-07-31 1989-03-01 Japan Immuno Research Laboratories Co., Ltd. Procédé pour déterminer des lymphocytes repondant irrégulièrement et réactif et composition pour cela
WO1989008711A1 (fr) * 1988-03-11 1989-09-21 The Biomembrane Institute Anticorps monoclonaux et mise au point d'un vaccin developpe contre des antigenes cancereux humains par immunisation avec de la mucine animale et humaine et avec des conjugues porteurs de glucides synthetiques

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0304663A1 (fr) * 1987-07-31 1989-03-01 Japan Immuno Research Laboratories Co., Ltd. Procédé pour déterminer des lymphocytes repondant irrégulièrement et réactif et composition pour cela
WO1989008711A1 (fr) * 1988-03-11 1989-09-21 The Biomembrane Institute Anticorps monoclonaux et mise au point d'un vaccin developpe contre des antigenes cancereux humains par immunisation avec de la mucine animale et humaine et avec des conjugues porteurs de glucides synthetiques

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Title
CANCER RESEARCH vol. 48, no. 8, 15 April 1988, PHILADELPHIA PA, US pages 2214 - 2220 T. KJELDSEN ET AL. 'Preparation and characterization of monoclonal antibodies directed to the tumor-associated O-linked sialosyl-2-6 alpha-N-acetylgalactosaminyl (sialosyl-Tn) epitope.' *
See also references of WO9111199A1 *
THE JOURNAL OF IMMUNOLOGY vol. 142, no. 9, 1 May 1989, BALTIMORE MD, US pages 3070 - 3075 S. PINCUS ET AL. 'Treatment of HIV tissue culture infection with monoclonal antibody-ricin A chain conjugates.' *
THE JOURNAL OF VIROLOGY vol. 64, no. 6, June 1990, BALTIMORE MD, US pages 2833 - 2840 J. HANSEN ET AL. 'Inhibition of human immunodeficiency virus (HIV) infection in vitro by anti-carbohydrate monoclonal antibodies: Peripheral glycosylation of HIV envelope glycoprotein gp120 may be a target for virus neutralization.' *

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