EP0493508A1 - Nouveaux peptides associes a la region de liaison cd4 de la gp120 et leur mode d'emploi - Google Patents

Nouveaux peptides associes a la region de liaison cd4 de la gp120 et leur mode d'emploi

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Publication number
EP0493508A1
EP0493508A1 EP90915005A EP90915005A EP0493508A1 EP 0493508 A1 EP0493508 A1 EP 0493508A1 EP 90915005 A EP90915005 A EP 90915005A EP 90915005 A EP90915005 A EP 90915005A EP 0493508 A1 EP0493508 A1 EP 0493508A1
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European Patent Office
Prior art keywords
antibodies
hiv
peptide
compound
antibody
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EP90915005A
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German (de)
English (en)
Inventor
Thomas Kieber-Emmons
William John Woodrow Morrow
William H. Rastetter
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Idec Pharmaceuticals Corp
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Idec Pharmaceuticals Corp
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Publication of EP0493508A1 publication Critical patent/EP0493508A1/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates generally to peptides and methods of selecting antibodies, more particularly, to novel peptides and analogs thereof associated with the CD4 binding domain or region of the gp120 segment of the HIV envelope protein and their use to generate HIV neutralizing antibodies for use in protective vaccination against AIDS or immunotherapy.
  • immunomodulating agents were due to the quantitative and qualitative deficiencies in the neutralizing antibodies which they elicit.
  • such antibodies are frequently associated with reagents or epitopes on the envelope which are constantly mutating, a feature which enables the virus to stay a step ahead of the immune system.
  • epitopes associated with gp120 molecule of HIV which will elicit broadly reactive or group-specific neutralizing responses which will afford protection against or
  • HIV human immunodeficiency virus
  • HTLV-III human immunodeficiency virus
  • LAV human immunodeficiency virus
  • ACV displays selective tropism for cells that express the CD4 receptor, primarily infecting the helper/inducer T cell
  • lymphocytes See, Klatzmann, D., et al., Nature , 312:767 (1984);
  • Proposed therapeutic strategies for HIV infection include the in vivo
  • soluble CD4 to compete with membrane-bound CD4 receptors for the HIV's gp120
  • the use of soluble CD4 coupled to toxic drugs to target infected cells see, Chaudhary, V.K., et al., Nature, 335:369 (1988)
  • anti-CD4 receptor antibodies as gp120 surrogates to boost or elicit an in vivo immune response (“active immunotherapy") that includes high titers of HIV neutralizing antibodies
  • active immunotherapy that includes high titers of HIV neutralizing antibodies
  • Prophylactic vaccination strategies also have focused on inducing anti-HIV, in particular, anti-gp120 antibodies.
  • amino acid sequence numbering scheme for gp120 can vary depending on the HIV strain or isolate sequenced. For example, segment 397 to 439 discussed by Lasky is based on the HTLVIIIB strain and corresponds to 415 to 456 for the BRU strain.
  • Anti-gp120 monoclonal antibodies developed by immunization with the entire gp120, that block gp120 binding to CD4 have been mapped to residues 422-432; it has also been shown that a deletion mutant of gp120 involving residues 426-437 and a mutation at position 433 abrogates CD4 binding.
  • Short peptide segments of the 415-456 region of gp120 have been suggested as T cell epitopes capable of eliciting cellular immunity, but these peptides have not been implicated as playing a role in humoral immunity.
  • the neutralizing epitope of gp120 is a complex peptide conformation with primary, secondary and tertiary structure where amino acids interact both with each other to maintain the epitope's conformation and with the CD4 receptor to facilitate binding. Elucidation of the molecular basis for these structural and functional relationships in the native gp120 amino acid sequences and their immunological significance remains a considerable scientific challenge. Duplication of these structural and functional relationships of the neutralizing epitope is an essential feature of a gp120 surrogate and is the focus of the present invention. Notwithstanding the designation of the "CD4 binding site" by Lasky,
  • the CD4 binding region may include the CD4 binding site identified by Lasky, Kowalski, et al., but also incorporates additional amino acid sequences on the N and/or C terminus sides of this site.
  • the present invention is based on the identification of peptides and analogs thereof within the CD4 binding region of gp120 that contain one or more neutralizing epitope(s) involved in the binding of gp120 to its cellular receptor, CD4. More specifically, the present invention is based on peptides and immunologically equivalent analogs thereof from the gp120 region from about 335 to about 517 (amino acid position numbers may vary depending on HIV isolate).
  • the present invention is directed to a compound characterized by the capability of eliciting and/or binding with HIV neutralizing antibodies, wherein the capability results from an amino acid sequence of at least 17 amino acid residues which: (a) is a neutralizing epitope(s) from the CD4 binding region of the gp120 envelope protein of an HIV isolate; (b) is a portion or segment of the epitope; or (c) is an immunological equivalent of the epitope or portion thereof.
  • the present invention further includes a method for generating antibodies for use as a vaccine or in immunotherapy. The first step of the method requires coating a solid support with an effective amount of a composition including the compound described above.
  • a serum sample containing polyclonal anti-gp120 or anti-compound antibodies is applied to the support, wherein a first plurality of the antibodies in the sample complex with the compound.
  • the serum sample may be, for example, HIV positive human sera or sera from a mammalian host immunized with gp120 or a compound of the present invention.
  • the solid support is then separated from the serum sample, and the first plurality of antibodies are eluted from the solid support.
  • An immunogenic composition including the first plurality of antibodies is then formulated and used to immunize a mammalian host.
  • a second plurality of polyclonal antibodies which are immunologically reactive with the first plurality of antibodies and immunologically competitive with gp120, is selected and purified from the sera of the host.
  • a second plurality of monoclonal antibodies is selected by sacrificing the host and generating hybridomas using an antibody producing organ of the host.
  • the second plurality of monoclonal antibodies are characterized as immunologically reactive with the first plurality of antibodies and immunologically competitive with gp120.
  • This second plurality of antibodies are gp120 surrogates that may be used, for example, in the treatment or prevention of AIDS.
  • the present invention also includes a second method for generating antibodies for use as a vaccine or in immunotherapy.
  • the first step of the method requires immunizing a first mammalian host with an immunogenic composition including a compound of the present invention. Then, a serum sample from the first host is applied to a solid support coated with gp120, whereby a first plurality of antibodies in the sample complex with the gp120. The solid support is separated from the serum sample, and the first plurality of antibodies eluted from the solid support.
  • a second mammalian host is immunized with an immunogenic formulation including the first plurality of antibodies.
  • a second plurality of polyclonal antibodies which are immunologically reactive with the first plurality of antibodies and are immunologically competitive with gp120, is selected and purified from the sera of the second host.
  • a second plurality of monoclonal antibodies may be selected by sacrificing the second host and generating
  • the second plurality of monoclonal antibodies are characterized as
  • This second plurality of antibodies are gp120 surrogates that may be used, for example, in the treatment or prevention of HIV infection.
  • FIG. 1 depicts the sequences of examples of peptides within the scope of the present invention. Where tested, the various peptides' antisyncytial potentials for a number of HTV strains are also shown.
  • FIG. 2 shows flow cytometry assay results illustrating the ability of the B138 peptide to inhibit the binding of HIV I ( ⁇ IB and RF isolates) to CD4 positive T cells (CEM line). Also shown is the lack of inhibition by a control peptide 1005-05.
  • FIG. 3 shows proliferative responses of mononuclear cells from both HIV seronegative and seropositive individuals in response to peptide B 138.
  • FIG. 4 shows further flow cytometric analysis depicting murine anti B-138 antibodies reacting with HIV infected T cells.
  • FIG. 5 shows ELISA assay results depicting the reactivity of sera from HIV seropositive and seronegative controls with peptide B138.
  • the conserved region of the CD4 binding region of gp120 is thought to be a potential target for controlling HIV infection.
  • Studies to characterize those interactions between CD4 and gp120 that can stimulate or inhibit immune responses toward HIV are critical to the design of agents to treat HIV infections. Seeking to identify a neutralizing epitope(s) that plays a role in this gp120/CD4 interaction, the applicants examined some of the in vitro binding and immunological characteristics of peptides derived from the region of gp120 extending from amino acid residue 335 to 517 of various HIV isolates. (The amino acid position numbers may vary slightly from isolate to isolate.) This region of interest is referred to by the applicants as the "CD4 binding region.”
  • spacer amino acids may have no impact on a peptides' immunological properties.
  • the present invention is not based on the precise delineation of amino acid sequences, but rather the present invention is based on the synthesis of peptides and compounds containing such peptides which share the immunological characteristic of being capable of eliciting and/or binding with the HTV neutralizing antibodies.
  • HIV neutralizing as used in the present application includes both the ability to prevent HIV infection in new cells or the ability to block syncytial formation between cells.
  • the present invention is directed to any compound which is capable of eliciting and/or binding with HIV neutralizing antibodies.
  • This characteristic capability results from an amino acid sequence which is a neutralizing epitope(s) from the CD4 binding region of the gp120 envelope protein of an HIV isolate, or a portion or segment of such an epitope.
  • the amino acid sequence may be an immunological equivalent of the epitope or portion thereof.
  • the present invention may include a compound, other than the naturally occurring HIV envelope protein, characterized by the capability of eliciting and/or binding with HIV neutralizing antibodies, wherein such compound includes the neutralizing epitope or segment thereof from the CD4 binding region of the gp120 envelope protein of an HIV isolate.
  • the amino acid sequence of gp120 which participates in formation of the HIV neutralizing epitope stretches from at or about amino acid position 335 to at or about amino acid position 517, depending which HTV isolate is being analyzed.
  • peptide sequences and analogs from the MN isolate, it is preferred most that whatever isolate is used results in peptides that elicit HIV neutralizing antibodies that are grouped rather than type-specific. Although it is conceivable that type-specific peptides or neutralizing antibodies could be formulated in a cocktail for vaccine or immunotherapy use, it is preferred that a single peptide or HIV neutralizing antibodies elicited thereby be effective against all isolates and represent a conserved epitope from the CD4 binding region of gp120.
  • conjugates of the peptide or peptide analog that may enhance its immunogenicity.
  • the peptide or analog may be conjugated to such carrier proteins as tetanus toxoid, keyhole limpet hemocyanin (KLH), bovine serine albumin (BSA), and hepatitis B virus core antigen.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serine albumin
  • the peptides of the present invention may also be conjugated to T cell epitopes designed to elicit a cellular immune response.
  • peptides of the present invention may be conjugated to viral fusion proteins or included in chimeric constructs, e.g., polio virus/peptide. Moreover, it is possible, using the basic methods reviewed by
  • fragment thereof e.g., chimeric antibodies.
  • peptide of the present invention represents an immunologically specific neutralizing epitope on the native gp120, it is imperative that the peptides, analogs, and conjugates thereof retain or be able to adopt the same three dimensional shape that contributes to the binding of CD4. Any derivative of the peptides of the present invention that meets this functional criteria is within the scope of the present invention.
  • Amino acids useful according to the present invention and their standard abbreviations are listed below in Table 1.
  • the compounds of the present invention typically include a peptide of at least 17 to about 100 amino acid residues.
  • the peptide may be selected from any one of the amino acid sequences listed in Table 2.
  • the peptide may be a peptide analog of the amino acid sequences listed in Table 2 where the amino acid residues are modified, substituted, deleted, inserted or added to the sequence, provided that the immunoreactivity of the analog to anti-peptide antibodies (which are also reactive with gp120) is preserved in the peptide analog.
  • the amino acid cysteine (“C") is highlighted by an asterisk (“*") to denote its special function.
  • cysteine residues In addition to forming a site for cross linking to carrier proteins, if a pair of cysteine residues are sufficiently far apart, they are capable of forming a disulfide bond between one another and thereby contributing a loop-like configuration to an amino acid sequence. Thus, the three dimensional conformation of peptide sequences can be modified by the addition or deletion of cysteine residues.
  • a single loop bracketed by two amino acid sequences is formed by a disulfide bond between the two C* with the amino acid sequences outside the loop (termed “tails") contributing to the conformational or binding properties necessary to mimic the neutralizing epitope.
  • tails the amino acid sequences outside the loop
  • two glycines have been substituted for the native cysteines and a substantial amino acid sequence has been deleted from the peptide. This deleted amino acid sequence represents a known
  • a skilled artisan may synthesize any of the peptides of the present invention by using an automated peptide synthesizer (for example, an Applied Biosystems model 430A) using standard t-Boc chemistry as reported by Carpino, L.A., J. Am Chem Soc, 79:4427 (1957), the disclosure of which is hereby incorporated by reference.
  • an automated peptide synthesizer for example, an Applied Biosystems model 430A
  • standard t-Boc chemistry as reported by Carpino, L.A., J. Am Chem Soc, 79:4427 (1957), the disclosure of which is hereby incorporated by reference.
  • Analogs of the preferred peptides can be prepared by varying the number, order, or type of amino acids used in the standard synthesis method.
  • the term "analog” as used herein encompassed any of the peptides in the present invention, segments thereof, peptides that contain substitutions or modifications of amino acid residues, and peptides that contain insertions or deletions of amino acids in the peptide or additions to the terminal amino acid residues of the peptide. Additional examples of peptide analogs are shown in FIG. 1 and include:
  • S is Serine and R is Arginine
  • C is Cysteine.
  • the peptide designated 1005/45 although depicted as a linear amino acid sequence above, is preferably cyclized by a disulfide bond between the terminal cysteines. All data, except the coupling of the peptide to the affinity column, that refers to the
  • 1005/45 peptide was developed with the cyclic form. Although applicants do not wish to be bound by any theory, it is postulated that the cyclic form of 1005/45 is a more rigid structure that locks in the conformational shape of the epitope more effectively than a linear peptide, e.g., B138.
  • Table 3 summarizes applicants' evaluation the biological properties of two of the peptides of the present invention, B138 and 1005/45.
  • Multi-strain Binds to HTLV IIIB Binds to HTLV I IIB, reactivity (FACS) LAV and SF33 LAV and SF33
  • CD4-gp120 Inhibits ⁇ 50% of Inhibits ⁇ 50% of inhibition assay gp120-CD4 reaction gp120-CD4
  • Peptides of the present invention are designed to contain a neutralizing epitope of gp120 and, therefore, may be used as gp120 surrogates in targeting the gp120/CD4 interaction.
  • the peptide may be used as a vaccine in uninfected individuals to induce protective titers of neutralizing anti-HIV antibodies.
  • the peptide may be used in active immunotherapy of HIV positive individuals to initiate or boost their immune response of neutralizing anti-HIV antibodies.
  • peptides of the present invention may be any amino acid sequence having the same or different amino acids.
  • the peptides of the present invention may be any amino acid sequence having the same or different amino acids.
  • the peptides of the present invention may also be used to generate antibodies that can be used as templates to generate antiidiotype antibodies having the internal image of the neutralizing epitope structure contained in the peptide sequence. These antibodies, polyclonal or monoclonal, can then be used in vaccine formulations or in active immunotherapy. Accordingly, the present invention also includes monoclonal or polyclonal antibodies that carry the internal image of the peptides, as well as methods for generating these antibodies. Like the peptides per se, these antibodies may be used as gp120 surrogates.
  • a first method for generating such antibodies for use as vaccines or in active immunotherapy starts with coating a solid support with an effective amount of a peptide of the present invention.
  • the process of coating or linking a peptide to a solid phase support potentially alters the peptides' three-dimensional conformation, therefore it may be necessary to explore several techniques in order to achieve the desired technical results.
  • a preferred solid support is Reacti-GelTM obtained from Pierce Biochemicals, Rockford, IL.
  • This support matrix comprises a cross-linked agarose gel that is activated by carbonyldiimadazole (CDI).
  • CDI carbonyldiimadazole
  • the imidazoyl-carbamate group couples to ligands containing free amino groups most efficiently at a pH of between 9-11, although sensitive proteins can be coupled at pH 8.5 in 0.1M borate buffer.
  • cyanogen bromide (CNBr)-activated agarose which also couples peptides or proteins via free amino groups (Hudson, L. and Hay, F.C., Practical Immunology, 3rd edition, Blackwell Scientific Publications, Oxford, 1989).
  • serum as used herein "serum” is intended to included both serum and plasma
  • Suitable serum samples include, for example, human serum from HIV positive
  • This first plurality of anti-gp120/anti-peptide antibodies is formulated in a immunogenic composition and used to immunize a mammalian host, preferably a rabbit or guinea pig. The host is bled and a second
  • a second plurality of monoclonal antibodies is then selected and purified, e.g., over an affinity column coated with the immunogen, from the sera of the host.
  • a second plurality of monoclonal antibodies may be produced by using the basic method developed by Köhler and Milstein, reported in Nature, 256:495-97 (1975), the disclosure of which is hereby incorporated by reference. Briefly, this procedure would include: sacrificing the mammalian host immunized with the first plurality of antibodies; harvesting an antibody producing organ, e.g., spleen, from the host; preparing a cellular homogenate from the
  • this second plurality of antibodies is characterized by their immunological reactivity with the first plurality of antibodies and immunological competition with gp120. Additionally, the second plurality of antibodies may be characterized by their ability to induce in mammals titers of neutralizing anti-HIV antibodies. The second plurality of antibodies contain the internal image of the neutralizing epitope and, therefore, are candidates for use as a vaccine or in immunotherapy.
  • a second method for generating such antibodies starts with the immunization of a mammal host, preferably a rabbit or guinea pig, with a peptide composition of the present invention.
  • the peptide is not sufficiently immunogenic in the selected host, it can be formulated with an adjuvant and/or, like other haptens, an immunogenic response achieved by linking the peptide to a carrier.
  • Suitable adjuvants include muramyl dipetide adjuvants, e.g., SAF (Syntex Adjuvant Formulation), Freund's adjuvant, and alum.
  • Methods of linking carriers to haptens are well known in the art; and numerous carrier proteins are available for coupling with the peptides, e.g., KLH and tetanus toxoid.
  • a serum sample from the host is then applied to a solid support coated with gp120, whereby a first plurality of antibodies in the sample complex with the support bound gp120.
  • the gp120 isolate that is selected is not critical. However, if a particular HIV isolate is prevalent in the patient population targeted for treatment, that gp120 isolate may be selected for use on the solid support, so that the antibodies selected by the method are tailored for the HIV epidemiology.
  • the support is separated from the serum sample and the complexed antibodies eluted from the gp120 coated support. This first plurality of antibodies is then used to immunize a second mammal host, preferably a mouse.
  • a second plurality of polyclonal antibodies may be selected and purified, e.g., using an affinity column, from the sera of the second host.
  • a second plurality monoclonal antibodies is selected by sacrificing the second host and generating monoclonal antibody producing hybridomas according to the procedure described above.
  • This second plurality of antibodies, whether polyclonal or monoclonal, is characterized by their immunological reactivity with the first plurality of antibodies and their immunological competition with gp120. Additionally, the second plurality of antibodies may be characterized by their ability to induce in mammals titers of neutralizing anti-HTV antibodies.
  • the second plurality of antibodies contains the internal image of the neutralizing epitope represented by the peptide, and, therefore, are candidates for use as a vaccine or in immunotherapy.
  • a third method for generating antibody surrogates for gp120 starts with the immunization of a first mammalian host with an immunogenic composition including a peptide composition of the present invention. Then, using the procedure previously described, a hybridoma is generated from an antibody producing organ of the first mammalian host, where the hybridoma is capable of producing a monoclonal antibody characterized by its immunological reactivity with the peptide immunogen and its immunological reactivity with gp120. Additionally, this first monoclonal antibody may be characterized by its ability to neutralize HIV infection of CD4 positive T cells in vitro. Then, a second mammalian host is immunized with an immunogenic composition including the first monoclonal antibody.
  • a plurality of polyclonal antibodies may be selected and purified from the sera of the second host, wherein the polyclonal antibodies are characterized by their immunological reactivity with the first monoclonal antibody and immunological competition with gp120. Additionally, the second plurality of antibodies may be characterized by their ability to induce in mammals titers of neutralizing anti-HIV antibodies.
  • monoclonal antibodies are produced by generating a second hybridoma from an antibody producing organ of the second mammalian host. The hybridoma is capable of producing a second monoclonal antibody characterized by its immunological reactivity with the first monoclonal antibody and immunological competition with gp120. Additionally, this second monoclonal antibody may be screened for its ability to block syncytia of HIV in vitro.
  • the monoclonal and polyclonal antibodies produced by this method also contain an internal image of the
  • neutralizing epitope represented by the peptide and, therefore, are candidates for use as a vaccine or immunotherapy.
  • An additional aspect of the present invention is the generation of monoclonal antibodies to the peptides of the invention.
  • a skilled artisan may develop hybridoma cell lines producing monoclonal antibodies specific for the peptides of the present invention.
  • these monoclonal antibodies would not contain an internal image of the neutralizing epitope but rather would contain the mirror image of the neutralizing epitope and, therefore, specifically bind to such epitope.
  • the murine monoclonals could be "humanized” by the some of the techniques described by Reichmann, L., et al., Nature, 332:(6162) 323 (1988) the disclosure of which is hereby incorporated by reference.
  • human monoclonals could be made by a variety of methods, including the EVB transformation of PBL's from HIV positive individuals who's sera can be shown by ELISA to be reactive with one of the peptides of the present invention. (See, Banapour, B., et al., J. Immunol, 139:4027 (1987), the disclosure of which is hereby incorporated by reference.) In any event, these murine or human monoclonal antibodies will bind specifically to the neutralizing epitope of gp120 and, thus, are suitable per se, or for linking to toxins or anti-viral reagents for targeting of HIV, in passive immunotherapy.
  • a further aspect of the present invention is the use of the peptide of the present invention in immunoassay methods for the detection of antiHIV antibodies for the diagnosis, prognosis, or therapeutic monitoring of AIDS, ARC or pre-AIDS conditions.
  • immunoassay methods include coating a solid support with an effective amount of a peptide of the present invention, which functions as an antigen.
  • a test sera diluted with buffers is then applied to the solid support, where anti-HIV antibodies in the test sera form peptide antibody complexes with the support bound peptide.
  • the mixture is then incubated at room
  • Such immunoassays are preferably enzyme linked
  • ELISA immunoassays
  • a second known anti-human antibody tracer capable of complexing with the anti-HIV antibody, is labeled with an enzyme and applied to the mixture forming a peptideantibody-antibody sandwich.
  • the second known antibody is, for example, goat anti-human IgG antibody labeled with horseradish peroxidase.
  • the sandwich is detected by adding a substrate for the enzyme to form a colored product.
  • a suitable substrate for horseradish peroxidase is a mixture about 0.04% by weight orthophenylenediamine and about 0.012% by volume hydrogen peroxide in sodium citrate buffer, pH 5.0.
  • An alternative embodiment is an immunoradiometric assay ("IRMA"), which utilizes a second known anti-human antibody labeled with a radioactive element, e.g., 125 I-labeled. Further, the peptide antibody complex may be detected as an agglutination in an agglutination assay.
  • IRMA immunoradiometric assay
  • Such assays may be manufactured and sold as test kits which would include: a solid support; an immunoadsorbant including a peptide composition of the present invention coated on the solid support; a sample of normal serum as a negative control; a sample of serum containing anti-HIV antibodies as a positive control; and buffers for diluting the serum samples.
  • the enzyme labeled antihuman antibody or radiolabeled anti-human antibody may be also included in the kit.
  • Peptides were synthesized on an Applied Biosystems 430A automated peptide synthesizer using pMBHA resin as a solid support (Matsueda, G.R., et al., Peptides, 2:45 (1981)). All amino acids were coupled by preformed symmetric anhydrides (Yamashiro & Li, J. Am Chem. Soc., 100:5174 (1978)) with the exception of asparagine and glutamine which were coupled as their hydroxybenzotriazole (“HOBT”) active esters.
  • HOBT hydroxybenzotriazole
  • Arginine was coupled via a DCC-mediated procedure.
  • the Kaiser test (Kaiser, Anal. Biochem., 34:595, (1970)) was used to monitor the efficiency of individual couplings with double-couplings performed, if necessary.
  • the N-terminal amino groups were actylated by the addition of a solution of 10% acetic anhydride, 5% diisopropylethylamine in CH 2 CI2.
  • Peptides were cleaved from resin with either HF/Anisole/ Ethanedithiol or the 'low-high' procedure of Tam, et al., J. Am. Chem. Soc ., 21:6442, (1983).
  • peptides were purified by preparative HPLC using a Vydac C-18 column (21 ⁇ 250 mm). Peptides were eluted from the column with a 0.05% trifluoroacetic acid buffer and a 25-45% acetonitrile gradient over 40 minutes. The flow rate was 10 ml/min, and a typical load was 50-100 mg. Fractions were isolated from the peptide eluant by U.V. detection at 235 nm. The resultant fractions were lyophilized and analyzed for purity by analytical HPLC (Beckman System Gold) with a 100 mm
  • Acid hydrolysates were prepared by treating peptides with 6N HCl in vacuo at 110°C for 24 hours. Cysteine content was determined after conversion to S-carboxy methyl cysteine, tryptophan content after hydrolysis with 4N methanesulphonic acid. Hydrolyzed samples were analyzed on a Beckman 6300 amino acid analyzer interfaced with a Nelson 3000k data system. Norleucine was used as an internal standard.
  • EXAMPLE 2 COUPLING OF PEPTIDE TO CARRIER
  • the reaction solution was allowed to stir at room temperature until a cloudy ppt began to form ( ⁇ 1.5 hrs.) at which time the reaction was quenched by the addition of 500 ⁇ l of 2M glycine per 10 ml of solution.
  • the conjugate was
  • Peptides 1005/45 and B119 were conjugated to KLH and BSA utilizing amino groups on the carrier and the terminal cysteines on the peptide.
  • the carrier in solution at a concentration of 1 ⁇ g/ml, was treated with a 200 fold molar excess of sulfosuccinimidyl 4-(N-maleinidomethyl) cyclohexane-1-carboxylate ("Sulfo-SMCC,” a hetrobifunctional reagent available from Pierce Biochemicals, Rockford, IL) in PBS, for two hours at room temperature with stirring (Hashida, S., et al., J. Applied
  • Peptide was reduced by the addition of 50 mM dithiothreitol ("DTT") and incubation at 37°C for 1 hour.
  • DTT dithiothreitol
  • the reduced peptide was separated from reductant by a Sep-Pak (Waters) apparatus and subsequently eluted with 95% CH 3 CN directly into dialyzed, derivatized carrier.
  • the reaction was achieved under argon blanket and argon saturated solutions for two hours at room temperature with stirring. Free peptide was removed from the conjugate by dialysis.
  • Additional methods include carbodiimide-mediated coupling through the carboxyl group of the peptide and the primary amines of the carrier (Staros, et al., Anal. Biochem, 156:220 (1986)). Tetanus toxoid was conjugated to peptide utilizing the aforementioned methods.
  • Cyclization of the 1005/45 peptide through its terminal cysteines may enhance its conformational rigidity. Therefore, after initial reverse phase HPLC purification as previously described, peptide 1005/45 was cyclized by the formation of a disulfide bond between the terminal cysteines. Cyclization was effected by first reducing the SH groups in the peptide with 50 mm DTT. The peptide was separated from
  • cyclic 1005/45 includes the introduction of acetomidomethyl groups to to the cysteine residues. Cyclization is achieved by the removal of the ACM group with NaI in solution (Stewart, J.M., et al., Solid Phase Peptide Synthesis 2nd
  • the disulfide bond may be replaced with either a peptide bond or a homobifunctional linker, e.g., bis maleimide.
  • a homobifunctional linker e.g., bis maleimide.
  • bioassays were utilized to evaluate peptides derived from this region of gp120 as potential inhibitors of CD4 dependent cellular functions.
  • the 18mer tract extending from residue 421 to 438 and several other synthetic peptides from the 413-456 region were first assayed for their ability to inhibit HIV-1 and HIV-2 infection of CD4 + cells.
  • Both CD4 + T cells (VB) and B cells (AA5) were used as target cells in the infectivity assay.
  • the BRU (B138) isolate 421-438 residue sequence was the most effective of the peptides assayed to block infectivity of the CD4+ cell lines by the HTV-1 isolate I ⁇ B, LAV-1, SF33, and HIV-2. Cells were greater than 95% viable as tested by
  • the 421-438 peptide (1005-34) derived from the IIIB isolate was less effective in inhibiting infection as determined by syncytial formation utilizing the HXB2 cell line. These two peptides (B138 and 1005-34) differ at positions 423 and 429.
  • An analogous SIV HIV-2 consensus 18 residue peptide (1017-33) from the SK6W isolate did not block infectivity of the cell lines by the I ⁇ B isolate. To what extent the substitutions at positions 421, 426, 428 and 433-434 in the consensus peptide effects the CD4 binding conformation available to this peptide remains to be examined.
  • MHC histocompatibility complex
  • sequence RFDS was also assayed for syncytial blocking and failed to block syncytia.
  • This peptide has exhibited immunosuppressive activity of lymphocytes stimulated by influenza hemagglutinin.
  • HLA human lymphocyte antigen
  • CD4 may interact through the charged residues of this tetrapeptide type since an analog of the sequence RADS is present in the NH2 terminal immunoglobulin V-like domain of CD4.
  • the RADS tract in CD4 is adjacent to the purported binding site on CD4 for gp120, however, as shown in FIGURE 1 the CD4 derived peptide (1005/78) that contains this tract also did not inhibit syncytia. Indeed, reports by others have determined that the gp120 contact sites on CD4 are distinct from those which bind MHC class II molecules.
  • Syncytial blocking activity has been equated with the blocking of gp120/CD4 binding (see, Lifson, J.D., et al., Nature, 323:725 (1986))
  • flow cytometry analysis indicated that B138 can inhibit the binding of the two virus isolates examined at a concentration equal to that for syncytial blocking.
  • B138 peptide at 100 ⁇ g/ml appears to inhibit binding of both IIIB and RF isolates to CEM cells.
  • the control peptide 1000-05 did not inhibit the fluorescence staining.
  • T lymphocyte responses to HIV antigens characterized by lymphocyte proliferation or interleukin release are only detectable to a modest degree in vitro in individuals or chimpanzees infected with the AIDS retrovirus. To a large extent these findings are probably
  • lymphocytes peripheral blood
  • PBMCs mononuclear cells
  • B138 inhibited lymphocyte proliferation in response to tetanus toxoid by an average of 65%.
  • B138 The immunosuppressive properties of B138 parallel those observed by others in the suppression of immune responses by glycosylated gp120, except B138 does not inhibit PHA lymphocyte proliferation.
  • non-glycosylated, yeast-expressed, gp120 has been shown by others not to inhibit PHA or antigen-driven lymphocyte proliferation.
  • Infection by viruses such as CMV or influenza characteristically induces lymphocytes capable of proliferating in vitro to the sensitizing antigens. These proliferative responses are generally associated with the activity of CD4+ helper T lymphocytes. Applicants' studies and others indicate that gp120 binding to CD4 interferes with these proliferative responses.
  • lymphocytes from nearly all AIDS patients as well as from some HIV-infected individuals are unable to proliferate in response to recall antigens and these responses cannot generally be reconstituted with rIL-2.
  • B138 in vitro effects are the result of binding to and modulating the CD4 receptor. Although applicants do not wish to be bound by any theory, it may be that B138 modulates the cellular metabolism of the target cells in an undefined way which deactivates or reduces cell proliferation, or the target cells susceptibility to cell-to-cell fusion by syncytia.
  • the immunogenicity of B138 was determined by subcutaneous
  • mice and rabbits were shown to preferentially bind to m ⁇ -infected H9 cells as assessed by FACS analysis. This rabbit sera could immunoprecipitate purified gp120.
  • EXAMPLE 5 SOLID SUPPORT PREPARATION FOR GENERATION OF ANTIBODIES
  • Reacti-GelTM to prepare an affinity column linked with the B138 peptide to purify antibodies from mice immunized with B138.
  • the peptide was coupled to Reacti-GelTM by the following procedure: 5.1 mg B138 was dissolved in 7ml 0.1M borate buffer, pH8.5. 10 ml of ReactiGelTM 6X was dried under vacuum on a sintered glass funnel and added to the peptide solution; the peptide containing the mixture was then rotated for 48 hours at 4°C. After this time the presence of amine groups on the reacted gel was determined with ninhydrin reagent. Affi-gel 401 Coupling of Peptide With Bis-Maleimide Linker
  • peptide 1005/45 was coupled to Affi-gel 401 affinity resin (BioRad).
  • the functional SH group on the support is designed to form a reducible disulfide bond with SH groups on the ligand.
  • Our intent was to replace the disulfide bond with a "spacer" to reduce steric inhibition of antibody binding, along with minimal restriction of the peptide in solution, enabling the peptide to adopt a favorable tertiary conformation.
  • incorporation of the bis-maleimide linker may facilitate the coupling of the ligand/support and allows dissociation of the antipeptide antibody by pH adjustment, the support can then be be re-used.
  • Incorporation of linker with support and peptide is summarized as follows. Affi-gel 401, containing 5.1 ⁇ M of functional groups per ml of gel was washed extensively in a sintered glass funnel. A 100 ⁇ molar excess of bis-maleimidomethy ether per mole of functional groups on the Affi-gel support was added to the Affi-gel solution in 200 ⁇ l CH 2 CI 2 . The reaction vessel was sealed under argon and allowed to react for two hours at room temperature. The reaction mixture was transferred to a sintered glass funnel and again washed with CH 2 Cl 2 extensively.
  • the derivatized Affi-gel in 5 ml of CH 2 CI 2 was transferred to a reaction vessel to which was added a 10 ⁇ molar excess of reduced peptide to functional groups on the resin.
  • the reaction solution was sealed under argon and allowed to react overnight at room temperature with stirring. Residual unreacted -SH groups were capped by the addition of 100 ⁇ molar excess of N-ethyl-maleimide. Capping was allowed to proceed for 1 hour at room temperature with stirring.
  • the resin/peptide was washed with PBS pH 7.2 and stored at 2-8°C.
  • One of the embodiments of the present invention is the use B138 or a related synthetic peptide coupled to a solid support as a means to isolate antibodies that may be used to generate anti-idiotypic antibodies that may ultimately be used to elicit active anti-HIV immune responses in individuals infected with the AIDS virus or even as a prophylactic vaccine.
  • the procedure for carrying out this method follows.
  • Antibodies are isolated from the sera or plasma of HIV-infected primates (including humans or chimpanzees) or small vertebrate animals such as rodents or rabbits which have been immunized with either the peptide described herein or the gp120 molecule or fragments thereof.
  • An affinity column comprising the HIV envelope-derived peptide coupled to a solid phase matrix or support is prepared according to the procedure described in Example 5.
  • the antibodies are isolated from the serum or plasma of HIV-infected primates (including humans or chimpanzees) or small vertebrate animals such as rodents or rabbits that have been immunized with either the peptides described herein or the gp120 molecule or fragments thereof.
  • an immunoglobulin fraction of the serum or plasma is precipitated in the presence of 50% saturated ammonium sulfate (Hudson L. and Hay, F.C., Practical Immunology, 3rd edition, Blackwell Scientific Publications, Oxford, 1989).
  • the precipitated fraction is washed with ammonium sulfate and redissolved in a convenient volume of a physiologically neutral buffer, such as phosphate buffered saline (PBS), pH7.2, and passed over the column.
  • a physiologically neutral buffer such as phosphate buffered saline (PBS), pH7.2
  • PBS phosphate buffered saline
  • Continuous recycling of the immunoglobulin fraction over the column for an extended period of time (18 hours or overnight) is preferred in order to achieve maximum levels of antibody binding to the peptide. Such a procedure is facilitated by the use of a peristaltic pump.
  • the column is then washed with several volumes of PBS in order to remove passively adsorbed, non -peptide specific antibodies, and serum proteins.
  • the anti-peptide antibodies bound to the column are eluted at low pH, for example by washing the column with 0.1M glycine-HCl buffer, pH 2.5 to 4.0. All column eluates
  • eluting agents include solutions of high salt concentration, e.g., 2M sodium chloride or chaotropic agents such as 6M guanadine
  • the pH of the antibody preparation (referred to as the first plurality of antibodies or Ab1) is neutralized with a basic reagent such as Tris.
  • anti-idiotypic antibodies also termed the second plurality of antibodies or Ab2
  • Ab2 anti-idiotypic antibodies
  • the preferred technique is to immunize mice with the antibody contained in an immunogenic carrier such as Freund's adjuvant according to the standard protocol. After 4-6 weeks, the mice are bled and the serum anti-idiotype titre determined by ELISA. If the concentrations of these antibodies is substantial, the mice are sacrificed, their spleens removed and fused with a suitable cell line, such as SP/2 (Shulman, M., Wilde, CD., Köhler, G., Nature, 276:
  • Selection of candidate internal image-bearing antibodies is performed by assessing the ability of either immunizing peptide or gp120 or both of these molecules to inhibit the binding of Ab2 to Ab1 in an ELISA.
  • Candidate Ab2 beta antibodies are characterized by their ability to induce anti-peptide and anti-HIV responses (including anti-gp120 neutralizing antibodies in another animal species, preferably rabbits).
  • the purified Ab1s described above are inoculated in an immunogenic formulation into rabbits according to the method of Kennedy and Dreesman (Kennedy R.C., et al., J. Virol. Meth., 7:103-15 (1983)).
  • the animals are hyperimmunized with selected antibodies as described above. Initially they are injected intramuscularly with Ab1 emulsified in Freund's complete adjuvant. This is followed by a second injection, approximately one week later, with the antigen in Freund's incomplete adjuvant. Subsequent booster inoculations are made with the antibody precipitated on alum to increase its immunogenicity and to improve the chances of obtaining internal image anti-idiotypes. As many as eight injections of idiotype (approximately 100 ⁇ g per inoculation, given at 7-10 day intervals may be necessary to produce anti-idiotypes) (see Kennedy and Dreesman, supra). After 6-8 weeks and appropriate test bleeds to determine the anti-idiotype titer, the rabbits are
  • anti-idiotype antibodies are isolated by extensive absorption on affinity columns (Sepharose 4B, covalently-linked with human or immunoglobulins, depending on the origin of the Ab1) to remove non-idiotypic specificities.
  • anti-idiotype antibodies will be recognized as antigen-inhibitable by the immunizing idiotype.
  • screen for and determine anti-idiotype activity using the following solid-phase inhibition assay that has been used to study other idiotype-anti-idiotype and viral antigen
  • the antiidiotypes generated according to the present invention recognize true internal images of a HIV epitopes, their ability to recognize crossreactive, interspecies idiotypes is examined. This characteristic is examined using the technique of Kennedy, R.C., et al., Eur. J. Immunol., 13: 232-5 (1983)).
  • the antiidiotype (Ab2) is coated onto wells of a microtiter plate and after the conventional washing and blocking steps, 125 I-labeled idiotype is added to the wells.
  • the source of the idiotype is rabbits and mice hyperimmunized with HIV according to conventional techniques, as well as sera from HIV-infected chimpanzees and humans. They are radiolabeled with 125 I using the iodogen method. Should an Ab2 bind to anti-HIV idiotypes from more than one species there is a strong likelihood that the anti-idiotype bears the internal image of the antigen. Rats are the preferred species for evaluating candidate Ab2 beta antibodies generated by the polyclonal technique.
  • At least 20 animals are needed to test any particular anti-idiotype preparation. Ten are on test and inoculated with anti-idiotype and ten serve as controls and are inoculated with the same quantity of
  • immunoglobulin from the same species as the anti-idiotype comprises at least 50 ⁇ g of immunoglobulin emulsified in adjuvants or adsorbed onto alum as discussed above.
  • Test bleeds are made four weeks after the initial inoculation and at weekly intervals thereafter.
  • the anti-idiotype inoculated animals are screened for the presence of anti-HIV antibodies by the gp120 ELISA and the specificity of the response is measured by immunoblotting or radioimmunoprecipitation. Neutralizing activity (as described previously) is determined against multiple HIV isolates.
  • EXAMPLE 7 GENERATION OF GP120 SURROGATE ANTIBODIES
  • An additional embodiment of the present invention is the use 1005/45 or B138 as an immunogen to elicit antibodies in an immunized animal species that may be purified on gp120 affinity columns and thus provide a source of Ab1s, which in turn be used to generate Ab2 antiidiotype antibodies, which are candidates for vaccines or
  • immunogenicity In preferred formulations, various combinations of carrier (specific- and glutaraldehyde cross-linked KLH) and adjuvant (Freund's complete and incomplete, as well as muramyl dipeptides, such as SAF) are utilized.
  • An affinity column comprising the gp120 portion of the HIV envelope is coupled to a solid phase matrix according to the method described in Example 5.
  • Antibodies are isolated from the serum or plasma of small vertebrate animals such as rodents or rabbits which have been
  • the antigp120 antibodies are isolated in essentially the same manner described in Example 6.
  • both monoclonal or polyclonal anti-idiotypic antibodies are prepared by the methods described in Example 6.

Abstract

Nouveaux peptides synthétiques et leurs analogues, représentant un épitope neutralisant dans la glycoprotéine d'enveloppe extérieure (gp120) du virus de l'immunodéficience humaine (VIH), laquelle est prétendument impliquée dans la liaison de cette molécule à son récepteur cellulaire (CD4). L'invention concerne également des procédés de production d'anticorps réagissant avec l'image intérieure de l'épitope neutralisant ou contenant ladite image. On peut utiliser les peptides ou anticorps dans des immunotitrages de diagnostic, en tant que vaccins ou en immunothérapie.
EP90915005A 1989-09-22 1990-09-21 Nouveaux peptides associes a la region de liaison cd4 de la gp120 et leur mode d'emploi Withdrawn EP0493508A1 (fr)

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CA2047033A1 (fr) * 1990-07-19 1992-01-20 Elizabeth E. Sugg Peptides cycliques neutralisant le vih
CA2047078A1 (fr) * 1990-07-19 1992-01-20 Steven S. Bondy Peptides cycliques neutralisant le vih
CA2047042A1 (fr) * 1990-07-19 1992-01-20 John Hannah Peptides cycliques neutralisant le vih
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JPH05262662A (ja) * 1991-07-15 1993-10-12 Calpis Food Ind Co Ltd:The Ｈｉｖ重感染防止剤、抗イディオタイプ抗体作製用抗原及び抗イディオタイプ抗体の製造法
AU679439B2 (en) * 1992-02-10 1997-07-03 Duke University Use of synthetic peptides to induce tolerance to pathogenic T and B cell epitopes of autoantigens
US5556744A (en) * 1992-05-29 1996-09-17 The Trustees Of The University Of Pennsylvania Methods and compositions for diagnosing and treating certain HIV infected patients
WO1994028929A1 (fr) 1993-06-07 1994-12-22 Genentech, Inc. Polypeptides d'enveloppe du vih
ATE177843T1 (de) * 1994-01-19 1999-04-15 Us Health Peptomere mit erhöhter immunogenizität
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US5691447A (en) * 1995-03-24 1997-11-25 Tanox Biosystems, Inc. GC1q receptor, HIV-1 gp120 region binding thereto, and related peptides and targeting antibodies
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