WO1998036087A1 - Tolerance immunologique aux epitopes du hiv - Google Patents

Tolerance immunologique aux epitopes du hiv Download PDF

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Publication number
WO1998036087A1
WO1998036087A1 PCT/US1998/002766 US9802766W WO9836087A1 WO 1998036087 A1 WO1998036087 A1 WO 1998036087A1 US 9802766 W US9802766 W US 9802766W WO 9836087 A1 WO9836087 A1 WO 9836087A1
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seq
cells
epitopes
cell
subject
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PCT/US1998/002766
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English (en)
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David Scott
Elias Zambidis
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American National Red Cross
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Priority to CA002279492A priority Critical patent/CA2279492A1/fr
Priority to EP98908538A priority patent/EP0973933A1/fr
Publication of WO1998036087A1 publication Critical patent/WO1998036087A1/fr

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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G01N2333/162—HIV-1, HIV-2 env, e.g. gp160, gp110/120, gp41, V3, peptid T, DC4-Binding site

Definitions

  • the invention in the fields of immunology, molecular biology and medicine relates to compositions, primarily fusion immunoglobulins, and methods useful for inducing a state of immunological tolerance to selected epitopes of human immunodeficiency virus (HIV) gpl20 or target epitopes assocaited with other diseases.
  • Administration of these composition will induce and maintain tolerance to the epitopes in a subject infected with (or at high risk for) HIV, or in whom an immune response to a different target epitope is deleterious.
  • Prevention of antibody responses to the selected HIV epitopes promotes survival of the host immune system and contributes to treatment of HIV disease.
  • the compositions are also useful as adjuncts to HIV or other virus vaccines in modulating the immune response to maximize induction of protective anti-viral T cell immunity.
  • Immunological tolerance (hereinafter "tolerance"), the basis of the lack of reactivity of the immune system to self components, can also be induced artificially by a wide variety of manipulations. Hence, an animals can be rendered tolerant to antigens which are foreign. Autoimmunity is thought to result in part from the breakdown of tolerance to previously tolerated antigens. A variety of experimental procedures are known for inducing antigen-specific tolerance in neonates and adults (Billingham, R.E. et al. (1953) Nature 172:603-606; Chiller, J.M. et al. (1970) Proc. Natl. Acad. Sci. USA. 55:551-556; Borel, Y. et al.
  • autoimmune diseases For autoimmune diseases, studies have focused on the acquired induction of tolerance to autoantigens to prevent and/or ameliorate disease. For example, in murine models of multiple sclerosis or diabetes, prevention of disease has been accomplished with intrathymic, oral, or intravenous administration of , high doses of target autoantigens (Tisch, R. et al. (1993) Nature 366:12-15; Higgins, P.J. et al. (1988) J.Immunol.
  • Ig immunoglobulin
  • Tolerogenic carriers or “tolerogens”
  • Igs of different origin may vary in their persistence in an animal after administration and/or in the mechanism by which they induce tolerance.
  • IgG carriers have been by far the most efficacious inducers in adult animals of tolerance to haptens, nucleosides and peptides (Borel, Y. (1980) Immunol. Rev. 50:11; Scott, D.W.
  • heterologous oligopeptide epitopes of immunological interest have been inserted in-frame into bacterial flagellin (Newton, S. et al, (1989) Science 244:10-12; Jennings et al, (1989) Protein Eng. 2:365), influenza virus nucleoprotein (Chimini, G. et al. (1989) J. Exp. Med. 169:91-302), hepatitis B surface antigen (Rutgers et al, (1988) Bio/Technology 5:1065) and in the complementarity determining regions (CDR) of immunoglobulins (Billetta, R.
  • a peptide immunoglobulin fusion Ig protein or referred to herein as a "fusion Ig” or "fig” has been used to induce immunity.
  • a fig was made which expressed in the CDR3 of its V H region the repetitive tetrapeptide Asn-Ala-Asn-Pro (SEQ ID NO:l), designated (NANP) n (in single letter amino acid code), of the circumsporozoite protein of Plasmodium falciparum, an etiologic agent of malaria (Billetta et al., supra).
  • a monoclonal antibody (mAb) specific for (NANP) n which was made against P. falciparum bound to the above fig and was blocked by a synthetic (NANP) 3 peptide.
  • Such antibodies efficiently inhibited the invasion of cultured liver cells by P. falciparum.
  • immunity to malaria was induced in the absence of the parasite using antibody V regions engineered to mimic the parasite's molecular structure.
  • the authors suggested that antibody (idiotype) mimicry of an exogenous antigen is possible and may only require a discrete stretch of identity for successful mimicry.
  • T cell-targeted peptides in the form of fig molecules produced by cells transfected with chimeric V genes, activated specific T cells.
  • Zanetti et al. (supra) and Bona et al. (supra) produced chimeric Ig molecules (which are figs as the term is used herein) for the purpose of immunization (vaccination), not tolerization.
  • WO90/09084 casually preferred a speculative notion, lacking any particularity or evidence, that this type of construct could be used for tolerization, the authors provided no scientific basis for such a utility. In fact, the way in which their exogenous epitope was inserted into the Ig framework region resulted only in immunogenic, not tolerogenic, constructs.
  • recombinant fusion proteins including fig proteins
  • fig proteins may be useful as immunogens to induce immune responses to the heterologous oligopeptide.
  • vectors that can introduce the target epitope to which tolerance is desired into a host cell or whole animal, such that the epitope (a) induces tolerance and (b) persists in vivo so that it maintains the tolerant state. It is essential that any tolerization protocol include a means to maintain the specific state of tolerance.
  • the present inventors were the first to discover an approach that not only could induce tolerance to an oligopeptide presented to the immune system in the form of a recombinant fig protein but also could maintain a tolerant state in the subject.
  • the Immune Response to HIV gp!20 and its Role in AIDS The immune response to HIV has been studied extensively. Early studies suggested a role for neutralizing antibodies in protection or containment of HIV infection. This is particularly true in the case of simian immunodeficiency virus (SIV), a relative of HIV, where a cloned virus could be employed (Burns, D. et al.
  • SIV simian immunodeficiency virus
  • the CD4 molecule on T lymphocyte serves as receptor for major histocompatibility complex (MHC) class II antigens and is referred to as "coreceptor” because its engagement synergizes with engagement of the T cell receptor for antigen (TCR) in activating the cells.
  • MHC major histocompatibility complex
  • TCR T cell receptor for antigen
  • CD4 molecules were engaged by antibody independently of the TCR (in murine studies), the T cells were induced to undergo apoptosis (Wang, Z.Q. et al. (1994) Eur. J. Immunol. 24:1549-1552).
  • CD4 has a function of its own in facilitating the induction of apoptosis.
  • CD4 also serves as a cellular binding site or receptor for the HIV gpl20.
  • transgenic mice expressing a human CD4 transgene appropriate crosslinking of gpl20 caused massive deletion of HIV-reactive T cells in vivo (Wang, Z.Q. et al. (1994) Europ. J. Immunol. 24:1553-1551). If T cells in which CD4 is engaged by anti-CD4 antibody administration are capable of expressing functional Fas protein on their surface, they degrade their DNA and disintegrate rapidly.
  • Antibodies to gpl20 can lead to enhancement of HIV entry into non-T cells via Fc receptors (Homsy, J. et al. (1989) Science 244:1357, supra). Uptake of complexes between HIV and anti gpl20 antibody by cells of the immune system, particularly monocytes, can result in establishment of a latent, subclinical infection and a virus reservoir susceptible to later activation(Kliks, S.C., (1993) Proc. Natl. Acad. Sci. USA 90:11518)). HIV-infected patient sera frequently contain antibodies against a peptide of the gpl20 C5 region which cross-react with HLA-C monomo ⁇ hic determinants (DeSantis, C. et al. (1993) J. Infec. Dis. 755:1396; Palker,
  • the antibodies are an example of non-protective antibodies produced during HIV disease.
  • an antibody response to a variant virus may end in more extensive disease (Cook, R. et al. (1995) J. Virology 6 :1493).
  • production of non- neutralizing anti-HIV antibodies may pre-empt the formation of antibodies to important, neutralizing epitopes.
  • T cell surface may prime T cells for apoptosis, perhaps via the upregulation of the Fas molecule, CD95 (Oyaizu, N. et al. (1994) Blood 84:2622; Desbarats, J. et al. (1996) Proc. Natl. Acad. Sci. USA 95:11014-11018. Even picomolar concentrations of gpl20 could prime T cells for such activation-induced death.
  • Apoptosis in normal, non-infected (“bystander") CD4 + T cells may be programmed by (1) allowing gpl20 proteins to bind to CD4 via their natural affinity, and then (2) adding anti-gpl20 antibodies to bind and crosslink the gpl20-CD4 complexes (Finkel et al, supra; Banda et al, supra).
  • gpl20 proteins to bind to CD4 via their natural affinity
  • anti-gpl20 antibodies to bind and crosslink the gpl20-CD4 complexes
  • cross-linking of CD4 molecules was sufficient to induce apoptosis in CD4+ T cells if cross-linking was performed in unfractionated blood mononuclear cells (but not in purified T cells). The accelerated cell death through apoptosis was concluded to play an important role in the pathogenesis of HIV- 1 infection, and crosslinking of CD4 in vivo contributed to this mechanism.
  • Cross-linking of CD4 molecules induced either by anti-CD4 monoclonal antibody (mAb) or by HIN-1 envelope protein gpl60 (which includes gpl20) upregulates Fas mR ⁇ A and Fas antigen expression in normal lymphocytes (Oyaizu et al. (1994) surpra).
  • the present inventors have concluded that the antibody response to gp 120 in an infected subject is an important pathway leading to AIDS progression due to the pathogenetic component of CD4 + T cell depletion through bystander apoptosis as described above. Therefore, they have developed novel compositions and methods based on their general, flexible approach to the induction and maintenance of epitope-specific tolerance to eliminate virus-specific immune responsiveness.
  • T helper cell and/or antibody responsiveness to one or more epitopes of viral gpl20 is prevented or inhibited through the induction and maintenance of immune tolerance in T helper cells, B cells or both that are specific for one or a number of selected HIV gpl20 epitopes.
  • the present inventors have extended this approach to the induction of tolerance to any antigen, be it an autoantigen, an antigen of a microorganism or a tumor antigen, against which an undesired antibody response or T helper cell response occurs in a disease setting and is pathogenic or otherwise deleterious to the host.
  • any antigen be it an autoantigen, an antigen of a microorganism or a tumor antigen, against which an undesired antibody response or T helper cell response occurs in a disease setting and is pathogenic or otherwise deleterious to the host.
  • the present inventors have devised novel fusion proteins and DNA constructs coding therefor.
  • the fusion protein includes a desired peptide epitope or several epitopes, toward which immune tolerance is to be established, inserted in particular sites of the immunoglobulin ("Ig") heavy (“H") chain.
  • Ig immunoglobulin
  • H immunoglobulin
  • This product is termed a "fusion immunoglobulin” and is abbreviated "fig” herein.
  • a preferred fig includes an epitope or epitopes of HIV- 1, most preferably from the gpl20 glycoprotein of HIV- 1.
  • DNA encoding the targeted epitope or epitopes is inserted "in frame" within a DNA construct encoding the Ig heavy (H) chain. Iftwo or more targeted epitopes are included, they exist as contiguous or non-contiguous sequences in the protein from which they are derived, and may be either linear or conformational epitopes.
  • This fusion protein construct is then transfected into a cell line, preferably a myeloma or other line of B lymphocyte lineage (such as a human cell line transformed by Epstein-Barr virus) that produces Ig light (L) chains but that cannot produce H chains due either to a spontaneous or induced mutation.
  • a cell line preferably a myeloma or other line of B lymphocyte lineage (such as a human cell line transformed by Epstein-Barr virus) that produces Ig light (L) chains but that cannot produce H chains due either to a spontaneous or induced mutation.
  • the transfected Ig H chains are synthesized, they combine naturally with the host cell's Ig L chains to form complete immunoglobulin molecules (H 2 L 2 ) which are secreted.
  • This resultant Ig fusion protein contains the desired target epitope (or epitopes) preferably in its N- terminal region and functions as a tolerogen for both B cells and T cells and induces tolerance in vivo.
  • Transgenic mice producing such a fusion protein are highly tolerant immunologically to the epitopes included in the fig.
  • the present inventors have found that Ig fusion proteins such as these can be presented to the immune system in a tolerogenic fashion, either as an fig preparation or in the form of transgenic hemopoietic precursor cells or B cells expressing the fig, to induce both B and T cell tolerance to the targeted HIV-1 gpl20 epitope..
  • the present inventors have conceived of an approach that is useful in producing improved and effective immunity against a virus, in particular, human immunodeficiency virus (HIV-1, HIV-2) by inducing tolerance to selected nonprotective viral epitopes as discussed above.
  • a virus in particular, human immunodeficiency virus (HIV-1, HIV-2)
  • HIV-1 human immunodeficiency virus
  • the present inventors have created a fig comprising one or more peptide epitopes and the Ig H chain using recombinant methods as described herein.
  • the invention specifically involves:
  • the invention provides polynucleotides encoding the fig in the form of recombinant DNA molecules in vehicles such as plasmid and retroviral vectors, capable of expression in a desired eukaryotic host cell as disclosed herein.
  • the invention also provides hosts transfected or transduced with the fig constructs which are capable of producing in culture or in vivo the fig molecules and secreting them or displaying them on the cell surface.
  • This invention is useful for the treatment of any disease in which immunologic reactions are pathologic.
  • infectious and autoimmune diseases In many types of infections, where the host response to the organism damages the host.
  • the T cell response is responsible for as much or more pathology than the virus itself.
  • Antibody responses and the interaction of the antibodies with complement is responsible for the hemorrhagic shock syndrome elicited by flaviviruses (in particular) dengue virus or arenaviruses, such as Junin virus which causes Argentinean hemorrhagic fever. In both the latter cases, an efficient immune response leads to disaster for the host.
  • diseases for which the present invention can be used include viral diseases wherein virus- antibody complexes damage the host.
  • infants congenitally infected with cytomegalovirus have such circulating complexes that are deposited in the kidney.
  • Patients with hepatitis B virus infection have circulating complexes that result in arthritis and glomerulonephritis.
  • Antibodies generated against a virus can also act as autoantibodies directed against normal tissues, even tissues not infected directly by the virus.
  • An example of this is the polyendocrinopathies that develop in newborn animals infected with reovirus type 1 in whom antibodies against antigens in pancreatic islets, the anterior pituitary and the gastric mucosa have been observed.
  • Such examples abound in the art and may be found in any comprehensive treatise on microbiology or infectious diseases.
  • More classical autoimmune diseases are also treatable by the present invention as either cell-mediated or antibody responses to organ-specific antigens or common or cross-reactive antigens are the known pathogenic agents.
  • Specific tolerance induced by an fig of this invention is a promising therapeutic approach to the treatment of many types of autoimmune disease.
  • the invention provides an individual fig H chain or fig H chain dimers.
  • an fig molecule comprising (i) two different H chains, one of which is a fusion protein having one or more HIV gpl20 epitopes included in the V region, preferably at the N-terminus of a framework region, most preferably of the first framework region, and (ii) native L chains.
  • both H chains of the fig molecule are the fused H chains.
  • the present invention is directed to a fusion immunoglobulin (fig) heavy (H) chain protein comprising a mammalian, preferably human, Ig H chain fused in frame after the leader in its N-terminal region to one or more HIV gpl20 epitopes, wherein the fig H chain is tolerogenic in a host with respect to the gpl20 epitopes.
  • the tolerogenic epitope(s) is or are fused to the variable region of the Ig H chain, preferably at the N terminus of a framework region of the variable region.
  • HIV gpl20 epitope or epitopes are fused to the N-terminal amino acid residue of the mammalian Ig H chain such that all amino acids encoding the gpl20 epitope or epitopes are N-terminal to the Ig-encoding amino acids.
  • an intact fig protein comprising two Ig H chains and two Ig L chains, wherein at least one of the H chains is the fig H chain described above.
  • both of the H chains are the above fig H chains.
  • a preferred Ig is one which fixes complement and has a longer serum half life.
  • the fig H chain is an Ig ⁇ chain, more preferably an Ig ⁇ , ⁇ 2 or ⁇ 3 chain.
  • the Ig is human IgG and preferred fig iso types are IgG, IgG 2 and
  • the one or more gpl20 epitopes comprises a full length gpl20 protein
  • the gpl20 epitopes are one or more peptides selected from the group consisting of the Cl region, the V3 loop and the C5 region.
  • the gpl20 epitope is a B cell epitope comprising a peptide selected from the group consisting of: VPVWKEATTTLFCASDAKAY (SEQ ID NO:2), EVHNVWATHACVPTD (SEQ ID NO:3), YDTEVHNVWA (SEQ ID NO:4), PQEWLVNVT (SEQ ID NO:5), PQEVVLVNVTENFDMWKNDM (SEQ ID NO:6), PNNNTRKSIR (SEQ ID NO:7), NNNTRKRIRIQRGPGR (SEQ ID NO:8), RKSIR (SEQ ID NO:9), IQRGPGRAFV (SEQ ID NO:2), VPVWKEATTTLFCASDAKAY (SEQ ID NO:2), EVHNVWATHACVPTD (SEQ ID NO:3), YDTEVHNVWA (SEQ ID NO:4), PQEWLVNVT (SEQ ID NO:5), PQEVVLVNVTENFDMWKND
  • PNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHC SEQ ID NO: 14
  • NNTRKSIRIQRG SEQ ID NO: 15
  • NKRKRIHIGPGRAFYTTKNIIGTIC SEQ ID NO: 16
  • RKSIRIQRGPGRAFV SEQ ID NO: 17
  • IRIQRGPGR SEQ ID NO: 18
  • KRIRIQRGPGRAFVTIG (SEQ ID NO: 19), QRGPGRAF (SEQ ID NO:20), RGPGRAFV (SEQ ID NO:21), RKRIHIGPGRAFYTT (SEQ ID NO:22), RGPGRAFVTIG (SEQ ID NO:23), SISGPGRAFYTG (SEQ ID NO:24), KRIHI (SEQ ID NO:25), KRIHIGP (SEQ ID NO:26), IHIGPGR (SEQ ID NO:27), HIGPGR (SEQ ID NO:28), HIGPGRA (SEQ ID NO:29), HIGP (SEQ ID NO:30), RIHIGPGRAFYTTG (SEQ ID NO:31), RIQRGPGRAF
  • the gpl20 epitope is a T helper cell epitope comprising a peptide selected from the group consisting of: EQLWVTVYYGVPV (SEQ ID NO:53), VYYGVPVWKEA (SEQ ID NO:54), GVPVWKEATTLFC (SEQ ID NO:55), AHKVWATHACV (SEQ ID NO:56), NVWATHACVPTD (SEQ ID NO:57), CVPTNPVPQEW (SEQ ID NO:58), VEQMHEDIISLW (SEQ ID NO:59), EQMHEDIISLWDQ (SEQ ID NO:60), EQMHEDIISLWDQSL (SEQ ID NO:61), HEDIISLWDQSLK (SEQ ID NO:62), VTVYYGVPVWKEATTTLFC (SEQ ID NO:63), WLVNVTENFNM (SEQ ID NO:64),
  • SLKPCVKLTPLCY (SEQ ID NO:65), CTRPNNNTRKSIRIQRGPG(Y) (SEQ ID NO:66), NTRKSIRIQRGPGR (SEQ ID NO:67), EQRGPGRAFVTIGKI (SEQ ID NO:68), RIQRGPGRAFVTIGK (SEQ ID NO:69), RIHIGPGRAFYTTKN (SEQ ID NO:70), GRAFVTIGKIGNMRQ (SEQ ID NO:71), QRGPGRAFVTIGKIGNMRQAH (SEQ ID NO:72), VGKAMYAPPISGQIR (SEQ ID NO:73), GNSNNESEIFRPGGG (SEQ ID NO:74),
  • the present invention is further directed to a DNA molecule comprising a nucleotide sequence encoding any fusion Ig H chain as described above.
  • an expression vector useful for producing the above fusion Ig product and for inducing and maintaining immunological tolerance to one or more epitopes of HIV gp 120 protein in a subject preferably a human.
  • the vector preferably comprises (a) a DNA molecule as above, operably linked to (b) transcriptional and translational control regions operable in a hematopoietic cell or lymphoid cell of the subject.
  • the transcriptional and translational control regions provide for constitutive expression of the DNA sequence in a lymphoid cell or a hematopoietic cell.
  • a preferred vector is a retroviral vector.
  • a naked DNA vector may also be used.
  • the present invention also provides a hemopoietic or lymphoid cell transformed by a vector as above, which cell stably expresses the fig protein.
  • Stable expression is expression which is not transient, and persists for weeks or even months, preferably for the in vivo lifespan of the cell in which the fig is expressed.
  • a cell is preferably a human bone marrow cell, a resting B lymphocyte or an activated B lymphocyte which has been activated by a mitogen or other polyclonal B cell activator.
  • a method for producing the fusion Ig of the invetnion by culturing the above transformed cell.
  • any cell type may be used which can express an Ig H chain gene as well as the DNA encoding the fig and secrete it into the culture medium.
  • the present invention includes a pharmaceutical composition comprising:
  • the fig is an isologous IgG molecule.
  • a method for immunologically tolerizing a subject to one or more HIV gpl20 epitopes comprising administering to the subject an effective amount of a fusion Ig pharmaceutical composition as described above.
  • a method for immunologically tolerizing a subject to one or more HIV g ⁇ l20 epitopes comprising introducing into the subject an effective amount of transformed cells as described above, thereby tolerizing the subject.
  • the invention is directed to a method for immunologically tolerizing a subject to one or more HIV gpl20 epitopes comprising introducing into the subject an effective amount of transformed cells as above, thereby tolerizing the subject.
  • the subject Prior to introducing the transformed hemopoietic cells into the subject, the subject may be treated to diminish the host's hemopoietic cells, although this may not be necessary in a patient with AIDS. Tolerance may also be achieved by a combination of treatment with transformed cells and a pharmaceutical composition comprising fig as described above.
  • This invention is also directed to a method of (i) inducing and (ii) maintaining immunological tolerance to an epitope or epitopes of HIV gpl20 protein in a subject, comprising:
  • the subjects are preferably humans and the transformed cells are human cells.
  • Figure 1 shows the amino acid sequence of HIV- 1 gpl20 (mature protein).
  • the boldface sequence, SEQ ID NO:83, (with position numbers in the right margin) is the consensus sequence of the protein from subtype B virus (the prevalent HJN-1 subtype in the United States).
  • the following characters are used in the consensus sequence: ( 1 ) single letter code; UPPER CASE letter indicates that the amino acid residue in that position is conserved for all known viral isolates of subtype B;
  • the concensus sequence is read left to right. Shown vertically below each position in the consensus sequence (where appropriate) are alternative amino residues that have been identified at that position in mutants or variants of subtype B. Residues which happen to be adjacent to one another below the consensus sequence line are NOT to be read left to right as they do NOT represent adjacent residues in an actual gpl20 sequence. (Note: all the variant residues below the consensus sequence line are UPPER CASE for clarity only.) All of the sequence information in Fig. 1 (and Fig. 2) was obtained from The Human Retroviruses and AIDS Genetic Sequence 1995 Compendium, published by the Los Alamos National Laboratory: Theoretical Biology and Biophysics Division, Los Alamos, NM.
  • Figure 2 shows the aligned consensus sequences for the major subtypes or "clades" of HIV- 1 as published in 77 e Human Retroviruses and AIDS Genetic Sequence 1995 Compendium (see Figure 1). HIV subtypes are defined and distinguished based on their nucleotide (and not amino acid) sequences. Certain "signature sequences" are characteristic of a subtype, for example, the GPGR consensus sequence at the tip of the V3 loop of the subtype B that appears as a GPGQ consensus for most other subtypes. The letter/symbol conventions are the same as those used in the consensus sequence in Figure 1.
  • the mature protein begins to the right of the "/" Other landmarks indicated include the V3 neutralization loop, the C terminus of gpl20 (indicated by a "/" on sheet 2/4) and the N-terminal segment (about 16 residues) of the HIV-1 gp41 protein.
  • Figure 3 shows the aligned amino acid sequences of gpl20 (including the signal sequence) from several strains or isolates of HJN-1.
  • the top line of each grouping is the subtype B consensus sequence (SEQ ID NO: 105; also appearing in Figures 1 and 2).
  • the footnotes describing each variant or isolate and the markings used in Figure 3 are as follows:
  • the first 27 - 30 amino acids left of the "//" mark comprise the signal sequence of gpl20.
  • the mature gp 120 protein begins to the right of the "//”.
  • a space appears after each 10 residues. To preserve alignment, spaces have sometimes been omitted and for the consensus sequence, additional residues have been placed above the main sequence line.
  • CON-B is the consensus sequence for gpl20 of subtype B (SEQ ID NO:105). UPPER or lower case letters are as described for Figs. 1 and 2. The presence of single letter amino acid codes or "?” above the consensus sequence line indicates the existence of additional residues in some subtype B isolates at approximately the positions indicated. In some locations, arrows appear in the sequence line as place indicators for such additional residues. Each arrow is not intended to correspond to a single residue and points to the known residues (usually "?") that may occupy that region in various isolates.. (3) BH10 isolate (SEQ ID NO: 106): Ratner, L. et al. Nature 3/3:277-284(1985) (Genbank
  • LAV-BRU isolate (SEQ ID NO:107): Wain-Hobson, S. et al, Cell 40:9-17(1985) (Genbank SWISS PROT Accession No. P03377 )
  • ARV2/SF2 isolate SEQ ID NO: 108: Sanchez-Pescador, R., et al. Science 227:484- 492(1985) (Genbank SWISS PROT Accession No. P03378)
  • MN isolate SEQ ID NO:109: Gurgo, C. et al, Virology 7 4:531-536(1988) (Genbank SWISS PROT Accession No. P05877)
  • 92US712.4 isolate SEQ ID NO:l 10
  • This sample was part of a set of sequences generated through the NIAID/NIH DAIDS HIV variation program.
  • the virus was derived from an asymptomatic individual from Baltimore, thought to be infected by parenteral i.v. drug user contact.
  • the env sequence clustered with HIV-1 B subtype sequences. Gao, F. et al., J. Virol. 70:1651-1667 (1996) (Genbank SWISS PROT Accession Number U08449). This sequence was randomly chosen as a subtype B isolate for illustrative purposes and for comparison with the more common variant sequences.
  • Figures 4A and 4B illustrate a preferred engineering strategy for inserting a foreign epitope at the N-terminus of an IgG ⁇ chain.
  • Figure 4A depicts the inco ⁇ oration of an oligonucleotide (SEQ ID NO:l 11) encoding the ⁇ phage Cl repressor peptide 12-26 (SEQ ID NO: 112) as described in Examples. This fig was expressed in murine J558 myeloma cells.
  • the present invention introduces an oligonucleotide or polynucleotide encoding one or more native or synthetic gpl20 peptide epitopes into an Ig H chain, preferably a human ⁇ chain ( Figure 4B).
  • FIGS. 5 A and 5B show strategy for the construction, expression, and epitope recognition of a fusion Ig gene by inserting a foreign epitope into a V H gene.
  • Fig. 5 A presents a scheme for constructing the fig.
  • a modified 12-26 nucleotide sequence was ligated into a Pstl site of a 1.3-kb murine V H (LVDJ) chain fragment.
  • the Pstl site appears at the coding sequence of the fifth amino acid of the FRl; therefore, a repeat of the first five FRl amino acids was designed to follow the coding sequence of the 15 amino acids of 12-26, so as not to perturb proper framework region folding after insertion.
  • Fig. 5B is a gel pattern showing recognition of epitopes by immunoblotting.
  • Purified control IgG (P6) of 12-26-IgG (Q3) samples were electrophoresed on SDS/10% polyacrylamide gels, transferred onto nitrocellulose, and probed with antimouse IgGl (left lanes) or with biotinylated anti- 12-26 mAb B3.11 (right lanes) plus AP-conjugated secondary reagents.
  • FIG. 6 shows in vivo effects of 12-26-IgG pretreatment on peptide-specific humoral immune responses.
  • Male BALB/c mice were injected i.v. with a single 1-mg dose of deaggregated protein G-purified P6 ( ⁇ ) or Q3 ( / ) IgG.
  • Mice were immunized and total or isotypic anti-peptide IgG titers were quantitated by ELISA 8 days after a secondary antigenic boost.
  • Isotyped anti-peptide titrations (IgG, and IgG 2b ) represent mean abso ⁇ tion values from assays of three individual mice in each group. 0 , Preimmunization sera
  • FIG 7 shows in vivo effects of 12-26-IgG pretreatment on peptide-specific cellular immune responses.
  • Tertiary cytokine (IL-2 and IL-4) responses of enriched splenic T cells (3 x 10 6 cells per ml) from mice displaying tolerized humoral immune secondary responses are shown.
  • IL-2 and IL-4 production in supernatants was determined in triplicate by CTLL and CT.4S assay, respectively. "Medium only" backgrounds were subtracted; these values ranged from 1 to 4 units/ml in all assays.
  • Figure 8 shows structure and genomic Southern blotting of transgenic mice expressing 12-26-IgG specifically in the B-lymphocyte lineage.
  • a murine IgG, b H chain construct containing endogenous immunoglobulin promoter and enhancer (E H ) sequences was modified to express 12-26 peptide and a repeat of perturbed framework region sequence (FRl) at the N-terminus. Fertilized embryos were injected with this linearized construct and transgenic mice were generated via standard procedures. Genomic DNA from tail biopsies was digested with BamYil and EcoRI to release a 1.3 kb V H fragment, fractionated on 0.8% agarose/TB ⁇ , and transferred onto nylon membranes via alkaline Southern transfer . Southern blots were probed with random- primed 32 P-labeled DNA sequence containing 3 tandem repeats of 12-26 nucleotide sequence.
  • E H endogenous immunoglobulin promoter and enhancer
  • FIG. 9 A presents titers of total anti-peptide IgG (open symbols), or IgG, isotype (closed symbols) for Line 5 transgenic mice measured after peptide immunizations and secondary boosts.
  • Fig. 9B presents splenic T cell cytokine responses from tolerant Line 5 trangenic (Tg) mice determined by CTLL assay. Error bars signify the standard error of the mean for 3-4 mice per group.
  • Figure 10 shows profound peptide-specific cellular and humoral immune tolerance in transgenic bone marrow chimeras expressing 12-26-IgG. Chimeras were prepared with 1:1 mixtures of Line 17 Tg and non- transgenic (NTg) bone marrow (/).
  • Antibody responses to peptide are shown. Anti-HEL specificity controls showed no differences between groups. Nonirradiated mice injected with saline (0) displayed immune responses similar to control chimeras reconstituted with 100% NTg bone marrow (0). Error bars signify standard error of the mean of 2-3 mice per group.
  • Figure 11 shows the induction of peptide-specific humoral immune tolerance in normal immunocompetent adults by intravenous injection of various preparations of 12-26-IgG-expressing lymphoid tissues.
  • Normal, nonirradiated BALB/c males were injected iv with 4xl0 7 sex-matched splenocytes, Percoll ® -gradient-purified (60- 10% fraction) resting B cells, 48-hour activated LPS blasts, or crude unfractionated bone marrow cells from Line 17 transgenic mice.
  • Recipients were rested for 7-10 days before immunization with 50 ⁇ g peptide in CFA (SC base of tail). Mice were boosted with an additional 50 ⁇ g in saline 2 weeks later and serum antibody titers determined 8 days later.
  • Figures 12A and 12B present an analysis of B-cell tolerance induction in tolerized transgenic or normal adult subjects.
  • Fig. 12A Nontransgenic ( 0 ), Line 5 transgenic ( V ), or line 17 transgenic ( / ) mice were immunized intraperitoneally with 50 ⁇ g 12-26-HEL conjugate in CFA, and boosted with the same in saline 2 weeks later.
  • Anti-peptide and anti-HEL (all >10 5 , not shown) titers were determined by ELISA as described in the text.
  • Fig. 12A Nontransgenic ( 0 ), Line 5 transgenic ( V ), or line 17 transgenic ( / ) mice were immunized intraperitoneally with 50 ⁇ g 12-26-HEL conjugate in CFA, and boosted with the same in saline 2 weeks later.
  • Anti-peptide and anti-HEL (all >10 5 , not shown) titers were determined by ELISA as described in the text.
  • Serum titers from adoptively transferred recipients boosted with 50 ⁇ g 12-26-HEL conjugate in IFA were similarly determined: BALB/c recipients were irradiated with 400 rads, and injected with 5 x 10 7 splenocytes from Line 17 Tg-tolerized donors (closed circles, various sources of lymphoid tissue) or non-transgenic injected, non-tolerized donors (open diamonds). Splenic donors had been previously primed and boosted with 12-26 peptide and HEL (at different subcutaneous locations), and had previously displayed tolerance (experiment from Figure 11).
  • FIGS 13A. 13B,and 13C summarize studies showing the induction of tolerance in previously-primed adult recipients by either resting, B cells, B cell blasts or chemically fixed B cells.
  • BALB/c mice were immunized SC with 50 ⁇ g 12-26 peptide in CFA 1-2 weeks before iv injection of 4x107 Line 17 transgenic (! or nontransgenic control ( 0 ) purified resting B cells (Fig. 13 A), LPS-activated B cell blasts (Fig. 13B), or carbodiimide- fixed B cells (Fig. 13C).
  • the mice were challenged IP with 50 ⁇ g soluble peptide 1-2 weeks following tolerizing injections, and antibody titers (ELIS A) determined 8 days later.
  • the graphs show peptide-specific total IgG or two IgG isotypes (IgG, and IgG 2b ),
  • Figures 14 and 15 show B cell expression, epitope recognition, and direct antigenic presentation of retro virally-synthesized peptide-IgG.
  • Fig. 14 shows the structure and proviral integration of murine Moloney leukemia retroviral construct MBAE.BAK.
  • Fig. 15 shows tissue expression of 12-26 mRNA in long-term ( ⁇ 3 months) recipients of gene- transferred ( + ) or mock-transduced ( — ) BM progenitors.
  • Figure 16 shows the induction of peptide-specific cellular immune tolerance in adult bone marrow chimeras infused with peptide-Ig-expressing progenitor cells.
  • mice were sublethally irradiated (600 rads) and injected iv with 1-2x10 ⁇ gene-modified or mock-transduced BM.
  • Recipients were immunized with peptide in CFA 2 months post-infusion and draining LN cells were restimulated in vitro with dilutions of synthetic peptide and 25-50 ⁇ g/ml purified protein derivative (PPD, Connaught) in RPMI 1640 with 0.5% heat-inactivated autologous serum.
  • Stimulation indices represent ratios of proliferation to medium alone backgrounds (5,609+2,271 cpm).
  • IL-2 and IFN- ⁇ were quantitated by CTLL and ELIS A assays, respectively (Gilbert, K.M., et al. (1994) J. Exp. Med. 779:249-258). Additional experiments also revealed a diminution of peptide-specific IL-4 release in LN of tolerized recipients. Error bars signify standard error of the mean for 3 individual mice per group. This experiment was done at least twice with 3-4 mice per group with similar results.
  • Figures 17 A, 17B and 17C show the induction of peptide-specific humoral immune tolerance in adult bone marrow chimeras infused with peptide-Ig-expressing progenitor cells.
  • BALB/c mice were sublethally irradiated with either (A) 200 rads
  • Fig. 17A or 600 rads (Fig. 17B,C) and infused with 1-2x10 ⁇ gene-transferred (triangles) or mock-transduced (circles) BM cells.
  • Mice were primed and boosted for humoral responses either (Fig. 17 A) one month, or (Fig. 17B,C) 2 months post- infusion with synthetic 12-26 peptide, and HEL as a specificity control.
  • Non- manipulated, immunized BALB/c always produced titers similar to recipients infused with mock-transduced BM cells (Fig. 17A, diamonds). Both total peptide-specific IgG (open symbols), or the main isotype IgGl (closed symbols) were diminished in all experiments.
  • mice were primed and boosted for humoral responses, and sacrificed 3 months later for analysis of splenic memory T cell responses. Cytokine release in individual splenic cultures was determined at 24 hours (IL-2) or 48 hours (IL-4); medium alone background values were less than 1-2 U/ml and were subtracted for clarity ( ⁇ U/ml).
  • Figure 19 shows persistence of gene- transferred B cells. Hybridomas generated from spleens of tolerized mice by PEG fusion of A20 cells with LPS-activated splenocytes (48 hours, 50 ⁇ g/ml LPS).
  • Hybridomas were selected in 1 mg/ml G418 and tested for their ability to activate T- cell hybrid 9C127 as above. Eight representative A20 hybridomas from each recipient (Mice #1-3) are shown.
  • Compendium The nucleotide and amino acid sequences of most known isolates of HIV- 1 are published in "The Human Retroviruses and AIDS Genetic Sequence 1995 Compendium” (hereinafter, “Compendium”), as well as earlier editions of the Compendium). This document is available in paper or electronic form from its publisher, the Los Alamos National Laboratory: Theoretical Biology and Biophysics
  • HAVMID Biophysics, Los Alamos, NM, 1995, (referred to herein as the "HIVMID”) provides T-cell epitope maps, alignments, and annotation (for T helpe epitopes and for CTL epitopes) , as well as a summary and map of linear B cell epitopes and monoclonal antibodies recognizing such epitopes.
  • This application inco ⁇ orates by reference the latest Compendium and HIVMID, but is also intended to include updates containing sequences of additional viral isolates as they are added and published.
  • the compendium (database) and HIVMID are publicly available on the World Wide Web at the address "http://hiv-web.lanl.gov".
  • the HF -1 env gene (which encodes the gpl60 precursor protein of both gp!20 and gp41 envelope proteins) consensus nucleotide sequences for 10 viral subtypes appear at pages I-A-358 to I-A-364 of the 1995 Compendium (NOV 1995).
  • Crosslinking of CD4 molecules on human T cells either by (a) HIV-1 virions bound to CD4 via viral gpl20, or (b) anti-gpl20 antibodies crosslinking of soluble gpl20 bound to CD4, primes or programs the T cells for apoptosis, as described herein.
  • an infected subject's antibody response to HIV-1 particularly to one or more epitopes of gpl20, contributes to the pathogenetic process by targeting bystander T cells to self-destruct.
  • the inventors have discovered an approach to modulate these responses by inducing selective immunological tolerance either at the level of B cells, T helper cells or both, resulting in diminished antibody responses to one or more gpl20 epitopes.
  • an improved HIV vaccine may include in addition to an HIV immunogenic preparation, a fig in accordance with this invention to reduce or prevent undesired antibodies.
  • tolerant or “tolerance” as used herein is defined functionally in terms of the immune response to an immunogenic challenge with an antigen.
  • a subject is tolerant if his response to an immunogenic challenge is reduced by at least about 50%, more preferably at least about 80% relative to a non-tolerant control subject.
  • Tolerance may be manifest by reduced reactivity in vivo such as antibody formation or in vitro, for example, by reduced lymphocyte proliferation.
  • a “tolerogen” is a form of antigen which, when it encounters the immune system, induces a state of immunological tolerance or hyporesponsiveness or anergy in the host. Such a state is tested by subsequent immunization or challenge of lymphocytes in vitro with the specific antigen in immunogenic form.
  • immunogenic with reference to an antigen or epitope is also a functional term which is dependent on the nature, form, dose and route of administration of the antigen (epitope) such that it has immunogenic properties, i.e., it induces immune reactivity resulting in antibodies or cellular immunity.
  • the same molecule e.g., a protein
  • antigens including low molecular weight haptens, can be rendered non-immunogenic and even tolerogenic by coupling them to homologous immunoglobulin molecules.
  • a key observation underlying this invention is that such "coupling" can be achieved by recombinant techniques in the form of a fig wherein a peptide epitope (or "antigenic determinant" or minimal antigenic structure) for which tolerance is desired is made part of the fig using methods described herein.
  • the present invention is useful as a therapeutic tolerogen, to curtail an ongoing immune response to a selected g ⁇ l20 epitope or epitopes during the course of HIV disease. In fact, that may be the more significant clinical utility of this invention.
  • the fig tolerogen is to modulate the response to an HIV vaccine such that the subject immunized with the vaccine and treated with the tolerogen responds to particular desired viral epitopes (expressed by the vaccine) and is prevented (or suppressed) in his response to other selected epitopes (expressed by the tolerogen)
  • the subject immunized with the vaccine and treated with the tolerogen responds to particular desired viral epitopes (expressed by the vaccine) and is prevented (or suppressed) in his response to other selected epitopes (expressed by the tolerogen)
  • HIV infection In the case of HIV infection, this would inhibit or prevent the production of antibodies that are of no benefit (e.g., non-neutralizing), and more importantly, are harmful via mechanisms such as bystander apoptosis or enhancing antibodies which promote infection of host cells. On its face, it might appear counterintuitive to inhibit an immune response to a virus which one wishes to eradicate. However, given the differences between epitopes of HIV recognized by antibodies and by cytotoxic T lymphocytes (CTL) (see HIVMID).
  • CTL cytotoxic T lymphocytes
  • B cell and/or T helper cell tolerance to one or more (even all) epitopes of gpl20 molecule may still permit an effective CTL response against other (non-tolerizing) gpl20 epitopes or, importantly, other non- envelope HIV proteins which are known to be immunogenic.
  • HIV T helper epitopes and CTL epitopes have been described in a number of publication, for example,
  • the present inventors have developed a flexible fusion protein approach for induction of unresponsiveness to defined B-cell and T-cell epitopes in vivo and in vitro. See, for example, Scott and Zambidis, co-pending application U.S.S.N. 08/195,874, PCT Publication WO 95/21926 and Zambidis, E.T. et al, (1996) Proc. Natl. Acad. Sci. USA 95:5019-5024, which references are hereby inco ⁇ orated by reference in their entirety. As described herein, this approach originally set forth for other antigens, is adapted for the production of compositions and methods useful for inducing unresponsiveness to one or more HIV gpl20 epitopes.
  • Epitope-specific tolerance is used to ablate undesired antibody responses while maintaining protective CTL responses.
  • B cell tolerance and T helper cell tolerance to all gpl20 epitopes, either by use of a fig into which a complete gpl20 sequence or one or more partial gpl20 sequences have been inserted, or by using a mixture of fusion Ig's each including a subset of gpl20 epitopes, anti-gpl20 antibody responsiveness can be prevented or diminished. Because the CD8 arm of the immune response is not affected, protective antiviral cell-mediated immunity, in particular CTL responses to HIV epitopes, remains intact.
  • IgG-g ⁇ l20 peptide fusion proteins are effective tolerogens which modulate anti-gpl20 responses. Furthermore, human hematopoietic precursor cells, whether from BM or other tissues, and their progeny B cells which express the IgG-gpl20 peptide fusion proteins are themselves tolerogenic agents which deliver or present on their surface the selected HIV peptides in tolerogenic form for induction and/or maintenance of the tolerant state.
  • the ongoing maintenance of tolerance is achieved by first transfecting bone marrow (BM) cells or peripheral hematopoietic stem cells from any tissue (for example, CD34 + peripheral blood stem cells in the human) with a DNA vector which includes a DNA sequence encoding a IgG-gpl20 fusion protein of the present invention.
  • the tolerogen is presented expressed in a myeloid cell (as determined in studies using SCID mouse BM).
  • the B cell expressing the tolerogenic fig may be a resting B cell, an activated B cell or B cell blast, or a transformed B cell (e.g., leukemia or lymphoma) which has been appropriately attenuated to ablate its oncogenic potential for use in human subjects. Long-lasting, even permanent tolerance can be induced by grafting transfected BM stem cells or peripheral stem cells. This approach is described in more detail in Example IV.
  • B cells are known to be capable of inducing tolerance by presentation of appropriate surface molecules in a tolerogenic fashion (Eynon, E.E. et al. (1992) J.
  • the present inventors discovered that resting B cells expressing a fig, after injection into a recipient subject, induce tolerance for natural epitope included in the fig, such as the phage ⁇ 12-26 epitope. Larger blast cells induced by stimulating such
  • LPS blasts B cells with bacterial hpopolysaccharide (LPS) (termed “LPS blasts”) also tolerize for this peptide.
  • Activated B cells are better tolerogenic vehicles in primed recipients than resting B cells. This is in contrast to the observations of Yuschenkoff et al.
  • splenic B cells stimulated with LPS are infected with a retro virus construct containing the desired epitope.
  • the 12-26 IgG fig has been used successfully in this way.
  • Such LPS blasts are tolerogenic for that epitope.
  • Hybridomas produced from the splenic B cells expressing the fig also express the fig transgene.
  • transgenic BM expressing 12-26 fig or normal (control) BM is injected into recipient mice irradiated with 200R, and the animals are immunized with the peptide in immunogenic form (in adjuvant), the following results have been obtained:
  • T cells in recipients of transgenic BM are tolerant, measured by T cell proliferation and production of cytokines (IL2, IL4, IFN- ⁇ ,. etc.).
  • cytokines IL2, IL4, IFN- ⁇ ,. etc.
  • Tolerance to a desired HIV gpl20 peptide epitope included in an fig construct is achieved using as a source of B cells expressing the fig on their surface any population of lymphocytes known to contain B cells or to differentiate into B cells. This may include an unfractionated population, a cell preparation enriched in B cells or their precursors, or a purified B cell population. Any conventional method for enriching or purifying B cells may be employed. Examples of tissue sources for B cells include BM, spleen, LN, peripheral blood or lymph. B cells may be resting or preferably are activated, for example, LPS blasts.
  • Human ⁇ globulin (HGG) (American Red Cross), a model tolerogenic carrier, is used as a carrier in these evaluations of a given peptide ("PEP") corresponding to one or a combination of epitopes of gpl20.
  • PEP a given peptide
  • MBS w-maleimodobenzoyl-N- hydroxysuccinimide ester
  • a known antigen hapten
  • FITC-coupled HGG may be used as a specificity control for tolerance, e.g., FITC-coupled HGG.
  • murine spleen cells are cultured for 24 hours with increasing concentrations of PEP-HGG, FITC-HGG or anti- ⁇ (positive control for tolerance); these cells are washed and then challenged with LPS in microculture for 4 days.
  • ELIS As for IgM and IgG anti-PEP, anti-gpl20, anti- HGG and anti-FITC are then performed by standard methodology.
  • This protocol allows for polyclonal stimulation that elicits measurable responses to all of these epitopes
  • the evaluation can be performed in PEP-primed subjects to verify that tolerance induction can be achieved in secondary B cells (Linton PJ, et al. (1991) J. Immunol. 146:4099). It is also helpful to perform dose response studies using PEP-HGG conjugates, as well as free peptide, administered intravenously. For example, groups of 4-5 mice are injected intravenously with 0.1, 0.3 or 1 mg of PEP alone, PEP-HGG, or FITC- HGG as a specificity control. Four to seven days later, mice are challenged with gpl20 in complete Freund's adjuvant (CFA).
  • CFA complete Freund's adjuvant
  • mice are bled on day -7 (before tolerance) and at 10 and 20 days after challenge; mice can then be boosted on day 20 and bled 7 days later to evaluate secondary IgG responsiveness.
  • Heterologous IgG's are known to be tolerogenic in vivo at ⁇ 10 "8 M (-0.1-1 mg/mouse).
  • Peptides for inducing T cell tolerance are commonly administered at higher concentrations (approximately 10 "7 M). It may also be advantageous to establish epitope density requirements for tolerance.
  • hapten-protein ratios of 5-10 are used with Ig conjugates. It would be desirable to control coupling reactions to achieve molar ratios (PEP:HGG) of 2,4,8, and 16.
  • the MBS cross-linker is cleavable, it is possible to quantitate ratios and create peptide-linker only controls. Primed recipients may require tolerogens with a higher epitope density. In the fig embodiment, higher epitope density is translated into inclusion of more copies of the DNA encoding the epitope, for example 2-10 copies, in the fig DNA construct if this is required to overcome a state of preexisting immunity in the subject.
  • the tolerogenic IgG-gp-120 peptide fusion proteins may include one or more peptides of gpl20, including the full-length gpl20 protein. If more than one peptide epitope is present, the different peptides may be arranged in the fusion protein in the same order and in contiguous form as they are in the native gpl20 protein. Alternatively, the peptides may be "reshuffled" in the fusion protein. Furthermore, one or more of the g ⁇ l20 peptides may be present in the fusion protein in two or more copies, either alone or with another gpl20 peptide.
  • the one or more epitopes selected for use in the tolerogenic fig is a linear epitope.
  • conformational epitopes become better defined, it will be possible to construct a fig having one or more epitopes which, in combination, yield the conformational determinant in the expressed fig. It is advantageous to use the largest fragment of the native gpl20 protein that
  • (a) can be fused with the Ig H chain while maintaining the required tertiary structure of the Ig portion of the fusion protein for tolerogenic activity and (b) can be accommodated by the vector used to transfer the fig-encoding DNA.
  • the advantage lies in the fact that the appropriate epitopes of such a fig are selected by the host MHC proteins (of antigen-presenting cells or, in this case, tolerogen-presenting cells) for presentation and tolerance induction. In humans, this would obviate the need to select a priori those epitopes of gpl20 which would interact with the HLA-DR molecules of a given subject to yield an active tolerogen for that subject.
  • Expression of the epitope on the fig can be tested using a conventional immunoassay with an antibody specific for the epitope (if it is a B cell epitope) or with lymphocyte proliferation or cytokine secretion assay (for a T helper cell epitope).
  • Antibodies recognizing such epitopes are available, and T cells can be prepared in vitro or long-term T cells lines of the appropriate specificity are available or can be prepared using conventional methods.
  • the Compendium, and in particular the HIVMID lists antibodies specific for each of the epitopes of Table II, for example.
  • the antibodies may be rodent mAbs, human polyclonal or mAbs or hybrid antibodies generated from such human or rodent mAbs.
  • the soluble fig can be administered in adjuvant to a host and tested for generation of peptide- specific T-cell responses in vivo, due to processing and presentation by endogenous APC, even in the context of an Ig scaffold (see Examples).
  • a gpl20 epitope of the present invention in particular a linear or “sequential" epitope, is preferably one comprising a "natural" sequence, defined as the sequence as it occurs in a consensus gpl20 sequence of a particular HIV subtype or a naturally occurring mutant thereof which has been isolated and characterized.
  • the epitope sequence may also be a variant of a natural sequence defined here as a sequence in which one or more amino acid residues has been replaced by a different residue, including substitutions not known to occur in natural viral isolates.
  • the only condition is that the variant sequence maintain the secondary and tertiary structure needed to create the desired the tolerogenic epitope when expressed in a fig protein either in solution or on a cell surface.
  • any variant maintain (a) the structure of the peptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the substitution site, or (c) the bulk of the side chain.
  • the types of substitutions which may be made in the gpl20 protein or peptide molecule of the present invention may be based on analysis of the frequencies of amino acid changes between a homologous protein of different species (e.g., Table 1-2 of Schulz et al. (supra) and Figure 3-9 of Creighton (supra). Base on such analysis, conservative substitutions are defined as exchanges within one of the following five groups:
  • Polar, negatively charged residues and their amides Asp, Asn, Glu, Gin; 3. Polar, positively charged residues: His, Arg, Lys;
  • deletions and insertions, and substitutions according to the present invention are those which do not produce radical changes in the structural or immunological characteristics of the gpl20 protein or peptide molecule when expressed as part of a fig.
  • substitutions, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays.
  • a variant typically is made by chemical synthesis or site-specific mutagenesis of the peptide-encoding nucleic acid, expression of the variant nucleic acid in an fig construct in recombinant cell culture, and, optionally, purification from the cell culture, for example, by immunoaffinity chromatography using an immobilized antibody specific for the natural (non-variant) epitope.
  • the presence of the desired epitope can be readily ascertained by one skilled in the art using an antibody, for example in an immunoassay using a mAb the binding of which defines the epitope.
  • a standard assay for example immunofluorescence of flow cytometry may be used to detect the variant epitope on the surface of a cell.
  • the presence of the desired epitope can be detected using a cellular assay, for example an assay which measures the stimulation of T lymphocytes to proliferate or to secrete cytokines.
  • a cellular assay for example an assay which measures the stimulation of T lymphocytes to proliferate or to secrete cytokines.
  • a tolerogenic gpl20-IgG fusion Ig protein modulates the responsiveness of B and/or T cells to non-neutralizing g ⁇ l20 epitopes, for example in the Cl region or in the C5 region which contains HLA-cross-reactive
  • the Cl region of gpl20 is noteworthy for its dominance in being a target for immune reactivity.
  • the V3 loop in particular the V3 region of the loop (see Tables II and III), is noteworthy as a target for neutralizing antibodies.
  • the C5 region is noteworthy for its cross reactivity with HL-A molecules and its stimulation of autoimmunity.
  • one or more epitopes of one of more of these regions would be useful as tolerogenic epitopes.
  • any gpl20 epitope which stimulates autoimmunity or against which an autoimmune host response is directed is a preferred epitope for use in a tolerogenic fig of this invention.
  • the host immune system will process any administered fig for presentation to T lymphocytes in conjunction with the host's MHC glycoproteins on the antigen-presenting cells ("APC")(or more appropriately, "tolerogen-presenting” cells), it is preferably to include a peptide of sufficient length for binding to host MHC molecules and subsequent presentation.
  • the gpl20 peptide may be as short as about 6 amino acids. Generally larger peptides are preferred, including those with more than one gpl20 epitope in the fig. For example, about 10-20 amino acids, preferably about 10-40 amino acids, more preferably about 10-60 amino acids are included in the fig.
  • T helper epitopes may be identified and selected using various published computer-based algorithms. It is preferable, though not required, to exclude cysteine from the tolerogenic fig because of the constraints this amino acid imposes on uncontrolled secondary structure.
  • the present inventors have developed a model system which utilizes mice made transgenic for human CD4 which is used to screen fig constructs for their efficacy and utility in humans. Administration to these mice of gpl20 and anti-gpl20 or HIV virions or crosslinked gpl20 leads to sensitization for subsequent apoptosis.
  • apoptosis occurs "spontaneously" in response to environmental exposure to antigens which engage the TCR and trigger the apoptotic process.
  • Specific antigens including peptides with defined epitopes are administered to more precisely activate T cell apoptosis. This can be evaluated by testing the animals for epitope-specific T cell unresponsiveness or hyporesponsiveness. The induction of B-cell tolerance and T helper cell tolerance to selected gpl20 epitopes can readily be tested in this model for its effect on the pathway of T-cell apoptosis.
  • the present inventors have utilized a peptide that contains both a T-cell and a B-cell epitope, created a fusion protein of this peptide with an IgG molecule serving as a "carrier” and have used it to induce epitope-specific T-cell and B-cell tolerance
  • a preferred peptide has the structural motif similar to that used earlier to form a 14-mer with a C-terminal cysteine for coupling ease using MBS ( -maleimodobenzoyl-N-hydroxysuccinimide ester) AAAFNMWKNDGGGC (SEQ ID NO:l 13). This peptide can be chemically conjugated to HGG for evaluation in vivo and in vitro for tolerogenicity. 37
  • Table IV above provides amino acids sequences of T helper cell epitopes of gpl20 that have been identified using either human or murine test systems and have been entered in the HIVMID published on the Los Alamos National Laboratory World Wide Web Site.
  • Preferred fig constructs include one or more of the epitopes presented in Table IV linked to the N-terminus of an Ig H chain as was described above for B cell epitopes (e.g., those in Table III).
  • the present inventors do not intend to be limited by this listing of sequences which are specifically based on the amino acids sequences of HIV subtype B viruses.
  • the art permits identification of other epitopic sequences derived from other HIV subtypes (discussed above) as well as viral isolates or "quasi species" thereof.
  • V3 MN RIHIG 42 V3 MN HIGPGRAF 43
  • T cell epitopes in particular T helper cell epitopes, for inclusion in a fig as described herein, utilize computer-based algorithms.
  • Several computer-driven algorithms have been devised in the art which exploit the alphabetic representation of amino acid sequence information to search for T cell epitopes by searching the amino acid sequence of a given protein for characteristics believed to be common to immunogenic peptides, and thereby locating regions that are likely to induce cellular immune response in vitro.
  • T cell epitopes within protein antigens employ a variety of methods, including the use of whole and fragmented native or recombinant antigenic protein, and the "overlapping peptide" method.
  • This approach involves synthesis of overlapping peptides which span the entire sequence of a given protein antigen, in the present case, gpl20. These overlapping peptides are then tested for their capacity to stimulate the relevant T cell responses in vitro, for example T cell proliferative responses (Vordermeier, H.M. et al. (1993) Immunology 80:6-12; Ashbridge, K.R. et al. (1992) J. Immunol. 148:2248-2255).
  • AMPHI searches a protein's primary structure for peptides with a high probability of folding as amphipathic structures (Margalit, H. et al. (1987) J. Immunol. 138:2213-2229;
  • OptiMer examines known amino acid sequences of proteins and generates a list of peptides that contain these motifs; the algorithm then identifies peptides that would be amphipathic if folded as a helix or twisted as a beta-strand, using the AMPHI algorithm. These potentially amphipathic peptides are compared to the list of MHC-binding motif matches. OptiMer extends the predicted amphipathic peptides, to maximize the density of MHC-binding motif matches per length of protein region.
  • the EpiMer algorithm searches protein amino acids sequences for MHC- binding motif matches, generating a list of matches for each protein. The relative density of these motif matches is determined along the length of the antigen, resulting in the generation of a motif-density histogram. Finally, the algorithm identifies protein regions in this histogram with a motif match density above an algorithm- defined cutoff density value, and produces a list of subsequences representing these clustered, or motif-rich regions.
  • the regions selected by EpiMer may be more likely to act as multi-determinant binding peptides than randomly chosen peptides from the same antigen, due to their concentration of MHC-binding motif matches.
  • OptiMer and EpiMer have been used to predict putative epitopes in five Mycobacterium tuberculosis (Mtb) protein antigens (14 kDa, 16 kDa, 19 kDa, 38 kDa, and 65 kDa) and three human immunodeficiency virus (HIV) protein antigens (nef, gpl60 which is the precursor of gpl20 and gp41, and reverse transcriptase (RT).
  • Mtb Mycobacterium tuberculosis
  • HAV human immunodeficiency virus
  • RT reverse transcriptase
  • EpiMer-predicted epitopes EpiMer-predicted epitopes, AMPHI-predicted epitopes, and peptides that would have been synthesized using the "overlapping peptide” method, to a selection of published T cell epitopes for the above proteins. These algorithms were used to predict T cell epitopes from within the published sequences of three HIV protein antigens. Epitopes published for the HIV protein antigens nef and gpl60 were almost exclusively class I
  • AMPHI generated 36 putative epitopes (totaling 666 amino acid residues), and 104 peptides (totaling over two thousand residues in length) would have been required by the overlapping peptide method.
  • the class I-restricted implementations of both OptiMer and EpiMer identified published epitopes with an efficiency comparable to that of AMPHI, and greater than that of the overlapping peptide method.
  • EpiMer's sensitivity per amino acid exceeds that of either OptiMer or AMPHI.
  • OptiMer For RT, the combined class I/class II implementation of OptiMer generated 18 putative epitopes (totaling 422 amino acids); the same implementation of EpiMer generated 22 putative epitopes (totaling 361 amino acids in length). These values compare with 23 putative epitopes generated by AMPHI (totaling 433 amino acids) and 55 peptides (totaling over one thousand residues) required by the overlapping peptide method. OptiMer and EpiMer predict published T cell epitopes for the HIV protein RT with both efficiency and sensitivity comparable to that of the AMPHI algorithm. EpiMer again attains the highest sensitivity per amino acid of these three algorithms.
  • EpiMatrix/HIV which predicts the sequences most likely to bind to MHC molecules when given a number of primary HIV protein sequences and which was developed by A.S. De Groot at Brown University and implemented for the Internet by AVX Design Inc., Buffalo, Rhode Island. Both a website and an online tool, EpiMatrix is located on the Internet at http://www.epimatrix.com hiv as of November 1, 1996.
  • the EpiMatrix algorithm yields a score for each peptide in a 10-mer frame. Scoring is a quantitative estimate of the likelihood (relative to other sequences) that a peptide will bind to a given HLA molecule. Two scoring methods are used: single-allele predictions score for specific HLA alleles and clustered predictions score peptides by the prevalence of MHC alleles in selected populations Matrices for all of the major (greater than 10% population prevalence)
  • MHC alleles representing world populations are included in the algorithm (B.M. Jesdale et al, Vaccines '97, Cold Spring Harbor Laboratory Press).
  • EpiMatrix reduces the total number of regions of HIV proteins to be evaluated in vitro, permitting more rapid identification of desired epitopes. (See, also AIDSWEEKLY Plus, 18 November 1996 issue).
  • Additional MHC binding motif-based algorithms have been described by K.C. Parker et al. (J. Immunol. (1994) 752:163-175) and Y. Altuvia et al. (1995) J. Mol Biol. 249:244-250).
  • binding to a given MHC molecule is predicted by a linear function of the residues at each position, based on empirically defined parameters, and in the case of Altuvia et al, known crystallographic structures are also taken into consideration, j. Hammer et al. (J. Exp. Med. (1994) 750:2353- 2358) described a technique known as "peptide side chain scanning" which is used to predict binding peptides for an MHC allele.
  • the EpiMer/EpiMatrix algorithm predicted putative T cell epitopes from protein sequences for HIV-1 nef, gpl60, gag p55, and tat that required fewer peptides and therefore fewer amino acid residues to be synthesized than either AMPHI-predicted peptides or overlapping peptides.
  • EpiMer predicted 43 peptide epitopes, AMPHI predicted 68 peptides , and the overlapping peptide method (20 amino acid long peptides overlapping by 10 amino acids) would have required 161 peptides. Details (amino acid start and stop, number of MHC binding motifs) of the predicted proteins are available36. Regions of HIV proteins that contain as many as 20 to 30 MHC binding motifs can be identified using this algorithm.
  • HIV protein regions that contain multiple overlapping class-II restricted epitopes also known as "multi-determinant” or multi-determinant peptides, have been identified in mice and humans. Such regions might be important to include in the synthesis of an fig having multiple tolerogenic T helper cell epitopes as described herein. This is particularly useful if a multi-determinant T cell epitope is involved in stimulating antibody responses (i.e., to B cell epitopes).
  • Table V presents a list of epitopes of gpl20 (and several N-terminal epitopes of gp41) which were identified using EpiMer (Roberts et al, supra). These sequences are from the BH10 strain of HIV- 1. The amino acid sequence of this HIV strain was obtained from the SWISS-PROT protein sequence data bank , Accession No. P03375 (EMBL Data Library, Heidelberg, Germany). The residue numbers shown in Table V are from this sequence bank. Those residues beyond amino acid 511 are part of gp41, not gpl20. In a preferred embodiment, the present invention provides a tolerogenic fig H chain or intact fig molecule which includes at the N- terminus of the H chain one or more of the HIV peptide epitopes listed in Table V.
  • HIV peptide epitopes which contain multiple MHC binding motifs, either conserved across HIV strains or derived from several different HIV strains, may be ideal candidates for targeting for T helper cell-directed tolerance induction, as it is assumed that the tolerogen will be presented in vivo by host MHC molecules.
  • epitopes with multiple MHC binding motifs or having an MHC binding motif present in the highest frequency in the subject population would be preferably selected for inclusion in a tolerogenic fig.
  • the EpiMer algorithm is particularly well suited for identifying and selecting such epitopes.
  • the present invention provides polynucleotides encoding the fig in the form of recombinant DNA molecules in vehicles such as plasmid and retroviral vectors, capable of expression in a desired eukaryotic host cell as disclosed herein.
  • the invention also provides hosts transfected or transduced with the fig constructs which are capable of producing in culture or in vivo the fig molecules and secreting them or displaying them on the cell surface.
  • FIG. 4 A depicts the inco ⁇ oration of an oligonucleotide, in this example encoding the ⁇ phage Cl repressor peptide 12-26.
  • Figure 4B depicts the same general scheme wherein a native or synthetic gpl20 peptide epitope is inserted in place of the 12-26 peptide.
  • Any Ig gene construct may be used for insertion of the tolerogenic epitope or epitopes.
  • a preferred Ig gene encodes human Ig, more preferably an Ig comprising a human ⁇ chain.
  • the DNA construct encodes an individual fig H chain, although the protein products of this invention include both the fig H chain and a complete assembled Ig molecules comprising the fig H chain having one or more HIV gpl 20 epitopes in combination with a native human Ig L chain.
  • the fig may also comprise two different H chains, one of which is a fusion protein having one or more HIV gpl 20 epitopes added to or included in the V region.
  • Genetic sequences, especially cDNA sequences, encoding either a complete fig H chains, the fig V regions or a human Ig C region of any Ig isotype, most preferably, an IgG isotype (i.e., a human C ⁇ chain) are also provided herein.
  • the invention also provides a genetic sequence, especially a cDNA sequence encoding an Ig V region fusion protein in which the V region encoding DNA has been combined in frame with one or more HIV gpl 20 epitopes.
  • a genetic sequence especially a cDNA sequence encoding an Ig V region fusion protein in which the V region encoding DNA has been combined in frame with one or more HIV gpl 20 epitopes.
  • genomic DNA sequences may also be used, cDNA sequences are particularly preferred.
  • One non-limiting approach to producing the fig comprises the steps of: 1. Selection of one or more gpl 20 epitopes as described below for which tolerance is desired; 2. Preparation of DNA encoding the epitope or epitopes selected above; this can be done by isolating HIV RNA and cloning an preparing cDNA corresponding to all or part of gpl 20, by isolating and cloning DNA from HIV-infected cell, or if the DNA is sufficiently short, synthesizing an oligonucleotide having the desired coding sequence.
  • the latter synthetic approach permits construction of artificial combinations of two or more gpl 20 epitopes or which are not contiguous in the native protein.
  • Oligonucleotides which can be used as primers for introducing useful restriction sites into the gpl 20 and human Ig DNA for subsequent linkage are well known in the art. See, for example, Sambrook et al, supra.
  • the gpl 20 DNA is linked to an Ig V gene cassette. Because the antibody specificity of the fig is not important, any V region DNA can be selected.
  • a preferred V gene is one which encodes a protein which, after fusion of a gpl 20 epitope or epitopes, even a full length gpl 20 protein, still maintains its ability to fold properly in an full Ig molecule (H 2 L 2 ).
  • variable (V) domain of an Ig chain includes hypervariable (HV) regions which are also known as complementarity-determining regions (CDRs) because they are important in "determining" the structure of the antibody combining site that is complementary the epitope bound.
  • HV hypervariable
  • CDRs complementarity-determining regions
  • FRs framework regions
  • the order of these regions in a V domain is as follows: FR1-HV1-FR2-HV2-FR3-HV3-FR4.
  • the framework regions form the ⁇ sheets that provide th structural framework of the domain, with the HV sequences corresponding to three loops at one edge o each sheet that are juxtaposed in the folded protein.
  • the HV loops from the V H and V L domains are brought together, creating a single HV site at the tip of the Fab fragment which forms the antigen binding site.
  • the first framework region is the most N-terminal of the V region. Eisen, H.N., GENERAL IMMUNOLOGY, (J. Lippincott Co., Philadelphia, 1990) at pages 57-59, in particular Figure 14-19 at page 58, shows the amino acid sequences of the first framework region of 5 different human H chains.
  • the first framework region includes the 30 N-terminal amino acids at which point the HV1 region follows.
  • a framework region of nine different human K L chains belonging to three different groups V ⁇ l, VKII and VKIII are shown in this textbook figure..
  • the FRs are about 30 residues, with a number of positions in each group serving as "framework residues" which serve to characterize each Vt group.
  • the heterologous epitope of the fig is preferably inserted immediately N terminal to the first framework region. In other embodiments, it may be fused "deeper" into the Ig sequence within the V region.
  • a spacer comprising between about 1 and 10 amino acids, preferably about 3- 5 residues, can be present between the C terminal residue of the heterologous epitope(s), preferably a gpl 20 epitope(s) and the N terminal residue of the Ig V region, provided that the protein can fold properly to present the gpl 20 epitope while maintaining its tolerogenic properties.
  • a repeat of the 5 N-terminal amino acids of the Ig H chain is inserted N- terminal from the added the gpl 20 peptide (or peptides) such that this pentapeptide sequence is repeated on either end of the inserted gpl 20 sequence.
  • a spacer as described herein may be linked to one or more of the added gpl 20 peptides.
  • a major pu ⁇ ose of the spacer is to permit unimpeded folding and proteolytic processing of the fig as if it were an normal Ig protein. This assures proper surface expression of the fig and association with MHC proteins on the surface of a tolerogen-presenting cell.
  • V region is a complete Ig H chain is constructed by combining the now altered V gene construct containing additional gpl 20 DNA with a C gene construct encoding a desired human C region, preferably a human C ⁇ protein.
  • the most preferred C region would be that encoding the ⁇ 3 isotype.
  • Ig H chain (or V H ) cDNA vectors are typically prepared from human cells and modified by site-directed mutagenesis to place a restriction site at the position in the human sequence in which the gpl 20 DNA is to be grafted. Preferably this is 5' to the nucleotide encoding the N-terminus of the Ig H chain or the V H protein.
  • Two coding DNA sequences are said to be "operably linked” if the linkage results in a continuously translatable sequence without alteration or interruption of the triplet reading frame.
  • a DNA coding sequence is operably linked to a gene expression element if the linkage results in the proper function of that gene expression element to result in expression of the coding sequence.
  • Expression vehicles include plasmids or other vectors, such as retroviral vectors.
  • a preferred vehicle carries a functionally complete human V H and C H having appropriate restriction sites engineered so that any gpl20-encoding nucleotide sequence with appropriate cohesive ends can be conveniently ligated thereto. These vehicles can be used as intermediates for propagation of DNA encoding any desired H chain (V H C H ) ready to receive a gpl 20 DNA sequence, and for the expression of the complete fig (gp!20-V H C H ).
  • Preferred hosts are mammalian cells, most preferably human cells, grown in vitro for prolonged periods, or taken from a host, cultured in vitro for pu ⁇ oses of transfection and then reintroduced into the host.
  • Mammalian cells provide post-translational modifications to the Ig protein molecules including leader peptide removal, folding and assembly of H and L chains, glycosylation of the protein chains and secretion of the complete functional fig protein.
  • Mammalian cells which may be useful as hosts for the production of fig proteins include cells of lymphoid origin, such as the hybridoma Sp2/O-Agl4 (ATCC CRL 1581) or the myeloma P3X63Ag8 (ATCC TIB 9), also abbreviated as P3, and its derivatives.
  • a preferred murine cell line for expressing the fig of this invention is J558L.
  • any cell line which allows for efficient expression and secretion of the fig constructs of the present invention and which promotes proper folding of the fig is preferred.
  • Known human lymphoid or hematopoietic cell lines may be used, including B lymphoblastoid lines, lymphomas, hybridomas or heterohybridomas. Examples of cell lines and approaches for expression of recombinant or chimeric or hybrid or modified Ig genes are described in Shin, S.U. et al, (1993) Int. Rev. Immunol. 70:177-186; Wright, A. et al, (1992) Crit.
  • H and L chain genes are available for the expression of cloned Ig H and L chain genes in mammalian cells (see Glover, D.M., ed.(1985) DNA Cloning, Vol. II, pp 143-238, IRL Press). Different approaches can be followed to obtain complete H 2 L 2 antibodies. It is possible to co-express H and L chains in the same cells to achieve intracellular association and linkage of H and L chains into complete tetrameric H 2 L 2 antibodies. The co-expression can occur by using either the same or different plasmids in the same host. Genes for both H and L chains can be placed into the same plasmid, which is then transfected into cells, thereby selecting directly for cells that express both chains.
  • cells may be transfected first with a plasmid encoding one chain, for example the L chain, followed by transfection of the resulting cell line with an H chain plasmid containing a second selectable marker.
  • Cell lines producing H 2 L 2 molecules via either route could be transfected with plasmids encoding additional copies of H, L, or H plus L chains in conjunction with additional selectable markers to generate cell lines with enhanced properties, such as higher production of assembled H 2 L 2 antibody molecules or enhanced stability of the transfected cell lines.
  • One particular strategy for inserting an HIV peptide sequence at or near the N- terminus of an Ig H chain is related to that described in Hebell, T. et al (1991) Science 254:102-105 and Ballard, D.W.
  • a first plasmid is constructed which preferably includes .a full genomic sequence of the Ig H chain and selectable markers, for example, neomycin and or/ampicillin resistance genes.
  • the source DNA encoding the HIV gpl 20 epitope or epitopes PCR is amplified to create the DNA encoding the desired single or multiple epitopes. Appropriate restriction sites are included on the primers so that the epitope-encoding DNA can be spliced into the Ig gene-containing vector.
  • the gpl 20 epitope sequence is subcloned into a site, preferably the V H site of the first plasmid. Recombinant clones are analyzed for proper orientation and polymerase induced errors by double stranded DNA sequencing methods (e.g., Sequenase® kit from U.S. Biochemical).
  • the promoter sequences useful for the DNA constructs of the of the present invention are any promoters which allow efficient expression of the fig DNA of the invention in a target cell of choice, for example a hematopoietic progenitor cell or a lymphoid cells, more preferably a B cell.
  • Preferred promoters are the promoters of the Ig gene into which the foreign epitope-encoding DNA is being inserted.
  • Other known promoters of either eukaryotic or viral origin may be used. Suitable promoters are inducible or repressible or, more preferably, constitutive. Examples of useful eukaryotic/viral promoters include the promoter of the mouse metallothionein I gene (Hamer, D., et al.
  • the fig construct into which the gpl 20 epitope(s) has been inserted is introduced ("gene transfer") into the appropriate target cells by conventional methods, e.g. , direct physical transfer of plasmid DNA, or preferably, by virus-mediated transfer, for example using a retroviral vector, as discussed below.
  • a number of means for transferring genes are known in the art and may be used herein, including, for example, electroporation and lipofection.
  • a preferred, and relatively efficient means for achieving transfer of genes is by retrovirus-mediated gene transfer (Gilboa, E. (1987) Bio-Essays 5:252-258; Williams, D.A. et al. (1984)
  • retroviruses recombinant amphotropic retroviruses have been used as vectors for the transfer of genes into human cells (Cone, R.D. et al. (1984) Proc. Natl. Acad. Sci. USA 57:6349- 6353; Danos, O. et al. (1988) Proc. Natl Acad. Sci. USA 55:6460-6464.
  • the targets for gene therapy are bone marrow or blood stem cells, for example, it may be advantageous to manipulate the cells in vitro with cytokines and then to infect them with the vector bearing the fig gene (Wilson, J.M. et al. (1990) Proc. Natl. Acad. Sci. USA 57:8437-8441).
  • Recombinant amphotropic retroviruses have been recognized as useful vectors for transferring genes efficiently into human cells, for example to correct enzyme deficiencies (Cone, R.D. et al. (1984) Proc. Natl. Acad. Sci. USA 57:6349-6353; Danos, O. et al, (1988) Proc. Natl. Acad. Sci.
  • a retroviral vector used for gene therapy be capable of infecting only desired cells and not cause generalized infection of cells throughout the body of the individual being treated. In the past, this has generally been accomplished by using helper-defective virus preparations, or mutants lacking the ⁇ packaging sequence, etc.
  • AAV adeno associated viral
  • ITRs inverted terminal repeats
  • AAV is a linear single stranded DNA parvo virus, and requires co-infection by a second unrelated virus in order to achieve productive infection.
  • AAV carries two sets of functional genes: rep genes, which are necessary for viral replication, and structural capsid protein genes (Hermonat, P.L., et al. (1984) J. Virol. 57:329-339).
  • the rep and capsid genes of AAV can be replaced by a desired DNA fragment to generate AAV plasmid DNA.
  • Transcomplementation of rep and capsid genes are required to create a recombinant virus stock. Upon transduction using such virus stock, one recombinant virus uncoats in the nucleus and integrates into the host genome by its molecular ends.
  • Liposomes may be used to encapsulate and deliver a variety of materials to cells, including nucleic acids and viral particles (Faller, D.V. et al. (1984) J. Virol. 49:269-212). Preformed liposomes that contain synthetic cationic lipids form stable complexes with polyanionic DNA (Feigner, P.L., et al. (1987) Proc. Natl. Acad. Sci. USA 54:7413-7417).
  • Cationic liposomes comprising some cationic lipid, that contained a membrane fusion-promoting lipid dioctadecyldimethyl-ammonium- bromide (DDAB) efficiently transfer heterologous genes into eukaryotic cells (Rose, J.K., et al. (1991) Biotechniques 70:520-525).
  • Cationic liposomes can mediate high level cellular expression of transgenes, or mRNA, by delivering them into cultured cell lines (Malone, R., et al. (1989) Proc. Natl. Acad. Sci. USA 5(5:60776081).
  • Gene transfer can also be achieved using "carrier mediated gene transfer” (Wu, CH. et al (1989) J. Biol. Chem. 264:16985; Wu, G.Y. et al. (1988) J. Biol. Chem. 263:14621 ; Soriano, P. et al. (1983) Proc. Natl. Acad. Sci. USA 50:7128; Wang, C-Y. et al (1982) Proc. Natl. Acad. Sci. USA 54:7851; Wilson, J.M. et al. (1992) J. Biol. Chem. 267:963).
  • Preferred carriers are targeted liposomes ( ⁇ icolau, C. et al. (1983) Proc.
  • gpl 20 peptides which are to be included in the tolerogenic fig. Any nucleotide sequence which encodes a chosen peptide epitope or series of epitopes may be used. Distinct gpl 20 epitopes may be combined in any order or combination provided that the coding nucleic acids provide an in-frame sequence both with respect to the gpl 20 epitopes and with respect to the Ig H gene utilized to construct the fig.
  • Treatment of an individual infected with HIV using the tolerogenic fig of this invention comprises parenterally administering a single or multiple doses of the fig to a subject, preferably a human.
  • the fig is preferably an isologous Ig, that is, of the same species as the subject.
  • a most preferred fig is fusion IgG molecule.
  • An effective tolerogenic dose is a function of the size and number of particular HIV gpl 20 epitopes included in a particular fig construct, the patient and his clinical status, and can vary from about 0.01 mg/kg body weight to about 1 g kg body weight.
  • a subject can be given this amount in a single dose or in multiple repeated doses.
  • Doses of hematopoietic cells or B cells expressing the fig are preferably administered at a dose between about 10 6 and 10 10 cells on one or several occasions.
  • the route of administration may include intravenous (iv) , subcutaneous (SC), intramuscular, intrapulmonary, intraperitoneal or other known routes.
  • iv intravenous
  • SC subcutaneous
  • intramuscular intrapulmonary
  • intraperitoneal intraperitoneal
  • the preferred route for administration of fig proteins or cells for tolerogenesis is by iv injection.
  • the fig of this invention may be advantageously utilized in combination with other therapeutic agents useful in the treatment or prevention of HIV disease, including prophylactic or therapeutic vaccine preparations, antiviral chemotherapeutic agents, immune response modulators including cytokines and hematopoietic growth factors, protective antibody reagents, etc.
  • the present inventors took advantage of the IgG molecule as a tolerogenic carrier, and created an engineered tolerogen with a grafted epitope at the N-terminus of an IgG heavy chain.
  • This engineered IgG was recognized by the immune system in a tolerogenic manner.
  • the model epitope chosen for this initial analysis is the well-characterized class-II MHC-restricted peptide sequence from the cl ⁇ repressor protein (pi -102), residues 12-26. This peptide contains both a B- and T- cell epitope. and is the immunodominant determinant in H-2 d mice immunized with the entire protein (26-30).
  • RPMI 1640 medium (GIBCO-BRL, Gaithersburg, MD) was supplemented with 5% FCS (Hyclone, Logan, UT), 2-ME, L-glutamine, penicillin, streptomycin, MEM nonessential amino acids, and sodium pyruvate.
  • FCS Hybridoma B3.11, which produces a monoclonal IgG, specific for the 12-26 peptide was a kind gift of Drs. Tom Briner and Malcolm Gefter (Immulogic,
  • B3.11 was affinity purified with goat anti-mouse IgG sepharose columns and biotinylated, or used as a neat culture supernatant. All alkaline- phosphatase (AP)-conjugated reagents were purchased from Southern Biotechnology Assoc. (Birmingham, AL). Synthetic peptide: The 12-26 15-mer LEDARRLKAIYEKKK (SEQ ID NO:l 12) was prepared with a solid-phase method and purified to >92% homogeneity using standard HPLC methods.
  • Peptide was conjugated to bovine albumin serum (BSA) rabbit gamma globulin (RGG), or keyhole limpet hemocyanin (KLH) as described (Roy, S. et al (1989) Science. 244:515-515).
  • BSA bovine albumin serum
  • RSG rabbit gamma globulin
  • KLH keyhole limpet hemocyanin
  • Oligonucleotides The following complementary synthetic oligonucleotides encoding the 12-26 sequence were designed with BamHI/Clal restriction ends, phosphorylated with T4 kinase and ATP, and cloned into the hypervariable region of flagellin construct pPX 1647: DWS1 : (SEQ ID NO: 195)
  • DWS2 (SEQ ID NO: 196)
  • PCR primers were also designed to amplify a modified 12-26 sequence from the chimeric 12-26-flagellin construct. This sequence includes 5' FRl V H sequence and
  • Ig-one 5'-TGATCTACTGCAGCTGGAGGACGCGCGGCG G-3' (SEQ ID NO:197)
  • ELISA determinations of serum peptide-specific IgG responses were done by coating ELISA plates with 50 ⁇ g/ml synthetic peptide. Antigen-coated plates were blocked with 1% gelatin/0.05% Tween 20 buffer, and duplicate serial dilutions of serum were incubated and probed with goat anti-mouse IgG isotype-specific secondary reagents. Titers are expressed as the geometric mean of the reciprocal dilution required to bring A 490 readings to prebleed levels or ⁇ 0.08 O.D. Protein Engineering Design:
  • Plasmid pSNR (Ballard, D.W. et al. (1986) Proc. Natl. Acad. Sci. USA. 55:9626-9630), which contains neo and amp resistance genes, as well as the full genomic sequence for a IgG, 0 H chain specific for the NP hapten, was obtained from Dr. Douglas Fearon (Cambridge University) and modified. A modified 12-26 sequence was created via PCR amplification of this sequence from the chimeric flagellin construct A29 (described in WO95/21926) utilizing PCR primers "Ig-one" and "Ig-two".
  • the modified 12-26 sequence was subcloned into the V H site of pSNR and recombinant clones were analyzed for proper orientation and Taq polymerase mutational errors by double-stranded DNA sequencing methods (USB Sequenase 2.0 kit). Expression, purification, and quantitation of transfected IgG:
  • Stably transfected clones were isolated in 1 mg/ml G418 (GIBCO-BRL), subcloned, and transfected IgG's from selected clones were purified from bulk supernatants or ascites with anti-mouse IgG-Sepharose or protein G columns. Since the original H chain binds with high affinity to the NIP (5-iodo-4-hydroxy-3-nitrophenylacetyl) hapten, purified or serum transfectoma IgG was quantitated using a modified NIP- gelatin binding ELISA , using anti-mouse IgG,-AP as a secondary reagent.
  • G418 1 mg/ml G418
  • Peptide-specific tolerance induction in adult recipients was accomplished by intravenous (“iv”) injection (in the lateral tail vein) of either 1 mg purified, deaggregated, chimeric (Q3) or control IgG (P6) diluted in saline, or by 3 repeated injections of mitomycin C-treated (50 ⁇ g/ml, SIGMA) P6- or Q3-secreting transfectomas.
  • iv intravenous
  • SC subcutaneously
  • ip intraperitoneally
  • HEL synthetic 12-26 peptide
  • CFA Freund's complete adjuvant
  • LN secondary (LN) responses following iv tolerization
  • animals were immunized in hind footpads with 20 ⁇ g peptide emulsified in CFA, and draining popliteal LNs were harvested 9 days later and restimulated in culture with dilutions of peptide and 50 ⁇ g/ml purified protein derivative (PPD, Connaught, Swiftwater, PA).
  • PPD Purified protein derivative
  • IL-2 and IL-4 secreted into the medium were determined from culture supernatants at 24 and 48 hours, respectively, in LN or splenic T-cell cultures using recombinant cytokines as standards.
  • the 12-26-IgG construct was prepared by modifying plasmid pSNR, which contains the genomic sequence encoding a murine IgG, b H chain.
  • Isologous IgG was chosen because of its documented activity as a tolerogenic "carrier" of potency equal to IgG 2 and greater than other Ig isotypes or other serum proteins.
  • the recombinant 12-26-IgG chimera is immunogenic and capable of priming 12-26-specific T and B cells in vivo.
  • Mice immunized with Q3 emulsified in CFA were able to prime 12-26-specif ⁇ c T cells comparable to the response elicited with synthetic peptide.
  • In vitro restimulation of LN cultures with synthetic peptide resulted in T-cell proliferation as well as IL-2 and IL-4 production in peptide- and Q3-primed, but not P6-primed LN cells. Immunization also led to a high serum anti- 12-26 IgG antibody titer detectable by peptide-specific ELISA.
  • Figure 6 shows that mice receiving pretreatments of Q3, but not control P6, were dramatically unresponsive to peptide challenge as assessed by ELISA of anti- peptide IgG, whereas control anti-HEL antibody titers were unaffected.
  • the predominant Ig isotype in this anti-peptide response in Balb/c mice is IgG
  • antibodies of all isotypes including IgG 2b were consistently diminished by the tolerogenic treatment with 12-26-IgG ( Figure 6).
  • mice received 3
  • transfectomas secreting Q3 or P6 control IgG
  • the cells had first been treated with mitomycin C.
  • This protocol resulted in transient appearance in serum of the transfected IgG's at levels reaching at least 10-500 ng/ml (assessed by NIP-gelatin ELISA).
  • This type of treatment resulted in diminution of peptide-specific humoral immune responses as well as reduction of LN cell proliferative responses.
  • Thl-type (IL-2) and Th2-type (IL-4) responses were absent in tolerized mice, a result consistent with the observed lack of anti-peptide IgG 2b and IgG, antibodies( Figure 6), which are dependent on these Th cell subsets.
  • the T cell response to peptide was diminished in 12-26-IgG pretreated animals when measured as short-term LN restimulation assays .
  • Mice tolerized with 1 mg of 12-26-IgG 10 days previous to peptide challenge had reduced LN IL-2 responses, but unaffected recall proliferative responses to the antigen PPD compared to control P6-injected animals.
  • a foreign immunogenic peptide genetically engrafted into an Ig scaffold can be very efficiently presented to the immune system in a tolerogenic manner when administered by the appropriate route and method.
  • pretreatment with peptide-Ig chimeras delivered either as single high doses or via slow release by transfected autologous B cells have utility in achieving efficient epitope-specific manipulation of undesired T-cell responses.
  • a concentration of 0.1 ⁇ g/ml was found to be minimally mitogenic (as assessed by anti-fluorescein [FITC] IgM ELISA's) and used for subsequent experiments. These results broaden the context in which the inserted epitope can be recognized: IgG and the polymerized flagellin molecule. In the latter context, the epitope readily stimulate B cells to produce epitope-specific IgM antibodies. We also tested the ability of 12-26-IgG to induce specific B-cell unresponsiveness. Enriched B cell populations were incubated in vitro with various doses of Q3 or P6 control IgG's, washed, and then cultured with either mitogenic LPS or 12-26-flagellin. Alternatively, BALB/c mice were injected iv with 1 mg of each protein, and splenic B cells were harvested and challenged in vitro 10 days later.
  • FITC anti-fluorescein
  • the fig construct is independently can induce epitope-specific unresponsiveness in B cells.
  • the magnitude of B cell tolerance was more modest in vivo than T cell tolerance, possibly reflecting either a requirement for higher epitope valency (the fig provides only a bivalent epitope, one on each arm of the H chain), or a higher dose requirement.
  • Exposure of mature B and T cells to antigen in an adult immune system may lead to either activation or tolerance depending on the route and method of exposure, as well as the availability of costimulatory signals from specialized APC. Since a major goal in clinical therapy in a variety of conditions (e.g., infection, autoimmunity, allergy, transplantation) is the induction of specific immune unresponsiveness in adult mature lymphocytes, a variety of approaches have exploited these pathways of exposure. Of these approaches, experimental tolerance induction with gamma-globulin carriers has been most extensively described. IV administration of soluble, deaggregated IgG's in the absence of adjuvants, induces both antigen-specific B-cell and T-cell tolerance even in the absence of a thymic environment. Mechanisms of specific clonal anergy/inactivation and deletion have been implicated in this type of experimental model.
  • N-terminus of an IgG heavy chain construct was tolerogenic in vivo and in vitro.
  • Conceptually similar approaches have been utilized to express immunogenic (rather than tolerogenic) malarial or viral peptides in the CDR3 loop of Ig H chains for the induction of enhanced anti-peptide immune responses, as described above.
  • the 12-26-IgG protein could act as an efficient immunogen when administered in an immunogenic manner (i.e., emulsified in CFA).
  • Zaghouani et al, 1993, supra showed that T-cell activation (for a class Il-restricted epitope) was enhanced 100-1000 fold when the epitope was part of an Ig-chimera, presented in vitro by stimulatory dendritic cells as APC.
  • the present results similarly show that an approximately 100-fold lower molar quantity of 12-26-IgG (as compared to free peptide) stimulated similar numbers of peptide-specific LN T cells from immunized mice.
  • the increased efficacy of the fig's of the present invention may indicate that common pathways are utilized.
  • the increased efficacy may directly result from (a) an increased half-life and (b) an
  • APC such as resting B cells
  • Fc receptor-mediated endocytosis or phagocytosis via the process of Fc receptor-mediated endocytosis or phagocytosis, and subsequently presented by these "non-professional" APC
  • IgG carriers can induce efficient B-cell unresponsiveness by mechanisms involving the crosslinking of surface IgM to Fc receptors.
  • One or more of the above mechanisms may be responsible for the enhanced tolerogenic efficiency of Ig carriers.
  • a fig specifically the 12-26-IgG fusion protein, can present an epitope in a tolerogenic fashion and induce both B- and T-cell tolerance.
  • a convenient property of this epitope allows simultaneous study of both cellular and humoral immune responses to a single immunodominant peptide.
  • the 12-26 peptide can induce a vigorous antibody response which is predominantly of the IgG, isotype, and can prime Th cells of both the Thl and Th2 phenotype. Tolerance induction with 12-26-IgG was globally effective in suppressing every type of immune response which can be elicited by this immunodominant peptide.
  • the inventors have therefore provided a powerful approach to determining the efficacy of inducing specific unresponsiveness to a defined antigens, particularly peptide antigens, for the modulation of undesired immune responses.
  • the present approach has advantages of that inserting heterologous epitopes into the H chain CDR3 because the N-terminus insertion does not restrict the size of the epitope or epitopes fused to the tolerogenic IgG carrier. Therefore, not only short peptides, but also larger, more complex foreign antigens may be fused in an fig construct for tolerogenic presentation.
  • the inventors initially chose the C-terminal peptide KYKVVKIEPLGVAPTKAKRRVVQREKR (SEQ ID NO: 199) (residues 485-51 in the BH10 variant ( Figure 3) and which ; corresponds approximately to positions 455-481 of the consensus sequence in Figure 1 ). This is in the conserved C5 C-terminal region of gpl20.
  • This peptide contains the B-cell epitope consisting of the KYK KAKRR (SEQ ID NO:200)motifs that are recognized by the M38 murine mAb (DeSantis et al, supra; Palker et al, supra).
  • the epitope recognized by M38 has been noted to be KYKVVKEIPLGVAPTKAKRR of SEQ ID NO: 199.
  • MAb M38 also binds to the C-terminus of gpl20, in a gp41 binding region. M38 also reacts with a common motif in the HLA-C heavy chain al region (KYKRQAQADRVNLRKLR; SEQ ID NO:201) that is mimicked in this C5 peptide. HIV-infected individuals have HLA class I-gpl20 cross-reactive antibodies.
  • the inventors first established that a 35-mer containing this M38-defined epitope was tolerogenic in vivo when chemically coupled to heterologous rabbit IgG. Since the C5 peptide was relatively large and not readily available. Shorter peptides containing the KYK and KAKRR sequences with different spacer residues and with a C-terminal cysteine for more controlled coupling to IgG carriers can be designed.
  • AAKYKGVAPTKAKRRGGC (SEQ IDNO:205) Control peptides (for example, available from the National Institute of Allergy and Infectious Diseases) encompassed the KYK, KAKRR (SEQ ID NO:200) motifs or the entire IEPTGVAPTKAKRR (SEQ ID NO:206)sequence recognized by the human mAbs.
  • peptide #3 above (“P3"), was similar in activity to the full sequence and that peptides containing only the KYK motif were noninhibitory, as expected, with these human anti-C5 mAbs.
  • P3-HGG Treatment with P3-HGG induced unresponsiveness for an anti-P3 response (and an anti-HGG response).
  • the present invention would require that the fig include one or more epitopes from these regions.
  • Resting and Activated B Lymphocytes Expressing fig are Tolerogenic Vehicles Since antigen-presenting B-lymphocytes are known to either augment or downregulate T-cell dependent immunity , it should be possible to modulate the immune response to a selected antigen (such as an autoantigen, a viral antigen or a tumor antigen) via gene-transfer of exogenous genes and constitutive expression in vivo by autologous APC. Such an approach would be advantageous for the induction of unresponsiveness, since tolerance to foreign antigens could be maintained indefinitely in vivo, especially if gene-transfer into long-lived lymphoid progenitors is achieved.
  • a selected antigen such as an autoantigen, a viral antigen or a tumor antigen
  • B cells can be either essential (Ron, Y. et al (1981) Eur. J. Immunol. 77:964-968; Janeway, C.J. et al. (1987) J. Immunol. 755:1051-1055; Constant, S. et al. (1995) J.
  • the present inventors generated a unique transgenic mouse system (see Example I) in which a foreign class Il-restricted immunodominant epitope is expressed as a self antigen specifically in the B cell compartment.
  • the foreign epitope, residues 12-26 from ⁇ cl repressor protein was grafted in-frame at the N- terminus of a murine IgG, heavy chain and is made endogenously as a transgene in the B-lymphocyte lineage.
  • the tolerogenic capabilities of this soluble engineered immunoglobulin in immunocompetent adult mice is described above.
  • mice Male and female B6D2 (H-2 b/d ) and B ALB/cByJ (H-2 d ) mice were purchased from the Jackson Laboratories (Bar Harbor, ME) at 3-8 weeks of age, and housed in pathogen-free, microisolater cages.
  • RPMI 1640 medium (GIBCO- BRL, Gaithersburg, MD) was supplemented with either heat-inactivated 5% FCS (Hyclone, Logan, UT), or heat-inactivated 0.5% autologous mouse serum (Jackson Immunochemicals), 2-ME, L-glutamine, penicillin, streptomycin, MEM nonessential amino acids, and sodium pyruvate.
  • Hybridoma B3.11 which produces an IgG, specific for the 12-26 peptide was from Drs. T. Briner and M.Gefter (Immulogic Co ⁇ ., Waltham, MA), and was originally derived by fusion with splenocytes from peptide-immunized BALB/c mice.
  • Monoclonal antibody (mAb) B3.11 was affinity purified from bulk-cultured supernatants with goat anti-mouse IgG sepharose columns and biotinylated. All alkaline-phosphatase (AP)-conjugated secondary reagents were purchased from Southern Biotechnology Assoc. (Birmingham, AL). The 12-26 15- mer LEDARRLKAIYEKKK (SEQ ID NO:l 12), or an N-terminal cysteine-modified 16-mer was prepared with a solid-phase method and purified to >92-95% homogeneity using standard HPLC methods.
  • AP alkaline-phosphatase
  • the cysteine-modified 12-26 peptide was covalently conjugated to hen egg white lysozyme (HEL) with Sulfo-MBS (Pierce, Rockford, Illinois), a sulfhydryl-specific crosslinking reagent.
  • Tg Transgenic mice.
  • Tg mice were derived by pronuclear injection of fertilized B6D2 eggs, and implantation into pseudopregnant females as described by Hogan et al. (Hogan, B. et al. (1986) Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Lab. Press, Plainview, N.Y. pp. 81-141, inco ⁇ orated by reference).
  • Tg mice Three original Tg founders were identified by genomic Southern blotting of tail DNA wid a 32 P-labeled probe containing 3 cloned, tandem copies of the 12-26 cDNA sequence. Two of these founders (Line 5 and Line 17) were selected for further analysis, bred onto the BALB/c background for at least 5-10 generations, and confirmed for H-2 d homozygosity via RFLP Southern blot analysis before use in BALB/c adoptive transfer experiments. Lines 5 and 17 were also rederived by Cesarean section (Taconic Labs) and thereafter housed in sterilized microisolater units at the Holland Laboratory to ensure healthy microorganism-free strains of Tg mice.
  • Tg offspring obtained via BALB/c matings were, in general, heterozygous for their transgene and distinguished from their nontransgenic (NTg) littermates by either 12-26 sequence Southern blotting of tail DNA, or serum NIP- binding IgG, ELISA.
  • NTg nontransgenic
  • ELISA serum NIP- binding IgG
  • Preparation of Bone Marrow Chimeras Eight week-old BALB/c recipients were sublethally irradiated (650 rads) with a 137 Cs source and injected iv with 10 7 cells consisting of a 1 :1 pooled mixture of non-Tg/Line 17 Tg bone marrow (BM) cells that had been depleted of erythrocytes.
  • BM bone marrow
  • mice were injected with NTg littermate BM cells(after 650 rads) or saline only (with no irradiation). All Tg/NTg donor BM was completely sex-matched and syngeneic with BALB/c recipients. Adoptively- transferred mice were rested for 7-8 weeks before immunization studies.
  • Bone marrow from both femurs and tibiae or spleen tissue was prepared in serum-free complete RPMI and depleted of erythrocytes.
  • Splenic B cells were obtained by depleting splenocytes of T cells by treatment with anti-T cell cocktail plus baby rabbit complement. Resting B cells were harvested by further fractionation on Percoll gradients and collecting the 60-70% layers as previously described (29).
  • purified B cells (4xl0 6 /ml) were incubated for 48 hrs in complete RPMI (5% FCS) in the presence of 50 ⁇ g/ml LPS (Sigma, St. Louis, MO), and washed 3 times before further use.
  • purified B cells were treated with carbodiimide (ECDI, Sigma) by incubating 10 8 cells in 0.5 ml of 75 mM ECDI (in saline) for 1 hour, on ice. All cells were washed extensively prior to iv injection.
  • ECDI carbodiimide
  • mice were also injected with 20 ⁇ g hen egg lysozyme (HEL) in CFA, intraperitoneally (ip). Two weeks later, mice received an additional antigenic boost of 50 ⁇ g peptide and 10 ⁇ g HEL in saline, injected ip. Antibody titers were determined from serum obtained 8 days after secondary boosts. Splenic memory T cell responses were measured in vitro 6-8 weeks following these secondary challenges.
  • HEL hen egg lysozyme
  • LN cells were restimulated in vitro with synthetic peptide or 25-50 ⁇ g/ml purified protein derivative (PPD, Connaught, Swiftwater, PA) in complete RPMI with 0.5% heat-inactivated autologous mouse serum (Jackson Immunochemicals, West Grove PA). On day 3, cultures were pulsed with 1 ⁇ Ci/well of [ 3 H]thymidine and incubated an additional 14-
  • Tg mice or adoptively transferred recipients were immunized with a chemical conjugate of cysteine-modified
  • Tg or NTg control mice were immunized ip with 50 ⁇ g 12-26-HEL emulsified 1 :1 in CFA and then boosted with 10 ⁇ g of the same conjugate in saline 2 weeks later.
  • Titers of IgG antibodies specific for the peptide- or HEL were determined by ELISA 8 days following this boost.
  • Irradiated (400 rad) BALB/c recipients were adoptively transferred (iv) with 5 x 10 7 splenocytes from previously tolerized BALB/c, and boosted ip with 100 ⁇ g 12-26-HEL conjugate in incomplete Freund's adjuvant (IF A). Serum bleeds were collected 8 days following this boost, and antibody titers determined by ELISA.
  • Tg B cells The ability of Tg B cells to directly present endogenous 12-26 peptide was assessed with T-cell hybridoma 9C127 which recognizes 12-26 peptide in the context of 1 A d .
  • Tg or control littermate B cell APC were purified as described above, and recultured in varying numbers in 200 ⁇ l microcultures with 10 4 9C127 cells/well in complete RPMI with 5% FCS. Supernatants were harvested 48 hours later, and multiple dilutions were assayed for IL-2 production as above.
  • ELISA determinations of serum peptide-specific or HEL-specific IgG responses were performed by coating plates with 50 ⁇ g/ml synthetic peptide or 5 ⁇ g/ml HEL and following standard ELISA protocols. Briefly, antigen-coated plates were blocked with 1% gelatin/0.05% Tween 20 buffer, and duplicate serial dilutions of serum were incubated and probed with goat anti-mouse
  • IgG isotype-specific secondary reagents conjugated to alkaline phosphatase. Titers are expressed as the geometric mean of the reciprocal dilution required to bring A 490 readings to prebleed levels or ⁇ 0.09 O.D.
  • 12-26-IgG H chain protein was detected in serum of Tg mice via its ability to bind to the NIP (5-iodo-4-hydroxy-3-nitrophenylacetyl) hapten using a modified NIP- binding ELISA (Grosschedl, R. et al. (1984) Cell. 55:647-658). Dilutions of sera from Tg mice were incubated on ELISA plates coated with NIP-gelatin or NIP-BSA conjugates (10 ⁇ g/ml), and subsequently probed with goat anti-mouse IgG,-AP as a secondary reagent. Detection of the 12-26 epitope in Tg sera could be demonstrated by similarly using NlP-sepharose beads (from Dr. T.
  • Tg mice expressing the engineered genomic (rearranged) H chain construct driven by its endogenous immunoglobulin promoter/enhancer sequences (Example I). Tg founders possessing 2-3 integrated copies were identified via Southern blotting of genomic tail biopsy
  • Serum expression of the NP-binding Tg H chain was detected as described by Grosschedl et al, supra. Since the Tg V H region binds with high affinity to the NIP hapten in combination with ⁇ l light chains, functional 12-26-IgG was detected indirectly with a NIP-binding IgG, ELISA Although probably representing a fraction (only ⁇ light chain-associated) of expressed Tg serum protein, NIP-binding IgG, assays revealed that Line 17 and 5 expressed between 1000-25000 ng/ml and 50-1000 ng/ml, respectively. The higher serum expression for Line 17 mice correlated with increased expression of surface IgG, in splenocytes as compared to Line 5 or NTg littermates.
  • Tg mice expressing foreign "neo" self-antigens have firmly established that tolerance induction can readily occur for membrane-bound and soluble proteins which are expressed ubiquitously, or in a tissue-specific manner during normal development (Goodnow, CC (1992) Ann. Rev. Immunol. 70:489-518; Miller, J.F.A.P. et al (1992) Ann. Rev. Immunol 70:51-69).
  • Tg animals expressing a model immunodominant epitope as part of serum IgG protein were similarly unresponsive to an immunogenic challenge with the epitope. Since the
  • 12-26 peptide contains both a T-cell and a B-cell epitope, we could measure both cellular and humoral immune responses to this relatively simple determinant with immunization assays. Draining LN cells from subjects who received SC injection of synthetic peptide in CFA and which were subsequently restimulated with antigen displayed a profound proliferative unresponsiveness and IL-2 production.
  • NTg mice (H-2 d ) primed with peptide in adjuvant and followed by a subsequent boost of peptide in saline 2 weeks later, developed an extremely high titer serum antibody response dominated by antibodies of the (Th2 -mediated) IgG, isotype (Soloway. et al, supra).
  • Profound humoral unresponsiveness was observed in Tg animals immunized in this manner (Fig. 9A). This could not be due to immune- complex binding with circulating serum fig (12-26-IgG) since these tolerant animals had diminished splenic memory T cell responses to 12-26 peptide (Figure 9B).
  • the extent of cellular and humoral unresponsiveness was comparable for both Line 5 and Line 17 suggesting that even lower levels of expression (Line 5) efficiently satisfied antigenic thresholds for tolerance induction.
  • Tg BM chimeras were constructed. Sublethally irradiated adult BALB/c mice were injected with 1 :1 mixtures of Line 17 Tg and NTg littermate BM, and the recipient's immune system was allowed to redevelop for 7-8 weeks in the presence of fig (12-26-IgG)-producing lymphoid tissue. Such treatment followed by immunogenic challenge with synthetic peptide revealed profoundly suppressed cellular and humoral ( Figure 10) peptide-specific immunity in these normal adult recipients.
  • Resting B cells are known to be competent in antigen processing and presentation functions, but have been described to possess defective costimulatory ability, in contrast to LPS- or surface Ig-activated B cell blasts which express abundant B7-1, B7-2, and CD40.
  • injection of a variety of different 12-26-IgG- expressing lymphoid preparations including Percoll gradient-purified resting B cells, LPS-activated blasts, crude BM , and even crude splenocyte preparations, were all highly effective in diminishing humoral (Figure 11) and cellular immune responses to the 12-26 peptide in adult recipients.
  • line 5 and 17 Tg mice were challenged with peptide conjugated to a different carrier, hen egg lysozyme (HEL), as a source of T cell help for potentially tolerized B cells.
  • HEL hen egg lysozyme
  • Potentially self-reactive anti-peptide B cells can receive foreign-reactive T cell help from HEL-specific T cells to produce autoantibodies.
  • Primed recipients received one of four preparations: (1) Percoll purified resting B cells, (2) crude BM cells, (3) LPS-activated B cell blasts, or (4) chemically-fixed B cells. One week later, they were boosted with peptide in saline, and humoral immune responses were subsequently determined. Although both resting B cells and crude BM cells produce specific unresponsiveness in antigen-naive recipients, both were ineffective in diminishing peptide-specific humoral immunity in previously primed subjects (Figure 13 A). LPS- activated Tg B cells completely reversed the ongoing immune response (Figure 16B). A significant reduction in anti-peptide antibody titers was also produced by treatment with fixed Tg B cells ( Figure 13C).
  • antigen-presenting B cell may be engineered to express immunoglobulins which contain within their structure tolerogens that can be employed to manipulate an undesired immune responses.
  • immunoglobulins which contain within their structure tolerogens that can be employed to manipulate an undesired immune responses.
  • Transgenic peptide-Ig chimeric molecules have the potential to be presented directly or secreted and re-presented, making it likely that tolerance induction by injected of peptide-expressing lymphoid tissue occurs via multiple pathways. This may also explain the potency of the fig tolerogens. Secretion of the fig tolerogen by activated transgenic B cells and re-presentation by non-transgenic APC may provide an additional tolerogenic pathway. This is supported by our observations that high doses of soluble peptide-IgG, or very low doses of fig from secreting transfected cells, upon injection d in vivo, are sufficient for inducing tolerance (Zambidis et al, supra). T-cell clonal deletion has been described in other transgenic models in which soluble self-Ig antigenic determinants were presented in the periphery or in the thymus (53,
  • a relatively low level of production of soluble and/or membrane fig capable of interacting with surface IgM molecules specific for the foreign epitope may explain why such cells were relatively less tolerogenic for the B cell compartment.
  • activated B cells with increased secretion of fig were more efficient B cell tolerogens, and were the only preparation we tested which could shut down an ongoing immune response.
  • Peripheral deletion of mature lymphocytes resulted from an exhaustive immune response ("propriocidal regulation") which was IL-2-dependent and mediated by the apoptosis-regulating surface molecules Fas and Fas ligand (Crispe, LN. (1994) Immunity. 7:347-349; Critchfield, J.M. et al. (1994) Science. 265:1139-1143; Singer, G.G. et al. (1994) Immunity. 7:365-371; Pulendran, B. et al. (1995) Nature 575:331-334; Shokat, K.M. et al. (1995) Nature 575:334-338; Lenardo, M.J. (1991) Nature 555:858-861).
  • a fig construct comprising a selected foreign epitope or epitopes in peripheral B cells using gene therapy strategies has great practical utility for modulating humoral and cellular immune responses.
  • genetic transfer and expression of tolerogens in lymphoid APC requires only knowledge of the DNA sequence encoding the target epitope towards which tolerance is desired.
  • the present method avoids the cumbersome antigen purification synthesis steps .
  • clinically useful tolerance would require that the antigen (tolerogen) persist, its genetic expression in long-lived APC or pluripotential hematopoietic stem cell precursors provides a means for achieving the requisite persistence.
  • the present inventors have also induced peptide-specific tolerance by expression a fig construct in peripheral B cells or hematopoietic stem cells using retroviral-mediated gene transfer.
  • Peripheral B Lymphocytes One potential strategy for the induction of clinically relevant tolerance is indirectly related to the original demonstration by Medawar's group of tolerance induction to foreign MHC antigens via injection of allogeneic hematopoietic cells into neonates (Billingham et al, supra). In adults, attempts to induce tolerance to foreign grafts by injecting accessory-cell depleted splenocytes (Ryan, J.J., et al. (1984) J. Immunology 755:2343-2350; Hori, S., et al. (1989) J. Immunology 745:1447-1452) or syngeneic transfected cells (Madsen, J.C, et al.
  • 12-26-IgG ⁇ H chain cDNA was derived by RT-PCR from J558L myeloma cells, transfected with the rearranged genomic construct (Examples I-III; Zambidis et al, supra) and subcloned into retroviral vector MBAE (Kang, J., et al. (1990) Proc.
  • Gl 3' primer TCGGAAGGGTCGACGGATCATTTACCAGGAGA (SEQ ID NO:208)
  • a high titer (10 ⁇ -10 ⁇ neomycin-resistant NIH 3T3 CFU/ml) ⁇ -2 packaging line (F6P) was prepared with recombinant plasmid MBAE.BAK, and assayed for helper virus via horizontal spread of neomycin resistance with NIH 3T3 cells.
  • Ecotropic F6P was prepared by "ping-pong" amplification using amphotropic line PA317. Producer lines were stored in liquid nitrogen and freshly thawed cells were utilized for each individual experiment.
  • B cell lines CH31, A20, J558L, and NS-1 (ATCC, Rockville, MD) were transduced with recombinant retrovirus via co-culture with adherent F6P cells for 24- 48 hours in the presence of 6 ⁇ g/ml polybrene (Sigma). Cells in suspension were washed and recultured in 1 mg/ml G418 for selection of stable transductants prior to genomic Southern blot, RT-PCR, ELISA, or antigen-presentation studies. Infection of BM progenitors and quantitation of G418-resistant colony-forming cells (CFC) has been described (Keller, G., et al.
  • CFC G418-resistant colony-forming cells
  • BM was harvested from femurs and tibiae of 6-8 week old BALB/c donors injected IV with 150 mg/kg 5-fluorouracil
  • Erythrocyte-depleted BM was co-cultured (5xl ⁇ 6/ml) with irradiated (2000 rads) F6P or ⁇ -2 parental cells (mock transduction).
  • Ten ml cultures in complete RPMI 1640 with 15% FCS were incubated at 37° C, 5% CO2 for 48 hours, and included 200 U/ml each of IL-3, IL-6, and IL-7 (Genzyme). 4 ⁇ g/ml polybrene was added to co-culture during the last 24 hours of infection.
  • Splenic B cells were similarly infected in vitro via co-culture with viral- producing F6P or parental ⁇ -2 (mock transduction).
  • Peripheral B cells were purified with anti-T cell antibody cocktail plus complement and Percoll density gradients (60- 70% layers). Purified B cells were pre-stimulated with 50 ⁇ g/ml bacterial
  • mice received an additional ip boost of 50 ⁇ g peptide and 10 ⁇ g HEL in saline.
  • Antibody titers were determined from serum bleeds 8 days after secondary boosts. Splenic memory T cell responses were measured in vitro 6-8 weeks following these secondary challenges by reculturing purified T cells (3xl0 ⁇ /ml) with irradiated (2500 rads) BALB/c splenocytes
  • Serum peptide-specific or HEL- specific IgG responses were determined by ELISA as described (supra). Cellular responses from draining popliteal and inguinal LN cells were assayed 9 days after SC immunization with 20 ⁇ g peptide in CFA. Cultures were pulsed with [ ⁇ Hjthymidine, harvested and counted as described above) IL-2 and IL-4 cytokine production was quantitated as above. Dilutions of anti-IL-2 mAb S4B6 and anti-IL-4 mAb 11B11 (ATCC) were included to confirm specificity. IFN- ⁇ was measured using a commercial ELISA kit (Intertest- ⁇ , Genzyme). 3. RT-PCR and immunologic methods
  • RNA from various tissue was reverse-transcribed (2 rounds) with AMV reverse transcriptase, dNTP's, and oligo dT and random hexamer primers
  • oligo Ig-one a g-32p-labeled oligonucleotide encoding 12-26 but which does not overlap with the 3' PCR primer: TGATCTACTGCAGCTGGAGGACGCGCGCGGCGG (SEQ ID NO:210).
  • Tissue RNA samples were compared via b-actin RT-PCR using commercially available primers (Stratagene).
  • 12-26-IgG H chain protein was detected in culture supernatants of transduced cell lines, or in sera of mice injected with gene-transferred cells, via its ability to bind to the NIP hapten using a modified NIP-binding ELISA as above. Briefly, dilutions of culture supernatants or sera were incubated on ELISA plates coated with NIP- gelatin conjugate (and subsequently probed with goat anti-mouse IgGi -AP. Standard curves with affinity-purified 12-26-IgG from supernatants of transfected J558L were used for quantitation.
  • a recombinant retroviral vector (Kang, J., et al, supra ) was modified by inserting a PCR-derived cDNA encoding the 12-26-1 gG H chain sequence ( Figure 14), and a high titer ecotropic packaging line (F6P) was generated for the in vitro infection of cell lines and hematopoietic tissue via co-culture methods (Keller, G., et al. (1985)
  • 12-26-IgG H chain can assemble with endogenous light chains in transduced B cell lines, to be expressed as a membrane surface protein, or secreted into cultured supernatants (50-80 ng/ml) inNS-1 and J558L myelomas.
  • Immunoprecipitation of secreted 12-26-IgG and immunoblot analysis with a peptide-specific monoclonal antibody (B3.l l) could directly demonstrate the expression of 12-26 peptide.
  • retrovirally-synthesized gene products are expected to give rise primarily to processed peptides presented by MHC class I molecules
  • endogenously- derived peptides can also be routed to endocytic class II MHC compartments in some cases (Weiss, S., et al. (1991) Cell 64:161-116).
  • Such a pathway should be enhanced for retrovirally-encoded 12-26-IgG H chain due to the efficient nature of the Ig secretory pathway in targeting the endosomal compartment.
  • BM chimeras were produced in sublethally irradiated (200-650 rads) BALB/c mice by infusing 5-fluorouracil (FU)-pretreated donor BM which had been co-cultured with F6P.
  • FU 5-fluorouracil
  • This protocol leads to newly developing lymphocytes and APC (lymphoid and non-lymphoid) that are derived from both the host, as well as the transplanted BM progenitors expressing 12-26-IgG. Mice were immunized 4-12 weeks post-infusion and specific immune responses were measured. Analysis of hematopoietic tissue
  • syngeneic BM from SCID mice was gene-transferred the tolerogenic activity of myeloid APC was analyzed. Although hematopoietic tissue from SCID mice is deficient in developing mature lymphoid cells, the APC function of cells of the myeloid (non-lymphoid) lineage remains intact (Dorshkind, K. et al.
  • the approach comprised stimulating Percoll® gradient-purified splenic B cells to proliferate with bacterial LPS, brief co-culture with F6P, and subsequent iv injection into normal, immunocompetent (non-irradiated) BALB/c recipients. This treatment resulted in an efficient suppression of peptide-specific humoral immunity comparable to that observed in the BM chimera experiments described above.
  • the bivalent nature of the secreted form of the tolerogenic epitope on the two H chains of the Ig-molecule can independently mediate effective peptide-specific B cell tolerance, probably via Fc-mediated antibody feedback mechanisms (Zambidis et al, supra).
  • the potency of tolerance induction using the present invention can exploit multiple pathways in the immune mechanism.
  • tolerogenic peptide-Ig constructs facilitates "tailor-designing" the immune response to a whole antigen by selectively inducing immunity (Zaghouani,
  • fusing an foreign antigenic sequences at the N-terminus is not limited by size restrictions, and can thus be adapted for expressing large multi-epitope antigens, for example, autoantigenic proteins such as factor VIII (Allain et al, supra), myelin basic protein (Higgins et al, supra; Critchfield et al, supra ), or glutamic acid decarboxylase (Tisch et al, supra).
  • factor VIII Allain et al, supra
  • myelin basic protein Higgins et al, supra; Critchfield et al, supra
  • glutamic acid decarboxylase Tisch et al, supra.
  • tolerogen as a gene sequence has many advantages over present tolerance induction protocols, since only the cDNA sequence of the target antigen, for example, one or more HIV gpl 20 epitopes, needs to be known. This avoids the need for a protein purification strategy. More importantly, since experimentally acquired tolerance eventually wanes, expression and persistence of the tolerogen in long-lived or multipotential hematopoietic tissue has the potential to modulate permanently a specific immune response.
  • an important application of the genetic tolerogenesis method of the present invention is to help eliminate genetically-altered cells encountered in gene therapy protocols.
  • Autologous cells genetically modified with adenoviral and retroviral vectors are known to induce immunity in a competent recipient due to immune recognition of vector-encoded products leading to subsequent elimination of transduced cells via both cellular and humoral immunity (Yang et al, supra).
  • immunity to low-level expression of viral proteins of first-generation El -deleted adenovirus can undoubtedly be reduced with further genetic manipulation of the vectors, rejection of the foreign transgenes expressed by such vectors remains an even more significant obstacle (Tripathy et al, supra).
  • lymphohematopoietic APC Although solid evidence exists for the tolerogenic role of lymphohematopoietic APC in irradiated bone marrow chimeras,
  • B7-2 (Hathcock, K.S., et al. (1994) J. Exp. Med. 750:631-640). Antigen-presentation by resting B cells has thus far been successful in inducting tolerance in naive recipients, but has proven ineffective in primed (Fuchs et al, supra) or allo-MHC- reactive recipients (Buhlmann et al, supra) unless an anti-gp39 (CD40-ligand) antibody was simultaneously injected to prevent upregulation of B cell costimulatory function.
  • costimulation-competent LPS blasts could serve as efficient tolerogenic APC in vivo in antigen-naive recipients, or could induce tolerance in vitro in previously activated T cell clones (Gilbert et al, supra).
  • transgenic mice expressing the fig construct specifically in the
  • mice transgenic for the human CD4 gene were immunized with 20 ⁇ g of gpl 20 in complete Freund's adjuvant, boosted with gpl 20 in incomplete adjuvant and then injected intravenously with 1 ⁇ g of gpl20 in PBS.
  • Peripheral blood lymphocytes were harvested at various times after the last injection (of soluble gpl 20). Total number of T cells in the peripheral blood were evaluated using flow cytometry to enumerate CD3 + cells. Table VI, below shows the results as percent of total blood lymphocytes which are CD3 +
  • HIV gpl 20 crosslinking induces apoptosis of CD3 cells in vivo in human CD4 transgenic mice
  • mice which had been immunized that is, mice expressing the human CD4 molecule which can bind gpl 20 or gpl20-anti-gpl20 complexes.
  • mice were immunized as above. Spleens were harvested 9 days after this last injection and were cultured with medium or with anti-CD3 mAb (145.2C1; 50 ⁇ g/ml coated wells) for 24 hours; cells were then harvested, fixed and assayed for apoptosis by propidium iodide uptake and flow cytometry. The percent of hypodiploid, apoptotic cells at 24 or 48 hours with anti-CD3 and at 24 hours with anti-IgM are shown in Table VII. Table VII
  • Bkgrnd Background values of apoptosis of freshly isolated cells (as opposed to cells cultured 24 or 48 hours).
  • the HIV retrovirus interacts with the host immune system in a puzzling way. Virtually everyone infected with the virus synthesizes antibodies directed against a number of the viral envelope epitopes. However, much of this humoral response has little if any protective value over the course of HIV pathogenesis [5-16]. Titers of neutralizing antibodies in AIDS patients are low [6], and the antibodies might cross- react with self-components due to molecular mimicry and structural/genetic similarities [7-9]. Furthermore, crosslinking CD4 by anti-CD4 antibodies or gpl 20 and anti-gpl20 antibodies can upregulate Fas expression and prime Th cells for activation- induced apoptosis [1, 10-16].
  • mice transgenic for huCD4 (“huCD4 Tg") and control mice with 20 ⁇ g gpl 20 in CFA led to titers exceeding 1 :10 5 within three weeks.
  • huCD4 Tg mice transgenic mice transgenic for huCD4
  • mice (CB6 FI) mice that had been immunized with rgpl20 SF2 mice (CB6 FI) mice that had been immunized with rgpl20 SF2 .
  • CB6 FI mice that had been immunized with rgpl20 SF2 .
  • the same amount of rgpl20 was also administered i.v. to unprimed huCD4 Tg mice and nontransgenic controls.
  • Boosting gpl20-primed control mice with gpl 20 gave rise to increased numbers of T and B cell as well as in the antibody titers.
  • boosting the primed huCD4 Tg mice (which express huCD4 on both T and B cells) with soluble gpl 20 resulted in lower secondary antibody titers than in controls.
  • the response to an irrelevant antigen, HEL was also reduced in the gpl 20 -primed and boosted huCD4 Tg mice.
  • the number of peripheral T cells and B cells in immunized huCD4 Tg decreased to 50% of the control levels.
  • mice BALB/cByJ and CB6 FI mice were purchased from the Jackson Laboratories (Bar Harbor, ME) at 6-10 weeks of age, and housed in pathogen-free, microisolater cages. Line 313 huCD4 Tg mice were obtained from Dr. Terri Finkel, Denver, CO). These transgenics were originally produced by Dr. Richard Flavell by injecting a huCD4 transgene into fertilized eggs and were maintained by repeated backcrosses on the C57B1/6 background [17].
  • the F, offspring between huCD4 Tg mice and BALB/cByJ are produced in our animal facility by crossbreeding female BALB/cByJ with male huCD4 Tg mice to yield huCD4 expressing mice histocompatible with CB6 FI mice.
  • the huCD4 molecule was shown to be functional in calcium signal transduction and in overcoming the block in positive selection induced by in vivo injection of mAbs to the endogenous mouse CD4 [17]. Since in these transgenic mice, expression of huCD4 is driven by CD2 regulatory elements, both B and T cells express huCD4.
  • RPMI 1640 medium Gibco-BRL, Gaithersburg, MD
  • heat-inactivated 5% fetal calf serum Hyclone, Logan, UT
  • 50 ⁇ M 2-mercaptoefhanol 2 mM L-glutamine
  • 100 U/ml penicillin 100 U/ml streptomycin
  • MEM nonessential amino acids 100 mM sodium pyruvate was used.
  • FITC- labeled anti-hamster IgG PE-labeled anti-mouse CD3, biotin-labeled anti -mouse CD 19 and anti-Fas antibody Jo2
  • FITC- labeled mouse anti-human CD4 mAb (FITC- Leu-3a) was obtained from Becton Dickinson (View Mountain, CA).
  • biotin- or FITC- labeled antibodies were purified in our lab by standard protocols: anti-mouse CD3 (145.2C11), anti-mouse CD4 (GK1.5), anti-mouse CD8 (53-6.72) and anti-mouse CD45R (RA36B2, B220).
  • Anti-gpl20 antibodies used for huCD4 crosslinking were obtained as follows: human monoclonal anti-gpl20 antibodies directed against gpl20 C-terminal peptide, (450-30D 100,100,1,1; abbreviated ' ⁇ SO-
  • HIV-1 SF2 gpl 20 in IF A intramuscularly after 2 weeks and 1 month of primary immunization, respectively.
  • the antibody titer was >1/10 5 as determined by ELISA using rgpl20 coated plates (1 ⁇ g/ml in Tris coating buffer, pH 9.0).
  • mice and Line 313 huCD4 transgenic mice were immunized intradermally with 20 ⁇ g HIV-1 SF2 rgpl20 emulsified in CFA and boosted intramuscularly with 20 ⁇ g rgpl20 in IF A 9-12 days later.
  • the percentages of human CD4+, mouse CD3+, CD8+ and B220+ cells in the peripheral blood were followed using dual color flow cytometry described below.
  • the spleens were harvested within 9 days after gpl 20 iv injection and the percentages of splenic CD3+ T cells were determined by flow cytometry.
  • the apoptotic cells in freshly harvested spleens were assessed by in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining (TUNEL). Spontaneous apoptosis and anti-CD3 activation- induced apoptosis was measured after anti-CD3 in vitro treatment for 24-72 hr.
  • the F, offspring between huCD4 Tg and BALB/cByJ mice, as well as CB6 FI control mice were immunized intradermally with 20 ⁇ g HIV-1 SF2 rgpl20 and 20 ⁇ g hen egg-white lysozyme (HEL, Sigma Chemical Co., St. Louis, MO) emulsified in CFA.
  • HEL HIV-1 SF2 rgpl20
  • hen egg-white lysozyme HEL, Sigma Chemical Co., St. Louis, MO
  • the CD3+ T cells were determined by flow cytometric analysis and the gpl 20 and HEL specific IgG responses were measured by ELISA by coating plates with 1 ⁇ g/ml gpl 20 or 1 ⁇ g/ml HEL, respectively.
  • Antibody titers were determined using CA-Cricket Graph software and were expressed as the serum dilution that would bring the OD to pre-immunization levels (OD 405 s. 0.04), assuming parallelism of curves.
  • mice were sacrificed on day 1, 4, 7, 11, and 20 after a single bolus of gpl 20 iv, and lymph nodes and spleens were harvested,and determinations made of cell phenotypes, spontaneous apoptosis and anti-CD3 stimulation index.
  • Flow cytometric analysis
  • the surface level of Fas expression on the splenocytes from Line 313 huCD4 transgenic mice after huCD4 crosslinking was measured by staining cells with hamster anti-Fas antibody (Jo2), followed by FITC-labeled anti-hamster IgG.
  • Jo2 hamster anti-Fas antibody
  • FITC-labeled anti-hamster IgG To determine the percentage of peripheral CD3+ T cells, blood was removed from the retroorbital plexus.
  • White blood cells were prepared by lysing red blood cells with Tris-buffered ammonium chloride buffer (pH 7.2).
  • PI fluorescence of individual cells was measured using flow cytometry.
  • Cell debris and clumps were excluded by gating for single cells by forward and side light scatter and by FL-2 area vs. FL-2 width.
  • a distinct cell cycle region of apoptosis (AQ) could be identified below the G Q /G, diploid peak and the percentage of cells in the A 0 region was quantitated.
  • crosslinking huCD4 by gpl 20 and anti-gpl20 antibody er se induced apoptosis in the huCD4 Tg splenocytes, though to a lesser extent than that with additional anti-CD3 treatment.
  • gpl 20 alone nor anti-gpl20 antibody alone had any effect on the priming and apoptosis induction, suggesting that anti-gpl20 antibody was required for huCD4 crosslinking-mediated apoptosis induction in vitro.
  • Apoptosis induction with anti-CD3 (%) refers to the percentages of apoptotic cells after crosslinking of huCD4 crosslinking with anti gpl 20 antibodies and after 24 hr of treatment with anti-CD3. Fas upregulation by huCD4 crosslinking for 45 min. was calculated as the % increase in median fluorescent channel over medium control.
  • the first bolus of gpl 20 induced a significant, though transient, loss of peripheral CD3+ T cells in huCD4 transgenic mice, but not in CB6 FI control mice.
  • repeated gpl 20 iv injections afterwards were not able to produce a state of long-lasting T cell loss, neither to induce the T cells to decline again after recovery from the first drop, though a slight lower level of CD3+ T cells were constantly observed after gpl 20 injections in the huCD4 transgenic mice than in the controls.
  • gpl20 i.v. injection also resulted in a loss of peripheral blood CD19+ B cells which express huCD4 driven by CD2 promoter but not the CD3-/CD 19-cells in the gpl20-immunized huCD4 transgenic mice, in the same pattern as the loss of
  • CD3+ T cells CD3+ T cells.
  • T cell depletion via apoptosis is not restricted to peripheral blood, but also occurs in spleen and lymph nodes.
  • T cells in spleen and lymph node are also deleted in immunized huCD4 transgenic mice receiving gpl 20 iv injection, we harvested spleens and lymph nodes 1 - 20 days after the first gpl20 i.v. injection and measured CD3+ T cells by flow cytometry. The numbers of splenic CD3+ T cells in all gpl20- immunized and -pretreated huCD4 Tg were decreased to 50-75% of those in control groups.
  • Boosting with gpl 20 not only boosts specific T and B cells for secondary responses, but the gpl 20 can also bind to huCD4 receptors to prime for apoptosis on all huCD4+ cells.
  • HEL the response to HEL as an irrelevant control response although HEL-specific T and B cells would be expected to bind gpl 20 to their huCD4 receptors like gpl 20 specific cells.
  • the results showed that huCD4 Tg mice primed with soluble gpl 20 had lower secondary titers than did controls, and the response to an irrelevant antigen, HEL, was also reduced in the gpl 20 - primed/boosted huCD4 Tg mice.
  • TCR ligation induces further apoptosis in the spleens of immunized huCD4 transgenic mice
  • CD3+ T cell population was depleted by 50%. Numbers of CD3+ cells then recovered to pre- injection levels within two weeks after a bolus of gpl 20 injection.
  • CD3+ T cells were primed for apoptosis by in vivo huCD4 crosslinking, we harvested spleens after a single bolus of gpl 20 and assayed in vitro the apoptosis stimulated by anti-CD3 antibody.
  • Anti-CD3 treatment resulted in lower stimulation in spleen cells from gpl 20 - immunized and -pretreated huCD4 Tg compared to CB6 FI control mice.
  • Anti-CD3 treatment also increased the percentage of apoptotic cells compared to the medium control treatment only in immunized huCD4 Tg mice receiving a bolus injection of gpl 20 .
  • huCD4 expression of huCD4 is driven in these mice by the CD2 promoter, which results in expression in both T and B cells (over 80% of splenocytes).
  • CD2 promoter results in expression in both T and B cells (over 80% of splenocytes).
  • Our results indicate that huCD4-expressing B cells may also be depleted in vivo.
  • a very small number of B cells (0.1-1%) express CD4 molecules annd their function in human is still unclear, as is their fate in HIV infection.
  • the present results suggest that huCD4 crosslinking- transmitted death signal might not necessarily require association with TCR/CD3 signaling pathway and that, under appropriate circumstances, huCD4 crosslinking is enough to send the death signal and induce the cells to die.
  • Annexin V binds to phosphatidylserine, which is translocated from the inner side of the plasma membrane to the outer layer and becomes exposed at the external surface on early apoptotic cells [41].
  • huCD4 transgenic mice produced a large amount of anti-gpl20 antibodies after the first bolus of gpl 20, circulating in the bloodstream, gpl 20 injected thereafter may not be able to compete for binding to huCD4 molecules, tthe relatively rapid recovery of CD3 T cells in vivo may reflect the small amount of available gpl 20 when delivered to the bloodstream of immunized mice as a bolus, in contrast to the small but steady production of viral gpl 20 by HIV in infected individuals. Recent studies [16] with non-tolerant gpl 20 transgenic mice are encouraging for the validity of this model.
  • mice were sublethally irradiated with 400 rads and injected with mock-transduced or 1-102 -IgG gene-transduced bone marrow cells. Mice were primed and boosted with pi -102 and HEL in CFA. Antibody responses were measured in ELISA by coating plates with 50 ⁇ g/ml synthetic peptides (peptides).
  • mice were sublethally irradiated as above and injected with mock-transduced or pi -102 transduced or pi -102 -IgG gene-transduced bone marrow cells. Mice were later primed and boosted with pl-102 and HEL in CFA. Antibody responses were assayed and analyzed as above.
  • mice that had been gene-transferred with MB AE- 1 - 102-IgG were specifically hyporesponsive to the pi -102 protein (and this occurred in a strain-specific manner). That is, Balb/c and C57B1/6 mice recognize epitopes contained in residues 12-26 and 73-88, respectively, whereas F, hybrid mice between these strains recognize epitopes at both sites. Gene-transfer produced tolerance in Fi mice to the whole pi -102 protein, as well as to the major determinants. No "epitope spreading" to minor epitopes was observed.
  • MHC-haplotype-specific manner to the immune system and induce profound tolerance This results is directly applicable to treatment of autoimmune diseases, as well as for creating a receptive environment for foreign or otherwise immunologically "unacceptable" proteins to be administered in the context of gene therapy.

Abstract

L'invention concerne des protéines d'immunoglobuline de fusion (fIg) comprenant un ou plusieurs épitopes hétérologues associés à des maladies pour lesquelles la réponse immunitaire est négative, lesdites protéines étant utiles pour induire une tolérance auxdits épitopes. Les épitopes gp120 du HIV-1 associés à l'intérieur d'un cadre à une chaîne lourde (H) d'Ig sont des produits de synthèse utiles pour induire une tolérance au HIV, spécifique aux épitopes. Le traitement d'un sujet avec ce type de produit de synthèse, ou bien avec des cellules lymphoïdes ou hématopoïétiques exprimant ou sécrétant ces molécules fIg, induit une tolérance immunologique spécifique aux épitopes considérés. En empêchant la production d'anticorps dirigés contre les épitopes gp120 déterminés, cette tolérance peut prévenir ou inhiber l'apoptose de 'voisinage' sur les cellules T hôtes non infectées ayant effectué une liaison, à leurs molécules CD4 de surface, avec la protéine gp 120 du HIV et subissant ensuite une réticulation sous l'effet d'anticorps parasites anti-gp120. Cela donne pour les cellules T en question une amorce à l'apoptose, en présence des antigènes qui les activent. On utilise de préférence les épitopes gp120 correspondant à des épitopes de lymphocites B non neutralisants ou certains épitopes de lymphocytes T auxiliaires pour élaborer les molécules fIg. Outre les chaînes H de fIg et les molécules d'Ig complètes, l'invention concerne l'ADN codant les chaînes H en question et les cellules transformées par ce type d'ADN.
PCT/US1998/002766 1997-02-13 1998-02-13 Tolerance immunologique aux epitopes du hiv WO1998036087A1 (fr)

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US8420099B2 (en) 1999-03-16 2013-04-16 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Chimeric protein for prevention and treatment of HIV infection
WO2001032714A1 (fr) * 1999-11-03 2001-05-10 King's College London Molécules hybrides de recombinaison
US8809290B2 (en) 2002-09-20 2014-08-19 Multicell Immunotherapeutics, Inc. Methods and compositions to generate and control the effector profile of T cells by simultaneous loading and activation of selected subsets of antigen presenting cells
WO2005097822A1 (fr) * 2004-04-09 2005-10-20 University Of Manitoba Identification de la sequence precise d'acides amines de l'epitope identifie par l'anticorps monoclonal igg1b12 puissant de neutralisation anti-vih 1 humain
EP1937300A2 (fr) * 2005-08-17 2008-07-02 Multicell Immunotherapeutics, Inc. Méthodes et compositions pour produire et contrôler le profil effecteur de lymphocytes t par chargement et activation simultanés de sous-ensembles sélectionnés de cellules présentatrices d'antigène
EP1937300A4 (fr) * 2005-08-17 2009-08-12 Multicell Immunotherapeutics I Méthodes et compositions pour produire et contrôler le profil effecteur de lymphocytes t par chargement et activation simultanés de sous-ensembles sélectionnés de cellules présentatrices d'antigène
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WO2015077789A2 (fr) 2013-11-25 2015-05-28 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Récepteurs d'antigènes chimériques pour lutter contre une infection par le vih
US10246505B2 (en) 2013-11-25 2019-04-02 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chimeric antigen receptors to control HIV infection
US11046959B2 (en) 2015-03-30 2021-06-29 The Board Of Regents Of The Nevada System Of Higher Education On Behalf Of The University Of Nevada, Las Vegas Compositions comprising TALENs and methods of treating HIV
US11957762B2 (en) 2015-05-29 2024-04-16 University Of Utah Research Foundation Immune tolerant and non-immune tolerant elastin-like recombinant peptides and methods of use
WO2016207782A1 (fr) * 2015-06-22 2016-12-29 STRELNIKOV, Evgeny Oligopeptides synthétiques immunogènes pour un vaccin contre le vih
US20190092818A1 (en) * 2017-09-22 2019-03-28 Kite Pharma, Inc. Chimeric polypeptides and uses thereof
US11572388B2 (en) 2017-09-22 2023-02-07 Kite Pharma, Inc. Chimeric polypeptides and uses thereof
US11198722B2 (en) 2017-10-06 2021-12-14 University Of Utah Research Foundation Immune tolerant elastin-like peptide tetramer guided nanoparticles and methods of use
CN113621032A (zh) * 2019-11-08 2021-11-09 贵州医科大学 一种具有seq id no.2序列的多肽以及具有强adcc效应的抗体和应用

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