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WO1996036357A1 - Couplage de peptides a des immunoglobulines par l'intermediaire de glucides - Google Patents

Couplage de peptides a des immunoglobulines par l'intermediaire de glucides Download PDF

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Publication number
WO1996036357A1
WO1996036357A1 PCT/US1996/006756 US9606756W WO9636357A1 WO 1996036357 A1 WO1996036357 A1 WO 1996036357A1 US 9606756 W US9606756 W US 9606756W WO 9636357 A1 WO9636357 A1 WO 9636357A1
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Prior art keywords
peptide
carbohydrate
linked
immunoglobulin
conjugate
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PCT/US1996/006756
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English (en)
Inventor
Constantin A. Bona
Y. C. Lee
Teodor-Doru Brumeanu
Philip Dehazya
Original Assignee
Bona Constantin A
Lee Y C
Brumeanu Teodor Doru
Philip Dehazya
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Application filed by Bona Constantin A, Lee Y C, Brumeanu Teodor Doru, Philip Dehazya filed Critical Bona Constantin A
Priority to AU59200/96A priority Critical patent/AU5920096A/en
Priority to EP96916458A priority patent/EP0833663A4/fr
Publication of WO1996036357A1 publication Critical patent/WO1996036357A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—Antibodies
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to methods for coupling peptides to immunoglobulin molecules via galactose or other sugar residues, and to immuno- globulin-carbohydrate-linked peptide ("ICLP") con- jugates produced by such methods.
  • ICLP conjugates have been found to be superior in eliciting an immune response when compared to unconjugated peptide.
  • Isolated peptides are frequently too small and/or too unstable to elicit an immune response by them ⁇ selves, and for this reason, a number of methods have been developed for linking peptides to larger "carrier" molecules. Enhancement of in vivo immunogenicity of such peptides depends upon the structure of the carrier to which they are coupled, as well as the type of intermolecular cross-linking between the peptide and its carrier.
  • One method for linking a peptide to a carrier is chemical conjugation. Chemical conjugates of pro- teinaceous carriers with synthetic peptides have been observed to elicit humoral and cellular anti-peptide immune responses in laboratory animals.
  • peroxidized sugars have been used to cross ⁇ link glycoproteins, including some enzymes.
  • the utility of this chemistry is restricted because cross- linking disturbs the biological functioning of most enzymes (Eyzaguirre, 1987, in “Chemical Modification of Enzymes: Active Site Studies", Ellis Horwood, Chichester, England) .
  • the present invention relates to methods for coupling peptides to carbohydrate moieties normally occurring in immunoglobulin molecules, and to immuno- globulin-carbohydrate-linked peptide ("ICLP") con ⁇ jugates produced by such methods.
  • ICLP immuno- globulin-carbohydrate-linked peptide
  • peptides are conjugated to carbohydrate residues of an immunoglobulin molecule by enzymatically oxidizing the carbohydrate residues to produce aldehyde groups, by reacting the oxidized carbohydrate residues with the peptides such that covalent attachments between the residues and peptides are formed, and by stabilizing the bond between the residues and peptides by reduction using appropriate reducing agents.
  • the enzymati ⁇ cally oxidized carbohydrate residues are galactose residues and galactose oxidase may be used for enzyma- tic oxidation.
  • enzymatic oxidation of galactose residues is preceded by enzyma ⁇ tic removal of terminal sialic acid residues attached to galactose residues.
  • Neuraminidase may be used for the enzymatic desialylation.
  • the present invention is based, at least in part, on the discovery that when a peptide corresponding to amino acid residues 110-120 of the hemagglutinin of influenza PR8 A virus (SEQ ID NO:7) was conjugated to immunoglobulin molecules via carbohydrate residues, the resulting ICLP conjugates were observed to activate HA 110-120 specific T cell hybridoma cells as efficiently as influenza PR8 virus, at levels 40-100 fold higher than the synthetic HA 110-120 peptide itself.
  • Utilizing immunoglobulin molecules as carriers for synthetic peptides offers a number of advantages.
  • Use of ICLP conjugates may not only prolong the half-life of the peptide, but may also, via binding of F c regions of the immunoglobulin molecule to cell surface recep ⁇ tors, recruit elements of the immune system so as to augment and improve the efficiency of the overall immune response.
  • the specificity of enzymes used to link peptide with immunoglobulin substantially reduces the generation of by-products and the creation of undesirable neodeterminants.
  • FIGURE 1 Protocol for the synthesis of immuno- globulin-carbohydrate-linked-peptide ("ICLP") con ⁇ jugates, which describes a preferred embodiment of the conjugation method of the present invention.
  • Sugar moieties of mouse and human immunoglobulins are depleted of N-acetylneuraminic acid (NANA, sialic acid) using neuraminidase from ArthroJa ⁇ ter ureafaciens (Powell and Varki, 1993, in "Sialidases", Green Publishing and John Wiley & Sons, New York, 17.12.1- 17.12.8) and Clostridium perfringens, and the adjacent galactose residues are subsequently oxidized at the carbon-6 position using galactose oxidase (Cleveland et al., 1975, Biochem.
  • the reductive alkyl- ation is favored between the aldehyde group generated enzymatically on the galactose and the ⁇ amino group of the synthetic peptide.
  • the synthetic peptide contains a N-terminal site for cathepsin E (Ala-Ala-Ala-Leu; SEQ ID NO:15) which has been artificially introduced to facilitate quick release of the peptides into the lyso- somal compartment of the antigen processing cells.
  • Schiff bases formed between galactose and peptides are stabilized by reduction with pyridine borane (Cabacungan et al., 1982, Anal. Biochem.124:272) .
  • FIGURE 2 Analysis of mouse IgG-carbohydrate- linked-HA conjugates by SDS -PAGE and Western blot.
  • Mouse monoclonal IgGl was (conjugated, via carbohydrate residues, to HA C 110-120 synthetic peptide, dialyzed against PBS in bags of 100,000 MWCO and aliquots were analyzed by SDS-PAGE under nonreducing and reducing conditions as described in Section 6.
  • Lanes 1 and 5 respectively, represent Coomassie staining of the non ⁇ reduced and reduced IgGl(control)
  • lanes 2 and 6, respectively show Coomassie staining of nonreduced and reduced IgG-carbohydrate-linked-HA conjugates.
  • FIGURE 3 Chromatographic removal of residual, unconjugated HA C 110-120 peptide from the ICLP con ⁇ jugates.
  • a Superose-6 gel filtration column was previously calibrated with molecular weight markers
  • mice IgG-carbohydrate-linked-HA and IgM-carbohydrate—linked-HA preparations were chromatographed as described in Section 6.
  • Major peaks represent either native mouse IgG and IgM or IgG-carbo- hydrate-linked-HA and IgM-carbohydrate-linked-HA con ⁇ jugates.
  • the peptide identification in the peak eluted at 80 minutes was confirmed by IRIA (competitive inhibition radio- im unoassay) and RP-HPLC (reverse-phase HPLC) .
  • FIGURE 4 Specificity of attachment of HA C 110-120 peptide to the sugar moiety of the immunoglobulins.
  • the lack of reactivity of rabbit anti-HA 110-120 antibodies to the heavy chains of the PGN-ase F treated conjugate indicates removal of the N-linked oligosaccharide/HA c 110-120 complex from the heavy chains of mouse IgG-carbohydrate-linked-HA conjugate (lane 2) .
  • FIGURE 5 Estimation of the degree of coupling of HA C 110-120 peptide to mouse IgG.
  • Mouse IgG-carbo- hydrate-linked-HA conjugate was prepared, purified and hydrolyzed with PGNase as described.
  • the amount of the enzymatically detached HA C 110-120 peptide from the con ⁇ jugate (open squares) was estimated by IRIA as a measure of percent inhibition of binding of rabbit anti-HA110-120 antibodies to plates coated with BSA-HA conjugate. Dotted lines show 50 percent inhibition obtained with either HA C 110-120 peptide detached from the conjugate or HA110-120 synthetic peptide (cali ⁇ bration) .
  • the amount of HA C 110-120 peptide (0.9ng) detected in 75 ⁇ l, as used in IRIA (competitive inhi ⁇ bition radioimmunoassay) was related to the cor ⁇ responding amount of mouse IgGl (86.4ng) found in 75 ⁇ l. Since 16.9:1 represents a molar ratio of 1:1 between heavy chain of IgG and HA C 110-120 peptide, and most of the peptides were found attached to the oligosaccharide chains of the heavy chain, the number of peptide units per heavy chain was calculated to be 5.68. This corresponds to an average of 11.4 peptides per molecule of IgG. Data points on the graph represent the mean of triplicate samples + SD.
  • FIGURE 6 Activation of HA110-120 specific, LD1- 24 T hybridoma cells by mouse IgG-carbohydrate-linked- HA conjugates.
  • 2PK3 (APC) cells were incubated with graded amounts of HA110-120 synthetic peptide, NP147- 161 synthetic peptide (control) , UV-inactivated PR8 virus, genetically antigenized Ig-HA, genetically antigenized Ig-NP (control) , enzymatically antigenized mouse IgG-carbohydrate-linked-HA and IgM-carbohydrate- linked-HA conjugates, and their appropriate controls.
  • IgG-carbohydrate-linked-HA and IgM-carbohydrate-linked- HA conjugates were previously rendered free of uncon- jugated HA C 110-120 peptide by extensive dialysis fol ⁇ lowed by size exclusion chromatography.
  • the ability of each of the antigens to specifically activate the LD1- 24 T hybridoma cells was compared at 50% activation as indicated by dotted lines. Data points on the graph represent the mean of quadruplicate samples + SD.
  • FIGURE 7 Western blot analysis of enzymatically antigenized mouse and human immunoglobulin.
  • Various isotypes of affinity purified mouse monoclonal and human myeloma IgG were glycosidically coupled with
  • HA C 110-120 antibodies as described. It should be noted that residual unconjugated HA C 110-120 peptide was not chromatographically removed from these particular con ⁇ jugates.
  • Mouse IgG-carbohydrate-linked-HA (lane 1) and human IgG-carbohydrate-linked-HA (lane 3) , mouse IgM- carbohydrate-linked-HA (lane 5) , human IgM-carbo- hydrate-linked-HA (lane 7) and human IgA-carbohydrate- linked-HA (lane 9) were analyzed in parallel with their appropriate controls: mouse IgG (lane 2) , human IgG (lane 4) , mouse IgM (lane 6) , human IgM (lane 8) , and human IgA (lane 10) .
  • FIGURE 8 T cell activation as a function of the antigen carrier molecule for HA 110-120.
  • Open circles represent HAllO-120 synthetic peptide; clear diamonds represent HA110-120 comprised in the CDR3 loop of an immunoglobulin; solid diamonds represent an IGP conjugate of the HA 110-120 peptide; and open triangles represent the HA110-120 peptide comprised in bromelain released HA protein,
  • HA110-120 immunopotency in the context of various antigen carriers symbols as set forth in (a) .
  • FIGURE 9 T cell activation indices for HA110-120 comprised in various carriers and exposed to (a) irradiated (empty bars) or fixed (solid bars) APCs; (b) chloroquine-treated APCs; or (c) fixed APCs together with anti-Fc gamma receptor and anti-class I inhibitors.
  • FIGURE 10 Diagram of model for antigen presentation by APC to a T helper cell.
  • peptide may be conjugated to an immunoglobulin according to the invention.
  • Peptides for use according to the invention comprise at least 2 and preferably at least five, amino acid residues and may comprise immunogenic epitopes of antigens; suitable antigens include, but are not limited to, antigens associated with pathogens, tumor cells, or "non-self" antigens with respect to a particular individual.
  • Peptides may be biologically active themselves (for example, growth factors, toxins, immune mediators, differentiation factors etc.) or be portions of biologically active proteins.
  • peptides which may be conjugated to immunoglobulins include B cell epitopes.
  • B cell epi- tope refers to a peptide, including a peptide comprised in a larger protein, which is able to bind to an immunoglobulin receptor of a B cell and participate in the induction of antibody production by the B cell.
  • V3 loop the hypervariable region 3 loop of the envelope protein of human immunodeficiency virus (“HIV") type 1 is known to be a B cell epitope.
  • sequence of this epitope varies, the following consensus sequence, corresponding to residues 301-319 of HIV-1 gpl20 protein, has been obtained: Arg-Lys-Ser-Ile-His- Ile-Gly-Pro-Gly-Arg-Ala-Phe-Tyr-Thr-Thr-Gly-Glu-Ile-Ile (SEQ ID NO:l) .
  • B cell epitopes which may be used according to the invention include, but are not limited to, epitopes associated with influenza virus strains, such as Trp-Leu-Thr-Lys-Lys-Gly-Asp-Ser-Tyr- Pro (SEQ ID NO:2) , which has been shown to be an immunodominant B cell epitope in site B of influenza HA1 hemagglutinin, the epitope Trp-Leu-Thr-Lys-Ser-Gly- Ser-Thr-Tyr-Pro (H3; SEQ ID NO:3), and the epitope Trp- Leu-Thr-Lys-Glu-Gly-Ser-Asp-Tyr-Pro (H2; SEQ ID NO:4) (Li et al., 1992, J.
  • Virol. 6 ⁇ :399-404 an epitope of F protein of measles virus (residues 404-414; Ile-Asn- Gln-Asp-Pro-Asp-Lys-Ile-Leu-Thr-Tyr; SEQ ID NO:5; Parlidos et al., 1992, Eur. J. Immunol. 22:2675-2680); an epitope of hepatitis virus pre-Sl region, from residues 132-145 (Leclerc, 1991, J. Immunol.
  • T cell epitopes which may be used are known or may be identified by methods known in the art, as set forth in Caton et al., 1982, Cell 31:417- 427.
  • peptides which may be conjugated to immunoglobulins may be T cell epitopes.
  • T cell epitope refers to a peptide, including a peptide com ⁇ prised in a larger protein, which is associated with MHC self antigens and recognized by a T cell and which functionally activates the T cell.
  • a T h epitope is recognized by a helper T cell and promotes the facilitation of B cell antibody production via the T h cell.
  • Such epitopes are believed to arise when antigen presenting cells ("APCs") have taken up and degraded a foreign protein.
  • APCs antigen presenting cells
  • a CTL epitope is recognized by a cyto- toxic T cell; when the CTL recognizes the CTL epitope on the surface of a cell, the CTL may induce lysis of the cell.
  • CTL epitopes are believed to arise when an APC has synthesized a protein which is then processed in the cell's cytoplasmic compartment, leading to the generation of peptides (including the CTL epitope) which associate with MHC class I antigens on the sur ⁇ face of the APC and are recognized by CD8 + cells, including CTL.
  • influ- enza A hemagglutinin (HA) protein bears, at amino acid residues 110-120, a T h epitope having the amino acid sequence Ser-Phe-Glu-Arg-Phe-Glu-Ile-Phe-Pro-Lys-Glu (SEQ ID N0:7) .
  • T cell epitopes include, but are not limited to, two promiscuous epitopes of tetanus toxoid, Asn-Ser-Val-Asp-Asp-Ala-Leu-Ile-Asn- Ser-Thr-Lys-Ile-Tyr-Ser-Tyr-Phe-Pro-Ser-Val (SEQ ID NO:8) and Pro-Glu-Ile-Asn-Gly-Lys-Ala-Ile-His-Leu-Val- Asn-Asn-Glu-Ser-Ser-Glu (SEQ ID N0:9; Ho et al. , 1990, Eur. J. Immunol.
  • cytochrome c from residues 88-103, Ala-Asn-Glu-Arg-Ala-Asp-Leu- Ile-Ala-Tyr-Leu-Gln-Ala-Thr-Lys (SEQ ID NO:10); an epitope of Mycobacteria heatshock protein, residues 350-369, Asp-Gln-Val-His-Phe-Gln-Pro-Leu-Pro-Pro-Ala- Val-Val-Lys-Leu-Ser-Asp-Ala-Leu-Ile (SEQ ID NO:11; Vordermir et al., Eur. J. Immunol.
  • T h epitopes which may be used are known or may be identified by methods known in the art.
  • PR8 influenza virus nucleoprotein bears, at amino acid residues 147-161, a CTL epitope having the amino acid sequence Thr-Tyr-Gln-Arg-Thr-Arg-Ala-Leu-Val-Arg-Thr- Gly-Met-Asp-Pro (SEQ ID NO:16).
  • CTL epitopes include, but are not limited to, those discussed in Rotzschke et al., 1991, Immunol. Today 12:447-455. Still further CTL epitopes which may be used are known or may be identi ⁇ fied by methods known in the art.
  • the peptide to be conjugated to immunoglobulin comprises a T cell epitope
  • APC antigen presenting cell
  • the lipo- philic quadruplet Ala-Ala-Ala-Leu SEQ ID NO:15
  • the lipo- philic quadruplet Ala-Ala-Ala-Leu that contains the cleavage site for cathepsins (Yonezawa et al., 1987, Arch. Biochem. Biophys 256:499) .
  • SEQ ID NO:15 the lipo- philic quadruplet Ala-Ala-Ala-Leu
  • class II antigens conjugated to immunoglobulin appear to elicit an immune response when exposed to fixed antigen presenting cells, consistent with immunogenicity in the absence of antigen processing (see, for example, Section 7, below) .
  • class I and class II antigen epitopes may be utilized according to the invention.
  • pep ⁇ tides conjugated to immunoglobulins may be cytokines.
  • IL-1 may be conjugated and delivered to the immune cells to provide stimulatory effect.
  • IL-2 may be conjugated to immuno- globulins having recognition sites for antigens present on tumor cells to effect delivery of the IL-2 to tumor cells for purpose of inhibiting their growth.
  • other pharmaceutical agents directed against various pathogens such as virus, bacteria, or fungi may be conjugated to immuno ⁇ globulins.
  • immunoglobulins having recognition sites for antigens present on the pathogens to which the agents are direc ⁇ ted against, targeted delivery of those agents may be effected.
  • Toxins such as tetanus toxoid, can also be conjugated to appropriate immunoglobulins for targeted delivery.
  • the conjugation method of the present invention may also be used to couple to an immunoglobulin molecule not just peptides, but other compounds.
  • the pharmaceutical agents to be coupled do not necessarily have to be peptides, as long as they possess certain reactive sites, whether they are present naturally or introduced into the agents for specific purpose of conjugation, for reacting with the aldehyde groups of the oxidized car ⁇ bohydrate residues to form covalent linkages.
  • One such reactive site is provided by the presence of an amino group.
  • Peptides for use according to the invention may be purified from natural sources, or may be recombinantly and/or chemically synthesized. They may contain amino acid analogs, and may be detectably labelled.
  • Immunoglobulins that may be used according to the invention include human and non-human mammalian immuno ⁇ globulins, as well as immunoglobulins prepared by com ⁇ bining portions of human and non-human mammalian immunoglobulin.
  • the immunoglobulins that may be used in the invention also include those engineered using recombinant technology.
  • Nonlimiting examples of such recombinant immunoglobulin may be a human immuno ⁇ globulin from which the CDR regions are removed and replaced with CDR regions of a murine antibody or a human immunoglobulin from which the variable regions are removed and replaced with the variable regions of a murine antibody.
  • the immunoglobulins may be of any class, including IgG, IgM, IgD, IgE, and IgA.
  • ICLP conjugates prepared using immunoglobulins of the IgM class may exhibit somewhat greater immunogenic activity than ICLP conjugates prepared using IgG class immuno ⁇ globulin.
  • Immunoglobulin may be monoclonal or poly- clonal, and may be obtained from cell cultures or from serum of a human or a non-human mammal.
  • immunoglobulin refers to molecules comprising both heavy and light chains.
  • conjugating methods of the invention may be used to link peptides to either heavy or light chains separately.
  • peptides may be linked to immunoglobulin molecules which have particular desirable properties or activities such as immunoglobulin molecules which are pinocytosed to the cytoplasm or which are transported to the nucleus.
  • specific nonlimiting embodiments of the present invention include conjugation of pharmaceutical agents with immunoglobulins having recognition sites for antigens present on cells or pathogens to which the pharma ⁇ ceutical agents are directed.
  • the immuno- globulins of the present invention include those having recognition sites for targeted cells or pathogens.
  • immunoglobulin may be treated with a neura- minidase to remove all or substantially all of the terminal sialic acid attached to carbohydrate residues on the immunoglobulin.
  • substantially all indicates that at least 90 percent of the sialic acid residues have been removed.
  • the neura- inidase may be obtained from, for example, Arthro- bacter ureafaciens, Clostridium perfringen ⁇ or Vibrio cholerae , or any source producing a neuraminidase having alpha 2-6 or alpha 2-3 specificity .
  • neuraminidases from Arthrobacter ureafaciens and Clo ⁇ tridium perfringen ⁇ may be incubated overnight at 37°C with lml of phosphate buffer, pH 6, containing mouse or human immunoglobulins (1 mg) and 5 mM of CaCl 2 .
  • phosphate buffer pH 6, containing mouse or human immunoglobulins (1 mg) and 5 mM of CaCl 2 .
  • the kinetics of the desialylation reac- tion may be monitored by determining free NANA released in solution as described in Warren, 1959, J. Biol. Chem. 234:1971.
  • neuraminidase treatment of the immunoglobulin molecule to remove terminal sialic acids before the enzymatic oxidation of carbohydrate residues is not necessary for the purposes of the invention, such treatment is preferred. This is especially so when the carbohydrate residues to be oxi ⁇ dized are galactose residues.
  • terminal sialic acids are removed, unmasking those galactose residues linked to terminal sialic acids and maximizing the number of sites available for conjugation.
  • NANA residues released by the desialyla- tion process may be substantially removed from the desialylated immunoglobulin composition. Such removal may be accomplished, for example, by dialysis, affinity chromatography, or gel filtration. In a specific, non ⁇ limiting embodiment, desialylated immunoglobulins may be dialyzed against PBS (pH 7) until substantially free NANA is removed.
  • carbohydrate residues such as, in particu ⁇ lar, galactose residues, unmasked by desialylation may be enzymatically oxidized at the C-6 position by galac- tose oxidase ("GAO") .
  • GAO galac- tose oxidase
  • carbohydrate resi ⁇ due to be oxidized may be either an internal or a terminal residue.
  • carbohydrate residues to be oxidized are galactose or galactosamine residues, which may be oxidized by galactose oxidase.
  • the glu- cose and glucosamine residues may also be oxidized, for example, by using glucose oxidase.
  • the Schiff bases formed between peptides and carbohydrate residues may be stabilized by reduc ⁇ tion.
  • reducing agents including but not limited to, pyridine borane, sodium boro- hydride, sodium cyanoborohydride and mercaptoethanol may be used.
  • GAO (10 U) a reducing agent pyridine borane ("PB"; 40 mM) and peptide (100 fold molar excess) may be added under continuous stirring for 48 hours at 37°C. Stabilization of Schiff bases formed between peptides and carbohydrate residues occurs upon reduction (Cabacungan et al., 1982, Anal. Biochem. 124:272) .
  • oxidation and reduction reactions may also be performed sequentially.
  • ICLP conjugates may be separated from unreacted reac- tants by methods such as dialysis, affinity chroma- tography, HPLC, etc.
  • the mixture may be dialyzed against PBS in bags with 100,000 MWCO (Spectrapor) , and then concen ⁇ trated to 0.1 ml in ultra concentrators of 100,000 MWCO (S&S) .
  • ICLP conjugates of the invention may be used in a number of commercial, diagnostic, and therapeutic applications.
  • ICLP conjugates may be used in affinity purification methods of a ligand of the ICLP-co prised peptide.
  • a ligand may be a molecule expressed by recombinant techniques in a bioreactor.
  • ICLP conjugates may be used in detecting or quantitating the presence or amount of the target antigen of the immunoglobulin comprised in the ICLP conjugate.
  • the pep- tides comprised in the ICLP conjugate may be detectably labelled, and the ICLP conjugate may be exposed to the target antigen under conditions that permit the binding of the immunoglobulin comprised in the ICLP conjugate to its target antigen.
  • the ICLP con- jugate may be exposed to the target antigen under con ⁇ ditions that permit the binding of the immunoglobulin comprised in the ICLP conjugate to its target antigen, and then the ICLP-target antigen complex may be further reacted to a detectably labelled secondary antibody directed toward the peptide comprised in the ICLP con ⁇ jugate. Since a number of peptides are comprised in each ICLP conjugate, the magnitude of the signal pro ⁇ quizd by the label would be multiplied.
  • ICLP conjugates where the peptide is a B-cell epitope, may further be used to label B cells.
  • the peptides comprised in the ICLP may be detectably labelled, and may be used to quantitate the number of B cells binding to the particular epitope in a sample of lymphocytes collected from the subject.
  • such an ICLP conjugate which need not be detectably labelled, may be used to test the ability of a subject to mount a humoral response to the particular B cell epitope. The inability of lymphocytes of a sub ⁇ ject to produce antibodies after exposure to the ICLP conjugate may indicate that the subject is not capable of developing humoral immunity to the epitope.
  • such an ICLP conjugate may be used to collect B cells which recognize the epitope; for example, the peptides comprised in the ICLP conjugate may be fluores- cently labelled, so that the B cells bound to ICLP con- jugate may be collected by fluorescence-activated cell sorting.
  • ICLP conjugates where the comprised peptide is a T h cell epitope, may further be used to test the ability of a subject to mount an immune response to the particular T h cell epitope.
  • peripheral blood lymphocytes may be collected from a test subject, and then, in a standard proliferative assay, may be exposed to the ICLP conjugate bearing the T h epitope. The amount of proliferation may then be determined, and may be compared to the degree of proliferation exhi ⁇ bited by peripheral blood lymphocytes from a control subject who has not been exposed to the epitope.
  • a result in which the amount of proliferation exhibited by the lymphocytes from the test subject is signifi- cantly greater than the amount of proliferation exhi ⁇ bited by the lymphocytes from the control subject, positively correlates with prior exposure of the test subject to the epitope, and may indicate that the test subject is or has been infected with a pathogen con- taining the epitope.
  • ICLP conjugates may be useful in the treatment of a wide variety of malignancies and viral infections. They are particularly well suited for treatment of infections by viruses which upon infection of the host cell cause expression of viral coat proteins prior to cell death. In most cases this cellular expression of viral coat proteins leads to a cell surface form of such proteins. Examples include but are not limited to the hemag- glutinin protein complex of influenza virus, the env proteins of murine leukemia virus, the env proteins of Rous sarcoma virus and the env proteins of HIV. Often the viral protein expressed by infected cells is the same viral coat protein which recognizes and binds to the cell receptor protein to initiate infection. This is true in the case of HIV.
  • the present invention provides for a method of treating a viral (or bacterial, protozoan, mycoplasmal, or fungal) infection comprising adminis ⁇ tering, to a subject in need of such treatment, an effective amount of a composition comprising ICLP conjugate.
  • treating refers to an amelioration in the clinical condition of the subject, and does not necessarily indicate that a complete cure has been achieved.
  • An amelioration in clinical condition refers to a prolonged survival, a decreased duration of illness, or a subjective improvement in the quality of life of the subject.
  • the present invention provides for a method of enhancing an immune response directed toward a viral, protozoan, mycoplasmal, bacterial or fungal pathogen, in a subject in need of such treatment, comprising administering, to a subject in need of such treatment, an effective amount of a composition comprising ICLP conjugate.
  • enhancing an immune response refers to an increase in cellular and/or humoral immunity.
  • the amount of cellular and/or humoral immunity is increased in the subject by at least 25 percent.
  • Such an enhanced immune response may be desirable during the course of infection, or before infection may have occurred (for example, in the context of a vaccine) .
  • the present invention also provides for a method of treating a malignancy or other neoplasm comprising administering, to a subject in need of such treatment, an effective amount of a composition comprising ICLP conjugate.
  • a method may utilize an antibody specific for a tumor associated antigen to which may be coupled a lymphokine such as GM-CSF (which increases susceptiblity of tumor cells to lysis by cytotoxic T lymphocytes) or interleukin-1, or a tumoricidal agent such as a toxin.
  • GM-CSF which increases susceptiblity of tumor cells to lysis by cytotoxic T lymphocytes
  • interleukin-1 interleukin-1
  • tumoricidal agent such as a toxin.
  • tumor regres ⁇ sion such as a decrease in tumor mass or in the number of metastases, of preferably at least 25 percent would be considered “treating", as would non-progression of disease.
  • the present invention provides for a method of enhancing an immune response directed toward a malignancy or other neoplasm, in a subject in need of such treatment, comprising administering, to a subject in need of such treatment, an effective amount of a composition comprising ICLP conjugate.
  • the present invention may be used to down-regulate the immune response.
  • the peptides comprised in the ICLP may be toleragenic or may be anti-idiotype relative to an undesirably overproduced antibody (for example, anti ⁇ body overproduced in the context of an autoimmune or allergic condition) .
  • An effective amount of such ICLP conjugate may be administered to a subject in need of such treatment.
  • Ig-HA and Ig-NP were genetically engineered as described in Zaghouani, et al., 1993, Science 259:224 and Zaghouani et al., 1992, J. Immunol. 148:3604.
  • Ig- HA is a chimeric BALB/c IgG2b molecule in which the CDR3 loop is replaced by a T cell epitope (HA110-120) from the HA of PR8 influenza A virus.
  • Ig-NP is a BALB/c IgG2b molecule in which the CDR3 loop is replaced by the NP147-161 CTL epitope.
  • the murine IgGl monoclonal antibody 7.21.2 is directed toward the pl85 neu gene product from rat, and was kindly provided by
  • Murine IgM monoclonal antibody L.59- 3 is directed toward toposiomerase I (Muryoi et al., 1991, Autoimmunity 9.:109) and was purified from culture supernatants on a rat anti-murine K chain-Sepharose column.
  • Human myeloma proteins IgGl, IgA and IgM were obtained as affinity purified proteins from Sigma.
  • SYNTHETIC PEPTIDES HA110-120 corresponds to the amino acid residues 110-120 of HA from influenza PR8 A virus (Zaghouani et al., 1993, Science 259:224) .
  • NP 147-161 corresponds to the amino acid residues 147-161 of NP from influenza PR8 A virus (Zaghouani et al. , 1992, J. Immunol.
  • ENZYMES Neuraminidases from Arthrobacter ureafaciens (90.8 U/mg protein, 10 U/ml) and Clo ⁇ tridium per- fringen ⁇ (4.8 U/mg protein, 48 U/ml) were obtained from Calbiochem.
  • Galactose oxidase (690 U/mg protein, 4.5 mg/ml) was obtained from Sigma, and N-glycosidase F (PGN ase F, 25,000 U/mg protein, 20 U/ml) was obtained from Boehringer Mannheim. Other chemicals were pur ⁇ chased from Sigma unless otherwise indicated.
  • PR8 INFLUENZA A VIRUS PR8 influenza A virus was prepared from allantoic fluid of embryonated eggs on a sucrose gradient. The viral preparation used in the following experiments was UV-inactivated.
  • ANTIBODIES Rabbit anti-HA110-120 antibodies were obtained by immunization of rabbits with KLH-HA110-120 conjugate and affinity purified on a BSA-HA110-120-Sepharose column as described in Brumeanu et al., 1993, J. Immunol. Methods 160:65. Rabbit anti-mouse ⁇ l and K chains and goat anti-rabbit antibodies were obtained from Boehringer Mannheim.
  • Neuraminidases from Arthrobacter ureafacien ⁇ and Clo ⁇ tridium perfringen ⁇ were first incubated overnight at 37°C with 1ml of phosphate buf ⁇ fer, pH 6, containing mouse or human immunoglobulins (1 mg) and 5 mM of CaCl 2 .
  • Our preliminary experiments using the combination of these two neuraminidases indi ⁇ cated complete desialylation of mouse IgG.
  • the kine- tics of the desialylation reaction were monitored by determining free NANA released in solution, as des ⁇ cribed in Warren, 1959, J. Biol. Chem. 234:1971.
  • Desialylated immunoglobulins were dialyzed against PBS (pH 7) to remove free NANA, and galactose oxidase ("GAO"; 10 U) , pyridine borane ("PB"; 40 mM) and HA C 110-120 synthetic peptide (100 fold molar excess) were added, while continuously stirring, for 48 hours at 37°C.
  • GAO galactose oxidase
  • PB pyridine borane
  • HA C 110-120 synthetic peptide 100 fold molar excess
  • Membranes were then washed extensively with PBS-0.05% Tween 20 and bound rabbit anti-peptide antibodies were revealed after 2 hours of incubation at room temperature with 2 x 10 5 cpm of 125 ⁇ - goat anti-rabbit antibodies in 1% BSA-PBS.
  • samples were analyzed by SDS-PAGE under reducing conditions. Gels were electrotransferred on PVDF membranes and the ⁇ l and K chains were revealed with 2 x 10 5 cpm of 125 I-rabbit anti-mouse ⁇ l and K chains antibodies.
  • Membranes were washed for 2 hours at room temperature with PBS-0.05% Tween 20, dried, and exposed onto Kodak X-OMAT films, overnight at -70°C.
  • ICLP conjugates were rendered free of unconjugated peptides using size exclusion chromatography on a Superose-6 HR 10/30 column (Pharmacia) . Briefly, ICLP conjugates were dialyzed against PBS, concentrated to 0.1 ml using ultraconcentrators with 100,000 MWCO and then applied onto the Superose-6 column equilibrated in PBS at a flow rate of 0.2 ml/min. Fractions were col- lected every minute, and the peak fractions containing conjugates were pooled, concentrated and used for fur ⁇ ther investigations. The chromatographic profile was monitored at 254nm since the synthetic peptide HA C 110- 120 peptide is detectable at that wavelength.
  • a standard inhibition curve was con ⁇ structed with synthetic HA110-120 peptide and the amount of HA C 110-120 peptide detached from IgG-car ⁇ bohydrate-linked-HA conjugate that showed 50% inhi- bition was estimated. Precisely, 0.9ng of HA C 110-120 peptide that was found in 75 ⁇ l of N-glycosidase digest, was able to inhibit 50% of the rabbit anti- HA110-120 antibody activity. To estimate the number of HA C 110-120 peptide units per molecule of Ig, the amount of Ig corresponding to 75 ⁇ l (86.4ng) was determined by Biuret micro assay.
  • T CELL ACTIVATION ASSAY 2PK3 B lymphoma cells were used as antigen pre ⁇ senting cells (APCs).
  • Irradiated (2,200 rads) APCs (10 4 ) were incubated for 48 hours in round bottom 96- well plates with graded amounts of IGPs as follows: murine IgG or IgG-carbohydrate-linked-HA, murine IgM or IgM-carbohydrate-linked-HA, human IgG-carbohydrate- linked-HA, genetically engineered Ig-HA or Ig-NP, HA110-120 and NP147-161 synthetic peptides and UV- inactivated influenza PR8 A virus.
  • the first step in the synthesis of ICLP conjugates consisted of enzymatic desialylation of terminal NANA residues of immunoglobulin-bound oligosaccharides. Although most of the oligosaccharide chains contain terminal NANA residues linked to adjacent Gal residues by ⁇ , (2-6) bonds, the presence of Gal- ⁇ , (2-3)-NANA linkages has also been reported (Kobata et al., 1989, Ciba Found. Symp. , 145(0) :224) .
  • HA C 110-120 peptide occurs on the sugar moiety of immuno ⁇ globulin
  • N-linked oligosaccharide chains of a chromatographically purified mouse IgG-carbohydrate- linked-HA conjugate were hydrolyzed with N-glycosidase (PGNase F) .
  • PGNase F N-glycosidase
  • Preparations of non-hydrolyzed and hydrol ⁇ yzed conjugate were analyzed in parallel for the pres ⁇ ence of HA C 110-120 peptide by Western blot developed with rabbit anti-HA110-120 antibodies.
  • HA C 110-120 peptide coupled to the sugar moiety of Ig we measured the pro- liferative response of the HA110-120-specific T cell hybridoma, LD1-24, to mouse and human ICLP conjugates. All ICLP conjugates were chromatographically purified prior to use in this assay. Data depicted in Figure 6 show the dose effect activation of T cells induced by various antigens containing HA110-120 epitope such as HA110-120 synthetic peptide, UV-inactivated PR8 virus, antigenized Ig-HA and peptidized ICLPs.
  • T cell activation was confirmed by con ⁇ trols such as: native IgG and IgM, NP 147-161 syn- thetic peptide, and genetically antigenized Ig-NP.
  • con ⁇ trols such as: native IgG and IgM, NP 147-161 syn- thetic peptide, and genetically antigenized Ig-NP.
  • Dose-dependent activation of the specific T helper cells was obtained with all HA110-120 related antigens.
  • mouse IgG-carbohydrate-linked-HA conjugate was as efficient as Ig-HA chimera and 60 fold higher than the HA110-120 synthetic peptide itself.
  • a similar response was obtained for the human IgG-carbo ⁇ hydrate-linked-HA conjugate.
  • a mouse IgM-carbohydrate- linked-HA preparation produced twice as much activation as the genetically antigenized Ig-HA, and 600 times greater activation than the HA110-120 synthetic peptide itself.
  • Chimeric molecules bearing short sequences of foreign genes represent a new tool in delivering specific epitopes to the immunocompetent cells. How ⁇ ever, the utilities of genetically antigenized immuno ⁇ globulins are somewhat limited by the number of pep ⁇ tides that can be expressed per molecule of immunoglobulin.
  • the present inven ⁇ tion was developed, which uses a novel coupling method ⁇ ology to create linkages between carbohydrate residues contained in self immunoglobulin and viral epitopes.
  • the conjugates produced by this methodology showed efficient delivery of the viral epitopes to the immuno ⁇ competent cells.
  • the conjugation of the viral peptide was enzymatically targeted to carbohydrate residues of the immunoglobulins.
  • galactose residues can be chemically oxidized with periodate (Morell et al.,
  • the molecular masses of the IgM-carbohydrate-linked-HA and IgA-carbohydrate- linked-HA populations did not correspond to the mole ⁇ cular masses of any cross-linked products between the heavy and light chains.
  • Molecular studies showed that a single B cell clone may encode for the synthesis of more than one species of glycosyltransferases, in con ⁇ trast to the unique protein structure of immunoglobulin (Harada et al., 1987, Anal. Biochem. 164.(2) :374) .
  • a variety of bianternary and complex oligo- saccharides were found on the same monoclonal immuno- globulin molecules (Kobata et al., 1989, Ciba Found.
  • HA110-120 peptide is recog ⁇ nized by CD4 + T cells in association with I-E d class II MHC alleles (Haberman et al., 1990 J. Immunol. 145:3087) .
  • Both engineered chimeric Ig-HA and IgG- carbohydrate-linked-HA were 40 to 60 fold more effi- cient than HA110-120 synthetic peptide ( Figure 6) .
  • IgM-carbohydrate-linked-HA was 2.5 times more efficient in stimulating T helper cells than the genetically antigenized Ig-HA and 100 fold more efficient than HA110-120 synthetic peptide itself.
  • HA110-120 peptide is released from viral HA as well as from antigenized Ig-HA within the lysosomal compartment (Bru eanu et al., 1993, J. Exp. Med. 178:1795) .
  • Investigations on the efficacy in stimulating specific T helper clones with synthetic peptides and synthetically glycosylated peptides at the amino terminus showed no significant difference (Ishioka et al., 1992, J. Immuonol.
  • HA110-120 peptide and more efficiently release the pep ⁇ tides into lysosomal vesicles we added a lipophilic quadruplet AAAL (SEQ ID NO:15), that contains the cleavage site for cathepsins to the ⁇ -amino end of the serine residue (Yonezawa et al., 1987, Arch. Biochem. Biophys 256(2) :499) . It is known that lysosomal cathepsins play an important role in the processing of exogenous molecules.
  • EXAMPLE DEFYING THE CONVENTIONAL CLASS II PATHWAY OF ANTIGEN PRESENTATION BY A NEW CLASS OF ANTIGENS: IMMUNO-GLYCO-PEPTIDE CONJUGATES Presentation of peptides in the context of MHC class II molecules for specific recognition by recep ⁇ tors on CD4 + T cells has been, prior to the present invention, possessed as the ultimate triggering event in T cell activation. Dogma demanded that prior to epi- tope presentation to T helper cells, antigen is intern ⁇ alized by antigen processing cells, fragmented in lyso- somes, assembled into complexes with the appropriate MHC alleles, and finally exported to the cell surface. Any deficiency in these events has been believed to be likely to result in T cell unresponsiveness to the antigen.
  • the particular IGP conjugates used in these experiments link the ⁇ -amino terminus of the HA110-120 immunodominant CD4 + T cell epitope of the hemagglutinin (HA) of influenza PR8 A virus, containing a cleavage site for cathepsin ("HAc") to the sixth carbon of galactose residues of immunoglobulins.
  • HAcllO-120 epitope was observed to elicit a specific T helper response in association with I-E d class II alleles.
  • the IGP conjugates were found to efficiently activate the HA110-120 specific T cell hybridoma LD1-24 cell line in vitro.
  • HAcllO-120 peptide such as (1) HAcllO-120 synthetic peptide; (2) HA110-120 expressed in the CDR3 loop of the V H gene of immunoglobulin (a genetically engineered Ig-HA chi ⁇ mera) ; (3) HA110-120 epitope as comprised in the bromelain released HA protein from PR8 influenza A virus (BHA) ; and (4) the HAcllO-120 peptide enzym ⁇ atically conjugated to galactose residues of immuno ⁇ globulin (mouse IgG2b-Gal-HAcllO-120 conjugate) .
  • immunoglobulin a genetically engineered Ig-HA chi ⁇ mera
  • the magnitude of T cell activation appears to depend upon the amount of epitope delivered to APCs. Indeed, we found that Ig-HA, for example, may charge I-E d class II molecules with a 40-fold greater amount of HA110-120 epitope than the synthetic peptide itself. It would seem that the IGP conjugate, which carries an average of four HAcllO-120 peptides per molecule of immunoglobulin, was more effete at stimulating T cells than Ig-HA expressing two HA110-120 peptides, or BHA which expresses one peptide per pro- tein unit. We further investigated the relationship between the immunopotency of the HA110-120 epitope and various antigen carrier moieties.
  • IgG2b-gal-HA110-120 conjugate was also able to activate LD1-24 T hybridoma cells in the presence of chloro- quine, although not to the same extent as in the absence of chloroquine ( Figure 9b) .
  • the presentation of IgG2b-gal-HA110-120 conjugate by fixed APCs was inhibited by anti-I-E d mAb 14-4-4S as well as rat anti-Fc ⁇ receptor mAb 24G-2 ( Figure 9c) . This indicates that antigen presentation may occur in an MHC-restricted manner with additional participation of the Fc ⁇ receptors on the surface of APCs.
  • the immunogenic epitopes linked to the sugar moiety of immunoglobulin are relatively free to assemble with class II antigens. As we found by inhibition of the T cell proliferation assay, this interaction appears to require cell surface stabilization of the immunoglobulin carrier by specific receptors, such as the Fc ⁇ receptors of APC 2PK3. Since these experiments were performed with APCs expressing, on their surface, a high density of class II antigens as well as Fc receptors, one may assume that the dis ⁇ tance between the two receptors may be important for charging the MHC antigen with the peptide.
  • the acces- sibility of the conjugated peptide to interact properly with the class II molecule may well depend on the con ⁇ formation of carbohydrates.
  • MOLECULE TYPE peptide (i ⁇ ) ORIGINAL SOURCE:
  • ORGANISM Human Immunodefficiency Virus Type 1
  • ORGANISM Measles Virus
  • MOLECULE TYPE peptide (i ⁇ ) ORIGINAL SOURCE:
  • Ala Asn Glu Arg Ala Asp Leu lie Ala Tyr Leu Gin Ala Thr Lys 1 5 10 15
  • ORGANISM Streptococcus A
  • ORGANISM Staphylococcus sp.
  • MOLECULE TYPE peptide
  • ORIGINAL SOURCE (A) ORGANISM:

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Abstract

Procédés de couplage enzymatique de peptides à des molécules d'immunoglobuline par l'intermédiaire de résidus glucide de molécules d'immunoglobuline. L'invention concerne également des conjugués peptide-immunoglobuline liés par glucide ('ICLP'), obtenus par ces procédés. Les conjugués ICLP apparaissent plus aptes à susciter une réponse immunitaire que les peptides non conjugués.
PCT/US1996/006756 1995-05-15 1996-05-13 Couplage de peptides a des immunoglobulines par l'intermediaire de glucides WO1996036357A1 (fr)

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AU59200/96A AU5920096A (en) 1995-05-15 1996-05-13 Carbohydrate-mediated coupling of peptides to immunoglobulins
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AU5920096A (en) 1996-11-29
CA2227326A1 (fr) 1996-11-21
EP0833663A4 (fr) 1999-04-07
EP0833663A1 (fr) 1998-04-08

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