AU5920096A - Carbohydrate-mediated coupling of peptides to immunoglobulins - Google Patents

Carbohydrate-mediated coupling of peptides to immunoglobulins

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Publication number
AU5920096A
AU5920096A AU59200/96A AU5920096A AU5920096A AU 5920096 A AU5920096 A AU 5920096A AU 59200/96 A AU59200/96 A AU 59200/96A AU 5920096 A AU5920096 A AU 5920096A AU 5920096 A AU5920096 A AU 5920096A
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peptide
carbohydrate
linked
conjugate
immunoglobulin
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AU59200/96A
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Constantin A. Bona
Teodor-Doru Brumeanu
Philip Dehazya
Y.C. Lee
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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

Description

Description
CARBOHYDRATE-MEDIATED COUPLING OF PEPTIDES TO
IMMUNOGLOBULINS
1. INTRODUCTION
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.
2. BACKGROUND OF THE INVENTION
2.1. COUPLING PEPTIDE TO CARRIER TO ENHANCE IMMUNOGENICITY
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. However, although more than 300 chemical cross- linkers with various reactivities have been developed over the past years, only a few are currently used to generate biologically active conjugates (Wong, 1991, in "Chemistry of Protein Conjugation And Cross-Linking", CRC Press, Inc., Boca Raton, Florida; Means and
Freeney, 1971, Bioconj. Chem. JL:2). A major drawback of chemical cross-linkers is the generation of new epitopes in the context of the carrier/cross- linker/peptide complex. Furthermore, because chemical cross-linkers typically lack specificity toward parti- cular groups, additional steps may be required to pro¬ tect cognate structures other than sites targeted for linkage (Freeney, 1987, Int. J. Peptide Prot. Res. 19:145) .
The advent of molecular biology has allowed for the preparation of chimeric molecules in which mini- genes encoding biologically active peptides are expres¬ sed in carrier genes, to result in recombinant expres¬ sion of peptide linked to carrier in a single molecule (Leclerc, 1994, Intern. Rev. Immunol. 11:103) . Because strong immune responses may be elicited against determinants borne by the carrier portion, rather than the incorporated peptide (Cammarota et al., 1992, Nature 356:799) , genes encoding self proteins have been genetically engineered to carry foreign epitopes (Lanza et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:11683) .
For example, expression of microbial B and T cell epitopes in the CDR3 loop of self immunoglobulins has been used to create chimeric molecules which were found to elicit epitope-specific cellular and humoral immune responses in vivo (Zaghouani et al., 1993, Science 259:224) . Genetically antigenized immunoglobulin ("Ig") carrying a CD4 epitope (HA 110-120) from hemag- glutinin ("HA") of influenza PR8 A virus, namely Ig-HA, was able to elicit specific and efficient immune responses in mice (Id. ) . This was correlated with high amounts of viral peptides released from the chimeric immunoglobulins into antigen presenting cells ("APCs") and presented efficiently to specific T helper cells (Brumeanu, 1993, J. Exp. Med.178:1795.. 2.2. CONJUGATION VIA GLYCOSIDIC LINKAGES Radioactive labelling of glycoproteins has been accomplished by conjugating radioactive cysteine methyl ester to aldehydes produced on penultimate galactose residues generated by treating glycoproteins with neura- minidase (to remove terminal sialic acid residues) and then with galactose oxidase (to generate aldehyde func¬ tional groups) (Mitchell et al., 1984, Arch. Biochem. Biophys. 229:544-554) . A similar method had previously been used to label proteins with tritium, wherein the aldehyde groups of the glycoproteins were reduced with tritiated borohydride (Morell and Ashwell, 1972, Methods Enzymol. 28:205) .
In another example of conjugation via carbohydrate residues, 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) .
3. SUMMARY OF THE INVENTION
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.
In particular non-limiting embodiments of the invention, 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. In a preferred embodiment of the invention, the enzymati¬ cally oxidized carbohydrate residues are galactose residues and galactose oxidase may be used for enzyma- tic oxidation. It is preferred that the 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 Fc 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.
As another advantage, the specificity of enzymes used to link peptide with immunoglobulin substantially reduces the generation of by-products and the creation of undesirable neodeterminants.
4. DESCRIPTION OF THE FIGURES
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 ArthroJaσter 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. 14.:1108). The reductive alkyl- ation is favored between the aldehyde group generated enzymatically on the galactose and the α 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 HAC110-120 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) , and lanes 2 and 6, respectively, show Coomassie staining of nonreduced and reduced IgG-carbohydrate-linked-HA conjugates. Western blots of the nonreduced IgG and IgG-carbohydrate- linked-HA that were developed with rabbit anti-HA 110- 120 antibodies are shown in lanes 3 and 4, respectively, and the reactivity of these with the reduced IgG and IgG-carbohydrate-linked-HA are shown in lanes 7 and 8, respectively. Identification of the light and heavy chains of the reduced IgG and IgG- carbohydrate-linked-HA conjugate was performed using Western blots developed with rabbit anti-murine γl and K chain antibodies; lanes 9 and 11 show, respectively, the heavy and light chains of the reduced IgG, and lanes 10 and 12 show, respectively, the heavy and light chains of the IgG-carbohydrate-linked-HA conjugate. As can be seen in lane 8, the heavy chains of mouse IgG were coupled to HAC110-120 peptide and their molecular weight was found to be slightly increased (lanes 6 and 8).
FIGURE 3: Chromatographic removal of residual, unconjugated HAC110-120 peptide from the ICLP con¬ jugates. A Superose-6 gel filtration column was previously calibrated with molecular weight markers
(Pharmacia) , and then mouse 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 late peak that eluted at 80 minutes, as indicated on the chromatograms of ICLP conjugates, represents residual free peptide. 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 HAC110-120 peptide to the sugar moiety of the immunoglobulins. Western blot analysis of the reduced IgG-carbohydrate- linked-HA conjugates, before and after treatment with PGNase F, lanes 1 and 2, respectively, was developed with rabbit anti-HA 110-120 antibodies as described in Section 6. 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/HAc110-120 complex from the heavy chains of mouse IgG-carbohydrate-linked-HA conjugate (lane 2) .
FIGURE 5: Estimation of the degree of coupling of HAC110-120 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 HAC110-120 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 HAC110-120 peptide detached from the conjugate or HA110-120 synthetic peptide (cali¬ bration) . The amount of HAC110-120 peptide (0.9ng) detected in 75 μl, as used in IRIA (competitive inhi¬ bition radioimmunoassay) , was related to the cor¬ responding amount of mouse IgGl (86.4ng) found in 75 μl. Since 16.9:1 represents a molar ratio of 1:1 between heavy chain of IgG and HAC110-120 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 HAC110-120 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
HAC110-120 antibodies as described. It should be noted that residual unconjugated HAC110-120 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. (a) 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, (b) 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.
5. DETAILED DESCRIPTION OF THE INVENTION
For purposes of clarity, and not by way of limitation, the detailed description of the invention is divided into the following subsections:
(i) peptides suitable for conjugation; (ii) immunoglobulins suitable for conjugation; (iii) enzymatic coupling of peptide to immunoglobulin; and (iv) utility of the invention.
5.1. PEPTIDES SUITABLE FOR CONJUGATION
Virtually any 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.
In non-limiting embodiments, peptides which may be conjugated to immunoglobulins, according to the inven- tion, include B cell epitopes. The term "B cell epi- tope", as used herein, 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.
For example, and not by way of limitation, the hypervariable region 3 loop ("V3 loop") of the envelope protein of human immunodeficiency virus ("HIV") type 1 is known to be a B cell epitope. Although the 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) .
Other examples of known 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. 147:3545- 3552) ; and an epitope of foot and mouth disease VP1 protein, residues 141-160, Met-Asn-Ser-Ala-Pro-Asn-Leu- Arg-Gly-Asp-Leu-Gln-Lys-Val-Ala-Arg-Thr-Leu-Pro (SEQ ID NO:6; Clarke et al., 1987, Nature 330:381-384) .
Still further B 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. In additional embodiments of the invention, peptides which may be conjugated to immunoglobulins may be T cell epitopes. The term "T cell epitope", as used herein, 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.
For example, a Th epitope is recognized by a helper T cell and promotes the facilitation of B cell antibody production via the Th cell. Such epitopes are believed to arise when antigen presenting cells ("APCs") have taken up and degraded a foreign protein. The peptide products of degradation, which include the Th epitope, appear on the surface of the APC in associ¬ ation with MHC class II antigens.
Further, 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.
For example, and not by way of limitation, influ- enza A hemagglutinin (HA) protein bears, at amino acid residues 110-120, a Th epitope having the amino acid sequence Ser-Phe-Glu-Arg-Phe-Glu-Ile-Phe-Pro-Lys-Glu (SEQ ID N0:7) .
Other examples of known 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. 20:477-483) ; an epitope of 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. 24...2061-2067) ; an epitope of hen egg white lysozy e, residues 48-61, Asp- Gly-Ser-Thr-Asp-Tyr-Gly-Ile-Leu-Gln-Ile-Asn-Ser-Arg (SEQ ID NO:12; Neilson et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89.: 7380-7383; an epitope of Streptococcus A M protein, residues 308-319, Gln-Val-Glu-Lys-Ala-Leu- Glu-Glu-Ala-Asn-Ser-Lys (SEQ ID NO:13; Rossiter et al., 1994, Eur. J. Immunol. 24.:1244-1247) ; and an epitope of Staphylococcus nuclease protein, residues 81-100, Arg- Thr-Asp-Lys-Tyr-Gly-Arg-Gly-Leu-Ala-Tyr-Ile-Tyr-Ala- Asp-Gly-Lys-Met-Val-Asn (SEQ ID NO:14; de Magistris, 1992, Cell 6jJ:l-20). Still further Th epitopes which may be used are known or may be identified by methods known in the art.
As another example, and not by way of limitation, 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).
Other examples of known 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.
If the peptide to be conjugated to immunoglobulin comprises a T cell epitope, it may be desirable to provide a means for releasing the T cell epitope from the immunoglobulin so as to facilitate appropriate pro¬ cessing by an antigen presenting cell ("APC") . For example, it may be desirable to incorporate the T cell epitope into a larger peptide which comprises a site susceptible to cleavage by an enzyme typically present in lysosomes of APCs. As a specific example, the lipo- philic quadruplet Ala-Ala-Ala-Leu (SEQ ID NO:15), that contains the cleavage site for cathepsins (Yonezawa et al., 1987, Arch. Biochem. Biophys 256:499) . may be added to the T cell epitope. It is known that lysosomal cathepsins play an important role in pro¬ cessing of exogenous molecules.
It has also been observed that 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) . Accordingly, class I and class II antigen epitopes may be utilized according to the invention. In additional embodiments of the invention, pep¬ tides conjugated to immunoglobulins may be cytokines. For example, IL-1 may be conjugated and delivered to the immune cells to provide stimulatory effect. In another example, 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.
In additional embodiments, other pharmaceutical agents directed against various pathogens such as virus, bacteria, or fungi may be conjugated to immuno¬ globulins. By conjugating the pharmaceutical agents to 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.
It should be noted that the conjugation method of the present invention may also be used to couple to an immunoglobulin molecule not just peptides, but other compounds. For example, 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.
5.2. IMMUNOGLOBULINS SUITABLE FOR CONJUGATION 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.
The term "immunoglobulin", as used herein, gen- erally refers to molecules comprising both heavy and light chains. However, if desirable, the conjugating methods of the invention may be used to link peptides to either heavy or light chains separately.
Further, 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.
As previously set forth in section 5.1, 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. Accordingly, the immuno- globulins of the present invention include those having recognition sites for targeted cells or pathogens.
5.3. ENZYMATIC COUPLING OF PEPTIDE TO IMMUNOGLOBULIN A schematic diagram depicting a preferred embodi¬ ment of the coupling method of the invention is set forth in Figure 1.
First, 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", as used in this paragraph, 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 perfringenε or Vibrio cholerae , or any source producing a neuraminidase having alpha 2-6 or alpha 2-3 specificity . In a pre¬ ferred, nonlimiting embodiment, neuraminidases from Arthrobacter ureafaciens and Cloεtridium perfringenε (50 mU of a 1:1 mixture of the neuraminidases obtained from the two sources) 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 CaCl2. Depending on the neuraminidase used, however, it may not be necessary to include CaCl2 in the reac¬ tion mixture. 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.
It should be noted that even though 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. By treating the immuno- globulin with neuraminidase, terminal sialic acids are removed, unmasking those galactose residues linked to terminal sialic acids and maximizing the number of sites available for conjugation.
Second, 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.
Third, 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") . Further, the carbohydrate resi¬ due to be oxidized may be either an internal or a terminal residue. Preferably, 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. Fourth, the Schiff bases formed between peptides and carbohydrate residues may be stabilized by reduc¬ tion. For this reaction, reducing agents, including but not limited to, pyridine borane, sodium boro- hydride, sodium cyanoborohydride and mercaptoethanol may be used. In a specific, nonlimiting embodiment, 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) . The oxidation and reduction reactions may also be performed sequentially. Fifth, after completion of the coupling reaction, ICLP conjugates may be separated from unreacted reac- tants by methods such as dialysis, affinity chroma- tography, HPLC, etc. In a specific, nonlimiting embodiment, 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) .
In particular embodiments, it may be desirable to conjugate a single species of peptide to an immuno- globulin molecule. In other embodiments, it may be desirable to conjugate a diversity of peptides to an immunoglobulin molecule.
A detailed description of one specific, non¬ limiting embodiment of the invention is set forth in section 6, below.
5.4. UTILITY OF THE INVENTION The ICLP conjugates of the invention may be used in a number of commercial, diagnostic, and therapeutic applications. In a first set of embodiments of the invention, ICLP conjugates may be used in affinity purification methods of a ligand of the ICLP-co prised peptide. For example, such a ligand may be a molecule expressed by recombinant techniques in a bioreactor.
In a second set of embodiments, 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. For example, 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. Alternatively, 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¬ duced by the label would be multiplied.
ICLP conjugates, where the peptide is a B-cell epitope, may further be used to label B cells. For example, 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. Similarly, 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. Fur- ther, 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 Th cell epitope, may further be used to test the ability of a subject to mount an immune response to the particular Th cell epitope. For example, 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 Th 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.
In further embodiments of the invention, 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.
Accordingly, 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. The term "treating" as used herein 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. The phrase "enhancing an immune response" refers to an increase in cellular and/or humoral immunity. In preferred embodiments, 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. For example, such 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. In addition to the definition of "treating" set forth above, 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.
Further, 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.
In still other embodiments, the present invention may be used to down-regulate the immune response. For example, 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.
In order that the invention described herein is better understood, the following examples are provided. These examples are for purposes of illustration only and are not intended to be construed in a limiting sense. EXAMPLE: ENZYME-MEDIATED CONJUGATION OF
IMMUNOGLOBULINS TO A PR8 INFLUENZA VIRAL EPITOPE VIA CARBOHYDRATE RESIDUES
6.1. MATERIALS AND METHODS 6.1.1. IMMUNOGLOBULINS
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. Similarly, 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
Dr. M. Greene, University of Pennsylvania; it was puri¬ fied from cell culture supernatants on a Protein A- Sepharose column. 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.
6.1.2. SYNTHETIC PEPTIDES HA110-120 (SFERFEIFPKE; SEQ ID NO:7) 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 (TYQRTRALVRTGMDP; SEQ ID NO:16) corresponds to the amino acid residues 147-161 of NP from influenza PR8 A virus (Zaghouani et al. , 1992, J. Immunol.
148:3604) . Peptides were synthesized at Biosynthesis Inc., Lewisville, TX, and were purified by reverse phase HPLC. The molecular mass of the peptides was confirmed by mass spectroscopy. KLH and BSA conjugates of HA110-120 were prepared as described in Liu et al., 1979, Biochemistry 18:690.
6.1.3. ENZYMES Neuraminidases from Arthrobacter ureafaciens (90.8 U/mg protein, 10 U/ml) and Cloεtridium per- fringenε (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.
6.1.4. 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.
6.1.5. 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 γl and K chains and goat anti-rabbit antibodies were obtained from Boehringer Mannheim.
6.1.6. ENZYMATIC SYNTHESIS OF ICLP CONJUGATES
Neuraminidases from Arthrobacter ureafacienε and Cloεtridium perfringenε (50 mU, 1:1 mixture) 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 CaCl2. 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 HAC110-120 synthetic peptide (100 fold molar excess) were added, while continuously stirring, for 48 hours at 37°C. The velocity of the enzymatic oxidation by GAO was studied in a peroxidase/o-tolidine coupled system by determining the increase in absorbance at 425nm resulting from the release of hydrogen peroxide (Avigad, 1985, Arch. Biochem. Biophys. 239:531) . Stabilization of Schiff bases between peptides and oxidized carbohydrate residues occurred upon reduction with PB as described in Cabacungan et al., 1982, Anal. Biochem. 124:272. After completion of the coupling reaction, the mixture was dialyzed against PBS in bags with 100,000 MWCO (Spectrapor) , concentrated to 0.1 ml in ultra concentrators of 100,000 MWCO (S&S) and fur¬ ther analyzed.
6.1.7. WESTERN BLOT ANALYSIS Murine IgG-carbohydrate-linked-HA, IgM-carbo- hydrate-linked-HA and human IgG-carbohydrate-linked-HA, IgM-carbohydrate-linked-HA and IgA-carbohydrate-linked- HA conjugates were analyzed by SDS-PAGE under reducing and non-reducing conditions (Wyckoff et al., 1977, Anal. Biochem. 7JB:459). The conjugates 10μg/20μl were electrophoresed on 10% polyacrylamide gels for 1 hour, at 150 volts using PROTEAN II mini-system apparatus
(BioRad) . Gels were either stained with Coomassie R- 250 or electrotransferred in semidry conditions onto PVDF membranes (0.22μ, Millipore, Waters Co.) for 30 minutes at 250 mAmps. Membranes were blocked overnight at 4°C with 5% fat free milk (Carnation) in PBS, washed with PBS and incubated overnight at 4°C with lμg/ml of affinity purified rabbit anti-HA110-120 antibodies in 1% BSA-PBS-0.05% Tween 20. 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 105 cpm of 125ι- goat anti-rabbit antibodies in 1% BSA-PBS. For the identification of the immunoglobulin heavy and light chains within the murine IgG-carbohydrate-linked-HA conjugate, samples were analyzed by SDS-PAGE under reducing conditions. Gels were electrotransferred on PVDF membranes and the γl and K chains were revealed with 2 x 105 cpm of 125I-rabbit anti-mouse γl 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.
6.1.8. SIZE EXCLUSION CHROMATOGRAPHY 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 HAC110- 120 peptide is detectable at that wavelength.
6.1.9. ESTIMATION OF THE DEGREE OF CONJUGATION To estimate the number of HAC110-120 peptides attached per immunoglobulin molecule, a batch of mouse IgG-carbohydrate-linked-HA conjugate (2 mg) was pre¬ pared as described above. The IgG-carbohydrate-linked- HA conjugate was then extensively dialyzed against PBS in bags of 100,000 MWCO and traces of residual uncon- jugated peptide were removed from ICLP conjugate by size exclusion chromatography as described. The pres- ence of HAC110-120 peptide coupled to the oligo- saccharide chains of the immunoglobulin was confirmed by Western blot developed with anti-HA110-120 anti¬ bodies. To estimate the number of peptides coupled per molecule of IgG, we first cleaved the sugar-peptide complex from the conjugate using PGNase F as described in Tarentino et al., 1989, Methods Cell Biol. 32:111. Briefly, a preparation of chromatographically purified ICLP conjugate (5 μg in 250 μl PBS) was boiled for five minutes in the presence of 10% mercaptoethanol and 0.1% SDS. The solution was then cooled on ice, and incu¬ bated overnight at 37°C with PGN ase F (0.04 U) and 0.5% Nonidet P40. The reaction mixture was dialyzed against PBS in bags with 1,000 MWCO and the amount of HAC110-120 peptide released from the conjugate was fur- ther determined by IRIA (Brumeanu et al. 1993, J.
Immunol. Methods 160:65) . Briefly, 96-well microtiter plates were coated overnight at 4°C with 5 μg/ml of BSA-HA110-120 in 0.1M carbonate buffer, pH 9.6 and blocked for 4 hours with 3% BSA. Mixtures of 2ng of rabbit anti-HA110-120 antibodies in 1% BSA-PBS and several dilutions of the solution containing N-glyco- sidase released peptides were then added into the plate and incubated overnight at 4°C. The plates were washed with PBS-0.05% Tween 20 and bound rabbit anti-peptide antibodies were revealed with 5 x 104 cpm of goat anti- rabbit antibodies. A standard inhibition curve was con¬ structed with synthetic HA110-120 peptide and the amount of HAC110-120 peptide detached from IgG-car¬ bohydrate-linked-HA conjugate that showed 50% inhi- bition was estimated. Precisely, 0.9ng of HAC110-120 peptide that was found in 75 μl of N-glycosidase digest, was able to inhibit 50% of the rabbit anti- HA110-120 antibody activity. To estimate the number of HAC110-120 peptide units per molecule of Ig, the amount of Ig corresponding to 75μl (86.4ng) was determined by Biuret micro assay. Since the immunoglobulin was pre¬ sent in the digest solution in the reduced form and the peptide was mostly attached to the oligosaccharide chains on the heavy chain of immunoglobulin, we cal¬ culated that 1:1 molar ratio between the heavy chain of IgG (50kDa) and HAC110-120 peptide (1.8 kDa) should correspond to 16.91:1. To this, the corresponding amounts of both heavy chains of the IgG and peptide, as found in the assays, were integrated and the estimated number of peptides per molecule of heavy chain of IgG was found to be 5.68. On the average, this corresponds to 11.4 peptide units coupled per molecule of IgG.
6.1.10. T CELL ACTIVATION ASSAY 2PK3 B lymphoma cells were used as antigen pre¬ senting cells (APCs). Irradiated (2,200 rads) APCs (104) 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. 2 x 104 HA110-120- specific LD1-24 T hybridoma cells (Haberman et al., 1990, J. Immunol 145. 3087) were then added. Culture supernatants were harvested and incubated for another 72 hours with 1.5 x 104 of IL-3 dependent DA-1 cells. IL-3 production was used in these experiments as a measure of the activation of HA110-120 specific T cells, as quantitated by MTT colorimetric assay (Mosmann, 1983, J. Immunol. Methods 65:55) . 6 . 2 . RESULTS
6.2.1. ENZYMATIC COUPLING OF HAC110-120 PEPTIDES TO IMMUNOGLOBULIN
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 α, (2-6) bonds, the presence of Gal-α, (2-3)-NANA linkages has also been reported (Kobata et al., 1989, Ciba Found. Symp. , 145(0) :224) . Using a (1:1) mixture of neuraminidase from Arthrobacter ureafacienε that is able to cleave Gal-α, (2-6)-NANA, and neuraminidase from Cloεtridium perfringenε, that preferentially cleaves Gal-α, (2-3)-NANA linkages (Corfield et al., 1983,
Biochim. Biophys. Acta. 744:121) - we were able to pro¬ duce greater than 95% yields of mouse and human asialo- immunogloublins.
The optimal parameters for enzymatic desialylation by neuraminidases, oxidation of Gal residues by GAO and reduction of the Gal-peptide bonds with PB were opti¬ mized with respect to time, pH, incubation temperature, and molar ratios. We found that a 1:100 molar ratio of Ig/peptide leads to saturation of the Gal residues with HAC110-120 peptides under the conditions described in the foregoing Materials and Methods section. Western blot analyses of the IGP conjugates developed with either anti-HA110-120 antibodies or anti-murine γl or K antibodies indicate that the coupling reaction occurred preferentially on the heavy chains of mouse IgGl (Fig¬ ure 2) .
6.2.2. PURIFICATION OF ICLP CONJUGATES Mouse IgG-carbohydrate-linked-HA and IgM-carbo- hydrate-linked-HA conjugates were purified by size exclusion chromatography on a Superose-6 column. It should be noted that dialysis of the conjugates was not sufficient to completely remove the unconjugated pep¬ tides used in excess in the coupling reaction. Small amounts of residual peptide eluted in the salt volume of the column (late peak corresponding to the elutiσn time of 80 minutes, Figure 3) . While the peak symmetry of the native immunoglobulins was in accord with their globular structure, the conjugates showed loss of peak symmetry that may be related to the attachment of lin- ear structures such as HAC110-120 peptide. This modification was most obvious in the case of IgM-carbo- hydrate-linked-HA conjugate, which showed a higher degree of coupling than the IgG-carbohydrate-linked-HA conjugate.
6.2.3. SPECIFICITY OF COUPLING HAC110-120 TO
THE SUGAR MOIETY OF IMMUNOGLOBULINS
To determine whether or not the coupling of HAC110-120 peptide occurs on the sugar moiety of immuno¬ globulin, the N-linked oligosaccharide chains of a chromatographically purified mouse IgG-carbohydrate- linked-HA conjugate were hydrolyzed with N-glycosidase (PGNase F) . Preparations of non-hydrolyzed and hydrol¬ yzed conjugate were analyzed in parallel for the pres¬ ence of HAC110-120 peptide by Western blot developed with rabbit anti-HA110-120 antibodies. Data depicted in Figure 4 indicate that the enzymatic detachment of the oligosaccharide chains from a mouse IgG-carbo¬ hydrate-linked-HA preparation occurred at the aspara- gine-N-linkage with subsequent removal of HAC110-120 peptide. This demonstrates that coupling of the pep¬ tide was specifically targeted to the sugar moiety of immunoglobulin. 6.2.4. EFFICIENCY OF ENZYMATIC COUPLING OF HAC110- 120 PEPTIDES TO THE SUGAR MOIETY OF
IMMUNOGLOBULINS
For the conditions of the coupling reaction estab- lished in our experiments, at 1:100 molar ratio between Ig and HAC110-120 peptide the Gal residues reached saturation. Higher ratios showed no significant increase in the amount of peptides attached per mole¬ cule of immunoglobulin. The average number of HAC110- 120 peptides coupled per molecule of mouse IgGl at 1:100 ratio was 11.5 (Figure 5).
It should be noted that incorporation into the HA110-120 peptide of the lipophilic quadruplet amino acid sequence AAAL corresponding to the cleavage site of the cathepsins did not interfere with the reactivity of rabbit anti-HA110-120 antibodies (Figures 2 and 5) .
6.2.5. IMMUNOGENICITY OF MOUSE ICLP CONJUGATES
To study the immunogenicity of HAC110-120 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. The specificity of 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. Dose-dependent activation of the specific T helper cells was obtained with all HA110-120 related antigens. At 50% activation, 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.
6.2.6. ENZYMATIC COUPLING OF HAC110-120 PEPTIDE TO MOUSE AND HUMAN IMMUNOGLOBULINS OF VARIOUS ISOTYPES To investigate the versatility of the enzymatic coupling procedure, we attempted to peptidize various isotypes of mouse and human immunoglobulins. Using similar reaction conditions as for the synthesis of mouse IgG-carbohydrate-linked-HA conjugates, we were able to generate other mouse and human ICLP conjugates (Figure 7) . It should be noted that mouse and human IgG-carbohydrate-linked-HA conjugates showed remarkable homogeneity indicating a lack of cross linking between heavy and heavy, or heavy and light chains of the immunoglobulins. However, a certain degree of hetero¬ geneity was detected among the mouse and human IgM- carbohydrate-linked-HA and IgA-carbohydrate-linked-HA conjugates.
6.3. DISCUSSION Coupling of non-immunogenic components such as haptens and synthetic peptides to protein carriers is an important means for studying the antigenicity of small size molecules. However, because the chemical coupling techniques are mostly used to generate protein conjugates, several disadvantages frequently occur when these conjugates are used to provoke specific immune responses in animals. One major complication pre¬ viously encountered has been the induction of an immune response against the carrier. A second is the gener- ation of neo-determinants introduced by the extrinsic chemical groups of the cross-linkers used to bridge the carrier to the peptides. Although "zero-length" cross- linkers, as compared to other homo- and heterobifunc- tional cross-linkers, do not introduce neodeterminants on the conjugates and preclude the risk of toxicity, targeting chemical cross-linkers toward particular active groups on proteins or peptides provides no absolute specificity.
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.
Based on these considerations, 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. Although galactose residues can be chemically oxidized with periodate (Morell et al.,
1972, Methods Enzymol. 2.8:205), this particular type of conjugation may involve other non-reducing termini of the sugar moiety and it may also damage the fine func¬ tional structures on the immunoglobulin molecules. Galactose oxidase (Malmstrδm et al., 1975, in "The
Enzymes XIIB", 3rd. Ed., Boyer P., ed. , Academic Press, NY, p. 527) , was used to generate a highly reactive C-6 aldehyde derivative on immunoglobulins. The C-6 alde¬ hyde group reacted selectively with the α-amino terminus of the peptides and formed Schiff bases that were stabilized upon mild reduction with pyridine borane. The ICLP conjugates were rendered free of unconjugated peptides by size exclusion chromatography.
Using this methodology we generated homogeneous conjugates between various isotypes of mouse or human immunoglobulins and a CD4+epitope from HA of influenza PR8 A virus. The IgG-carbohydrate-linked-HA conjugates showed no cross-linkage between heavy and light chains of the immunoglobulins. The remarkable homogeneity of these conjugates may correlate with the ability of α amino group of the peptide in excess to compete effi¬ ciently for galactose residues with other primary amines of the immunoglobulin, such as the e amino groups of lysine residues. The e amino groups of lysine residues require higher pH (>9.3) to become reactive relative to the pH used to couple the HAC110- 120 peptide to galactose (pH 7) . However, a restrained heterogeneity was observed in the case of IgM-carbo- hydrate-linked-HA and IgA-carbohydrate-linked-HA conju¬ gates. It is unlikely that the presence of two to three populations of conjugates, as revealed by Western blot analysis, may represent primarily heteropolymers between heavy and light chains since these patterns were not observed from any of the IgG-carbohydrate- linked-HA conjugates. Moreover, 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) . Indeed, a variety of bianternary and complex oligo- saccharides were found on the same monoclonal immuno- globulin molecules (Kobata et al., 1989, Ciba Found. Symp. 4_5:224). This suggests that the heterogeneity of IgM-carbohydrate-linked-HA and IgA-carbohydrate- linked-HA conjugates may be related to the micro- heterogeneity of the sugar moiety on these particular immunoglobulins. The coupling efficiency of this enzymatic method was evaluated using the average number of HAC110-120 peptides per molecule of immunoglobulin. With mono¬ clonal antibody 7.21.2, an average of 11.4 peptides were coupled per molecule of murine IgGl (Figure 4) . Deglycosylation of the ICLP conjugates with N-glyco- sidase showed that most of the peptide acceptors were located on asparagine-N-linked oligosaccharides. Western blots of the conjugates developed with anti- peptide, anti-γl and anti-κ antibodies revealed that the oligosaccharide-peptide complexes were formed pre¬ ferentially on the heavy chain of the immunoglobulin molecules (Figure 2) .
The coupling of peptide to the sugar moiety of immunoglobulin enhanced significantly the immuno- genicity of the peptide. HA110-120 peptide is recog¬ nized by CD4+ T cells in association with I-Ed 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) . It is worth noting that 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. We demonstrated that 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. 148:2446) . Paral¬ lel studies using 1H NMR spectroscopy of synthetic α- amino glycosylated peptides indicated that the carbo¬ hydrate moiety did not change the α-helical con- formation of the peptide (Elofsson et al., 1993, Carbohydrate Research 246:89.31) . Indeed, the α- helical conformation that is required for the recog¬ nition by specific T helper cells was preserved in a synthetic N-glycosylated peptide HEL51-61 even after association with the corresponding MHC class II allele (Allen et al., 1987, Nature (London) 327:713) . More¬ over, it was shown that carbohydrates do not themselves associate with MHC molecules and are not presented to T helper cells (Ishioka et al., 1992, J. Immunol. 148:2446; Harding et al. , 1991, Proc. Natl. Acad. Sci. U.S.A. 8j$:2740). This suggests that small carbohydrate fragments attached to the N terminus of the T helper stimulating peptides do not exhibit down-regulating effects on the cellular immune response. However, to facilitate specific cleavage at the N terminus of the
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 α-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.
Previous experiments have shown that eleven-mer HA110-120 peptides, but not truncated HA110-117 pep¬ tide, stimulated LD1-24 specific T helper cells (Brumeanu et al., 1993, J. Exp. Med. 128.:1795). Thus, it is likely that the peptides delivered by the IGP conjugates into the lysosomal compartment were intact sequences of the HA110-120 peptides. 7. 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, acclaimed 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.
Although processing events were believed to neces¬ sarily precede presentation, some denatured antigens, such as ovalbumin, were shown to stimulate CD4+ T cells prior to cell processing. This phenomenon was hypo¬ thesized to result from the ability of certain T cell subsets to recognize immunodominant epitopes made accessible by the denaturing process, in association with class II antigens. In this section, a cell-free processing pathway for the presentation of native proteins to CD4+ T cells is described. The pathway was identified in studies involving enzymatically engineered Immuno-Glyco-Peptide conjugates (IGP) . The particular IGP conjugates used in these experiments link the α-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. The HAcllO-120 epitope was observed to elicit a specific T helper response in association with I-Ed class II alleles. The IGP conjugates were found to efficiently activate the HA110-120 specific T cell hybridoma LD1-24 cell line in vitro.
Different indices of T cell activation were observed for various protein carriers for HAcllO-120 peptide, such as (1) HAcllO-120 synthetic peptide; (2) HA110-120 expressed in the CDR3 loop of the VH 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) . All these protein carriers activated the specific HA110-120 T cell hybridoma LD1-24 to different extents when pre¬ sented by APC 2PK3 B cell lymphoma cells (Figure 8a) . The efficacy in stimulating LD1-24 T cells at a 50% index of activation for both IgG2b-Gal-HAcllO-120 con¬ jugate and BHA was found to be ten-fold higher than the efficacy of either Ig-HA or the HAcllO-120 synthetic peptide itself. IgG2b-Gal-HAcllO-120 conjugate showed a slightly higher T cell activation index than BHA when administered at similar molar doses with respect to the protein carrier. At first, 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-Ed 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 efective 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. We normalized the amount of HA110-120 peptide contained in each carrier as nano- moles of peptide per molecule of carrier. The nor¬ malized amount of HA110-120 epitope was then integrated against the index of T cell activation. In contrast to the T cell activation indexes, the epitope immuno¬ potency of BHA was slightly higher than the immuno- potency of the IGP conjugate. Among all these protein carriers, BHA was found to be the most potent carrier for the HA110-120 epitope. This result may be explained by the theory that additional co-stimulatory signals provided by the foreign boundaries of an immunodominant epitope encased in viral proteins may increase the immunopotency of the epitope. Both BHA and IGP con¬ jugates showed higher immunopotency than Ig-HA and the HAcllO-120 synthetic peptide itself (Figure 8b) . These results suggest that both the amount of epitope delivered to APCs, as well as the epitope boundaries, may be important in determining the amount of T cell stimulation. Indeed, it has been demonstrated that sugar moieties may endow increased immunogenicity to the peptide determinants. For example, various sugar polymers, such as dextrans, were successfully used as carriers to induce elevated anti-protein antibody titers. However, the inner mechanism(s) of triggering efficient immune responses by sugar carriers has remained ambiguous. In order to determine whether the immunogenicity of an epitope, surrounded by a sugar moiety (such as occur in an IGP conjugate) , depends only on proteolytic degradation in the lysosomal compartment of APCs, we compared the indexes of T cell activation obtained with IGP conjugates carrying HA peptides with (HAc) or with¬ out (HA) a cleavage site for cathepsins. We found neither significant differences between the T cell activation index of IgG2b-gal-HAcllO-120 and IgG2b-gal- HA110-120 conjugates, nor differences in epitope immunopotency of these two IGP conjugates. Further investigations showed that among all car¬ rier systems used in our experiments, including an IgM- gal-HAcllO-120 conjugate, only IgG2b-gal-HA110-120 con¬ jugate was able to activate LD1-24 T hybridoma cells when they were presented by paraformaldehyde fixed antigen presenting cells ("APCs"; Figure 9a). The
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) . Moreover, it was observed that the presentation of IgG2b-gal-HA110-120 conjugate by fixed APCs was inhibited by anti-I-Ed mAb 14-4-4S as well as rat anti-Fcγ receptor mAb 24G-2 (Figure 9c) . This indicates that antigen presentation may occur in an MHC-restricted manner with additional participation of the Fcγ receptors on the surface of APCs.
Preliminary experiments in our laboratory using NP147-155 peptide (a CTL epitope) of nucleoprotein of PR8 influenza virus coupled on the galactose residues of MOPC 141 showed insignificant killing by specific CTLs of 2PK3 used as target cells. The length of pep¬ tide used in these experiments was the minimum required for class I presentation (9-10 amino acid residues) . Without being bound to any particular theory, we hypothesize a new antigen presenting cell-free pro¬ cessing model as illustrated in Figure 10. In this model, the activation of the CD4+ T cell occurs throughout the recognition of a MHC-peptide complex formed in particular embodiments. 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 Fcγ 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. We found that different immunoglobulins appear to expose the carbohydrate moiety differently on the surface. Thus, a mouse IgGl/k known to contain four galactose residues per molecule of immunoglobulin was found to expose only one reactive galactose when tested in a galactose oxidase assay. Since the galactose residues are located in a sub- terminal position on the carbohydrate moieties of the immunoglobulins, one may assume that the accessibility of these residues may play a role in the extent of exposure of the coupled peptides on the surface of IGP conjugates.
Various publications are cited herein which are hereby incorporated by reference in their entireties.
SEQUENCE LISTING (1) GENERAL INFORMATION
(i) APPLICANT: Bona, Constantin A. Lee, Y.C.
Brumeanu, Teodor-Doru Dehazya, Philip
(ii) TITLE OF THE INVENTION: CARBOHYDRATE-MEDIATED COUPLING
OF
PEPTIDES TO IMMUNOGLOBULINS (iϋ) NUMBER OF SEQUENCES: 16
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Brumbaugh, Graves, Donohue & Raymond
(B) STREET: 30 Rockefeller Plaza
(C) CITY: New York (D) STATE: NY
(E) COUNTRY: USA
(F) ZIP: 10112-0228
(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS ( D) SOFTWARE: FastSEQ Version 1.5
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/477,424 (B) FILING DATE: 07-JUNE-1995
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Clark, Richard S
(B) REGISTRATION NUMBER: 26,154
(C) REFERENCE/DOCKET NUMBER: 29889-A-165/31384
(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 212-408-2558
(B) TELEFAX: 212-765-2519
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iϋ) ORIGINAL SOURCE:
(A) ORGANISM: Human Immunodefficiency Virus Type 1
(iv) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 301...319 (C) OTHER INFORMATION: Envelope Protein gpl20
(v) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Arg Lys Ser lie His lie Gly Pro Gly Arg Ala Phe Tyr Thr Thr Gly 1 5 10 15 Glu lie lie
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: ea ino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) ORIGINAL SOURCE:
(A) ORGANISM: Influenza Virus (iv) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) OTHER INFORMATION: HA1 hemagglutinin protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Trp Leu Thr Lys Lys Gly Asp Ser Tyr Pro 1 5 10
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) ORIGINAL SOURCE: (A) ORGANISM: Influenza Virus
(iv) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) OTHER INFORMATION: H3 protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Trp Leu Thr Lys Ser Gly Ser Thr Tyr Pro 1 5 10
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) ORIGINAL SOURCE:
(A) ORGANISM: Influenza Virus
(iv) FEATURE:
(A) NAME/KEY: (B) LOCATION:
(C) OTHER INFORMATION: H2 protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: :
Trp Leu Thr Lys Glu Gly Ser Asp Tyr Pro 1 5 10 (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) ORIGINAL SOURCE:
(A) ORGANISM: Measles Virus
(iv) FEATURE: (A) NAME/KEY:
(B) LOCATION: 404...414
(C) OTHER INFORMATION: F protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: lie Asn Gin Asp Pro Asp Lys lie Leu Thr Tyr 1 5 10
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) ORIGINAL SOURCE:
(A) ORGANISM: Foot and Mouth Disease Virus (iv) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 141...160
(C) OTHER INFORMATION: VP1 protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Asn Ser Ala Pro Asn Leu Arg Gly Asp Leu Gin Lys Val Ala Arg 1 5 10 15
Thr Leu Pro
(2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iϋ) ORIGINAL SOURCE:
(A) ORGANISM: Influenza PR8A Virus
(iv) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 110...120 (C) OTHER INFORMATION: Hemagglutinin Protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ser Phe Glu Arg Phe Glu lie Phe Pro Lys Glu 1 5 10
(2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(iii) ORIGINAL SOURCE: (A) ORGANISM:
(iv) FEATURE:
(A) NAME/KEY: (B) LOCATION:
(C) OTHER INFORMATION: Tetanus Toxoid Protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Asn Ser Val Asp Asp Ala Leu lie Asn Ser Thr Lys lie Tyr Ser Tyr 1 5 10 15 Phe Pro Ser Val
20
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: eamino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) ORIGINAL SOURCE: (A) ORGANISM:
(iv) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) OTHER INFORMATION: Tetanus Toxoid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Pro Glu lie Asn Gly Lys Ala lie His Leu Val Asn Asn Glu Ser Ser
1 5 10 15
Glu
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: .amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) ORIGINAL SOURCE:
(A) ORGANISM:
(iv) FEATURE: (A) NAME/KEY:
(B) LOCATION: 88...103
(C) OTHER INFORMATION: Cytochro e C Protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ala Asn Glu Arg Ala Asp Leu lie Ala Tyr Leu Gin Ala Thr Lys 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacteria (iv) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 350...369
(C) OTHER INFORMATION: Heat Shock Protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Asp Gin Val His Phe Gin Pro Leu Pro Pro Ala Val Val Lys Leu Ser 1 5 10 15
Asp Ala Leu lie 20
(2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: e-uieino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(iii) ORIGINAL SOURCE: (A) ORGANISM: Hen
(iv) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 48...61 (C) OTHER INFORMATION: Egg White Lysozyme
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Asp Gly Ser Thr Asp Tyr Gly lie Leu Gin lie Asn Ser Arg 1 5 10
(2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(iii) ORIGINAL SOURCE:
(A) ORGANISM: Streptococcus A
(iv) FEATURE:
(A) NAME/KEY: (B) LOCATION: 308...319
(C) OTHER INFORMATION: M Protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Gin Val Glu Lys Ala Leu Glu Glu Ala Asn Ser Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: .amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) ORIGINAL SOURCE:
(A) ORGANISM: Staphylococcus sp.
(iv) FEATURE: (A) NAME/KEY:
(B) LOCATION: 81...100
(C) OTHER INFORMATION: Nuclease Protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Arg Thr Asp Lys Tyr Gly Arg Gly Leu Ala Tyr lie Tyr Ala Asp Gly 1 5 10 15
Lys Met Val Asn 20
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iϋ) ORIGINAL SOURCE: (A) ORGANISM:
(iv) FEATURE:
(A) NAME/KEY:
(B) LOCATION: (C) OTHER INFORMATION: Synthetic Peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Ala Ala Ala Leu 1
(2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(iii) ORIGINAL SOURCE:
(A) ORGANISM: Influenza PR8A Virus
(iv) FEATURE:
(A) NAME/KEY: (B) LOCATION: 147...161
(C) OTHER INFORMATION: NP Protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Thr Tyr Gin Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp Pro 1 5 10 15

Claims (26)

WE CLAIM :
1. A method of conjugating a peptide to an immunoglobulin molecule via a carbohydrate residue of the immunoglobulin molecule which comprises the steps of: (a) enzymatically oxidizing the carbohydrate residue of the immunoglobulin molecule; (b) reacting the oxidized carbohydrate residue with an amino group of the peptide; and (c) stabilizing the reaction product of step (b) by reaction with a reducing agent.
2. The method of claim 1, wherein the carbo- hydrate residue is galactose or galactosamine, the galactose or galactosamine being oxidized by galactose oxidase.
3. The method of claim 2, wherein the carbo- hydrate residue is galactose.
4. The method of claim 3, wherein the galactose is covalently linked to a sialic acid residue.
5. The method of claim 4, further comprising a step of enzymatically removing the sialic acid residue linked to the galactose residue prior to the enzymatic oxidation of the galactose residue.
6. The method of claim 5, wherein the sialic acid is enzymatically removed by neuraminidase.
7. The method of claim 6, wherein the peptide comprises an immunogenic peptide.
8. The method of claim 7, wherein the immuno- genie peptide comprises a B cell epitope.
9. The method of claim 7, wherein the immuno- genie peptide comprises a helper T cell epitope.
10. The method of claim 6, wherein the peptide comprises the IL-1 epitope.
11. The method of claim 6, wherein the peptide comprises the tetanus toxoid epitope.
12. The method of claim 9, wherein the peptide is covalently linked at an amino group to the amino acid sequence Ala-Ala-Ala-Leu before it is conjugated to the carbohydrate residue, such that the Ala-Ala-Ala-Leu sequence occurs between the peptide and the carbo- hydrate residue upon conjugation.
13. The method of claim 6, wherein the reducing agent comprises pyridine borane.
14. The method of claim 1, wherein the peptide comprises an immunogenic peptide.
15. The method of claim 14, wherein the immuno- genie peptide is a B cell epitope.
16. The method of claim 14, wherein the immuno- genie peptide is a helper T cell epitope.
17. A purified immunoglobulin-carbohydrate- linked-peptide conjugate derivable from the method of claim 1.
18. A purified immunoglobulin-carbohydrate- linked-peptide conjugate derivable from the method of claim 6.
19. The purified immunoglobulin-carbohydrate- linked peptide conjugate of claim 18, wherein the pep- tide is a B cell epitope.
20. The purified immunoglobulin-carbohydrate- linked peptide conjugate of claim 18, wherein the peptide is a helper T cell epitope.
21. The purified immunoglobulin-carbohydrate- linked peptide conjugate of claim 18, wherein the pep- tide is the IL-1 epitope.
22. The purified immunoglobulin-carbohydrate- linked peptide conjugate of claim 18, wherein the pep- tide is the tetanus toxoid epitope.
23. The purified immunoglobulin-carbohydrate- linked peptide conjugate of claim 17, wherein the pep- tide is a B cell epitope or a helper T cell epitope.
24. A purified immunoglobulin molecule comprising (a) an immunoglobulin molecule and (b) a peptide, wherein the immunoglobulin is linked to the peptide via a carbohydrate residue of the immunoglobulin molecule and wherein the peptide comprises a B cell epitope or a helper T cell epitope.
25. The purified immunoglobulin molecule of claim 24, wherein the peptide is a B cell epitope.
26. The purified immunoglobulin molecule of claim 24, wherein the peptide is a helper T cell epitope. 81 27. A vaccine comprising an effective amount of
82 the purified immunoglobulin-carbohydrate-linked peptide
83 conjugate of claim 19 and a suitable carrier.
84 28. A vaccine comprising an effective amount of
85 the purified immunoglobulin-carbohydrate-linked peptide
86 conjugate of claim 20 and a suitable carrier.
87 29. A vaccine comprising an effective amount of
88 the purified immunoglobulin-carbohydrate-linked peptide
89 conjugate of claim 23 and a suitable carrier.
90 30. A vaccine comprising an effective amount of
91 the purified immunoglobulin molecule of claim 24 and a
92 suitable carrier.
93 31. A method of enhancing an immune response to a
94 pathogen comprising administering an effective amount
95 of the purified immunoglobulin-carbohydrate-linked-
96 peptide conjugate of claim 19 and a suitable carrier.
97 32. A method of enhancing an immune response to a
98 pathogen comprising administering an effective amount
99 of the purified im unoglobulin-carbohydrate-linked-
100 peptide conjugate of claim 20 and a suitable carrier.
101 33. A method of enhancing an immune response to a
102 pathogen comprising administering an effective amount
103 of the purified immunoglobulin-carbohydrate-linked-
104 peptide conjugate of claim 23 and a suitable carrier.
105 34. The method of claim 7, wherein the immuno-
106 genie peptide comprises a cytotoxic T cell epitope. 107 35. The method of claim 14, wherein the immuno-
108 genie peptide is a cytotoxic T cell epitope.
109 36. The purified immunoglobulin-carbohydrate-
110 linked peptide conjugate of claim 18, wherein the
111 peptide is a cytotoxic T cell epitope.
112 37. The purified immunoglobulin-carbohydrate-
113 linked peptide conjugate of claim 17, wherein the pep-
114 tide is a cytotoxic T cell epitope.
115 38. A purified immunoglobulin molecule comprising
116 (a) an immunoglobulin molecule and (b) a peptide,
117 wherein the immunoglobulin is linked to the peptide via
118 a carbohydrate residue of the immunoglobulin molecule
119 and wherein the peptide comprises a cytotoxic T cell
120 epitope.
AU59200/96A 1995-05-15 1996-05-13 Carbohydrate-mediated coupling of peptides to immunoglobulins Abandoned AU5920096A (en)

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US477424 1995-06-07
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CA2227326A1 (en) 1996-11-21

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