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WO1998015286A1 - Lymphocytes t a cytolyse specifique des glucides - Google Patents

Lymphocytes t a cytolyse specifique des glucides Download PDF

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Publication number
WO1998015286A1
WO1998015286A1 PCT/US1997/018146 US9718146W WO9815286A1 WO 1998015286 A1 WO1998015286 A1 WO 1998015286A1 US 9718146 W US9718146 W US 9718146W WO 9815286 A1 WO9815286 A1 WO 9815286A1
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Prior art keywords
polypeptide
immunogenic composition
carbohydrate moiety
mhc
cell
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Howard Grey
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La Jolla Institute for Allergy and Immunology
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La Jolla Institute for Allergy and Immunology
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    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001169—Tumor associated carbohydrates
    • 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
    • A61K2039/605—MHC molecules or ligands thereof

Definitions

  • CTL cytolytic T cells
  • CTL Histocompatibity Complex
  • peptides derived from processed cellular proteins are transported into the endoplasmic reticulum, where they associate with newly assembled class I molecules that are eventually transported to and expressed on the cell surface as peptide-MHC complexes (Townsend et al., 1989, Annu. Rev. Immunol. 7:601).
  • the polypeptide length of naturally processed peptides analyzed following elution from class I MHC molecules is generally 9 ⁇ 1 amino acids.
  • the peptide length was found to be 8 amino acids (Fal et al., 1991, Nature 351:290), whereas in all other cases (i.e., all other murine alleles and all human alleles tested) , the peptide length was found to be 9 amino acids.
  • CTL may play an important role in anti-tumor immunity.
  • a number of tumor-associated antigens have been identified. Many of these antigens are carbohydrate structures that are not normally found on differentiated cells, but are expressed at early stages of differentiation, and are generated in tumors because of defects in enzymatic glycosylation steps that operate in normal differentiated somatic cells. Although carbohydrates activate B cells for a humoral response, anti-carbohydrate T cell responses have been difficult to achieve.
  • T cells stimulated according to the invention recognize the carbohydrate moiety of the immunogenic composition with substantially no recognition of the polypeptide backbone portion to which the carbohydrate moiety is linked, i.e., there is little or no crossreactivity between target cells bearing the polypeptide (in the absence of a linked carbohydrate moiety) on their surface and those bearing the glycopeptide.
  • the carbohydrate- specific CTL generated according to the invention can lyse tumor cells bearing the carbohydrate on their surface regardless of whether the carbohydrate antigen is a component of a glycopeptide or glycolipid.
  • the immunogenic composition includes a synthetic polypeptide of at least eight amino acids at least two of which are anchor residues, and a carbohydrate moiety linked to an internal amino acid of the polypeptide.
  • the polypeptide binds with high affinity to a polypeptide binding groove of a MHC class I molecule with the carbohydrate moiety extending beyond the polypeptide binding groove of the MHC molecule to contact and stimulate the T cell to specifically lyse a target cell expressing the carbohydrate moiety its surface.
  • the method of stimulating a carbohydrate-specific cytotoxic T cell to lytic activity includes the steps of (a) providing a class I MHC molecule, (b) contacting the class I MHC molecule with a synthetic polypeptide of at least eight amino acids at least two of which are anchor residues and a carbohydrate moiety linked to an internal amino acid, and (c) contacting the carbohydrate moiety with a CD8-positive T cell.
  • the polypeptide binds with high affinity to a polypeptide binding groove of the MHC molecule with the carbohydrate moiety extending beyond the polypeptide binding groove of the MHC molecule, and the CD8-positive T cell is stimulated to specifically lyse a target cell with the carbohydrate moiety its surface.
  • Figs. 1-4 are line graphs showing hapten-specific cell lysis of EL-4 target cells by splenocytes derived from a mouse immunized with the polypeptide L174 (AIIAKFAAL; SEQ ID NO:l) haptenated with trinitrophenol (TNP) at position 5.
  • Each of Figs. 1-4 represents data from splenocytes derived from a different mouse.
  • FIGs. 5 and 6 are line graphs showing hapten- specific cell lysis of EL-4 target cells by splenocytes derived from a mouse immunized with the polypeptide L175 (AIIAFAKAAL; SEQ ID NO: 2) haptenated with TNP at position 7.
  • Figs. 5 and 6 are line graphs showing hapten- specific cell lysis of EL-4 target cells by splenocytes derived from a mouse immunized with the polypeptide L175 (AIIAFAKAAL; SEQ ID NO: 2) haptenated with TNP at position 7.
  • 5 and 6 represents data from splenocytes derived from a different mouse.
  • Open symbols represent data from an experiment in which EL-4 target cells were incubated with the haptenated polypeptide antigen, L175; closed symbols represent data from a control experiment in which EL-4 target cells incubated with the corresponding non-haptenated polypeptide antigen, L148; and a dotted line represents data from a control experiment in which EL-4 target cells were incubated without antigen.
  • FIG. 7 and 8 are line graphs showing hapten- specific cell lysis of EL-4 target cells by splenocytes derived from a mouse immunized with the polypeptide L176 (AIIAFAAAKL; SEQ ID NO: 3) haptenated with TNP at position 9.
  • Each of Figs. 7 and 8 represents data from splenocytes derived from a different mouse.
  • Open symbols represent data from an experiment in which EL-4 target cells were incubated with the haptenated polypeptide antigen, L176; closed symbols represent data from a control experiment in which EL-4 target cells were incubated with the corresponding non-haptenated polypeptide antigen, L150; and a dotted line represents data from a control experiment in which EL-4 target cells incubated without antigen.
  • Fig. 9 is a line graph showing hapten-specific cell lysis of EL-4 target cells by restimulated splenocytes derived from two different mice immunized with the polypeptide L174. As above, control experiments were carried out in which EL-4 target cells were incubated without antigen and with the non-glycosylated L146 polypeptide.
  • Fig. 10 is a line graph showing hapten-specific cell lysis of EL-4 target cells by restimulated splenocytes derived from two different mice immunized with the polypeptide L174. As above, control experiments were carried out in which EL-4 target cells were incubated without antigen and with the non-glycosylated L146 polypeptide.
  • Fig. 11 is a line graph showing carbohydrate- specific cell lysis of EL-4 target cells by splenocytes derived from a mouse immunized with the polypeptide 2IB, a glycopeptide containing the TF antigen (Gal / 01-3GalNAc ⁇ - 0-serine oligosaccharide) linked via a serine linkage to the same polypeptide backbone as used in the TNP experiments described above (AIIASFAAL; SEQ ID NO: 4). Control experiments were carried out in which EL-4 target cells were incubated without antigen and with the corresponding unglycosylated polypeptide, L205.
  • Fig. 12 is a diagram of the structure of a HLA-A3- like MHC class I-binding polypeptide showing the location and identity of primary anchor residues, secondary anchor residues, and detrimental amino acids.
  • the invention features an immunogenic composition which stimulates CD8 + T cells to specifically lyse a target cell with a specific carbohydrate moiety on its surface.
  • the method of the invention involves contacting a MHC class I molecule with the immunogenic composition to form a MHC-glycopeptide complex and contacting the complex with a CD8 + T cell.
  • the immunogenic composition contains a polypeptide portion characterized by at least two anchor residues and a carbohydrate moiety linked to an internal amino acid of the polypeptide. When bound to an MHC molecule, the carbohydrate moiety of the polypeptide is extended beyond the confines of the polypeptide-binding groove of the MHC molecule.
  • polypeptide is meant any chain of amino acids without posttranslational modification (e.g., glycosylation or phosphorylation) .
  • a polypeptide is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state.
  • a polypeptide which is chemically synthesized, i.e., a synthetic polypeptide, or produced in a cellular system different from the cell from which it naturally originates, e.g., a recombinant polypeptide will be substantially free from its naturally associated components .
  • the polypeptide is at least 8 amino acids in length, more preferably at least 9 amino acids in length, and most preferably the length of the polypeptide exceeds the length of canonical polypeptides for binding to a class I MHC molecule, e.g., 10, 11, 12, 13, 14, 15 or more amino acids in length.
  • a canonical polypeptide is meant a polypeptide which has been processed by the antigen processing pathways of a living cell and is bound to a class I MHC molecule on the surface of the living cell. After elution of the naturally-processed polypeptide from the class I MHC molecule, the size and amino acid sequence can be determined using standard methods.
  • the length of a naturally-processed polypeptide for a given MHC class I allele is readily determined by elution of bound polypeptides from class I MHC molecules on cells.
  • a canonical peptide has the optimal length for binding to a class I MHC molecule.
  • the canonical polypeptide length is 9 ⁇ 1 amino acids, typically 8 or 9 amino acids.
  • the canonical polypeptide length is 8 amino acids.
  • the preferred polypeptide is elongated, i.e., the polypeptide contains one or more amino acids in excess of the canonical length for a given class I MHC allele.
  • the additional amino acids are located in the region between the anchor residues.
  • the region of polypeptide containing the additional amino acids forms a bulge in the polypeptide backbone onto which a carbohydrate moiety can be linked.
  • the carbohydrate moiety is linked to a polypeptide having the canonical length for a given MHC class I allele.
  • the carbohydrate moiety is linked to an internal amino acid via a linker group, e.g., a long side chain of an internal amino acid, e.g., lysine, which serves to position the carbohydrate moiety outside the polypeptide-binding groove of the MHC molecule.
  • a variable length linker group may be utilized to customize the position of the carbohydrate moiety with respect to the polypeptide-binding groove, e.g. , the polypeptide may be positioned closer or further away from the polypeptide-binding groove to skew the immune response to the carbohydrate moiety and not the polypeptide to which it is linked.
  • the linker group is an ⁇ -amino alkanoic acid.
  • the polypeptide portion of the glycopeptide may include three or more anchor residues, e.g., four anchor residues, to stabilize the MHC-glycopeptide complex and contains an amino acid with a short side chain (or no side chain) in a non-anchor residue position.
  • the polypeptide preferably contains such an amino acid in each non-anchor residue position.
  • the amino acid in the non-anchor position is a neutral amino acid such as serine, glycine or an alanine.
  • the amino acid to which the carbohydrate moiety is linked is preferably a serine, threonine, asparagine, or lysine.
  • the stimulated CTL lacks cross reactivity between a given glycosylated polypeptide and the corresponding unglycosylated polypeptide. Most preferably, the stimulated CTL also lacks reactivity to the class I MHC molecule.
  • the class I MHC molecule is cell-associated, and the cell has the same haplotype as the CD8-positive T cell.
  • the length of the polypeptide exceeds the canonical polypeptide length for binding to a class I MHC molecule (e.g., 9 ⁇ 1 amino acids for most MHC class I alleles) .
  • a class I MHC molecule e.g. 9 ⁇ 1 amino acids for most MHC class I alleles
  • Such elongation may cause the polypeptide to bulge out in regions of the polypeptide not directly involved in polypeptide-MHC binding.
  • the carbohydrate moiety is linked to an amino acid, e.g., a serine, threonine, asparagine, or lysine, located in the protruding portion, i.e., the bulge region, of the polypeptide.
  • the polypeptide is designed to bind to the MHC molecule with high affinity.
  • high affinity polypeptide is meant a polypeptide the MHC binding affinity of which is 500 nM or less. Preferably, the binding affinity is 50 nM or less.
  • the preferred polypeptide has two properly positioned anchor residues.
  • the binding affinity of a polypeptide may also be enhanced by introducing a secondary anchor residue resulting in enhanced immunogenic potential.
  • anchor residue is meant an amino acid which is important in promoting binding to a particular allele of a class I MHC molecule.
  • an anchor residue may be an amino acid with side chains that occupy a major binding pocket in the class I MHC polypeptide binding site.
  • a polypeptide of canonical length typically has two primary anchor residues which mediate binding to the MHC class I molecule. Primary anchor residues are typically located at or near the N- or
  • a preferred polypeptide has anchor residues in positions 5 (or 6) and 8 (or 9) of an 8 or 9-amino acid polypeptide, respectively) .
  • secondary anchor residue is meant an additional residue (in excess of the two primary anchor residues) which stabilize polypeptide-MHC class I binding.
  • a polypeptide which binds to a class I MHC molecule with high affinity has at least two anchor residues (i.e., primary anchor residues, and may contain at least three anchor residues (i.e., two primary anchor residues and one secondary anchor residue) , or four or more anchor residues. These secondary anchor residues function to strengthen the polypeptide-MHC binding interaction, e.g., improve the binding affinity of a MHC class I binding polypeptide.
  • MHC class I molecule-binding polypeptides The structural characteristics of a MHC class I molecule-binding polypeptides is determined using standard methods for eluting and determining the size and sequence of the polypeptide, e.g., methods described in Van Bleek et al. , 1990, Nature 348:213-216, Falk et al., 1991, Nature 351:290-296, or Paul, W. , ed. , Fundamental Immunology, 3rd edition, Raven Press, New York, pp. 641- 643. As discussed above, anchor residue positions for many MHC class have been determined. Table 1 shows the positions of anchor residues for allele-specific polypeptide binding to human and mouse MHC molecules determined by direct peptide sequencing. Human MHC class I molecules have been further classified into HLA supertypes based on shared polypeptide binding characteristics.
  • peptide motifs recognized by products of five common HLA-A alleles are characterized by a positively charged amino acid (R or K) at the C-termini and a hydroxyl-containing (S or T) or hydrophobic (L, V, I, or M) residue at position 2.
  • Peptide motifs that bind to the B7-like supertype are characterized by a P at position 2 and hydrophobic/aromatic residues at the C- terminus.
  • Peptides with broad binding specificities e.g., peptides that bind to A2-like, A3-like, B7-like, B44-like supertypes are known in the art (e.g., as described in Sidney et al., 1995, J. Immunol. 154:247- 259; del Guercio et al. , 1995, J. Immunol. 154:685-693; Sidney et al., 1996, J. Immunol. 157:3480-3490; and Sidney et al.
  • polyalanine (or polyserine or polyglycine) peptides of 9 or 10 residues in length are synthesized. These synthetic peptides have the appropriate major and minor MHC contact residues that allow them to bind with high affinity to all or most of the alleles within the following supertypes: A2-like, A3-like, B7-like, B44-like. Carbohydrate conjugation to these four peptide backbones will result in a vaccine capable of eliciting TACA-specific CTL in the vast majority of immunocompetent humans .
  • Primary anchor residues of preferred polypeptides which bind to the HLA-A2-like supertype are a small or aliphatic amino acid (L, I, V, M, A, or T) in position 2 and also in the C-terminal position.
  • Primary anchor residues of preferred polypeptides which bind to the HLA- B7-like supertype are a P in position 2 and a hydrophobic or aliphatic amino acid (L, I, V, M, A, F, W, and Y) in the C-terminal position.
  • Primary anchor residues of preferred polypeptides which bind to the HLA-A3 supertype are a A, L, I, V, M, S, or T in position 2 and a positively-charged amino acid such as a R or K in the C-terminal position.
  • secondary anchor residues and their preferred positions can also be determined.
  • detrimental amino acids i.e., those which reduce the affinity of polypeptide-MHC binding, are also determined.
  • detrimental amino acids are to be avoided.
  • the primary anchor residues (in position 2 and the C-terminal position) , secondary anchor residues (in position 3, 6, 7, and 8), as well as detrimental amino acids (in position 1 and 3) have been determined for polypeptides which bind to the HLA-A3-like alleles of human MHC class I molecules (see Fig. 12).
  • the polypeptide binds to the MHC molecule with an inhibitory capacity of no greater than 500 nM, preferably with an inhibitory capacity of no greater than 50 nM, more preferably with an inhibitory capacity of no greater than 25 nM, and most preferably with an inhibitory capacity of no greater than 1 nM.
  • Inhibitory capacity is, in general, inversely related to immunogenicity.
  • the carbohydrate moiety of the polypeptide is linked to an internal amino acid.
  • internal amino acid is meant an amino acid located in or near the middle of a given polypeptide, i.e., neither in the N- nor the C-terminal positions of the polypeptide.
  • the internal amino acid is located between the two primary anchor residues.
  • the carbohydrate moiety is linked to an amino acid in position 4, 5, or 6 of a 9-residue polypeptide.
  • Internal amino acids may form a central core structure important in binding of the polypeptide to the class I MHC molecule.
  • the carbohydrate is preferably linked to an amino acid in position 5 of a 9-residue polypeptide.
  • the carbohydrate moiety may be associated with a tumor, such as an adenocarcinoma, lymphoma, melanoma, or neuroblastoma, or other carcinomas characterized as having an unique or upregulated carbohydrate antigen, e.g., lung, stomach, colon, or pancreatic cancers.
  • a tumor such as an adenocarcinoma, lymphoma, melanoma, or neuroblastoma, or other carcinomas characterized as having an unique or upregulated carbohydrate antigen, e.g., lung, stomach, colon, or pancreatic cancers.
  • the carbohydrate moiety is an immunodominant antigen, e.g., the tumor-associated carbohydrate antigens TF, Tn, STn GD 2 , and GD 3 , GM 2 , GM 3 , and LE a ' b ' or x .
  • TF is an antigen expressed on human adenocarcinomas.
  • Tn is meant a monosaccharide, GalNAc, O-linked to a serine or threonine on the polypeptide.
  • STn is meant the sialated form of Tn.
  • GD 3 is a tetrasaccharide with two sialic acid residues, which is the precursor to the pentasaccharide GD 2 .
  • hapten is meant a functional group corresponding to a single antigenic determinant which is not immunogenic unless attached to larger molecule, i.e., the carrier.
  • a hapten can be an organic molecule such as TNP or a carbohydrate moiety, whereas the larger molecule, i.e., the carrier, is typically a polypeptide.
  • the carrier may also be a lipid.
  • the invention also includes a vaccine containing a glycopeptide which elicits a carbohydrate-specific cytolytic T cell immune response, and a method of generating a therapeutic immune response or protective immunity against a tumor by administering such a glycopeptide to a mammal, e.g., a human patient.
  • the immunogenic composition may be administered to patients at risk of developing cancer or already suffering from cancer to generate tumor cell-specific CTL.
  • compositions of the invention in which the carbohydrate moiety is one that is associated with an infectious agent, e.g., a pathogenic bacteria or virus, may be administered to patients as a vaccine against or to treat bacterial infections.
  • Suitable carbohydrate moieties are associated with bacteria which cause tuberculosis, leprosy, and mycobacterial infections.
  • arabino annan is a suitable carbohydrate moiety which can be used to generate CTL to treat or prevent mycobacterial infections.
  • a lipopeptide of the following general structure (PAM) 2 KSS-TH-CTL, in which two palmitic acid residues (PAM) are linked through a KSS spacer to a T helper epitope (TH) , which in turn is linked to the CTL epitope.
  • PAM palmitic acid residues
  • TH T helper epitope
  • Such antigen preparations when administered subcutaneously without any additional adjuvant, have been found to be highly immunogenic in mice as well as humans.
  • lipopeptide compositions offer an alternative antigenic construct for elicitation of carbohydrate-specific CTL responses.
  • HBV Pol 635-643 GLYSSTVPV (SEQ ID 33 9.8 NO: 6)
  • HPV Pol 1076-1084 HLYSHPIIL SEQ ID 38 10.2 NO: 7
  • HBV Pol 1344-1352 WILRGTSFV (SEQ ID 278 _a NO: 10)
  • HBV Pol 996-1004 NLSWLSLDV (SEQ ID 385 6.5 NO: 11)
  • HPV 16 E6 29-38 TTHDIILECV (SEQ ID 238 37.5 NO: 14) HBV Pol 992-1000 LLSSNLSWL (SEQ ID 1087 - NO: 15)
  • HBV Pol 985-993 NLQSLTNLL (SEQ ID 2000 - NO: 16)
  • HBV Pol 43-51 HLLVGSSGL (SEQ ID 2778 - NO: 17)
  • HBV Pol 28-36 LLDDEAGPL (SEQ ID .b - NO:20)
  • HBV Pol 594-602 PLEEELPRL (SEQ ID • - NO:21)
  • HBV Pol 42-50 DLNLGNLNV (SEQ ID • - NO:25)
  • HBV Pol 724-732 PLPIHTAEL (SEQ ID • - NO: 27)
  • HLA-A2.1 motif-containing polypeptides were split into four groups based on their HLA-A2 binding capacity; high affinity (KD ⁇ 50nM) ; intermediate affinity (KD 50-500 nM) ; low affinity (KD 500-5000 nM) ; and non-binders. Under the assay conditions described herein, the KD approximates the IC50% determinations. When the immunogenicity of thesepolypeptides was determined in HLA-A2 transgenic mice, it was found that 5 of 5 high affinity polypeptides and 3 of 5 intermediate affinity polypeptides were immunogenic, whereas 0 to 5 low affinity polypeptides and 0 of 8 non-binders were immunogenic.
  • the ability to quantitatively measure MHC binding affinity and relate that affinity directly with immunogenicity in vitro is useful to predict suitable haptenated polypeptide compositions for in vivo immunizations.
  • the binding affinity of polypeptides that bind weakly to MHC can be enhanced by introducing more amino acids at the critical anchor residue positions which stabilize polypeptide/MHC binding resulting in a high affinity polypeptide with enhanced immunogenic potential.
  • a similar strategy can be used to change the allele-specificity of a given polypeptide, e.g., K b binding haptenated polypeptides can be converted to D binding polypeptides by introducing amino acid substitutions at the anchor residues.
  • Peptide size restrictions for MHC binding can arise from two non-mutually exclusive mechanisms: 1) high affinity binding to class I MHC molecules requires a certain length to optimize the interaction of polypeptide backbone and side chains to MHC; and/or 2) antigen processing and transport into the endoplasmic reticulum (perhaps involving intermediary polypeptide binding chaperones) imposes a size restriction.
  • polypeptides that are at least 6 amino acids longer than the 9-mer are capable of binding
  • polypeptides 2-3 amino acids longer than the canonical length are capable of binding.
  • Peptides were synthesized by Fmoc chemistry using a multiple peptide synthesizer (Symphony/Multiplex, Protein Technologies, Inc.). Peptides were cleaved automatically on the synthesizer using trifluoroacetic acid (TFA) as a cleavage reagent. Peptides were purified to greater than 90% purity by C18 reverse phase high pressure liquid chro atography (HPLC) and were characterized by amino acid composition analysis and/or mass spectroscopy. TNP modifications were introduced by using e-N-TNP lysine derivatives for peptide synthesis (custom synthesized by Bachem Bioscience Inc. , King of Prussia, PA) . Oligosaccharide modifications were introduced using standard methods as described below.
  • MHC purification EL-4 cells were used as a source of K b and D b molecules.
  • NP40 cell lysates from large-scale (10 10 to 10 11 cells/ml) cell cultures were filtered through 0.45 micron filters and purified by affinity chromatography using the Y3 antibody for purification of K b molecules and 281485 monoclonal antibody for purification of D b .
  • the MHC molecules were eluted with diethylamine, 1% N-octylglucoside, pH 11.5, and immediately neutralized with 1 M Tris pH 6.8, concentrated by ultrafiltration and stored at 4°C. Protein purity and concentration were monitored by SDS- PAGE analysis.
  • MHC binding assay Previously identified MHC binding polypeptides for K b and D b were iodinated by the Chloramine T method. MHC concentrations previously determined to yield approximately 15% of bound polypeptide were used in the assays.
  • an assay of antigen-dependent CTL degranulation can be used.
  • release of the granule enzyme serine esterase into the medium is measured.
  • 10 5 CTL and 10 5 target cells or insolubilized antigen are placed in flat bottom wells in 0.1 ml RPMI media, incubated at 37°C for varying periods of time, supernatant removed and mixed with a reaction mixture which contains the substrate N-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT, CalBiochem, San Diego, CA) and 5, 5-dithiobis (2- Nitrobenzoic acid) (Sigma Chemical Co., St.
  • CTL lines are established by stimulating 2 x 10 5 T cells from the initial in vitro cultures with 5 x 10 6 irradiated spleen cells as source of antigen presenting cells (APC) in individual wells of a 24-well plate. Each well contains 2 ml of medium supplemented with 20 ⁇ g of peptide antigen and 20% final concentration of con-A supernatant as a source of ly phokines.
  • Con-A supernatants are prepared by stimulating rat spleen cells with Con-A and harvesting the culture supernatant. T cells are stimulated on a 7 day cycle and fed with complete medium containing 20% Con-A supernatant on the fourth day of the propagation cycle.
  • CTL clones are isolated by culturing propagated lines at limiting dilution (10, 1, 0.33 cells per well) in RPMI complete medium containing 20 ⁇ g of peptide, 20% Con-A supernatant, and 7.5 x 10 5 irradiated spleen cells. Wells with observable cell growth are picked, replated, and maintained in 24 well plates under identical culture conditions. When sufficient numbers of cells are available, they are assayed for cytolytic activity against target cells in the presence of peptide. Hapten specificity of the anti-TNP CTL response
  • the invention provides an immunogenic composition, e.g., a hapten-peptide conjugate, e.g., a synthetic polypeptide to which a carbohydrate moiety is linked that: 1) binds with high affinity to a MHC class I molecule; and 2) is of a size and amino acid composition such that the T cell response is skewed toward recognition of the hapten, i.e., the carbohydrate moiety, rather than either the peptide or the MHC molecule.
  • a hapten-peptide conjugate e.g., a synthetic polypeptide to which a carbohydrate moiety is linked that: 1) binds with high affinity to a MHC class I molecule; and 2) is of a size and amino acid composition such that the T cell response is skewed toward recognition of the hapten, i.e., the carbohydrate moiety, rather than either the peptide or the MHC molecule.
  • the murine MHC class I K b molecule were chosen as a model system because: 1) the crystal structure of peptide/MHC complexes with naturally-processed polypeptides is known; 2) the K b -restricted CTL recognition of the hapten, TNP, has been characterized; and 3) a K b -restricted tumor model system is available.
  • Detrimental amino acids i.e., those which reduce the affinity of polypeptide-MHC binding, tend to be charged amino acids, especially negatively charged ones.
  • Detrimental residues at non-anchor positions interfere with the "docking" of peptides in the MHC binding groove.
  • detrimental amino acids are substituted with neutral amino acids having short side chains or no side chains.
  • an octapeptide with a polyalanine (or glycine) backbone into which an anchor residue F or Y at position 5 and an anchor residue L or M at position 8 are inserted was synthesized.
  • Such poly-alanine or poly-glycine peptides with the appropriate anchor residues bind with high affinity binding to MHC class I molecules.
  • Poly-alanine or poly-glycine anchor residue- containing peptides were synthesized and tested for their ability to bind detergent-solubilized and purified K b by determining their capacity to inhibit the binding of a known radioodinated ligand to K b .
  • the quantity of polypeptide required to achieve 50% inhibition of binding of the radiolabeled ligand approximates the affinity (KD) of the polypeptide for MHC.
  • TCR interaction with such a haptenated peptide-MHC complex is more dependent on binding of the haptenic moiety and less on interaction with the MHC itself.
  • X- ray crystallographic analysis can be used to confirm the structure of the haptenated polypeptide-MHC class I molecule complexes.
  • An increase in polypeptide length was found to be compatible with K b binding.
  • the binding of an elongated polypeptide is associated with a conformational change in which the polypeptide tends to bulge further out from the MHC polypeptide binding groove compared to the conformation of the canonical-sized polypeptide, i.e., a polypeptide having the length of a naturally-processed polypeptide.
  • the extent to which such a protrusion from the binding groove occurs is dictated by the length of the polypeptide and the extent that the increase in length is still compatible with high affinity MHC binding.
  • some MHC molecules such as the human MHC class I alleles, HLA-All, can accommodate polypeptides as long as 15 amino acids.
  • Peptides of different lengths were tested for their K b binding capacity. Increased length was accomplished by insertion of extra amino aids between the N-terminus and the first anchor position (F/Y) , or between the first anchor and the C-terminus. Polypeptide lengths ranged from 8 to 13 amino acids.
  • Poly-alanine peptides which have the canonical length (i.e., 8 or 9 amino acids) and elongated polyalanine peptides (i.e., longer than 8 or 9 amino acids) and having appropriate anchor residues were evaluated for their ability to bind to either K b or D b molecules. These data are shown in Table 4.
  • AAAAFAAL (SEQ ID NO: 35) 75
  • AAAAAFAAL (SEQ ID NO: 37) 610
  • AIAAAFAAL (SEQ ID NO: 38) 240
  • AAIAAFAAL (SEQ ID NO: 39) 90
  • AIIAAFAAL SEQ ID NO: 40
  • AIIAAAFAAL (SEQ ID NO: 41) 65
  • Three K b motif-containing peptides were made: one of the canonical 8 residue length and two 10 residue peptides, with two extra alanines being inserted either between the N-terminus and the first anchor position or between the first anchor and the C-terminal residue.
  • the canonical D b peptide bound with high affinity (17 nM) , while the two longer peptide bound with 20- to 70-fold lower affinity.
  • the canonical 8 residue peptide bound with an intermediate affinity of about 350 nM, and the two longer peptides bound with either a slightly lower affinity (KD 700 nM) or, in the case of the 10 residue peptide with the extra alanines between the anchor position and the C-terminus, with somewhat higher affinity (200 nM) .
  • the peptide backbone can be changed from alanine to glycine and alternative anchor residues inserted, i.e., F at position 5 and L at the C-terminus.
  • alternative anchor residues i.e., F at position 5 and L at the C-terminus.
  • secondary anchor residues were inserted into the poly-alanine (glycine) backbone. These changes can also be made simultaneously. The insertion of secondary anchor residues, singly or in combination, was found to improve the binding capacity of the canonical peptide.
  • a complementary, independent strategy to accomplish the goal of having a haptenic group of a peptide extend beyond the confines of the MHC binding groove is to attach the hapten to an amino acid having a long side chain.
  • This can readily be accomplished by introducing a "linker” between the e-amino group of a lysine residue and the hapten.
  • the end result of this type of modification would have the following formula:
  • polypeptide derivatives are prepared according to standard procedures, e.g., that described in Bodanski et al., 1984, The Practice of Peptide Synthesis, Springer Verlag, pp.275, and incorporated into a standard solid phase peptide synthesis.
  • a lysine derivative with appropriate orthogonal protecting groups is incorporated into the peptide.
  • the side chain protecting group of lysine Prior to cleavage of the peptide from the solid support, the side chain protecting group of lysine is selectively deblocked and a hapten-linker construct such as (TNP-NH- (CH 2 ) - n -C0 2 H) is added.
  • Suitable lysine derivatives include Fmoc-Lys (Alloc) -OH and Fmoc-Lys (Dde) -OH, whose side chain protecting groups can be selectively removed using standard methods, e.g., those described in Kunz et al. , 1989, Chem. Pept. Proteins 4:119 or Bycroft et al., 1993, Chem. Commun. 9:778).
  • This strategy allows the preparation of analogs having different linker lengths from a single batch of resin-linked peptide.
  • TNP haptenated peptides ranging in size from the canonical 8-mer to the longest peptides that are compatible with high affinity K b binding were synthesized.
  • the TNP-lysyl (or TNP-1inker-lysyl) moiety was placed at the position (s) which by computer modeling suggests they might be at the top of the peptide bulge, e.g, at position 5
  • TNP peptide-MHC complexes are made and tested for the capacity of the MHC/TNP peptide complexes to react with anti-TNP antibodies.
  • RMA-S cells are used as the source of K b . These cells, when pre-incubated at room temperature, express empty MHC that can be loaded and stabilized by the K b binding TNP peptides. The complexes are double stained with fluorescent anti-K b and anti-TNP antibodies, and then analyzed using fluorescence- activated cell sorting (FACS) analysis under limiting conditions of time and anti-TNP concentration. Data from these analyses indicate how accessible the TNP group is for reactivity with antibody when bound to K b as a haptenic moiety conjugated to peptides and side chains of varying length. Accessibility for antibody binding is predictive of accessibility to the TCR on the CTL.
  • FACS fluorescence- activated cell sorting
  • TNP peptides all capable of high affinity binding to K b and differing in peptide length and side chain length to which the TNP is attached, was chosen to immunize mice for the generation of hapten-specific CTL.
  • hapten-specific CTL and the characterization of the specificity of the CTL for hapten, peptide and MHC i
  • Immunization On the basis of the experiments described above, a series of haptenated peptides were chosen as immunogens for the generation of class I MHC restricted T cell responses. H-2 b mice were immunized subcutaneously with haptenated peptide in IFA. Two weeks after priming, spleen cells were re-stimulated in vitro with the haptenated peptide, and effector functions measured 2-6 days later.
  • the recognition of the haptenated peptide can be assessed by: 1) the capacity of TNP peptide to sensitize H-2 b target cells for killing by the immune T cells; 2) the induction of a proliferative response (with or without the addition of IL-2) ; and 3) the stimulation of IFN- ⁇ production.
  • the carbohydrate antigens are components of glycolipids or glycoproteins that are very different than the poly-alanine or poly-glycine peptide backbones discussed above.
  • the immunogenic compositions of the invention are preferably hapten (i.e., carbohydrate) specific, with little or no crossreactivity with the peptide, hapten- peptide conjugate or the MHC class I molecule itself.
  • MHC molecules serve at lease two functions in the recognition of antigen by TCR.
  • the second function is to directly contribute to the interaction with the TCR through the participation of residues in the ⁇ helices of the peptide binding groove that are located on the top of the helix and point up toward the solvent, thus being in a position to engage the TCR.
  • the immunogenic compositions of the invention were tested to evaluate the role of MHC molecules in the recognition of a hapten by a TCR.
  • TACA haptens for the induction of carbohydrate- specific CTL
  • carbohydrate antigens There are several tumor-associated carbohydrate antigens that have been identified by virtue of their reactivity with well-defined anti-oligosaccharide antibodies.
  • the simplest structure, the Tn antigen is a monosaccharide, GalNAc, O-linked to a serine or threonine on the peptide.
  • Stn is a disaccharide that is the sialated version of Tn.
  • GD 3 is a tetrasaccharide with two sialic acid residues, which is the precursor to the pentasaccharide GD 2 (Table 6) .
  • T cell response will be increasingly specific for the saccharide moiety of the glycopeptides with increasing complexity of the carbohydrate.
  • Neutral saccharides due to their lack of hydrophobicity and charge, are limited to hydrogen bonding as a means of interaction with immune receptors.
  • introduction of one (STn) or two (GD 2 and GD 3 ) sialic acid residues to a synthetic polypeptide can contribute to a more potent, higher affinity response, due to the capacity to form salt bridges between the sialic acid and basic residues in the TCR.
  • STn antigens are prepared using procedures known in the art, e.g., Garg et al., 1994, Adv. Carbo. Chem. Biochem. 50:277. Serine or threonine is incorporated via an ⁇ -glycosidic linkage using the activated forms of 2- azido galactose (Tn) .
  • Tn 2- azido galactose
  • the glycoamino acids are introduced into the desired peptides utilizing solid phase peptide synthesis, e.g., using Fmoc chemistry.
  • the polypeptides are then deprotected using standard methods to yield the desired products.
  • STn derivatives are prepared using a combined che o-enzymatic synthetic strategy known in the art (Braun et al., 1993, Bioorg. Med. Chem. 1:197).
  • the sialic acid is introduced enzymatically in either of two ways: 1) before incorporation of the glyco-amino acid into the peptide; or 2) after the completed synthesis and deprotection of the glycopeptide.
  • An ⁇ 2 , 6-sialyl transferase is then used to introduce the sialic acid onto the deprotected Tn-glycopeptides, in combination with a regenerative sugar nucleotide cycle for CMP-sialic acid, utilizing reaction conditions commonly used for sialylated sugars.
  • oligosaccharides are synthesized and linked to a polypeptide backbone using known methods.
  • GD 3 and GD 2 moieties are linked as follows.
  • the carbohydrate portions GD 3 and GD 2 gangliosides are linked to a ceramide residue through an O-glycosidic bond in the ⁇ configuration.
  • these carbohydrate structures are prepared in a form which is O-linked to either serine or threonine residue.
  • tBOC or Fmoc peptide chemistries are effective in solid phase peptide synthesis, with Fmoc chemistry being the preferred method.
  • the synthesis of many carbohydrate structures can begin from a common intermediate, NANAc.2, 3Gal / -31,4Glcj9-0- protected amino acid.
  • This intermediate is prepared by first coupling either trichloroacetimidate or halide peracetylated lactose with an appropriately protected serine or threonine residue, using one of several standard procedures (e.g., Garg et al., 1994, Adv. Carbo. Chem. Biochem. 50:277 or Polt et al., 1992, J. Am. Chem. Soc. 114:10249).
  • the lactoside is deacetylated and then enzymatically sialated, using conditions commonly used to manufacture sialosides with an ⁇ 2,3-Sialyl transferase and a CMP-sialic acid regeneration cycle, thereby providing the key intermediate, NANAc.2, 3Gal?l,4Glc3-0- protected amino acid.
  • the synthesis of the oligosaccharide portion of GD 3 begins by reacting
  • Synthesis of GM 2 oligosaccharide, a precursor for GD 2 begins by reacting the NANAc.2, 3Gal/?l,4Glc/3-0- protected amino acid with a /Jl,4-GalNAc transferase and UDP-GalNAc at stoichiometric amounts.
  • the enzymes required for the synthesis of the UDP-GalNAc are commercially available.
  • GD 2 is then made by the use of ⁇ 2,8 sialyl transferase.
  • the GM 2 structure GalNAc, ⁇ l,4- (NANA ⁇ 2, 3) Gal/31, 4Glc/3-0-protected amino acid is used to prepare this material, using enzymatic sialylation as described and yielding the GalNAc?l,4- (NANAc.2, 8NANA ⁇ 2, 3) Gal / 3l,4Glc;3-0-protected amino acid.
  • these compounds are acetylated. Protecting groups are then modified and incorporated into the various peptides, as described above.
  • the oligosaccharide portions of GD 2 and GD 3 can be obtained from the ganglioside GD l , which can be isolated easily and in reasonable yields from bovine brain. This material can then be converted with the appropriate glycosidases into GD 2 or GD 3 and the newly created oligosaccharides removed from the ceramide lipids with ceramide glycanase. The O-glycosides of these sugars are formed by acetylating the isolated carbohydrates, as described above, activating the carbohydrate and then coupling with the appropriate acid.
  • linker groups e.g., ⁇ -hydroxy alkanoic acids of varying lengths, OH-(CH 2 ) n -C0 2 H, can be used.
  • the oligosaccharide-linker moiety is then coupled to the peptide via the eNH 2 group of lysine in an identical manner to that described for TNP.
  • the invention provides an immunogenic composition and a vaccine containing the immunogenic composition, e.g., a glycopeptide which elicits a carbohydrate- specific cytolytic T cell immune response, and a method of generating protective immunity against a tumor by administering such a glycopeptide to a mammal, e.g., a human patient.
  • a vaccine containing the immunogenic composition e.g., a glycopeptide which elicits a carbohydrate- specific cytolytic T cell immune response
  • a method of generating protective immunity against a tumor by administering such a glycopeptide to a mammal, e.g., a human patient.
  • Immunogenic compositions with high affinity binding for human MHC class I molecules i.e. HLA alleles
  • HLA alleles The lengths of naturally-processed HLA-binding polypeptides and the location of critical anchor residues for several human MHC class I molecules, i.e., HLA alleles, have been determined (Sidney et al., 1996, Human Immunology 45:79).
  • polypeptides containing anchor residues in known critical positions and in which non-anchor residue positions are preferably occupied by neutral amino acids with short (or no) side chains can be synthesized.
  • Carbohydrate moieties e.g., TACA are linked to such synthetic polypeptides as described above, i.e., to a protuding portion of an elongated polypeptide or via a linker group, to extend the carbohydrate moiety of the immunogenic composition beyond the polypeptide binding groove when bound to an HLA molecule.
  • the immunogenic compositions of the invention can be administered to a mammal, e.g., a human patient needing treatment for cancer, using standard methods, e.g., those described in Vitiello et al., 1995, J. Clin. Invest. 95:341.
  • the compositions are administered to the patient subcutaneously, intramuscularly, or intravenously in a pharmaceutically acceptable carrier such as physiological saline or DMSO. It is expected that a dosage of approximately 0.1 to 100 ⁇ moles of the polypeptide of the invention would be administered per kg of body weight per day.
  • dosage for any given patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Determination of optimal dosage is well within the abilities of a pharmacologist of ordinary skill.
  • immune cells e.g. , peripheral blood mononuclear cells or bone marrow cells
  • immune cells can be obtained from a patient or an appropriate donor and activated ex vivo with the immunogenic compositions, and then returned to the patient.
  • the stimulated CTL are administered to the patient intravenously.
  • EXAMPLE 1 IMMUNOGENIC COMPOSITION TO STIMULATE TNP- SPECIFIC CTL
  • K b murine MHC molecule
  • the canonical K b binding motif is an 8-amino acid peptide with tyrosine or phenylalanine at position 5, and leucine or methionine at position 8.
  • the best combinations of those four amino acids on a simple polyamino acid backbone which had minimal side chains to engage the T cell receptor was determined.
  • the peptide was also extended yielding polypeptides of 9 to 11 amino acids.
  • Polypeptides containing glycine, alanine, and serine in non-anchor residue positions were made and tested. Glycine was found not to be compatible with high affinity binding. Various combinations of amino acids in anchor residue positions were also tested. Phenylalanine and leucine provided the highest affinity interaction, but even with these two anchor residues in place, the affinity was not as high as natural peptides that have been previously demonstrated to bind with high affinity to K b . Therefore, one or two "secondary" anchor residues were added to the polypeptide.
  • This high affinity peptide was then elongated either near the N-terminus, i.e., between the N-terminus and the first anchor residue, or near the C-terminus between the first and second anchor residues. Although significant binding was observed with peptides as long as 11 amino acids, the binding affinity was found to decline sharply with peptides longer than 9 amino acids.
  • the first series of immunization experiments utilized peptides in which TNP lysine was placed at positions 5 (of a 9-mer) , and positions 7 and 9 (of 10- mer peptides).
  • the K b binding data is shown in Table 7.
  • Peptides which bind with an IC50% in the range of 50 nM or less are almost always (>90%) immunogenic, and peptides that bind with IC50% in the range of 50-500 nM are immunogenic about 50% of the time.
  • all peptides tested are predicted to be immunogenic.
  • C57B1/6 mice were immunized with 50 ⁇ g of each of these peptides subcutaneously in incomplete Freund's adjuvant.
  • splenocytes were plated in culture flasks (3 x 10 7 cells per flask) together with lipopolysaccharide (LPS) -activated normal C57B1/6 splenocytes as accessory cells and 10 ⁇ g of peptide.
  • LPS lipopolysaccharide
  • IL-2 lipopolysaccharide
  • TACA TF antigen (Gal/?l-3GalNAcc.-0-serine oligosaccharide) ) attached via a serine linkage to the same peptide backbone as used in the TNP experiments (AIIASFAAL; SEQ ID NO: 4) was made and tested.
  • the carbohydrate moiety i.e., the hapten
  • Mice were immunized and splenocytes harvested as described in Example 1. The splenocytes were tested for carbohydrate-specific CTL activity. Fig.
  • any carbohydrate moiety can be linked to position 5 of the cartridge and used to immunize a mammal to generate a CTL response specific for that particular carbohydrate moiety.
  • the TACA associated with that particular type of cancer is linked to position 5 of the cartridge (or a predetermined position of a polypeptide cartridge known to bind to the HLA allele of the patient) for the purpose of treating the patient's cancer, i.e., generating CTL to kill tumor cells in the patient.

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Abstract

La présente invention concerne une composition immunogène contenant une fraction de glucide liée à un acide aminé interne d'un polypeptide. Ce polypeptide se lie avec une grande affinité à un sillon de liaison d'un polypeptide d'une molécule MHC de classe I. En l'occurrence, la fraction glucide dépasse du sillon de liaison du polypeptide de la molécule MHC de façon à stimuler la lyse spécifique, par la lymphocyte T, d'une cellule cible, dont la surface porte la fraction glucide. L'invention concerne également un procédé de stimulation de la lyse, par un lymphocyte T CB8+, de cellules cibles de façon spécifique aux glucides.
PCT/US1997/018146 1996-10-08 1997-10-08 Lymphocytes t a cytolyse specifique des glucides Ceased WO1998015286A1 (fr)

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US6919318B1 (en) 1998-04-22 2005-07-19 Chiron Corporation Enhancing immune responses to genetic immunization by using a chemokine
WO2004085461A3 (fr) * 2003-03-24 2005-09-15 Immatics Biotechnologies Gmbh Peptide associe a une tumeur et se liant a des molecules mhc

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WO1993021948A1 (fr) * 1992-04-28 1993-11-11 Astra Aktiebolag Produits de conjugaison peptides-glucides suscitant une immunite due aux lymphocytes t

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WO1993021948A1 (fr) * 1992-04-28 1993-11-11 Astra Aktiebolag Produits de conjugaison peptides-glucides suscitant une immunite due aux lymphocytes t

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DEL GUERCIO ET AL: "BINDING OF A PEPTIDE ANTIGEN TO MULTIPLE HLA ALLELES ALLOWS DEFINITION OF AN A2-LIKE SUPERTYPE", THE JOURNAL OF IMMUNOLOGY, vol. 154, 1995, pages 685 - 693, XP002055570 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6919318B1 (en) 1998-04-22 2005-07-19 Chiron Corporation Enhancing immune responses to genetic immunization by using a chemokine
WO2004085461A3 (fr) * 2003-03-24 2005-09-15 Immatics Biotechnologies Gmbh Peptide associe a une tumeur et se liant a des molecules mhc
AU2004224159B2 (en) * 2003-03-24 2011-06-02 Immatics Biotechnologies Gmbh Tumour-associated peptides binding to MHC molecules

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