Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70539—MHC-molecules, e.g. HLA-molecules
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/14011—Baculoviridae
  • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
  • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • 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

Definitions

• the present invention relates to immunologically active molecules comprising an epitope of interest, more than one major histocompatibility complex class II element, and an immunoglobulin constant region element.
• the function of the immune system is to remove pathogens and eliminate diseased cells; this process is only effective if antigens associated with offending cells and microbes are distinguishable from antigens normally present in the body.
• the distinction between self and non-self is effected, at least in part, by the interaction of classes of molecules present on cellular elements of the immune system, including B lymphocytes, T lymphocytes, and professional antigen presenting cells ("APCs") such as macrophages and dendritic cells.
• APCs professional antigen presenting cells
• TCR T cell receptor
• MHC major histocompatibility complex
• Cytotoxic T lymphocytes which bear CD8 surface antigen, and are referred to as “CD8 + T lymphocytes" will only kill cells bearing a foreign antigen in the context of a self MHC class I molecule.
• helper T lymphocytes which bear CD4 surface antigen, and are referred to as “CD4 + T lymphocytes" will only proliferate in response to a foreign antigen in the context of a self MHC class II molecule on the surface of an APC (for review, see Davies, H., 1997, Introductory Immunobiology, Chapman & Hall, New York, pp. 177-223).
• class I molecules are comprised of a heavy chain and a ⁇ 2 -microglobulin light chain
• class II molecules are heterodimers comprised of and ⁇ chains, each having two domains and being of approximately the same length.
• the DNA regions containing MHC genes have been well characterized for mouse and man, the mouse MHC being referred to as the "H-2 complex” and the human MHC being referred to as the "HLA complex” (for Human Leukocyte Antigen).
• Class I molecules are encoded at the A, B and C loci in man and the K, D, and L loci in mouse.
• Class II molecules are encoded at the DP, DQ and DR regions in man and the I-A and I-E regions in mouse. At each region, a multitude of alleles have been identified.
• T cell activation While the combined interaction between T cells, MHC-peptide complexes and costimulatory molecules on the surface of APCs induces T cell activation, the absence of costimulatory molecules induces T cell unresponsiveness. The latter seems to account for the peripheral self-tolerance of tissue-specific antigens (Guerder et al., 1995, Int. Rev. Immunol. 13:135-146). Based on this knowledge, soluble antigen presenting molecules such as (i) MHC molecules extracted from cell membranes and subjected to peptide elution followed by exchange for specific peptides in vitro (Nag et al, 1996, Cell.
• MHC class I /peptide complexes in order to improve binding to the TCR.
• a human HLA- A2 heavy chain MHC molecule expressing the BirA-dependent biotinylation site at its carboxy terminus was tetramerized by streptavidin and used to phenotype low frequency HlV-specific T cells in the blood of HIV-infected individuals (Altman et al., 1996, Science 274:94-96).
• a similar approach showed that soluble MHC class I/peptide complexes dimerized by a polyclonal anti-class I MHC antibody (but not the corresponding monomers) were able to bind with high affinity to antigen-specific T cells (Abastado et al., 1995, J. Exp. Med.
• the present invention relates to immunologically active molecules comprising an epitope of interest, more than one MHC class II element, and an immunoglobulin constant region element, wherein the epitope is comprised in a fusion protein which comprises a MHC element, wherein each MHC class II element comprises two non-covalently associated chains comprising extracellular domains of a MHC class II protein, and wherein the MHC class II elements are covalently joined by one or more disulfide linkages present in the immunoglobulin constant region element.
• the molecules of the invention may be used to selectively eliminate T cells bearing TCRs which react with the epitope of interest in the context of the major histocompatibility complex class II element, and therefore may be used to eliminate or reduce specific T cell populations, for example, but not by way of limitation, in the treatment of an autoimmune disease and/or a graft-versus host disease.
• FIGURE 1 Gene fragments of the HA110-120/IE d ⁇ /Fc ⁇ 2a chimeric gene after cloning in the p2Bac vector. Gene fragments are shown by digestion with the restriction enzymes used for cloning and analyzed in a 1% agarose gel.
• Lanes 1 and 2 show low and high molecular weight markers ( ⁇ Xl 74/HaeIII and ⁇ /Hindlll, respectively); lane 3 shows a band of 194 bp which corresponds to the cloned fragment containing sequences of the I-E d ⁇ l-F leader/HAl 10-120/linker, obtained by amplification with I-E d ⁇ l-F and I-E d ⁇ l-R primers and digested with EcoRI/BamHI enzymes; lane 4 shows a band of 609 bp corresponding to the cloned fragment containing sequences of linker/I-E d ⁇ 1/I-E d ⁇ domains obtained by amplification with I-E d ⁇ 2-F and I-E d ⁇ 2-R primers and digested with BamHI/Apal enzymes.
• the lower band corresponds to the fragment present in lane 1 due to the existence of a BamHI site in the multiple cloning site of p2Bac upstream to the cloned sequences;
• lane 5 shows a band of 699 bp corresponding to a cloned fragment containing sequences of the hinge-CHI -CH2 domains of the Ig-Fc region obtained by amplification with Fc-F and Fc-R primers and digested with Apal/Hindlll enzymes;
• lane 6 shows a band of 1502 bp corresponding to the entire HA110-120/I-E d ⁇ /Fc ⁇ a chimeric gene, cloned in the p2Bac vector and digested with EcoRI/Hindlll enzymes,
• FIGURE 2A-B Scheme for the genetic construction of a DEF chimeric molecule.
• A sets forth the nucleotide sequence (with gaps, SEQ ID NO: 1-6) of the
• FIG. 1 diagrams the cloning of I-E d ⁇ and HA110- 120/I-E d ⁇ /FC ⁇ 2a genes under the pi 0 and pPolH promoters, respectively, of a p2Bac baculovirus vector.
• FIGURE 3A-B Chromatographic analyses of the DEF molecule.
• A depicts the results of size exclusion chromatography on a Superose 6 column of affinity purified DEF molecules.
• (B) represents the Western blot analysis after transferring the gels onto PVDF membranes: lane 1 and 2, DEF molecule under no reducing/no boiling conditions and under reducing and boiling conditions, respectively, as revealed with 125 I-goat anti-mouse ⁇ 2a Abs; lanes 3 and 4, DEF molecule under no reducing/no boiling conditions and under reducing/boiling conditions, respectively, as revealed with 125 I-14-4-4 mAb.
• FIGURE 5 A L Cytofluorometric analyses of DEF molecule. Panels A,
• D and G indicate the fluorescence background of the developing antibody (goat anti- ⁇ 2a- FITC conjugate);
• panel B indicates binding of DEF molecule (lO ⁇ g/ml) to 14.3d TCR of 14-3-1 TcH as revealed by the goat anti- ⁇ 2a-FITC conjugate;
• panel C depicts inhibition of binding of DEF molecule (10 ⁇ g/ml) to B long melanoma cells as revealed by goat anti- ⁇ 2a-FITC conjugate;
• panel F shows inhibition of binding of DEF molecule (10 ⁇ g/ml) by
• FIGURE 6 The genes in the major histocompatibility complex in the mouse.
• FIGURE 7 The genes in the major histocompatibility complex of the human on chromosome 6.
• the present invention relates to immunologically active molecules comprising an epitope of interest, more than one major histocompatibility complex class II element, and an immunoglobulin constant region element.
• the molecules of the invention are referred to herein as "DEF” molecules, as they are Disulfide-linked and comprise an Epitope as well as an immunoglobulin constant region (Fc) element.
• the DEF molecules of the invention comprise at least one subunit wherein the epitope of interest is comprised in a fusion protein which also comprises a MHC element (i.e., the epitope of interest and the MHC element are expressed as part of the same protein, and are translated from the same RNA molecule).
• epitope of interest refers to a molecular structure which serves as the part of an antigen which is recognized by an antigen receptor, such as a TCR.
• the epitope is "of interest” if it is part of an antigen associated with an infectious agent, a self antigen relevant to an autoimmune disease or alloreactive epitopes associated with graft rejection or graft versus host (GVH) disease.
• the epitopes which may be used according to the invention, provided that the epitope of interest is able to associate with the MHC elements of the construct so as to be able to bind to a T cell receptor (such binding may be evaluated, for example, by fluorescence activated cell sorting analysis ("FACS";see Section 6.1, below). Accordingly, issues such as electrostatic charge, hydrophobicity, hydrophilicity, and steric hindrance should be considered when designing the molecule. In certain cases, variation of peptide linkers and other regions of the DEF molecule may be used to optimize orientation of the epitope of interest and MHC elements.
• FACS fluorescence activated cell sorting analysis
• epitopes of interest which may be incorporated into DEF molecules according to the invention include those derived from glutamic acid decarboxylase 65 (associated with insulin dependent diabetes mellitus); myelin basic protein (associated with multiple sclerosis); human cartilage glucoprotein 39 (associated with rheumatoid arthritis); wheat gliadin (associated with celiac disease); and acetyl choline receptor (associated with myasthenia gravis).
• MHC class II element refers to a MHC class II molecule or portions thereof which is a functional antigen presenting molecule; as such, the MHC class II element preferably comprises an chain component and a ⁇ chain component.
• the and ⁇ chain components preferably comprise all or part of the extracellular domains of the complete ⁇ and ⁇ chain proteins.
• the MHC class II element may be human or non-human, and is selected to be of the same species as the intended recipient of the DEF molecule. Examples of human MHC class II elements include DP, DQ and DR molecules and portions thereof, for which numerous alleles are known.
• MHC class II elements associated with particular autoimmune conditions for example, DR3, DQw2 and DR4, DQw3 (associated with insulin dependent diabetes mellitus (IDDM)); DR4, DQw3 and DR1, DQwl (associated with rheumatoid arthritis); DR2, DQwl (associated with multiple sclerosis); DR3, DQw2 and DR7, DQw2 (associated with celiac disease); DR4, DQw3, and DR6, DQw (associated with pemphigus vulgaris); DR8 and DR5 (associated with pauciarticular juvenile rheumatoid arthritis); DR3, Dqw2, and DR2, DQwl (associated with systemic lupus erythematosis);
• IDDM insulin dependent diabetes mellitus
• DR4 DQw3 and DR1 associated with rheumatoid arthritis
• DR2, DQwl associated with multiple sclerosis
• DR3 associated with Sjogren's syndrome
• DR2, DQwl associated with narcolepsy
• DR3, DQw2 associated with Graves' disease
• DR3,DQw2 associated with dermatitis herpetiformis
• immunoglobulin constant region element refers to a portion of an immunoglobulin molecule comprising all or part of the C-terminal portion of both heavy chains wherein one heavy chain constant region component is joined to the other by one or more disulfide bond.
• the immunoglobulin constant region element is alternatively referred to herein as an Fc element, even though the term is not herein restricted to the product resulting from papain digestion of an immunoglobulin molecule.
• the immunoglobulin constant region element comprises all or a portion of the hinge region, as the hinge region may comprise the required disulfide bond.
• the DEF molecule of the invention comprises four protein chains, wherein two heterodimers are joined together via one or more disulfide bond in the immunoglobulin constant region element.
• Each heterodimer comprises (i) a first protein chain which itself comprises the immunoglobulin constant region element linked to a MHC class II or ⁇ chain element and (ii) a second protein chain which comprises the complementary ( ⁇ or ⁇ ) chain element; the first or second chain or both may comprise the epitope of interest.
• the DEF molecule of the invention is preferably glycosylated.
• the DEF molecule of the invention comprises (a) two MHC class II elements; (b) an immunoglobulin constant region element comprising two protein chains covalently joined by a disulfide linkage; and (c) an epitope of interest, wherein each of the protein chains of the immunoglobulin constant region element is covalently joined to one of the two MHC class II elements by a peptide bond.
• nucleic acids encoding a DEF molecule may be prepared as follows.
• the B DEF subunit is encoded by a nucleic acid sequence comprising regions encoding both MHC subunit and Fc elements; to produce such a nucleic acid construct, nucleic acids encoding (i) one or more external domains of a first subunit of an MHC molecule and optionally an epitope of interest and (ii) a constant (Fc) region of an immunoglobulin molecule may be prepared separately and then ligated together.
• the other DEF-encoding construct needed to produce the complete DEF molecule (the A DEF subunit) may comprise sequence encoding one or more external domains of a second MHC subunit, and may optionally further comprise sequence encoding an epitope of interest.
• Constructs encoding the A and B DEF subunits may then be translated into the corresponding proteins in an appropriate expression system.
• oligonucleotide primers may be designed so that nucleic acid sequence encoding one or more external domains of a first MHC subunit may be obtained by reverse transcription - polymerase chain reaction ("RT-PCR") of mRNA obtained from cells expressing the MHC class II gene (for example, a lymphoma cell line).
• RT-PCR reverse transcription - polymerase chain reaction
• oligonucleotide primers used in the RT-PCR reaction may be designed to (optionally) incorporate nucleic acid sequence encoding the epitope of interest, nucleic acid sequence encoding other peptide linkers, where appropriate, and/or nucleic acid sequence containing one or more restriction enzyme cleavage site(s) that facilitate the splicing together of MHC and Fc regions.
• Nucleic acid encoding at least a portion of the Fc hinge region may also be included in a primer, particularly where such nucleic acid encodes a restriction enzyme cleavage site.
• the primers should also be designed such that nucleic acid and/or encoded peptide sequences necessary or desirable for the proper processing of the final DEF product are included, such as, but not limited to, a leader sequence and polyadenilation sequences.
• nucleic acid encoding the epitope of interest may be introduced into constructs encoding the A and/or B DEF subunit using genetic engineering techniques, at any point within the MHC gene according to the invention; incorporation into an oligonucleotide primer used in RT-PCR is suggested above because first, it is most convenient, and second, because it results in a DEF molecule having the epitope in a position where it has greater flexibility to associate with the MHC domains. Such flexibility may be further improved by providing for a peptide linker between the epitope and the MHC region; adjusting the length of the peptide linker may optimize epitope/MHC interactions.
• nucleic acid encoding all or a portion of an Fc region of an immunoglobulin molecule may be prepared.
• Such nucleic acid preferably comprises a region encoding the hinge portion of an immunoglobulin molecule.
• nucleic acid encoding such Fc regions may be obtained by RT-PCR of mRNA obtained from cell lines expressing an immunoglobulin comprising the desired Fc region. Primers for performing such a RT-PCR reaction may be designed using known Fc sequences.
• the foregoing GENBANK and Kabat references include a series of references providing the sequences of various Fc regions, as well as cell lines expressing said Fc regions.
• the primers may preferably be designed to comprise one or more restriction enzyme cleavage site to facilitate ligation to nucleic acid encoding the MHC portion of the molecule and incorporation into a suitable vector.
• nucleic acid sequences encoding the MHC and Fc portions of the desired DEF molecule may be joined (for example, by cleavage at specific sites using one or more restriction enzymes followed by ligation) and introduced into an appropriate expression vector.
• Suitable expression vectors include prokaryotic expression vectors such as pRSET, pTrcHis, pSE420, pSE380, and PSE280; baculovirus expression vectors such as pVL1392/3, pBlueBacHis, p2Bac, pAC360, and pBluBacIII; eucaryotic expression vectors such as pCMV/EBNA, ⁇ PopTM6, pREP4, pCEP4, pREP7, pREP8, pREP9, pREP 10, pEBVHis, PRC/CMV, pRC/RSV, pcDNA3, pcDNAI/Amp, pcDNAI, pCDM8; and yeast expression vectors such as pYES2; suitable expression systems include mammalian cells such as COS cells, insect cells (e.g.
• a particularly preferred expression vector is p2Bac (Invitrogen) for use in the baculovirus infection of SF9 insect cells in culture.
• the expression vector preferably comprises elements necessary and/or desirable for the expression of the DEF subunit, including promoter/enhancer sequences, appropriate transcription and translation signals, polyadenylation sites, Shine Delgarno sequences, etc..
• a DEF construct encoding the A DEF subunit, which comprises one or more external domains of a second (complementary) MHC subunit may be prepared using similar techniques, which may or may not comprise an epitope of interest.
• the two DEF constructs used to produce the complete DEF molecule may either be contained in the same or in separate expression vectors.
• both A and B DEF subunit encoding nucleic acid constructs are incorporated into the same expression vector, so that they are produced simultaneously, thereby allowing the DEF molecule to assemble.
• separate expression vectors may either be cointroduced into a cell for protein synthesis (for example, by cotransfection where markers allow for the selection of cells containing both constructs), or the A and B DEF subunits may be produced separately and then allowed to associate into the desired tetramers.
• Molecular weight, degree of glycosylation, and disulfide linkage of the resulting DEF molecule may be evaluated and confirmed by standard techniques (see, for example, Example Section 6, below).
• a DEF molecule may be produced using methods similar to those set forth above except that the epitope of interest may not be produced as part of one or both A and B DEF subunits during transcription and translation of the molecule. Therefore, the nucleic acids encoding the A and B DEF subunits do not encode the epitope of interest; instead, the epitope of interest may be added to the molecule later, for example, by chemical conjugation or by association in vitro or in vivo.
• the epitope of interest may be conjugated to a parent DEF molecule (which lacks the epitope of interest (the "E" component) but is nonetheless referred to herein as a DEF molecule to avoid overly burdensome nomenclature) by linkage to amino acid or carbohydrate components (see, for example, Brumeanu et al, 1995, J. Immunol. Methods 183:185-197; Brumeanu et al, 1996, Nature Biotech. 14:722-725; Brumeanu et al., 1997, Eur. J. Immunol.
• DEF molecules of the invention may be further modified to adjust their biological properties according to their intended purpose. For example, but not by way of limitation, half-life may be increased by pegylation of either amino acid or carbohydrate residues (see, for example, Brumeanu et al., 1995, J. Immunol. Methods 183:185-197; Brumeanu et al., 1996, Nature Biotech. 14:722-725; Brumeanu et al., 1997, Eur.
• the DEF molecule may be covalently or noncovalently linked to another molecule having biological activity which may act as a cytokine or as a toxin; exemplary cytokines include TGF ⁇ , IL-4, IL-5, IL- 10 and exemplary toxins include cyclosporin, steroids or activators of apoptosis such as ICE or FASL.
• the present invention further provides for a method of treating an autoimmune disease or of treating or preventing a graft versus host disease comprising administering, to a subject in need of such treatment, an effective amount of a DEF molecule according to the invention.
• autoimmune diseases include, but are not limited to, rheumatoid arthritis including juvenile and adult forms, diabetes, multiple sclerosis, systemic lupus erythematosis, scleroderma, sjogren's syndrome, celiac
• treating does not necessarily imply that the method achieves a complete cure, but rather that the subject exhibits a significant degree of clinical improvement as judged by symptoms and/or clinical signs.
• the choice of MHC class II element comprised in the DEF molecule may be such that the MHC class II element is syngeneic with an MHC class II protein expressed on the APCs of the subject to which it will be administered; for example, DEF comprising an MHC class II element corresponding to the HLA-DR2 allele may be administered to a subject whose APCs express HLA-DR2, etc..
• the MHC class II element comprised in the DEF molecule may be such that the MHC class II element is syngeneic with an MHC class II protein expressed by APCs of the graft donor.
• the invention provides for the manufacturing of pharmaceutical compositions for use in the treatment of the foregoing conditions.
• the genes encoding for the I-E d ⁇ and Ig-Fc ⁇ 2a chain fragments were obtained by reverse transcription - polymerase chain reaction ("RT-PCR") from total RNA isolated from the 2PK3 B lymphoma cell line (obtained from the American Type Culture Collection (“ATCC”), accession number ATCC TIB 203) and 14-44-4 hybridoma cells (producing IgG2a, obtained from the ATCC, accession number ATCC HB32), respectively.
• the gene encoding the I-E d ⁇ chain fragment was obtained from the I-E d gene by PCR.
• oligonucleotides IE d ⁇ -RT and Fc-RT were used for retrotranscription of IE d ⁇ and IgG2a mRNA, respectively. Pairs IE d ⁇ l-F and I-E d ⁇ l-R were used for PCR amplification of the leader and the first four codons of the ⁇ l domain of the I-E d ⁇ gene
• amino acids -26 through +4 amino acids -26 through +4.
• the senqence encoding the eleven amino acids of the HA110-120 peptide and five amino acids of peptide linker containing a BamHI site was introduced into the 3' end of the oligonucleotide IE d ⁇ -R. Pairs IE d ⁇ 2-F and IE d ⁇ 2-R were used for amplification of codons 5-190 of the IE d ⁇ gene. Sequence encoding for twelve amino acids of peptide linker containing a BamHI site was included at the 5' end of IE d ⁇ 2-F.
• the sequence encoding the amino acids 228-232 of Fc ⁇ 2a-hinge region was included at the 3' end of the IE d ⁇ 2-R. Pairs Fc-F and Fc-R were used for amplification of the sequence encoding the secreted hinge, CH2 and CH3 of the IgG2a gene (amino acids 228-478).
• a single gene made of the three amplified gene fragments was obtained by digestion with restriction enzymes at sites included in the primers used for amplification and cloned under the polyhedrin promoter of the p2Bac vector (obtained from Invitrogen) at the EcoRI and Hindlll sites of the multiple cloning site (FIGURE 1 and FIGURE 2).
• the p2Bac vector was previously modified at the multiple cloning site by elimination of the nucleotides 2764-2791 containing an ATG codon. The new multiple cloning site became BamHI-EcoRI-Smal-Hindlll.
• Pairs LE d ⁇ -F and IE d -R were used for amplification of the sequence encoding the leader, ⁇ l and ⁇ 2 regions of the IE d ⁇ chain. Sequence encoding a Stul site was included at the 5' end of IE d -F and sequence encoding a stop codon and a Spel site was included at the 3' end of IE d ⁇ -R. The amplified gene was cloned under the plO promoter of p2Bac in the Stul, Spel sites. Sequencing of the cloned genes showed in- frame assembling of the genes and the absence of mutations.
• the baculovirus/insect cell system was used to generate the HA110-120/I-E d ⁇ /Fc ⁇ 2a dimeric molecule.
• Recombinant baculoviruses were obtained by cotransfection of linear AcMNPV DNA and DEF-p2Bac vector in SF9 insect cells following manufacturer's instructions (the manufacturer being Invitrogen). Screening and titration of recombinant viruses were done by detection of protein production. Briefly, cell culture supematants were incubated in 96-well microtiter plates coated with 5 ⁇ g/ml goat anti-murine ⁇ 2a antibodies and bound DEF molecules were revealed with 125 I-goat anti- ⁇ 2a antibodies.
• the eluted material was collected in Tris 1M, pH 8, dialyzed against PBS, and concentrated on Carbowax 20,000 Da (Sigma Chemicals) in dialysis bags of 1 ,000 dalton MWCO (Spectrapor). The eventual aggregated material was removed by ultracentrifugation and the protein concentration was estimated by RIA, using a standard curve developed using murine 14- 4-4 monoclonal antibody by a previously established protocol (Brumeanu et al., 1996, Immunotechnol. 2:85-95). Chromatographic characterization.
• a Superose 6 column (30 x 1 cm, Pharmacia, LKB) was first equilibrated with ammonium bicarbonate 0.1M, pH 8.0, and then a mixture of MW standard proteins including mouse IgG, chicken egg ovalbumin and cytochrome c was applied to the column at a 1 ml/min flow rate. After standardization, 100 ⁇ g DEF in 200 ⁇ l PBS was applied to the column under identical running conditions and the elution time of the peak-tubes was plotted against the elution time of the MW standard proteins. Individual tubes were also analyzed for the presence of DEF molecule by SDS-PAGE and Western blot.
• affinity purified DEF was chromatographed on a MONO Q anion exchange column (5/30 HR,
• the column was equilibrated with 20 mM Tris/HCl, pH 7.5, and 100 ⁇ g DEF in 200 ⁇ l equilibrating buffer was applied at a 1 ml/min flow rate over 30 minutes using a 0 to 0.5M NaCl gradient. Tubes were collected at one minute intervals and the peak-tubes were pooled together to be analyzed for the presence of DEF molecule by SDS-PAGE and Western blot.
• SDS-PAGE and Western blot analysis were performed in 4- 12% gradient gels (Bio-Rad) according to the manufacturer's instruction. Briefly, 5 ⁇ g of the affinity-purified DEF molecule was incubated either at room temperature or boiled in Tris/HCl/SDS 0.1% buffer for 5 min, in the presence or absence of reducing agent ( ⁇ - mercaptoethanol, 2-ME). Samples were electrophoresed for 45 minutes at 150 volts, and the gels were either silver-stained or electrotransfered in semidry conditions for 45 minutes at 200 mAmps/gel onto PVDF membranes (0.2 ⁇ ).
• Membranes were blocked overnight at 4°C with 5% fat-free milk (Carnation) in PBS, incubated for 2 hours at room temperature with ,25 I-14-4-4 monoclonal antibody in PBS/BSA 1% at 10 7 cpm/membrane, then washed extensively with 0.05% Tween 20 in PBS, and exposed overnight on Kodak X-OMAT film at -80°C.
• Transgenic T cells were purified from the spleens of transgenic mice on Ficoll-Hypaque followed by enrichment on nylon-wool columns as described in Brumeanu et al., 1996, Nature Biotechnology 14:722-725. Purified T cells were incubated for 30 minutes at room temperature with various concentrations of DEF or 14-4-4 mAb as control (2 ⁇ g-50 ng). Rabbit complement (Sigma Chemicals) was added for 45 minutes and then the cells were stained with eosin and fixed with formaldehyde according to a standard protocol (Thompson, R.A. (ed.), 1981, Techniques in Clinical Immunology, Blackwell Scientific Publications, Oxford). The percentage of lysed cells was determined microscopically in an inverted Zeiss microscope. 6.2. RESULTS
• the minor bands of 55 and 80 kDa correspond to the molecular size of the HAl 10-120/I-E d ⁇ /Fc ⁇ 2a monomer and HAl 10-120/I-E d ⁇ /Fc ⁇ 2a dimer, respectively. These monomers were generated under boiling conditions, by dissociation of the dimeric
• the DEF molecule Under reducing and non-boiling conditions, the DEF molecule migrated as three major bands of 50, 55 and 80 kDa which correspond to HAl 10-120/I-E d ⁇ /Fc ⁇ 2a and HAl 10-120/I-E d ⁇ /FC ⁇ 2a monomers respectively, and a minor band of 30 kDa which corresponds to I-E ⁇ chain (FIGURE 4A, lane 3). Under reducing and boiling conditions, DEF molecule migrated as two bands of 50 and 55 kDa as well as 30 kDa, which correspond to the HAl 10-120/I-E d ⁇ /FC ⁇ 2a monomer and I-E ⁇ chain, respectively.
• the two bands of 50 and 55 kDa of the HAl 10-120/I-E d ⁇ /Fc ⁇ 2a may represent two major forms of glycosylation as also found by anion exchange chromatography.
• Western blot analysis developed with goat anti-mouse ⁇ 2a antibodies revealed also the 170 kDa band under nonreducing and no boiling conditions, and two bands of 50 and 55 kDa under reducing and boiling conditions. This indicates that the dimeric DEF was correctly folded and preserved the antigenicity of the Fc ⁇ 2a fragment (FIGURE 4B, lanes 1 and 2).
• Dimeric DEF molecule was also revealed under nonreducing and no boiling conditions by 14-4-4 monoclonal antibody which recognizes a conformational epitope of I-E d ⁇ (Ozato et al., 1980, J. Immunol. 124:533-540; FIGURE 4B, lane 3), but not under reducing and boiling conditions (FIGURE 4B, lane 4). Similar results on the antigenicity of DEF molecule were obtained in RIA using plates coated with goat anti- ⁇ 2a antibodies and revealed with either 125 I-goat anti- ⁇ 2a antibodies or 125 I- 14-4-4 mAb.
• FITC FACS analysis showed that 68% of T cells bound DEF (FIGURES 5B and 5 A, respectively). Similar results were obtained by staining with the 6.5.2 mAb specific for the 14.3d TCR. The binding of DEF to TCR was inhibited by 6.5.2 mAb (FIGURE 5C). When T cells purified from transgenic mice were stained with DEF molecules, we detected 32% positive T cells. The same percentage was obtained by staining the purified transgenic T cells with 6.5.2 mAb-FITC conjugate.
• DISCUSSION We have generated a disulfi de-stabilized dimer of the CD4 T cell immunodominant epitope of HA of the A/PR/8/34 influenza virus, covalently linked to a murine I-E d -Ig chimeric molecule (DEF). Nucleotide sequencing of the chimeric genes indicated that the gene fragments were assembled in frame, and that no mutations occurred during the process of genetic constraction. The recombinant DEF molecule was secreted by the transfected SF9 insect cells as a 170 kDa soluble dimer.
• the dimer was stable in the presence of detergent (0.1% SDS), which indicated that the HAl 10-120 peptide was correctly assembled in the groove of the DEF molecule, being able to stabilize the MHC heterodimer. Indeed, it was previously shown that only the I-E d class II molecules loaded in vitro with peptide, but not the empty class II molecules, migrate in SDS-PAGE under nonreducing/no boiling conditions as a single protein component (Germain et al., 1996, Immunol. Rev. 151:5-30). The recombinant DEF dimer preserved the antigenicity of both MHC and

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