WO2013125738A1 - Polypeptides derived from swine leukocyte antigen and use thereof - Google Patents

Abstract

The present invention relates to polypeptides derived from swine leukocyte antigen (SLA) which T cells indirectly recognize via human leukocyte antigen (HLA). The present invention also relates to nucleic acids coding for the polypeptides and to a method for using the polypeptides and nucleic acids. The polypeptides derived from swine leukocyte antigen (SLA) may exhibit an activity of inhibiting binding of HA_306-318, which are peptides derived from the influenza virus, to HLA-DR4, and therefore, can be evaluated as peptides having strong antigenicity presented via HLA-DR4 molecules. Accordingly, the peptides of the present invention may induce peptide-specific CD4 T cell reaction, or induce a production of peptide-specific regulatory T cells, and thus can be used in inhibiting a heterogeneous immune rejection response.

Description

Polypeptides derived from porcine major histocompatibility and uses thereof

The present invention relates to a pig major histocompatibility antigen (SLA) -derived polypeptide that is indirectly recognized by T cells via human HLA, a nucleic acid encoding the polypeptide, and a method of using the same.

Organ transplantation is the only ultimate treatment for end-stage organ failure, but there is a risk of rejection after xenotransplantation. Transplant rejection occurs in the order of hyperacute rejection, acute vascular rejection, acute cellular xenograft rejection, and chronic rejection. The problem of immunorejection is that pigs knocked out of α 1,3-galactosyltransferase (α1,3-GalT), which makes α1,3 Gal, a major antigen that causes acute immune rejection in 2002 (GTKO) It is being solved to some extent by the production of A, and a lot of researches have been made on the problem of acute vascular rejection by strategies such as regulation of complement regulatory proteins (CD55, CD46, CD59), activation of vascular endothelial cells, control of coagulation. In fact, even the mechanism of T cell-mediated rejection is not well understood. Thus far, super acute rejection and acute vascular rejection occurring in xenotransplantation have emerged as an urgent problem to be solved first, but from now on, studies to overcome T cell mediated rejection, which is the next step, are actively studied. It needs to be promoted.

The paths by which T cells recognize heterologous antigens during xenotransplantation include direct recognition and indirect recognition. Direct recognition is that human T cells recognize and activate swine leukocyte antigen (SLA) directly. Indirect recognition is that human antigen presenting cells (APCs) detect pig SLA. The human T cell recognizes that the SLA-derived heterologous antigen peptide (peptide) is presented through its main histocompatibility antigen (HLA) through intracellular treatment (FIG. 1). Indirect recognition is much more important than direct recognition in the rejection of human T cells when transplanting porcine cells or organs to humans, because not only is the porcine SLA molecule different from the human HLA molecule, The interaction between pig co-stimulatory molecules and human co-stimulatory factors is much weaker than in the allogenic response. Therefore, it is necessary to identify what porcine SLA antigen-derived peptides (ie, peptides involved in the indirect recognition process) presented by human HLA antigens, and to develop a technique for inducing this peptide specific immunotolerance. It is very important in terms of control of xenotransplant rejection by T cells. In humans, foreign antigens, such as SLA antigens, are phagocytosed by antigen presenting cells and then presented to T cells through HLA class I molecules (HLA-A, B, C) and class II molecules (DP, DQ, DR). do. However, since HLA class I and class II molecules are so diverse (polymorphic), the peptides derived from SLA presented through HLA molecules can be different depending on the human HLA haplotype, so any peptide derived from SLA has a T cell response. Whether the main peptide (immunodominant peptide) of induction is inevitably different from person to person.

For the regulation of cell mediated rejection, it is essential to regulate the immune response of T cells, which play a major role in the natural immune response. For the regulation of human T cells, it is basically the identification of a pig SLA-derived epitope, which is present on the surface of pig cells and is known as a major antigen for organ transplant rejection, to determine which is the main epitope of the immune rejection reaction. Required. Once antigenic peptides have been identified, studies related to the induction of identified peptide-specific regulatory T cells (Tregs) should be supported. Induction of antigen-specific Tregs may be a major means of overcoming chronic immune rejection as well as acute cell mediated immunorejection. Nevertheless, the identity of porcine SLA-derived peptide antigens presented through human HLA, particularly HLA-DRB1 (35.0% of Koreans), the allele most commonly found in Koreans, has not yet been identified. There is a need to secure a source technology capable of suppressing the transplant rejection response to porcine cells and pig organs transplanted by a method of inducing immune tolerance.

Therefore, the present inventors are most commonly present in Koreans using transgenic mice (HLA-DRA / H2-Ea, HLA-DRB1 * 0401 / H2-Eb) expressing human HLA-DRB1 * 0401 molecules. The present invention was completed by finding an SLA-derived peptide presented through HLA-DRB1 (35.0%), which is an HLA-DR allele.

It is an object of the present invention to provide a swine major histocompatibility antigen (SLA) -derived polypeptide capable of regulating or controlling an immune rejection response following xenotransplantation. In addition, another object of the present invention is to provide a protein, a polynucleotide, a vector comprising the same, and a host cell capable of regulating or controlling an immune rejection response following xenotransplantation. Another object of the present invention to provide a pharmaceutical composition that can be used for the prevention or treatment of immune rejection reactions following xenotransplantation.

In order to achieve the above object, the present invention (a) SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 20 and SEQ ID NO: 32, and (b) one or a plurality of in the sequence of (a) An isolated or recombinant polypeptide is provided comprising any one or more polypeptide sequences selected from the group consisting of sequences whose amino acids have been modified by substitution, deletion or addition. The polypeptide is derived from swine leukocyte antigen (SLA) and can be recognized in T cells through human major histocompatibility antigen (HLA).

In one embodiment of the present invention, a sequence in which one or a plurality of amino acids in the sequence of (a) is modified by substitution, deletion or addition maintains the same function as the original polypeptide sequence, but is not limited thereto. However, it has at least 50%, preferably at least 70%, more preferably at least 80%, more preferably at least 90% sequence homology with the original polypeptide sequence.

In one embodiment of the invention, the polypeptide may be used to activate T cells or to inhibit the proliferation of T cells.

In one embodiment of the invention, the polypeptide may be used to suppress an immune response.

The present invention also provides an isolated or recombinant protein comprising the polypeptide according to the present invention and an Ig Fc polypeptide. The protein may perform a function of regulating or controlling an immune response.

The present invention also provides an isolated or recombinant protein comprising the polypeptide according to the present invention and a peptide that facilitates secretion of the polypeptide. Although not limited thereto, in one embodiment of the present invention, the peptide that facilitates secretion may be a signal peptide.

The present invention also provides a polynucleotide encoding the polypeptide or protein according to the present invention.

The present invention also provides a vector comprising the polynucleotide sequence according to the present invention.

The present invention also provides a host cell comprising the vector according to the present invention.

The present invention also provides a pharmaceutical composition comprising the polypeptide according to the present invention.

In one embodiment of the present invention, the pharmaceutical composition according to the present invention can be used for the prevention or treatment of immune rejection reaction following xenotransplantation.

The pig major histocompatibility antigen (SLA) -derived polypeptide of the present invention exhibited the activity of inhibiting the binding of human influenza virus-derived peptide HA _306-318 to human HLA-DR4. It can be evaluated with this powerful peptide. Accordingly, the peptides of the present invention can be used to induce a peptide specific CD4 T cell response or to inhibit heteroimmune rejection by inducing the production of peptide specific regulatory T cells.

1 is a diagram showing a path for T cells to recognize heterologous antigen during xenotransplantation. A) direct recognition, B) indirect recognition.

Figure 2 is a graph showing the binding of influenza virus derived peptide (HA _306-318 ) to human HLA-DR4 (DRB1 * 0401).

3 is a graph showing the ability of the polypeptide of SEQ ID NO: 10 to inhibit binding of human influenza virus derived peptide (HA _306-318 ) to human HLA-DR4 (DRB1 * 0401) at a concentration of 1 μM.

Figure 4 is a graph showing an ability to inhibit binding to the human HLA-DR4 (DRB1 * 0401) of the polypeptide of SEQ ID NO: 11 1 μM concentrations of influenza virus-derived peptide (HA _306-318).

5 is a graph showing the ability of the polypeptide of SEQ ID NO: 17 to inhibit the binding of human influenza virus derived peptide (HA _306-318 ) to human HLA-DR4 (DRB1 * 0401) at a concentration of 1 μM.

6 is a graph showing the ability of the polypeptide of SEQ ID NO: 20 to inhibit binding of human influenza virus derived peptide (HA _306-318 ) to human HLA-DR4 (DRB1 * 0401) at a concentration of 1 μM.

7 is a graph showing the ability of the polypeptide of SEQ ID NO: 32 to inhibit the binding of influenza virus derived peptide (HA _306-318 ) to human HLA-DR4 (DRB1 * 0401) at a concentration of 1 μM.

The present invention relates to a polypeptide derived from porcine major histocompatibility antigen (SLA), which may be recognized by T cells via human HLA. The inventors were able to identify human CD4 T cell activating epitopes among swine SLA-derived polypeptides while developing a technique to induce heterologous antigen specific immunotolerance. Human CD4 T cells recognize only antigens presented through HLA class II molecules, which are human antigen presenting cells. Thus, when transplanting porcine cells or organs into humans, porcine SLA molecules are phagocytosed by human antigen presenting cells and then presented to human T cells through human HLA class II molecules through intracellular processing and CD4 T cells are activated, resulting in heteroimmune rejection. The inventors have found, through computer modeling and in vitro experiments, peptides derived from porcine SLA that are highly antigenic peptides presented through human HLA-DR4 molecules (Table 1). Since the polypeptide of the present invention exhibited the activity of inhibiting the binding of human influenza virus-derived peptide HA _306-318 to human HLA-DR4, the antigenicity presented through the HLA-DR4 molecule can be evaluated as a strong peptide.

Accordingly, the present invention can provide an isolated or recombinant polypeptide comprising a peptide derived from porcine major histocompatibility. Isolated means when the nucleic acid, protein or other component is partially or completely separated from the component to which it is normally associated (other protein, nucleic acid, cell, synthetic reagent, etc.). The term recombinant refers to when the nucleic acid or polypeptide is derived from a nucleic acid or protein that is artificial or process, or artificial or process. The polypeptides, peptides and proteins are used interchangeably to refer to polymers of amino acid residues and are used in both naturally occurring and non-naturally occurring amino acid polymers. The term includes amino acid chains of any length, including full length proteins (ie, antigens), wherein the amino acid residues are linked by covalent peptide bonds.

Polypeptides of the invention include polypeptides having the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 20, or SEQ ID NO: 32, as well as variants that perform the same function. Such "variant" polypeptides include polypeptide sequences that differ by at least one amino acid residue from the sequence of the original polypeptide. In one embodiment of the invention, but not limited to, the variant polypeptide may comprise about 1%, 2%, 3%, 4%, 5%, 6%, of the original polypeptide sequence and the total number of polypeptide sequence residues, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 30% 40%, 50% or more different polypeptide sequences. In another embodiment, the variant polypeptide comprises at least about 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91% relative to the original polypeptide sequence. , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% polypeptide sequence having sequence identity. Variant polypeptides are polypeptide sequences of the original polypeptide by deletion, addition or substitution of one or more amino acid residues of the original polypeptide, or any combination of such deletion (s), addition (s) and / or substitution (s). And polypeptide sequences different from.

Accordingly, the present invention provides novel polypeptides, collectively referred to as polypeptides of the present invention, which are intended to include variants and / or derivatives of the polypeptide sequences disclosed herein. Polypeptides of the invention include recombinant or variant polypeptides that bind to human HLA. Polypeptides of the invention include recombinant fusion proteins comprising the variant polypeptides of the invention and include monomeric and dimeric forms of such fusion proteins. Polypeptides of the invention include multimers comprising one or more variant polypeptides of the invention. The invention also includes conjugates comprising one or more variant polypeptides of the invention. Some polypeptides of the invention are soluble polypeptides. For example, the present invention includes soluble fusion proteins comprising variant polypeptides linked to different polypeptides (eg, immunoglobulin polypeptides such as Ig Fc polypeptides) that enhance the solubility of the variant polypeptides.

The present invention can be used to treat diseases, disorders and abnormalities of the immune system, including the regulation of T cell dependent immune responses. Polypeptides, proteins, or nucleic acids encoding such polypeptides or proteins of the invention include, for example, autoimmune diseases, disorders and disorders, immunoproliferative diseases, graft-related disorders, treatment of immune system diseases, disorders and disorders in which immunosuppression is desired, donor tissue Can be useful for treatment methods involving transplantation of tissues, cells, organs or grafts from a donor to a recipient in which the suppression of an immune response in a cell, organ or graft resistant recipient is desirable. It can be used for the prevention or treatment of the immune rejection reaction.

In one embodiment of the present invention, the polypeptide of the present invention comprises a variety of mutagenesis, screening methods, etc. in order to suppress the immune response by T cells, including the function or binding activity abnormalities of human HLA-DR4 molecules Can be used to make novel variant molecules. The molecule may inhibit or block signaling pathways important for T cell activation, such that T cells may not be selectively activated but have reduced proliferative capacity. In other words, variant molecules using the polypeptides of the invention can function as immunosuppressive agents by inhibiting or blocking T cell-dependent immune responses as antagonists of signaling pathways for T cells to function.

Polypeptides of the invention can provide isolated or recombinant proteins comprising peptides that facilitate their secretion. The peptide that facilitates the secretion may be a signal peptide. The signal peptide is generally a peptide (or amino acid) sequence preceding the subject polypeptide, which is bound to and translated with the subject polypeptide, and serves to direct the polypeptide to the secretory system or to facilitate it. Signal peptides are generally covalently attached or fused to the amino terminus of the subject polypeptide and facilitate the secretion of the subject polypeptide from the host cell. Signal peptides are generally cleaved from the polypeptide of interest after translation.

The present invention also provides a polynucleotide encoding an polypeptide or protein of the invention. The amino acid sequence may be encoded in any one of six different reading frames provided by the polynucleotide sequence and its complement. Nucleic acids and polynucleotides are used interchangeably to refer to polymers of nucleic acid residues in single- or double-stranded form (eg, deoxyribonucleotides or ribonucleotides). Unless specifically limited, the term includes nucleic acids comprising known analogues of natural nucleotides that have similar binding properties as the original nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless expressly indicated otherwise, certain nucleic acid sequences also implicitly include obviously indicated sequences as well as their conservatively modified variants (eg, degenerate codon substitutions) and complementary nucleic acid sequences. In particular, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19 : 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605 2608 (1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes 8:91 98 (1994)). The variant nucleic acid comprises a nucleotide sequence that differs from the nucleotide sequence of the original nucleic acid (such as a WT nucleic acid) by one or more nucleic acid residues. In one embodiment, the variant nucleic acid is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 of the total number of nucleotide sequences of the original nucleic acid. %, 12%, 13%, 14%, 15%, 20%, 30% 40%, 50% or more different nucleotide sequences. In other embodiments, the variant nucleic acid is at least about 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, relative to the nucleotide sequence of the original nucleic acid. Nucleotide sequences having 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. Variant nucleic acids may be those of the original nucleic acid, for example, by deletion, addition or substitution of one or more nucleotide residues of the original nucleic acid, or any combination of such deletion (s), addition (s) and / or substitution (s). It may comprise different nucleotide sequences. Variations in nucleic acids may arise from alternative splicing or truncation of nucleotides, or errors in processing or cleavage of nucleotides. The reference or parent nucleic acid may itself be a variant nucleic acid.

The present invention also provides a vector comprising the polynucleotide sequence of the present invention. The vector can be any agent capable of delivering or maintaining nucleic acid in a host cell, for example, a nucleic acid complexed with a plasmid (eg, a DNA plasmid), a naked nucleic acid, a viral vector, a virus, one or more polypeptides or other molecules. Of course, it includes, but is not limited to, nucleic acids immobilized on solid state particles. Vectors can be useful as agents for delivering or maintaining exogenous genes and / or proteins in host cells. Vectors may be used to transduce, transfect, transform, or replicate a nucleic acid and / or protein other than those inherent in the cell or in a manner that is not inherent to the cell. To express. Vectors may include substances that help introduce nucleic acids into cells, such as viral particles, liposomes, protein coatings, and the like. Any method of delivering nucleic acid into a cell can be used. Unless otherwise indicated, a vector means any particular method of delivering nucleic acid into a cell or does not mean that any particular cell type is the subject of transduction. In one embodiment of the invention, the vector may be an expression vector. An expression vector generally refers to a nucleic acid construct or sequence that is produced recombinantly or synthetically, with a series of specific nucleic acid components that allow transcription of a particular nucleic acid in a host cell. Expression vectors generally comprise a nucleic acid to be transcribed that is operably linked to a promoter. The expression includes any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and / or secretion. For example, as the expression vector of the present invention, an expression vector compatible with a prokaryotic host cell may be used. Such prokaryotic expression vectors include a BLUESCRIPT vector (Stratagene), a T7 expression vector (Invitrogen), a pET vector (Novagen), and the like. This includes. Expression vectors that are compatible with eukaryotic host cells include, for example, pCMV vector (Invitrogen), pIRES vector (Clontech), pSG5 vector (Stratagene), pCDNA3.1 (Invitrogen Life Technologies), pCDNA3 (Invitrogen Life Technologies), Ubiquitous Chromatin Opening Element (UCOE ™) expression vector (Millipore).

The vector of the present invention may include a suitable promoter or regulatory sequence. Expression control sequences are generally linked to and / or operably linked to the nucleic acid sequences of the present invention. Expression control sequences are nucleotide sequences that promote, enhance, or control the expression of other nucleotide sequences. Suitable expression control sequences that may be employed include promoters including constitutive promoters, inducible promoters and / or inhibitory promoters, enhancers that amplify expression, initiation sequences, termination translation sequences, splicing control sequences, and the like. When nucleic acids of the invention are included in a vector, the nucleic acids are generally functionally linked to appropriate transcriptional regulatory sequences (promoters) to induce mRNA synthesis. Promoters have a particularly important effect on the level of recombinant polypeptide expression. Any suitable promoter can be used. Examples of suitable promoters include cytomegalovirus (CMV) promoters with or without the first intron A, HIV long terminal repeat promoters, phosphoglycerate kinase (PGK) Promoter, Ross Granulomatous Virus

(Rous sarcoma virus, RSV) promoters, such as the RSV long terminal repeat (LTR) promoter, SV40 promoter, mouse mammary tumor virus (MMTV) promoter, HSV promoter such as Lap2 promoter or herpes thymidine kinase (herpes) thymidine kinase promoters (such as described in Wagner et al. (1981) Proc. Natl. Acad. Sci. 78: 144-145), promoters derived from SV40 or the Epstein Barr virus, adeno- Associated virus (adeno-associated viral, AAV) promoters, such as the p5 promoter, metallothionein promoters (eg, both metallothionein promoters or mouse metallothionein promoters (Palmiter et al. (1983) Science 222) : 809-814)), human ubiquitin C promoter, E. coli promoters such as the lac and trp promoters, phage lambda PL promoters, and prokaryotic or eukaryotes Other promoters known to control the expression of a gene in a cell comprises (from virus infected cells directly, or in a cell).

The expression level of a nucleic acid of the invention (or a corresponding polypeptide of the invention) can be assessed by techniques known in the art. Northern Blot analysis (e.g., McMaster et al., Proc. Natl. Acad. Sci. USA 74 (11): 4835-38 (1977) and Sambrook), reverse transcriptase- polymerase chain

reaction, RT-PCR) (US Pat. No. 5,601,820 and Zaheer et al., Neurochem. Res. 20: 1457-63 (1995)), and in situ hybridization techniques (eg US Pat. No. 5,750,340) And 5,506,098). In addition, quantification of proteins can be found in Lowry assays and other protein quantitative assays (see, eg, Bradford, Anal. Biochem. 72: 248-254 (1976); Lowry et al., J. Biol. Chem. 193: 265 (1951)). Can be achieved. Western blot analysis of the recombinant polypeptides of the invention obtained from lysates of cells transfected with polynucleotides encoding the recombinant polypeptide is another suitable technique for assessing the level of recombinant polypeptide expression.

Vectors of the invention, such as expression vectors or polynucleotides, may comprise ribosomal-binding sites for translation initiation and transcription-termination regions. For example, suitable transcription-termination regions are polyadenylation sequences that facilitate cleavage and polyadenylation of RNA transcripts produced from DNA sequences. Synthetic optimization sequences, as well as the polyadenylation sequence of BGH (Bovine Growth Hormone), human growth hormone gene, polyoma virus, TK (Thymidine Kinase), EBV (Epstein) Any, including Papstein Barr Virus, rabbit beta globin, and papillomaviruses, including human papillomaviruses and BPV (Bovine Papilloma Virus) Suitable polyadenylation sequences can be used The vectors or polynucleotides of the invention further comprise site-specific recombination sites, which can be used to modulate the transcription of the nucleotide sequence of interest. In addition, the vector or polynucleotide of the present invention can target polypeptide expression to a desired cell compartment, membrane, or organelle. Or, in order to induce polypeptide secretion into the periplasmic space or cell culture medium, a nucleic acid encoding a secretion / position shift sequence may be included. Or an amino acid sequence corresponding to the relocation sequence (s) In addition, dihydrofolate reductase resistance, neomycin resistance, or tetracycline in E. coli For the selection of transformed host cells, such as) or ampicillin resistance, the vectors or polynucleotides of the invention may comprise one or more selection marker nucleotide sequences or genes. Nucleotides may contain an origin of replication useful for propagation in microorganisms. There.

The present invention also provides engineered host cells transduced, transfected, or transformed with the vector of the present invention or the nucleic acid of the present invention. Host cell herein refers to any cell that is easy to transform with the nucleic acid. The engineered host cell can be cultured in a conventional nutrient medium modified to be suitable for activating a promoter, selecting a transformant, or amplifying a nucleic acid of interest. Culture conditions, such as temperature, pH, etc., are those previously used with a host cell selected for expression, and to those skilled in the art and to, for example, Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, 3rd edition, It will be apparent from the references cited herein, including Wiley-Liss, New York and references cited therein. Polypeptides of the invention encoded by such vectors or nucleic acids of the invention can be expressed in such host cells and isolated by standard techniques. For example, a polypeptide released into a cell culture can be isolated from the culture by ultracentrifugation or similar techniques. Polypeptides of the invention can be used in various expression hosts, including but not limited to animal cells, such as mammalian cells (eg, CHO cells), including human and non-human primate cells, and in plants, yeast, fungi, bacteria, And can be produced in non-animal cells such as the like. Examples of suitable expression hosts include bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium; Fungal cells such as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; Insect cells such as Drosophila and Spodoptera frugiperda; Mammalian cells, such as CHO (eg CHO-K1), COS (eg COS-1, COS-7), BHK, and HEK (eg HEK 293) cells, Bowes melanoma cells, And plant cells.

The present invention provides a cell comprising any one or more of the nucleic acids, vectors, or other constructs of the invention or any combination thereof. Also included are cells comprising one or more of the polypeptides, fusion proteins, or other constructs described herein, or any combination of one or more thereof. Cells of the invention are generally isolated or recombinant cells and may include host cells. Such cells, such as recombinant cells, can be modified by transformation, transfection, and / or infection by at least one nucleic acid, vector, or other construct of the invention.

Such cells may be eukaryotic cells (eg mammalian, yeast or plant cells) or prokaryotic cells (eg bacterial cells), such as calcium phosphate transfection (see eg calcium phosphate co-precipitation method), DEAE- Including dextran-mediated transfection, electroporation (Irving et al., Cell 64: 891-901 (1991)), gene or vaccine guns, injections, lipid infections and biolistics or other conventional techniques well-known above. Various known methods can be used to transform into any such construct of the invention. Host cell strains are optionally selected according to their ability to regulate the expression of the inserted sequence or to process the expressed protein in a desired manner. Such modifications of proteins include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Different host cells, such as E. coli, Bacillus sp., Yeast, or mammalian cells such as CHO, HeLa, BHK, MDCK, HEK 293, WI38, etc., have specific cellular machinery and characteristic mechanisms for such post-translational activity. It can be chosen to ensure accurate modification and processing of the introduced foreign protein.

Nucleic acids of the invention can be inserted into appropriate host cells (in culture or in host organisms) to allow the host to express the protein of interest. Any suitable host cell is transformed and / or transduced with the nucleic acid of the invention. Examples of suitable expression hosts include: bacterial cells such as E. coli, Streptomyces, Bacillus sp., And Salmonella typhimurium; Fungal cells such as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; Insect cells such as Drosophila and Spodoptera frugiperda; Vero cells, HeLa cells, CHO cells (eg CHO-K1), COS cells, WI38 cells, NIH-3T3 cells (and other fibroblasts such as MRC-5 cells), MDCK cells, KB cells, SW-13 cells , Mammalian cells such as MCF7 cells, BHK cells, HEK-293 cells, Bowes melanoma cells and plant cells.

The invention also provides host cells that are transduced, transformed or transfected with at least one nucleic acid or vector of the invention. As discussed above, the vectors of the present invention generally comprise the nucleic acids of the present invention. Host cells are engineered (eg, transduced, transformed, infected or transfected) using a vector of the invention, which can be, for example, a cloning vector or an expression vector. The vector may be in the form of a plasmid, viral particles, phage, attenuated bacteria, or any other suitable type of vector. Suitable host cells for the production of recombinant polypeptides of the invention and / or for transduction and / or infection with the viral vectors of the invention for replication of the viral vectors of the invention include the above-described cells. Examples of cells proven to be suitable for packaging viral vector particles are described, for example, in Polo et al., Proc. Natl. Acad. Sci. 96 (8): 4598-603 (1999), Farson et al., J. Gene Med. 1 (3): 195-209 (1999), Sheridan et al., Mol. Ther. 2 (3): 262-75 (2000), Chen et al., Gene Ther. 8 (9): 697-703 (2001), and Pizzaro et al., Gene Ther. 8 (10): 737-745 (2001). In replication-deficient viral vectors such as AAV vectors, complementary cell lines, helper virus transformed cell lines, or cell lines transformed with plasmids encoding essential genes are required for replication of the viral vector.

Engineered host cells can be cultured in conventional nutrient media modified to be suitable for activating a promoter, selecting a transformant, or amplifying a nucleic acid of interest. Host cells can be cultured in serum-containing medium or serum-free medium.

The present invention includes immortalized cells or cell lines comprising one or more polypeptides of the invention (eg, including dimeric or monomeric fusion proteins and multimeric polypeptides), conjugates, nucleic acids or vectors.

The present invention also provides a pharmaceutical composition comprising the polypeptide of the present invention. The pharmaceutical composition may be used for the prevention or treatment of an immune rejection reaction following xenotransplantation. The pharmaceutical composition refers to a composition suitable for pharmaceutical use in a subject, including an animal or a human. Pharmaceutical compositions generally comprise an effective amount of an activator and a carrier, excipient or diluent. The carrier, excipient or diluent is generally a pharmaceutically acceptable carrier, excipient or diluent, respectively. The prophylaxis is intended to prevent or reduce the risk of a subject developing a pathology, disease or disorder when the agent is administered to a subject that does not exhibit signs or symptoms of the pathology, disease or disorder or exhibits only its initial signs or symptoms. It is an activity. Prophylactically useful agents (eg, nucleic acids or polypeptides) refer to agents that are useful for preventing the development of a disease, pathology or disorder, or which inhibit or inhibit further development or enhancement of a disease, pathology or disorder. The treatment is performed on a subject who exhibits the symptoms or signs of a pathology, disease or disorder, which treatment is performed on the subject for the purpose of reducing or eliminating such signs or symptoms. A therapeutic activity is the activity of an agent that, when administered to a subject suffering from such signs or symptoms, eliminates or reduces the signs or symptoms of a pathology, disease or disorder.

Molecules or components of the invention (eg, polypeptides, nucleic acids, vectors, and / or cells of the invention) can be administered to a subject as a composition. The composition generally comprises at least one such molecule or component and an excipient, carrier, or diluent. The composition may comprise a pharmaceutical composition comprising at least one such molecule or component and a pharmaceutically acceptable excipient, carrier, or diluent (eg, PBS). The pH of the compositions of the present invention generally ranges from about pH 6.0 to about pH 9.0, including, for example, about pH 6.5 to about pH 8.5, and usually ranges from about pH 7.0 to about pH 8.0. Some compositions of the invention include one or more salts (e.g., sodium chloride, sodium phosphate, calcium chloride, etc.), one or more buffers (e.g., HEPES, sodium citrate, sodium phosphate (e.g., Na2HPO4 / Na3PO4), succinate, tartrate, fumarate , Gluconate, oxalate, lactate, acetate, tris (hydroxymethyl) aminomethane (Tris), etc., one or more sugars or sugars (e.g. sucrose, mannose, maltose, trehalose, dextrose) Os, etc.), and / or one or more polyalcohol or sugar alcohols (eg, mannitol, sorbitol, glycol, glycerol, arabitol, erythritol, xylitol, ribitol, lactitol and the like). One or more monosaccharides, disaccharides and / or polysaccharides may be included in the composition. The composition of the present invention may comprise any concentration of said molecule or component effective to inhibit an immune response when administered to a subject.

An effective amount or dose of a molecule of the invention administered to a particular subject can be, for example, the disease, disorder, or condition being treated, the efficacy (ie, efficacy) of the particular variant of the invention to be administered, the mode of administration of the subject, and the particular It may vary depending on the individual's ability of the subject to withstand a specific amount of molecule. In any of the methods described herein, molecules or components of the invention (eg, polypeptides, nucleic acids, vectors, compositions, and / or cells of the invention) can be administered parenterally, subcutaneously, or intravenously. The molecules or components of the present invention may be administered in a therapeutically effective amount once, twice, three or four times a month, twice a week, once every two weeks, or once every two months.

Any of the methods described herein may further comprise administering to the subject an effective amount of at least one additional therapeutic or immunosuppressive agent or compound. Thus, for example, the invention relates to (1) an effective amount of at least one first immunosuppressive agent, wherein each such first immunosuppressive agent is directed to a subject in need of suppressing an immune response. , A nucleic acid, a vector, a composition, and / or a cell, and (2) administering an effective amount of at least one second immunosuppressive agent, wherein the method comprises: The reaction is suppressed.

Various additional therapeutic or immunosuppressive agents can be used or administered with molecules of the invention (eg, polypeptides, nucleic acids, vectors, compositions, and / or cells of the invention). Such agents are, for example, disease-modifying anti-rheumatic drugs (DMARDs) (e.g. methotrexate (MTX), cytokine antagonists (e.g. IL-2 or IL-6) Antagonists), steroidal compounds (e.g., corticosteroids, glucosteroids, e.g. prednisone or methylprednisone), nonsteroidal compounds, sodium salicylate, magnesium salicylate, ibuprofen, acetylsalicylic acid, acetaminophen, antibodies, anti-inflammatory cytokines Biological agents that block synthesis or production, Raptiva efalizumab, anti-inflammatory agents or compounds, and non-steroidal anti-inflammatory drugs (NSAIDs). Red or immunosuppressive agents can be administered to a subject in a pharmaceutical composition, including additional agents and pharmaceutically acceptable excipients or carriers. The effective amount and dose of the agent to be administered will vary depending on the specific agent Some such agents are currently used for the treatment of immunosuppression and the appropriate dosages are determined by the subject's ability to withstand the specific amount or dose and the disease, disorder or condition to be treated, And the immunosuppressive efficacy of the agent.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples. However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Experimental Examples.

<Example 1>

Binding Measurement of Influenza Virus-Derived Peptides on Human HLA-DR4

<1-1> Production of L243, a HLA-DR4 Specific Monoclonal Antibody

Since a large amount of HLA molecule-specific monoclonal antibody is required for the measurement of the binding force between the HLA molecule and the peptide, 500 μl pristane (Sigma Aldrich), which is about 1 week ago, was used for L243 (ATCC), a hybridoma producing HLA molecule-specific monoclonal antibody. ) in which the the Balb / c (Orient bio) treated by 10 ⁷ (cells) in the mouse peritoneal cavity by intraperitoneal injection causes inflammation following, peritoneal fluid samples were collected in a month using a 18G needle.

The HLA-specific monoclonal antibody (IgG2a) present in the intraperitoneal solution was added to the column filled with a bead containing protein A-bound beads in agarose and bound only to the HLA-specific monoclonal antibody. HLA-specific monoclonal antibody was eluted using elution buffer (pH3), and then buffer exchange was performed with PBS. The total protein content of each elution fraction was measured using a BCA assay kit (Thermo science), and the final purified antibody was identified as a single band through SDS-PAGE.

<1-2> Preparation of PRIESS Cell Lysate

Lysis buffer (1% NP-40 detergent) immediately added with a protease inhibitor (2 mM phenylmethylsulfonyl fluoride, 10 μg / ml aprotinin, 25 mM indoacetamide) to PRIESS cells expressing HLA molecules on the cell surface. , 0.15 M NaCl, 0.02% sodium azide in 0.05 M Sodium phosphate buffer, pH7) was added to 1 ml per 1 × 10 ⁸ cells to destroy the cells at 4 ° C. for 1 hour, centrifuged at 4000 g for 10 minutes, and then cell debris. Supernatant was removed. Add 5% sodium deoxycholate by 1/5 of the volume of the supernatant and mix for 10 minutes at 4 ℃ to increase the solubility of the HLA molecules, then perform ultracentrifugation for 100 minutes at 17,000 rpm and filter out the debris with a 0.45 μm filter. Cell lysates were obtained.

<1-3> Binding of Influenza Virus-Derived Peptides (HA306-318) to Human HLA-DR4

To a ELISA plate coated with L243, a HLA-DR4 specific monoclonal antibody, PRIESS cell lysate, a cell line expressing human HLA-DR4 (DRB1 * 0401) molecules, was added, incubated for 2 hours, washed with physiological saline, and bound. After removing all of the proteins that are not present, biotin-coupled influenza virus-derived peptide HA _306-318 (PKYVKQNTLKLAT, SEQ ID NO: 33) (synthesized by peptron) was added to each concentration and incubated overnight, followed by streptavidin. (streptavidin) attached alkaline phosphatase (Sigma Aldrich) and substrate 4-MUP (4-methylumbelliferyl phosphate) (Sigma Aldrich), respectively, followed by excitation wavelength 440 nm, emission Fluorescence values were measured at a wavelength of 365 nm. As a result, influenza virus-derived peptide HA _306-318 binds to HLA-DR4 in a concentration-dependent manner, and the binding is almost saturated at a concentration of about 1000 nM (FIG. 2).

<Example 2>

Identification of Porcine Major Tissue-Compatible Antigen (SLA) -Derived Peptides Presented Through Human HLA-DR4

A peptide derived from the extracellular domain present in the cell membranes of swine leukocyte antigen (SLA) class I (SLA-1,2,3) and class II (SLA-DR, DQ) molecules. Computer modeling of a peptide consisting of 15 amino acids that is expected to have good binding to the antigen-binding crefts of a human leukocyte antigen (HLA) class II molecule, HLA-DR4 (or more precisely, DRB1 * 0401) molecule (program name: SYFPEITHI program) Then, 32 peptides having excellent binding strength to HLA-DR4 and high covalentity with various SLA alleles among the predicted peptides were synthesized by using peptron (Table 1).

The binding _ability of the _synthesized peptides to HLA-DR4 is a method of relatively comparing the extent to which the influenza virus-derived peptide HA _306-318 described in Example 1 _inhibits HLA-DR4 binding (FIG. 2). Measured. That is, PRIESS cell lysate, a cell line expressing human HLA-DR4 molecules, was added to an ELISA plate coated with L243, an HLA-DR4-specific monoclonal antibody, incubated for 2 hours, and then washed with physiological saline to remove all unbound proteins. Next, HA _306-318 1000 nM, a biotin-bound influenza virus-derived peptide, and synthesized SLA-derived peptide were added at different concentrations, followed by incubation overnight, and then streptavidin-attached alkaline phosphatase and substrate 4 After adding -MUP (4-methylumbelliferyl phosphate), the fluorescence value was measured at an excitation wavelength of 440 nm and an emission wavelength of 365 nm.

Table 1

Peptide Name	Peptide sequence	IC 50 (nM)
SLA-1,2,3 Epitope (a1 & 2)
pep1	PHSLSYFYTAVSRPD (SEQ ID NO: 1)	500
pep2	EPRVPWIQQEGQDYW (SEQ ID NO: 2)	500
pep3	GQDYWDEETRKVKDN (SEQ ID NO: 3)	600
pep4	QDYWDEETRKVKDNA (SEQ ID NO: 4)	1000 <
pep5	PEYWDRETQISKETA (SEQ ID NO: 5)	1000 <
pep6	QEYWDRETQISKDNA (SEQ ID NO: 6)	1000 <
pep7	RNAMGSAQTFRVNLN (SEQ ID NO: 7)	500
Pep8	QNAMGSAQTFRVNLK (SEQ ID NO: 8)	650
pep9	YIALNEDLRSWTAAD (SEQ ID NO: 9)	800
pep10	LRSWTAADTAAQITK (SEQ ID NO: 10)	300
pep11	LRSWTAADTAAQISK (SEQ ID NO: 11)	50
SLA-1,2,3 Epitope (a3)
pep12	SLTWQREGQDQSQDM (SEQ ID NO: 12)	1000 <
pep13	SLTWQREGQDHSQDM (SEQ ID NO: 13)	1000 <
pep14	SLSWQREGQDQSQDM (SEQ ID NO: 14)	1000 <
pep15	SQDMELVETRPSGDG (SEQ ID NO: 15)	1000 <
SLA-DQA Epitope
pep16	THEFDGDEQFYVDLE (SEQ ID NO: 16)	1000 <
pep17	LRNIATLKHNLNIVT (SEQ ID NO: 17)	400
pep18	LRNIATAKHNLNILI (SEQ ID NO: 18)	1000 <
pep19	VINITWLKNGHSVKG (SEQ ID NO: 19)	550
pep20	VINITWLKNGHSVTE (SEQ ID NO: 20)	250
SLA-DQB1 Epitope
pep21	YRAVTPLGRPDADYL (SEQ ID NO: 21)	450
pep22	YRAVTPLGRPDADYW (SEQ ID NO: 22)	450
SLA-DRA Epitope
pep23	IFHVDMEKRETVWRL (SEQ ID NO: 23)	1000 <
pep24	VWRLEEFGHFASFEA (SEQ ID NO: 24)	1000 <
pep25	LEEFGHFASFEAQGA (SEQ ID NO: 25)	1000 <
SLA-DRB1 Epitope
pep26	YRAVTELGRPDAKYW (SEQ ID NO 26)	1000 <
pep27	FREVTEFGRPDAKYW (SEQ ID NO: 27)	1000 <
pep28	QKDILEDSRASVDTY (SEQ ID NO: 28)	1000 <
pep29	TVTVYPAKTQPLQHH (SEQ ID NO 29)	500
pep30	RVTVYPAKTQPLQHH (SEQ ID NO: 30)	1000 <
pep31	QEEVAGVVSTGLIPN (SEQ ID NO: 31)	1000 <
pep32	FQTMVMLETVPQSGE (SEQ ID NO: 32)	50

IC ₅₀ of Table 1 alone when added to the HA _306-318 peptide of influenza virus-derived fluorescence when a value of 100 indicates, the binding to the HLA-DR4 of the HA of influenza virus-derived peptide _306-318 50% inhibition The concentration of SLA derived peptides is shown.

In conclusion, the strong affinity with HLA-DR4 was shown to be pep11 (SEQ ID NO: 11) derived from SLA class I (IC ₅₀ : 50 nM) and pep32 (SEQ ID NO: 32) derived from SLA class II (IC ₅₀ : 50). nM), as well as pep10 (SEQ ID NO: 10) (IC ₅₀ : 300 nM), pep17 (SEQ ID NO: 17) (IC ₅₀ : 400 nM), pep20 (SEQ ID NO: 20) (IC ₅₀ : 250 nM) 3-7.

So far I looked at the center of the preferred embodiment for the present invention.

Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (7)

1. (a) SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 20, and SEQ ID NO: 32, and (b) wherein one or more amino acids in the sequence of (a) are modified by substitution, deletion or addition An isolated or recombinant polypeptide comprising any one or more polypeptide sequences selected from the group consisting of sequences, wherein the polypeptide is derived from a swine leukocyte antigen (SLA) and forms a human major histocompatibility antigen (HLA). Polypeptide, characterized in that it is recognized in T cells.
2. The method of claim 1,

   A sequence wherein one or more amino acids in the sequence of (a) is modified by substitution, deletion or addition is a polypeptide having at least 85% or more sequence homology to the original polypeptide sequence.
3. The method of claim 1,

   Said polypeptide is used to inhibit or enhance an immune response.
4. A isolated or recombinant protein comprising (a) the polypeptide of any one of claims 1 to 3, and (b) an Ig Fc polypeptide, the protein having the function of regulating or controlling an immune response.
5. A polynucleotide encoding the polypeptide of any one of claims 1 to 3.
6. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 3.
7. The method of claim 5,

   The pharmaceutical composition is a pharmaceutical composition, characterized in that it is used for the prevention or treatment of immune rejection reaction following xenotransplantation.

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