Patent application title: MODULATION OF ANTIGEN IMMUNOGENICITY BY DELETING EPITOPES RECOGNIZED BY NKT CELLS
Inventors:
Jean-Marie Saint-Remy (Grez-Doiceau, BE)
IPC8 Class: AC07K200FI
USPC Class:
1 1
Class name:
Publication date: 2021-11-18
Patent application number: 20210355162
Abstract:
The invention describes a method and compounds for the prevention of
immune responses towards allofactors, towards viral vectors used for gene
therapy and gene vaccination, towards proteins to which subjects are
naturally exposed, towards genetically-modified organisms and towards
undesirable effects related to vaccine administration for allergic or
infectious diseases.Claims:
1.-14: (canceled)
15. An engineered peptide derived from a peptide selected from the group consisting of an allofactor, a viral vector, a genetically-modified organism and a vaccine for an allergen, the peptide naturally comprising an epitope comprising hydrophobic amino acid residues in position P1 and/or P7 and the peptide being capable of binding to CD1d molecule and/or activating NKT cells, the engineered peptide having been modified to eliminate said epitope by substituting hydrophobic amino acid residues in position P1 and/or P7 with a non-hydrophobic residue, so that the binding to CD1d molecule is reduced in the engineered peptide as compared with the peptide.
16. The engineered peptide of claim 15, wherein the peptide comprises one or more motif consisting of [FWTHY]-X2X3-[ILMV]-X5X6-[FWTHY] and wherein the peptide has been modified to eliminate the [FWTHY] amino acids at positions P1 and/or P7 of the motif comprised in the peptide by substitution of the [FWTHY] amino acid by an amino acid different from [FWTHY].
17. The engineered peptide of claim 15, further comprising a substitution of the amino acid [ILMV] at position P4.
18. The engineered peptide of claim 15, wherein the engineered peptide is derived from human adenovirus 5.
19. The engineered peptide of claim 18, comprising a variant of one or more or all of the sequences: FIGLMYY, FDSICLY, FKKVAIT, FTRLKTK, WFLVQM, FMSMGAL and FDVVRVH.
20. The engineered peptide of claim 19, wherein the engineered peptide is derived from Mal d 1.
21. The engineered peptide of claim 19, comprising a variant of the sequence FKLIESY.
22. The engineered peptide of claim 15, wherein the engineered peptide is derived from alpha-gliadin.
23. The engineered peptide of claim 22, comprising a variant of the sequence YLQLQPF and/or the sequence FEEIRNL.
24. The engineered peptide of claim 15, wherein the engineered peptide is derived from Derlp.
25. The engineered peptide of claim 24, comprising a variant of one or more or all of the sequences: FSGVAAT, HSAIAVI and YPYVVIL.
26. The engineered peptide of claim 15, wherein the engineered peptide is derived from an IgG.
27. The engineered peptide of claim 26, comprising a variant of one or more or all of the sequences: YRVVSVL, FRVVSVL and HEALHNH.
Description:
FIELD OF THE INVENTION
[0001] The present invention relates to peptides or polypeptides to decrease natural immunogenicity or induce tolerance and their use in preventing immune responses elicited towards allofactors, towards viral vectors used for gene therapy or gene vaccination, towards environmental antigens used in food or feed or to which subjects are exposed by inhalation or contact, or to decrease side effects associated with vaccination by allergens or infectious agents.
BACKGROUND OF THE INVENTION
[0002] In many instances, administration of proteins for therapeutic purposes results in immune response against the therapeutic agents, which precludes any further administration of said therapeutic agent. Examples of this are provided by administration of factor VIII of the coagulation pathway in subjects affected by hemophilia A: about a third of treated subjects produce anti-factor VIII antibodies inhibiting the function of factor VIII. Administration of enzymes such as alpha-galactosidase in subjects suffering from deficiency in glycogen metabolism is likewise followed by an immune response against the therapeutic agent precluding any further administration. A third example is provided by antibodies used as therapeutic agents and directed towards lymphocyte surface molecule, cytokines or cytokine receptors. Overall, it is highly desirable to find new therapeutic approaches which would prevent immunization against therapeutic agents.
[0003] The use of viral vectors for gene therapy or gene vaccination is severely restricted by the fact that such viral vectors elicit an immune response which results in rapid elimination of cells transduced by the vector and rapid loss of transgene expression. Viral vectors elicit activation of the innate and adaptive immune response with variable degree of intensity depending of the nature of the viral vector. Thus, adenovirus vectors strongly stimulate innate immune responses followed by an adaptive immune response. Adeno-associated viral vectors trigger a weaker innate response but elicit antibody production. The potential of both gene therapy and gene vaccination is huge. A therapeutic approach by which the innate and/or the adaptive response towards viral vectors would represent a highly significant progress in the field.
[0004] Many subjects suffer from response elicited against proteins to which they are naturally exposed. Allergens, either airborne or from food, elicit reactions such as allergic rhinitis and asthma, urticaria, eczema and anaphylactic reaction. The reason as to why said subjects present such reactions is only partially elucidated. It would be of much benefit to reduce the natural immunogenicity of proteins a diverse as food allergens, wheat causing celiac disease or products such as enzymes to which subjects are exposed for professional reasons.
[0005] In vaccination strategies for either allergic diseases or against infectious agents, the therapeutic efficacy is often limited by side effects which are elicited by the proinflammatory properties of allergens or infectious agents. Such inflammatory effects preclude the use of higher doses of vaccines, and therefore of vaccine efficacy, and orientate the immune response towards unwanted cellular response, such as delayed-type reaction, and production of antibodies of an isotype which is not optimal for the condition considered. A better control of inflammation in vaccination would offer a much more efficient modulation of the immune response whilst reducing side effects related to inflammation.
[0006] Natural killer T (NKT) cells constitute a distinct lineage of non-conventional T lymphocytes that recognize antigens presented by the non-classical MHC complex molecule CD1d. Two subsets of NKT cells are presently described. Type 1 NKT cells, also called invariant NKT cells (iNKT), are the most abundant. They are characterized by the presence of an alpha-beta T cell receptor (TCR) made of an invariant alpha chain, Valpha14 in the mouse and Valpha24 in humans. This alpha chain is associated to a variable though limited number of beta chains. Type 2 NKT cells have an alpha-beta TCR but with a polymorphic alpha chain. However, it is apparent that other subsets of NKT cells exist, the phenotype of which is still incompletely defined, but which share the characteristics of being activated by glycolipids presented in the context of the CD1d molecule.
[0007] NKT cells typically express a combination of natural killer (NK) cell receptor, including NKG2D and NK1.1. NKT cells are part of the innate immune system, which can be distinguished from the adaptive immune system by the fact that they do not require expansion before acquiring full effector capacity. Activation of NKT cells results in various effects. Such cells release preformed mediators, including a large array of cytokines (including interleukin (IL)-4, interferon (IFN)-gamma, IL-21 and IFN-alpha) which provide help to B cells for the production of antibodies and it has been suggested that the release of cytokines could also influence CD4+ T cells (Burrows et al Nature Immunology 2009, 10: 669-671). In the context of the present invention, the prevention of both B cell activation and of major histocompatibility (MHC) class II-restricted CD4+ T cells activation is deemed to play a role in reducing or abolishing protein immunogenicity.
[0008] The recognition unit for NKT cells, the CD1d molecule, has a structure closely resembling that of the MHC class I molecule, including the presence of beta-2 microglobulin. It is characterized by a deep cleft bordered by two alpha chains and containing highly hydrophobic residues, which accepts lipid chains. The cleft is open at both extremities, allowing to accommodate longer chains. The canonical ligand for CD1d is the synthetic alpha galactosylceramide (alpha GalCer). However, many natural alternative ligands have been described, including glyco- and phospholipids, the natural lipid sulfatide found in myelin, microbial phosphoinositol mannoside and alpha-glucuronosylceramide. The present consensus (see reviews, such as Matsuda et al, Current Opinion in Immunology 2008, 20:358-368 and Godfrey et al, Nature reviews Immunology 2010, 11: 197-206) is that CD1d binds only ligands containing lipid chains, or in general a common structure made of a lipid tail which is buried into CD1d and a sugar residue head group that protrudes out of CD1d.
[0009] Peptides are not deemed to be able to activate NKT cells through presentation by CD1d. It was, however, suggested that long hydrophobic peptides containing bulky aminoacid residues could bind to CD1d (Castano et al, Science 1995, 269: 223-226). Observations carried out using phage display libraries expressing random sequence peptides with no defined physiological relevance, allowed establishing a theoretical consensus motif (Castano et al, Science 1995, 269: 223-226 and see below).
[0010] In fact, Castano et al show that the cells which are activated are CD8+ T cells, namely MHC class I restricted cells, and not NKT cells. These findings teach the one skilled in the art that there is no evidence that hydrophobic peptides are presented by CD1d molecules. The physiological relevance of the claims made by Castano et al was further questioned due to the inability to elicit NKT cells under conventional immunization protocols (Matsuda et al, Current Opinion in Immunology 2008, 20:358-368 and Brutkiewicz Journal of Immunology 2006, 177: 769-775). Artificial systems such as immunization with cells transfected to overexpress CD1d and loaded in vitro with an ovalbumin-derived peptide were able to elicit NKT cells. Likewise, intradermal immunization with plasmid DNA together with murine CD1d and costimulatory molecules induce cytolytic CD1d-restricted T cells (Lee et al, Journal of Experimental Medicine 1998, 187: 433-438). Hydrophobic peptides containing a structural motif made of an aromatic residue in position P1 and P7, and an aliphatic chain in position P4 were claimed by Castano et al (Science 269: 223, 1995) to contain a core motif for CD1d binding epitopes. As described above, the conclusions reached by Castano et al are not supported by data.
[0011] We made the unexpected finding that peptides encompassing a hydrophobic aminoacid sequence are in fact capable of eliciting activation of NKT cells.
[0012] If epitopes from proteins administrated for therapeutic purposes, or to which subjects are normally exposed, or when gene therapy or gene vaccination is carried out, or administered in the context of vaccination for allergic or infectious diseases bind to CD1d and thereby activate NKT cells, then alteration of said proteins by mutations and/or deletions to eliminate said epitopes would be highly desirable to prevent immunogenicity.
[0013] Identification of such epitopes followed by mutation, addition or deletion of aminoacids to prevent activation of NKT cells forms the basis of the present invention.
SUMMARY OF THE INVENTION
[0014] The present invention relates to the use of peptides or polypeptides for the treatment of immune responses elicited towards allofactors in a subject by preventing such immune response towards said allofactors.
[0015] The present invention also relates to the use of peptides or polypeptides for the treatment of immune responses elicited towards viral vectors used for gene therapy or gene vaccination in a subject by preventing such immune response towards said viral vectors.
[0016] The present invention also relates to the use of peptides or polypeptides made by genetically-modified organisms for the prevention in a subject of immune responses elicited by exposure to natural proteins.
[0017] The present invention further relates to the use of peptides or polypeptides for vaccination purposes when activation of innate immunity is detrimental.
[0018] The present invention also relates to methods to identify proteins which carry CD1d binding epitopes and to eliminate such epitopes by aminoacid substitution or deletion.
[0019] We made the unexpected finding that a significant proportion of peptides or polypeptides carried aminoacid sequences which allow them to bind and to be presented by CD1d determinants for activation of natural killer T (NKT) cells. Activation of such cells results in release of cytokines and, in some cases, in acquisition or increase of cytolytic properties.
[0020] The present invention relates in one aspect to the use of at least one isolated peptide or polypeptide used as an allofactor, which has been modified to eliminate at least one hydrophobic amino acid residue involved in the formation of an epitope recognized by NKT cells, as a medicament for preventing in a subject immune responses to said allofactor.
[0021] The present invention also relates in one aspect to the use of at least one isolated peptide or polypeptide used as a viral vector for gene therapy or gene vaccination, which has been modified to eliminate at least one hydrophobic amino acid residue involved in the formation of an epitope recognized by NKT cells, as a medicament for preventing in a subject immune responses to said viral vectors.
[0022] The present invention also relates in one aspect to the use of at least one isolated peptide or polypeptide produced by a genetically-modified organism, said peptide or polypeptide being modified to eliminate at least one hydrophobic amino acid residue involved in the formation of an epitope recognized by NKT cells, as a medicament for preventing in a subject immune responses to natural exposure to said peptides or polypeptides.
[0023] The present invention further relates in one aspect to the use of at least one isolated peptide or polypeptide used as a vaccine, which has been modified to eliminate at least one hydrophobic amino acid residue involved in the formation of an epitope recognized by NKT cells, as a medicament for preventing in a subject an unwanted or inappropriate immune response to said vaccine.
[0024] In a further aspect, the invention also covers the use of at least one isolated peptide or polypeptide used as an allofactor, a viral vector, a genetically-modified organism or a vaccine, which has been modified to eliminate at least one hydrophobic amino acid residue involved in the formation of an epitope recognized by NKT cells, as a medicament for preventing in a subject activation, cytokine production, cytolytic activity and suppressive activity on adaptive immune responses carried by CD4+ NKT cells in said subject.
[0025] The present invention relates to hydrophobic peptides or polypeptides encompassing at least one CD1d-restricted T cell epitope, in which aminoacids positioned as anchoring residues to CD1d are replaced by alternative aminoacids, or deleted, which results in a loss or significant reduction of binding to CD1d and thereby of NKT cell activation.
[0026] The structure of the CD1d molecule indicates that hydrophobic aminoacid residues are required to occupy the two hydrophobic pockets located at the extremities of the CD1d cleft and that an aliphatic residue should occupy the position in the middle of the cleft. Therefore, as a general example of CD1d binding sequence, the motif [FW]-xx-[ILM]-xx-[FWTH] can be used in which [FW] indicates that either F or W can occupy the first anchoring residue (P1), that the P4 position can be occupied by either I, L or M and that P7 can be occupied by F, W, T or H. x in this general model motif stands for any aminoacid. It should be clear for the one skilled in the art that various combinations of these aminoacid residues are possible. In a particular embodiment the general model motif can be presented as a reverted sequence such as [FWTH]-xx-[ILM]-xx-[FW]. In yet another particular embodiment at least one aminoacid is added within the CD1d binding motif, which disrupts the motif, prevents its capacity to bind to CD1d and thereby its capacity to activate NKT cells.
[0027] The present invention further relates more particularly to peptides or polypeptides wherein F, W, T, H or Y in positions P1 and P7 are replaced by a non-natural amino acid (for example a D-aminoacid) or by an organic compound.
[0028] In any of the above uses said allofactor may be any peptide or polypeptide used: (1) for replacement therapy for coagulation defects or fibrinolytic defects, including factor VIII, factor IX and staphylokinase; (2) hormones such as growth hormone or insulin; (3) cytokines and growth factors, such as interferon-alpha, interferon-gamma, GM-CSF and G-CSF; (4) antibodies for the modulation of immune responses, including anti-IgE antibodies in allergic diseases, anti-CD3 and anti-CD4 antibodies in graft rejection and a variety of autoimmune diseases, anti-CD20 antibodies in non-Hodgkin lymphomas; (5) erythropoietin in renal insufficiency and; (6) genetically modified antigens.
[0029] In any of the above uses said viral vector may be any peptide or polypeptide of RNA viruses (gamma-retroviruses and lentiviruses) or DNA viruses (adenoviruses, adeno-associated viruses, herpes viruses and poxviruses).
[0030] In any of the above uses said genetically-modified organism may be any organism of plant or animal origin, which is used as food or feed, for producing crops or manufacture material, or for producing transgenic animals for food or feed, or stock breeding.
[0031] In any of the above, said peptide or polypeptide used for vaccination may be from allergens or from infectious agents, including viruses, bacteria and parasites. Allergens may be airborne allergens such as those derived from house dust mite, from pollens or from domestic animals, food allergens such as peanut, ovalbumin, cereals, fruits and legumes, and contact antigens such as latex. Diseases characterizing allergen sensitization include allergic asthma, allergic rhino-sinusitis, anaphylactic shock, urticaria, atopic dermatitis and contact dermatitis.
[0032] The present invention also relates to methods for identifying peptides or polypeptides activating NKT cells and eliminates such activation by altering CD1d binding epitopes by substitution, addition or deletion of aminoacids. Said methods comprise the steps of incubating said peptide or polypeptide with cells carrying CD1d, followed by addition of a population of polyclonal NKT cells and determination of activation of said NKT cells.
[0033] The invention further encompasses isolated viral vectors characterized in that they comprise at least one peptide or polypeptide of an allofactor modified by substitution or deletion of at least one hydrophobic aminoacid, or at least one peptide or polypeptide from an allergen or from a infectious agent modified by substitution or deletion of at least one hydrophobic aminoacid residue. It should be understood that the viral vector itself may also be modified by substitution or deletion of hydrophobic aminoacid residues.
Definitions
[0034] The term "peptide" when used herein refers to a molecule comprising an amino acid sequence of between 2 and 200 amino acids, connected by peptide bonds, but which can in a particular embodiment comprise non-amino acid structures (like for example a linking organic compound). Peptides according to the invention can contain any of the conventional 20 amino acids or modified versions thereof, or can contain non-naturally occurring amino acids incorporated by chemical peptide synthesis or by chemical or enzymatic modification. The term "polypeptide" when used herein refers to generally longer peptides or proteins.
[0035] The term "epitope" when used herein refers to one or several portions (which may define a conformational epitope) of a protein which is/are specifically recognized and bound by an antibody or a portion thereof (Fab', Fab2', etc.) or a receptor presented at the cell surface of a B or T cell lymphocyte, and which is able, by said binding, to induce an immune response.
[0036] The term "antigen" when used herein refers to a structure of a macromolecule comprising one or more hapten(s) and/or comprising one or more T cell epitopes. Typically, said macromolecule is a protein or peptide (with or without polysaccharides) or made of proteic composition and comprises one or more epitopes; said macromolecule can herein alternatively be referred to as "antigenic protein" or "antigenic peptide".
[0037] The term "allergen" refers to a specific subset of antigen characterized by its capacity to elicit antibodies of the IgE isotype in predisposed individuals.
[0038] The term "T cell epitope" or "T-cell epitope" in the context of the present invention refers to a dominant, sub-dominant or minor T cell epitope, i.e., a part of an antigenic protein that is specifically recognized and bound by a receptor at the cell surface of a T lymphocyte. Whether an epitope is dominant, sub-dominant or minor depends on the immune reaction elicited against the epitope. Dominance depends on the frequency at which such epitopes are recognized by T cells and able to activate them, among all the possible T cell epitopes of a protein. In particular, a T cell epitope is an epitope bound by MHC class I or MHC class II molecules.
[0039] The term "NKT cell epitope" refers to a part of an antigenic protein that is specifically recognized and bound by a receptor at the cell surface of a T lymphocyte. In particular, a NKT cell epitope is an epitope bound by CD1d molecules.
[0040] The term "CD4+ effector cells" refers to cells belonging to the CD4-positive subset of T-cells whose function is to provide help to other cells, such as, for example B-cells. These effector cells are conventionally reported as Th cells (for T helper cells), with different subsets such as Th0, Th1, Th2, and Th17 cells.
[0041] The term "NKT cells" refers to cells of the innate immune system characterized by the fact that they carry receptors such as NK1.1 and NKG2D, and recognize epitopes presented by the CD1d molecule. In the context of the present invention, NKT cells can belong to either the type 1 (invariant) or the type 2 subset.
[0042] The "CD1d molecule" refers to a non-MHC derived molecule made of 3 alpha chains and an anti-parallel set of beta chains arranged into a deep hydrophobic groove opened on both sides and capable of presenting lipids, glycolipids or hydrophobic peptides to NKT cells.
[0043] The term "immune disorders" or "immune diseases" refers to diseases wherein a reaction of the immune system is responsible for or sustains a malfunction or non-physiological situation in an organism. Immune disorders in the context of the present invention refer to pathology induced by infectious agents and tumor surveillance.
[0044] The term "allofactor" refers to a protein, peptide or factor (i.e. any molecule) displaying polymorphism when compared between two individuals of the same species, and, more in general, any protein, peptide or factor that induces an (alloreactive) immune response in the subject receiving the allofactor. By extension, allofactors also include genetically-modified proteins used for feeding.
[0045] The term "alloantigen" or "allograft antigen" when used herein refer to an antigen derived from (shed from and/or present in) a cell or tissue which, when transferred from a donor to a recipient, can be recognized and bound by an antibody of B or T-cell receptor of the recipient. Alloantigens are typically products of polymorphic genes. An alloantigen is a protein or peptide which, when compared between donor and recipient (belonging to the same species), displays slight structural differences. The presence of such a donor antigen in the body of a recipient can elicit an immune response in the recipient. Such alloreactive immune response is specific for the alloantigen.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The present invention provides ways to prevent, in a subject, an immune response towards allofactors, towards viral vectors used for gene therapy or gene vaccination, towards proteins used for food or feed, towards proteins to which said subject is exposed by inhalation or by stings, or to prevent, in a subject, an undesirable activation of innate immunity in the use of vaccines towards allergens or infectious agents.
[0047] In particular, the invention provides ways to prevent the expansion and functional activity of CD4+ NKT cells. Such cells are usually classified into two distinct subsets, namely type 1 for NKT cells carrying an invariant TCR alpha chain (Valpha14 in the mouse, Valpha24 in humans), or type 2 NKT cells which present with a diverse alpha chain repertoire. However, recent evidence has suggested that alternative subsets of NKT cells which do not fit in the type 1 or type 2 category. It is the purpose of the present invention to include these non conventional NKT cells, provided they carry the CD4 co-receptor. Upon presentation of an antigen bound to CD1d, NKT cells are rapidly activated and secrete a number of cytokines thought to be determinant to influence other cells from both the innate and adaptive immune systems. In some circumstances, said activated NKT cells acquire or increase cytotoxic properties. In yet additional circumstances, said activated NKT cells suppress or reduce the elicitation of an adaptive immune response by interaction with class II-restricted CD4+ T cells.
[0048] In the context of the present invention, we made the unexpected observation that peptides can be presented by the CD1d molecule. A characteristic of the CD1d molecule is that it is made of two anti-parallel alpha chains forming a cleft sitting atop of a platform made of two anti-parallel beta chains. The cleft is narrow and deep and accept only hydrophobic residues, classically deemed to be only lipids. The cleft can accommodate a sequence of 7 aminoacids characterized as a hydrophobic residue in position (P)1 and 7, and an aliphatic residue in P4. P1 is an obligate hydrophobic residue, such as F, W, H or Y. However, P7 is permissive and can contain alternative residues provided they are not polar. Residues in P4 are preferably aliphatic but are optional. A general sequence for a CD1d binding motif is therefore [FWTHY]-X.sub.2X.sub.3-[ILMV]-X.sub.5X.sub.6-[FWTHY]. It should however be clear for those skilled in the art that the motif is symmetrical and that P7 can be considered as P1, and P1 can be considered as P7. The general sequence of a CD1d binding motif is provided here as a general indication without any limiting intention. Peptides and polypeptides considered for application of the present invention are defined according to their capacity to activate NKT cells by presentation into CD1d molecule.
[0049] Hydrophobic peptides or polypeptides capable of activating NKT cells and, consequently, carrying a CD1d-binding motif are found in allofactors, viral vectors, proteins used for food or feed, proteins to which said subject is exposed by inhalation or by stings, genetically-modified proteins and allergens, thereby endowing said allofactor, viral vector, genetically-modified protein or allergen with the capacity to activate CD4+ NKT cells.
[0050] The present invention relates to the production of peptides or polypeptides containing CD1d binding motif(s), which confer them with the capacity to activate NKT cells and which are modified by substitution of hydrophobic residues in P1 and/or P7, with, optionally, substitution or deletion of aliphatic residues in P4, or any combination of these, which results in a loss or significant reduction of the capacity of peptides or polypeptides to bind to CD1d and thereby results in a loss or significant reduction of said peptides or polypeptides to activate NKT cells.
[0051] In a more particular embodiment, F, W, T, H or Y in positions P1 and/or P7 are replaced by a non-hydrophobic aminoacid residue, or, optionally, I, L, M or V in position P4 is replaced by a non-aliphatic residue, or any combination of these.
[0052] In yet another particular embodiment, hydrophobic residues located in position P1 and/or P7, or, optionally, aliphatic residues located in P4, or any combination of these, are replaced by at least one non-natural aminoacid different from non-natural F, W, T, H, Y, or by a non-aromatic organic compound.
[0053] In yet another particular embodiment at least one aminoacid is added within the CD1d binding motif, in any location within the P1 to P7 sequence, which disrupts the motif, prevents its capacity to bind to CD1d and thereby its capacity to activate NKT cells.
[0054] In a preferred embodiment, non-natural aminoacids are D-aminoacids.
[0055] The present invention also relates to the production of peptides or polypeptides containing CD1d binding motif(s), which confer them with the capacity of activate NKT cells, and which are modified by deletion of hydrophobic residues in P1 and/or P7, or, optionally, by deletion of aliphatic residues in P4, or any combination of these, which results in a loss or significant reduction of the capacity of peptides or polypeptides to bind to CD1d and thereby results in a loss or significant reduction of said peptides or polypeptides to activate NKT cells.
[0056] Upon administration to a subject, such peptides or polypeptides are not loaded on CD1d and thereby are prevented from activating NKT cells.
[0057] In a further aspect, the invention also covers the use of at least one isolated peptide or polypeptide comprising at least one substitution or deletion of F, W, T, H or Y in positions P1 or P7 for preventing in a subject an immune response towards allofactor administration, viral vector administration, proteins to which said subject is exposed by food, feed, systemic or inhalation route, or allergens or infectious agents used for vaccination purposes.
[0058] In yet a further aspect, the invention covers the use of at least one isolated peptide or polypeptide comprising at least one substitution or deletion of F, W, T, H or Y in positions P1 or P7 for preventing in a subject the activation of NKT cells towards allofactor administration, viral vector administration, proteins to which said subject is exposed by food, feed, systemic or inhalation route, or some allergens or infectious agents used for vaccination purposes.
[0059] In yet a further aspect, the invention also covers the use of at least one isolated peptide or polypeptide comprising at least one substitution or deletion of F, W, T, H or Y in positions P1 or P7 as a medicament for preventing in a subject an immune response towards allofactor administration, viral vector administration, proteins to which said subject is exposed by food, feed, systemic or inhalation route, genetically modified peptides or polypeptides or some allergens or infectious agents used for vaccination purposes.
[0060] The number of CD1d binding motifs when present in a peptide or polypeptide, is very limited. Examples of such peptides or polypeptides are provided below. Typically a polypeptide presents one to five of these motifs.
[0061] An additional advantage of the present invention is that the CD1d molecule presents a very limited degree of polymorphism. It is therefore obvious for the one skilled in the art that the same aminoacid substitutions, addition or deletions according to the present invention provide peptides or polypeptides useful for all or a large majority of subjects. This is in sharp contrast with peptide or polypeptide motifs binding to MHC class II molecules, wherein a large number of peptides can be delineated which contain the appropriate sequence. This is due to the minimum constraints imposed to MHC class II binding peptides and to the large polymorphism of class II molecules.
[0062] Peptides and polypeptides which are the object of the present invention are identified as follows:
[0063] (1) a peptide or polypeptide aminoacid sequence is, optionally, evaluated for the presence of at least one CD1d motif containing an hydrophobic residue in P1 and P7, and an aliphatic residue in P4. A general sequence such as [FWTHY]-X.sub.2X.sub.3-[ILMV]-X.sub.5X.sub.6-[FWTHY] can be used for using algorithms well known in the art such as http://expasy.org/tools/scanprosite/
[0064] This general sequence should be considered as a tool to help identifying which sequence(s) in said peptide or polypeptide contain a motif which could enable said peptide or polypeptide to activate NKT cells.
[0065] (2) the capacity of the peptide or polypeptide to bind to CD1d and to activate NKT cells is tested in vitro using a cell line expressing the CD1d molecule. Examples of such cell lines are known in the art (for instance JAWS2 cells). In a preferred embodiment, the cell line is not presenting MHC class II molecules and is transduced for hyperexpression of CD1d using a viral vector containing the DNA sequence of CD1d or any other means known in the art to introduce a gene in a cell. Methods for cell transduction are known in the art. The cell line is loaded in culture with the peptide or polypeptide, or with a synthetic peptide encompassing the corresponding sequence. Such synthetic peptides are easily produced by synthesis, using for instance the fmoc solid phase synthesis well known in the art. Efficient presentation of the peptide, polypeptide or corresponding synthetic peptide by the CD1d molecule is then evaluated by measuring the activation of NKT cells. Such cells can be obtained from peripheral blood by, for instance, magnetic sorting and maintained in culture with stimulants such as alpha-gal-ceramide, in the presence of cytokines such as IL-2, IL-15 or IL-7. These methods are described in the art (see for instance Godfrey et al, Nature Reviews. Immunology 2010, 11: 197-206). Activation of NKT cells is assessed using methods such as evaluation of cytokine production.
[0066] Alternatively, peptides actually presented by APC in CD1d molecules can be eluted and separated by various chromatography methods. Full description of such methodology will be found in Scott et al, Immunity, 12: 711-720, 2000. Said peptides are then sequenced to identify which aminoacid residues are located in P1 and P7.
[0067] Alternatively, said synthetic peptides can be loaded on tetramers of the CD1d molecule to detect NKT cells specific for such peptide. One possibility is to use fluorescence-labeled tetramers and detection using a fluorescence-activated cell sorting system (facs).
[0068] (3) the aminoacid sequences identified as being able to activate NKT cells and, optionally, identified by algorithms, are then modified by either substitution or deletion. In a preferred embodiment, F, W, T, H or Y in positions P1 and/or P7 are replaced by at least one aminoacid different from F, W, T, H, Y. Natural aminoacids can be modified by post-transcriptional modifications or substituted with chemical groups such as methyl groups. In another preferred embodiment, F, W, T, H or Y in positions P1 and/or P7 are replaced by any suitable alternative non-natural aminoacid. Examples of non-natural aminoacid residues are D-aminoacids. In yet another embodiment, F, W, T, H or Y in positions P1 and/or P7 are replaced by at least one aminoacid different from F, W, T, H, Y. In another preferred embodiment, F, W, T, H or Y in position P1 is replaced by at least one aminoacid different from F, W, T, H, Y, by any suitable alternative non-natural aminoacid or by a non-aromatic organic compound. Such aminoacid substitution is obtained using methods well known in the art. In yet a further preferred embodiment, F, W, T, H or Y in position P1 is deleted. In yet another embodiment, F, W, T, H or Y in positions P1 and P7 are deleted. Methods to carry out said deletions are well known in the art. In yet another particular embodiment at least one aminoacid is added within the CD1d binding motif, in any location within the P1 to P7 sequence.
[0069] According to the present invention medicaments are envisaged for the treatment of diseases wherein administration of allofactors are required, such as in:
[0070] (1) congenital or acquired deficiency in factors associated with coagulation (such as factor VIII, factor IX or factor X) or fibrinolysis, with defect in enzymes associated with the metabolism of polysaccharides or glycogen (such as in Pompe disease), or with defect in hormone production (such as insulin in diabetes or growth hormone in nanism)
[0071] (2) acute or chronic situations wherein it is advantageous to administer a curative agent, such as thrombolytic agents including staphylokinase and microplasmin,
[0072] (3) disorders of the immune system in which it is required to administer cytokines (or their receptor) or growth factors (such as interferon-alpha, interferon-beta, interferon-gamma, G-CSF, GM-GSF, KGF or erythropoietin)
[0073] (4) diseases characterized by chronic inflammation or inappropriate immune responses, wherein therapeutic antibodies should be administered, including anti-tumor necrosing factor, anti-CD3 or anti-CD4 antibodies in autoimmune diseases and graft rejection, antibodies to lymphocyte surface markers (such as anti-CD20 antibodies in non-Hodgkin lymphomas), or antibodies to factor VIII in the prevention of thrombosis. The list of therapeutic antibodies is growing fast and the present invention intends to cover the use of any antibodies used for therapeutic purposes in general.
[0074] According to the present invention medicaments are also envisaged for use in gene therapy and gene vaccination, wherein viral vectors are utilized and wherein the immune response against said vectors precludes transgene expression.
[0075] According to the present invention medicaments are also envisaged for diseases elicited by exposure to environmental proteins, such as:
[0076] (1) proteins to which said subject is exposed by food or feed. Examples of these are cereals such as wheat, maize, rice, soybean and colza, vegetables such as potato and beetroot, fruits such as rosacea, nuts, and avocado, enzymes, anti-viral or anti-bacterial drugs.
[0077] (2) proteins towards which the subject is exposed by inhalation, systemic route or by stinging. Examples of these are allergic reactions to pollens, contact reaction to latex or hymenoptera stings.
[0078] According to the present invention medicaments are also envisaged for immunization (vaccination) such as:
[0079] (1) vaccination against allergens
[0080] (2) vaccination against infectious agents, including viruses, bacteria and parasites
[0081] In both these circumstances it may be advantageous to prevent an activation of the innate immune system so as to prevent excess of inflammation and its detrimental consequences on the result of said vaccination. Another advantage in the setting of vaccination to allergens or infectious agents is that the elimination of NKT cell activation prevents the suppressive effect of activated NKT cells on the development of an adaptive response against said allergens or said infectious agents.
[0082] It should be recognized that the above list is not exhaustive and that the invention intends to cover newly-introduced products such as antibodies, cytokines, growth factors or peptides and polypeptides used for replacement in congenital or acquired deficiencies, and genetically-modified proteins.
[0083] It should be understood that any of the peptides or polypeptides listed above may be administered in the form of gene for transgenesis, which may be carried out using viral vectors or other means known by those skilled in the art. In such a case, the viral vector itself may be modified according to the present invention by eliminating CD1d binding motifs.
[0084] The medicament of the invention is usually, though not necessarily, a (pharmaceutical) formulation comprising as active ingredient at least one of the peptides or polypeptides of the invention or a gene therapeutic vector capable of expressing said peptides or polypeptides.
[0085] Apart from the active ingredient(s), such formulation will comprise at least one of a (pharmaceutically acceptable) diluent.
[0086] A notable exception to this rule is the use of proteins from genetically-modified organisms for food or feed, exposure by inhalation or by the systemic route.
[0087] In general, administration of peptides or polypeptides of the invention prevents activation of the innate immune system, more particularly activation of NKT cells, more particularly the production of cytokines associated with NKT cell activation.
[0088] The route of administration for peptides or polypeptides of the present invention may vary according to the indication and/or the nature of the peptides or polypeptides. Examples are intravenous injection of coagulation factors, subcutaneous injection of insulin and oral administration of genetically-modified proteins. The present invention intends to cover all other possible routes of administration such as intranasal, sublingual, percutaneous, intramuscular, intrarectal or intravaginal.
[0089] As explained in detail further on, the peptides or polypeptides of the present invention can be made by chemical synthesis, which further allows the incorporation of non-natural amino acids. The peptides or polypeptides of the present invention can also be produced using methods know in the art for the production of recombinant proteins using expression systems such as bacterial cells, yeast cells, insect cells, plant cells or mammalian cells.
[0090] Another aspect of the present invention relates to methods for generating peptides and polypeptides of the present invention described herein. Such methods include the identification of NKT-cell epitopes from allofactors, viral vectors, proteins to which said subject is exposed by food, feed, systemic or inhalation route, allergens or specific infectious agents. Ways for in vitro and in silico identification NKT-cell epitopes are amply known in the art and some aspects are elaborated upon hereafter.
[0091] The identification of a NKT-cell epitope in the context of the present invention is known to a person skilled in the art. For instance, peptide sequences isolated from allofactors, viral vectors, proteins to which said subject is exposed by food, feed, systemic or inhalation route, genetically-modified proteins, allergens or specific infectious agents are tested by, for example, NKT cell biology techniques, to determine whether the peptide sequences elicit a NKT cell response. Those peptide sequences found to elicit a NKT cell response are defined as having NKT cell stimulating activity. Mammal NKT cell stimulating activity can further be tested by culturing NKT cells obtained from an individual sensitized to an allofactor, viral vector, proteins to which said subject is exposed by food, feed, systemic or inhalation route, genetically-modified protein, allergen or specific infectious agent, and determining whether proliferation of NKT cells occurs in response to the peptide/epitope as measured, e.g., by cellular uptake of tritiated thymidine. Stimulation indices for responses by NKT cells to peptides/epitopes can be calculated as the maximum CPM in response to a peptide/epitope divided by the control CPM. A NKT cell stimulation index (S.I.) equal to or greater than two times the background level is considered "positive." Positive results are used to calculate the mean stimulation index for each peptide/epitope for the group of peptides/epitopes tested. Non-natural NKT-cell epitopes can further optionally be tested for their binding affinity to CD1d molecules. The binding of non-natural NKT-cell epitopes to CD1d molecules can be performed in different ways. For instance, soluble CD1d molecules are obtained and made tetrameric by synthesis and/or chemical coupling. The CD1d molecule is purified by affinity chromatography. Soluble CD1d molecules are incubated with a biotin-labeled reference peptide produced according to its strong binding affinity for said CD1d molecule. Peptides to be assessed for CD1d binding are then incubated at different concentrations and their capacity to displace the reference peptide from its CD1d binding site is calculated by addition of neutravidin. Methods can be found in for instance in Texier et al., (2000) J. Immunology 164, 3177-3184) for peptides presented by the MHC class II determinants, but the method can easily be applied to CD1d-restricted NKT cell epitopes. The immunogenic peptides or polypeptides of the invention have a mean NKT cell stimulation index of greater than or equal to 2. An immunogenic peptide having a NKT cell stimulation index of greater than or equal to 2 is considered useful as a candidate to carry out the substitution or deletion of hydrophobic aminoacid residues, or addition of aminoacids within the sequence of the CD1d binding motif, as described in the present invention.
[0092] If two or more aminoacid sequences which share an area of overlap in the native peptide or polypeptide sequence are found to have human NKT cell stimulating activity, as determined by T cell biology techniques, mutation or deletion of hydrophobic aminoacid residues may be carried out for residues belonging to one or to both of the sequences.
[0093] The peptides or polypeptides of the invention can be produced by recombinant expression in, e.g., bacterial cells (e.g. Escherichia coli), yeast cells (e.g., Pichia species, Hansenula species, Saccharomyces or Schizosaccharomyces species), insect cells (e.g. from Spodoptera frugiperda or Trichoplusia ni), plant cells or mammalian cells (e.g., CHO, COS cells). The construction of the therefore required suitable expression vectors (including further information such as promoter and termination sequences) involves meanwhile standard recombinant DNA techniques. Recombinantly produced peptides or polypeptides of the invention can be derived from a larger precursor protein, e.g., via enzymatic cleavage of enzyme cleavage sites inserted adjacent to the N- and/or C-terminus of the peptide or polypeptide, followed by suitable purification.
[0094] In view of the limited length of some of the peptides of the invention, they can be prepared by chemical peptide synthesis, wherein peptides are prepared by coupling the different amino acids to each other. Chemical synthesis is particularly suitable for the inclusion of e.g. D-amino acids, amino acids with non-naturally occurring side chains or natural amino acids with modified side chains such as methylated cysteine. Chemical peptide synthesis methods are well described and peptides can be ordered from companies such as Applied Biosystems and other companies. Peptide synthesis can be performed as either solid phase peptide synthesis (SPPS) or contrary to solution phase peptide synthesis. The best-known SPPS methods are t-Boc and Fmoc solid phase chemistry which is amply known to the skilled person. In addition, peptides can be linked to each other to form longer peptides using a ligation strategy (chemoselective coupling of two unprotected peptide fragments) as originally described by Kent (Schnolzer & Kent (1992) Int. J. Pept. Protein Res. 40, 180-193) and reviewed for example in Tam et al. (2001) Biopolymers 60, 194-205. This provides the potential to achieve protein synthesis which is beyond the scope of SPPS. Many proteins with the size of 100-300 residues have been synthesized successfully by this method.
[0095] The physical and chemical properties of a peptide or polypeptide of the invention (e.g. solubility, stability) is examined to determine whether the peptide is/would be suitable for use in therapeutic compositions. Typically this is optimized by adjusting the sequence of the peptide. Optionally, the peptide can be modified after synthesis (chemical modifications e.g. adding/deleting functional groups) using techniques known in the art.
[0096] The production of genetically-modified organisms relies on methods well known for those skilled in the art, including cloning, site-directed mutagenesis and growth.
[0097] The present invention also relates to nucleic acid sequences encoding the peptides or polypeptides of the present invention and methods for their use, e.g., for recombinant expression or in gene therapy. In particular, said nucleic acid sequences are capable of expressing peptides of the invention.
[0098] In gene therapy, recombinant nucleic acid molecules encoding the peptides or polypeptides of the present invention can be used as naked DNA or in liposomes or other lipid systems for delivery to target cells. Other methods for the direct transfer of plasmid DNA into cells are well known to those skilled in the art for use in human gene therapy and involve targeting the DNA to receptors on cells by complexing the plasmid DNA to proteins. In its simplest form, gene transfer can be performed by simply injecting minute amounts of DNA into the nucleus of a cell, through a process of microinjection. Once recombinant genes are introduced into a cell, they can be recognized by the cell normal mechanisms for transcription and translation, and a gene product will be expressed. Other methods have also been attempted for introducing DNA into larger numbers of cells. These methods include: transfection, wherein DNA is precipitated with calcium phosphate and taken into cells by pinocytosis; electroporation, wherein cells are exposed to large voltage pulses to introduce holes into the membrane); lipofection/liposome fusion, wherein DNA is packed into lipophilic vesicles which fuse with a target cell; and particle bombardment using DNA bound to small projectiles. Another method for introducing DNA into cells is to couple the DNA to chemically modified proteins. Adenovirus proteins are capable of destabilizing endosomes and enhancing the uptake of DNA into cells. Mixing adenovirus to solutions containing DNA complexes, or the binding of DNA to polylysine covalently attached to adenovirus using protein crosslinking agents substantially improves the uptake and expression of the recombinant gene. Adeno-associated virus vectors may also be used for gene delivery into vascular cells. As used herein, "gene transfer" means the process of introducing a foreign nucleic acid molecule into a cell, which is commonly performed to enable the expression of a particular product encoded by the gene. The said product may include a protein, polypeptide, anti-sense DNA or RNA, or enzymatically active RNA. Gene transfer can be performed in cultured cells or by direct administration into mammals. In another embodiment, a vector comprising a nucleic acid molecule sequence encoding a peptide according to the invention is provided. In particular embodiments, the vector is generated such that the nucleic acid molecule sequence is expressed only in a specific tissue. Methods of achieving tissue-specific gene expression are well known in the art, e.g., by placing the sequence encoding an immunogenic peptide of the invention under control of a promoter, which directs expression of the peptide specifically in one or more tissue(s) or organ(s). Expression vectors derived from viruses such as retroviruses, vaccinia virus, adenovirus, adeno-associated virus, herpes viruses, RNA viruses or bovine papilloma virus, may be used for delivery of nucleotide sequences (e.g., cDNA) encoding peptides, homologues or derivatives thereof according to the invention into the targeted tissues or cell population. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors containing such coding sequences. Alternatively, engineered cells containing a nucleic acid molecule coding for a peptide or polypeptide according to the invention may be used in gene therapy.
[0099] The medicament of the invention is usually, but not necessarily (for instance proteins produced by genetically-modified organisms which are used for food, feed, or to which said subject is exposed to systemic or inhalation route), a (pharmaceutical) formulation comprising as active ingredient at least one of the peptides or polypeptides of the invention, a gene therapeutic vector capable of expressing said peptide or polypeptide. Apart from the active ingredient(s), such formulation will comprise at least one of a (pharmaceutically acceptable) diluent. Typically, pharmaceutically acceptable compounds can be found in, e.g., a Pharmacopeia handbook (e.g. US-, European- or International Pharmacopeia). The medicament or pharmaceutical composition of the invention normally comprises a (prophylactically or therapeutically) effective amount of the active ingredient(s) wherein the effectiveness is relative to the condition or disorder to be prevented or treated.
[0100] The medicament or pharmaceutical composition of the invention may need to be administered to a subject in need as part of a prophylactic or therapeutic regimen comprising multiple administrations of said medicament or composition. Said multiple administrations usual occur sequentially and the time-interval between two administrations can vary and will be adjusted to the nature of the active ingredient and the nature of the condition to be prevented or treated. The amount of active ingredient given to a subject in need of a single administration can also vary and will depend on factors such as the physical status of the subject (as for instance weight and age), the status of the condition to be prevented or treated, and the experience of the treating doctor, physician or nurse.
[0101] The term "diluents" refers for instance to physiological saline solutions. The term "pharmaceutically acceptable carrier" means any material or substance with which the active ingredient is formulated in order to facilitate its application or dissemination to the locus to be treated, for instance by dissolving, dispersing or diffusing the said composition, and/or to facilitate its storage, transport or handling without impairing its effectiveness. They include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents (for example phenol, sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodium chloride) and the like. Additional ingredients may be included in order to control the duration of action of the active ingredient in the composition. The pharmaceutically acceptable carrier may be a solid or a liquid or a gas which has been compressed to form a liquid, i.e. the compositions of this invention can suitably be used as concentrates, emulsions, solutions, granulates, dusts, sprays, aerosols, suspensions, ointments, creams, tablets, pellets or powders. Suitable pharmaceutical carriers for use in said pharmaceutical compositions and their formulation are well known to those skilled in the art, and there is no particular restriction to their selection within the present invention. They may also include additives such as wetting agents, dispersing agents, stickers, adhesives, emulsifying agents, solvents, coatings, antibacterial and antifungal agents (for example phenol, sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodium chloride) and the like, provided the same are consistent with pharmaceutical practice, i.e. carriers and additives which do not create permanent damage to mammals. The pharmaceutical compositions of the present invention may be prepared in any known manner, for instance by homogeneously mixing, coating and/or grinding the active ingredients, in a one-step or multi-steps procedure, with the selected carrier material and, where appropriate, the other additives such as surface-active agents. They may also be prepared by micronisation, for instance in view to obtain them in the form of microspheres usually having a diameter of about 1 to 10 .mu.m, namely for the manufacture of microcapsules for controlled or sustained release of the active ingredients.
[0102] Peptides or polypeptides, homologues or derivatives thereof according to the invention (and their physiologically acceptable salts or pharmaceutical compositions all included in the term "active ingredients") may be administered by any route appropriate to the condition to be prevented or treated and appropriate for the compounds, here the peptide or polypeptide to be administered. Possible routes include regional, systemic, oral (solid form or inhalation), rectal, nasal, topical (including ocular, buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intraarterial, intrathecal and epidural). The preferred route of administration may vary with for example the condition of the recipient or with the condition to be prevented or treated.
[0103] The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.
[0104] Viral vectors for the purpose of gene therapy or gene vaccination are highly amenable to modifications by means of recombinant nucleic acid technology. In view of the above, a skilled person will further easily envisage that the elimination of the viral vector NKT-cell epitope as applied in the peptides or polypeptides and their uses according to the invention can be introduced immediately in the viral vector itself. Hence, the invention further encompasses modified viral vectors defined as isolated viral vectors characterized in that CD1d binding motifs have been eliminated by aminoacid substitution or deletion.
[0105] The present invention will now be illustrated by means of the following examples, which are provided without any limiting intention. Furthermore, all references described herein are explicitly included herein by reference.
EXAMPLES
Example 1: Coagulation Factor VIII
[0106] Patients suffering from hemophilia A lack sufficient amounts of factor VIII (FVIII), which is the reason for uncontrolled bleeding tendency. Such patients are treated by infusions of FVIII purified from plasma source or produced by recombinant technology. Administration of FVIII results in the formation of specific antibodies, which in more or less 30% of the cases inhibit the function of FVIII as a coagulation cofactor.
[0107] Using an algorithm, we identified within the sequence of the FVIII molecule 3 sequences bearing a CD1d binding sequence, which matched the [FWTHY]-X.sub.2X.sub.3-[ILMV]-X.sub.5X.sub.6-[FWTHY] sequence motif. These motifs are located in the A1 and A3 domains, respectively:
[0108] FCHISSH in A1 domain (aminoacids 309-315, SEQ ID1)
[0109] FWKVQHH in A3 domain (aminoacids 1816-1822, SEQ ID2)
[0110] FHAINGY in A3 domain (aminoacids 1918-1924, SEQ ID3)
[0111] These sequences have in common (underlined) an aromatic residue (phenylalanine, F) in position 1, an aliphatic residue (isoleucine, I, or valine, V) in position 4, and an aromatic residue (histidine, H, or tyrosine, Y) in position 7.
[0112] To determine whether these sequences could activate NKT cells in vivo, FVIII (2 IU) was injected intravenously to hemophilia A mice on 4 occasions separated by a 1-week interval. Hemophilia A mice produce no FVIII due to a stop codon introduced in the FVIII gene in exon 16.
[0113] Mice were sacrificed 10 days after the last injection, the spleen was removed and CD4+ T cells were prepared by magnetic bead sorting. NKT cells are characterized by expression of CD4 and recognition of antigen presented by CD1d molecule. A tetramer of CD1d was obtained from a commercial supplier and loaded with 15 aminoacid long FVIII peptides, which included peptides containing SEQ ID1, SEQ ID2 and SEQ ID3. Significant binding of CD1d tetramers loaded with these peptides was observed, indicating that these 3 peptides were able to bind to CD1d and that injection of FVIII elicited activation of NKT cells. Representative results are given in FIG. 1.
[0114] Further, direct immunization with peptides containing a CD1d motif (peptides of SEQ ID1, SEQ ID2 or SEQ ID3) was sufficient as to elicit the activation of NKT cells and production of antibodies to FVIII. Prominent activation was observed with peptide of SEQ Representative results are given in FIG. 2.
[0115] Bone marrow chimeras were constructed in which hemophilia A mice were first irradiated and reconstituted with the bone marrow of mice lacking NKT cells, namely CD1d knocked-out mice. In the absence of NKT cells, mice were unable to produce significant amounts of antibodies to FVIII, and virtually no antibodies inhibiting the function of FVIII (FIG. 3).
[0116] The A1 domain of FVIII was produced by recombinant technology in its natural sequence or with a substitution of F309 and H315 by serine (polypeptide of SEQ ID4). FVIII A1 domains in natural sequence or SEQ ID4 were used to immunize separate groups of mice. The results showed that substitution of F309 and H315 by S (SEQ ID4) was sufficient to prevent activation of NKT cells as assessed from spleen CD4+ T cells as described above.
[0117] The A3 domain of FVIII was produced by recombinant technology in its natural sequence or with a substitution of F1816 and H1822 by serine (polypeptide of SEQ ID 5). FVIII A3 domains in natural sequence or SEQ ID5 were used to immunize separate groups of mice. The results showed that substitution of F1816 and H1822 by S (SEQ ID5) was sufficient to prevent activation of NKT cells as assessed from spleen CD4+ T cells as described above.
[0118] A B domain-deleted FVIII molecule in its natural sequence elicited activation of NKT cells (see above). A FVIII molecule with 4 aminoacid substitutions was prepared containing F3095, H315S, F18165 and F1918S (SEQ ID6). Intravenous injections of such mutated FVIII in hemophilia A mice did not result in the formation of antibodies to FVIII and, consequently, no antibodies inhibiting the function of FVIII.
[0119] It was therefore concluded that:
[0120] (1) FVIII naturally contains several CD1d binding motifs;
[0121] (2) these CD1d motifs are functional and elicit activation of NKT cells;
[0122] (3) activation of NKT cells is a requisite for the production of antibodies to FVIII;
[0123] (4) elimination of the CD1d motifs by aminoacid substitution is sufficient to eliminate the production of anti-FVIII antibodies
[0124] It should be clear for those skilled in the art that the invention also applies to animals made transgenic for the production of coagulation factors such as factor IX.
DETAILED DESCRIPTION OF THE DRAWINGS
[0125] FIG. 1
[0126] NKT cells recognize factor VIII epitopes presented by CD1d
[0127] Hemophilia A mice were immunized with 2 IU Factor VIII on 4 occasions separated by one week. The spleen was then removed and CD4+ T cells were prepared by magnetic bead sorting. A fluorochrome-labeled tetramer of CD1d was obtained from a commercial supplier and loaded with 15 aminoacid long FVIII peptides, which included peptides containing SEQ ID2 and ID3. Loading was carried out at room temperature overnight in the dark. Tetramers were then incubated for 30 minutes at 4.degree. C. with the CD4+ T cell population and the cell suspension was analyzed by Facs.
[0128] The figure shows that CD1d tetramers loaded with peptide of SEQ ID1 (44pept in the figure) are recognized by NKT cells. CD1d ctl(-) shows the % of NKT cells recognizing unloaded tetramers. CD1d ctl(+) shows the % of NKT cells recognizing tetramers loaded with alpha-gal ceramide, which recruits all NKT cells. Up to 45% of NKT cells recognize 44pept, which is compatible with the absence of polymorphism at CD1d level and very limited polymorphism at the level of the NKT T cell receptor.
[0129] FIG. 2
[0130] Immunization of hemophilia A mice with a CD1 d-restricted peptides elicits anti-factor VIII antibodies
[0131] Hemophilia A mice were immunized 3 times subcutaneously with 50 .mu.g of an equimolar mixture of peptides of SEQ IDI (44pept) and peptide of SEQ ID2 (256pept) adsorbed on aluminum hydroxide. A control group received physiological serum instead of peptides. Plasma was taken 10 days after the last immunization and assessed for the presence of anti-Factor VIII antibodies, using a direct binding assay. Briefly, Factor VIII (10 IU/ml) was insolubilized on polystyrene plates, which were washed and incubated with a 1/10 dilution of plasma. After a further washing, a HRP-labeled goat anti-mouse antiserum was added, followed by an enzyme substrate. Colour development was read as OD.
[0132] The figure shows that hemophilia A mice immunized with peptides of SEQ ID1 and of SEQ ID2 develop antibodies to Factor VIII.
[0133] FIG. 3
[0134] Hemophilia A mice reconstituted with bone marrow from CD1d KO mice do not produce antibodies to Factor VIII
[0135] Hemophilia A mice were lethally irradiated and reconstituted with the bone marrow of CD1d KO mice (5.times.10.sup.6/mouse), which lack NKT cells. Six weeks after bone marrow reconstitution the mice received 4 IV injections of 2 IU/ml separated by one week. Mice were bled 10 days after the last immunization and the plasma was assayed for the presence of anti-Factor VIII antibodies using a direct binding assay as described in the legend of FIG. 2. A control group of irradiated hemophilia A mice was reconstituted with a normal bone marrow.
[0136] The figure indicates that, although control mice produce high concentrations of anti-FVIII antibodies after the fourth injection of Factor VIII (left panel), mice reconstituted with the bone marrow of NKT cell deficient mice did not (right panel).
Example 2: Adenovirus 5 Viral Vectors
[0137] Viral vectors are commonly used for gene therapy and gene vaccination. One of the most common of these viral vectors is derived from adenovirus, serotype 5. Adenoviruses (Ad) are non-enveloped viruses possessing a linear, double-stranded DNA genome of about 35 kb. Human Ad5 has a capsid consisting of 3 major structural proteins: hexon, penton, and fiber. Neutralizing antibodies are raised towards hexon proteins. Such antibodies are very common in humans as a consequence of viral infection. The presence of such antibodies blocks the entry of the viral vector and, consequently, prevents expression of the transgene protein carried by the vector. Anti-Ad5 antibodies are generated in the course of an adaptive response, which depends on activation of CD4+ T cells specific for epitopes presented in the context of MHC class II molecules.
[0138] It is known that Ad5 activates the innate immune system, though the precise mechanism by which it occurs and the location where it takes place remain unclear. Yet, activation of the innate immune system could be a required step for neutralizing antibodies to be formed.
[0139] Using algorithms, we identified 7 aminoacid sequences matching with the general motif [FWTHY]-X.sub.2X.sub.3-[ILMV]-X.sub.5X.sub.6-[FWTHY] of a CD1d binding sequence (SEQ ID7, with motifs underlined) in hexon 6.
[0140] Mice were injected intravenously with 10.sup.9 PFU Ad5 vector on 3 occasions at 10-day intervals. CD4+ T cells were then prepared from the spleen by magnetic bead sorting. CD4+ T cells were incubated with CD1d tetramers loaded with peptides corresponding to each of the 7 sequences identified. It showed that a significant proportion (.+-.10%) of CD4+ NKT cells were labeled by tetramers, indicating that Ad5 vector injections activated NKT cells specific for the peptide of SEQ ID7. In addition, such mice produced specific antibodies of the IgG2a isotype, characteristic of neutralizing antibodies in the mouse.
[0141] A viral vector was prepared which contained a substitution of [FW] by serine S for each of the 7 aminoacid sequences identified. This mutated viral vector (SEQ ID8, with underlined motifs) was used to immunize animals according to the same protocol as described above for the natural sequence. The proportion of NKT cells as assessed using tetramers loaded with the peptide in natural sequence (SEQ ID7) was <1% and the concentration of Ad5 virus specific antibodies was significantly reduced (up to 10-fold).
[0142] It was therefore concluded that substitution of F to S in each P1 location of CD1d binding motifs was sufficient as to reduce NKT cell activation and thereby reduce the production of anti-Ad5 antibodies.
Example 3: Genetically-Modified Proteins
[0143] Proteins to which subjects are exposed by way of inhalation or ingestion are frequently eliciting unwanted reactions in predisposed subjects. Allergic asthma affects millions of people across the world. Food allergy on the other hand has an overall prevalence of .+-.2.5% in the general population. Allergens either airborne, ingested or penetrating the skin could share properties by which they activate NKT cells.
[0144] One of the most common food allergen is apple (Malus domesticus), and allergenicity is almost exclusively borne by the Mal d 1 protein, a 159 aminoacid long protein, which protects the plant against infectious agents. A sequence motif was identified using computer algorithms, which corresponds to the general motif [FWTHY]-X.sub.2X.sub.3-[ILMV]-X.sub.5X.sub.6-[FWTHY] of a CD1d binding sequence.
[0145] FKLIESY corresponding to aminoacids 144-150 of Mal d 1 (SEQ ID9)
[0146] A recombinant form of Mal d 1, in which F144 and Y150 were mutated in S was produced by genetic engineering. The recombinant form of Mal d 1 therefore encompasses peptide of sequence:
[0147] SKLIESS (SEQ ID10)
[0148] Synthetic peptides corresponding to SEQ ID9 and SEQ ID10 were produced. Their capacity to activate NKT cells was determined in vitro using human dendritic cells derived from peripheral blood monocytes of an individual sensitized to Mal d 1. Dendritic cells loaded with each one of the two peptides were incubated in the presence of NKT cells obtained from the same individual by sorting peripheral lymphocytes using specific markers such as CD4 and NKG2D. It was observed that NKT cells incubated with peptide of SEQ ID9 activated a significant proportion of NKT cells, while the mutated peptide of SEQ ID10 did not. Additionally, human CD1d tetramers loaded with peptides of SEQ ID9 were recognized by a significant proportion of NKT cells, but tetramers loaded with the mutated peptide of SEQ ID were recognized by less than 1% of NKT cells.
[0149] The two F144S and Y1505 mutations are introduced directly in clonal cells by site-directed mutagenesis. The full organism is then produced by conventional growth strategies. Apples produced by this GMO do not elicit allergic reactions.
[0150] One specific application of the peptides or polypeptides of the present invention is celiac disease (gluten intolerance). This disease is among the most commons in human beings and is related to T cell activation to gliadin epitopes which are presented in the context of MHC class II determinants. A genetic susceptibility has been described, with human beings carrying the HLA-DQ2 or DQ8 class II determinant being predisposed to disease. These class II determinants present peptides which have been submitted to deamidation by transglutaminase. However, these events are the results of intestinal inflammatory reaction, likely related to the innate immune system.
[0151] Gliadins are monomers of 250-300 aminoacid residues. A search for the general motif [FW]-XX-[ILM]-XX-[FWTHY] of a CD1d binding sequence using computer algorithms identified such sequence (SEQ ID11, see listing of sequences) in alpha-gliadin. A mutated form of alpha-gliadin was then produced in which the F residue of the motif was substituted by a S residue (SEQ ID12, see addendum).
[0152] The same procedure as for Mal d 1 was followed to show that, although polypeptide of SEQ ID11 activated a significant proportion of NKT cells when presented by antigen-presenting dendritic cells, the mutated form of the polypeptide (SEQ ID12) failed to do so. As for Mal d 1, human CD1d tetramers loaded with a synthetic peptide representing the motif identified in the polypeptide of
[0153] SEQ ID11 were recognized by NKT cells, while tetramers loaded with the mutated form of the motif as shown in SEQ ID12 were not.
[0154] The mutation was introduced directly in clonal cells by site-directed mutagenesis. The full organism was then produced by conventional growth strategies. Cereals containing the mutated form of gliadin do not elicit reactions of intolerance.
[0155] It should be obvious for those skilled in the art that the present invention can also be applied to proteins which are added to, for instance, genetically-modified organisms to increase their resistance to insecticides, pesticides or any other modifications judged to be beneficial. Such modifications carry the risk of creating new CD1d binding motifs.
[0156] Additional examples of genetically-modified proteins with reduced allergenicity/immunogenicity are:
[0157] food allergens such as soybean, peanut and fruits of the Rosaceous family
[0158] milk proteins
[0159] airborne allergens such as latex (Hevea brasiliensis), pollens of grasses such as Rye grass (Lolium perenne), Timothy (Phleum pratense) or Kentucky blue grass (Poa pratensis)
[0160] fish parvalbumin
[0161] honey bee phospholipase A2
[0162] It should also be clear for the one skilled in the art that the invention extends to methods by which peptides or polypeptides of the invention are produced, including the production of transgenic plants and animals.
Example 4: Allergen Der p 1
[0163] Der p 1 is a cysteine protease which is the main allergen of the so-called house dust mite (HDM), D. pteronyssinus. Sensitization to HDM is by far the commonest trigger of allergic asthma and rhinitis worldwide. Der p 1 contains 3 motifs matching the general CD1d binding motif [FWTHY]-X.sub.2X.sub.3-[ILMV]-X.sub.5X.sub.6-[FWTHY], as identified using computer algorithms and which are:
[0164] SEQ ID13: FSGVAAT aminoacids 38-44 of Der p 1
[0165] SEQ ID14: HSAIAAVI aminoacids 135-141 of Der p 1
[0166] SEQ ID15: YPYVVIL aminoacids 216-222 of Der p 1
[0167] Peptides of SEQ ID13, SEQ ID14 and SEQ ID15 were synthesized and used to load CD1d tetramers.
[0168] BALB/c mice were submitted to intranasal administration of Der p 1, using 50 .mu.l of saline containing 100 .mu.s of Der p 1. This challenge procedure was repeated twice on three consecutive days at one-week interval. The mice were sacrificed 5 days after the last nasal instillation and the spleen was removed. CD4+ T cells were purified by magnetic bead sorting and incubated in the presence of the CD1d tetramers loaded with peptides of SEQ ID13, SEQ ID or SEQ ID15. By fluorescence-activated cell sorter (facs) determination, it was observed that a significant percentage of cells (.+-.10%) were stained with the tetramers, identifying them as CD4+ NKT cells. It was therefore concluded that peptides of SEQ ID13, SEQ ID14 and SEQ ID15 were functional in binding to CD1d and in being recognized by NKT cells.
[0169] CD4+ T cells obtained from the above experiments were incubated in culture medium in the presence of an antigen-presenting cell which expresses the CD1d molecule. Such cells are commercially available, as for instance the JAWS2 cells, which do not express MHC class II determinants. JAWS2 cells were loaded with Der p 1 and presentation of Der p 1-derived epitopes by CD1d was evaluated by measuring the production of cytokines such as IFN-gamma and IL-4 as markers of NKT activation. It could be observed that a significant production of cytokines was present, confirming that Der p 1 contained epitopes presented by CD1d molecules.
[0170] Next, a mutated form of Der p 1 was prepared by genetic engineering, in which the 3 aminoacid residues predicted to be in position P1 for CD1d binding of peptides of SEQ ID13, SEQ ID14 and SEQ ID15 were substituted by serine. The mutated Der p 1 (SEQ ID16) was used for nasal instillation as described above with Der p 1 in natural sequence (SEQ ID17). In such a case, no significant binding of CD4+ T cell splenocytes was observed when incubated with the tetramers loaded with peptide of SEQ ID13, peptide of SEQ ID14 or peptide of SEQ ID15, indicating that the mutated Der p 1 had lost its capacity to activate NKT cells specific for these peptides.
[0171] Further, mutated Der p 1 (SEQ ID16) was used to load JAWS2 cells and tested for its capacity to activate NKT cells. For this experiment, NKT cells were used as obtained from mice immunized with Der p 1 in either natural or mutated configuration. The production of IFN-gamma and IL-4 was taken as an indication of NKT activation. It was observed that NKT cells obtained from mice immunized with natural sequence Der p 1 failed to be activated when incubated in the presence of JAWS2 cells loaded with mutated Der p 1.
[0172] It was therefore concluded that Der p 1 in natural sequence contained functional CD1d restricted T cell epitopes activating NKT cells. Further, elimination of such functional CD1d-restricted epitopes by mutation was sufficient to eliminate NKT cell activation.
Example 5: Antibodies
[0173] Antibodies are used as therapeutic agents in a large number of indications, from chronic inflammatory diseases such as rheumatoid arthritis (e.g., anti-TNF-alpha antibodies) or allergic asthma (e.g. anti-IgE antibodies), to tumors (e.g., anti-CD20 antibodies). More than 120 therapeutic antibodies are presently used for clinical applications at various stages from preclinical to phase III trials and accepted for routine clinical practice.
[0174] Therapeutic antibodies are either chimeric or fully humanized, which contains sequence of foreign origin only in the complementarity determining regions of the variable parts. A minority of such antibodies are derived from the human repertoire and, as such, considered as poorly immunogenic. However, antibodies towards the therapeutic antibody, even when directly derived from the human repertoire, are produced by a majority of the patients under treatment, with, in a significant proportion of the cases, the production of antibodies neutralizing the activity of the therapeutic agent.
[0175] A search for epitopes matching the CD1d binding motif in human IgG antibody sequence was carried out using computer algorithms. One of such motif was identified in the CH2 region (second domain of the heavy chain constant part) of each of the 4 IgG subclass (IgG1, IgG2, IgG3 and IgG4) and a second motif was identified in the CH3 loop of IgG1, IgG2 and IgG4:
[0176] SEQ ID18: YRVVSVL (CH2 of IgG1 and IgG4)
[0177] SEQ ID19: FRVVSVL (CH2 of IgG2 and IgG3)
[0178] SEQ ID20: HEALHNH (CH3 loop of IgG1, IgG2 and IgG4)
[0179] Synthetic peptides corresponding to SEQ ID18, SEQ ID19 and SEQ ID20 were produced and used to load human CD1d tetramers as for the examples above (see for instance example 4 for allergen Der p 1). Peripheral blood cells were obtained by venous puncture of patients who had received an injection of a therapeutic antibody during the previous 5 days. CD4+ T cells were purified by magnetic bead sorting. The cells were then incubated with tetramers loaded with peptides of SEQ ID18, SEQ ID19 or SEQ ID20. Analysis by facs identifies a significant proportion of NKT cells (.+-.10%) labeled by tetramers.
[0180] Monoclonal human antibodies of the IgG4 isotype were derived from the peripheral blood B lymphocytes by transformation with the Epstein-Barr virus. The genomic sequence of such antibodies was obtained from transformed B cells. A viral vector containing the corresponding cDNA sequence was constructed and used for transfection of CHO cells. All these methods are known in the art (see for instance, Jacquemin et al Blood 92: 496-506, 1998).
[0181] The hydrophobic aminoacid residues located in position 1 in the peptides of SEQ ID18 and SEQ ID20 were mutated to a serine and the mutated antibody produced by transfected CHO cells.
[0182] Peripheral blood CD4+ T cells obtained as above were exposed in culture medium to human dendritic cells (derived from human peripheral blood monocytes by methods known in the art) and loaded with either the antibody in natural configuration (SEQ ID21) or its mutated counterpart (SEQ ID22). After culturing the cells with CD4+ T cells for 5 to 7 days, the population of CD4+ T cells activated by either natural or mutated antibody was evaluated. CD4+ NKT cells were separated from CD4+ T cells using an antibody to NKG2D, a surface marker associated with NK or NKT cells only.
[0183] It was observed that CD4+ T cells and NKT cells were activated when the antibody in natural sequence was used (SEQ ID21), while the mutated form of the antibody (SEQ ID22) only activate class II restricted CD4+ T cells and not NKT cells.
[0184] It was concluded that human IgG antibodies contained epitopes corresponding to the [FWTHY]-X.sub.2X.sub.3-[ILMV]-X.sub.5X.sub.6-[FWTHY] motif, having the capacity to be recognized by and to activate NKT cells. Further, mutation of key hydrophobic aminoacid residues within such motif was sufficient to prevent activation of NKT cells.
[0185] It should be understood that the examples provided here are not exhaustive and that combinations of proteins or peptides containing various numbers of aminoacid substitutions or deletions can be envisioned. For instance, in example 1, various combinations of substitution of hydrophobic aminoacids can be delineated.
TABLE-US-00001 Sequence listings Factor VIII aminoacids 309-315 (human) SEQ ID1 FCHISSH Factor VIII aminoacids 1816-1822 (human) SEQ ID2 FWKVQHH Factor VIII aminoacids 1918-1924 (human) SEQ ID3 FHAINGY Factor A1 domain (mutations F309S and H315S underlined) (human) SEQ ID4 1 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPF 41 NTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEV 81 YDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ 121 REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLS 161 HVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFA 201 VFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNR 241 SLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRN 281 HRQASLEISPITFLTAQTLLMDLGQFLLSCHISSSQHDGM 321 EAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF 361 DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVL 401 APDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTRE 441 AIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGI 481 TDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDG 521 PTKSDPRCLTRYYSSFVNMERDLASG Factor VIII A3 domain (mutations F1816S and H18225 underlined) (human) SEQ ID5 1637 SQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKED 1677 FDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPH 1717 VLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL 1757 GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQR 1798 QGAEPRKNFVKPNETKTYSWKVQHSMAPTKDEFDCKAW 1836 AYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQE 1876 FALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKEN 1916 YRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHS 1956 IHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGI 1996 WRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDF 2036 QITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDL 2076 LAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTY 2116 RGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTH 2156 YSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSY 2196 FTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQ 2236 KTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFF 2276 QNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH 2316 QIALRMEVLGCEAQDLY* Factor VIII (mutations F309S, H315S, F1816S and F1918S underlined) (human) SEQ ID6 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHA VGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASD PLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFA VFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL MDLGQFLLSCHISSSQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDL TDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVL APDDRSYKSQYLNNGPQRIGRKYKKVREMAYTDETEKTREAIQUESGILG PLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKD FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSEVNMERDLASGLIGP LLICYKESVDQRGNQIMSDKRNVILFSVEDENRSWYLTENIQRFLPNPAG VQLEDPEFQASNIMHSINGYVEDSLQLSVCLHEVAYWYILSIGAQTDFLS VFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNR GMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPS TRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTP HGLSLSDLQEAKYETESDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFT PESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDN TSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLES GLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKT NKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRM LMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKML FLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKV VVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEK KETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYEGAYAPVLQD FRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPN TSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPS TLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIR PIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTL EMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHI YQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVA TESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILS LNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREI TRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFI AAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRG ELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGA EPRKNFVKPNETKTYSWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSG LIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCR APCNIQMEDPTFKENYRSHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSN ENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVEC LIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKL ARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQ FIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIR LHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMF ATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKS LLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPP LLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY*GWPLQHLPLPSPLPPQL QGSVPPWLAFYLCAKS*QTLP*SLL Hexon, Human adenovirus 5, (CD1d binding motifs underlined): (virus) SEQ ID7 MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTHDV TTDRSQRLTLRFIPVDREDTA YSYKARFTLAVGDNRVLDMASTSFDIRGVLDRGPTFKPYSGTAYNALAPKGAPNPCE WDEAATALEINLEEEDDDNE DEVDEQAEQQKTHVFGQAPYSGINITKEGIQIGVEGQTPKYADKTFQPEPQIGESQWY ETEINHAAGRVLKKTTPMK PCYGSYAKPTNENGGQGILVKQQNGKLESQVEMQFFSTTEAAAGNGDNLTPKVVLY SEDVDIETPDTHISYMPTIKE GNSRELMGQQSMPNRPNYIAFRDNFIGLMYYNSTGNMGVLAGQASQLNAVVDLQD RNTELSYQLLLDSIGDRTRYFS MWKQAVDSYDPDVRIIENHGTEDELPNYCFPLGGVINTETLTKVKPKTGQENGWEK DATEFSDKNEIRVGNNFAMEI NLNANLWRNFLYSNIALYLPDKLKYSPSNVKISDNPNTYDYMNKRVVAPGLVDCYI NLGARWSLDYMDNVNPFNHHR NAGLRYRSMLLGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSS LGNDLRVDGASIKFDSICLY ATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNWA AFRGWAFTRLKTKETPSLGS GYDPYYTYSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDG EGYNVAQCNMTKDWFLVQM LANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDYQQVGILHQHNNS GFVGYLAPTMREGQAYPANFP YPLIGKTAVDSITQKKELCDRTLWRIPFSSNFMSMGALTDLGQNLLYANSAHALDMT FEVDPMDEPTLLYVLFEVFD VVRVHRPHRGVIETVYLRTPFSAGNATT Hexon, Human adenovirus 5 (mutations of P1 anchoring residue underlined): (virus) SEQ ID8 MATPSMNIPQWSYMHISGQDASEYLSPGLVQFARATETSFSLNNKFRNPTVAPTHDV TTDRSQRLTLRFIPVDREDTA YSYKARFTLAVGDNRVLDMASTSFDIRGVLDRGPTEKPYSGTASNALAPKGAPNPCE WDEAATALEINLEEEDDDNE DEVDEQAEQQKTHVFGQAPSSGINITKEGIQIGVEGQTPKYADKTFQPEPQIGESQWY ETEINHAAGRVLKKTTPMK
PCYGSYAKPTNENGGQGILVKQQNGKLESQVEMQFFSTTEAAAGNGDNLTPKVVLY SEDVDIETPDTHISYMPTIKE GNSRELMGQQSMPNRPNYIAFRDNSIGLMYYNSTGNMGVLAGQASQLNAVVDLQD RNTELSYQLLLDSIGDRTRYFS MWKQAVDSYDPDVRIIENHGTEDELPNYCFPLGGVINTETLTKVKPKTGQENGWEK DATEFSDKNEIRVGNNFAMEI NLNANLWRNFLSSNIALYLPDKLKYSPSNVKISDNPNTYDYMNKRVVAPGLVDCYIN LGARWSLDYMDNVNPFNHHR NAGLRSRSMLLGNGRYVPFSIQVPQKSFAIKNLLLLPGSYTYEWNFRKDVNMVLQSS LGNDLRVDGASIKSDSICLY ATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNWA AFRGWASTRLKTKETPSLGS GYDPYYTYSGSIPYLDGTFYLNHTSKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDG EGYNVAQCNMTKDSFLVQM LANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDSQQVGILHQHNNS GFVGYLAPTMREGQAYPANFP SPLIGKTAVDSITQKKFLCDRTLWRIPFSSNSMSMGALTDLGQNLLYANSAHALDMT FEVDPMDEPTLLYVLFEVSD VVRVHRPSRGVIETVYLRTPFSAGNATT Mal d 1, malus domesticus, aminoacids 144-150 SEQ ID9 FKLIESY Mal d 1, malus domesticus, F144S and Y150S mutations underlined (vegetal) SEQ ID10 SKLIESS Alpha-Gliadin (CD1d binding motif underlined) SEQ ID11 MVRVPVPQLQPQNPSQQQPQEQVPLVQQQQFPGQQQPFPPQQPYPQPQPFPSQQPYL QLQPFPQPQLPYPQPQLPY PQPQLPYPQPQPFRPQQPYPQSQPQYSQPQQPISQQQQQQQQQQQQKQQQQQQQQIL QQILQQQLIPCRDVVLQQH SIAYGSSQVLQQSTYQLVQQLCCQQLWQIPEQSRCQAIHNVVHAIILHQQQQQQQQQ QQQPLSQVSFQQPQQQYPS GQGSFQPSQQNPQAQGSVQPQQLPQFEEIRNLALETLPAMCNVYIPPYCTIAPVGIFGT NYR Alpha-Gliadin (mutation underlined) SEQ ID12 MVRVPVPQLQPQNPSQQQPQEQVPLVQQQQFPGQQQPFPPQQPYPQPQPFPSQQPSL QLQPFPQPQLPYPQPQLPY PQPQLPYPQPQPFRPQQPYPQSQPQYSQPQQPISQQQQQQQQQQQQKQQQQQQQQIL QQILQQQLIPCRDVVLQQH SIAYGSSQVLQQSTSQLVQQLCCQQLWQIPEQSRCQAISNVVHAIILHQQQQQQQQQ QQQPLSQVSFQQPQQQYPS GQGSFQPSQQNPQAQGSVQPQQLPQSEEIRNLALETLPAMCNVYIPPSCTIAPVGIFGT NYR D. pteronyssinus Der p 1, aminoacids 38-44 (pyroglyphidae, Dermatophagoides pteronyssinus, European house dust mite) SEQ ID13 FSGVAAT D. pteronyssinus Der p 1, aminoacids 135-141 (pyroglyphidae, Dermatophagoides pteronyssinus, European house dust mite) SEQ ID14 HSAIAAVI D. pteronyssinus Der p 1, aminoacids 216-222 (pyroglyphidae, Dermatophagoides pteronyssinus, European house dust mite) SEQ ID15 YPYVVIL Mature Der p 1 (mutations of P1 anchoring residues F38S, H135S and Y216S underlined) (pyroglyphidae, Dermatophagoides pteronyssinus, European house dust mite) SEQ ID16 ETNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWASSGVAATESAYLAYRNQSLD LAEQELVDCASQHGCHGDTI PRGIEYIQHNGVVQESYYRYVAREQSCRRPNAQRFGISNYCQTYPPNVNKIREALAQT SSAIAVIIGIKDLDAFRHY DGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAANI DLMMIEESPYVVIL Mature Der p 1 (CD1d epitopes underlined): (pyroglyphidae, Dermatophagoides pteronyssinus, European house dust mite) SEQ ID17 ETNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLD LAEQELVDCASQHGCHGDTI PRGIEYIQHNGVVQESYYRYVAREQSCRRPNAQRFGISNYCQTYPPNVNKIREALAQT HSAIAVIIGIKDLDAFRHY DGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAANI DLMMIEEYPYVVIL IgG antibody, CH2 domain of IgG1 and IgG4 (human) SEQ ID18 YRVVSVL IgG antibody, CH2 domain of IgG2 and IgG3 (human) SEQ ID19 FRVVSVL IgG antibody, CH3 domain of IgGl, IgG2 and IgG4 (human) SEQ ID20 HEALHNH Human IgG4 FC fragment (CD1d epitopes underlined) (human) SEQ ID21 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSL GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSLGK Human IgG4 FC fragment (mutated aminoacids underlined) (human) SEQ ID22 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSL GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTSRVVSVLTVLHQDWLNGKEYKCKVSNKG LPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFS CSVMSEALHNHYTQKSLSLSLGK
Sequence CWU
1
1
2317PRTHomo sapiens 1Phe Cys His Ile Ser Ser His1 527PRTHomo
sapiens 2Phe Trp Lys Val Gln His His1 537PRTHomo sapiens
3Phe His Ala Ile Asn Gly Tyr1 54546PRTHomo sapiens 4Ala Thr
Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5
10 15Met Gln Ser Asp Leu Gly Glu Leu
Pro Val Asp Ala Arg Phe Pro Pro 20 25
30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys
Lys 35 40 45Thr Leu Phe Val Glu
Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro 50 55
60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala
Glu Val65 70 75 80Tyr
Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val
85 90 95Ser Leu His Ala Val Gly Val
Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105
110Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp
Lys Val 115 120 125Phe Pro Gly Gly
Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130
135 140Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr
Ser Tyr Leu Ser145 150 155
160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu
165 170 175Leu Val Cys Arg Glu
Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180
185 190His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu
Gly Lys Ser Trp 195 200 205His Ser
Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210
215 220Ala Arg Ala Trp Pro Lys Met His Thr Val Asn
Gly Tyr Val Asn Arg225 230 235
240Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His
245 250 255Val Ile Gly Met
Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260
265 270Gly His Thr Phe Leu Val Arg Asn His Arg Gln
Ala Ser Leu Glu Ile 275 280 285Ser
Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290
295 300Gln Phe Leu Leu Ser Cys His Ile Ser Ser
Ser Gln His Asp Gly Met305 310 315
320Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu
Arg 325 330 335Met Lys Asn
Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340
345 350Ser Glu Met Asp Val Val Arg Phe Asp Asp
Asp Asn Ser Pro Ser Phe 355 360
365Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370
375 380Tyr Ile Ala Ala Glu Glu Glu Asp
Trp Asp Tyr Ala Pro Leu Val Leu385 390
395 400Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu
Asn Asn Gly Pro 405 410
415Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr
420 425 430Asp Glu Thr Phe Lys Thr
Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440
445Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu
Ile Ile 450 455 460Phe Lys Asn Gln Ala
Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile465 470
475 480Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg
Leu Pro Lys Gly Val Lys 485 490
495His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys
500 505 510Trp Thr Val Thr Val
Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515
520 525Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu
Arg Asp Leu Ala 530 535 540Ser
Gly5455696PRTHomo sapiens 5Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln
Arg Glu Ile Thr Arg1 5 10
15Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile
20 25 30Ser Val Glu Met Lys Lys Glu
Asp Phe Asp Ile Tyr Asp Glu Asp Glu 35 40
45Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe
Ile 50 55 60Ala Ala Val Glu Arg Leu
Trp Asp Tyr Gly Met Ser Ser Ser Pro His65 70
75 80Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val
Pro Gln Phe Lys Lys 85 90
95Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr
100 105 110Arg Gly Glu Leu Asn Glu
His Leu Gly Leu Leu Gly Pro Tyr Ile Arg 115 120
125Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln
Ala Ser 130 135 140Arg Pro Tyr Ser Phe
Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln145 150
155 160Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe
Val Lys Pro Asn Glu Thr 165 170
175Lys Thr Tyr Ser Trp Lys Val Gln His Ser Met Ala Pro Thr Lys Asp
180 185 190Glu Phe Asp Cys Lys
Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu 195
200 205Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu
Val Cys His Thr 210 215 220Asn Thr Leu
Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe225
230 235 240Ala Leu Phe Phe Thr Ile Phe
Asp Glu Thr Lys Ser Trp Tyr Phe Thr 245
250 255Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn
Ile Gln Met Glu 260 265 270Asp
Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr 275
280 285Ile Met Asp Thr Leu Pro Gly Leu Val
Met Ala Gln Asp Gln Arg Ile 290 295
300Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile305
310 315 320His Phe Ser Gly
His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys 325
330 335Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val
Phe Glu Thr Val Glu Met 340 345
350Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu
355 360 365His Leu His Ala Gly Met Ser
Thr Leu Phe Leu Val Tyr Ser Asn Lys 370 375
380Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe
Gln385 390 395 400Ile Thr
Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg
405 410 415Leu His Tyr Ser Gly Ser Ile
Asn Ala Trp Ser Thr Lys Glu Pro Phe 420 425
430Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His
Gly Ile 435 440 445Lys Thr Gln Gly
Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln 450
455 460Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp
Gln Thr Tyr Arg465 470 475
480Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser
485 490 495Ser Gly Ile Lys His
Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr 500
505 510Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser
Thr Leu Arg Met 515 520 525Glu Leu
Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met 530
535 540Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr
Ala Ser Ser Tyr Phe545 550 555
560Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu
565 570 575Gln Gly Arg Ser
Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu 580
585 590Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys
Val Thr Gly Val Thr 595 600 605Thr
Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe 610
615 620Leu Ile Ser Ser Ser Gln Asp Gly His Gln
Trp Thr Leu Phe Phe Gln625 630 635
640Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr
Pro 645 650 655Val Val Asn
Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile 660
665 670His Pro Gln Ser Trp Val His Gln Ile Ala
Leu Arg Met Glu Val Leu 675 680
685Gly Cys Glu Ala Gln Asp Leu Tyr 690 69562372PRTHomo
sapiens 6Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1
5 10 15Met Gln Ser Asp
Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20
25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser
Val Val Tyr Lys Lys 35 40 45Thr
Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro 50
55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro
Thr Ile Gln Ala Glu Val65 70 75
80Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro
Val 85 90 95Ser Leu His
Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100
105 110Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu
Lys Glu Asp Asp Lys Val 115 120
125Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130
135 140Gly Pro Met Ala Ser Asp Pro Leu
Cys Leu Thr Tyr Ser Tyr Leu Ser145 150
155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu
Ile Gly Ala Leu 165 170
175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu
180 185 190His Lys Phe Ile Leu Leu
Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200
205His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala
Ala Ser 210 215 220Ala Arg Ala Trp Pro
Lys Met His Thr Val Asn Gly Tyr Val Asn Arg225 230
235 240Ser Leu Pro Gly Leu Ile Gly Cys His Arg
Lys Ser Val Tyr Trp His 245 250
255Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu
260 265 270Gly His Thr Phe Leu
Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 275
280 285Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu
Met Asp Leu Gly 290 295 300Gln Phe Leu
Leu Ser Cys His Ile Ser Ser Ser Gln His Asp Gly Met305
310 315 320Glu Ala Tyr Val Lys Val Asp
Ser Cys Pro Glu Glu Pro Gln Leu Arg 325
330 335Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp
Asp Leu Thr Asp 340 345 350Ser
Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355
360 365Ile Gln Ile Arg Ser Val Ala Lys Lys
His Pro Lys Thr Trp Val His 370 375
380Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu385
390 395 400Ala Pro Asp Asp
Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405
410 415Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val
Arg Phe Met Ala Tyr Thr 420 425
430Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile
435 440 445Leu Gly Pro Leu Leu Tyr Gly
Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455
460Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly
Ile465 470 475 480Thr Asp
Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys
485 490 495His Leu Lys Asp Phe Pro Ile
Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505
510Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro
Arg Cys 515 520 525Leu Thr Arg Tyr
Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530
535 540Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys
Glu Ser Val Asp545 550 555
560Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe
565 570 575Ser Val Phe Asp Glu
Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580
585 590Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu
Asp Pro Glu Phe 595 600 605Gln Ala
Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610
615 620Leu Gln Leu Ser Val Cys Leu His Glu Val Ala
Tyr Trp Tyr Ile Leu625 630 635
640Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr
645 650 655Thr Phe Lys His
Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660
665 670Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu
Asn Pro Gly Leu Trp 675 680 685Ile
Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690
695 700Leu Leu Lys Val Ser Ser Cys Asp Lys Asn
Thr Gly Asp Tyr Tyr Glu705 710 715
720Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn
Ala 725 730 735Ile Glu Pro
Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg 740
745 750Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro
Glu Asn Asp Ile Glu Lys 755 760
765Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn 770
775 780Val Ser Ser Ser Asp Leu Leu Met
Leu Leu Arg Gln Ser Pro Thr Pro785 790
795 800His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys
Tyr Glu Thr Phe 805 810
815Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser
820 825 830Glu Met Thr His Phe Arg
Pro Gln Leu His His Ser Gly Asp Met Val 835 840
845Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys
Leu Gly 850 855 860Thr Thr Ala Ala Thr
Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser865 870
875 880Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro
Ser Asp Asn Leu Ala Ala 885 890
895Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His
900 905 910Tyr Asp Ser Gln Leu
Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 915
920 925Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu
Glu Asn Asn Asp 930 935 940Ser Lys Leu
Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp945
950 955 960Gly Lys Asn Val Ser Ser Thr
Glu Ser Gly Arg Leu Phe Lys Gly Lys 965
970 975Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn
Ala Leu Phe Lys 980 985 990Val
Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala 995
1000 1005Thr Asn Arg Lys Thr His Ile Asp
Gly Pro Ser Leu Leu Ile Glu 1010 1015
1020Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu
1025 1030 1035Phe Lys Lys Val Thr Pro
Leu Ile His Asp Arg Met Leu Met Asp 1040 1045
1050Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys
Thr 1055 1060 1065Thr Ser Ser Lys Asn
Met Glu Met Val Gln Gln Lys Lys Glu Gly 1070 1075
1080Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe
Phe Lys 1085 1090 1095Met Leu Phe Leu
Pro Glu Ser Ala Arg Trp Ile Gln Arg Thr His 1100
1105 1110Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro
Ser Pro Lys Gln 1115 1120 1125Leu Val
Ser Leu Gly Pro Glu Lys Ser Val Glu Gly Gln Asn Phe 1130
1135 1140Leu Ser Glu Lys Asn Lys Val Val Val Gly
Lys Gly Glu Phe Thr 1145 1150 1155Lys
Asp Val Gly Leu Lys Glu Met Val Phe Pro Ser Ser Arg Asn 1160
1165 1170Leu Phe Leu Thr Asn Leu Asp Asn Leu
His Glu Asn Asn Thr His 1175 1180
1185Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys Lys Glu Thr
1190 1195 1200Leu Ile Gln Glu Asn Val
Val Leu Pro Gln Ile His Thr Val Thr 1205 1210
1215Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr
Arg 1220 1225 1230Gln Asn Val Glu Gly
Ser Tyr Glu Gly Ala Tyr Ala Pro Val Leu 1235 1240
1245Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr
Lys Lys 1250 1255 1260His Thr Ala His
Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu 1265
1270 1275Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu
Lys Tyr Ala Cys 1280 1285 1290Thr Thr
Arg Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr 1295
1300 1305Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe
Arg Leu Pro Leu Glu 1310 1315 1320Glu
Thr Glu Leu Glu Lys Arg Ile Ile Val Asp Asp Thr Ser Thr 1325
1330 1335Gln Trp Ser Lys Asn Met Lys His Leu
Thr Pro Ser Thr Leu Thr 1340 1345
1350Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln Ser
1355 1360 1365Pro Leu Ser Asp Cys Leu
Thr Arg Ser His Ser Ile Pro Gln Ala 1370 1375
1380Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe Pro
Ser 1385 1390 1395Ile Arg Pro Ile Tyr
Leu Thr Arg Val Leu Phe Gln Asp Asn Ser 1400 1405
1410Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys Asp Ser
Gly Val 1415 1420 1425Gln Glu Ser Ser
His Phe Leu Gln Gly Ala Lys Lys Asn Asn Leu 1430
1435 1440Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly
Asp Gln Arg Glu 1445 1450 1455Val Gly
Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr Lys 1460
1465 1470Lys Val Glu Asn Thr Val Leu Pro Lys Pro
Asp Leu Pro Lys Thr 1475 1480 1485Ser
Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys 1490
1495 1500Asp Leu Phe Pro Thr Glu Thr Ser Asn
Gly Ser Pro Gly His Leu 1505 1510
1515Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile
1520 1525 1530Lys Trp Asn Glu Ala Asn
Arg Pro Gly Lys Val Pro Phe Leu Arg 1535 1540
1545Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu
Asp 1550 1555 1560Pro Leu Ala Trp Asp
Asn His Tyr Gly Thr Gln Ile Pro Lys Glu 1565 1570
1575Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys Thr Ala
Phe Lys 1580 1585 1590Lys Lys Asp Thr
Ile Leu Ser Leu Asn Ala Cys Glu Ser Asn His 1595
1600 1605Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys
Pro Glu Ile Glu 1610 1615 1620Val Thr
Trp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys Ser Gln 1625
1630 1635Asn Pro Pro Val Leu Lys Arg His Gln Arg
Glu Ile Thr Arg Thr 1640 1645 1650Thr
Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile 1655
1660 1665Ser Val Glu Met Lys Lys Glu Asp Phe
Asp Ile Tyr Asp Glu Asp 1670 1675
1680Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr
1685 1690 1695Phe Ile Ala Ala Val Glu
Arg Leu Trp Asp Tyr Gly Met Ser Ser 1700 1705
1710Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val
Pro 1715 1720 1725Gln Phe Lys Lys Val
Val Phe Gln Glu Phe Thr Asp Gly Ser Phe 1730 1735
1740Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu
Gly Leu 1745 1750 1755Leu Gly Pro Tyr
Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val 1760
1765 1770Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser
Phe Tyr Ser Ser 1775 1780 1785Leu Ile
Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 1790
1795 1800Lys Asn Phe Val Lys Pro Asn Glu Thr Lys
Thr Tyr Ser Trp Lys 1805 1810 1815Val
Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys 1820
1825 1830Ala Trp Ala Tyr Phe Ser Asp Val Asp
Leu Glu Lys Asp Val His 1835 1840
1845Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu
1850 1855 1860Asn Pro Ala His Gly Arg
Gln Val Thr Val Gln Glu Phe Ala Leu 1865 1870
1875Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr
Glu 1880 1885 1890Asn Met Glu Arg Asn
Cys Arg Ala Pro Cys Asn Ile Gln Met Glu 1895 1900
1905Asp Pro Thr Phe Lys Glu Asn Tyr Arg Ser His Ala Ile
Asn Gly 1910 1915 1920Tyr Ile Met Asp
Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln 1925
1930 1935Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser
Asn Glu Asn Ile 1940 1945 1950His Ser
Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 1955
1960 1965Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu
Tyr Pro Gly Val Phe 1970 1975 1980Glu
Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val 1985
1990 1995Glu Cys Leu Ile Gly Glu His Leu His
Ala Gly Met Ser Thr Leu 2000 2005
2010Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala
2015 2020 2025Ser Gly His Ile Arg Asp
Phe Gln Ile Thr Ala Ser Gly Gln Tyr 2030 2035
2040Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly
Ser 2045 2050 2055Ile Asn Ala Trp Ser
Thr Lys Glu Pro Phe Ser Trp Ile Lys Val 2060 2065
2070Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr
Gln Gly 2075 2080 2085Ala Arg Gln Lys
Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile 2090
2095 2100Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr
Tyr Arg Gly Asn 2105 2110 2115Ser Thr
Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser 2120
2125 2130Gly Ile Lys His Asn Ile Phe Asn Pro Pro
Ile Ile Ala Arg Tyr 2135 2140 2145Ile
Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg 2150
2155 2160Met Glu Leu Met Gly Cys Asp Leu Asn
Ser Cys Ser Met Pro Leu 2165 2170
2175Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser
2180 2185 2190Ser Tyr Phe Thr Asn Met
Phe Ala Thr Trp Ser Pro Ser Lys Ala 2195 2200
2205Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln
Val 2210 2215 2220Asn Asn Pro Lys Glu
Trp Leu Gln Val Asp Phe Gln Lys Thr Met 2225 2230
2235Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu
Leu Thr 2240 2245 2250Ser Met Tyr Val
Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly 2255
2260 2265His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys
Val Lys Val Phe 2270 2275 2280Gln Gly
Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp 2285
2290 2295Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile
His Pro Gln Ser Trp 2300 2305 2310Val
His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala 2315
2320 2325Gln Asp Leu Tyr Gly Trp Pro Leu Gln
His Leu Pro Leu Pro Ser 2330 2335
2340Pro Leu Pro Pro Gln Leu Gln Gly Ser Val Pro Pro Trp Leu Ala
2345 2350 2355Phe Tyr Leu Cys Ala Lys
Ser Gln Thr Leu Pro Ser Leu Leu 2360 2365
23707952PRTHuman adenovirus type 5 7Met Ala Thr Pro Ser Met Met Pro
Gln Trp Ser Tyr Met His Ile Ser1 5 10
15Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln
Phe Ala 20 25 30Arg Ala Thr
Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35
40 45Thr Val Ala Pro Thr His Asp Val Thr Thr Asp
Arg Ser Gln Arg Leu 50 55 60Thr Leu
Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr65
70 75 80Lys Ala Arg Phe Thr Leu Ala
Val Gly Asp Asn Arg Val Leu Asp Met 85 90
95Ala Ser Thr Ser Phe Asp Ile Arg Gly Val Leu Asp Arg
Gly Pro Thr 100 105 110Phe Lys
Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115
120 125Ala Pro Asn Pro Cys Glu Trp Asp Glu Ala
Ala Thr Ala Leu Glu Ile 130 135 140Asn
Leu Glu Glu Glu Asp Asp Asp Asn Glu Asp Glu Val Asp Glu Gln145
150 155 160Ala Glu Gln Gln Lys Thr
His Val Phe Gly Gln Ala Pro Tyr Ser Gly 165
170 175Ile Asn Ile Thr Lys Glu Gly Ile Gln Ile Gly Val
Glu Gly Gln Thr 180 185 190Pro
Lys Tyr Ala Asp Lys Thr Phe Gln Pro Glu Pro Gln Ile Gly Glu 195
200 205Ser Gln Trp Tyr Glu Thr Glu Ile Asn
His Ala Ala Gly Arg Val Leu 210 215
220Lys Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Lys Pro225
230 235 240Thr Asn Glu Asn
Gly Gly Gln Gly Ile Leu Val Lys Gln Gln Asn Gly 245
250 255Lys Leu Glu Ser Gln Val Glu Met Gln Phe
Phe Ser Thr Thr Glu Ala 260 265
270Ala Ala Gly Asn Gly Asp Asn Leu Thr Pro Lys Val Val Leu Tyr Ser
275 280 285Glu Asp Val Asp Ile Glu Thr
Pro Asp Thr His Ile Ser Tyr Met Pro 290 295
300Thr Ile Lys Glu Gly Asn Ser Arg Glu Leu Met Gly Gln Gln Ser
Met305 310 315 320Pro Asn
Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu
325 330 335Met Tyr Tyr Asn Ser Thr Gly
Asn Met Gly Val Leu Ala Gly Gln Ala 340 345
350Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr
Glu Leu 355 360 365Ser Tyr Gln Leu
Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe 370
375 380Ser Met Trp Lys Gln Ala Val Asp Ser Tyr Asp Pro
Asp Val Arg Ile385 390 395
400Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro
405 410 415Leu Gly Gly Val Ile
Asn Thr Glu Thr Leu Thr Lys Val Lys Pro Lys 420
425 430Thr Gly Gln Glu Asn Gly Trp Glu Lys Asp Ala Thr
Glu Phe Ser Asp 435 440 445Lys Asn
Glu Ile Arg Val Gly Asn Asn Phe Ala Met Glu Ile Asn Leu 450
455 460Asn Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser
Asn Ile Ala Leu Tyr465 470 475
480Leu Pro Asp Lys Leu Lys Tyr Ser Pro Ser Asn Val Lys Ile Ser Asp
485 490 495Asn Pro Asn Thr
Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro Gly 500
505 510Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg
Trp Ser Leu Asp Tyr 515 520 525Met
Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala Gly Leu Arg 530
535 540Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg
Tyr Val Pro Phe His Ile545 550 555
560Gln Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu
Pro 565 570 575Gly Ser Tyr
Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met Val 580
585 590Leu Gln Ser Ser Leu Gly Asn Asp Leu Arg
Val Asp Gly Ala Ser Ile 595 600
605Lys Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro Met Ala His 610
615 620Asn Thr Ala Ser Thr Leu Glu Ala
Met Leu Arg Asn Asp Thr Asn Asp625 630
635 640Gln Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met
Leu Tyr Pro Ile 645 650
655Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser Arg Asn Trp
660 665 670Ala Ala Phe Arg Gly Trp
Ala Phe Thr Arg Leu Lys Thr Lys Glu Thr 675 680
685Pro Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr Ser
Gly Ser 690 695 700Ile Pro Tyr Leu Asp
Gly Thr Phe Tyr Leu Asn His Thr Phe Lys Lys705 710
715 720Val Ala Ile Thr Phe Asp Ser Ser Val Ser
Trp Pro Gly Asn Asp Arg 725 730
735Leu Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val Asp Gly Glu
740 745 750Gly Tyr Asn Val Ala
Gln Cys Asn Met Thr Lys Asp Trp Phe Leu Val 755
760 765Gln Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly
Phe Tyr Ile Pro 770 775 780Glu Ser Tyr
Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro785
790 795 800Met Ser Arg Gln Val Val Asp
Asp Thr Lys Tyr Lys Asp Tyr Gln Gln 805
810 815Val Gly Ile Leu His Gln His Asn Asn Ser Gly Phe
Val Gly Tyr Leu 820 825 830Ala
Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn Phe Pro Tyr 835
840 845Pro Leu Ile Gly Lys Thr Ala Val Asp
Ser Ile Thr Gln Lys Lys Phe 850 855
860Leu Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser Asn Phe Met865
870 875 880Ser Met Gly Ala
Leu Thr Asp Leu Gly Gln Asn Leu Leu Tyr Ala Asn 885
890 895Ser Ala His Ala Leu Asp Met Thr Phe Glu
Val Asp Pro Met Asp Glu 900 905
910Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg Val
915 920 925His Arg Pro His Arg Gly Val
Ile Glu Thr Val Tyr Leu Arg Thr Pro 930 935
940Phe Ser Ala Gly Asn Ala Thr Thr945
9508952PRTHuman adenovirus type 5 8Met Ala Thr Pro Ser Met Met Pro Gln
Trp Ser Tyr Met His Ile Ser1 5 10
15Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe
Ala 20 25 30Arg Ala Thr Glu
Thr Ser Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35
40 45Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg
Ser Gln Arg Leu 50 55 60Thr Leu Arg
Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr65 70
75 80Lys Ala Arg Phe Thr Leu Ala Val
Gly Asp Asn Arg Val Leu Asp Met 85 90
95Ala Ser Thr Ser Phe Asp Ile Arg Gly Val Leu Asp Arg Gly
Pro Thr 100 105 110Phe Lys Pro
Tyr Ser Gly Thr Ala Ser Asn Ala Leu Ala Pro Lys Gly 115
120 125Ala Pro Asn Pro Cys Glu Trp Asp Glu Ala Ala
Thr Ala Leu Glu Ile 130 135 140Asn Leu
Glu Glu Glu Asp Asp Asp Asn Glu Asp Glu Val Asp Glu Gln145
150 155 160Ala Glu Gln Gln Lys Thr His
Val Phe Gly Gln Ala Pro Ser Ser Gly 165
170 175Ile Asn Ile Thr Lys Glu Gly Ile Gln Ile Gly Val
Glu Gly Gln Thr 180 185 190Pro
Lys Tyr Ala Asp Lys Thr Phe Gln Pro Glu Pro Gln Ile Gly Glu 195
200 205Ser Gln Trp Tyr Glu Thr Glu Ile Asn
His Ala Ala Gly Arg Val Leu 210 215
220Lys Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Lys Pro225
230 235 240Thr Asn Glu Asn
Gly Gly Gln Gly Ile Leu Val Lys Gln Gln Asn Gly 245
250 255Lys Leu Glu Ser Gln Val Glu Met Gln Phe
Phe Ser Thr Thr Glu Ala 260 265
270Ala Ala Gly Asn Gly Asp Asn Leu Thr Pro Lys Val Val Leu Tyr Ser
275 280 285Glu Asp Val Asp Ile Glu Thr
Pro Asp Thr His Ile Ser Tyr Met Pro 290 295
300Thr Ile Lys Glu Gly Asn Ser Arg Glu Leu Met Gly Gln Gln Ser
Met305 310 315 320Pro Asn
Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Ser Ile Gly Leu
325 330 335Met Tyr Tyr Asn Ser Thr Gly
Asn Met Gly Val Leu Ala Gly Gln Ala 340 345
350Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr
Glu Leu 355 360 365Ser Tyr Gln Leu
Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe 370
375 380Ser Met Trp Lys Gln Ala Val Asp Ser Tyr Asp Pro
Asp Val Arg Ile385 390 395
400Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro
405 410 415Leu Gly Gly Val Ile
Asn Thr Glu Thr Leu Thr Lys Val Lys Pro Lys 420
425 430Thr Gly Gln Glu Asn Gly Trp Glu Lys Asp Ala Thr
Glu Phe Ser Asp 435 440 445Lys Asn
Glu Ile Arg Val Gly Asn Asn Phe Ala Met Glu Ile Asn Leu 450
455 460Asn Ala Asn Leu Trp Arg Asn Phe Leu Ser Ser
Asn Ile Ala Leu Tyr465 470 475
480Leu Pro Asp Lys Leu Lys Tyr Ser Pro Ser Asn Val Lys Ile Ser Asp
485 490 495Asn Pro Asn Thr
Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro Gly 500
505 510Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg
Trp Ser Leu Asp Tyr 515 520 525Met
Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala Gly Leu Arg 530
535 540Ser Arg Ser Met Leu Leu Gly Asn Gly Arg
Tyr Val Pro Phe Ser Ile545 550 555
560Gln Val Pro Gln Lys Ser Phe Ala Ile Lys Asn Leu Leu Leu Leu
Pro 565 570 575Gly Ser Tyr
Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met Val 580
585 590Leu Gln Ser Ser Leu Gly Asn Asp Leu Arg
Val Asp Gly Ala Ser Ile 595 600
605Lys Ser Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro Met Ala His 610
615 620Asn Thr Ala Ser Thr Leu Glu Ala
Met Leu Arg Asn Asp Thr Asn Asp625 630
635 640Gln Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met
Leu Tyr Pro Ile 645 650
655Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser Arg Asn Trp
660 665 670Ala Ala Phe Arg Gly Trp
Ala Ser Thr Arg Leu Lys Thr Lys Glu Thr 675 680
685Pro Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr Ser
Gly Ser 690 695 700Ile Pro Tyr Leu Asp
Gly Thr Phe Tyr Leu Asn His Thr Ser Lys Lys705 710
715 720Val Ala Ile Thr Phe Asp Ser Ser Val Ser
Trp Pro Gly Asn Asp Arg 725 730
735Leu Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val Asp Gly Glu
740 745 750Gly Tyr Asn Val Ala
Gln Cys Asn Met Thr Lys Asp Ser Phe Leu Val 755
760 765Gln Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly
Phe Tyr Ile Pro 770 775 780Glu Ser Tyr
Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro785
790 795 800Met Ser Arg Gln Val Val Asp
Asp Thr Lys Tyr Lys Asp Ser Gln Gln 805
810 815Val Gly Ile Leu His Gln His Asn Asn Ser Gly Phe
Val Gly Tyr Leu 820 825 830Ala
Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn Phe Pro Ser 835
840 845Pro Leu Ile Gly Lys Thr Ala Val Asp
Ser Ile Thr Gln Lys Lys Phe 850 855
860Leu Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser Asn Ser Met865
870 875 880Ser Met Gly Ala
Leu Thr Asp Leu Gly Gln Asn Leu Leu Tyr Ala Asn 885
890 895Ser Ala His Ala Leu Asp Met Thr Phe Glu
Val Asp Pro Met Asp Glu 900 905
910Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Ser Asp Val Val Arg Val
915 920 925His Arg Pro Ser Arg Gly Val
Ile Glu Thr Val Tyr Leu Arg Thr Pro 930 935
940Phe Ser Ala Gly Asn Ala Thr Thr945 95097PRTMalus
domestica 9Phe Lys Leu Ile Glu Ser Tyr1 5107PRTMalus
domestica 10Ser Lys Leu Ile Glu Ser Ser1 511290PRTHomo
sapiens 11Met Val Arg Val Pro Val Pro Gln Leu Gln Pro Gln Asn Pro Ser
Gln1 5 10 15Gln Gln Pro
Gln Glu Gln Val Pro Leu Val Gln Gln Gln Gln Phe Pro 20
25 30Gly Gln Gln Gln Pro Phe Pro Pro Gln Gln
Pro Tyr Pro Gln Pro Gln 35 40
45Pro Phe Pro Ser Gln Gln Pro Tyr Leu Gln Leu Gln Pro Phe Pro Gln 50
55 60Pro Gln Leu Pro Tyr Pro Gln Pro Gln
Leu Pro Tyr Pro Gln Pro Gln65 70 75
80Leu Pro Tyr Pro Gln Pro Gln Pro Phe Arg Pro Gln Gln Pro
Tyr Pro 85 90 95Gln Ser
Gln Pro Gln Tyr Ser Gln Pro Gln Gln Pro Ile Ser Gln Gln 100
105 110Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln Lys Gln Gln Gln Gln Gln 115 120
125Gln Gln Gln Ile Leu Gln Gln Ile Leu Gln Gln Gln Leu Ile Pro Cys
130 135 140Arg Asp Val Val Leu Gln Gln
His Ser Ile Ala Tyr Gly Ser Ser Gln145 150
155 160Val Leu Gln Gln Ser Thr Tyr Gln Leu Val Gln Gln
Leu Cys Cys Gln 165 170
175Gln Leu Trp Gln Ile Pro Glu Gln Ser Arg Cys Gln Ala Ile His Asn
180 185 190Val Val His Ala Ile Ile
Leu His Gln Gln Gln Gln Gln Gln Gln Gln 195 200
205Gln Gln Gln Gln Pro Leu Ser Gln Val Ser Phe Gln Gln Pro
Gln Gln 210 215 220Gln Tyr Pro Ser Gly
Gln Gly Ser Phe Gln Pro Ser Gln Gln Asn Pro225 230
235 240Gln Ala Gln Gly Ser Val Gln Pro Gln Gln
Leu Pro Gln Phe Glu Glu 245 250
255Ile Arg Asn Leu Ala Leu Glu Thr Leu Pro Ala Met Cys Asn Val Tyr
260 265 270Ile Pro Pro Tyr Cys
Thr Ile Ala Pro Val Gly Ile Phe Gly Thr Asn 275
280 285Tyr Arg 29012290PRTHomo sapiens 12Met Val Arg
Val Pro Val Pro Gln Leu Gln Pro Gln Asn Pro Ser Gln1 5
10 15Gln Gln Pro Gln Glu Gln Val Pro Leu
Val Gln Gln Gln Gln Phe Pro 20 25
30Gly Gln Gln Gln Pro Phe Pro Pro Gln Gln Pro Tyr Pro Gln Pro Gln
35 40 45Pro Phe Pro Ser Gln Gln Pro
Ser Leu Gln Leu Gln Pro Phe Pro Gln 50 55
60Pro Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln65
70 75 80Leu Pro Tyr Pro
Gln Pro Gln Pro Phe Arg Pro Gln Gln Pro Tyr Pro 85
90 95Gln Ser Gln Pro Gln Tyr Ser Gln Pro Gln
Gln Pro Ile Ser Gln Gln 100 105
110Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Lys Gln Gln Gln Gln Gln
115 120 125Gln Gln Gln Ile Leu Gln Gln
Ile Leu Gln Gln Gln Leu Ile Pro Cys 130 135
140Arg Asp Val Val Leu Gln Gln His Ser Ile Ala Tyr Gly Ser Ser
Gln145 150 155 160Val Leu
Gln Gln Ser Thr Ser Gln Leu Val Gln Gln Leu Cys Cys Gln
165 170 175Gln Leu Trp Gln Ile Pro Glu
Gln Ser Arg Cys Gln Ala Ile Ser Asn 180 185
190Val Val His Ala Ile Ile Leu His Gln Gln Gln Gln Gln Gln
Gln Gln 195 200 205Gln Gln Gln Gln
Pro Leu Ser Gln Val Ser Phe Gln Gln Pro Gln Gln 210
215 220Gln Tyr Pro Ser Gly Gln Gly Ser Phe Gln Pro Ser
Gln Gln Asn Pro225 230 235
240Gln Ala Gln Gly Ser Val Gln Pro Gln Gln Leu Pro Gln Ser Glu Glu
245 250 255Ile Arg Asn Leu Ala
Leu Glu Thr Leu Pro Ala Met Cys Asn Val Tyr 260
265 270Ile Pro Pro Ser Cys Thr Ile Ala Pro Val Gly Ile
Phe Gly Thr Asn 275 280 285Tyr Arg
290137PRTDermatophagoides pteronyssinus 13Phe Ser Gly Val Ala Ala Thr1
5148PRTDermatophagoides pteronyssinus 14His Ser Ala Ile Ala
Ala Val Ile1 5157PRTDermatophagoides pteronyssinus 15Tyr
Pro Tyr Val Val Ile Leu1 516223PRTDermatophagoides
pteronyssinus 16Glu Thr Asn Ala Cys Ser Ile Asn Gly Asn Ala Pro Ala Glu
Ile Asp1 5 10 15Leu Arg
Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys 20
25 30Gly Ser Cys Trp Ala Ser Ser Gly Val
Ala Ala Thr Glu Ser Ala Tyr 35 40
45Leu Ala Tyr Arg Asn Gln Ser Leu Asp Leu Ala Glu Gln Glu Leu Val 50
55 60Asp Cys Ala Ser Gln His Gly Cys His
Gly Asp Thr Ile Pro Arg Gly65 70 75
80Ile Glu Tyr Ile Gln His Asn Gly Val Val Gln Glu Ser Tyr
Tyr Arg 85 90 95Tyr Val
Ala Arg Glu Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg Phe 100
105 110Gly Ile Ser Asn Tyr Cys Gln Ile Tyr
Pro Pro Asn Val Asn Lys Ile 115 120
125Arg Glu Ala Leu Ala Gln Thr Ser Ser Ala Ile Ala Val Ile Ile Gly
130 135 140Ile Lys Asp Leu Asp Ala Phe
Arg His Tyr Asp Gly Arg Thr Ile Ile145 150
155 160Gln Arg Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala
Val Asn Ile Val 165 170
175Gly Tyr Ser Asn Ala Gln Gly Val Asp Tyr Trp Ile Val Arg Asn Ser
180 185 190Trp Asp Thr Asn Trp Gly
Asp Asn Gly Tyr Gly Tyr Phe Ala Ala Asn 195 200
205Ile Asp Leu Met Met Ile Glu Glu Ser Pro Tyr Val Val Ile
Leu 210 215
22017223PRTDermatophagoides pteronyssinus 17Glu Thr Asn Ala Cys Ser Ile
Asn Gly Asn Ala Pro Ala Glu Ile Asp1 5 10
15Leu Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln
Gly Gly Cys 20 25 30Gly Ser
Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr 35
40 45Leu Ala Tyr Arg Asn Gln Ser Leu Asp Leu
Ala Glu Gln Glu Leu Val 50 55 60Asp
Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly65
70 75 80Ile Glu Tyr Ile Gln His
Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg 85
90 95Tyr Val Ala Arg Glu Gln Ser Cys Arg Arg Pro Asn
Ala Gln Arg Phe 100 105 110Gly
Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asn Val Asn Lys Ile 115
120 125Arg Glu Ala Leu Ala Gln Thr His Ser
Ala Ile Ala Val Ile Ile Gly 130 135
140Ile Lys Asp Leu Asp Ala Phe Arg His Tyr Asp Gly Arg Thr Ile Ile145
150 155 160Gln Arg Asp Asn
Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val 165
170 175Gly Tyr Ser Asn Ala Gln Gly Val Asp Tyr
Trp Ile Val Arg Asn Ser 180 185
190Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr Phe Ala Ala Asn
195 200 205Ile Asp Leu Met Met Ile Glu
Glu Tyr Pro Tyr Val Val Ile Leu 210 215
220187PRTHomo sapiens 18Tyr Arg Val Val Ser Val Leu1
5197PRTHomo sapiens 19Phe Arg Val Val Ser Val Leu1
5207PRTHomo sapiens 20His Glu Ala Leu His Asn His1
521327PRTHomo sapiens 21Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser Arg1 5 10
15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70
75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90
95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro
100 105 110Glu Phe Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120
125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 130 135 140Asp Val Ser Gln Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150
155 160Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe 165 170
175Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195
200 205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg 210 215 220Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225
230 235 240Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp 245
250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys 260 265 270Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275
280 285Arg Leu Thr Val Asp Lys Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser 290 295
300Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305
310 315 320Leu Ser Leu Ser
Leu Gly Lys 32522327PRTHomo sapiens 22Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5
10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65
70 75 80Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro
Ser Cys Pro Ala Pro 100 105
110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val 130 135
140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
Asp145 150 155 160Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175Asn Ser Thr Ser Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp 180 185
190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Gly Leu 195 200 205Pro Ser Ser Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210
215 220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
Glu Met Thr Lys225 230 235
240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260
265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 275 280 285Arg Leu
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290
295 300Cys Ser Val Met Ser Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser305 310 315
320Leu Ser Leu Ser Leu Gly Lys 325237PRTArtificial
SequenceCD1d ConsensusMISC_FEATURE(1)..(1)Xaa is Phe, Trp, Thr, His, or
TyrMISC_FEATURE(2)..(2)Any amino acidMISC_FEATURE(3)..(3)Any amino
acidMISC_FEATURE(4)..(4)Xaa is Ile, Leu, Met, or
ValMISC_FEATURE(5)..(5)Any amino acidMISC_FEATURE(6)..(6)Any amino
acidMISC_FEATURE(7)..(7)Xaa is Phe, Trp, Thr, His, or Tyr 23Xaa Xaa Xaa
Xaa Xaa Xaa Xaa1 5
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