DNA VACCINE AGAINST AMYLOID-BETA AND TAU

Abstract

An object of the present invention is to provide a vaccine that can simultaneously reduce A.beta. deposition and tau deposition in the brain by means of a single molecule. The present invention provides a recombinant vector comprising DNA encoding amyloid-.beta., DNA encoding an immunoglobulin Fc sequence, and DNA encoding tau.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Continuation of copending application Ser. No. 15/578,123, filed on Nov. 29, 2017, which is a 35 U.S.C. 371 National Stage of PCT International Application No. PCT/JP2016/081038 on Oct. 20, 2016, which claims the benefit under 35 U.S.C. .sctn. 119(a) to Patent Application No. 2015-207888, filed in Japan on Oct. 22, 2015, all of which are hereby expressly incorporated by reference into the present application.

TECHNICAL FIELD

[0002] The present invention relates to DNA vaccines against amyloid-.beta. and tau.

BACKGROUND ART

[0003] Alzheimer's disease is a disease in which moderate to severe cerebral atrophy is grossly visible in the frontal association cortex, temporal lobe, and hippocampal region, and is characterized by three major microscopic findings, i.e., senile plaques (amyloid-.beta. (A.beta.) deposition), neurofibrillary tangles (hyperphosphorylated tau deposition), and neuronal loss.

[0004] There are many reports that amyloid accumulation precedes tau deposition and neuronal changes, and amyloid accumulation has also been observed in autopsy brains of non-dementia elderly individuals and Down's syndrome individuals. In recent years, the "amyloid hypothesis" has come to be accepted, which assumes that amyloid deposition is positioned uppermost in this pathology, and the prevention of amyloid accumulation would allow subsequent events, such as intraneuronal tau accumulation and neuronal loss, to be prevented to a certain extent.

[0005] From pathological examination, however, it has been reported that although anti-immunotherapy effectively reduces A.beta. deposition, its effect of reducing tau deposition is extremely weak (Non Patent Literature 1: Boche, D. et al., Acta Neuropathol 120, 13-20).

[0006] At present, DNA vaccines against A.beta. are known (Patent Literature 1: WO 2010/110408); however, no DNA vaccine is known that can reduce A.beta. deposition and tau deposition simultaneously by means of a single molecule.

PRIOR ART DOCUMENTS

Patent Literature

[0007] Patent Literature 1: WO 2010/110408

Non Patent Literature

[0007]

[0008] Non Patent Literature 1: Boche, D. et al., Acta Neuropathol 120, 13-20

SUMMARY OF INVENTION

Problems to be Solved by the Invention

[0009] The present invention has been made in view of the aforementioned circumstances, and the problem to be solved by the invention is to provide a vaccine that can reduce A.beta. deposition and tau deposition in the brain simultaneously by means of a single molecule (single construct).

Means for Solving the Problem

[0010] As a result of extensive research to solve the aforementioned problem, the present inventors succeeded in reducing A.beta. deposition and tau deposition in the brain simultaneously by means of a single molecule, through the use of a recombinant vector comprising DNA encoding amyloid-.beta., DNA encoding an immunoglobulin Fc sequence, and DNA encoding tau, thereby completing the present invention.

[0011] In summary, the present invention is as set forth below.

[0012] (1) A recombinant vector comprising DNA encoding amyloid-.beta., DNA encoding an immunoglobulin Fc sequence, and DNA encoding tau.

[0013] (2) The vector according to (1) above, wherein the DNA encoding amyloid-.beta. is DNA encoding repeats of amyloid-.beta. sequence.

[0014] (3) The vector according to (1) or (2) above, wherein the DNA encoding tau is DNA encoding repeats of tau sequence.

[0015] (4) The vector according to any of (1) to (3) above, wherein the amyloid-.beta. is A.beta.1-42.

[0016] (5) A DNA vaccine for prevention or treatment of Alzheimer's disease, comprising the recombinant vector according to any of (1) to (4) above.

[0017] (6) A DNA vaccine for reducing brain A.beta. and brain tau, comprising the recombinant vector according to any of (1) to (4) above.

[0018] (7) An inducer of anti-A.beta. antibody and anti-tau antibody, comprising the recombinant vector according to any of (1) to (4) above.

[0019] (8) A polypeptide comprising amino acid sequences of amyloid-.beta., an immunoglobulin Fc sequence, and tau.

[0020] (9) The polypeptide according to (8) above, wherein the amyloid-.beta. consists of repeats of amyloid-.beta. sequence.

[0021] (10) The polypeptide according to (8) or (9) above, wherein the tau consists of repeats of tau sequence.

[0022] (11) The polypeptide according to any of (8) to (10) above, wherein the amyloid-.beta. is A.beta.1-42.

[0023] (12) A polypeptide expressed from the recombinant vector according to any of (1) to (4) above.

[0024] (13) A vaccine for prevention or treatment of Alzheimer's disease, comprising the polypeptide according to any of (8) to (12) above.

[0025] (14) A vaccine for reducing brain A.beta. and brain tau, comprising the polypeptide according to any of (8) to (12) above.

[0026] (15) An inducer of anti-A.beta. antibody and anti-tau antibody, comprising the polypeptide according to any of (8) to (12) above.

Advantageous Effects of Invention

[0027] According to the present invention, A.beta. deposition and tau deposition in the brain can be reduced simultaneously by means of a single molecule.

BRIEF DESCRIPTION OF DRAWINGS

[0028] FIG. 1 shows a schematic diagram of the structure of YM7555.

[0029] FIG. 2 shows the results of Western blotting detection of protein expression by the recombinant vector of the present invention.

[0030] FIG. 3 shows a graph showing the results of ELISA measurement of protein expression by the recombinant vector of the present invention.

[0031] FIG. 4 shows the schedule of immunization with YM7555 and blood collection.

[0032] FIG. 5 shows the results of induction of anti-A.beta. antibody and anti-tau antibody in Tg mice.

[0033] FIG. 6 shows the results of induction of anti-A.beta. antibody and anti-tau antibody in wild-type mice.

[0034] FIG. 7 shows graphs showing the effects of reducing brain A.beta. and brain tau according to the present invention.

[0035] FIG. 8 shows a schematic diagram of the structure of YM7555P.

DESCRIPTION OF EMBODIMENTS

[0036] The present invention will be hereinafter described in detail. The following embodiments are illustrative of the present invention, and are not intended to limit the present invention. The present invention can be carried out in various modes, without departing from the gist of the invention.

[0037] 1. Summary

[0038] At present, a construct comprising DNA encoding A.beta. and a DNA vaccine comprising such a construct are known (Patent Literature 1: WO 2010/110408); however, a DNA vaccine that comprises both DNA encoding A.beta. and DNA encoding tau, in particular, a DNA vaccine that comprises DNAs encoding repeats of A.beta. sequence and repeats of tau sequence, is unknown. One reason for this is that a skilled artisan predicts the possibility that if repeats of A.beta. sequence and repeats of tau sequence were combined, the respective sequences would conformationally interfere with each other in the expressed polypeptide, leading to a failure to induce antibodies against A.beta. or tau. Even if the skilled artisan attempts to prepare a construct comprising both DNA encoding A.beta. and DNA encoding tau, the preparation of such a construct is technically difficult, which is considered to be one reason why such a construct has never been prepared in the past. Specifically, because each of the DNA encoding A.beta. and the DNA encoding tau has high hydrophobicity, and the DNA encoding repeats of A.beta. sequence and the DNA encoding repeats of tau sequence comprise the repeats, these DNAs are likely to form a conformation due to self-ligation when ligated using a generally known cloning method, and their ligation to a vector or the ligation reaction between the constructs is unlikely to proceed. Thus, it has been technically difficult for the skilled artisan to prepare a construct comprising both the DNA encoding A.beta. and the DNA encoding tau, by using a general method.

[0039] Even if such a construct can be prepared, it has been extremely difficult to prepare a DNA vaccine that can actually express in vivo a polypeptide comprising A.beta. and tau as a single molecule, which polypeptide stimulates the immune system to induce the production of antibodies against A.beta., tau, and their related substances, which antibodies, in turn, can reduce A.beta. and tau in the brain simultaneously.

[0040] Even if a skilled artisan considers in vivo expression of A.beta. and tau, the skilled artisan would generally conceive of combined administration of two types of molecules, i.e., a recombinant vector comprising DNA encoding A.beta. and a recombinant vector comprising DNA encoding tau.

[0041] In contrast, the present inventors conducted extensive research based on the idea that a DNA vaccine that can reduce A.beta. deposition and tau deposition in the brain simultaneously by means of a single molecule would be useful for treatment or prevention of Alzheimer's disease. As a result, the present inventors succeeded in reducing A.beta. deposition and tau deposition in the brain simultaneously by means of a single molecule, through the use of a recombinant vector comprising DNA encoding A.beta., DNA encoding an immunoglobulin Fc sequence, and DNA encoding tau, as a DNA vaccine, by employing the unique techniques described in the Examples of the present specification, thereby completing the present invention.

[0042] In particular, the recombinant vector of the present invention, by virtue of a synergistic effect of its components, can reduce phosphorylated tau that is strongly neurotoxic in vivo, and thus, is extremely effective for treating or preventing Alzheimer's disease.

[0043] The term "simultaneously" as used herein is not necessarily intended to mean simultaneously in time, but also mean both at the same site (cell population).

[0044] 2. Recombinant Vector and Polypeptide

[0045] (1) Amyloid-.beta. (A.beta.)

[0046] Amyloid-.beta. (A.beta.) is a polypeptide consisting of 40-43 amino acids cleaved from the precursor protein (APP: amyloid-.beta. protein precursor) by the action of .beta.- and .gamma.-secretases.

[0047] The term "A.beta." as used herein refers to a polypeptide comprising 15 or more contiguous amino acids, preferably 20 or more contiguous amino acids, and more preferably refers to a polypeptide having an amino acid sequence consisting of amino acids 1-42 at the N-terminus of A.beta.(A.beta.1-42), in the native A.beta. amino acid sequence.

[0048] Nucleotide sequences of DNAs encoding A.beta. are available from given databases. For example, the nucleotide sequence as set forth in Genbank accession no. NC_000021.7 can be used as a nucleotide sequence of DNA encoding human A.beta., and the nucleotide sequence as set forth in accession no. NC_000082.5 can be used as a nucleotide sequence of DNA encoding mouse A.beta..

[0049] In the present invention, DNAs comprising nucleotide sequences of various regions of A.beta. can be prepared by performing PCR using DNA encoding A.beta. as a template, and using primers for amplifying desired regions. Examples of such DNAs include DNA encoding a polypeptide having an amino acid sequence consisting of 43 amino acids cleaved by .gamma.-secretase (designated as "A.beta.1-43"); DNA encoding a polypeptide having an amino acid sequence consisting of amino acids 1-20 at the N-terminus of A.beta. (designated as "A.beta.1-20"); DNA encoding a polypeptide having an amino acid sequence consisting of amino acids 1-40 at the N-terminus of A.beta. (designated as "A.beta.1-40"); and DNA encoding a polypeptide having an amino acid sequence consisting of amino acids 1-42 at the N-terminus of A.beta. (designated as "A.beta.1-42"). Preferred is the DNA encoding A.beta.1-42.

[0050] In the present invention, examples of A.beta. used in the polypeptide comprising amino acid sequences of amyloid-.beta. (A.beta.), an immunoglobulin Fc sequence, and tau (hereinafter also referred to as the "polypeptide of the present invention") include A.beta.1-43, A.beta.1-20, A.beta.1-40, and A.beta.1-42, with A.beta.1-42 being preferred.

[0051] The amino acid sequences of human A.beta.1-43, A.beta.1-20, A.beta.1-40, and A.beta.1-42 are shown in SEQ ID NOS: 2, 4, 6, and 8, respectively, and the amino acid sequences of mouse A.beta.1-43, A.beta.1-20, A.beta.1-40, and A.beta.1-42 are shown in SEQ ID NOS: 10, 12, 14, and 16, respectively. The nucleotide sequences of DNAs encoding human A.beta.1-43, A.beta.1-20, A.beta.1-40, and A.beta.1-42 are shown in SEQ ID NOS: 1, 3, 5, and 7, respectively, and the nucleotide sequences of DNAs encoding mouse A.beta.1-43, A.beta.1-20, A.beta.1-40, and A.beta.1-42 are shown in SEQ ID NOS: 9, 11, 13, and 15, respectively.

[0052] The DNA encoding human or mouse A.beta.1-20, A.beta.1-40, or A.beta.1-42 can be prepared from the DNA encoding human or mouse A.beta.1-43 by using PCR.

[0053] In addition to the DNAs encoding human or mouse A.beta.1-43, A.beta.1-20, A.beta.1-40, and A.beta.1-42 described above, the following DNAs can be used in the recombinant vector of the present invention:

[0054] DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1, and encoding a protein having human A.beta. activity;

[0055] DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 3, and encoding a protein having human A.beta. activity;

[0056] DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5, and encoding a protein having human A.beta. activity;

[0057] DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 7, and encoding a protein having human A.beta. activity;

[0058] DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 9, and encoding a protein having mouse A.beta. activity;

[0059] DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 11, and encoding a protein having mouse A.beta. activity;

[0060] DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 13, and encoding a protein having mouse A.beta. activity; and

[0061] DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 15, and encoding a protein having mouse A.beta. activity.

[0062] Each of human A.beta.1-43, human A.beta.1-20, human A.beta.1-40, human A.beta.1-42, mouse A.beta.1-43, mouse A.beta.1-20, mouse A.beta.1-40, and mouse A.beta.1-42 has its own A.beta. activity. Therefore, for example, in the case of a protein expressed from the DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1, and encoding a protein having human A.beta. activity, it is sufficient for this protein to have A.beta. activity equivalent to that of human A.beta.1-43. The same also applies to proteins expressed from DNAs hybridizing under stringent conditions to DNAs consisting of nucleotide sequences complementary to the nucleotide sequences shown in other SEQ ID NOS, and encoding proteins having A.beta. activity.

[0063] The term "stringent conditions" as used herein may refer to any of low-stringency conditions, moderate-stringency conditions, and high-stringency conditions. The "low-stringency conditions" refers to, for example, conditions of 5.times.SSC, 5.times.Denhardt's solution, 0.5% SDS, 50% formamide, and 32.degree. C. The "moderate-stringency conditions" refers to, for example, conditions of 5.times.SSC, 5.times.Denhardt's solution, 0.5% SDS, 50% formamide, and 42.degree. C. The "high-stringency conditions" refers to, for example, conditions of 5.times.SSC, 5.times.Denhardt's solution, 0.5% SDS, 50% formamide, and 50.degree. C. Under these conditions, it is expected that DNA having a higher homology will be efficiently obtained at a higher temperature. A plurality of factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration are considered to affect the stringency of hybridization, and a skilled artisan can achieve the same stringency by selecting these factors as appropriate. For detailed hybridization procedures, reference may be made to "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)) and the like.

[0064] As the DNA encoding A.beta. in the recombinant vector of the present invention, DNA having 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology (identity) to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, or 7, and encoding a protein having human A.beta. activity, can be used. Moreover, DNA having 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology to the nucleotide sequence shown in SEQ ID NO: 9, 11, 13, or 15, and encoding a protein having mouse A.beta. activity, can also be used.

[0065] As A.beta. in the polypeptide of the present invention, a polypeptide having 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology (identity) to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 35, and having human A.beta. activity, can be used. Preferred as such A.beta. is a polypeptide having 85% or more homology to the amino acid sequence shown in SEQ ID NO: 8 or 35 and having human A.beta. activity, and more preferred is a polypeptide having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 8 or 35 and having human A.beta. activity. Furthermore, as A.beta. in the polypeptide of the present invention, a polypeptide having 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology to the amino acid sequence shown in SEQ ID NO: 10, 12, 14, or 16, and having mouse A.beta. activity, can be used. Preferred as such A.beta. is a polypeptide having 85% or more homology to the amino acid sequence shown in SEQ ID NO: 16 and having mouse A.beta. activity, and more preferred is a polypeptide having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 16 and having mouse A.beta. activity.

[0066] In the polypeptide of the present invention, A.beta. includes, in addition to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 35, a polypeptide consisting of an amino acid sequence mutated by deletion, substitution, or addition, or a combination thereof, of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 35, and having human A.beta. activity.

[0067] Examples of the above-described amino acid sequence mutated by deletion, substitution, or addition, or a combination thereof, of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 35 include the following:

[0068] (i) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 35 have been deleted;

[0069] (ii) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 35 have been substituted with other amino acids;

[0070] (iii) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids have been added to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 35; and

[0071] (iv) an amino acid sequence mutated by a combination of (i) to (iii) above.

[0072] In the polypeptide of the present invention, A.beta. includes, in addition to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10, 12, 14, or 16, a polypeptide consisting of an amino acid sequence mutated by deletion, substitution, or addition, or a combination thereof, of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 10, 12, 14, or 16, and having mouse A.beta. activity.

[0073] Examples of the above-described amino acid sequence mutated by deletion, substitution, or addition, or a combination thereof, of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 10, 12, 14, or 16 include the following:

[0074] (i) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids in the amino acid sequence shown in SEQ ID NO: 10, 12, 14, or 16 have been deleted;

[0075] (ii) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids in the amino acid sequence shown in SEQ ID NO: 10, 12, 14, or 16 have been substituted with other amino acids;

[0076] (iii) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids have been added to the amino acid sequence shown in SEQ ID NO: 10, 12, 14, or 16; and

[0077] (iv) an amino acid sequence mutated by a combination of (i) to (iii) above.

[0078] The term "A.beta. activity" as used herein is intended to mean the activity of A.beta. to be produced, accumulate, and/or aggregate in the brain of a subject (such as a human or a mouse) to form A.beta. deposition (senile plaques). The A.beta. activity can be measured by an immunological technique such as immunohistological staining or ELISA. With immunohistological staining and the like, the A.beta. activity can be measured by expressing the protein to be evaluated in the brain of a test animal (such as a mouse), performing immunostaining using anti-A.beta. antibody on tissue sections in which the protein has been expressed, and detecting A.beta. production, accumulation, aggregation, and/or deposition and the like.

[0079] The phrase "having A.beta. activity" as used herein is intended to mean having an activity of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, and preferably 90% or more, compared to the A.beta. activity taken as 100% of a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 35, or SEQ ID NO: 10, 12, 14, or 16.

[0080] For the preparation of polypeptides having the above-described mutations, mutations into polynucleotides can be introduced using a mutagenesis kit that utilizes a site-directed mutagenesis method such as the Kunkel method or Gapped duplex method, for example, QuikChange.TM. Site-Directed Mutagenesis Kit (Stratagene), GeneTailor.TM. Site-Directed Mutagenesis System (Invitrogen), or TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, and the like; Takara Bio). Furthermore, methods such as site-directed mutagenesis methods as described in the following literatures can be used: "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)); "Current Protocols in Molecular Biology" (John Wiley & Sons (1987-1997)); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488-92; Kramer and Fritz (1987) Method. Enzymol. 154: 350-67; Kunkel (1988) Method. Enzymol. 85: 2763-6; and the like.

[0081] In the present invention, the DNA encoding A.beta. may be DNA encoding repeats of A.beta. sequence. A vector comprising the DNA encoding repeats of A.beta. sequence can express a plurality of A.beta. or a polypeptide comprising the plurality of A. Furthermore, in the polypeptide of the present invention, A.beta. may consist of repeats of A.beta. sequence. The plurality of A.beta. or the polypeptide comprising the plurality of A.beta. forms an A.beta. oligomer extracellularly. This oligomer stimulates the immune system to induce antibodies against the A.beta. oligomer. This is expected to reduce the A.beta. oligomer, which is more neurotoxic than the A.beta. monomers.

[0082] The present invention can exert an enhanced effect of reducing brain A.beta., by comprising repeats of A.beta. sequence or DNA encoding the same. The present invention can also induce antibodies against various molecules (such as pEA.beta.3-42, ABri, and ADan) having neurotoxicity and high amyloid aggregation propensity, by comprising repeats of A.beta. sequence or DNA encoding the same.

[0083] The range of the number of repeats of A.beta. sequence is not limited as long as A.beta. forms a folded structure to achieve improved antigenicity over A.beta. monomers; the number of repeats of A.beta. sequence is preferably 2 to 4, more preferably 3 to 4, and still more preferably 4.

[0084] (2) Tau

[0085] The recombinant vector of the present invention comprises DNA encoding tau. The polypeptide of the present invention also comprises an amino acid sequence of tau.

[0086] Tau is a protein with a molecular weight of about 50,000 that is present in nerve axons or the like, and contributes to microtubular stability.

[0087] In the present invention, tau may be either human-derived tau (human tau) or mouse-derived tau (mouse tau), and is preferably human tau. There are six isoforms of tau (0N3R, 1N3R, 0N3R, 2N3R, 1N4R, and 2N4R); however, in the present invention, tau is not limited to any of these isoforms.

[0088] Nucleotide sequences of DNAs encoding tau are available from given databases. Examples of the available nucleotide sequence of DNA encoding human tau include, but not limited to, the nucleotide sequence of the DNA encoding isoform 2N4R of tau (Genbank accession no. NM_005910.5). The nucleotide sequence of the DNA encoding human tau (2N4R, full-length) is shown in SEQ ID NO: 17. The amino acid sequence of human tau (2N4R, full-length) is shown in SEQ ID NO: 18.

[0089] In the present invention, DNAs comprising nucleotide sequences of various regions of tau can be prepared by performing PCR using DNA encoding tau as a template, and using primers for amplifying desired regions. Examples of such DNAs include DNA encoding a polypeptide (partial polypeptide) comprising/consisting of an amino acid sequence consisting of at least 6 or more, 8 or more, 10 or more, 20 or more, or 30 or more contiguous amino acid residues in the full-length amino acid sequence of tau. Specific examples of the DNA encoding the partial polypeptide include DNA encoding a polypeptide comprising an amino acid sequence consisting of amino acid residues 295 to 305 at the N-terminus of tau (designated as "tau295-305"); and DNA encoding a polypeptide having an amino acid sequence consisting of amino acid residues 379 to 408 at the N-terminus of tau (designated as "tau379-408").

[0090] The nucleotide sequences of the DNA encoding human tau295-305 and the DNA encoding human tau379-408 are shown in SEQ ID NOS: 19 and 21, respectively. The amino acid sequences of human tau295-305 and human tau379-408 are shown in SEQ ID NOS: 20 and 22, respectively.

[0091] In addition to the DNAs encoding human tau described above, the following DNAs can be used as the DNA encoding tau in the present invention:

[0092] DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 17, and encoding a protein having tau activity;

[0093] DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 19, and encoding a protein having tau activity; and

[0094] DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 21, and encoding a protein having tau activity.

[0095] Each of human tau, human tau295-305, and human tau379-408 has its own tau activity. Therefore, for example, in the case of a protein expressed from the DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 17, and encoding a protein having tau activity, it is sufficient for this protein to have tau activity equivalent to that of human tau. The same also applies to proteins expressed from DNAs hybridizing under stringent conditions to DNAs consisting of nucleotide sequences complementary to the nucleotide sequences shown in other SEQ ID NOS, and encoding proteins having tau activity.

[0096] The term "stringent conditions" is as defined above.

[0097] As the DNA encoding tau in the recombinant vector of the present invention, DNA having 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology (identity) to the nucleotide sequence shown in SEQ ID NO: 17, 19, or 21, and encoding a protein having human tau activity, can be used.

[0098] As tau in the polypeptide of the present invention, a polypeptide having 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology (identity) to the amino acid sequence shown in SEQ ID NO: 18, 20, 22, or 24, and having human tau activity, can be used. Preferred as such tau is a polypeptide having 85% or more homology to the amino acid sequence shown in SEQ ID NO: 18, 20, or 22 and having human tau activity, and more preferred is a polypeptide having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 18, 20, 22, or 24 and having human tau activity.

[0099] In the polypeptide of the present invention, tau includes, in addition to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 18, 20, 22, or 24, a polypeptide consisting of an amino acid sequence mutated by deletion, substitution, or addition, or a combination thereof, of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 18, 20, 22, or 24, and having human tau activity.

[0100] Examples of the above-described amino acid sequence mutated by deletion, substitution, or addition, or a combination thereof, of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 18, 20, 22, or 24 include the following:

[0101] (i) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids in the amino acid sequence shown in SEQ ID NO: 18, 20, 22, or 24 have been deleted;

[0102] (ii) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids in the amino acid sequence shown in SEQ ID NO: 18, 20, 22, or 24 have been substituted with other amino acids;

[0103] (iii) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids have been added to the amino acid sequence shown in SEQ ID NO: 18, 20, 22, or 24; and

[0104] (iv) an amino acid sequence mutated by a combination of (i) to (iii) above.

[0105] The term "tau activity" as used herein is intended to mean the activity of tau to be produced, accumulate, and/or aggregate in the brain of a subject (such as a human or a mouse). The tau activity can be measured by an immunological technique such as immunohistological staining or ELISA. With immunohistological staining, for example, the tau activity can be measured by expressing the protein to be evaluated in the brain of a test animal (such as a mouse), performing immunostaining using anti-tau antibody on tissue sections in which the protein has been expressed, and detecting tau production, accumulation, aggregation, and/or deposition and the like.

[0106] The phrase "having human tau activity" as used herein is intended to mean having an activity of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, and preferably 90% or more, compared to the human tau activity taken as 100% of a polypeptide having the amino acid sequence shown in SEQ ID NO: 18, 20, 22, or 24.

[0107] The method for preparing polypeptides having such mutations is as described above.

[0108] In the present invention, the DNA encoding tau may be DNA encoding repeats of tau sequence. Furthermore, in the polypeptide of the present invention, tau may consist of repeats of tau sequence. A vector comprising the DNA encoding repeats of tau sequence can express a plurality of tau or a polypeptide comprising the plurality of tau. Examples of the DNA encoding repeats of tau sequence include DNA encoding repeats of human tau295-305 sequence or human tau379-408 sequence. Examples of polypeptides comprising amino acid sequences of repeats of tau sequence include a polypeptide comprising an amino acid sequence of the repeats of human tau295-305 or human tau379-408.

[0109] The range of the number of repeats of tau sequence is not limited as long as tau forms a folded structure to achieve improved antigenicity over tau; the number of repeats of tau sequence is preferably 2 to 4, more preferably 3 to 4, and still more preferably 4.

[0110] Phosphorylated tau molecules lose their microtubule-binding ability, and are bound together to form aggregates. The recombinant vector and polypeptide of the present invention, by virtue of a synergistic effect of components thereof, can reduce phosphorylated tau that is strongly neurotoxic in vivo, and thus, is extremely effective for treating or preventing Alzheimer's disease.

[0111] (3) Immunoglobulin Fc (IgFc) Sequence

[0112] The recombinant vector of the present invention comprises DNA encoding an immunoglobulin Fc (IgFc) sequence. The polypeptide of the present invention also comprises an amino acid sequence of the IgFc sequence. Introduction of a gene encoding the IgFc sequence into the living body can promote intracellular transcription and translation of the polypeptides comprising A.beta. and tau, and can further promote extracellular release of these polypeptides to more strongly stimulate immune responses against A.beta. and tau.

[0113] Examples of the IgFc sequence used in the present invention include a human IgFc sequence and a mouse IgFc sequence. The nucleotide sequence as set forth in Genbank accession no. BC014258 can be used as a nucleotide sequence of DNA encoding a human IgFc sequence, and the nucleotide sequence as set forth in accession no. XM_484178.3 can be used as a nucleotide sequence of DNA encoding a mouse IgFc sequence.

[0114] SEQ ID NOS of the nucleotide sequences of the DNAs encoding the human and mouse IgFc sequences used in the present invention, as well as the amino acid sequences of these human and mouse IgFc sequences, are shown below:

[0115] The nucleotide sequence of the DNA encoding the human IgFc sequence: SEQ ID NO: 25

[0116] The amino acid sequence of the human IgFc sequence: SEQ ID NO: 26 The nucleotide sequence of the DNA encoding the mouse IgFc sequence: SEQ ID NO: 27

[0117] The amino acid sequence of the mouse IgFc sequence: SEQ ID NO: 28

[0118] Each of the nucleotide sequences shown in SEQ ID NOS: 25 and 27 is a nucleotide sequence into which mutations have been introduced to substitute cysteine residues in the original IgFc sequence with serine residues. This is intended to avoid the formation of disulfide bonds.

[0119] The following DNAs can also be used as the DNAs encoding the human and mouse IgFc sequences used in the present invention:

[0120] DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 25, and encoding a protein having human IgFc activity; and

[0121] DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 27, and encoding a protein having mouse IgFc activity.

[0122] The term "IgFc activity" as used herein is intended to mean the activity that promotes intracellular production and extracellular release of the polypeptide comprising the IgFc sequence. For example, the IgFc activity of a certain protein can be measured by expressing a fusion protein of IgFc and a polypeptide of interest in cultured cells, and quantifying an increase in the amount of the polypeptide present in the cultured cells or culture supernatant. The polypeptide can be quantified using an immunological technique such as ELISA or EIA.

[0123] As the DNA encoding the IgFc sequence in the recombinant vector of the present invention, DNA having 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology to the nucleotide sequence shown in SEQ ID NO: 25, and encoding a protein having human IgFc activity, can be used. Moreover, DNA having 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology to the nucleotide sequence shown in SEQ ID NO: 27, and encoding a protein having mouse IgFc activity, can also be used.

[0124] The IgFc activity is as described above.

[0125] As the IgFc sequence in the polypeptide of the present invention, a polypeptide having 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology (identity) to the amino acid sequence shown in SEQ ID NO: 26, and having human IgFc activity, can be used. Preferred as such an IgFc sequence is a polypeptide having 85% or more homology to the amino acid sequence shown in SEQ ID NO: 26 and having human IgFc activity, and more preferred is a polypeptide having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 26 and having human IgFc activity. Furthermore, as the IgFc sequence in the polypeptide of the present invention, a polypeptide having 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology to the amino acid sequence shown in SEQ ID NO: 28, and having mouse IgFc activity, can be used. Preferred as such an IgFc sequence is a polypeptide having 85% or more homology to the amino acid sequence shown in SEQ ID NO: 28 and having mouse IgFc activity, and more preferred is a polypeptide having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 28 and having mouse IgFc activity.

[0126] In the polypeptide of the present invention, the IgFc sequence includes, in addition to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 26, a polypeptide consisting of an amino acid sequence mutated by deletion, substitution, or addition, or a combination thereof, of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 26, and having human IgFc activity.

[0127] Examples of the above-described amino acid sequence mutated by deletion, substitution, or addition, or a combination thereof, of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 26 include the following:

[0128] (i) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids in the amino acid sequence shown in SEQ ID NO: 26 have been deleted;

[0129] (ii) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids in the amino acid sequence shown in SEQ ID NO: 26 have been substituted with other amino acids;

[0130] (iii) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids have been added to the amino acid sequence shown in SEQ ID NO: 26; and

[0131] (iv) an amino acid sequence mutated by a combination of (i) to (iii) above.

[0132] In the polypeptide of the present invention, the IgFc sequence includes, in addition to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 28, a polypeptide consisting of an amino acid sequence mutated by deletion, substitution, or addition, or a combination thereof, of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 28, and having mouse IgFc activity.

[0133] Examples of the above-described amino acid sequence mutated by deletion, substitution, or addition, or a combination thereof, of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 28 include the following:

[0134] (i) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids in the amino acid sequence shown in SEQ ID NO: 28 have been deleted;

[0135] (ii) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids in the amino acid sequence shown in SEQ ID NO: 28 have been substituted with other amino acids;

[0136] (iii) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids have been added to the amino acid sequence shown in SEQ ID NO: 28; and

[0137] (iv) an amino acid sequence mutated by a combination of (i) to (iii) above.

[0138] In another aspect, the recombinant vector of the present invention comprises a polynucleotide comprising DNA encoding amyloid-.beta., DNA encoding an immunoglobulin Fc sequence, and DNA encoding tau.

[0139] Examples of such polynucleotides include, although not limited to, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 30 (DNA encoding YM7555P). In the recombinant vector of the present invention, in addition to the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 30, the following polynucleotides can be used:

[0140] a polynucleotide hybridizing under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 30, and encoding a polypeptide having an activity to induce anti-A.beta. antibody and/or anti-tau antibody; and

[0141] a polynucleotide having 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more (preferably 90% or more) homology (identity) to the nucleotide sequence shown in SEQ ID NO: 30, and encoding a polypeptide having an activity to induce anti-A.beta. antibody and/or anti-tau antibody.

[0142] The phrase "activity to induce anti-A.beta. antibody and/or anti-tau antibody" as used herein is intended to mean an activity to induce anti-A.beta. antibody and/or anti-tau antibody in vivo in a mammal serving as a subject (such as a mouse or a human). This activity can be measured by an immunological technique such as immunohistological staining, Western blotting, or ELISA. For example, this activity can be measured by administering the polypeptide of the present invention or the recombinant vector expressing the same to a mammal, collecting blood from the mammal, and measuring the antibody titer of the anti-A.beta. antibody and/or the anti-tau antibody in the blood using ELISA.

[0143] The phrase "having an activity to induce anti-A.beta. antibody and/or anti-tau antibody" is intended to mean having an activity of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, and preferably 90% or more, compared to the activity taken as 100% of the polypeptide having the amino acid sequence shown in SEQ ID NO: 31.

[0144] The term "stringent conditions" is as defined above.

[0145] Examples of the polypeptide of the present invention include, although not limited to, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 31 (YM7555P). In addition to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 31, the following polypeptides can be used as the polypeptide of the present invention:

[0146] a polypeptide having 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more (preferably 90% or more) homology (identity) to the amino acid sequence shown in SEQ ID NO: 31, and having an activity to induce anti-A.beta. antibody and/or anti-tau antibody; and

[0147] a polypeptide consisting of an amino acid sequence mutated by deletion, substitution, or addition, or a combination thereof, of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 31, and having an activity to induce anti-A.beta. antibody and/or anti-tau antibody.

[0148] Examples of the above-described amino acid sequence mutated by deletion, substitution, or addition, or a combination thereof, of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 31 include the following:

[0149] (i) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids in the amino acid sequence shown in SEQ ID NO: 31 have been deleted;

[0150] (ii) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids in the amino acid sequence shown in SEQ ID NO: 31 have been substituted with other amino acids;

[0151] (iii) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and still more preferably 1) amino acids have been added to the amino acid sequence shown in SEQ ID NO: 31; and

[0152] (iv) an amino acid sequence mutated by a combination of (i) to (iii) above.

[0153] (4) Preparation of Recombinant Vector

[0154] The recombinant vector of the present invention comprises DNA encoding A.beta., DNA encoding an immunoglobulin Fc (IgFc) sequence, and DNA encoding tau.

[0155] The DNA encoding A.beta., tau, or the IgFc sequence may be derived from the same animal species as or different animal species from the animal serving as a subject that will receive the administration of the DNA vaccine; however, it is preferred to use DNA encoding A.beta., tau, or an IgFc sequence derived from the same animal species.

[0156] Each of the DNA encoding A.beta., the DNA encoding the IgFc sequence, and the DNA encoding tau has already been cloned. Thus, the DNAs comprised in the vector of the present invention can be obtained using a general genetic engineering technique. For example, a nucleic acid synthesis method using a DNA synthesizer, which is commonly used as a genetic engineering technique, can be used. Moreover, a PCR method that involves isolating or synthesizing DNA sequences for use as templates, designing primers specific for each of the DNAs, and amplifying the gene sequence using a PCR system, or a gene amplification method using a cloning vector, can be used. Furthermore, each of the DNAs can be obtained from a cDNA library or genomic library by performing a known hybridization method such as colony hybridization, plaque hybridization, or Southern blotting, using, as a probe, DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 18, 19, 21, 25, or 27, or a fragment thereof. The above-described methods can be readily performed by a skilled artisan in accordance with "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)) and the like. A known method can be used to purify the resulting PCR products.

[0157] A general genetic engineering technique can be adopted to prepare the recombinant vector. For example, samples of the DNAs of interest encoding A.beta., tau, and the IgFc sequence are prepared by PCR and the like. PCR can be performed by a general method using KOD polymerase or another DNA polymerase a. The amplified fragments of interest are digested with restriction enzymes, and then inserted into restriction enzyme sites or a multi-cloning site in a plasmid vector such as pCR (registered trademark)-Blunt II-TOPO (registered trademark) vector (Invitrogen). The resulting PCR products are confirmed for their nucleotide sequences with a sequencer to select a plasmid comprising the proper sequence. It is preferred that such a DNA sample can be confirmed as an electrophoretically single plasmid.

[0158] As a promoter comprised in the recombinant vector of the present invention, actin promoter, EF1 promoter, CMV promoter, CAG promoter or the like can be used. These promoters may each be ligated to an appropriate plasmid.

[0159] The term "stringent conditions" is as defined above. The phrase "having promoter activity" is intended to mean having the transcriptional activity of a gene encoding a structural protein or a non-structural protein.

[0160] In the vector of the present invention, the above-described A.beta., tau, and IgFc sequence are comprised in operable form to allow their expression. Specifically, the transgenes (DNAs) are inserted into the vector in a manner that allows expression of the transgenes under the control of appropriate regulatory elements. The DNA encoding A.beta., the DNA encoding the IgFc sequence, and the DNA encoding tau may be individually inserted into different sites in the same vector, or may be inserted contiguously in tandem. The term "regulatory elements" as used herein refers to, for example, promoters, enhancers, and transcription terminators.

[0161] The vector of the present invention may carry an additional foreign gene at a position different from the regions into which the DNA encoding A.beta., the DNA encoding the IgFc sequence, and the DNA encoding tau have been inserted. Such a foreign gene may be, for example, although not particularly limited to, a marker gene for vector monitoring, a regulatory gene for the immune system such as a cytokine or hormone, or a signal sequence (leader sequence).

[0162] Examples of the recombinant vector of the present invention include the following:

[0163] (i) pVAX1 comprising DNA encoding A.beta.1-42, DNA encoding the IgFc sequence, and DNA encoding tau, downstream of the CMV promoter;

[0164] (ii) pVAX1 comprising DNA encoding repeats of A.beta.1-42 sequence, DNA encoding the IgFc sequence, and DNA encoding tau, downstream of the CMV promoter;

[0165] (iii) pVAX1 comprising DNA encoding A.beta.1-42, DNA encoding the IgFc sequence, and DNA encoding repeats of tau sequence, downstream of the CMV promoter; and

[0166] (iv) pVAX1 comprising DNA encoding repeats of A.beta.1-42 sequence, DNA encoding the IgFc sequence, and DNA encoding repeats of tau sequence, downstream of the CMV promoter.

[0167] Examples of "tau" used herein include human tau (full-length), human tau295-305, and human tau379-408, with human tau379-408 being preferred.

[0168] Preferably, pVAX1 comprising DNA encoding A.beta.1-42, DNA encoding the IgFc sequence, and DNA encoding tau downstream of the CMV promoter comprises an Ig leader (IgL) sequence between the CMV promoter and the DNA encoding A.beta.1-42, and comprises a spacer sequence (also referred to as the "linker") between the DNA encoding the IgFc sequence and the DNA encoding tau. More preferably, such pVAX1 comprises the CMV promoter, the Ig leader sequence, the DNA encoding A.beta.1-42, the DNA encoding the IgFc sequence, the spacer sequence, and the DNA encoding tau in this order.

[0169] Preferably, pVAX comprising DNA encoding repeats of A.beta.1-42 sequence, DNA encoding the IgFc sequence, and DNA encoding repeats of tau sequence downstream of the CMV promoter comprises an Ig leader (IgL) sequence between the CMV promoter and the DNA encoding repeats of A.beta.1-42 sequence, and comprises spacer sequences between individual "DNAs encoding A.beta.1-42" of the DNA encoding repeats of A.beta.1-42 sequence, between the DNA encoding the IgFc sequence and the DNA encoding repeats of tau sequence, and between individual "DNAs encoding tau" of the DNA encoding repeats of tau sequence. More preferably, such pVAX1 comprises the CMV promoter, the Ig leader sequence, the DNA encoding repeats of A.beta.1-42 sequence, the DNA encoding the IgFc sequence, the spacer sequence, and the DNA encoding repeats of tau sequence in this order (FIG. 1).

[0170] The above-described recombinant vectors can comprise DNAs of mouse or human origin. Vectors comprising DNAs of mouse origin can be used in preclinical trials or reagents, while vectors comprising DNAs of human origin can be used in pharmaceutical compositions or reagents.

[0171] In accordance with the same technique as described above, a vector that does not comprise the DNA encoding A.beta.1-42 but comprises the Ig leader (IgL) sequence, the DNA encoding tau, and the DNA encoding the IgFc sequence can be prepared. Examples of such vectors include a vector comprising IgL-tau.times.1-huFc (the Ig leader (IgL) sequence, DNA encoding one tau sequence, and DNA encoding the IgFc sequence); and a vector comprising IgL-Tau.times.4-huFc (the Ig leader (IgL) sequence, DNA encoding four repeats of tau sequence, and DNA encoding the IgFc sequence; also designated herein as "tau.times.4-IgFc").

[0172] (5) Preparation of Polypeptide

[0173] The polypeptide of the present invention comprises amino acid sequences of amyloid-.beta., an immunoglobulin Fc sequence, and tau.

[0174] The polypeptide of the present invention can be prepared using a known technique, and can be specifically produced as follows.

[0175] (i) Preparation of Expression Vector

[0176] The vector for expressing the polypeptide of the present invention is not limited to particular vectors as long as host cells can carry the vector for expression, and examples thereof include plasmid DNAs and bacteriophages. Examples of plasmid DNAs include, although not limited to, plasmid vectors such as pCR (registered trademark)-Blunt II-TOPO (registered trademark) vector (Invitrogen).

[0177] As the vector for expressing the polypeptide of the present invention, the recombinant vector prepared in accordance with "(4) Preparation of Recombinant Vector" above can be used. Specifically, the polypeptide of the present invention may be a polypeptide expressed from the recombinant vector comprising DNA encoding amyloid-.beta., DNA encoding the immunoglobulin Fc sequence, and DNA encoding tau.

[0178] (ii) Transformation

[0179] Examples of hosts for producing the polypeptide of the present invention include, although not limited to, mammalian cells, bacteria such as Bifidobacterium bifidum, lactobacilli, and E. coli, insect cells, yeasts, and molds.

[0180] The recombinant DNA can be introduced into a host using a known method. Examples of methods for introducing the above-described vector into a host include the calcium phosphate method, the DEAE-dextran method, electroporation, and the cationic lipid method.

[0181] The introduction of DNA can be confirmed using selection marker genes (such as ampicillin resistance gene, neomycin resistance gene, hygromycin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, kanamycin resistant gene, zeocin resistance gene, and blasticidin resistance gene).

[0182] (iii) Production of Polypeptide

[0183] The polypeptide of the present invention can be obtained by culturing the above-described transformant comprising the polynucleotide encoding the polypeptide or a variant thereof, and collecting the polypeptide from the culture.

[0184] The term "culture" refers to any of culture supernatant, cultured cells, cultured bacteria, or cell or bacterial homogenates. The transformant of the present invention is cultured in accordance with a general method used for culturing of a host.

[0185] For culturing of a recombinant transfected with an expression vector comprising an inducible transcription promoter as a promoter, an inducer may be added to the medium, as required. When IPTG is used as an inducer, IPTG is added in an amount of 0.1 to 1.0 mM. IPTG is added 2 to 12 hours after the start of culture, and, after the addition, the culture is continued for additional 1 to 12 hours.

[0186] If the polypeptide of the present invention accumulates in the bacteria or cells after the culture, the polypeptide of interest is collected by homogenizing the bacteria or cells with a homogenizer and the like. When the polypeptide of the present invention is produced outside the bacteria or cells, the culture is used as is, or the bacteria or cells is removed by, for example, centrifugation. Then, the polypeptide is collected from the culture using ammonium sulfate precipitation procedures and the like, and then isolated and purified, as required, using any of various types of chromatography and the like.

[0187] As the cell extract, extracts from eukaryotic or prokaryotic cells can be used, which include, for example, extracts of wheat germ, rabbit reticulocytes, mouse L-cells, HeLa cells, CHO cells, budding yeast, and E. coli. These cell extracts may be concentrated or not concentrated.

[0188] (iv) Peptide Synthesis

[0189] The polypeptide of the present invention can be obtained by chemical synthesis. Peptide synthesis can be performed by an existing method with a synthesizer. Alternatively, the polypeptide of the present invention can be obtained by requesting the peptide synthesis to and purchasing from a custom peptide synthesis service company.

[0190] 3. Vaccines (DNA Vaccine Comprising Recombinant Vector and Vaccine Comprising Polypeptide) and Inducer of Anti-A.beta. Antibody and Anti-Tau Antibody

[0191] The present invention provides a DNA vaccine (pharmaceutical composition) for prevention or treatment of Alzheimer's disease, which comprises the above-described recombinant vector. In another aspect, the present invention provides a DNA vaccine for reducing brain A.beta. and brain tau, which comprises the above-described vector.

[0192] Such a DNA vaccine is intended to induce antibodies against the proteins of interest in a living body by: integrating DNAs encoding the proteins of interest into a vector (a plasmid or virus); administering the vector to the living body; and expressing the proteins of interest in the living body in which the vector has been administered to stimulate the immune system. The DNA vaccine remains in the body for a long time after the administration, and continues to slowly produce the encoded proteins. Thus, excessive immune responses can be avoided. The DNA vaccine can also be modified using a genetic engineering technique.

[0193] The present invention also provides a vaccine (pharmaceutical composition) comprising the polypeptide comprising amino acid sequences of amyloid-.beta., the immunoglobulin Fc sequence, and tau. In another aspect, the present invention provides a vaccine for reducing brain A.beta. and brain tau, which comprises the above-described polypeptide.

[0194] The term "treatment", "treat", or "treating" as used herein refers to contacting (for example, administering) the vaccine of the present invention with a subject after the onset of a disease, thereby alleviating symptoms of the disease, compared to when the subject is not contacted with the vaccine, and does not completely suppressing the symptoms of the disease. The "onset of a disease" refers to the manifestation of symptoms of the disease in the body.

[0195] The term "prevention", "prevent", or "preventing" as used herein refers to contacting (for example, administering) the vaccine of the present invention with a subject before the onset of a disease, thereby alleviating symptoms of the disease after the onset of the disease, compared to when the subject is not contacted with the vaccine, and does not refer to completely suppressing the onset of the disease.

[0196] The term "reduction", "reduce", or "reducing" as used herein refers to decreasing the amount of A.beta. and/or tau present in the brain, which encompasses decreasing the amount of brain A.beta. and/or brain tau accumulated, aggregated, or deposited in the brain. Furthermore, the DNA vaccine of the present invention comprising the above-described vector can also be used as a vaccine for suppressing an increase in the amount of brain A.beta. and/or brain tau.

[0197] In another aspect, the present invention provides an inducer of anti-A.beta. antibody and anti-tau antibody, which comprises the above-described vector or polypeptide.

[0198] The presence of various subspecies of neurotoxic A.beta. has been recently revealed. A.beta. oligomers, which are most intensively analyzed, can be divided into those of low-molecular-weight type formed by aggregation of 2, 3, or 4 molecules, and those of high-molecular-weight type formed by aggregation of 12 or more molecules. Moreover, strong neurotoxicity has also been observed in pEA.beta.3-42, which is N-terminally truncated and pyrrole-modified by post-translational modification (Saido et al., Neurosci Lett, 215, 173-176, 1996; Schlenzig et al., Biochemistry, 48, 7072-7078, 2009). Furthermore, some findings have been obtained which suggest that molecules such as ABri (Ghiso et al., Brain Pathol, 16, 71-79, 2006) and ADan, which have high amyloid aggregation propensity although completely differing in amino acid sequence from A.beta., are also implicated in the onset of Alzheimer's disease.

[0199] The recombinant vector and the polypeptide of the present invention can also induce antibodies against various molecules (such as pEA.beta.3-42, ABri, and ADan) having neurotoxicity and high amyloid aggregation propensity.

[0200] pEA.beta.3-42 is a molecule produced from A.beta.1-42 by N-terminal truncation with glutaminyl cyclase (QC) and post-translational modification (pyrrole modification). pEA.beta.3-42 is highly neurotoxic, and this molecule itself has a high tendency to aggregate. pEA.beta.3-42 also acts to enhance the aggregation propensity of unmodified A.beta., and is one of the major factors responsible for lesion formation in Alzheimer's disease.

[0201] ABri is a causative molecule for familial British dementia, while ADan is a causative molecule for familial Danish dementia. ABri and ADan are molecules produced by being cleaved as long molecules from their precursor proteins, due to a gene mutation in the stop codon of each precursor protein. ABri and ADan have high amyloid aggregation propensity (amyloid collectively refers to the phenomenon in which small molecules are aggregated and deposited), and are key molecules in the disease progression. ABri and ADan are thus considered to play some role in the lesion formation in Alzheimer's disease.

[0202] When the vector of the present invention is used as a DNA vaccine or an inducer of anti-A.beta. antibody and anti-tau antibody, gene transfer can be accomplished either by directly administering the vector to a target site of a subject, or by indirectly administering the vector by infecting the vector into the subject-derived cells or other cells for gene transfer, and then injecting the infected cells into a target site. Likewise, when the polypeptide of the present invention is used as a DNA vaccine or an inducer of anti-A.beta. antibody and anti-tau antibody, it can be directly administered to a target site of a subject.

[0203] Furthermore, the vector or polypeptide of the present invention may be introduced into phospholipid vesicles such as liposomes, for administration of the vesicles. The vesicles carrying the vector or polypeptide of the present invention may be introduced by lipofection into given cells. And then, the resulting cells may be administered systemically, for example, intravenously or intraarterially. The resulting cells may be administered locally to the brain and the like.

[0204] Examples of lipids for forming liposome structures include phospholipids, cholesterols, and nitrogen-containing lipids. Phospholipids are generally suitable, which include natural phospholipids such as phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, cardiolipin, sphingomyelin, egg yolk lecithin, soybean lecithin, and lysolecithin, as well as hydrogenated products thereof obtained in accordance with a standard method. Synthetic phospholipids can also be used, which include dicetyl phosphate, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylserine, eleostearoylphosphatidylcholine, and eleostearoylphosphatidylethanolamine.

[0205] Liposomes can be produced using any method that allows the liposomes to carry the DNA or polypeptide, including, for example, conventional methods such as reverse phase evaporation, ether injection, and surfactant-based techniques.

[0206] Lipids including these phospholipids can be used singly or in combinations of two or more. In this case, a lipid containing an atomic group having a cationic group in the molecule, such as ethanolamine or choline, may be used to increase the binding rate of electrically negative DNA. In addition to these major phospholipids for liposome formation, other additives such as cholesterols, stearyl amine, and .alpha.-tocopherol, which are generally known as liposome-forming additives, can also be used. The liposomes thus obtained can further contain a membrane fusion promoter, such as polyethylene glycol, in order to enhance their uptake into cells in the affected area or target tissue.

[0207] The vaccine, the inducer of anti-A.beta. antibody and anti-tau antibody, or the like of the present invention can be formulated in accordance with a routine method, and may comprise pharmaceutically acceptable carriers. Such carriers may be additives, including water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, carboxymethylcellulose sodium, sodium polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerine, glycerine, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, and surfactants acceptable as pharmaceutical additives.

[0208] The above-described additives can be selected singly or as an appropriate combination from among those listed above, according to the dosage form of the vaccine or the inducer of anti-A.beta. antibody and anti-tau antibody of the present invention. For example, for use as injectable formulations, the purified vector can be dissolved in a solvent (for example, physiological saline, buffer, or glucose solution), and then supplemented with Tween 80, Tween 20, gelatin, human serum albumin, or the like. Alternatively, the vaccine or the inducer may be lyophilized for use as dosage forms that are dissolved before use. Examples of excipients for lyophilization include saccharides such as mannitol, glucose, lactose, sucrose, and sorbitol; starches such as those derived from corn, wheat, rice, potato, and other plants; celluloses such as methylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose sodium; gums such as gum arabic and gum tragacanth; gelatin; and collagen.

[0209] Examples of subjects to receive the administration of the vaccine, the inducer of anti-A.beta. antibody and anti-tau antibody, or the like of the present invention include mammals, including, for example, humans; as well as non-human primates such as monkeys; rodents such as mice and rats; and all other mammals such as rabbits, goats, sheep, pigs, cattle, and dogs; with humans being preferred. Animals (subjects) to receive the administration are, for example, those suffering from Alzheimer's disease, those suspected to have Alzheimer's disease, those showing increased A.beta. deposition, those showing increased tau deposition, or those showing neuronal loss. Subjects to receive the administration may also be subjects (patients) in need of treatment and/or prevention of Alzheimer's disease, reduction in brain A.beta. and brain tau, and/or induction of anti-A.beta. antibody and anti-tau antibody.

[0210] The dosage of the vaccine, the inducer of anti-A.beta. antibody and anti-tau antibody, or the like of the present invention will vary depending on the age, sex, symptoms, route of administration, frequency of administration, and dosage form. The mode of administration can be selected as appropriate for the age and symptoms of the patient. The effective dosage of the vaccine is an amount of the vaccine required to alleviate the signs or condition of the disease. The therapeutic effect and toxicity of this vaccine can be determined by standard pharmacological procedures in cell culture or in laboratory animals, for example, using ED50 (therapeutically effective dose in 50% of the population) or LD50 (lethal dose for 50% of the population). Likewise, the effective dosage of the inducer of anti-A.beta. antibody and anti-tau antibody is an amount of the inducer required to induce detectable levels of anti-A.beta. antibody and/or anti-tau antibody in a biological sample (including blood, cells, and tissue) collected from the patient. Anti-A.beta. antibody and/or anti-tau antibody can be detected by an immunological technique such as ELISA or immunostaining. A skilled artisan would be able to determine an appropriate dosage for the vaccine or the inducer of anti-A.beta. antibody and anti-tau antibody.

[0211] The route of administration can be selected as appropriate, and examples of routes of administration include, although not limited to, percutaneous, intranasal, transbronchial, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Particularly preferred are intramuscular administration and subcutaneous administration. Inoculation may be made at a single site or multiple sites.

[0212] The dose ratio between therapeutic and toxic effects is a therapeutic index, and can be expressed as ED50/LD50.

[0213] For humans, the vaccine or the inducer of anti-A.beta. antibody and anti-tau antibody of the present invention is administered at a single dosage of about 1 to 1000 .mu.g, preferably about 10 to 500 .mu.g, and more preferably about 50 to 250 .mu.g. The frequency of administration may be once or more as long as the side effects are within a clinically acceptable range.

[0214] In the previous development of vaccines for Alzheimer's disease, studies have been conducted focusing on antibodies against A.beta. and Th2 activity. Thus, it is desirable to measure in advance the antibody titer or cellular immune activity as a vaccine.

[0215] For example, the cellular immune activity can be evaluated by separating and culturing lymphocytes from the body, and measuring their .sup.3H-thymidine uptake.

[0216] Likewise, the Th2 activity can be evaluated by separating plasma from peripheral blood, and measuring its antibody titer by ELISA.

[0217] Once the vaccine of the present invention is administered to an animal subject, immune responses against A.beta. and/or tau are induced. Specifically, because the above-described amino acid sequence of A.beta.1-43, A.beta.1-20, A.beta.1-40, A.beta.1-42, or tau comprises an epitope, antibody production is induced upon administration of the vaccine of the present invention.

[0218] Immune responses against A.beta. and tau can be detected by measuring the amount of anti-A.beta. antibody and anti-tau antibody produced. The amount of the antibodies produced can be measured by a general immunological technique such as ELISA (enzyme-linked immunosorbent assay). Likewise, the therapeutic effect of the vaccine can be confirmed, for example, as a reduction in the amount of A.beta. and tau in brain tissues or as a decrease in A.beta. deposition (senile plaques). The amount or the state of deposition of A.beta. and tau in brain tissues can be observed by immunohistochemistry and the like.

[0219] 4. Method for Treating and Preventing Alzheimer's Disease, Method for Reducing Brain A.beta. and Brain Tau, and Method for Inducing Anti-A.beta. Antibody and Anti-Tau Antibody

[0220] The recombinant vector, polypeptide, and vaccine of the present invention can be used in a method for treating or preventing Alzheimer's disease, a method for reducing brain A.beta. and/or brain tau, and a method for inducing anti-A.beta. antibody and/or anti-tau antibody. Specifically, the present invention provides a method for treating or preventing Alzheimer's disease, a method for reducing brain A.beta. and/or brain tau, and a method for inducing anti-A.beta. antibody and/or anti-tau antibody, each comprising administering the recombinant vector or polypeptide of the present invention to a subject.

[0221] In the methods of the present invention, the terms "treating", "preventing", and "reducing", as well as the mode of administration, the method of formulation, the dosage form, the target (subject) to receive the administration, the dosage, the route of administration, and the like are as described in the "3." section above.

[0222] The present invention will be hereinafter described in detail with reference to Examples; however, the invention is not limited to these Examples.

Example 1

[0223] 1. Construction of Recombinant Vector (Plasmid) Comprising DNAs Encoding IgL Sequence, A.beta., IgFc Sequence, and Tau

[0224] (1) Amplification and Cloning of DNAs Encoding IgL Sequence and IgFc Sequence

[0225] To clone DNAs encoding an immunoglobulin .kappa. leader (hereinafter "IgL") sequence and an immunoglobulin Fc (hereinafter "Fc" or "IgFc") sequence, human peripheral blood-derived mRNA was used as a material to synthesize cDNAs using ReverTra Ace-.alpha.- (TOYOBO, Tokyo, Japan). Primers comprising the 5' or 3' end of the nucleotide sequence encoding each sequence and having an appropriate restriction enzyme site (IgL: Bam HI or Xho I; IgFc: Kpn I or Not I) were designed and used to amplify DNAs encoding (or DNAs comprising DNAs encoding) human IgL sequence (SEQ ID NO: 32) and human IgFc sequence using KOD-plus- (Toyobo, Tokyo, Japan). Although the original human IgFc sequence comprises three codons each encoding a cysteine residue near the 5' end, these codons were each modified to encode a serine residue (TGT.fwdarw.TCT or TGC.fwdarw.TCC) during primer design so as to avoid S--S bonding, and the primers thus designed were used to obtain amplification products.

[0226] (2) Amplification and Cloning of DNA Encoding A.beta.

[0227] DNA encoding amyloid-.beta.1-42 (hereinafter "A.beta." or "A.beta.1-42") was prepared by oligonucleotide synthesis, provided that two oligonucleotides were first synthesized, which comprised the 5' or 3' end of the nucleotide sequence encoding A.beta. and were partially complementary to each other (24 bp in the middle of the A.beta. sequence), because a sequence covering the full-length (126 bp) was difficult to synthesize. An appropriate restriction enzyme site (Xho I, Kpn I) was added to each end. After these oligonucleotides were annealed, the entire duplex was prepared by polymerase reaction.

[0228] A construct comprising DNA encoding four repeats of A.beta.1-42 sequence (hereinafter also referred to as "A.beta..times.4") (SEQ ID NO: 34) was prepared by ligating four units of A.beta.1-42 via three linker sequences (GGTGGCGGTGGCTCG: SEQ ID NO: 29). First, two constructs, i.e., "A.beta.1-42+linker sequence+A.beta.1-6" and "A.beta.37-42+linker sequence+A.beta.1-42", were prepared by PCR amplification. Next, both constructs were mixed together and used as a template for PCR amplification with a sense primer designed to have a restriction enzyme site Xho I on the 5'-side of the nucleotide sequence of A.beta.1-42 and an antisense primer designed to have a restriction enzyme site Kpn I on the 3'-side of A.beta.1-42. The amplification products were electrophoresed on an agarose gel. Among bands that appeared in a ladder pattern, a band of about 560 bp corresponding to the molecular weight of A.beta..times.4 was excised and purified.

[0229] (3) Amplification and Cloning of DNA Encoding Tau

[0230] DNA encoding tau379-408 (hereinafter "tau" or "tau379-408") was prepared by preparing a sense strand and an antisense strand by oligonucleotide synthesis, and annealing the strands to form a duplex.

[0231] A construct comprising DNA encoding four repeats of tau sequence (hereinafter "tau.times.4") (SEQ ID NO: 23) was prepared by ligating four units of tau via three linker sequences (GGTGGCGGTGGCTCG: SEQ ID NO: 29). First, two constructs, i.e., "tau379-408+linker sequence+tau1-3" and "tau406-408+linker sequence+tau379-408", were prepared by PCR amplification. Next, both constructs were mixed together and used as a template for PCR amplification with a sense primer designed to have a restriction enzyme site Sal I on the 5'-side of the nucleotide sequence of tau379-408 and an antisense primer designed to have a stop codon and a restriction enzyme site Not I on the 3'-side of tau379-408. The amplification products were electrophoresed on an agarose gel. Among bands that appeared in a ladder pattern, a band of about 420 bp corresponding to the molecular weight of (tau379-408).times.4 was excised and purified.

[0232] Each of the products obtained in (1), (2), and (3) was inserted into a cloning vector (Zero Blunt TOPO PCR Kit for Sequencing, Invitrogen, Tokyo, Japan), followed by sequence analysis to select a clone having the sequence of interest.

[0233] 2. Ligation of Four Constructs Comprising DNAs Encoding IgL, A.beta..times.4, IgFc, and tau.times.4

[0234] Insertion of DNAs Encoding IgL Sequence, A.beta..times.4, IgFc Sequence, and tau.times.4 into Expression Vector

[0235] DNAs encoding the IgL sequence, (A.beta.1-42).times.4, and the IgFc sequence were excised from the cloning vectors at the restriction enzyme sites previously added to each DNA, and then purified from an agarose gel (MinElute Gel Extraction Kit, Qiagen, Tokyo, Japan). First, the three constructs of IgL, A.beta..times.4, and IgFc were ligated together using Ligation High (Toyobo, Tokyo, Japan). To compensate for low ligation efficiency due to the self-ligation of A.beta..times.4 and the low yield of the ligation product, this ligation product was used as a template for PCR amplification of the IgL-A.beta..times.4-IgFc ligation sequence, using the same sense primer as that used for the amplification of the IgL sequence (primer comprising the restriction enzyme site BamH I and the 5' end portion of the IgL sequence) and the same antisense primer as that used for the amplification of the IgFc sequence (primer comprising the restriction enzyme site Sal I and the 3' end portion of the IgFc sequence). Without using the TA cloning site, the pTarget vector was cleaved at the two restriction enzyme sites BamH I and Sal I located upstream and downstream, respectively, of the TA cloning site to give the same overhangs as those of the IgL-A.beta..times.4-IgFc ligation sequence. The IgL-A.beta..times.4-IgFc ligation sequence and the pTarget vector were electrophoresed on an agarose gel and purified (MinElute Gel Extraction Kit), and then ligated (Ligation High) to give pTarget/IgL-A.beta..times.4-IgFc. Sequence analysis confirmed that the proper sequence was obtained.

[0236] Next, the two restriction enzyme sites Sal I and Not I immediately downstream of the pTarget/IgL-A.beta..times.4-IgFc insert were cleaved to give the same overhangs as those of the tau.times.4 construct. Using Ligation High, tau.times.4 was integrated to complete the IgL-A.beta..times.4-IgFc-tau.times.4 ligation sequence within the pTarget vector (pTarget/IgL-A.beta..times.4-IgFc-tau.times.4).

[0237] 3. Insertion of DNA Encoding IgL-A.beta..times.4-IgFc-tau.times.4 into Expression Vector

[0238] The IgL-A.beta..times.4-IgFc-tau.times.4 ligation sequence was cleaved at the restriction enzyme sites BamH I at the 5' end portion and Not I at the 3' end portion, and excised from the pTarget vector. The pVAX1 vector (Life Technologies Japan, Tokyo, Japan) was cleaved at the same BamH I and Not I as those of the insert within the cloning site. The IgL-A.beta..times.4-IgFc-tau.times.4 ligation sequence and the pVAX1 vector were each electrophoresed on an agarose gel and then purified, and the resulting products were ligated (Ligation High) to complete a pVAX1/IgL-A.beta..times.4-IgFc-tau.times.4 plasmid. The pVAX1/IgL-A.beta..times.4-IgFc-tau.times.4 plasmid was produced on a large scale in E. coli, and final sequence analysis confirmed that the proper sequence was obtained.

[0239] pVAX1/IgL-A.beta..times.4-IgFc-tau.times.4 (hereinafter also referred to as "YM7555") was thus obtained as an example recombinant vector comprising DNAs encoding A.beta., the IgFc sequence, and tau. The nucleotide sequence of the obtained pVAX1/IgL-A.beta..times.4-IgFc-tau.times.4 insert is shown in SEQ ID NO: 30. The amino acid sequence of the expression product (polypeptide) (YM7555P) of IgL-A.beta..times.4-IgFc-tau.times.4 is shown in SEQ ID NO: 31. The amino acid sequences of the IgL sequence, A.beta..times.4, and tau.times.4 in YM7555P are shown in SEQ ID NOS: 33, 35, and 24, respectively. FIG. 1 shows a schematic diagram of the structure of IgL-A.beta..times.4-IgFc-tau.times.4.

[0240] Because the DNA encoding four repeats of A.beta.1-42 sequence (denoted as "A.beta..times.4" or "(A.beta.1-42).times.4") and the DNA encoding four repeats of tau sequence (denoted as "tau.times.4" or "(tau379-408).times.4") have high hydrophobicity, and comprise the repeats of sequence, these DNAs have a problem that they are likely to form a conformation due to self-ligation when ligated using a generally known cloning method, and their ligation to a vector or the ligation reaction between the constructs is unlikely to proceed.

[0241] To solve this problem, in the present Examples, the recombinant vector of the present invention was completed using a combination of the following unique methods developed by the present inventors, i.e., (a) large excess insert method, (b) amplification and ligation method, and (c) 100 colonies method and the like:

[0242] (a) Large Excess Insert Method

[0243] A general ligation method involves mixing a vector and an insert in a ratio of 1:1 to 1:10; in the present Examples, however, the vector and the insert were mixed in a ratio of 1:10 to 1:1000. For the ligation in the present Examples, an optimum mixing ratio from ratios of 1:10 to 1:1000 was adopted. To prevent self-ligation, the samples were heated at 95.degree. C. for 5 minutes immediately before the ligation.

[0244] (b) Amplification and Ligation Method

[0245] Appropriate restriction enzyme sites were added to both ends of a construct, and the construct was simultaneously amplified and ligated by PCR with primers having the 5' and 3' terminal nucleotide sequences of the final product.

[0246] (c) 100 Colonies Method

[0247] Because the above-described insert having repeats is susceptible to self-ligation and mutation, 100 colonies of clones were picked up and stored. Then, plasmids were purified from 12-24 colonies, and sequenced until clones having the plasmid comprising the proper nucleotide sequence were obtained.

[0248] 4. Recombinant Peptide and the Like

[0249] The A.beta.1-42 synthetic peptide was purchased from Peptide Institute, Inc. The expression product (YM3711P) of the recombinant vector pVAX1/IgL-(A.beta.1-42).times.4-huIgFc-huIL-4 (YM3711) (WO 2010/110408) and the expression product (YM7555P) of YM7555 were purified from serum-free culture supernatants of FreeStyle (trademark) 293-F cells (Invitrogen) transfected with YM3711 and YM7555, respectively. Specifically, the culture supernatants were obtained 4 days after the start of culturing the cells transfected with each of YM3711 and YM7555, and filtered. Because YM3711P and YM7555P comprised IgFc, these products were further purified using HiTrap Protein G column (GE Healthcare). Eluates were collected, and confirmed to have strong A.beta. immunoreactivity at O.D.280 nm with an anti-A.beta. monoclonal antibody, 6E10.

[0250] The recombinant tau protein was purchased from Wako Pure Chemical Industries, Ltd.

[0251] 5. Confirmation of Protein Expression in Cultured Cells

[0252] To confirm whether YM7555 induces the protein of interest, the YM7555 plasmid was transfected into HEK293 cells, and the properties of the protein (YM7555P) secreted into the culture supernatants were analyzed by Western blotting with anti-A.beta. antibody or anti-tau antibody. YM7555P was also quantitatively analyzed with a commercial sandwich ELISA kit for A.beta. quantification.

[0253] The results are shown in FIGS. 2 and 3. Western blotting showed that the expression product of YM7555 (YM7555P) was detected with both anti-A.beta. antibody and anti-tau antibody, as expected (FIGS. 2A and 2B). Although the estimated molecular weight of the monomer is about 60 kDa, a major band of 120 kDa appeared because the expressed YM7555P tends to form a dimer to multimer. Because the expression product of YM3711 (YM3711P) (monomer: about 60 kDa) and the (A.beta.1-42).times.4-IgFc product (A.beta..times.4-Fc) (monomer: about 40 kDa) detected with the anti-A.beta. antibody demonstrated similar properties (FIG. 2B), this phenomenon was assumed to be due to the tendency of the repeated structure to form a complex. The expression product of tau.times.4-IgFc (tau.times.4-Fc) (monomer: about 40 kDa) was detected only with the anti-tau antibody (FIG. 2A), which confirmed that all the searched products were detected in an antibody-specific manner. Furthermore, a molecule with a molecular weight smaller than about 60 kDa was not detected, which showed that the A.beta.1-42 molecule and tau molecule were not separately formed, and an A.beta.1-42-tau molecular complex was expressed as a single molecule and released extracellularly.

[0254] Since there is no commercial sandwich ELISA kit that recognizes the tau 379-408 sequence, each product was quantified with an ELISA kit for A.beta. quantification. As a result, sufficient levels of expression were observed for all the YM3711P, YM7555P, and A.beta..times.4-Fc products comprising the amino acid sequence of A.beta. (FIG. 3). The expression level of YM7555P was relatively lower than that of YM3711P, which was extremely high. This phenomenon was assumed to be due to high hydrophobicity of the translated tau.times.4 sequence, which causes a burden on the cells, or makes it difficult for the product to be secreted into the culture supernatant as a soluble protein.

[0255] In summary, the experiments showed that the polypeptide comprising both A.beta. and tau is expressed as a single molecule from the recombinant vector of the present invention.

[0256] FIG. 8 shows a schematic diagram of the structure of YM7555P.

[0257] 6. Induction of Anti-A.beta.1-42 Antibody and Anti-Tau Antibody in Tg Mice

[0258] New Zealand albino rabbits or 3.times.Tg mice carrying three-types of familial Alzheimer's disease-related mutated genes (B6; 129-Psen1.sup.tm1Mpm Tg(APPSwe,tauP301L)1Lfa/Mmjax) were regularly intramuscularly injected with YM7555, blood was collected with time, and variations in anti-A.beta. antibody and anti-tau antibody titers were measured by ELISA. The 3.times.Tg mice were sacrificed at 2 weeks after the final administration to collect the brains, and the therapeutic effect according to the present invention was evaluated by pathological and immunohistochemical analysis and quantification of proteins in the brain extracts by ELISA.

[0259] Initially, the titers of the anti-A.beta. antibody and the anti-tau antibody in the 3.times.Tg mice were measured.

[0260] FIG. 4 shows the schedule of immunization with YM7555 and blood collection. Blood was collected from the model mice (Tg) and wild-type mice before the immunization with YM7555 (S0; circles), and the mice were immunized with YM7555 at weeks 0, 2, 4, and 6 (black rhombuses). Blood was collected every 2 weeks after the initial immunization, and antibody titers were measured (white circles; S1, S2, S3, and S4).

[0261] The results are shown in FIG. 5. The results of induction of the anti-A.beta. antibody in the three Tg mice (changes in titer) are each shown in FIGS. 5A-C, and the results of induction of the anti-tau antibody in the three Tg mice are each shown in FIGS. 5D-F. For both antibodies, the antibody titer began to increase at weeks 4 to 8 after the initial immunization, and showed the maximum value at week 12. The OD value increased about 10-fold compared to the initial value. In FIG. 5, the symbol .diamond-solid. indicates the results obtained with 16-fold diluted plasma, and the symbol .box-solid. indicates the results obtained with 32-fold diluted plasma. The temporary decrease in antibody titer for the S2 plasma shown in FIG. 5C is considered to be due to a search error. In FIG. 5, "S0" indicates blood collection before the immunization, and "S1", "S2", "S3", and "S4" indicate blood collection at weeks 2, 4, 6, and 8, respectively. "S3f" indicates that the blood collection at week 6 is the final blood collection.

[0262] These results showed that the polypeptide in the form of a single molecule expressed from the recombinant vector of the present invention simultaneously induces the anti-A.beta. antibody and the anti-tau antibody at high titers in vivo.

[0263] In summary, the recombinant vector of the present invention was shown to be useful as an inducer of the anti-A.beta. antibody and the anti-tau antibody.

[0264] 7. Induction of Anti-A.beta.1-42 Antibody and Anti-Tau Antibody in Wild-Type Mice

[0265] The similar tests to those in the "6." section above were conducted using wild-type mice instead of the Tg mice.

[0266] As a result, the level of increase in the antibody titers of the anti-A.beta. antibody and the anti-tau antibody was comparable to that with the Tg mice.

[0267] The results are shown in FIG. 6. The results of induction of the anti-A.beta. antibody in the three wild-type mice (changes in titer) are each shown in FIGS. 6A-C, and the results of induction of the anti-tau antibody in the three wild-type mice are each shown in FIGS. 6D-F.

[0268] 8. Reduction in A.beta. Deposition and Tau Deposition in Tg Mice

[0269] To confirm whether the recombinant vector of the present invention reduces A.beta. deposition and tau deposition as intended, 3.times.Tg mice were regularly administered YM7555, and A.beta. and tau in the cerebral cortex (frontal lobe cortex) were quantified by sandwich ELISA. More specifically, the 3.times.Tg mice were sacrificed at 2 weeks after the final administration of YM7555, the brains were collected, and each of the proteins, i.e., (A) A.beta. (A.beta.1-42), (B) total tau (Total tau), and (C) phosphorylated tau (pTau), in the brain extracts was quantified by ELISA.

[0270] The results are shown in FIG. 7. In FIG. 7, "un Tx" indicates the amount of each protein in Tg mice without the administration of YM7555, and "YM7555 Tx" indicates the amount of each protein in the Tg mice that received the administration of YM7555.

[0271] As shown in FIG. 7, the amount of each of the proteins, i.e., (A) A.beta. (A.beta.1-42), (B) total tau (Total tau), and (C) phosphorylated tau (pTau), was clearly decreased in the YM7555-treated group. In particular, phosphorylated tau (C) having strong neurotoxicity was not detected at all in the YM7555-treated group, which was an unexpected result.

[0272] These results showed that the recombinant vector of the present invention is useful as a DNA vaccine for reducing brain A.beta. and brain tau.

[0273] The recombinant vector of the present invention was also shown to be extremely useful as a DNA vaccine for the prevention or treatment of Alzheimer's disease, because it reduced brain A.beta. and brain tau, which are considered to be causative substances for Alzheimer's disease, and in particular, reduced phosphorylated tau with strong toxicity.

SEQUENCE LISTING FREE TEXT

[0274] SEQ ID NO: 19: synthetic DNA

[0275] SEQ ID NO: 20: synthetic peptide

[0276] SEQ ID NO: 21: synthetic DNA

[0277] SEQ ID NO: 22: synthetic peptide

[0278] SEQ ID NO: 23: synthetic DNA

[0279] SEQ ID NO: 24: synthetic peptide

[0280] SEQ ID NO: 29: synthetic DNA

[0281] SEQ ID NO: 30: synthetic DNA

[0282] SEQ ID NO: 31: synthetic peptide

[0283] SEQ ID NO: 34: synthetic DNA

[0284] SEQ ID NO: 35: synthetic peptide

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

[0285] This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled "2017-11-29_SequenceListing_4456-0239PUS1.txt" created on Nov. 29, 2017 and is 61,056 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.

Sequence CWU 1

1

351129DNAHomo sapiensCDS(1)..(129) 1gat gca gaa ttc cga cat gac tca gga tat gaa gtt cat cat caa aaa 48Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15ttg gtg ttc ttt gca gaa gat gtg ggt tca aac aaa ggt gca atc att 96Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30gga ctc atg gtg ggc ggt gtt gtc ata gcg aca 129Gly Leu Met Val Gly Gly Val Val Ile Ala Thr 35 40243PRTHomo sapiens 2Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val Ile Ala Thr 35 40360DNAHomo sapiensCDS(1)..(60) 3gat gca gaa ttc cga cat gac tca gga tat gaa gtt cat cat caa aaa 48Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15ttg gtg ttc ttt 60Leu Val Phe Phe 20420PRTHomo sapiens 4Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe 205120DNAHomo sapiensCDS(1)..(120) 5gat gca gaa ttc cga cat gac tca gga tat gaa gtt cat cat caa aaa 48Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15ttg gtg ttc ttt gca gaa gat gtg ggt tca aac aaa ggt gca atc att 96Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30gga ctc atg gtg ggc ggt gtt gtc 120Gly Leu Met Val Gly Gly Val Val 35 40640PRTHomo sapiens 6Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val 35 407126DNAHomo sapiensCDS(1)..(126) 7gat gca gaa ttc cga cat gac tca gga tat gaa gtt cat cat caa aaa 48Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15ttg gtg ttc ttt gca gaa gat gtg ggt tca aac aaa ggt gca atc att 96Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30gga ctc atg gtg ggc ggt gtt gtc ata gcg 126Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40842PRTHomo sapiens 8Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val Ile Ala 35 409129DNAMus musculusCDS(1)..(129) 9gat gca gaa ttc gga cat gat tca gga ttt gaa gtc cgc cat caa aaa 48Asp Ala Glu Phe Gly His Asp Ser Gly Phe Glu Val Arg His Gln Lys1 5 10 15ctg gtg ttc ttt gct gaa gat gtg ggt tcg aac aaa ggc gcc atc atc 96Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30gga ctc atg gtg ggc ggc gtt gtc ata gca acc 129Gly Leu Met Val Gly Gly Val Val Ile Ala Thr 35 401043PRTMus musculus 10Asp Ala Glu Phe Gly His Asp Ser Gly Phe Glu Val Arg His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val Ile Ala Thr 35 401160DNAMus musculusCDS(1)..(60) 11gat gca gaa ttc gga cat gat tca gga ttt gaa gtc cgc cat caa aaa 48Asp Ala Glu Phe Gly His Asp Ser Gly Phe Glu Val Arg His Gln Lys1 5 10 15ctg gtg ttc ttt 60Leu Val Phe Phe 201220PRTMus musculus 12Asp Ala Glu Phe Gly His Asp Ser Gly Phe Glu Val Arg His Gln Lys1 5 10 15Leu Val Phe Phe 2013120DNAMus musculusCDS(1)..(120) 13gat gca gaa ttc gga cat gat tca gga ttt gaa gtc cgc cat caa aaa 48Asp Ala Glu Phe Gly His Asp Ser Gly Phe Glu Val Arg His Gln Lys1 5 10 15ctg gtg ttc ttt gct gaa gat gtg ggt tcg aac aaa ggc gcc atc atc 96Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30gga ctc atg gtg ggc ggc gtt gtc 120Gly Leu Met Val Gly Gly Val Val 35 401440PRTMus musculus 14Asp Ala Glu Phe Gly His Asp Ser Gly Phe Glu Val Arg His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val 35 4015126DNAMus musculusCDS(1)..(126) 15gat gca gaa ttc gga cat gat tca gga ttt gaa gtc cgc cat caa aaa 48Asp Ala Glu Phe Gly His Asp Ser Gly Phe Glu Val Arg His Gln Lys1 5 10 15ctg gtg ttc ttt gct gaa gat gtg ggt tcg aac aaa ggc gcc atc atc 96Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30gga ctc atg gtg ggc ggc gtt gtc ata gca 126Gly Leu Met Val Gly Gly Val Val Ile Ala 35 401642PRTMus musculus 16Asp Ala Glu Phe Gly His Asp Ser Gly Phe Glu Val Arg His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40175811DNAHomo sapiensCDS(323)..(1648) 17ggacggccga gcggcagggc gctcgcgcgc gcccactagt ggccggagga gaaggctccc 60gcggaggccg cgctgcccgc cccctcccct ggggaggctc gcgttcccgc tgctcgcgcc 120tgcgccgccc gccggcctca ggaacgcgcc ctcttcgccg gcgcgcgccc tcgcagtcac 180cgccacccac cagctccggc accaacagca gcgccgctgc caccgcccac cttctgccgc 240cgccaccaca gccaccttct cctcctccgc tgtcctctcc cgtcctcgcc tctgtcgact 300atcaggtgaa ctttgaacca gg atg gct gag ccc cgc cag gag ttc gaa gtg 352 Met Ala Glu Pro Arg Gln Glu Phe Glu Val 1 5 10atg gaa gat cac gct ggg acg tac ggg ttg ggg gac agg aaa gat cag 400Met Glu Asp His Ala Gly Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln 15 20 25ggg ggc tac acc atg cac caa gac caa gag ggt gac acg gac gct ggc 448Gly Gly Tyr Thr Met His Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly 30 35 40ctg aaa gaa tct ccc ctg cag acc ccc act gag gac gga tct gag gaa 496Leu Lys Glu Ser Pro Leu Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu 45 50 55ccg ggc tct gaa acc tct gat gct aag agc act cca aca gcg gaa gat 544Pro Gly Ser Glu Thr Ser Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp 60 65 70gtg aca gca ccc tta gtg gat gag gga gct ccc ggc aag cag gct gcc 592Val Thr Ala Pro Leu Val Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala75 80 85 90gcg cag ccc cac acg gag atc cca gaa gga acc aca gct gaa gaa gca 640Ala Gln Pro His Thr Glu Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala 95 100 105ggc att gga gac acc ccc agc ctg gaa gac gaa gct gct ggt cac gtg 688Gly Ile Gly Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val 110 115 120acc caa gct cgc atg gtc agt aaa agc aaa gac ggg act gga agc gat 736Thr Gln Ala Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp 125 130 135gac aaa aaa gcc aag ggg gct gat ggt aaa acg aag atc gcc aca ccg 784Asp Lys Lys Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro 140 145 150cgg gga gca gcc cct cca ggc cag aag ggc cag gcc aac gcc acc agg 832Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg155 160 165 170att cca gca aaa acc ccg ccc gct cca aag aca cca ccc agc tct ggt 880Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly 175 180 185gaa cct cca aaa tca ggg gat cgc agc ggc tac agc agc ccc ggc tcc 928Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser 190 195 200cca ggc act ccc ggc agc cgc tcc cgc acc ccg tcc ctt cca acc cca 976Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro 205 210 215ccc acc cgg gag ccc aag aag gtg gca gtg gtc cgt act cca ccc aag 1024Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys 220 225 230tcg ccg tct tcc gcc aag agc cgc ctg cag aca gcc ccc gtg ccc atg 1072Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met235 240 245 250cca gac ctg aag aat gtc aag tcc aag atc ggc tcc act gag aac ctg 1120Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu 255 260 265aag cac cag ccg gga ggc ggg aag gtg cag ata att aat aag aag ctg 1168Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Ile Asn Lys Lys Leu 270 275 280gat ctt agc aac gtc cag tcc aag tgt ggc tca aag gat aat atc aaa 1216Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys 285 290 295cac gtc ccg gga ggc ggc agt gtg caa ata gtc tac aaa cca gtt gac 1264His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Pro Val Asp 300 305 310ctg agc aag gtg acc tcc aag tgt ggc tca tta ggc aac atc cat cat 1312Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His315 320 325 330aaa cca gga ggt ggc cag gtg gaa gta aaa tct gag aag ctt gac ttc 1360Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe 335 340 345aag gac aga gtc cag tcg aag att ggg tcc ctg gac aat atc acc cac 1408Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His 350 355 360gtc cct ggc gga gga aat aaa aag att gaa acc cac aag ctg acc ttc 1456Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe 365 370 375cgc gag aac gcc aaa gcc aag aca gac cac ggg gcg gag atc gtg tac 1504Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr 380 385 390aag tcg cca gtg gtg tct ggg gac acg tct cca cgg cat ctc agc aat 1552Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn395 400 405 410gtc tcc tcc acc ggc agc atc gac atg gta gac tcg ccc cag ctc gcc 1600Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala 415 420 425acg cta gct gac gag gtg tct gcc tcc ctg gcc aag cag ggt ttg tga 1648Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 430 435 440tcaggcccct ggggcggtca ataattgtgg agaggagaga atgagagagt gtggaaaaaa 1708aaagaataat gacccggccc ccgccctctg cccccagctg ctcctcgcag ttcggttaat 1768tggttaatca cttaacctgc ttttgtcact cggctttggc tcgggacttc aaaatcagtg 1828atgggagtaa gagcaaattt catctttcca aattgatggg tgggctagta ataaaatatt 1888taaaaaaaaa cattcaaaaa catggccaca tccaacattt cctcaggcaa ttccttttga 1948ttcttttttc ttccccctcc atgtagaaga gggagaagga gaggctctga aagctgcttc 2008tgggggattt caagggactg ggggtgccaa ccacctctgg ccctgttgtg ggggtgtcac 2068agaggcagtg gcagcaacaa aggatttgaa acttggtgtg ttcgtggagc cacaggcaga 2128cgatgtcaac cttgtgtgag tgtgacgggg gttggggtgg ggcgggaggc cacgggggag 2188gccgaggcag gggctgggca gaggggagag gaagcacaag aagtgggagt gggagaggaa 2248gccacgtgct ggagagtaga catccccctc cttgccgctg ggagagccaa ggcctatgcc 2308acctgcagcg tctgagcggc cgcctgtcct tggtggccgg gggtgggggc ctgctgtggg 2368tcagtgtgcc accctctgca gggcagcctg tgggagaagg gacagcgggt aaaaagagaa 2428ggcaagctgg caggagggtg gcacttcgtg gatgacctcc ttagaaaaga ctgaccttga 2488tgtcttgaga gcgctggcct cttcctccct ccctgcaggg tagggggcct gagttgaggg 2548gcttccctct gctccacaga aaccctgttt tattgagttc tgaaggttgg aactgctgcc 2608atgattttgg ccactttgca gacctgggac tttagggcta accagttctc tttgtaagga 2668cttgtgcctc ttgggagacg tccacccgtt tccaagcctg ggccactggc atctctggag 2728tgtgtggggg tctgggaggc aggtcccgag ccccctgtcc ttcccacggc cactgcagtc 2788accccgtctg cgccgctgtg ctgttgtctg ccgtgagagc ccaatcactg cctatacccc 2848tcatcacacg tcacaatgtc ccgaattccc agcctcacca ccccttctca gtaatgaccc 2908tggttggttg caggaggtac ctactccata ctgagggtga aattaaggga aggcaaagtc 2968caggcacaag agtgggaccc cagcctctca ctctcagttc cactcatcca actgggaccc 3028tcaccacgaa tctcatgatc tgattcggtt ccctgtctcc tcctcccgtc acagatgtga 3088gccagggcac tgctcagctg tgaccctagg tgtttctgcc ttgttgacat ggagagagcc 3148ctttcccctg agaaggcctg gccccttcct gtgctgagcc cacagcagca ggctgggtgt 3208cttggttgtc agtggtggca ccaggatgga agggcaaggc acccagggca ggcccacagt 3268cccgctgtcc cccacttgca ccctagcttg tagctgccaa cctcccagac agcccagccc 3328gctgctcagc tccacatgca tagtatcagc cctccacacc cgacaaaggg gaacacaccc 3388ccttggaaat ggttcttttc ccccagtccc agctggaagc catgctgtct gttctgctgg 3448agcagctgaa catatacata gatgttgccc tgccctcccc atctgcaccc tgttgagttg 3508tagttggatt tgtctgttta tgcttggatt caccagagtg actatgatag tgaaaagaaa 3568aaaaaaaaaa aaaaaggacg catgtatctt gaaatgcttg taaagaggtt tctaacccac 3628cctcacgagg tgtctctcac ccccacactg ggactcgtgt ggcctgtgtg gtgccaccct 3688gctggggcct cccaagtttt gaaaggcttt cctcagcacc tgggacccaa cagagaccag 3748cttctagcag ctaaggaggc cgttcagctg tgacgaaggc ctgaagcaca ggattaggac 3808tgaagcgatg atgtcccctt ccctacttcc ccttggggct ccctgtgtca gggcacagac 3868taggtcttgt ggctggtctg gcttgcggcg cgaggatggt tctctctggt catagcccga 3928agtctcatgg cagtcccaaa ggaggcttac aactcctgca tcacaagaaa aaggaagcca 3988ctgccagctg gggggatctg cagctcccag aagctccgtg agcctcagcc acccctcaga 4048ctgggttcct ctccaagctc gccctctgga ggggcagcgc agcctcccac caagggccct 4108gcgaccacag cagggattgg gatgaattgc ctgtcctgga tctgctctag aggcccaagc 4168tgcctgcctg aggaaggatg acttgacaag tcaggagaca ctgttcccaa agccttgacc 4228agagcacctc agcccgctga ccttgcacaa actccatctg ctgccatgag aaaagggaag 4288ccgcctttgc aaaacattgc tgcctaaaga aactcagcag cctcaggccc aattctgcca 4348cttctggttt gggtacagtt aaaggcaacc ctgagggact tggcagtaga aatccagggc 4408ctcccctggg gctggcagct tcgtgtgcag ctagagcttt acctgaaagg aagtctctgg 4468gcccagaact ctccaccaag agcctccctg ccgttcgctg agtcccagca attctcctaa 4528gttgaaggga tctgagaagg agaaggaaat gtggggtaga tttggtggtg gttagagata 4588tgcccccctc attactgcca acagtttcgg ctgcatttct tcacgcacct cggttcctct 4648tcctgaagtt cttgtgccct gctcttcagc accatgggcc ttcttatacg gaaggctctg 4708ggatctcccc cttgtggggc aggctcttgg ggccagccta agatcatggt ttagggtgat 4768cagtgctggc agataaattg aaaaggcacg ctggcttgtg atcttaaatg aggacaatcc 4828ccccagggct gggcactcct cccctcccct cacttctccc acctgcagag ccagtgtcct 4888tgggtgggct agataggata tactgtatgc cggctccttc aagctgctga ctcactttat 4948caatagttcc atttaaattg acttcagtgg tgagactgta tcctgtttgc tattgcttgt 5008tgtgctatgg ggggaggggg gaggaatgtg taagatagtt aacatgggca aagggagatc 5068ttggggtgca gcacttaaac tgcctcgtaa cccttttcat gatttcaacc acatttgcta 5128gagggaggga gcagccacgg agttagaggc ccttggggtt tctcttttcc actgacaggc 5188tttcccaggc agctggctag ttcattccct ccccagccag gtgcaggcgt aggaatatgg 5248acatctggtt gctttggcct gctgccctct ttcaggggtc ctaagcccac aatcatgcct 5308ccctaagacc ttggcatcct tccctctaag ccgttggcac ctctgtgcca cctctcacac 5368tggctccaga cacacagcct gtgcttttgg agctgagatc actcgcttca ccctcctcat 5428ctttgttctc caagtaaagc cacgaggtcg gggcgagggc agaggtgatc acctgcgtgt 5488cccatctaca gacctgcagc ttcataaaac ttctgatttc tcttcagctt tgaaaagggt 5548taccctgggc actggcctag agcctcacct cctaatagac ttagccccat gagtttgcca 5608tgttgagcag gactatttct ggcacttgca agtcccatga tttcttcggt aattctgagg 5668gtggggggag ggacatgaaa tcatcttagc ttagctttct gtctgtgaat gtctatatag 5728tgtattgtgt gttttaacaa atgatttaca ctgactgttg ctgtaaaagt gaatttggaa 5788ataaagttat tactctgatt aaa 581118441PRTHomo sapiens 18Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly1 5 10 15Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val65 70 75 80Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu 85 90 95Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro 100 105 110Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val 115 120 125Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly 130 135 140Ala Asp Gly Lys Thr

Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro145 150 155 160Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro 165 170 175Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly 180 185 190Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser 195 200 205Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys 210 215 220Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys225 230 235 240Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val 245 250 255Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly 260 265 270Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln 275 280 285Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly 290 295 300Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser305 310 315 320Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln 325 330 335Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser 340 345 350Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn 355 360 365Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala 370 375 380Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser385 390 395 400Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser 405 410 415Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val 420 425 430Ser Ala Ser Leu Ala Lys Gln Gly Leu 435 4401933DNAArtificialSynthetic DNACDS(1)..(33) 19gat aat atc aaa cac gtc ccg gga ggc ggc agt 33Asp Asn Ile Lys His Val Pro Gly Gly Gly Ser1 5 102011PRTArtificialSynthetic Construct 20Asp Asn Ile Lys His Val Pro Gly Gly Gly Ser1 5 102190DNAArtificialSynthetic DNACDS(1)..(90) 21cgc gag aac gcc aaa gcc aag aca gac cac ggg gcg gag atc gtg tac 48Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr1 5 10 15aag tcg cca gtg gtg tct ggg gac acg tct cca cgg cat ctc 90Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu 20 25 302230PRTArtificialSynthetic Construct 22Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr1 5 10 15Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu 20 25 3023408DNAArtificialSynthetic DNACDS(1)..(408) 23cgc gag aac gcc aaa gcc aag aca gac cac ggg gcg gag atc gtg tac 48Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr1 5 10 15aag tcg cca gtg gtg tct ggg gac acg tct cca cgg cat ctc ggt ggc 96Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Gly Gly 20 25 30ggt ggc tcg cgc gag aac gcc aaa gcc aag aca gac cac ggg gcg gag 144Gly Gly Ser Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu 35 40 45atc gtg tac aag tcg cca gtg gtg tct ggg gac acg tct cca cgg cat 192Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His 50 55 60ctc ggt ggc ggt ggc tcg cgc gag aac gcc aaa gcc aag aca gac cac 240Leu Gly Gly Gly Gly Ser Arg Glu Asn Ala Lys Ala Lys Thr Asp His65 70 75 80ggg gcg gag atc gtg tac aag tcg cca gtg gtg tct ggg gac acg tct 288Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser 85 90 95cca cgg cat ctc ggt ggc ggt ggc tcg cgc gag aac gcc aaa gcc aag 336Pro Arg His Leu Gly Gly Gly Gly Ser Arg Glu Asn Ala Lys Ala Lys 100 105 110aca gac cac ggg gcg gag atc gtg tac aag tcg cca gtg gtg tct ggg 384Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly 115 120 125gac acg tct cca cgg cat ctc tga 408Asp Thr Ser Pro Arg His Leu 130 13524135PRTArtificialSynthetic Construct 24Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr1 5 10 15Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Gly Gly 20 25 30Gly Gly Ser Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu 35 40 45Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His 50 55 60Leu Gly Gly Gly Gly Ser Arg Glu Asn Ala Lys Ala Lys Thr Asp His65 70 75 80Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser 85 90 95Pro Arg His Leu Gly Gly Gly Gly Ser Arg Glu Asn Ala Lys Ala Lys 100 105 110Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly 115 120 125Asp Thr Ser Pro Arg His Leu 130 13525696DNAHomo sapiensCDS(1)..(696) 25gag ccc aaa tct tct gac aaa act cac aca tcc cca ccg tcc cca gca 48Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala1 5 10 15cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc 96Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg 144Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg 192Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag 240Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70 75 80tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag 288Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc 336Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc 384Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc 432Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc 480Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser145 150 155 160gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac 528Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac 576Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc 624Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag 672Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220agc ctc tcc ctg tct ccg ggt aaa 696Ser Leu Ser Leu Ser Pro Gly Lys225 23026232PRTHomo sapiens 26Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala1 5 10 15Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70 75 80Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser145 150 155 160Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220Ser Leu Ser Leu Ser Pro Gly Lys225 23027711DNAMus musculusCDS(1)..(711) 27gag ccc cgg gtg ccc atc acc cag aac ccc agc ccc cca ctg aaa gag 48Glu Pro Arg Val Pro Ile Thr Gln Asn Pro Ser Pro Pro Leu Lys Glu1 5 10 15agc ccc ccc tct gcc gct cct gat ctg ctg ggc gga ccc agc gtg ttc 96Ser Pro Pro Ser Ala Ala Pro Asp Leu Leu Gly Gly Pro Ser Val Phe 20 25 30atc ttc cca ccc aag atc aag gac gtg ctg atg atc agc ctg agc ccc 144Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro 35 40 45atg gtg acc agc gtg gtg gtg gac gtg tcc gag gac gac ccc gac gtg 192Met Val Thr Ser Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val 50 55 60cag atc agt tgg ttc gtg aac aac gtg gag gtg cac acc gcc cag acc 240Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr65 70 75 80cag acc cac cgg gag gac tac aac agc acc ctg aga gtg gtg tcc gcc 288Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala 85 90 95ctg ccc atc cag cac cag gac tgg atg agc ggc aaa gaa ttc aag agc 336Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Ser 100 105 110aaa gtg aac aac cgg gcc ctg ccc agc ccc atc gag aaa acc atc agc 384Lys Val Asn Asn Arg Ala Leu Pro Ser Pro Ile Glu Lys Thr Ile Ser 115 120 125aag ccc aga ggc cct gtg cgg gct cct cag gtg tac gtg ctg ccc cca 432Lys Pro Arg Gly Pro Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro 130 135 140ccc gcc gag gaa atg acc aag aaa gag ttc agc ctg acc agc atg atc 480Pro Ala Glu Glu Met Thr Lys Lys Glu Phe Ser Leu Thr Ser Met Ile145 150 155 160acc ggc ttt ctg ccc gcc gag atc gcc gtg gac tgg acc agc aac ggc 528Thr Gly Phe Leu Pro Ala Glu Ile Ala Val Asp Trp Thr Ser Asn Gly 165 170 175cgg acc gag cag aac tac aag aac acc gcc acc gtg ctg gac agc gac 576Arg Thr Glu Gln Asn Tyr Lys Asn Thr Ala Thr Val Leu Asp Ser Asp 180 185 190ggc agc tac ttc atg tac agc aag ctg cgg gtg cag aag tcc acc tgg 624Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Gln Lys Ser Thr Trp 195 200 205gag aga ggc agc ctg ttc gcc agc agc gtg gtg cac gag ggc ctg cac 672Glu Arg Gly Ser Leu Phe Ala Ser Ser Val Val His Glu Gly Leu His 210 215 220aac cac ctg acc acc aag acc atc agc cgg tcc ctg gga 711Asn His Leu Thr Thr Lys Thr Ile Ser Arg Ser Leu Gly225 230 23528237PRTMus musculus 28Glu Pro Arg Val Pro Ile Thr Gln Asn Pro Ser Pro Pro Leu Lys Glu1 5 10 15Ser Pro Pro Ser Ala Ala Pro Asp Leu Leu Gly Gly Pro Ser Val Phe 20 25 30Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro 35 40 45Met Val Thr Ser Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val 50 55 60Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr65 70 75 80Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala 85 90 95Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Ser 100 105 110Lys Val Asn Asn Arg Ala Leu Pro Ser Pro Ile Glu Lys Thr Ile Ser 115 120 125Lys Pro Arg Gly Pro Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro 130 135 140Pro Ala Glu Glu Met Thr Lys Lys Glu Phe Ser Leu Thr Ser Met Ile145 150 155 160Thr Gly Phe Leu Pro Ala Glu Ile Ala Val Asp Trp Thr Ser Asn Gly 165 170 175Arg Thr Glu Gln Asn Tyr Lys Asn Thr Ala Thr Val Leu Asp Ser Asp 180 185 190Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Gln Lys Ser Thr Trp 195 200 205Glu Arg Gly Ser Leu Phe Ala Ser Ser Val Val His Glu Gly Leu His 210 215 220Asn His Leu Thr Thr Lys Thr Ile Ser Arg Ser Leu Gly225 230 2352915DNAArtificialSynthetic DNA 29ggtggcggtg gctcg 15301740DNAArtificialSynthetic DNACDS(1)..(1740) 30atg gag aca gac aca ctc ctg cta tgg gta ctg ctg ctc tgg gtt cca 48Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15ggt tcc act ggt gac gcg gcc ctc gag gat gca gaa ttc cga cat gac 96Gly Ser Thr Gly Asp Ala Ala Leu Glu Asp Ala Glu Phe Arg His Asp 20 25 30tca gga tat gaa gtt cat cat caa aaa ttg gtg ttc ttt gca gaa gat 144Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp 35 40 45gtg ggt tca aac aaa ggt gca atc att gga ctc atg gtg ggc ggt gtt 192Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val 50 55 60gtc ata gcg ggt ggc ggt ggc tcg gat gca gaa ttc cga cat gac tca 240Val Ile Ala Gly Gly Gly Gly Ser Asp Ala Glu Phe Arg His Asp Ser65 70 75 80gga tat gaa gtt cat cat caa aaa ttg gtg ttc ttt gca gaa gat gtg 288Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val 85 90 95ggt tca aac aaa ggt gca atc att gga ctc atg gtg ggc ggt gtt gtc 336Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val 100 105 110ata gcg ggt ggc ggt ggc tcg gat gca gaa ttc cga cat gac tca gga 384Ile Ala Gly Gly Gly Gly Ser Asp Ala Glu Phe Arg His Asp Ser Gly 115 120 125tat gaa gtt cat cat caa aaa ttg gtg ttc ttt gca gaa gat gtg ggt 432Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly 130 135 140tca aac aaa ggt gca atc att gga ctc atg gtg ggc ggt gtt gtc ata 480Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile145 150 155 160gcg ggt ggc ggt ggc tcg gat gca gaa ttc cga cat gac tca gga tat 528Ala Gly Gly Gly Gly Ser Asp Ala Glu Phe Arg His Asp Ser Gly Tyr 165 170 175gaa gtt cat cat caa aaa ttg gtg ttc ttt gca gaa gat gtg ggt tca 576Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser 180 185 190aac aaa ggt gca atc att gga ctc atg gtg ggc ggt gtt gtc ata gcg 624Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala 195 200 205ggt acc gag ccc aaa tct tct gac aaa act cac aca tcc cca ccg tcc 672Gly Thr Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser 210 215 220cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca 720Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro225 230 235 240aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc 768Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

Thr Pro Glu Val Thr Cys 245 250 255gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg 816Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag 864Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg 912Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac 960His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn305 310 315 320aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg 1008Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335cag ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg gat gag 1056Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 340 345 350ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat 1104Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac 1152Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc 1200Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe385 390 395 400ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac 1248Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg 1296Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430cag aag agc ctc tcc ctg tct ccg ggt aaa gtc gac cgc gag aac gcc 1344Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Val Asp Arg Glu Asn Ala 435 440 445aaa gcc aag aca gac cac ggg gcg gag atc gtg tac aag tcg cca gtg 1392Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val 450 455 460gtg tct ggg gac acg tct cca cgg cat ctc ggt ggc ggt ggc tcg cgc 1440Val Ser Gly Asp Thr Ser Pro Arg His Leu Gly Gly Gly Gly Ser Arg465 470 475 480gag aac gcc aaa gcc aag aca gac cac ggg gcg gag atc gtg tac aag 1488Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys 485 490 495tcg cca gtg gtg tct ggg gac acg tct cca cgg cat ctc ggt ggc ggt 1536Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Gly Gly Gly 500 505 510ggc tcg cgc gag aac gcc aaa gcc aag aca gac cac ggg gcg gag atc 1584Gly Ser Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile 515 520 525gtg tac aag tcg cca gtg gtg tct ggg gac acg tct cca cgg cat ctc 1632Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu 530 535 540ggt ggc ggt ggc tcg cgc gag aac gcc aaa gcc aag aca gac cac ggg 1680Gly Gly Gly Gly Ser Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly545 550 555 560gcg gag atc gtg tac aag tcg cca gtg gtg tct ggg gac acg tct cca 1728Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro 565 570 575cgg cat ctc tga 1740Arg His Leu31579PRTArtificialSynthetic Construct 31Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Asp Ala Ala Leu Glu Asp Ala Glu Phe Arg His Asp 20 25 30Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp 35 40 45Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val 50 55 60Val Ile Ala Gly Gly Gly Gly Ser Asp Ala Glu Phe Arg His Asp Ser65 70 75 80Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val 85 90 95Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val 100 105 110Ile Ala Gly Gly Gly Gly Ser Asp Ala Glu Phe Arg His Asp Ser Gly 115 120 125Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly 130 135 140Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile145 150 155 160Ala Gly Gly Gly Gly Ser Asp Ala Glu Phe Arg His Asp Ser Gly Tyr 165 170 175Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser 180 185 190Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala 195 200 205Gly Thr Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser 210 215 220Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro225 230 235 240Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn305 310 315 320Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 340 345 350Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe385 390 395 400Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Val Asp Arg Glu Asn Ala 435 440 445Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val 450 455 460Val Ser Gly Asp Thr Ser Pro Arg His Leu Gly Gly Gly Gly Ser Arg465 470 475 480Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys 485 490 495Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Gly Gly Gly 500 505 510Gly Ser Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile 515 520 525Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu 530 535 540Gly Gly Gly Gly Ser Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly545 550 555 560Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro 565 570 575Arg His Leu3269DNAHomo sapiensCDS(1)..(69) 32atg gag aca gac aca ctc ctg cta tgg gta ctg ctg ctc tgg gtt cca 48Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15ggt tcc act ggt gac gcg gcc 69Gly Ser Thr Gly Asp Ala Ala 203323PRTHomo sapiens 33Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Asp Ala Ala 2034549DNAArtificialSynthetic DNACDS(1)..(549) 34gat gca gaa ttc cga cat gac tca gga tat gaa gtt cat cat caa aaa 48Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15ttg gtg ttc ttt gca gaa gat gtg ggt tca aac aaa ggt gca atc att 96Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30gga ctc atg gtg ggc ggt gtt gtc ata gcg ggt ggc ggt ggc tcg gat 144Gly Leu Met Val Gly Gly Val Val Ile Ala Gly Gly Gly Gly Ser Asp 35 40 45gca gaa ttc cga cat gac tca gga tat gaa gtt cat cat caa aaa ttg 192Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu 50 55 60gtg ttc ttt gca gaa gat gtg ggt tca aac aaa ggt gca atc att gga 240Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly65 70 75 80ctc atg gtg ggc ggt gtt gtc ata gcg ggt ggc ggt ggc tcg gat gca 288Leu Met Val Gly Gly Val Val Ile Ala Gly Gly Gly Gly Ser Asp Ala 85 90 95gaa ttc cga cat gac tca gga tat gaa gtt cat cat caa aaa ttg gtg 336Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val 100 105 110ttc ttt gca gaa gat gtg ggt tca aac aaa ggt gca atc att gga ctc 384Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu 115 120 125atg gtg ggc ggt gtt gtc ata gcg ggt ggc ggt ggc tcg gat gca gaa 432Met Val Gly Gly Val Val Ile Ala Gly Gly Gly Gly Ser Asp Ala Glu 130 135 140ttc cga cat gac tca gga tat gaa gtt cat cat caa aaa ttg gtg ttc 480Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe145 150 155 160ttt gca gaa gat gtg ggt tca aac aaa ggt gca atc att gga ctc atg 528Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met 165 170 175gtg ggc ggt gtt gtc ata gcg 549Val Gly Gly Val Val Ile Ala 18035183PRTArtificialSynthetic Construct 35Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val Ile Ala Gly Gly Gly Gly Ser Asp 35 40 45Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu 50 55 60Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly65 70 75 80Leu Met Val Gly Gly Val Val Ile Ala Gly Gly Gly Gly Ser Asp Ala 85 90 95Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val 100 105 110Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu 115 120 125Met Val Gly Gly Val Val Ile Ala Gly Gly Gly Gly Ser Asp Ala Glu 130 135 140Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe145 150 155 160Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met 165 170 175Val Gly Gly Val Val Ile Ala 180

Claims

1. A polypeptide comprising a fusion protein having a region consisting of (i)-(vi): (i) four (4) repeats of A.beta.1-42 or A.beta.1-43 sequence, (ii) an immunoglobulin Fc, and (iii) four (4) repeats of tau sequence, (iv) optionally an Ig leader sequence, (v) optionally peptide linker/s, and (vi) optionally amino acid sequence/s encoded by restriction enzyme site/s, wherein the four (4) repeats of tau sequence comprises four (4) repeats of the amino acid sequences as set forth in SEQ ID NO: 22.

2. The polypeptide according to claim 1, wherein the amyloid 13 is A.beta.1-42.

3. The polypeptide according to claim 1, wherein the polypeptide does not comprise a portion containing IL-4.

4. The polypeptide according to claim 1, wherein the repeats of A.beta.1-42 or A.beta.1-43, an immunoglobulin Fc sequence, and repeats of tau sequence of the fusion protein are contained in said order in the fusion protein.

5. The polypeptide according to claim 1, wherein each repeat of A.beta.1-42 comprises the amino acid sequence of SEQ ID NO: 8.

6. The polypeptide according to claim 1, wherein the four (4) repeats of tau sequence comprises the amino acid sequence as set forth in SEQ ID NO: 24.

7. A vaccine for prevention or treatment of Alzheimer's disease, comprising the polypeptide according to claim 1.

8. A vaccine for reducing brain A.beta. and brain tau, comprising the polypeptide according to claim 1.

9. An inducer of anti-A.beta. antibody and anti-tau antibody, comprising the polypeptide according claim 1.

10. A recombinant vector comprising DNA having a region consisting of (i)-(vi): (i) DNA encoding four (4) repeats of A.beta.1-42 or A.beta.1-43 sequence, (ii) DNA encoding an immunoglobulin Fc sequence, (iii) DNA encoding four (4) repeats of tau sequence, (iv) optionally DNA encoding an Ig leader sequence, (v) optionally DNA encoding peptide linker/s, and (vi) optionally restriction enzyme sites, wherein the four (4) repeats of tau sequence comprises four (4) repeats of the amino acid sequences as set forth in SEQ ID NO: 22.

11. A DNA vaccine for prevention or treatment of Alzheimer's disease, comprising the recombinant vector according to claim 10.

12. A DNA vaccine for reducing brain A.beta. and brain tau, comprising the recombinant vector according to claim 10.

13. An inducer of anti-A.beta. antibody and anti-tau antibody, comprising the recombinant vector according to claim 10.