WO2020096046A1

WO2020096046A1 - Senescent t cell-targeting vaccine for preventing or treating abnormal sugar metabolism

Info

Publication number: WO2020096046A1
Application number: PCT/JP2019/043886
Authority: WO
Inventors: 啓徳中神; 翔太吉田
Original assignee: 国立大学法人大阪大学
Priority date: 2018-11-09
Filing date: 2019-11-08
Publication date: 2020-05-14

Abstract

Provided is a vaccine for preventing or treating abnormal sugar metabolism, said vaccine comprising any of (1) to (3): (1) a polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 or a nonhuman mammal-derived amino acid sequence corresponding to SEQ ID NO: 2; (2) a polypeptide containing an amino acid sequence which is derived from the amino acid sequence represented by SEQ ID NO: 2 or from a nonhuman mammal-derived amino acid sequence corresponding to SEQ ID NO: 2 by substitution, deletion, insertion or addition of one to several amino acid residues; and (3) an expression vector capable of expressing the aforesaid polypeptide (1) or (2).

Description

Vaccine for targeting aging-related T cells to prevent or treat abnormal glucose metabolism

The present invention includes a vaccine for preventing or treating abnormal glucose metabolism, which comprises a specific partial amino acid sequence of CD153 as an immunogen, and an antibody that inhibits senescence-related T cells by recognizing the partial amino acid sequence of CD153, a sugar. The present invention relates to a preventive or therapeutic agent for metabolic disorders.

Glucose metabolism abnormality represented by diabetes is caused by insufficient amount of insulin or lack of action of insulin in the body, resulting in an increase in blood glucose as compared with that of a healthy person, and as a result, microangiopathy and arteries in the kidney, retina, nerves, etc. It is a metabolic disease that significantly impairs a healthy life due to macrovascular disorders such as hardening. Until now, hypoglycemic agents such as insulin, insulin secretagogues, insulin sensitizers, and α-glucosidase inhibitors have been widely applied as clinical treatments. However, although these hypoglycemic agents have been found to be useful, each has many problems. For example, insulin has the risk of causing hypoglycemia when administered in an inappropriate manner and dose. In addition, the effectiveness of insulin secretagogues and insulin sensitizers decreases in patients whose pancreatic insulin secretory capacity is markedly reduced. Alternatively, the effectiveness of insulin and insulin secretagogues decreases in patients with marked insulin resistance.

The adaptive immune system plays a role of protecting the living body by distinguishing self-originating substances from non-self-originating substances, preventing and eliminating infections such as bacteria and viruses. There are T cells corresponding to various antigens in the body, and the corresponding T cells stimulated by the infection of the antigens eliminate the antigens, and part of them remain in the body as memory T cells, so that Be able to respond quickly to infections. However, it has recently been known that the function of the adaptive immune system decreases with age (immune senescence). Immune aging is characterized by reduced adaptive immune response, increased inflammatory predisposition, and increased autoimmune risk. Thus, like other cellular senescence-associated secretory traits (SASPs) in other senescent cells, immune senescence is also based on the inflammatory-promoting trait, which causes chronic inflammation and metabolic disorders such as arteriosclerosis and diabetes. .. In a recent study, a subpopulation of CD4-positive T cells that play a major role in the adaptive immune system that is PD-1-positive and CD153-positive (aging-related T cells (SA-T cells)) gradually becomes systemic with aging. It has been reported to increase and has been suggested to be a substance of immune senescence. More recently, it has been reported that senescence-related T cells are also significantly increased in the visceral adipose tissue of mice loaded with a high-fat diet and contribute to the formation of pathological conditions of type 2 diabetes such as insulin resistance by causing chronic inflammation. (Non-Patent Document 1). These reports suggest that senescence-related T cells are involved in abnormal glucose metabolism. However, there has been no report to date that provides a method for preventing or treating abnormal glucose metabolism by targeting senescence-related T cells.

The present invention provides a vaccine for use in the prevention or treatment of dysmetabolism related to aging-related T cells, and an agent for preventing or treating dysmetabolism, which comprises an antibody that inhibits aging-related T cells. It is an issue.

The inventors predicted the three-dimensional structure of CD153 expressed in senescence-related T cells, deduced multiple partial amino acid sequences predicted to have high antigenicity from the amino acid sequence of CD153, and determined the amino acid sequence from senescence-related T It was designed as an antigen candidate capable of inducing an antibody that inhibits cells. When a plurality of the synthesized antigen candidates were conjugated to a carrier protein and administered to mice with an adjuvant, one kind of antigen candidate capable of inducing an antibody that strongly binds to CD153 was identified. Then, the inventors have found an effect of improving obesity and glucose metabolism in mice immunized with the antigen.
The present inventors have completed the present invention as a result of further studies based on these findings.

That is, the present invention provides the following.
[1] A vaccine for preventing or treating abnormal glucose metabolism, which comprises any of the following (1) to (3):
(1) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence derived from a non-human mammal corresponding to SEQ ID NO: 2;
(2) Amino acid in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence derived from non-human mammal corresponding to SEQ ID NO: 2 A polypeptide comprising a sequence,
(3) An expression vector capable of expressing the polypeptide of (1) or (2) above.
[2] The vaccine according to [1], which comprises the following (1 ′) or (2 ′):
(1 ') a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2,
(2 ′) An expression vector capable of expressing the polypeptide of (1 ′) above.
[3] The vaccine according to [1], which comprises the following (1 ″) or (2 ″):
(1 '') a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4,
(2 ″) An expression vector capable of expressing the polypeptide of (1 ″) above.
[4] The vaccine according to any one of [1] to [3], which further comprises a carrier protein.
[5] The vaccine according to [4], wherein the carrier protein is keyhole limpet hemocyanin.
[6] The vaccine according to any one of [1] to [5], which further comprises an adjuvant.
[7] The vaccine according to [6], wherein the adjuvant is CpG oligodeoxynucleotide.
[8] The abnormal glucose metabolism is selected from the group consisting of diabetes, impaired glucose tolerance, obesity, hyperinsulinemia, diabetic neuropathy, diabetic nephropathy and diabetic retinopathy, [1] to [7] ] Vaccine according to any one of.
[9] A prophylactic or therapeutic agent for glucose metabolism disorder, which comprises an antibody that recognizes the following polypeptide (1) or (2) and inhibits senescence-related T cells:
(1) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence derived from a non-human mammal corresponding to SEQ ID NO: 2;
(2) Amino acid in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence derived from non-human mammal corresponding to SEQ ID NO: 2 A polypeptide comprising a sequence.
[10] The prophylactic or therapeutic agent according to [9], which comprises an antibody that recognizes the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 and inhibits senescence-related T cells.
[11] The prophylactic or therapeutic agent according to [9], which comprises an antibody that recognizes the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 and inhibits senescence-related T cells.
[12] The preventive or therapeutic agent according to any one of [9] to [11], wherein the senescence-related T cells are CD4 positive PD-1 positive CD153 positive T cells.
[13] The abnormal glucose metabolism is selected from the group consisting of diabetes, impaired glucose tolerance, obesity, hyperinsulinemia, diabetic neuropathy, diabetic nephropathy, and diabetic retinopathy, [9] to [12]. ] The preventive or therapeutic agent as described in any one of these.

By using the partial amino acid sequence of CD153 of the present invention as a vaccine, an antibody that inhibits senescence-related T cells expressing CD153 on the cell surface is induced, and thereby an inflammatory protein or inflammatory protein from senescence-related T cells is induced. It can reduce chemokine secretion and improve glucose metabolism disorders. In addition, the above effects can be directly obtained by using an antibody that recognizes the partial amino acid sequence of CD153. Furthermore, since the half-life of the antibody is longer than that of conventional hypoglycemic agents, it can be expected to reduce the frequency of drug administration to patients.

Evaluation of antibodies induced by the CD153 vaccine. 7-week-old C57BL / 6J mice (n = 3-6, each group) were treated with CD153-alum (Alum) vaccine (#A, #B, #C, #D and #E) on

days

0 and 14 or Immunized with the KLH-alum vaccine. The CD153-Alum vaccine consisted of 30 μg of CD153 peptide (# A- # E) and 200-300 μg of KLH, and the KLH-Alum vaccine consisted of 200-300 μg of KLH. (A) The titer to CD153-BSA was expressed as the dilution ratio (OD 50%) of the serum that gave half the maximum binding on

days

14 and 28 after immunization. (B) The titer against recombinant mouse CD153 was expressed as OD 450 nm at 28 days after immunization. (C) Western blot analysis of the ability of the antibody induced on day 28 by the CD153 # D-alum vaccine to recognize the rmCD153 protein. Serum from mice immunized with CD153 # D-alum vaccine or KLH vaccine was diluted 500-fold. A commercially available anti-CD153 antibody was diluted to 0.05 μg / ml. 100 ng of rmOPN was applied to lane 1 as a negative control. 30ng rmCD153 and 100ng rmCD153 were applied to

lanes

2 and 3, respectively. Precision Plus protein two-color standard (Bio-Rad) was applied as a marker to both ends of each membrane. (D) IgG153 (Th2 response) and IgG2b on

day

0, 14 and 28 in mice immunized with CD153 # D-alum vaccine (n = 3) or CD153 # D-CpG vaccine (n = 6). , IgG2c and IgG3 (Th1 response) levels. Serum was collected on day 42. The titer against CD153-BSA is expressed as OD50%. All data are expressed as mean ± standard error. N.D., not detected. Effect of CD153 # D-CpG vaccine on CD153-positive senescence-associated T cells in the spleen induced by TLR7 ligand administration. (A) Percentage of senescence-related T cells in spleen tissue and VAT at 20 and 3 months of age. Mice were fed a normal diet. PD-1 ⁺ , CD153 ⁺ cells in CD4 ⁺ , CD44 ⁺ , CD62L ⁻ cells were defined as senescence-associated T cells. (B) Time course of R848 (TLR7 ligand) administration and injection of vaccine. R848 was intraperitoneally administered to 12-week-old female C57BL / 6N mice (N = 3, each group) three times a week. Mice treated with 5 μg of R848 for 4 weeks were sacrificed at 16 weeks of age, and mice treated with 10 μg of R848 for 2 weeks were sacrificed at 18 weeks of age. All immunized mice were vaccinated with CD153 # D-CpG vaccine or KLH-CpG vaccine at 8, 10 and 12 weeks of age. The PBS control group was not vaccinated and PBS was administered instead of R848. The R848 control group was not vaccinated, and R848 was administered as in the vaccine group. (C) Titer against CD153-BSA during R848 administration in mice immunized with the CD153 # D-CpG vaccine (n = 6; 8, 10, 12, 14 weeks old; n = 3; 16, 18 weeks old) ). Titers were expressed as the dilution of serum that gave half maximal binding (OD 50%). (D) Percentage of senescence-related T cells induced by R848 administration in spleen tissue in the presence or absence of CD153 # D-CpG vaccine or KLH-CpG vaccine. (E) Spleen weight of mice administered with R848 at 18 weeks of age in the presence or absence of CD153 # D-CpG vaccine or KLH-CpG vaccine. All data are expressed as mean ± standard error. * p <0.05; ** p <0.01; *** p <0.001. Metabolic analysis in HFD-loaded mice immunized with CD153 # D-alum vaccine or CD153 # D-CpG vaccine. (A) HFD (high fat diet) loading and vaccination time course. Seven-week-old male C57BL / 6J mice were vaccinated multiple times with the alum conjugate vaccine or the CpG conjugate vaccine. Mice (n = 3, each group) immunized with the CD153 # D-alum vaccine or the CD153 # D-CpG vaccine were vaccinated at 7, 9, 11, 13, and 15 weeks of age, and HFD was started at 15 weeks of age. Was given. Mice immunized with the CD153 # D-CpG vaccine or the KLH-CpG vaccine (n = 6, each group) were vaccinated at 7, 9, 11, and 16 weeks of age, and were given HFD from 11 weeks of age. The ND (normal diet) control group (n = 5) was not vaccinated and continued to receive ND. The HFD control group (n = 6) was not vaccinated and was given HFD from 11 weeks of age. The ND control group and the HFD control group were subjected to metabolic analysis and sacrificed in the same manner as the CpG conjugate vaccine group. (B) Titer against CD153-BSA during HFD challenge in mice immunized with CD153 # D-alum vaccine and CD153 # D-CpG vaccine. Titers were expressed as the dilution of serum that gave half maximal binding (OD 50%). (C) Pre-metabolic body weight of mice immunized with CD153 # D-CpG vaccine or KLH-CpG vaccine. All mice except the ND control group were given HFD from the age of 11 weeks. *; for CD153 # D-CpG group versus KLH-CpG group. (D) Weekly average amount of HFD uptake per mouse (g / week / mouse) during the HFD challenge period in mice immunized with CD153 # D-CpG vaccine or KLH-CpG vaccine. The period was 11 to 19 weeks old. (E) Light period (8: 00-20: 00) and dark period (20: 00-8: 00) at 21-22 weeks of age in mice immunized with the CD153 # D-CpG vaccine or the KLH-CpG vaccine. VO ₂ (g / week / mouse) in. All data are expressed as mean ± standard error. * p <0.05; ** p <0.01; *** p <0.001. Assessment of glucose tolerance and insulin sensitivity in HFD-loaded mice immunized with the CD153 # D-alum vaccine or the CD153 # D-CpG vaccine. (A and B) Alum conjugate vaccine (CD153 # D-Alum or KLH-Alum) (A; n = 3, Each group) or CpG conjugate vaccine (CD153 # D-CpG or KLH-CpG) (B; n) = 6, 6, each group), blood glucose concentration and basal glucose ratio during the intraperitoneal insulin tolerance test (ipITT). The ND control group (n = 5) and the HFD (high fat diet) control group (n = 6) were treated in the same manner as the vaccination group. *; CD153 # D-CpG group vs. KLH-CpG group. (C) Area under the curve (AUC) of blood glucose level against ITT. The AUC was estimated using the trapezoidal formula. (D and E) Blood glucose concentration during oral glucose tolerance test (OGTT) in mice immunized with alum conjugate vaccine (D) or CpG conjugate vaccine (E). *; CD153 # D-CpG group vs. KLH-CpG group. †; CD153 # D-CpG group versus HFD control group. (F) AUC of blood glucose for OGTT. The AUC was estimated using the trapezoidal formula. †; versus HFD control group. (G) HOMA-IR index 6 hours after fasting. All data are expressed as mean ± standard error. * p <0.05; ** p <0.01; *** p <0.001; †††† p <0.0001. Effect of CD153 # D-CpG vaccine on CD153-positive senescence-related T cells induced by HFD loading. (A and B) Percentage of age-related T cells induced by HFD loading in spleen tissues (A) and VAT (B) of mice immunized with alum conjugate vaccine or CpG conjugate vaccine. PD-1 ⁺ , CD153 ⁺ cells in CD4 ⁺ , CD44 ⁺ , CD62L ⁻ cells were defined as senescence-associated T cells. †; versus HFD control group. (C) CDC activity of CD153 # D vaccine-induced antibody was assessed in 2 x 10 ⁴ LPS-stimulated RAW 264.7 cells in the presence of 10% complement serum (n = 3, each sample). Purification of used vaccine-derived IgG antibody from serum at 22-25 weeks of age in HFD-loaded mice immunized with CD153 # D-alum vaccine, CD153 # D-CpG vaccine, KLH-alum vaccine or KLH-CpG vaccine did. N, No antibody stimulation; P, 100 μg / ml anti-mouse MHC Class I (H-2Kd / H-2Dd) antibody; filled right triangle, concentration of purified IgG antibody (30, 100, 300 μg / ml); Open triangles, anti-mouse CD153 antibody concentration (10, 30, 100 μg / ml). All data are expressed as mean ± standard error. * p <0.05; † p <0.05. Histological analysis of VAT, kidney and lung tissues in HFD-loaded mice immunized with the CD153 # D-CpG vaccine. From 4 groups of mice (ND (normal diet) control group, HFD (high fat diet) control group, KLH-CpG vaccine group, and CD153 # D-CpG vaccine group, each = 3 and each group), VAT, kidney tissue and Lung tissue was collected and followed the HFD challenge and vaccination time course. (A) VAT weight between 4 groups. (B) Photomicrograph of mean surface area of adipocytes between four groups and fat accumulation stained with hematoxylin and eosin (HE). Scale bar: 100 μm. (C) Quantification of macrophages in crown-like structures among 4 groups. The average of the ND control group was set to 100%. Scale bar: 100 μm. All data are expressed as mean ± standard error. * p <0.05; ** p <0.01. (D) Quantification of CD153-positive senescence-related cells in the crown-like structure among the 4 groups. The average of the ND control group was set to 100%. Scale bar: 100 μm. (E) Micrographs of fat accumulation stained with IgG among 4 groups, kidney tissue stained with HE or IgG, and lung tissue stained with HE or IgG. Scale bar: 100 μm. All data are expressed as mean ± standard error. * p <0.05; ** p <0.01. Seven-week-old C57BL / 6J mice (n = 3-4, each group) were immunized with mouse CD153 # D-alum vaccine or human CD153 # D-alum vaccine on

days

0, 14 and 28. Mouse CD153 # D-alum vaccine and human CD153 # D-alum vaccine were conjugated with KLH (200-300 μg) and consisted of 30 μg of CD153 peptide mixed with alum. (A) Amino acid sequence of mouse CD153 # D peptide (76-85aa) and human CD153 # D peptide (71-80aa). (B) The titer of the human CD153 antibody against human or mouse CD153-BSA was expressed as the dilution ratio (OD50%) of the serum that gave half the maximum binding at 42 days after immunization. (C) The titer of human CD153 antibody against recombinant human (rh) or recombinant mouse (rm) CD153 was expressed as OD 450 nm at 42 days after immunization. (D) The titer of mouse CD153 antibody against rhCD153 was expressed as OD 450 nm at 42 days after immunization. All data are expressed as mean ± standard error. N.D., not detected.

1. Vaccine for preventing or treating abnormal glucose metabolism The present invention provides a vaccine for preventing or treating abnormal glucose metabolism (hereinafter referred to as the vaccine of the present invention).

Glucose metabolism disorder that is prevented or treated by the vaccine of the present invention is a condition in which glucose uptake into muscle, liver and adipose tissue and conversion to glycogen are reduced due to decreased insulin secretion or insulin sensitivity, and blood glucose is high. There is no particular limitation as long as the condition is maintained and the disease induced by the condition. Examples of abnormal glucose metabolism include diabetes (type 1 diabetes, type 2 diabetes), impaired glucose tolerance, obesity, hyperinsulinemia, diabetic neuropathy, diabetic nephropathy and diabetic retinopathy.

The subject of administration of the vaccine of the present invention may be any mammal, but it is a mammal that develops glucose metabolism abnormality or a mammal that may develop glucose metabolism abnormality. As mammals, for example, mice, rats, hamsters, experimental animals such as rodents and rabbits such as guinea pigs, pets such as dogs and cats, livestock such as cattle, pigs, goats, horses and sheep, humans, monkeys, Examples thereof include primates such as orangutans and chimpanzees, and humans are particularly preferable. The administration subject may or may not be treated for glucose metabolism abnormality. When the vaccine of the present invention contains a polypeptide, the polypeptide is a substance derived from an administration subject (that is, when administered to humans, the vaccine is a human-derived polypeptide, and when administered to mice, the vaccine is Is a mouse-derived polypeptide).

The vaccine of the present invention comprises the following (1) to (3):
(1) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence derived from a non-human mammal corresponding to SEQ ID NO: 2;
(2) Amino acid in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence derived from non-human mammal corresponding to SEQ ID NO: 2 A polypeptide comprising a sequence,
(3) It includes either an expression vector capable of expressing the polypeptide of (1) or (2) above.
The vaccine of the present invention preferably has the following (1 ′) or (2 ′):
(1 ') a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2,
(2 ′) An expression vector capable of expressing the polypeptide of (1 ′) above, or
The following (1 '') or (2 ''):
(1 '') a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4,
(2 ″) An expression vector capable of expressing the polypeptide of (1 ″) above is included.

The above-mentioned polypeptide (1) contained in the vaccine of the present invention is a partial sequence of the amino acid sequence constituting CD153. CD153 is a known gene, and its nucleotide sequence and amino acid sequence are also known. For example, the amino acid sequence represented by SEQ ID NO: 2 contained in the above polypeptide (1) corresponds to amino acid numbers 71 to 80 of human CD153 (SEQ ID NO: 5). The partial sequence is encoded by the nucleotide sequence shown in SEQ ID NO: 1, for example. Furthermore, the non-human mammal-derived amino acid sequence corresponding to SEQ ID NO: 2 contained in the above-mentioned polypeptide (1) includes information on the amino acid sequence represented by SEQ ID NO: 2 in the present specification and known sequences. Using the nucleotide sequence information of the genome or mRNA of non-human mammals disclosed in the database, design appropriate primers and probes derived from the nucleotide sequence information of non-human mammals, such as RT-PCR and plaque hybridization It can be easily obtained using ordinary genetic engineering techniques. For example, the partial sequence of the mouse CD153 amino acid sequence corresponding to SEQ ID NO: 2 which is a partial sequence of human CD153 is shown in SEQ ID NO: 4 corresponding to amino acid numbers 76 to 85 of mouse CD153 (SEQ ID NO: 6). An amino acid sequence can be mentioned, and the partial sequence is encoded by, for example, the nucleotide sequence shown in SEQ ID NO: 3. The term “non-human mammal” as used herein means a mammal excluding humans from the above-mentioned mammals.

The above-mentioned polypeptide (1) contained in the vaccine of the present invention is preferably a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 for human and the amino acid sequence represented by SEQ ID NO: 4 for mouse. (The polypeptides of (1 ′) and (1 ″) above).

In the polypeptide of (2) contained in the vaccine of the present invention, one or several (preferably 1 to several (2 to 5)) amino acids are deleted or substituted in the partial sequence of the CD153 amino acid sequence, The amino acid sequence inserted or added. As such a polypeptide, in the case of human, one or several (preferably 1 to several (2 to 5)) amino acids in the amino acid sequence shown in SEQ ID NO: 2 are deleted, substituted, inserted or added. Included amino acid sequences. Examples of the amino acid sequence include (1) an amino acid sequence in which one or several (preferably 1 to several (2 to 5)) amino acids in the amino acid sequence represented by SEQ ID NO: 2 are deleted, (2 ) An amino acid sequence having 1 or several (preferably 1 to several (2 to 5)) amino acids added to the amino acid sequence represented by SEQ ID NO: 2, (3) the amino acid sequence represented by SEQ ID NO: 2. An amino acid sequence in which one or several (preferably 1 to several (2 to 5)) amino acids are inserted, (4) 1 or several (preferably 1 to several) in the amino acid sequence shown in SEQ ID NO: 2. An amino acid sequence in which (2 to 5) amino acids are substituted with other amino acids, or (5) an amino acid sequence in which the mutations in (1) to (4) above are combined (in this case, the sum of the mutated amino acids is 1 or several (preferably 1 to several (2 to 5)).
The amino acid sequence contained in the polypeptide of (2) above is 1 or several (preferably 1 to several (2 to 5)) in the amino acid sequence derived from the non-human mammal corresponding to SEQ ID NO: 2. An amino acid sequence in which the amino acid of is deleted, substituted, inserted or added can also be preferably mentioned. As such a polypeptide, in the case of mouse, one or several (preferably 1 to several (2 to 5)) amino acids in the amino acid sequence shown in SEQ ID NO: 4 are deleted, substituted, inserted or added. Included amino acid sequences. Examples of the amino acid sequence include (1) an amino acid sequence in which one or several (preferably 1 to several (2 to 5)) amino acids in the amino acid sequence represented by SEQ ID NO: 4 are deleted, (2 ) An amino acid sequence having 1 or several (preferably 1 to several (2 to 5)) amino acids added to the amino acid sequence shown in SEQ ID NO: 4, (3) to the amino acid sequence shown in SEQ ID NO: 4. An amino acid sequence in which 1 or several (preferably 1 to several (2 to 5)) amino acids are inserted, (4) 1 or several (preferably 1 to several) in the amino acid sequence shown in SEQ ID NO: 4. An amino acid sequence in which (2 to 5) amino acids are substituted with other amino acids, or (5) an amino acid sequence in which the mutations in (1) to (4) above are combined (in this case, the sum of the mutated amino acids is 1 or several (preferably 1 to several (2 to 5)).

“Conservative amino acid substitutions” are examples of “substitution of amino acid residues”. The conservative amino acid substitution means substitution of a specific amino acid with an amino acid having a side chain having the same property as that of the amino acid. Specifically, in a conservative amino acid substitution, a particular amino acid is replaced with another amino acid that belongs to the same group as that amino acid. Groups of amino acids with side chains of similar nature are known in the art. For example, such groups of amino acids include amino acids having basic side chains (eg, lysine, arginine, histidine), amino acids having acidic side chains (eg, aspartic acid, glutamic acid), amino acids having neutral side chains. (For example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan). Further, an amino acid having a neutral side chain, further, an amino acid having a polar side chain (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), and an amino acid having a non-polar side chain (for example, alanine, Valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan). As another group, for example, an amino acid having an aromatic side chain (eg, phenylalanine, tryptophan, tyrosine), an amino acid having a side chain containing a hydroxyl group (alcoholic hydroxyl group, phenolic hydroxyl group) (eg, serine, threonine, Tyrosine) can also be mentioned.

The “deletion of amino acid residue” includes, for example, selecting and deleting an arbitrary amino acid residue from the amino acid sequence shown in SEQ ID NO: 2.

“Insertion of amino acid residue” or “addition of amino acid residue” means, for example, insertion or addition of an amino acid residue inside, N-terminal side or C-terminal side of the amino acid sequence shown in SEQ ID NO: 2. can give. In order to enhance the water solubility of the peptide, one or two residues of a basic amino acid, arginine (Arg) or lysine (Lys), may be added to the N-terminal side or C-terminal side of the amino acid sequence.

The above-mentioned polypeptide (2) contained in the vaccine of the present invention preferably has one or several amino acid residues substituted in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 4. , A polypeptide consisting of a deleted, inserted, or added amino acid sequence.

The above-mentioned polypeptides (1), (2), (1 '), and (1' ') (hereinafter referred to as the polypeptide of the present invention) may contain additional amino acids. Such amino acid additions are permissible as long as the polypeptide induces a specific immune response against CD153. The amino acid sequence to be added is not particularly limited, and examples thereof include a tag for facilitating detection and purification of the polypeptide. As the tag, Flag tag, histidine tag, c-Myc tag, HA tag, AU1 tag, GST tag, MBP tag, fluorescent protein tag (for example, GFP, YFP, RFP, CFP, BFP, etc.), immunoglobulin Fc tag, etc. It can be illustrated. The position where the amino acid sequence is added is the N-terminal and / or the C-terminal of the polypeptide of the present invention.

The amino acids used in the polypeptide of the present invention include L-form, D-form and DL-form, but usually L-form is preferred. These polypeptides can be used in the present invention after being synthesized by an ordinary polypeptide synthesis method, but the production method, the synthesis method, the procurement method and the like are not particularly limited in the present invention.

In the expression vector of (3), (2 ′) or (2 ″), the polynucleotide (DNA encoding the polypeptide of (1), (2), (1 ′) or (1 ″) above is used. Alternatively, RNA (preferably DNA) is operably linked to the downstream of a promoter capable of exhibiting promoter activity in the cells of the mammal to be administered. That is, the expression vector of (3), (2 ′) or (2 ″) is a transcription product of (1), (2), (1 ′) or (1 ″) under the control of a promoter. Can be expressed. By administering the expression vector of (3), (2 ′) or (2 ″) to a mammal, the above-mentioned (1), (2), (1 ′) or (1 ′) in the body of the mammal. The polypeptide of ') is produced, and a specific immune response to the polypeptide of (1), (2), (1') or (1 '') is induced in the mammal.
The promoter used is not particularly limited as long as it can function in the cells of the mammal to be administered. As the promoter, a polI type promoter, a polII type promoter, a polIII type promoter and the like can be used. Specifically, SV40-derived early promoters, viral promoters such as cytomegalovirus LTR, and mammalian constitutive protein gene promoters such as β-actin gene promoter are used.

The expression vector of (3), (2 ′) or (2 ″) is preferably a polynucleotide encoding the polypeptide of (1), (2), (1 ′) or (1 ″) above. It contains a transcription termination signal, that is, a terminator region, downstream. Furthermore, a selectable marker gene for selecting transformed cells (a gene that imparts resistance to drugs such as tetracycline, ampicillin, and kanamycin, a gene that complements an auxotrophic mutation, and the like) can be included.

The type of vector used as an expression vector in the present invention is not particularly limited, but examples of vectors suitable for administration to mammals such as humans include viral vectors and plasmid vectors. Examples of viral vectors include retrovirus, adenovirus, adeno-associated virus and the like. A plasmid vector is preferably used in consideration of easiness of production and handling and economical efficiency.

The vaccine of the present invention is encoded by the polypeptide of (1), (2), (1 ′) or (1 ″) above or the expression vector of (3), (2 ′) or (2 ″) above. In order to enhance the immunogenicity of the polypeptide, it is preferable to further include a carrier protein. The carrier protein is a substance that imparts immunogenicity by binding to a molecule (hapten) that does not have immunogenicity due to its small molecular weight, and is known in the art. Preferable examples of carrier proteins include bovine serum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA), keyhole limpet hemocyanin (KLH), thyroglobulin (TG), immunoglobulin and the like. A particularly preferred carrier protein is keyhole limpet hemocyanin (KLH). When the vaccine of the present invention is the polypeptide of (1), (2), (1 ′) or (1 ″), the carrier protein is (1), (2), (1 ′) or ( 1 ″) may be conjugated to the N-terminus or C-terminus of the polypeptide. As a method for conjugating, a cysteine residue is introduced into the polypeptide of (1), (2), (1 ′) or (1 ″), and the carrier is introduced via the SH group which is the side chain of the cysteine. It can be conjugated by binding to an amino group of a protein (MBS method). Alternatively, conjugation can also be achieved by linking the ε-amino group of the lysine residue of the protein and the amino groups such as α-amino group (glutaraldehyde method). When the vaccine of the present invention is the expression vector of (3), (2 ′) or (2 ″) above, the polypeptide of (1), (2), (1 ′) or (1 ″) above The polynucleotide encoding the carrier protein may be linked to the 5'side or 3'side of the polynucleotide encoding the carrier protein.

The vaccine of the present invention preferably further contains an adjuvant which is pharmaceutically acceptable and compatible with the active ingredient. Adjuvants are generally substances that nonspecifically enhance the immune response of the host and numerous adjuvants are known in the art. The adjuvant used in the vaccine of the present invention is not particularly limited as long as it can nonspecifically enhance the immune response, but includes, for example, alum, CpG oligodeoxynucleotide, dsRNA, montanide, cervarix, etc., and is preferable. Is a CpG oligodeoxynucleotide.

The vaccine of the present invention comprises the polypeptide of (1), (2), (1 ′) or (1 ″) or the expression vector of (3), (2 ′) or (2 ″) , And can be provided as a pharmaceutical composition containing any carrier, for example, a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may be appropriately selected depending on the dosage form, for example, sucrose, an excipient such as starch, cellulose, a binder such as methyl cellulose, starch, a disintegrating agent such as carboxymethyl cellulose, magnesium stearate, Lubricants such as Aerosil, fragrances such as citric acid and menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinylpyrrolide, surface active agents Examples thereof include, but are not limited to, dispersants such as agents, diluents such as water and physiological saline, base waxes, and the like.

Furthermore, when the vaccine of the present invention is the expression vector of (3), (2 ′) or (2 ″) above, the vaccine of the present invention further comprises a nucleic acid in order to promote introduction of the expression vector into cells. A transfer reagent can be included. When a viral vector is used as the expression vector, retronectin, fibronectin, polybrene or the like can be used as the gene transfer reagent. When a plasmid vector is used as the expression vector, lipofectin, lipofectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, poly (ethyleneimine) (PEI), etc. Cationic lipids can be used.

The vaccine of the present invention can be orally or parenterally administered to mammals. The polypeptide and expression vector are preferably administered parenterally because they can be degraded in the stomach. Suitable formulations for oral administration include solutions, capsules, sachets, tablets, suspensions, emulsions and the like. Suitable formulations for parenteral administration (eg subcutaneous injection, intramuscular injection, local infusion, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants. , A buffer solution, a bacteriostatic agent, a tonicity agent, etc. may be contained. Aqueous and non-aqueous sterile suspensions are also included, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The preparation can be enclosed in a container such as an ampoule or a vial in a unit dose or multiple doses. Alternatively, the active ingredient and a pharmaceutically acceptable carrier may be lyophilized and stored in a state in which they may be dissolved or suspended in an appropriate sterile vehicle immediately before use.

The content of the active ingredient in the pharmaceutical composition is usually about 0.1 to 100% by weight, preferably about 1 to 99% by weight, more preferably about 10 to 90% by weight, based on the whole pharmaceutical composition.
The dose of the vaccine of the present invention varies depending on the administration subject, administration method, administration form, etc., but when the active ingredient is the above-mentioned polypeptide (1), (2), (1 ′) or (1 ″) Is usually administered in the range of 1 μg to 1000 μg per adult, preferably 20 μg to 100 μg per adult, usually 2 to 3 times over 4 to 12 weeks, and the antibody titer is decreased. If this is the case, an additional dose will be given each time. When the active ingredient is the expression vector of (3), (2 ′) or (2 ″), the expression vector per adult is usually in the range of 1 μg to 1000 μg, preferably 20 μg to 100 μg. Usually, it is administered 2 to 3 times over 4 to 12 weeks, and if the antibody titer decreases, additional administration is performed once each time.

By administering the vaccine of the present invention to a mammal, a specific immune response against CD153 (specific antibody production, specific T cell proliferation, etc.) is induced, and the mammal acquires a neutralizing antibody against CD153, Senescence-related T cells that express CD153 on the cell surface are inhibited. Senescence-related T cells have the characteristic of not proliferating or producing cytokines in response to stimulation of T cell receptors, and secreting large amounts of inflammatory proteins and chemokines, and increase systemically with age. Is. Chronic inflammation is caused by an increase in age-related T cells, and abnormal glucose metabolism such as insulin resistance and impaired glucose tolerance is induced. Therefore, as a result of the inhibition of senescence-related T cells by the vaccine of the present invention, a preventive or therapeutic effect on abnormal glucose metabolism is exerted. The senescence-related T cells are not particularly limited as long as they are T cells having the above characteristics, for example, CD4 positive PD-1 positive CD153 positive T cells, preferably, CD4 positive CD44 positive CD62L negative PD-1 positive CD153 positive. Examples include T cells.

The present invention also provides a kit comprising one or more containers containing one or more components of the vaccine of the present invention. By using the kit of the present invention, abnormal glucose metabolism can be prevented, or its symptoms can be treated or reduced.

2. Antibody for Preventing or Treating Glucose Metabolism Disorders The present invention also provides a preventive or therapeutic agent for glucose metabolism disorders (the preventive or therapeutic agent of the present invention).
Glucose metabolism disorders that are prevented or treated by the prophylactic or therapeutic agent of the present invention, administration targets of the prophylactic or therapeutic agent of the present invention are glucose metabolism disorders that are prevented or treated by the vaccine of the present invention, and administration targets of the vaccine of the present invention May be similar to.

The preventive or therapeutic agent of the present invention has the following (1) or (2):
(1) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence derived from a non-human mammal corresponding to SEQ ID NO: 2;
(2) Amino acid in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence derived from non-human mammal corresponding to SEQ ID NO: 2 It includes an antibody that recognizes a polypeptide comprising the sequence and inhibits senescence-associated T cells.
The preventive or therapeutic agent of the present invention is preferably
(1 ′) an antibody that recognizes the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 and inhibits senescence-related T cells, or
(1 ″) It includes an antibody that recognizes the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 and inhibits senescence-related T cells.

The above polypeptide (1) is preferably a polypeptide having the amino acid sequence represented by SEQ ID NO: 2 for human and the amino acid sequence represented by SEQ ID NO: 4 for mouse.

An antibody that recognizes the polypeptide of (1), (2), (1 ′) or (1 ″) (hereinafter, the antibody of the present invention) binds to CD153 expressed on the surface of senescence-related T cells. Since it can inhibit aging-related T cells and reduce the secretion of inflammatory proteins and chemokines, it can be an effective preventive or therapeutic agent for glucose metabolism abnormality. That is, administration of the antibody can be expected to have a therapeutic effect on a patient who has developed a glucose metabolism disorder and a preventive effect on a subject who may develop the disorder.

As the antibody of the present invention, a natural antibody such as a polyclonal antibody or a monoclonal antibody, a chimeric antibody that can be produced by using a transgenic mouse or gene recombination technology, a humanized antibody and a single chain antibody, and a human antibody-producing gene are introduced. Human antibodies produced by the mouse and phage display, and fragments thereof are included. The antibody of the present invention is not particularly limited as long as it is an antibody that recognizes the polypeptide of the present invention and inhibits senescence-related T cells, but is preferably a monoclonal antibody from the viewpoint of specificity for the polypeptide of the present invention. Alternatively, from the viewpoint of human clinical application, the antibody of the present invention is preferably a humanized antibody or a human antibody.

The above antibody fragment means a region of a part of the above-mentioned antibody, and specifically, for example, F (ab ') ₂ , Fab', Fab, an antibody fragment containing an Fc region, Fv (variable fragment of antibody), Examples include sFv, dsFv (disulphide stabilized Fv), dAb (single domain antibody) and the like (Exp. Opin. Ther. Patents, Vol. 6, No. 5, p. 441-456, 1996).

The above-mentioned humanized antibody refers to an antibody produced by gene recombination technology in which only the antigen recognition site is derived from a gene other than human and the remaining site is derived from a human gene. In addition, the above-mentioned human antibody is a human antibody produced by a transgenic mouse into which a human antibody-producing gene has been introduced (eg, TransChromoMouse (trademark)), VH gene and VL gene derived from human B lymphocyte mRNA or genome. It refers to an antibody prepared from a library constructed by randomly combining the above-mentioned antibodies based on a human antibody library expressing an antibody variable region by a display technique such as a phage display method.

The class of antibody is not particularly limited, and the antibody of the present invention includes antibodies having any isotype such as IgG, IgM, IgA, IgD or IgE. IgG or IgM is preferable, and IgG is more preferable in view of easiness of antibody purification.

Next, a method for producing an antibody will be described.
A polyclonal antibody or a monoclonal antibody can be produced by a method known per se. That is, the immunogen (polypeptide of the present invention), optionally with Freund's adjuvant (Freund's Adjuvant), mammals, for example, in the case of a polyclonal antibody, mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig, Immunize preferably a mouse, rat, hamster, guinea pig, goat, horse or rabbit, such as goat, horse or cow. In the case of a monoclonal antibody, mice, rats, hamsters, etc. are immunized in the same manner.

The polypeptide of the present invention can be used as an immunogen as it is, but it is desirable to immunize it as a complex with a high molecular weight compound having a molecular weight of 10,000 or more. Therefore, when used as an immunogen, the polypeptide of the present invention may be formed into a complex with a high molecular compound (eg, carrier protein etc.) by a method known per se. For example, a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 was synthesized according to the method described above, and bovine serum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA), keyhole limpet hemocyanin (KLH). ), Thyroglobulin (TG), immunoglobulins and other carrier proteins. The complex can then be used as the preferred immunogen. As the complex, a complex with keyhole limpet hemocyanin is preferably used.

1 to 2, preferably 1 amino acid can be added to the polypeptide of the present invention for the purpose of forming a complex between the polypeptide and a carrier protein. The position of the added amino acid may be any position of the polypeptide and is not particularly limited, but the N-terminal or C-terminal of the polypeptide is preferable.

In forming the complex, known methods can be applied without limitation as long as the antigenicity of the polypeptide of the present invention can be maintained. For example, it is also possible to introduce a cysteine residue into the polypeptide of the present invention and bind it to the amino group of the polymer compound (carrier protein) via the SH group which is the side chain of the cysteine (MBS method). In addition, ε-amino groups of lysine residues of proteins and amino groups such as α-amino groups can also be bound to each other (glutaraldehyde method).

Specifically, the polyclonal antibody can be produced as follows. That is, the immunogen is administered to a mouse, rat, hamster, guinea pig, goat, horse or rabbit, preferably goat, horse or rabbit, more preferably rabbit subcutaneously, intramuscularly, intravenously, in a hood pad or intraperitoneally. Immunizations are given by several injections. Usually, about 1 to 14 days after the initial immunization, immunization is performed 1 to 5 times, and serum is obtained from the immunized mammal about 1 to 5 days after the final immunization.

Although serum itself can be used as a polyclonal antibody, ultrafiltration, ammonium sulfate fractionation, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), anti-immunoglobulin column Alternatively, the purified antibody obtained by isolating and / or purifying the antibody by affinity column chromatography using a protein A / G column, a column obtained by crosslinking an immunogen, or the like can be used.

Examples of the method for producing a monoclonal antibody include the following methods. First, a hybridoma is prepared from the antibody-producing cells obtained from the immunized animal and myeloma cells (myeloma cells) having no autoantibody-producing ability, and the hybridoma is cloned. That is, using a culture supernatant of a hybridoma as a sample, an immunological method is used to produce a monoclonal antibody that exhibits a specific affinity for the peptide of the present invention used for immunization of mammals and does not show cross-reactivity with a carrier protein. Select a clone. Then, an antibody can be produced from the culture supernatant of the hybridoma by a method known per se.

Specifically, a monoclonal antibody can be produced as follows. That is, the immunogen is subcutaneously, intramuscularly, or intravenously in a mouse, rat, or hamster (including a transgenic animal produced to produce an antibody derived from another animal such as a human antibody-producing transgenic mouse). Immunization is carried out by injecting 1 to several times in the foot pad or intraperitoneal cavity, or by transplantation. Usually, about 1 to 14 days after the first immunization, immunization is performed 1 to 4 times, and antibody-producing cells are obtained from the spleen of the immunized mammal about 1 to 5 days after the final immunization.

Hybridomas (fused cells) that secrete monoclonal antibodies can be prepared according to the method of Kohler and Milstein et al. (Nature, Vol. 256, p. 495-497, 1975) and modification methods similar to them. That is, antibody-producing cells contained in spleen, lymph node, bone marrow or tonsil, etc., preferably spleen, obtained from a mammal immunized as described above, and preferably mouse, rat, guinea pig, hamster, rabbit or human. A hybridoma is obtained by cell fusion with a myeloma cell having no autoantibody-producing ability derived from a mammal such as, for example, a mouse, rat or human.

Examples of myeloma cells used for cell fusion include mouse-derived myeloma P3 / X63-AG8.653 (653; ATCC No. CRL1580), P3 / NSI / 1-Ag4-1 (NS-1), P3 / X63-Ag8. .U1 (P3U1), SP2 / 0-Ag14 (Sp2 / 0, Sp2), PAI, F0 or BW5147, rat-derived myeloma 210RCY3-Ag.2.3., Human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15.

The screening of hybridomas producing a monoclonal antibody is carried out by culturing the obtained hybridomas in, for example, a microtiter plate, and the culture supernatant of the wells in which the proliferation is observed. And the carrier protein of the supernatant can be measured and compared by an immunoassay such as ELISA.

Hybridomas cloned by screening are cultured in a medium (eg, DMEM containing 10% fetal bovine serum). Then, the centrifugation supernatant of the culture can be used as a monoclonal antibody solution. Further, the hybridoma can be injected into the abdominal cavity of an animal derived from the hybridoma to cause ascites in the animal, and the ascites obtained from the animal can be used as a monoclonal antibody solution. Monoclonal antibodies are preferably isolated and / or purified in a manner similar to the polyclonal antibodies described above.

Chimeric antibodies are, for example, “Experimental Medicine (Extra special issue), Vol. 6, No. 10, 1988”, Japanese Patent Publication No. 3-73280, and humanized antibodies are, for example, Japanese Patent Publication No. 4-506458. Publication, JP-A-62-296890, etc., human antibodies, for example, "Nature Genetics, Vol.15, P.146-156, 1997", "Nature Genetics, Vol.7, P.13-21, 1994 , Special Publication No. 4-504365, International Publication WO94 / 25585, "Nikkei Science, June issue, 40 to 50 pages, 1995", "Nature, Vol.368, p.856-859, 1994 ”and Japanese Patent Publication No. 6-500233.

Antibody production by phage display can be carried out, for example, by recovering and concentrating phage having an affinity for an antigen by biopanning from a phage library prepared for human antibody screening, thereby producing antibodies such as Fab. Can be easily obtained. In this case, it is preferable to screen the antibody library using the polypeptide of the present invention as an antigen. For preferable antibody libraries and antibody screening methods, see `` Science, 228: 4075, p.1315-1317 (1985), '' `` Nature, 348 :, p.552-554, (1990), '' `` Curr. Protein Pept.Sci. ., Sep; 1 (2): 155-169 (2000) ”, international publication WO01 / 062907, etc. can be referred to. The antibody can be prepared by using the antibody fragment thus obtained or by utilizing the DNA of the phage.

The amount of the antibody contained in the prophylactic or therapeutic agent of the present invention is not particularly limited as long as the above effects are exhibited, but is usually 0.001 to 90% by weight of the total prophylactic or therapeutic agent of the present invention. , Preferably 0.005 to 50% by weight, more preferably 0.01 to 10% by weight.

The prophylactic or therapeutic agent of the present invention may contain a pharmaceutically acceptable carrier in addition to the antibody as the active ingredient. As such a carrier, a carrier usually used in the field of formulation can be used. For example, excipients such as sucrose, starch, mannitol, sorbit, lactose, glucose, calcium phosphate, calcium carbonate, sodium benzoate, and sulfite. Preservatives such as sodium hydrogen, methylparaben, propylparaben, stabilizers such as citric acid, sodium citrate, acetic acid, suspending agents such as methylcellulose, polyvinylpyrrolidone, aluminum stearate, dispersants such as surfactants, water, physiological Examples thereof include diluents such as saline, base waxes such as glycerin and polyethylene glycol, but are not limited thereto.

Examples of the dosage form of the prophylactic or therapeutic agent of the present invention include, but are not limited to, solutions and injection preparations. The prophylactic or therapeutic agent of the present invention may be in the form of controlled release preparation such as immediate release preparation or sustained release preparation. Since an antibody is generally soluble in an aqueous solvent, it is easily absorbed by any of the above dosage forms. Furthermore, the solubility of the antibody can be increased by a method known per se.

The preventive or therapeutic agent of the present invention, which can be used for the prevention, treatment or alleviation of glucose metabolism abnormality, can be produced by using the above-mentioned antibody as an active ingredient according to a means known per se as a pharmaceutical production method. ..

For example, the prophylactic or therapeutic agent of the present invention suitable for systemic administration may be produced by dissolving an effective amount of the antibody of the present invention in an aqueous or non-aqueous isotonic sterile injection solution (eg, injection preparation). it can. It may be produced by freeze-drying the antibody of the present invention (eg, freeze-dried preparation) and dissolving it in an aqueous or non-aqueous isotonic sterile diluent. Moreover, the prophylactic or therapeutic agent of the present invention suitable for topical administration can be produced by dissolving the antibody of the present invention in a diluent such as water or physiological saline (eg, liquid preparation). The liquid agent can also be used by inhalation therapy to the bronchus or lungs using a nebulizer. In addition, these agents may contain an antioxidant, a buffer solution, a bacteriostatic agent, an isotonicity agent and the like. These prophylactic or therapeutic agents of the present invention, like ampoules and vials, can be enclosed in a container in a unit dose or multiple doses.

The dose of the prophylactic or therapeutic agent of the present invention can be appropriately set depending on the activity, type or amount of the antibody contained as an active ingredient, administration subject, administration route, age and weight of administration subject, and the like. (60 kg body weight) The daily dose (effective amount) is 0.1 mg to 1000 mg, preferably 0.1 mg to 500 mg, and more preferably 0.1 mg to 300 mg as the amount of antibody. The prophylactic or therapeutic agent of the present invention can be administered once or in several divided doses per day as needed, or can be administered in divided doses for several days.

The preventive or therapeutic agent of the present invention can be used in combination with a known preventive or therapeutic agent for abnormal glucose metabolism. Examples of such known preventive or therapeutic agents for abnormal glucose metabolism include antidiabetic agents such as insulin, tolbutamide, glyclopyramide, glibenclamide, metformin, epalrestat, voglibose, acarbose, troglitazone. These may be used alone or in combination of a plurality of types. In the present specification, the term “combination” means that the prophylactic or therapeutic agent of the present invention is used in combination with the known prophylactic or therapeutic agent for abnormal glucose metabolism, and its form of use is not particularly limited. For example, administration as a pharmaceutical composition containing both the prophylactic or therapeutic agent of the present invention and the known prophylactic or therapeutic agent for abnormal glucose metabolism, or separate formulation without mixing, and simultaneous or staggered administration Including both.

The present invention will be described in more detail below with reference to examples, but it goes without saying that the present invention is not limited thereto.

Vaccine Design and Peptide Synthesis Five different antigenic peptides were selected from the amino acid sequence of mouse CD153 based on three-dimensional predicted structure and high antigenicity analysis of epitope information (Table 1). The N-terminal of the peptide was conjugated to KLH (Enzo Life Sciences Inc., Farmingdale, NY, USA) as a carrier protein, and the synthetic peptide was purified by reverse phase HPLC (> 98% purity) (Peptide Institute Inc., Osaka, Japan). The CD153 peptide vaccine was reconstituted with 0.5-1 mg / ml CD153 peptide and 5-10 mg / ml KLH in sterile phosphate buffered saline (PBS).

Animals All animal experiment procedures have been evaluated and approved by the Animal Experiment Committee of Osaka University. Basic guidelines for animal experiments in research facilities (MEXT, Japan), Basic guidelines for animal experiments in institutions (Ministry of Health, Labor and Welfare, Japan), animals Followed the recommendations of the guidelines (Science Council of Japan, Japan) for the proper implementation of experiments. Seven-week-old male C57BL / 6J mice and eight-week-old female C57BL / 6N mice were purchased from CLEA Japan Inc. and kept in a temperature and light cycle control facility. The mice had free access to food and water. C57BL / 6J mice were fed with normal diet (ND) (MF, 12.8 kcal% fat; Oriental Yeast Co., LTD.) Or high-fat diet (HFD) (D12492, 60 kcal% fat; Research Diets Inc.) to give C57BL. / 6N mice received ND.

Vaccination Schedule A single dose of CD153 vaccine was prepared as a mixture of CD153-KLH peptide solution (30 μg CD153 peptide and 200-300 μg KLH) and adjuvant solution. Prepared as a single dose KLH vaccine as a mixture of 200-300 μg KLH and adjuvant solution. The adjuvant solution contained 30 μl Alhydrogel (CD153-Alum, KLH-Alum; Invivogen) or 30 μg CpG ODN 1585 (CD153-CpG, KLH-CpG; Invivogen). In studies of TLR7 ligand administration, female C57BL / 6N mice were vaccinated subcutaneously with the CD153-CpG vaccine or the KLH-CpG vaccine at 8, 10 and 12 weeks of age. In studies of HFD burden, males were given D153-alum vaccine or KLH-alum vaccine at 7, 9, 11, 13, 15 weeks of age, or D153-CpG vaccine or KLH-CpG vaccine at 7, 9, 11, 16 weeks of age. C57BL / 6J mice were vaccinated subcutaneously. Serum was collected from the tail vein and anti-CD153 antibody titer was measured by enzyme-linked immunosorbent assay (ELISA).

TLR7 ligand administration R848 (TLR7 ligand; InvivoGen) was intraperitoneally injected into 12-week-old female C57BL / 6N mice three times a week. Mice sacrificed at 16 weeks of age were injected with 5 μg of R848 for 4 weeks. Mice sacrificed at 18 weeks of age were injected with 5 μg of R848 for 4 weeks and 10 μg of R848 for another 2 weeks.

Cell line and culture conditions Mouse macrophage cell line RAW 264.7 was obtained from American Type Culture Collection (ATCC). RAW 264.7 was grown in Dulbecco's modified Eagle medium (DMEM; Nacalai Tesque, Kyoto, Japan) supplemented with 10% (v / v) heat-inactivated FBS (Sigma Aldrich, MO, USA), and RAW 264.7 was grown before recovery. The cells were stimulated with μg / ml of LPS (from L4391, E. coli O111: B4; Sigma Aldrich, MO, USA) for 24 hours. The cell line was cultured at 37 ° C in 5% CO ₂ according to the ATCC animal cell culture guide.

Serum anti-CD153 antibody titers were quantified by enzyme-linked immunosorbent assay ELISA. 96 wells with CD153-BSA conjugate (Peptide Institute Inc., Osaka, Japan) or recombinant mouse CD153 protein (no carrier; R & D Systems, Minneapolis, MN, USA) diluted in 50 mM carbonate buffer at 10 μg / ml. ELISA plates were coated overnight at 4 ° C. After blocking with PBS containing 5% skim milk, serum was serially diluted 100 to 325,000-fold in blocking buffer, added to each well, and incubated overnight at 4 ° C. After washing each well with 0.05% PBS Tween-20 (PBS-T), the mouse IgG-specific horseradish peroxidase (HRP) -conjugated antibody (1: 1000; GE Healthcare, UK) for 3 hours at room temperature. Each well was incubated. For the IgG subclass determination assay, anti-mouse IgG subclass specific HRP conjugated antibody (1: 1000; IgG1, IgG2b, IgG2c and IgG3, Abcam) was used. After washing with PBS-T, the color was developed with the peroxidase

chromogenic substrate

3,3 ′, 5,5′-tetramethylbenzidine (TMB; Sigma Aldrich, MO, USA), and the reaction was stopped with 0.5 N sulfuric acid. All plates were measured at 450 nm absorbance using an iMark Microplate Absorbance Reader (Bio-Rad, CA, USA). The half maximal antibody titer was determined according to the maximum in the dilution range for each sample.

Western blot analysis

30 or 100 ng of recombinant mouse CD153 (rmCD153; R & D Systems) and 100 ng of recombinant mouse OPN (rmOPN; used as negative control; R & D Systems) were electrophoretically separated by SDS / PAGE and washed with water. Blotted on wet Immobilon-P PVDF transfer membrane (Merck Millipore Ltd.). Membranes blotted with 500-fold diluted serum from mice immunized with CD153 vaccine or KLH vaccine or 0.05 μg / ml commercial anti-CD153 antibody (R & D Systems) were incubated overnight at 4 ° C. After incubating for 1 hour with 2000-fold diluted HRP-conjugated IgG antibody (GE Healthcare), the chemiluminescence signal visualized with Chemi-Lumi One L (Nacalai Tesque) was detected with LAS 1000 (Fuji Film), and It was analyzed with Gauge software version 3.2.

Complement-dependent cytotoxicity assay Total IgG from pooled sera from immunized mice using 50% saturated ammonium sulfate, Zeba spin desalting columns and Melon gel IgG spin purification kit (Thermo Scientific, Rockford, IL, USA). The antibody was purified. For complement-dependent cytotoxicity (CDC) assay, LPS-stimulated RAW 264.7 cell suspension containing 2 x 10 ⁴ cells in DMEM supplemented with 1% FBS at various concentrations (30, 100, 300 μg). (ml / ml) was mixed with purified IgG antibody or positive control antibody and incubated at 4 ° C for 1 hour. The positive control antibodies used were anti-mouse CD153 antibody (functional grade, RM153; eBioscience) and anti-mouse MHC Class I (H-2Kd / H-2Dd) antibody (functional grade, 34-1-2S; eBioscience). .. Low-Tox-M rabbit complement (Cedarlane, Hornby, Canada) was then added to the resuspension. Cell death was assessed as a percentage of 7-AAD positive cells using flow cytometric analysis.

Metabolic measurement Insulin sensitivity was assessed by an intraperitoneal insulin tolerance test (ipITT) after a 4-hour fast. Blood glucose levels were measured before, 30, 60, and 120 minutes after intraperitoneal injection of 0.75 U / kg of recombinant human insulin (Humulin R; Eli Lilly Japan KK, Japan). Glucose tolerance was assessed by the oral glucose tolerance test (OGTT) after a 6-hour fast. Blood glucose levels were measured before, 15, 30, 60, and 120 minutes after oral administration of 2.0 g / kg glucose. A homeostatic model assessment of insulin resistance (HOMA-IR) was calculated as an indicator of insulin resistance: fasting serum glucose (mmol / L) x fasting serum insulin (pmol / L) /22.5. Oxygen consumption was measured with an O ₂ / CO ₂ metabolism measuring system (MK-5000RQ; Muromachi Kikai, Tokyo, Japan) for small animals. Each mouse was placed in an airtight chamber maintained at 25 ° C with an airflow of 0.50 L / min for 24 hours or more, and oxygen consumption was standardized by a kilogram ^0.75 body weight.

Flow cytometric analysis Spleen samples from immunized mice were disrupted and suspended in staining buffer (BD Biosciences, San Diego, CA, USA). Shred VAT into fine pieces, 37 ° C in a digestion buffer consisting of HBSS containing 10% FBS, 100 μg / ml DNase I and 200 U / ml collagenase type I (Worthington, Lakewood, NJ, USA), Shaking culture was carried out for 1 hour. The digested tissue was centrifuged at 1000 xg, 4 ° C for 10 minutes and resuspended in staining buffer. Red blood cells were removed from a suspension of splenocytes and the interstitial vascular fraction of fat (SVF) using ACK red blood cell lysis buffer (Gibco, Grand Island, NY, USA). After red blood cell lysis, suspended splenocytes and SVF were filtered through a 70-μm filter, centrifuged at 1000 xg, 4 ° C for 10 minutes and resuspended in staining buffer. Block the Fc receptor with anti-mouse CD16 / 32 antibody (mouse Fc receptor inhibitor; BD Biosciences) at 4 ° C for 20 minutes, and then stain the cells with a mixture of fluorescent-labeled antibodies at 4 ° C for 40 minutes in the dark. did. The antibodies used were specific for CD4 (RM4-4), CD44 (IM7), CD153 (RM153) (BD Biosciences), CD62L (MEL-14) and PD-1 (29F.1A12) (Bio Legend). It was 7-AAD viability staining solution was added to exclude dead cells. Flow cytometric analysis was performed using BD FACS Canto II (BD Biosciences) and analyzed using BD FACS Diva software and FCAP Array software (BD Biosciences).

Immunohistochemistry For paraffin sections, mouse VAT, kidney and lung samples were fixed with 10% neutral buffered formalin and embedded in paraffin. Hematoxylin and eosin staining was performed in VAT, kidney tissue and lung tissue. After deparaffinization and rehydration, immunohistochemical staining for F4 / 80 (CI: A3-1, Abcam) and CD153 (Biorbyt) was performed on VAT and immunohistochemistry for IgG (only secondary antibody used). Staining was performed on VAT, kidney tissue and lung tissue. For antigen retrieval, F4 / 80 and IgG staining were incubated with proteinase K (Agilent Technologies) for 5 minutes, and CD153 staining was incubated with HistoVT One (Nacalai Tesque) at 90 ° C. for 30 minutes. For enzymatic detection, use the polymer-based detection kit (MAX-PO (M), Nichirei Bioscience) for F4 / 80 and IgG staining and avidin-biotin for CD153 staining according to the manufacturer's recommendations. A complex kit (VECTASTAIN Elite ABC kit, Vector Laboratories) was used. Inhibition of endogenous peroxidase was performed with 0.3-0.6% H ₂ O ₂ in methanol prior to enzymatic detection.

Statistical analysis All data were expressed as mean ± standard error. The statistical significance of the difference between the two groups was verified by a two-tailed Student's t-test. Differences between multiple groups were analyzed by analysis of variance (ANOVA) and verified by Tukey's multiple comparison test. Differences were considered statistically significant when P <0.05. Statistical analysis was performed using Prism GraphPad version 6.07 (GraphPad Software).

Example 1 CD153-specific IgG induced by the CD153 # D vaccine recognized the rmCD153 protein, and CD153-CpG vaccination tended to induce a Th1 immune response.
The CD153 vaccine consists of an antigenic peptide and a carrier protein and induces specific antibodies against CD153. According to the epitope information of mouse CD153, the present inventors have developed five peptides as antigens (#A, 116-125 aa; #B, 182-189 aa; #C, 101-108 aa; #D, 76-85 aa; # E, 234-239 aa; Table 1) was designed. Five peptide vaccines (30 μg dose CD153 peptide / mouse) were conjugated with keyhole limpet hemocyanin (KLH) as a carrier protein and administered to 7-week-old male C57BL / 6J mice twice at 2 week intervals together with alum adjuvant. In the antibody production evaluation by ELISA, the titer against mouse CD153-BSA first increased on day 14 in mice immunized with #A or #C vaccine, and on day 28 immunized with each of the 5 vaccines. It was successfully increased in mice (Fig. 1A). In the evaluation of ELISA using recombinant mouse CD153 (rmCD153), the antibody induced by the CD153 # D vaccine strongly reacted with rmCD153 (FIG. 1B). Western blot analysis also showed that the antibodies induced by the CD153 # D vaccine reacted specifically and strongly with rmCD153. The commercially available anti-CD153 antibody and CD153 # D vaccine-derived antibody used as the positive control antibody recognized 30 ng of rmCD153 protein and 100 ng of rmCD153 protein, but the recombinant mouse OPN protein (rmOPN; as a negative control protein). Use) was not recognized. On the other hand, the antibody induced by the KLH control vaccine did not recognize the rmCD153 protein or the rmOPN protein (Fig. 1C). These results demonstrate that the CD153 # D vaccine-derived antibody efficiently recognizes the rmCD153 protein. Therefore, the inventors selected the CD153 # D vaccine as the vaccine to be used in Examples 2 to 5 below. Moreover, alum adjuvant can induce Th2 immune response and CpG adjuvant can induce Th1 immune response. Therefore, the inventors have evaluated serum IgG subclass antibody against CD153 (IgG1; Th2 immune response, IgG2b, IgG2c and IgG3; Th1 immune response) by ELISA, CD153 # D-CpG vaccination is CD153 # D-Alum vaccine. It was confirmed whether the Th1 immune response can be further induced by inoculation. As a result, in IgG subclass analysis, CD153 # D-CpG vaccination induced more IgG2b, IgG2c and IgG3 than CD153 # D-alum vaccination (FIG. 1D). These results show that CD153 # D-CpG vaccination efficiently induces a Th1 immune response.

Example 2 CD153 # D-CpG Vaccine-Induced Antibody Reduces Spleen CD153-Positive Senescence-Associated T Cells Induced by In Vivo Administration of TLR7 Ligand
It has been reported that CD153-positive senescence-related T cells gradually increase systemically with age. The inventors also confirmed that CD153-positive senescence-related T cells in spleen tissue of 20-month-old male C57BL / 6J mice were increased compared to 3 months of age (18.7 ± 0.5% versus 3.8 ± 0.2). %; p <0.0001; n = 3, each group). In VAT, CD153-positive senescence-related T cells tended to increase in old mice compared with young mice (8.2 ± 1.0% versus 4.3 ± 1.4%; p = 0.08; n = 3, each group) ( (Figure 2A). Several studies report that the development of CD153-positive senescence-associated T cells is dependent on autoreactive B cells, especially spontaneous germinal centers (GCs), and that the GC response is driven by continuous administration of TLR7 ligands Has been done. Since CD153-positive senescence-related T cells are efficiently induced by TLR7-driven autoreactive B cells, we examined CD153-positive senescence-related T cells under R848-administered conditions and tested for CD153 # D-CpG vaccination. Estimated effect. Following the experimental R848 administration protocol (FIG. 2B), the CD153 # D-CpG vaccine was administered to 8-week-old female C57BL / 6N mice three times at 2-week intervals, and then R848 three times a week for 4-6 weeks. .. The anti-CD153 antibody titer induced by the CD153 # D-CpG vaccine was maintained at a high level during the administration period of R848 (FIG. 2C). In addition, by flow cytometry analysis, the proportion of CD153-positive senescence-related T cells in the pancreas showed almost no difference between the PBS control group and the R848-administered group at 16 weeks of age, but compared with the PBS control group of 18 weeks of age. It was shown that the increase was observed in the 18-week-old R848-administered group. Furthermore, the inventors confirmed that the proportion of CD153-positive senescence-related T cells in the pancreas was reduced in mice immunized with the CD153 # D-CpG vaccine compared to the R848-administered group and the KLH-CpG vaccinated group. (Fig. 2D). Furthermore, continued TLR7 stimulation induces chronic immune activation and is known to cause splenomegaly, so CD153 # D-CpG vaccination improves splenomegaly associated with R848 stimulation. In order to evaluate whether or not the present inventors further examined the spleen weight of R848-treated mice. The inventors found that spleen weight was significantly increased by continuous R848 stimulation, and spleen weight was decreased in mice immunized with the CD153 # D-CpG vaccine as compared to the R848-administered group (FIG. 2E). In addition, spleen weight tended to decrease in mice immunized with the KLH-CpG vaccine (p = 0.07, versus R848 control group). These results suggested that CD153 # D-CpG vaccination ameliorates CD153-positive senescence-associated T cells in the spleen and splenomegaly stimulated by chronic immune activation.

Example 3 HFD-induced insulin resistance and impaired glucose tolerance are ameliorated by CD153 # D-CpG vaccination.
Recent studies have reported that CD153-positive senescence-associated T cells accumulate in adipose tissue of HFD-fed mice, exacerbating glucose and insulin resistance. Therefore, the inventors next investigated whether CD153 # D-CpG vaccination of HFD-loaded mice could reduce CD153-positive senescence-associated T cells in VAT and improve glucose and insulin resistance. CD153 # D-Alum vaccine or CD153 # D-CpG vaccine was administered multiple times to 7-week-old male C57BL / 6J mice according to the experimental HFD challenge study protocol (Figure 3A), followed by 10-11 weeks of HFD challenge. went. Both anti-CD153 antibody titers induced by the CD153 # D-alum vaccine and the CD153 # D-CpG vaccine remained high during HFD challenge (Fig. 3B). Body weights did not differ significantly between the CD153 # D-alum vaccinated and CD153 # D-CpG vaccinated groups (data not shown). However, the body weight of mice immunized with the CD153 # D-CpG vaccine was significantly lower than that of mice immunized with the KLH-CpG vaccine and tended to be lower than that of the HFD control group (Fig. 3C). Also, the weekly average HFD intake was decreased in mice immunized with the CD153 # D-CpG vaccine as compared to the HFD control group and the KLH-CpG vaccine group (Fig. 3D). In addition, oxygen consumption was estimated as an indirect measure of metabolism. Mice immunized with the CD153 # D-CpG vaccine tended to have increased oxygen consumption in the dark phase compared to mice immunized with the KLH-CpG vaccine (p = 0.08) (Fig. 3E). The inventors next evaluated the intraperitoneal insulin tolerance test (ipITT) and the oral glucose tolerance test (OGTT). Insulin sensitivity did not differ between the CD153 # D-alum vaccinated and KLH-alum vaccinated groups (Fig. 4A), but the CD153 # D-CpG vaccinated group had higher insulin sensitivity than the KLH-CpG vaccinated group. And the area under the curve (AUC) was improved (Figures 4B and 4C). Although glucose tolerance did not differ between the CD153 # D-alum vaccinated group and the KLH-alum vaccinated group (Fig.4D), in the CD153 # D-CpG vaccinated group, the KLH-CpG vaccinated group and the HFD control Glucose tolerance and AUC were significantly improved compared to the group (Figures 4E and 4F). Further, in the CD153 # D-CpG vaccinated group, HOMA-IR, which is an index of insulin resistance, was significantly improved as compared with the KLH-CpG vaccinated group (Fig. 4G). These results indicate that CD153-CpG vaccination ameliorates obesity-induced metabolic abnormalities such as glucose tolerance and insulin resistance.

Example 4 CD153 # D-CpG Vaccine-Induced Antibody Reduces CD153-Positive Senescence-Associated T Cells Induced by HFD Loading.
The inventors analyzed the proportion of obesity-induced CD153-positive senescence-related T cells by flow cytometry. Flow cytometric analysis showed that the proportion of CD153-positive senescence-related T cells in the pancreas did not change between the ND control group and the HFD control group. The inventors also found that neither CD153 # D-alum vaccination nor CD153 # D-CpG vaccination affected the proportion of CD153-positive senescence-associated T cells in the pancreas (FIG. 5A). On the other hand, the proportion of CD153-positive senescence-related T cells in VAT in the HFD control group was significantly increased compared to that in the ND control group. Furthermore, the inventors found that, unlike CD153 # D-alum vaccination, CD153 # D-CpG vaccination reduced the proportion of CD153-positive senescence-associated T cells in VAT (Fig. 5B). These results show that in HFD-loaded obese mice, CD153 # D-CpG vaccination locally improves, if not systemically, CD153-positive senescence-related T cells. An antibody-mediated complement dependent cytotoxicity (CDC) assay was performed to determine whether the CD153 # D-CpG vaccine-induced antibody was able to eliminate immune cells that overexpress CD153. Since CD153 expression of LPS-stimulated RAW264.7 cells was higher (40-60%) than that of mouse splenocytes or adipocytes (5-20%) in this example, LPS-stimulated RAW264.7 cells were subjected to CDC. Selected as target cells for the assay. The CDC assay was supplemented with purified IgG (30, 100, 300 μg / ml) from mice immunized with the CD153 # D-CpG vaccine, similar to the commercially available anti-mouse CD153 antibody (10, 30, 100 μg / ml). Induced body-dependent cell death, whereas purified IgG (30, 100, 300 μg / ml) from mice immunized with CD153 # D-alum vaccine, KLH-alum vaccine or KLH-CpG vaccine did not (Fig. 5C).

Example 5 CD153 # D-CpG Vaccine Reduces Accumulation of Macrophages and CD153 Positive Senescence-Associated T Cells in Adipose Crown-Like Structures
Histological analysis of VAT, kidney tissue and lung tissue was performed. VAT weight (Figure 6-1A) and mean adipocyte surface area (Figure 6-1B) are decreased in mice immunized with the CD153 # D-CpG vaccine compared to the HFD control group and the KLH-CpG vaccinated group. There was a tendency. Obesity results in the accumulation of macrophages in the crown-like structure (CLS) surrounding adipocytes, which is associated with chronic inflammation of VAT. In addition, CD153-positive senescence-associated T cells are also localized in CLS. Immunohistochemical staining for F4 / 80 showed less accumulation of F4 / 80 ⁺ cells in the CLS of mice immunized with the CD153 # D-CpG vaccine than in HFD control mice (Figure 6-1C). Immunohistochemical staining for CD153 also showed that the accumulation of CD153-positive senescence-related T cells in the CLS of F4 / 80 ⁺ cells in the CD153 # D-CpG vaccinated group was improved over the HFD control group (Fig. 6-2D). Furthermore, the inventors stained VAT, kidney tissue and lung tissue with an anti-mouse IgG antibody and evaluated whether or not autoreactive antibody was present (FIG. 6-2E). VAT had no positive staining for IgG except for a slight positive staining for IgG in CLD of mice immunized with the CD153 # D-CpG vaccine. In lung tissue, IgG antibody deposition on the alveolar wall was present in mice immunized with KLH-CpG vaccine or CD153 # D-CpG vaccine, but alveolar wall thickness and inflammatory cells among 4 groups There was no obvious difference in the infiltration of the. Similarly, in renal tissues, there was IgG antibody deposition to the glomerulus in mice immunized with the KLH-CpG vaccine or the CD153 # D-CpG vaccine, but glomerular capillary loop thickness and fragility among the 4 groups was present. There was no obvious difference.

Example 6 Anti-human CD153 antibody is not cross-reactive with mouse CD153 and anti-mouse CD153 antibody is not cross-reactive with human CD153.
The inventors designed a human CD153 # D vaccine homologous to the CD153 # D vaccine (mouse CD153 # D vaccine) from the human CD153 amino acid sequence in order to verify the cross-reactivity of the anti-human CD153 antibody and the anti-mouse CD153 antibody. (Fig. 7A). The mouse CD153 # D-alum vaccine and the human CD153 # D-alum vaccine were each administered to 7-week-old male C57BL / 6J mice three times for two weeks, and the production of mouse or human CD153 antibody was evaluated by ELISA. The inventors found that antibodies induced with the human CD153 # D-alum vaccine strongly reacted with human CD153 # D-BSA and recombinant human CD153 (rhCD153) (Fig. 7B, C). On the other hand, the antibody did not react with mouse CD153 # D-BSA and recombinant mouse CD153 (rmCD153) (Fig. 7B, C). In addition, the antibody induced by the mouse CD153 # D-alum vaccine reacted with rmCD153 but hardly with rhCD153 (Fig. 7D).

By using the partial amino acid sequence of CD153 of the present invention as a vaccine, an antibody that inhibits senescence-related T cells expressing CD153 on the cell surface is induced, and thereby an inflammatory protein or inflammatory protein from senescence-related T cells is induced. It can reduce chemokine secretion and improve glucose metabolism disorders. Since the antibody induced by the vaccine has a long half-life, it does not require frequent administration unlike conventional therapeutic agents for abnormal glucose metabolism. This application is based on Japanese Patent Application No. 2018-211771 (filed on: Nov. 9, 2018) filed in Japan, the contents of which are incorporated in full herein.

Claims

A vaccine for preventing or treating abnormal glucose metabolism, which comprises any of the following (1) to (3):
(1) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence derived from a non-human mammal corresponding to SEQ ID NO: 2;
(2) Amino acid in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence derived from non-human mammal corresponding to SEQ ID NO: 2 A polypeptide comprising a sequence,
(3) An expression vector capable of expressing the polypeptide of (1) or (2) above.
The vaccine according to claim 1, comprising the following (1 ') or (2'):
(1 ') a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2,
(2 ′) An expression vector capable of expressing the polypeptide of (1 ′) above.
The vaccine according to claim 1, comprising the following (1 '') or (2 ''):
(1 '') a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4,
(2 ″) An expression vector capable of expressing the polypeptide of (1 ″) above.
The vaccine according to any one of claims 1 to 3, further comprising a carrier protein.
The vaccine according to claim 4, wherein the carrier protein is keyhole limpet hemocyanin.
The vaccine according to any one of claims 1 to 5, further comprising an adjuvant.
The vaccine according to claim 6, wherein the adjuvant is CpG oligodeoxynucleotide.
The glucose metabolism disorder is selected from the group consisting of diabetes, glucose intolerance, obesity, hyperinsulinemia, diabetic neuropathy, diabetic nephropathy and diabetic retinopathy. Vaccine according to paragraph.
A prophylactic or therapeutic agent for glucose metabolism disorder, which comprises an antibody that recognizes the following polypeptide (1) or (2) and inhibits senescence-related T cells:
(1) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence derived from a non-human mammal corresponding to SEQ ID NO: 2;
(2) Amino acid in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence derived from non-human mammal corresponding to SEQ ID NO: 2 A polypeptide comprising a sequence.
The preventive or therapeutic agent according to claim 9, which comprises an antibody that recognizes the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 and inhibits senescence-related T cells.
The preventive or therapeutic agent according to claim 9, which comprises an antibody that recognizes the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 and inhibits senescence-related T cells.
The preventive or therapeutic agent according to any one of claims 9 to 11, wherein the senescence-related T cells are CD4 positive PD-1 positive CD153 positive T cells.
The glucose metabolism disorder is selected from the group consisting of diabetes, glucose intolerance, obesity, hyperinsulinemia, diabetic neuropathy, diabetic nephropathy and diabetic retinopathy. The preventive or therapeutic agent according to the item.