JP2008161074A - CAVEOLIN-1-Fcgamma1 FUSION PROTEIN AND ITS USE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an endogenous ligand of CD26-mediated T-cell costimulation. <P>SOLUTION: It is shown that caveolin-1 is the endogenous ligand of CD26-mediated T-cell costimulation. Since a caveolin-1-Fcγ1 fusion protein inhibits the CD26-mediated T-cell costimulation caused by caveolin-1, a drug composition comprising the fusion protein can be used as an immunosuppressant agent or the like. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a caveolin-1-Fcγ1 fusion protein and a method for using the fusion protein.

  The immune system function provided in the living body is one of the most important functions for maintaining a life phenomenon. The immune system is originally responsible for defense against foreign substances by distinguishing between self and non-self. In addition, the immune system does not take an organ structure and controls the maintenance of the living body by a complicated and dynamic process involving 1 trillion cells. The immune system ultimately protects itself from non-self through an inflammatory response. Therefore, for example, understand the inflammatory response in infectious diseases, the inflammatory response accompanied by organ destruction in autoimmune diseases, and the immune response in transplantation treatment aimed at restoring organ function by organ replacement and the inflammatory response that is the final aspect This is also very important for maximizing the desired therapeutic effect.

  The entry of antigens into cells in the first stage of inflammation occurs non-specifically or via antibodies or complement receptors. The antigen taken up by the cell is processed into an antigen presenting cell (APC) and converted into a peptide fragment that is presented to the T cell as a complex with a major histocompatibility complex (MHC) class II molecule. The APC includes dendritic cells, macrophages, B cells, Langerhans cells, interdigitating cells, peripheral blood monocytes, etc. (so-called professional APC). Simultaneously with antigen presentation to T cells, receptor stimulation called co-stimulation, which is an auxiliary signal, triggers and enhances T cell activation. Activation of T cells requires both antigen-specific and costimulatory stimulation provided by the MHC peptide complex.

Differences in the expression levels of the auxiliary signal receptor molecules CD80 and CD86 expressed on APC, or the types of cytokines produced, cause bias in subsequent differentiation of helper T cells ( _TH cells). For example, under the influence of interleukin-12 (IL-12), differentiation into the T _H 1 type involved in cellular immunity is dominant, whereas under the influence of IL-4, T involved in humoral immunity is dominant. Differentiation into _H 2 type predominates. Thus, sensitization is established when primary effector cells are activated or antibody production is induced. At the same time, memory T cells and memory B cells are generated. Examples of primary effector cells include T _H 1 cells, cytotoxic T cells (CTL), mast cells, monocytes, macrophages, basophils, neutrophils, NK cells, and platelets.

  Next, inflammatory endothelial cells are activated by the action of cytokines, chemokines, chemical mediators, etc. generated by the activation of primary effector cells, and secondary cells such as monocytes, macrophages, neutrophils, eosinophils, etc. Effector cell activation is triggered to cause inflammation. Inflammatory responses include phagocytosis of causative substances in the local area of inflammation, apoptosis of effector cells by Fas / Fas-L, immunoreceptor tyrosine-based inhibition motif (immunoreceptor tyrosine) by CTLA 4 (cytotoxic T-lymphocyte-associated antigen 4) and FcγRIIB -based inhibitory motif) is finally suppressed by the transmission of inhibitory signals.

  Immunological disorders such as autoimmune diseases or rejection reactions, graft-versus-host disease (GVHD), etc. show inflammatory reactions accompanied by organ destruction, and it has been clarified that the various effector mechanisms described above are involved. Nevertheless, a detailed understanding of the immune pathology of these diseases remains inadequate and complete control of these diseases has not yet been achieved.

  In the treatment of autoimmune diseases and transplantation complications, various immunosuppressive therapies have been tried to control the above-mentioned effector mechanisms of inflammation. The point for controlling immune disorders is thought to be suppression of effector T cells. In fact, many of the major immunosuppressive drugs target T cells. Therefore, studying T cells as effector cells of inflammation can be said to be very important for overcoming immune disorders.

In view of such circumstances, the present inventors have conducted research on effector T cells, and in particular, discovered the CD26 molecule, which is a T cell activation antigen, and analyzed its function. The CD26 molecule is an important molecule for CD4 positive T cells to exert effector functions, and its expression increases with activation of T cells and stimulates activation from CD3 / TCR (T cell receptor complex). It has been clarified by an experimental system using a specific antibody of CD26 that it is a costimulatory molecule to be enhanced. Moreover, the CD26-positive T cells, is very easily moved T _H 1-type cells, a subset of the sites of inflammation, and contributes to the immune disorders such as autoimmune diseases, and rejection or GVHD, such as rheumatoid arthritis, also It is known to accumulate in the affected area. Therefore, a deeper understanding of CD26-positive T cells will enable the establishment of more pathological treatments.

The CD26 molecule was reported as a human peripheral blood T cell surface antigen that reacts with a mouse monoclonal antibody named Ta1, and was subsequently established as a T cell activation antigen because it is strongly expressed on activated T cells. On the other hand, dipeptidyl peptidase IV (DPPIV) has been known as a peptidase enzyme present on the surface of liver and intestinal mucosal cells. Recent gene cloning showed that DPPIV and CD26 are identical.
In other words, it was revealed that CD26 is expressed not only in lymphocytes but also in vascular endothelial cells, endometrium, etc. in addition to epithelium such as kidney, bile duct, pancreatic duct, intestinal tract and prostate.

  The human CD26 gene encodes a 110 kDa membrane protein consisting of 766 amino acids. CD26 deduced from cDNA is a type II membrane glycoprotein, and its structure is present in the cytoplasm at the N-terminal side and extracellularly at the C-terminal side. The amino acid in the intracellular region of CD26 is only 6 residues, the transmembrane portion is 22 residues, the extracellular portion is 738 residues, and it can be said that most of the portion exists outside the cell. The extracellular region contains a consensus sequence of serine proteases with an active center at the 630th serine residue in a portion close to the C-terminal side. There is a cysteine-rich domain immediately on the N-terminal side, and there are binding sites for adenosine deaminase (ADA), fibronectin, collagen, and the like. The homology between human CD26, rat DPPIV and mouse CD26 is 85.5% and 89%, respectively.

CD26 molecule is expressed on memory T cells in peripheral blood lymphocytes. When the expression of CD26 on quiescent T cells is examined by flow cytometry, the expression intensity shows a triphasic pattern, among which the CD26 ^high population is known to play an important role in the immune system. . The CD26 ^high population belongs to memory T cells that express CD45RO, reacts to memory antigens such as tetanus toxoid, induces B cell antibody production, and induces MHC class I specific killer T cells. it can.
As described above, this CD26-positive T cell population is a T _H 1 type cell that secretes cytokines such as IL-2 and IFN (interferon) -γ. Furthermore, this cell population has the ability to migrate between vascular endothelial cells, migrates and accumulates at the inflammatory site, and is thought to play an important role in the inflammatory site.

As described above, the CD26 molecule is a so-called costimulatory molecule, and by stimulating with a CD26-specific antibody simultaneously with the antigen-specific primary signal from the TCR, a non-antigen-specific secondary signal is transmitted to the T cell, Induces T cell activation.
Moreover, by itself stimulate peripheral blood T cells and CD26 transgenic Jurkat cells with anti-CD26 antibody, CD3 zeta ^{chain, p56 lck, p59 fyn, ZAP} -70, Mitogen activated protein kinase (MAP kinase), c-Cbl, Phospholipase It has also been shown that tyrosine phosphorylation of signal transduction proteins such as Cγ and that tyrosine phosphorylation produced by CD3 single stimulation is enhanced and prolonged by co-crosslinking of CD3 and CD26.

  Thus, CD26 is directly involved in the T cell activation signal transduction mechanism. The present inventor has found that caveolin-1 on monocytes treated with tetanus toxoid binds to CD26 (Patent Document 1). In addition, after caveolin-1 stimulated by CD26 was phosphorylated, it was shown that it activates NF-κB and ultimately enhances the expression of CD86 on APC. These studies reveal one aspect of the mechanism by which CD26-positive T cells are activated in response to memory antigens such as tetanus toxoid.

However, it has not yet been clarified whether the interaction between APC and T cells via caveolin-1 leads to T cell costimulatory activity by CD26, and the intrinsic cause of T cell costimulation via CD26 The sex ligand was unknown.
International Publication No. 2005/063170 Pamphlet

As described above, CD26-mediated activation of T cells has been known only when a CD26-specific antibody is used. For identification of endogenous costimulatory ligands corresponding to the so-called CD26 molecule, It was not reached.
Accordingly, an object of the present invention is to identify and provide a ligand for CD26-mediated T cell costimulation.
Another object of the present invention is to provide a method for using the identified CD26 endogenous ligand.
Another object of the present invention is to provide a pharmaceutical composition for controlling an immune response mediated by T cells.

  In order to solve the above-mentioned problems, the present inventor conducted intensive studies and identified caveolin-1 as an endogenous ligand for T26 costimulation via CD26. Specifically, it was found that caveolin-1 expressed as a fusion protein with an immunoglobulin protein binds to CD26 and activates T cells in the presence of an anti-CD3 antibody. In addition, the present inventor has shown that a fusion protein of caveolin-1 and immunoglobulin protein (caveolin-1-Fcγ1 fusion protein) inhibits the binding of CD26 and caveolin-1 and inhibits T cell costimulation. I found it. As described above, the present inventor has completed the present invention.

That is, the present invention relates to the following.
(1) Contains a first region consisting of all or part of the caveolin-1 skeleton region and a second region consisting of the hinge, C _H 2 and C _H 3 regions of an immunoglobulin molecule, and has a binding activity to CD26. A caveolin-1-Fcγ1 fusion protein.
Examples of the caveolin-1 backbone region include the following polypeptides (a) or (b).
(a) a polypeptide comprising the amino acid sequence from the 82nd position to the 101st position in the amino acid sequence represented by SEQ ID NO: 2
(b) in the amino acid sequence from the 82nd to the 101st amino acid sequence shown in SEQ ID NO: 2, consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and Polypeptide having binding activity (2) DNA encoding the fusion protein according to (1) above.
(3) A caveolin-1-Fcγ1 fusion protein expression vector containing the DNA of (2) above.
(4) A pharmaceutical composition comprising the fusion protein according to (1) above.
The above pharmaceutical composition is used for autoimmune disease, rejection, graft-versus-host disease, rheumatoid arthritis, vasculitis syndrome, malignant tumor, malignant mesothelioma, acute or chronic graft-versus-host disease, rejection, obstructive arteriosclerosis Can be used to treat symptom, myocardial infarction or cerebral infarction.
(5) A CD26-mediated T cell costimulatory ligand containing all or part of the caveolin-1 skeletal region.
(6) CD26-mediated T cells comprising a first region consisting of all or part of the caveolin-1 skeletal region and a second region consisting of the hinge, C _H 2 and C _H 3 regions of an immunoglobulin molecule Costimulatory ligand.
(7) A method of costimulation of T cells, which comprises contacting the fusion protein according to (1) with an isolated T cell in the presence of an anti-CD3 antibody.
(8) A screening method for a substance that controls T26 costimulatory activity via CD26 by caveolin-1,
(i) When the fusion protein described in (1) is contacted with CD26, and (ii) When the fusion protein described in (1) and the test substance are contacted with CD26, CD26 and the fusion protein Measuring the binding amount or T cell costimulatory activity.
In this screening method, the measurement of T cell costimulatory activity includes, for example, measurement of IL-2 production from T cells or measurement of T cell proliferation activity.
(9) A method for evaluating T cell costimulatory activity via CD26 possessed by an evaluation target substance,
(i) When the fusion protein described in (1) is contacted with CD26, and (ii) When the fusion protein described in (1) and the test substance are contacted with CD26, CD26 and the fusion protein Measuring the binding amount or T cell costimulatory activity.
In this evaluation method, the substance to be evaluated includes a substance having dipeptidyl peptidase IV inhibitory activity.

  According to the present invention, a soluble caveolin-1-Fcγ1 fusion protein, a DNA encoding the protein, and an expression vector for the protein are provided. Since the fusion protein of the present invention functions as a T26 costimulatory ligand via CD26, the fusion protein can costimulate isolated T cells via CD26 in the presence of anti-CD3 antibodies.

  Moreover, according to another aspect of the present invention, there is provided a pharmaceutical composition containing a caveolin-1-Fcγ1 fusion protein. The pharmaceutical composition of the present invention includes, for example, autoimmune disease, rejection, graft-versus-host disease, rheumatoid arthritis, vasculitis syndrome, malignant tumor, malignant mesothelioma, acute or chronic graft-versus-host disease, rejection, Useful for the treatment of obstructive arteriosclerosis, myocardial infarction or cerebral infarction.

  According to another aspect of the present invention, a substance that controls T26 costimulatory activity via CD26 can be screened using the fusion protein of the present invention. Since the substance identified by the screening method of the present invention can control T cell costimulatory activity, it is useful as an agent for controlling immune activity.

  According to another aspect of the present invention, the T cell costimulatory activity of the substance to be evaluated can be evaluated using the fusion protein of the present invention. By the evaluation method of the present invention, for example, the immunosuppressive action of a substance having DPPIV inhibitory activity can be evaluated.

The T cell receptor (TCR) is expressed on T cells and recognizes the antigenic peptides presented by the major histocompatibility complex (MHC) on antigen presenting cells (APC). It has been clarified that activation of T cells requires not only signals by antigen recognition of T cell receptors but also co-stimulatory signals at the same time.
CD26 expressed on the T cell surface has been shown to function as a costimulatory molecule for T cells from studies using anti-CD26 antibodies, but the endogenous ligand for CD26-mediated costimulation is unknown.
Therefore, the present inventor identified the endogenous ligand of T cell costimulation via CD26 as follows. First, we focused on caveolin-1 expressed on APC. In order to stably produce caveolin-1, which is a membrane protein, a soluble protein (caveolin-1-Fcγ1 fusion protein) in which the heavy chain constant region (Fc) of immunoglobulin G (IgG) is fused to caveolin-1 Produced. Next, we examined whether caveolin-1-Fcγ1 fusion protein could bind to CD26 and activate T cells and function as a costimulatory ligand. As a result, it was revealed that caveolin-1 is an endogenous ligand for T cell costimulation via CD26.

In addition, it was revealed that the caveolin-1-Fcγ1 fusion protein functions as a costimulatory ligand for costimulating isolated T cells in the presence of an anti-CD3 antibody that stimulates TCR.
On the other hand, as shown in FIG. 12, it was revealed that caveolin-1-Fcγ1 fusion protein inhibits CD26-mediated costimulation of C cells by caveolin-1 and inhibits activation of T cells.
The present inventor has completed the present invention based on the above new findings.
Hereinafter, the present invention will be described in detail.

1. Fusion protein (1) caveolin-1
In the present invention, caveolin-1 is a membrane protein forming the caveola skeleton, and is known to have a function as a signal transduction molecule and a lipid transport function in cells.
In the present invention, caveolin-1 may be a protein derived from a mammalian tissue, cell or body fluid (blood, digestive fluid, spinal fluid, etc.), or may be a synthetic protein. Examples of mammals include humans, guinea pigs, rats, mice, hamsters, rabbits, cats, dogs, goats, pigs, sheep, cows, horses, monkeys, and the like.

Caveolin-1 in the present invention is, for example, a protein comprising the amino acid sequence represented by SEQ ID NO: 2, and preferably a protein comprising the amino acid sequence represented by SEQ ID NO: 2. SEQ ID NO: 2 represents the amino acid sequence of caveolin-1 derived from human.
The protein containing the amino acid sequence shown by SEQ ID NO: 2 is encoded by DNA containing the base sequence shown by SEQ ID NO: 1, for example. The base sequence represented by SEQ ID NO: 1 is described in GenBank accession number: NM_001753, and the amino acid sequence represented by SEQ ID NO: 2 is described in GenBank accession number: NP_001744.

(2) Skeletal region of caveolin-1
caveolin-1 has a scaffolding domain (SCD: scaffolding domain) about 20 amino acids in the center of the molecule.
All or part of the caveolin-1 backbone region of the present invention can also be used as a constituent part of the fusion protein of the present invention. In addition, all or part of the caveolin-1 skeleton region of the present invention has a binding activity to CD26, and can further co-stimulate T cells via CD26.

  As the skeleton region of caveolin-1 in the present invention, the amino acid sequence from the 82nd to the 101st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2 (hereinafter referred to as “amino acid sequence represented by SEQ ID NO: 2 (82 to 101)”) Polypeptides consisting of (also referred to as).

The caveolin-1 skeletal region of the present invention has 90% or more, preferably about 95% or more, more preferably about 98% or more identity to the amino acid sequence represented by SEQ ID NO: 2 (82 to 101) ( A protein having an amino acid sequence having homology) and having a binding activity to CD26 is also included.
Here, “binding activity to CD26” means an activity of binding to the extracellular region of CD26. The strength of binding activity (binding affinity) to CD26 possessed by the protein is not particularly limited. For example, the binding affinity possessed by the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (82 to 101) is about 10%. Above, preferably 20% or more, more preferably 30% or more.
The binding activity of the protein to CD26 can be measured by a known binding assay. For example, an immunoprecipitation method, an analysis method using BIAcore, an immunochemical method such as ELISA (enzyme linked immunosolvent assay), Western blot, and the like can be mentioned.

Further, in the present invention, one or several amino acids in the amino acid sequence represented by SEQ ID NO: 2 (82 to 101) are deleted, substituted, or added to the skeleton region of caveolin-1 or a combination thereof. A protein having a mutated amino acid sequence and having a binding activity to CD26 is also included.
In the amino acid sequence represented by SEQ ID NO: 2 (82 to 101), as an amino acid sequence in which one or several amino acids are deleted, substituted, or added, or mutated by a combination thereof, for example, (i) a sequence 1 to 9 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) amino acids in the amino acid sequence represented by No. 2 (82 to 101) A deleted amino acid sequence, (ii) 1 to 9 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2) in the amino acid sequence represented by SEQ ID NO: 2 (82 to 101) , More preferably 1) an amino acid sequence in which an amino acid is substituted with another amino acid, (iii) 1 to 9 (for example, 1 to 5) in the amino acid sequence represented by SEQ ID NO: 2 (82 to 101), Preferably 1-3, more preferred Ku is 1-2, more preferably amino acids are added in one), to the amino acid sequence mutated by a combination of (iv) above (i) ~ (iii).

When one or several amino acids are substituted in the amino acid sequence represented by SEQ ID NO: 2 (82 to 101), it is desirable to substitute with an amino acid having properties similar to those of the amino acid before substitution.
The properties of amino acids include, for example, acidic amino acids (Asp, Glu), basic amino acids (Lys, Arg, His), neutral amino acids (Gly, Ala, Val, Leu, Ile, Ser, Thr, Cys, Met, Asn, Gln, Pro, Phe, Tyr, Trp), aliphatic amino acids (Gly, Ala, Val, Leu, Ile, Ser, Thr, Cys, Met, Asn, Gln), aromatic amino acids (Phe, Tyr, Trp), min It can be classified into branched amino acids (Val, Leu, Ile), sulfur-containing amino acids (Cys, Met), acid amide amino acids (Asn, Gln) and the like.
Therefore, for example, substitution between amino acids having similar properties, such as acidic amino acids or basic amino acids, is preferred as a substitution that retains the properties of the protein. The number and position of amino acids to be substituted are not particularly limited.

  In the amino acid sequence represented by SEQ ID NO: 2 (82 to 101), a DNA encoding an amino acid sequence in which mutation such as deletion, substitution or addition has been made in one or several amino acids is described in “Molecular Cloning, A Laboratory Manual 2nd”. ed. "(Cold Spring Harbor Press (1989))," Current Protocols in Molecular Biology "(John Wiley & Sons (1987-1997)), Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488-92 , Kunkel (1988) Method. Enzymol. 85: 2763-6 and the like.

In order to introduce a mutation into DNA in order to prepare a protein having a mutation as described above, a mutation introduction kit using a site-directed mutagenesis method such as Kunkel method or Gapped duplex method, for example, QuikChange ^™ Site-Directed Mutagenesis Kit (Stratagene), GeneTailor ^TM Site-Directed Mutagenesis System (Invitrogen), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc .: Takara Bio) Can be used.

  In the present invention, the caveolin-1 skeleton region may contain a portion continuing from the caveolin-1 skeleton region to the N-terminal side and / or the C-terminal side as long as it has CD26 binding activity. The length of the N-terminal side and / or C-terminal side part contained in the skeleton region of caveolin-1 is not particularly limited. As such a protein, for example, a protein consisting of the amino acid sequence from the 75th to the 101st amino acid sequence or a protein consisting of the amino acid sequence from the 2nd to the 101st amino acid sequence shown in SEQ ID NO: 2 Can do.

  Furthermore, in the present invention, the caveolin-1 backbone region may be a fusion protein added by another peptide sequence. Peptide sequences added to the Caveolin-1 backbone region include influenza agglutinin (HA), glutathione S-transferase (GST), multiple histidine tags (6 × His, 10 × His, etc.), maltose binding protein (MBP), c Sequences that facilitate identification of proteins such as -myc fragment, V5 tag, and FLAG can be selected. Further, the caveolin-1 skeleton region of the present invention may contain a human E-cadherin signal peptide (hu-ECDSP).

  The caveolin-1 skeletal region of the present invention can be produced by standard purification methods from the mammalian tissues or cells described above. It can also be produced by culturing a transformant containing DNA encoding the caveolin-1 skeleton region of the present invention. In the case of producing from a mammalian tissue or cell, it can be isolated by a known method such as chromatography after homogenizing the mammalian tissue or cell.

Further, in the present invention, the caveolin-1 skeleton region has nucleotide sequences from the 244th to the 303rd of the nucleotide sequence represented by SEQ ID NO: 1 (hereinafter referred to as “SEQ ID NO: 1 (244 to 303)”. And a protein encoded by a DNA containing a nucleotide sequence ”. In addition, the caveolin-1 skeleton region is a protein encoded by DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 (244 to 303). Thus, a protein having a binding activity to CD26 is also included.
Such a DNA is obtained by a known hybridization method such as colony hybridization, plaque hybridization, Southern blotting, etc. using a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 (244 to 303) or a fragment thereof as a probe. And a genomic library. For the method for preparing the library, “Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Press (1989)) and the like can be referred to. Commercially available cDNA libraries and genomic libraries may also be used.

In the present specification, stringent conditions include, for example, “2 × SSC, 0.1% SDS, 42 ° C.”, “1 × SSC, 0.1% SDS, 37 ° C.”, and more stringent as washing conditions after hybridization. Examples of such conditions include “1 × SSC, 0.1% SDS, 65 ° C.”, “0.5 × SSC, 0.1% SDS, 50 ° C.”, and the like.
Hybridization can be performed by a known method. Hybridization methods include, for example, “Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Laboratory Press (1989)), “Current Protocols in Molecular Biology” (John Wiley & Sons (1987-1997)), etc. You can refer to it.

  In the present specification, the DNA hybridizing under stringent conditions includes, for example, at least 80% or more, preferably 90% or more, more preferably 95% or more, and the base sequence represented by SEQ ID NO: 1. Preferably, DNA containing a base sequence having 97% or more identity (homology) is included. A value indicating identity can be calculated by using a known program such as BLAST.

In addition, DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1 (244 to 303) is, for example, 1 or 2 in the base sequence represented by SEQ ID NO: 1. Examples thereof include DNA containing a nucleotide sequence in which mutation such as deletion, substitution or addition has occurred in several nucleic acids.
Here, in the base sequence represented by SEQ ID NO: 1 (244 to 303), for example, (a) SEQ ID NO: 1 (SEQ ID NO: 1 (SEQ ID NO: 1) 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) of the nucleic acid in the base sequence represented by 244 to 303) has been deleted. (B) 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) in the nucleotide sequence represented by SEQ ID NO: 1 (244 to 303) Is a base sequence in which one nucleic acid is replaced with another nucleic acid, (c) 1 to 10 (eg, 1 to 5, preferably 1 to the base sequence represented by SEQ ID NO: 1 (244 to 303)) Three, more preferably 1-2, and even more preferably 1) nucleic acids were added Group sequence, and a nucleotide sequence mutated by a combination of (d) above (a) ~ (c).

  In the present invention, the DNA encoding the skeletal region of caveolin-1 is designed, for example, by designing a primer or probe based on the base sequence represented by SEQ ID NO: 1, and gene amplification techniques such as PCR from cDNA libraries and genomic libraries. (Current Protocols in Molecular Biology, John Wiley & Sons (1987) Section 6.1-6.4) or a hybridization method.

  In the present invention, the nucleotide sequence can be confirmed by sequencing by a conventional method. For example, the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463) can be used. It is also possible to analyze the sequence using an appropriate DNA sequencer.

  A part of the caveolin-1 skeleton region of the present invention may be any peptide as long as it is a partial peptide of the caveolin-1 skeleton region described above and has a binding activity to CD26. For example, a protein comprising, for example, 19 amino acids or 18 amino acids in the amino acid sequence of the caveolin-1 skeleton region can be used.

(3) Fcγ1
In the present invention, Fcγ1 means a region consisting of the hinge region, C _H 2 region, and C _H 3 region of an immunoglobulin molecule.
It is known that when Fcγ1 is fused to the carboxyl terminus of a given protein, the fusion protein is expressed as a soluble protein. That is, Fcγ1 has the property of expressing a protein as a soluble protein. In the present specification, this property of Fcγ1 is sometimes referred to as “soluble protein expression effect”.

  In the present invention, Fcγ1 may be a protein derived from a mammalian tissue, cell or body fluid (blood, digestive fluid, spinal fluid, etc.), or may be a synthetic protein.

In the present invention, Fcγ1 is, for example, a protein comprising the amino acid sequence represented by SEQ ID NO: 4, and preferably a protein comprising the amino acid sequence represented by SEQ ID NO: 4. SEQ ID NO: 4 represents the amino acid sequence of human-derived Fcγ1.
The protein consisting of the amino acid sequence represented by SEQ ID NO: 4 is encoded by DNA containing the base sequence represented by SEQ ID NO: 3, for example.

  Fcγ1 in the present invention consists of an amino acid sequence having 90%, preferably about 95% or more, more preferably about 98% or more identity (homology) to the amino acid sequence represented by SEQ ID NO: 4, and Also included are proteins having a soluble protein expression effect.

Here, the “soluble protein expression action” refers to culturing a transformant containing a DNA encoding a fusion protein in which Fcγ1 is fused to the carboxyl terminal side of a predetermined protein, and the target protein in the soluble fraction or culture supernatant. This can be confirmed by detecting the expression. For example, when the target fusion protein is expressed in the soluble fraction (for example, cytoplasmic fraction) or culture supernatant of animal cells containing the DNA, it can be said that Fcγ1 has a soluble protein expression action. Similarly, when the transformant is Escherichia coli, it can be said that Fcγ1 has a soluble protein expression action when the fusion protein is expressed in the soluble fraction or the culture supernatant. The soluble fraction is obtained, for example, by cultivating the cultured transformant by a known method such as ultrasonic disruption, disruption with a cell lysate, disruption with a homogenizer, and the disrupted solution is obtained as a supernatant fraction obtained by centrifugation. be able to.
The protein expression level can be measured by known methods such as SDS-PAGE, protein quantification, and immunochemical methods such as ELISA and Western blot.

In addition, Fcγ1 in the present invention comprises an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 4, or are mutated by a combination thereof, and is soluble. Proteins having protein expression action are also included.
The mode of mutation such as deletion, substitution or addition in the amino acid sequence is as described above.

  In the amino acid sequence represented by SEQ ID NO: 4, when one or several amino acids are substituted, it is desirable to substitute an amino acid having properties similar to those of the amino acid before substitution. The mode of substitution is as described above. Further, the number and position of amino acids to be substituted are not particularly limited.

  In the amino acid sequence represented by SEQ ID NO: 4, a DNA encoding an amino acid sequence in which mutation such as deletion, substitution or addition has been made in one or several amino acids can be prepared as described above.

  Furthermore, in the present invention, Fcγ1 may be a fusion protein added by another peptide sequence. Peptide sequences added to Fcγ1 include influenza agglutinin (HA), glutathione S transferase (GST), multiple histidine tags (6 × His, 10 × His, etc.), maltose binding protein (MBP), V5 tag, c-myc Fragments, sequences that facilitate identification of proteins such as FLAG, and the like can be selected.

  The Fcγ1 of the present invention can be produced from the aforementioned mammalian tissues or cells by standard purification methods. It can also be produced by culturing a transformant containing DNA encoding Fcγ1 of the present invention. In the case of producing from a mammalian tissue or cell, it can be isolated by a known method such as chromatography after homogenizing the mammalian tissue or cell.

In the present invention, Fcγ1 includes a protein encoded by a DNA containing the base sequence represented by SEQ ID NO: 3. Fcγ1 is a protein encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 3, and has a soluble protein expression effect Is also included.
Such DNA can be obtained in the same manner as described above for obtaining DNA encoding the caveolin-1 skeleton region. The stringent conditions are the same as described above.

  In the present specification, the DNA hybridizing under stringent conditions includes, for example, at least 80% or more, preferably 90% or more, more preferably 95% or more, and the base sequence represented by SEQ ID NO: 3, Preferably, DNA containing a base sequence having 97% or more identity (homology) is included.

Further, a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 3 is, for example, one or several nucleic acids in the base sequence represented by SEQ ID NO: 3. Examples thereof include DNA containing a base sequence having a mutation such as deletion, substitution or addition.
The mode of mutation such as deletion, substitution, or addition in the base sequence is as described above.

  In the present invention, for DNA encoding Fcγ1, for example, a primer or probe is designed based on the nucleotide sequence represented by SEQ ID NO: 3, and gene amplification technology (Current Protocols in Molecular) such as PCR is used from a cDNA library and a genomic library. Biology, John Wiley & Sons (1987) Section 6.1-6.4) or a hybridization method.

(4) Fusion protein The present invention provides a fusion protein containing a first region consisting of all or part of the caveolin-1 skeleton region and a second region consisting of Fcγ1.
As the fusion protein of the present invention, for example, a protein comprising the amino acid sequence represented by SEQ ID NO: 5 (hereinafter also referred to as “NT-Fc” or “NT-Fcγ1”), or the amino acid sequence represented by SEQ ID NO: 6 is used. Examples thereof include proteins (hereinafter also referred to as “SCD-Fc” or “SCD-Fcγ1”).

The fusion protein of the present invention is expressed as a soluble protein and has a binding activity to CD26. It also functions as a T cell costimulatory ligand via CD26 for isolated T cells.
Furthermore, since the fusion protein of the present invention has a T cell activity inhibitory action as described later, it is also useful as an active ingredient of a pharmaceutical composition.

  The fusion protein of the present invention can be expressed using a recombinant vector containing a DNA encoding the fusion protein (hereinafter also referred to as “expression vector of the fusion protein of the present invention”). In the fusion protein expression vector of the present invention, for example, a target DNA fragment is excised from the DNA encoding the first region or the second region, and the obtained DNA fragment is ligated downstream of the promoter in an appropriate expression vector. Can be manufactured.

  In order to prepare an expression vector for the fusion protein of the present invention, DNA encoding the second region may be inserted into a vector incorporating the DNA encoding the first region in consideration of the reading frame of the first region. . Alternatively, the DNA encoding the first region may be inserted into the vector in which the DNA encoding the second region is incorporated in consideration of the reading frame of the second region. Alternatively, the DNA encoding the first region and the DNA encoding the second region may be simultaneously inserted into the vector. Here, taking into account the reading frame means adjusting the position and length of the DNA inserted into the vector so that the first region and the second region are expressed as a fusion protein without being frame-shifted. .

  For insertion of DNA into the vector, ligase reaction, topoisomerase reaction and the like can be used. For example, a method in which the purified DNA is cleaved with an appropriate restriction enzyme and the resulting fragment is inserted into an appropriate restriction enzyme site or a multicloning site in the vector to be ligated to the vector is employed.

  The expression vector of the fusion protein of the present invention is not limited to the origin of the basic vector as long as the fusion protein can be expressed. For example, a plasmid derived from E. coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, λ Bacteriophages such as phages, detoxified animal or insect viruses such as retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses or baculoviruses can be used. In addition, for example, a commercially available vector such as a pEB-CAG vector can also be used.

  The expression vector for the fusion protein of the present invention may contain a multicloning site, a promoter, an enhancer, a terminator, a signal peptide cassette, a selection marker cassette and the like as long as the fusion protein can be expressed. Moreover, if necessary when inserting DNA, a linker may be added as appropriate.

A promoter can be incorporated upstream of the DNA encoding the fusion protein. The promoter is not particularly limited as long as it can appropriately express the fusion protein of the present invention in the host. For example, trp promoter, lac promoter, CMV promoter, EF1α promoter, SRα promoter, SV40 promoter, CAG promoter, etc. are used. can do.
A terminator can be incorporated downstream of the DNA encoding the fusion protein.

As a signal peptide contained in the fusion protein of the present invention, for example, a human E-cadherin signal peptide (hu-ECDSP) (SEQ ID NO: 7) can be used.
hu-ECDSP (SEQ ID NO: 7): MGPWSRSLSALLLLLQVSSWLCQEPKL

Selectable marker cassettes include ampicillin resistance genes, kanamycin resistance genes, neomycin resistance genes, drug resistance genes such as hygromycin resistance genes, dihydrofolate reductase genes, or genes encoding tag proteins such as fluorescent proteins and myc be able to.
In addition, the fusion protein expression vector of the present invention may contain an SD sequence and a Kozak sequence. These sequences may be inserted into the vector by being inserted into the 5 ′ end of the DNA encoding the fusion protein of the present invention, or may be added to the DNA by PCR and incorporated into the vector.

(6) Transformant A transformant can be produced by introducing the expression vector for the fusion protein of the present invention into a host. Such a transformant is also included in the scope of the present invention.
The host used in the present invention is not particularly limited as long as the target fusion protein can be expressed after the introduction of the vector of the present invention. For example, E. coli, Bacillus subtilis, yeast, animal cells Insect cells, plant cells and the like. A person skilled in the art can select a suitable host for the expression vector of the fusion protein of the present invention.
In the screening method of the present invention and the evaluation method of the present invention (described later), it is preferable to use animal cells as host cells.

  Examples of E. coli include K12 strain, JM109 strain, XL1-Blue strain and the like. These strains can be easily obtained from, for example, the American Type Culture Collection (ATCC). Examples of Bacillus subtilis include Bacillus subtilis. Examples of yeast that can be used include Schizosaccharomyces pombe and Pichia pastoris. As animal cells, COS-7, CHO cells, Hela cells, 293 cells, HEK293FT cells, Jurkat T cells, 300-19 mouse precursor B cells, and the like are used. As insect cells, Sf9 cells, Sf21 cells and the like are used.

The method for introducing the vector into the host is not particularly limited. For example, known methods such as electroporation method, calcium phosphate method, lipofection method, DEAE dextran method, spheroplast method, and lithium acetate method can be used. Can be mentioned. When the vector is a viral vector, the gene can be introduced by culturing host cells, adding the viral vector to the culture medium, and further culturing.
For example, when animal cells are used as host cells, it is preferable to introduce genes by lipofection.

  By the above method, a transformant transformed with an expression vector containing DNA encoding the fusion protein of the present invention can be obtained.

The method for culturing the transformant of the present invention can be carried out according to a usual method used for culturing a host. The culture medium is preferably a medium capable of efficiently cultivating the transformant, and can be appropriately selected from known media by those skilled in the art.
For example, when the host is an animal cell, serum, amino acids, glucose, antibiotics, etc. can be added using a medium such as DMEM, MEM, RPMI, F12, etc. if necessary. In particular, when the animal cells are HEK293FT cells, it is preferable to use FreeStyle ^™ 293 Expression Medium as the culture medium.
The culture condition of the transformant is not particularly limited, but is carried out at a temperature of 10 ° C to 45 ° C, preferably 10 ° C to 40 ° C for 5 to 120 hours, preferably about 5 to 100 hours. In addition, if necessary, the medium can be exchanged, aerated, and stirred. In particular, when culturing floating animal cells, shaking culture may be performed.

  As described above, the fusion protein of the present invention can be produced inside or outside the transformant. The fusion protein of the present invention can be obtained from the culture of the transformant of the present invention. Since the fusion protein of the present invention is expressed as a soluble protein, it can be obtained from a soluble fraction or culture supernatant derived from a culture.

For example, when the fusion protein of the present invention is extracted from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and then subjected to ultrasonication, freeze disruption, or the like. Cells or cells are destroyed, and a soluble fraction can be obtained by a method such as centrifugation or filtration. Then, the fusion protein of the present invention can be purified from the obtained soluble fraction by appropriately combining known protein purification methods. Known protein purification methods include, for example, dialysis, ammonium sulfate precipitation, ultrafiltration, gel filtration, SDS-PAGE, or chromatography such as ion chromatography, affinity chromatography, or reverse phase chromatography. The method that I did.
For example, also when acquiring the fusion protein of this invention from a culture supernatant, it can refine | purify with said protein purification method.

2. CD26 mediated T cell costimulatory activity The inventor has shown that caveolin-1 is an endogenous ligand for CD26 mediated T cell costimulation. Specifically, it was found that T cells are costimulated by bringing the fusion protein of the present invention into contact with isolated T cells in the presence of an anti-CD3 antibody. That is, all or part of the caveolin-1 skeleton region or the fusion protein of the present invention is useful as a T cell costimulatory ligand via CD26 for isolated T cells.
Accordingly, the present invention provides a T26 costimulatory ligand via CD26 containing all or part of the caveolin-1 backbone region, or the fusion protein of the present invention. The present invention also provides a method of costimulation of T cells, which comprises contacting the fusion protein of the present invention with isolated T cells in the presence of an anti-CD3 antibody.
The fusion protein of the present invention co-stimulates isolated T cells via CD26 instead of caveolin-1 on APC, while acting suppressively on T cell costimulation in the presence of APC (Example 6) Therefore, the fusion protein of the present invention can costimulate T cells in the absence of APC or in the presence of activated APC, and functions as a costimulatory ligand.

  As the T cell costimulatory ligand of the present invention, all or part of the caveolin-1 skeleton region or the fusion protein of the present invention may be used as it is, or may be used in a state expressed in a transformant. Good. Moreover, what was formulated with a suitable additive can also be used (refer the term of the pharmaceutical composition below).

  The isolated T cells used in the present invention are CD26 positive T cells. CD26 positive T cells can be isolated from peripheral blood collected from a living body by flow cytometry using CD3 and CD26 as markers, for example. In addition, monocyte cells can be collected from peripheral blood collected from a living body, and peripheral blood T cells can be isolated using a commercially available kit such as MACS Pan T cell isolation kit II.

  Anti-CD3 antibodies that stimulate isolated T cells mimic antigen-specific primary signals to TCR. The anti-CD3 antibody used in the present invention is not particularly limited as long as it can bind to the TCR / CD3 complex. For example, OKT3 which is an anti-CD3 monoclonal antibody can be used. An anti-CD3 antibody to which a tag such as alkaline phosphatase is bound can also be used.

  Co-stimulation of T cells can be performed, for example, by adding the isolated T cells and the fusion protein of the present invention to a solid phase on which an anti-CD3 antibody is immobilized. It can also be performed by adding isolated T cells to a solid phase on which an anti-CD3 antibody and the fusion protein of the present invention are immobilized. Alternatively, it can be performed by culturing isolated T cells with an anti-CD3 antibody and the fusion protein of the present invention. For example, when the fusion protein of the present invention is expressed in CHO cells, the T cells can be co-stimulated by co-culturing isolated T cells and CHO cells in the presence of an anti-CD3 antibody.

Co-stimulation of T cells via CD26 causes IL-2 (interleukin 2) production and T cell proliferation. Therefore, the T cell costimulatory activity of all or part of the caveolin-1 skeletal region or the fusion protein of the present invention can be measured by IL-2 production activity or T cell proliferation activity.
The production activity of IL-2 can be confirmed by measuring the amount of IL-2 secreted from T cells into the culture supernatant by ELISA, Western blotting, MS spectrum method or the like.
The proliferative activity of T cells can be confirmed by measuring the amount of radiolabeled thymidine (eg, [ ³ H] -TdR) taken up by a scintillation counter.

3. Pharmaceutical Composition The fusion protein of the present invention inhibits T cell costimulation when APC and CD4 positive T cells are stimulated with tetanus toxoid as shown in Example 6 or FIG. That is, the fusion protein of the present invention has an action of inhibiting c26-mediated T cell costimulation by caveolin-1. Therefore, the fusion protein of the present invention is useful for suppressing an immune response mediated by T cells. Since CD26 positive T cells are T _H 1 type cells, the fusion protein of the present invention is preferably useful for suppressing an immune response via T _H 1 type T cells.
The present invention provides a pharmaceutical composition containing the fusion protein of the present invention. The pharmaceutical compositions of the present invention, T cells, since preferably capable of suppressing the immune response through the T _H 1 type T cells, T cells, diseases preferably via T _H 1-type T cells, e.g. , Autoimmune disease, rejection, graft-versus-host disease, rheumatoid arthritis, vasculitis syndrome, malignant tumor, malignant mesothelioma, acute or chronic graft-versus-host disease, rejection, obstructive arteriosclerosis, myocardial infarction or It is useful for the treatment or prevention of diseases such as cerebral infarction.
The pharmaceutical composition of the present invention can be administered to mammals.

  The dose of the fusion protein contained in the pharmaceutical composition of the present invention varies depending on the patient's age, sex or body weight, administration method, administration period, type of preparation, etc., but can be appropriately determined by those skilled in the art. . For example, 1 to 1000 mg, preferably 10 to 500 mg, more preferably 50 to 100 mg per day can be administered to an adult (body weight 60 kg), but is not limited thereto. The administration may be divided into 1 to 3 times per day.

In addition, in the pharmaceutical composition containing the fusion protein of the present invention as an active ingredient, the fusion protein may be used as it is, or a formulated protein may be used.
Additives used in formulation include excipients, binders, wetting agents, lubricants, emulsifiers, disintegrating agents, flavoring agents, surfactants, buffers, stabilizers, preservatives, preservatives, preservatives Agents, tonicity agents and the like. These can be used alone or in appropriate combination to produce a preparation by a conventional method.
As preparations, oral preparations such as tablets, powders, granules, fine granules, capsules, syrups, lozenges, external preparations such as suppositories, ointments, eye drops, nasal drops, poultices, or injections Agents (intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal injection, infusion, etc.) can be mentioned.

Further, the present invention is an effective amount of a fusion protein of the present invention, characterized by administering to a patient, T cells, preferably mediated diseases T _H 1-type T cells, for example, autoimmune diseases, rejection, Graft-versus-host disease, rheumatoid arthritis, vasculitis syndrome, malignant tumor, malignant mesothelioma, acute or chronic graft-versus-host disease, rejection, obstructive arteriosclerosis, myocardial infarction or cerebral infarction I will provide a.
Furthermore, the present invention relates to diseases mediated by T cells, preferably T _H1 type T cells, such as autoimmune diseases, rejection, graft-versus-host disease, rheumatoid arthritis, vasculitis syndrome, malignant tumors, malignant mesothelioma It also relates to the use of the fusion protein of the invention for the manufacture of a pharmaceutical composition for the treatment of tumors, acute or chronic graft-versus-host disease, rejection, obstructive arteriosclerosis, myocardial or cerebral infarction.

4). Screening Method The present invention provides a method for screening a substance that regulates C26-mediated T cell costimulatory activity by caveolin-1.
Screening for substances that control CD26-mediated T cell costimulatory activity by caveolin-1 by changing the binding between CD26 and caveolin-1 by conducting a binding assay between CD26 and the fusion protein of the present invention can do.

The screening of the present invention comprises: (i) a case where the fusion protein of the present invention is contacted with CD26; and (ii) a case where the fusion protein of the present invention and a test substance are contacted with CD26. This is performed by comparing the binding amount or T cell costimulatory activity.
Here, as CD26, a cell in which CD26 is stably or transiently expressed (for example, Jurkat T cell, 300-19 mouse precursor B cell, etc.), CD26 positive T cell or a membrane fraction thereof should be used. Can do.
Moreover, the fusion protein itself may be used as the fusion protein of the present invention, or a protein expressed in the transformant of the present invention may be used.

In the screening method of the present invention, the amount of binding can be quantified by immunoprecipitation such as immunoprecipitation, surface plasmon sensor method, ELISA and Western blot.
The screening method by measuring the amount of binding can be performed as follows, for example. First, a suspension of cells expressing CD26 is added to a culture plate on which the fusion protein of the present invention is immobilized. Next, after culturing the cells for a predetermined time in the presence or absence of the test substance, the plate is washed to remove cells that have not bound to the fusion protein. Next, by detecting CD26 in the plate, the amount of binding between CD26 and the fusion protein is measured using an anti-CD26 antibody.

Screening by measuring T cell costimulatory activity can be performed, for example, as follows. First, a suspension of T cells expressing CD26 is added to a culture plate on which the fusion protein of the present invention is immobilized. Next, after culturing T cells for a predetermined time in the presence or absence of the test substance, the cells are collected and a cell lysate is prepared. T cell costimulatory activity in the lysate of this cell is measured.
As mentioned above, T cells secrete IL-2 when CD26 positive T cells are costimulated. Therefore, T cell costimulatory activity can be confirmed by measuring the amount of secreted IL-2 production. The measurement of IL-2 production is preferably used in screening using CD26-expressing T cells (for example, CD26-positive T cells or T cells forcibly expressing CD26).
Furthermore, when CD26 positive T cells are costimulated, the T cells are activated and the proliferation rate is increased. Therefore, T cell costimulatory activity can be confirmed by measuring T cell proliferating activity. The measurement of T cell proliferating activity is preferably used in screening using T cells expressing CD26 (for example, CD26 positive T cells or T cells forcibly expressing CD26).
IL-2 production activity and T cell proliferation activity can be measured as described above.

In the screening method of the present invention, the substance that promotes the binding between CD26 and the fusion protein of the present invention or the substance that enhances T cell costimulatory activity enhances the T cell costimulatory signal compared to the absence of the test substance. Useful as a substance. And this substance can be used as an active ingredient of an immunostimulant in order to activate the immune response via T _H 1 type T cells.
On the other hand, in the screening method of the present invention, the substance that inhibits the binding between CD26 and the fusion protein of the present invention or the substance that suppresses T cell costimulatory activity compared to the absence of the test substance is a T _H 1 type T cell. Can be used as an active ingredient of an immunosuppressive agent.

5. Evaluation methods
CD26 is known to be the same protein as DPPIV, a serine protease, and the active center of this serine protease activity has been clarified to be the 630th serine. In addition, DPPIV is a degrading enzyme of GLP-1 (glucagon-like peptide-1), a hormone that enhances insulin secretion, and since inhibitors of DPPIV have a hypoglycemic action, they can be used as therapeutic agents for diabetes. Development is underway.
The CD26 region (caveolin-binding domain) required for binding to the caveolin-1 skeleton region contains the 630th serine necessary for DPPIV enzyme activity (WO 2005/063170 pamphlet). That is, the binding region of c26olin-1 in CD26 and the DPPIV enzyme active region are partly in common. Furthermore, in Example 7, the caveolin-1 backbone region that binds to CD26 is shown to inhibit DPPIV enzyme activity.
Therefore, DPPIV inhibitors not only inhibit DPPIV enzyme activity but also potentially inhibit the binding of CD26 (DPPIV) to caveolin-1 and potentially inhibit CD26-mediated T cell costimulation. It can be said that it has.

Therefore, the present invention provides a method for evaluating T cell costimulatory activity via CD26 possessed by a substance to be evaluated (for example, a DPPIV inhibitor).
Since it is possible to examine whether a substance inhibits the binding between CD26 and caveolin-1 by using a binding assay of CD26 and the fusion protein of the present invention, inhibition of T cell costimulation via CD26 possessed by the substance The degree of activity can be evaluated.
For example, if a DPPIV inhibitor is evaluated to have T cell costimulatory inhibitory activity, the substance may have an immunosuppressive effect in addition to a hypoglycemic effect by inhibiting DPPIV. It can be judged that it is expensive.

Evaluation of the substance is performed by (i) when the fusion protein of the present invention is contacted with CD26, and (ii) when the fusion protein of the present invention and the test substance are contacted with CD26. This is done by comparing the amount of binding or T cell costimulatory activity.
CD26 used in the evaluation method of the present invention, the fusion protein of the present invention, and the measurement method are the same as those used in the screening method of the present invention.

  Hereinafter, the present invention will be illustrated by specific examples, but the present invention is not limited thereto.

1. Cells, antibodies and reagents used (1) Cells
HEK293FT (human embryonal kidney), Jurkat T cell line (JKTwt), and Jurkat T cells stably transfected with human CD26 (J.CD26wt) or 300-19 mouse precursor B cells (300-19-CD26wt) The culture was performed by the method described in Ohnuma et al., 2004; Tanaka et al., 1992; Tanaka et al., 1993.
Human peripheral blood T cells were collected from healthy volunteers and purified from cultured peripheral blood monocytic cells (PBMC) using MACS Pan T cell isolation kit II (Miltenyi, Auburn, CA). Informed consent was obtained from healthy volunteers.

(2) Antibody The anti-CD26 monoclonal antibody (mAb) IF7 and the anti-CD3 mAb OKT3 described in Kung et al 1979 and Morimoto et al., 1989 were used. 4B10, an anti-CD28 mAb, was obtained from ATCC. APC-conjugated anti-CD3 mAb, PE (phycoerythrin) -conjugated anti-CD26 mAb and FITC-conjugated streptavidin were purchased from BD Biosciences (San Jose, Calif.). HRP (horseradish peroxidase) -conjugated anti-goat IgG was purchased from Abcam (Cambridge, UK). HRP-conjugated anti-human IgG was purchased from Amersham Biosciences (Piscataway, NJ), and anti-Xpress mAb and HRP-conjugated anti-Xpress mAb were purchased from Invitrogen (Carlsbad, CA).

(3) Reagent Biotinylation of the reconstituted protein or antibody was performed according to the instruction manual using the EZ-Link ^™ Sulfo-NHS-LC-Biotin reagent (PIERCE, Rockford, IL).
Protease inhibitor cocktail, phosphatase inhibitor cocktail and poly-L-lysine were purchased from Sigma-Aldrich (St. Louis, MO).

2. Flow cytometry (FCM)
In the analysis of J. CD26wt or 300-19-CD26wt binding to biotinylated NT-Fc or NTΔSCD-Fc, 1 × 10 ⁶ cells were washed with ice-cold PBS (phosphate buffered saline), Fcγ1 and mouse Ig Incubation with isotype (1 μg / ml) inhibited non-specific binding. Subsequently, it was reacted with biotinylated NT-Fc or NTΔSCD-Fc (1 μg / ml) and stained with FITC-conjugated streptavidin (1: 500).

  In inhibition experiments, unlabeled mouse IgG (20 μg / ml) or unlabeled anti-CD26 mAb (20 μg / ml) was incubated with cells and then reacted with biotinylated NT-Fc or NTΔSCD-Fc.

In the cytokine production analysis experiment, first, purification was performed in the presence or absence of anti-CD3 (0.05 μg / ml) and NT-Fc (5 μg / ml) or NTΔSCD-Fc (5 μg / ml) bound to the plate. T cells (1 × 10 ⁶ cells / well) were stimulated in AIM-V culture medium (GIBCO, Grand Island, NY) for 72 hours.

Flow cytometric analysis of 10,000 visualized cells was performed with FACS calibur ^™ (Becton-Dickinson, La Jolla, Calif.). Each experiment was repeated at least 3 times, and the results were expressed in the form of a histogram or a dot plot of a representative experimental example.

3. T cell proliferation assay and IL-2 production assay
1 × 10 ⁵ purified T cells were cultured in 96 μL AIM-V medium (GIBCO, Grand Island, NY) in 96 well flat bottom plates (COSTAR, Corning, NY).
For solid phase stimulation, anti-CD3 mAb (OKT3, 0.05 μg / ml) and / or anti-CD26 mAb (5 μg / ml), anti-CD28 mAb (4B10, 5 μg / ml) or Fc fusion protein (5 μg / ml) were bound to the plate.
For stimulation with CHO cells transfected with Caveolin-1, purified T cells (1 × 10 ⁵ cells / well) were prepared in various amounts in the presence of soluble anti-CD3 mAb (OKT3, 0.05 μg / ml). Cultured with CHO cell transformants (T cells: CHO cells = 800, 400, 200, 100, 50, 25: 1, or no CHO cells). Prior to costimulation of T cells, CHO transformants were fixed with 0.05% glutaraldehyde for 30 seconds at room temperature and then washed 3 times with PBS.
T cell proliferation was measured by [ ³ H] -TdR (ICN Radiochemical, Irvin, Calif.) Uptake. The cells were incubated for 96 hours, pulsed with 1 μCi / well of [ ³ H] -TdR for 16 hours, and harvested with a glass fiber filter. The incorporated radioactivity was quantified with a liquid scintillation counter.

For IL-2 production assays, anti-CD3 mAb (1.0 μg / ml) and / or anti-CD26 mAb (10 μg / ml) with 5 × 10 ⁵ native Jurkat cells (JKTwt) or J.CD26 cells bound to the plate The cells were cultured in the presence of anti-CD28 mAb (10 μg / ml) or Fc fusion protein (10 μg / ml). After 48 hours of incubation, culture supernatants were collected from 3 wells and IL-2 content was measured using Biotrack ELISA Kit (Amersham Biosciences) according to the instructions.

  In inhibition experiments, cells were treated with soluble anti-CD26 mAb (IF7), anti-CD28 mAb (4B10) or control mouse Ig (20 μg / ml each) in plates coated with stimulatory antibodies and / or Fc proteins. Cultured.

4). Cell lysate preparation, immunoprecipitation and Western blot Stimulated cells (1 x 10 ⁸ cells) are pelleted and TBSD buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.1% Digitonin, 10 ² fold diluted protease inhibitor cocktail (Sigma), 10 ² fold diluted phosphatase inhibitor cocktail (Sigma)) and immunoprecipitated. Thereafter, SDS-PAGE and Western blot analysis were performed.

5. statistics
Student's t test was used to test the significance of differences between controls and samples. P <0.005 was considered significant.

Production of caveolin-1-Fcγ1 fusion protein The purpose of this example is to produce a soluble fusion protein of the extracellular region containing the caveolin-1 backbone region and the Fcγ1 region. Specifically, a soluble fusion protein (NT-Fc) composed of human caveolin-1 N-terminal region and human IgG ₁ Fcγ1, or N-terminal region excluding caveolin-1 skeleton region (SCD) and human IgG ₁ An object is to produce a soluble fusion protein (NTΔSCD-Fc) consisting of Fcγ1 (FIG. 1). Another object of the present invention is to produce a soluble fusion protein (SCD-Fc) consisting of the caveolin-1 backbone region and Fcγ1.

(1) Construction of expression plasmid for caveolin-1-Fcγ1 fusion protein
In constructing a caveolin-1-Fcγ1 fusion protein expression plasmid, a cassette vector for expressing an Fcγ1 fusion protein expressing only the Fcγ1 portion not fused with caveolin-1 was first constructed (FIG. 2). That is, the human E-cadherin signal peptide (huECDSP) and the human IgG heavy chain hinge, C _H 2 and C _H 3 part (Fcγ1) expression cassettes were inserted into the multi-cloning site of the pEB-CAG vector (pEB6- CAG-huECDSP-Fcγ ₁ ).

Next, an expression plasmid for caveolin-1-Fcγ1 fusion protein in which the extracellular portion of caveolin-1 was fused to Fcγ1 protein was prepared as follows.
First, between huECDSP-Fcγ ₁ of Fc.gamma. ₁ protein expression cassette vector produced (pEB6-CAG-huECDSP-Fcγ 1), was cloned extracellular region of Caveolin-1. That is, the gene encoding the N-terminal 2-101 amino acid of Caveolin-1 was cloned into pEB6-CAG-huECDSP-Fcγ ₁ vector by genetic recombination (Cav-1-NT-Fcγ 1). A vector (Cav-1-NT-ΔSCD-Fcγ ₁ ) incorporating a gene encoding the N-terminal 2 to 80th amino acid was also prepared in the same manner (FIG. 1). Moreover, the vector which integrated the gene which codes the 75th-101st amino acid of caveolin-1 as a skeleton region was produced similarly.

(2) Expression of caveolin-1-Fcγ1 fusion protein
The Caveolin-1-Fc fusion protein was first expressed by introducing a plasmid into HEK293T. That is, using Lipofectamine 2000 reagent (Invitrogen), 5 μg of the plasmid construct was introduced into 1.0 × 10 ⁶ HEK293T seeded on a 6-well plate, and the culture supernatant was collected 48 hours later.
In addition, large-scale purification can be performed by introducing a gene constructed in suspension-type 293 cells FreeStyle ^TM 293-F cells and then shaking culture, so that a sufficient amount of Caveolin-1-Fc fusion protein is secreted into the culture supernatant. Study was carried out. 1 x 10 ⁷ 293-F cells were shake-cultured in 30 ml of serum-free medium in a 125 ml Erlenmyere flask, and 20 μg of pEB6-CAG-hu ECDSP-Caveolin-1-Fcγ ₁ vector was introduced using 293fectin ^TM reagent did. After 48 hours, 20 ml of medium was added and transferred to a 250 ml Erlenmyere flask. After further culturing for 24 hours, 50 ml of culture supernatant was collected. Thereafter, the supernatant was collected every 72 hours, and at the same time, the cells were passaged so that the cell concentration became 1.0 × 10 ⁶ / ml, and the supernatant was collected and stored frozen for one month after the introduction of the plasmid.
The Caveolin-1-Fc fusion protein was purified from the collected supernatant by affinity purification using a Protein A column (Pierce).
Quantification of the Fc protein concentration in the culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA) (BETHYL LABORATORIES Inc.) for human IgG.

Measurement of the human IgG concentration by human ELISA, whereas culture supernatant of vector control transduced cells is below the detection sensitivity, the pEB6-CAG-hu ECDSP-Caveolin -1-Fcγ 1 vector transduced cells supernatants It was found that human IgG components of NT-Fc of about 10 μg / ml, NTΔSCD-Fc of about 15 μg / ml, and SCD-Fc of about 20 μg / ml were contained (FIG. 3A). Therefore, when Fc fusion protein was purified from 50 ml of the culture supernatant using a Protein A column, 500 μg of NT-Fc, 900 μg of NTΔSCD-Fc, and 500 μg of SCD-Fc were purified.

  FIG. 3B shows the result of SDS-PAGE of the purified protein and Western blotting with anti-human IgG. The electrophoresis sample was prepared under non-reducing conditions (-2ME: lanes 1-5) and reducing conditions (+ 2ME: lanes 6-9). Further, FIG. 3C shows the result of SDS-PAGE of the purified protein and CBB staining of the gel. Lanes 1 and 6 were purified mouse IgG, and electrophoresis samples were prepared under reducing conditions (+ 2ME: lane 6-9) and non-reducing conditions (-2ME: lane 6-10).

Binding activity of caveolin-1-Fc fusion protein to CD26 (1) In this example, whether or not CD26 binds to the NT-Fc fusion protein prepared in Example 2 was examined.
For the examination, a Jurkat T cell line (J.CD26wt) and a mouse precursor B cell line 300-19 (300-19-CD26wt) stably expressing full-length human CD26 were used.
When J.CD26wt cell lysate was used, CD26 co-precipitated with NT-Fc (lane 2) as shown in FIG. 4, but both Fcγ ₁ (lane 1) and NTΔSCD-Fc (lane 3). It did not precipitate.

Next, the binding of NT-Fc to CD26 on the cell surface was examined using flow cytometry.
As shown in FIG. 5A, J.CD26wt expressing CD26 on the cell surface was stained with anti-CD26-FITC mAb (panel b (2)). This staining with anti-CD26-FITC mAb was inhibited by unlabeled anti-CD26 mAb (panel b (3)) but not by control IgG (panel b (4)).
J.CD26wt was also stained with biotinylated NT-Fc followed by streptavidin-conjugated FITC ((2) in FIG. 5A panel c). This staining with NT-Fc was inhibited by unlabeled anti-CD26 mAb ((3) in FIG. 5A panel c) but not by control IgG ((4) in FIG. 5A panel c). On the other hand, J.CD26wt was not stained by NTΔSCD-Fc (FIG. 5A, panel d).

In order to further confirm the binding between CD26 expressed on the cell surface and NT-Fc, flow cytometry analysis was performed using 300-19-CD26wt.
As shown in FIG. 5B panel b, 300-19-CD26 wt expressing CD26 on the cell surface was stained with anti-CD26-FITCmAb (panel b (2)). This staining with anti-CD26-FITCmAb was inhibited by unlabeled anti-CD26 mAb (panel b (3)) but not by control IgG (panel b (4)).
300-19-CD26wt was also stained with biotinylated NT-Fc followed by streptavidin-conjugated FITC ((2) in FIG. 5B panel c). This staining with NT-Fc was inhibited by unlabeled anti-CD26 mAb ((3) of FIG. 5B panel c) but not by control IgG ((4) of FIG. 5B panel c). On the other hand, 300-19-CD26wt was not stained by NTΔSCD-Fc (FIG. 5B, panel d).

  Native 300-19 cells or Jurkat T cells were not stained with anti-CD26 mAb or NT-Fc.

  From the above, it was shown that the soluble N-terminal region of caveolin-1 binds to CD26 expressed on the cell surface. It was also shown that caveolin-1 skeletal region (SCD) is required for binding to CD26.

(2) Next, in order to examine the binding characteristics between NT-Fc and CD26, the binding affinity was measured using BIAcore.
Measurement was performed using BIAcore ^TM J (Biacore JAPAN, Tokyo, Japan) using HBS buffer (25 mM HEPES (pH 7.4), 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20) obtained from Biacore AB (Uppsala, Sweden). )
Fcγ1 (Fig. 6 (a)), NT-Fc (Fig. 6 (b)) or NTΔSCD-Fc (Fig. 6 (c)) was prepared by using Amine Coupling Kit (Biacore AB) in 10 mM sodium acetate (pH 5.0). Used to bind to a research grade CM5 sensor chip (Biacore AB) by reacting for 5 minutes. This resulted in an immobilized chip of about 5,000 to about 6,000 reaction units (RU).
After bonding, the chip surface was washed with 5 mM NaOH. This 5 mM NaOH was also used to regenerate Fcγ1, NT-Fc or NTΔSCD-Fc immobilized chip.

Reconstituted soluble CD26 (rsCD26) contains the extracellular region of human CD26 (ohnuma et al., 2001; Tanaka et al., 1994). Various concentrations of rsCD26 (50 nM, 25 nM, 12.5 nM, 6.3 nM, 3.2 nM or 1.6 nM) were injected onto Fcγ1, NT-Fc or NTΔSCD-Fc immobilized chips for 120 seconds.
The equilibrium binding analysis was performed using BIA evaluation software version 2.1 (BIAcore AB).

As a result, the Kd value between NT-Fc and rsCD26 was ˜2 × 10 ⁻⁵ M by equilibrium binding analysis at each concentration of rsCD26 (FIG. 6 (b)).
This result clearly shows that the N-terminal region of caveolin-1 binds directly to CD26.

(3) Next, in T cell costimulation via CD26, it was examined whether NT-Fc had the same costimulatory effect as anti-CD-26 mAb.
The present inventors have previously shown that, when an antibody is bound to CD26 in J.CD26wt cells, T cells are costimulated in a TCR / CD3-dependent manner and IL-2 production is enhanced ( Tanaka et al., 1992).
As shown in FIG. 7A, IL-2 production was induced by anti-CD3 mAb and NT-Fc bound to the plate in J.CD26wt cells. This induction was at the same level as that induced by anti-CD3 mAb and anti-CD28 mAb, or anti-CD3 mAb and anti-CD26 mAb. CD28 and CD26 are the same T cell costimulatory molecules as CD26.
On the other hand, IL-2 production was not observed even when JKTwt cells not expressing CD26 were stimulated with anti-CD3 mAb and anti-CD26 mAb, anti-CD3 mAb and NT-Fc. In addition, IL-2 production in J.CD26wt or JKTwt cells was not observed using Fcγ1 or NTΔSCD-Fc as controls.

Furthermore, in order to investigate that the costimulatory activity of T cells by NT-Fc is mediated by CD26, inhibition experiments were conducted using mAb specific for CD26 that inhibits the binding between NT-Fc and CD26. Went.
As shown in FIG. 7B, IL-2 production by anti-CD3 mAb and NT-Fc bound to the plate was inhibited by soluble anti-CD26 mAb, but not by soluble anti-CD28 mAb. Moreover, Fcγ1 or NTΔSCD-Fc, which is a controll, did not show T cell costimulatory activity.

  The results of Examples 1 and 2 (FIGS. 4-7) showed that caveolin-1 bound directly to CD26 and induced CD26-mediated T cell costimulation.

The purpose of this example is to confirm that NT-Fc can reproduce the effect of anti-CD26 antibody in CD26-mediated T cell costimulation. It was.
(1) First, solid-phase stimulation was performed using peripheral blood T cells isolated from blood provided by a healthy person.
As shown in FIG. 8A, T cell proliferation is induced by anti-CD3 mAb and NT-Fc immobilized on a solid phase, and the degree of induction is induced by anti-CD3 mAb and anti-CD28 mAb, or anti-CD3 mAb and anti-CD26 mAb. It was the same level as the induction by. On the other hand, when Fcγ1 or NTΔSCD-Fc as a control was used, T cell proliferation was not observed.

Further, as shown in FIG. 8B, NT-Fc induced costimulation of T cells in a concentration-dependent manner.
In J. CD26wt, proliferation was induced by stimulation with anti-CD3 mAb and NT-Fc, similar to the results of peripheral blood T cells. On the other hand, proliferation was not induced in Jurkat cells not expressing CD26.

  The above indicates that NT-Fc is a protein that binds functionally to CD26 and specifically binds to CD26.

(2) Furthermore, in order to examine the costimulatory activity of caveolin-1, the present inventor prepared CHO cells that stably express human caveolin-1.
First, a vector expressing a GFP fusion protein of full-length human caveolin-1 (Cav-wt + CHO), a vector expressing a GFP fusion protein obtained by removing SCD from human caveolin-1 (Cav-ΔSCD + CHO) or insert Each of the vectors (mock + CHO) into which was inserted was prepared. CHO cells were transfected with each prepared vector.
As shown in FIG. 8C, Cav-wt + CHO cells expressed T cell costimulatory activity in the presence of anti-CD3 mAb. However, such costimulatory activity was not observed in Cav-ΔSCD + CHO cells or mock + CHO cells.

  From the above, it was shown that caveolin-1 has T cell costimulatory activity via the TCR / CD3 pathway and can proliferate T cells.

Inhibition of T cell costimulation by caveolin-1
To further investigate whether the T-cell costimulatory activity of NT-Fc is mediated by CD26, a CD26-specific mAb that inhibits the binding of NT-Fc to J.CD26 or 300-19-CD26wt (Fig. Inhibition experiments with the antibodies used in 5A and B) were performed.
First, as shown in FIG. 9A, proliferation of T cells by anti-CD3 mAb and anti-CD26 mAb bound to the plate was inhibited by soluble anti-CD26 mAb, but not by soluble anti-CD28 mAb. On the other hand, T cell proliferation by anti-CD3 mAb and anti-CD28 mAb bound to the plate was inhibited by soluble anti-CD28 mAb, but not by soluble anti-CD26 mAb.

Under such experimental conditions, proliferation of T cells by plate-bound anti-CD3 mAb and NT-Fc was inhibited by soluble anti-CD26 mAb but not by soluble anti-CD28 mAb (FIG. 9A).
Furthermore, costimulation with NT-Fc was inhibited in a concentration-dependent manner by CD26 specific mAb (FIG. 9B). Moreover, IgG or anti-CD28 mAb as a control did not inhibit the costimulatory activity of NT-Fc at a concentration of 0 to 50 μg / ml.

  The results shown in FIGS. 4 to 9 clearly show that caveolin-1 directly binds to CD26 and induces T cell costimulation via CD26.

Inhibition of CD26-mediated T cell costimulation by Caveolin-1-Fcγ1 fusion protein
CD26 positive cells are known to be activated in response to memory antigens such as tetanus toxoid. Therefore, a remixing experiment of APC / CD4 + T cells by tetanus toxoid (TT) stimulation was performed as follows (FIG. 10A). Specifically, monocytes (0.5 × 10 ³ ) purified from PBMC were cultured in Macrophage-SFM medium supplemented with 0.5 μg / ml tetanus toxoid (Calbiochem), and 24 hours later, purified CD4 + T cells derived from the same donor ( 1 × 10 ⁴ ), seeded in 96 well-round bottom multiplate (Costar), and cultured in AIM-V serum-free medium for 96 hours.

In the inhibition experiment, monocytes immediately before mixing were treated with 1, 5, 10, 20 μg / ml of CD86 antibody (clone IT2.2, BD PharMingen), CTLA4-Ig (Ancell), and control Ig (Ancell). Similarly, CD4 + T cells were treated with anti-CD26 antibody (1F7) and anti-CD28 antibody (4B10) immediately before mixing, and inhibition experiments were performed.
Here, CTLA4, also called CD152, is a protein that appears on T cells upon activation of T cells and binds to CD80 and CD86 on APC as a ligand. The interaction between CTLA4 and its ligand is thought to suppress the T cell immune response.
1 μCi / well of ³ H-thymidine was added in the last 16 hours of the culture, the cells were collected on a glass filter with a harvester, and the radioactivity was measured with a liquid scintillation counter.
FIG. 10A schematically shows a protocol for the reconstruction experiment.

  The expression pattern of CD3, CD4, CD26, CD28 in purified CD4 + cells is shown in FIG. 10B. The purity of CD4 is 98% or more.

First, when tetanus toxoid was added, CD4 + T cells showed a strong proliferative response (* in FIG. 10C).
Then, when CD4 + T cells were treated with CD26 antibody (1F7) immediately before remixing, proliferation was suppressed (bar graph 3 in FIG. 10C). The T cell proliferation inhibitory effect of 1F7 in the liquid layer has been shown in the previous report, and the results of this Example are equivalent (Ohnuma, et al., 2002).
On the other hand, when treated with CD28 antibody (4B10), the proliferation response by tetanus toxoid was enhanced (bar graph 2 in FIG. 10C). This is interpreted that CD28 antibody (4B10) acts as an agonistic antibody, unlike CD26 antibody 1F7, even in the presence of APC (Gimmi, et al., 1991).
Further, when monocytes pulsed with tetanus toxoid were treated with CTLA4-Ig or CD86 antibody (IT2.2) immediately before remixing and reconstitution experiments were performed, proliferation of T cells was suppressed (bar graph 5 in FIG. 10C). , 6).
Furthermore, when CD4 + T cells immediately before remixing were treated with Fc fusion protein and a reconstitution experiment was performed, the T cell proliferation reaction was suppressed by NT-Fc and SCD-Fc (bar graphs 8, 10 in FIG. 10C). However, no change was observed in NTΔSCD-Fc (bar graph 9 in FIG. 10C).

In order to confirm the above-described inhibitory effect, it was examined whether the concentration dependency had an inhibitory effect. As shown in FIG. 10D, CTLA4-Ig inhibited tetanus toxoid-reactive CD4 + T cell proliferation in a concentration-dependent manner (* in FIG. 10D), and the same results as previously reported were observed (Ohnuma, et al ., 2001).
Furthermore, NT-Fc and SCD-Fc also showed an inhibitory effect in a concentration-dependent manner (**, *** in FIG. 10D).
On the other hand, with NTΔSCD-Fc treatment, no inhibitory effect was observed even when added to 1-20 μg / ml (# in FIG. 10D).

  From this Example, it was shown that caveolin-1-Fcγ1 fusion protein suppresses the proliferation reaction of T cells. Therefore, it was shown that the caveolin-1-Fcγ1 fusion protein blocks the interaction of CD26-caveolin-1 in the antigen-specific immune response and may have a T cell suppressive action or a cancer suppressive action.

Inhibitory effect on DPPIV enzyme activity by caveolin-1-Fcγ1 fusion protein Production and purification of soluble CD26 (rsCD26) were performed according to the method established by the inventors (Ohnuma et al., 2001, Tanaka et al., 1994). . Add 1 μM rsCD26 to each concentration of DPPIV inhibitor Valine-pyrrolidide (Val-pyr, 1 mM stock solution in DMSO) or Fc fusion protein so that the final volume is 50 μl per sample. After mixing for 5 minutes at ° C, the DPPIV enzyme activity was measured. DPPIV-Glo ^™ Protease Assay (Promega) was used for quantification of DPPIV enzyme activity. The enzyme activity was measured by measuring the luminol luminescence produced from Gly-Pro-aminoluciferin by DPPIV activity using a luminometer, and the relative luminescence intensity (RLU ) The measurement results are shown as the average value (RLU) of triplicate and standard error.

  When DPPIV inhibitor Valine-pyrrolidide (Val-Pyr) was added to soluble CD26 and DPPIV enzyme activity was measured, as shown in FIG. 11A, DPPIV enzyme activity was inhibited depending on the concentration of Val-Pyr. (▲). At this time, the 50% inhibitory concentration IC50 was calculated to be about 3 μM, which was almost the same as the previous report (2.4 μM) (Senten et al., 2002).

  Next, when NT-Fc was added to soluble CD26 and the DPPIV enzyme activity was examined, as with Val-Pyr, the DPPIV enzyme activity was suppressed depending on the concentration of NT-Fc, and the IC50 was calculated to be 15 μM. (O in FIG. 11B). Similarly, the DPPIV enzyme activity of soluble CD26 was also suppressed by SCD-Fc (X in FIG. 11B), but not suppressed by NTΔSCD-Fc (▲ in FIG. 11B).

  From the above results, it was shown that NT-Fc and SCD-Fc bind to CD26 and further suppress the DPPIV enzyme activity of CD26. Therefore, it is expected that the binding site between caveolin-1 and CD26 and the DPPIV enzyme inhibitory site are partly common, and DPPIV inhibitors have the potential to potentially suppress T cell immune responses. It was. It was also shown that caveolin-1-Fcγ1 fusion protein could be used as a therapeutic agent for diabetes by inhibiting DPPIV.

  The above examples show that caveolin-1 is a CD26 costimulatory ligand. It was also shown that caveolin-1 binding to CD26 induces T cell proliferation and NF-κB activation along with TCR / CD3 costimulation.

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It is a figure which shows caveolin-1-Fcγ1 fusion protein. 1 is a schematic diagram of a pEB6-CAG-hu ECDSP-Fcγ1 vector. It is a figure which shows the result of SDS-PAGE and the Western blot using the cell which expressed caveolin-1-Fcγ1 fusion protein. It is a figure which shows the coupling | bonding to CD26 of NT-Fc by an immunoprecipitation experiment. 500 μg of J. CDwt cell lysate was preabsorbed with Fcγ1 and protein A sepharose beads and immunoprecipitated (IP) with 2 μg of Fcγ1 (lane 1), NT-Fc (lane 2) or NTΔSCD (lane 3). The immunoprecipitated complexes were then separated using 5-20% SDS-PAGE, Western blotted and immunoblotted with anti-CD26 mAb (upper panel). The 50 μg cell lysate used was also analyzed (lane 4). Anti-CD26 mAb was peeled off from the membrane, and HRP-conjugated anti-human IgG was bound (lower panel). Results similar to those shown in FIG. 4 were obtained in three independent experiments. It is a figure which shows the coupling | bonding to CD26 of NT-Fc by flow cytometry. (A): J.CD26wt cells were used for Fc fusion protein binding activity. Panel (a): shows forward and side scatter diagrams of the analyzed cells. The black circle indicates the analyzed gate region. Panel (b): Cells were stained with FITC-conjugated control mouse IgG (1) or FITC-conjugated anti-CD26 mAb (2). For inhibition assays, cells were first reacted with unlabeled anti-CD26 mAb (3) or unlabeled control mouse IgG (4) and then stained as in (2). Panel (c): Cells were stained with biotinylated Fcγ1 (control) (1) or biotinylated NT-Fc (2) and reacted with FITC-conjugated streptavidin. For inhibition assays, cells were first reacted with unlabeled anti-CD26 mAb (3) or unlabeled control IgG (4) and then stained as in (2). Panel (d): Cells were stained with biotinylated Fcγ1 (control) (1) or biotinylated NTΔSCD-Fc (2) and reacted with FITC-conjugated streptavidin. For inhibition assays, cells were first reacted with unlabeled anti-CD26 mAb (3) or unlabeled control IgG (4) and then stained as in (2). All four graphs shown in panel (d) overlapped at the same position. (B): Fc fusion protein binding assay similar to (A) was performed using 300-19-CD26wt cells. It is a figure which shows the coupling | bonding to CD26 of NT-Fc by the experiment using Biacore. The affinity of the Fc fusion protein for soluble CD26 (rsCD26) was measured by equilibrium binding. rsCD26 is diluted from 50 nM to 2-fold dilution (50 nM, 25 nM, 12.5 nM, 6.3 nM, 3.2 nM and 1.6 nM) at 25 ° C. on Fcγ1 (a), NT-Fc (b) or NTΔSCD- Flowed over Fc (c). These proteins are immobilized at 6032 RU (response unit), 4996 RU, or 4852 RU, respectively. The curve shown in FIG. 5 represents specific binding after subtracting the background response observed in control cells. It is a figure which shows the IL-2 production activity of NT-Fc. (A): Native Jurkat (JKTwt) or J.CD26wt (5 × 10 ⁵ / well) was cultured in a 96-well flat bottom plate, and the antibody and / or Fc fusion protein (anti-CD3 antibody, 10 μg shown below the graph) anti-CD28 antibody, anti-CD26 antibody, Fcγ1, NT-Fc, NTΔSCD-Fc, 10 μg / ml each). After culturing for 48 hours, the culture supernatant was collected from 3 wells and the IL-2 content was measured. The values in the graph represent the mean ± SE determined from 3 cultures of 3 independent experiments. “*” And “***” indicate a significant increase (p <0.05), and “**” and “#” indicate no significant change compared to the control. (B): After inhibition with anti-CD26 antibody, anti-CD28 antibody or control mouse IgG, J.CD26wt cells were stimulated as described in (A) and IL2 was measured. The values in the graph represent the mean ± SE determined from 3 cultures of 3 independent experiments. “*” And “**” indicate significant inhibition results obtained by inhibition with anti-CD26 mAb (p <0.05). “#” And “##” indicate significant inhibition results obtained by inhibition with anti-CD28 mAb (p <0.05). It is a figure which shows that a peripheral blood T cell proliferates by costimulation of NT-Fc and anti-CD3mAb. (A): 1 × 10 ⁵ / well of T cells were treated with the antibody and / or Fc fusion protein (anti-CD3 antibody, 0.05 μg / ml; anti-CD28 antibody, anti-CD26 antibody, Fcγ1, NT− Fc, NTΔSCD-Fc, 5 μg / ml each) were cultured in a 96-well flat bottom plate. After 96 hours of culture, proliferation was measured by [ ³ H] -thymidine (TdR) uptake for 16 hours. The values in the graph show the mean ± SE determined from 3 cultures of 5 donors. “*” Indicates a significant increase (p <0.05), and “**” indicates no significant change compared to the control. (B): 1 × 10 ⁵ / well T cells were cultured in a 96-well flat-bottom plate in which Fc fusion protein at the concentration described below was immobilized in the presence of anti-CD3 antibody (0.05 μg / ml). . Proliferation was measured as shown in (A). The values in the graph show the mean ± SE determined from 3 cultures of 5 donors. “*” Indicates a significant increase compared to the control (p <0.05). (C): 1 × 10 ⁵ / well T cells were cultured in solution with various amounts of CHO transformants immobilized with 0.05% glutaraldehyde in the presence of anti-CD3 antibody (0.05 μg / ml). . “Cav-wt ⁺ CHO”, “Cav-ΔSCD ⁺ CHO” or “mock ⁺ CHO” were transfected with GFP-full length caveolin-1, caveolin-1 excluding GFP-backbone region, or GFP expression vector, respectively. Means CHO cells. Proliferation was measured as shown in (A). The values in the graph show the mean ± SE determined from 3 cultures of 5 donors. “*” Indicates a significant increase compared to the control (p <0.05). It is a figure which shows that the proliferation of the T cell induced | guided | derived by NT-Fc is inhibited by anti-CD26mAb. (A): After blocking T cells with soluble anti-CD26 antibody, anti-CD28 antibody or control mouse IgG, T cells were stimulated by the same method as in FIG. 8A, and proliferation was measured. The values in the graph show the mean value ± S.E. obtained from 3 cultures of 5 donors. “*” Or “***” indicates a significant inhibition result obtained by inhibition with anti-CD26 mAb or NT-Fc (p <0.05). “**” indicates a significant inhibition result obtained by inhibition with anti-CD28 mAb (p <0.05). (B): After blocking by incubating T cells with soluble anti-CD26 mAb, anti-CD28 mAb or control mouse IgG (0, 0.5, 5.0, 10.0, 20.0 and 50 μg / ml), T Cells were stimulated with anti-CD3 antibody (0.05 μg / ml) and NT-Fc (5 μg / ml) bound to the plate, and proliferation was measured in the same manner as in FIG. 8A. The values in the graph show the mean value ± S.E. obtained from 3 cultures of 5 donors. “*” Indicates a significant increase compared to the control (p <0.05). It is a figure which shows the inhibitory effect of caveolin-1-Fcγ1 fusion protein on the proliferation response of CD4 + T cells by tetanus toxoid stimulation. It is a figure which shows the result of the inhibition experiment of DPPIV enzyme activity. It is a schematic diagram which shows the inhibitory effect of CD26 by caveolin-1-Fcγ1 fusion protein in antigen-specific T cell activation.

SEQ ID NO: 1 shows the base sequence of DNA encoding human caveolin-1 shown by SEQ ID NO: 2.
SEQ ID NO: 2 This shows the amino acid sequence of human caveolin-1.
SEQ ID NO: 3 This shows the base sequence of DNA encoding Fcγ1 shown in SEQ ID NO: 4.
SEQ ID NO: 4 This shows the amino acid sequence of Fcγ.
SEQ ID NO: 5 This shows the amino acid sequence of NT-Fcγ1.
SEQ ID NO: 6: This shows the amino acid sequence of SCD-Fcγ1.
SEQ ID NO: 7 This shows the amino acid sequence of hu-ECDSP.

Claims (13)

caveolin-1, which has a first region consisting of all or part of the skeleton region of caveolin-1 and a second region consisting of the hinge, C _H 2 and C _H 3 regions of an immunoglobulin molecule, and has a binding activity to CD26 -1-Fcγ1 fusion protein. The fusion protein according to claim 1, wherein the caveolin-1 backbone region is the following polypeptide (a) or (b).
(a) a polypeptide comprising the amino acid sequence from the 82nd position to the 101st position in the amino acid sequence represented by SEQ ID NO: 2
(b) in the amino acid sequence from the 82nd to the 101st amino acid sequence shown in SEQ ID NO: 2, consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and Polypeptide having binding activity   DNA encoding the fusion protein according to claim 1 or 2.   A caveolin-1-Fcγ1 fusion protein expression vector containing the DNA according to claim 3.   A pharmaceutical composition comprising the fusion protein according to claim 1 or 2.   Autoimmune disease, rejection, graft-versus-host disease, rheumatoid arthritis, vasculitis syndrome, malignant tumor, malignant mesothelioma, acute or chronic graft-versus-host disease, rejection, obstructive arteriosclerosis, myocardial infarction or brain The pharmaceutical composition according to claim 5 for treating infarction.   CD26-mediated T cell costimulatory ligand containing all or part of the caveolin-1 skeletal region. CD26-mediated T cell costimulatory ligand containing a first region consisting of all or part of the caveolin-1 skeleton region and a second region consisting of the hinge, C _H 2 and C _H 3 regions of an immunoglobulin molecule .   A method for co-stimulation of T cells, comprising contacting the fusion protein according to claim 1 or 2 with isolated T cells in the presence of an anti-CD3 antibody. A method for screening a substance that regulates CD26-mediated T cell costimulatory activity by caveolin-1,
(i) When the fusion protein according to claim 1 or 2 is brought into contact with CD26, and (ii) When the fusion protein according to claim 1 or 2 and the test substance are brought into contact with CD26, fusion with CD26 Measuring the amount of protein binding or T cell costimulatory activity.   11. The method according to claim 10, wherein the measurement of T cell costimulatory activity is measurement of IL-2 production from T cells or measurement of T cell proliferation activity. A method for evaluating T cell costimulatory activity via CD26 of a substance to be evaluated,
(i) When the fusion protein according to claim 1 or 2 is brought into contact with CD26, and (ii) When the fusion protein according to claim 1 or 2 and the test substance are brought into contact with CD26, fusion with CD26 Measuring the amount of protein binding or T cell costimulatory activity.   The method according to claim 12, wherein the substance to be evaluated is a substance having dipeptidyl peptidase IV inhibitory activity.

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