# Patent

## Title
Antibody specifically recognizing an epitope for switching to TH1 cell

## Abstract

The present invention relates to a novel epitope that converts T cell to type 1 helper T (TH1) cell. Specifically, the present invention relates to an epitope constituting the 56th to 65th amino acids (SEQ ID No. 2) of extracellular domain (ECD) of activation-inducible tumor necrosis factor receptor (AITR), an antibody recognizing the epitope, a polynucleotide encoding the epitope, a polynucleotide encoding the antibody, an expression vector comprising the polynucleotide encoding the epitope or antibody, a transformant introduced with the vector, a composition comprising the antibody for converting T cell to TH1 cell and a method for converting T cell to TH1 cell, a pharmaceutical composition comprising the antibody for preventing or treating cancer, a method for treating cancer using the antibody, a composition comprising the antibody for enhancing immunity, and a method for enhancing immunity using the antibody.

## Background
RELATED APPLICATIONS
This application claim priority to Korean Application No. 10-2012-0061791 field on Jun. 8, 2012 and Korean Application No. 10-2012-0088645 filed on Aug. 13, 2012, both of which are incorporated herein by reference in their entireties.
TECHNICAL FIELD
The present invention relates to a novel epitope that converts T cell to type 1 helper T (TH1) cell. Specifically, the present invention relates to an epitope constituting the 56th to 65th amino acids (SEQ ID No. 2) of extracellular domain (ECD) of activation-inducible tumor necrosis factor receptor (AITR), an antibody recognizing the epitope, a polynucleotide encoding the epitope, a polynucleotide encoding the antibody, an expression vector comprising the polynucleotide encoding the epitope or antibody, a transformant introduced with the vector, a composition comprising the antibody for converting T cell to TH1 cell and a method of conversion for the same, a pharmaceutical composition comprising the antibody for preventing or treating cancer, a method for treating cancer using the antibody, a composition for enhancing immunity comprising the antibody, and a method for enhancing immunity using the antibody.
BACKGROUND ART
Various types of receptors belonging to the tumor necrosis factor receptor superfamily (TNFR) superfamily are involved in the regulation of cell proliferation, cell differentiation, and cell death. Also, TNFR receptors play an important role in development of immune response, tumor necrosis, and protection against autoimmune diseases. Examples of the TNFR receptors include CD28, 4-1BB, OX40, CD40, or CD27.
Human activation-inducible tumor necrosis factor receptor (AITR) is a member of TNFR superfamily, and is also called glucocorticoid-induced TNFR-related protein (GITR), tumor necrosis factor receptor superfamily member 18 (TNFRSF18), or CD357. AITR is a type I transmembrane protein of TNFR superfamily. Immunoregulatory T cell (Treg cell or CD4+CD25high T cell) constitutively expresses GITR at high level, and when peripheral blood mononuclear cell (PBMC) is activated, expression of GITR is rapidly up-regulated. In particular, a signal transduction through GITR is known to inhibit a suppressive function of Treg cells, which in turn increase a resistance of CD4+ and CD8+ T cells to the suppression by Treg cells, thereby activating the CD4+ and CD8+ T cells. Also, a monoclonal antibody (mAb, e.g., DTA-1) that specifically binds to GITR or a physiological ligand thereof i.e., GITRL is known to enhance antitumor immunity through various in vivo models. On the other hand, activation of GITR inhibits Treg cell which is capable of suppressing the autoreactive T cells, thereby promoting autoimmune response.
Although there has not been much information found about the biological functions of AITR and AITR ligand (AITRL), it is known that AITR is expressed at basal level in a resting T cell, but its expression level rapidly increases upon activation of T cell. Also, it has been reported that AITR expression level was increased in herniated disc tissue cell isolated from lumber disc herniation patients as well as in the Treg cells from active systemic lupus erythematosus patients.
AITR binds with TNFR-associated factors (TRAFs) such as TRAF1, TRAF2 and TRAF3. When AITR interacts with its ligand, this recruits TRAF2 and ultimately activates NF-κB/NIK (Kwon, B. et al., J Biol Chem 274, 6056-6061, 1999). TRAF protein is a type of cytoplasmic adapter protein, and is involved in signal transduction for extracellular proliferation, differentiation, activation and migration. The N-terminal sites of TRAF proteins vary a lot, and all of the TRAF proteins except for TRAF1 possess a RING-finger motif at the N-terminal site thereof. A RING-finger is essential for the TRAF-mediated signal transduction. In the previous study, when the genes encoding all six types of TRAF proteins were knockout in mouse by gene targeting, various phenotypes were observed, suggesting that TRAFs have significantly various biological functions (Ha, H., Curr Protoc Immunol. Chapter 11: Unit11.9D, 2009).
A Ca2+-dependent transcription factor known as nuclear factor of activated T cells (NFATs) regulates not only T cells but also various types of growth factors and cytokines. Also, it regulates cell-to-cell interaction molecules essential for morphogenesis, development, and other functions in various types of cells and organs. Recently, NFAT is shown to be an important factor in induction of specific genetic programs that regulate cell differentiation to TH lineage and effector or regulatory functions of TH cells, via the transcriptional regulation of TH lineage-specific transcription factors such as T-bet (TH1), GATA-3 (TH2), RORγt (TH17), and Foxp3 (Treg). In addition, it has been reported that NFAT family regulates transcription of various cytokines and cytokine receptors thereof (Macian, F. Nat Rev Immunol. 5, 472-484, 2005).
One of the most important factors determining the fate of CD4+ T cell is cytokine milieu during the TCR-mediated activation of naïve CD4+ T cell. The major signaling pathway triggered by cytokines is an activation of the signaling transducer and activator of transcription (STAT) family proteins. The STAT proteins play an essential role in differentiation and expansion of TH cells. In particular, IFN-γ/NFAT1/STAT-1 pathway activates T-bet which is the master transcription factor specifying the TH1 lineage, while NFAT1/STAT-3 pathway activates RORγ which is the master transcription factor specifying the TH17 lineage. Also, IL-4/NFAT2/STAT-6 pathway activates GATA-3 which is the master transcription factor specifying the TH2 lineage, and NFAT1/STAT-5 pathway activates Foxp3 which is the master transcription factor specifying the Treg lineage. These complex intracellular signaling cascades play an important role in determining the differentiation of TH cell and functions thereof.
Naive CD4 T cell can differentiate into four different types of T cells such as TH1, TH2, TH17, and induced Treg cells. Among them, TH1 cell regulates an immune response to pathogen in the cell. Particularly in humans, it is known to play an important role in resisting to Mycobacterial infection. The major cytokines of TH1 cell are interferon-γ (IFN-γ), lymphotoxin α (LTα), and IL-2, and IFN-γ is known to play a critical role in activation of macrophage. IFN-γ is a dimeric water-soluble cytokine, and plays an important role in congenital and acquired immunity. Specifically, IFN-γ is known to suppress tumor growth in diseases like cancer (Ikeda H, et al., Cytokine Growth Factor Rev. 13(2), 95-109, 2002).
Treg cells are present at high level in tumor environment and can suppress effector function of T cell. Therefore, when an effector T cell is transferred together with Treg cell in adoptive transfer, the therapeutic effect of effector T cells in adoptive transfer is diminished. Also, TGF-β secreted by Treg cells can suppress cytotoxic function of T cells. Thus, development of a therapeutic drug that can reduce the number of Treg cells by inducing differentiation of Treg cell in tumor is required.
DISCLOSURE
Technical Problem
In an effort to investigate a method for specifically differentiating T cell, the present inventors have developed an AITR-specific antibody that specifically binds to a sequence of 56th to 65th amino acids of extracellular domain (ECD) of AITR present in a regulatory T (Treg) cell and confirmed that the AITR-specific antibody can bind to the regulatory T cell and convert it to TH1 cell that is an effector T cell secreting IFN-γ, which in turn significantly reduces the number of the regulatory T cell within tumor demonstrating antitumor activity, thereby completing the present invention.
Technical Solution
One object of the present invention is to provide a polypeptide which is an epitope, represented by an amino acid sequence of SEQ ID No. 2, of activation-inducible tumor necrosis factor receptor (AITR) for converting T cells to type 1 helper T (TH1) cell.
Another object of the present invention is to provide an antibody specifically recognizing the epitope of AITR, represented by SEQ ID No. 2.
Another object of the present invention is to provide a method for preparing the antibody.
Another object of the present invention is to provide a polynucleotide encoding the epitope or antibody, an expression vector comprising the polynucleotide, and a transformant introduced with the vector.
Another object of the present invention is to provide a composition comprising the antibody for converting T cell to type 1 helper T (TH1) cell.
Another object of the present invention is to provide a method for converting T cell to TH1 cell, comprising treating T cell with the antibody.
Another object of the present invention is to provide a pharmaceutical composition comprising the antibody for preventing or treating cancer.
Another object of the present invention is to provide a method for treating cancer using the antibody.
Another object of the present invention is to provide a composition for enhancing immunity, comprising the antibody.
Another object of the present invention is to provide a method for enhancing immunity by using the antibody.
Advantageous Effects
The present invention has identified a new epitope of AITR, represented by SEQ ID No. 2, which can convert T cell to type 1 helper T (TH1) cell. Also, it was confirmed that an antibody that can specifically recognize the epitope can effectively convert T cell to TH1 cell. Therefore, as the antibody of the present invention effectively converts T cell to TH1 cell, it can be used for preventing or treating a disease like cancer which requires suppression of Treg cell and increase of TH1 cell.



DESCRIPTION OF DRAWINGS
FIG. 1 demonstrates the process of generating anti-AITR mAbs from the human antibody library. FIG. 1A shows the Coomassie blue-stained gel showing the presence of anti-AITR Fab clones selected by phage displaying; and bivalent forms of anti-AITR mAbs that were generated from transiently transfected HEK293. FIG. 1B shows flow cytometry analysis results demonstrating the binding of anti-AITR mAbs to AITR in AITR-overexpressed Jurkat cells that were stained with PE-conjugated anti-AITR mAbs. FIG. 1C shows the proportion of AITR-expressing cells (%) in a population of CD4+, CD8+, and CD19+ cells. PBMCs were stimulated with anti-CD3/IL-2 or lipopolysaccharide (LPS) for 2 days and stained with PE-conjugated anti-AITR mAb (clone A41). FIG. 1D shows the expression of AITR in activated CD4+ T cells. The purified CD4+ T cells were stimulated with anti-CD3/IL-2 for 3 days. AITR expression in the activated CD4+ T cells was detected with PE-conjugated anti-AITR mAbs.
FIG. 2 shows the expression of AITR in CD4+ T cells in an activation-dependent manner. Isolated PBMCs were stimulated with anti-CD3/IL-2 or LPS for 2 days. FIG. 2A shows the stimulated cells stained with PE-conjugated anti-AITR mAb (clone A41) in a test group of CD4+, CD8+, and CD19+ cells. Expressions of AITR (arrow pointing) in CD4+ T cells (upper panels), CD8+ T cells (middle panels), and CD19+ cells (down panels) were detected by staining both of the non-treated group or activated group of cells. FIG. 2B shows the stimulated cells stained with PE-conjugated anti-AITR mAb in a group of CD11c+CD14+, BDCA-2+ and CD56+ cells. Expressions of AITR were detected by staining the non-treated group (left panels) or activated group (right panels). The representative flow cytometric profiles are shown as dot plot diagrams. The proportion of AITR-expressing cells in each cell population is indicated. One of the results from three independent experiments is shown in this figure.
FIG. 3 shows the epitope mapping of the anti-AITR mAbs of the present invention. FIG. 3A shows a schematic diagram of full-length AITR and recombinant GST protein lysates used for antibody production and epitope mapping. Top: an intracellular domain (ICD) and an extracellular domain (ECD) of AITR. Bottom: twelve AITR recombinants R1, R2, and R3 to R12 (R3-R12). FIG. 3B shows western blotting analysis demonstrating a specific binding of anti-AITR mAbs to each fragment of AITR-ECD. Fragments of AITR-GST proteins are shown by Coomassie blue staining (Top and left panel). Molecular weight marker and recombinant GST protein size are indicated on the left of panel. FIG. 3C shows the position of each epitope for anti-AITR antibodies (A27, A35, A41, B32, and B62) within the amino acid sequence of AITR-extracellular domain (ECD) (SEQ ID No. 1).
FIG. 4 demonstrates the induction of differentiation of CD4+ T cells into different phenotypes by anti-AITR mAbs which recognize different fragments of AITR-ECDs of the present invention. FIG. 4A shows the analysis of cytokine secretion through human TH1/TH2 cytokine bead array and IL-17A ELISA assay. The purified CD4+ T cells were stimulated with anti-CD3/anti-AITR mAbs (clones A27, A35 or A41) for 3 days. FIG. 4B shows the proportions of cytokine-secreting cells, i.e., IL-4+, IFN-γ, and IL-17A+ cells (%). Purified CD4+ T cells were stimulated with anti-CD3/IL-2 for 3 days and treated with anti-AITR mAb or AITR ligand for additional 7 days. The cultured cells were stained for endogenous IL-4, IFN-γ, and IL-17A, which were then analyzed by flow cytometry. Then the proportion of IL-4+, IFN-γ+, and IL-17A+ cells (%) were calculated. The bar graphs (mean±SD) show the average value of data from three independent experiments, single asterisk, *p<0.05.
FIG. 5 shows the induction of cell division in CD4+ T cells by anti-AITR mAbs. CFSE-labeled CD4+ T cells were seeded in a flat-bottomed 96-well tissue culture plate coated with anti-CD3/IL-2 and treated with anti-AITR mAbs for 3 days. Histograms are shown in a resolution able to show up to five cycles of cell divisions by flow cytometry. The experimental result shown is based on one of the three independent experiments.
FIG. 6 shows the induction of expression of TH lineage-specific marker in CD4+ T cells. Purified CD4+ T cells were stimulated with anti-CD3/IL-2 for 3 days (A), and treated with anti-AITR mAbs (B) or AITR ligand (C) for additional 7 days. The cultured cells were stained for endogenous IL-4, IFN-γ, and IL-17A, and the surfaces thereof were stained with PE-conjugated anti-AITR mAb (clone 41). Then the cells were analyzed by flow cytometry. The experimental result shown is based on one of the three independent experiments.
FIG. 7 demonstrates that the anti-AITR monoclonal antibodies of the present invention recognizing different ECDs of AITR recruit different signaling proteins in CD4+ T cells, thereby demonstrating different activities. Purified CD4+ T cells were stimulated with anti-CD3/IL-2 for 3 days and were further treated with anti-AITR mAbs for 2, 6, 12, and 24 hr. FIG. 7A shows the western blotting analysis demonstrating the expression of TRAFs. After 24 hr of culturing, the cultured cells were lysed and immunoprecipitated with anti-AITR mAbs (clones A27, A35 and A41) and Protein G. Then the immunoprecipitates were immunoblotted with anti-TRAF1, anti-TRAF2, anti-TRAF3, anti-TRAF5, anti-TRAF6, or anti-AITR mAb (clones A27, A35 and A41). FIG. 7B shows the western blotting analysis demonstrating the expression of NFAT1/2, p-p38, p-ERK1/2, p-JNK1/2, and p-NF-kB in the cells which were treated with anti-AITR monoclonal antibodies in a time-dependent manner. The cells were treated with anti-AITR mAbs for the indicated times and lysed. Then the cell lysates were analyzed by western blotting using the antibodies against NFAT1/2, p-p38, p-ERK1/2, p-JNK1/2, and p-NF-κB. The expression level of the target proteins was normalized to β-actin expression level. FIG. 7C shows the flow cytometry analysis showing the expression of STATs. After 24 hours of culturing, the cells were analyzed by flow cytometry using the antibodies against p-STAT-1, p-STAT-3, p-STAT-4, p-STAT-5 and p-STAT-6. FIG. 7D shows the flow cytometry analysis demonstrating the expression of STAT proteins and master transcription factors. After 24 hr of culturing, the cells were stained with T-bet, GATA-3, or RORγt antibody and analyzed by flow cytometry. Expression of STAT proteins and master transcription factors (filled histograms, arrow pointing) are shown. Numerical values indicated in each panel represent an average of the data (mean±SD) from three independent experiments (compared with control, open histograms).
FIG. 8 shows the recruitment of TRAF protein and activation of signal transduction molecule by anti-AITR mAb in CD4+ T cell and Treg cell.
FIG. 9 shows the activation of STAT protein and master transcription factor by anti-AITR mAb in CD4+ T cell and Treg cell.
FIG. 10 shows the switching effect of anti-AITR mAbs in CD4+ T cells. Purified CD4+ T cells were stimulated with anti-CD3/IL-2 for 3 days for activation. Then the cells were further treated with anti-AITR mAbs for additional 7 days. Subsequently, the treated cells were washed and re-stimulated with media comprising the antibodies having different or same epitopes for 7 days. The cells were stained for endogenous cytokines IFN-γ, IL-4, and IL-17A. Then the effector phenotypes were examined. Also, the number of cell populations was calculated. Data shown are the average of three independent experimental results (mean±SD), single asterisk, *p<0.05 (compared to other groups).
FIG. 11 shows the switching effect of anti-AITR mAbs in CD4+ T cell.
FIG. 12 shows the AITR-mediated conversion of human Treg to TH1 or TH17. The sample of CD4+ T cells isolated from healthy subjects and cancer patients were treated with anti-CD3/IL-2 for 2 days for activation. FIG. 12A shows the analysis of the effector phenotypes of cytokines. CD4+CD25high T cells isolated from healthy subjects were treated with anti-AITR mAbs for 7 days. The cultured cells were stained for endogenous cytokines IFN-γ, IL-4, and IL-17A, and the effector phenotypes were examined by flow cytometry. FIG. 12B shows the measurements of the expression level of cytokines. The cell cultures were collected at the indicated times and the level of TGF-β, IFN-γ, IL-4, and IL-17A cytokines therein was measured. FIG. 12C shows the measurements of the expression level and mean fluorescence intensity (MFI) of master transcription factors. The isolated CD4+CD25high T cells were stimulated with anti-AITR mAbs. The cell cultures were collected at the indicated times and stained with the antibodies against T-bet, RORγt, GATA-3 and Foxp3. The expression level and mean fluorescence intensity (MFI) of these transcription factors were measured by using flow cytometry. FIG. 12D shows the analysis of the effector phenotypes of endogenous cytokines. The CD4+CD25high T cells isolated from cancer patients were stimulated with anti-AITR mAbs for 7 days. Subsequently, the treated cells were stained for endogenous cytokines IFN-γ, IL-4, and IL-17A, and the effector phenotypes thereof were analyzed by flow cytometry. The cells were sorted by using flow cytometry, which were then treated with anti-AITR mAbs for 7 days. The level of endogenous cytokines IFN-γ, IL-4, and IL-17A in the cultured cell was measured by using flow cytometry. The number of cell populations was calculated. The data represents the results from three independent experiments (mean±SD). *p<0.05 (compared to other groups)
FIG. 13 shows the AITR-mediated conversion of human Treg cells isolated from healthy subjects to TH1 or TH17. CD4+ T cells purified from healthy subjects were stimulated with anti-CD3/IL-2 for 5 days. FIG. 13A shows the expression of AITR and Foxp3 in CD4+CD25high T cells (R1) and CD4+CD25− T cells (R2). CD4+CD25high T cells (R1) and CD4+CD25− T cells (R2) were sorted by FACS sorter. Then the expression of AITR and Foxp3 was detected in the sorted cells. FIG. 13B shows the analysis of effector phenotype of CD4+CD25high T cells and CD4+CD25− T cells. The sorted CD4+CD25high T cells and CD4+CD25− T cells were stimulated with anti-AITR mAbs. The stimulated cells were stained for endogenous cytokines IL-4, IFN-γ, and IL-17A and were examined for effector phenotype by flow cytometry. When the cells were treated with anti-AITR mAbs and examined for Foxp3 expression, it was observed that Foxp3 expression was reduced by A27. One of the three independent experimental results is shown.
FIG. 14 shows the AITR-mediated conversion of human Treg cells isolated from cancer patients to TH1 cells. The CD4+ T cells purified from cancer patients (n=10 in each case) were stimulated with anti-CD3/IL-2 for 5 days. FIG. 14A shows the analysis of AITR and Foxp3 expression in CD4+CD25high T cells. CD4+CD25high T cells were sorted by FACS sorter and the expression of AITR and Foxp3 was examined in the sorted cells. Data represent the average result of three independent experiments (mean±SD). FIG. 14B shows the analysis of effector phenotype in CD4+CD25high T cells. The sorted CD4+CD25high T cells were stimulated with anti-AITR mAb (clone A27) for 7 days. The cultured cells were stained for endogenous cytokines IL-4, IFN-γ, and IL-17A and were examined for effector phenotype by flow cytometry. One of the three independent experimental results is shown.
FIG. 15 shows the recruitment of TRAFs and activation of other signal molecules in CD4+CD25high T cells by anti-AITR mAbs. The sorted CD4+CD25high T cells were stimulated with anti-AITR mAbs for 6, 12, or 24 hours. FIG. 15A shows the western blotting analysis of CD4+CD25high T cells for the expression of TRAF proteins. After 24 hours of culturing, cell lysates were prepared, which were then immunoprecipitated with protein G. The immunoprecipitates were immunoblotted with anti-TRAF1, 2, 3, 5 and 6, or anti-AITR mAb. FIG. 15B shows the western blotting analysis of CD4+CD25high T cell lysates prepared at the indicated times for the expression of NFAT1/2, p-p38, p-ERK1/2, p-JNK1/2, p-NF-κB, or β-actin. The results were normalized according to the level of β-actin expression. Data shown here are representative of three independent experiments.
FIGS. 16A and B show the induction and activation of STAT proteins and master transcription factors in CD4+CD25highT cells by anti-AITR mAbs. The sorted CD4+CD25highT cells were stimulated with anti-AITR mAbs. FIG. 16A shows the analysis of STAT protein expression. After 24 hours of culturing, the cultured cells were analyzed by flow cytometry using the antibodies against p-STAT-1, p-STAT-3, p-STAT-4, p-STAT-5 and p-STAT-6. Expressions of STAT proteins are shown as filled histograms (arrow pointing) in comparison to the control group (unfilled histogram). Data are representative of three independent experiments (mean ±SD). FIG. 16B shows the flow cytometry analysis of T-bet, GATA-3, RORγt and Foxp3 expression. Culture cells were collected at the indicated times and stained with T-bet, GATA-3, RORγt and Foxp3 antibodies, and then analyzed by flow cytometry. One of the three independent experimental results is shown.
FIG. 17 shows the comparison of anti-tumor activity of anti-AITR mAbs of the present invention each recognizing different fragments of AITR-ECD. Hu-PBL-SCID mice were challenged subcutaneously (s.c.) with 1×106 of H441 lung carcinoma cells. When the size of tumor reached 1 cm, anti-AITR mAbs were injected intraperitoneally to tumor-challenged mice. FIG. 17A shows the flow cytometry analysis of T cell expression in PBMCs. Severe combined immunodeficiency mice were injected with human peripheral blood leukocytes (hu-PBL-SCID) and after 4 to 5 weeks, peripheral blood mononuclear cells (PBMCs) were collected from hu-PBL-SCID mice and examined for human T cell expression by flow cytometry. FIG. 17B shows the measurement of tumor volume (mm3) in hu-PBL-SCID mice at the indicated time in each test group (n=5 to 8). FIG. 17C shows the proportion of the cells expressing human CD4, Foxp3, CD8, CD56 and mCD45. After 12 days of anti-AITR mAbs (clone A27) injection, four types of cells including PBMCs, tumor, tumor-draining lymph nodes (DLN), and spleen cells were collected from mouse and stained for human CD4, human CD8, human Foxp3, human CD56, and mouse CD45. Human populations were gated as mouse CD45-negative population. The number of cell groups was calculated. Data represent the average value of three independent experimental results (mean±SD). *p<0.05 (compared to other groups).
FIG. 18 shows the generation of hu-PBL-SCID mouse. FIG. 18A shows the flow cytometry analysis of mouse spleen cells from hu-PBL-SCID mouse. After 4 to 5 weeks of PBMCs injection, spleen cells were isolated from hu-PBL-SCID mouse and examined for the expression of human cells (mouse CD45-negative population) or mouse cells (mouse CD45-positive population) by flow cytometry. FIG. 18B shows the flow cytometry analysis of mouse cells (gated on R1, upper panels) and human cells (gated on R2, lower panels) in each group of lymphocyte populations (hCD3, hCD4, hCD8 and hCD19) and non-lymphocyte populations (hCD11c, hCD14 and hCD56). One of the three independent experimental results is shown.
FIG. 19 demonstrates that A27 which is anti-AITR antibody of the present invention recognizing AA 56-65 among AITR-ECDs suppresses the accumulation of Treg cell in tumor and enhances the activity of CD8+ T cell and CD8+CD56+ T cell in tumor. Hu-PBL-SCID mice were challenged subcutaneously (s.c.) with 1×106 of H441 lung carcinoma cells and treated with human IgG (A) or anti-AITR mAb (clone A27) (B). On the twelfth day of antibody injection, PBMCs, tumors, tumor-draining lymph nodes (DLN), and spleen cells were isolated and stained for hCD4, hCD8, hFoxp3, hCD56, and mCD45. Human population was gated as mouse CD45-negative population. One of the three independent experimental results is shown.



BEST MODE
As one aspect, the present invention provides a polypeptide defined by an amino acid sequence of SEQ ID No. 2, which is an epitope of activation-inducible tumor necrosis factor receptor (AITR), for converting T cell to type 1 helper T (TH1) cell.
As used herein, “activation-inducible tumor necrosis factor receptor (AITR)” refers to a type of receptor belonging to TNFR superfamily, and can be interchangeably used with glucocorticoid-induced TNFR-related protein (GITR), tumor necrosis factor receptor superfamily member 18 (TNFRSF18), or CD357. The AITR may preferably be human AITR, but is not limited thereto. Information on AITR can be obtained from a well-known database such as NCBI GenBank. For instance, the information on AITR may be found by searching with NCBI Reference Sequence: NP—004186, but is not limited thereto. The AITR is expressed constitutively at high level in activated T cell or Treg cell.
As used herein, the term “epitope” refers to a site on antigen determining the antigen specificity, and can be interchangeable with antigen determining group or antigen determining site. For the purpose of the present invention, the epitope refers to the site corresponding to 56th to 65th amino acids of AITR-ECD, which can convert T cell to TH1 cell, but is not limited thereto as long as the sequence has the same converting activity. Also, the scope of the epitope may include the sequence having at least 80%, 90%, 95%, 98%, or 99% sequence homology to the sequence of 56th to 65th amino acids of AITR-ECD without limitation, as long as it has the same activity as the above-specified sequence. The sequence of 56th to 65th amino acids of AITR-ECD is shown in FIG. 3 and Table 2, and named as SEQ ID No. 2. The present inventors identified for the first time that T cell can be converted to TH1 cell by using an epitope represented by an amino acid sequence of SEQ ID No. 2. In an effort to identify the site that can specifically convert T cell to TH1 cell in AITR-ECD constituted of 139 amino acids (SEQ ID No. 1), the present inventors have confirmed that when the sequence of 56th to 65th amino acids represented by SEQ ID No. 2 is used as an epitope, it can specifically convert T cell to TH1 cell. On the other hand, other sequences adjacent to 56th to 65th amino acids did not show the converting activity to TH1 cell, and thus it was identified that the sequence of SEQ ID No. 2 is highly specific for T cell conversion to TH1 cell (Experimental Examples 2 and 4 to 8).
As used herein, “conversion of T cell to type 1 helper T (TH1) cell” refers to converting T cell to TH1 cell through antibody binding to a specific epitope of AITR represented by SEQ ID No. 2 or non-antibody protein specifically binding to the same. Preferably the conversion refers to the conversion of T cell to TH1 cell secreting IFN-γ through an antibody binding to the epitope of AITR in T cell represented by SEQ ID No. 2, but is not limited thereto. The conversion of T cell to TH1 can be identified by observing the expression of TH1 cell-specific marker, for example cytokines such as IFN-γ or IL-2; activation of IFN-γ/NFAT/STAT-1 signal transduction pathway; and expression of transcription factor such as T-bet. The T cell refers to a T cell expressing AITR, and for example it may be the activated T cell or regulatory T cell. In one example of the present invention, it was identified that CD4+ T cell and regulatory T cell expressed AITR when stimulated by anti-CD3/IL-2 or LPS. The TH1 cell is an immune cell secreting interferon-γ (IFN-γ). TH1 cell mediates immune response to intracellular pathogen, and is known to prevent tumor from developing into cancer. Also, it has been reported that inflammation caused by TH1 cell in tumor does not promote development of cancer, but rather prevents it from growing (Haabeth O A et al., Nat Commun 2011; 2:240). Therefore, a polypeptide of the present invention which is an epitope of AITR represented by SEQ ID No. 2 that can convert T cell to TH1 cell can be effectively used in the development of antibody for preventing or treating cancer.
In the conversion of T cell to TH1 cell, T cell may be a regulatory T cell (Treg cell).
As used herein, “Treg cell” is a subpopulation of T cells, and refers to a type of T cell that can down regulate immune system. Also, a regulatory T cell can be interchangeably used with Treg cell, CD4+CD25high cell, or CD4+CD25highFoxp3+ cell. The Treg cell comprises both of natural or constitutive regulatory T cell and adaptive or inducible regulatory T cell. With regard to tumor development, Treg cell promotes local tumor growth. Therefore, Treg cell is regarded as a big obstacle in immune therapy for cancer. In addition, with regard to tumor immunity, suppression of tumor regulatory T cell is an important part of cancer treatment (Liu Z et al., J Immunol 182(10), 6160-7, 2009). Therefore, conversion of Treg cell to TH1 cell could be a significantly useful method for preventing or treating cancer. In the conversion of Treg cell to TH1 cell, a signal transduction pathway and transcription factors associated with TH1 differentiation take important roles. In particular, a transcription factor Foxp3 is a Treg cell-specific endogenous marker. When Treg cell is differentiated into TH1 cell, the intracellular level of Foxp3 gets reduced. On the other hand, the level of T-bet which is a transcription factor involved in TH1 cell differentiation is increased through IFN-γ/NFAT1/STAT-1 pathway.
In one example of the present invention, it was confirmed that Treg cell was differentiated into TH1 cell by A27 which is a representative antibody of the present invention that specifically recognizes the epitope of SEQ ID No. 2. However, other AITR-specific antibody recognizing the site other than the epitope of SEQ ID No. 2 did not show the above converting activity. To be specific, A35 antibody recognizing the sequence of 41st to 50th amino acids and A41 antibody recognizing the sequence of 20th to 30th amino acids could not differentiate Treg cell to TH1 cell, even though these epitope sites were significantly close to the sequence of 56th to 65th amino acids of AITR-ECD (FIGS. 3, 4, and 6 to 17). In addition, even after the T cell was converted to the cells that secrete a specific cytokine through treatment with A35 or A41, the cell could be re-converted to IFN-γ-secreting cell, when treated with A27 antibody recognizing the epitope of SEQ ID No. 2 of the present invention. These results suggest that the epitope of SEQ ID No. 2 is significantly important site in cell conversion to TH1 cell (FIGS. 10 and 11). Also, in one example of the present invention, a molecular mechanism behind the conversion to TH1 cell through an epitope of SEQ ID No. 2 was investigated. To be specific, NFAT1 is involved in the conversion to TH1 cell and it increases phosphorylation level of STAT-1 protein (FIGS. 7 to 9; and FIGS. 15 and 16). In addition, it was observed that the level of Foxp3 which is a Treg cell-specific marker was reduced while the level of T-bet which is a TH1 cell-specific marker was increased, thereby confirming that Treg cell could be efficiently converted to TH1 cell (FIGS. 12 and 13; and FIGS. 16 to 19).
As another aspect, the present invention provides an antibody specifically recognizing an epitope of activation-inducible tumor necrosis factor receptor (AITR), wherein the epitope is represented by SEQ ID No. 2.
As used herein, the term “antibody” refers to a protein molecule that acts as a receptor recognizing an antigen specifically, comprising an immunoglobulin molecule that responses to a specific antigen immunologically. Also, the antibody comprises all of polyclonal antibody, monoclonal antibody, whole antibody, and antibody fragment. In addition, the scope of antibody includes a chimeric antibody (for example, humanized murine antibody) and bivalent or bispecific molecule (for example, bispecific antibody), diabody, triabody, and tetrabody. The whole antibody is constituted of two full-length light chains and two full-length heavy chains, wherein each of the light chains is connected with heavy chains through disulfide binding. The whole antibody comprises IgA, IgD, IgE, IgM, and IgG. IgG comprises IgG1, IgG2, IgG3, and IgG4 as subtypes thereof. The antibody fragment refers to a fragment capable of binding to antigen, and it comprises Fab, Fab′, F(ab′)2, and Fv. The Fab is constituted of variable regions of light chain and heavy chain, constant region of light chain, and the first constant region of heavy chain (CH1 domain), having one antigen-binding site. Fab′ is different from Fab in that it has a hinge region which comprises more than one cysteine residue at C-terminal of heavy chain CH1 domain. F(ab′)2 antibody is formed through disulfide bonding of cysteine residues in the hinge region of Fab′. A variable fragment (Fv) refers to a minimized antibody fragment having heavy chain variable region and light chain variable region. In a double stranded Fv (dsFv), heavy chain variable region and light chain variable region are connected through disulfide bonding while in a single stranded Fv (scFv), heavy chain variable region and light chain variable region are covalently bonded through peptide linker. These antibody fragments can be obtained by treating the antibody with protease (for example, when a whole antibody is restriction digested with papain, Fab is obtained and when restriction digested with pepsin, F(ab′)2 fragment can be obtained). Preferably, the antibody fragment can be generated by using genetic recombination technique.
As used herein, the term “monoclonal antibody” refers to an antibody molecule that has been obtained from a substantially identical antibody clone, which shows single-binding specificity and affinity for a specific epitope.
Typically, immunoglobulin has heavy chain and light chain and each of the heavy chain and light chain possesses constant region and variable region (also known as domains). A variable region of light chain and heavy chain comprises three variable regions and four framework regions called complementarity-determining region (hereinafter referred to as “CDR”). The CDR mainly acts to bind to an epitope of antigen. CDRs of each chain is conventionally called CDR1, CDR2, and CDR3 successively starting from the N-terminal, and also they are distinguished by the chain at which they are located.
As used herein, the term “human antibody” refers to a molecule derived from human immunoglobulin, in which all of the amino acid sequences constituting the antibody including complementarity-determining regions and framework regions are composed of amino acid sequences of human immunoglobulin. Human antibody is conventionally used for treating human diseases, which has more than three potential advantages. First, human antibody interacts more favorably with human immune system, and thus it can destroy the target cell more efficiently, for example through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Secondly, human immune system does not recognize the human antibody as a foreign substance. Thirdly, when smaller dose of drug was administered at lower frequency, half-life of human antibody in human circulation is similar to that of naturally-occurring antibodies. In one example of the present invention, it was confirmed that a human monoclonal antibody specifically recognizing the epitope of SEQ ID No. 2 demonstrates a strong affinity toward AITR and thus can effectively convert T cell to TH1 cell (FIGS. 1, 3, 4, and 6 to 17). The heavy chain and light chain domains of the antibody capable of converting T cell to TH1 cell effectively are all human-derived showing lower rate of immunogenicity, and thus the antibody can be effectively used for treating cancer.
As used herein, “antibody specifically recognizing an epitope of activation-inducible tumor necrosis factor receptor (AITR) represented by SEQ ID No. 2” refers to an antibody that can convert T cell to TH1 cell by specifically recognizing an epitope of SEQ ID No. 2, but is not limited thereto. The antibody particularly converts AITR-expressing T cells, for example activated T cell or Treg cell, to TH1 cell, and thus it can be effectively used for preventing or treating cancer; and for enhancing immunity.
The antibody specifically recognizing the epitope of SEQ ID No. 2 may preferably comprise a heavy chain variable region comprising heavy chain complementarity-determining region 1 (CDR1) represented by SEQ ID No. 6; heavy chain CDR2 represented by SEQ ID No. 7; and heavy chain CDR3 represented by SEQ ID No. 8, and a light chain variable region comprising light chain CDR1 represented by SEQ ID No. 10; light chain CDR2 represented by SEQ ID No. 11; and light chain CDR3 represented by SEQ ID No. 12. More preferably, the antibody may comprise an amino acid sequence of heavy chain variable region represented by SEQ ID No. 5 and an amino acid sequence of light chain variable region represented by SEQ ID No. 9, but is not limited thereto. In one example of the present invention, a human monoclonal antibody comprising the amino acid sequence of heavy chain variable region represented by SEQ ID No. 5 and the amino acid sequence of light chain variable region represented by SEQ ID No. 9 was named as A27. The nucleic acids encoding the antibody that specifically recognizes the epitope represented by SEQ ID No. 2 may comprise a nucleotide sequence encoding a heavy chain variable region represented by SEQ ID No. 13; and a nucleotide sequence encoding a light chain variable region represented by SEQ ID No. 14, but is not limited thereto.
The AITR-specific human monoclonal antibody recognizing the epitope represented by SEQ ID No. 2 may preferably comprise a heavy chain variable region comprising heavy chain complementarity-determining region 1 (CDR1) represented by SEQ ID No. 16; heavy chain CDR2 represented by SEQ ID No. 17; and a heavy chain CDR3 represented by SEQ ID No. 18, and a light chain variable region comprising light chain CDR1 represented by SEQ ID No. 20; light chain CDR2 represented by SEQ ID No. 21; and light chain CDR3 represented by SEQ ID No. 22. More preferably, the antibody may comprise an amino acid sequence of heavy chain variable region represented by SEQ ID No. 15 and an amino acid sequence of light chain variable region represented by SEQ ID No. 19, but is not limited thereto. In one example of the present invention, the antibody comprising the amino acid sequence of heavy chain variable region represented by SEQ ID No. 15 and the amino acid sequence of light chain variable region represented by SEQ ID No. 19 was named as B32. The nucleic acids encoding the antibody that specifically recognizes the epitope represented by SEQ ID No. 2 may comprise a nucleotide sequence encoding a heavy chain variable region represented by SEQ ID No. 23; and a nucleotide sequence encoding a light chain variable region represented by SEQ ID No. 24, but is not limited thereto.
Likewise, in the present invention it was confirmed that even though the antibodies comprise different amino acid sequences, as long as they can specifically recognize the epitope of SEQ ID No. 2 for specific conversion of T cell to TH1 cell, those antibodies can be effectively used for preventing or treating cancer, and for enhancing immunity.
In addition, if the antibody of the present invention comprises a constant region, the constant region may be derived from IgG, IgA, IgD, IgE, and IgM or a combination or hybrid thereof.
As used herein, the term “combination” refers to, when forming a dimer or polymer, a combination of a polypeptide coding for single-chain immunoglobulin constant region of same origin with single-chain polypeptide of different origin. For example, the dimer or polymer can be formed with more than two constant regions selected from the group consisting of constant regions of IgG, IgA, IgD, IgE, and IgM.
As used herein, the term “hybrid” refers to the presence of more than two immunoglobulin heavy chain constant regions of different origins within a single-chain immunoglobulin heavy chain constant region. For example, a hybrid of domains can be formed with 1 to 4 domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG, IgA, IgD, IgE, and IgM. Meanwhile, a combination or hybrid of heavy chain constant regions of the subtypes of IgG such as IgG1, IgG2, IgG3, and IgG4 is also possible. The combination and hybrid are the same as described above.
Also, if the antibody of the present invention comprises a light chain constant region, the light chain constant region may be derived from lambda (λ) or kappa (κ) light chain.
In one example of the present invention, A27 and B32 antibodies that specifically bind to AITR were selected from a human antibody library (Experimental Example 1), and their specific binding to AITR was confirmed (FIG. 1). In addition, the antibody recognition sites on AITR-ECD were identified (FIG. 3 and Table 2). Also, it was confirmed that unlike other antibodies that recognize other sites of AITR, the above-described antibodies can convert T cell to TH1 cell (FIGS. 3, 4, and 6 to 17), and furthermore, even the T cells that were already differentiated into specific T cell, the antibodies can re-convert the cell to TH1 cell (Experimental Examples 7 and 8, and FIGS. 10 and 11). These results support that the AITR-specific antibody of the present invention that recognizes an epitope of SEQ ID No. 2 can efficiently convert T cell, especially Treg cell, to TH1 cell, and thus the antibody can be used for prevention or treatment of cancer, and enhancement of immunity.
As another aspect, the present invention provides a method for preparing the antibody.
The antibody of the present invention can be easily prepared by a conventional method for production of antibody. For example, a monoclonal antibody can be prepared by generating hybridoma with B lymphocyte obtained from immunized animals (Koeher and Milstein, 1976, Nature, 256:495), and phage display technique, but is not limited thereto. A polyclonal antibody can be easily prepared by a conventional method for production of antibody, and also it can be prepared by using an antigen comprising the epitope of the present invention.
Production of antibody library by using phage display technique is done by obtaining an antibody gene directly from B lymphocyte without producing hybridoma and expressing the antibody on the phage surface. By using the phage display technique, many limitations associated with the production of monoclonal antibody can be overcome by B-cell immortalization. In general, a phage display technique consists of the steps of (1) inserting oligonucleotides of random sequences to the nucleotide sequence corresponding to N-terminal of phage coat protein pIII (or pIV); (2) expressing a fusion protein of a partial naïve coat protein and a polypeptide coded by the oligonucleotide of random sequence; (3) treating a receptor material that can bind to the polypeptide coded by the oligonucleotide; (4) eluting the peptide-phage particles bound to receptors by lowering pH or using competitive molecules for binding to the receptors; (5) amplifying a phage sample eluted by panning within the host cells; (6) repeating the above process to obtain desired amount of phage products; and 7) determining the sequence of active peptide among the DNA sequences of phage clones selected by panning.
Preferably, production of a monoclonal antibody of the present invention can be done by using phage display technique. Those skilled in the art can easily perform each step of the preparation method of the present invention according to a conventional phage display technique, for example a method described in Barbas et al. (METHODS: A Companion to Methods in Enzymology 2:119, 1991; J. Virol. 2001 July; 75(14):6692-9) and Winter et al. (Ann. Rev. Immunol. 12:433, 1994). A type of phage that can be used for generating antibody library includes filamentous phage such as fd, M13, f1, If1, Ike, Zj/Z, Ff, Xf, Pf1, or Pf3 phage, but is not limited thereto. In addition, a type of vector that can be used for expressing heterologous gene on filamentous phage surface includes a phage vector such as fUSE5, fAFF1, fd-CAT1, or fdtetDOG or a phagemid vector such as pHEN1, pComb3, pComb8, or pSEX, but is not limited thereto. Also, a type of helper phage that can be used for providing a wild-type coat protein which is required for the successful reinfection of recombinant phage for amplification includes M13K07 or VSCM13 helper phage, but is not limited thereto.
The hybridoma-derived monoclonal antibody or polynucleotide encoding phage display clones of the present invention can be easily isolated and analyzed for the sequence thereof through a conventional process. For example, oligonucleotide primers designed to specifically amplify the heavy chain and light chain coding region from hybridoma or phage template DNA can be used. Once the polynucleotide is isolated, it can be inserted to an expression vector, which can be subsequently introduced to a suitable host cell, thereby producing a desired monoclonal antibody from the transformed host cell (i.e., transformant). Therefore, the method for preparing the antibody of the present invention may be a method for preparing antibody comprising the step of amplifying the expression vector comprising polynucleotide encoding the antibody, but is not limited thereto.
As another aspect, the present invention provides a polynucleotide encoding the epitope or antibody, an expression vector comprising the polynucleotide, and a transformant introduced with the vector.
The antibody is the same as described above.
The expression vector of the present invention comprising a polynucleotide encoding the epitope or antibody is not limited to, but may be a vector that can replicate and/or express the polynucleotide in eukaryotic cells including animal cells (for example, human, monkey, rabbit, rat, hamster, and mouse cell etc), plant cell, yeast cell, insect cell, or bacterial cell (for example, E. coli), or prokaryotic cells. Preferably, the vector may be a vector wherein the nucleotide is operably linked to a suitable promoter for the expression thereof in a host cell and at least one selection marker is included. For example, the expression vector of the present invention may be in a form of a phage vector, plasmid vector, cosmid vector, mini-chromosome vector, viral vector, or retroviral vector wherein the polynucleotide is introduced.
The expression vector comprising polynucleotide encoding the antibody may be an expression vector comprising each of polynucleotides encoding the heavy chain or light chain of the antibody, or an expression vector comprising all of polynucleotides encoding heavy chain or light chain.
The transformant of the present invention wherein the expression vector is introduced is not limited to, but may be prepared from bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium; yeast cells; fungal cells such as Pichia pastoris; insect cells such as Drosophila, Spodoptera Sf9 cell; animal cells such as Chinese hamster ovary (CHO) cells, mouse bone marrow cell SP2/0, human lymphoblastoid, COS, mouse bone marrow cell NSO, 293T, BOW melanoma cell lines, HT-1080, baby hamster kidney (BHK) cells, human embryonic kidney (HEK) cells, human retinal cell PERC.6; or plant cells by transforming the same with the expression vector.
As used herein, the term “introduction” refers to a method of transferring a vector comprising the polynucleotide encoding epitope or antibody to a host cell. Such introduction can be done by using various methods known in the art such as Calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofectamine, and protoplast fusion. Also, transduction refers to a transfer of a target molecule into the cell by using viral particles through infection. Furthermore, a vector can be introduced to the host cell through gene bombardment. In the present invention, the term introduction can be interchangeably used with transfection.
As another aspect, the present invention provides a composition comprising the antibody for converting T cell to type 1 helper T (TH1) cell.
The antibody, T cell, TH1 cell, and conversion of T cell to TH1 cell are the same as described above. The antibody of the present invention specifically recognizing the epitope of AITR represented by SEQ ID No. 2 can convert T cell to TH1 cell specifically, and thus a composition comprising the antibody can be used for the conversion of T cell to TH1 cell. Especially, since the composition of the present invention can convert the already differentiated cell to TH1 cell, it can be diversely used depending on the purpose.
As another aspect, the present invention provides a method for converting T cell to type 1 helper T (TH1) cell, comprising treating T cell with the antibody.
The antibody, T cell, TH1 cell, and conversion of T cell to TH1 cell are the same as described above. When the T cell, especially regulatory T cell, is treated with the antibody of the present invention, it can be effectively converted to TH1 cell, and thus the antibody can be effectively used in the treatment of diseases that need the function of TH1 cell.
As another aspect, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the antibody.
As used herein, the term “cancer” refers to any type of cancer as long as it can be treated by the antibody of the present invention, and examples of such cancer include esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, oral cancer, pharynx cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, renal cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin disease, lymphoma, and multiple myeloma blood cancer.
As used herein, the term “prevention” refers to all actions that suppress or delay the onset of cancer by administering a composition.
As used herein, the term “treatment” refers to all actions that can alleviate or beneficially change the symptoms of cancer by administering a composition.
The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier.
As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not inhibit a biological activity and feature of the administered compound without irritating an organism. Types of pharmaceutical carriers suitable for a composition of liquid formulation include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a combination thereof, as a sterilized and acceptable carrier to the organism. If necessary, other conventional additives such as antioxidant, buffer solution, and bacteriostatic agent can be added. Also, the pharmaceutical composition may be formulated into a formulation for injection such as aqueous solution, suspension, emulsion; pill, capsule, granule, and tablet by further adding a diluent, dispersant, surfactant, binder, and lubricant.
The pharmaceutical composition may be in a form of various oral or parenteral formulations. The composition is formulated with conventional diluents or excipients, including fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include tablets, pills, powders, granules, and capsules. These solid formulations are prepared by mixing one or more compounds with at least one of excipients, for example, starch, calcium carbonate, sucrose, lactose, and gelatin. Also, other than the simple excipients, lubricants such as magnesium stearate and talc are used. In addition, types of liquid formulation for oral administration include a suspension, solution, emulsion and syrup. To this liquid formulation, not only a commonly used diluent such as water and liquid paraffin may be added, but also various types of excipients such as wetting agents, sweetening agents, flavors, and preservatives can be added. Types of formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulation, and suppositories. As non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. The base composition for suppository may include witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerinated gelatin.
The pharmaceutical composition of the present invention may be formulated in any one of the forms selected from the group consisting of tablets, pills, powders, granules, capsules, suspension, solutions, emulsions, syrups, sterilized aqueous solution, non-aqueous solution, lyophilized formulation and suppository.
The composition of the present invention is administered in a pharmaceutically effective amount.
As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to treat a disease, at a reasonable benefit/risk ratio applicable to any medical treatment. The effective dosage level of the composition may be determined by the factors including a type of subject, severity of condition, an age and sex of subject, a type of cancer, an activity of drug, a sensitivity towards drug, duration and route of administration, excretion rate, duration of treatment, a type of drugs used in combination with the composition, and other factors well-known in the medical field. The composition of the present invention may be administered alone or in combination with other medicines, and it may be administered sequentially or simultaneously with conventional medicines. Also, the present composition may be administered in single or multiple doses. In view of all of these factors, it is important to administer the composition in a minimum amount yielding the maximum possible effect without causing side effects, which can be easily determined by those skilled in the art.
In one example of the present invention, it was confirmed that the antibody of the present invention binding to the epitope of SEQ ID No. 2 can efficiently convert T cell to IFN-γ-producing TH1 cell. As described above, the antibody of the present invention can significantly reduce the number of Treg cells which pose obstacles to prevention or treatment of cancer, while increasing the number of TH1 cells that help preventing or treating cancer, and therefore it can be effectively used in anticancer treatment (FIGS. 4, and 12 to 19). Also, it was confirmed that especially A27 which is a representative antibody binding to the epitope of SEQ ID No. 2 demonstrated a significant anticancer effect compared to a control group in vivo (FIG. 17), thereby suggesting that A27 can be effectively used in the treatment of cancer.
As another aspect, the present invention provides a method for treating cancer, comprising administering the antibody to a subject in need thereof.
The antibody and cancer are the same as described above.
The method for treating cancer may be a method comprising the step of administering the pharmaceutical composition comprising the antibody and pharmaceutically acceptable carrier in addition to a subject having cancer or suspected of having cancer. The pharmaceutically acceptable carrier is the same as described above. The method for treating cancer may preferably be a method comprising the step of administering the composition comprising the antibody to a subject having cancer.
The type of subject includes mammals and birds such as cows, pigs, sheep, chickens, dogs, and human, and is not limited as long as cancer in the subject can be treated by administration of the composition of the present invention.
The composition may be administered in single or multiple doses in a pharmaceutically effective amount. In this regard, the composition may be administered in a form of solutions, powders, aerosols, capsules, enteric-coated tablets or capsules or suppositories. The routes for administration include intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary and intrarectal administration, but are not limited thereto. However, since peptides are digestible when orally administered, the active ingredients of a composition for oral administration need to be coated or formulated such that they can be protected against degradation in stomach. In addition, a pharmaceutical composition may be administered through a certain apparatus capable of transporting the active ingredients into a target cell.
The pharmaceutical composition of the present invention comprises an antibody that specifically binds to the epitope of SEQ ID No. 2. As described above, the antibody of the present invention efficiently converts regulatory T cells to IFN-γ-producing TH1 cells, and thus it can be effectively used for treatment of cancer. Therefore, when the pharmaceutical composition comprising the antibody is administered into the body, the onset, proliferation, or metastasis of cancer can be suppressed or prevented from progressing, thereby treating cancer.
As another aspect, the present invention provides a method for enhancing immunity, comprising administering the antibody to a subject in need thereof.
The antibody, subject, and administration are the same as described above. The antibody of the present invention recognizing the epitope of SEQ ID No. 2 converts Treg cell, which suppresses immune response, to TH1 cell which induces immune response by secreting IFN-γ or IL-2, and thus by administering the antibodies to a subject, the immune response can be enhanced. Also, in one example of the present invention, it was observed that cancer patients had 3 times higher proportion of Treg cells compared to normal subjects (Experimental Example 8 and FIGS. 13 and 14), and thus if the antibody is administered to a subject having cancer or suspected of having cancer, the immune response can be enhanced even more effectively.
As another aspect, the present invention provides a composition for enhancing immunity, comprising the antibody.
The composition may be in a form of a pharmaceutical composition or a food composition, but is not limited thereto. The pharmaceutical composition is the same as described above.
The food composition comprises the components that are conventionally included in the preparation of food product, for example, protein, carbohydrate, fat, nutrient, seasoning, and flavoring. Examples of such carbohydrate include conventional sugars such as monosaccharide (e.g. glucose and fructose), disaccharide (e.g. maltose, sucrose, oligosaccharide) and polysaccharide (e.g. dextrin, cyclodextrin) and sugar alcohol such as xylitol, sorbitol, and erythritol. As for flavoring, a natural flavoring (e.g. stevia extract such as thaumatin, rebaudioside A, and glycyrrhizin) and a synthetic flavoring (e.g. saccharine and aspartame) may be used.
[Mode for Invention]
Hereinafter, the present invention is described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.
Example 1
Cell Isolation
Peripheral venous blood samples newly collected from healthy individuals and cancer patients were treated with heparin and peripheral-blood mononuclear cells (PBMCs) were isolated by Ficoll-paque gradient centrifugation (GE Healthcare, Piscataway, N.J.). The isolated PBMCs were resuspended in RPMI 1640 medium containing 1% FBS for 2 days. Subsequently, naive CD4+ T cell subsets were collected through positive selection using anti-CD4+ antibodies (Miltenyi Biotec, Gladbach, Germany). The purity of the selected CD4+ T cell fractions was approximately 97%.
In order to obtain high purity of CD4+CD25high T cells, the isolated CD4+ T cells were stimulated with 0.1 μg/ml anti-CD3 antibodies (HIT3a; BD PharMingen, San Diego, Calif.) for 7 days, and 100 U/ml IL-2 (Interleukin-2; PeproTech, Rocky Hill, N.J.) was added every 2 days. Subsequently, the cultured CD4+ T cells were stained with the PE-cy5-conjugated anti-CD25 antibody and FITC-conjugated anti-CD4 antibody. Finally, the CD4+CD25high T cells or CD4+CD25− T cells were isolated by using FACS Aria (BD Biosciences, San Jose, Calif.).
Example 2
Antibodies and Flow Cytometry
In the flow cytometry experiment, the present inventors used fluorochrome-conjugated antibodies against CD4, CD8, CD11c, IFN-γ, Foxp3, CTLA-4, CD62L, CD45RO, CD45RA (BD Bioscience), CD19, CD56, CD14, CD127, IL-4, IL-17A, GATA-3, T-bet, RORγt (ebioscience, San Diego, Calif.), and BDCA-2 (Miltenyi Biotec). Also, for anti-AITR antibody, biotinylated AITR-specific antibodies (A27, A35, A41, B32, and B62 clones) were used. Intracellular level of cytokines was measured after 5 hour-long pre-incubation with 50 ng/ml phorbol 12-myristate 13-acetate (PMA), 10 μg/ml ionomycin, and 10 μg/ml Brefeldin A (Sigma-Aldrich, St Louis. MO). FACSCalibur flow cytometer (BD Bioscience) was used for all of the flow cytometry analysis and WinMDI software (Ver. 2.9, Scripps. Institute) was used for data analysis.
Example 3
Preparation of Soluble AITR and AITR-Overexpressing Cell
Specific binding of human anti-AITR mAbs was measured by using Enzyme-linked immunosorbent assay (ELISA) or FACS. In order to generate a fusion protein of a water-soluble AITR-ECD (26th to 139th amino acids) and GST protein, a GST-tagged AITR-ECD expression vector (pGEX-6p-1/AITR-ECD) was constructed. First, AITR-coding sequence was amplified by PCR using a sense primer and antisense primer shown in Table 1 and using cDNA extracted from the activated CD4+ T cells as a PCR template.
Then the amplified AITR-coding sequence was restriction digested with BamHI/XhoI, and cloned into a vector pGEX-6p-1 to generate a GST-tagged AITR-ECD expression vector (pGEX-6p-1/AITR-ECD).









TABLE 1






Sequence (5′→3′)
SEQ ID No.







Forward
AAGCTTGGTCAGCGCCCCACCGGG
49


Primer




 


Reverse
CCGGCAGAGCCGCCTTAACTCGAG
50


Primer









The pGEX-6p-1/AITR-ECD was transformed into E. coli (BL21-DE3-pLyss) and the protein expression was induced by adding 0.20 mM IPTG. The recombinant AITR-GST proteins expressed in E. coli were purified by running them through a glutathione agarose 4B bead column.
For generating AITR-overexpressing cells, the gene fragment encoding AITR-transmembrane domain (TM)-ECD was inserted in-frame with CD5L sequence in a mammalian vector pcDNA3.1(+), i.e. pcDNA3.1(+)/CD5L-AITR-TM-ECD. Then pcDNA3.1(+)/Empty vector or pcDNA3.1(+)/CD5L-AITR-TM-ECD vector was transient transfected into Jurkat cells by electroporation. AITR expression on cell surface was detected by staining with biotin-conjugated anti-AITR mAb.
Example 4
Preparation of Anti-AITR Monoclonal Antibodies (Anti-AITR mAbs)
The pCANTAB5E phagemid vector comprising human Fab antibody cDNA library was transformed into TG1 E. coli cells. Subsequently, the transformants were cultured in 2×YT broth containing 100 μg/ml ampicillin and 50 μg/ml kanamycin, and then superinfected with M13K07 helper phage. After culturing the cells at 30° C. for 24 hours, the cell culture was centrifuged down to collect culture supernatant only. Then the supernatant was added with 3.3% (w/w) polyethylene glycol (Sigma) and 2.3% (w/w) NaCl to precipitate recombinant phage particles obtained. The precipitated phage particles were resuspended in 2×YT broth.
Biopanning was performed using the above-prepared suspension as a library in order to select the phage specific to a human AITR-GST fusion protein by phage display (1 to 4 times biopanning). Among the recombinant phage particles from the culture supernatant of each clone, a positive clone of human AITR-GST fusion protein was selected through ELISA.
In addition, to generate a bivalent form of antibody, Fab genes (VH and VL) obtained from phage displaying were prepared by PCR, and then cloned into a restriction enzyme site of the expression vector pDCMV-DHFR. As a result, an antibody gene and construct thereof of five different clones (A27, A35, A41, B62, and B32) were obtained. To generate a bivalent form of anti-human AITR mAbs, the construct of antibody gene was transiently transfected into HEK293 and CHO cells, and the protein products were purified from the culture supernatant by using a protein G column (GE Healthcare).
Example 5
Epitope Mapping of Anti-AITR Monoclonal Antibodies (Anti-AITR mAbs)
For identifying a precise epitope recognized by the anti-AITR mAbs of the present invention, twelve different fragments (R1 to R12) corresponding to AITR-ECD were prepared and cloned into a GST-vector, pGEX-6p-1 (FIG. 3A).
The pGEX-6p-1/R1-R12 was transformed into E. coli cell line, BL21-DE3-pLyss, and the protein expression was induced by adding 0.20 mM IPTG. The recombinant R1- GST to R12-GST fusion proteins expressed in E. coli were purified by running them through a glutathione agarose 4B bead column (Peptron). At this time, horseradish peroxidase-coupled anti-human IgG was diluted 10,000 times (Sigma Aldrich, St Louis, Mo., USA). Also, chemiluminescence was monitored by using SuperSignal West Pico Chemiluminescence Substrate (Pierce, Rockford, Ill.).
Example 6
Cell Division and Cytokine Analysis
In the present invention, CD4+ T cells were labeled with CFSE (Molecular Probes, Invitrogen). The proliferation of CFSE-labeled CD4+ T cells (5×105 cells/well) was stimulated by treating the cells with anti-CD3 antibody (0.1 g/ml)/IL-2 (100 U/ml) and 5 μg/ml anti-AITR mAb for 72 hr. After 72 hours of culturing, the cell-free supernatant was separated and analyzed for cytokine level by using Th1/Th2 cytokines bead array kit (BD Bioscience), human TGF-β1 Quantikine ELISA kit (R&D system, Minneapolis, Minn.), and human IL-17A ELISA kit (Abcam, Cambridge, UK).
Example 7
Analysis of Differentiated Effector in CD4+ T Cell
Purified CD4+ T cells (5×105 cells/well) were stimulated with 0.1 μg/ml anti-CD3 antibody and 100 U/ml IL-2 for 3 days. Then, the cultured cells were treated with 5 μg/ml anti-AITR mAbs or 0.1 μg/ml AITR ligand (AITRL) for another 7 days.
Subsequently, the differentiated effector T cell subsets were washed with 1×PBS, and then cultured with a fresh medium containing 5 μg/ml anti-AITR antibody recognizing different epitope for another 7 days while adding 100 U/ml IL-2 for every 2 days. Then the cultured cells were harvested and endogenous IFN-γ, IL-4 and IL-17A were stained for three-color flow cytometry analysis.
Example 8
Analysis of Interaction Between AITR and TRAFs
Purified CD4+ T cells (5×105 cells/well) were stimulated with 0.1 μg/ml anti-CD3 antibody and 100 U/ml IL-2 for 2 days. The cultured cells were treated with 5 μg/ml anti-AITR mAbs for 24 hr. Then the cells were lysed with 100 μl RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.25% Na-deoxycholate, 1 mM PMSF, protease inhibitors, and phosphatase inhibitors).
For immunoprecipitation, cell lysates were incubated with 20 μl of 1:1 slurry of protein G-sepharose for 1 hour. Then, the precipitate was washed with 1×PBS, and protein complexes were eluted. Western blot analysis of eluate was performed by using anti-TPRAFs (TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6) (Santa Cruz Biotechnology, Santa Cruz, Calif.) or anti-AITR mAbs and using horseradish peroxidase-conjugated goat anti-rabbit IgG or anti-human IgG as a secondary antibody.
In a separate experiment, the sorted CD4+CD25high T cells (5×105 cells/well) were stimulated with 5 μg/ml anti-AITR mAbs for 24 hr and the interaction between AITR and TRAF was analyzed by using the same method described above.
Example 9
Analysis of Phosphorylation of Endogenous Signaling Pathway Protein
Purified CD4+ T cells (1×106 cells/well) were stimulated with anti-CD3 antibody and IL-2 for 3 days. The cultured cells were treated with anti-AITR mAbs for 2, 6, 12 and 24 hours. Then the cells were harvested and the suspension thereof was prepared. Levels of phospho-STATs (STAT1, STAT2, STAT3, STAT4, STAT5, and STAT6) and master transcription factors (T-bet, GATA-3, RORγt and Foxp3) were analyzed by flow cytometry. The cells in the culture were lysed with RIPA buffer. Subsequently, total protein extract was resolved on 8% to 12% SDS-polyacrylamide gel electrophoresis and immunoblotted with antibodies against NFAT1/2, p-p38, p-ERK1/2, p-JNK1/2, and p-NF-κB (Cell Signaling, Danvers, Mass., USA). The same blot was re-probed with an anti-R-actin antibody as a control for protein loading.
In a separate experiment, the sorted CD4+CD25high T cells (5×105 cells/well) were stimulated with 5 μg/ml anti-AITR mAbs and phosphorylation of the proteins involved in intracellular signaling pathway was analyzed by the same method described above.
Example 10
Phenotype Conversion of Human CD4+CD25highFoxp3+ (Treg) Cell to Different Subset
Human Treg cells isolated from healthy subjects and cancer patients were sorted by the method described in Example 1. The cells were stimulated with 5 μg/ml anti-AITR mAbs for 7 days while adding 100 U/ml IL-2 every 2 days. Then the cultured cells were harvested and the expression of AITR, CTLA-4, CD62L, CD127, CD45RO and CD45RA on cell surface was confirmed. Also, endogenous IFN-γ, IL-4 and IL-17A and master transcription factors were stained and the cells were analyzed by flow cytometry. In addition, culture supernatants were collected and the expression of IFN-γ, IL-4, IL-17A, and TGF-β was confirmed.
In a separate experiment, the sorted CD4+CD25− (non-Treg) cells (5×106 cells/well) were stimulated with 5 μg/ml anti-AITR mAbs. Culture cells were stained for endogenous IFN-γ, IL-4 and IL-17A and then analyzed by three-color flow cytometry.
Example 11
NOD-SCID Mouse Engraftment and Anti-Tumor Activity
First, PBMCs were obtained from HLA-A*0201-type healthy donors using a method described in Example 1. Five- to seven-week old NOD-SCID mice (Jackson Laboratory, Barharbor, Me.) were grown under specific-pathogen-free (SPF) conditions in the animal facility of National Cancer center, Korea. All animal experiments were reviewed and approved by Animal Care and Use Committee of National Cancer center, and performed in accordance with the Guide for Care and Use of Laboratory Animals. Human mononuclear cells were purified by Ficoll-paque gradient centrifugation. Then within 3 hours of recovery, 50×106 human PBMCs contained in RPMI were intraperitoneally (i.p.) injected into NOD-SCID mice. The engrafted mice were analyzed after 4 to 5 weeks of injecting human PBMCs (HLA-A*0201 type).
To analyze the anti-tumor activity of anti-AITR mAbs, hu-PBL-SCID mice were subcutaneously (s.c.) injected with (HLA-A*02 type, 1×107 cells/mice) human lung carcinoma cell line H441. When a tumor size reached about 1 cm in diameter, anti-AITR mAbs were intraperitoneally injected three times a day (200 μg/i.p. injection). Human IgG was used as control. Tumor volume of mouse was measured at the indicated time in each group. Also, after 12 days of injecting antibody, the PBMCs, tumor cells, tumor-draining lymph nodes (DLN), and spleen cells were isolated from the mouse and stained for hCD4, hCD8, hFoxp3, hCD56, and mCD45, and then analyzed by flow cytometry.
Example 12
Statistical Analysis
All experimental data were analyzed with a statistical program called Prism 5.0 GraphPad (San Diego, Calif.). Student's t-test was performed to determine the statistical significance of the difference between test groups.
Experimental Example 1
Generation of Anti-AITR Monoclonal Antibodies
As described in Example 4, five Fab antibodies against AITR were selected from a human Fab antibody library and named as A27, A35, A41, B32, and B62. The antibody A27 comprises a heavy chain variable region of SEQ ID No. 5 and a light chain variable region of SEQ ID No. 9, and the antibody B32 comprises a heavy chain variable region of SEQ ID No. 15 and a light chain variable region of SEQ ID No. 19. The antibody A35 comprises a heavy chain variable region of SEQ ID No. 25 and a light chain variable region of SEQ ID No. 29, and the antibody B62 comprises a heavy chain variable region of SEQ ID No. 35 and a light chain variable region of SEQ ID No. 36. The antibody A41 comprises a heavy chain variable region of SEQ ID No. 39 and a light chain variable region of SEQ ID No. 43.
In addition, anti-AITR genes were generated by grafting VH and VL DNA sequences into a human IgG, backbone. Anti-AITR mAbs A27, A35, A41, B32, and B62, were produced by transient transfection of the antibody constructs into HEK293 cells (FIG. 1A). These antibodies showed similar affinity to AITR in the AITR-overexpressed Jurkat cells (FIG. 1B).
Experimental Example 2
Epitope Mapping of Anti-AITR Monoclonal Antibodies
In this experiment, epitopes recognized by five human anti-AITR mAbs selected in Experimental Example 1 were identified. To confirm the epitope, AITR-GST fusion proteins named as R1 to R12 were used (FIG. 3). Immunoblotting results of R0-GST to R12-GST proteins with anti-AITR mAbs demonstrated that the five antibodies were classified into 3 groups based on their recognition site of AITR, i.e., epitope.
A27 and B32 specifically bound to R1 and R2 sites; A35 and B62 specifically bound to R1 to R4 and R8 to R11; and A41 specifically bound to R1 to R6, R8, and R9 (FIG. 3B). As a result of investigating the epitope sites more precisely, it was confirmed that A27 and B32 specifically recognized the sequence of 56th to 65th amino acids (AA 56-65) of AITR-ECD; A35 and B62 specifically recognized the sequence of 41st to 50th amino acids (AA 41-50) of AITR-ECD; and A41 specifically recognized the sequence of 20th to 30th amino acids (AA 20-30) of AITR-ECD (FIG. 3C and Table 2). The epitope site recognized by A27 and B32 was named as SEQ ID No. 2; the epitope site recognized by A35 and B62 was named as SEQ ID No. 3, and the epitope site recognized by A41 was named as SEQ ID No. 4.









TABLE 2





Antibody
Epitope
SEQ ID No.







A27&B32
HCGDPCCTTC (AA 56-65)
2


 


A35&B62
ECCSEWDCMC (AA 41-50)
3


 


A41
GTDARCCRVHT (AA 20-30)
4









Experimental Example 3
Expression of AITR in Immune Cell
The expression of AITR was up-regulated after stimulation of PBMCs, but when PBMCs were not stimulated, mRNA of AITR was not detected. Also, the expression of AITR was significantly up-regulated after CD3 stimulation in T cells. Furthermore, the expression of AITR was analyzed in T cells, B cells, NK cells, and monocytes by flow cytometry. The results demonstrated that the human anti-AITR mAbs of the present invention mainly recognized AITR in the activated CD4 T cells which had nearly 40-fold higher expression of AITR. When the antibodies were used in CD8+ T cells, B cells, NK cells, and monocytes, neither of resting nor activated AITR was detected (FIG. 1C).
These results demonstrate that the anti-AITR mAbs of the present invention recognize rapidly up-regulated AITR expression in the activated CD4+ T cell population, through TCR-mediated activated signal.
Experimental Example 4
Co-Stimulation of Cell Division And Cytokine Production in Differentiated CD4+ T Cell Population by AITR Signaling
After identifying that AITR is expressed mainly in the activated CD4+ T cells (FIG. 1C), the role of AITR was investigated as a co-stimulatory molecule in proliferation and cytokine secretion.
Purified CFSE-labeled CD4+ T cells were stimulated with immobilized anti-CD3 and cultured in the presence of anti-AITR mAbs. Unlike when cultured with anti-CD3 only, when the cells were co-cultured with anti-AITR mAbs and anti-CD3, the proliferation of activated CD4+ T cells was significantly increased (FIG. 5).
Subsequently, the culture supernatant was analyzed to identify a type of cytokine secreted depending on the type of anti-AITR mAbs. The results demonstrated that the antibodies of the present invention recognizing different sites of AITR changed the level of different cytokines. To be specific, cell treatment with A27 increased the level of TH1 cytokines IL-2 and IFN-γ, whereas treatment with A35, unlike with A27, increased the level of TH17 cytokine IL-17A. On the other hand, cell treatment with A41 significantly increased the level of TH2 cytokines IL-2, IL-4, and IL-5 (FIG. 4A).
In addition, the markers of phenotypic CD4+ T cells for the expression of IFN-γ, IL-17A, and IL-4 in the activated CD4+ T cells were analyzed when the CD4+ T cells were treated with the anti-AITR mAbs or AITRL of the present invention. The results show that when treated with A27, the number of IFN-γ-producing CD4+ T cells was increased (90.2±0.50%), and when treated with A35, the number of IL-17A-producing CD4+ T cells was increased (37.8±0.11%). Likewise, A41 increased the number of IL-4-producing CD4+ T cells (91.5±6.21%). Furthermore, the ligand thereof, i.e. AITRL promoted the production of IFN-γ in TH1 CD4+ T cells (FIGS. 4B and 6).
Taken together, these results demonstrate that anti-AITR mAbs of the present invention can act as a co-stimulatory signal in CD4+ T cells and promote the cell differentiation to a specific TH cell and cytokine secretion. In particular, these results show that the antibody of the present invention can induce different effects depending on the recognition sites of AITR. For instance, as A27 and B32 antibodies of the present invention recognize the epitope of SEQ ID No. 2 among many different sites of AITR in CD4+ T cell, they can induce the secretion of IL-2 and IFN-γ, which are TH1 cell cytokines, among different cytokines.
Experimental Example 5
Interaction Between AITR-Family Proteins and TRAF-Family Proteins and Downstream Signal Transduction
Previous studies have reported that tumor necrosis factor receptor-associated factor (TRAF) proteins are involved in activation of NF-κB and that downstream signaling molecules such as ERK1/2, JNK1/2, and p38 can be activated by TNFR family members. Also, cytoplasmic domain of AITR contains acidic residues that are conserved in mouse or human and are involved in association of AITR with TRAF protein. AITR is known to interact with TRAF1, TRAF2, and TRAF3, but not with TRAF5 and TRAF6 (Kwon B et al., J Biol Chem 274, 6056-6061, 1999; Ha, H et al., Curr Protoc Immunol. Chapter 11: Unit11.9D, 2009). Hence, the present inventors investigated the type of TRAF protein that AITR interacts with when AITR is activated by the anti-AITR mAbs of the present invention in the activated CD4+ T cells.
AITR-TRAF complexes were immunoprecipitated with Protein G, from the lysate of activated CD4+ T cell by using anti-AITR mAbs and the immunoprecipitates were immunoblotted with anti-TRAF antibody and anti-AITR mAbs.
The results demonstrate that the antibodies of the present invention recognizing different sites of AITR recruit different types of TRAF protein to AITR (FIG. 7A). To be specific, A27 which recognizes the epitope of SEQ ID No. 2 of the present invention recruited TRAF1 and TRAF2 to AITR, whereas A35 which recognizes the epitope of SEQ ID No. 3 recruited TRAF6 to AITR. A41 which recognizes the epitope of SEQ ID No. 4 recruited TRAF3 and TRAF5 to AITR. These results suggest that depending on the site of AITR recognized by each antibody, different cytoplasmic signaling can be induced.
Furthermore, A27 antibody of the present invention that recruits TRAF1 and TRAF2 could form heterooligomers, which then activated p-JNK1/2 and p-NF-κB. On the other hand, a comparative antibody A35 which recruits TRAF6 activated p-p38 and p-NF-κB. A41 which recruits TRAF3 and TRAF5 activated p-ERK1/2 (FIGS. 7A, 7B, and 8).
T cells express NFAT1, NFAT2, and NFAT3, and NFAT proteins are essential for producing effector cytokine when TCR is activated in TH cells. NFAT proteins also play important roles in regulating TH cell differentiation. For this reason, the present inventors investigated whether NFAT1 or NFAT2 can be activated when CD4+ T cells are treated with anti-AITR mAbs of the present invention.
As a result, it was found that A27 recognizing the epitope of SEQ ID No. 2 and A35 recognizing the epitope of SEQ ID No. 3 of the present invention increased the number of activated NFAT1 in a time-dependent manner. Unlike A27 and A35, a comparative antibody A41 increased the expression of NFAT2, but not NFAT1 (FIG. 7B).
These results suggest that AITR contains a binding domain for TRAFs or adaptor cytoplasmic molecules that can activate downstream signaling. Also, the results demonstrate that depending on the site of AITR recognized by anti-AITR mAbs, different types of TRAF proteins can be recruited, which then activates different cytoplasmic signaling pathways. Moreover, the AITR antibodies specific to the epitope of SEQ ID No. 2 of the present invention not only activate specific TRAF proteins, but also show specific NFAT activities. This result suggests that depending on the site of AITR recognized by AITR antibody, different NFAT translocates into the nucleus, which then induces different NFAT-dependent gene expression.
These results support that particularly A27 and B32 of the present invention recognize the epitope of SEQ ID No. 2 among many sites of AITR and recruit TRAF1 and TRAF2 among many different types of TRAFs, which then induce p-JNK1/2- and p-NF-kB-mediated cytoplasmic signaling pathways, thereby converting the cell into IFN-γ-producing cell through NFAT1, wherein IFN-γ is a cytokine of TH1 cell.
Experimental Example 6
Transcription Factors and STATs Protein Activated Differentiation of CD4+ T Cells with Anti-AITR mAbs
Previous studies have reported that signaling transducer and activation of transcription (STAT) proteins and master transcription factors are essential in fate determination of TH cell and cytokine production (Hermann-Kleiter, N. & Baier G, Blood. 15, 2989-2997, 2010). Hence, the present inventors investigated the activities of STAT proteins and master transcription factors in the CD4+ T cells activated by anti-AITR mAbs of the present invention.
A flow cytometric analysis was performed using the antibodies against p-STATs (p-STAT-1, p-STAT-3, p-STAT-4, p-STAT-5, and p-STAT-6) and T-bet, GATA-3, and RORγt. As a result, compared to the group treated with anti-CD4 and IL-2 only without anti-AITR antibody, the group treated with A27 showed increased number of activated p-STAT-1 (52.8±0.25%), p-STAT-4 (54.2±0.19%), and T-bet (80.2±0.08%). The group treated with A35 showed increased number of activated p-STAT-3 (63.1±1.24%) and RORγt (53.4±2.18%), and the group treated with A41 showed increased number of activated p-STAT-5 (85.1±0.38%), p-STAT-6 (79.2±0.12%), and GATA-3 (76.5±0.11%) (FIGS. 7C, 7D, and 9).
These results suggest that the anti-AITR mAbs of the present invention recognizing different sites of AITR can induce different signaling cascades mediated by AITR in CD4+ T cells. Especially, these results support that the A27 and B32 antibodies of the present invention recognizing the epitope of SEQ ID No. 2 mediates the cell fate determination of TH cell via a signaling pathway mediated by STAT-1, STAT-4, and T-bet.
Furthermore, these results suggest that the antibody of the present invention recognizing the epitope of SEQ ID No. 2 can induce cell conversion to TH1 cell which is regulated by STAT-1 signaling and T-bet transcription factor.
Experimental Example 7
Conversion of Treg Cell to TH1 Cell by Anti-AITR mAbs
Experimental Examples 5 and 6 showed that the anti-AITR mAbs of the present invention specifically binding to the epitope of SEQ ID No. 2 regulate a specific signal transduction factor involved in TH cell fate determination in CD4+ T cells. That is, according to the epitope sites within AITR, T cell can be converted to a totally different type of TH cell. Hence, the present inventors hypothesized that by targeting different epitope sites recognized by anti-AITR mAbs of the present invention, functional phenotype of CD4+ T cells can be converted. To confirm this, CD4+ T cells were treated with the representative antibodies A27, A35, and A41 which recognize three different sites of AITR to determine the converting activity thereof.
The results showed that a comparative antibody A35 converted IFN-γ-producing cells (99.2±0.21%) that were initially induced by A27, an antibody specific to the epitope of SEQ ID No. 2 of the present invention, to IL-17A-producing cells (18.8±0.23%). Also, a comparative antibody A41 converted IFN-γ-producing cells (99.2±0.21%) to IL-4-producing cells (38.7±0.32%). In addition, A35-induced IL-17A-producing cells (35.2±0.60%) were converted to IFN-γ-producing cells (82.6±1.21%) by A27 antibody of the present invention, and converted to IL-4-producing cells (70.5±0.62%) by a comparative antibody A41. A41-induced IL-4-producing cells (98.2±0.29%) were converted to IFN-γ-producing cells (90.1±0.42%) by A27 antibody of the present invention, and converted to IL-17A-producing cells (16.9±0.58%) by a comparative antibody A35 (FIGS. 10 and 11).
These results demonstrate that the antibody of the present invention that is specific to the AITR epitope of SEQ ID No. 2 can convert the T cell to a specific cytokine-producing cell by recognizing the specific site of AITR, i.e. epitope of SEQ ID No. 2 (AA 56-65). Furthermore, these results support that the A27 and B32 of the present invention recognizing the epitope of SEQ ID No. 2 can convert the T cell specifically to IFN-γ-producing TH1 cell, unlike the comparative groups such as A35 which induces cell conversion to IL-17A-producing TH17 cell and A41 which induces cell conversion to IL-4-producing TH2 cell.
Experimental Example 8
Converting Activity of Anti-AITR Antibody in Conversion of CD4+CD25highFoxp3+ (Treg) Cell
It is known that GITR or AITR acts as a co-stimulatory molecule in Treg cell, and the expression thereof remains high intrinsically in Treg cell even without activation. Hence, the present inventors investigated the role of AITR in Treg cells when stimulated by anti-AITR mAbs.
First, after stimulation with anti-CD3 and IL-2, Treg cells (CD4+CD25high cells) and non-Treg cells (CD4+CD25− cells) were isolated from healthy subjects and cancer patients by FACS-sorter. Both of Treg cells isolated from healthy subjects and cancer patients showed constitutive expression of Foxp3 and AITR. Non-Treg cells showed a lower expression of AITR than in Treg cells. The number of Treg cells isolated from cancer patients was three times higher than that from healthy subjects (FIGS. 13 and 14). Based on the results from the experimental example, it was determined whether the anti-AITR mAbs of the present invention can convert Treg cells.
The results showed that A27 recognizing the epitope of SEQ ID No. 2 of the present invention converted both of the Treg cells isolated from healthy subjects and cancer patients to IFN-γ-producing cells (93.9±0.12% and 88.2±0.43% respectively). On the other hand, a comparative group A35 recognizing the epitope of SEQ ID No. 3 converted Treg cells from healthy subjects to IL-17A-producing cells (10.4±0.32%), but not the Treg cells from cancer patients. Also, another comparative group A41 recognizing the epitope of SEQ ID No. 4 could not convert the Treg cells from both healthy subjects and cancer patients (FIGS. 12A, 12D, 13B, and 14B).
In addition, when the secreted cytokines were analyzed, A27 and A35 decreased TGF-1 secretion, but increased IFN-γ and IL-17A secretion. On the other hand, a comparative group A41 increased TGF-R secretion in a time-dependent manner (FIG. 12B). Furthermore, as a result of identifying the mechanism behind signal transduction, it was observed that, as shown in Experimental Example 5, A27 recruited TRAF1 and TRAF2 and increased the expression of NFAT1, thereby activating p-JNK1/2 and p-NF-kB signaling pathways. A35 recruited TRAF6 and activated p-p38 and p-NF-kB signaling pathways (FIG. 15). These results demonstrate that A27 and A35 reduce the expression of Foxp3 but increase the expression of T-bet or RORγt through the above signaling pathways. Through these mechanisms, A27 can convert Treg cell to TH1 cell and A35 can convert Treg cell to TH17 cell (FIGS. 9, 12C, and 16). However, A41 did not change the expression level of Foxp3 in Treg cell (FIGS. 9, 12C, and 16). Based on these, it was determined that when A41 recruits TRAF protein to AITR, it recruits TRAF3 more strongly than TRAF5, and activates ERK1/2, NF-kB, and STAT5, thereby inducing different signaling cascade in Treg cells from A27 or A35 through NFAT1 transcription factor, etc (FIGS. 9 and 15).
These results support that the antibody of the present invention recognizing the epitope of SEQ ID No. 2 can differentiate the T cell specifically into TH1 cell, through different signaling pathway from those of other epitopes.
Experimental Example 9
Anti-Tumor Activity of Anti-AITR mAbs in Human Tumor Xenograft Model
To examine the anti-tumor activity of anti-AITR mAbs of the present invention in vivo, Hu-PBL-SCID mouse model was used. Human PBMCs (HLA-A*0201 type) isolated from healthy subjects were intraperitoneally injected into NOD-SCID mouse. Between 28th day and 35th day after injection, the spleen cells and blood cells isolated from the Hu-PBL-SCID mice were analyzed and human immune cells were observed in the gated mouse CD45 negative population (FIGS. 17A and 18). These mice (eight or five subjects per each group) were challenged s.c. with 1×107 cells/mouse of human lung tumor cell line H441 (HLA-A*02 type). Once the tumor size reached about 1 cm in diameter, the tumor-induced mouse was administered intraperitoneally with an anti-AITR mAb of the present invention A27, or a comparative group A35 or A41 three times a day and a dose was 200 μg per injection.
As a result, A27 significantly suppressed the growth of tumor compared to the comparative groups, i.e. A35- and A41-treated groups, demonstrating a high anti-tumor activity compared to other groups. On the other hand, the anti-AITR antibodies A35 and A41 which recognize different epitope from A27 could not suppress the growth of tumor (A35), or showed significantly lower rate of suppressing tumor growth than A27 (A41) (FIG. 17).
In addition, after 12 days of injecting anti-AITR mAbs, A27 significantly reduced the number of intra-tumor Treg cells (CD4+Foxp3+) compared to CD4+TIL (A27:2%, CD4+TIL: 27%). On the other hand, A27 significantly increased the number of CD4+IFN-γ+ cells, CD8+CD56− cells, and CD8+CD56− cells (FIGS. 17 to 19). These results support that A27 of the present invention recognizes AA 56-65 site of AITR-ECD in Treg cells and effectively converts Treg cell to CD4+IFN-γ+ cell, thereby inducing anti-tumor activity.
Overall, these results demonstrate that among many anti-AITR antibodies, only when the antibody of the present invention recognizing the epitope of SEQ ID No. 2 in AITR is used, the anti-tumor activity can be induced. Therefore, the experimental results suggest that when the antibody recognizing the epitope of SEQ ID No. 2 is used, Treg cell can be converted to IFN-γ-producing cell, thereby inducing anti-tumor activity. Based on the above description, it should be understood by those skilled in the art that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention without departing from the technical idea or essential features of the invention as defined in the following claims. In this regard, the above-described examples are for illustrative purposes only, and the invention is not intended to be limited by these examples. The scope of the present invention should be understood to include all of the modifications or modified form derived from the meaning and scope of the following claims or its equivalent concepts.

## Claims

1. An antibody specifically recognizing on epitope of activation-inducible tumor necrosis factor receptor (AITR), wherein the epitope is represented by SEQ ID No. 2 and wherein the antibody comprises a heavy chain variable region comprising heavy chain complementarity-determining region (CDR) 1 represented by SEQ ID No. 6; heavy chain CDR2 represented by SEQ ID No. 7; and heavy chain CDR3 represented by SEQ ID No. 8, and a light chain variable region comprising light chain CDR1 represented by SEQ ID No. 10; light chain CDR2 represented by SEQ ID No. 11; and light chain CDR3 represented by SEQ ID No. 12.

2. The antibody of claim 1, which comprises an amino acid sequence of heavy chain variable region represented by SEQ ID No. 5 and an amino acid sequence of light chain variable region represented by SEQ ID No. 9.

3. An antibody specifically recognizing an epitope of activation-inducible tumor necrosis factor receptor (AITR), wherein the epitope is represented by SEQ ID No. 2 and wherein the antibody comprises a heavy chain variable region comprising heavy chain complementarity-determining region (CDR)1 represented by SEQ ID No. 16; heavy chain CDR2 represented by SEQ ID No. 17; and heavy chain CDR3 represented by SEQ ID No. 18, and a light chain variable region comprising light chain CDR1 represented by SEQ ID No. 20; light chain CDR2 represented by SEQ ID No. 21; and light chain CDR3 represented by 22.

4. The antibody of claim 3, which comprises an amino acid sequence of heavy chain variable region represented by SEQ ID No. 15 and an amino acid sequence of light chain variable region represented by SEQ ID No. 19.

5. A polynucleotide encoding the antibody of claim 1.

6. An expression vector comprising the polynucleotide of claim 5.

7. A transformant introduced with the vector of claim 6.

8. A method for treating cancer, comprising administering the antibody of claim 1 to a subject in need thereof.