JP5552630B2 - Medicament for treating or preventing HTLV-I associated myelopathy, and method for testing the efficacy of antibody therapy for patients with HTLV-I associated myelopathy - Google Patents

Description

The present invention relates to a pharmaceutical agent for treating or preventing HTLV-I-associated myelopathy, and a method for testing the effect of antibody therapy on patients with HTLV-I-associated myelopathy.

Currently, there are approximately 1.2 million people in Japan infected with human T-cell leukemia virus type-1 (hereafter abbreviated as "HTLV-I"). Some of these infected people develop HTLV-I associated myelopathy (hereafter abbreviated as "HAM"), and another portion of these infected people develop adult T-cell leukemia (hereafter abbreviated as "ATL").

Because the number of people affected by HAM is very small, research into elucidating the cause and developing treatments has been slow to progress. Currently, there is no established effective treatment for HAM, and there is a strong desire for a treatment to be developed as soon as possible.

The pathology of HAM is characterized by a very large number of HTLV-I-infected cells in peripheral blood and cerebrospinal fluid, which induces an excessive immune response in proportion to the amount of HTLV-I-infected cells, resulting in an abnormal increase in HTLV-I-specific cytotoxic T lymphocytes. In spinal lesions of HAM, no viral infection of nerve tissue is observed, but cellular infiltration of HTLV-I-infected CD4 ⁺ T cells and surrounding CD8 ⁺ cytotoxic T lymphocytes, as well as excessive production of inflammatory cytokines, are observed. Based on these findings, the pathology of HAM is considered to be nerve tissue damage caused by an excessive immune response due to infected cells.

The treatment strategies for HAM based on these pathological conditions include (1) elimination of HTLV-I-infected cells, (2) calming of the excessive immune response (spinal inflammation), and (3) regeneration of the damaged spinal cord. To cure HAM, spinal cord regeneration therapy is ultimately necessary. However, in order to make regenerative therapy successful, it is essential to first achieve (1) and (2). To date, steroids and interferon have been used to treat HAM in order to (2) calm the excessive immune response. However, these treatment methods have serious problems in that while they are effective in the short term, they have side effects and weakened therapeutic effects in the long term, the disease progresses even if treatment is continued, and they are poorly effective in reducing infected cells, which are the main cause of the disease. Furthermore, because HTLV-I has a low expression level of viral genes, the therapeutic effects of reverse transcriptase inhibitors or protease inhibitors are also poor. As a treatment to solve these problems, (1) antibody therapy that specifically attacks and kills HTLV-I-infected cells in order to eliminate them is considered.

The present inventor has been conducting research focusing on HTLV-I-infected cells. As a result, it was found that in HAM patients, HTLV-I selectively infects CD4 ⁺ CD25 ⁺ T cells, and furthermore, these infected cells excessively stimulate the immune response (see Non-Patent Document 1). It is known that CD4 ⁺ CD25 ⁺ T cells contain regulatory T cells that suppress the onset of autoimmune diseases. As a result of further research, the present inventor found that the quantity and function of regulatory T cells are decreased in CD4 ⁺ CD25 ⁺ T cells of HAM patients, and that the expression of the tax gene of HTLV-I suppresses the expression of FoxP3, a marker transcription factor of regulatory T cells (see Non-Patent Document 2).

On the other hand, in ATL caused by the same HTLV-I, the acquired immune system surrounding HTLV-I-infected cells is completely different from that of HAM. It has been reported that the number of HTLV-I-specific CD8 ⁺ cytotoxic T lymphocytes in peripheral blood is very low in ATL. It has also been reported that HTLV-I selectively infects CD4 ⁺ CD25 ⁺ T cells in ATL patients, but most of the HTLV-I-infected cells (CD4 ⁺ CD25 ⁺ ) in ATL patients are FoxP3 positive. From these findings, it is considered that in ATL, HTLV-I-infected CD4 ⁺ CD25 ⁺ regulatory T cells proliferate in a neoplastic manner and exert their original regulatory function, causing a low immune response state in the host.

Based on the above, it is believed that the differences in HTLV-I-infected cells determine the phenotypes of two completely different diseases caused by the same virus: autoimmune disease (HAM) and leukemia (ATL).

In recent years, it has been reported that CC chemokine receptor 4 (hereinafter abbreviated as "CCR4") is expressed in HTLV-I-infected cells of ATL patients. Accordingly, anti-CCR4 antibodies that can be used in antibody therapy for ATL have been developed (Patent Documents 1 to 4). Currently, phase I clinical trials using humanized anti-CCR4 antibodies targeting ATL are being conducted.

CCR4 is known to be expressed in T helper type 2 (hereinafter abbreviated as "Th2") cells and regulatory T cells in healthy individuals. Based on this, it was predicted that HTLV-I-infected cells would not express CCR4 in HAM patients, who are characterized by a decrease in regulatory T cells.
International Publication No. 01/64754 International Publication No. 03/018635 International Publication No. 2005/035582 International Publication No. 2005/053741 Yamano Y., Cohen CJ, Takenouchi N., YaoK., Tomaru U., Li HC, Reiter Y., Jacobson S., "Increased Expression of Human T Lymphocyte Virus Type I (HTLV-I) Tax11-19 Peptide-Human Histocompatibility Leukocyte Antigen A*201 Complexes on CD4+ CD25+ T Cells Detected by Peptide-specific, Major HistocompatibilityComplex-restricted Antibodies in Patients with HTLV-I-associated Neurologic Disease", J Exp Med., 199(10), 1367-1377, 2004. Yamano Y., Takenouchi N., Li HC, TomaruU., Yao K., Grant CW, MaricD. A., Jacobson S., "Virus-induced dysfunction of CD4+CD25+ T cells in patients with HTLV-I-associated neuroimmunologicaldisease", J Clin Invest., 115(5), 1361-1368, 2005.

As mentioned above, in order to effectively treat HAM, it is necessary to develop an antibody therapy that can eliminate HTLV-I-infected cells, but no medicine that can be used for antibody therapy against HAM has yet been developed.

The present invention has been made in consideration of these points, and aims to provide a pharmaceutical agent that can be used in antibody therapy for HAM, and a method for predicting the efficacy of such antibody therapy.

The inventors conducted extensive research and surprisingly found that HTLV-I-infected cells (pathogenic cells) in HAM patients express CCR4, and further investigation led to the completion of the present invention.

That is, a first aspect of the present invention relates to the following medicine.
[1] A pharmaceutical agent for treating or preventing HTLV-I-associated myelopathy, comprising an anti-human CCR4 antibody that specifically binds to human CCR4 or a fragment of the antibody.
[2] The pharmaceutical according to [1], wherein the antibody specifically binds to the extracellular domain of human CCR4.

The second aspect of the present invention relates to the following prediction method.
[3] A method for predicting the effectiveness of antibody therapy using an anti-human CCR4 antibody or a fragment of said antibody for a patient with HTLV-I-associated myelopathy, comprising the steps of: preparing peripheral blood mononuclear cells collected from the patient; preparing a cell population by removing cells expressing human CCR4 from the peripheral blood mononuclear cells; measuring the proliferation activity of the peripheral blood mononuclear cells and the proliferation activity of the cell population; and predicting the effectiveness of the antibody therapy using the proliferation activity of the peripheral blood mononuclear cells and the proliferation activity of the cell population as indicators.
[4] The prediction method described in [3], which predicts that the antibody therapy will be effective for the patient when the proliferation activity of the cell group is lower than the proliferation activity of the peripheral blood mononuclear cells.

The medicine of the present invention can efficiently eliminate HTLV-I infected cells (pathogenic cells) in HAM patients, and therefore can effectively treat HAM. In addition, the medicine of the present invention can reduce the number of HTLV-I infected cells in the bodies of HTLV-I infected but asymptomatic individuals, and therefore can prevent the onset of HTLV-I related diseases such as HAM.

Furthermore, according to the prediction method of the present invention, it is possible to predict whether antibody therapy will be effective for a patient before antibody therapy is administered, so that antibody therapy can be administered only to patients for whom antibody therapy is effective.

1. Medicament of the Present Invention The medicament of the present invention is a medicament for treating HAM or preventing the onset of HAM, and is characterized in that it comprises an anti-human CCR4 antibody or a fragment thereof that specifically binds to human CCR4.

The present inventors have examined whether various cell surface molecules can be markers for HTLV-I-infected cells (pathogenic cells) in HAM patients, and have surprisingly found that CCR4 can be a useful marker for infected cells. As shown in the Examples (described later), the HTLV-I infection rate of CCR4-positive (CCR4 ⁺ ) cells in peripheral blood mononuclear cells (hereinafter abbreviated as "PBMCs") of HAM patients is extremely higher than the HTLV-I infection rate of CCR4-negative (CCR4 ^- ) cells. Based on this, the present inventors considered that antibody therapy using anti-CCR4 antibodies could be a treatment for HAM.

PBMCs from healthy individuals rarely proliferate spontaneously in vitro without stimulation, but PBMCs from HAM patients are known to proliferate spontaneously in vitro without stimulation. This spontaneous proliferation is thought to reflect the proliferation of HTLV-I-infected cells (pathogenic cells) and the excessive immune response caused by HTLV-I-infected cells. The present inventors have demonstrated that the spontaneous proliferation in vitro is suppressed when CCR4 ⁺ cells are removed from PBMCs from HAM patients using an anti-human CCR4 antibody (see Examples). Based on this, the present inventors have determined that antibody therapy using an anti-CCR4 antibody can be a treatment for HAM.

The most important point in determining whether antibody therapy using anti-CCR4 antibodies can be used to treat HAM is whether it causes side effects. To address this issue, it is necessary to clarify the role of CCR4 ⁺ cells in the pathology of HAM.

The HTLV-I infection rate of CD4 ⁺ CD25 ⁺ CCR4 ⁺ T cells of HAM patients is extremely high (see Examples). As mentioned above, it has been reported that CCR4 is expressed in Th2 cells and regulatory T cells in healthy individuals. The present inventors investigated the expression of FoxP3 (a marker molecule of regulatory T cells) in CD4 ⁺ CD25 ⁺ CCR4 ⁺ T cells. As a result, strong expression of FoxP3 was observed in CD4 ⁺ CD25 ⁺ CCR4 ⁺ T cells of ATL patients, but almost no expression of FoxP3 was observed in CD4 ⁺ CD25 ⁺ CCR4 ⁺ T cells of HAM patients. From this, it is considered that the CD4 ⁺ CD25 ⁺ CCR4 ⁺ T cells of HAM patients hardly contain regulatory T cells.

The present inventors also investigated cytokines (interferon-γ, interleukin 2, interleukin 17, etc.) produced by CD4 ⁺ CD25 ⁺ CCR4 ⁺ T cells. As a result, production of interferon-γ, an inflammatory cytokine, was hardly observed in CD4 ⁺ CD25 ⁺ CCR4 ⁺ T cells of ATL patients, but excessive production of interferon-γ was observed in CD4 ⁺ CD25 ⁺ CCR4 ⁺ T cells of HAM patients. This suggests that CD4 ⁺ CD25 ⁺ CCR4 ⁺ T cells of HAM patients do not exhibit the functions of suppressive T cells such as Th2 cells and regulatory T cells, and are rather dysfunctional.

Based on the above, it is predicted that removal of dysfunctional CCR4 ⁺ cells from HAM patients will be less harmful, and antibody therapy using anti-CCR4 antibodies is currently unlikely to have significant side effects.

The anti-human CCR4 antibody or fragment thereof contained in the pharmaceutical of the present invention is not particularly limited as long as it is an antibody or fragment thereof that can specifically bind to cells expressing human CCR4 (hereinafter referred to as "human CCR4-expressing cells"), but an antibody or fragment thereof that has cytotoxic activity against human CCR4-expressing cells is preferred. This is because the cytotoxic activity of the antibody can remove human CCR4-expressing cells, including the causative cells of HAM. Furthermore, from the viewpoint of specifically binding to human CCR4-expressing cells, the anti-human CCR4 antibody or fragment thereof contained in the pharmaceutical of the present invention is preferably an antibody that can specifically bind to the extracellular domain of human CCR4.

Examples of human CCR4-expressing cells include CD4 ⁺ CD25 ⁺ CCR4 ⁺ T cells. Examples of cytotoxic activity include complement-dependent cytotoxicity (hereinafter abbreviated as "CDC") and antibody-dependent cell-mediated cytotoxicity (hereinafter abbreviated as "ADCC").

Examples of anti-human CCR4 antibodies included in the pharmaceutical of the present invention include human chimeric antibodies, human CDR-grafted antibodies, and human antibodies.

A human chimeric antibody is an antibody consisting of a heavy chain variable region (hereinafter abbreviated as "VH") and a light chain variable region (hereinafter abbreviated as "VL") of an antibody of an animal other than human, and a heavy chain constant region (hereinafter abbreviated as "CH") and a light chain constant region (hereinafter abbreviated as "CL") of a human antibody. The type of animal for the variable region is not particularly limited as long as it is an animal capable of producing a hybridoma, such as a mouse, rat, hamster, or rabbit. A human chimeric antibody can be produced by obtaining cDNA encoding the VH and VL of an antibody of an animal other than human that specifically binds to human CCR4, inserting them into an expression vector having genes encoding the CH and CL of a human antibody, constructing a human chimeric antibody expression vector, and introducing the cDNA into an animal cell for expression. The CH of a human chimeric antibody is not particularly limited as long as it is a human immunoglobulin (hereinafter abbreviated as "hIg"), but is preferably of the hIgG class. The CL of a human chimeric antibody is not particularly limited as long as it belongs to the hIg class.

A human CDR-grafted antibody is an antibody in which the complementarity determining regions (hereinafter abbreviated as "CDR") of the VH and VL of an antibody of an animal other than human are grafted to appropriate positions of the VH and VL of a human antibody. A human CDR-grafted antibody can be produced by constructing a cDNA encoding a V region in which the CDRs of the VH and VL of an antibody of an animal other than human that specifically binds to CCR4 are grafted onto the framework (hereinafter abbreviated as "FR") of the VH and VL of any human antibody, inserting the cDNA into an expression vector having DNA encoding the CH and CL of a human antibody to construct a human CDR-grafted antibody expression vector, and introducing the vector into an animal cell for expression. The amino acid sequences of the FRs of the VH and VL of a human antibody are not particularly limited as long as they are amino acid sequences derived from a human antibody. The CH of a human CDR-grafted antibody is not particularly limited as long as it is a hIg, but is preferably of the hIgG class. The CL of a human CDR-grafted antibody is not particularly limited as long as it belongs to the hIg class.

Examples of the anti-human CCR4 antibody fragment contained in the pharmaceutical of the present invention include the above-mentioned antibody fragments. The type of the antibody fragment is not particularly limited, and examples thereof include Fab, Fab', F(ab') ₂ , scFv, diabody, dsFv, and peptides containing CDR.

Fab is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity, obtained by treating IgG with papain (a protease). Fab of an anti-human CCR4 antibody can be produced by treating the anti-human CCR4 antibody with papain, or by inserting DNA encoding the Fab of the antibody into an expression vector and introducing the vector into a prokaryote or eukaryote for expression.

F(ab') ₂ is an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity, among fragments obtained by treating IgG with pepsin (a protease). F(ab') ₂ of an anti-human CCR4 antibody can be prepared by treating an anti-human CCR4 antibody with pepsin or by binding Fab' (described below) via a thioether bond or disulfide bond.

Fab' is an antibody fragment having an antigen-binding activity and a molecular weight of about 50,000, obtained by cleaving the disulfide bond in the hinge region of F(ab') _2. Fab' of an anti-human CCR4 antibody can be prepared by treating F(ab') ₂ of the anti-human CCR4 antibody with dithiothreitol, or by inserting DNA encoding the Fab' of the antibody into an expression vector and introducing the vector into a prokaryote or eukaryote for expression.

An scFv is an antibody fragment with antigen-binding activity in which one VH and one VL are linked using an appropriate peptide linker. An scFv of an anti-human CCR4 antibody can be produced by obtaining cDNA encoding the VH and VL of an anti-human CCR4 antibody, constructing DNA encoding the scFv, inserting this DNA into an expression vector, and introducing this expression vector into a prokaryote or eukaryote for expression.

A diabody is an antibody fragment formed by dimerization of scFv and has bivalent antigen-binding activity. An anti-human CCR4 antibody diabody can be produced by obtaining cDNA encoding the VH and VL of an anti-human CCR4 antibody, constructing DNA encoding the diabody, inserting this DNA into an expression vector, and introducing this expression vector into a prokaryote or eukaryote for expression.

A dsFv is an antibody fragment in which polypeptides in which one amino acid residue in each of VH and VL has been replaced with a cysteine residue are linked via a disulfide bond between the cysteine residues. A dsFv of an anti-human CCR4 antibody can be produced by obtaining cDNA encoding the VH and VL of an anti-human CCR4 antibody, constructing DNA encoding a dsFv, inserting this DNA into an expression vector, and introducing this expression vector into a prokaryote or eukaryote for expression.

A peptide containing a CDR is a peptide that contains at least one region of the CDR of VH or VL. A peptide containing a CDR of an anti-human CCR4 antibody can be produced by constructing DNA encoding the CDRs of the VH and VL of an anti-human CCR4 antibody, inserting this DNA into an expression vector, and introducing this expression vector into a prokaryote or eukaryote for expression. A peptide containing a CDR of an anti-human CCR4 antibody can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).

As described above, the anti-human CCR4 antibody or fragment thereof contained in the pharmaceutical of the present invention is preferably an antibody or fragment thereof having cytotoxic activity against human CCR4-expressing cells. Examples of antibodies having cytotoxic activity against human CCR4-expressing cells include anti-human CCR4 antibodies having an N-glycoside-linked complex glycan in the Fc region, in which fucose is not bound to the N-acetylglucosamine at the reducing end of the N-glycoside-linked complex glycan. Antibodies having N-glycoside-linked complex glycans in which fucose is not bound to the N-acetylglucosamine at the reducing end are known to have strong ADCC activity (see Patent Document 3). Furthermore, examples of antibody fragments having cytotoxic activity against human CCR4-expressing cells include fragments of anti-human CCR4 antibodies having an antibody Fc region that contains an N-glycoside-linked complex-type glycan, in which fucose is not bound to the N-acetylglucosamine at the reducing end of the N-glycoside-linked complex-type glycan.

The anti-human CCR4 antibody or a fragment thereof contained in the pharmaceutical of the present invention is, for example, the anti-human CCR4 antibody or a fragment thereof described in the above-mentioned Patent Documents 1 to 4.

The pharmaceutical agent containing the anti-human CCR4 antibody or a fragment thereof of the present invention may be administered alone as a therapeutic agent for HAM, but is preferably provided as a pharmaceutical formulation prepared by mixing it with one or more pharmacologically acceptable carriers and by any method well known in the technical field of pharmaceuticals.

There are no particular limitations on the administration route, and the most effective route for treating HAM can be selected as appropriate. Examples of administration routes include oral administration, and parenteral administration such as oral administration, airway administration, rectal administration, subcutaneous administration, intramuscular administration, and intravenous administration. Intravenous administration is usually preferred.

The dosage form is not particularly limited and can be appropriately selected depending on the route of administration, etc. Examples of dosage forms include injections, sprays, capsules, tablets, granules, syrups, suppositories, ointments, tapes, etc. Preparations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules, etc. Preparations suitable for parenteral administration include injections, suppositories, sprays, etc. In general, injections are preferred.

Injectables can be prepared using a carrier consisting of a salt solution, a glucose solution, or a mixture of the two. In addition, powdered injectables can be produced by lyophilizing an anti-human CCR4 antibody or a fragment thereof in accordance with a conventional method and adding sodium chloride thereto.

The dosage and frequency of administration will vary depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but the amount of active ingredient is usually around 10 μg/kg to 20 mg/kg per day for adults.

The pharmaceutical of the present invention can suppress excessive immune responses by removing HTLV-I-infected cells (pathogenic cells) in the bodies of HAM patients, and can therefore be used in antibody therapy using anti-CCR4 antibodies against HAM. In other words, the pharmaceutical of the present invention can be used as a therapeutic agent for HAM.

The pharmaceutical of the present invention can also be used as a preventive agent for HTLV-I-infected asymptomatic individuals. It is known that groups of HTLV-I-infected asymptomatic individuals with a high number of virus-infected cells are at high risk of developing HTLV-I-related diseases. Therefore, reducing the number of virus-infected cells is effective in preventing the onset of the disease. Since the pharmaceutical of the present invention can reduce the number of HTLV-I-infected cells in the body, it can also be used as a preventive agent for the onset of HTLV-I-related diseases such as HAM.

2. The prediction method of the present invention The prediction method of the present invention is a method for predicting the effect of antibody therapy using an anti-human CCR4 antibody or a fragment thereof on a HAM patient (subject).

The prediction method of the present invention includes: (1) a first step of preparing PBMCs collected from a HAM patient; (2) a second step of preparing a cell group by removing cells expressing human CCR4 from the PBMCs prepared in the first step; (3) a third step of measuring the proliferation activity of the PBMCs prepared in the first step and the proliferation activity of the cell group prepared in the second step; and (4) a fourth step of predicting the effect of the antibody therapy using the proliferation activity of the PBMCs and the proliferation activity of the cell group measured in the third step as indicators.

In the first step, PBMCs collected from a HAM patient are prepared. These PBMCs contain HTLV-I infected cells (pathogenic cells) (see Examples). There are no particular limitations on the method for preparing PBMCs. For example, peripheral blood mononuclear cells may be isolated from the patient's blood by a conventional method.

In the second step, cells expressing human CCR4 are removed from the PBMCs prepared in the first step to prepare a cell group. This process mimics the action of anti-CCR4 antibodies in antibody therapy.

As mentioned above, many HTLV-I-infected cells (pathogenic cells) express CCR4. Therefore, it is believed that this process can remove many HTLV-I-infected cells from PBMCs. The method for removing cells expressing CCR4 from PBMCs is not particularly limited, and may be appropriately selected from methods known to those skilled in the art, such as the magnetic bead method or column method using an anti-CCR4 antibody.

In the third step, the proliferation activity of the PBMCs prepared in the first step and the proliferation activity of the cell population prepared in the second step are measured.

The method for measuring the proliferation activity of cells is not particularly limited, and may be appropriately selected from methods known to those skilled in the art, such as the ^3H -thymidine incorporation method. In HTLV-I infected cells (pathogenic cells), the proliferation activity is highest on the 6th to 7th day after the start of culture. Therefore, in the prediction method of the present invention, it is preferable to measure the proliferation activity of cells on the 6th to 7th day after the start of culture. The method for culturing cells may be appropriately selected from methods known to those skilled in the art.

In the fourth step, the efficacy of the antibody therapy is predicted using the proliferation activity of the PBMCs and the proliferation activity of the cell population measured in the third step as indicators.

As mentioned above, peripheral blood mononuclear cells from healthy individuals rarely proliferate spontaneously in a test tube without stimulation, but peripheral blood mononuclear cells from HAM patients are known to proliferate spontaneously in a test tube even without stimulation. This spontaneous proliferation is thought to reflect the proliferation of HTLV-I-infected cells (pathogenic cells) and the excessive immune response caused by HTLV-I-infected cells. Therefore, although there are individual differences between patients, the proliferation activity of the PBMCs prepared in the first step is usually high. On the other hand, if this spontaneous proliferation is caused by HTLV-I-infected cells, the proliferation activity of the cell group prepared in the second step should be lower than that of the PBMCs prepared in the first step. Therefore, when the proliferation activity of the cell group prepared in the second step is lower than that of the peripheral blood mononuclear cells prepared in the first step, it is predicted that the antibody therapy is effective for the patient.

The prediction method of the present invention can predict whether antibody therapy will be effective for a patient before antibody therapy is administered.

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[Example 1]
In this example, in order to demonstrate that antibody therapy using anti-CCR4 antibody is applicable to HAM, it is shown that many of the HTLV-I-infected cells contained in PBMCs from HAM patients express CCR4.

1. Preparation of PBMCs and cell separation PBMCs were separated from the blood of HAM patients by Ficoll centrifugation density method. CD4 ⁺ CD25 ^- cells and CD4 ⁺ CD25 ⁺ cells were separated from the obtained PBMCs using a flow cytometer (FACSCalibur; Becton Dickinson). FITC-labeled mouse anti-human CD4 antibody (eBiosciences) was used as the anti-CD4 antibody. APC-labeled mouse anti-human CD25 antibody (Becton Dickinson) was used as the anti-CD25 antibody.

Figure 1 (A) is a two-parameter dot plot showing the expression patterns of CD4 and CD25 in PBMCs from a HAM patient. Cells in the area indicated by "B" were separated as CD4 ⁺ CD25 ^- cells, and cells in the area indicated by "C" were separated as CD4 ⁺ CD25 ⁺ cells.

Next, CCR4- and CCR4 ⁺ cells were separated from the CD4 ^{+ CD25-} cell population using a flow cytometer ⁽ JSAN; Bay Biosciences). Similarly, CCR4- and CCR4 ⁺ cells were separated from the CD4 ⁺ CD25 ⁺ cell population. As the anti-CCR4 antibody, a PE-Cy7-labeled mouse anti-human CCR4 antibody (1G1; Becton Dickinson) was used. As a result, four ^cell populations were obtained from the PBMCs of the HAM patient: (1) CD4 ⁺ CD25 ^{- CCR4-} , (2) CD4 ⁺ CD25 ^- CCR4 ⁺ , (3) CD4 ⁺ CD25 ^{+ CCR4-} , and (4) CD4 ⁺ CD25 ⁺ CCR4 ⁺ .

Fig. 1(B) is a histogram showing the expression pattern of CCR4 in the CD4 ^{+ CD25-} cell population (cells contained in the region indicated by "B" in Fig. 1(A)). In the figure, cells contained in the region to the left of the dashed line were separated as (1) CD4 ⁺ CD25 ^{- CCR4-} cells, and cells contained in the region to the right of the dashed line were separated as (2) CD4 ⁺ CD25 ^- CCR4 ⁺ cells.

Fig. 1(C) is a histogram showing the expression pattern of CCR4 in the CD4 ⁺ CD25 ⁺ cell population (cells contained in the region indicated by "C" in Fig. 1(A)). Cells contained in the region to the left of the dashed line in the figure were separated as (3) CD4 ⁺ CD25 ^{+ CCR4-} cells, and cells contained in the region to the right of the dashed line were separated as (4) CD4 ⁺ CD25 ⁺ CCR4 ⁺ cells.

2. Measurement of HTLV-I viral load For each of the cell populations (1) to (4) above, the number of provirus copies per 100 cells was measured by real-time PCR.

2 is a graph (n=8) showing the results of measuring the number of provirus copies in each cell population. As shown in this graph, the number of HTLV-I infecting (4) CD4 ⁺ CD25 ⁺ CCR4 ⁺ T cells was significantly higher than the number of HTLV-I infecting (3) CD4 ⁺ CD25 ⁺ CCR4 ^- T cells. In addition, the number of HTLV-I infecting (2) CD4 ⁺ CD25 ^- CCR4 ⁺ T cells was also significantly higher than the number of HTLV-I infecting (1) CD4 ⁺ CD25 ^- CCR4 ^- T cells.

These results indicate that CCR4 is expressed in HTLV-I-infected cells in PBMCs from HAM patients. This suggests that antibody therapy using anti-CCR4 antibodies may be applicable to HAM.

[Example 2]
In this example, to demonstrate the applicability of antibody therapy using anti-CCR4 antibody to HAM, we demonstrate that the number of HTLV-I-infected cells in PBMCs can be reduced by removing CCR4 ⁺ cells using anti-CCR4 antibody.

1. Preparation of PBMCs and cell separation PBMCs were separated from the blood of HAM patients by Ficoll centrifugation density method. CCR4 ⁺ cell populations and ^CCR4- cell populations were separated from the obtained PBMCs using a kit containing magnetic beads (Rat Anti-Mouse IgG1 MicroBeads; Miltenyi Biotec). Mouse anti-human CCR4 antibody (KM2160; Kyowa Hakko Kirin Co., Ltd.) was used as the anti-CCR4 antibody. Hereinafter, the cell population bound to the KM2160 antibody (CCR4 ⁺ cell population) is abbreviated as KM2160(+), and the cell population not bound to the KM2160 antibody (CCR4- ^cell population) is abbreviated as KM2160(-).

2. Cell Culture RPMI medium (containing 5% human serum, 1% penicillin, and 1% streptomycin) was dispensed into each well of a round-bottom 96-well microplate. 5 x ¹⁰⁴ KM2160(-) cells were seeded into the wells containing RPMI medium and cultured at 37°C and 5% _CO2 for 7 days. As a control, 5 x ¹⁰⁴ PBMCs were seeded into the wells containing RPMI medium and cultured at 37°C and 5% _CO2 for 7 days.

3. Measurement of HTLV-I viral load For KM2160(+) and KM2160(-), immediately after isolation from PBMCs, the proviral copy numbers per 100 cells were measured by real-time PCR.

In addition, the number of provirus copies per 100 cells was measured by real-time PCR for KM2160(-) and PBMCs after 7 days of culture.

Figure 3 is a graph showing the results of measuring the proviral copy numbers for KM2160(+) and KM2160(-) immediately after isolation from PBMCs. As shown in this graph, the number of HTLV-I infecting KM2160(-) was significantly lower than the number of HTLV-I infecting KM2160(+). This suggests that anti-human CCR4 antibodies bind to HTLV-I-infected cells with some degree of specificity, and that anti-human CCR4 antibodies can be used to reduce HTLV-I-infected cells in the body.

Figure 4 is a graph showing the results of measuring the number of proviral copies in KM2160(-) and PBMCs after culture. As shown in this graph, the number of HTLV-I infecting KM2160(-) was significantly lower than the number of HTLV-I infecting PBMCs even after 7 days of culture. This suggests that by reducing HTLV-I-infected cells in the body using anti-human CCR4 antibodies, it is possible to maintain a low number of HTLV-I-infected cells for a certain period of time.

These results show that the number of HTLV-I-infected cells in PBMCs from HAM patients can be reduced using anti-human CCR4 antibodies. This suggests that antibody therapy using anti-CCR4 antibodies may be applicable to HAM.

[Example 3]
In this example, an example is shown in which the prediction method of the present invention was used to predict whether antibody therapy using an anti-human CCR4 antibody would be effective for a HAM patient.

1. Preparation of PBMCs and cell separation PBMCs were separated from the blood of HAM patients (n=3) by the Ficoll centrifugation density method. KM2160(+) and KM2160(-) were separated from the obtained PBMCs by the same procedure as in Example 2.

2. Cell Culture RPMI medium (containing 5% human serum, 1% penicillin, and 1% streptomycin) was dispensed into each well of a round-bottom 96-well microplate. PBMCs, KM2160(+) and KM2160(-) were seeded at 5×10 ⁴ cells per well into which RPMI medium was dispensed, and cultured at 37° C. in a 5% CO ₂ atmosphere for 6 days.

3. Measurement of Cell Proliferation Activity After culture, PBMCs, KM2160(+) and KM2160(-) were allowed to incorporate ^3H -thymidine for 16 hours, and the cell proliferation activity was measured.

Figure 5 is a graph showing the cell proliferation activity of PBMCs and KM2160(-) for each patient. Figure 6 is a graph showing the results of three HAM patients in total. As shown in these graphs, the proliferation activity of KM2160(-) was significantly reduced compared to the proliferation activity of PBMCs in all HAM patients, but the degree of reduction in proliferation activity differed from patient to patient. This suggests that the effect of antibody therapy using anti-human CCR4 antibodies differs for each HAM patient. In this case, it is predicted that antibody therapy will be more effective for HAM1 and HAM3 patients than for HAM2 patients.

In addition, in Figures 5 and 6, the cell proliferation activity is also observed in KM2160(-), which is thought to be due to the fact that HTLV-I-infected cells cannot be completely removed by the KM2160 antibody, and that cell groups that are not removed by the KM2160 antibody also have proliferation activity. In addition, although the results are not shown here, the proliferation activity of KM2160(+) in HAM patients was significantly higher than that of KM2160(+) in healthy individuals.

Based on the above results, it is believed that the prediction method of the present invention can predict whether antibody therapy using anti-human CCR4 antibodies will be effective for HAM patients.

The pharmaceutical of the present invention is useful, for example, as a therapeutic drug for HAM. Furthermore, the prediction method of the present invention is useful, for example, as a test for predicting the therapeutic effect before antibody therapy is administered to HAM patients.

(A) is a two-parameter dot plot showing the expression patterns of CD4 and CD25 in PBMCs from a HAM patient. (B) is a histogram showing the expression pattern of CCR4 in the CD4 ^{+ CD25-} cell population. (C) is a histogram showing the expression pattern of CCR4 in the CD4 ⁺ CD25 ⁺ cell population. 1 is a graph showing the results of measuring proviral copy numbers in five cell populations. 1 is a graph showing the results of measuring the proviral copy numbers in KM2160(+) and KM2160(-) immediately after isolation. 13 is a graph showing the proviral copy numbers in KM2160(-) and PBMCs after culture. 1 is a graph showing the cell proliferation activity of PBMCs and KM2160(-). 1 is a graph showing the cell proliferation activity of PBMCs and KM2160(-).

Claims (3)

A pharmaceutical for treating or preventing HTLV-I-associated myelopathy, comprising an anti-human CCR4 antibody that specifically binds to human CCR4 or a fragment of said antibody. The pharmaceutical according to claim 1, wherein the antibody specifically binds to the extracellular domain of human CCR4. A method for testing the degree of effect of antibody therapy using an anti-human CCR4 antibody or a fragment of said antibody on a patient with HTLV-I-associated myelopathy prior to administering said antibody therapy , comprising:
Providing peripheral blood mononuclear cells collected from the patient;
preparing a cell population by removing cells expressing human CCR4 from the peripheral blood mononuclear cells;
measuring the proliferation activity of the peripheral blood mononuclear cells and the proliferation activity of the cell population;
analyzing the efficacy of the antibody therapy using the proliferation activity of the peripheral blood mononuclear cells and the proliferation activity of the cell group by comparing with a criterion that the antibody therapy is effective for the patient when the proliferation activity of the cell group is lower than the proliferation activity of the peripheral blood mononuclear cells;
A test method having the following characteristics.

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