JP2018024615A - Pharmaceutical composition for treating inflammatory disease related to htlv-1 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pharmaceutical composition for treating inflammatory disease related to HTLV-1, particularly, HTLV-1 related myelopathy (HAM) or HTLV-1 related uveitis (HU).SOLUTION: A pharmaceutical composition for treating inflammatory disease related to HTLV-1 contains, as an active ingredient, an anti-TfR antibody that specifically binds to human transferrin receptor (TfR).SELECTED DRAWING: None

Description

  The present invention relates to a pharmaceutical composition for treating inflammatory diseases associated with HTLV-1, in particular HTLV-1-related myelopathy (HAM) or HTLV-1-associated uveitis (HU).

  Human T-cell leukemia virus type I (Human T-cell leukemia virus type-1; hereinafter abbreviated as “HTLV-1”) is a virus that infects human T lymphocytes. According to a 2008 survey by the Ministry of Health, Labor and Welfare, there are around 1.08 million people infected with HTLV-1. Many HTLV-1 infected individuals become asymptomatic carriers for life, but about 4-5% of infected individuals develop adult T-cell leukemia (hereinafter abbreviated as “ATL”). In addition, some infected individuals have HTLV-1-related inflammatory diseases such as HTLV-1-associated myelopathy (hereinafter abbreviated as “HAM”) or HTLV-1-related uveitis (HTLV). -1-associated eutis (hereinafter abbreviated as “HU”) (Non-patent Documents 1 and 2).

  Inflammatory diseases related to HTLV-1 have a very small number of patients, and research for elucidating the etiology and developing therapeutic agents is difficult to proceed. Currently, no effective treatment has been established for inflammatory diseases associated with HTLV-1.

As a characteristic of the pathological condition of HAM, the number of HTLV-1 infected cells in the peripheral blood and cerebrospinal fluid is greatly increased, and an excessive immune response is induced in proportion to the number of cells, resulting in HTLV-1 specific cytotoxicity. It is mentioned that the number of sex T lymphocytes increases abnormally. In HAM spinal cord lesions, no viral tissue infection is observed, but HTLV-1-infected CD4 ⁺ T cells and the surrounding CD8 ⁺ cytotoxic T lymphocytes are infiltrated, and overproduction of inflammatory cytokines is observed Is done. From these, it is considered that the pathological condition of HAM is a neurological tissue disorder due to an excessive immune response caused by infected cells (Non-patent Document 2). In HU, it is considered that inflammation is caused by infection with HTLV-1.

  Treatment strategies for HAM based on these pathological conditions include (1) elimination of HTLV-1-infected cells, (2) suppression of hyperimmune response, that is, suppression of spinal cord inflammation, and (3) regeneration of damaged spinal cord. . In order to completely cure HAM, regenerative treatment of the spinal cord is ultimately necessary. However, currently no spinal cord regeneration therapy has been developed. At present, the administration of steroids and interferons effective in suppressing spinal cord inflammation is used as a treatment method for HAM. However, although these treatments improve temporary symptoms and suppress the progression of symptoms, the effects are not sustained because the effect of reducing HTLV-1-infected cells is poor. Steroids and interferons have various side effects such as induction of infection, osteoporosis, and liver damage, and long-term administration for symptom improvement is difficult. The elimination of HTLV-1 infected cells is considered to be the most effective treatment for solving these problems. Regarding the treatment of HU, steroid drug administration is currently being performed, but since it is likely to recur, removal of infected cells is still considered the most effective treatment. One method of specifically attacking and killing infected cells is molecular targeted therapy.

The inventors of the present invention have so far proceeded with research focusing on HTLV-1-infected cells. As a result, it was found that HTLV-1 was selectively infected with CD4 ⁺ CD25 ⁺ T cells in HAM patients, and that the infected cells excessively stimulated the immune response (Non-patent Document 3, 4). Moreover, it was found by DNA microarray analysis that CD4 ⁺ CD25 ⁺ T cells infected with HTLV-1 expressed CC chemokine receptor 4 (CCR4) at a higher level than HTLV-1 non-infected cells. A therapeutic method for HAM using an antibody was developed and patent applications were filed (Patent Documents 1 and 2). However, in clinical use of anti-CCR4 antibodies, side effects such as severe skin disorders, infections, and liver dysfunction frequently occur, and attention should be paid to treatment of HAM using anti-CCR4 antibodies.

Japanese Patent No. 5552630 International Publication No. 2014/007303

Percher F, Jeannin P, Martin-Latil S, Gessain A, Afonso PV, Vidy-Roche A, Ceccaldi PE., “Mother-to-Child Transmission of HTLV-1 Epidemiological Aspects, Mechanisms and Determinants of Mother-to-Child Transmission ”Viruses. 2016 Feb 3; 8 (2) Matsuura E, Yamano Y, Jacobson S., “Neuroimmunity of HTLV-1 Infection” J Neuroimmune Pharmacol. 2010 Sep; 5 (3): 310-25 Yamano Y., Cohen CJ, Takenouchi N., Yao K., Tomaru U., Li HC, Reiter Y., Jacobson S., "Increased Expression of Human T Lymphocyte Virus Type I (HTLV-1) Tax11-19 Peptide- Human Histocompatibility Leukocyte Antigen A * 201 Complexes on CD4 + CD25 + T Cells Detected by Peptide-specific, Major Histocompatibility Complex-restricted Antibodies in Patients with HTLV-1-associated Neurologic Disease ", J Exp Med., 199 (10), 1367-1377 , 2004 Yamano Y., Takenouchi N., Li HC, Tomaru U., YaoK., Grant CW, Maric DA, Jacobson S., "Virus-induced dysfunction of CD4 + CD25 + T cells in patients with HTLV-1-associated neuroimmunological disease" , J Clin Invest., 115 (5), 1361-1368, 2005

  As mentioned above, no effective treatment has been developed for inflammatory diseases associated with HTLV-1 due to HTLV-1 infection, such as HAM or HU. An object of the present invention is to provide a pharmaceutical composition that can be used for effective antibody therapy against inflammatory diseases related to HTLV-1, particularly HAM or HU.

  The present inventors have conducted extensive studies to solve the above problems, and surprisingly, HTLV-1 infected cells (pathogenic cells) of HAM or HU patients have transferredrin receptor (TfR). We found that it was highly expressed, and examined the effect of HAM or HU patients on HTLV-1-infected cells using an antibody against TfR. As a result, the anti-TfR antibody significantly suppressed the growth of HTLV-1-infected cells (pathogenic cells) derived from HAM or HU patients, and also suppressed the secretion of cytokines associated with the pathological condition. From these results, it was found that a pharmaceutical composition containing an anti-TfR antibody is useful for the treatment of inflammatory diseases related to HTLV-1, particularly HAM or HU, and completed the present invention.

That is, the present invention provides the following [1] to [11].
[1] A pharmaceutical composition for treating an inflammatory disease related to HTLV-1, comprising as an active ingredient an anti-TfR antibody or a fragment thereof that specifically binds to a human transferrin receptor (TfR).
[2] The first complementarity determining region (CDR1), the second complementarity determining region (CDR2) and the third complementarity determining region (CDR3) of the heavy chain variable region (VH) of the anti-TfR antibody are SEQ ID NO: 1, An amino acid sequence represented by 25 and 3, or an amino acid sequence in which one or several amino acids are deleted, added, substituted and / or inserted in the amino acid sequence represented by SEQ ID NOs: 1, 25 and 3, and the light chain variable An amino acid sequence in which the first complementarity determining region (CDR1), the second complementarity determining region (CDR2) and the third complementarity determining region (CDR3) of the region (VL) are represented by SEQ ID NOs: 4, 5 and 6, respectively, or The pharmaceutical composition according to [1], which is an amino acid sequence in which one or several amino acids are deleted, added, substituted and / or inserted in the amino acid sequences represented by SEQ ID NOs: 4, 5, and 6.
[3] The first complementarity determining region (CDR1), the second complementarity determining region (CDR2) and the third complementarity determining region (CDR3) of the heavy chain variable region (VH) of the anti-TfR antibody are respectively SEQ ID NO: 7 8 or 9, or an amino acid sequence in which one or several amino acids are deleted, added, substituted and / or inserted in the amino acid sequence shown in SEQ ID NOs: 7, 8 and 9. The first complementarity determining region (CDR1), light chain second complementarity determining region (CDR2) and light chain third complementarity determining region (CDR3) of the variable region (VL) are represented by SEQ ID NOs: 10, 11, and 12, respectively. Or an amino acid sequence in which one or several amino acids are deleted, added, substituted and / or inserted in the amino acid sequence shown in SEQ ID NOs: 10, 11 and 12 in [1] The pharmaceutical compositions of the mounting.
[4] The first complementarity determining region (CDR1), the second complementarity determining region (CDR2) and the third complementarity determining region (CDR3) of the heavy chain variable region (VH) of the anti-TfR antibody are respectively SEQ ID NO: 13 , 14 and 15, or an amino acid sequence in which one or several amino acids are deleted, added, substituted and / or inserted in the amino acid sequence shown in SEQ ID NOs: 13, 14 and 15. The first complementarity determining region (CDR1), the light chain second complementarity determining region (CDR2) and the light chain third complementarity determining region (CDR3) of the variable region (VL) are represented by SEQ ID NOs: 16, 17, and 18, respectively. Or an amino acid sequence in which one or several amino acids are deleted, added, substituted and / or inserted in the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18. That the pharmaceutical composition according to [1].
[5] The pharmaceutical composition according to any one of [1] to [4], wherein the anti-TfR antibody is a human antibody.
[6] The anti-TfR antibody comprises Fab, Fab ′, F (ab ′) ₂ , single chain antibody (scFv), dimerized V region (Diabody), disulfide stabilized V region (dsFv) and CDR The pharmaceutical composition according to any one of [1] to [5], which is an antibody fragment selected from the group consisting of peptides.
[7] The pharmaceutical composition according to any one of [1] to [6], wherein the inflammatory disease associated with HTLV-1 is HTLV-1-related myelopathy.
[8] The pharmaceutical composition according to any one of [1] to [6], wherein the inflammatory disease associated with HTLV-1 is HTLV-1-associated uveitis.
[9] Use of an anti-TfR antibody that specifically binds to TfR for producing a pharmaceutical composition for the treatment of inflammatory diseases related to HTLV-1.
[10] An anti-TfR antibody that specifically binds to TfR for the treatment of inflammatory diseases associated with HTLV-1.
[11] A method for treating an inflammatory disease associated with HTLV-1, which comprises administering an effective amount of an anti-TfR antibody that specifically binds to TfR.

  According to the pharmaceutical composition of the present invention, HTLV-1-infected cells (pathogenic cells) of HTLV-1-related inflammatory disease patients, particularly HAM or HU patients, can be efficiently eliminated, and cytokines involved in the pathological condition Can be effectively treated for inflammatory diseases associated with HTLV-1, particularly HAM or HU.

It is a figure which shows the antigen reactivity of an anti- TfR antibody. It is a figure which shows the HTLV-1 provirus amount (infected cell rate) inhibitory effect of the anti- TfR antibody TSP-A74 with respect to PBMC derived from a HAM patient. It is a figure which shows the proliferation inhibitory effect of the anti- TfR antibody TSP-A74 with respect to PBMC derived from a HAM patient. It is a figure which shows the cytokine production inhibitory effect of the anti- TfR antibody TSP-A74 with respect to PBMC derived from a HAM patient. It is a figure which shows the HTLV-1 provirus amount (infection cell rate) suppression effect of the anti- TfR antibody TSP-A02 and TSP-A17 with respect to PBMC derived from a HAM patient. It is a figure which shows the proliferation inhibitory effect of anti- TfR antibody TSP-A02 and TSP-A17 with respect to PBMC derived from a HAM patient. It is a figure which shows the cytokine production inhibitory effect of the anti- TfR antibody TSP-A02 and TSP-A17 with respect to HAM patient origin PBMC. It is a figure which shows the HTLV-1 provirus amount (infected cell rate) inhibitory effect of the anti- TfR antibody TSP-A74 with respect to HU patient origin PBMC. It is a figure which shows the proliferation inhibitory effect of the anti- TfR antibody TSP-A74 with respect to HU patient origin PBMC. It is a figure which shows the cytokine production inhibitory effect of the anti- TfR antibody TSP-A74 with respect to HU patient origin PBMC.

Next, the present invention will be described in more detail.
Definitions and General Techniques Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. In general, the nomenclature and techniques used for cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are well known in the art. And are usually used.

  The methods and techniques of the present invention are generally described in various general and more specific references cited and discussed throughout this specification, according to conventional methods well known in the art, unless otherwise indicated. Carry out as it is.

TfR
The human transferrin receptor (TfR) is a single-transmembrane protein encoded by human chromosome 3 and composed of 760 amino acids shown in SEQ ID NO: 27. This protein, also known as the CD71 antigen, is involved in cellular iron uptake and is thought to be involved in cell proliferation. In the present invention, human TfR is not particularly limited in terms of structure, and monomers, multimers, intact forms expressed in the cell membrane, solubilized forms, truncated forms, gene mutations and deletions formed in the extracellular region Also included are mutated forms generated by the above, forms that have been post-translationally modified by phosphorylation and the like.

Antibody In the present specification, the following abbreviations (in parentheses) are used in accordance with the convention as necessary.
Heavy chain (H chain), light chain (L chain), heavy chain variable region (VH), light chain variable region (VL), complementarity determining region (CDR), first complementarity determining region (CDR1), second Complementarity determining region (CDR2), third complementarity determining region (CDR3), heavy chain first complementarity determining region (VH CDR1), heavy chain second complementarity determining region (VH CDR2), heavy chain first 3 complementarity determining region (VH CDR3), light chain first complementarity determining region (VL CDR1), light chain second complementarity determining region (VL CDR2), light chain third complementarity determining region (VL CDR3) ).

  As used herein, the term “antibody” is synonymous with immunoglobulin and should be understood as commonly known in the art. Specifically, the antibody is not limited by any method for producing the antibody. Antibodies include, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.

  As used herein, the term “human antibody” means any antibody in which the variable region and constant region sequences are human sequences. Human antibodies have sequences derived from human genes, but have been altered, for example, to reduce possible immunogenicity, increase affinity, remove cysteines that can cause undesirable folding, etc. Includes antibodies. Human antibodies also include those antibodies that can be glycosylated that are not characteristic of human cells and are produced recombinantly in non-human cells. These antibodies can be prepared in various forms by known means.

  In the variable region of the antibody constituting the pharmaceutical composition of the present invention, the sequence of the framework region (FR) is not particularly limited as long as it does not substantially affect the specific binding to the corresponding antigen. Although the FR region of a human antibody is preferably used, the FR region of an animal species other than human (for example, mouse or rat) can also be used.

  In one embodiment of the antibody constituting the pharmaceutical composition of the present invention, a constant region is included in addition to the variable region (for example, an IgG type antibody). The sequence of the constant region is not particularly limited, and for example, a known human antibody constant region can be used. The heavy chain constant region (CH) of a human antibody may be any as long as it belongs to human immunoglobulin (hereinafter referred to as hIg), but is preferably of the hIgG class, more specifically, the hIgG class. Subclasses such as hIgG1, hIgG2, hIgG3, and hIgG4 belonging to the above can be used. The light chain constant region (CL) may be any as long as it belongs to hIg, and those of κ class or λ class can be used. Moreover, the constant region of animal species other than humans (for example, mouse and rat) can also be used.

  The amino acid sequence of the CDR, FR or constant region used for the antibody constituting the pharmaceutical composition of the present invention may be the original CDR, FR or constant region amino acid sequence from which it is derived, or one or several. (For example, 1 to 8, preferably 1 to 5, more preferably 1 to 3, particularly preferably 1 or 2) amino acids are deleted, added, substituted and / or inserted into different amino acid sequences. May be used. Here, “an amino acid sequence in which one or several amino acids are deleted, added, substituted and / or inserted” means at least 90% or more, preferably 95% or more, more preferably 98% or more with the amino acid sequence. An amino acid sequence having sequence identity.

  The antibody constituting the pharmaceutical composition of the present invention may differ in amino acid sequence, molecular weight, isoelectric point, presence / absence of sugar chain, form, etc., depending on the cell or host producing the antibody described later or the purification method. As long as the obtained antibody has an activity equivalent to that of the antibody of the present invention, it is included in the present invention. For example, an antibody in which the amino acid sequence described in the present invention has been modified after translation is also included in the present invention. Furthermore, antibodies that have undergone post-translational modifications to sites other than known post-translational modifications are also included in the present invention as long as they have the same activity as the antibodies of the present invention. When the antibody of the present invention is expressed in prokaryotic cells such as E. coli, a methionine residue is added to the N-terminus of the original antibody amino acid sequence. The antibody of the present invention also includes such an antibody.

  The antibody constituting the pharmaceutical composition of the present invention is an anti-TfR antibody or a fragment thereof that specifically binds to human TfR. Among these, the first complementarity determining region (CDR1), the second complementarity determining region (CDR2) and the third complementarity determining region (CDR3) of the heavy chain variable region (VH) are SEQ ID NOS: 1, 25 and 3, respectively. The amino acid sequence shown or the amino acid sequence shown in SEQ ID NOs: 1, 25 and 3, wherein one or several amino acids are deleted, added, substituted and / or inserted, and the light chain variable region (VL) The first complementarity determining region (CDR1), the second complementarity determining region (CDR2) and the third complementarity determining region (CDR3) of SEQ ID NOs: 4, 5 and 6, respectively, or SEQ ID NO: 4, An anti-TfR antibody having an amino acid sequence in which one or several amino acids are deleted, added, substituted and / or inserted in the amino acid sequences shown by 5 and 6; heavy chain variable region (VH) first The amino acid sequences in which the complementarity determining region (CDR1), the second complementarity determining region (CDR2) and the third complementarity determining region (CDR3) are represented by SEQ ID NOs: 7, 8 and 9, respectively, or SEQ ID NOs: 7, 8 and 9 In which one or several amino acids are deleted, added, substituted and / or inserted, and the light chain variable region (VL) first complementarity determining region (CDR1) and second complement The sex determining region (CDR2) and the third complementarity determining region (CDR3) have one or several amino acid sequences represented by SEQ ID NOs: 10, 11, and 12, or amino acid sequences represented by SEQ ID NOs: 10, 11, and 12, respectively. An anti-TfR antibody whose amino acid sequence is deleted, added, substituted and / or inserted; and a heavy chain variable region (VH) first complementarity determining region (CDR1) , The second complementarity determining region (CDR2) and the third complementarity determining region (CDR3) are 1 in the amino acid sequence represented by SEQ ID NOs: 13, 14, and 15 or the amino acid sequences represented by SEQ ID NOs: 13, 14, and 15, respectively. Or an amino acid sequence in which several amino acids are deleted, added, substituted and / or inserted, the light chain variable region (VL) first complementarity determining region (CDR1), second complementarity determining region (CDR2) and The third complementarity determining region (CDR3) is deleted or added in the amino acid sequence represented by SEQ ID NOs: 16, 17 and 18, respectively, or in the amino acid sequence represented by SEQ ID NOs: 16, 17 and 18; Anti-TfR antibodies that are substituted and / or inserted amino acid sequences are preferred. Here, “the amino acid sequence obtained by deleting, adding, substituting and / or inserting one or several amino acids in the amino acid sequence represented by SEQ ID NOs: 1 to 18” is the sequence of the CDRs shown in SEQ ID NOs: 1 to 18 It means a CDR sequence having a sequence identity of at least 90% or more, preferably 95% or more, more preferably 98% or more.

Preparation of antibodies (1) Acquisition of scFv that reacts with antigen using phage display library Acquisition of antibodies constituting the pharmaceutical composition of the present invention should be prepared according to several methods known in the art. Can do. For example, phage display technology can be used to provide a library containing a repertoire of antibodies with varying affinities for TfR. These libraries can then be screened to identify and isolate antibodies to TfR. Preferably, the phage library is a scFv phage display library generated using human VL and VH cDNA prepared from mRNA isolated from human B cells. Methods for preparing and screening such libraries are known in the art. Genetic material is recovered from a phage clone showing reactivity that was screened using human TfR as an antigen. By analyzing the gene of the selected phage, it is possible to determine the VH and VL DNA sequences encoding the variable region of the human antibody that binds to the antigen. A human antibody can be obtained by converting the scFv into IgG using this scFv sequence.

(2) IgG conversion of scFv (production of human antibody)
A human antibody can be obtained by preparing an expression vector for H chain or L chain, expressing it in a host cell, and collecting and purifying the supernatant. Alternatively, human antibodies can be obtained by expressing VH and VL in the same vector (tandem type). These methods are well known, for example, WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, Reference can be made to International Publication No. 95/01438, International Publication No. 95/15388, International Publication No. 97/10354, and the like.

  Specifically, a full-length heavy chain gene can be obtained by ligating DNA encoding VH with other DNA molecules encoding heavy chain constant regions (CH1, CH2 and CH3). The sequence of the human heavy chain constant region gene is known in the art (see, eg, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and. Human Services, NIH Publication No. 91-3242), DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgG1 or IgG2 constant region. An IgG1 constant region sequence can be any of a variety of allele or allotype sequences known to occur between different individuals, such as Gm (1), Gm (2), Gm (3), Gm (17). May be. These allotypes correspond to substitutions of naturally occurring amino acids in the IgG1 constant region.

  A full-length light chain gene (and Fab light chain gene) can be obtained by ligating DNA encoding VL with other DNA molecules encoding light chain constant region (CL). The sequence of the human light chain constant region gene is known in the art (see, eg, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and. Human Services, NIH Publication No. 91-3242), DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region. The kappa constant region may be any of a variety of alleles known to occur between different individuals, such as Inv (1), Inv (2), Inv (3). The λ constant region may be derived from any of the three λ genes.

  By inserting the H-chain or L-chain-encoding DNA obtained as described above into an expression vector, an expression vector is prepared, expressed in a host cell, and the supernatant is recovered and purified, whereby a human antibody is obtained. You can get it. Examples of the expression vector include plasmid vectors, retrovirus vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, plant virus vectors such as cauliflower mosaic virus vectors and tobacco mosaic virus vectors, cosmids, YACs, EBV-derived episomes, and the like. . The expression vector and the expression regulatory sequence may be appropriately selected so as to be compatible with the expression host cell to be used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or both genes can be inserted into the same expression vector. Antibody genes can be obtained by standard methods (eg, ligation of restriction sites on antibody gene fragments and complementary restriction sites on vectors, or blunt end ligation of antibody gene fragments and vectors if no restriction sites are present). Can be inserted into an expression vector.

  Suitable vectors are functionally complete human CH or CL immunogens with appropriate restriction sites engineered to allow easy insertion and expression of any VH or VL sequence as described above. It encodes a globulin sequence. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C domain, and also in the splice region present in the human CH exon. Polyadenylation and transcription termination occur at natural chromosomal sites downstream of the coding region. The recombinant expression vector can also encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide derived from a non-immunoglobulin protein).

  The antibody expression vector constituting the pharmaceutical composition of the present invention comprises, in addition to the antibody gene and control sequence, a sequence that controls replication of the vector in the host cell (for example, an origin of replication) and a further sequence such as a selectable marker gene. You may have. The selectable marker gene facilitates selection of host cells into which the vector has been introduced. For example, usually the selectable marker gene confers resistance to drugs such as G418, hygromycin and methotrexate on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification), the neomycin phosphotransferase gene (for G418 selection), and the glutamate synthase gene.

  A host cell is transformed with the antibody gene expression vector produced by the above method. The host cell may be any cell that can produce the antibody of the present invention, such as bacteria, yeast, animal cells, insect cells, and plant cells, but animal cells are preferred. Examples of animal cells include dhfr gene-deficient Chinese hamster ovary cells CHO / DG44 cells, monkey-derived cells COS cells (A. Wright & SL Morrison, J. Immunol., 160, 3393-3402, 1998), mouse myeloma Sp2 / 0. Cells (K. Motmans et al., Eur. J. Cancer Prev., 5, 512-519, 1996; RP Junghans et al., Cancer Res., 50, 1495-1502, 1990). For transformation, the lipofectin method (RWMalone et al., Proc. Natl. Acad. Sci. USA, 86, 6077-6081, 1989; PLFelgner et al., Proc. Natl. Acad. Sci. USA, 84, 7413-7417, 1987), electroporation method, calcium phosphate method (FL Graham & AJ van der Eb, Virology, 52, 456-467, 1973), DEAE-Dextran method and the like are preferably used.

  After culturing the transformant, the human antibody can be separated from the cells of the transformant or from the culture solution. For separation and purification of antibodies, methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, affinity chromatography, ion exchange chromatography, gel filtration chromatography and the like can be used in appropriate combination.

Antibody Fragments Antibody fragments can be prepared based on the antibody of the present invention or based on the sequence information of the gene encoding the antibody of the present invention. Examples of antibody fragments include Fab, Fab ′, F (ab ′) ₂ , scFv, and dsFv antibodies.

  Fab is a fragment having a molecular weight of about 50,000, which is obtained by digesting IgG with papain in the presence of cysteine and consisting of an L chain and an H chain fragment consisting of the H chain variable region, CH1 domain and part of the hinge region. is there. In the present invention, the antibody can be obtained by digesting with papain. Alternatively, Fab can be prepared from a transformant obtained by incorporating a part of the H chain of the antibody and DNA encoding the L chain into an appropriate vector and transforming using the vector.

Fab ′ is a fragment having a molecular weight of about 50,000 obtained by cleaving a disulfide bond between the H chains of F (ab ′) ₂ described later. In the present invention, the antibody is obtained by digesting with pepsin and cleaving disulfide bonds using a reducing agent. Similarly to Fab, it can also be prepared by genetic engineering using DNA encoding Fab ′.

F (ab ′) ₂ is a fragment (Fab ′) composed of an L chain obtained by pepsin digestion of IgG and an H chain fragment consisting of the H chain variable region, the CH1 domain, and a part of the hinge part. Is a fragment having a molecular weight of about 100,000 linked by a disulfide bond. In the present invention, the antibody is obtained by pepsin digestion. Similarly to Fab, it can be prepared by genetic engineering using DNA encoding F (ab ′) ₂ .

scFv is an antibody fragment in which an Fv comprising an H chain variable region and an L chain variable region is made into a single chain by linking the C terminus of one chain and the N terminus of the other chain with an appropriate peptide linker. As the peptide linker, for example, (GGGGGS) ₃ having high flexibility can be used. For example, a DNA encoding the scFv antibody is constructed using DNA encoding the H chain variable region and L chain variable region of the above antibody and DNA encoding a peptide linker, and this is incorporated into an appropriate vector, and the vector is used. ScFv can be prepared from the transformant transformed as described above.

  dsFv is an Fv fragment in which a Cys residue is introduced at an appropriate position in the H chain variable region and the L chain variable region, and the H chain variable region and the L chain variable region are stabilized by disulfide bonds. The position of Cys residue introduction in each chain can be determined based on the three-dimensional structure predicted by molecular modeling. In the present invention, for example, three-dimensional structures are predicted from the amino acid sequences of the H chain variable region and the L chain variable region of the antibody, and DNAs encoding the H chain variable region and the L chain variable region into which mutations are introduced based on the prediction, Is incorporated into an appropriate vector, and dsFv can be prepared from a transformant transformed with the vector.

  The antibody fragments can also be multimerized by linking scFv antibody, dcFv antibody, etc. using an appropriate linker, or by fusing streptavidin.

Inflammatory disease associated with HTLV-1 An inflammatory disease associated with HTLV-1 refers to an inflammatory disease caused by infection with HTLV-1. Examples of the inflammatory disease include HTLV-1-related myelopathy (HAM), HTLV-1-related uveitis (HU), HTLV-1-related arthritis, dermatitis, myositis and the like.

Pharmaceutical Composition According to the present invention, there is provided a pharmaceutical composition containing the anti-TfR antibody of the present invention. In one embodiment of the invention, the pharmaceutical composition containing an anti-TfR antibody is used for the treatment of inflammatory diseases associated with HTLV-1. In a further preferred embodiment, the pharmaceutical composition containing the anti-TfR antibody is used for the treatment of HAM or HU.

  As one aspect of the pharmaceutical composition of the present invention, the anti-TfR antibody of the present invention is used as an active ingredient. The pharmaceutical composition of the present invention exhibits therapeutic effects on inflammatory diseases related to HTLV-1 by utilizing the cell growth inhibitory activity, cell death inducing activity, ADCC activity, CDC activity, etc. of the antibody. The antibody may have only one of the above activities or may have a plurality of activities at the same time.

Formulations with a pharmaceutical composition containing the anti-human TfR antibody of the present invention are also included within the scope of the present invention. Such a formulation preferably comprises a physiologically acceptable diluent or carrier in addition to the pharmaceutical composition containing the anti-TfR antibody, and other agents such as other antibodies or anticancer agents. And a mixture thereof. Suitable carriers include, but are not limited to, physiological saline, phosphate buffered saline, phosphate buffered saline glucose solution, and buffered saline. Alternatively, the anti-TfR antibody may be lyophilized (freeze-dried) and reconstituted by adding a buffered aqueous solution as described above when needed.
The dosage forms include tablets, capsules, granules, powders, suspensions, syrups, etc., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, transdermal, transmucosal, nasal administration And parenteral administration by pulmonary, suppository and the like. The pharmaceutical preparation of the present invention may be administered alone or in combination with other drugs.

  The dosage of the pharmaceutical composition of the present invention varies depending on the subject's symptoms, age, body weight, dosage form, etc., but in general, in oral administration, the amount of antibody is about 0.01 mg to 1000 mg per day for an adult. The dose is preferably 0.1 mg to 500 mg, more preferably 1 mg to 100 mg, and these can be administered once or divided into several times. In parenteral administration, about 0.01 mg to 1000 mg, preferably 0.1 mg to 500 mg, more preferably 1 mg to 100 mg can be administered once by subcutaneous injection, intramuscular injection or intravenous injection.

The method for measuring the proliferation activity of the cells is not particularly limited, and may be appropriately selected from methods known to those skilled in the art such as ³ H-thymidine incorporation method. In HTLV-1 infected cells (pathogenic cells), the proliferation activity is highest 6 to 7 days after the start of culture. Therefore, it is preferable to measure the proliferation activity of the cells 6 to 7 days after the start of the culture. The cell culture method may be appropriately selected from methods known to those skilled in the art.

  The following examples further illustrate the present invention in detail but are not to be construed to limit the scope thereof.

  The therapeutic effect of anti-human TfR antibody was examined using peripheral blood mononuclear cells (PBMC) isolated from peripheral blood of patients with HAM or HU, which is an HTLV-1-related inflammatory disease. The action of the anti-TfR antibody was evaluated based on (1) the HTLV-1-infected cell rate of PBMC, (2) spontaneous growth activity, and (3) changes in the amount of various cytokines produced.

Reference Example 1 Phage antibody screening using cancer cell line (1) Screening of phage antibody binding to cancer cells (hepatoma cell line HepG2)
HepG2 cells were cultured in a 15 cm dish and detached from the dish with 2 mg / mL collagenase I / cell lysis buffer (Gibco BRL). Cells were collected and washed with chilled PBS, then mixed with 1 × 10 ¹³ cfu of human antibody phage library, and the reaction solution (1% BSA, 0.1% NaN ₃ , MEM) was added and the reaction was allowed to rotate slowly at 4 ° C. for 4 hours. After completion of the reaction, the reaction solution was divided into two, 0.6 mL of an organic solution (dibutyl phthalate cycloheximide 9: 1) prepared in advance for each was layered, and centrifuged for 2 minutes (3000 rpm) with a microcentrifuge. The supernatant was discarded, and the cells that settled at the bottom of the tube were suspended in 0.7 mL of 1% BSA / MEM, and 0.7 mL of an organic solvent was layered thereon. Similarly, the supernatant was discarded by centrifugation, and the cells were suspended in 0.3 mL of PBS and frozen in liquid nitrogen.

Frozen cells were thawed at 37 ° C. and infected with 20 mL of E. coli DH12S (OD0.5) in 1 hour. Escherichia coli infected with the phage was placed in 600 mL of 2 × YTGA medium (2 × YT, 200 μg / mL amplisulfate, 1% Glucose) and cultured at 30 ° C. overnight. Thereafter, 10 mL was taken and placed in 200 mL 2 × YTA medium (2 × YT, 200 μg / mL ampiculate) and cultured at 37 ° C. for 1.5 hours. 1 × 10 ¹¹ helper phage KO7 was added and further cultured at 37 ° C. for 1 hour. 800 mL of 2 × YTGAK medium (2 × YT, 200 μg / mL amphisulfate, 0.05% Glucose, 50 μg / mL kanamylcin) was added and cultured at 30 ° C. overnight. The supernatant was collected by centrifugation (8000 rpm) for 10 minutes. 200 mL of PEG solution (20% polyethyleneglycol 6000, 2.5 M NaCl) was added to the collected supernatant and stirred well, and then phages were precipitated by centrifugation (8000 rpm) for 10 minutes. This was suspended in 10 mL of PBS and used as the 1st screening phage.

Next, 2nd screening was performed. 2 × 10 ⁷ cultured cells and 1 × 10 ¹⁰ 1st screening phage were mixed, and the reaction solution (1% BSA, 0.1% NaN 3, MEM) was added to a final volume of 0.8 mL. Thereafter, the same method as the 1st screening was performed to obtain 2nd screening phage.

The 3rd screening was performed in the same manner as described above using 1 × 10 ⁹ 2nd screening phages.

(2) Phage antibody analysis 3rd-screened phages are collected, DNA sequences are analyzed by existing methods, incomplete antibodies retaining defective regions and antibodies with overlapping sequences are excluded, and phages having independent antibody sequences Antibody was obtained.

  In the same manner, 21 types of cancer cells shown in Table 1 below were used to screen for phage antibodies that react with cancer antigens. As a result, as shown in Table 1, 1863 phage antibodies having independent sequences were obtained.

Reference Example 2 Screening of phages that react with soluble human TfR (1) Preparation of soluble TfR antigen-producing cells TfR cDNA was prepared by PCR using cancer cell lines MIAPaCa2 and SKOV-3. A cDNA of TfR extracellular domain was prepared by a conventional method and inserted into pCMV-Script (Clontech) to prepare a soluble TfR antigen expression vector. This expression vector was introduced into cell line 293T to produce expression cells that produce soluble TfR antigens.

(2) Screening for positive phages by ELISA The supernatant of the soluble TfR-producing cells was collected, and the soluble TfR antigen was obtained by purification. Using this soluble TfR antigen, the reactivity of the antigen antibody by ELISA was examined. That is, soluble TfR antigen was prepared to 10 μg / mL with PBS, added to Immuno Module / Stripe Plates (NUNK) at 50 μL / well, and allowed to stand at 37 ° C. for 2 hours. Thereafter, the soluble TfR antigen was discarded, Blocking solution (5% skim milk / 0.05% NaN ₃ / PBS) was added at 200 μL / well, and blocking was performed at 37 ° C. for 2 hours. The blocking solution was removed, the plate was washed with PBS, 100 μL / well of the phage culture supernatant shown in Table 1 was added, and the mixture was reacted at 37 ° C. for 1 hour. After washing 5 times with PBS, 100 μL / well of 1 μg / mL Rabbit anti-cp3 diluted with PBS / 0.05% Tween 20 was added and reacted at 37 ° C. for 1 hour. After washing 5 times with PBS, anti-Rabbit IgG (H + L) -HRP diluted 2000 times with PBS / 0.05% Tween 20 was further added at 100 μL / well and reacted at 37 ° C. for 1 hour. After washing 5 times with PBS, 100 μL / well of OPD in 0.1 M citrate phosphate buffer (pH 5.1) + 0.01% H ₂ O ₂ was added and allowed to react at room temperature for 5 minutes. 2N H ₂ SO ₂ was added at 100 μL / well to stop the color reaction. Thereafter, the absorbance at 492 nm was measured with SPECTRAmax340PC (Molecular Devices). As a result, 20 of the 1863 phages showed significant positive reaction with the soluble TfR antigen. Analysis of the DNA sequences of these 20 strains of phages confirmed that each CDR sequence was novel. Among them, the CDR sequences of TSP-A01, TSP-A02, and TSP-A17 antibodies, and the sequences of VH and VL are shown in Tables 2-7.

Reference Example 3 Acquisition of TSP-A74 antibody For the purpose of improving the productivity and function of TSP-A01 antibody, two amino acids of CDR2 of the variable region of TSP-A01 antibody were modified and optimized for clinical application. A TSP-A74 antibody was prepared. The CDR sequences of TSP-A74 antibody and the sequences of VH and VL are shown in Tables 8 and 9.

Example 1 Antigen reactivity study of anti-TfR antibody Antigen reaction of anti-TfR antibodies of TSP-A01, TSP-A02, TSP-A17, and TSP-A74 using TfR-expressing cells K562 (ATCC CCL-243: CML) The sex was examined. K562 cells were collected by centrifugation and washed once with PBS. Thereafter, the cells were suspended at 1 × 10 ⁶ / mL with FACS Buffer (1% BSA, 2 mM EDTA, 0.1% NaN _{3 in} PBS). Dispense 100 μL of this cell suspension into a 96-well V bottom plate (Costar 3897), and further prepare each antibody to 0.2-2 μg / mL with FACS Buffer, add to the cells, and add 1 to 4 ° C. Incubated for hours. After the cells were washed twice with FACS Buffer, 100 μL of Alexa488-anti-human IgG (invitrogen) solution diluted 750 times with FACS Buffer was added, stirred, and further incubated at 4 ° C. for 1 hour. After washing twice with a FACS Buffer by centrifugation, it was set in HTS of FACS Calibur (BD), and the FL1 fluorescence intensity of each well was measured. As shown in FIG. 1, each antibody showed strong reactivity with K562 cells.

Example 2 Examination of therapeutic effect of anti-TfR antibody TSP-A74 for HAM patients (1) Inhibitory effect of HTLV-1 proviral DNA in PBMC of HAM patients by TSP-A74 antibody was cryopreserved in liquid nitrogen RPMI-1640 (WAKO: 189-) containing 6% Fetal Bovine Serum (FBS) (GIBCO: 10437-028) was dissolved in 6 cases of HAM patient PBMC (HAM-PBMC) in a 37 ° C. water bath. 02025) After washing with medium, cells were prepared to 1 × 10 ⁶ cells / mL using the same medium. Then, each HAM-PBMC was seed | inoculated by 10 < ⁵ > cells / 100 microliter / well to U bottom (round bottom) 96-Well Plate (FALCON). TSP-A74 or isotype control antibody Pab1400 is prepared in a medium to 0.02, 0.2, 2 and 20 μg / mL, and 100 μL is added to each well to give a final concentration of 0, 0.001, 0.01, 0.1, 1 (10 ⁻³ , 10 ⁻² , 10 ⁻¹ , 1) μg / mL. As a control, 1 μg / mL steroidal anti-inflammatory drug Prednisolone Sodium Phosphate (PSL) (Funakoshi: P7012) was used. Thereafter, the cells were cultured in a 37 ° C. CO ₂ incubator.
Seven days after the start of the culture, 10-well cell suspensions (1 × 10 ⁶ cells) were collected for each culture condition, centrifuged, the supernatant was removed, the cell pellet was recovered, and genomic DNA extraction was performed. The amount of HTLV-1 provirus (infected cell rate) was measured by real-time PCR using the extracted genomic DNA as a template (Yamano et al. Blood. 2002, 99 (1): 88-94).
HTLV-1 provirus of cells cultured with addition of TSP-A74, Pab1400, and PSL, with the amount of HTLV-1 provirus in each HAM patient PBMC cultured in medium alone without adding antibody or reagent as 100% The relative value of the amount was calculated, and the average value of 6 HAM patients was determined. The significant difference test was performed using One-way ANOVA of GraphPad Prism 6 (MDF).

  The amount of HTLV-1 provirus in HAM-PBMC was significantly reduced by the addition of TSP-A74. At that time, under the condition of adding Pab1400, which is an isotype control, an effect of reducing the amount of HTLV-1 provirus in HAM-PBMC was not observed (FIG. 2). Therefore, it was suggested that the anti-TfR antibody has an effect of eliminating HTLV-1 infected cells in HAM-PBMC.

(2) Examination of action of TSP-A74 antibody on spontaneous growth activity of HAM-PBMC HAM-PBMC grows spontaneously in culture under unstimulated conditions and has the characteristic of showing the spread of HTLV-1 infection. Such spontaneous growth activity of HAM-PBMC reflects the hyperimmune response characteristic of HAM. Therefore, it was examined whether TSP-A74 suppresses the spontaneous growth activity of HAM-PBMC.
HAM-PBMC cells were seeded at 1 × 10 ⁵ cells / well on a U-bottom (round bottom) 96-well plate. TSP-A74 or isotype control antibody Pab1400 is prepared in a medium to 0.02, 0.2, 2 and 20 μg / mL, and 100 μL is added to each well to give a final concentration of 0, 0.001, 0.01, 0.1, 1 (10 ⁻³ , 10 ⁻² , 10 ⁻¹ , 1) μg / mL. As a control, 1 μg / mL PSL was used. Thereafter, the cells were cultured in a CO ₂ incubator at 37 ° C., and 6 days later, H ³ -Thymidine was added to 1 μCi / well and further incubated for 16 hours. Thereafter, the cultured cells were adsorbed on a glass filter (Printed Filtermat A, PerkinElmer) using a cell harvester (Tomtec MH3, PerkinElmer), dried sufficiently, and then impregnated with solid scintillator MeltiLex-A (PerkinElmer), MicroB The amount of ³ H-thymidine incorporated into cells was measured using WALLAC MicroBeta TriLux 1450-021) (Yamano et al. PLoS One. 2009, 4 (8): e6517). Relative to the count of cells cultured with addition of TSP-A74, Pab1400, and PSL, assuming that the average of the count of ³ H-Thymidine in each HAM patient PBMC cultured with only medium without adding antibody or reagent is 100% The value was calculated and the average value of 6 HAM patients was determined. The significant difference test was performed using One-way ANOVA of GraphPad Prism 6 (MDF).

  As a result, the spontaneous growth of HAM-PBMC was remarkably suppressed by the addition of TSP-A74 at a concentration of 0.1 μg / mL or higher. At that time, no significant effect on the spontaneous growth activity was observed under the condition of adding Pab1400, which is an isotype control (FIG. 3). Therefore, it was suggested that the anti-TfR antibody has the effect of normalizing the hyperimmune response of PBMC derived from HAM patients.

(3) Examination of the effect of TSP-A74 on cytokine production of HAM-PBMC PBMCs of 6 HAM patients that had been cryopreserved in liquid nitrogen were dissolved in a 37 ° C. water bath and contained 10% FBS After washing with RPMI-1640 medium, cells were prepared to 1 × 10 ⁶ cells / mL using the same medium. Then, each HAM-PBMC was seed | inoculated at 10 < ⁵ > cells / 100 microliter / well to U bottom (round bottom) 96-Well Plate. TSP-A74 or isotype control antibody Pab1400 is prepared in a medium to 0.02, 0.2, 2 and 20 μg / mL, and 100 μL is added to each well to give a final concentration of 0, 0.001, 0.01, 0.1, 1 (10 ⁻³ , 10 ⁻² , 10 ⁻¹ , 1) μg / mL. As a control, 1 μg / mL PSL was used. Thereafter, the cells were cultured in a 37 ° C. CO ₂ incubator. Seven days later, the cultured cells were collected and centrifuged to collect the culture supernatant.
The amount of cytokine in the culture supernatant was measured with a flow cytometer BD FACSCanto II (BD Biosciences) using a Cytokine Bead Array kit (BD Biosciences) (IFN-γ: BD Biosciences, 560111, IL-6: BD Biosciences). 558276) (Yamauchi et al. J Infect Dis. 2015, 211 (2): 238-48).
Cytokine concentration in the culture solution when each concentration of TSP-A74 or Pab1400 was added and cultured, using as a control the concentration of cytokine in the culture solution of cells cultured with only medium without adding either TSP-A74 or Pab1400 The changes were analyzed comparatively. The significant difference test was performed using One-way ANOVA of GraphPad Prism 6 (MDF).

  As a result, in the presence of TSP-A74, the production amounts of IFN-γ and IL-6 were significantly reduced (FIG. 4). It was suggested that the anti-TfR antibody has an action of suppressing the production of inflammatory cytokines IFN-γ and IL-6 produced from HAM-PBMC.

Example 3 Examination of therapeutic effect of anti-TfR antibody TSP-A02 or TSP-A17 on HAM patients In order to confirm the usefulness of anti-TfR antibody in the treatment of HAM, other anti-TfR antibodies (TSP-A02, TSP-A17) Was used to examine the effect on HAM patient-derived cells.

  In the same manner as in Example 2 (1) to (3) above, PBMCs separated from the peripheral blood of 5 HAM patients were seeded in a 96-well plate, and 1 μg / mL TSP-A02 or TSP-A17 was added. Wells added with 1 μg / mL Pab1400 and wells added with 1 μg / mL PSL were prepared and cultured. After cultivation, the amount of HTLV-1 proviral DNA (FIG. 5), spontaneous growth activity (FIG. 6), and the production amounts of IFN-γ and IL-6 in the culture supernatant (FIG. 7) were measured.

As a result, as shown in FIG. 5, the amount of HTLV-1 proviral DNA in PBMCs of HAM patients was significantly reduced when both anti-TfR antibodies TSP-A02 and TSP-A17 were added, compared to when Pub1400 was added.
In addition, as shown in FIG. 6, both anti-TfR antibodies TSP-A02 and TSP-A17 markedly suppressed the spontaneous growth activity of HAM patient PBMC as compared to Pab1400.
Furthermore, as shown in FIG. 7, the concentrations in the culture supernatant of IFN-γ and IL-6 produced from HAM patient PBMC were significantly higher when both anti-TfR antibodies of TSP-A02 and TSP-A17 were added compared to Pab1400. Diminished. Therefore, it was revealed that the anti-TfR antibody inhibits IFN-γ and IL-6 production from HAM patient PBMC.

Example 4 Examination of therapeutic effect of anti-TfR antibody on HU patients Furthermore, the possibility of treatment of HU, which is an HTLV-1-related inflammatory disease, by anti-TfR antibody was examined. In the same manner as described in Example 2, PUCs derived from HU patients (2 cases) were cultured, and TSP-A74, isotype control antibody Pad 1400 or PSL was added to PBMC cells to a final concentration of 1 μg / mL, Cultivation was performed in a 37 ° C. CO ₂ incubator. After the culture, the HTLV-1 proviral DNA level of the cells, the spontaneous growth activity of the cells, and changes in IFN-γ and IL-6 concentrations in the culture supernatant were analyzed in the same manner as in Examples 2 (1) to (3). .

  As a result, as shown in FIGS. 8 to 10, TSP-A74 significantly suppressed the amount of HTLV-1 provirus, cell proliferation, and production of IFN-γ and IL-6 in PBMC of HU patients. Therefore, the therapeutic potential of HU, which is an HTLV-1-related inflammatory disease of anti-TfR antibody, was also suggested.

Claims (8)

  A pharmaceutical composition for treating an inflammatory disease associated with HTLV-1, comprising as an active ingredient an anti-TfR antibody that specifically binds to a human transferrin receptor (TfR) or a fragment thereof.   The first complementarity determining region (CDR1), the second complementarity determining region (CDR2) and the third complementarity determining region (CDR3) of the heavy chain variable region (VH) of the anti-TfR antibody are SEQ ID NOS: 1, 25 and 3 respectively. Or an amino acid sequence in which one or several amino acids are deleted, added, substituted and / or inserted in the amino acid sequence shown in SEQ ID NOs: 1, 25 and 3, and the light chain variable region (VL ) In which the first complementarity determining region (CDR1), the second complementarity determining region (CDR2) and the third complementarity determining region (CDR3) are represented by SEQ ID NOs: 4, 5 and 6, respectively, or SEQ ID NO: 4 The pharmaceutical composition according to claim 1, which is an amino acid sequence in which one or several amino acids are deleted, added, substituted and / or inserted in the amino acid sequences represented by 5 and 6.   The first complementarity determining region (CDR1), the second complementarity determining region (CDR2), and the third complementarity determining region (CDR3) of the heavy chain variable region (VH) of the anti-TfR antibody are SEQ ID NOs: 7, 8, and 9, respectively. Or an amino acid sequence in which one or several amino acids are deleted, added, substituted and / or inserted in the amino acid sequence shown in SEQ ID NOs: 7, 8 and 9, and the light chain variable region (VL ) In which the first complementarity determining region (CDR1), the second complementarity determining region (CDR2) and the third complementarity determining region (CDR3) are represented by SEQ ID NOs: 10, 11 and 12, respectively, or SEQ ID NO: 10 The pharmaceutical composition according to claim 1, wherein one or several amino acids are deleted, added, substituted and / or inserted in the amino acid sequences represented by 11 and 12 .   The first complementarity determining region (CDR1), the second complementarity determining region (CDR2) and the third complementarity determining region (CDR3) of the heavy chain variable region (VH) of the anti-TfR antibody are SEQ ID NOS: 13, 14 and 15 respectively. Or an amino acid sequence in which one or several amino acids are deleted, added, substituted and / or inserted in the amino acid sequence shown in SEQ ID NOs: 13, 14 and 15, and the light chain variable region (VL ) In which the first complementarity determining region (CDR1), the second complementarity determining region (CDR2) and the third complementarity determining region (CDR3) are represented by SEQ ID NOs: 16, 17 and 18, respectively, or SEQ ID NO: 16 17 or 18, wherein one or several amino acids are deleted, added, substituted and / or inserted. The pharmaceutical composition according.   The pharmaceutical composition according to any one of claims 1 to 4, wherein the anti-TfR antibody is a human antibody. The anti-TfR antibody consists of a peptide comprising Fab, Fab ′, F (ab ′) ₂ , single chain antibody (scFv), dimerized V region (Diabody), disulfide stabilized V region (dsFv) and CDR. The pharmaceutical composition according to any one of claims 1 to 5, which is an antibody fragment selected from the group.   The pharmaceutical composition according to any one of claims 1 to 6, wherein the inflammatory disease associated with HTLV-1 is HTLV-1 associated myelopathy.   The pharmaceutical composition according to any one of claims 1 to 6, wherein the inflammatory disease associated with HTLV-1 is HTLV-1-associated uveitis.