WO2015020193A1 - 新規抗ヒトtslp受容体抗体 - Google Patents
新規抗ヒトtslp受容体抗体 Download PDFInfo
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- WO2015020193A1 WO2015020193A1 PCT/JP2014/071008 JP2014071008W WO2015020193A1 WO 2015020193 A1 WO2015020193 A1 WO 2015020193A1 JP 2014071008 W JP2014071008 W JP 2014071008W WO 2015020193 A1 WO2015020193 A1 WO 2015020193A1
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- tslp receptor
- antibody
- human tslp
- human
- receptor antibody
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Definitions
- the present invention relates to a novel anti-human TSLP receptor antibody.
- Thymic stromal lymphopoietin is an epithelial cell-derived cytokine produced in response to pro-inflammatory stimuli.
- TSLP Thymic stromal lymphopoietin
- Dendritic cells express the hematopoietin receptor family members TSLP receptor and IL-7 receptor ⁇ chain, and TSLP accepts a heterodimer consisting of TSLP receptor and IL-7 receptor ⁇ chain as a receptor. Binds and activates dendritic cells.
- Dendritic cells activated by TSLP are thymus and activation regulated chemokine (TARC (CCL17)), macrophage-derived chemokine (MDC (CCL22)), etc. It is expressed (Nat. Immunol., 2002, Vol. 7, p. 673-680).
- TARC and MDC are Th2 chemokines and are known to attract Th2 cells to sites of inflammation (Int. Immunol., 1999, Vol. 11, p. 81-88).
- dendritic cells activated by TSLP strongly induce naive T cells to differentiate into Th2 type, and these Th2 cells produce IL-4, IL-5, IL-13, and TNF ⁇ , and have an inflammatory response (Nat. Immunol., 2002, Vol. 7, p. 673-680).
- TSLP is overexpressed in the skin of patients with systemic scleroderma (Arthritis Rheum., 2013, Vol. 65, p. 1335-1346), and TSLP receptor
- a bleomycin-induced scleroderma model test using knockout mice the expression of IL-13 and IL-17 is almost completely suppressed in the local area of skin inflammation, and the proportion of collagen in the pathological image is significantly improved.
- IL-13 and IL-17 has been reported (Ann. Rheum. Dis., 2013, Vol. 72, p. 2018-23).
- the antibodies to human TSLP receptor that have been studied so far include mouse monoclonal antibody 13H5 and its humanized antibody hu13H5 (Patent Document 1), and mouse monoclonal antibody 1D6.
- TSLPR-012_141 has the highest neutralizing activity among the antibodies described in these documents (Patent Documents 2 to 4). TSLPR-012_141 has been evaluated in various neutralization activity tests.
- TSLP-stimulated human TSLP receptor stable expression Ba / F3 cell proliferation assay system For example, TSLP-stimulated human peripheral blood-derived dendritic cell TARC, MDC and IL-8 production assay system, and TSLP-stimulated human peripheral blood-derived tree It has been confirmed that TSLPR-012 — 141 exhibits neutralizing activity in a Th2 cytokine production assay system in dendritic cells and naive T cell co-culture systems (Patent Document 4). However, antibodies with higher neutralizing activity are desirable for use as antibody drugs.
- the main factors that define the effective dose of antibody drugs include binding activity and neutralization activity to the antigen of the antibody, and the amount of antigen present in the body, but improving the binding activity and neutralization activity It can be said that this is a very beneficial improvement that leads to a reduction in dose and as a result also reduces the patient's economic burden and medical costs.
- An object of the present invention is to provide an anti-human TSLP receptor antibody that specifically binds to a human TSLP receptor and inhibits the action of human TSLP via the human TSLP receptor.
- an anti-human TSLP receptor antibody comprising a light chain variable region consisting of the amino acid sequence of Nos. 1 to 108 was provided.
- the anti-human TSLP receptor antibody inhibits the expression of TARC mRNA and the production of MDC protein induced by TSLP (Examples 5 and 6), and further suppresses allergic reactions in a simian ascaris antigen sensitization model (Examples) 7) It was clarified that the present invention was completed.
- this invention includes the following invention as a medically or industrially useful substance or method.
- Anti-human TSLP receptor comprising a heavy chain variable region comprising the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 3
- Antibody or antigen-binding fragment thereof
- the anti-human TSLP receptor antibody according to (1) above comprising a heavy chain consisting of the amino acid sequence of amino acid numbers 1 to 447 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 3. .
- a polynucleotide comprising a base sequence encoding the heavy chain variable region of the antibody according to (1) above.
- a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody according to (1) above.
- An expression vector comprising the polynucleotide according to (8) and / or (9) above.
- (11) A host cell transformed with the expression vector according to (10) above, which is selected from the group consisting of the following (a) to (d).
- a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TSLP receptor antibody described in (1) above and a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody A host cell transformed with an expression vector;
- B an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TSLP receptor antibody according to (1) above and a poly comprising the base sequence encoding the light chain variable region of the antibody
- C a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TSLP receptor antibody according to (1) above; and
- D (1) above A host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of the anti-
- a method for producing an anti-human TSLP receptor antibody or an antigen-binding fragment thereof comprising a step of culturing the host cell according to (11) and expressing an anti-human TSLP receptor antibody or an antigen-binding fragment thereof.
- a method for producing an anti-human TSLP receptor antibody comprising culturing the host cell according to (12) above and expressing the anti-human TSLP receptor antibody.
- a fusion with another peptide or protein or a modifying agent is bound. Modifications are also included.
- the anti-human TSLP receptor antibody of the present invention binds to the human TSLP receptor and has a neutralizing activity on the action of human TSLP via the human TSLP receptor, and is suitable for allergic inflammatory diseases such as asthma and systemic It can be used as a preventive or therapeutic agent for scleroderma.
- FIG. 6 shows the effect of reducing the concentration of Ascaris antigen-specific IgE in a fully human T7-27 monkey sensitization model.
- the vertical axis indicates the relative plasma Ascaris antigen-specific IgE concentration of each sample when the plasma Ascaris antigen-specific IgE concentration in Day 22 of one individual in the Vehicle group is 2000 U / mL.
- the IgE concentration of Day 1 (before administration of Ascaris antigen solution suspended in aluminum hydroxide gel) in the Vehicle group and the antibody administration group, the IgE concentration of Day 22 in the Vehicle group, and the IgE concentration of Day 22 in the antibody administration group are shown.
- FIG. 1 before administration of Ascaris antigen solution suspended in aluminum hydroxide gel
- FIG. 5 shows the action of reducing Ascaris antigen-specific skin reaction in a fully human T7-27 sensitized model of S. acaris antigen.
- the results when phosphate buffered saline (PBS) and 100 ⁇ g / mL Ascaris antigen solution were administered to Day 22 are shown.
- the vertical axis represents the value (delta cm) obtained by subtracting the diameter of the skin reaction after administration of PBS from the diameter of the skin reaction after administration of Ascaris antigen solution.
- mouth asthma model of an anti- TSLP receptor antibody is shown.
- the vertical axis indicates the value of PenH was used as an indicator of respiratory function ( ⁇ p ⁇ 0.01, ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01, ⁇ p ⁇ 0.05).
- 2 shows the infiltration inhibitory effect of eosinophils in a mouse asthma model of an anti-TSLP receptor antibody.
- the vertical axis represents the number of eosinophils in the BALF cell suspension ( *** p ⁇ 0.01).
- 2 shows TARC mRNA expression inhibitory action in a mouse asthma model of anti-TSLP receptor antibody.
- the vertical axis shows the expression level of TARC mRNA.
- the basic structure of antibody molecules is the same for each class, with a heavy chain with a molecular weight of 50,000 to 70,000 and a light chain with 20,000 to 30,000. Consists of The heavy chain is usually composed of a polypeptide chain containing about 440 amino acids, has a characteristic structure for each class, and corresponds to IgG, IgM, IgA, IgD, IgE, Ig ⁇ , Ig ⁇ , Ig ⁇ , Ig ⁇ . It is called.
- IgG has IgG1, IgG2, IgG3, and IgG4 subclasses, and the corresponding heavy chains are called Ig ⁇ 1, Ig ⁇ 2, Ig ⁇ 3, and Ig ⁇ 4.
- the light chain is usually composed of a polypeptide chain containing about 220 amino acids, and two types of L-type and K-type are known, and are called Ig ⁇ and Ig ⁇ , respectively.
- the peptide structure of the basic structure of an antibody molecule has a molecular weight of 150,000 to 190,000, in which two heavy chains and two light chains that are homologous are linked by a disulfide bond (SS bond) and a non-covalent bond, respectively. .
- the two light chains can be paired with any heavy chain.
- Each antibody molecule always consists of two identical light chains and two identical heavy chains.
- a domain located on the amino-terminal (N-terminal) side of both heavy chain and light chain is called a variable region because its amino acid sequence is not constant even if it is a specimen from the same class (subclass) of the same species.
- Each domain is called a heavy chain variable region (V H ) and a light chain variable region (V L ).
- V H heavy chain variable region
- V L light chain variable region
- the amino acid sequence on the carboxy terminus (C terminus) side of the variable region is almost constant for each class or subclass and is called a constant region (each domain has C H 1, C H 2, C H 3, or C L ).
- the antigenic determinant site of an antibody is composed of V H and V L , and the specificity of binding depends on the amino acid sequence of this site.
- biological activities such as binding to complement and various cells reflect differences in the structure of the constant region of each class Ig. It has been found that the variability of the light chain and heavy chain variable regions is almost limited to the three small hypervariable regions present in both chains, these regions being complementarity determining regions (CDRs; each N-terminal). From the side, it is called CDR1, CDR2, CDR3). The remaining part of the variable region is called the framework region (FR) and is relatively constant.
- CDRs complementarity determining regions
- Various antigen-binding fragments including V H and V L of antibodies also have antigen-binding activity.
- typical antigen-binding fragments single-chain variable region fragments (scFv), Fab, Fab ′, F (ab ') 2 is mentioned.
- Fab is a light chain, VH, composed of heavy chain fragments comprising a portion of the C H 1 domain and hinge region, an antibody, a monovalent fragment.
- Fab ′ is a monovalent antibody fragment composed of a heavy chain fragment comprising a light chain, a VH, C H 1 domain, and a part of the hinge region. Includes the cysteine residues that made up the -S bond.
- An F (ab ′) 2 fragment is a bivalent antibody fragment in which two Fab ′ fragments are joined by an inter-heavy chain SS bond in the hinge region.
- scFv is a monovalent antibody fragment composed of V H and V L linked by a linker.
- the anti-human TSLP receptor antibody or antigen-binding fragment thereof of the present invention is an anti-human TSLP receptor antibody or antigen-binding fragment thereof having the following characteristics.
- An anti-human TSLP receptor antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1 from amino acid number 1 to 118 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 3 to amino acid number 1 to 108; Its antigen-binding fragment.
- the anti-human TSLP receptor antibody of the present invention or a binding fragment thereof has the above characteristics and further comprises a heavy chain constant region and a light chain constant region.
- a heavy chain constant region any subclass constant region (eg, Ig ⁇ 1, Ig ⁇ 2, Ig ⁇ 3, or Ig ⁇ 4 constant region as a heavy chain constant region, and Ig ⁇ or Ig ⁇ constant region as a light chain constant region) may be selectable.
- the human Ig ⁇ 1 constant region is preferred as the heavy chain constant region, and the human Ig ⁇ constant region is preferred as the light chain constant region.
- human Ig ⁇ 1 constant region examples include a human Ig ⁇ 1 constant region comprising the amino acid sequence of amino acid numbers 119 to 448 of SEQ ID NO: 1.
- human Ig ⁇ constant region examples include a human Ig ⁇ constant region consisting of the amino acid sequence from amino acid numbers 109 to 214 of SEQ ID NO: 3.
- the anti-human TSLP receptor antibody or antigen-binding fragment thereof of the present invention includes the above heavy chain variable region and light chain variable region, the heavy chain constant region is a human Ig ⁇ 1 constant region, and the light chain constant region is human Ig ⁇ . More preferred are anti-human TSLP receptor antibodies or antigen-binding fragments thereof that are constant regions.
- the antigen-binding fragment of the invention is scFv, Fab, Fab ′, or F (ab ′) 2 .
- a person skilled in the art can produce a fusion of an antibody or antigen-binding fragment thereof with another peptide or protein using a method known in the art, or a modified form in which a modifying agent is bound. Is also possible.
- the antibody or antigen-binding fragment thereof of the present invention includes such a fusion or modified form of the antibody or antigen-binding fragment thereof.
- an anti-human TSLP receptor comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1 from amino acid number 1 to 118 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 3 to amino acid number 1 to 108
- the antibody or antigen-binding fragment thereof includes a fusion of the anti-human TSLP receptor antibody or binding fragment thereof with another peptide or protein, or a modified form in which a modifying agent is bound.
- Other peptides and proteins used for the fusion are not particularly limited as long as they do not reduce the binding activity of the antibody or antigen-binding fragment thereof.
- human serum albumin various tag peptides, artificial helix motif peptide, maltose binding protein , Glutathione S transferase, various toxins, and other peptides or proteins that can promote multimerization.
- the modifying agent used for the modification is not particularly limited as long as it does not reduce the binding activity of the antibody or antigen-binding fragment thereof, and examples thereof include polyethylene glycol, sugar chain, phospholipid, liposome, and low molecular compound.
- the anti-human TSLP receptor antibody of the present invention is an anti-human TSLP receptor antibody having the following characteristics.
- An anti-human TSLP receptor antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3.
- the antibody of the present invention includes not only an antibody having a full-length heavy chain, but also an antibody having a heavy chain lacking the C-terminal lysine, glutamine or glutamic acid at the N-terminal heavy chain modified to pyroglutamic acid by pyroglutamylation. Also included are antibodies that have been post-translationally modified upon expression in the cell, such as received antibodies.
- the antigen-binding fragment of the present invention is subject to post-translational modification upon expression in the cell, such as an antigen-binding fragment in which glutamine or glutamic acid at the N-terminus of the antigen-binding fragment is modified to pyroglutamate by pyroglutamylation. Also included are antigen-binding fragments.
- the anti-human TSLP receptor antibody of the present invention includes the anti-human TSLP receptor antibody described below.
- a heavy chain consisting of an amino acid sequence in which the glutamic acid of amino acid number 1 of SEQ ID NO: 1 is modified with pyroglutamic acid and / or lacking the lysine of amino acid number 448 of SEQ ID NO: 1, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3 An anti-human TSLP receptor antibody.
- the present invention also includes an anti-human TSLP receptor antibody or antigen-binding fragment thereof having the following characteristics.
- CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 1
- CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 1
- a heavy chain variable region comprising CDR3, CDR1 comprising the amino acid sequence of amino acid numbers 24 to 34 of SEQ ID NO: 3, CDR2 comprising the amino acid sequence of amino acid numbers 50 to 56 of SEQ ID NO: 3, and SEQ ID NO: 3
- the anti-human TSLP receptor antibody or antigen-binding fragment thereof of the present invention binds to human TSLP receptor. Whether an antibody or an antigen-binding fragment binds to a human TSLP receptor can be confirmed using a known binding activity measurement method. Examples of the method for measuring the binding activity include methods such as Enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR).
- ELISA Enzyme-linked immunosorbent assay
- SPR surface plasmon resonance
- a protein obtained by fusing human TSLP receptor and human Fc human TSLP receptor-human Fc fusion protein (encoded by the base sequence of SEQ ID NO: 5)
- the test antibody is added thereto and reacted.
- a secondary antibody such as anti-IgG antibody labeled with an enzyme such as horseradish peroxidase (HRP) is reacted, washed, and then a reagent for detecting its activity (for example, in the case of HRP labeling, BM-Chemiluminescence ELISA Substrate). Whether or not the test antibody binds to the human TSLP receptor can be confirmed by measuring the activity using (POD) (Roche Diagnostics).
- POD horseradish peroxidase
- SPR for example, Biacore (registered trademark) 2000 (GE Healthcare Japan) can be used.
- a test antibody is immobilized on the surface of a sensor chip, and a human TSLP receptor and mouse Fc fusion protein (human TSLP receptor-mouse Fc fusion protein (encoded by the base sequence of SEQ ID NO: 6) is used. To be added to the flow path. By analyzing the association rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD) between the antibody and the human TSLP receptor, it is confirmed whether or not the test antibody binds to the human TSLP receptor. be able to.
- ka association rate constant
- kd dissociation rate constant
- KD dissociation constant
- the antibody or antigen-binding fragment of the present invention includes an antibody or antigen-binding fragment thereof that also binds to TSLP receptors derived from other animals (for example, monkey TSLP receptor).
- the binding activity to the receptor can be measured.
- the anti-human TSLP receptor antibody or antigen-binding fragment thereof of the present invention binds to human TSLP receptor and has neutralizing activity against human TSLP receptor.
- the neutralizing activity for human TSLP receptor means an activity that inhibits any biological activity caused by binding of human TSLP to human TSLP receptor by binding to human TSLP receptor.
- One or more biological activities of human TSLP through the body can be assessed as an indicator. Examples of such neutralizing activity include TSLP-induced TARC mRNA expression inhibitory activity and TSLP-induced MDC protein production inhibitory activity using human peripheral blood mononuclear cells (PBMC).
- PBMC peripheral blood mononuclear cells
- In vivo tests can also be used to evaluate the effects of the anti-human TSLP receptor antibody or antigen-binding fragment thereof of the present invention in more detail.
- the in vivo efficacy of the anti-human TSLP receptor antibody can be evaluated using an anti-allergic reaction test using a simian squalis antigen sensitization model.
- the anti-human TSLP receptor antibody or antigen-binding fragment thereof of the present invention is based on the sequence information of the heavy chain variable region and light chain variable region of the anti-human TSLP receptor antibody of the present invention disclosed in the present specification. It can be readily made by one skilled in the art using methods known in the art.
- the anti-human TSLP receptor antibody or antigen-binding fragment thereof of the present invention is not particularly limited. For example, it is produced according to the method described in ⁇ Method for producing anti-human TSLP receptor antibody of the present invention> described later. can do.
- the anti-human TSLP receptor antibody or antigen-binding fragment thereof of the present invention is further purified if necessary, and then formulated in accordance with a conventional method, and human TSLP such as allergic inflammatory diseases such as asthma and systemic scleroderma and
- human TSLP receptor can be used for the prevention or treatment of diseases associated with pathogenesis.
- the polynucleotide of the present invention encodes a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TSLP receptor antibody of the present invention, and the light chain variable region of the anti-human TSLP receptor antibody of the present invention.
- a polynucleotide containing a base sequence to be included is included.
- the polynucleotide comprising the base sequence encoding the heavy chain variable region of the anti-human TSLP receptor antibody of the present invention is a heavy chain variable region comprising the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 1.
- polynucleotide containing the base sequence encoding the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 1 from amino acid Nos. 1 to 118 include, for example, a polynucleotide comprising the base sequence of SEQ ID NO: 2 from base No. 1 to 354 Nucleotides are mentioned.
- the polynucleotide comprising the base sequence encoding the heavy chain variable region of the anti-human TSLP receptor antibody of the present invention is a polynucleotide comprising the base sequence encoding the heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1. It is a nucleotide.
- Examples of the polynucleotide containing the base sequence encoding the heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 include the polynucleotide containing the base sequence shown in SEQ ID NO: 2.
- the polynucleotide comprising the base sequence encoding the light chain variable region of the anti-human TSLP receptor antibody of the present invention is a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 3.
- polynucleotide containing the base sequence encoding the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 3 from amino acid number 1 to 108 include, for example, the polynucleotide comprising the base sequence of base number 1 to 324 of SEQ ID NO: 4 Is mentioned.
- the polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human TSLP receptor antibody of the present invention comprises a base sequence encoding a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3. It is a nucleotide.
- Examples of the polynucleotide containing the base sequence encoding the light chain consisting of the amino acid sequence shown in SEQ ID NO: 3 include the polynucleotide containing the base sequence shown in SEQ ID NO: 4.
- the polynucleotide of the present invention can be easily prepared by those skilled in the art using a method known in the art based on the base sequence.
- the polynucleotide of the present invention can be synthesized using gene synthesis methods known in the art. Examples of such gene synthesis methods include various methods known to those skilled in the art, such as antibody gene synthesis methods described in WO90 / 07861.
- the expression vector of the present invention encodes a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TSLP receptor antibody of the present invention and / or the light chain variable region of the anti-human TSLP receptor antibody of the present invention.
- Preferred expression vectors of the present invention include an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TSLP receptor antibody of the present invention, and encodes the light chain of the anti-human TSLP receptor antibody of the present invention.
- An expression vector comprising a polynucleotide comprising a base sequence, or a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TSLP receptor antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody And an expression vector containing.
- Expression vectors used for expressing the polynucleotide of the present invention include various host cells such as eukaryotic cells (eg, animal cells, insect cells, plant cells, yeast) and / or prokaryotic cells (eg, E. coli).
- eukaryotic cells eg, animal cells, insect cells, plant cells, yeast
- prokaryotic cells eg, E. coli
- expression vectors examples include plasmid vectors and viral vectors (eg, adenovirus, retrovirus), and preferably, pEE6.4 or pEE12.4 (Lonza) can be used.
- viral vectors eg, adenovirus, retrovirus
- pEE6.4 or pEE12.4 Longza
- an antibody gene can be expressed by introducing a variable region gene fragment into an expression vector having a human Ig constant region gene in advance, such as AG- ⁇ 1 and AG- ⁇ (for example, see WO94 / 20632).
- the expression vector of the present invention may contain a promoter operably linked to the polynucleotide of the present invention.
- promoters for expressing the polynucleotide of the present invention in animal cells include virus-derived promoters such as CMV, RSV, SV40, actin promoter, EF (longation factor) 1 ⁇ promoter, heat shock promoter, and the like.
- the promoter for expression in bacteria include trp promoter, lac promoter, ⁇ PL promoter, tac promoter, and the like.
- promoters for expression in yeast include GAL1 promoter, GAL10 promoter, PH05 promoter, PGK promoter, GAP promoter, and ADH promoter.
- the transformed host cell of the present invention includes a host cell transformed with the expression vector of the present invention selected from the group consisting of the following (a) to (d).
- A an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TSLP receptor antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody Transformed host cells
- B an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TSLP receptor antibody of the present invention and an expression comprising a polynucleotide comprising the base sequence encoding the light chain variable region of the antibody
- C a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TSLP receptor antibody of the present invention; and (d) the
- the transformed host cell of the present invention is a host cell transformed with the expression vector of the present invention selected from the group consisting of the following (a) to (d).
- B Transformation with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TSLP receptor antibody of the present invention and an expression vector comprising a polynucleotide comprising the base sequence encoding the light chain of the antibody Host cells;
- C a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TSLP receptor antibody of the present invention; and (d) the anti-human TS
- the expression vector of the present invention may contain a start codon and a stop codon.
- the expression vector of the present invention comprises an enhancer sequence, the antibody of the present invention or the 5 ′ and 3 ′ untranslated regions of the gene encoding the heavy chain variable region or light chain variable region thereof, secretory signal sequence, splicing It may contain a junction, a polyadenylation site, or a replicable unit.
- the expression vector of the present invention may contain a start codon, a stop codon, a terminator region, and a replicable unit.
- the expression vector of the present invention may contain a selection marker (for example, tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene, neomycin resistance gene, dihydrofolate reductase gene) that is usually used depending on the purpose. .
- a selection marker for example, tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene, neomycin resistance gene, dihydrofolate reductase gene
- Preferred transformed host cells of the present invention include a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TSLP receptor antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody;
- a host cell transformed with an expression vector comprising a nucleotide sequence and an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding the heavy chain of the anti-human TSLP receptor antibody of the present invention and a nucleotide sequence encoding the light chain of the antibody
- the host cell to be transformed is not particularly limited as long as it is compatible with the expression vector to be used and can be transformed with the expression vector to express the antibody.
- Examples of host cells to be transformed include various cells (for example, animal cells (for example, CHO-K1SV cells), insect cells, etc., such as natural cells or artificially established cells usually used in the technical field of the present invention. (For example, Sf9), bacteria (such as Escherichia), yeast (such as Saccharomyces, Pichia), and the like.
- cultured cells such as CHOK1SV cells, CHO-DG44 cells, 293 cells, and NS0 cells are used. Can be used.
- the method for transforming the host cell is not particularly limited, and for example, a calcium phosphate method, an electroporation method, or the like can be used.
- the method for producing the anti-human TSLP receptor antibody or antigen-binding fragment thereof of the present invention comprises the steps of culturing the transformed host cell of the present invention and expressing the anti-human TSLP receptor antibody or antigen-binding fragment thereof. Methods of producing an anti-human TSLP receptor antibody or antigen-binding fragment thereof are included.
- the anti-human TSLP receptor antibody or antigen-binding fragment thereof of the present invention includes an anti-human TSLP receptor antibody or antigen-binding fragment thereof produced by the method of producing the anti-human TSLP receptor antibody or antigen-binding fragment of the present invention. Is also included.
- the method for producing the anti-human TSLP receptor antibody or antigen-binding fragment thereof of the present invention comprises culturing the transformed host cell of the present invention and expressing the anti-human TSLP receptor antibody or antigen-binding fragment thereof. As long as it is, it is not particularly limited.
- Preferred host cells used in the method include the above-described preferred transformed host cells of the present invention.
- the cultured host cells can be cultured by a known method.
- the culture conditions such as temperature, medium pH, and culture time are appropriately selected.
- the medium include MEM medium containing about 5 to 20% fetal bovine serum (Science, 1959, Vol. 130, No. 3373, p. 432-7), DMEM medium ( Virology, 1959, Vol. 8, p. 396), RPMI 1640 medium (J. Am. Med. Assoc., 1967, Vol. 199, p. 519), 199 medium (Exp. Biol. Med., 1950, Vol. 73, p.1-8) and the like can be used.
- the pH of the medium is preferably about 6 to 8, and the culture is usually carried out at about 30 to 40 ° C. for about 15 to 72 hours with aeration and stirring as necessary.
- the host cell is an insect cell, for example, Grace's medium containing fetal bovine serum (Proc. Natl. Acad. Sci. USA, 1985, Vol. 82, p. 8404) can be used as the medium. .
- the pH of the medium is preferably about 5 to 8, and the culture is usually carried out at about 20 to 40 ° C. for about 15 to 100 hours with aeration and agitation as necessary.
- a liquid medium containing a nutrient source is appropriate.
- the nutrient medium preferably contains a carbon source, inorganic nitrogen source or organic nitrogen source necessary for the growth of the transformed host cell.
- the carbon source include glucose, dextran, soluble starch, and sucrose.
- the inorganic nitrogen source or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, and meat extract. , Soybean meal, potato extract and the like.
- other nutrients eg, inorganic salts (eg, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, etc.), antibiotics (eg, tetracycline, neomycin, ampicillin, kanamycin, etc.) Good.
- the pH of the medium is preferably about 5-8.
- preferred media include LB medium, M9 medium (Mol. Clo., Cold Spring Harbor Laboratory, Vol. 3, A2.2) and the like. Cultivation is usually carried out at about 14 to 43 ° C. for about 3 to 24 hours with aeration or agitation as necessary.
- yeast for example, a Burkholder minimum medium (Proc. Natl. Acad. Sci. USA, 1980, Vol. 77, p. 4505) can be used as the medium. Cultivation is usually carried out at about 20 to 35 ° C. for about 14 to 144 hours with aeration or agitation as necessary.
- the anti-human TSLP receptor antibody of the present invention or an antigen-binding fragment thereof can be expressed.
- the method of producing the anti-human TSLP receptor antibody or antigen-binding fragment thereof of the present invention includes the step of culturing the transformed host cell of the present invention and expressing the anti-human TSLP receptor antibody or antigen-binding fragment thereof. In addition, it may further comprise a step of recovering, preferably isolating or purifying the anti-human TSLP receptor antibody or antigen-binding fragment thereof from the transformed host cell. Isolation or purification methods include, for example, methods using solubility such as salting out and solvent precipitation, methods using differences in molecular weight such as dialysis, ultrafiltration and gel filtration, ion exchange chromatography and hydroxylapatite chromatography.
- the antibody accumulated in the culture supernatant can be purified by various chromatography, for example, column chromatography using a protein A column or a protein G column.
- the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising the anti-human TSLP receptor antibody of the present invention or an antigen-binding fragment thereof and a pharmaceutically acceptable excipient.
- the pharmaceutical composition of the present invention can be prepared by an ordinarily used method using an excipient usually used in the art, that is, a pharmaceutical excipient or a pharmaceutical carrier.
- Examples of dosage forms of these pharmaceutical compositions include parenteral agents such as injections and infusions, and can be administered by intravenous administration, subcutaneous administration, or the like.
- excipients, carriers, additives and the like corresponding to these dosage forms can be used within a pharmaceutically acceptable range.
- the pharmaceutical composition of the present invention may contain a plurality of types of anti-human TSLP receptor antibodies of the present invention or antigen-binding fragments thereof.
- a pharmaceutical composition containing an antibody having a chain C-terminal lysine and not subjected to N-terminal post-translational modification is also included in the present invention.
- the pharmaceutical composition of the present invention containing the anti-human TSLP receptor antibody of the present invention includes the pharmaceutical composition containing two or more anti-human TSLP receptor antibodies of the following (1) to (4): Is also included.
- An anti-human TSLP receptor antibody comprising a heavy chain comprising the amino acid sequence of amino acid numbers 1 to 447 of SEQ ID NO: 1 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 3.
- An anti-human TSLP receptor comprising an amino acid sequence represented by SEQ ID NO: 1 and comprising a heavy chain in which glutamic acid of amino acid number 1 is modified with pyroglutamic acid and a light chain comprising the amino acid sequence represented by SEQ ID NO: 3. antibody.
- the amount of the anti-human TSLP receptor antibody or antigen-binding fragment thereof of the present invention in the formulation varies depending on the degree and age of the patient's symptoms, the dosage form of the formulation to be used, the binding titer of the antibody, etc. About 0.001 mg / kg to 100 mg / kg can be used.
- the pharmaceutical composition of the present invention can be used as a pharmaceutical composition for preventing or treating diseases in which human TSLP and human TSLP receptor are involved in pathogenesis, such as asthma.
- the present invention includes a pharmaceutical composition for preventing or treating asthma comprising the anti-human TSLP receptor antibody of the present invention or an antigen-binding fragment thereof and a pharmaceutically acceptable excipient.
- the present invention also includes a method for preventing or treating asthma comprising the step of administering a therapeutically effective amount of the anti-human TSLP receptor antibody or antigen-binding fragment thereof of the present invention.
- the present invention also includes the anti-human TSLP receptor antibody of the present invention or an antigen-binding fragment thereof for use in the prevention or treatment of asthma.
- the present invention also includes the use of the anti-human TSLP receptor antibody of the present invention or an antigen-binding fragment thereof in the manufacture of a pharmaceutical composition for preventing or treating asthma.
- concentration mol / L is expressed as M.
- 1M sodium hydroxide aqueous solution means 1 mol / L sodium hydroxide aqueous solution.
- Example 1 Acquisition of TSLP receptor-Fc fusion protein
- human TSLP receptor-human Fc fusion protein a protein in which human TSLP receptor and human Fc are fused
- human TSLP human TSLP receptor and mouse Fc are fused
- human TSLP human TSLP Receptor-mouse Fc fusion protein
- human TSLP monkey TSLP receptor and human Fc
- Human TSLP receptor-human Fc fusion gene (SEQ ID NO: 5), human TSLP receptor-mouse Fc fusion gene (SEQ ID NO: 6) and monkey TSLP receptor-human Fc fusion gene (SEQ ID NO: 7) vector for mammalian cell expression Recombined with GS vector (Lonza) pEE12.4.
- the prepared vector was transfected into FreeStyle 293 cells (Life technologies) using a gene introduction reagent 293fectin (Life technologies), and the cells were transferred to FreeStyle 293 Expression medium (without using Life Technologies serum system).
- culture supernatants containing human TSLP receptor-human Fc fusion protein, human TSLP receptor-mouse Fc fusion protein or monkey TSLP receptor-human Fc fusion protein were obtained.
- Each TSLP receptor-Fc fusion protein was purified from the obtained culture supernatant using a protein purification column Protein G column (GE Healthcare Japan).
- Example 2 Acquisition of TSLP mutant-Flag protein
- Human or monkey TSLP variant-Flag gene (SEQ ID NO: 8 or 9 (in order to prevent loss of activity due to cleavage by furin protease, the amino acid sequence of human or monkey wild-type TSLP having a mutation inserted at its cleavage site) Respectively encoded)) was recombined into GS vector pEE12.4.
- the prepared vector was transfected into FreeStyle 293 cells using 293fectin, and the cells were cultured in a serum-free culture system using FreeStyle 293 Expression medium for 1 week, and then human TSLP mutant-Flag protein or monkey TSLP mutant. -Each culture supernatant containing Flag protein was obtained. Each TSLP mutant-Flag protein was purified from the obtained culture supernatant using an anti-FLAG M2 antibody affinity gel (Sigma).
- Example 3 Production of fully human anti-human TSLP receptor antibody
- Human monoclonal antibody development technology "Velosimune technology: Regeneron (US Pat. No. 6,596,541)"
- / F3 cells vectors encoding human TSLP receptor gene (SEQ ID NO: 10) and human IL-7 receptor ⁇ chain gene (SEQ ID NO: 11) are introduced into mouse Ba / F3 cells (RIKEN: RCB0805) Immunized).
- the spleen and lymph nodes of the immunized mouse were removed, lymphocytes were collected, and this was cell-fused with mouse myeloma cells SP2 / 0 (ATCC CRL-1581) to prepare a hybridoma.
- the hybridoma was monocloned and cultured in CD hybridoma medium (Life technologies), which is a serum-free medium.
- the antibody was purified from the obtained culture supernatant using an antibody purification kit Protein G Purification kit (Proteus).
- ELISA using the human TSLP receptor-human Fc fusion protein and monkey TSLP receptor-human Fc fusion protein prepared in Example 1 was performed. Further, in order to evaluate the neutralizing activity of the antibody, the cell growth inhibition assay of human TSLP receptor-expressing Ba / F3 cells stimulated with the monkey TSLP mutant-Flag protein prepared in Example 2, and the antibody prepared in Example 2 were used. The monkey TSLP mutant-Flag protein stimulated MDC protein production inhibition assay of monkey whole blood was performed.
- T7-27 (chimeric antibody) has binding activity and neutralizing activity for human and monkey TSLP receptors.
- Genes encoding the heavy and light chains of the antibody were cloned from the hybridoma producing T7-27 and sequenced.
- the aforementioned antibody is an antibody whose variable region is derived from human and whose constant region is derived from mouse. Therefore, an expression vector containing both heavy chain and light chain genes was constructed using a GS vector, and a fully human antibody was prepared. Specifically, a gene encoding a signal sequence (Nigel Whittle et al., Protein Engineering 1987; 1 (6): 499-505.) On the 5 ′ side of the heavy chain variable region gene of the T7-27 antibody, and 3 The human Ig ⁇ 1 constant region gene (consisting of the nucleotide sequence from nucleotide numbers 355 to 1344 of SEQ ID NO: 2) was linked to the 'side, and this heavy chain gene was inserted into the GS vector pEE6.4.
- a gene encoding a signal sequence is located 5 ′ of the light chain variable region gene of the antibody, and a human Ig ⁇ constant region gene (from base number 325 of SEQ ID NO: 4) on the 3 ′ side.
- Each light chain gene was inserted into the GS vector pEE12.4.
- the base sequence encoding the heavy chain of the prepared T7-27 fully human antibody (fully human T7-27) is shown in SEQ ID NO: 2, the amino acid sequence encoded thereby is shown in SEQ ID NO: 1, and the light chain of the antibody
- the nucleotide sequence encoding is shown in SEQ ID NO: 4, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 3.
- the heavy chain variable region of fully human T7-27 consists of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 1, and the heavy chain CDR1, CDR2, CDR3 are amino acid numbers 31 to 35 of SEQ ID NO: 1, respectively. It consists of an amino acid sequence from 50 to 66, 99 to 107.
- the light chain variable region of fully human T7-27 consists of amino acid sequences from amino acid numbers 1 to 108 of SEQ ID NO: 3, and the CDR1, CDR2, and CDR3 of the light chain are amino acid numbers 24 to 34 of SEQ ID NO: 3, It consists of an amino acid sequence from 50 to 56, 89 to 97.
- Antibody expression was performed by two methods, transient expression and constant expression, using the above-described GS vector into which the heavy chain and light chain genes of the antibody were inserted.
- transient expression FreeStyle 293 cells cultured at about 1 million cells / mL in FreeStyle 293 Expression medium were transfected with both the above heavy chain and light chain expression vectors using 293fectin. Cultured for days.
- CHO-K1SV cells Longza
- CD-CHO medium Life technologies
- the culture supernatant was purified using a protein A column or protein G column (GE Healthcare Japan) to obtain a purified antibody of a fully human antibody.
- the above-mentioned GS vector into which the antibody heavy chain and light chain genes were inserted was digested with NotI and PvuI, and ligation kit Ligation-Convenience Kit (NIPPONGENE) or ligation reagent Ligation- Ligation was performed using high (TOYOBO) to construct a GS vector in which both heavy chain and light chain genes were inserted.
- This expression vector encodes full-length heavy and light chains, and antibodies were expressed by transfection in CHO-K1SV cells.
- the culture supernatant was purified with a protein A column or a protein G column (GE Healthcare Japan) to obtain a purified antibody of a fully human antibody.
- Analysis of the amino acid modification of purified fully human T7-27 revealed that most of the purified antibody had a heavy chain C-terminal lysine deletion.
- Example 4 Evaluation of binding activity by SPR analysis
- an anti-human TSLP receptor antibody TSLPR-012_141 Patent Document 4
- TSLPR-012_141 Patent Document 4
- Example 5 TSLP-induced TARC mRNA expression inhibition evaluation using human PBMC
- Human PBMC also includes dendritic cells expressing TSLP receptor, and can be used for evaluation of anti-human TSLP receptor antibodies.
- TSLPR-012 — 141 was used as a comparative antibody. From the results of Test Example 1 described later, there was a correlation between the improvement of the asthma model by anti-TSLP receptor antibody and the inhibition of the expression of TARC mRNA in blood. It is a system.
- 200,000 human PBMC (AllCells) were seeded in a 96-well plate (Gleiner) with 160 ⁇ L of RPMI1640 medium (Life technologies).
- a dilution series of human TSLP variant-Flag protein prepared in Example 2 (7 steps in a final concentration range of 0.1 ng / mL to 100 ng / mL) was prepared in RPMI1640 medium and added to the culture solution at 20 ⁇ L. .
- each anti-human TSLP receptor antibody was prepared in RPMI 1640 medium to a final concentration of 0.3 ⁇ g / mL, and 20 ⁇ L was added to the culture solution.
- Taq's TaqMan probe (Ccl17, Hs00171074, Life technologies), ⁇ -actin TaqMan probe (Actb, Hs99999993, Life technologies), Express qPCR SuperMix (A10313, Lif)
- the expression level of TARC mRNA was measured by TaqMan PCR method. Each antibody was tested in duplicate, and the measurement results were analyzed by a comparative CT method to calculate the expression level of TARC mRNA. From there, the inhibition rate of the antibody at each TSLP concentration was calculated.
- the inhibition rate of wells to which RPMI1640 medium was added instead of human TSLP variant-Flag protein was set as 100%, and the average value of wells to which human TSLP variant-Flag protein was added at 30 and 100 ng / ml was the inhibition rate of 0. Set as%.
- the calculated inhibition rate was analyzed, and a TSLP concentration that could inhibit 50% of 0.3 ⁇ g / mL antibody was calculated by 3-parameter logistic curve fitting (Table 2). It means that the neutralization activity with respect to TSLP of a test antibody is so strong that this TSLP density
- Table 2 TSLP-induced TARC mRNA expression inhibitory activity using human PBMC
- Example 6 TSLP-induced MDC protein production inhibition evaluation using human PBMC
- TSLPR-012 — 141 TSLPR-012 — 141 was used as a comparative antibody.
- Human PBMC is prepared by diluting human blood with an equal volume of PBS, layering it on an equal volume of Ficoll-Paque PLUS (GE Healthcare Japan), and centrifuging at room temperature at 400 xg for 30 minutes. did. About 300,000 human PBMCs were seeded in a 96-well plate (Gleiner) with 100 ⁇ L of RPMI 1640 medium (Life technologies). The human TSLP variant-Flag protein prepared in Example 2 was prepared in RPMI 1640 medium to a final concentration of 5 ng / mL, and 10 ⁇ L was added to the culture solution. After incubating in a CO 2 incubator set at 37 ° C.
- dilution series of each anti-human TSLP receptor antibody (5 steps in the final concentration range from 0.1 ng / mL to 10 ⁇ g / mL) were added to RPMI1640 medium. 10 ⁇ L was prepared and added to the culture, and further incubated for 5 days.
- a well to which RPMI1640 medium was added instead of human TSLP mutant-Flag protein and a well to which RPMI1640 medium was added instead of antibody were prepared. Thereafter, the culture supernatant was collected, and the amount of MDC produced was evaluated using Human CCL22 / MDC Quantikine ELISA Kit (R & D) using the supernatant diluted 20-fold with PBS (Life technologies).
- T7-27 has about 9 times higher TSLP-induced MDC protein production inhibitory activity than TSLPR-012_141.
- Ascaris antigen-Alum solution Suspended antigen solution (hereinafter referred to as Ascaris antigen-Alum solution) was sensitized by administering 3.6 mL / kg intraperitoneally and 0.4 mL / kg intramuscularly.
- a Vehicle group a solvent (20 mM sodium citrate buffer / 120 mM NaCl (pH 6.0)
- n 3
- Alum was produced by the following method.
- Aluminum sulfate (14-18 hydrate) (Wako) was dissolved in ultrapure water to prepare a 1M solution, passed through a 0.22 ⁇ m filter, and 1M sodium hydroxide (Nacalai Tesque) was white. It was added until no precipitation occurred. The supernatant was removed, washed 5 times with ultrapure water, and further washed 3 times with PBS (WAKO). The washed white precipitate was finely pulverized with a homogenizer (KINEMATICA, CH-6010) under ice cooling. Next, after centrifugation at 2000 rpm for 5 minutes at 4 ° C. with a centrifuge (Hitachi, himac CR21), the supernatant was removed and washed twice with PBS. The obtained precipitate was suspended in PBS to obtain Alum.
- KINEMATICA homogenizer
- the solution was removed.
- 100 ⁇ L of each of the collected plasma and calibration curve sample was added.
- the plasma Ascalis antigen-specific IgE concentration in Day 22 of one individual in the Vehicle group was 2000 U / mL, and a dilution series (2000 U / mL to 16 U) prepared with a diluted solution (PBS containing 5% Blocking One) was used. / ML) was used.
- the value obtained by subtracting the diameter of the skin reaction after administration of PBS from the diameter of the skin reaction after administration of the Ascaris antigen solution was defined as delta cm.
- the results at the time of 100 ⁇ g / mL Ascaris antigen solution administration are shown in FIG. When 1 and 10 ⁇ g / mL Ascaris antigen solution was administered, a skin reaction sufficient to evaluate the test antibody was not induced.
- the fully human T7-27 decreased the Ascaris antigen-specific skin reaction when administered with a 100 ⁇ g / mL Ascaris antigen solution in the monkey Ascaris antigen sensitization model as compared with the Vehicle group.
- mice (Charles River) are intraperitoneally administered with Day 0 (first sensitization) and Day 5 with mite antigen (Dp) (LSL) at a dose of 100 ⁇ g Dp / 0.5 mL saline / mouse. The sensitization was carried out.
- Airway inflammation was induced by nasally administering Day 12 and Day 19 at a dose of 100 ⁇ g Dp / 50 ⁇ L physiological saline / mouse.
- PBS WAKO
- Antibody administration day was administered once a day at Day-1, Day2, Day5, Day8, Day11, Day14, and Day18 (antibody administration day) at each dose (0.1, 1, 10 mg / kg).
- Anti-mouse TSLP receptor antibody WAKO was dissolved subcutaneously.
- a dexamethasone administration group (Dex group) was provided as a positive control group.
- dexamethasone dissolved in PBS at a dose of 3 mg / kg was administered intraperitoneally once a day from Day 12 to Day 19.
- the set treatment groups are as follows.
- Dex group (n 10): Mite antigen was administered intraperitoneally to Day 0 and Day 5, mite antigen was administered nasally to Day 12 and Day 19, and dexamethasone was administered intraperiperitone
- the mouse was placed in Day 20 in a dedicated unconstrained chamber.
- a transducer using a respiratory anemometer attached to the chamber was connected to a respiratory function analyzer BioSystem XA (Buxco), and a change in the chamber internal pressure with respect to atmospheric pressure was detected by the transducer.
- BioSystem XA BioSystem XA
- acetyl- ⁇ -methylcholine chloride (Sigma) (0.25, 0.5, 1, 2, 4 mg / mL) dissolved in physiological saline was added. Inhalation exposure was performed while increasing the concentration in order.
- PenH calculated mechanically using changes in chamber pressure, was used as an indicator of respiratory function. The measurement results are shown in FIG.
- the blood obtained above was diluted 10-fold with RPMI 1640 medium (Life technologies) and seeded on a plate (IWAKI) (1 mL / well).
- Mouse TSLP R & D
- PBS PBS
- 100 ⁇ L was added.
- total RNA was extracted with 30 ⁇ L of water using RNeasy 96 kit (Qiagen).
- reverse transcription reaction of 10 ⁇ L of RNA was performed using High Capacity cDNA Reverse Transcription Kit (Life technologies).
- TARC's TaqMan probe (Ccl17, Mm01244826 g1, Life technologies), ⁇ -actin TaqMan probe (Actb, Mm006079939 s1, Life technologies Oligotech2L)
- the expression level of TARC mRNA was measured by TaqMan PCR method.
- the measurement results were analyzed by the comparative CT method, and the expression level was calculated.
- the results when 10 ng / mL TSLP was added are shown in FIG.
- the average value and standard error of each group were obtained.
- the Student-t test was used for a significant difference test between the PBS group, the Normal group, the Saline group, and the Dex group.
- Dunnett's multiple comparison was used for a significant difference test between the PBS group and the antibody administration group. In all cases, the case of p ⁇ 0.05 was considered significant.
- the antibody-administered group showed an inhibitory effect on the infiltration of eosinophils into the bronchoalveolar lung compared with the PBS group.
- the TARC mRNA expression inhibitory action was observed in the antibody-administered group compared to the PBS group.
- the anti-human TSLP receptor antibody of the present invention is useful for the prevention or treatment of various diseases in which human TSLP and human TSLP receptor are involved in pathogenesis.
- the polynucleotide, expression vector, host cell and production method of the present invention are useful for producing the anti-human TSLP receptor antibody.
- the base sequences shown in SEQ ID NOs: 2 and 4 in the sequence listing are the heavy and light chain base sequences of fully human T7-27, respectively, and the amino acid sequences shown in SEQ ID NOs: 1 and 3 are Are the heavy and light chain amino acid sequences encoded by SEQ ID NOs: 2 and 4, respectively.
- the nucleotide sequences represented by SEQ ID Nos. 5, 6, 7, 8 and 9 in the sequence listing are respectively human TSLP receptor-human Fc fusion protein, human TSLP receptor-mouse Fc fusion protein, and monkey TSLP receptor-human Fc fusion. It is a base sequence encoding protein, human TSLP mutant-Flag protein, and monkey TSLP mutant-Flag protein.
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Abstract
Description
(1)配列番号1のアミノ酸番号1から118までのアミノ酸配列からなる重鎖可変領域、及び配列番号3のアミノ酸番号1から108までのアミノ酸配列からなる軽鎖可変領域を含む、抗ヒトTSLP受容体抗体又はその抗原結合フラグメント。
(2)前記抗体の重鎖定常領域がヒトIgγ1定常領域である、上記(1)に記載の抗ヒトTSLP受容体抗体又はその抗原結合フラグメント。
(3)前記抗体の軽鎖定常領域がヒトIgκ定常領域である、上記(1)に記載の抗ヒトTSLP受容体抗体又はその抗原結合フラグメント。
(4)前記抗体の重鎖定常領域がヒトIgγ1定常領域であり、前記抗体の軽鎖定常領域がヒトIgκ定常領域である、上記(1)に記載の抗ヒトTSLP受容体抗体又はその抗原結合フラグメント。
(5)上記抗原結合フラグメントが一本鎖可変領域フラグメント、Fab、Fab’、又はF(ab’)2である、上記(1)~(4)に記載の抗原結合フラグメント。
(6)配列番号1に示されるアミノ酸配列からなる重鎖、及び配列番号3に示されるアミノ酸配列からなる軽鎖を含む、上記(1)に記載の抗ヒトTSLP受容体抗体。
(7)配列番号1のアミノ酸番号1から447までのアミノ酸配列からなる重鎖、及び配列番号3に示されるアミノ酸配列からなる軽鎖を含む、上記(1)に記載の抗ヒトTSLP受容体抗体。
(8)上記(1)に記載の抗体の重鎖可変領域をコードする塩基配列を含む、ポリヌクレオチド。
(9)上記(1)に記載の抗体の軽鎖可変領域をコードする塩基配列を含む、ポリヌクレオチド。
(10)上記(8)及び/又は(9)に記載のポリヌクレオチドを含む発現ベクター。
(11)以下の(a)~(d)からなる群より選択される、上記(10)に記載の発現ベクターで形質転換された宿主細胞。
(a)上記(1)に記載の抗ヒトTSLP受容体抗体の重鎖可変領域をコードする塩基配列を含むポリヌクレオチドと該抗体の軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)上記(1)に記載の抗ヒトTSLP受容体抗体の重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体の軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;
(c)上記(1)に記載の抗ヒトTSLP受容体抗体の重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;及び
(d)上記(1)に記載の抗ヒトTSLP受容体抗体の軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。
(12)以下の(a)~(d)からなる群より選択される、上記(10)に記載の発現ベクターで形質転換された宿主細胞。
(a)上記(6)に記載の抗ヒトTSLP受容体抗体の重鎖をコードする塩基配列を含むポリヌクレオチドと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)上記(6)に記載の抗ヒトTSLP受容体抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;
(c)上記(6)に記載の抗ヒトTSLP受容体抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;及び
(d)上記(6)に記載の抗ヒトTSLP受容体抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。
(13)上記(11)に記載の宿主細胞を培養し、抗ヒトTSLP受容体抗体又はその抗原結合フラグメントを発現させる工程を包含する、抗ヒトTSLP受容体抗体又はその抗原結合フラグメントを生産する方法。
(14)上記(12)に記載の宿主細胞を培養し、抗ヒトTSLP受容体抗体を発現させる工程を包含する、抗ヒトTSLP受容体抗体を生産する方法。
(15)上記(13)に記載の方法で生産された抗ヒトTSLP受容体抗体又はその抗原結合フラグメント。
(16)上記(14)に記載の方法で生産された抗ヒトTSLP受容体抗体。
(17)上記(1)~(7)、(15)、及び(16)のいずれかに記載の抗ヒトTSLP受容体抗体又はその抗原結合フラグメント及び薬学的に許容される賦形剤を含む、医薬組成物。
(18)喘息の予防又は治療用医薬組成物である、上記(17)に記載の医薬組成物。
(19)上記(1)~(7)、(15)、及び(16)のいずれかに記載の抗ヒトTSLP受容体抗体又はその抗原結合フラグメントの治療有効量を投与する工程を包含する、喘息を予防又は治療する方法。
(20)喘息の予防又は治療に使用するための、上記(1)~(7)、(15)、及び(16)のいずれかに記載の抗ヒトTSLP受容体抗体又はその抗原結合フラグメント。
(21)喘息の予防又は治療用医薬組成物の製造における、上記(1)~(7)、(15)、及び(16)のいずれかに記載の抗ヒトTSLP受容体抗体又はその抗原結合フラグメントの使用。
上記(1)~(7)、(15)、及び(16)に記載の抗ヒトTSLP受容体抗体又はその抗原結合フラグメントには、他のペプチド及び蛋白質との融合体や、修飾剤を結合させた修飾体も含まれる。
本発明の抗ヒトTSLP受容体抗体又はその抗原結合フラグメントは、以下の特徴を有する抗ヒトTSLP受容体抗体又はその抗原結合フラグメントである。
配列番号1のアミノ酸番号1から118までのアミノ酸配列からなる重鎖可変領域、及び配列番号3のアミノ酸番号1から108までのアミノ酸配列からなる軽鎖可変領域を含む、抗ヒトTSLP受容体抗体又はその抗原結合フラグメント。
配列番号1に示されるアミノ酸配列からなる重鎖、及び配列番号3に示されるアミノ酸配列からなる軽鎖を含む、抗ヒトTSLP受容体抗体。
配列番号1のアミノ酸番号1のグルタミン酸がピログルタミン酸に修飾され及び/又は配列番号1のアミノ酸番号448のリジンを欠くアミノ酸配列からなる重鎖、並びに配列番号3に示されるアミノ酸配列からなる軽鎖を含む、抗ヒトTSLP受容体抗体。
配列番号1のアミノ酸番号31から35までのアミノ酸配列からなるCDR1、配列番号1のアミノ酸番号50から66までのアミノ酸配列からなるCDR2、及び配列番号1のアミノ酸番号99から107までのアミノ酸配列からなるCDR3とを含む重鎖可変領域、並びに、配列番号3のアミノ酸番号24から34までのアミノ酸配列からなるCDR1、配列番号3のアミノ酸番号50から56までのアミノ酸配列からなるCDR2、及び配列番号3のアミノ酸番号89から97までのアミノ酸配列からなるCDR3とを含む軽鎖可変領域を含む、抗ヒトTSLP受容体抗体又はその抗原結合フラグメント。
本発明のポリヌクレオチドには、本発明の抗ヒトTSLP受容体抗体の重鎖可変領域をコードする塩基配列を含むポリヌクレオチド、及び、本発明の抗ヒトTSLP受容体抗体の軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドが含まれる。
本発明の発現ベクターには、本発明の抗ヒトTSLP受容体抗体の重鎖可変領域をコードする塩基配列を含むポリヌクレオチド及び/又は本発明の抗ヒトTSLP受容体抗体の軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターが含まれる。
(a)本発明の抗ヒトTSLP受容体抗体の重鎖可変領域をコードする塩基配列を含むポリヌクレオチドと該抗体の軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)本発明の抗ヒトTSLP受容体抗体の重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体の軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;
(c)本発明の抗ヒトTSLP受容体抗体の重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;及び
(d)本発明の抗ヒトTSLP受容体抗体の軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。
(a)本発明の抗ヒトTSLP受容体抗体の重鎖をコードする塩基配列を含むポリヌクレオチドと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)本発明の抗ヒトTSLP受容体抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;
(c)本発明の抗ヒトTSLP受容体抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;及び
(d)本発明の抗ヒトTSLP受容体抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。
本発明の医薬組成物には、本発明の抗ヒトTSLP受容体抗体又はその抗原結合フラグメント及び薬学的に許容される賦形剤を含む医薬組成物が含まれる。本発明の医薬組成物は、当該分野において通常用いられている賦形剤、即ち、薬剤用賦形剤や薬剤用担体等を用いて、通常使用される方法によって調製することができる。これら医薬組成物の剤型の例としては、例えば、注射剤、点滴用剤等の非経口剤が挙げられ、静脈内投与、皮下投与等により投与することができる。製剤化にあたっては、薬学的に許容される範囲で、これら剤型に応じた賦形剤、担体、添加剤等を使用することができる。
(1)配列番号1のアミノ酸番号1から447までのアミノ酸配列からなる重鎖、及び配列番号3に示されるアミノ酸配列からなる軽鎖を含む、抗ヒトTSLP受容体抗体。
(2)配列番号1に示されるアミノ酸配列からなり、アミノ酸番号1のグルタミン酸がピログルタミン酸に修飾された重鎖、及び配列番号3に示されるアミノ酸配列からなる軽鎖を含む、抗ヒトTSLP受容体抗体。
(3)配列番号1のアミノ酸番号1から447までのアミノ酸配列からなり、アミノ酸番号1のグルタミン酸がピログルタミン酸に修飾された重鎖、及び配列番号3に示されるアミノ酸配列からなる軽鎖を含む、抗ヒトTSLP受容体抗体。
(4)配列番号1に示されるアミノ酸配列からなる重鎖、及び配列番号3に示されるアミノ酸配列からなる軽鎖を含む、抗ヒトTSLP受容体抗体。
抗体の結合活性の評価に用いるために、ヒトTSLP受容体とヒトFcを融合させた蛋白質(ヒトTSLP受容体-ヒトFc融合蛋白質)、ヒトTSLP受容体とマウスFcを融合させた蛋白質(ヒトTSLP受容体-マウスFc融合蛋白質)及びサルTSLP受容体とヒトFcを融合させた蛋白質(サルTSLP受容体-ヒトFc融合蛋白質)を取得した。ヒトTSLP受容体-ヒトFc融合遺伝子(配列番号5)、ヒトTSLP受容体-マウスFc融合遺伝子(配列番号6)及びサルTSLP受容体-ヒトFc融合遺伝子(配列番号7)を哺乳細胞発現用ベクターGSベクター(Lonza社)pEE12.4にそれぞれ組み換えた。作製したベクターを、遺伝子導入試薬293フェクチン(Life technologies社)を用いてFreeStyle 293細胞(Life technologies社)へ遺伝子導入し、この細胞をFreeStyle 293 Expression medium(Life technologies社)を用いた無血清培養系で1週間培養後、ヒトTSLP受容体-ヒトFc融合蛋白質、ヒトTSLP受容体-マウスFc融合蛋白質又はサルTSLP受容体-ヒトFc融合蛋白質を含む培養上清をそれぞれ取得した。取得した培養上清から蛋白質精製カラムプロテインGカラム(GEヘルスケアジャパン社)を用いて各TSLP受容体-Fc融合蛋白質を精製した。
抗体の中和活性の評価に用いるために、ヒトTSLP変異体にFlagタグを結合させた蛋白質(ヒトTSLP変異体-Flag蛋白質)及びサルTSLP変異体にFlagタグを結合させた蛋白質(サルTSLP変異体-Flag蛋白質)を取得した。ヒト又はサルTSLP変異体-Flag遺伝子(配列番号8又は9(フューリンプロテアーゼにより切断されて活性が失われるのを防ぐために、その切断部位に変異を挿入したヒト又はサル野生型TSLPのアミノ酸配列をそれぞれコードする))をGSベクターpEE12.4にそれぞれ組み換えた。作製したベクターを、293フェクチンを用いてFreeStyle 293細胞へ遺伝子導入し、この細胞をFreeStyle 293 Expression mediumを用いた無血清培養系で1週間培養後、ヒトTSLP変異体-Flag蛋白質又はサルTSLP変異体-Flag蛋白質を含む培養上清をそれぞれ取得した。取得した培養上清から抗FLAG M2抗体アフィニティーゲル(Sigma社)を用いて、各TSLP変異体-Flag蛋白質を精製した。
ヒトモノクローナル抗体開発技術「ベロシミューン」(VelocImmune antibody technology:Regeneron社(米国特許6596541号))マウスに、免疫反応を惹起するアジュバントと共に、ヒトTSLP受容体-Fc(R&D社)及びヒトTSLP受容体発現Ba/F3細胞(ヒトTSLP受容体遺伝子(配列番号10)及びヒトIL-7受容体α鎖遺伝子(配列番号11)をコードするベクターをマウスBa/F3細胞(理化学研究所:RCB0805)へ導入することで作製)を免疫した。常法に従い、免疫したマウスの脾臓やリンパ節を摘出しリンパ球を収集し、これをマウスミエローマ細胞SP2/0(ATCC CRL-1581)と細胞融合することでハイブリドーマを作製した。ハイブリドーマのモノクローン化を行い、無血清培地であるCDハイブリドーマメディウム(Life technologies社)で培養した。得られた培養上清から抗体精製キットProtein G Purification kit(Proteus社)を用いて抗体を精製した。
完全ヒト型T7-27の結合活性を詳細に測定するために、SPR解析を行った。本実施例では、比較抗体として、抗ヒトTSLP受容体抗体 TSLPR-012_141(特許文献4)を用いた。
表1:SPR解析によるヒトTSLP受容体に対する結合活性
完全ヒト型T7-27の中和活性を評価するために、ヒト末梢血単核細胞(PBMC)におけるTSLP誘発のTARC mRNA発現阻害を評価した。ヒトPBMCには、TSLP受容体を発現している樹状細胞も含まれるため、抗ヒトTSLP受容体抗体の評価に用いることができる。比較抗体として、TSLPR-012_141を用いた。後述の試験例1の結果から、抗TSLP受容体抗体による喘息モデルの病態改善と血中TARC mRNAの発現阻害に相関が認められたことから、本評価系は病態への有効性を示唆する評価系である。
表2:ヒトPBMCを用いたTSLP誘発TARC mRNA発現阻害活性
完全ヒト型T7-27の中和活性を評価するために、ヒトPBMCにおけるTSLP誘発のMDC蛋白質産生阻害を評価した。比較抗体として、TSLPR-012_141を用いた。
表3:ヒトPBMCを用いたTSLP誘発MDC蛋白質産生阻害評価
サルをアスカリス抗原で感作させることで、アスカリス抗原特異的IgEが誘導され、アレルギー反応として皮膚反応が惹起される。
雄性カニクイザルにDay1、Day8及びDay15に水酸化アルミニウムゲル(以下、Alumと称する)で懸濁したアスカリス抗原液(0.5mg/mL DNP-Ascaris(LSL社)、50mg/mL Alumの濃度でPBSに懸濁した抗原液。以下、アスカリス抗原-Alum液と称する。)を、腹腔内に3.6mL/kg、筋肉内に0.4mL/kgを投与することで感作した。なお、処置群として、Normal群(無処置群。n=2)、Vehicle群(溶媒(20mM クエン酸ナトリウム緩衝液/120mM NaCl(pH6.0))を感作1日前に静脈内投与した群。n=3)及び抗体投与群(10mg/kgの完全ヒト型T7-27(溶媒で希釈)を感作1日前に静脈内投与した群。n=3)をそれぞれ設定した。
なお、Alumは、以下の方法で作製した。
硫酸アルミニウム(14~18水和物)(Wako社)を超純水に溶解して1Mの溶液を調製し、0.22μmのフィルターに通した後、1M 水酸化ナトリウム(ナカライテスク社)を白色沈殿が生じなくなるまで添加した。上清を除去し、超純水で5回洗浄後、さらにPBS(WAKO社)で3回洗浄した。洗浄した白色沈殿を、氷冷下においてホモジナイザー(KINEMATICA社、CH-6010)により細かく砕いた。次に、遠心機(日立社、himac CR21)で2000rpm、5分、4℃にて遠心後、上清を除去し、PBSで2回洗浄した。得られた沈殿物をPBSに懸濁してAlumを得た。
上記カニクイザルよりDay1(アスカリス抗原-Alum液を投与する前)、Day8、Day15及びDay22と継時的に採血し、遠心(1800×g、4℃、10分)後、血漿を回収した。血漿中のアスカリス抗原特異的IgEの産生量は以下の方法を用いて測定した。
DNP-Ascarisを100μg/mLの濃度になるようにPBSで調製し、Nunc-ImmunoTM MicroWellTM 96ウェルソリッドプレート(Nunc社)に100μL添加して室温で一晩固相化した。ブロッキング剤(Blocking One:ナカライテスク社)を200μL添加し、室温で30分静置した後、その溶液を除去した。次に、回収した血漿及び検量線用サンプルをそれぞれ100μL添加した。検量線用サンプルには、Vehicle群の1個体のDay22における血漿中アスカリス抗原特異的IgE濃度を2000U/mLとし、希釈溶液(5%Blocking One含有PBS)により調製した希釈系列(2000U/mL~16U/mL)を用いた。室温にて1時間静置した後、T-PBS(0.05% Tween-20含有PBS)にて5回洗浄し、希釈溶液で10000倍に希釈したHRP標識ヒトIgE検出抗体(A80-108P:Bethyl社)を100μL添加した。再び室温にて1時間静置した後、T-PBSで5回洗浄した。最後にペルオキシダーゼ発色キット(ML-1120T:SUMILON社)を用いて測定した。吸光度はSpectraMax(モレキュラーデバイス社)で測定した。Day1及びDay22の結果を、図1に示す。
Day22に、Vehicle群及び抗体投与群の各個体の皮内にPBS並びに1、10及び100μg/mL アスカリス抗原溶液(DNP-AscarisをPBSで懸濁した溶液)を、それぞれ100μL、同一個体の剃毛した腹部各2か所(1匹あたり合計8カ所)に投与した。Normal群には、各個体の皮内にPBS及び100μg/mL アスカリス抗原溶液を、それぞれ100μL、同一個体の剃毛した腹部各4か所(1匹あたり合計8カ所)に投与した。皮膚感作20分後に、ノギスを用いて直径を測定することで皮膚反応を観察した。それぞれの個体において、アスカリス抗原溶液投与後の皮膚反応の直径からPBS投与後の皮膚反応の直径を引いた値をdelta cmとした。100μg/mL アスカリス抗原溶液投与時の結果を、図2に示す。なお、1及び10μg/mL アスカリス抗原溶液を投与した場合、被験抗体を評価するのに十分な皮膚反応は惹起されなかった。
マウスダニ抗原感作モデルは喘息モデルとして知られており、その病態としては、気道反応性の亢進及び気管支肺胞への好酸球の浸潤等が挙げられる。
NC/Nga マウス(チャールスリバー社)に、Day0(初回感作時)及びDay5に、ダニ抗原(Dp)(LSL社)を100μg Dp/0.5mL生理食塩液/マウスの用量で腹腔内投与することで感作を実施した。Day12及びDay19に、ダニ抗原を100μg Dp/50μL生理食塩液/マウスの用量で点鼻投与することで気道炎症を惹起した。抗体投与群には、Day-1、Day2、Day5、Day8、Day11、Day14、及びDay18(抗体投与日)に1日1回、各用量(0.1、1、10mg/kg)でPBS(WAKO社)に溶解した抗マウスTSLP受容体抗体(WAKO社)を皮下投与した。陽性対照群としてデキサメタゾン投与群(Dex群)を設けた。Dex群には、Day12からDay19まで1日1回、3mg/kgの用量でPBSに溶解したデキサメタゾンを腹腔内投与した。設定した処置群は以下のとおりである。
[処置群]
Normal群(n=10):
無処置
Saline群(n=6):
Day0及びDay5にダニ抗原を腹腔内投与、Day12及びDay19に生理食塩液を点鼻投与、抗体投与日にPBSを皮下投与
PBS群(n=10):
Day0及びDay5にダニ抗原を腹腔内投与、Day12及びDay19にダニ抗原を点鼻投与、抗体投与日にPBSを皮下投与
抗体投与群(0.1、1、10mg/kg)(各用量につきn=10):
Day0及びDay5にダニ抗原を腹腔内投与、Day12及びDay19にダニ抗原を点鼻投与、抗体投与日に各用量の抗体を皮下投与
Dex群(n=10):
Day0及びDay5にダニ抗原を腹腔内投与、Day12及びDay19にダニ抗原を点鼻投与、Day12からDay19にデキサメタゾンを腹腔内投与
Claims (21)
- 配列番号1のアミノ酸番号1から118までのアミノ酸配列からなる重鎖可変領域、及び配列番号3のアミノ酸番号1から108までのアミノ酸配列からなる軽鎖可変領域を含む、抗ヒトTSLP受容体抗体又はその抗原結合フラグメント。
- 前記抗体の重鎖定常領域がヒトIgγ1定常領域である、請求項1に記載の抗ヒトTSLP受容体抗体又はその抗原結合フラグメント。
- 前記抗体の軽鎖定常領域がヒトIgκ定常領域である、請求項1に記載の抗ヒトTSLP受容体抗体又はその抗原結合フラグメント。
- 前記抗体の重鎖定常領域がヒトIgγ1定常領域であり、前記抗体の軽鎖定常領域がヒトIgκ定常領域である、請求項1に記載の抗ヒトTSLP受容体抗体又はその抗原結合フラグメント。
- 上記抗原結合フラグメントが一本鎖可変領域フラグメント、Fab、Fab’、又はF(ab’)2である、請求項1に記載の抗原結合フラグメント。
- 配列番号1に示されるアミノ酸配列からなる重鎖、及び配列番号3に示されるアミノ酸配列からなる軽鎖を含む、請求項1に記載の抗ヒトTSLP受容体抗体。
- 配列番号1のアミノ酸番号1から447までのアミノ酸番号からなる重鎖、及び配列番号3に示されるアミノ酸配列からなる軽鎖を含む、請求項1に記載の抗ヒトTSLP受容体抗体。
- 請求項1に記載の抗体の重鎖可変領域をコードする塩基配列を含む、ポリヌクレオチド。
- 請求項1に記載の抗体の軽鎖可変領域をコードする塩基配列を含む、ポリヌクレオチド。
- 請求項8及び/又は9に記載のポリヌクレオチドを含む発現ベクター。
- 以下の(a)~(d)からなる群より選択される、請求項10に記載の発現ベクターで形質転換された宿主細胞。
(a)請求項1に記載の抗ヒトTSLP受容体抗体の重鎖可変領域をコードする塩基配列を含むポリヌクレオチドと該抗体の軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)請求項1に記載の抗ヒトTSLP受容体抗体の重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体の軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;
(c)請求項1に記載の抗ヒトTSLP受容体抗体の重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;及び
(d)請求項1に記載の抗ヒトTSLP受容体抗体の軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。 - 以下の(a)~(d)からなる群より選択される、請求項10に記載の発現ベクターで形質転換された宿主細胞。
(a)請求項6に記載の抗ヒトTSLP受容体抗体の重鎖をコードする塩基配列を含むポリヌクレオチドと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)請求項6に記載の抗ヒトTSLP受容体抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;
(c)請求項6に記載の抗ヒトTSLP受容体抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;及び
(d)請求項6に記載の抗ヒトTSLP受容体抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。 - 請求項11に記載の宿主細胞を培養し、抗ヒトTSLP受容体抗体又はその抗原結合フラグメントを発現させる工程を包含する、抗ヒトTSLP受容体抗体又はその抗原結合フラグメントを生産する方法。
- 請求項12に記載の宿主細胞を培養し、抗ヒトTSLP受容体抗体を発現させる工程を包含する、抗ヒトTSLP受容体抗体を生産する方法。
- 請求項13に記載の方法で生産された抗ヒトTSLP受容体抗体又はその抗原結合フラグメント。
- 請求項14に記載の方法で生産された抗ヒトTSLP受容体抗体。
- 請求項1~7、15、及び16のいずれかに記載の抗ヒトTSLP受容体抗体又はその抗原結合フラグメント及び薬学的に許容される賦形剤を含む、医薬組成物。
- 喘息の予防又は治療用医薬組成物である、請求項17に記載の医薬組成物。
- 請求項1~7、15、及び16のいずれかに記載の抗ヒトTSLP受容体抗体又はその抗原結合フラグメントの治療有効量を投与する工程を包含する、喘息を予防又は治療する方法。
- 喘息の予防又は治療に使用するための、請求項1~7、15、及び16のいずれかに記載の抗ヒトTSLP受容体抗体又はその抗原結合フラグメント。
- 喘息の予防又は治療用医薬組成物の製造における、請求項1~7、15、及び16のいずれかに記載の抗ヒトTSLP受容体抗体又はその抗原結合フラグメントの使用。
Priority Applications (23)
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AU2014303356A AU2014303356B2 (en) | 2013-08-09 | 2014-08-08 | Novel anti-human TSLP receptor antibody |
CA2920484A CA2920484C (en) | 2013-08-09 | 2014-08-08 | Novel anti-human tslp receptor antibody |
RU2016107814A RU2689528C2 (ru) | 2013-08-09 | 2014-08-08 | Новое антитело к tslp-рецептору человека |
UAA201602101A UA119328C2 (uk) | 2013-08-09 | 2014-08-08 | Антитіло до тslp-рецептора людини |
DK14833804.9T DK3031913T3 (da) | 2013-08-09 | 2014-08-08 | Hidtil ukendt antistof mod humane tslp-receptorer |
BR112016002743-4A BR112016002743B1 (pt) | 2013-08-09 | 2014-08-08 | anticorpo anti-receptor da tslp humana ou um fragmento de ligação ao antígeno deste, polinucleotídeo, vetor de expressão, método para produzir o dito anticorpo, composição farmacêutica e uso do dito anticorpo para prevenção ou tratamento de asma |
MX2016001796A MX369208B (es) | 2013-08-09 | 2014-08-08 | Anticuerpo contra el receptor de tslp humana novedoso. |
RS20190580A RS58696B1 (sr) | 2013-08-09 | 2014-08-08 | Novo antitelo protiv humanog tslp receptora |
LTEP14833804.9T LT3031913T (lt) | 2013-08-09 | 2014-08-08 | Naujas antikūnas, atpažįstantis žmogaus tslp receptorių |
JP2015530978A JP6380394B2 (ja) | 2013-08-09 | 2014-08-08 | 新規抗ヒトtslp受容体抗体 |
SI201431208T SI3031913T1 (sl) | 2013-08-09 | 2014-08-08 | Novo protitelo anti-humanega TSLP receptorja |
KR1020167005884A KR102260680B1 (ko) | 2013-08-09 | 2014-08-08 | 신규 항 인간 tslp 수용체 항체 |
PL14833804T PL3031913T3 (pl) | 2013-08-09 | 2014-08-08 | Nowe przeciwciało przeciwko receptorowi ludzkiej tslp |
ES14833804T ES2730978T3 (es) | 2013-08-09 | 2014-08-08 | Anticuerpo anti-receptor de TSLP humana nuevo |
EP14833804.9A EP3031913B1 (en) | 2013-08-09 | 2014-08-08 | Novel anti-human tslp receptor antibody |
SG11201600935SA SG11201600935SA (en) | 2013-08-09 | 2014-08-08 | Novel anti-human tslp receptor antibody |
US14/927,007 US9328171B2 (en) | 2013-08-09 | 2015-10-29 | Anti-human TSLP receptor antibody |
IL24385116A IL243851B (en) | 2013-08-09 | 2016-01-28 | A new anti-human tslp receptor antibody |
PH12016500237A PH12016500237B1 (en) | 2013-08-09 | 2016-02-03 | Novel anti-human tslp receptor antibody |
US15/090,051 US9908941B2 (en) | 2013-08-09 | 2016-04-04 | Anti-human TSLP receptor antibody |
HK16111216.7A HK1223123A1 (zh) | 2013-08-09 | 2016-09-23 | 新穎抗人類 受體抗體 |
HRP20190752TT HRP20190752T1 (hr) | 2013-08-09 | 2019-04-23 | Novo protutijelo protiv ljudskog receptora tslp |
CY20191100496T CY1121678T1 (el) | 2013-08-09 | 2019-05-08 | Νεο αντισωμα εναντι του ανθρωπινου υποδοχεα tslp |
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WO2017104778A1 (ja) * | 2015-12-18 | 2017-06-22 | アステラス製薬株式会社 | 抗ヒトtslp受容体抗体含有医薬組成物 |
RU2794148C2 (ru) * | 2015-12-18 | 2023-04-12 | Апстрим Байо, Инк. | Фармацевтическая композиция, содержащая антитело к рецептору tslp человека |
CN116217724A (zh) * | 2021-12-02 | 2023-06-06 | 北京东方百泰生物科技股份有限公司 | 一种抗tslp的单克隆抗体、其抗原结合片段及其应用 |
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KR20230058434A (ko) | 2020-08-26 | 2023-05-03 | 플래그쉽 파이어니어링 이노베이션스 브이아이, 엘엘씨 | SARS-CoV-2를 표적으로 하는 항원 결합 분자 |
CN114853888B (zh) * | 2021-02-05 | 2023-11-03 | 上海洛启生物医药技术有限公司 | 抗tslp纳米抗体及其应用 |
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WO2023028612A2 (en) * | 2021-08-27 | 2023-03-02 | Board Of Regents, The University Of Texas System | Anti-tslpr (crlf2) antibodies |
CN114028562B (zh) * | 2021-11-01 | 2022-05-24 | 江苏荃信生物医药股份有限公司 | 包含抗人胸腺基质淋巴细胞生成素(tslp)单克隆抗体的液体制剂 |
CN116217725B (zh) * | 2021-12-02 | 2023-09-22 | 北京东方百泰生物科技股份有限公司 | 一种抗tslp单克隆抗体的纯化方法 |
WO2023215910A1 (en) | 2022-05-06 | 2023-11-09 | Generate Biomedicines, Inc. | Antigen binding molecules targeting sars-cov-2 |
US20240173405A1 (en) * | 2022-11-07 | 2024-05-30 | Upstream Bio, Inc. | Pharmaceutical compositions comprising anti-human tslp receptor antibodies and methods of using the same |
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