JP6962514B2 - Monoclonal antibody that specifically binds to yeast-derived recombinant human GM-CSF - Google Patents

Description

The present invention relates to a monoclonal antibody that specifically binds to yeast-derived recombinant human GM-CSF.

Pulmonary alveolar proteinosis (PAP) is a disorder in the process of production or degradation of surfactants (biosurfactants composed of 90% phospholipids and 10% protein), which is the cause. It is a rare disease in which surfactants accumulate in the alveolar space or in the terminal bronchiole. PAPs (hereinafter, estimated frequency of occurrence in parentheses) are autoimmune PAPs (about 90%), secondary PAPs (about 9%), congenital PAPs (about 1% or less), and unclassified PAPs (extremely). It is classified as rare).

Among PAP patients, autoimmune PAP, which accounts for the majority, has neutralized autoantibodies against granulocyte macrophage colony-stimulating factor (GM-CSF), and functions of alveolar macrophages and neutrophils. Disorders are involved in the pathology. GM-CSF is a glycoprotein produced by macrophages, lymphocytes, epithelial cells or vascular endothelial cells, which acts on granulocytes or macrophage progenitor cells to promote their proliferation and differentiation.
In the lungs of healthy individuals, GM-CSF released from type II alveolar epithelial cells, one of the cells that make up the respiratory epithelium of the alveolar, binds to the GM-CSF receptor of immature alveolar macrophages. By transmitting a signal by binding between GM-CSF and a receptor, PU. 1 is expressed and terminal differentiation of alveolar macrophages occurs, resulting in mature macrophages. Mature macrophages maintain alveolar homeostasis by incorporating and degrading surfactant. On the other hand, in the lungs of autoimmune PAP patients, GM-CSF released from type II alveolar epithelial cells is neutralized by anti-GM-CSF autoantibodies abundant in the alveoli, and GM-CSF is immature lung. Prevents binding to alveolar macrophage receptors. Therefore, terminal differentiation of alveolar macrophages does not occur, and the degradation of surfactant is impaired.

Symptoms of autoimmune PAP include, for example, dyspnea on exertion, cough, sputum, weight loss, fever, etc., but about 30% of patients are asymptomatic. It is a characteristic of autoimmune PAP that the symptoms are relatively minor for the imaging findings. The 5-year survival rate of autoimmune PAP is 96%, and the 10-year survival rate is 88%, but patients often require repeated treatments such as total lung lavage during this period.

Examples of the treatment method for autoimmune PAP include lavage therapy (whole lung lavage), inhalation therapy of GM-CSF as a trial treatment method, subcutaneous injection of GM-CSF (see, for example, Non-Patent Document 1) and the like. ..

The GM-CSF used for administration by inhalation or subcutaneous injection is a yeast-derived recombinant human GM-CSF in which the 23rd arginine from the N-terminal is replaced with leucine, but the function is maintained and the genzyme is maintained. It is sold by the company under the trade name of Leucine (registered trademark).

On the other hand, GM-CSF is known to be a cause of various disease states.
There are a wide variety of diseases caused by GM-CSF, but the typical ones are (1) allergic diseases such as asthma, atopy, and pollinosis, (2) graft rejection, and graft-versus-host disease. Host disease; GVHD), (3) autoimmune diseases such as rheumatoid arthritis, and the like. For example, overexpressed human GM-CSF is detected in the lungs of allergic individuals and joints of patients with rheumatoid arthritis, and human GM-CSF mRNA is excessively detected in the skin of allergic individuals. In addition, it has been reported that monocytes, which are inflammation-inducing cells of atopic dermatitis, have an extended lifespan due to the production of GM-CSF. In addition, GM-CSF has been shown to stimulate the proliferation of leukemia cells and is considered to be a causative factor of leukemia.

From these reports, in order to treat various diseases caused by human GM-CSF, it has strong affinity and neutralizing ability for overexpressed human GM-CSF and shows rejection reaction. It has been proposed to administer no anti-human GM-CSF antibody (including anti-human GM-CSF autoantibodies) (see, eg, Patent Documents 1 and 2 and Non-Patent Document 2).

Japanese Unexamined Patent Publication No. 2000-198799 Japanese Unexamined Patent Publication No. 2007-116947

Mani S. Kavuru et al., “Exogenous Granulocyte-Macrophage Colony-Stimulating Factor Administration for Pulmonary Alveolar Proteinosis”, Am. J. Respir. Crit. Care Med., Vol.161, pp1143-1148, 2000. K. Uchida et al., “Granulocyte / macrophage-colony-stimulating factor autoantibodies and myeloid cell immune functions in healthy subjects”, Blood, vol.113, no.11, 2009.

In the treatment method of yeast-derived recombinant human GM-CSF for autoimmune PAP patients by inhalation therapy or subcutaneous injection, in order to calculate the maximum blood concentration and half-life of yeast-derived recombinant human GM-CSF in the patient's body. There is a need for a method for distinguishing between the human GM-CSF produced in the above and the yeast-derived recombinant human GM-CSF that has entered the body by inhalation.
In the anti-human GM-CSF antibodies described in Patent Documents 1 and 2 and Non-Patent Document 2, the human GM-CSF produced in the body of the patient and the yeast-derived recombinant human GM-CSF are both human GM-CSF. Since it is CSF, it cannot be detected separately.

The present invention has been made in view of the above circumstances, and provides a monoclonal antibody that specifically binds only to yeast-derived recombinant human GM-CSF.

The present invention includes the following aspects.
The anti-yeast-derived recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) antibody or the antigen-binding fragment thereof according to the first aspect of the present invention is (a) the amino acid shown in SEQ ID NO: 1. CDR-L1 containing an amino acid sequence in which one to several amino acids are deleted, substituted or added in the sequence or the amino acid sequence shown in SEQ ID NO: 1 and (b) the amino acid sequence shown in SEQ ID NO: 2. Or, in the amino acid sequence shown in SEQ ID NO: 2, CDR-L2 containing an amino acid sequence in which one to several amino acids are deleted, substituted or added, and (c) the amino acid sequence shown in SEQ ID NO: 3. Alternatively, in the amino acid sequence shown in SEQ ID NO: 3, a light chain variable domain containing CDR-L3 containing an amino acid sequence in which one to several amino acids are deleted, substituted or added, and / or (d). ) CDR-H1 containing an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence shown in SEQ ID NO: 4 and (e). CDR-H2 containing an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5 or the amino acid sequence shown in SEQ ID NO: 5, and (f) sequence. Variable chain including CDR-H3 containing the amino acid sequence shown in No. 6 or the amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 6. Has a domain.

The anti-yeast-derived recombinant human GM-CSF antibody or an antigen-binding fragment thereof according to the above aspect comprises CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 1 and CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 2. , CDR-L3 containing the amino acid sequence set forth in SEQ ID NO: 3, a light chain variable domain containing, and / or CDR-H1 containing the amino acid sequence set forth in SEQ ID NO: 4, and the amino acid sequence set forth in SEQ ID NO: 5. May have a heavy chain variable domain comprising CDR-H2 comprising, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 6.

The hybridoma that produces a monoclonal antibody that specifically binds to the yeast-derived recombinant human GM-CSF according to the second aspect of the present invention is a hybridoma having accession number NITE P-02224.

The method for producing a monoclonal antibody that specifically binds to yeast-derived recombinant human GM-CSF according to the third aspect of the present invention includes the step of culturing the hybridoma according to the second aspect and the antibody produced from the hybridoma. A method comprising a step of isolation.

The kit for detecting yeast-derived recombinant human GM-CSF in the blood sample of a subject to which yeast-derived recombinant human GM-CSF according to the fourth aspect of the present invention is administered is an anti-yeast according to the first aspect. Derived Recombinant Human GM-CSF antibody or an antigen-binding fragment thereof.

The detection kit according to the above embodiment may further contain an antibody against the labeled anti-yeast-derived recombinant human GM-CSF antibody or an antigen-binding fragment thereof.

The method for detecting the yeast-derived recombinant human GM-CSF in the blood sample of the subject to which the yeast-derived recombinant human GM-CSF according to the fifth aspect of the present invention is administered is the blood sample and the first. A method comprising a primary antigen-antibody reaction step in which an antigen-antibody reaction is carried out by contacting an anti-yeast-derived recombinant human GM-CSF antibody or an antigen-binding fragment thereof according to the embodiment.

In the detection method according to the above aspect, the antigen-antibody reaction is further carried out by contacting the labeled anti-yeast-derived recombinant human GM-CSF antibody or an antibody against the antigen-binding fragment thereof after the primary antigen-antibody reaction step. It may be provided with a secondary antigen-antibody reaction step to be carried out.

According to the above aspect, only yeast-derived recombinant human GM-CSF can be specifically detected. In addition, according to the detection kit according to the above aspect, the accurate blood concentration of yeast-derived recombinant human GM-CSF can be measured in a pharmacokinetic test on a healthy body, and the maximum blood concentration and half-life can be measured. The period can be calculated. Furthermore, in a physician-initiated clinical trial of autoimmune PAP patients, accurate trough values (minimum blood drug concentration in steady state after repeated doses of the drug) can be measured, and the patient's systemic clearance (drug) can be measured. The ability to excrete outside the body) can be estimated.

No. 1 in Test Example 1. It is a schematic diagram which shows each well on which the synthetic peptide of 1-4 and the full-length yeast-derived recombinant human GM-CSF (No. 5) (also known as sargramostim, manufactured by Genzyme) was immobilized. (A) No. 1 immediately after adding the reaction terminator in Test Example 1. It is an image showing the state of color development of each well on which 1 to 4 synthetic peptides and full-length yeast-derived recombinant human GM-CSF (No. 5) (also known as sargramostim, manufactured by Genzyme) are immobilized. .. (B) No. 1 in Test Example 1. It is a graph which shows the result of having measured the absorbance at 450 nm in each well on which the synthetic peptides 1 to 5 were solid-phased. Denatured yeast-derived recombinant human GM-CSF, unmodified yeast-derived recombinant human GM-CSF, Escherichia coli-derived recombinant human GM-CSF, CHO (Chinese Hamster Ovaly) cell-producing recombinant human GM-CSF in Test Example 2. It is a graph which shows the result of having measured the absorbance at 450 nm in each well which solidified bovine serum albumin (BSA) and bovine serum albumin (BSA). It is a graph which shows the time-dependent change of the blood concentration of the yeast-derived recombinant human GM-CSF in the healthy cynomolgus monkey to which 50 mg of yeast-derived recombinant human GM-CSF was administered by inhalation in Test Example 4.

≪Monclonal antibody≫
In one embodiment, the present invention
(A) CDR-L1 containing an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1.
(B) CDR-L2 containing an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2.
(C) CDR-L3 containing an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 3.
Light chain variable domain containing, and / or
(D) CDR-H1 containing an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence shown in SEQ ID NO: 4.
(E) CDR-H2 containing an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5 or the amino acid sequence shown in SEQ ID NO: 5.
(F) CDR-H3 containing an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 6 or the amino acid sequence shown in SEQ ID NO: 6.
Provided is an anti-yeast-derived recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) antibody having a heavy chain variable domain containing, or an antigen-binding fragment thereof.

The antibody or antigen-binding fragment thereof of the present embodiment specifically detects only yeast-derived recombinant human GM-CSF by specifically binding to leucine 23 residue of yeast-derived recombinant human GM-CSF. Can be done. Furthermore, by using the antibody of the present embodiment or an antigen-binding fragment thereof, the concentration of yeast-derived recombinant human GM-CSF in the blood sample of the subject to which yeast-derived recombinant human GM-CSF has been administered is accurately measured. be able to.

As used herein, the term "granulocyte macrophage colony-stimulating factor (GM-CSF)" is a glycoprotein produced by macrophages, lymphocytes, epithelial cells or vascular endothelial cells, and is a granulocyte. Alternatively, it means one that acts on the precursor cells of macrophages and promotes their proliferation and differentiation.
In the amino acid sequence (SEQ ID NO: 7) of the yeast-derived recombinant human GM-CSF in the present embodiment, the 23rd amino acid residue from the N-terminal is replaced with arginine to leucine. The present inventors have found a monoclonal antibody that specifically binds to the 23 residues of leucine, and have completed the present invention.
In the present specification, the Xth amino acid from the N-terminal is referred to as "amino acid X residue". For example, when the 23rd amino acid from the N-terminal is leucine, it is called "leucine 23 residue".

As used herein, the term "monoclonal antibody" means an antibody having substantially uniform specificity. Also, as used herein, monoclonal antibodies include all classes and subclasses of immunoglobulins.
As used herein, the "antigen-binding fragment" of a monoclonal antibody means a part (partial fragment) of the antibody that specifically recognizes a target protein. Specifically, Fab, Fab', F (ab') 2, variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc (Fv) 2, diabody, multispecific antibody, And these polymers and the like.

As used herein, "specific binding" means that an antibody binds only to a target protein (antigen), eg, an in vitro assay, preferably a plasmon resonance assay using a purified wild antigen (eg,). , BIAcore, GE-Healthcare Uppsala, Sweden, etc.), and can be quantified by binding of the antibody to the epitope of the antigen. The affinity of binding can be defined by ka (rate constant for antibody binding from antibody-antigen complex), kD (dissociation constant), and KD (kD / ka). The binding affinity (KD) when the antibody is specifically bound to the antigen ^{is preferably 10-8} mol / L or less, preferably ^10-13 mol / L or more and ^10-9 mol / L or less. Is more preferable.

The antibody of the present embodiment or an antigen-binding fragment thereof is shown in SEQ ID NO: 1, among them, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 2, and SEQ ID NO: 3. A light chain variable domain containing an amino acid sequence, CDR-H1 containing the amino acid sequence set forth in SEQ ID NO: 4, and CDR-H2 containing the amino acid sequence set forth in SEQ ID NO: 5. It preferably comprises a heavy chain variable domain comprising CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 6.
As used herein, "CDR" means complementarity-determining region.

The antibody of the present embodiment or an antigen-binding fragment thereof specifically recognizes only yeast-derived recombinant human GM-CSF by specifically binding to leucine 23 residue of yeast-derived recombinant human GM-CSF. Is. Therefore, if the yeast-derived recombinant human GM-CSF retains the specific recognition ability of the leucine 23 residue, 1 to 1 in the amino acid sequences shown in SEQ ID NOs: 1 to 6 of the above (a) to (f). Several amino acids may be deleted, substituted or added.
Here, the number of amino acids that may be deleted, substituted or added is preferably 1 to 3, more preferably 1 to 2, and even more preferably 1.

As used herein, "substitution" means substituting with another amino acid residue having a chemically similar side chain. A group of amino acid residues having chemically similar amino acid side chains is well known in the art to which the present invention belongs. For example, acidic amino acids (aspartic acid and glutamic acid), basic amino acids (ricin, arginine, histidine), and neutral amino acids, amino acids having a hydrocarbon chain (glycine, alanine, valine, leucine, isoleucine, proline), hydroxy groups Amino acids with (serine / threonine), sulfur-containing amino acids (cysteine / methionine), amino acids with amide groups (asparagin / glutamine), amino acids with imino groups (proline), amino acids with aromatic groups (phenylalanine / tyrosine / It can be classified by tryptophan) and the like.
An amino acid sequence variant in which one to several amino acids are deleted, substituted or added in the antibody of the present embodiment or an antigen-binding fragment thereof has a control antibody (for example, conventional anti-human GM-CSF) having an antigen-binding activity. It is preferably higher than (antibodies, etc.).

Further, since expression purification is easy, the antibody of the present embodiment or an antigen-binding fragment thereof uses a flexible peptide linker to form a heavy chain variable domain and a light chain variable domain, which are the minimum units required for recognizing an antigen. It is preferably a combined univariable domain fragment. That is, it is preferably an scFv antibody.
Specifically, the antibody of the present embodiment or an antigen-binding fragment thereof has the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence shown in SEQ ID NO: 5, the amino acid sequence shown in SEQ ID NO: 6, and SEQ ID NO: 1. It is preferable to have the amino acid sequence shown in FIG. 2, the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 3 in this order.

The antibody of the present embodiment or an antigen-binding fragment thereof can be prepared, for example, by a hybridoma method, a recombinant DNA method, or the like.

Examples of the hybridoma method include a method developed by Kohler and Milstein (see, for example, Kohler & Milstein, Nature, 256: 495, 1975.). The antibody-producing cells used in the cell fusion step in this method include, for example, immunization with an antigen (for example, yeast-derived recombinant human GM-CSF, a peptide fragment containing 23 residues of leucine thereof, or a cell expressing these). Examples thereof include spleen cells, lymph node cells, peripheral blood leukocytes, etc. of animals (eg, mice, rats, hamsters, rabbits, monkeys, goats, etc.). Further, antibody-producing cells obtained by allowing an antigen to act on the above-mentioned cells or lymphocytes isolated from a non-immune animal in a medium can also be used. As the myeloma cells, various known cell lines can be used. The antibody-producing cells and myeloma cells may be of different animal species origin as long as they can be fused, but are preferably of the same animal species origin. As a method for obtaining a hybridoma, for example, a monoclonal antibody specific to a target protein is obtained by cell fusion between spleen cells obtained from an antigen-immunized mouse and mouse myeloma cells and then screened. Examples thereof include a method for obtaining a hybridoma to be produced. Examples of the method for obtaining the monoclonal antibody produced by the hybridoma include a method for obtaining the monoclonal antibody against the target protein by culturing the hybridoma and from the ascites of the mammal transplanted with the hybridoma.

As a recombinant DNA method, for example, the antibody of the present embodiment or the DNA encoding the functional fragment of the antibody is cloned from a hybridoma, B cell, or the like, incorporated into an appropriate vector, and this is incorporated into a host cell (for example, a mammalian cell line). , Escherichia coli, yeast cells, insect cells, plant cells, etc.) to produce the antibody of this embodiment as a recombinant antibody (for example, "PJ Delves, Antibody Production: Essential Techniques, 1997 WILEY"). , "P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS", "Vandamme AM et al., Eur. J. Biochem. 192: 767-775 (1990)").
In the expression of the DNA encoding the antibody of the present embodiment, the DNA encoding the heavy chain or the light chain may be separately incorporated into the expression vector to transform the host cell, and the heavy chain and the light chain are encoded. The DNA may be integrated into a single expression vector to transform the host cell (see, eg, International Patent Application No. 94/11523). The antibody of the present embodiment can be obtained in a substantially pure and uniform form by culturing the host cell, separating and purifying it in the host cell or from the culture medium. For the separation and purification of the antibody, the method used in the conventional purification of a polypeptide can be used. In the method using the transgenic animal production technique, for example, a transgenic animal in which the antibody gene is incorporated (for example, cow, goat, sheep, pig, etc.) is prepared, and the antibody gene is derived from the milk of the transgenic animal. Examples thereof include a method of obtaining a large amount of monoclonal antibody to be used.

The antibody of the present embodiment or an antigen-binding fragment thereof may be an amino acid sequence variant as described above as long as it can specifically bind to the leucine 23 residue of yeast-derived recombinant human GM-CSF. ..
Amino acid sequence variants can be made by mutagenesis into the DNA encoding the antibody strand or by peptide synthesis. The site where the amino acid sequence of the antibody is modified may be the constant region of the heavy chain or light chain of the antibody as long as it has the same activity as the antibody before modification, and the variable region (framework region and). It may be CDR). In addition, a method of modifying the amino acid of the CDR to screen for an antibody having an enhanced affinity for the antigen may be used (for example, "PNAS, 102: 8466-8471 (2005)", "Protein Engineering, Design &Selection". , 21: 485-493 (2008) ", International Publication No. 2002/051870," J. Biol. Chem., 280: 24880-24887 (2005) "," Protein Engineering, Design & Selection, 21: 345-351 (2008) )"reference).

The antigen-binding activity of the antibody of the present embodiment or its antigen-binding fragment (including the above-mentioned amino acid sequence variant and the like) can be determined by, for example, ELISA method, Western blotting method, flow cytometry method, Western blotting method, dot blot method. , Radioimunoassay method, immunoprecipitation method, immunostaining method and the like.

≪Hybridoma≫
In one embodiment, the present invention provides a hybridoma that produces a monoclonal antibody that specifically binds to a yeast-derived recombinant human GM-CSF, wherein the hybridoma is accession number NITE P-02224.

According to the hybridoma of the present embodiment, a monoclonal antibody that specifically binds to the leucine 23 residue of yeast-derived recombinant human GM-CSF can be obtained. The monoclonal antibody produced by the hybridoma of the present embodiment is shown in SEQ ID NO: 1, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 2, and SEQ ID NO: 3. A light chain variable domain containing an amino acid sequence, CDR-H1 containing an amino acid sequence set forth in SEQ ID NO: 4, and CDR-H2 containing an amino acid sequence set forth in SEQ ID NO: 5. It is an antibody containing a heavy chain variable domain containing CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 6.

The hybridoma in the present embodiment can be produced by using the hybridoma method exemplified in the above-mentioned << monoclonal antibody >>.
Specifically, yeast-derived recombinant human GM-CSF, a peptide fragment containing 23 residues of leucine thereof, or cells expressing these are diluted and subjected to an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline, or the like. It becomes turbid, and an appropriate amount of a usual adjuvant (for example, Freund's complete adjuvant, Freund's incomplete adjuvant, etc.) is mixed, emulsified, and administered subcutaneously or intraperitoneally to mice every 4 to 21 days a total of 2 to 10 times. Moreover, a suitable carrier may be used at the time of antigen immunization. After confirming that the antibody level in the serum of the mouse is increased by immunization, final immunization is performed, and the spleen or lymph node of the immunized animal is collected 2 to 5 days after the surface system. Subsequently, the collected cells are fused with mouse myeloma cells to prepare monoclonal antibody-producing mouse hybridoma cells.

Cell fusion can be performed by known methods. For example, antibody-producing cells (cells such as spleen and lymph nodes) collected from immunized animals and myeloma cells are well mixed in a culture medium at a ratio of about 1: 1 to 10: 1 and PEG solution (for example, average). (Molecular weight of about 1,000 to 6,000) is heated to 37 ° C., PEG is added to both cells at a concentration of 30 to 60% (w / v), mixed, and the two cells are fused. After removing the PEG, the fused cells are seeded at a predetermined concentration.

Examples of mouse-derived myeloma cell lines include P3 (P3-X63Ag8), P3U1 (P3-X63Ag8U1), X63.653 (X63Ag8.653), SP2 (Sp2 / 0-Ag14), FO, NS-1 (NSI). / 1-Ag4-1), NSO / 1, FOX-NY and the like are examples of rat-derived myeloma cell lines such as Y3-Ag12.3, YB2 / 0 and IR983F.

Next, for example, the cells are cultured in a HAT selective culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine) for several days to several weeks to kill cells other than the hybridoma and selectively culture the hybridoma.

Next, hybridomas that produce the desired antibody are screened from among the hybridomas that survived in the selective culture medium. The screening method is not particularly limited. For example, in the above-mentioned << monoclonal antibody >>, the target hybridoma is examined by examining the presence or absence of the binding property between the antigen and the antibody in the hybridoma culture supernatant by using the method exemplified as the method for evaluating the binding activity to the antigen. You can select stocks. Then, a hybridoma that produces a target antibody can be selected and monocloned as necessary to obtain a monoclonal antibody-producing hybridoma.

The hybridoma of this embodiment was stipulated in the Putabest Treaty on March 22, 2016 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD) (2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture). It has been deposited domestically under the accession number NITE P-02224.

<< Method for producing a monoclonal antibody that specifically binds to yeast-derived recombinant human GM-CSF >>
In one embodiment, the present invention is a method for producing a monoclonal antibody that specifically binds to a yeast-derived recombinant human GM-CSF, wherein the above-mentioned step of culturing a hybridoma and an antibody produced from the hybridoma are simply used. Provided is a manufacturing method comprising a step of separating.

According to the production method of the present embodiment, a monoclonal antibody that specifically binds to the leucine 23 residue of yeast-derived recombinant human GM-CSF can be obtained. The monoclonal antibody obtained by the production method of the present embodiment is shown in SEQ ID NO: 1, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 2, and SEQ ID NO: 3. CDR-L3 containing the amino acid sequence, and / or CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 4 and CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 5. , CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 6 and an antibody comprising a heavy chain variable domain comprising.

<Culture process>
First, the above-mentioned hybridoma is cultured. As the medium, for example, RPMI1640 containing 10% fetal bovine serum is generally used, and in addition, a medium developed for hybridomas (for example, GIT medium (manufactured by Far East Science Co., Ltd., manufactured by Wako Pure Chemical Industries, Ltd.)). Etc.), etc. Further, examples of the medium used for selecting a hybridoma include a HAT selective culture medium (a culture medium containing hypoxanthin, aminopterin, and thymidine), and the culture temperature is, for example, 30. The temperature may be ℃ or more and 40 ℃ or less, and the culture time can be appropriately set depending on the number of hybridomas required.
The monoclonal antibody can be obtained from the hybridoma by culturing the hybridoma and obtaining it as a culture supernatant thereof, or by administering the hybridoma to the peritoneum of a mammal to proliferate it and obtain it as ascites. ..

<Isolation process>
Isolation and purification of the monoclonal antibody can be carried out by a usual method for separating and purifying immunoglobulin. For example, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, adsorption / desorption method by ion exchanger (for example, DEAE, etc.), ultracentrifugation method, gel filtration method, antigen-binding solid phase or active adsorbent. A monoclonal antibody can be isolated and purified by a method using (for example, protein A, protein G, etc.).

The antigen-binding activity of the obtained monoclonal antibody can be evaluated using the method exemplified as the method for evaluating the antigen-binding activity in the above-mentioned << monoclonal antibody >>.

<< Kit for detecting yeast-derived recombinant human GM-CSF in the blood sample of a subject to which yeast-derived recombinant human GM-CSF was administered >>
In one embodiment, the present invention is a kit for detecting yeast-derived recombinant human GM-CSF in a blood sample of a subject to which yeast-derived recombinant human GM-CSF has been administered, and is derived from the above-mentioned anti-yeast. A detection kit containing a recombinant human GM-CSF antibody or an antigen-binding fragment thereof is provided.

According to the detection kit of the present embodiment, only yeast-derived recombinant human GM-CSF can be specifically detected. In addition, in a pharmacokinetic test on a healthy body, the accurate blood concentration of yeast-derived recombinant human GM-CSF can be measured, and the maximum blood concentration and half-life can be calculated. Furthermore, in a physician-initiated clinical trial of autoimmune PAP patients, accurate trough values can be measured and the patient's systemic clearance can be estimated.

<Antibody against the labeled anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment>
In addition to the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or antigen-binding fragment thereof, the detection kit of the present embodiment further comprises the labeled anti-yeast-derived recombinant human GM-CSF antibody or antigen-binding fragment thereof. It may contain an antibody against. The labeled antibody against the anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment is an antibody against an animal (for example, mouse, rat, hamster, rabbit, monkey, goat, etc.) from which the above-mentioned monoclonal antibody is derived. It is preferable to have. For example, in the case of the antibody against the above-mentioned hybridoma, since it is a monochrome antibody derived from a mouse, it is preferable to use an anti-mouse antibody. In addition, the labeled antibody against the anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment includes all classes and subclasses of immunoglobulins.

Examples of the substance that labels the antibody against the anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment include stable isotopes, radioactive isotopes, fluorescent substances, enzymes, magnetic substances, and the like. Is preferable to be a fluorescent substance because it is easy and highly sensitive. By providing the above-mentioned labeling substance, it is possible to easily and highly sensitively confirm whether or not yeast-derived recombinant human GM-CSF is bound.

Examples of stable isotopes include ¹³ C, ¹⁵ N, ² H, ¹⁷ O, and ¹⁸ O. Radioisotopes include, for example, ³ H, ¹⁴ C, ¹³ N, ³² P, ³³ P, and ³⁵ S.

Examples of the fluorescent substance include cyanine dyes (for example, Cy3, Cy5, etc.), rhodamine 6G reagents, and other known fluorescent dyes (for example, GFP, FITC (Fluorescein), TAMRA, etc.).

Examples of the enzyme include alkaline phosphatase and peroxidase (HRP). When the labeling substance is an enzyme, it is preferable to use an enzyme substrate. In the case of alkaline phosphatase, p-nitrophenyl phosphate (pNPP), 4-methylumbelliferyl phosphate (4-MUP) and the like can be used as the enzyme substrate. When the enzyme is peroxidase, 3,3'-diaminobenzidine (DAB), 3,3', 5,5'-tetramethylbenzine (TMB), o-phenylenediamine (OPD), 2,2-azinodine- (3). -Ethylbenzothiazolin-6-sulfonic acid) (ABTS), 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) and the like can be used.

Examples of the magnetic material include gadolinium, Gd-DTPA, Gd-DTPA-BMA, Gd-HP-DO3A, iodine, iron, iron oxide, chromium, manganese, or a complex or chelate complex thereof.

<Support>
In the detection kit of the present embodiment, the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment may be immobilized on a support.

The form of the support can be appropriately selected from those known in the art, for example, microtiter plates, tubes, capillary tubes, beads, particles, filter papers, filters, membranes, discs, sticks, strips, slide glasses, chips and the like. Can be mentioned.

As the material of the support, any material known in the art can be used, and for example, glass, resin (for example, polystyrene, polyester, polyacrylamide, polyacrylate, polymethacrylate, polypropylene, polyvinylbutyrate, polyvinyl chloride, etc.) can be used. Examples thereof include polytetrafluoroethylene, polyvinylidene fluoride, nylon, rayon, etc.), cellulose and derivatives thereof (for example, nitrocellulose, etc.).
The method for immobilizing the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or antigen-binding fragment thereof on a support is, for example, physical adsorption, covalent bond or non-covalent bond (eg, ionic bond, electrostatic bond, hydrophobicity). Interaction, etc.) and the like.

The support is preferably blocked to prevent non-specific adsorption. Any of the blocking agents known in the art can be used, for example, serum albumin of mammals other than the mammal to be detected (for example, when the subject is human, for example, bovine serum albumin (BSA)), skim milk. Examples include powdered milk and casein.
The surface of the support on which the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment is immobilized and treated with blocking is a water-soluble polymer (for example, polyvinylpyrrolidone, polyvinyl alcohol, or hydroxypropylmethylcellulose and hydroxyethylcellulose). Such as cellulose ester), or non-reducing polysaccharides (eg, sucrose, trehalose, raffinose, etc.) and the like. With such coding, the solid phase containing the above-mentioned monoclonal antibody on the support is highly stabilized and suitable for long-term storage.

<Others>
The detection kit of the present embodiment may further contain a reaction terminator, if necessary. Examples of the reaction terminator include sulfuric acid, sodium hydroxide and the like.

The detection kit of this embodiment may further contain a buffer solution. As the buffer solution, the buffer solution can be appropriately selected from those known in the art, and for example, a phosphate buffer solution, a Tris hydrochloric acid buffer solution, a citrate buffer solution, a carbon dioxide buffer solution, a borate buffer solution, and a succinic acid buffer solution. , Acetate buffer and the like. The buffer may optionally contain at least one of NaCl, a surfactant (eg, Tween 20, Triton X-100, etc.) and a preservative (eg, sodium azide, etc.). Specific examples of the buffer solution include Phosphate Buffered Saline (PBS), PBS-T (PBS-Tween 20), Tris Buffered Saline (TBS), or TBS-T (TBS). TBS-Tween 20) and the like can be mentioned.

In the detection kit of the present embodiment, the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or antigen-binding fragment thereof and the labeled antibody against the anti-yeast-derived recombinant human GM-CSF antibody or antigen-binding fragment thereof are dried. It may be in a state or may be dissolved in the above-mentioned buffer solution. Above all, these antibodies are preferably in a dry state from the viewpoint of storage stability.

The detection kit of the present embodiment may further include a standard solution for creating a standard curve (calibration curve), a standard solution diluent (for example, the above-mentioned buffer solution, etc.).
The detection kit of this embodiment further comprises a cleaning solution (eg, buffer as described above) and a sealant (eg, plate seal if the support is a plate) that seals the support during incubation. You may be.

The detection kit of the present embodiment may further include a detection device. The detection device can be appropriately selected from those known in the art, and examples thereof include a microplate reader, a fluorescence scanner, a two-photon excitation scanner, and the like.

<< Method for detecting yeast-derived recombinant human GM-CSF in the blood sample of a subject to which yeast-derived recombinant human GM-CSF was administered >>
In one embodiment, the present invention is a method for detecting yeast-derived recombinant human GM-CSF in a blood sample of a subject to which yeast-derived recombinant human GM-CSF has been administered. Provided is a method comprising a primary antigen-antibody reaction step in which an antigen-antibody reaction is carried out by contacting the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or an antigen-binding fragment thereof.

According to the detection method of the present embodiment, only yeast-derived recombinant human GM-CSF can be specifically detected. In addition, in a pharmacokinetic test on a healthy body, the accurate blood concentration of yeast-derived recombinant human GM-CSF can be measured, and the maximum blood concentration and half-life can be calculated. Furthermore, in a physician-initiated clinical trial of autoimmune PAP patients, accurate trough values can be measured and the patient's systemic clearance can be estimated.

<Primary antigen-antibody reaction>
First, a blood sample is collected from a subject to which yeast-derived recombinant human GM-CSF has been administered. When measuring the peak concentration of yeast-derived recombinant human GM-CSF in blood, a blood sample may be collected, for example, 15 to 30 minutes after administration of yeast-derived recombinant human GM-CSF.

In the present specification, examples of the "blood sample" include blood, serum, plasma and the like.
In the present specification, examples of the "healthy body" and the "subject" include various mammals including humans and non-human animals, and among them, humans are preferable.

In addition, as a method of administering yeast-derived recombinant human GM-CSF to a subject, for example, a method of intravenously injecting a preparation containing yeast-derived recombinant human GM-CSF, or spray administration into the trachea using a microspray. Or a method of administering via airway using a membrane-type nebulizer or a jet nebulizer.

The preparation containing the yeast-derived recombinant human GM-CSF may contain a pharmaceutically acceptable carrier or diluent as other components. Pharmaceutically acceptable carriers or diluents include, for example, excipients, dilutants, bulking agents, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, viscous agents. Examples include agents, flavoring agents, solubilizing agents, additives and the like. By using one or more of these carriers, a pharmaceutical composition in the form of an injection, a liquid, a suspension, an emulsion, a syrup, or the like can be prepared.

A colloidal dispersion system can also be used as the carrier. The colloidal dispersion system is expected to have the effect of enhancing the in vivo stability of yeast-derived recombinant human GM-CSF and the effect of enhancing the transferability of yeast-derived recombinant human GM-CSF to specific organs, tissues, or cells. NS. Examples of the colloidal dispersion system include polyethylene glycol, polymer composites, polymer aggregates, nanocapsules, microspheres, beads, oil-in-water emulsifiers, micelles, mixed micelles, and lipids including liposomes. can.

As pharmacologically acceptable carriers or diluents, more specifically, sterile water, physiological saline, vegetable oils, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives. Examples thereof include those formulated by appropriately combining with an agent, a binder and the like and mixing in a unit dose form required for generally accepted pharmaceutical practice.

When the preparation containing the yeast-derived recombinant human GM-CSF is an injection, the sterile composition can be formulated according to the usual preparation practice using a vehicle such as distilled water for injection. Examples of the aqueous solution for injection include a physiological saline solution, an isotonic solution containing glucose and other auxiliary agents, for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride and the like, which are appropriately dissolved. With auxiliary agents (eg, alcohol (specifically, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol, etc.)), or nonionic surfactants (eg, polysorbate 80 (TM), HCO-50, etc.) It may be used together.

Examples of the oily liquid include sesame oil and soybean oil, and examples of the solubilizing agent may be used in combination with, for example, benzyl benzoate, benzyl alcohol and the like. Further, a buffer (for example, phosphate buffer, sodium acetate buffer, etc.), a soothing agent (for example, procaine hydrochloride, etc.), a stabilizer (for example, benzyl alcohol, phenol, etc.), or an antioxidant is further added. You may. The prepared injection solution is usually filled in a suitable ampoule.

Injectables can also be prepared as non-aqueous diluents (eg, vegetable oils such as polyethylene glycol and olive oil, alcohols such as ethanol), suspensions, or emulsions. Such sterilization of the injection can be performed by filtration sterilization with a filter, blending of a bactericidal agent, or the like. Injections can be produced in the form of errand preparation. That is, a sterile solid composition can be prepared by a freeze-drying method or the like, and the composition can be dissolved in distilled water for injection or another solvent before use.

The dose of the yeast-derived recombinant human GM-CSF is appropriately adjusted in consideration of the age, sex, body weight, symptom, treatment method, administration method, treatment time, etc. of the subject.
For example, when a subject (human) is inhaled using a microspray, a membrane nebulizer or a jet nebulizer, the daily dose to the subject is, for example, 1 ng to 5 μg of yeast-derived recombinant human per kg body weight. An amount of GM-CSF may be administered, for example, an amount of 10 ng to 1 μg of yeast-derived recombinant human GM-CSF may be administered, for example, an amount of 50 ng to 500 ng of yeast-derived recombinant human GM-CSF may be administered. Just do it. The number of administrations may be one to several times per day on average.

The peak blood concentration of yeast-derived recombinant human GM-CSF may be, for example, 200 pg / mL to 2 ng / mL, for example, 400 pg / mL to 1.5 ng / mL, for example, 500 pg / mL. It may be ~ 1 ng / mL.

Subsequently, the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment is brought into contact with a blood sample collected from a subject, and the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or its antigen is brought into contact with each other. A primary antigen-antibody reaction is carried out between the binding fragment and the antigen (yeast-derived recombinant human GM-CSF) in the blood sample. This antigen-antibody reaction is preferably carried out at 4 ° C. or higher and 37 ° C. or lower. The reaction time may be appropriately adjusted according to the antibody titer of the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment and the amount of antigen (yeast-derived recombinant human GM-CSF) in the blood sample. can.

The above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment may be dissolved or diluted with, for example, a buffer solution or the like. The buffer solution to be used is the same as that exemplified in the above-mentioned << Kit for detecting yeast-derived recombinant human GM-CSF in the blood sample of the subject to which yeast-derived recombinant human GM-CSF was administered >>. Things can be mentioned.

When the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment is immobilized on a support such as a microtiter plate, tube, slide glass or chip, the anti-yeast-derived recombinant human GM-CSF antibody or A blood sample may be dropped and brought into contact with the support on which the antigen-binding fragment is immobilized. When the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment is immobilized on a support such as beads or particles, the anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment can be used. The immobilized support is suspended in a buffer solution or the like, and a solution containing the anti-yeast-derived recombinant human GM-CSF antibody or the support on which the antigen-binding fragment thereof is immobilized is mixed and contacted with a blood sample. Just do it. The buffer solution to be used is the same as that exemplified in the above-mentioned << Kit for detecting yeast-derived recombinant human GM-CSF in the blood sample of the subject to which yeast-derived recombinant human GM-CSF was administered >>. Things can be mentioned.

Further, a pretreatment step for denaturing the yeast-derived recombinant human GM-CSF contained in the blood sample may be provided before the primary antigen-antibody reaction. By denatured yeast-derived recombinant human GM-CSF, it is possible to prevent the anti-human GM-CSF autoantibody contained in the blood sample from binding to yeast-derived recombinant human GM-CSF to form a complex. .. Examples of the method of denaturation include a method of heating in the presence of a denaturing agent or a surfactant at a temperature of, for example, 90 ° C. or higher and 95 ° C. or lower, for example, 3 minutes or more and 10 minutes or less. Examples of the denaturing agent used include dithiothreitol (DTT) and the like. Examples of the surfactant to be used include Sodium dodecyl sulfate (SDS) and the like.

The primary antigen-antibody reaction may be detected by using the above-mentioned labeled anti-yeast-derived recombinant human GM-CSF antibody or an antigen-binding fragment thereof. Further, when the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment is immobilized on a support such as a microtiter plate, tube, slide glass or chip, the yeast-derived recombinant human GM-CSF can be used. When bound to the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment, the primary antigen-antibody reaction may be detected by a sensor or the like. From the viewpoint of detection sensitivity and reaction specificity, it is preferable to carry out the subsequent secondary antigen-antibody reaction step to detect yeast-derived recombinant human GM-CSF.

<Secondary antigen-antibody reaction>
Subsequently, the blood sample is removed and washed. The washing may be performed using, for example, a buffer solution or the like. The buffer solution to be used is the same as that exemplified in the above-mentioned << Kit for detecting yeast-derived recombinant human GM-CSF in the blood sample of the subject to which yeast-derived recombinant human GM-CSF was administered >>. Things can be mentioned.

Subsequently, the labeled antibody against the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment is brought into contact with the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment. This antigen-antibody reaction is also preferably carried out at 4 ° C. or higher and 37 ° C. or lower. The reaction time can be appropriately adjusted depending on the antibody titer of the labeled anti-yeast-derived recombinant human GM-CSF antibody used or the antibody against the antigen-binding fragment thereof.

As the antibody against the above-mentioned labeled anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment to be used, the above-mentioned << yeast-derived recombinant human GM-CSF-derived from yeast in the blood sample of the subject to which the above-mentioned yeast-derived recombinant human GM-CSF was administered. The same as those exemplified in the kit for detecting recombinant human GM-CSF >> can be mentioned.

The labeled antibody against the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment may be dissolved or diluted with, for example, a buffer solution or the like. The buffer solution to be used is the same as that exemplified in the above-mentioned << Kit for detecting yeast-derived recombinant human GM-CSF in the blood sample of the subject to which yeast-derived recombinant human GM-CSF was administered >>. Things can be mentioned.

When the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment is immobilized on a support such as a microtiter plate, tube, slide glass or chip, the above-mentioned anti-yeast-derived recombinant human is labeled. A solution containing the GM-CSF antibody or an antibody against the antigen-binding fragment thereof may be added dropwise and contacted. When the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment is immobilized on a support such as beads or particles, the anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment can be used. The solution containing the immobilized support may be mixed and contacted with the labeled solution containing the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or an antibody against an antigen-binding fragment thereof.

Further, after the secondary antigen-antibody reaction, washing may be performed in the same manner as after the primary antigen-antibody reaction. When the labeled substance of the labeled anti-yeast-derived recombinant human GM-CSF antibody or the antibody-labeled substance thereof against the antigen-binding fragment thereof is an enzyme, the step of contacting with the enzyme substrate and the reaction stop solution are added to stop the reaction. It may be further provided with a step of causing the reaction.

Subsequently, the presence or absence of the yeast-derived recombinant human GM-CSF can be determined by detecting the labeled anti-yeast-derived recombinant human GM-CSF antibody or an antibody against the antigen-binding fragment thereof. Furthermore, by detecting the labeled anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment, the yeast-derived recombinant human GM-CSF contained in the blood sample can be quantified. The detection method can be appropriately selected by those skilled in the art depending on the type of labeling substance. For example, when detecting an anti-human antibody labeled with a fluorescent substance, it can be detected by a fluorescent scanner, a two-photon excitation scanner, or the like.

The detection method of the present embodiment is a detection mechanism when the above-mentioned anti-yeast-derived recombinant human GM-CSF antibody or its antigen-binding fragment is immobilized on a support such as a microtiter plate, tube, slide glass or chip. By combining with a peripheral device necessary for analysis such as a sample dispensing mechanism, it can be provided as a detection system for yeast-derived recombinant human GM-CSF in a fully automatic or semi-automatic blood sample. According to this detection system, the presence or absence of yeast-derived recombinant human GM-CSF contained in a blood sample can be easily determined fully or semi-automatically. Furthermore, the yeast-derived recombinant human GM-CSF contained in the blood sample can be quantified, and the accurate blood concentration of the yeast-derived recombinant human GM-CSF can be measured.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the following Examples.

[Example 1] Establishment of hybridoma The establishment of the hybridoma and the production of the monoclonal antibody shown below were outsourced to ITM. In addition, ITM used the iliac lymph node method (see Japanese Patent No. 4098796, which was carried out with a license from Shigei Medical Research Institute) to establish a hybridoma and to produce a monoclonal antibody.

(1) Immunity to Mice First, yeast-derived recombinant human GM-CSF (manufactured by GENZYME) was denatured by heating at 95 ° C. for 5 minutes in the presence of 0.1% dithiothreitol (DTT). Subsequently, this denatured yeast-derived recombinant human GM-CSF was injected into the mouse ridge and immunized. The injection was prepared to 200 μg / injection and immunized only once.

(2) Preparation of hybridomas Subsequently, the spleen was excised from the immune mouse, and the excised spleen cells and myeloma cells (P3Ag3.653 strain, manufactured by ATCC) were fused with polyethylene glycol and sorted by HAT medium. .. Subsequently, an anti-yeast-derived recombinant human GM-CSF antibody hybridoma was screened. Screening was performed by the ELISA method using a denatured yeast-derived recombinant human GM-CSF as an antigen.

The strains that were positive in the screening were monocloned, and finally, multiple anti-yeast-derived recombinant human GM-CSF antibody acidic hybridomas were established. One of the hybridoma strains was deposited because it was found by performing Test Examples 1 and 2 described later that a monoclonal antibody that specifically binds only to yeast-derived recombinant human GM-CSF was produced (NITE P). -02224).

Subsequently, the above deposited hybridoma was cultured, and IgG was bound to a protein G column (manufactured by GE Bioscience) using an immunoglobulin fraction precipitated and concentrated with 50% ammonium sulfate, and the mixture was used at 100 mM glycine (pH 2.7). It was eluted and separated from the culture supernatant to obtain a purified antibody.

Hereinafter, the antibody obtained from NITE P-02224 will be referred to as "40-1H antibody".

[Test Example 1] Examination of antibody specificity 1
(1) Peptide Immobilization Four types of peptide fragments (No. 1 to 4) consisting of the partial amino acid sequences of yeast-derived recombinant human GM-CSF shown in Table 1 below were commissioned by GenScript to synthesize them. Subsequently, No. 1 to 4 synthetic peptides and full-length yeast-derived recombinant human GM-CSF (No. 5) (also known as sargramostim and Genzyme) were prepared in solutions having a concentration of 1 μg / mL, respectively, and immobilized. (See Fig. 1).

(2) Detection by ELISA (Enzyme-linked immuno-sorbent assay) Subsequently, 50 μL of 40-1H antibody dissolved in PBS was added to each well and reacted at room temperature for 45 minutes. Subsequently, it was washed 3 times with 200 μL of PBS-Tween. Subsequently, as a secondary antibody, an anti-mouse IgG antibody (manufactured by Sigma-Aldrich) labeled with horseradish peroxidase (HRP) was used. 50 μL of secondary antibody was added to each well and reacted at room temperature for 30 minutes. Subsequently, it was washed 3 times with 200 μL of PBS-Tween. Subsequently, 50 μL of TMB (3,3', 5,5'-tetramethylbenzidine) was added as an enzyme substrate and reacted at room temperature for 15 minutes. Subsequently, 50 μL of 0.5 M sulfuric acid was added as a reaction terminator, and immediately, the absorbance at 450 nm was measured using a microplate reader (manufactured by Bio-Rad). The results are shown in FIG. 2 and Table 2. FIG. 2 (A) is an image showing the state of color development of each well immediately after the addition of the reaction terminator, and FIG. 2 (B) is a graph showing the result of measuring the absorbance at 450 nm in each well.

From FIGS. 2 (A) and 2 (B), No. 1 and No. At 5, color development was detected. Further, the absorbance of 1.0 or more was detected, but hardly detected in the other wells. No. 1 and No. In 5, from this, the 40-1H antibody was No. It was inferred that it specifically binds to the peptide fragment of 1.

[Test Example 2] Examination of antibody specificity 2
(1) Immobilization of Peptide Denatured yeast-derived recombinant human GM-CSF using the same method as in Example 1, unmodified yeast-derived recombinant human GM-CSF (manufactured by Genzyme), E. coli-derived group Known methods for replacement human GM-CSF (also known as Morgramostim, manufactured by Amytop), CHO (Chinese Hamster Ovaly) cell-producing recombinant human GM-CSF (manufactured by Humanzyme), and bovine serum albumin (BSA). Was prepared using. Subsequently, these five types of recombinant human GM-CSF were prepared in a solution having a concentration of 1 μg / mL in a 96-well plate, and solid-phased.

(2) Detection by ELISA Using the same method as in (2) of Test Example 1, the absorbance at 450 nm was measured. The results are shown in FIG.

From FIG. 3, No. 1 and No. Absorbance was detected in 2, but hardly detected in the other wells. From this, it was inferred that the 40-1H antibody specifically binds to yeast-derived recombinant human GM-CSF.

[Test Example 3] Examination of antibody specificity 3
(1) Peptide Immobilization 13 types of peptide fragments (No. 6, 8-19) consisting of partial amino acid sequences of yeast-derived recombinant human GM-CSF shown in Table 2 below and Escherichia coli-derived recombinant human GM-CSF. A peptide fragment (No. 7) consisting of the partial amino acid sequence of No. 7 was commissioned by GenScript to synthesize it. Subsequently, No. Each of 6 to 19 synthetic peptides was prepared in a solution having a concentration of 1 μg / mL and immobilized.

(2) Detection by ELISA Using the same method as in (2) of Test Example 1, the absorbance at 450 nm was measured. The results are shown in Table 2 above. When the absorbance in the wells where the peptide was not immobilized was used as the control, those with significantly higher absorbance than the control were marked with "○", and those with no significant difference in absorbance were marked with "x". Was judged.

From Table 2, the amino acid sequence (that is, epitope) of the yeast-derived recombinant human GM-CSF recognized by the 40-1H antibody was 18 amino acid residues from the 10th to the 27th from the N-terminal. The 18 amino acid residue contains the 23rd leucine residue from the N-terminal. Therefore, it was confirmed that the 40-1H antibody does not react with the natural GM-CSF having the 23rd arginine residue from the N-terminal.
In addition, since the 18 amino acid residues include the entire first α-helix in yeast-derived recombinant human GM-CSF, it was inferred that the 40-1H antibody recognizes this tertiary structure. ..

[Test Example 4] Pharmacokinetic test of yeast-derived recombinant human GM-CSF (1) Inhalation administration of yeast-derived recombinant human GM-CSF to healthy cynomolgus monkeys First, 50 mg of yeast-derived recombinant human GM-CSF was administered to healthy cynomolgus monkeys. It was administered by inhalation. Then, immediately after inhalation, blood was collected 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours later, and serum was separated.

(2) Detection by ELISA Next, an alkaline phosphatase (ALP) -labeled mouse monoclonal antibody (33-8F: IgG1) (hereinafter, referred to as "33-8F antibody" may be used, using the 40-1H antibody as a capture antibody. ), The quantitative sandwich ELISA method of yeast-derived recombinant human GM-CSF was used for detection.
Specifically, first, the sera obtained in (1) were heat-denatured at 95 ° C. for 30 minutes in the presence of SDS, respectively. Then, the serum after heat denaturation and the 33-8F antibody were mixed and allowed to stand at 4 ° C. overnight. This mixed sample was then added to a microplate on which the 40-1H antibody was immobilized and hatched at room temperature for 1 hour. Then, after washing the microplate, a CDP-Star (registered trademark) alkaline phosphatase substrate (Tropix (registered trademark) CDP-Star (registered trademark) Sapphire II: Applied Biosystems) was added. Next, the luminescence was measured using a fluorescence / luminescence microplate reader (manufactured by Bersold). The results are shown in FIG. In FIG. 4, "LEUKINE®" represents yeast-derived recombinant human GM-CSF.

From FIG. 4, the blood concentration of the yeast-derived recombinant human GM-CSF increased sharply immediately after inhalation, and a peak was observed 15 minutes after inhalation. After that, the blood concentration of yeast-derived recombinant human GM-CSF gradually decreased, but did not disappear during the observation period. The half time of the yeast-derived recombinant human GM-CSF was 20.1 minutes.
From the above, it was clarified that the yeast-derived recombinant human GM-CSF administered into the body can be specifically detected by using the 40-1H antibody.

According to the anti-yeast-derived recombinant human GM-CSF antibody of the present embodiment or an antigen-binding fragment thereof, only yeast-derived recombinant human GM-CSF can be specifically detected. In addition, according to the detection kit of the present embodiment, it is possible to accurately measure the accurate blood concentration of yeast-derived recombinant human GM-CSF in a pharmacokinetic test on a healthy body, and the maximum blood concentration and half-life. The period can be calculated. Furthermore, in a physician-initiated clinical trial of autoimmune PAP patients, accurate trough values (minimum blood drug concentration in steady state after repeated doses of the drug) can be measured, and the patient's systemic clearance (drug) can be measured. The ability to excrete outside the body) can be estimated.

Claims (8)

A hybridoma that produces a monoclonal antibody that specifically binds to a yeast-derived recombinant human GM-CSF, wherein the hybridoma has accession number NITE P-02224. A method for producing a monoclonal antibody that specifically binds to yeast-derived recombinant human GM-CSF.
The step of culturing the hybridoma according to claim 1 and
The step of isolating the antibody produced from the hybridoma and
A manufacturing method characterized by comprising. A light chain variable containing CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 2, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 3. Domain and
Variable chain including CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 6. An anti-yeast-derived recombinant human GM-CSF antibody having a domain. An antibody produced from the hybridoma according to claim 1. A kit for detecting yeast-derived recombinant human GM-CSF in a blood sample of a subject to which yeast-derived recombinant human GM-CSF has been administered, which comprises the antibody according to claim 3 or 4. Detection kit. The detection kit according to claim 5, further comprising the labeled anti-yeast-derived recombinant human GM-CSF antibody. A method for detecting yeast-derived recombinant human GM-CSF in a blood sample of a subject to which yeast-derived recombinant human GM-CSF has been administered.
A method comprising a primary antigen-antibody reaction step in which the blood sample is brought into contact with the antibody according to claim 3 or 4 to carry out an antigen-antibody reaction. The second aspect of claim 7, further comprising a secondary antigen-antibody reaction step of contacting the labeled anti-yeast-derived recombinant human GM-CSF antibody to carry out an antigen-antibody reaction after the primary antigen-antibody reaction step. Method.

Images