JP6952295B2 - Method for producing human anti-HLA monoclonal antibody - Google Patents

Description

The present invention relates to a method for producing a human anti-HLA monoclonal antibody and the use of the antibody.

HLA (Human Leukocyte Antigen), which is a major histocompatibility complex molecule in humans, includes Class I antigens (A, B, C, E, F, G) and Class II antigens (DR, DQ, DP). ) Etc., and there are numerous polymorphisms in each antigen. Each human has two types of coexistence inherited from his parents, and the HLA type differs from person to person. In hematopoietic stem cell transplantation and the like, it is generally important that at least A, B and DR types are compatible.

It has also been found that the likelihood of disease morbidity varies depending on the type of HLA. For example, the relationship between diseases such as diabetes, rheumatoid arthritis, and multiple sclerosis and specific HLA is known, and in recent years, the possibility of diagnosing and treating diseases using anti-HLA antibodies has been reported (for example,). Patent Documents 1 and 2, Non-Patent Document 1). As an example, an attempt was made to treat lymphoid tumors with anti-HLA class II antibodies, and the 2015 American Society of Hematology reported the results of a clinical trial (Phase I) that the therapeutic effect was remarkable and the side effects were minor. (Non-Patent Document 2). Furthermore, for example, it has been attempted to improve the treatment results by easily monitoring the recovery of normal hematopoiesis after allogeneic hematopoietic stem cell transplantation, early recurrence of hematopoietic tumors, and minimal residual disease by flow cytometry examination (non-patent). Document 3).

Conventionally, antibodies, particularly monoclonal antibodies, have been produced by a method using a hybridoma in which an antigen is administered to an animal such as a mouse and the obtained antibody-producing cells are fused with myeloma cells. The monoclonal antibody produced by this method is originally a non-human monoclonal antibody, but for in vivo use in humans, it should be made into a humanized antibody or even a human antibody by utilizing genetic recombination technology. Is known to be suitable.

U.S. Patent Application Publication No. 2014/0227178 Japanese Patent Application Laid-Open No. 2015-180607

Cancer Science, June 2007, vol.98, no.6, 921-928 2015 American Society of Hematology, abstract # 2740 Watanabe N et al. BBMT 2008, 14 (6): 693-701

As mentioned above, while the usefulness of anti-HLA antibodies has been reported, monoclonal antibodies (mAbs) against HLA molecules are known to be difficult to prepare due to their polymorphisms. Even if a transgenic mouse having a human HLA gene is immunized with a purified HLA antigen, it is very difficult to obtain the desired antibody. Therefore, there is a need for a method for rapidly obtaining an anti-HLA monoclonal antibody having binding specificity for each HLA type that differs from human to human as a fully human antibody.

In the clinical setting of hematology, there is an opportunity to diagnose anti-HLA antibody-positive patients among multiparous women and those who have experienced blood transfusion. In such anti-HLA antibody-positive patients, it is known that the transfused platelets are quickly destroyed by the anti-HLA antibody and the transfusion effect cannot be obtained, although the antibody-positive does not cause symptoms. ing. From these findings, lymphocytes that produce anti-HLA antibody exist in the bodies of some healthy subjects and patients who have undergone blood transfusion / transplantation, and the anti-HLA antibody produced by the lymphocytes becomes cells that express HLA antigen. It is known to combine and destroy it.

On the other hand, the group of the present inventors is developing a method for obtaining antibody-producing cells specific for a specific antigen from human peripheral blood-derived cells in a short period of time using a chip (Nature Medicine, Vol. .15, No.9, September 2009, 1088-1092, Patent No. 4148637).

Based on the above findings and techniques, the present inventors have cloned immunoglobulin genes from anti-HLA antibody-producing cells in the peripheral blood or bone marrow of anti-HLA antibody-positive patients, thereby providing anti-HLA useful for clinical examinations and the like. We have found that it is possible to produce monoclonal antibodies.

That is, the present invention provides the following.
(1) A method for isolating cells producing a human anti-HLA monoclonal antibody having specificity for a specific HLA type.
(a) Steps to identify the presence of anti-HLA antibodies in mononucleosis derived from human subjects,
(b) A step of culturing mononucleosis containing cells capable of producing the desired anti-HLA monoclonal antibody, and
(c) The method described above comprising the step of isolating antibody-producing cells from cultured mononucleosis.
(2) A method for producing a human anti-HLA monoclonal antibody having specificity for a specific HLA type.
(d) A step of cloning a gene encoding an antibody produced by an antibody-producing cell obtained according to the method described in (1) above.
(e) Steps to introduce the cloned gene into cells in vitro, and
(f) The method described above comprising culturing the cells, proliferating them and recovering the antibody.
(3) The method according to (1) or (2) above, wherein mononuclear cells are obtained from cells derived from peripheral blood or bone marrow.
(4) The method according to any one of (1) to (3) above, wherein the human subject is a female who has experience of pregnancy or a subject who has experience of blood transfusion, hematopoietic stem cell transplantation, or organ transplantation.
(5) The above-mentioned (1) to (4), wherein the HLA type belongs to any of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP. the method of.
(6) The method according to any one of claims (1) to (5), wherein the HLA type is HLA-B61.
(7) An isolated fully human anti-HLA monoclonal antibody obtained by the method according to any one of (2) to (6) above.
(8) A monoclonal antibody that specifically binds to the human HLA-B61 antigen, and the following:
(a) A heavy chain variable region polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 or a polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence.
(b) A light chain variable region polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4 or a polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence. The above monoclonal antibody.
(9) A monoclonal antibody that specifically binds to the human HLA-B61 antigen, and the following:
(a) A heavy chain variable region polypeptide which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence, and
(b) The above, which comprises a light chain variable region polypeptide which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5 or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence. Monoclonal antibody.
(10) A monoclonal antibody that specifically binds to the human HLA-B61 antigen, CDH1 consisting of the amino acid sequence shown in SEQ ID NO: 6, CDH2 consisting of the amino acid sequence shown in SEQ ID NO: 7, and SEQ ID NO: 8. The above-mentioned monoclonal antibody comprising CDH3 consisting of the amino acid sequence shown, CDL1 consisting of the amino acid sequence shown in SEQ ID NO: 9, CDL2 consisting of the amino acid sequence shown in SEQ ID NO: 10, and CDL3 consisting of the amino acid sequence shown in SEQ ID NO: 11.
(11) An anti-HLA monoclonal antibody having the same binding specificity as the monoclonal antibody according to any one of (7) to (10) above.
(12) A cytotoxic activator for a specific HLA type, which comprises the monoclonal antibody according to any one of (7) to (10) above.
(13) Includes contacting a sample collected from a patient with aplastic anemia with the monoclonal antibody according to any one of (7) to (11) above to detect the expression of the HLA-B61 molecule in the sample. , A method of predicting the effectiveness of immunosuppressive therapy for the patient.

INDUSTRIAL APPLICABILITY The present invention provides a novel method for producing a monoclonal antibody against an HLA molecule, which has been known to be difficult to produce. Since mononucleosis obtained from a human subject is used, the obtained antibody is a fully human antibody, and it is considered that the risk of developing side effects is extremely low when administered to humans.

Further, in the production of a monoclonal antibody by gene recombination, it is easy to partially modify the antibody at the time of gene recombination. For example, when an anti-HLA antibody is used as a test reagent, the Fc portion of the antibody is human type, so when testing using human peripheral blood, non-specific binding occurs in the Fc portion, and the test results show There is a risk of impact. However, by converting the Fc moiety to, for example, a mouse type, this non-specific binding can be avoided and can be properly tested.

Furthermore, aplastic anemia cases in which HLA-B61-deficient blood cells for which immunosuppressive therapy is easily responded can be diagnosed by the anti-HLA-B61 antibody obtained by the method of the present invention.

The outline of the ISAAC (Immunospot Array Assay on a Chip) method used for obtaining the anti-HLA antibody of the present invention is shown. The detection result of HLA-B61 antigen by anti-HLA-B61 antibody is shown. A: Shows binding to a control antibody or anti-HLA-B61 antibody of an HLA-B61 positive B-LCL (B-lymphoblastoid cell lines) cell line. B: Shows binding of HLA-B61 negative B-LCL cell line to control antibody or anti-HLA-B61 antibody. The binding to the control antibody or anti-HLA-B61 antibody of the peripheral blood CD3 + fraction of the HLA-B61 negative patient (A) or the HLA-B61 positive patient (B) is shown. The binding of various antibodies or antibody-positive donor sera to the HLA antigen (HLA-A24 or HLA-B61) is shown. The binding to the control antibody or anti-HLA-A24 antibody of the peripheral blood CD3 + fraction of the HLA-A24 negative patient (A) or the HLA-A24 positive patient (B) is shown. Shows HLA-dependent complement-dependent cytotoxic activity (CDC) by anti-HLA-B61 antibody. The anti-HLA-B61 antibody exhibits specific cytotoxic activity against cells from donors 1 and 2 carrying the HLA-B 40:02 allele in the presence of complement. On the other hand, the isotype control antibody (anti-influenza virus antibody) showed no cytotoxic activity against any donor. It shows antibody-dependent cellular cytotoxicity (ADCC) against HLA-B61 positive B-LCL cell line by anti-HLA-B61 antibody. Most cells remain alive with the isotype control antibody, but an increased proportion of dead cells is shown with the anti-HLA-B61 antibody.

[Method of isolating human anti-HLA monoclonal antibody-producing cells]
The present invention provides a method for isolating cells that produce human anti-HLA monoclonal antibodies that have specificity for a particular HLA type.

Specifically, the method of the present invention is described below.
(a) Steps to identify the presence of anti-HLA antibodies in mononucleosis derived from human subjects,
(b) A step of culturing mononucleosis containing cells capable of producing the desired anti-HLA monoclonal antibody, and
(c) Including the step of isolating antibody-producing cells from cultured mononucleosis.

In the method of the present invention, the human subject may be a healthy person or a patient having a specific disease, and is not particularly limited. However, subjects who are likely to have anti-HLA antibodies include, for example, women who have had pregnancy or who have had blood transfusions, hematopoietic stem cell transplants, or organ transplants.

Mononuclear cells are a group of cells that include lymphocytes and monocytes, and thus include antibody-producing cells and other cells. Mononuclear cells derived from human subjects can be obtained from cells derived from peripheral blood or bone marrow derived from subjects. Alternatively, mononucleosis from other organs such as the spleen and lymph nodes from the subject can be utilized (Hum Antibodies Hybridomas. 1: 160-165, 1990).

The mononuclear sphere can be prepared, for example, as follows. Peripheral blood or the like is layered on a mononuclear cell separation solution (specific gravity = about 1.077), and after centrifugation (400xg, about 30 minutes), up to 0.5 cm above the opaque intermediate layer containing mononuclear cells. Remove the upper layer. Then, phosphate buffered saline is added to the opaque intermediate layer, mixed slowly, centrifuged (250 xg, 10 minutes), and then the cells that have settled are mononuclear cells.

As used herein, the "specific HLA type" is an HLA type that belongs to any of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP, for example. Intended for HLA antigens such as HLA-A2, HLA-A24, HLA-B61, HLA-B62, HLA-DR15, HLA-DR4. Therefore, "having specificity for a specific HLA type" means, for example, having specificity for a specific HLA antigen and not having specificity for another HLA antigen. "Specificity" in the binding of antigens and antibodies is as generally understood in the art. However, it is possible, for example, that an anti-HLA monoclonal antibody may cross-react with an HLA antigen having a similar epitope. Thus, anti-HLA monoclonal antibodies can bind to one or more HLA antigens. In this case, the multiple HLA antigens to which one monoclonal antibody can bind can be antigens at the same locus (eg HLA-B) or antigens at different loci (eg HLA-A and HLA-B). .. For example, an anti-HLA-A24 monoclonal antibody may recognize Bw4, which is called a public epitope, in which case the antibody may also recognize some HLA-B antigens.

The HLA type targeted in the method of the present invention is not particularly limited, but is preferably any of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP. The type to which it belongs. For example, the HLA type is HLA-A24, HLA-B61, etc.

The method of the present invention first carries out a step of identifying the presence of an anti-HLA antibody in mononucleosis derived from a human subject.

The presence of anti-HLA antibody in mononucleosis allows, for example, a sample containing mononucleosis, such as serum from a subject, or mononucleosis obtained as described above to be contacted and bound to various HLA antigens. It can be identified by determining the presence or absence of. Specifically, but not limited to, a serum containing antibody-producing cells was reacted with microbeads (for example, FlowPRA, ONE LAMBDA) on which an HLA antigen was immobilized, and further labeled with a fluorescent label or the like. The presence of the anti-HLA antibody can be identified by reacting the anti-human antibody as a secondary antibody (ONE LAMBDA) and then measuring the fluorescent intensity of the beads by flow cytometry.

The method of the invention then carries out the step of culturing mononucleosis containing cells capable of producing the anti-HLA monoclonal antibody of interest. Culturing is preferably carried out in the presence of cytokines.

Addition of cytokines in cell culture is generally performed in the art, but the cytokines used in the method of the present invention include one or more cytokines individually or as a cocktail, and in particular. For example, but not limited to, for 1 to 3x10 ⁶ / ml mononuclear cells, human interleukin 17 (hIL17) (about 2 ng / ml) and / or human interleukin 21 (hIL21) (about 100 ng). / ml) is preferably included. Preferably, the following components: R848 (resikimod, about 5 μg / ml), hIL2 (human interleukin 2, about 1000 IU / ml), CpG2006 (about 2.5 μg / ml), anti-CD40 antibody (about 5 μg / ml) , HIL21 (about 100 ng / ml), hIL17 (about 2 ng / ml), hIL4 (human interleukin 4, about 10 ng / ml), hBAFF (B cell activating factor) (about 100 ng) It contains one or more of / ml) (each concentration is the final concentration), and particularly preferably all of these components are added to the culture solution.

The culture is not particularly limited, but is preferably carried out at 37 ° C. for 1 to 4 weeks, for example. At this stage, cytokine stimulation can efficiently proliferate mononuclear cells.

The method of the invention then carries out the steps of isolating antibody-producing cells from cultured mononucleosis.

Methods for isolating antibody-producing cells include a method using microchips (Nature Medicine 2009; 15: 1088-92), a method using flow cytometry (BMC Biology 2012; 10:80), and antibody-producing cells. After separating CD138-positive cells, which are markers, with beads or a cell sorter, the cells are marginally diluted on a 96-well plate, and the antibody in the culture supernatant is detected by ELISA or the like to detect antibody-producing cells. HLA antigen-expressing cell line Can be appropriately used by seeding each well and identifying antibody-producing cells using the damage to the cells as an index (International Publication WO 2012/046745).

In one embodiment, this step follows the ISAAC (Immunospot Array Assay on a Chip) method (Nature Medicine, Vol.15, No.9, September 2009, 1088-1092) developed by a group of the present inventors. It can be carried out. The procedure of this method will be schematically described with reference to FIG.

Specifically, in this method, the cultured mononuclear cells are applied to a microarray so as to have one cell per well, and further cultured.

The microarray used in this method may be any one as long as it can hold and culture one cell per well, and is not particularly limited, but for example, one available from Valneva is preferably used. Can be used. Suitable microarray configurations are disclosed, for example, in Japanese Patent No. 4069171.

In the above method, as shown in FIG. 1, an antigen or a secondary antibody capable of specifically binding to the target antibody is previously coated on the surface of the microarray. The substance to be coated can be appropriately selected depending on the means used for the subsequent antibody detection.

Cell culture on a microarray can be, for example, at 37 ° C. for about 3 hours, without limitation.

After culturing, cells are collected for each well to identify antibody-producing cells.
Identification of anti-HLA antibody-producing cells can be performed, for example, by adding a target HLA-type antigen or a secondary antibody and measuring the presence or absence of binding. The binding can be detected by adding, for example, an antigen labeled with a fluorescent dye or the like or a secondary antibody, and using the presence or absence of fluorescence as an index.

In the above ISAAC method, for example, in the embodiment shown in FIG. 1, since the antigen pre-coated on the surface of the microarray and the antibody secreted from the antibody-producing cells specifically bind to each other, the secondary is further fluorescently labeled. By adding the antibody, the well in which the target anti-HLA antibody-producing cell is present can be identified, and the cell can be isolated.

Devices such as a fluorescence microscope (Olympus BX51WI) and a micromanipulator (Eppendorf, TransferMan® NK2) can be used to carry out the method of the present invention.

Fully human anti-HLA monoclonal antibody-producing cells can be isolated by the method including the above steps (a) to (c). The obtained antibody-producing cells can be used as they are for producing a monoclonal antibody, but can be stored as cells, for example, frozen until use.

[Method for producing human anti-HLA monoclonal antibody]
The present invention also provides a method for producing a human anti-HLA monoclonal antibody having specificity for a specific HLA type. Specifically, this method
(d) Cloning the gene encoding the antibody produced by the antibody-producing cells isolated according to the above method,
(e) Steps to introduce the cloned gene into cells in vitro, and
(f) Includes the steps of culturing the cells, proliferating them and recovering the antibody. That is, the anti-HLA monoclonal antibody can be obtained as a recombinant protein by introducing a polynucleotide encoding the antibody into an appropriate host cell and expressing it.

In the antibody production method of the present invention, first, a step of cloning a gene encoding an antibody produced by an antibody-producing cell obtained according to the above method is carried out. More specifically, although not particularly limited, the antibody gene expressed by the isolated antibody-producing cells can be amplified by, for example, a one-cell RT-PCR method and obtained as a polynucleotide such as cDNA. The polynucleotide to be amplified and obtained may be DNA or RNA.

The method of the invention then carries out the steps of introducing the cloned gene into cells in vitro.

Specifically, the polynucleotide such as cDNA obtained in the above step is incorporated into an appropriate expression vector such as pcDNA3.4 (Life Technologies) and introduced into a host cell such as HEK293 cells. In this case, the gene encoding the heavy chain and the gene encoding the light chain of the antibody can be integrated into separate vectors and introduced into cells. As the means for introduction into the host cell, those used in the art can be appropriately used. For example, a polynucleotide can be incorporated into a vector such as a viral vector, a plasmid vector, a phage vector, etc. together with a regulatory sequence such as a signal sequence, a poly A sequence, a promoter sequence, and a selection marker, and this can be appropriately incorporated into a bacterium such as Escherichia coli, yeast, or insect. It can be introduced into a suitable host cell such as a cell or an animal cell. As the host cell, it is particularly preferable to use a mammalian cell line, and for example, Expi293F cells (Life Technologies) can be preferably used.

The method of the invention then carries out the steps of culturing the cells, proliferating them and recovering the antibody. Whether or not the cells are producing the antibody of interest can be confirmed by ELISA.

By carrying out the steps (d) to (f) above, the desired human anti-HLA monoclonal antibody can be obtained as a recombinant protein. Cloning of a gene is a technique usually performed in the art, and a person skilled in the art can appropriately carry out the method of the present invention based on the description of the present specification and common general knowledge in the art.

[Anti-HLA monoclonal antibody]
Using the method of the present invention described above, human anti-HLA monoclonal antibody-producing cells that can be contained in the mononucleosis of a human subject can be rapidly isolated and proliferated to obtain the desired anti-HLA monoclonal antibody. can. Since the monoclonal antibody obtained by the method of the present invention is a fully human antibody, it can be used for therapeutic purposes against humans. Therefore, the present invention provides an isolated fully human anti-HLA monoclonal antibody obtained by the above method.

Furthermore, by applying the method of the present invention, a monoclonal antibody having the same binding specificity as the anti-HLA antibody contained in a mononuclear cell derived from a subject can be prepared as a humanized antibody or a chimeric antibody. By "having the same binding specificity", for example, it is intended to recognize and bind to the same epitope, and specifically, it may have the same CDR sequence.

In the present specification, an antibody is a complete antibody or an antigen-binding fragment, for example, Fab, Fab', F (ab') ₂ fragments, and a heavy chain variable region and a light chain variable region with a linker. It may be a linked single-chain antibody fragment (scFv fragment) or the like. The linker in the scFv fragment may be any one commonly used in the art, and is not particularly limited, but is, for example, a peptide linker consisting of 5 to 25 amino acid residues, preferably 10 to 20 amino acid residues. Can be mentioned. These fragments and derivatives that can be understood by those skilled in the art to the extent that they do not affect antigen binding, such as modifications to facilitate antibody purification, increase stability, or reduce toxicity when used in vivo. Derivatives that have been subjected to the above are also included in "antibodies" in the present specification in contexts other than "fully human antibodies".

The anti-HLA monoclonal antibody of the present invention can be used in vitro for the presence or absence of engraftment after allogeneic hematopoietic stem cell transplantation, recovery of normal hematopoiesis, and easy monitoring of early recurrence and minimal residual disease of hematopoietic tumors. can. In addition, anti-HLA monoclonal antibodies against class II antigens can be promising therapeutic agents for B cell lineage tumors (malignant lymphoma, multiple myeloma, leukemia).

A specific embodiment of the present invention is a human anti-HLA-B61 monoclonal antibody.
Alleles of B * 40: 02, B * 40: 06, and B * 40: 03 are known for HLA-B61, and in HLA-B61 positive subjects, one or both HLAs are the above alleles. It is thought that it is. It is known that the frequency of B * 40: 02 alleles is high in patients with aplastic anemia (Blood, 2011, December 15, 118 (25) 6601-9).

On the other hand, in the group of the present inventors, some patients with aplastic anemia have mutations in the gene encoding HLA-B61, and as a result, HLA-B61 cannot be expressed, and immunosuppression in such patients. We have found that the therapy works.

Therefore, anti-HLA-B61 monoclonal antibodies can be used to pre-evaluate the effectiveness of immunosuppressive therapy in patients with aplastic anemia. In this case, since the evaluation can be performed in vitro, the anti-HLA-B61 monoclonal antibody is a non-human antibody, eg, humanized, even if it is a human antibody, as long as it has specific binding to the HLA-B61 antigen. It may be an antibody or a chimeric antibody, and as described above, it may be an antibody fragment or derivative having antigen-binding property in addition to the complete antibody.

Monoclonal antibodies that specifically bind to the human HLA-B61 antigen include, for example:
(a) A heavy chain variable region polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 or a polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence.
(b) A light chain variable region polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4 or a polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence. Can be.

Monoclonal antibodies that specifically bind to the human HLA-B61 antigen are also:
(a) A heavy chain variable region polypeptide which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence, and
(b) A light chain variable region polypeptide which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5 or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence. could be.

The monoclonal antibody that specifically binds to the human HLA-B61 antigen further includes CDH1 consisting of the amino acid sequence shown in SEQ ID NO: 6, CDH2 consisting of the amino acid sequence shown in SEQ ID NO: 7, and the amino acid sequence shown in SEQ ID NO: 8. It may include CDH3 consisting of CDH3 consisting of, CDL1 consisting of the amino acid sequence represented by SEQ ID NO: 9, CDL2 consisting of the amino acid sequence represented by SEQ ID NO: 10, and CDL3 consisting of the amino acid sequence represented by SEQ ID NO: 11.

A monoclonal antibody having the above-mentioned specific sequence can be obtained by carrying out the above-mentioned method of the present invention, but based on the sequence information, techniques usually used in this field such as chemical synthesis and gene recombination can be used. It can also be obtained by using.

The above-mentioned specifically described sequences have been actually found by the present inventors, but those skilled in the art can use the above-mentioned methods of the present invention to bind anti-HLA to the same or different epitopes. -B61 monoclonal antibody can be obtained. When the antibody of the present invention is produced by synthesis or genetic recombination, the sequences of the heavy chain and light chain constant regions or the sequences of the framework region can be the sequences usually found in human antibodies. If intended for in vitro use, these sequences may be of non-human origin and may be in the form of chimeric antibodies. Furthermore, when intended for in vivo use, modifications can be made for the purpose of reducing toxicity, improving stability, etc. For example, the sequence of the Fc region can be considered.

The present inventors have found that the obtained monoclonal antibody of the present invention can exhibit cytotoxic activity specifically for HLA type. Accordingly, the present invention also provides cytotoxic activators for specific HLA types, including the human anti-HLA monoclonal antibodies described above. The cytotoxic activator of the present invention can be suitably used for the treatment of diseases that may occur depending on the HLA type, either alone or in combination with other agents such as anticancer agents and anti-inflammatory agents.

The present invention further comprises contacting a sample taken from a patient with aplastic anemia with a monoclonal antibody according to any of the above to detect the expression of HLA-B61 molecule in the sample. Provided is a method for predicting the effectiveness of suppressive therapy. The sample may be any sample as long as the expression of the HLA-B61 antigen can be confirmed, and is not particularly limited, and examples thereof include peripheral blood and bone marrow cells.

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

[Example 1 Preparation of anti-HLA-B61 monoclonal antibody using peripheral blood of anti-HLA-B61 antibody positive donor]
(Method)
In this example, antibody-producing cells were isolated and antibodies were prepared according to the method described in Nature Medicine 2009, 15: 1088-92 by A. Jin et al.

The serum obtained from the donor is reacted with latex beads (microbeads (Flow PRA, ONE LAMBDA)) immobilized with various HLA antigens, and further, a fluorescence (fluorescein isothiocyanate: FITC) -labeled anti-human antibody is used as a secondary antibody. After the reaction as (ONE LAMBDA), the presence or absence of anti-HLA antibody was identified by measuring the fluorescence intensity of the beads by flow cytometry.

Peripheral blood of a donor positive for anti-HLA-B61 antibody was ^{layered on a mononuclear cell separation solution (Lymphoprep TM} , Alere Technologies AS, specific density = about 1.077) and centrifuged (400xg, about 30 minutes). went. After centrifugation, the upper layer was carefully sucked up with a Pasteur pipette up to 0.5 cm above the opaque intermediate layer containing mononuclear cells and removed. The opaque intermediate layer was transferred to a new centrifuge tube with a Pasteur pipette, phosphate buffered saline was added, mixed slowly, and then centrifuged (250 xg, 10 minutes). The supernatant was removed, and the precipitated cells were separated as mononuclear cells and temporarily cryopreserved.

The cryopreserved mononuclear cells were thawed, and 1.38x10 ⁷ mononuclear cells were cultured at 37 ° C. for 6 days in RPMI medium containing 10% FCS supplemented with cytokines having the following composition.

After culturing, wash with PBS, remove unnecessary cells, add CD138 microbeads (manufactured by Miltenyi Biotec) to increase the frequency of target cells, react at 4 ° C for 15 minutes, and 8.2x10 ⁶ mononuclear cells. ^Five 7.3x10 CD138-positive mononuclear cells were isolated from the spheres.

CD138-positive mononuclear cells were seeded on four cell arrays (Varneva) coated with solubilized HLA-B61 antigen (Fig. 1). Here, one cell was filled per well, and the filling rate to the well was 65%. Cells on the wells were further cultured at 37 ° C. for about 3 hours in RPMI 1640 medium containing 10% FCS.

After culturing, the presence of a specific antibody was detected using a fluorescently labeled anti-human IgG antibody (anti-Human IgG-Fc Cy3, Millipore). On the other hand, ^{cells were stained with Oregon Green (CellTrace TM} Oregon Green (Life Technologies)). Therefore, the wells in which both Cy3 and Oregon green fluorescence are detected are cells producing the desired anti-HLA-B61 antibody.

The number of spots (wells) that could be detected was 14, and cells could be recovered from 3 of these wells. Furthermore, when the anti-HLA-B61 antibody gene was cloned from the recovered cell-derived gene, the target anti-HLA-B61 antibody gene could be cloned from one well-derived cell.

The anti-HLA-B61 antibody gene was introduced into Expi293F cells (Life Technologies) to produce an anti-HLA-B61 antibody.

The results of analyzing the base sequences of the variable regions of the heavy chain and the light chain of the obtained antibody are shown below. The underline shows each base sequence that encodes the CDR.

The amino acid sequences of the heavy chain and light chain variable regions of the antibody obtained based on the above nucleotide sequence, and the amino acid sequences of each CDR are shown in Table 2 and the sequence table below. The determination of these variable regions and CDR regions can be investigated using, for example, the ImMunoGeneTics (IMGT) V-Quest tool (http://www.imgt.org/) (Giudicelli V et al., IMGT / V-QUEST,). an integrated software program for immunoglobulin and T cell receptor VJ and VDJ rearrangement analysis. Nucleic Acids Res 2004; 32: W435-40).

[Example 2 Detection of HLA-B61 antigen-positive blood cells and HLA-B61 antigen-deficient blood cells using an anti-HLA-B61 monoclonal antibody]
The anti-HLA-B61 monoclonal antibody (anti-HLA-B61mAbs) prepared in Example 1 and the control monoclonal antibody (anti-influenza virus antibody, prepared by the Department of Immunology, Toyama University) are labeled with fluorescein isothiocyanate (FITC). Then, HLA-B61 antigen positive or negative B-LCL (B-lymphoblastoid cell lines) cells (derived from healthy donors) were detected by flow cytometry. As shown in FIG. 2A, the antibody of the present invention was able to clearly detect HLA-B61 antigen-positive B-LCL, whereas the control antibody could not detect it (FIG. 2A). On the other hand, when HLA-B61 antigen-negative B-LCL was used, no difference was observed between the control antibody and the anti-HLA-B61 antibody (Fig. 2B).

In addition, as a result of attempting to detect HLA-B61 antigen-positive or negative cells using peripheral blood derived from human subjects, HLA-B61 antigen-positive T lymphocytes could be clearly detected by flow cytometry (Fig. 3A). And B).

Furthermore, when ELISA was performed using a plate on which the HLA-B61 antigen (manufactured by Wakunaga Pharmaceutical Co., Ltd.) was immobilized, a positive reaction was observed with anti-HLA-B61 positive serum, and significant binding was observed with the anti-HLA-B61 monoclonal antibody. Was observed (Fig. 4). This binding was not observed when HLA-A24 antigen (manufactured by Wakunaga Pharmaceutical Co., Ltd.) was used as the immobilized antigen, and it was clear that it was a specific binding to HLA-B61.

[Example 3 Preparation of anti-HLA-A24 monoclonal antibody]
In the same manner as in Example 1, an anti-HLA-A24 monoclonal antibody was isolated from the peripheral blood of an anti-HLA-A24 antibody-positive donor, and an anti-HLA-A24 monoclonal antibody was prepared using gene cloning technology. The results of analyzing the base sequences of the variable regions of the heavy chain and the light chain of the obtained antibody are shown below. The underline shows each base sequence that encodes the CDR in the heavy chain.

The amino acid sequences of the heavy chain and light chain variable regions of the antibody obtained based on the above base sequence, and the amino acid sequences of each CDR are shown in Table 3 and the sequence table below.

[Example 4 Detection of HLA-A24 antigen-positive blood cells and HLA-A24 antigen-deficient blood cells using an anti-HLA-A24 monoclonal antibody]
The anti-HLA-A24 monoclonal antibody prepared in Example 3 and the control monoclonal antibody (anti-influenza virus antibody, prepared by the Department of Immunology, Toyama University) were labeled with fluorescein isothiocyanate (FITC) and used in Example 2. As a result of attempting to detect HLA-A24 antigen-positive or negative cells using peripheral blood derived from human subjects in the same manner as above, HLA-A24 antigen-positive T lymphocytes could be clearly detected by flow cytometry ( FIG. 5).

[Example 5 complement-dependent cytotoxicity (CDC)]
The function of the anti-HLA-B61 monoclonal antibody prepared in Example 1 was examined.

2x10 ⁵ HLA-B61 positive B-LCL cells (HLA type B35: 01/40: 02 or B40: 02/40: 06) or HLA-B61 negative B-LCL cells (HLA type 51: 01/52: 06), respectively. To 01 or B44: 03/5 2:01), an anti-HLA-B61 monoclonal antibody (10 μg / ml) was added and incubated at 37 ° C. for 20 minutes. After centrifugation (600 x g, 5 minutes), cells are added to RPMI containing 10% human serum (healthy donor serum) as a complement source and incubated at 37 ° C for 60 minutes for 7-aminoactinomycin D. After adding (7-AAD) and incubating at 4 ° C. for 10 minutes, the cytotoxic activity was examined by flow cytometry.

FIG. 6 shows the viability of HLA-B61 positive or negative cells after incubation for 30 or 60 minutes. As a result, cytotoxic activity was observed for HLA-B61-positive B-LCL cells, but not for HLA-B61-negative B-LCL cells, and antibody-induced cytotoxic activity was observed for HLA. Demonstrated to be type-specific.

[Example 6 HLA-type specific antibody-dependent cellular cytotoxicity (ADCC)]
5x10 ⁴ HLA-B61 positive B-LCL cells (target (T)) were added to the RPMI medium, and the anti-HLA-B61 monoclonal antibody 20 μg / ml prepared in Example 1 and human as effector cells (effector (E)) Peripheral blood mononuclear cells (E / T ratio 40) and CFSE (5- (6) -carboxyfluorescein diacetate succinimidyl ester) 2.5 μM were added and reacted at 37 ° C for 2 hours, followed by 7-AAD. The cytotoxic activity was examined by flow cytometry. As a result, the antibody of the present invention showed clear cytotoxic activity as shown in FIG. 7A. No cytotoxic activity was observed with the negative control isotype monoclonal antibody (Fig. 7B).

A human anti-HLA monoclonal antibody, which is currently difficult to produce, can be rapidly produced using mononucleosis derived from a human subject and used for examination, treatment, and the like. The production of a conventional monoclonal antibody requires a great deal of time and cost, but according to the method of the present invention, it can be produced in about 1-2 months.

Using the antibody produced by the method of the present invention, it is possible to easily monitor the recovery of normal hematopoiesis after allogeneic hematopoietic stem cell transplantation, early recurrence of hematopoietic tumors, and minimal residual disease, and improve the treatment results. Since the number of allogeneic hematopoietic stem cell transplants in Japan is about 3500 / year and the number of allogeneic hematopoietic stem cell transplants in the world is about 25000 / year, the utility of the present invention is considered to be very large. In addition, even when a blood product containing an anti-HLA antibody is transfused, no major side effects occur in most cases, so it is considered that there is no major problem in terms of safety.

Moreover, since the HLA class II antigen is expressed only in some blood cells such as B cell lineage, the anti-HLA class II monoclonal antibody that can be produced by the method of the present invention is a B cell lineage tumor (malignant). It can be a promising therapeutic agent for lymphoma, multiple myeloma, leukemia).

Furthermore, it has become possible to rapidly identify aplastic anemia cases in which HLA-B61-deficient blood cells for which immunosuppressive therapy is effective are detected using the anti-HLA-B61 monoclonal antibody of the present invention, and immunosuppressive therapy can be used. You can get started properly.

The method of the present invention can also be applied to the production of monoclonal antibodies of autoantibodies detected in autoimmune diseases, and there is a possibility that the monoclonal antibodies can be used for elucidating the pathophysiology of autoimmune diseases and developing therapeutic methods. be.

Claims (11)

A method for producing a human anti-HLA monoclonal antibody having specificity for a specific HLA type.
Mononucleosis from human subjects whose presence of anti-HLA antibody was identified was isolated and
The mononuclear cells were applied to a microarray with one cell per well and cultured.
Identify wells that have antibodies that specifically bind to the HLA-type antigen of interest.
Cells producing the desired anti-HLA monoclonal antibody were isolated from the wells.
Cloning the gene encoding the antibody produced by the isolated cells
The method described above comprising introducing the cloned gene into a cell in vitro and then culturing and propagating the cell into which the gene has been introduced to recover the antibody. The method of claim 1, wherein the mononucleosis is obtained from cells derived from peripheral blood, spleen, lymph nodes or bone marrow. The method according to claim 1 or 2, wherein the human subject is a female who has experience of pregnancy, or a subject who has experience of blood transfusion, hematopoietic stem cell transplantation, or organ transplantation. The method according to any one of claims 1 to 3, wherein the HLA type is a type belonging to any one of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP. The method according to any one of claims 1 to 4, wherein the HLA type is HLA-B61. A monoclonal antibody that specifically binds to the human HLA-B61 antigen, including:
(a) A heavy chain variable region polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 or a polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence. , A heavy chain variable region polypeptide containing the CDR1 region consisting of the amino acid sequence shown by SEQ ID NO: 6, the CDR2 region consisting of the amino acid sequence shown by SEQ ID NO: 7, and the CDR3 region consisting of the amino acid sequence shown by SEQ ID NO: 8.
(b) A light chain variable region polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4 or a polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence. Includes a light chain variable region polypeptide comprising the CDR1 region consisting of the amino acid sequence set forth in SEQ ID NO: 9, the CDR2 region consisting of the amino acid sequence set forth in SEQ ID NO: 10, and the CDR3 region consisting of the amino acid sequence set forth in SEQ ID NO: 11. , The above monoclonal antibody. A monoclonal antibody that specifically binds to the human HLA-B61 antigen, including:
(a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3, or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence, and the amino acid sequence shown in SEQ ID NO: 6. A heavy chain variable region polypeptide comprising a CDR1 region consisting of, a CDR2 region consisting of the amino acid sequence represented by SEQ ID NO: 7, and a CDR3 region consisting of the amino acid sequence represented by SEQ ID NO: 8.
(b) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5, or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence, and the amino acid sequence shown in SEQ ID NO: 9. The monoclonal antibody comprising a light chain variable region polypeptide comprising a CDR1 region consisting of, a CDR2 region consisting of the amino acid sequence represented by SEQ ID NO: 10, and a CDR3 region consisting of the amino acid sequence represented by SEQ ID NO: 11. A monoclonal antibody that specifically binds to the human HLA-B61 antigen, which is represented by CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 6, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 7, and SEQ ID NO: 8. A heavy chain containing CDR3 consisting of an amino acid sequence, CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 9, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 10, and light containing CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 11. The monoclonal antibody described above, which comprises a chain. An anti-HLA monoclonal antibody having the same binding specificity as the monoclonal antibody according to any one of claims 6 to 8. A cytotoxic activator for a specific HLA type, which comprises the monoclonal antibody according to any one of claims 6 to 9. The expression of the HLA-B61 molecule in the sample is detected by contacting a sample collected from an aplastic anemia patient with the monoclonal antibody according to any one of claims 6 to 9, or claim 5. To prepare a human anti-HLA monoclonal antibody by the method of The above method, which is a method for predicting the effectiveness of immunosuppressive therapy, in which immunosuppressive therapy is predicted to be effective when the expression of HLA-B61 molecule is not detected .

Images