JP2010222255A - Method for screening antibody and antibody obtained by the method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for screening an antibody, to provide an antibody obtained by the method, and to provide a pharmaceutical composition containing the antibody as an active ingredient. <P>SOLUTION: There are provided the new method for screening the antibody which can control the function of B-cell at a relatively low dose to prevent and treat autoimmune diseases and the like without disappearing the B-cells of peripheral circulation; the antibody obtained by the method; and the pharmaceutical composition containing the antibody as an active ingredient. The new method for screening the antibody is achieved by screening an antibody against the B-cell-specific surface antigen and having actions for controlling the interleukine-6 production by the B-cell and/or for controlling the interleukine-10 production by the B-cell. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention is an antibody against a B cell-specific surface antigen and regulates interleukine (hereinafter sometimes referred to as “IL”)-6 production by B cells and / or inhibits IL-10 production by B cells. The present invention relates to a method for screening an antibody having a regulating action, an antibody obtained thereby, and a pharmaceutical composition containing the antibody as an active ingredient.

  In recent years, it has been recognized that antibody drugs targeting B cells are effective in treating cancers such as non-Hodgkin lymphoma and related B cell lymphomas. Among them, there are anti-CD20 antibodies or anti-CD22 antibodies targeting the B cell-specific surface antigen CD20 or CD22, and it has been reported that a method of selectively depleting B cells with these antibodies is effective. . For example, Rituxan (registered trademark) is a chimeric monoclonal antibody specific for CD20 antigen, which is a surface molecule of B cells (Non-patent Document 1), and selectively eliminates B cells for a long period of time (Non-patent Document 1). Reference 2). This antibody has already been used for the treatment of non-Hodgkin lymphoma (Non-patent Document 3).

  Recently, the results of clinical trials showing that antibody drugs targeting B cells for cancer treatment are effective against autoimmune diseases. For example, in a clinical trial for patients with refractory rheumatoid arthritis, the Rituxan and methotrexate combination group showed an improvement effect far superior to the methotrexate single administration group, and it has been reported that the effect persists (Non-patent Document) Four). On the other hand, regarding anti-CD22 antibody preparations, the results of clinical trials in which Epratuzumab is effective against primary Sjogren's syndrome have been reported (Non-patent Document 5). These effects are thought to be due to the calming of the autoimmune reaction because IL-6 production is blocked by the disappearance of pathogenic B cells. However, regarding the mechanism of sedation of the autoimmune response of epratuzumab, the possibility of providing an inhibitory signal to B cells, that is, the phosphorylation-inducing activity of CD22 was examined (Non-patent Document 6), but the mechanism of action is still unknown. is there. In addition, methods for treating autoimmune diseases using novel anti-CD22 monoclonal antibodies have been reported (Patent Documents 1 and 2).

  Although the results of clinical trials have been shown as described above, these antibody drugs eliminate B cells circulating in the periphery for a long time. As a result, “B cells that play a role in biological defense functions” and “B cells that control autoimmunity in the direction of healing through the production of Interleukine-10” (Non-Patent Documents 7 and 8) are used for a long time. The risk of infectious diseases and cancer increases, and at the same time the natural healing power decreases.

Furthermore, since these antibody drugs are very expensive treatment methods, the cost of the drugs is an obstacle to use. For example, Rituxan has been used in a clinical trial for patients with rheumatoid arthritis at a high dose of administering 1 g twice (Non-patent Document 4). Epratuzumab is also a clinical trial for primary Sjogren's syndrome, in which 360 mg / m ² is administered four times every other week (Non-patent Document 5). It becomes.

  In view of the above, an antibody drug targeting B cells that exerts an effect by controlling the function of pathogenic B cells at a relatively low dose without completely eliminating peripheral circulating B cells is ideal. . This is thought to be achieved in particular by antibody drugs that reduce the ability of pathogenic B cells to produce IL-6 and / or induce or maintain B cells that produce IL-10 that is important for natural healing. The mechanism is the inhibitory receptor (CD22, Fc gamma RIIB (CD32), CD72, and paired immunoglobulin-like receptor (PIR)) present on the B cell membrane in the pathogenic B cell in an abnormally activated state. -B and the like) is expected to induce B cells to a normal state by providing an inhibitory signal (Non-patent Document 9).

However, as described above, antibodies that non-selectively eliminate or reduce the function of B cells by binding to a B cell-specific surface antigen have been reported so far, but in the treatment of autoimmune diseases, IL- Use anti-B cell surface antigen antibodies with 6 and / or IL-10 production as an index, and screen for effective antibodies with IL-6 and / or IL-10 production as an index No method has been reported. In addition, there is no report on agonist antibodies that treat autoimmune diseases by providing inhibitory signals to pathogenic B cells in an abnormally activated state, and screening using a certain stage of the inhibitory signaling pathway as an index There are no reports on how to do this.
JP 2005-526044 gazette JP 2005-526501 gazette Blood 83, 435-445 (1994) Arthritis Rheum 54, 613-620 (2006) Curr Opin Oncol 10, 548-551 (1998) N Engl J Med 350, 2572-2581 (2004) 2005 American College of Rheumatology (Title No. 681, Abstracts of Society: S277) Clin Cancer Res 9, 3982s-3990s, (2003) J Exp Med 184, 2271-2278 (1996) Nat Immunol 3, 944-950 (2002) Current Opinion in Immunology 17, 290-297 (2005)

  An object of the present invention is to screen for an antibody against a B cell-specific surface antigen and having an action of regulating IL-6 production by B cells and / or regulating IL-10 production by B cells It is providing a method, providing the antibody, and providing the pharmaceutical composition which uses the antibody obtained by this as an active ingredient.

  As a result of conducting research to search for antibodies targeting B cells that are effective for autoimmune diseases and the like, antibodies having IL-6 production regulating action and / or IL-10 production regulating action have been obtained. The present inventors have found that B cell function is controlled at a relatively low dose without losing peripheral circulating B cells, and is useful for preventing and treating autoimmune diseases and B cell lymphoma.

That is, the gist of the present invention is as follows.
(1) Screening method of antibody against B cell specific surface antigen, i) Step of adding antibody against B cell specific surface antigen to B cell, ii) Next, Interleukine-6 production by B cell changed And / or a method for screening an antibody, comprising the step of confirming that the production of Interleukine-10 by B cells has changed.
(2) The screening method according to (1), wherein the B cell-specific surface antigen is any one or more selected from CD22α, CD22β, CD19, CD20, CD79α, and CD79β.
(3) The screening method according to (1) or (2), wherein the B cell-specific surface antigen is CD22α and / or CD22β.
(4) B cells are selected from established B cells, rodent peripheral blood-derived cells, rodent lymph node-derived cells, human peripheral blood-derived cells, human lymph node-derived cells and human tonsil-derived cells, The screening method according to any one of (3) to (3).
(5) The screening method according to any one of (1) to (4), wherein the production of Interleukine-6 by B cells is induction and / or enhancement by activation of B cells.
(6) B cell activation is anti-IgM antibody, anti-IgM antibody bead, anti-IgG antibody, anti-IgG antibody bead, anti-Ig antibody, anti-Ig antibody bead, Staphylococcus aureus Cowan I strain cell component, Lipopolysaccharide, Pokeweed mitogen The screening method according to (5), wherein the activation is any one or more selected from Epstein-Barr virus, CpG oligodeoxynucleotides and Toll like receptor ligand.
(7) The step (ii) is one in which the production of Interleukine-6 by B cells is suppressed and / or the production of Interleukine-10 by B cells is enhanced (1) to (6) The screening method in any one of.
(8) Suppression of Interleukine-6 production by B cells causes only B cells producing Interleukine-6 to disappear and / or causes inhibition of Interleukine-6 production only to B cells producing Interleukine-6 The screening method according to (7), wherein
(9) The screening method according to any one of (1) to (8), wherein the change in Interleukine-6 production is suppressed by 5% or more and 100% or less.
(10) The screening method according to any one of (1) to (8), wherein a change in production of Interleukine-6 is suppressed by 25% or more and 100% or less.
(11) The screening method according to any one of (1) to (8), wherein the change in production of Interleukine-6 is confirmed to be suppressed by 40% or more and 100% or less.
(12) The screening method according to any one of (1) to (11), wherein the change in production of Interleukine-10 is enhanced by 150% or more.
(13) A method for producing an antibody, wherein the antibody obtained by the method according to (1) is produced by a genetic recombination technique.
(14) An antibody obtained by the method according to any one of (1) to (13).
(15) The antibody according to (14), wherein the antibody is a monoclonal antibody.
(16) The antibody according to (14) or (15), wherein the antibody is a human monoclonal antibody.
(17) A pharmaceutical composition comprising the antibody according to any one of (14) to (16) as an active ingredient.
(18) The pharmaceutical composition according to (17), which is used for treatment and / or prevention of a disease involving Interleukine-6 and / or Interleukine-10 and a disease selected from B lymphoma.
(19) The pharmaceutical composition according to (17), which is used for prevention and / or treatment of an autoimmune disease or Castleman's disease.
(20) The pharmaceutical composition according to (17), which is used for prevention and / or treatment of rheumatoid arthritis, multiple sclerosis or systemic lupus erythematosus.

  According to the present invention, a novel antibody screening method that can control B cell function and prevent or treat autoimmune diseases at a relatively low dose without losing peripheral circulating B cells, The antibody obtained thereby and a pharmaceutical composition containing the antibody as an active ingredient can be provided.

  The present invention will be described in detail below.

  One embodiment of the present invention is a method for screening an antibody against a B cell-specific surface antigen, and has the purpose of regulating IL-6 production by B cells, and / or IL-10 by B cells. Examples thereof include a method for screening an antibody having an action of regulating production.

(1) Acquisition of antibody As the antibody against the B cell-specific surface antigen used in the present invention, any antibody known as an antibody against the B cell-specific surface antigen can be used. Preferred are anti-CD22α antibody, anti-CD22β antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD79α antibody or anti-CD79β antibody, and most preferred is anti-CD22α antibody or anti-CD22β antibody. In the present invention, unless otherwise specified, anti-CD22 antibody means anti-CD22α antibody and anti-CD22β antibody, and CD22 means CD22α and CD22β.

  Antibodies against B cell-specific surface antigens can be prepared commercially or by known techniques. For example, a method for preparing a high-titer preparation by collecting only positive plasma of autoimmune disease patients, a method for obtaining antiserum from an animal immunized with a B cell-specific surface antigen protein or a B cell-specific surface antigen-expressing cell (Current Protocols In Immunology, Unit 2.4, (1991)), a method for preparing monoclonal antibodies by culturing antibody-producing cells (hybridomas) obtained by cell fusion (Current Protocols In Immunology, Unit 2.6-2.7, (1991)), Methods for preparing antibodies by vector immunization (Neurobiol Dis 14, 365-379 (2003)), methods for preparing recombinant antibodies by genetic engineering techniques (Mol. Cells 20, 17-29 (2005), J Immunother 29, 1-9 (2006)).

  Here, the method for obtaining the B cell-specific antigen protein includes, for example, using a commercially available product or using a gene recombination product. B cell-specific antigen-expressing cells include cells that originally express B-cell-specific antigens, or cells that have increased B-cell-specific antigen expression levels by introducing a B-cell-specific antigen expression plasmid into the cells. Can be used.

  B cell-specific antigen expression plasmids are cDNA or genomic DNA prepared from appropriate cells, B cell-specific antigen cDNA cloned from a cDNA library, genomic DNA library, etc., or a known sequence such as a document or database. It can be constructed by inserting a B cell-specific antigen cDNA obtained using information such as DNA chemically synthesized from information into an appropriate expression vector. In this case, the B cell-specific antigen cDNA may be full-length, a cDNA fragment lacking a base, or a chimeric cDNA such as mouse and human.

The antibody of the present invention is not particularly limited as long as it binds to a B cell-specific antigen, and human antibodies, mouse antibodies, rat antibodies, rabbit antibodies, sheep antibodies, goat antibodies, camel antibodies, etc. can be used as appropriate. In order to reduce the heteroantigenicity, a chimeric antibody, a humanized antibody, a human antibody and the like are preferable. The antibody of the present invention may be either a full-length antibody or an antibody fragment as long as it has the ability to bind to a B cell-specific antigen. Specific examples of antibody fragments include Fab, Fab ′, F (ab ′) ² , Fv, scFv and the like. The antibody of the present invention may be a conjugated antibody bound to various molecules such as polyethylene glycol, radioactive substance, and toxin according to a known method.

(2) Types of B cells The B cells used in the present invention may be established B cells or primary cultured B cells. The established B cell may be a known cell line having IL-6 production ability and / or IL-10 production ability, preferably Ramos (ATCC NO. CRL-1596), Ramos.2G6.4C10 (ATCC NO. CRL-1923), RPMI1788 (ATCC NO. CCL-156) and Raji (ATCC NO. CCL-86) are used. As primary cultured B cells, B cells derived from rodent peripheral blood, rodent lymph nodes, human peripheral blood, human lymph nodes or human tonsils are used. Most preferred are human peripheral blood-derived B cells. Whether or not the cells are IL-6 or IL-10-producing cells can be examined by the Enzyme Linked Immunosorbent Assay (ELISA) method or the like as shown in Examples described later.

(3) IL-6
IL-6 in the present invention is abnormally expressed in autoimmune diseases such as rheumatoid arthritis (RA) and multiple myeloma, and is one of the causative substances in these pathologies (Eur J Immunol 18, 1797- 1801 (1988)).

  As a method for measuring the amount of IL-6 production, there is a method using ELISA as shown in Examples described later. Other methods include Cytometric Bead Array Kit (BD Pharmingen) using Flow Cytometer, mouse B cell hybridoma strain, Bioassay using B9 cells (Current Protocols In Immunology, Unit 6.6.1-6.6.4, (1991) ) And the like can be used to measure IL-6 production.

(4) IL-10
IL-10 in the present invention has been identified as a factor that suppresses cytokine production from Th1 cells (J Exp Med 170, 2081-2095 (1989)), such as multiple sclerosis (MS) and RA. It is a substance known to have a preventive and therapeutic effect on autoimmune diseases (Eur J Immunol 23, 1745-1751 (1993), J Immunol 164, 1576-1581 (2000)).

  As a method for measuring IL-10, it can be carried out by a method using Cytometric Bead Array Kit (BD Pharmingen) using a Flow Cytometer in the same manner as the above IL-6 production method.

(5) Changes in IL-6 production In the present invention, changes in IL-6 production include both suppression of IL-6 production and induction and / or enhancement of IL-6 production. IL-6 production specifically means induction / enhancement by B cell activation, which includes anti-IgM antibodies, anti-IgM antibody beads, anti-IgG antibodies, anti-IgG antibody beads, anti-Ig antibodies, anti-Ig antibody beads, Staphylococcus aureus Cowan I strains (SAC), Lipopolysaccharide (LPS), Pokeweed mitogen (PWM), viruses such as Epstein-Barr virus (EBV), Ion channels activator (A23187 ionophore, Ionomycin, etc.), Protein kinase C activator ( Phorbol esters, etc.), B cell surface molecule antibodies (anti-CD20 antibody, anti-CD40 antibody, etc.), B cell surface molecule ligands (CD40L, etc.) expressed cells, CpG oligodeoxynucleotides, Toll like receptor ligands, various lymphokines (IL- 1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IFN-γ, TNF-α, TNF-β, TGF-β, etc.), 8-Mercaptoguanosine (8 -MG), Dextran Sulfate, Poly (IC), Indolactam, etc. Any of these may be a commercially available product.

  Preferably, the activation of B cell is anti-IgM antibody, anti-IgM antibody bead, anti-IgG antibody, anti-IgG antibody bead, anti-Ig antibody, anti-Ig antibody bead, virus such as SAC, LPS, PWM, EBV, Ion channels It means that it can be performed by activator (A23187 ionophore, Ionomycin, etc.), Protein kinase C activator (Phorbol esters, etc.), CpG oligodeoxynucleotides and / or Toll like receptor ligand.

  Most preferably, the activation of B cells is anti-IgM antibody, anti-IgM antibody bead, anti-IgG antibody, anti-IgG antibody bead, anti-Ig antibody, anti-Ig antibody bead, SAC, LPS, PWM, EBV etc. virus, CpG It means that it can be done by oligodeoxynucleotides and / or Toll like receptor ligands.

Anti-IgM antibodies include goat, rabbit, sheep, cow, horse, camel, chicken, pig, monkey, guinea pig, hamster, mouse or rat anti-human IgM polyclonal antibody, or mouse, rat, hamster, rabbit, goat Sheep or monkey anti-human IgM monoclonal antibodies are used. Most preferably, mouse anti-human IgM F (ab ′) ² is used as the anti-IgM antibody. CpG oligodeoxynucleotides are sequences frequently found in bacterial-derived DNA, and by stimulating B cell receptor via Toll Like Receptor 9 (J Exp Med 195, 1507-1512 (2002)), IL -6 induces and / or enhances production.

  As a method for activating B cells, a suitable means is selected depending on the concentration of the stimulant and the number of cells. For example, a method including adding a solution containing the stimulant to the B cell culture solution is performed. Can do.

  In the present invention, it is preferable that IL-6 production by B cells is suppressed by an antibody against a B cell-specific surface antigen, and in particular, suppression of IL-6 production occurs only in B cells that produce IL-6. Alternatively, IL-6 production is preferably suppressed by eliminating only B cells that produce IL-6. Suppression of IL-6 production is realized by suppressing IL-6 secretion from B cells. These include suppression of IL-6 gene transcription activity, suppression of IL-6 protein synthesis, and suppression of IL-6 protein secretion from cells. The elimination of only B cells producing IL-6 is realized by the elimination of B cells producing IL-6 by necrosis or apoptosis.

  Necrosis is necrosis caused by exogenous (antibody-dependent cellular cytotoxicity (ADCC), complement, toxin, anoxia, etc.), and membrane changes are first seen due to cell swelling and autolysis. .

  Apoptosis is a form of cell death induced by a self-destructive program originally written in a gene, and is characterized by degradation of nuclear DNA, degeneration and condensation of the nucleus, and phagocytosis of cell debris.

  Necrosis or apoptosis can be detected with Annexin V, which uses fluorescent or biotin-labeled changes in the distribution of phosphatidyl serine in the cell membrane observed in the early stage of apoptosis using Annexin V, Biotin Apoptosis Detection Kit (R & D Systems, Inc.), etc. After staining the cells that have undergone apoptosis, the cells that have undergone necrosis are stained with the DNA fluorescent staining agent Propidium Iodide that stains red fluorescence, and each cell can be detected by analyzing with a Flow Cytometer.

  In the present invention, it is desirable that the IL-6 production amount before the antibody against the B cell-specific surface antigen is added to the B cell is inhibited by 5% or more and 100% or less after the antibody is added. This means that when the IL-6 production amount by B cells before adding the antibody is 100%, the production amount decreases from 95% to 0% after the antibody addition. Desirably, the IL-6 production amount before adding the antibody to B cells is desirably inhibited by 25% or more and 100% or less after the antibody is added. This means that when the IL-6 production amount by B cells before adding the antibody is 100%, the production amount is reduced from 75% to 0% after the antibody addition. More desirably, the amount of IL-6 production before the antibody is added to B cells is desirably inhibited by 40% or more and 100% or less after the antibody is added. This means that when the IL-6 production amount by B cells before addition of the antibody is 100%, the production amount decreases from 60% to 0% after the antibody addition.

(6) Change in IL-10 production In the present invention, the change in IL-10 production includes both suppression of IL-10 production and induction and / or enhancement of IL-10 production.

  In the present invention, IL-10 production by B cells is preferably induced and / or enhanced by an antibody against a B cell-specific surface antigen. Induction and / or enhancement of IL-10 production is achieved by inducing and / or enhancing IL-10 secretion from B cells. These include induction and / or enhancement of IL-10 gene transcriptional activity, induction and / or enhancement of IL-10 protein synthesis, and enhancement of IL-10 protein secretion from cells.

  In the present invention, it is desirable that the IL-10 production amount before the antibody against the B cell-specific surface antigen is added to the B cell is enhanced 1.5 times or more after the antibody is added. The greater the increase in IL-10 production, the better. For example, the increase in IL-10 production by the B cells before addition of the antibody may be 2 times, 10 times, 50 times, or 100 times.

  In addition, “having a step of confirming that IL-6 production by B cells has changed and / or a step of confirming that IL-10 production by B cells has changed” means that IL-6 by B cells is changed. Confirming that production has changed, confirming that IL-10 production by B cells has changed, or confirming that IL-6 production has changed by B cells and changing IL-10 production It also means having a step of confirming that it has been done. Preferably, the method comprises confirming suppression of IL-6 production by B cells, comprising confirming induction and / or enhancement of IL-10 production by B cells, or IL-6 production by B cells. And the step of confirming that IL-10 production is induced and / or enhanced. Most preferably, it means having a step of confirming that IL-6 production by B cells is suppressed and IL-10 production is induced and / or enhanced.

(7) Confirmation method
In order to confirm that IL-6 production or IL-10 production has changed, a method for confirming a change in the transcriptional activity of each gene, a method for confirming a change in the amount of each protein synthesis, And a method for confirming a change in the amount secreted from the blood.

  Examples of the method for confirming the change in transcription activity include a confirmation method using a reporter gene assay according to a conventional method. Specifically, by creating a vector in which an appropriate reporter factor such as luciferase is connected downstream of the promoter region of IL-6 gene or IL-10 gene and introducing it into B cells, the luciferase activity is measured. is there.

  A method for confirming the change in the amount of protein synthesis is a method in which B cells are homogenized and the amount of IL-6 or IL-10 contained therein is measured by an enzyme linked immunosorbent assay (ELISA).

  As a method for confirming a change in the amount of protein secreted from cells, a culture supernatant of B cells is collected, and the amount of IL-6 or IL-10 contained therein is measured by ELISA. As a specific method for measuring by ELISA, for example, there is a method as shown in Example 10.

(8) Screening method In the present invention, a method of screening for the target antibody is realized, for example, as follows. That is, various human B cell specific antibodies are added to human peripheral blood mononuclear cells (PBMC), and an appropriate amount of anti-human IgM F (ab ') ² is added, and the mixture is incubated at 37 ° C for 48 hours in a CO ₂ incubator. Culture is performed, and IL-6 production and IL-10 production in the supernatant are measured.

  As a result of measuring the IL-6 concentration and / or IL-10 concentration in this way, when the IL-6 production is regulated and / or the IL-10 production is regulated, the antibody of the present invention It is determined that

  The antibody selected by the screening according to the present invention has an effect of treating and / or preventing diseases selected from IL-6 and / or IL-10-related diseases and B-cell lymphoma, and is an autoimmune disease or B-cell lymphoma It is effective for the prevention and / or treatment of the above. Examples of autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS), and systemic lupus erythematosus (SLE).

(9) Antibody production method In the present invention, once an antibody is selected by screening, the antibody can be produced by a conventional method. That is, methods such as production of antibodies by culturing hybridomas that produce the antibodies, production of recombinant antibodies by genetic engineering techniques, and the like. Specifically, by inserting all or part of the cDNA having the base sequence of the antibody selected by the screening method of the present invention into a recombinant vector, creating a transformant introduced with the recombinant vector, This is a method for producing a target antibody.

  The recombinant vector used for the production of the antibody in the present invention may be a vector that can be expressed in prokaryotic cells such as Escherichia coli (for example, pBR322, pUC119, or a derivative thereof). Examples of vectors that can be expressed in mammalian cells include plasmid vectors as described in the Examples, pcDNA3.1 (Invitrogen), pDON-AI DNA (Takara Bio Inc.), and the like. It is done.

  The transformant used for the production of the antibody in the present invention may be a prokaryotic cell such as E. coli, but is preferably a eukaryotic cell, more preferably a mammal-derived cell. Examples of mammalian cells include Chinese hamster ovary cells (CHO cells).

(10) Types of antibodies The antibodies selected and produced by screening in the present invention include both polyclonal antibodies and monoclonal antibodies. A monoclonal antibody is preferable, and a human monoclonal antibody is more preferable.

(11) Pharmaceutical composition When the antibody obtained by the method according to the present invention is used as a prophylactic and / or therapeutic agent, it can be formulated according to a known method and administered. For example, it can be orally or parenterally administered to humans or mammals as a liquid or as a pharmaceutical composition of an appropriate dosage form. Examples of the dose of the antibody obtained by the method according to the present invention to humans include 0.01 mg / kg to 50 mg / kg.

  The dose depends on age, body weight, general health, sex, diet, administration period, administration method, excretion rate, combination of drugs, and the condition of the condition when the patient is being treated. Determined by considering factors.

  According to the present invention, it is possible to provide a screening method for effectively selecting an antibody effective for a disease associated with IL-6 and / or IL-10 and a disease selected from B cell lymphoma. In addition, an antibody selected by the method can be used to provide a therapeutic and / or prophylactic agent for a disease involving effective IL-6 and / or IL-10 and a disease selected from B-cell lymphoma.

  Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to the examples as long as the gist thereof is not exceeded.

Example 1 Preparation of Recombinant Mouse CD22 High-Expressing Cells Mouse CD22-expressing cells were prepared by genetic engineering techniques as follows. First, mRNA was extracted from mouse B lymphoma cell line WEHI231 cell (ATCC NO. CRL-1702) using Quick Prep Micro mRNA Purification Kit (manufactured by Amersham Pharmacia Biotech). RT-PCR was performed using RNA PCR Kit (AMV) Ver.2.1 (manufactured by Takara Bio Inc.) using the Random 9 mers attached to the kit. Thereafter, a PCR reaction was performed using mouse CD22 sequence-specific primers (SEQ ID NOS: 1 and 2 in the sequence listing) and Pyrobest DNA polymerase (manufactured by Takara Bio Inc.), and an amplified fragment of about 2.8 kb was separated, and then a DNA sequencer ( Amersham Pharmacia) was used to determine the nucleotide sequence by the dideoxy method using fluorescently labeled primers (SEQ ID NOS: 3 and 4 in the sequence listing). This fragment was cloned as mouse CD22 cDNA into pCR2.1-TOPO of TOPO TA Cloning Kit (Invitrogen) and named pNT48. Further, the mouse CD22 cDNA fragment was inserted into an animal cell expression vector to construct pCD22 / mouse. Using Lipofectamine 2000 (Invitrogen), pCD22 / mouse was transfected into Chinese hamster ovary cell line CHO-K1 cells to obtain transformants. Mouse CD22 highly expressing cells were selected by Cell ELISA (Current Protocols In Immunology Unit 2.1), recloned by limiting dilution, and the most stably highly expressing strain was named rCD22-CHO # 34.

Example 2: Preparation of anti-mouse CD22 antibody For initial immunization, a suspension of recombinant mouse CD22 high-expressing cells (rCD22-CHO # 34) and adjuvant (FCA) mixed 1: 1 was prepared. Subcutaneous administration was performed at 1 × 10 ⁷ cells / animal. From the second time onward, the antigen was diluted with physiological saline and immunized several times. After the antibody titer measurement, the booster was performed by intraperitoneal administration of rCD22-CHO # 34 to the individual with increased antibody titer. Armenian hamster spleen cells were collected and fused with the mouse myeloma cell line P3U1 cells by the polyethylene glycol (PEG) method (Kohler G, Milstein C. Nature 256: 495-497, 1975). Screening was performed using the culture supernatant of cells subjected to HAT selection after fusion, and anti-mouse CD22 monoclonal antibody-producing cells (hybridomas) were selected (Current Protocols In Immunology Unit 2.5). Finally, 7 types (18 strains as subclones) of anti-mouse CD22 monoclonal antibody-producing hybridomas that specifically react with mouse CD22 were established. Various anti-mouse CD22 monoclonal antibodies were prepared from the culture supernatant obtained by culturing the hybridoma by conventional purification techniques.

Example 3 Production of Recombinant Human CD22 High-Expressing Cells Human CD22-expressing cells were produced by genetic engineering techniques as follows. First, PCR was performed from a commercially available cDNA library derived from human lymphoma cells (manufactured by Invitrogen) using human CD22 sequence-specific primers (SEQ ID NOs: 5 and 6 in the sequence listing) and Pyrobest DNA polymerase (manufactured by Takara Bio) and amplified. After separating the approximately 2.6 kb fragment, the nucleotide sequence is determined by the dideoxy method or dye terminator method using a fluorescently labeled primer using a DNA sequencer (Amersham Pharmacia or Applied Biosystems), and is a known human CD22 sequence This was confirmed (SEQ ID NO: 7 and 8 in the sequence listing). This fragment was cloned as human CD22 into ZeroCRnt TOPO PCR Cloning Kit (Invitrogen) pCR-BluntII-TOPO and named pNT62. Further, the human CD22 cDNA was inserted into an animal cell expression vector pcDNA3.1Zeo (+) (manufactured by Invitrogen) to construct pTM025. PTM025 was transfected into mouse myeloma cell line NS0 cells using Lipofectamine 2000 (manufactured by Invitrogen), and transformants were obtained using DMEM medium containing 10% FBS and 250 μg / mL zeocin as a selection medium. Human CD22 highly expressing cells were selected in the same manner as in Example 1 and recloned by limiting dilution, and the most stably highly expressing strain was named G11-14. Human CD22 expressed by this clone is a fully human type (FIG. 1 (1)).

  Next, the first mouse that binds domains 1 and 2 of mouse CD22 (J Immunol. 149: 2641-2649, 1992) and domains 3 to 7 of human CD22 (J Exp Med. 173: 137-146, 1991) -Human-hybrid CD22-expressing cells were prepared as follows. Using the primer shown in SEQ ID NO: 9 in the Sequence Listing and the primer shown in SEQ ID NO: 10 in the Sequence Listing with a phosphate group on the 5 ′ side, a PCR reaction was performed using the plasmid pNT48 having mouse CD22 as a template, and mouse CD22 A DNA fragment containing domains 1 and 2 was obtained and digested with HindIII. Also, PCR was performed using pTM025 as a template using the primers shown in SEQ ID NO: 11 and SEQ ID NO: 12 in the sequence listing to obtain a DNA fragment containing domains 3 to 7 of human CD22 and digested with PstI . Combine these two fragments with a vector fragment obtained by digesting pcDNA3.1Zeo (+) (Invitrogen) with HindIII and PstI to bind domains 1 and 2 of mouse CD22 and domains 3 to 7 of human CD22 The first mouse-human-hybrid CD22 expression plasmid pTM029 was constructed. Next, pTM029 was transfected into mouse myeloma cell line NS0 cells using Lipofectamine 2000 (Invitrogen) to obtain transformants. The target first mouse / human-hybrid CD22 high-expressing cells were selected in the same manner as in Example 1 and recloned by limiting dilution to obtain two strains that were most stably highly expressed. / 30 and B4-152. This clone-expressed CD22 is a hybrid type of mouse-type domains 1 and 2 and human-type domains 3 to 7 (FIG. 1 (2)).

    Furthermore, second mouse-human-hybrid CD22-expressing cells in which domains 1 and 2 of mouse CD22 and domains 5 to 7 of human CD22 were bound were prepared as follows. A DNA fragment containing mouse CD22 domains 1 and 2 by performing a PCR reaction using the primer shown in SEQ ID NO: 9 in the sequence listing and the primer shown in SEQ ID NO: 10 in the sequence listing with a phosphate group on the 5 ′ side. And digested with HindIII. Further, a PCR reaction was performed using the primers shown in SEQ ID NO: 13 in the sequence listing and SEQ ID NO: 14 in the sequence listing to obtain a DNA fragment containing human CD22 domains 5 to 7 and digested with PstI. These fragments were combined with vector fragments obtained by digesting pcDNA3.1Zeo (+) (manufactured by Invitrogen) with HindIII and PstI to bind domains 1 and 2 of mouse CD22 and domains 5 to 7 of human CD22. A second mouse-human-hybrid CD22 expression plasmid pTM027 was constructed. Next, pTM027 was transfected into mouse myeloma cell line NS0 cells using Lipofectamine 2000 (Invitrogen) to obtain transformants. The target second mouse / human-hybrid CD22 high-expressing cell was selected in the same manner as in Example 1, and recloning was performed by limiting dilution. The strain that highly stably expressed high was named B9-4. This clone-expressed CD22 is a hybrid of mouse-type domains 1 and 2 and human-type domains 5 to 7 (FIG. 1 (3)).

Example 4: Preparation of anti-human CD22 antibody Recombinant human CD22 highly expressing cells G11-14 were suspended in physiological saline at 1 × 10 ⁷ cells / 100 μL, and FcgRIIb-deficient BALB / c mice (5 weeks old, female) (Nature. 379: 346-349 1996) was immunized 4 times subcutaneously. Recombinant human CD22-expressing cells 34-1 / 30 and B9-4 were suspended in 1 × 10 ⁷ cells / 100 μL with mitomycin C treatment (150 μg / mL, 37 ° C. for 60 minutes), and FcgRIIb-deficient BALB / c mice (5 weeks old, female) were immunized subcutaneously 11 times or 15 times, respectively. After measuring the antibody titer of the immunized mice, boosters were obtained by intraperitoneal administration of human CD22 highly expressing cells (G11-14, 34-1 / 30, B9-4) to the individuals with increased antibody titers. Mouse spleen cells were collected and fused with the mouse myeloma cell line X63 • Ag8 cells or P3U1 cells by the PEG method (Nature 256: 495-497, 1975). In the same manner as in Example 2, screening was performed using the culture supernatant of cells subjected to HAT selection after fusion, and anti-human CD22 or anti-human-mouse hybrid type monoclonal antibody-producing cells (hybridoma) were selected. Hereinafter, these are collectively referred to as “anti-human CD22 monoclonal antibody”. Finally, 126 types of anti-human CD22 monoclonal antibody-producing hybridomas that specifically react with human CD22 were established. Various anti-human CD22 monoclonal antibodies were prepared from the culture supernatant obtained by culturing the hybridoma by a conventional purification method (Current Protocols In Immunology Unit 2.6-2.7).

Example 5: Screening system for anti-mouse CD22 antibody using IL-6 production-inhibiting activity and IL-10 production-inducing activity as an index using local lymph node cells of mouse experimental autoimmune encephalomyelitis (EAE) model
(1) Construction of mouse experimental autoimmune encephalomyelitis (EAE) model As mouse experimental autoimmune encephalomyelitis (EAE) model is used as a disease model of multiple sclerosis (MS), This model was made according to the report and used for experiments (Nature 317: 355-358, 1985). In other words, PLP / JX SJL / J F1 mice (The Jackson Laboratory, 10 weeks old, female), MBP Ac1-11 peptide (peptide) consisting of amino acids 1 to 11 of mouse myelin basic protein (MBP) Laboratory) and Adjuvant complete H37RA (Difco) suspension were immunized once subcutaneously, and EAE was induced by intravenous injection of pertussis toxin (200 ng) (List Pharmaceuticals) on day 0 and day 2.

(2) Administration method Anti-mouse CD22 antibody A, B or C was administered to the EAE model animal at a dose of 0.2 to 200 μg / head once a day from the day of immunization for 5 days (N = 4). . The antibody was used after diluting with physiological saline.

(3) Cell preparation method
Measurement of IL-6: Local lymph nodes of each group were collected on day 10 after the start of immunization, and the cell suspension was adjusted (N = 4). Antigen (MBP Ac1-11, 30 μg / mL) was added to lymph node cells (5 × 10 ⁶ cells / mL) in each group, and IL-6 in the culture supernatant 48 hours after the start of stimulation was determined by ELISA ( Mouse IL-6 BD OptEIA ELISA set, BD).

Measurement of IL-10: Local lymph nodes of each group were collected on the 10th day after the start of immunization, and the cell suspension was adjusted (N = 4). Anti-Mu (10 μg / mL) + Anti-CD40 (1 μg / mL) was added to each group of lymph node cells (5 × 10 ⁶ cells / mL) to selectively stimulate B cells. IL-10 in the culture supernatant 48 hours after the start of stimulation was measured by ELISA (Mouse IL-10 BD OptEIA ELISA set, BD).

(4) Results IL-6 production produced from local lymph node cells by antigen stimulation was strongly suppressed in a dose-dependent manner by anti-mouse CD22 antibody A, B or C (FIG. 2). On the other hand, IL-10 production from local lymph node cells was enhanced several times in a dose-dependent manner by anti-mouse CD22 antibody B (FIG. 3 (2)). From the above, it was found that by using this screening system, an anti-mouse CD22 antibody exhibiting IL-6 production inhibitory activity and an anti-mouse CD22 antibody exhibiting IL-10 production inducing activity can be selected.

Example 6: Effect of anti-mouse CD22 antibody on mouse EAE model Both anti-mouse CD22 antibody A having IL-6 production inhibitory ability and both IL-6 production inhibitory ability and IL-10 production inducing ability shown in Example 5 Each of the anti-mouse CD22 antibody Bs having the above was administered to an EAE model mouse, and the therapeutic effect was examined using the survival rate as an index.

(1) Construction of mouse EAE model EAE-onset mice were constructed in the same manner as in Example 5.

(2) Administration method To the EAE model animal, the anti-mouse CD22 antibody A or B shown in Example 5 was administered from the tail vein once daily from the day of immunization at a dose of 2 to 200 μg / head for 5 days. (N = 10). The antibody was used after diluting with physiological saline.

(3) Results The results are shown in FIG. Both anti-mouse CD22 antibodies A and B, which exert a similar strong inhibitory action on IL-6 production, exhibited the same effect on EAE. The persistence of the effect on EAE by anti-mouse CD22 antibody administration was significant compared to the control group. Anti-mouse CD22 antibody B (FIG. 4 (2)), which has both functions of inhibiting IL-6 production and inducing IL-10 production, is anti-mouse CD22 antibody A (FIG. 4 (4) Compared with 1)), it was shown that a significant effect was exerted on EAE from a lower dose.

Example 7: Effect of anti-mouse CD22 antibody on lymphocyte subsets in mouse EAE model For the purpose of examining whether anti-mouse CD22 antibody exerts its effect by eliminating B cells, lymphocyte subsets of anti-CD22 antibody The effects on the

(1) Construction of mouse EAE model EAE-onset mice were constructed in the same manner as in Example 5.

(2) Administration method Anti-mouse CD22 antibody C having IL-6 production inhibitory action shown in Example 5 was administered to the EAE model animal once daily at a dose of 200 μg / head for 5 days from the tail vein. (N = 4). The antibody was used after diluting in physiological saline.

(3) Flow cytometry analysis Prepare a suspension of local lymph node cells in each group on the 10th day of immunization, and use phycoerythrin (PE) -labeled rat anti-mouse B220 monoclonal antibody (BD Pharmingen) for B cell marker detection. For detection of T cell markers, fluorescein-isothiocyanate (FITC) labeled rat anti-mouse CD3 monoclonal antibody (BD Pharmingen) was used and stained at 4 ° C. for 30 minutes. After washing, each group of lymphocyte subsets was analyzed using a FACS Calibur (Becton Dickinson).

  In order to exclude the influence of dead cells on flow cytometry analysis, dead cells were stained with propidium iodide (2 mg / ml; Sigma), and propidium iodide positive cells were excluded and analyzed.

(4) Results Table 1 shows the results of flow cytometry analysis. It has been found that the CD22 antibody screened in the present invention does not significantly affect B cell survival. Therefore, it is expected that B cells important for biological defense will not be lost and serious infections will not be induced.

  table 1

Example 8: Effect of anti-mouse CD22 antibody on spontaneous mouse systemic lupus erythematosus (SLE) model Anti-mouse CD22 antibody C having the ability to suppress IL-6 production shown in Example 5 was administered to SLE model mice and the treatment The effect was examined using the urine occult blood score as an index.

(1) Spontaneous mouse SLE model
NZW X BXSB F1 mice (Japan SLC) that spontaneously develop SLE-like pathology were used. The animals were raised to 8 weeks of age when the disease had already started to progress and were used for experiments. This SLE-like condition is said to be caused by pathological B1 cells as in human SLE (Autoimmunity 15, 99-105 (1993)).

(2) Urine occult blood score
Fresh urine was collected every 2 weeks and occult blood was examined by using Ames urine test paper (Hemacombisics, Bayer) to obtain an occult blood score.

(3) Administration method Anti-mouse CD22 antibody C having the IL-6 production inhibitory action shown in Example 5 was applied to the SLE model animal at a dose of 200 μg / head once a day at 8 weeks of age for only 5 days. Administration was via the tail vein (N = 10). The antibody was used after diluting in physiological saline.

(4) Results FIG. 5 shows the degree of progression of SLE-like pathology after administration of anti-mouse CD22 antibody. The anti-mouse CD22 antibody-administered group significantly suppressed the progression of lupus nephritis that developed early from the control group. This inhibitory effect lasted for more than 3 months after the end of administration.

Example 9: Effect of anti-mouse CD22 antibody on mouse type II collagen-induced arthritis (CIA) model Anti-mouse CD22 antibody B having IL-6 production-inhibiting ability and IL-10 production-inducing ability shown in Example 5 was treated as CIA. It was administered to model mice, and the therapeutic effect was examined using the arthritis score as an index.

(1) Construction of mouse type II collagen-induced arthritis (CIA) model The establishment of the CIA model, which is an animal model of rheumatoid arthritis (RA), is similar to human rheumatoid arthritis. This was carried out using FcgRIIb-deficient mice in which Jp was observed (J Exp Med. 146: 857-868 (1977)). Specifically, FcgRIIb-deficient C57BL / 6 mice (10 weeks old, female) were immunized once subcutaneously with bovine type II collagen (collagen technology workshop) and Adjuvant complete H37RA (Difco), and CIA Triggered.

(2) Arthritis score Based on the following criteria, the severity was determined for each limb and scored from 0 to 4 points. The arthritis score of each mouse was the sum of the limb scores (maximum: 16 points) (Int Arch Allergy Appl Immunol. 35: 456-467 (1969)).

Judgment criteria: 0 points: No symptoms, 1 point: Redness of only one small joint such as extremities, 2 points: Redness or swelling of 2 or more small joints, or relatively large joints such as wrists and ankles, 3 points: Redness and swelling of one hand and the entire leg, 4 points: Furthermore, the overall swelling of one hand and the leg reaches the maximum (3) Antibody administration method
CIA model animals were treated with the anti-mouse CD22 antibody B or anti-mouse IL-6 Receptor (IL-6R) antibody (Clone D7715A7, BD Pharmingen) shown in Example 5 at a dose of 200 μg / head from the day of immunization (day 0). It was administered intraperitoneally once a day for 5 days (N = 10). The antibody was used after diluting in physiological saline.

(4) Results FIG. 6 shows the degree of progression of arthritis after administration of anti-mouse CD22 antibody. Anti-mouse CD22 antibody B significantly suppressed the progression of arthritis as compared to the control group. This inhibitory effect lasted for 2 months after the end of the administration, which was the observation period.

  Since the anti-mouse CD22 antibody B obtained by the present invention was found to strongly suppress IL-6 production from the results of Example 5, the efficacy was compared with the anti-IL-6R antibody that inhibits IL-6 signal. . As a result, anti-CD22 antibody B showed a long-lasting effect as compared with anti-IL-6R antibody.

Example 10: Screening system for anti-human CD22 antibody using human peripheral blood mononuclear cells (PBMC) (1) Blood collection method As an anticoagulant during blood collection, heparin sodium (Mitsubishi Pharma; final concentration 50 units) or EDTA ( GIBCO; final concentration 5 mM) was collected from healthy volunteers.

(2) Sorting of peripheral blood mononuclear cells (PBMC)
Using Lymphoprep (AXIS-SHIELD), PBMCs were isolated from blood collected from healthy volunteers according to a conventional method (Scand. J. Clin Lab. Invest, 21, Suppl. 97.). The obtained PBMC was adjusted to a predetermined cell concentration with a medium.

(3) Culture method
PBMCs are plated at 10 ⁵ / well in a round-bottom 96-well plate, added with various anti-human CD22 antibodies, cultured for several hours, and then added with anti-human IgM F (ab ') ² at a concentration of 10 mg / mL, Cells were stimulated. After culturing at 37 ° C. for 48 hours in a CO ₂ incubator, the culture supernatant was collected by centrifugation.

(4) Method for measuring IL-6 production and IL-10 production The ELISA supernatant (Human IL-6 BD OptEIA) is prepared by appropriately diluting the culture supernatant and corresponding IL-6 production and IL-10 production. ELISA set, Human IL-10 BD OptEIA ELISA set, BD Biosciences).

(5) Screening method for anti-human CD22 antibody The anti-human CD22 antibody obtained in Example 4 was evaluated for IL-6 production inhibitory activity and IL-10 production induction activity. In other words, various anti-human CD22 antibodies were added to PBMC, and after culturing for about 6 hours, anti-human IgM F (ab ') ² was further added at a concentration of 10 mg / mL, and cultured at 37 ° C for 48 hours in a CO ₂ incubator. went. IL-6 production and IL-10 production in the supernatant were measured.

(6) Results As shown in FIG. 7, anti-human CD22 antibodies having an IL-6 production inhibitory activity of 50% or more compared to the control were selected. In addition, as shown in FIG. 8, anti-human CD22 antibodies having an IL-10 production-inducing activity of 200% or more compared to the control were selected.

FIG. 3 shows a schematic diagram of the three types of recombinant human CD22-expressing cell CD22 molecules obtained in Example 3. (1) Recombinant CD22-expressing cell line G11-14, (2) Schematic diagram of recombinant CD22-expressing cell line B9-4, (3) Recombinant CD22-expressing cell lines 34-1 / 30 and B4-152 Indicates. FIG. 5 shows the measurement results of IL-6 production inhibitory activity of anti-mouse CD22 antibody in Example 5. FIG. 6 shows the measurement results of IL-10 production-inducing activity of anti-mouse CD22 antibody in Example 5. FIG. 6 shows the effect of anti-mouse CD22 antibody administration on the mouse EAE model of Example 6 using survival rate as an index. (1) shows the survival rate when anti-CD22 antibody A is administered, and (2) shows the survival rate when anti-CD22 antibody B is administered. FIG. 9 shows the effect of anti-mouse CD22 antibody administration on the spontaneous mouse systemic lupus erythematosus (SLE) model of Example 8 using occult blood as an index. FIG. 9 is a graph showing the effect of anti-mouse CD22 antibody administration on the mouse type II collagen-induced arthritis (CIA) model of Example 9, using the arthritis score as an index. FIG. 4 shows the measurement results of IL-6 production inhibitory activity of the anti-human CD22 antibody of Example 10. Column “0” indicates the amount of IL-6 produced when PBMC is not stimulated. The column “anti-IgM” shows the amount of IL-6 produced when anti-human IgM F (ab ′) ² was added to stimulate PBMC. The column “control” shows the amount of IL-6 produced when a control antibody is added under stimulation with anti-human IgM F (ab ′) ² . Column “1-26” shows IL-6 production when various anti-human CD22 antibodies (NO. 1-26) were added under anti-human IgM F (ab ′) ² stimulation. FIG. 10 shows the results of IL-10 production-inducing activity of the anti-human CD22 antibody of Example 10. Column “0” indicates IL-10 production when PBMC is not stimulated. The column “anti-IgM” shows the amount of IL-10 produced when anti-human IgM F (ab ′) ² was added to stimulate PBMC. The column “control” shows the amount of IL-10 produced when a control antibody is added under stimulation with anti-human IgM F (ab ′) ² . Column “1-26” shows IL-10 production when various anti-human CD22 antibodies (NO. 1-26) were added under anti-human IgM F (ab ′) ² stimulation.

SEQUENCE LISTING

<110> Mitsubishi Pharma Corporation

<120> Methods for antibody screening

<130> 2007P00042
<160> 14

<170> PatentIn version 3.1

<210> 1
<211> 30
<212> DNA
<213> Artificial

<220>
<223> Inventor: Okamoto, Tadao; Miura, Masami

<220>
<221> misc_feature
<223> Primer based on murine CD22


<400> 1
gggaattcgc cacaccaccc ccaaccgtgg 30


<210> 2
<211> 30
<212> DNA
<213> Artificial

<220>
<221> misc_feature
<223> Primer based on murine CD22


<400> 2
tttctagatc aagaacctga aacagcagct 30


<210> 3
<211> 2607
<212> DNA
<213> Mus musculus

<220>
<221> CDS
<222> (1) .. (2607)
<223> cDNA based on murine CD22


<400> 3
atg cgc gtc cat tac ctg tgg ctc ctc ttg atc cta gga cat gtg gct 48
Met Arg Val His Tyr Leu Trp Leu Leu Leu Ile Leu Gly His Val Ala
1 5 10 15

tcg gct cgg tac agc tca gca aat gat tgg acc gtt gac cat ccc caa 96
Ser Ala Arg Tyr Ser Ser Ala Asn Asp Trp Thr Val Asp His Pro Gln
20 25 30

acc ctc ttt gcc tgg gag gga gcc tgc atc agg att cct tgc aag tac 144
Thr Leu Phe Ala Trp Glu Gly Ala Cys Ile Arg Ile Pro Cys Lys Tyr
35 40 45

aaa act cca cta ccc aag gca cgt ctg gac aac atc ctc ctt ttt cag 192
Lys Thr Pro Leu Pro Lys Ala Arg Leu Asp Asn Ile Leu Leu Phe Gln
50 55 60

aac tat gag ttt gac aag gcc acc aag aaa ttc aca gga act gtc ctg 240
Asn Tyr Glu Phe Asp Lys Ala Thr Lys Lys Phe Thr Gly Thr Val Leu
65 70 75 80

tac aac gcc aca aag act gag aag gac cca gag tct gag ctg tac ctt 288
Tyr Asn Ala Thr Lys Thr Glu Lys Asp Pro Glu Ser Glu Leu Tyr Leu
85 90 95

tct aag caa ggg aga gta aca ttt ctg ggg aac aga ata gac aat tgt 336
Ser Lys Gln Gly Arg Val Thr Phe Leu Gly Asn Arg Ile Asp Asn Cys
100 105 110

acc ctg aaa atc cac ccg ata cgt gcc aat gac agt ggg aat ctg ggg 384
Thr Leu Lys Ile His Pro Ile Arg Ala Asn Asp Ser Gly Asn Leu Gly
115 120 125

ttg agg atg acc gca ggg act gaa cga tgg atg gag ccc att cac ctc 432
Leu Arg Met Thr Ala Gly Thr Glu Arg Trp Met Glu Pro Ile His Leu
130 135 140

aat gtc tcg gag aaa ccg ttc caa cct tac atc cag atg cca tca gaa 480
Asn Val Ser Glu Lys Pro Phe Gln Pro Tyr Ile Gln Met Pro Ser Glu
145 150 155 160

atc cgg gaa tcc caa agt gtc act ctg acc tgt gga ctg aat ttc tcc 528
Ile Arg Glu Ser Gln Ser Val Thr Leu Thr Cys Gly Leu Asn Phe Ser
165 170 175

tgc ttt ggg tat gac atc ctc ttg aag tgg ttc cta gaa gat tct gag 576
Cys Phe Gly Tyr Asp Ile Leu Leu Lys Trp Phe Leu Glu Asp Ser Glu
180 185 190

atc acc tcc atc acc tct tct gtc acc tcc atc acc tct tct gtc acc 624
Ile Thr Ser Ile Thr Ser Ser Val Thr Ser Ile Thr Ser Ser Val Thr
195 200 205

tcc tcc att aag aat gtc tat aca gag agc aag ctc acc ttc caa cca 672
Ser Ser Ile Lys Asn Val Tyr Thr Glu Ser Lys Leu Thr Phe Gln Pro
210 215 220

aag tgg acg gac cac gga aag agt gtg aag tgc caa gtc cag cac tcc 720
Lys Trp Thr Asp His Gly Lys Ser Val Lys Cys Gln Val Gln His Ser
225 230 235 240

tcc aaa gtg ctc tca gag tgc act gtg cat ctg gat gtt aag tat acc 768
Ser Lys Val Leu Ser Glu Cys Thr Val His Leu Asp Val Lys Tyr Thr
245 250 255

ccg aag ctg gag atc aag gtc aat ccc aca gag gtg gaa aag aac aat 816
Pro Lys Leu Glu Ile Lys Val Asn Pro Thr Glu Val Glu Lys Asn Asn
260 265 270

tct gtg acc atg aca tgc cgg gtt aac agc agc aac ccg aaa ctc agg 864
Ser Val Thr Met Thr Cys Arg Val Asn Ser Ser Asn Pro Lys Leu Arg
275 280 285

acc gtg gcg gtg tct tgg ttc aag gat ggg cgc ccc cta gag gat cag 912
Thr Val Ala Val Ser Trp Phe Lys Asp Gly Arg Pro Leu Glu Asp Gln
290 295 300

gaa ctg gaa cag gaa caa cag atg tcc aag cta att ctg cat tca gtg 960
Glu Leu Glu Gln Glu Gln Gln Met Ser Lys Leu Ile Leu His Ser Val
305 310 315 320

acc aag gac atg aga ggg aaa tac cgg tgc cag gct tcc aac gac ata 1008
Thr Lys Asp Met Arg Gly Lys Tyr Arg Cys Gln Ala Ser Asn Asp Ile
325 330 335

ggc cca gga gag tcg gaa gaa gtg gaa ctc acg gtg cac tat gct cca 1056
Gly Pro Gly Glu Ser Glu Glu Val Glu Leu Thr Val His Tyr Ala Pro
340 345 350

gaa ccc tcc agg gtt cac atc tac cct tcc ccc gct gaa gag gga cag 1104
Glu Pro Ser Arg Val His Ile Tyr Pro Ser Pro Ala Glu Glu Gly Gln
355 360 365

tca gta gag ctg att tgt gag tca ctg gcc agt cca agt gca aca aac 1152
Ser Val Glu Leu Ile Cys Glu Ser Leu Ala Ser Pro Ser Ala Thr Asn
370 375 380

tac acc tgg tat cac aac agg aaa cct ata cct gga gac acc caa gag 1200
Tyr Thr Trp Tyr His Asn Arg Lys Pro Ile Pro Gly Asp Thr Gln Glu
385 390 395 400

aag ctc cgc atc cct aaa gtc tcc ccg tgg cat gct ggg aat tac tct 1248
Lys Leu Arg Ile Pro Lys Val Ser Pro Trp His Ala Gly Asn Tyr Ser
405 410 415

tgc ttg gca gag aac cgt ctg ggt cat gga aag ata gac cag gaa gct 1296
Cys Leu Ala Glu Asn Arg Leu Gly His Gly Lys Ile Asp Gln Glu Ala
420 425 430

aag ctg gat gtc cat tat gct ccc aag gcg gtg acc aca gtg att cag 1344
Lys Leu Asp Val His Tyr Ala Pro Lys Ala Val Thr Thr Val Ile Gln
435 440 445

agc ttc aca cca atc ctg gaa gga gac agt gtg acc ctg gtc tgt agg 1392
Ser Phe Thr Pro Ile Leu Glu Gly Asp Ser Val Thr Leu Val Cys Arg
450 455 460

tac aac tcc agc aat cca gac gtc acc tcc tac aga tgg aac cct caa 1440
Tyr Asn Ser Ser Asn Pro Asp Val Thr Ser Tyr Arg Trp Asn Pro Gln
465 470 475 480

ggt tct ggg agt gtg ctc aaa ccc gga gtg ctg agg att cag aaa gtg 1488
Gly Ser Gly Ser Val Leu Lys Pro Gly Val Leu Arg Ile Gln Lys Val
485 490 495

aca tgg gat tcc atg cct gtc agc tgt gct gcc tgc aac cac aag tgt 1536
Thr Trp Asp Ser Met Pro Val Ser Cys Ala Ala Cys Asn His Lys Cys
500 505 510

tcg tgg gcc ctc cct gtc atc ctg aat gtc cac tac gcc ccc aga gac 1584
Ser Trp Ala Leu Pro Val Ile Leu Asn Val His Tyr Ala Pro Arg Asp
515 520 525

gtg aag gta ctg aag gta agc ccc gca tca gag atc cgc gct ggg cag 1632
Val Lys Val Leu Lys Val Ser Pro Ala Ser Glu Ile Arg Ala Gly Gln
530 535 540

cgt gtc ctc ctc caa tgc gac ttc gca gag agc aac ccg gca gag gtc 1680
Arg Val Leu Leu Gln Cys Asp Phe Ala Glu Ser Asn Pro Ala Glu Val
545 550 555 560

cgc ttc ttc tgg aag aag aat ggg agt ctc gtg cag gaa ggg agg tac 1728
Arg Phe Phe Trp Lys Lys Asn Gly Ser Leu Val Gln Glu Gly Arg Tyr
565 570 575

ctg agc ttc ggc tcc gtc tcc cca gaa gat tct gga aat tat aac tgc 1776
Leu Ser Phe Gly Ser Val Ser Pro Glu Asp Ser Gly Asn Tyr Asn Cys
580 585 590

atg gtc aac aac tcc atc gga gag acc ttg tca cag gcc tgg aac ctc 1824
Met Val Asn Asn Ser Ile Gly Glu Thr Leu Ser Gln Ala Trp Asn Leu
595 600 605

caa gtg ctg tat gct cct cgg agg cta cgt gtg tcc atc agc cct ggg 1872
Gln Val Leu Tyr Ala Pro Arg Arg Leu Arg Val Ser Ile Ser Pro Gly
610 615 620

gac cat gtg atg gag ggg aag aag gcc acc ttg tcc tgt gag agt gat 1920
Asp His Val Met Glu Gly Lys Lys Ala Thr Leu Ser Cys Glu Ser Asp
625 630 635 640

gcc aat ccg ccc atc tca cag tac acc tgg ttt gac tcc agt ggc caa 1968
Ala Asn Pro Pro Ile Ser Gln Tyr Thr Trp Phe Asp Ser Ser Gly Gln
645 650 655

gac ctc cac tcc tca ggc cag aaa ctg aga ctg gaa ccc ctg gag gtc 2016
Asp Leu His Ser Ser Gly Gln Lys Leu Arg Leu Glu Pro Leu Glu Val
660 665 670

caa cac acg ggt tcc tac cgc tgc aaa ggg acc aat ggg ata ggc aca 2064
Gln His Thr Gly Ser Tyr Arg Cys Lys Gly Thr Asn Gly Ile Gly Thr
675 680 685

gga gag tca cca ccc agc acc ctc act gtc tac tac agt cca gag acc 2112
Gly Glu Ser Pro Pro Ser Thr Leu Thr Val Tyr Tyr Ser Pro Glu Thr
690 695 700

atc ggc aag cgg gtc gcc ttg gga cta ggc ttc tgc ctg act atc ttc 2160
Ile Gly Lys Arg Val Ala Leu Gly Leu Gly Phe Cys Leu Thr Ile Phe
705 710 715 720

atc ctg gcc atc tgg ggg atg aaa atc cag aaa aaa tgg aag caa aac 2208
Ile Leu Ala Ile Trp Gly Met Lys Ile Gln Lys Lys Trp Lys Gln Asn
725 730 735

cgc agc cag cag ggg ctt cag gaa aat tcc agt ggc cag agc ttt ttt 2256
Arg Ser Gln Gln Gly Leu Gln Glu Asn Ser Ser Gly Gln Ser Phe Phe
740 745 750

gtg agg aac aaa aag gct agg agg acc cct ctc tca gaa ggc ccc caa 2304
Val Arg Asn Lys Lys Ala Arg Arg Thr Pro Leu Ser Glu Gly Pro Gln
755 760 765

tcc cag gga tgc tac aat ccg gca atg gat gac act gtt agt tat gcc 2352
Ser Gln Gly Cys Tyr Asn Pro Ala Met Asp Asp Thr Val Ser Tyr Ala
770 775 780

atc ttg cgc ttt cca gag agt gac acg cac aat gct gga gat gca ggg 2400
Ile Leu Arg Phe Pro Glu Ser Asp Thr His Asn Ala Gly Asp Ala Gly
785 790 795 800

acc cca gca aca cag gct cct cct cca aac aac agc gac acg gtc act 2448
Thr Pro Ala Thr Gln Ala Pro Pro Pro Asn Asn Ser Asp Thr Val Thr
805 810 815

tac tcg gtg ata cag aag cgg cct atg ggg gat tat gag aat gtg aat 2496
Tyr Ser Val Ile Gln Lys Arg Pro Met Gly Asp Tyr Glu Asn Val Asn
820 825 830

ccg agc tgc ccg gag gat gag agc atc cat tac tca gag ctg gtt cag 2544
Pro Ser Cys Pro Glu Asp Glu Ser Ile His Tyr Ser Glu Leu Val Gln
835 840 845

ttt ggg gct ggt aag cgg ccc cag gca aag gaa gac gta gac tat gtg 2592
Phe Gly Ala Gly Lys Arg Pro Gln Ala Lys Glu Asp Val Asp Tyr Val
850 855 860

acc ctc aag cac tga 2607
Thr Leu Lys His
865


<210> 4
<211> 868
<212> PRT
<213> Mus musculus

<220>
<223> Amino-acid sequence of murine CD22

<400> 4

Met Arg Val His Tyr Leu Trp Leu Leu Leu Ile Leu Gly His Val Ala
1 5 10 15


Ser Ala Arg Tyr Ser Ser Ala Asn Asp Trp Thr Val Asp His Pro Gln
20 25 30


Thr Leu Phe Ala Trp Glu Gly Ala Cys Ile Arg Ile Pro Cys Lys Tyr
35 40 45


Lys Thr Pro Leu Pro Lys Ala Arg Leu Asp Asn Ile Leu Leu Phe Gln
50 55 60


Asn Tyr Glu Phe Asp Lys Ala Thr Lys Lys Phe Thr Gly Thr Val Leu
65 70 75 80


Tyr Asn Ala Thr Lys Thr Glu Lys Asp Pro Glu Ser Glu Leu Tyr Leu
85 90 95


Ser Lys Gln Gly Arg Val Thr Phe Leu Gly Asn Arg Ile Asp Asn Cys
100 105 110


Thr Leu Lys Ile His Pro Ile Arg Ala Asn Asp Ser Gly Asn Leu Gly
115 120 125


Leu Arg Met Thr Ala Gly Thr Glu Arg Trp Met Glu Pro Ile His Leu
130 135 140


Asn Val Ser Glu Lys Pro Phe Gln Pro Tyr Ile Gln Met Pro Ser Glu
145 150 155 160


Ile Arg Glu Ser Gln Ser Val Thr Leu Thr Cys Gly Leu Asn Phe Ser
165 170 175


Cys Phe Gly Tyr Asp Ile Leu Leu Lys Trp Phe Leu Glu Asp Ser Glu
180 185 190


Ile Thr Ser Ile Thr Ser Ser Val Thr Ser Ile Thr Ser Ser Val Thr
195 200 205


Ser Ser Ile Lys Asn Val Tyr Thr Glu Ser Lys Leu Thr Phe Gln Pro
210 215 220


Lys Trp Thr Asp His Gly Lys Ser Val Lys Cys Gln Val Gln His Ser
225 230 235 240


Ser Lys Val Leu Ser Glu Cys Thr Val His Leu Asp Val Lys Tyr Thr
245 250 255


Pro Lys Leu Glu Ile Lys Val Asn Pro Thr Glu Val Glu Lys Asn Asn
260 265 270


Ser Val Thr Met Thr Cys Arg Val Asn Ser Ser Asn Pro Lys Leu Arg
275 280 285


Thr Val Ala Val Ser Trp Phe Lys Asp Gly Arg Pro Leu Glu Asp Gln
290 295 300


Glu Leu Glu Gln Glu Gln Gln Met Ser Lys Leu Ile Leu His Ser Val
305 310 315 320


Thr Lys Asp Met Arg Gly Lys Tyr Arg Cys Gln Ala Ser Asn Asp Ile
325 330 335


Gly Pro Gly Glu Ser Glu Glu Val Glu Leu Thr Val His Tyr Ala Pro
340 345 350


Glu Pro Ser Arg Val His Ile Tyr Pro Ser Pro Ala Glu Glu Gly Gln
355 360 365


Ser Val Glu Leu Ile Cys Glu Ser Leu Ala Ser Pro Ser Ala Thr Asn
370 375 380


Tyr Thr Trp Tyr His Asn Arg Lys Pro Ile Pro Gly Asp Thr Gln Glu
385 390 395 400


Lys Leu Arg Ile Pro Lys Val Ser Pro Trp His Ala Gly Asn Tyr Ser
405 410 415


Cys Leu Ala Glu Asn Arg Leu Gly His Gly Lys Ile Asp Gln Glu Ala
420 425 430


Lys Leu Asp Val His Tyr Ala Pro Lys Ala Val Thr Thr Val Ile Gln
435 440 445


Ser Phe Thr Pro Ile Leu Glu Gly Asp Ser Val Thr Leu Val Cys Arg
450 455 460


Tyr Asn Ser Ser Asn Pro Asp Val Thr Ser Tyr Arg Trp Asn Pro Gln
465 470 475 480


Gly Ser Gly Ser Val Leu Lys Pro Gly Val Leu Arg Ile Gln Lys Val
485 490 495


Thr Trp Asp Ser Met Pro Val Ser Cys Ala Ala Cys Asn His Lys Cys
500 505 510


Ser Trp Ala Leu Pro Val Ile Leu Asn Val His Tyr Ala Pro Arg Asp
515 520 525


Val Lys Val Leu Lys Val Ser Pro Ala Ser Glu Ile Arg Ala Gly Gln
530 535 540


Arg Val Leu Leu Gln Cys Asp Phe Ala Glu Ser Asn Pro Ala Glu Val
545 550 555 560


Arg Phe Phe Trp Lys Lys Asn Gly Ser Leu Val Gln Glu Gly Arg Tyr
565 570 575


Leu Ser Phe Gly Ser Val Ser Pro Glu Asp Ser Gly Asn Tyr Asn Cys
580 585 590


Met Val Asn Asn Ser Ile Gly Glu Thr Leu Ser Gln Ala Trp Asn Leu
595 600 605


Gln Val Leu Tyr Ala Pro Arg Arg Leu Arg Val Ser Ile Ser Pro Gly
610 615 620


Asp His Val Met Glu Gly Lys Lys Ala Thr Leu Ser Cys Glu Ser Asp
625 630 635 640


Ala Asn Pro Pro Ile Ser Gln Tyr Thr Trp Phe Asp Ser Ser Gly Gln
645 650 655


Asp Leu His Ser Ser Gly Gln Lys Leu Arg Leu Glu Pro Leu Glu Val
660 665 670


Gln His Thr Gly Ser Tyr Arg Cys Lys Gly Thr Asn Gly Ile Gly Thr
675 680 685


Gly Glu Ser Pro Pro Ser Thr Leu Thr Val Tyr Tyr Ser Pro Glu Thr
690 695 700


Ile Gly Lys Arg Val Ala Leu Gly Leu Gly Phe Cys Leu Thr Ile Phe
705 710 715 720


Ile Leu Ala Ile Trp Gly Met Lys Ile Gln Lys Lys Trp Lys Gln Asn
725 730 735


Arg Ser Gln Gln Gly Leu Gln Glu Asn Ser Ser Gly Gln Ser Phe Phe
740 745 750


Val Arg Asn Lys Lys Ala Arg Arg Thr Pro Leu Ser Glu Gly Pro Gln
755 760 765


Ser Gln Gly Cys Tyr Asn Pro Ala Met Asp Asp Thr Val Ser Tyr Ala
770 775 780


Ile Leu Arg Phe Pro Glu Ser Asp Thr His Asn Ala Gly Asp Ala Gly
785 790 795 800


Thr Pro Ala Thr Gln Ala Pro Pro Pro Asn Asn Ser Asp Thr Val Thr
805 810 815


Tyr Ser Val Ile Gln Lys Arg Pro Met Gly Asp Tyr Glu Asn Val Asn
820 825 830


Pro Ser Cys Pro Glu Asp Glu Ser Ile His Tyr Ser Glu Leu Val Gln
835 840 845


Phe Gly Ala Gly Lys Arg Pro Gln Ala Lys Glu Asp Val Asp Tyr Val
850 855 860


Thr Leu Lys His
865


<210> 5
<211> 20
<212> DNA
<213> Artificial

<220>
<221> misc_feature
<223> Primer based on human CD22


<400> 5
gcttgcaccc agacacgaca 20


<210> 6
<211> 20
<212> DNA
<213> Artificial


<220>
<221> misc_feature
<223> Primer besed on human CD22


<400> 6
cctctgctgc agcccatcca 20


<210> 7
<211> 2544
<212> DNA
<213> Homo sapiens


<220>
<221> CDS
<222> (1) .. (2544)
<223> cDNA besed on human CD22


<400> 7
atg cat ctc ctc ggc ccc tgg ctc ctg ctc ctg gtt cta gaa tac ttg 48
Met His Leu Leu Gly Pro Trp Leu Leu Leu Leu Val Leu Glu Tyr Leu
1 5 10 15

gct ttc tct gac tca agt aaa tgg gtt ttt gag cac cct gaa acc ctc 96
Ala Phe Ser Asp Ser Ser Lys Trp Val Phe Glu His Pro Glu Thr Leu
20 25 30

tac gcc tgg gag ggg gcc tgc gtc tgg atc ccc tgc acc tac aga gcc 144
Tyr Ala Trp Glu Gly Ala Cys Val Trp Ile Pro Cys Thr Tyr Arg Ala
35 40 45

cta gat ggt gac ctg gaa agc ttc atc ctg ttc cac aat cct gag tat 192
Leu Asp Gly Asp Leu Glu Ser Phe Ile Leu Phe His Asn Pro Glu Tyr
50 55 60

aac aag aac acc tcg aag ttt gat ggg aca aga ctc tat gaa agc aca 240
Asn Lys Asn Thr Ser Lys Phe Asp Gly Thr Arg Leu Tyr Glu Ser Thr
65 70 75 80

aag gat ggg aag gtt cct tct gag cag aaa agg gtg caa ttc ctg gga 288
Lys Asp Gly Lys Val Pro Ser Glu Gln Lys Arg Val Gln Phe Leu Gly
85 90 95

gac aag aat aag aac tgc aca ctg agt atc cac ccg gtg cac ctc aat 336
Asp Lys Asn Lys Asn Cys Thr Leu Ser Ile His Pro Val His Leu Asn
100 105 110

gac agt ggt cag ctg ggg ctg agg atg gag tcc aag act gag aaa tgg 384
Asp Ser Gly Gln Leu Gly Leu Arg Met Glu Ser Lys Thr Glu Lys Trp
115 120 125

atg gaa cga ata cac ctc aat gtc tct gaa agg cct ttt cca cct cat 432
Met Glu Arg Ile His Leu Asn Val Ser Glu Arg Pro Phe Pro Pro His
130 135 140

atc cag ctc cct cca gaa att caa gag tcc cag gaa gtc act ctg acc 480
Ile Gln Leu Pro Pro Glu Ile Gln Glu Ser Gln Glu Val Thr Leu Thr
145 150 155 160

tgc ttg ctg aat ttc tcc tgc tat ggg tat ccg atc caa ttg cag tgg 528
Cys Leu Leu Asn Phe Ser Cys Tyr Gly Tyr Pro Ile Gln Leu Gln Trp
165 170 175

ctc cta gag ggg gtt cca atg agg cag gct gct gtc acc tcg acc tcc 576
Leu Leu Glu Gly Val Pro Met Arg Gln Ala Ala Val Thr Ser Thr Ser
180 185 190

ttg acc atc aag tct gtc ttc acc cgg agc gag ctc aag ttc tcc cca 624
Leu Thr Ile Lys Ser Val Phe Thr Arg Ser Glu Leu Lys Phe Ser Pro
195 200 205

cag tgg agt cac cat ggg aag att gtg acc tgc cag ctt cag gat gca 672
Gln Trp Ser His His Gly Lys Ile Val Thr Cys Gln Leu Gln Asp Ala
210 215 220

gat ggg aag ttc ctc tcc aat gac acg gtg cag ctg aac gtg aag cac 720
Asp Gly Lys Phe Leu Ser Asn Asp Thr Val Gln Leu Asn Val Lys His
225 230 235 240

acc ccg aag ttg gag atc aag gtc act ccc agt gat gcc ata gtg agg 768
Thr Pro Lys Leu Glu Ile Lys Val Thr Pro Ser Asp Ala Ile Val Arg
245 250 255

gag ggg gac tct gtg acc atg acc tgc gag gtc agc agc agc aac ccg 816
Glu Gly Asp Ser Val Thr Met Thr Cys Glu Val Ser Ser Ser Asn Pro
260 265 270

gag tac acg acg gta tcc tgg ctc aag gat ggg acc tcg ctg aag aag 864
Glu Tyr Thr Thr Val Ser Trp Leu Lys Asp Gly Thr Ser Leu Lys Lys
275 280 285

cag aat aca ttc acg cta aac ctg cgc gaa gtg acc aag gac cag agt 912
Gln Asn Thr Phe Thr Leu Asn Leu Arg Glu Val Thr Lys Asp Gln Ser
290 295 300

ggg aag tac tgc tgt cag gtc tcc aat gac gtg ggc ccg gga agg tcg 960
Gly Lys Tyr Cys Cys Gln Val Ser Asn Asp Val Gly Pro Gly Arg Ser
305 310 315 320

gaa gaa gtg ttc ctg caa gtg cag tat gcc ccg gaa cct tcc acg gtt 1008
Glu Glu Val Phe Leu Gln Val Gln Tyr Ala Pro Glu Pro Ser Thr Val
325 330 335

cag atc ctc cac tca ccg gct gtg gag gga agt caa gtc gag ttt ctt 1056
Gln Ile Leu His Ser Pro Ala Val Glu Gly Ser Gln Val Glu Phe Leu
340 345 350

tgc atg tca ctg gcc aat cct ctt cca aca aat tac acg tgg tac cac 1104
Cys Met Ser Leu Ala Asn Pro Leu Pro Thr Asn Tyr Thr Trp Tyr His
355 360 365

aat ggg aaa gaa atg cag gga agg aca gag gag aaa gtc cac atc cca 1152
Asn Gly Lys Glu Met Gln Gly Arg Thr Glu Glu Lys Val His Ile Pro
370 375 380

aag atc ctc ccc tgg cac gct ggg act tat tcc tgt gtg gca gaa aac 1200
Lys Ile Leu Pro Trp His Ala Gly Thr Tyr Ser Cys Val Ala Glu Asn
385 390 395 400

att ctt ggt act gga cag agg ggc ccg gga gct gag ctg gat gtc cag 1248
Ile Leu Gly Thr Gly Gln Arg Gly Pro Gly Ala Glu Leu Asp Val Gln
405 410 415

tat cct ccc aag aag gtg acc aca gtg att caa aac ccc atg ccg att 1296
Tyr Pro Pro Lys Lys Val Thr Thr Val Ile Gln Asn Pro Met Pro Ile
420 425 430

cga gaa gga gac aca gtg acc ctt tcc tgt aac tac aat tcc agt aac 1344
Arg Glu Gly Asp Thr Val Thr Leu Ser Cys Asn Tyr Asn Ser Ser Asn
435 440 445

ccc agt gtt acc cgg tat gaa tgg aaa ccc cat ggc gcc tgg gag gag 1392
Pro Ser Val Thr Arg Tyr Glu Trp Lys Pro His Gly Ala Trp Glu Glu
450 455 460

cca tcg ctt ggg gtg ctg aag atc caa aac gtt ggc tgg gac aac aca 1440
Pro Ser Leu Gly Val Leu Lys Ile Gln Asn Val Gly Trp Asp Asn Thr
465 470 475 480

acc atc gcc tgc gca cgt tgt aat agt tgg tgc tcg tgg gcc tcc cct 1488
Thr Ile Ala Cys Ala Arg Cys Asn Ser Trp Cys Ser Trp Ala Ser Pro
485 490 495

gtc gcc ctg aat gtc cag tat gcc ccc cga gac gtg agg gtc cgg aaa 1536
Val Ala Leu Asn Val Gln Tyr Ala Pro Arg Asp Val Arg Val Arg Lys
500 505 510

atc aag ccc ctt tcc gag att cac tct gga aac tcg gtc agc ctc caa 1584
Ile Lys Pro Leu Ser Glu Ile His Ser Gly Asn Ser Val Ser Leu Gln
515 520 525

tgt gac ttc tca agc agc cac ccc aaa gaa gtc cag ttc ttc tgg gag 1632
Cys Asp Phe Ser Ser Ser His Pro Lys Glu Val Gln Phe Phe Trp Glu
530 535 540

aaa aat ggc agg ctt ctg ggg aaa gaa agc cag ctg aat ttt gac tcc 1680
Lys Asn Gly Arg Leu Leu Gly Lys Glu Ser Gln Leu Asn Phe Asp Ser
545 550 555 560

atc tcc cca gaa gat gct ggg agt tac agc tgc tgg gtg aac aac tcc 1728
Ile Ser Pro Glu Asp Ala Gly Ser Tyr Ser Cys Trp Val Asn Asn Ser
565 570 575

ata gga cag aca gcg tcc aag gcc tgg aca ctt gaa gtg ctg tat gca 1776
Ile Gly Gln Thr Ala Ser Lys Ala Trp Thr Leu Glu Val Leu Tyr Ala
580 585 590

ccc agg agg ctg cgt gtg tcc atg agc ccg ggg gac caa gtg atg gag 1824
Pro Arg Arg Leu Arg Val Ser Met Ser Pro Gly Asp Gln Val Met Glu
595 600 605

ggg aag agt gca acc ctg acc tgt gag agc gac gcc aac cct ccc gtc 1872
Gly Lys Ser Ala Thr Leu Thr Cys Glu Ser Asp Ala Asn Pro Pro Val
610 615 620

tcc cac tac acc tgg ttt gac tgg aat aac caa agc ctc ccc tac cac 1920
Ser His Tyr Thr Trp Phe Asp Trp Asn Asn Gln Ser Leu Pro Tyr His
625 630 635 640

agc cag aag ctg aga ttg gag ccg gtg aag gtc cag cac tcg ggt gcc 1968
Ser Gln Lys Leu Arg Leu Glu Pro Val Lys Val Gln His Ser Gly Ala
645 650 655

tac tgg tgc cag ggg acc aac agt gtg ggc aag ggc cgt tcg cct ctc 2016
Tyr Trp Cys Gln Gly Thr Asn Ser Val Gly Lys Gly Arg Ser Pro Leu
660 665 670

agc acc ctt act gtc tac tat agc ccg gag acc atc ggc agg cga gtg 2064
Ser Thr Leu Thr Val Tyr Tyr Ser Pro Glu Thr Ile Gly Arg Arg Val
675 680 685

gct gtg gga ctc ggg tcc tgc ctc gcc atc ctc atc ctg gca atc tgt 2112
Ala Val Gly Leu Gly Ser Cys Leu Ala Ile Leu Ile Leu Ala Ile Cys
690 695 700

ggg ctc aag ctc cag cga cgt tgg aag agg aca cag agc cag cag ggg 2160
Gly Leu Lys Leu Gln Arg Arg Trp Lys Arg Thr Gln Ser Gln Gln Gly
705 710 715 720

ctt cag gag aat tcc agc ggc cag agc ttc ttt gtg agg aat aaa aag 2208
Leu Gln Glu Asn Ser Ser Gly Gln Ser Phe Phe Val Arg Asn Lys Lys
725 730 735

gtt aga agg gcc ccc ctc tct gaa gac ccc cac tcc ctg gga tgc tac 2256
Val Arg Arg Ala Pro Leu Ser Glu Asp Pro His Ser Leu Gly Cys Tyr
740 745 750

aat cca atg atg gaa gat ggc att agc tac acc acc ctg cgc ttt ccc 2304
Asn Pro Met Met Glu Asp Gly Ile Ser Tyr Thr Thr Leu Arg Phe Pro
755 760 765

gag atg aac ata cca cga act gga gat gca gag tcc tca gag atg cag 2352
Glu Met Asn Ile Pro Arg Thr Gly Asp Ala Glu Ser Ser Glu Met Gln
770 775 780

aga cct ccc cgg acc tgc gat gac acg gtc act tat tca gca ttg cac 2400
Arg Pro Pro Arg Thr Cys Asp Asp Thr Val Thr Tyr Ser Ala Leu His
785 790 795 800

aag cgc caa gtg ggc gac tat gag aac gtc att cca gat ttt cca gaa 2448
Lys Arg Gln Val Gly Asp Tyr Glu Asn Val Ile Pro Asp Phe Pro Glu
805 810 815

gat gag ggg att cat tac tca gag ctg atc cag ttt ggg gtc ggg gag 2496
Asp Glu Gly Ile His Tyr Ser Glu Leu Ile Gln Phe Gly Val Gly Glu
820 825 830

cgg cct cag gca caa gaa aat gtg gac tat gtg atc ctc aaa cat tga 2544
Arg Pro Gln Ala Gln Glu Asn Val Asp Tyr Val Ile Leu Lys His
835 840 845


<210> 8
<211> 847
<212> PRT
<213> Homo sapiens

<220>
<223> Amino-acid sequence of human CD22

<400> 8

Met His Leu Leu Gly Pro Trp Leu Leu Leu Leu Val Leu Glu Tyr Leu
1 5 10 15


Ala Phe Ser Asp Ser Ser Lys Trp Val Phe Glu His Pro Glu Thr Leu
20 25 30


Tyr Ala Trp Glu Gly Ala Cys Val Trp Ile Pro Cys Thr Tyr Arg Ala
35 40 45


Leu Asp Gly Asp Leu Glu Ser Phe Ile Leu Phe His Asn Pro Glu Tyr
50 55 60


Asn Lys Asn Thr Ser Lys Phe Asp Gly Thr Arg Leu Tyr Glu Ser Thr
65 70 75 80


Lys Asp Gly Lys Val Pro Ser Glu Gln Lys Arg Val Gln Phe Leu Gly
85 90 95


Asp Lys Asn Lys Asn Cys Thr Leu Ser Ile His Pro Val His Leu Asn
100 105 110


Asp Ser Gly Gln Leu Gly Leu Arg Met Glu Ser Lys Thr Glu Lys Trp
115 120 125


Met Glu Arg Ile His Leu Asn Val Ser Glu Arg Pro Phe Pro Pro His
130 135 140


Ile Gln Leu Pro Pro Glu Ile Gln Glu Ser Gln Glu Val Thr Leu Thr
145 150 155 160


Cys Leu Leu Asn Phe Ser Cys Tyr Gly Tyr Pro Ile Gln Leu Gln Trp
165 170 175


Leu Leu Glu Gly Val Pro Met Arg Gln Ala Ala Val Thr Ser Thr Ser
180 185 190


Leu Thr Ile Lys Ser Val Phe Thr Arg Ser Glu Leu Lys Phe Ser Pro
195 200 205


Gln Trp Ser His His Gly Lys Ile Val Thr Cys Gln Leu Gln Asp Ala
210 215 220


Asp Gly Lys Phe Leu Ser Asn Asp Thr Val Gln Leu Asn Val Lys His
225 230 235 240


Thr Pro Lys Leu Glu Ile Lys Val Thr Pro Ser Asp Ala Ile Val Arg
245 250 255


Glu Gly Asp Ser Val Thr Met Thr Cys Glu Val Ser Ser Ser Asn Pro
260 265 270


Glu Tyr Thr Thr Val Ser Trp Leu Lys Asp Gly Thr Ser Leu Lys Lys
275 280 285


Gln Asn Thr Phe Thr Leu Asn Leu Arg Glu Val Thr Lys Asp Gln Ser
290 295 300


Gly Lys Tyr Cys Cys Gln Val Ser Asn Asp Val Gly Pro Gly Arg Ser
305 310 315 320


Glu Glu Val Phe Leu Gln Val Gln Tyr Ala Pro Glu Pro Ser Thr Val
325 330 335


Gln Ile Leu His Ser Pro Ala Val Glu Gly Ser Gln Val Glu Phe Leu
340 345 350


Cys Met Ser Leu Ala Asn Pro Leu Pro Thr Asn Tyr Thr Trp Tyr His
355 360 365


Asn Gly Lys Glu Met Gln Gly Arg Thr Glu Glu Lys Val His Ile Pro
370 375 380


Lys Ile Leu Pro Trp His Ala Gly Thr Tyr Ser Cys Val Ala Glu Asn
385 390 395 400


Ile Leu Gly Thr Gly Gln Arg Gly Pro Gly Ala Glu Leu Asp Val Gln
405 410 415


Tyr Pro Pro Lys Lys Val Thr Thr Val Ile Gln Asn Pro Met Pro Ile
420 425 430


Arg Glu Gly Asp Thr Val Thr Leu Ser Cys Asn Tyr Asn Ser Ser Asn
435 440 445


Pro Ser Val Thr Arg Tyr Glu Trp Lys Pro His Gly Ala Trp Glu Glu
450 455 460


Pro Ser Leu Gly Val Leu Lys Ile Gln Asn Val Gly Trp Asp Asn Thr
465 470 475 480


Thr Ile Ala Cys Ala Arg Cys Asn Ser Trp Cys Ser Trp Ala Ser Pro
485 490 495


Val Ala Leu Asn Val Gln Tyr Ala Pro Arg Asp Val Arg Val Arg Lys
500 505 510


Ile Lys Pro Leu Ser Glu Ile His Ser Gly Asn Ser Val Ser Leu Gln
515 520 525


Cys Asp Phe Ser Ser Ser His Pro Lys Glu Val Gln Phe Phe Trp Glu
530 535 540


Lys Asn Gly Arg Leu Leu Gly Lys Glu Ser Gln Leu Asn Phe Asp Ser
545 550 555 560


Ile Ser Pro Glu Asp Ala Gly Ser Tyr Ser Cys Trp Val Asn Asn Ser
565 570 575


Ile Gly Gln Thr Ala Ser Lys Ala Trp Thr Leu Glu Val Leu Tyr Ala
580 585 590


Pro Arg Arg Leu Arg Val Ser Met Ser Pro Gly Asp Gln Val Met Glu
595 600 605


Gly Lys Ser Ala Thr Leu Thr Cys Glu Ser Asp Ala Asn Pro Pro Val
610 615 620


Ser His Tyr Thr Trp Phe Asp Trp Asn Asn Gln Ser Leu Pro Tyr His
625 630 635 640


Ser Gln Lys Leu Arg Leu Glu Pro Val Lys Val Gln His Ser Gly Ala
645 650 655


Tyr Trp Cys Gln Gly Thr Asn Ser Val Gly Lys Gly Arg Ser Pro Leu
660 665 670


Ser Thr Leu Thr Val Tyr Tyr Ser Pro Glu Thr Ile Gly Arg Arg Val
675 680 685


Ala Val Gly Leu Gly Ser Cys Leu Ala Ile Leu Ile Leu Ala Ile Cys
690 695 700


Gly Leu Lys Leu Gln Arg Arg Trp Lys Arg Thr Gln Ser Gln Gln Gly
705 710 715 720


Leu Gln Glu Asn Ser Ser Gly Gln Ser Phe Phe Val Arg Asn Lys Lys
725 730 735


Val Arg Arg Ala Pro Leu Ser Glu Asp Pro His Ser Leu Gly Cys Tyr
740 745 750


Asn Pro Met Met Glu Asp Gly Ile Ser Tyr Thr Thr Leu Arg Phe Pro
755 760 765


Glu Met Asn Ile Pro Arg Thr Gly Asp Ala Glu Ser Ser Glu Met Gln
770 775 780


Arg Pro Pro Arg Thr Cys Asp Asp Thr Val Thr Tyr Ser Ala Leu His
785 790 795 800


Lys Arg Gln Val Gly Asp Tyr Glu Asn Val Ile Pro Asp Phe Pro Glu
805 810 815


Asp Glu Gly Ile His Tyr Ser Glu Leu Ile Gln Phe Gly Val Gly Glu
820 825 830


Arg Pro Gln Ala Gln Glu Asn Val Asp Tyr Val Ile Leu Lys His
835 840 845


<210> 9
<211> 26
<212> DNA
<213> Artificial

<220>
<221> misc_feature
<223> Primer besed on murine CD22


<400> 9
ccaagcttgc caccatgcgc gtccat 26


<210> 10
<211> 20
<212> DNA
<213> Artificial


<220>
<221> misc_feature
<223> Primer besed on murine CD22


<400> 10
atacttaaca tccagatgca 20


<210> 11
<211> 24
<212> DNA
<213> Artificial

<220>
<221> misc_feature
<223> Primer besed on human CD22


<400> 11
accccgaagt tggagatcaa ggtc 24


<210> 12
<211> 20
<212> DNA
<213> Artificial


<220>
<221> misc_feature
<223> Primer besed on human CD22


<400> 12
cttttctgct cagaaggaac 20


<210> 13
<211> 20
<212> DNA
<213> Artificial

<220>
<221> misc_feature
<223> Primer besed on human CD22


<400> 13
cctcccaaga aggtgaccac 20


<210> 14
<211> 26
<212> DNA
<213> Artificial

<220>
<221> misc_feature
<223> Primer besed on human CD22


<400> 14
gcctgcaggt gtcaatgttt gaggat 26

Claims (20)

This is a screening method for antibodies to B cell-specific surface antigens, i) the step of adding antibodies to B cell-specific surface antigens to B cells, ii) and then confirming that the production of Interleukine-6 by B cells has changed An antibody screening method comprising the steps of: and / or confirming that production of Interleukine-10 by B cells has changed. The screening method according to claim 1, wherein the B cell-specific surface antigen is one or more selected from CD22α, CD22β, CD19, CD20, CD79α and CD79β. The screening method according to claim 1 or 2, wherein the B cell-specific surface antigen is CD22α and / or CD22β. The B cell is selected from an established B cell, a rodent peripheral blood-derived cell, a rodent lymph node-derived cell, a human peripheral blood-derived cell, a human lymph node-derived cell, and a human tonsil-derived cell. The screening method in any one of. The screening method according to any one of claims 1 to 4, wherein the production of Interleukine-6 by B cells is induction and / or enhancement by activation of B cells. Activation of B cells is anti-IgM antibody, anti-IgM antibody bead, anti-IgG antibody, anti-IgG antibody bead, anti-Ig antibody, anti-Ig antibody bead, Staphylococcus aureus Cowan I strain cell component, Lipopolysaccharide, Pokeweed mitogen, Epstein- The screening method according to claim 5, wherein the screening method is activation by any one or more selected from Barr virus, CpG oligodeoxynucleotides and Toll like receptor ligand. The process of ii) is one in which production of Interleukine-6 by B cells is suppressed and / or production of Interleukine-10 by B cells is enhanced. Screening method. Inhibition of Interleukine-6 production by B cells is to eliminate only B cells that produce Interleukine-6 and / or to cause production inhibition of Interleukine-6 production only to B cells that produce Interleukine-6. The screening method according to claim 7. The screening method according to any one of claims 1 to 8, wherein a change in production of Interleukine-6 is suppressed by 5% or more and 100% or less. The screening method according to any one of claims 1 to 8, wherein a change in production of Interleukine-6 is suppressed by 25% or more and 100% or less. The screening method according to any one of claims 1 to 8, wherein the change in production of Interleukine-6 is suppressed by 40% or more and 100% or less. The screening method according to any one of claims 1 to 11, wherein the change in production of Interleukine-10 is enhanced by 150% or more. A method for producing an antibody, wherein the antibody obtained by the method according to claim 1 is produced by a genetic recombination technique. An antibody obtained by the method according to any one of claims 1 to 13. 15. The antibody of claim 14, wherein the antibody is a monoclonal antibody. The antibody according to claim 14 or 15, wherein the antibody is a human monoclonal antibody. A pharmaceutical composition comprising the antibody according to any one of claims 14 to 16 as an active ingredient. 18. The pharmaceutical composition according to claim 17, which is used for treatment and / or prevention of a disease involving Interleukine-6 and / or Interleukine-10 and a disease selected from B lymphoma. 18. The pharmaceutical composition according to claim 17, which is used for prevention and / or treatment of an autoimmune disease or Castleman's disease. 18. The pharmaceutical composition according to claim 17, which is used for prevention and / or treatment of rheumatoid arthritis, multiple sclerosis or systemic lupus erythematosus.