WO2006003999A1

WO2006003999A1 - Human antihuman b7rp-1 antibody and antibody fragment thereof

Info

Publication number: WO2006003999A1
Application number: PCT/JP2005/012094
Authority: WO
Inventors: Kazuhisa Sugimura; Yuji Ito; Toshihiro Nakashima; Masaharu Torikai
Original assignee: Juridical Foundation The Chemo-Sero-Therapeutic Research Institute
Priority date: 2004-07-05
Filing date: 2005-06-30
Publication date: 2006-01-12
Also published as: JPWO2006003999A1

Abstract

It is intended to provide a human antihuman B7 related protein 1 (hereinafter referred to as B7RP-1) antibody, its antibody fragment and a method of using the same. By using the phage antibody method, a human antihuman B7RP-1 antibody having a high affinity for human B7RP-1 and its antibody fragment are obtained. The antibody and its antibody fragment thus obtained are expected as useful as remedies for inflammation and immune abnormality.

Description

Human anti-human B7RP-1 antibody and antibody fragments thereof

Technical field

[0001] The present invention relates to a human anti-human B7RP-1 antibody that binds to human B7 Related Protein 1 (also known as B7h, GL50, ICOS ligand, hereinafter referred to as "B7R Pl") and inhibits its biological activity, Relates to antibody fragments. These antibodies and antibody fragments suppress the excessive activity of T cells induced by binding of B7RP-1 to ICOS (Inducible costimulator), which is a receptor on the T cells, to prevent the transplantation during organ transplantation. It is expected to be used as an immunosuppressant and a therapeutic agent for immune abnormalities such as allergies and autoimmunity.

Background art

[0002] ICOS was discovered as the third molecule of the T28 co-stimulatory receptor CD28 family, and is a membrane-bound protein that forms a homodimer. It is induced on the surface (Non-Patent Document 1). Co-stimulation via this molecule increases the proliferation of both CD4 and CD8 T cells, and induces the expression of CD154 (CD40 ligand) in T cells, along with the secretion of cytoplasmic ins such as IL_4 and IL-10. . This suggests that ICOS is particularly important for various effector functions in activated T cells. In fact, in mice that knocked out ICOS, T cell proliferation and IL-4 production ability by antigen stimulation disappear (Non-patent Document 2).

[0003] B7RP-1, identified as a ligand for ICOS, belongs to the family of B7 molecules (CD80, CD86) that are ligands for CD28, another costimulatory molecule, and is stimulated by B cells, macrophages, and LPS. It is a membrane-bound protein expressed in non-immune cells (Non-patent Document 3). B7 RP-1 is the only currently known ligand for ICOS and does not show cross-linking with CD28 or CTLA4 (CD154) under physiological conditions. The binding between ICOS and B7RP-1 is very important for the helper function of T cells. In ICOS knockout mice, the antibody class switch due to the helper function is remarkably suppressed (Non-patent Document 2). However, it is known that this suppression is partially recovered by B cell stimulation from CD40 using an anti-CD40 antibody (Non-patent Document 4). [0004] Pre-treatment of signal block between ICOS / B7RP-1 with anti-ICOS inhibitory antibody or ICOS-Fc in mouse tracheal inflammation model experiment by inhalation of OVA using mice immunized with ovalbumin (OVA) In mice, invasion of lymphocytes and neutrophils in bronchoalveolar lavage (BAL) was reduced, and production of cytoplasmic ins such as IL-4 and IL-10 was also suppressed. Such suppression was also observed by blocking the signal between CD28 / B7 by CTLA4-FC, but in particular, the pattern of IL-4 production was significantly different with the timing of administration of these inhibitors . Because of this difference in the roles of costimulatory CD28 / B7 and ICOS / B 7RP-1, the former plays an important role in the initiation of T cell activity, whereas the latter It has been shown that it plays a major role in controlling the effector function after activation (Non-patent Document 5).

[0005] It has been clarified that the cause of human unclassifiable immunodeficiency (CVID) is a gene deficiency caused by ICOS homozygote. This indicates the importance of ICOS in helper T cell function leading to maturation and antibody production of human B cells, and strongly suggests that ICOS is a causative gene of various immune diseases (non-) Patent Document 6).

[0006] In allogeneic heart transplantation using mice, administration of an anti-ICOS inhibitor antibody only delayed CD4, CD8-positive T cells, and macrophages into transplanted fragments that only delayed acute rejection. Infiltration was also suppressed. Such an effect by this anti-ICOS inhibitory antibody was used as an immunosuppressive agent, showed an immunosuppressive effect synergistic with cyclosporin A, and delayed the time until rejection (Non-patent Document 7). Inhibition of CD28 / B7 signal by CTLA4-Ig and protein inhibition of CD40 / CD40 ligand (CD154) by anti-CD40 ligand antibody as a protein preparation for immunosuppression ICOS / B7RP The suppression effect due to the inhibition between -1 has the same or much better results

[0007] In an experiment with mice using the experimental allergic encephalomyelitis (EAE) model, administration of an anti-ICOS inhibitor antibody could prolong the time to onset of EAE. However, administration of anti-ICOS inhibitor antibody at the same time as antigen peptide sensitization for EAE induction resulted in a dramatic exacerbation of the symptoms (non-patented Reference 8). This suggests that inhibition of signals with anti-ICOS antibodies in clinical practice has a risk of exacerbating allergic symptoms.

[0008] On the other hand, there have been few reports on the use of monoclonal antibodies against B7RP-1, but rat monoclonal antibodies against mouse B7RP-1 have not been successfully used in mouse collagen-induced arthritis (CIA). ) Has been reported to have an effect by therapeutic administration, suggesting that the signal between ICOS / B7RP-1 also plays an important role in the pathogenesis of autoimmune diseases, (Non-patent document 9).

[0009] Non-Patent Document 1: Hutloff, A. et al. (1999) Nature, 397 (6716), p.263-266

Non-Patent Document 2: Dong, C. et al. (2001) Nature, 409 (6816), p.97-101

Non-Patent Document 3: Yoshinaga, S. K. et al. (1999) Nature, 402 (6763), p.827-832

Non-Patent Document 4: McAdam, A. J. et al. (2001) Nature, 409 (6816), p.102-105

Non-Patent Document 5: Gonzalo, J. A. et al. (2001) Nat. Immunol, 2 (7), p.597-604

Non-Patent Document 6: Grimbacher, B. et al. (2003) Nat. Immunol, 4 (3), p.261-268

Non-Patent Document 7: Ozkaynak, E. et al. (2001) Nat. Immunol, 2 (7), p.591-596

Non-Patent Document 8: Rottman, J.B., et al. (2001) Nat. Immunol, 2 (7), p.605-611

Non-Patent Document 9: Iwai, H. et al. (2002) J. Immunol, 169 (8), p.4332- 4339

Disclosure of the invention

Problems to be solved by the invention

[0010] T cell proliferation, infiltration into the affected area, and effector function are critically involved in exacerbation of symptoms of immune abnormal diseases such as organ transplant rejection and allergies. Drugs that inhibit or control the signal between ICOS / B7RP-1 involved in such functions are expected as therapeutic agents for immune abnormalities such as immunosuppression and allergy during organ transplantation.

[0011] If a specific monoclonal antibody that blocks the signal between ICOS / B7RP-1 can be developed, it will be an effective treatment for many diseases that develop and exacerbate due to the signal from ICOS / B7RP-1. There is expected.

[0012] Anti-ICOS antibody and anti-B7RP-1 antibody are considered as antibodies that block the auxiliary signal of ICOS / B7RP-1. The antigen-binding portion of an antibody is bivalent, and therefore the antibody and target molecule There is a possibility that a cell is signaled by binding, and in particular, in the case of an anti-ICOS antibody, the administration for the purpose of inhibiting the signal of ICOS also reverses the possibility of promoting the proliferation of T cells by sending a signal in reverse. Can not. In this sense, inhibition of the signal between ICOS / B7RP-1 by anti-B7RP-1 antibody is unlikely to promote T cell proliferation activity via ICOS. It is known that the expression of B7RP-1 is decreased by ICOS / B7RP-1 signal even if the signal is sent to B cell by binding of anti-B7RP-1 antibody to B7RP-1 on B cell. Therefore, the anti-B7R P-1 antibody is considered to be superior as an inhibitory antibody because it works to suppress the effector function of T cells.

[0013] Currently, there are only a few monoclonal antibodies derived from mice and rats as antibodies against human B7RP-1 (anti-human B7RP-1 antibodies). Such conventional anti-human B7 RP-1 antibodies are derived from non-humans and are therefore recognized and excluded as foreign substances when administered to humans due to their high immunogenicity. Therefore, it is difficult to use conventional anti-human B7RP-1 antibody as a therapeutic agent for diseases.

[0014] As a method for solving this problem, a mouse monoclonal antibody against human B7RP-1 is considered to be humanized using protein engineering techniques. Administration or long-term administration can produce antibodies that inhibit the activity of the humanized anti-B7RP-1 antibody to be administered, and may cause serious side effects that are not only significantly attenuated. In addition, a great deal of labor and cost are required for construction in which activity is often reduced by humanization.

[0015] The present invention provides a human anti-human B7RP-1 antibody and a fragment thereof having both safety and therapeutic effects, and proposes a method for using them.

Means for solving the problem

[0016] As a result of studying the above problems, the present inventor expressed genes encoding immunoglobulin H chain and L chain variable regions (VH, VL) prepared from peripheral blood B lymphocytes of healthy individuals. An anti-human B7RP-1 single-chain Fv antibody molecule (antibody fragment) was obtained from a human single-chain Fv antibody phage display library, and its amino acid sequence and the base sequence of the gene encoding it were revealed. Furthermore, the present inventors have found that this single chain Fv antibody inhibits the physiological activity of human B7RP-1, and have completed the present invention. [0017] The antibody according to the present invention has been isolated using an antibody phage library derived from a human antibody gene, and these antibodies have completely human-derived sequences, and as they are for use in human therapy. Even if it is used, there is no problem as immunogenicity. In that sense, the conventional mouse monoclonal antibody strength has also been resolved with respect to the immunogenicity of mouse-derived sequences that are feared in humanized antibodies produced by humanization technology. In addition, its development cost is very low compared to humanized antibodies. On the other hand, in recent years, human antibodies have been produced by transchromosome mice having human chromosomes. However, the technology for producing human antibodies having an inhibitory action on human molecules is completely animal-like as in the present invention. The fact that it can be manufactured without using is a great advantage.

That is, the present invention includes the following 1) to 29) as medically or industrially useful methods' substances.

[0019] 1) A human anti-human B7RP-1 antibody having binding property to human B7 Related Protein 1 (hereinafter referred to as B7RP-1).

[0020] 2) The complementarity determining region (CDR) of the H chain has the following amino acid sequence (a) or (b), and the complementarity determining region (CDR) of the L chain is the following (c) or ( The human anti-human B7RP-1 antibody according to 1) having the amino acid sequence of d):

(a) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 as CDR1, any one of SEQ ID NOs: 4 to 6 as CDR2, and any one of SEQ ID NOs: 7 to 9 as CDR3;

(b) as the amino acid sequence of CDR1-3, SEQ ID NO: 1-3, 4-6, or 7-9, respectively, or one or more amino acids in these amino acid sequences are substituted, deleted, inserted, and / or An added amino acid sequence that can serve as a complementarity determining region of the heavy chain to human B7RP-1;

(c) the amino acid sequence represented by any one of SEQ ID NOs: 10 to 12 as CDR1, any one of SEQ ID NOs: 13 to 15 as CDR2, and any one of SEQ ID NOs: 16 to 18 as CDR3;

(d) As the amino acid sequences of CDR1 to CDR3, SEQ ID NOs: 10 to 12, 13 to 15, or 16 to 18, or one or more amino acids in these amino acid sequences are substituted or missing, respectively. A deleted, inserted, and Z or added amino acid sequence that can serve as the complementarity determining region of the light chain for human B7RP-1.

[0021] 3) Amino acid sequence power of CDRs 1 to 3 of H chain SEQ ID NO: 1, 4 and 7, SEQ ID NO: 2, 5 and 8, or combinatorial power of SEQ ID NOs: 3, 6 and 9 Amino acid sequence power of CDRs 1 to 3 of the light chain SEQ ID NO: 10, 13 and 16, SEQ ID NO: 11, 14 and 17, or SEQ ID NO: 12, 15 and 18 is also an amino acid sequence in which the combinatorial power is also selected 1) Or a human anti-human B7RP-1 antibody according to 2).

[0022] 4) Amino acid sequence ability of the combination of CDRs 1 to 3 of the H chain and CDRs 1 to 3 of the L chain SEQ ID NOS: 1, 4 and 7 and SEQ ID NOS: 10, 13 and 16, SEQ ID NOS: 2, 5 and 8 The human anti-human B7RP-1 antibody according to 3) above, which is any one of SEQ ID NOs: 11, 14 and 17, or SEQ ID NOs: 3, 6 and 9 and SEQ ID NOs: 12, 15 and 18.

[0023] 5) The heavy chain variable region has the following amino acid sequence (e) or (f), and the light chain variable region has the following amino acid sequence (g) or (h): The human anti-human B7RP-1 antibody described in any one of 4)

(e) the amino acid sequence ability of any one of SEQ ID NOS: 19 to 21; selected amino acid sequence;

(f) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and Z or added in the amino acid sequence shown in SEQ ID NOs: 19 to 21, and the heavy chain variable region for human B7RP-1 Can be;

(g) SEQ ID NOS: 22 to 24 !, any one amino acid sequence ability Selected amino acid sequence;

(h) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and Z or added in the amino acid sequences shown in SEQ ID NOs: 22 to 24, and the light chain variable region for human B7RP-1 What can be.

[0024] 6) Amino acid sequence ability of the combination of the H chain variable region and the L chain variable region SEQ ID NO: 19 and SEQ ID NO: 22, SEQ ID NO: 20 and SEQ ID NO: 23, or SEQ ID NO: 21 and SEQ ID NO: 24 The human anti-human B7RP-1 antibody according to any one of 1) to 5) above, which is an amino acid sequence represented by:

[0025] 7) A heavy chain variable region fragment of a human anti-human B7RP-1 antibody having binding ability to human B7RP-1.

[0026] 8) The complementarity determining region (CDR) has the following amino acid sequence (a) or (b): H chain variable region fragment of human anti-human B7RP-1 antibody according to:

(b) as the amino acid sequence of CDR1-3, SEQ ID NO: 1-3, 4-6, or 7-9, respectively, or one or more amino acids in these amino acid sequences are substituted, deleted, inserted, and / or An added amino acid sequence that can serve as a complementarity-determining region of H chain for human B7RP-1.

[0027] 9) Amino acid sequence ability of CDR1 to CDR3, wherein the combination ability of SEQ ID NOs: 1, 4, and 7, SEQ ID NOs: 2, 5, and 8, or SEQ ID NOs: 3, 6, and 9 is also selected. A heavy chain variable region fragment of the human anti-human B7RP-1 antibody according to 8).

[0028] 10) The heavy chain variable region fragment of the human anti-human B7RP-1 antibody according to any one of 7) and 9) having the following amino acid sequence (e) or (f):

(f) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and Z or added in the amino acid sequence shown in SEQ ID NOs: 19 to 21, and the heavy chain variable region for human B7RP-1 What can be.

[0029] 11) Human anti-human B7RP-1 antibody L chain variable region fragment having binding to human B7RP-1

[0030] 12) The light chain variable region fragment of the human anti-human B7RP-1 antibody according to 11) above, wherein the complementarity determining region (CDR) has the following amino acid sequence (c) or (d):

(c) The amino acid sequence shown by any one of SEQ ID NOs: 10 to 12 as CDR1, any one of SEQ ID NOs: 13 to 15 as CDR2, and any one of SEQ ID NOs: 16 to 18 as CDR3 ;

(d) As the amino acid sequences of CDR1 to CDR3, any one of SEQ ID NOs: 10 to 18, 13 to 15, or 16 to 18, or one or more amino acids in these amino acid sequences are substituted. , Deleted, inserted, and / or added amino acid sequence that can serve as the complementarity determining region of the L chain of human anti-human B7RP-1 antibody. [0031] 13) The above, wherein the amino acid sequences of CDR1 to CDR3 are amino acid sequences in which SEQ ID NOs: 10, 13, and 16, SEQ ID NOs: 11, 14, and 17, or the combination powers of SEQ ID NOs: 12, 15, and 18 are also selected. A light chain variable region fragment of the human anti-human B7RP-1 antibody according to 12).

[0032] 14) The light chain variable region fragment of the human anti-human B7RP-1 antibody according to any one of 11) and 13) having the following amino acid sequence (g) or (h):

[0033] 15) The heavy chain variable region fragment of the human anti-human B7RP-1 antibody according to any one of 7) and 7) above, and the human anti-human B7RP- A single-chain variable region fragment of a human-derived antibody against human B7RP-1, which is formed by linking one antibody L-chain variable region fragment.

[0034] 16) From the above 7) to 10) !, the human anti-human B7RP-1 antibody heavy chain variable region fragment described in any one of the above, and Z or the human described in any of 11) above force 14) A human-derived antibody against B7RP-1 or an antibody fragment thereof, comprising a human-derived antibody constant region linked to an L-chain variable region fragment of an anti-human B7RP-1 antibody.

[0035] 17) The above antibody fragment power Fab, Fab ', F (ab'), scAb, or scFv-Fc

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The antibody fragment according to 16).

[0036] 18) A fusion antibody or fragment thereof obtained by fusing the antibody or fragment thereof according to any one of 1) to 17) above with a peptide or another protein.

[0037] 19) A modified antibody or fragment thereof obtained by binding a modifying agent to the antibody or fusion antibody according to any one of 1) to 18) above or a fragment thereof.

[0038] 20) A gene encoding the antibody or fusion antibody or fragment thereof according to any one of 1) to 19) above.

[0039] 21) A recombinant expression vector comprising the gene according to 20) above.

[0040] 22) A transformant into which the gene according to 20) above is introduced.

[0041] 23) A human anti-B7RP-1 antibody by expressing the gene described in 20) above in a host Or a method of producing fragments thereof.

[0042] 24) The antibody or fragment thereof described in any one of 1) to 17) above, the fusion antibody or fragment thereof described in 18) above, or the modified antibody or fragment thereof described in 19) above B7RP-1 detection reagent used.

[0043] 25) A gene therapy agent comprising the gene according to 20) above.

[0044] 26) The antibody or fragment thereof described in any one of 1) to 17) above, the fusion antibody or fragment thereof described in 18) above, or the modified antibody or fragment thereof described in 19) above Used binding inhibitor of B7RP-1 and ICOS.

[0045] 27) The antibody or fragment thereof described in any one of 1) to 17) above, the fusion antibody or fragment thereof described in 18) above, or the modified antibody or fragment thereof described in 19) above Recognized A low molecular weight compound or its derivative designed based on the antigenic determinant region on B7RP-1.

[0046] 28) A binding activity inhibitor of B7RP-1 and ICOS using the low molecular weight compound or the derivative thereof according to 27) above.

[0047] 29) A preventive or therapeutic agent for inflammation and immune abnormal diseases caused by the interaction between B7RP-1 and ICOS, using the binding activity inhibitor described in 26) or 28) above. The invention's effect

[0048] The human monoclonal antibody and the antibody fragment molecule of the present invention have the variable region of a human-derived anti-human B7 RP-1 antibody, and react strongly with human B7RP-1 to inhibit its interaction with ICOS. To do. From this, the antibody and the antibody fragment of the present invention can be used as a preventive or therapeutic agent for inflammation and immune abnormal diseases caused by the binding of B7RP-1 and ICOS.

Brief Description of Drawings

[0049] FIG. 1 is a diagram showing the results of an ELISA for evaluating the binding specificity of a cloned single-chain Fv antibody phage to B7RP-1-FC.

[0050] [Fig.2] ELI evaluated for binding specificity of cloned single-chain Fv antibody to B7RP-1-FC

The figure which shows the result of SA.

[0051] [FIG. 3] The isolated anti-B7RP-1 single chain Fv antibody and B7RP1 immobilized on the sensor chip. The figure which shows the result of having evaluated the kinetic analysis of coupling | bonding by the surface plasmon resonance.

[0052] FIG. 4 is a diagram showing the results of an ELISA for evaluating the binding inhibitory activity between ICOS-Fc and B7RP-1-FC by an isolated anti-B7RP-1 single chain Fv antibody.

[0053] [FIG. 5] A diagram showing the results of evaluating the binding of an isolated anti-B7RP-1 single-chain Fv antibody to B cells using a flow cytometer (the group stained with FITC and PE is boxed) Is indicated).

[0054] FIG. 6 is a graph showing the results of evaluating T cell proliferation inhibitory activity by blocking ICOS costimulatory signals using an isolated anti-B7RP-1 single chain Fv antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

[0055] The scFv used for the antibodies and antibody fragments of the present invention was obtained as follows.

[0056] From the peripheral blood B lymphocytes of 20 healthy individuals, RT-PCR method was used to amplify immunoglobulin heavy (H) chain and light (L) chain cDNAs, and then bind them together with linker DNA. The scFv DNA was prepared by random combination of the heavy chain variable region (VH chain or VH) and the light chain variable region (VL chain or VL) derived from healthy lymphocytes.

[0057] The scFv DNA was incorporated into a phagemid vector pCANTAB5E to prepare a healthy person-derived scFv display phage library consisting of 10 ⁹ clones. This library was recovered by binding to human B7RP-1 immobilized on a solid phase, concentrated and screened for anti-human B7RP-1 scFv display phage clones. As a result, each screened screen produced scFv that binds to human B7RP-1.

[0058] As an expression method of scFv, for example, it can be expressed in E. coli. In the case of Escherichia coli, it can be expressed by functionally binding scFv to be expressed such as a commonly used useful promoter and a signal sequence for antibody secretion. Examples of promoters include lacZ promoter and araB promoter. As a signal sequence for secretion of scFv, a pelB signal sequence (Lei, SP. et al., J. BacterioL, 1987, 169: 4379-4383) is preferably used when expressed in the periplasm of E. coli. For secretion into the culture supernatant, the signal sequence of the g13 protein of M13 phage can also be used.

[0059] The expressed scFv can be purified to homogeneity by separating the inside and outside of the cell and the host force. Book Since the scFv expressed in the invention has an E tag sequence added to its C-terminus, it can be easily purified in a short time using affinity chromatography using an anti-E tag antibody. In addition, it can be purified by a combination of separation and purification methods used in normal proteins. For example, antibodies can be separated and purified by combining column chromatography such as ultrafiltration, salting out, gel filtration, Z ion exchange, and Z hydrophobic chromatography.

[0060] Methods for measuring the binding activity of the obtained antibody or antibody fragment to human B7RP-1 include methods such as ELISA and BIAcore. For example, when ELISA is used, a sample containing the target antibody or antibody fragment, for example, E. coli culture supernatant or purified antibody, is added to a 96-well plate on which human B7RP-1-Fc is immobilized. Next, a secondary antibody labeled with an enzyme such as peroxidase is added, the plate is incubated and washed, and then the chromogenic substrate TMBZ is added and the absorbance is measured to evaluate the antigen-binding activity. In addition, when using BIAcore, the binding dissociation constant of the target sample can be obtained by immobilizing B7-RP1-FC on the sensor chip or capturing B7-RP1-FC on the anti-human Fc F (ab ′) antibody. Measure

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be able to.

[0061] As a method for measuring the B7RP-1 / ICOS binding inhibitory activity of the obtained antibody or antibody fragment, there are methods such as ELISA and BIAcore. For example, when using ELISA, prepare a sample containing the target antibody or antibody fragment in a 96-well plate with human B7R-1-Fc immobilized and a mixture of biotin-labeled ICOS-Fc. Next, add streptavidin labeled with an enzyme such as peroxidase, incubate and wash the plate, then add chromogenic substrate TMBZ and measure the absorbance to evaluate B7RP-1 / ICOS binding inhibitory activity. be able to.

[0062] Further, as a method for examining the binding of the obtained antibody or antibody fragment to B7RP-1 on peripheral blood B lymphocytes, there is a method using a flow cytometer. For example, when analyzing human peripheral blood lymphocytes with a fluorescent flow cytometer, peripheral blood lymphocytes are purified from human peripheral blood, stimulated with PMA and PHA, and then the HgG antibody is added to the HgG Fe. Block the y-receptor, and add the sample containing the target antibody or antibody fragment (if scFv with E tag is added, the mixture of the sample containing scFv and the anti-E tag antibody) to react. cell After washing, PE-labeled streptavidin and FITC-labeled anti-CD19 antibody as a human B cell-specific marker are reacted and fluorescently labeled. After washing, a two-dimensional flow cytometric analysis of PE and FITC channels can be performed using a fluorescent flow cytometer to evaluate the binding to B7RP-1 on peripheral blood B lymphocytes.

[0063] As a method for examining the inhibitory activity against the T cell co-stimulation signal for the above-mentioned antibodies and antibody fragments, there is a T cell proliferation stimulation assay. For example, after coating a 96-well plate with a mixture of anti-CD3 antibody and anti-HgG Fc fragment F (ab ') antibody,

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7RP-1-FC is added and reacted. After washing the plate again, a sample containing the target antibody or antibody fragment is added and reacted, and then peripheral blood lymphocytes prepared from human peripheral blood are added and cultured. By adding tritium thymidine during the culture and measuring the amount of tritium thymidine taken up by the cells, the inhibitory activity against T cell costimulatory signals can be evaluated.

[0064] As a result of the separation and evaluation of anti-human B7RP-1 scFv by the above method, it specifically binds to B7RP-1, and its affinity is high enough to be comparable to the receptor ICOS. T7 cells can bind to B7RP-1 expressed on antigen-presenting cells, inhibit the binding of B7RP-1 to ICOS, and can stimulate T cells by co-stimulation via B7RP-1 / ICOS. It has been shown to suppress growth. Therefore, these antibodies have similar effects in vivo, and are considered useful as drugs that inhibit B7RP-1 / ICOS binding and T cell proliferation.

[0065] The amino acid sequences of the VH and VL chains of the three types of scFv (223, 323, 325) having the inhibitory activity and the base sequences encoding them are as follows.

(1) Clone 223

The amino acid sequence of the VH chain of clone 223 is shown in SEQ ID NO: 19. The amino acid sequences of CDR1 to CDR3 of the VH chain are shown in SEQ ID NOs: 1, 4 and 7. That is, in the amino acid sequence of the VH chain shown in SEQ ID NO: 19, the 31st to 35th amino acid sequences are CDR1 (SEQ ID NO: 1), the 50th to 66th amino acid sequences are CDR2 (SEQ ID NO: 4), 99th to The 109th amino acid sequence corresponds to CDR3 (SEQ ID NO: 7). The base sequence of the gene encoding the VH chain is shown in SEQ ID NO: 25. [0066] The amino acid sequence of the VL chain of clone 223 is shown in SEQ ID NO: 22. The amino acid sequences of CDR1-3 of the VL chain are shown in SEQ ID NOs: 10, 13, and 16. That is, in the amino acid sequence of the VL chain shown in SEQ ID NO: 22, the 24th to 35th amino acid sequence is CDR1 (SEQ ID NO: 10), the 51st to 57th amino acid sequence is CDR2 (SEQ ID NO: 13), and the 90th to 98th. The second amino acid sequence corresponds to CDR3 (SEQ ID NO: 16). The base sequence of the gene encoding the VL chain is shown in SEQ ID NO: 28.

[0067] (2) Clone 323

The amino acid sequence of the VH chain of clone 323 is shown in SEQ ID NO: 20. The amino acid sequences of CDR1 to CDR3 of the VH chain are shown in SEQ ID NOs: 2, 5, and 8. That is, in the amino acid sequence of the VH chain shown in SEQ ID NO: 20, the 30th to 34th amino acid sequences are CDR1 (SEQ ID NO: 2), the 49th to 65th amino acid sequences are CDR2 (SEQ ID NO: 5), and the 98th to The 108th amino acid sequence corresponds to CDR3 (SEQ ID NO: 8). The base sequence of the gene encoding the VH chain is shown in SEQ ID NO: 26.

[0068] The amino acid sequence of the VL chain of clone 323 is shown in SEQ ID NO: 23. The amino acid sequences of CDR1-3 of the VL chain are shown in SEQ ID NOs: 11, 14, and 17. That is, in the VL chain amino acid sequence shown in SEQ ID NO: 23, the 23rd to 33rd amino acid sequence is CDR1 (SEQ ID NO: 11), the 49th to 55th amino acid sequence is CDR2 (SEQ ID NO: 14), and the 88th to 98th. The second amino acid sequence corresponds to CDR3 (SEQ ID NO: 17). The base sequence of the gene encoding the VL chain is shown in SEQ ID NO: 29.

[0069] (3) Clone 325

The amino acid sequence of the VH chain of clone 325 is shown in SEQ ID NO: 21. The amino acid sequences of CDR1 to CDR3 of the VH chain are shown in SEQ ID NOs: 3, 9, and 15. That is, in the amino acid sequence of the VH chain shown in SEQ ID NO: 21, the 31st to 35th amino acid sequence is CDR1 (SEQ ID NO: 3), the 50th to 66th amino acid sequence is CDR2 (SEQ ID NO: 9), and the 99th to The 108th amino acid sequence corresponds to CDR3 (SEQ ID NO: 15). The base sequence of the gene encoding the VH chain is shown in SEQ ID NO: 27.

[0070] The amino acid sequence of the VL chain of clone 325 is shown in SEQ ID NO: 24. The amino acid sequences of CDR1-3 of the VL chain are shown in SEQ ID NOs: 12, 15, and 18. Ie, SEQ ID NO In the amino acid sequence of the VL chain shown in 24, the 23rd to 33rd amino acid sequence is CDR1 (SEQ ID NO: 12), the 49th to 55th amino acid sequence is CDR2 (SEQ ID NO: 15), and the 88th to 98th amino acid sequence. Corresponds to CDR3 (SEQ ID NO: 18). The base sequence of the gene encoding the VL chain is shown in SEQ ID NO: 30.

[0071] Based on each amino acid sequence information (VH chain, VL chain, CDR1 to 3) described in the force sequence table described above for the amino acid sequence and base sequence for each clone obtained by the present invention, alone or It is also possible to use a combination of a plurality of sequences as appropriate.

[0072] The antibody of the present invention and the antibody fragment thereof are mutations in which part of the VH chain and VL chain, and their CDRs, are not limited to the sequence shown in the above SEQ ID NO. It may be a polypeptide.

[0073] That is, in the amino acid sequence described in each SEQ ID NO: 1 or more amino acids are substituted, deleted, inserted, and Z or added amino acid sequences, and H for human B7RP-1 Also included are polypeptides that serve as chain or L chain complementarity-determining regions and H or L chain variable regions.

[0074] Here, "one or more amino acids are substituted, deleted, inserted, and Z or added" means substitution or deletion by a known mutant protein production method such as site-directed mutagenesis. It means that as many amino acids as can be deleted, inserted, and Z or added, for example, about 1 to 6 amino acids are substituted, deleted, inserted, and Z or added. Such “mutation” mainly means a mutation artificially introduced by a known method for producing a mutant protein, but a similar mutant polypeptide existing in nature (eg, human) is isolated and purified. There may be.

[0075] The above-mentioned "mutation" means that when the antibody of the present invention or an antibody fragment thereof is used as a therapeutic agent (when administered to a human), it retains a human-derived structure or causes an immune reaction by the human. The range is not particularly limited when it is used within the range where it does not occur and is used as a detection instrument or diagnostic kit (when not administered to humans).

[0076] The VH chain and Z or VL chain disclosed in the present invention are mainly obtained in the form of scFv using the phage antibody method. In principle, the application is limited to scFv. There is no. For example, the disclosed VH chain and Z or VL chain can be Fab, Fab 'or F (ab') combined with part of the constant region of human immunoglobulin, and scFv combined with the constant region of the light chain of human immunoglobulin.

2

Other antibody fragments such as single-chain antibodies (scAb) and scFv-Fc in which scFv is bound to the constant region of the heavy chain of human immunoglobulin are also included in the scope of application.

[0077] The antibody or fragment thereof of the present invention can also be a fusion antibody or fragment thereof fused to the antibody or fragment with a peptide or other protein.

[0078] These antibodies and antibody fragments, or the above-mentioned fusion antibodies or fragments thereof, can also be modified antibodies or fragments thereof in which a polymer modifying agent such as polyethylene glycol is bound.

[0079] When preparing an scFv in which the Fv of the H chain and the L chain are linked with an appropriate linker, for example, any single chain peptide having an amino acid strength of 10 to 25 residues is used as the peptide linker. .

[0080] The antibody or antibody fragment of the present invention is prepared from an appropriate host (for example, based on the gene sequence information encoding the VH chain and VL chain of each clone obtained by the present invention shown in SEQ ID NOs: 25 to 30). The antibody of the present invention or a fragment thereof can be expressed.

[0081] The gene of the present invention can also be used as an adjuvant for gene therapy for regulating the interaction between B7RP-1 and ICOS.

[0082] The antibody of the present invention or an antibody fragment thereof can be used as an inhibitor of binding activity between B7RP-1 and ICOS.

[0083] In addition, the molecular design based on the antigenic determinant region on human B7RP-1 recognized by these antibodies or fragments thereof is important for the development of small molecules that act on B7RP-1 / ICOS signaling. Provide a simple means.

[0084] The low molecular weight compound includes a peptide having an amino acid sequence that can be recognized by the antibody of the present invention or a fragment thereof, and a compound that mimics the three-dimensional structure thereof. A modified peptide obtained by adding an unnatural amino acid to the amino acid sequence of the peptide can also be used. The same applies to fusion proteins obtained by fusing these peptides with other proteins. Above pep The same applies to a modified molecule in which a polymer modifier such as polyethylene glycol is bound to a tide or a compound.

[0085] These low molecular weight compounds or their derivatives can also be used as inhibitors of the binding activity between B7RP-1 and ICOS.

[0086] Furthermore, the binding activity inhibitor comprising the antibody of the present invention or an antibody fragment thereof, and a low molecular weight compound or a derivative thereof is used for the inflammation and immune abnormal diseases caused by the interaction between B7RP-1 and ICOS. It is effective as a preventive or therapeutic agent.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

Example

[0088] <Example 1: Construction of phage library from healthy subjects>

The phage library was constructed using peripheral blood lymphocytes from 20 healthy individuals as a starting material with reference to the method reported by JD Marks et al. (J. Mol. Biol, 222: 581-597, 1991). ,It was constructed. The constructed VH (γ) —V κ, VH (γ) —V, VH () —V κ, VH () V sub-libraries are 1.1 X 10 ⁸ , 2.1 X 10 ⁸ , 8.4 X 10 ^{7, respectively.} It was evaluated as having a diversity of 5.3 x 10 ⁷ clones.

[0089] << Example 2: Panning >>

Human B7RP- 1- Fc lO / z g) is dissolved in 1 ml of 0.1 M NaHCO solution, and Imunotube (Nunc)

Three

It was coated at 4 ° C. After washing once with PBS containing 0.1% Tween 20, it was blocked with 0.5% gelatin ZPBS for 2 hours at room temperature. After washing 6 times with 0.1% TweenZPBS, 1 ml of an antibody phage library derived from a healthy person (single-chain antibody-displayed phage solution, 10 ¹² TUZml) was prepared and reacted at room temperature for 2 hours.

[0090] After removing non-specific phage by washing 20 times with 0.1% Tween20 / PBS, 0.17 glycine containing lmg / ml BSA (usi serum albumin) was added to the phage clone bound to B7RP-1-Fc. -Eluted with HCl (pH 2.2) and immediately neutralized with 1.0 M Tris-HCl (pH 9.1). The neutralized phage solution was added to an immunotube coated with human IgG and blocked with 0.5% gelatin ZPBS, and allowed to react at room temperature for 2 hours. The supernatant was added to E. coli TGI (20 ml) in logarithmic growth phase, left at 30 ° C for 30 minutes, and then partly spread on a SOBAG plate. The medium was changed to 30 ml of 2 XYTAG, and then cultured at 30 ° C. After centrifuging the culture solution at 2200 rpm × 10 minutes, the precipitated Escherichia coli was suspended in 3 ml of 2 XYTAG, and used as the primary pan-Jung coli library. This TG1 solution was planted in 2 XYTAG medium, rescued using helper phage, and a phage library after screening was prepared.

[0091] The above biopanning operation was performed three times in total, and the phage solutions obtained by the secondary and tertiary panning were used as the secondary phage library and the tertiary phage library, respectively. . However, in secondary panning and tertiary panning, 5% skim milk and 0.5% BSA were used as blocking solutions, respectively.

[0092] Clone was arbitrarily extracted from SOBAG plate after secondary panning and tertiary panning, and short chain Fv antibody phage clone was prepared to confirm specificity for B7RP-1-FC and sequence of antibody gene Analysis was performed.

[0093] << Example 3: Screening B7RP-1 ELISA))

Confirmation of the binding specificity of the separated single chain Fv antibody phage by ELISA was carried out by the following method. An ELISA plate (Nunc) was coated with B7RP-1-Fc (80 ng / well) at 4 ° C and blocked with 0.5% gelatin / PBS. After washing with 0.1% Tween20 / PBS, an isolated single-chain Fv phage clone (1 × 10 ¹³ pfo / ml) 40 1 was added and reacted at room temperature for 2 hours. The detection of the binding phage was carried out by reacting with the addition of piotinylated anti-M13 phage antibody (Pharmacia) as the primary antibody and AP labeled streptavidin as the secondary antibody, followed by reaction with the substrate solution (10% 2,2-iminodiethanol) lmg / ml PNP-phosphate in PBS) was measured, and the absorbance at 405 nm was measured with a multiplate auto reader NJ-2001 (Nunc). As a result, it was proved that all clones finally evaluated were specific for the force B7RP-1 (Fig. 1).

[0094] << Example 4: Sequence analysis of clones >>

Determine the DNA sequence of VH and VL of the scFv gene of the isolated clone using the Dye terminate cycle sequencing FS Ready Reaction kit (Applied Biosystems). As a result of E LISA and sequence analysis, the isolated clones were classified into 4 types of scFv.

[Example 5: Expression and purification of human-derived anti-human B7RP-IscFv]

The scFv clone that reacts with human B7RP-1 isolated in Examples 2 and 3 was also recovered from plasmid DNA and transformed into E. coli HB2151 according to a conventional method. 2% glucose and 1 These E. coli cells are pre-cultured overnight in 2 XYT medium containing 00 g / mL ampicillin, and then partially transplanted into dalcose-free 2 ΧΥΤ medium, and final concentration of ImM IPTG and 100 g / mL ampicillin is added. The scFv expression was induced by incubation for a period of time. After completion of the culture, the cells were collected by centrifugation, suspended in PBS containing ImM EDTA, and left on ice for 30 minutes. Subsequently, the mixture was centrifuged at 8,90 OX g for 30 minutes, and the supernatant was filtered through a 0.45 μm filter to obtain a periplasm fraction, which was used as a starting material for scFv purification.

[0096] The purification starting material thus prepared was purified according to a conventional method by affinity chromatography using an anti-E tag antibody. After dialysis with PBS, endotoxin was removed with an endotoxin removal column Detoxi-gel (PIERCE) according to the attached protocol! Concentrated with Centricon (Amicon) with a molecular weight of 10,000, filtered through a 0.45 μm filter to obtain a purified sample. Storage was performed at -20 ° C.

<< Example 6: Binding of purified scFv to human B7RP-1 >>

The measurement of the binding activity of the purified single chain Fv antibody to B7RP-1 was performed by the following method. ELISA plate (Nunc) was coated with B7RP-1-Fc (80 ng / well) at 4 ° C, blocked with 0.5% gelatin / PBS, washed with 0.1% Tween20 / PBS, and isolated A mixture of single chain Fv antibody (g / ml) and anti-E-tag antibody was added and reacted for 2 hours. For detection, AP-labeled anti-mouse IgG Fey antibody as a secondary antibody was added and reacted for 60 minutes, and then a substrate solution was added, and the absorbance at 405 nm was measured with a multiplate auto reader NJ-2001. As a result, all the clones finally evaluated were found to be specific for B7RP-1 (Fig. 2).

[0098] For Clone 223, the binding dissociation constant between the single-chain Fv antibody and B7RP-1 was examined using BIAcore. B7RP-1-Fc, ICOS-Fc, CT LA-4-Fc diluted with 10 mM acetate buffer (pH 4.0) at 20 ° C in 4 flow cells on sensor chip CM5 (Biacore AB, Uppsala, Sweden) , Anti-human Fc F (ab ') antibody (each 10 μg / mL) was immobilized at a flow rate of 5 μL / min.

2

It was. After washing the sensor chip with HBS buffer (lOOmM HEPES / 5M NaCl / 0.5M EDTA / 0.005% Tween20 (Pharmacia a biotech)), react with B 7RP-1-Fc diluted with HBS buffer / 0.01% BSA. Anti-human Fc F (ab ′) antibody was captulated. Continue with each scF

2

V sample (adjusted to 100 nM) is injected and reacted at a flow rate of 10 L / min, binding per sample The dissociation constant was measured. Further, before the step of reacting each sample, scFv was eluted with 200 mM glycine-HC1 buffer (pH 2.2) containing 200 mM NaCl and washed. BIAevaluation software was used for data analysis. As a result, B7RP-1-Fc first reacted only to the chip to which ICOS-Fc and anti-human Fc F (ab ') antibody were immobilized. From the results, B7RP-1 and FOS

2

The bond dissociation constant was about 7 nM. Subsequently, when 233 was reacted, the chip with B7-RPl-Fc immobilized and the anti-human Fc F (ab ') antibody were allowed to capture B7-RPl-Fc.

2

It was estimated that 233 binds to the binding site with ICOS, because it binds only to the chip and binds to ICOS and does not bind to B7-RP and Fc. The binding dissociation constant with B7 RP-1 was calculated to be about 13-15 nM, confirming that it was almost equivalent to the receptor ICOS (Fig. 3).

Example 7: Inhibition of binding between ICOS-Fc and B7RP-1-FC by anti-B7RP-1 single chain Fv antibody

A 96-well plate was coated with B7RP-1-Fc (40 ng / well) at 4 ° C, blocked with 0.5% gelatin / PBS, and washed with 0.1% Tween20 / PBS. A mixture of 125ηΜ to 2M anti-B7RP-1 single chain Fv antibody solution and piotin-labeled ICOS-Fc was added and reacted for 90 minutes. After washing, AP-labeled streptavidin was added and reacted for 30 minutes, and then the substrate solution was added and the absorbance at 405 nm was measured. As a result, clones 223, 323, and 325 were found to inhibit the binding between ICOS and B7RP-1 in a dose-dependent manner with the added single-chain Fv antibody (Fig. 4).

Example 8: Analysis of single-chain Fv antibody binding to B7RP-1 on peripheral blood B lymphocytes by flow cytometry

In order to confirm the binding activity of isolated human anti-B7RP-1 single chain Fv antibody to B7RP-1 molecule expressed on cells, analysis was performed using human peripheral blood lymphocytes using a fluorescence flow cytometer. did. PBMCs were purified from human peripheral blood using a conventional method using ficoll. After stimulation with PMA (5 ng / ml) and PHA (2 / zg / ml) for 41 hours, the human IgG antibody is blocked by covering with human IgG antibody, and single-chain Fv antibody and anti-E tag antibody The mixture was allowed to react for 90 minutes. After washing the cells, they were reacted with streptavidin labeled with PE and anti-CD19 antibody labeled with FITC for 30 minutes as a human B cell-specific marker, and labeled with fluorescence. After cleaning, Coulter EPICS XL (Coulter Miami, FL) flow cytometry Two-dimensional flow cytometry analysis was performed on PE and FITC channels. As a result, it was confirmed that the clone name, 223, 323, 325 [koo! In the long run, it was shown to have binding activity to B7RP-1 on B cells (FIG. 5). Example 9: T cell proliferation inhibitory activity against T cell co-stimulation signal by human anti-B7RP-1 single chain Fv antibody

The inhibitory activity of the obtained single-chain Fv antibody on the transmission of auxiliary signals between B7RP-1 on antigen-presenting cells and ICOS on T cells was analyzed using T cell proliferation activity as an indicator. Anti-CD3 antibody (2 μg / ml) and anti-HgG Fc fragment F (ab ') antibody (2.4 / z g / ml) on 96-well round bottom plate

2

The mixture was coated at 37 ° C for 90 minutes. After the plate was washed with PBS, B7RP-1-Fc (l μg / ml) was added and reacted at 37 ° C. for 2 hours. After washing the plate again, the 1ηΜ to 500ηΜ single-chain Fv antibody and anti-B7RP-1 antibody prepared with RPMI1640 were reacted for 30 minutes, and then peripheral blood lymphocytes (1 X 10 ⁵ cells / well) were added and cultured at 37 ° C. Tritium thymidine (0.5 Ci / well) was added 48 hours later, and the culture was further cultured for 18 hours. The cell DNA was adsorbed on a glass filter, dissolved in a liquid scintillator, and the amount of tritium thymidine in the DNA was measured with a scintillation counter. In all clones, the dose-dependent inhibition of T cell proliferation by single-chain Fv antibody was almost the same as when HCOS-Fc was added, and the IC OS signal of these single-chain Fv clones 223, 323, 325 Inhibitory activity was shown (Fig. 6).

Claims

The scope of the claims

[1] A human anti-human B7RP-1 antibody having binding property to human B7 Related Protein 1 (hereinafter referred to as B7RP-1).

[2] The complementarity determining region (CDR) of the H chain has the following amino acid sequence (a) or (b), and the complementarity determining region (CDR) of the L chain is the following (c) or (d) Having the amino acid sequence of

1. Human anti-human B7RP-1 antibody according to 1:

(d) As the amino acid sequences of CDR1 to CDR3, SEQ ID NOs: 10 to 12, 13 to 15, or 16 to 18, or one or more amino acids in these amino acid sequences are substituted, deleted, inserted, and Z or An added amino acid sequence that can serve as a complementarity determining region of L chain to human B7RP-1.

[3] Amino acid sequence power of CDRs 1 to 3 of H chain SEQ ID NO: 1, 4 and 7, SEQ ID NO: 2, 5, and

8, or the combinatorial force of SEQ ID NOs: 3, 6 and 9 is the amino acid sequence selected, and the amino acid sequence power of CDRs 1-3 of the light chain SEQ ID NOs: 10, 13, and 16, SEQ ID NOs: 11, 14, and 17, The human anti-human B7RP-1 antibody according to claim 1 or 2, wherein the combination power of Nos. 12, 15, and 18 is also selected.

[4] The amino acid sequence of the combination of H chain CDR1-3 and L chain CDR1-3 is SEQ ID NO: 1.

, 4 and 7 and SEQ ID NO: 10, 13 and 16, SEQ ID NO: 2, 5 and 8 and SEQ ID NO: 11, 14 and 17, or SEQ ID NO: 3, 6 and 9 and SEQ ID NO: 12, 15 and 18 The human anti-human B7RP-1 antibody according to claim 3, which is a combination of the two.

[5] The heavy chain variable region has the following amino acid sequence (e) or (f), and the light chain variable region has the following amino acid sequence (g) or (h): Any of the human anti-human B7RP-1 antibodies described in:

[6] Amino acid sequence ability of the combination of the H chain variable region and the L chain variable region SEQ ID NO: 19 and SEQ ID NO: 22, SEQ ID NO: 20 and SEQ ID NO: 23, or SEQ ID NO: 21 and SEQ ID NO: 24 6. The human anti-human B7RP-1 antibody according to any one of claims 1 to 5, which has an amino acid sequence.

[7] A heavy chain variable region fragment of a human anti-human B7RP-1 antibody having binding ability to human B7RP-1.

[8] The heavy chain variable region fragment of the human anti-human B7RP-1 antibody according to claim 7, wherein the complementarity determining region (CDR) has the following amino acid sequence (a) or (b):

[9] Amino acid sequence power of CDRs 1 to 3 SEQ ID NO: 1, 4, and 7, SEQ ID NO: 2, 5, and 8, or combination power of SEQ ID NOs: 3, 6, and 9 A heavy chain variable region fragment of the described human anti-human B7RP-1 antibody.

[10] The heavy chain variable region fragment of a human anti-human B7RP-1 antibody according to any one of claims 7 to 9, which has the following amino acid sequence (e) or (f):

[11] A light chain variable region fragment of a human anti-human B7RP-1 antibody capable of binding to human B7RP-1.

[12] The light chain variable region fragment of the human anti-human B7RP-1 antibody according to claim 11, wherein the complementarity determining region (CDR) has the following amino acid sequence (c) or (d):

(d) As the amino acid sequences of CDR1 to CDR3, any one of SEQ ID NOs: 10 to 18, 13 to 15, or 16 to 18, or one or more amino acids in these amino acid sequences are substituted. , Deleted, inserted, and / or added amino acid sequence that can serve as the complementarity determining region of the L chain of human anti-human B7RP-1 antibody.

[13] Amino acid sequence power of CDRs 1 to 3 SEQ ID NO: 10, 13 and 16, SEQ ID NO: 11, 14 and 17, or SEQ ID NO: 12, 15 and 18 combinatorial power A light chain variable region fragment of the described human anti-human B7RP-1 antibody.

[14] The light chain variable region fragment of the human anti-human B7RP-1 antibody according to any one of claims 11 to 13, which has the following amino acid sequence (g) or (h):

[15] The heavy chain variable region fragment of the human anti-human B7RP-1 antibody according to any one of claims 7 to 10 and the L of the human anti-human B7RP-1 antibody according to any of claims 11 to 14. Single-chain variable region of a human-derived antibody against human B7RP-1, formed by linking a chain variable region fragment Fragment.

[16] The heavy chain variable region fragment of the human anti-human B7RP-1 antibody according to any one of claims 7 to 10, and the human anti-human B7RP-1 antibody according to any one of Z or claim 11 to 14. A human-derived antibody against human B7RP-1 or an antibody fragment thereof, comprising a human-derived antibody constant region linked to the L chain variable region fragment of.

[17] The antibody fragment is Fab, Fab ′, F (ab ′), scAb, or scFv-Fc.

2

The antibody fragment according to 6.

[18] A fusion antibody or fragment thereof obtained by fusing the antibody or fragment thereof according to any one of claims 1 to 17 with a peptide or another protein.

[19] A modified antibody or fragment thereof, wherein a modifying agent is bound to the antibody or fusion antibody or fragment thereof according to any one of claims 1 to 18.

[20] A gene encoding the antibody or fusion antibody or fragment thereof according to any one of claims 1 to 19.

[21] A recombinant expression vector comprising the gene according to claim 20.

[22] A transformant into which the gene according to claim 20 has been introduced.

[23] A method for producing a human anti-B7RP-1 antibody or a fragment thereof by expressing the gene according to claim 20 in a host.

[24] B7RP- using the antibody or fragment thereof according to any one of claims 1 to 17, or the fusion antibody or fragment thereof according to claim 18, or the modified antibody or fragment thereof according to claim 19. 1 detection reagent.

[25] A gene therapy agent comprising the gene according to claim 20.

[26] B7RP- using the antibody or fragment thereof according to any one of claims 1 to 17, or the fusion antibody or fragment thereof according to claim 18, or the modified antibody or fragment thereof according to claim 19. Inhibitor of binding activity between 1 and ICOS.

[27] The B7RP- recognized by the antibody or fragment thereof according to any one of claims 1 to 17, or the fusion antibody or fragment thereof according to claim 18, or the modified antibody or fragment thereof according to claim 19. A low molecular weight compound or its derivative designed based on the antigenic determinant region above 1. A binding activity inhibitor of B7RP-1 and ICOS using the low molecular weight compound or derivative thereof according to claim 27.

29. A prophylactic or therapeutic agent for inflammation and immune abnormalities caused by the interaction between B7RP-1 and ICOS using the binding activity inhibitor according to claim 26 or claim 28.