JPWO2006003999A1 - Human anti-human B7RP-1 antibody and antibody fragment thereof - Google Patents

Abstract

  Provided are a human anti-human B7 Related Protein 1 (hereinafter, B7RP-1) antibody, the antibody fragment, and a method of using the same. By using the phage antibody method, a human anti-human B7RP-1 antibody having high affinity for human B7RP-1 and the antibody fragment thereof were obtained. The obtained antibody and the antibody fragment are expected as therapeutic agents for inflammation caused by B7RP-1 and immunological disorders.

Description

  The present invention relates to a human anti-human B7RP-1 antibody or an antibody fragment thereof that binds to human B7 Related Protein 1 (also known as B7h, GL50, ICOS ligand, hereinafter referred to as “B7RP-1”) and inhibits its biological activity. Regarding The antibody and the antibody fragment suppress the excessive activation of T cells caused by the binding of B7RP-1 to ICOS (Inducible costimulator), which is a receptor on T cells, and thus immunity at the time of organ transplantation. It is expected to be used as a suppressive agent and a therapeutic agent for immune disorders such as allergies and autoimmunity.

  ICOS was discovered as the third molecule of the co-stimulatory receptor CD28 family of T cells, and is a membrane-bound protein that forms a homodimer. By stimulating T cells with a signal via TCR, ICOS is present on the surface of T cells. Induced (Non-Patent Document 1). Co-stimulation via this molecule increases the proliferation of both CD4 and CD8 T cells, induces the secretion of cytokines such as IL-4 and IL-10, and induces the expression of T154 CD154 (CD40 ligand). From this, it is considered that ICOS is particularly important in various effector functions in activated T cells. In fact, in mice knocking out ICOS, T cell proliferation and IL-4 producing ability upon antigen stimulation disappear (Non-patent Document 2).

  B7RP-1, which was identified as a ligand for ICOS, belongs to the family of B7 molecules (CD80, CD86) that are ligands for CD28, another co-stimulatory molecule, and is non-immunized stimulated by B cells, macrophages, LPS, etc. It is a membrane-bound protein expressed in cells (Non-Patent Document 3). B7RP-1 is the only currently known ligand for ICOS, and under physiological conditions, does not show cross-linking properties with CD28 and CTLA4 (CD154). The binding between ICOS and B7RP-1 is very important for the helper function of T cells, and in ICOS knockout mice, antibody class switching by the helper function is significantly suppressed (Non-patent Document 2). However, it is known that this suppression is partially restored by B cell stimulation from CD40 using an anti-CD40 antibody (Non-patent Document 4).

  In a mouse bronchial inflammation model experiment by inhalation of OVA using a mouse immunized with ovalbumin (OVA), in a mouse pretreated with a signal block between ICOS/B7RP-1 by anti-ICOS inhibitory antibody or ICOS-Fc, Invasion of lymphocytes and neutrophils in alveolar lavage (BAL) was reduced, and production of cytokines such as IL-4 and IL-10 was suppressed. Such suppression was also seen by blocking the signal between CD28/B7 by CTLA4-Fc, but in particular, the pattern of IL-4 production was remarkably different with the administration timing of these inhibitors. .. From this, the difference between the roles of costimulatory CD28/B7 and ICOS/B7RP-1 is that the former plays an important role in the initiation of T cell activation, whereas the latter controls the effector function after activation. It has been shown to play a major role in doing so (Non-Patent Document 5).

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

  In cardiac allograft transplantation in mice, administration of anti-ICOS inhibitory antibody not only delays acute rejection but also suppresses infiltration of CD4, CD8-positive T cells and macrophages into transplanted fragments. did. Such an effect of this anti-ICOS inhibitory antibody showed a synergistic immunosuppressive effect with cyclosporin A used as an immunosuppressant, and delayed the time until rejection (Non-patent Document 7). As protein preparations for immunosuppression, inhibition of CD28/B7 signal by CTLA4-Ig and inhibition of CD40/CD40 ligand (CD154) signal by anti-CD40 ligand antibody have been investigated. The inhibitory effect due to inhibition between B7RP-1 is equivalent to or significantly superior to those.

  In a mouse experiment using an experimental allergic encephalomyelitis (EAE) model, administration of an anti-ICOS inhibitory antibody could prolong the time until the onset of EAE. However, administration of the anti-ICOS inhibitory antibody at the same time as the sensitization with the antigen peptide for EAE induction resulted in a dramatic increase in symptoms (Non-Patent Document 8). This suggests that clinically, inhibition of signals with anti-ICOS antibody poses a risk of exacerbating allergic symptoms and the like.

  On the other hand, the use of a monoclonal antibody against B7RP-1 has not been reported so far, but a rat monoclonal antibody against mouse B7RP-1 against collagen type II-induced arthritis (CIA) in mice. It has been reported that therapeutic administration showed an effect, and it is suggested that the signal between ICOS/B7RP-1 plays an important contribution to the pathogenesis of autoimmune diseases (Non-Patent Document 9). ..

Hutloff, A. et al. (1999) Nature, 397(6716), p.263-266. Dong, C. et al. (2001) Nature, 409(6816), p.97-101. Yoshinaga, S. K. et al. (1999) Nature, 402(6763), p.827-832. McAdam, A. J. et al. (2001) Nature, 409(6816), p.102-105. Gonzalo, J. A. et al. (2001) Nat. Immunol., 2(7), p. 597-604. Grimbacher, B. et al. (2003) Nat. Immunol., 4(3), p.261-268. Ozkaynak, E. et al. (2001) Nat. Immunol., 2(7), p.591-596. Rottman, J.B. et al. (2001) Nat. Immunol., 2(7), p.605-611. Iwai, H. et al. (2002) J. Immunol., 169(8), p.4332-4339.

  Proliferation of T cells, infiltration into affected areas, and effector function play a decisive role in exacerbation of symptoms of immunological disorders such as rejection of organ transplantation and allergies. A drug that inhibits or controls a signal between ICOS/B7RP-1 involved in such a function is expected as a therapeutic drug for immunosuppressive diseases at the time of organ transplantation, immunological disorders such as allergies.

  If a specific monoclonal antibody that blocks the signal between ICOS/B7RP-1 can be developed, it is expected to be an effective therapeutic means for many diseases caused and exacerbated by the ICOS/B7RP-1 signal. ..

  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 the antibody is divalent, and therefore, there is a possibility that a signal may be introduced into cells by the binding between the antibody and the target molecule. Especially, in the case of anti-ICOS antibody, administration for the purpose of inhibiting the signal of ICOS is possible. On the contrary, the possibility of promoting T cell proliferation by sending a signal cannot be excluded. In that sense, inhibition of the signal between ICOS/B7RP-1 by the anti-B7RP-1 antibody is unlikely to promote T cell proliferation activation 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-B7RP-1 antibody is considered to be excellent as an inhibitory antibody because it acts to suppress the effector function of T cells.

  Currently, as antibodies against human B7RP-1 (anti-human B7RP-1 antibodies), only a few monoclonal antibodies derived from mouse or rat have been obtained. Since such a conventional anti-human B7RP-1 antibody is of non-human origin, it is recognized and eliminated as a foreign substance when administered to human due to its high immunogenicity. Therefore, it is difficult to use the conventional anti-human B7RP-1 antibody as a therapeutic drug for diseases.

  As a method for solving this problem, it is considered that a mouse monoclonal antibody against human B7RP-1 is humanized by using a protein engineering method. Upon administration, an antibody that inhibits the activity of the humanized anti-B7RP-1 antibody to be administered is produced, and not only the effect thereof is significantly attenuated, but also serious side effects may be caused. In addition, humanization often causes a decrease in activity, which requires a great deal of labor and cost for construction.

  The present invention provides a human anti-human B7RP-1 antibody and a fragment thereof having both safety and therapeutic effect, and proposes a method of using them.

  The present inventor, as a result of studying the above-mentioned problems, revealed that a human single chain expressing a gene encoding a variable region (VH, VL) of immunoglobulin H chain and L chain prepared from peripheral blood B lymphocytes of a healthy person. An anti-human B7RP-1 single chain Fv antibody molecule (antibody fragment) was obtained from the Fv antibody phage display library, and its amino acid sequence and the nucleotide sequence of the gene encoding it were clarified. Furthermore, they have found that this single-chain Fv antibody inhibits the physiological activity of human B7RP-1 and completed the present invention.

  The antibody according to the present invention is isolated using an antibody phage library derived from a human antibody gene, and these antibodies have completely human-derived sequences, and can be used as they are for therapeutic use in humans. , It has no problem as immunogenicity. In that sense, the immunogenicity against a mouse-derived sequence which is feared in a humanized antibody prepared by a humanization technique from a conventional mouse monoclonal antibody has been solved. Also, its development cost is much lower than that of humanized antibody. On the other hand, in recent years, human antibodies have been produced by transchromosome mice having human chromosomes, but the technique for producing human antibodies having an inhibitory effect on human molecules is completely different from the use of animals as in the present invention. It is a great merit that it can be manufactured without using it.

  That is, the present invention includes the following inventions 1) to 29) as a method or substance useful medically or industrially.

  1) A human anti-human B7RP-1 antibody having a binding property to human B7 Related Protein 1 (hereinafter, B7RP-1).

2) The complementarity determining region (CDR) of H chain has the amino acid sequence of (a) or (b) below, and the complementarity determining region (CDR) of L chain of the following (c) or (d) The human anti-human B7RP-1 antibody described in 1) above, which has an amino acid sequence:
(A) an amino acid sequence represented by any one of SEQ ID NOS: 1 to 3 as CDR1, 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 sequences of CDR1 to 3, respectively, SEQ ID NOs: 1 to 3, 4 to 6, or 7 to 9, or one or more amino acids in these amino acid sequences are substituted, deleted, inserted, and/or An added amino acid sequence which can be the complementarity determining region of the H chain to human B7RP-1;
(C) the amino acid sequence represented by any one of SEQ ID NOS: 10-12 as CDR1, any one of SEQ ID NOS: 13-15 as CDR2, and any one of SEQ ID NOS: 16-18 as CDR3;
(D) As the amino acid sequences of CDR1 to 3, SEQ ID NOS: 10 to 12, 13 to 15, or 16 to 18, 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 the complementarity determining region of the L chain to human B7RP-1.

  3) The amino acid sequence of CDR1 to 3 of the H chain is an amino acid sequence selected from the combination of SEQ ID NOS: 1, 4 and 7, SEQ ID NOS: 2, 5 and 8, or SEQ ID NOS: 3, 6 and 9, and the L chain. The amino acid sequences of CDR1 to 3 are the amino acid sequences selected from the combination of SEQ ID NOS: 10, 13 and 16, SEQ ID NOS: 11, 14 and 17, or SEQ ID NOS: 12, 15 and 18, respectively. The described human anti-human B7RP-1 antibody.

  4) The amino acid sequences of the combination of CDR 1 to 3 of H chain and CDR 1 to 3 of L chain are SEQ ID NOs: 1, 4 and 7 and SEQ ID NOs: 10, 13 and 16, SEQ ID NOs: 2, 5 and 8 and SEQ ID NO: 11 , 14 and 17, or the combination of any one of SEQ ID NOS: 3, 6 and 9 and SEQ ID NOS: 12, 15 and 18, the human anti-human B7RP-1 antibody according to 3) above.

5) The above 1) to 4), wherein the H chain variable region has the following amino acid sequence (e) or (f) and the L chain variable region has the following amino acid sequence (g) or (h): The human anti-human B7RP-1 antibody described in any of the following:
(E) an amino acid sequence selected from the amino acid sequences of any one of SEQ ID NOs: 19 to 21;
(F) An amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and/or added in the amino acid sequences shown in SEQ ID NOs: 19 to 21, which serves as an H chain variable region for human B7RP-1. Possible things;
(G) an amino acid sequence selected from the amino acid sequences of any one of SEQ ID NOs: 22 to 24;
(H) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and/or added in the amino acid sequences shown in SEQ ID NOs: 22 to 24, which is an L chain variable region for human B7RP-1. Urumono.

  6) The amino acid sequences of the combination of the H chain variable region and the L chain variable region are amino acids represented by the combinations of 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 a sequence.

  7) H chain variable region fragment of human anti-human B7RP-1 antibody having human B7RP-1 binding property.

8) The H chain variable region fragment of the human anti-human B7RP-1 antibody described in 7) above, wherein the complementarity determining region (CDR) has the following amino acid sequence (a) or (b):
(A) an amino acid sequence represented by any one of SEQ ID NOS: 1 to 3 as CDR1, 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 sequences of CDR1 to 3, respectively, SEQ ID NOs: 1 to 3, 4 to 6, or 7 to 9, 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 the complementarity determining region of the H chain to human B7RP-1.

  9) The above 8), wherein the amino acid sequences of CDR1 to 3 are amino acid sequences selected from the combination of SEQ ID NOS: 1, 4 and 7, SEQ ID NOS: 2, 5 and 8, or SEQ ID NOS: 3, 6 and 9. H-chain variable region fragment of human anti-human B7RP-1 antibody.

10) The H chain variable region fragment of the human anti-human B7RP-1 antibody according to any of 7) to 9) above, which has the following amino acid sequence (e) or (f):
(E) an amino acid sequence selected from the amino acid sequences of any one of SEQ ID NOs: 19 to 21;
(F) An amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and/or added in the amino acid sequences shown in SEQ ID NOs: 19 to 21, which serves as an H chain variable region for human B7RP-1. Urumono.

  11) A human anti-human B7RP-1 antibody L chain variable region fragment having human B7RP-1 binding properties.

12) The L chain variable region fragment of the human anti-human B7RP-1 antibody described in 11) above, wherein the complementarity determining region (CDR) has the following amino acid sequence (c) or (d):
(C) the amino acid sequence represented by any one of SEQ ID NOS: 10-12 as CDR1, any one of SEQ ID NOS: 13-15 as CDR2, and any one of SEQ ID NOS: 16-18 as CDR3;
(D) As the amino acid sequence of CDR1 to 3, any one of SEQ ID NOs: 10 to 18 and 13 to 15, or 16 to 18, or substitution or deletion of one or more amino acids in these amino acid sequences, An inserted and/or added amino acid sequence which can be the complementarity determining region of the L chain of the human anti-human B7RP-1 antibody.

  13) The above 12), wherein the amino acid sequences of CDR1 to 3 are amino acid sequences selected from the combination of SEQ ID NOS: 10, 13 and 16, SEQ ID NOS: 11, 14 and 17, or SEQ ID NOS: 12, 15 and 18. L-chain variable region fragment of human anti-human B7RP-1 antibody.

14) The L chain variable region fragment of the human anti-human B7RP-1 antibody according to any of 11) to 13) above, which has the following amino acid sequence (g) or (h):
(G) an amino acid sequence selected from the amino acid sequences of any one of SEQ ID NOs: 22 to 24;
(H) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and/or added in the amino acid sequences shown in SEQ ID NOs: 22 to 24, which is an L chain variable region for human B7RP-1. Urumono.

  15) The H chain variable region fragment of the human anti-human B7RP-1 antibody described in any of 7) to 10) above and the L of the human anti-human B7RP-1 antibody described in any of 11) to 14) above. A single-chain variable region fragment of a human-derived antibody against human B7RP-1, which is formed by linking with a chain variable region fragment.

  16) The H chain variable region fragment of the human anti-human B7RP-1 antibody described in any of 7) to 10) above, and/or the human anti-human B7RP-1 antibody described in any of 11) to 14) above. A human-derived antibody against B7RP-1, or an antibody fragment thereof, which comprises a human-derived antibody constant region linked to the L chain variable region fragment of.

17) The antibody fragment according to 16) above, wherein the antibody fragment is Fab, Fab′, F(ab′) ₂ , scAb, or scFv-Fc.

  18) A fusion antibody or a 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.

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

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

  21) A recombinant expression vector containing the gene according to 20) above.

  22) A transformant in which the gene according to 20) above is introduced.

  23) A method for producing a human anti-B7RP-1 antibody or a fragment thereof by expressing the gene described in 20) above in a host.

  24) B7RP-1 using the antibody or fragment thereof described in any 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. Detection reagent.

  25) A gene therapeutic agent containing the gene according to 20) above.

  26) B7RP-1 using the antibody or fragment thereof described in any 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. And ICOS binding activity inhibitor.

  27) B7RP-1 which is recognized by the antibody or fragment thereof described in any 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. A low-molecular-weight compound or a derivative thereof, which has been molecularly designed based on the above antigen-determining region.

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

  29) A preventive or therapeutic agent for inflammation and immunological disorders caused by the interaction between B7RP-1 and ICOS, which uses the binding activity inhibitor according to 26) or 28) above.

  The human monoclonal antibody and the antibody fragment molecule of the present invention have the variable region of human-derived anti-human B7RP-1 antibody, and strongly react with human B7RP-1 to inhibit its interaction with ICOS. From this, the antibody and the antibody fragment of the present invention can be used as a prophylactic or therapeutic drug for inflammation and immunological disorders caused by the binding of B7RP-1 and ICOS.

The figure which shows the result of ELISA which evaluated the binding specificity with respect to B7RP-1-Fc of the cloned single chain Fv antibody phage.

The figure which shows the result of ELISA which evaluated the binding specificity with respect to B7RP-1-Fc of the cloned single chain Fv antibody.

The figure which shows the result of having evaluated the kinetic analysis of the binding of the isolated anti-B7RP-1 single chain Fv antibody and B7RP1 immobilized on the sensor chip by surface plasmon resonance.

The figure which shows the result of ELISA which evaluated the binding inhibitory activity between ICOS-Fc and B7RP-1-Fc by the isolated anti-B7RP-1 single chain Fv antibody.

The figure which shows the result of having evaluated the binding property to the B cell of the isolated anti-B7RP-1 single chain Fv antibody which used the flow cytometer (the population which was stained with both FITC and PE is shown by the box).

The figure which shows the result of having evaluated the T cell proliferation inhibitory activity by the block of the ICOS costimulatory signal using the isolated anti-B7RP-1 single chain Fv antibody.

  The scFv used for the antibody and antibody fragment of the present invention was obtained as follows.

  Immunoglobulin heavy (H) chain and light (L) chain cDNAs were amplified by RT-PCR from peripheral blood B lymphocytes of 20 healthy subjects, and both were linked with a linker DNA to obtain healthy lymphocytes. ScFv DNA was prepared by randomly combining the H chain variable region (VH chain or VH) and the L chain variable region (VL chain or VL) from the origin.

This scFv DNA was incorporated into a phagemid vector pCANTAB5E to prepare a scFv display phage library derived from healthy individuals consisting of 10 ⁹ clones. This library was bound to human B7RP-1 immobilized on a solid phase, recovered, concentrated, and screened for an anti-human B7RP-1 scFv display phage clone. As a result, each clone screened produced an scFv that binds to human B7RP-1.

  The scFv can be expressed in E. coli, for example. In the case of Escherichia coli, the scFv to be expressed, such as a commonly used useful promoter and signal sequence for antibody secretion, can be functionally linked and expressed. 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) may be used when expressed in the periplasm of Escherichia coli. The signal sequence of the g3 protein of M13 phage can also be used for secretion into the culture supernatant.

  The expressed scFv can be isolated intracellularly or extracellularly from the host and purified to homogeneity. Since the scFv expressed in the present 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 is also possible to purify by combining the separation and purification methods used for ordinary proteins. For example, an antibody can be separated and purified by combining column chromatography such as ultrafiltration, salting out, gel filtration/ion exchange/hydrophobic chromatography.

As a method of measuring the binding activity of the obtained antibody or antibody fragment to human B7RP-1, there are methods such as ELISA and BIAcore. For example, when using ELISA, a sample containing a target antibody or antibody fragment, such as culture supernatant of E. coli 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 to measure the absorbance, whereby the antigen-binding activity can be evaluated. When BIAcore is used, B7-RP1-Fc is immobilized on the sensor chip, or B7-RP1-Fc is captured by anti-human Fc F(ab') ₂ antibody, and the binding dissociation constant of the target sample is measured. Can be measured.

  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 ELISA is used, a mixture of a sample containing a target antibody or antibody fragment and biotin-labeled ICOS-Fc is added to a 96-well plate on which human B7RP-1-Fc is immobilized. Next, streptavidin labeled with an enzyme such as peroxidase is added, and after the plate is incubated and washed, the chromogenic substrate TMBZ is added and the absorbance is measured, whereby the B7RP-1/ICOS binding inhibitory activity can be evaluated. ..

  Further, as a method for examining the binding property 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 using a human peripheral blood lymphocyte to analyze with a fluorescence flow cytometer, human peripheral blood lymphocytes are purified from human peripheral blood, stimulated with PMA and PHA, and then human IgG antibody is added to the human IgG Fcγ receptor. Blocking is performed, and a sample containing the target antibody or antibody fragment (a mixture of a sample containing scFv and an anti-E tag antibody in the case of scFv added with E tag) is added and reacted. After washing the cells, PE-labeled streptavidin and an anti-CD19 antibody labeled with FITC as a human B cell-specific marker are reacted and fluorescently labeled. After washing, using a fluorescent flow cytometer, two-dimensional flow cytometric analysis on PE and FITC channels can be performed to evaluate the binding property to B7RP-1 on peripheral blood B lymphocytes.

As a method for examining the inhibitory activity against the T cell costimulatory signal of the above antibodies and antibody fragments, there is a T cell proliferation stimulation assay. For example, a 96-well plate is coated with a mixed solution of an anti-CD3 antibody and an anti-human IgG Fc fragment F(ab') ₂ antibody, washed, and then B7RP-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 the T cell costimulatory signal can be evaluated.

  The anti-human B7RP-1 scFv was isolated and evaluated by the method described above, and as a result, it was found that it specifically binds to B7RP-1, and that its affinity is high enough to be comparable to that of the ICOS receptor. Capable of binding to B7RP-1 expressed on cells, inhibiting the binding between B7RP-1 and ICOS, and suppressing the proliferation of T cells by co-stimulation via B7RP-1/ICOS It has been shown. Therefore, these antibodies show similar effects in vivo and are considered to be useful as agents for suppressing B7RP-1/ICOS binding and T cell proliferation.

The amino acid sequences of the VH chains and VL chains of the three types of scFv (223, 323, 325) having the above-mentioned inhibitory activity and the nucleotide 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 109th. The th amino acid sequence corresponds to CDR3 (SEQ ID NO: 7). The nucleotide sequence of the gene encoding the VH chain is shown in SEQ ID NO:25.

  The amino acid sequence of the VL chain of clone 223 is shown in SEQ ID NO:22. The amino acid sequences of CDR1 to CDR3 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 sequences are CDR1 (SEQ ID NO: 10), the 51st to 57th amino acid sequences are CDR2 (SEQ ID NO: 13), 90th to 98th. The th amino acid sequence corresponds to CDR3 (SEQ ID NO: 16). The nucleotide sequence of the gene encoding the VL chain is shown in SEQ ID NO:28.

(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), 98th to 108th. The th amino acid sequence corresponds to CDR3 (SEQ ID NO: 8). The nucleotide sequence of the gene encoding the VH chain is shown in SEQ ID NO:26.

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

(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 sequences are CDR1 (SEQ ID NO: 3), the 50th to 66th amino acid sequences are CDR2 (SEQ ID NO: 9), 99th to 108th. The th amino acid sequence corresponds to CDR3 (SEQ ID NO: 15). The nucleotide sequence of the gene encoding the VH chain is shown in SEQ ID NO:27.

  The amino acid sequence of the VL chain of clone 325 is shown in SEQ ID NO:24. The amino acid sequences of CDR1 to CDR3 of the VL chain are shown in SEQ ID NOs: 12, 15 and 18. That is, in the amino acid sequence of the VL chain shown in SEQ ID NO: 24, the 23rd to 33rd amino acid sequences are CDR1 (SEQ ID NO: 12), the 49th to 55th amino acid sequences are CDR2 (SEQ ID NO: 15), 88th to 98th. The th amino acid sequence corresponds to CDR3 (SEQ ID NO: 18). The nucleotide sequence of the gene encoding the VL chain is shown in SEQ ID NO:30.

  Although the amino acid sequence and the nucleotide sequence of each clone obtained according to the present invention have been described above, based on each amino acid sequence information (VH chain, VL chain, each CDR1 to 3) described in the sequence listing, single or multiple It is also possible to use the sequences in appropriate combination.

  The antibody of the present invention and the antibody fragment thereof are not limited to the sequences shown in the above SEQ ID NOs as the above VH chain and VL chain, and their CDRs, and they are mutant polypeptides in which a part of them is modified. It may be.

  That is, in the amino acid sequence described in each SEQ ID NO:, consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and/or added, and the H chain or L chain of human B7RP-1 Also included are polypeptides that are the complementarity determining regions, the variable regions of the H or L chains.

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

  It should be noted that the above-mentioned “mutation” means that when the antibody of the present invention or an antibody fragment thereof is used as a therapeutic drug (when administered to human), it has a human-derived structure or does not cause human immune reaction. When used as a detection instrument or diagnostic kit (when not administered to humans), the range is not particularly limited.

The VH chain and/or VL chain disclosed in the present invention was obtained mainly by using the phage antibody method in the form of scFv, but its application is not limited to scFv in principle. For example, a complete molecular form in which the disclosed VH chain and/or VL chain is linked to a constant region of human immunoglobulin, or Fab, Fab′ or F(ab′) ₂ combined with a part of the constant region of human immunoglobulin, Further, other antibody fragments such as single-chain antibody (scAb) in which scFv is bound to the constant region of human immunoglobulin L chain, and scFv-Fc in which scFv is bound to the constant region of human immunoglobulin H chain are also included. It is included in the applicable range.

  Further, the antibody or fragment thereof of the present invention may be a fusion antibody or fragment thereof obtained by fusing the antibody or fragment with a peptide or another protein.

  In addition, a modified antibody or a fragment thereof in which a polymer modifier such as polyethylene glycol is bound to these antibodies and antibody fragments or the above fusion antibody or a fragment thereof can also be used.

  When preparing scFv in which Fvs of H chain and L chain are linked with an appropriate linker, an arbitrary single chain peptide consisting of, for example, 10 to 25 amino acid residues is used as the peptide linker.

  The antibody or antibody fragment of the present invention is suitable for a suitable host (for example, bacteria, 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). (Yeast) to express the antibody or fragment thereof of the present invention.

  The gene of the present invention can also be used as an adjunct to gene therapy for controlling the interaction between B7RP-1 and ICOS.

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

  In addition, molecular design based on the antigen-determining region on human B7RP-1 recognized by these antibodies or fragments thereof provides an important means for developing low-molecular compounds that act on B7RP-1/ICOS signaling. ..

  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 imitating its three-dimensional structure. A modified peptide obtained by adding an unnatural amino acid to the amino acid sequence of the peptide can be used as well. The same applies to fusion proteins obtained by fusing these peptides with other proteins. The same applies to a modified molecule in which a polymer modifier such as polyethylene glycol is bound to the above peptide or compound.

  These low molecular weight compounds or derivatives thereof can also be used as inhibitors of the binding activity of B7RP-1 and ICOS.

  Furthermore, a binding activity inhibitor comprising the antibody or antibody fragment thereof of the present invention, and a low molecular weight compound or a derivative thereof is a preventive or therapeutic agent for inflammation and immunological disorders caused by the interaction between B7RP-1 and ICOS. Is effective as.

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

<<Example 1: Construction of phage library from healthy person>>
The phage library was constructed by using peripheral blood-derived lymphocytes derived from 20 healthy subjects 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)-Vκ, VH(γ)-Vλ, VH(μ)-Vκ, and VH(μ)-Vλ sub-libraries are 1.1×10 ⁸ , 2.1×10 ⁸ , and 8.4×10 ^{7, respectively.} , 5.3×10 ⁷ clones were evaluated as having a diversity.

<<Example 2: Panning>>
Human B7RP-1-Fc (10 μg) was dissolved in 1 ml of 0.1 M NaHCO ₃ solution, and coated on an immunotube (Nunc) overnight at 4° C. After washing once with PBS containing 0.1% Tween 20, it was blocked with 0.5% gelatin/PBS for 2 hours at room temperature. After washing 6 times with 0.1% Tween/PBS, 1 ml of a healthy person-derived antibody phage library (single chain antibody-displaying phage solution, 10 ¹² TU/ml) was added and reacted for 2 hours at room temperature.

  After non-specific phages were removed by washing with 0.1% Tween20/PBS for 20 times, phage clones bound to B7RP-1-Fc were added with 0.1 M glycine-HCl (pH 2) containing 1 mg/ml BSA (bovine serum albumin). .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/PBS and reacted for 2 hours at room temperature. The supernatant was added to E. coli TG1 (20 ml) in the logarithmic growth phase, allowed to stand at 30° C. for 30 minutes, then partly spread on a SOBAG plate, and the rest was replaced with 30 ml of 2×YTAG The cells were cultured overnight at 30°C. The culture solution was centrifuged at 2200 rpm×10 minutes, and the precipitated Escherichia coli was suspended in 3 ml of 2×YTAG to prepare a primary panning Escherichia coli library. This TG1 solution was planted in 2×YTAG medium and rescued with helper phage to prepare a phage library after screening.

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

  A clone was arbitrarily extracted from the SOBAG plate after the second panning and the third panning to prepare a short chain Fv antibody phage clone, and the specificity to B7RP-1-Fc was confirmed and the sequence of the antibody gene was analyzed. ..

<<Example 3: Screening B7RP-1 ELISA>>
The binding specificity of the separated single-chain Fv antibody phage was confirmed by ELISA by the following method. B7RP-1-Fc (80 ng/well) was coated on an ELISA plate (Nunc) at 4° C. overnight, and blocked with 0.5% gelatin/PBS. After washing with 0.1% Tween 20/PBS, 40 μl of the isolated single-chain Fv phage clone (1×10 ¹³ pfu/ml) was added, and the mixture was reacted at room temperature for 2 hours. To detect the bound phage, biotinylated anti-M13 phage antibody (Pharmacia) was used as the primary antibody, and AP-labeled streptavidin was added as the secondary antibody to react, and then the substrate solution (1% containing 10% 2,2-iminodiethanol) was used. /ml PNP-phosphate in PBS) was added, and the absorbance at 405 nm was measured with a multi-plate auto reader NJ-2001 (Nunc). As a result, all of the finally evaluated clones were found to be specific to B7RP-1 (Fig. 1).

<<Example 4: Sequence analysis of clones>>
The DNA base sequences of VH and VL of the scFv gene of the isolated clones were determined using Dye terminator cycle sequencing FS Ready Reaction kit (Applied Biosystems). As a result of ELISA and sequence analysis, the isolated clones were classified into 4 types of scFv.

<Example 5: Expression and purification of human-derived anti-human B7RP-1scFv>
Plasmid DNA was recovered from the scFv clone that reacts with human B7RP-1 isolated in Examples 2 and 3 above, and Escherichia coli HB2151 was transformed by a conventional method. After overnight culture of these E. coli in 2×YT medium containing 2% glucose and 100 μg/mL ampicillin, they were partially transplanted to glucose-free 2×YT medium and added with a final concentration of 1 mM IPTG and 100 μg/mL ampicillin. The cells were cultured for 4 hours to induce scFv expression. After completion of the culture, the cells were collected by centrifugation, suspended in PBS containing 1 mM EDTA, and left in ice for 30 minutes. Then, the mixture was centrifuged at 8,900×g for 30 minutes, and the supernatant was filtered through a 0.45 μm filter to give a periplasmic fraction, which was used as a scFv-purified starting material.

  The purified starting material thus prepared was purified by affinity chromatography using an anti-E tag antibody according to a conventional method. After dialysis with PBS, endotoxin was removed with an endotoxin removal column Detoxi-gel (PIERCE) according to the attached protocol. After concentrating with Centricon (Amicon) with a molecular weight cut of 10,000, it was 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 binding activity of the purified single chain Fv antibody to B7RP-1 was measured by the following method. ELISA plate (Nunc) was coated with B7RP-1-Fc (80ng/well) at 4°C overnight, blocked with 0.5% gelatin/PBS, washed with 0.1% Tween20/PBS, and isolated single chain 40 μl of a mixture of Fv antibody (20 μg/ml) and anti-E-tag antibody was added and reacted for 2 hours. For detection, AP-labeled anti-mouse IgG Fcγ antibody was added as a secondary antibody and reacted for 60 minutes, then a substrate solution was added, and the absorbance at 405 nm was measured with a multi-plate auto reader NJ-2001. As a result, all of the finally evaluated clones were found to be specific to B7RP-1 (Fig. 2).

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, CTLA-4-Fc, anti-human diluted in 10 mM acetate buffer (pH 4.0) at 20°C in 4 flow cells on sensor chip CM5 (Biacore AB, Uppsala, Sweden) Fc F(ab′) ₂ antibody (10 μg/mL each) was immobilized at a flow rate of 5 μL/min. After washing the sensor chip with HBS buffer (100 mM HEPES/5M NaCl/0.5M EDTA/0.005% Tween20 (Pharmacia biotech)), B7RP-1-Fc diluted with HBS buffer/0.01% BSA was reacted to Captured with human Fc F(ab') ₂ antibody. Subsequently, each scFv sample (adjusted to 100 nM) was injected at a flow rate of 10 μL/min to cause a reaction, and the binding/dissociation constant of each sample was measured. Further, before the step of reacting each sample, the scFv was eluted and washed with a 200 mM glycine-HCl buffer solution (pH 2.2) containing 200 mM NaCl. BIAevaluation software was used for data analysis. As a result, B7RP-1-Fc first showed a reaction only on the chip on which ICOS-Fc and anti-human Fc F(ab') ₂ antibody were immobilized, and as a result, the binding dissociation constant between B7RP-1 and ICOS was about It was found to be 7 nM. Subsequently, when 233 was reacted, it showed binding only to the chip on which B7-RP1-Fc was immobilized and the chip on which B7-RP1-Fc was captured by the anti-human Fc F(ab') ₂ antibody, and ICOS Since B7-RP1-Fc bound thereto did not show binding properties, it was presumed that 233 binds to the binding site with ICOS. The binding dissociation constant with B7RP-1 was calculated to be about 13 to 15 nM, which confirmed that the binding dissociation constant was almost the same as that of 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. overnight, blocked with 0.5% gelatin/PBS, and washed with 0.1% Tween20/PBS. A mixture of 125 nM to 2 μM anti-B7RP-1 single-chain Fv antibody solution and biotin-labeled ICOS-Fc was added and reacted for 90 minutes. After washing, AP-labeled streptavidin was added, and after reacting for 30 minutes, a substrate solution was added and the absorbance at 405 nm was measured. As a result, it was found that clones 223, 323, and 325 inhibited 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 binding of single-chain Fv antibody to B7RP-1 on peripheral blood B lymphocytes by flow cytometer>>
In order to confirm the binding activity of the isolated human anti-B7RP-1 single chain Fv antibody to the B7RP-1 molecule expressed on the cells, human peripheral blood lymphocytes were used and analyzed using a fluorescence flow cytometer. .. PBMCs were purified from human heparin peripheral blood by a standard method using Ficoll. After stimulation with PMA (5 ng/ml) and PHA (2 μg/ml) for 41 hours, human IgG Fcγ receptor was blocked by adding human IgG antibody, and a mixture of single-chain Fv antibody and anti-E tag antibody was added. It was made to react. After washing the cells, they were reacted with PE-labeled streptavidin and anti-CD19 antibody labeled with FITC as a human B cell-specific marker for 30 minutes for fluorescent labeling. After washing, two-dimensional flow cytometric analysis was performed on PE and FITC channels using a Coulter EPICS XL (Coulter Miami, FL) flow cytometer. As a result, in the clone names, 223, 323, and 325, staining of the FITC-labeled B cell population with PE was confirmed in the clone names, 223, 323, and 325. It was shown to have binding activity (Figure 5).

<Example 9: T cell proliferation inhibitory activity against T cell costimulatory signal by human anti-B7RP-1 single chain Fv antibody>
The inhibitory activity of the obtained single-chain Fv antibody against the transmission of an auxiliary signal between B7RP-1 on the antigen-presenting cells and ICOS on the T cells was analyzed using the T cell proliferation activity as an index. A 96-well round bottom plate was coated with a mixed solution of anti-CD3 antibody (2 μg/ml) and anti-human IgG Fc fragment F(ab′) ₂ antibody (2.4 μg/ml) at 37° C. for 90 minutes. After washing the plate with PBS, B7RP-1-Fc (1 μg/ml) was added and reacted at 37° C. for 2 hours. After washing the plate again, 1 nM to 500 nM single-chain Fv antibody prepared by RPMI1640 and anti-B7RP-1 antibody were added and reacted for 30 minutes, and then peripheral blood lymphocytes (1×10 ⁵ cells/cell prepared from human peripheral blood were prepared. well) was added and the cells were cultured at 37°C. After 48 hours from the start of culture, tritium thymidine (0.5 μCi/well) was added, and the cells were further cultured for 18 hours. 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 the clones, almost the same dose-dependent inhibition of T cell proliferation by the single-chain Fv antibody as in the case of adding human ICOS-Fc occurred, and the ICOS signals of these single-chain Fv clones 223, 323, and 325 were detected. Inhibitory activity was shown (Figure 6).

Claims (29)

  A human anti-human B7RP-1 antibody having binding properties to human B7 Related Protein 1 (hereinafter, B7RP-1). The H chain complementarity determining region (CDR) has the following amino acid sequence (a) or (b), and the L chain complementarity determining region (CDR) has the following amino acid sequence (c) or (d): The human anti-human B7RP-1 antibody according to claim 1, which comprises:
(A) an amino acid sequence represented by any one of SEQ ID NOS: 1 to 3 as CDR1, 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 sequences of CDR1 to 3, respectively, SEQ ID NOs: 1 to 3, 4 to 6, or 7 to 9, or one or more amino acids in these amino acid sequences are substituted, deleted, inserted, and/or An added amino acid sequence which can be the complementarity determining region of the H chain to human B7RP-1;
(C) the amino acid sequence represented by any one of SEQ ID NOS: 10-12 as CDR1, any one of SEQ ID NOS: 13-15 as CDR2, and any one of SEQ ID NOS: 16-18 as CDR3;
(D) As the amino acid sequences of CDR1 to 3, SEQ ID NOS: 10 to 12, 13 to 15, or 16 to 18, 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 the complementarity determining region of the L chain to human B7RP-1.   CDR 1 to 3 of the H chain is an amino acid sequence selected from a combination of SEQ ID NOs: 1, 4 and 7, SEQ ID NOs: 2, 5 and 8, or SEQ ID NOs: 3, 6 and 9, and CDR 1 of the L chain. The human according to claim 1 or 2, wherein the amino acid sequence of ~3 is an amino acid sequence selected from the combination of SEQ ID NOS: 10, 13 and 16, SEQ ID NOS: 11, 14 and 17, or SEQ ID NOS: 12, 15 and 18. Anti-human B7RP-1 antibody.   The amino acid sequences of the combinations of CDR 1 to 3 of H chain and CDR 1 to 3 of L chain are SEQ ID NOs: 1, 4 and 7 and SEQ ID NOs: 10, 13 and 16, SEQ ID NOs: 2, 5 and 8 and SEQ ID NOs: 11 and 14, respectively. And 17, or the combination of any one of SEQ ID NOS: 3, 6 and 9 and SEQ ID NOS: 12, 15 and 18, The human anti-human B7RP-1 antibody according to claim 3. 5. The H chain variable region has the following amino acid sequence (e) or (f), and the L chain variable region has the following amino acid sequence (g) or (h). The described human anti-human B7RP-1 antibody:
(E) an amino acid sequence selected from the amino acid sequences of any one of SEQ ID NOs: 19 to 21;
(F) An amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and/or added in the amino acid sequences shown in SEQ ID NOs: 19 to 21, which serves as an H chain variable region for human B7RP-1. Possible things;
(G) an amino acid sequence selected from the amino acid sequences of any one of SEQ ID NOs: 22 to 24;
(H) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and/or added in the amino acid sequences shown in SEQ ID NOs: 22 to 24, which is an L chain variable region for human B7RP-1. Urumono.   The amino acid sequences of the combination of the H chain variable region and the L chain variable region are the amino acid sequences represented by the combinations of 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 claims 1 to 5.   A variable region of an H chain of a human anti-human B7RP-1 antibody having human B7RP-1 binding property. The human anti-human B7RP-1 antibody heavy chain variable region fragment according to claim 7, wherein the complementarity determining region (CDR) has the following amino acid sequence (a) or (b):
(A) an amino acid sequence represented by any one of SEQ ID NOS: 1 to 3 as CDR1, 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 sequences of CDR1 to 3, respectively, SEQ ID NOs: 1 to 3, 4 to 6, or 7 to 9, or one or more amino acids in these amino acid sequences are substituted, deleted, inserted, and/or An added amino acid sequence which can serve as a complementarity determining region of the H chain to human B7RP-1.   The human according to claim 8, wherein the amino acid sequence of CDR1 to 3 is an amino acid sequence selected from the combination of SEQ ID NOs: 1, 4 and 7, SEQ ID NOs: 2, 5 and 8, or SEQ ID NOs: 3, 6 and 9. H chain variable region fragment of anti-human B7RP-1 antibody. The H chain variable region fragment of the human anti-human B7RP-1 antibody according to claim 7, which has the following amino acid sequence (e) or (f):
(E) an amino acid sequence selected from the amino acid sequences of any one of SEQ ID NOs: 19 to 21;
(F) An amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and/or added in the amino acid sequences shown in SEQ ID NOs: 19 to 21, which serves as an H chain variable region for human B7RP-1. Urumono.   An L chain variable region fragment of a human anti-human B7RP-1 antibody having binding properties to human B7RP-1. The human anti-human B7RP-1 antibody L chain variable region fragment according to claim 11, wherein the complementarity determining region (CDR) has the following amino acid sequence (c) or (d):
(C) the amino acid sequence represented by any one of SEQ ID NOS: 10-12 as CDR1, any one of SEQ ID NOS: 13-15 as CDR2, and any one of SEQ ID NOS: 16-18 as CDR3;
(D) As the amino acid sequence of CDR1 to 3, any one of SEQ ID NOs: 10 to 18 and 13 to 15, or 16 to 18, or substitution or deletion of one or more amino acids in these amino acid sequences, An inserted and/or added amino acid sequence which can be the complementarity determining region of the L chain of the human anti-human B7RP-1 antibody.   The human according to claim 12, wherein the amino acid sequence of CDR1 to 3 is an amino acid sequence selected from the combination of SEQ ID NOS: 10, 13 and 16, SEQ ID NOS: 11, 14 and 17, or SEQ ID NOS: 12, 15 and 18. L chain variable region fragment of anti-human B7RP-1 antibody. The L chain variable region fragment of the human anti-human B7RP-1 antibody according to claim 11, which has the following amino acid sequence (g) or (h):
(G) an amino acid sequence selected from the amino acid sequences of any one of SEQ ID NOs: 22 to 24;
(H) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and/or added in the amino acid sequences shown in SEQ ID NOs: 22 to 24, which is an L chain variable region for human B7RP-1. Urumono.   A human anti-human B7RP-1 antibody H chain variable region fragment according to any one of claims 7 to 10, and a human anti-human B7RP-1 antibody L chain variable region fragment according to any one of claims 11 to 14. A single-chain variable region fragment of a human-derived antibody against human B7RP-1, which is formed by linking   The H chain variable region fragment of the human anti-human B7RP-1 antibody according to any one of claims 7 to 10, and/or the L chain variable region of the human anti-human B7RP-1 antibody according to any one of claims 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 a region fragment. The antibody fragment according to claim 16, wherein the antibody fragment is Fab, Fab', F(ab') ₂ , scAb, or scFv-Fc.   A fusion antibody or a 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.   A modified antibody or a fragment thereof, which is obtained by binding a modifying agent to the antibody or the fusion antibody or the fragment thereof according to any one of claims 1 to 18.   A gene encoding the antibody or the fusion antibody or the fragment thereof according to any one of claims 1 to 19.   A recombinant expression vector comprising the gene according to claim 20.   A transformant having the gene according to claim 20 introduced therein.   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.   A reagent for detecting B7RP-1 using the antibody or fragment thereof according to any one of claims 1 to 17, the fusion antibody or fragment thereof according to claim 18, or the modified antibody or fragment thereof according to claim 19. ..   A gene therapy agent comprising the gene according to claim 20.   B7RP-1 and ICOS using the antibody or fragment thereof according to any one of claims 1 to 17, the fusion antibody or fragment thereof according to claim 18, or the modified antibody or fragment thereof according to claim 19. A binding activity inhibitor.   An antigen on B7RP-1 recognized by the antibody or fragment thereof according to any one of claims 1 to 17, the fusion antibody or fragment thereof according to claim 18, or the modified antibody or fragment thereof according to claim 19. A low-molecular compound or its derivative whose molecule is designed based on the determinant region.   An inhibitor of the binding activity between B7RP-1 and ICOS, which comprises the low molecular weight compound according to claim 27 or a derivative thereof.   A preventive or therapeutic agent for inflammation and immunological disorders caused by the interaction between B7RP-1 and ICOS, which uses the binding activity inhibitor according to claim 26 or 28.

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