WO2006003999A1 - Human antihuman b7rp-1 antibody and antibody fragment thereof - Google Patents
Human antihuman b7rp-1 antibody and antibody fragment thereof Download PDFInfo
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- WO2006003999A1 WO2006003999A1 PCT/JP2005/012094 JP2005012094W WO2006003999A1 WO 2006003999 A1 WO2006003999 A1 WO 2006003999A1 JP 2005012094 W JP2005012094 W JP 2005012094W WO 2006003999 A1 WO2006003999 A1 WO 2006003999A1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Definitions
- 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 Plโ) and inhibits its biological activity, Relates to antibody fragments.
- B7R Pl human B7 Related Protein 1
- 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.
- 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).
- 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).
- Non-patent Document 4 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.
- OVA ovalbumin
- 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
- 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.
- 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.
- 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.
- inhibition of the signal between ICOS / B7RP-1 by anti-B7RP-1 antibody is unlikely to promote T cell proliferation activity via ICOS.
- 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.
- 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.
- a great deal of labor and cost are required for construction in which activity is often reduced by humanization.
- 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.
- 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.
- 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.
- 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.
- the present invention includes the following 1) to 29) as medically or industrially useful methods' substances.
- B7RP-1 A human anti-human B7RP-1 antibody having binding property to human B7 Related Protein 1 (hereinafter referred to as B7RP-1).
- 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):
- 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).
- 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 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:
- 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.
- 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.
- a gene therapy agent comprising the gene according to 20) above.
- 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.
- 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.
- 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
- 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.
- 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).
- 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.
- the scFv used for the antibodies and antibody fragments of the present invention was obtained as follows.
- 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.
- 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.
- scFv 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.
- 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.
- the signal sequence of the g13 protein of M13 phage can also be used.
- the expressed scFv can be purified to homogeneity by separating the inside and outside of the cell and the host force.
- 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.
- Methods for measuring the binding activity of the obtained antibody or antibody fragment to human B7RP-1 include methods such as ELISA and BIAcore.
- ELISA 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.
- 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.
- 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 โฒ) antibody.
- ELISA 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.
- an enzyme such as peroxidase
- 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.
- PE-labeled streptavidin and FITC-labeled anti-CD19 antibody as a human B cell-specific marker are reacted and fluorescently labeled.
- 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.
- T cell proliferation stimulation assay 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,
- 7RP-1-FC 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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
- 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)
- 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.
- 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.
- 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
- polypeptides that serve as chain or L chain complementarity-determining regions and H or L chain variable regions.
- Such โmutationโ 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.
- the above-mentioned โmutationโ 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).
- 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.
- the application is limited to scFv.
- 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.
- 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.
- scAb single-chain antibodies
- 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.
- 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.
- antibodies and antibody fragments can also be modified antibodies or fragments thereof in which a polymer modifying agent such as polyethylene glycol is bound.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 ( โ ) โV โ , VH ( โ ) โV, VH () โV โ , 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.
- coli TGI (20 ml) in logarithmic growth phase, left at 30 ยฐ 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 ยฐ C. After centrifuging the culture solution at 2200 rpm โ 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.
- 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).
- 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 โ 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.
- the mixture was centrifuged at 8,90 OX g for 30 minutes, and the supernatant was filtered through a 0.45 โ m filter to obtain a periplasm fraction, which was used as a starting material for scFv purification.
- 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 โ m filter to obtain a purified sample. Storage was performed at -20 ยฐ C.
- 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 ยฐ 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.
- 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.
- all the clones finally evaluated were found to be specific for B7RP-1 (Fig. 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
- 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.
- 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 ยฐ C, blocked with 0.5% gelatin / PBS, and washed with 0.1% Tween20 / PBS.
- a mixture of 125 โ to 2M anti-B7RP-1 single chain Fv antibody solution and piotin-labeled ICOS-Fc was added and reacted for 90 minutes.
- 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.
- 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
- 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.
- 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.
- the mixture was coated at 37 ยฐ C for 90 minutes. After the plate was washed with PBS, B7RP-1-Fc (l โ g / ml) was added and reacted at 37 ยฐ C. for 2 hours. After washing the plate again, the 1 โ to 500 โ 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 ยฐ 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.
- 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).
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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
ใใๆใใ B7RP-1ๆไฝใใใณใใฎๆไฝใใฉใฐใกใณใ ย Human anti-human B7RP-1 antibody and antibody fragments thereof
ๆ่กๅ้ ย Technical field
[0001] ๆฌ็บๆใฏใใใ B7 Related Protein 1 (ๅฅๅใ B7hใ GL50ใ ICOSใชใฌใณใใไปฅไธใใB7R P-lใใจใใ๏ผใซ็ตๅใใใใฎ็็ฉๆดปๆงใ้ปๅฎณใใใใๆใใ B7RP-1ๆไฝใพใใฏ่ฉฒๆไฝ ใใฉใฐใกใณใใซ้ขใใใๅฝ่ฉฒๆไฝใใใณๆไฝใใฉใฐใกใณใใฏใ B7RP-1ใใใใฎ T็ดฐ่ไธ ใฎใฌใปใใฟใผใงใใ ICOS (Inducible costimulator)ใจ็ตๅใใใใจใซใใฃใฆๆน่ตทใใใ T็ดฐ่ใฎ้ๅบฆใชๆดปๆงใฃๅใๆใใใใจใงใ่ๅจ็งปๆคๆใฎๅ
็ซๆๅถๅคใใขใฌใซใฎใผ'่ชๅทฑ ๅ
็ซใชใฉใฎๅ
็ซ็ฐๅธธๆง็พๆฃใฎๆฒป็่ฌใจใใฆๆๅพ
ใใใใ ย [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.
่ๆฏๆ่ก ย Background art
[0002] ICOSใฏใ T็ดฐ่ใฎ่ฃๅฉๅบๆฟใฌใปใใฟใผ CD28ใใกใใชใผใฎ็ฌฌ 3็ช็ฎใฎๅๅญใจใใฆ็บ่ฆ ใใใใใขใใคใใผใๅฝขๆใใ่็ตๅๅ่็ฝใงใใใ TCRใไปใใใทใฐใใซใซใใฃใฆ T็ดฐ่ใๅบๆฟใใใใจใง T็ดฐ่่กจ้ขไธใซ่ชๅฐใใใ๏ผ้็น่จฑๆ็ฎ 1)ใใใฎๅๅญใไปใ ใ่ฃๅฉๅบๆฟใฏใ CD4ใ CD8ๅๆนใฎ T็ดฐ่ใฎๅขๆฎใๅขๅคงใใใ IL_4ใ IL-10ใชใฉใฎใตใคใ ๅใคใณใฎๅๆณใจใจใใซใ T็ดฐ่ใฎ CD154 (CD40ใชใฌใณใ๏ผใฎ็บ็พใ่ชๅฐใใใใใฎใใจใ ใใ ICOSใฏ็นใซใๆดปๆงๅ T็ดฐ่ใซใใใๆงใ
ใชใจใใงใฏใฟใผๆฉ่ฝใซใใใฆ้่ฆใงใ ใใจ่ใใใใใๅฎ้ใซใ ICOSใใใใฏใขใฆใใใใใฆในใงใฏใๆๅๅบๆฟใซใใ T็ดฐ่ใฎ ๅขๆฎใ IL-4็ฃ็่ฝใๆถๅคฑใใ (้็น่จฑๆ็ฎ 2)ใ ย [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] ICOSใฎใชใฌใณใใจใใฆๅๅฎใใใ B7RP-1ใฏใใใใฒใจใคใฎ่ฃๅฉๅบๆฟๅๅญใงใใ CD28 ใฎใชใฌใณใใงใใ B7ๅๅญ๏ผCD80ใ CD86)ใฎใใกใใชใผใซๅฑใใ B็ดฐ่ใใใฏใญใใกใผใธใ LPSใชใฉใงๅบๆฟใใใ้ๅ
็ซ็ดฐ่ใซ็บ็พใใ่็ตๅ่็ฝใงใใ๏ผ้็น่จฑๆ็ฎ 3)ใ B7 RP-1ใฏใ็พๅจ็ฅใใใฆใใๅฏไธใฎ ICOSใฎใชใฌใณใใงใใใ็็ๅญฆ็ใชๆกไปถไธใงใฏใ C D28ใ CTLA4 (CD154)ใชใฉใจใฎไบคๅทฎ็ตๅๆงใฏ็คบใใชใใ ICOSใจ B7RP-1้ใฎ็ตๅใฏ ใ T็ดฐ่ใฎใใซใใผๆฉ่ฝใซ้ๅธธใซ้่ฆใงใใใ ICOSใฎใใใฏใขใฆใใใฆในใงใฏใใใซ ใใผๆฉ่ฝใซใใๆไฝใฎใฏใฉในในใฃใใใ่ใใๆๅถใใใ๏ผ้็น่จฑๆ็ฎ 2)ใใใใใใ ใฎๆๅถใฏๆ CD40ๆไฝใ็จใใ CD40ใใใฎ B็ดฐ่ๅบๆฟใซใใฃใฆใ้จๅ็ใซๅๅพฉใใ ใใจใ็ฅใใใฆ ใฝใ (้็น่จฑๆ็ฎ 4)ใ
[0004] ใชใใขใซใใใณ๏ผOVA)ใซใใๅ
็ซใใใใฆในใ็จใใ OVAใฎๅธๆฐใซใใใใฆในๆฐ็ฎก ๆฏ็็ใขใใซๅฎ้จใงใๆ ICOS้ปๅฎณๆไฝใใใใฏ ICOS-Fcใซใใ ICOS/B7RP-1้ใฎ ใทใฐใใซใใญใใฏใฎๅๅฆ็ใใใใใฆในใงใฏใๆฐ็ฎกๆฏ่บ่ๆดๆตๆถฒ (BAL๏ผ bronchoalveo lar lavage)ไธญใงใฎใชใณใ็ใๅฅฝไธญ็ใฎไพตๅ
ฅใๆธๅฐใใ IL- 4ใ IL- 10ใชใฉใฎใตใคใๅใคใณ ใฎ็ฃ็ใๆใใใใใใใฎใใใชๆๅถใฏใ CTLA4-FCใซใใ CD28/B7้ใฎใทใฐใใซใฎ ใใญใใฏใซใใฃใฆใใฟใใใใใ็นใซใใใใใฎ้ปๅฎณๅคใฎๆไธๆๆใฎ้ใใซไผดใใ IL- 4็ฃ็ใฎใใฟใผใณใ้ก่ใซ็ฐใชใฃใฆใใใใใฎใใจๅใใ่ฃๅฉๅบๆฟ CD28/B7ใจ ICOS/B 7RP-1ใฎๅฝนๅฒใฎ้ ใใจใใฆใๅ่
ใฏ T็ดฐ่ใฎๆดปๆงใฃๅ้ๅงใซใ ใใฆ้่ฆใชๅฝนๅฒใๆใ ใใฎใซๅฏพใใๅพ่
ใฏใใฎๆดปๆงใฃๅๅพใฎใจใใงใฏใฟใผๆฉ่ฝใๅถๅพกใใไธใงไธป่ฆใชๅฝนๅฒใ ๆ
ใฃใฆ ใฝใใใจใ็คบใใใ (้็น่จฑๆ็ฎ 5)ใ [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] ใใใฎๅ้กไธ่ฝๅๅ
็ซไธๅ
จ (CVID๏ผ common variable immunodeficiency)ใฎๅๅ ใใ ICOSใฎใใขใถใฃใดใผใใซใใ้บไผๅญๆฌ ๆใซใใใใจใๆใใใซใใใฆใใใใใฎใใจใฏ ใใใ B็ดฐ่ใฎๆ็ใๆไฝ็ฃ็ใธใจๅฐใใใซใใผ T็ดฐ่ๆฉ่ฝใซใใใ ICOSใฎ้่ฆๆง ใ็คบใใฆใใใจใจใใซใ ICOSใ็จฎใ
ใฎๅ
็ซ็พๆฃใฎๅๅ ้บไผๅญใจใชใใใจใๅผทใ็คบๅใ ใฆใใ๏ผ้็น่จฑๆ็ฎ 6)ใ ย [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] ใใฆในใ็จใใๅฟ่ใฎๅ็จฎ็ฐๅไฝ (ใกๅฃ๏ผ็งปๆคใซใใใฆใๆ ICOS้ปๅฎณๆไฝใฎๆไธ ใฏใๆฅๆงใฎๆ็ตถๅๅฟใ้
ๅปถใใใใ ใใงใชใ็งปๆคๆญ็ใธใฎ CD4ใ CD8้ฝๆง T็ดฐ่ใ ใใฏใญใใกใผใธใฎๆตธๆฝคใๆๅถใใใใใฎๆ ICOS้ปๅฎณๆไฝใซใใใใฎใใใชๅนๆใฏใๅ
็ซๆๅถๅคใจใใฆไฝฟ็จใใใฆ ใใใทใฏใญในใใชใณ Aใจ็ธไน็ใชๅ
็ซๆๅถๅนๆใ็คบใใๆ ็ตถใพใงใฎๆๆใ้
ๅปถใใใ (้็น่จฑๆ็ฎ 7)ใๅ
็ซๆๅถใ็ฎ็ใจใใ่็ฝ่ฃฝๅคใจใใฆใ CTLA4- Igใซใใ CD28/B7้ใฎใทใฐใใซใฎ้ปๅฎณใใๆ CD40ใชใฌใณใๆไฝใซใใ CD40 /CD40ใชใฌใณใ๏ผCD154)้ใฎใทใฐใใซใฎ้ปๅฎณใๆค่จใใใฆใใๅ ICOS/B7RP-1้ ใฎ้ปๅฎณใซใใๆๅถๅนๆใฏใใใใใจๅ็ญใใใใฏใๆ ผๆฎตใซๅชใใ็ตๆใใใใใใฆใใ ย [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] ๅฎ้จ็ใขใฌใซใฎใผๆง่ณ่้ซ็๏ผEAE๏ผ experimental allergic encephalomyelitis)ใขใ ใซใ็จใใใใฆในใฎๅฎ้จใงใฏใๆ ICOS้ปๅฎณๆไฝใฎๆไธใซใใใ EAEใฎ็บ็ใพใงใฎๆ ้ใๅปถใฐใใใจใใงใใใใๅใใชใใใ EAE่ชๅฐใฎใใใฎๆๅใใใใๆไฝใจๅๆ ๆใซๆ ICOS้ปๅฎณๆไฝใฎๆไธใ่กใใจใ็็ถใๅ็ใซๅขๆชใใ็ตๆใจใชใฃใ (้็น่จฑ
ๆ็ฎ 8)ใใใฎใใจใฏใ่จๅบใซใใใฆๆ ICOSๆไฝใงใทใฐใใซใ้ปๅฎณใใใใจใฏใใขใฌใซ ใฎใผ็็ถใชใฉใๅขๆชใใๅฑ้บๆงใๅญใใใจใ็คบๅใใฆ ใใใ [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] ไธๆนใ B7RP-1ใซๅฏพใใใขใใฏใญใผใใซๆไฝใฎไฝฟ็จไพใฏใใใใพใงใใพใๅ ฑๅใใใฆ ใใชใใใใใฆใน B7RP-1ใซๅฏพใใใฉใใใขใใฏใญใผใใซๆไฝใใใใฆในใฎใณใฉใผใฒใณ่ช ๅฐ้ข็ฏ็๏ผCIA: collagen type II- induced arthritis)ใซๅฏพใใฆๆฒป็็ๆไธใซใใๅนๆใ ็คบใใใใจใๅ ฑๅใใใฆใใใ่ชๅทฑๅ
็ซ็พๆฃใฎ็
ๆ
ๅฝขๆใซใ ICOS/B7RP-1้ใฎใทใฐ ใใซใ้่ฆใชๅฏไธใๆใใใฆ ใใใใจใ็คบๅใใใฆ ใใ๏ผ้็น่จฑๆ็ฎ 9)ใ ย [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] ้็น่จฑๆ็ฎ 1 : Hutloff, A.ใใ (1999) Nature, 397(6716), p.263- 266 ย [0009] Non-Patent Document 1: Hutloff, A. et al. (1999) Nature, 397 (6716), p.263-266
้็น่จฑๆ็ฎ 2 : Dong, C.ใใ (2001) Nature, 409(6816), p.97- 101 ย Non-Patent Document 2: Dong, C. et al. (2001) Nature, 409 (6816), p.97-101
้็น่จฑๆ็ฎ 3 :Yoshinaga, S. K.ใใ (1999) Nature, 402(6763), p.827-832 ย Non-Patent Document 3: Yoshinaga, S. K. et al. (1999) Nature, 402 (6763), p.827-832
้็น่จฑๆ็ฎ 4:McAdam, A. J.ใใ (2001) Nature, 409(6816), p.102- 105 ย Non-Patent Document 4: McAdam, A. J. et al. (2001) Nature, 409 (6816), p.102-105
้็น่จฑๆ็ฎ 5 : Gonzalo, J. A.ใใ (2001) Nat. Immunol, 2(7), p.597- 604 ย Non-Patent Document 5: Gonzalo, J. A. et al. (2001) Nat. Immunol, 2 (7), p.597-604
้็น่จฑๆ็ฎ 6 : Grimbacher, B.ใใ (2003) Nat. Immunol, 4(3), p.261-268 ย Non-Patent Document 6: Grimbacher, B. et al. (2003) Nat. Immunol, 4 (3), p.261-268
้็น่จฑๆ็ฎ 7 : Ozkaynak, E.ใใ (2001) Nat. Immunol, 2(7), p.591- 596 ย Non-Patent Document 7: Ozkaynak, E. et al. (2001) Nat. Immunol, 2 (7), p.591-596
้็น่จฑๆ็ฎ 8 : Rottman, J. B.ใใ (2001) Nat. Immunol, 2(7), p.605- 611 ย Non-Patent Document 8: Rottman, J.B., et al. (2001) Nat. Immunol, 2 (7), p.605-611
้็น่จฑๆ็ฎ 9 : Iwai, H.ใใ (2002) J. Immunol, 169(8), p.4332- 4339 ย Non-Patent Document 9: Iwai, H. et al. (2002) J. Immunol, 169 (8), p.4332- 4339
็บๆใฎ้็คบ ย Disclosure of the invention
็บๆใ่งฃๆฑบใใใใจใใ่ชฒ้ก ย Problems to be solved by the invention
[0010] ่ๅจ็งปๆคใฎๆ็ตถๅๅฟใใขใฌใซใฎใผใชใฉใฎๅ
็ซ็ฐๅธธๆง็พๆฃใฎ็็ถใฎๅขๆชใซใฏใ T็ดฐ ่ใฎๅขๆฎใๆฃ้จใธใฎๆตธๆฝคใใจใใงใฏใฟใผๆฉ่ฝใๆฑบๅฎ็ใช้ขไธใใใฆใใใใใฎใใใช ๆฉ่ฝใซ้ขไธใใ ICOS/B7RP-1้ใฎใทใฐใใซใฎ้ปๅฎณใใใใใฏๅถๅพกใ่กใๅป่ฌๅใฏใ ่ๅจ็งปๆคๆใฎๅ
็ซๆๅถใใขใฌใซใฎใผใชใฉใฎๅ
็ซ็ฐๅธธๆง็พๆฃใฎๆฒป็่ฌใจใใฆๆๅพ
ใ ใใใ [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] ICOS/B7RP-1้ใฎใทใฐใใซใใใญใใฏใใ็น็ฐ็ใชใขใใฏใญใผใใซๆไฝใ้็บใใ ใใจใใงใใใฐใ ICOS/B7RP-1ใฎใทใฐใใซใซใใฃใฆ็บ็ใๅขๆชใใๅคใใฎ็พๆฃใฎๆ ๅนใชๆฒป็ๆๆฎตใซใชใใใจใๆๅพ
ใใใใ ย [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] ICOS/B7RP-1ใฎ่ฃๅฉใทใฐใใซใใใญใใฏใใๆไฝใจใใฆใๆ ICOSๆไฝใจๆ B7RP-1 ๆไฝใ่ใใใใใๆไฝใฎๆๅ็ตๅ้จใฏ 2ไพกใงใใใใใใใใๆไฝใจๆจ็ๅๅญใจใฎ
็ตๅใซใใฃใฆ็ดฐ่ใซใทใฐใใซใๅ
ฅใใๅฏ่ฝๆงใใใใ็นใซๆ ICOSๆไฝใฎๅ ดๅใ ICO Sใฎใทใฐใใซใ้ปๅฎณใใ็ฎ็ใงใฎๆไธใใ้ใซใทใฐใใซใ้ใใใจใงใ T็ดฐ่ใฎๅขๆฎ ใไฟใๅฏ่ฝๆงใ้คๅคใงใใชใใใใฎๆๅณใงใๆ B7RP-1ๆไฝใซใใ ICOS/B7RP-1้ ใฎใทใฐใใซ้ปๅฎณใฏใ ICOSใไปใใฆ T็ดฐ่ใฎๅขๆฎๆดปๆงใฃๅใไฟใใใจใฏ่ใใใใช ใใ ใใจใ B็ดฐ่ไธใฎ B7RP-1ใธใฎๆ B7RP-1ๆไฝใฎ็ตๅใซใใ B็ดฐ่ใธใทใฐใใซใ้ใ ใใใจใใฆใใ ICOS/B7RP-1ใทใฐใใซใซใใใ B7RP-1ใฏ็บ็พใๆธๅฐใใใใจใ็ฅใใ ใฆใใใ T็ดฐ่ใฎใจใใงใฏใฟใผๆฉ่ฝใๆๅถใใๆนๅใซๅใใใใ้ปๅฎณๆไฝใจใใฆๆ B7R P-1ๆไฝใฏๅชใใฆใใใจ่ใใใใใ [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] ็พๅจใใใ B7RP-1ใซๅฏพใใๆไฝ (ๆใใ B7RP-1ๆไฝ๏ผใจใใฆใฏใใใฆในใใฉใใ็ฑๆฅใฎ ใขใใฏใญใผใใซๆไฝใ ใใใคใๅๅพใใใฆ ใใใซ้ใใชใใใใใฎใใใชๅพๆฅใฎๆใใ B7 RP-1ๆไฝใฏใ้ใใ็ฑๆฅใฎใใใใใฎ้ซใๅ
็ซๅๆงใซใใฃใฆใใใใซๅฏพใใฆๆไธใใ ๅ ดๅใ็ฐ็ฉใจใใฆ่ช่ญ'ๆ้คใใใใใใใใฃใฆใๅพๆฅใฎๆใใ B7RP-1ๆไฝใใ็พๆฃใฎ ๆฒป็่ฌๅคใจใใฆ็จใใใใจใฏๅฐ้ฃใงใใใ [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] ใใฎๅ้กใ่งฃๆฑบใใๆนๆณใจใใฆใใใ B7RP-1ใซๅฏพใใใใฆในใขใใฏใญใผใใซๆไฝใใ ่็ฝๅทฅๅญฆ็ๆๆณใ็จใใฆใใๅใใใใจใ่ใใใใๅ ใใฆในใขใใฏใญใผใใซๆไฝ็ฑ ๆฅใฎ้
ๅใไธ้จๅซใใใใๅๅพฉๆไธใ้ทๆๆไธใซใใใๆไธใใใใๅๆ B7RP-1ๆ ไฝใฎๆดปๆงใ้ปๅฎณใใใใใชๆไฝใไฝใใใใใฎๅนๆใ่ใใๆธๅผฑใใใ ใใงใชใ้ ็ฏคใชๅฏไฝ็จใๆใๅฏ่ฝๆงใใใใใพใใใใๅใซใใๆดปๆงใไฝไธใใใใจใๅคใๆง็ฏใซ ใฏๅคๅคงใชๅดๅใจใณในใใ่ฆใใใ ย [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] ๆฌ็บๆใฏใๅฎๅ
จๆงใจๆฒป็ๅนๆใๅ
ผใญๅใใใใๆใใ B7RP-1ๆไฝใใใณใใฎๆญ็ใ ๆไพใใใจใจใใซใใใใใฎๅฉ็จๆนๆณใๆๆกใใใใฎใงใใใ ย [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.
่ชฒ้กใ่งฃๆฑบใใใใใฎๆๆฎต ย Means for solving the problem
[0016] ๆฌ็บๆ่
ใฏใไธ่จ่ชฒ้กใซๅฏพใใฆๆค่จใใ็ตๆใๅฅๅธธไบบใฎๆซๆขข่ก Bใชใณใ็ใใ่ชฟ่ฃฝ ใใๅ
็ซใฐใญใใชใณ H้ใใใณ L้ใฎๅฏๅค้ ๅ (VH,VL)ใใณใผใใใ้บไผๅญใ็บ็พ ใใใใๅ้ Fvๆไฝใใกใผใธใใฃในใใฌใคใฉใคใใฉใชใผใใใๆใใ B7RP-1ๅ้ Fvๆไฝ ๅๅญ (ๆไฝๆญ็๏ผใๅๅพใใใใฎใขใใ้
ธ้
ๅใใใณใใใใณใผใใใ้บไผๅญใฎๅกฉๅบ ้
ๅใๆใใใซใใใใใใซใใใฎๅ้ Fvๆไฝใใใใใฎ B7RP-1ใฎ็็ๆดปๆงใ้ปๅฎณใ ใใใจใ่ฆๅบใใๆฌ็บๆใๅฎๆใใใใซ่ณใฃใใ
[0017] ๆฌ็บๆใซใใๆไฝใฏใใใๆไฝ้บไผๅญใซ็ฑๆฅใใๆไฝใใกใผใธใฉใคใใฉใชใผใ็จใใฆ ๅ้ขใใใใฎใงใใใใใใใฎๆไฝใฏๅฎๅ
จใซใใ็ฑๆฅใฎ้
ๅใใใกใใใฎใพใพใใใธใฎๆฒป ็็จใจใใฆๅฉ็จใใฆใใๅ
็ซๅๆงใจใใฆใฏๅ้กใชใใใฎใงใใใใใฎๆๅณใงใฏใๅพๆฅ ใฎใใฆในใขใใฏใญใผใใซๆไฝๅใใใๅๆ่กใซใใฃใฆไฝ่ฃฝใใใใใๅๆไฝใซใใใฆๅฑ ๆงใใใใใฆใน็ฑๆฅใฎ้
ๅใซๅฏพใใๅ
็ซๅๆงใซใคใใฆใ่งฃๆฑบใใใฆใใใใพใใใใฎ ้็บใณในใใฏใใใๅๆไฝใซๆฏในใฆ้ๅธธใซไฝใใไธๆนใงใ่ฟๅนดใใใใฎๆ่ฒไฝใใใคใใฉ ใณในใฏใญใขใฝใผใ ใใฆในใซใใใใๆไฝ็ฃ็ใ่กใใใฆ ใใใใใใใฎๅๅญใซๅฏพใใ้ป ๅฎณไฝ็จใๆใใใใๆไฝใไฝ่ฃฝใใๆ่กใใๆฌ็บๆใฎใใใซใพใฃใใๅ็ฉใไฝฟ็จใใ ใซไฝ่ฃฝใงใใใใจใฏใๅคงใใชใกใชใใใงใใใ [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.
[0018] ใใชใใกใๆฌ็บๆใฏใๅปๅญฆไธใพใใฏ็ฃๆฅญไธๆ็จใชๆนๆณ'็ฉ่ณชใจใใฆไธ่จ 1)ใ29) ใฎ็บๆใๅซใใใฎใงใใใ ย That is, the present invention includes the following 1) to 29) as medically or industrially useful methods' substances.
[0019] 1)ใใ B7 Related Protein 1 (ไปฅไธใ B7RP- 1)ใซ็ตๅๆงใๆใใใใๆใใ B7RP- 1ๆ ไฝใ ย [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) H้ใฎ็ธ่ฃๆงๆฑบๅฎ้ ๅ (CDR)ใไปฅไธใฎ๏ผa)ใพใใฏ (b)ใฎใขใใ้
ธ้
ๅใๆใใ L ้ใฎ็ธ่ฃๆงๆฑบๅฎ้ ๅ (CDR)ใไปฅไธใฎ๏ผc)ใพใใฏ (d)ใฎใขใใ้
ธ้
ๅใๆใใใไธ่จ 1)ใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝ๏ผ ย [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) CDRlใจใใฆ้
ๅ็ชๅท 1ใ3ใฎใใใใไธใคใ CDR2ใจใใฆ้
ๅ็ชๅท 4ใ6ใฎใ ใใใไธใคใใใใณ CDR3ใจใใฆ้
ๅ็ชๅท 7ใ9ใฎใใใใไธใคใซใใใใใใ็คบใ ใใใขใใ้
ธ้
ๅ๏ผ ย (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) CDRlใ3ใฎใขใใ้
ธ้
ๅใจใใฆใใใใใใ้
ๅ็ชๅท 1ใ3ใ 4ใ6ใใพใใฏ 7ใ9 ใใพใใฏใใใใขใใ้
ธ้
ๅใซใใใฆ 1ใพใใฏใใไปฅไธใฎใขใใ้
ธใ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅ ใใใใณ/ใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใงใใฃใฆใใใ B7RP-1ใซๅฏพใใ H้ใฎ็ธ่ฃ ๆงๆฑบๅฎ้ ๅใจใชใใใใใฎ๏ผ ย (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) CDRlใจใใฆ้
ๅ็ชๅท 10ใ12ใฎใใใใไธใคใ CDR2ใจใใฆ้
ๅ็ชๅท 13ใ15 ใฎใใใใไธใคใ CDR3ใจใใฆ้
ๅ็ชๅท 16ใ18ใฎใใใใไธใคใซใใใใใใ็คบ ใใใใขใใ้
ธ้
ๅ๏ผ ย (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) CDRlใ3ใฎใขใใ้
ธ้
ๅใจใใฆใใใใใใ้
ๅ็ชๅท 10ใ12ใ 13ใ15ใใพใใฏ 16ใ18ใใพใใฏใใใใขใใ้
ธ้
ๅใซใใใฆ 1ใพใใฏใใไปฅไธใฎใขใใ้
ธใ็ฝฎๆใๆฌ
ๅคฑใๆฟๅ
ฅใใใใณ Zใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใงใใฃใฆใใใ B7RP-1ใซๅฏพใใ L้ ใฎ็ธ่ฃๆงๆฑบๅฎ้ ๅใจใชใใใใใฎใ (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) H้ใฎ CDR1ใ3ใฎใขใใ้
ธ้
ๅๅ ้
ๅ็ชๅท 1ใ 4ใใใณ 7ใ้
ๅ็ชๅท 2ใ 5ใใ ใณ 8ใใพใใฏ้
ๅ็ชๅท 3ใ 6ใใใณ 9ใฎ็ตใฟๅใใๅ ้ธๆใใใใขใใ้
ธ้
ๅใงใใใ L้ใฎ CDR1ใ3ใฎใขใใ้
ธ้
ๅๅ ้
ๅ็ชๅท 10ใ 13ใใใณ 16ใ้
ๅ็ชๅท 11ใ 14ใ ใใณ 17ใใพใใฏ้
ๅ็ชๅท 12ใ 15ใใใณ 18ใฎ็ตใฟๅใใๅใ้ธๆใใใใขใใ้
ธ้
ๅ ใงใใไธ่จ 1)ใพใใฏ 2)ใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใ ย [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) H้ใฎ CDR1ใ3ใจ L้ใฎ CDR1ใ3ใจใฎ็ตใฟๅใใใฎใขใใ้
ธ้
ๅๅ ้
ๅ็ช ๅท 1ใ 4ใใใณ 7ใจ้
ๅ็ชๅท 10ใ 13ใใใณ 16ใ้
ๅ็ชๅท 2ใ 5ใใใณ 8ใจ้
ๅ็ชๅท 11ใ 14ใใใณ 17ใใพใใฏ้
ๅ็ชๅท 3ใ 6ใใใณ 9ใจ้
ๅ็ชๅท 12ใ 15ใใใณ 18ใจใฎใใใ ใไธใคใฎ็ตใฟๅใใใงใใใไธ่จ 3)ใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใ ย [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) H้ๅฏๅค้ ๅใไปฅไธใฎ๏ผe)ใพใใฏ (f)ใฎใขใใ้
ธ้
ๅใๆใใ L้ๅฏๅค้ ๅใไปฅ ไธใฎ๏ผg)ใพใใฏ๏ผh)ใฎใขใใ้
ธ้
ๅใๆใใใไธ่จ 1)ๅใ 4)ใฎ ใใใใใซ่จ่ผใฎใใ ๆใใ B7RP- 1ๆไฝ๏ผ ย [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)้
ๅ็ชๅท 19ใ21ใฎใใใใไธใคใฎใขใใ้
ธ้
ๅๅ ้ธๆใใใใขใใ้
ธ้
ๅ๏ผย (e) the amino acid sequence ability of any one of SEQ ID NOS: 19 to 21; selected amino acid sequence;
(f)้
ๅ็ชๅท 19ใ21ใซ็คบใใใใขใใ้
ธ้
ๅใซใใใฆ 1ใพใใฏใใไปฅไธใฎใขใใ้
ธใ ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅใใใใณ Zใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใงใใฃใฆใใใ B7RP-1ใซ ๅฏพใใ H้ๅฏๅค้ ๅใจใชใใใใใฎ๏ผ (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)้
ๅ็ชๅท 22ใ24ใฎ!ใใใใไธใคใฎใขใใ้
ธ้
ๅๅ ้ธๆใใใใขใใ้
ธ้
ๅ๏ผย (g) SEQ ID NOS: 22 to 24 !, any one amino acid sequence ability Selected amino acid sequence;
(h)้
ๅ็ชๅท 22ใ24ใซ็คบใใใใขใใ้
ธ้
ๅใซใใใฆ 1ใพใใฏใใไปฅไธใฎใขใใ้
ธใ ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅใใใใณ Zใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใงใใฃใฆใใใ B7RP-1ใซ ๅฏพใใ L้ๅฏๅค้ ๅใจใชใใใใใฎใ (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) H้ๅฏๅค้ ๅใจ L้ๅฏๅค้ ๅใจใฎ็ตใฟๅใใใฎใขใใ้
ธ้
ๅๅ ้
ๅ็ชๅท 19ใจ ้
ๅ็ชๅท 22ใ้
ๅ็ชๅท 20ใจ้
ๅ็ชๅท 23ใใพใใฏ้
ๅ็ชๅท 21ใจ้
ๅ็ชๅท 24ใจใฎ็ต ใฟๅใใใซใใ็คบใใใใขใใ้
ธ้
ๅใงใใใไธ่จ 1)ใใ 5)ใฎใใใใใซ่จ่ผใฎใใ ๆใใ B7RP- 1ๆไฝใ ย [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)ใใ B7RP-1ใซ็ตๅๆงใๆใใใใๆใใ B7RP-1ๆไฝใฎ H้ๅฏๅค้ ๅใใฉใฐใกใณใใ ย [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)็ธ่ฃๆงๆฑบๅฎ้ ๅ (CDR)ใไปฅไธใฎ๏ผa)ใพใใฏ (b)ใฎใขใใ้
ธ้
ๅใๆใใใไธ่จ 7
)ใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ H้ๅฏๅค้ ๅใใฉใฐใกใณใ๏ผ [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:
(a) CDRlใจใใฆ้
ๅ็ชๅท 1ใ3ใฎใใใใไธใคใ CDR2ใจใใฆ้
ๅ็ชๅท 4ใ6ใฎใ ใใใไธใคใใใใณ CDR3ใจใใฆ้
ๅ็ชๅท 7ใ9ใฎใใใใไธใคใซใใใใใใ็คบใ ใใใขใใ้
ธ้
ๅ๏ผ ย (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) CDRlใ3ใฎใขใใ้
ธ้
ๅใจใใฆใใใใใใ้
ๅ็ชๅท 1ใ3ใ 4ใ6ใใพใใฏ 7ใ9 ใใพใใฏใใใใขใใ้
ธ้
ๅใซใใใฆ 1ใพใใฏใใไปฅไธใฎใขใใ้
ธใ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅ ใใใใณ/ใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใงใใฃใฆใใใ B7RP-1ใซๅฏพใใ H้ใฎ็ธ่ฃ ๆงๆฑบๅฎ้ ๅใจใชใใใใใฎใ ย (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) CDR1ใ3ใฎใขใใ้
ธ้
ๅๅ ้
ๅ็ชๅท 1ใ 4ใใใณ 7ใ้
ๅ็ชๅท 2ใ 5ใใใณ 8ใใพ ใใฏ้
ๅ็ชๅท 3ใ 6ใใใณ 9ใฎ็ตใฟๅใใๅใ้ธๆใใใใขใใ้
ธ้
ๅใงใใใไธ่จ 8) ใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ H้ๅฏๅค้ ๅใใฉใฐใกใณใใ ย [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)ไปฅไธใฎ๏ผe)ใพใใฏ (f)ใฎใขใใ้
ธ้
ๅใๆใใใไธ่จ 7)ๅใ 9)ใฎ ใใใใใซ่จ ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ H้ๅฏๅค้ ๅใใฉใฐใกใณใ๏ผ ย [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):
(e)้
ๅ็ชๅท 19ใ21ใฎใใใใไธใคใฎใขใใ้
ธ้
ๅๅ ้ธๆใใใใขใใ้
ธ้
ๅ๏ผย (e) the amino acid sequence ability of any one of SEQ ID NOS: 19 to 21; selected amino acid sequence;
(f)้
ๅ็ชๅท 19ใ21ใซ็คบใใใใขใใ้
ธ้
ๅใซใใใฆ 1ใพใใฏใใไปฅไธใฎใขใใ้
ธใ ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅใใใใณ Zใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใงใใฃใฆใใใ B7RP-1ใซ ๅฏพใใ H้ๅฏๅค้ ๅใจใชใใใใใฎใ (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)ใใ B7RP-1ใซ็ตๅๆงใๆใใใใๆใใ B7RP-1ๆไฝใฎ L้ๅฏๅค้ ๅใใฉใฐใกใณใ ย [0029] 11) Human anti-human B7RP-1 antibody L chain variable region fragment having binding to human B7RP-1
[0030] 12)็ธ่ฃๆงๆฑบๅฎ้ ๅ (CDR)ใไปฅไธใฎ๏ผc)ใพใใฏ (d)ใฎใขใใ้
ธ้
ๅใๆใใใไธ่จ 11)ใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ L้ๅฏๅค้ ๅใใฉใฐใกใณใ๏ผ [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) CDRlใจใใฆ้
ๅ็ชๅท 10ใ12ใฎใใใใไธใคใ CDR2ใจใใฆ้
ๅ็ชๅท 13ใ15 ใฎใใใใไธใคใใใใณ CDR3ใจใใฆ้
ๅ็ชๅท 16ใ18ใฎใใใใไธใคใซใใใใ ใใ็คบใใใใขใใ้
ธ้
ๅ๏ผ ย (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) CDRlใ3ใฎใขใใ้
ธ้
ๅใจใใฆใใใใใใ้
ๅ็ชๅท 10ใ18ใ 13ใ15ใฎใใ ใใไธใคใใพใใฏ 16ใ18ใใพใใฏใใใใขใใ้
ธ้
ๅใซใใใฆ 1ใพใใฏใใไปฅไธใฎใก ใใ้
ธใ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅใใใใณ/ใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใงใใฃใฆใใใๆใ ใ B7RP-1ๆไฝใฎ L้ใฎ็ธ่ฃๆงๆฑบๅฎ้ ๅใจใชใใใใใฎใ
[0031] 13) CDR1ใ3ใฎใขใใ้
ธ้
ๅใใ้
ๅ็ชๅท 10ใ 13ใใใณ 16ใ้
ๅ็ชๅท 11ใ 14ใ ใใณ 17ใใพใใฏ้
ๅ็ชๅท 12ใ 15ใใใณ 18ใฎ็ตใฟๅใใๅใ้ธๆใใใใขใใ้
ธ้
ๅ ใงใใใไธ่จ 12)ใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ L้ๅฏๅค้ ๅใใฉใฐใกใณใใ (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)ไปฅไธใฎ๏ผg)ใพใใฏ (h)ใฎใขใใ้
ธ้
ๅใๆใใใไธ่จ 11)ๅใ 13)ใฎใใใใใซ ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ L้ๅฏๅค้ ๅใใฉใฐใกใณใ๏ผ ย [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):
(g)้
ๅ็ชๅท 22ใ24ใฎ!ใใใใไธใคใฎใขใใ้
ธ้
ๅๅ ้ธๆใใใใขใใ้
ธ้
ๅ๏ผย (g) SEQ ID NOS: 22 to 24 !, any one amino acid sequence ability Selected amino acid sequence;
(h)้
ๅ็ชๅท 22ใ24ใซ็คบใใใใขใใ้
ธ้
ๅใซใใใฆ 1ใพใใฏใใไปฅไธใฎใขใใ้
ธใ ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅใใใใณ Zใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใงใใฃใฆใใใ B7RP-1ใซ ๅฏพใใ L้ๅฏๅค้ ๅใจใชใใใใใฎใ (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.
[0033] 15)ไธ่จ 7)ๅใ 10)ใฎใใใใใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ H้ๅฏๅค้ ๅใใฉ ใฐใกใณใใจใไธ่จ 11)ๅ 14)ใฎใใใใใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ L้ๅฏๅค้ ๅใใฉใฐใกใณใใจใ้ฃ็ตใใฆใชใใใใ B7RP-1ใซๅฏพใใใใ็ฑๆฅใฎๆไฝใฎไธๆฌ้ๅฏๅค้ ๅใใฉใฐใกใณใใ ย [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)ไธ่จ 7)ใใ 10)ใฎ!ใใใใใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ H้ๅฏๅค้ ๅใใฉ ใฐใกใณใใใใใณ Zใพใใฏไธ่จ 11)ๅ 14)ใฎใใใใใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝ ใฎ L้ๅฏๅค้ ๅใใฉใฐใกใณใใซใใใ็ฑๆฅใฎๆไฝๅฎๅธธ้ ๅใ้ฃ็ตใใฆใชใใ B7RP-1ใซ ๅฏพใใใใ็ฑๆฅใฎๆไฝใพใใฏใใฎๆไฝใใฉใฐใกใณใใ ย [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)ๅฝ่ฉฒๆไฝใใฉใฐใกใณใๅ Fabใ Fab'ใ F(ab')ใ scAbใใพใใฏ scFv-Fcใงใใไธ่จ ย [0035] 17) The above antibody fragment power Fab, Fab ', F (ab'), scAb, or scFv-Fc
2 ย 2
16)ใซ่จ่ผใฎๆไฝใใฉใฐใกใณใใ ย The antibody fragment according to 16).
[0036] 18)ไธ่จ 1)ใใ 17)ใฎใใใใใซ่จ่ผใฎๆไฝใพใใฏใใฎใใฉใฐใกใณใใจใใใใๆ ใใฏไปใฎใฟใณใใฏ่ณชใจใ่ๅใใใ่ๅๆไฝใพใใฏใใฎใใฉใฐใกใณใใ [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)ไธ่จ 1)ใใ 18)ใฎใใใใใซ่จ่ผใฎๆไฝใใใใฏ่ๅๆไฝใพใใฏใใฎใใฉใฐใก ใณใใซไฟฎ้ฃพๅคใ็ตๅใใใฆใชใไฟฎ้ฃพๆไฝใพใใฏใใฎใใฉใฐใกใณใใ [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)ไธ่จ 1)ใใ 19)ใฎใใใใใซ่จ่ผใฎๆไฝใใใใฏ่ๅๆไฝใพใใฏใใฎใใฉใฐใก ใณใใใณใผใใใ้บไผๅญใ [0038] 20) A gene encoding the antibody or fusion antibody or fragment thereof according to any one of 1) to 19) above.
[0039] 21)ไธ่จ 20)ใซ่จ่ผใฎ้บไผๅญใๅซใ็ตๆใ็บ็พใใฏใฟใผใ [0039] 21) A recombinant expression vector comprising the gene according to 20) above.
[0040] 22)ไธ่จ 20)ใซ่จ่ผใฎ้บไผๅญใๅฐๅ
ฅใใใๅฝข่ณช่ปขๆไฝใ [0040] 22) A transformant into which the gene according to 20) above is introduced.
[0041] 23)ไธ่จ 20)ใซ่จ่ผใฎ้บไผๅญใๅฎฟไธปใซ็บ็พใใใใใจใซใใฃใฆใใใๆ B7RP-1ๆไฝ
ใพใใฏใใฎๆญ็ใ็็ฃใใๆนๆณใ [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)ไธ่จ 1)ใใ 17)ใฎใใใใใซ่จ่ผใฎๆไฝใพใใฏใใฎใใฉใฐใกใณใใใพใใฏไธ่จ 1 8)ใซ่จ่ผใฎ่ๅๆไฝใพใใฏใใฎใใฉใฐใกใณใใใพใใฏไธ่จ 19)ใซ่จ่ผใฎไฟฎ้ฃพๆไฝใพใ ใฏใใฎใใฉใฐใกใณใใ็จใใ B7RP-1ใฎๆคๅบ่ฉฆ่ฌใ ย [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)ไธ่จ 20)ใซ่จ่ผใฎ้บไผๅญใๅซใ้บไผๅญๆฒป็ๅคใ ย [0043] 25) A gene therapy agent comprising the gene according to 20) above.
[0044] 26)ไธ่จ 1)ใใ 17)ใฎใใใใใซ่จ่ผใฎๆไฝใพใใฏใใฎใใฉใฐใกใณใใใพใใฏไธ่จ 1 8)ใซ่จ่ผใฎ่ๅๆไฝใพใใฏใใฎใใฉใฐใกใณใใใพใใฏไธ่จ 19)ใซ่จ่ผใฎไฟฎ้ฃพๆไฝใพใ ใฏใใฎใใฉใฐใกใณใใ็จใใ B7RP-1ใจ ICOSใจใฎ็ตๅๆดปๆง้ปๅฎณๅคใ ย [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)ไธ่จ 1)ใใ 17)ใฎใใใใใซ่จ่ผใฎๆไฝใพใใฏใใฎใใฉใฐใกใณใใใพใใฏไธ่จ 1 8)ใซ่จ่ผใฎ่ๅๆไฝใพใใฏใใฎใใฉใฐใกใณใใใพใใฏไธ่จ 19)ใซ่จ่ผใฎไฟฎ้ฃพๆไฝใพใ ใฏใใฎใใฉใฐใกใณใใ่ช่ญใใ B7RP-1ไธใฎๆๅๆฑบๅฎ้ ๅใซๅบใฅใใฆๅๅญ่จญ่จใใใ ไฝๅๅญๅๅ็ฉใพใใฏใใฎ่ชๅฐไฝใ ย [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)ไธ่จ 27)ใซ่จ่ผใฎไฝๅๅญๅๅ็ฉใพใใฏใใฎ่ชๅฐไฝใ็จใใ B7RP-1ใจ ICOSใจ ใฎ็ตๅๆดปๆง้ปๅฎณๅคใ ย [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)ไธ่จ 26)ใพใใฏไธ่จ 28)ใซ่จ่ผใฎ็ตๅๆดปๆง้ปๅฎณๅคใ็จใใ B7RP-1ใจ ICOSใจ ใฎ็ธไบไฝ็จใซใใๆน่ตทใใใ็็ใใใณๅ
็ซ็ฐๅธธๆง็พๆฃใฎไบ้ฒใพใใฏๆฒป็่ฌใ ็บๆใฎๅนๆ ย [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] ๆฌ็บๆใฎใใใขใใฏใญใผใใซๆไฝใใใณ่ฉฒๆไฝใใฉใฐใกใณใๅๅญใฏใใใ็ฑๆฅๆใใ B7 RP-1ๆไฝใฎๅฏๅค้ ๅใๆใใใใ B7RP-1ใจๅผทใๅๅฟใใฆใใใฎ ICOSใจใฎ็ธไบไฝ็จใ ้ปๅฎณใใใใใฎใใจใใใๆฌ็บๆใฎๆไฝใใใณ่ฉฒๆไฝใใฉใฐใกใณใใฏ B7RP-1ใจ ICOSใจ ใฎ็ตๅใซใใๆน่ตทใใใ็็ใใใณๅ
็ซ็ฐๅธธๆง็พๆฃใฎไบ้ฒใพใใฏๆฒป็่ฌใจใใฆไฝฟ็จ ใใใใจใใงใใใ ย [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.
ๅณ้ขใฎ็ฐกๅใช่ชฌๆ ย Brief Description of Drawings
[0049] [ๅณ 1]ใฏใญใผใณใฃๅใใๅ้ Fvๆไฝใใกใผใธใฎ B7RP-1-FCใซๅฏพใใ็ตๅ็น็ฐๆงใ่ฉไพก ใใ ELISAใฎ็ตๆใ็คบใๅณใ ย [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] [ๅณ 2]ใฏใญใผใณใฃๅใใๅ้ Fvๆไฝใฎ B7RP-1-FCใซๅฏพใใ็ตๅ็น็ฐๆงใ่ฉไพกใใ ELI[0050] [Fig.2] ELI evaluated for binding specificity of cloned single-chain Fv antibody to B7RP-1-FC
SAใฎ็ตๆใ็คบใๅณใ The figure which shows the result of SA.
[0051] [ๅณ 3]ๅ้ขใใๆ B7RP-1ๅ้ Fvๆไฝใจใปใณใตใผใใใไธใซๅบๅฎๅใใใ B7RP1ใจใฎ
็ตๅใฎ้ๅบฆ่ซ็่งฃๆใ่กจ้ขใใฉใบใขใณๅ
ฑ้ณดใซใใฃใฆ่ฉไพกใใ็ตๆใ็คบใๅณใ [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] [ๅณ 4]ๅ้ขใใใๆ B7RP-1ๅ้ Fvๆไฝใซใใ ICOS-Fcใจ B7RP-1-FC้ใฎ็ตๅ้ปๅฎณ ๆดปๆงใ่ฉไพกใใ ELISAใฎ็ตๆใ็คบใๅณใ [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] [ๅณ 5]ใใญใผใตใคใใกใผใฟใ็จใใๅ้ขใใๆ B7RP-1ๅ้ Fvๆไฝใฎ B็ดฐ่ใธใฎ็ตๅ ๆงใ่ฉไพกใใ็ตๆใ็คบใๅณ๏ผFITCไธฆใณใซ PEใงใจใใซๆ่ฒใใใ้ๅฃใใใใฏในใง็คบ ใใฆใใ๏ผใ [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] [ๅณ 6]ๅ้ขใใๆ B7RP-1ๅ้ Fvๆไฝใ็จใใ ICOS่ฃๅฉๅบๆฟใทใฐใใซใฎใใญใใฏใซใ ใ T็ดฐ่ๅขๆฎ้ปๅฎณๆดปๆงใ่ฉไพกใใ็ตๆใ็คบใๅณใ ย [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.
็บๆใๅฎๆฝใใใใใฎๆ่ฏใฎๅฝขๆ
ย BEST MODE FOR CARRYING OUT THE INVENTION
[0055] ๆฌ็บๆใฎๆไฝใใใณๆไฝใใฉใฐใกใณใใซไฝฟ็จใใใ scFvใฏไปฅไธใฎใใใซใใฆๅพใใ ใใ [0055] The scFv used for the antibodies and antibody fragments of the present invention was obtained as follows.
[0056] ๅฅๅธธ่
20ๅๅใฎๆซๆขข่ก Bใชใณใ็ใใใ RTโ PCRๆณใซใฆใๅ
็ซใฐใญใใชใณ้ (H) ้ใ่ปฝ (L)้ cDNAใๅขๅน
ใๆดใซไธก่
ใใชใณใซไธ DNAใง็ตๅใใๅฅๅธธ่
ใชใณใ็็ฑ ๆฅใฎ H้ๅฏๅค้ ๅ (VH้ใพใใฏ VH)ใจ L้ๅฏๅค้ ๅ (VL้ใพใใฏ VL)ใฎใฉใณใใ ใช็ตใฟๅใใใซใใ scFv DNAใไฝ่ฃฝใใใ ย [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] ใใฎ scFv DNAใใใกใผใธใใใใฏใฟใผ pCANTAB5Eใซ็ต่พผใฟใ 109ใฏใญใผใณใใใชใ ๅฅๅธธ่
็ฑๆฅ scFvใใฃในใใฌใคใใกใผใธใฉใคใใฉใชใผใไฝ่ฃฝใใใใใฎใฉใคใใฉใชใผใใๅบ ็ธใซๅบๅฎใฃๅใใใใใ B7RP-1ใจ็ตๅใใใฆๅๅใๆฟ็ธฎใใๆใใ B7RP-1 scFvใใคใน ใใฌใคใใกใผใธใฏใญใผใณใในใฏใชใผใใณใฐใใใใใฎ็ตๆใในใฏใชใผใใณใฐใใใๅใฏใญ ใผใณใฏใใใ B7RP- 1ใจ็ตๅใใ scFvใ็ฃ็ใใใ [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] scFvใฎ็บ็พๆนๆณใจใใฆใฏใไพใใฐใๅคง่
ธ่ใง็บ็พใใใใใจใใงใใใๅคง่
ธ่ใฎๅ ด ๅใๅธธ็จใใใๆ็จใชใใญใขใผใฟใผใๆไฝๅๆณใฎใใใฎใทใฐใใซ้
ๅ็ญใ็บ็พใใใ scFvใๆฉ่ฝ็ใซ็ตๅใใใฆ็บ็พใใใใใจใใงใใใไพใใฐใใญใขใผใฟใผใจใใฆใฏใ la cZใใญใขใผใฟใผใ araBใใญใขใผใฟใผ็ญใๆใใใใจใใงใใใ scFvใฎๅๆณใฎใใใฎใท ใฐใใซ้
ๅใจใใฆใฏใๅคง่
ธ่ใฎใใชใใฉใบใ ใซ็บ็พใใใๅ ดๅใ pelBใทใฐใใซ้
ๅ๏ผL ei, SP.ใใ J. BacterioL, 1987, 169๏ผ 4379- 4383)ใ็จใใใจใใใๅน้คไธๆธ
ไธญใซๅๆณ ใใใใซใฏ M13ใใกใผใธใฎ g3่็ฝใฎใทใฐใใซ้
ๅใ็จใใใใจใใงใใใ ย [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] ็บ็พใใใ scFvใฏ็ดฐ่ๅ
ๅคใๅฎฟไธปๅใๅ้ขใๅไธใซใพใง็ฒพ่ฃฝใใใใจใใงใใใๆฌ
็บๆใง็บ็พใใใ scFvใฏใใใฎ Cๆซ็ซฏใซ E tag้
ๅใไปๅ ใใใฆใใใฎใงใๆ E tag ๆไฝใ็จใใใกใใฃใฆใใฃใผใฏใญใใใฐใฉใใฃใผใ็จใใฆใๅฎนๆใซ็ญๆ้ใง็ฒพ่ฃฝใใใ ใจใใงใใใใใฎไปใ้ๅธธใฎใฟใณใใฏ่ณชใงไฝฟ็จใใใฆใใๅ้ขใ็ฒพ่ฃฝๆนๆณใ็ตใฟๅใ ใใฆ็ฒพ่ฃฝใใใใจใๅฏ่ฝใงใใใไพใใฐใ้ๅคๆฟพ้ใๅกฉๆใใฒใซๆฟพ้ Zใคใชใณไบคๆ Z ็ๆฐดใฏใญใใ็ญใฎใซใฉใ ใฏใญใใใฐใฉใใฃใผใ็ตใฟๅใใใใฐๆไฝใๅ้ข.็ฒพ่ฃฝใใใ ใจใใงใใใ [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] ๅพใใใๆไฝใๆไฝใใฉใฐใกใณใใฎใใ B7RP-1ใซๅฏพใใ็ตๅๆดปๆงใๆธฌๅฎใใๆนๆณใจ ใใฆใฏใ ELISAใ BIAcore็ญใฎๆนๆณใใใใไพใใฐ ELISAใ็จใใๅ ดๅใใใ B7RP- 1 -Fcใๅบ็ธๅใใ 96็ฉดใใฌใผใใซ็ฎ็ใฎๆไฝใๆไฝใใฉใฐใกใณใใๅซใ่ฉฆๆใไพใใฐ ๅคง่
ธ่ใฎๅน้คไธๆธ
ใ็ฒพ่ฃฝๆไฝใๅ ใใใๆฌกใซใใผใฉใญใทใใผใผ็ญใฎ้
ต็ด ใงๆจ่ญใ ใไบๆฌกๆไฝใๆทปๅ ใใใใฌใผใใใคใณใญใฅใใผใทใงใณใๆดๆตใใๅพใ็บ่ฒๅบ่ณช TMBZ ใๅ ใใฆๅธๅ
ๅบฆใๆธฌๅฎใใใใจใงๆๅ็ตๅๆดปๆงใ่ฉไพกใใใใจใใงใใใใพใใ BIAco reใ็จใใๅ ดๅใใปใณใตใผใใใใซ B7-RP1-FCใๅบๅฎๅใใๅใใพใใฏๆใใ Fc F(ab ')ๆไฝใซ B7-RP1-FCใใญใคใใใคใผใใใฆใ็ฎ็ใฎ่ฉฆๆใฎ็ตๅ่งฃ้ขๅฎๆฐใๆธฌๅฎใใย [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 โฒ) antibody. Measure
2 2
ใใจใใงใใใ ย be able to.
[0061] ใพใใๅพใใใๆไฝใๆไฝใใฉใฐใกใณใใฎ B7RP-1/ICOS็ตๅ้ปๅฎณๆดปๆงใๆธฌๅฎใใ ๆนๆณใจใใฆใ ELISA, BIAcore็ญใฎๆนๆณใใใใไพใใฐ ELISAใ็จใใๅ ดๅใใใ B7R ฮก-1-Fcใๅบ็ธๅใใ 96็ฉดใใฌใผใใซ็ฎ็ใฎๆไฝใๆไฝใใฉใฐใกใณใใๅซใ่ฉฆๆใจใใ ใฉใใณๆจ่ญใใ ICOS-Fcใๆททๅใใใใฎใใซๅฉใใใๆฌกใซใใผใฉใญใทใใผใผ็ญใฎ้
ต็ด ใงๆจ่ญใใในใใฌใใใขใใธใณใๆทปใซใญใใใใฌใผใใใคใณใญใฅใใผใทใงใณใๆดๆตใใๅพใ ็บ่ฒๅบ่ณช TMBZใๅ ใใฆๅธๅ
ๅบฆใๆธฌๅฎใใใใจใง B7RP-1/ICOS็ตๅ้ปๅฎณๆดปๆงใ่ฉ ไพกใใใใจใใงใใใ ย [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] ๆดใซใๅพใใใๆไฝใๆไฝใใฉใฐใกใณใใซใคใใฆใๆซๆขข่ก Bใชใณใ็ไธใฎ B7RP-1ใธ ใฎ็ตๅๆงใ่ชฟในใๆนๆณใจใใฆใใใญใผใตใคใใกใผใฟใ็จใใๆนๆณใใใใไพใใฐใใใๆซ ๆขข่กใชใณใ็ใ็จใใฆใ่ๅ
ใใญใผใตใคใใกใผใฟใง่งฃๆใใๅ ดๅใใใๆซๆขข่กใใๆซๆขข ่กใชใณใ็ใ็ฒพ่ฃฝใใ PMAใจ PHAใงๅบๆฟใใๅพใใ HgGๆไฝใใซใญใใฆใ HgG Fe yใฌใป ใใฟใผใฎใใญใใฏใ่ก ใใ็ฎ็ใฎๆไฝใๆไฝใใฉใฐใกใณใใๅซใ่ฉฆๆ๏ผE tagใไปใซใญใ ใ scFvใงใใใฐ scFvใๅซใ่ฉฆๆใจๆ E tagๆไฝใฎๆททๅ็ฉ๏ผใๅ ใๅๅฟใใใใ็ดฐ่
ใๆดๆตๅพใ PEใฉใใซใคๅใใในใใฌใใใขใใธใณใชใใณใซใใใ B็ดฐ่็น็ฐ็ใใผใซใผใจใ ใฆใ FITCใงใฉใใซใคๅใใๆ CD19ๆไฝใๅๅฟใใใ่ๅ
ใฉใใซๅใใใๆดๆตๅพใ่ๅ
ใใญใผใตใคใใกใผใฟใ็จใใฆใ PEใชใใณใซ FITCใใฃใณใใซใงใฎ 2ๆฌกๅ
ใใญใผใตใคใใกใใช ไธ่งฃๆใ่ก ใฝใๆซๆขข่ก Bใชใณใ็ไธใฎ B7RP-1ใธใฎ็ตๅๆงใ่ฉไพกใใใใจใใงใใใ [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] ไธ่จใฎๆไฝใๆไฝใใฉใฐใกใณใใซใคใใฆใ T็ดฐ่่ฃๅฉๅบๆฟใทใฐใใซใซๅฏพใใ้ปๅฎณๆดป ๆงใ่ชฟในใๆนๆณใจใใฆใฏใ T็ดฐ่ๅขๆฎๅบๆฟใขใใปใฃใใใใไพใใฐใ 96็ฉดใใฌใผใใซใ ๆ CD3ๆไฝใจๆใ HgG Fcใใฉใฐใกใณใ F(ab')ๆไฝใฎๆททๅๆถฒใใณใผใใๆดๆตใใใฎใกใ B [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,
2 ย 2
7RP-1-FCใๅ ใใฆๅๅฟใใใใๅๅบฆใใฌใผใใๆดๆตใใๅพใ ็ฎ็ใฎๆไฝใๆไฝใใฉ ใฐใกใณใใๅซใ่ฉฆๆใๅ ใๅๅฟใใใใใฎๅพใใๆซๆขข่กใใ่ชฟ่ฃฝใใๆซๆขข่กใชใณใ็ใ ๅ ใใๅน้คใใใๅน้คไธญใซใใชใใฆใ ใใใธใณใๆทปๅ ใใ็ดฐ่ใๅใ่พผใใ ใใชใใฆใ ใ ใใธใณ้ใๆธฌๅฎใใใใจใงใ T็ดฐ่่ฃๅฉๅบๆฟใทใฐใใซใซๅฏพใใ้ปๅฎณๆดปๆงใ่ฉไพกใใใ ใจใใงใใใ ย 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] ไธ่จใฎๆนๆณใซใใใๆใใ B7RP-1 scFvใๅ้ขใ่ฉไพกใใ็ตๆใ B7RP-1ใซ็น็ฐ็ใซ ็ตๅใใใใจใใๅใใใฎ่ฆชๅๆงใฏใฌใปใใฟใผใงใใ ICOSใซๅนๆตใใ็จ้ซใใใฎใงใ ใใใจใๆๅๆ็คบ็ดฐ่ไธใซ็บ็พใใใฆใใ B7RP-1ใซใ็ตๅใใใใใจใ B7RP-1ใจ ICOS ใจใฎ็ตๅใ้ปๅฎณใใใใจใใใใณ B7RP-1/ICOSใไปใใ่ฃๅฉๅบๆฟใซใใ T็ดฐ่ใฎๅข ๆฎใๆๅถใใใใจใ็คบใใใใๅพใฃใฆใใใใฎๆไฝใฏใ็ไฝๅ
ใงใๅๆงใฎๅนๆใ็คบใ ใB7RP-l/ICOSใฎ็ตๅใใใณ T็ดฐ่ใฎๅขๆฎใๆๅถใใ่ฌๅคใจใใฆๆ็จใงใใใจ่ใ ใใใใ ย [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] ไธ่จ้ปๅฎณๆดปๆงใๆใใ 3็จฎ้กใฎ scFv (223ใ 323ใ 325)ใฎ VH้ใใใณ VL้ใฎใก ใใ้
ธ้
ๅใใใณใใใใณใผใใใๅกฉๅบ้
ๅใฏใไธ่จใฎ้ใใงใใใ ย [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)ใฏใญใผใณ 223 ย (1) Clone 223
ใฏใญใผใณ 223ใฎ VH้ใฎใขใใ้
ธ้
ๅใ้
ๅ็ชๅท 19ใซ็คบใใใๅฝ่ฉฒ VH้ใฎ CDR1 ใ3ใฎใขใใ้
ธ้
ๅใ้
ๅ็ชๅท 1ใ 4ใใใณ 7ใซ็คบใใใใใชใใกใ้
ๅ็ชๅท 19ใซ็คบใ VH้ใฎใขใใ้
ธ้
ๅใซใใใฆใ 31็ช็ฎใ35็ช็ฎใฎใขใใ้
ธ้
ๅใ CDR1 (้
ๅ็ช ๅท 1)ใ 50็ช็ฎใ66็ช็ฎใฎใขใใ้
ธ้
ๅใ CDR2 (้
ๅ็ชๅท 4)ใ 99็ช็ฎใ109็ช็ฎ ใฎใขใใ้
ธ้
ๅใ CDR3 (้
ๅ็ชๅท 7)ใซๅฏพๅฟใใฆใใใใพใใๅฝ่ฉฒ VH้ใใณใผใใใ ้บไผๅญใฎๅกฉๅบ้
ๅใ้
ๅ็ชๅท 25ใซ็คบใใใ
[0066] ใพใใใฏใญใผใณ 223ใฎ VL้ใฎใขใใ้
ธ้
ๅใ้
ๅ็ชๅท 22ใซ็คบใใใๅฝ่ฉฒ VL้ใฎ C DR1ใ3ใฎใขใใ้
ธ้
ๅใ้
ๅ็ชๅท 10ใ 13ใใใณ 16ใซ็คบใใใใใชใใกใ้
ๅ็ชๅท 22ใซ็คบใ VL้ใฎใขใใ้
ธ้
ๅใซใใใฆใ 24็ช็ฎใ35็ช็ฎใฎใขใใ้
ธ้
ๅใ CDR1 (้
ๅ็ชๅท 10)ใ 51็ช็ฎใ57็ช็ฎใฎใขใใ้
ธ้
ๅใ CDR2 (้
ๅ็ชๅท 13)ใ 90็ช็ฎ ใ98็ช็ฎใฎใขใใ้
ธ้
ๅใ CDR3 (้
ๅ็ชๅท 16)ใซๅฏพๅฟใใฆใใใใพใใๅฝ่ฉฒ VL้ ใใณใผใใใ้บไผๅญใฎๅกฉๅบ้
ๅใ้
ๅ็ชๅท 28ใซ็คบใใใ 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)ใฏใญใผใณ 323 ย [0067] (2) Clone 323
ใฏใญใผใณ 323ใฎ VH้ใฎใขใใ้
ธ้
ๅใ้
ๅ็ชๅท 20ใซ็คบใใใๅฝ่ฉฒ VH้ใฎ CDR1 ใ3ใฎใขใใ้
ธ้
ๅใ้
ๅ็ชๅท 2ใ 5ใใใณ 8ใซ็คบใใใใใชใใกใ้
ๅ็ชๅท 20ใซ็คบใ VH้ใฎใขใใ้
ธ้
ๅใซใใใฆใ 30็ช็ฎใ34็ช็ฎใฎใขใใ้
ธ้
ๅใ CDR1 (้
ๅ็ช ๅท 2)ใ 49็ช็ฎใ65็ช็ฎใฎใขใใ้
ธ้
ๅใ CDR2 (้
ๅ็ชๅท 5)ใ 98็ช็ฎใ108็ช็ฎ ใฎใขใใ้
ธ้
ๅใ CDR3 (้
ๅ็ชๅท 8)ใซๅฏพๅฟใใฆใใใใพใใๅฝ่ฉฒ VH้ใใณใผใใใ ้บไผๅญใฎๅกฉๅบ้
ๅใ้
ๅ็ชๅท 26ใซ็คบใใใ ย 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] ใพใใใฏใญใผใณ 323ใฎ VL้ใฎใขใใ้
ธ้
ๅใ้
ๅ็ชๅท 23ใซ็คบใใใๅฝ่ฉฒ VL้ใฎ C DR1ใ3ใฎใขใใ้
ธ้
ๅใ้
ๅ็ชๅท 11ใ 14ใใใณ 17ใซ็คบใใใใใชใใกใ้
ๅ็ชๅท 23ใซ็คบใ VL้ใฎใขใใ้
ธ้
ๅใซใใใฆใ 23็ช็ฎใ33็ช็ฎใฎใขใใ้
ธ้
ๅใ CDR1 (้
ๅ็ชๅท 11)ใ 49็ช็ฎใ55็ช็ฎใฎใขใใ้
ธ้
ๅใ CDR2 (้
ๅ็ชๅท 14)ใ 88็ช็ฎ ใ98็ช็ฎใฎใขใใ้
ธ้
ๅใ CDR3 (้
ๅ็ชๅท 17)ใซๅฏพๅฟใใฆใใใใพใใๅฝ่ฉฒ VL้ ใใณใผใใใ้บไผๅญใฎๅกฉๅบ้
ๅใ้
ๅ็ชๅท 29ใซ็คบใใใ ย [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)ใฏใญใผใณ 325 ย [0069] (3) Clone 325
ใฏใญใผใณ 325ใฎ VH้ใฎใขใใ้
ธ้
ๅใ้
ๅ็ชๅท 21ใซ็คบใใใๅฝ่ฉฒ VH้ใฎ CDR1 ใ3ใฎใขใใ้
ธ้
ๅใ้
ๅ็ชๅท 3ใ 9ใใใณ 15ใซ็คบใใใใใชใใกใ้
ๅ็ชๅท 21ใซ็คบ ใ VH้ใฎใขใใ้
ธ้
ๅใซใใใฆใ 31็ช็ฎใ35็ช็ฎใฎใขใใ้
ธ้
ๅใ CDR1 (้
ๅ ็ชๅท 3)ใ 50็ช็ฎใ66็ช็ฎใฎใขใใ้
ธ้
ๅใ CDR2 (้
ๅ็ชๅท 9)ใ 99็ช็ฎใ108็ช ็ฎใฎใขใใ้
ธ้
ๅใ CDR3 (้
ๅ็ชๅท 15)ใซๅฏพๅฟใใฆใใใใพใใๅฝ่ฉฒ VH้ใใณใผใ ใใ้บไผๅญใฎๅกฉๅบ้
ๅใ้
ๅ็ชๅท 27ใซ็คบใใใ ย 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] ใพใใใฏใญใผใณ 325ใฎ VL้ใฎใขใใ้
ธ้
ๅใ้
ๅ็ชๅท 24ใซ็คบใใใๅฝ่ฉฒ VL้ใฎ C DR1ใ3ใฎใขใใ้
ธ้
ๅใ้
ๅ็ชๅท 12ใ 15ใใใณ 18ใซ็คบใใใใใชใใกใ้
ๅ็ชๅท
24ใซ็คบใ VL้ใฎใขใใ้
ธ้
ๅใซใใใฆใ 23็ช็ฎใ33็ช็ฎใฎใขใใ้
ธ้
ๅใ CDR1 (้
ๅ็ชๅท 12)ใ 49็ช็ฎใ55็ช็ฎใฎใขใใ้
ธ้
ๅใ CDR2 (้
ๅ็ชๅท 15)ใ 88็ช็ฎ ใ98็ช็ฎใฎใขใใ้
ธ้
ๅใ CDR3 (้
ๅ็ชๅท 18)ใซๅฏพๅฟใใฆใใใใพใใๅฝ่ฉฒ VL้ ใใณใผใใใ้บไผๅญใฎๅกฉๅบ้
ๅใ้
ๅ็ชๅท 30ใซ็คบใใใ [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] ๆฌ็บๆใซใใๅพใใใใฏใญใผใณๆฏใซใขใใ้
ธ้
ๅใใใณๅกฉๅบ้
ๅใซใคใใฆไธ่ฟฐใใ ๅ ้
ๅ่กจใซ่จ่ผใใใๅใขใใ้
ธ้
ๅๆ
ๅ ฑ (VH้ใ VL้ใๅ CDR1ใ3)ใใใจใซ ใๅ็ฌใพใใฏ่คๆฐใฎ้
ๅใ้ฉๅฎ็ตใฟๅใใใฆไฝฟ็จใใใใจใๅฏ่ฝใงใใใ ย [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] ๆฌ็บๆใฎๆไฝใใใณใใฎๆไฝใใฉใฐใกใณใใฏใไธ่จ VH้ใใใณ VL้ใไธฆใณใซใใ ใใฎ CDRใจใใฆใไธ่จ้
ๅ็ชๅทใซ็คบใใใ้
ๅใซ้ๅฎใใใใใฎใงใฏใชใใใใใฎ ไธ้จใๆนๅคใใใๅค็ฐใใชใใใใใงใใฃใฆใใใใ ย [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] ใใชใใกใๅ้
ๅ็ชๅทใซ่จ่ผใใใใขใใ้
ธ้
ๅใซใใใฆใ 1ใพใใฏใใไปฅไธใฎใก ใใ้
ธใ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅใใใใณ Zใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใใใชใใใใคใใ B7RP-1ใซๅฏพใใ H้ใพใใฏ L้ใฎ็ธ่ฃๆงๆฑบๅฎ้ ๅใ H้ใพใใฏ L้ใฎๅฏๅค้ ๅใจใช ใใใชใใใใใๅซใพใใใ ย [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] ใใใงใใ1ใพใใฏใใไปฅไธใฎใขใใ้
ธใ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅใใใใณ Zใพใใฏไปๅ ใใ ใใใจใฏใ้จไฝ็น็ฐ็็ช็ถๅค็ฐ่ช็บๆณ็ญใฎๅ
ฌ็ฅใฎๅค็ฐใฟใณใใฏ่ณชไฝ่ฃฝๆณใซใใ็ฝฎๆ ใๆฌ ๅคฑใๆฟๅ
ฅใใใใณ Zๅใฏไปๅ ใงใใ็จๅบฆใฎๆฐใไพใใฐใ 1ใชใใ 6็จๅบฆใฎๆฐใฎใขใ ใ้
ธใ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅใใใใณ Zๅใฏไปๅ ใใใใใจใๆๅณใใใใใฎใใใชใๅค็ฐใ ใจใใฆใฏใไธปใจใใฆใๅ
ฌ็ฅใฎๅค็ฐใฟใณใใฏ่ณชไฝ่ฃฝๆณใซใใไบบ็บ็ใซๅฐๅ
ฅใใใๅค็ฐใ ๆๅณใใใใๅคฉ็ถ (ไพใใฐใใ๏ผใซๅญๅจใใๅๆงใฎๅค็ฐใใชใใใใใๅ้ข็ฒพ่ฃฝใใใ ใฎใงใใฃใฆใใใใ ย [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 โmutationโ 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] ใชใใไธ่จใๅค็ฐใใฏใๆฌ็บๆใฎๆไฝใพใใฏใใฎๆไฝใใฉใฐใกใณใใใๆฒป็่ฌใจใใฆๅฉ ็จใใๅ ดๅ (ใใใซๆไธใใๅ ดๅ๏ผใซใฏใใใ็ฑๆฅใฎๆง้ ใไฟๆใพใใฏใใใๅ
็ซๅๅฟ ใ่ตทใใใชใ็ฏๅฒใง่กใใๆคๅบๅจๅ
ทใ่จบๆญใญใใใชใฉใจใใฆไฝฟ็จใใๅ ดๅ (ใใใซๆไธ ใใชใๅ ดๅ๏ผใซใฏใใใฎ็ฏๅฒใฏ็นใซๅถ้ใใใชใใ ย [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] ๆฌ็บๆใง้็คบใใใ VH้ใใใณ Zใพใใฏ VL้ใฏใใใกใผใธๆไฝๆณใ็จใใฆไธปใจ ใใฆ scFvใฎๅฝขใงๅพใใใใใฎใงใใๅ ๅๅใจใใฆใใฎ้ฉ็จใฏ scFvใซ้ๅฎใใใใใจ ใฏใชใใไพใใฐใ้็คบใใ VH้ใใใณ Zใพใใฏ VL้ใใใๅ
็ซใฐใญใใชใณใฎๅฎๅธธ้
ๅใจ้ฃ็ตใใๅฎๅ
จๅๅญๅใใพใใใๅ
็ซใฐใญใใชใณใฎๅฎๅธธ้ ๅใฎไธ้จใจ็ตใฟๅใใใ Fab, Fab'ใพใใฏ F(ab')ใใใใซ scFvใใใๅ
็ซใฐใญใใชใณใฎ L้ใฎๅฎๅธธ้ ๅใจ็ตๅใ [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 ย 2
ใใไธๆฌ้ๆไฝ (scAb)ใใ scFvใใใๅ
็ซใฐใญใใชใณใฎ H้ใฎๅฎๅธธ้ ๅใจ็ตๅใใ ใ scFv-Fcใชใฉใฎไปใฎๆไฝใใฉใฐใกใณใใใใฎ้ฉ็จ็ฏๅฒใซๅซใพใใใ ย 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] ใพใใๆฌ็บๆใฎๆไฝใพใใฏใใฎใใฉใฐใกใณใใฏใๅฝ่ฉฒๆไฝใพใใฏใใฉใฐใกใณใใซใบใ ใใๆใใฏไปใฎใฟใณใใฏ่ณชใจ่ๅใใใ่ๅๆไฝใพใใฏใใฎใใฉใฐใกใณใใจใใใใจใ ใงใใใ [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] ใพใใใใใใฎๆไฝใใใณๆไฝใใฉใฐใกใณใใใใใฏไธ่จ่ๅๆไฝใพใใฏใใฎใใฉใฐ ใกใณใใซใใใชใจใใฌใณใฐใชใณใผใซใชใฉใฎ้ซๅๅญไฟฎ้ฃพๅคใ็ตๅใใใไฟฎ้ฃพๆไฝใพใใฏใ ใฎใใฉใฐใกใณใใจใใใใจใใงใใใ ย [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] H้ใจ L้ใฎ Fvใ้ฉๅฝใชใชใณใซไธใง้ฃ็ตใใใ scFvใ่ชฟ่ฃฝใใๅ ดๅใใใใใใชใณ ใซใผใจใใฆใฏใไพใใฐใขใใ้
ธ 10ใ25ๆฎๅบๅใใชใไปปๆใฎไธๆฌ้ใใใใใ็จใใ ใใใ [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] ๆฌ็บๆใฎๆไฝใพใใฏๆไฝใใฉใฐใกใณใใฏใ้
ๅ็ชๅท 25ใใ 30ใซ็คบใใๆฌ็บๆใซใใ ๅพใใใๅใฏใญใผใณใฎ VH้ใใใณ VL้ใใณใผใใใ้บไผๅญ้
ๅๆ
ๅ ฑใใใจใซใ้ฉๅฝ ใชๅฎฟไธป (ไพใใฐใ็ดฐ่ใ้
ตๆฏ๏ผใซๅฐๅ
ฅใใฆใๆฌ็บๆใฎๆไฝใพใใฏใใฎใใฉใฐใกใณใใ็บ ็พใใใใใจใใงใใใ ย [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] ใพใๆฌ็บๆใฎ้บไผๅญใฏใ B7RP-1ใจ ICOSใจใฎ็ธไบไฝ็จใ่ชฟ็ฏใใใใใฎใ้บไผๅญ ๆฒป็ใฎ่ฃๅฉๅคใจใใฆใๅฉ็จใงใใใ ย [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] ๆฌ็บๆใฎๆไฝใพใใฏใใฎๆไฝใใฉใฐใกใณใใฏใ B7RP-1ใจ ICOSใจใฎ็ตๅๆดปๆง้ปๅฎณๅค ใจใใฆๅฉ็จๅฏ่ฝใงใใใ [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] ใพใใใใใใฎๆไฝใพใใฏใใฎใใฉใฐใกใณใใ่ช่ญใใใใ B7RP-1ไธใฎๆๅๆฑบๅฎ้ ๅ ใซๅบใฅใๅๅญ่จญ่จใใใใจใซใใใ B7RP-1/ICOSใฎใทใฐใใชใณใฐใซไฝ็จใใไฝๅๅญๅ ๅ็ฉใฎ้็บใซ้่ฆใชๆๆฎตใๆไพใใใ ย [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] ใใฎไฝๅๅญๅๅ็ฉใซใฏใๆฌ็บๆใฎๆไฝใพใใฏใใฎใใฉใฐใกใณใใ่ช่ญใใใใขใใ้
ธ ้
ๅใใใชใใใใใใใใณใใฎ็ซไฝๆง้ ใๆจกใใใฃใๅ็ฉใๅซใพใใใใใฎใใใใ ใฎใขใใ้
ธ้
ๅใซ้ๅคฉ็ถใขใใ้
ธใๅ ใใๆนๅคใใใใใๅๆงใซไฝฟ็จใงใใใใพใใใ ใใใฎใใใใใไปใฎใฟใณใใฏ่ณชใจ่ๅใใใ่ๅ่็ฝ่ณชใๅๆงใงใใใไธ่จใฎใบใ
ใใใพใใฏๅๅ็ฉใซใใใชใจใใฌใณใฐใชใณใผใซใชใฉใฎ้ซๅๅญไฟฎ้ฃพๅคใ็ตๅใใใไฟฎ้ฃพ ๅๅญใๅๆงใงใใใ [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] ใใใใฎไฝๅๅญๅๅ็ฉใพใใฏใใฎ่ชๅฐไฝใใ B7RP-1ใจ ICOSใจใฎ็ตๅๆดปๆง้ปๅฎณๅคใจ ใใฆๅฉ็จๅฏ่ฝใงใใใ ย [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] ใใใซใๆฌ็บๆใฎๆไฝใพใใฏใใฎๆไฝใใฉใฐใกใณใใไธฆใณใซไฝๅๅญๅๅ็ฉใพใใฏใ ใฎ่ชๅฐไฝใใใชใ็ตๅๆดปๆง้ปๅฎณๅคใฏใ B7RP-1ใจ ICOSใจใฎ็ธไบไฝ็จใซใใๆน่ตทใใใ ็็ใใใณๅ
็ซ็ฐๅธธๆง็พๆฃใฎไบ้ฒใพใใฏๆฒป็่ฌใจใใฆๆๅนใงใใใ ย [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.
[0087] ไปฅไธใๆฌ็บๆใๅฎๆฝไพใซๅบใฅใ่ฉณ็ดฐใซ่ชฌๆใใใใๆฌ็บๆใฏไฝใใใใใซ้ๅฎใ ใใใใฎใงใฏใชใใ ย Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.
ๅฎๆฝไพ ย Example
[0088] ใๅฎๆฝไพ 1๏ผๅฅๅธธ่
ใใใฎใใกใผใธใฉใคใใฉใชใผใฎๆง็ฏใ ย [0088] <Example 1: Construction of phage library from healthy subjects>
ใใกใผใธใฉใคใใฉใชใผใฎๆง็ฏใฏใ J. D. Marksใ๏ผJ. Mol. Biol, 222: 581-597, 1991) ใซใใๅ ฑๅใใใฆใใๆนๆณใๅ่ใซใๅฅๅธธ่
20ๅ็ฑๆฅๆซๆขข่ก็ฑๆฅใชใณใ็ใๅบ็บๆ ๆใซใๆง็ฏใใใๆง็ฏใใ VH( ฮณ )โ V ฮบใ VH( ฮณ )โ Vใใ VH( ๏ผโ V ฮบใ VH ( ๏ผ V ใฎๅใตใใฉใคใใฉใชไธใฏใใใใ 1.1 X 108ใ 2.1 X 108ใ 8.4 X 107ใ 5.3 X 107ใฏใญใผ ใณใฎๅคๆงๆงใๆใใใจ่ฉไพกใใใใ 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 (ฮณ) โV ฮบ, VH (ฮณ) โV, VH () โV ฮบ, 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] ใๅฎๆฝไพ 2 :ใใณใใณใฐใ [0089] << Example 2: Panning >>
ใใ B7RP- 1- Fc lO /z g)ใ 0.1M NaHCOๆบถๆถฒ lmlใซๆบถ่งฃใใใฃใ ใใใฅใผใ๏ผNunc) ย Human B7RP- 1- Fc lO / z g) is dissolved in 1 ml of 0.1 M NaHCO solution, and Imunotube (Nunc)
3 ย Three
ใซใผๆใ 4ยฐCใซใฆใณใผใใใใ 0.1%Tween20ใๅซใ PBSใง 1ๅๆดๆตๅพใ 0.5%ใผใฉใใณ ZPBSใงใ 2ๆ้ใๅฎคๆธฉใซใฆใใญใใฏใใใ 0.1%TweenZPBSใงใ 6ๅๆดๆตใใๅพใๅฅ ๅธธไบบ็ฑๆฅใฎๆไฝใใกใผใธใฉใคใใฉใชใผ๏ผไธๆฌ้ๆไฝๆ็คบใใกใผใธๆถฒใ 1012 TUZml)ใ lmlใซๅฉใใ 2ๆ้ใๅฎคๆธฉใงๅๅฟใใใใ It was coated at 4 ยฐ 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] ้็น็ฐ็ใชใใกใผใธใ 0.1%Tween20/PBSใง 20ๅใฎๆดๆตใซใใฃใฆ้คใใๅพใ B7RP - 1-Fcใซ็ตๅใใใใกใผใธใฏใญใผใณใ lmg/ml BSA (ใฅใท่กๆธ
ใขใซใใใณ๏ผใๅซใ 0.1M ใฐใชใทใณ- HCl (pH2.2)ใงๆบถๅบใใ็ดใกใซ 1.0M Tris- HCl (pH9.1)ใงไธญๅใใใไธญๅใใ ใใกใผใธๆบถๆถฒใใใใ IgGใใณใผใใ 0.5%ใผใฉใใณ ZPBSใงใใญใใฏใใใฃใ ใใใฅใผใ ใซๅ ใใ 2ๆ้ใๅฎคๆธฉใงๅๅฟใใใใใใฎไธๆธ
ใใๅฏพๆฐๅขๆฎๆใฎๅคง่
ธ่ TGI (20ml) ใซๅ ใใ 30ยฐCใซใฆใ 30ๅใๆพ็ฝฎๅพใไธ้จใ SOBAGใใฌใผใใซๆใใๆฎใใซใคใใฆใฏ
ๅนๅฐใ 30mlใฎ 2 XYTAGใซๆใใๅพใ 30ยฐCใซใฆใผๆๅน้คใใใๅน้คๆถฒใ 2200rpm X 10ๅใง้ ๅฟๅพใๆฒๆฎฟใใๅคง่
ธ่ใ 3mlใฎ 2 XYTAGใซๆธๆฟใใ 1ๆฌกใใณใฆใณใฐใฎๅคง่
ธ ่ใฉใคใใฉใชไธใจใใใใใฎ TG1ๆถฒใใ 2 XYTAGๅนๅฐใซๆคใใใใซใใผใใกใผใธใ็จ ใใฆใฌในใญใฅใผใใในใฏใชใผใใณใฐๅพใฎใใกใผใธใฉใคใใฉใชใผใ่ชฟ่ฃฝใใใ [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 ยฐ 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 ยฐ C. After centrifuging the culture solution at 2200 rpm ร 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] ไธ่จใฎใใคใชใใณใฆใณใฐใฎๆไฝใใๅ่จใง 3ๅ่กใใใใใใใ 2ๆฌกใ 3ๆฌกใฎใใณ- ใณใฐใงๅพใใใใใกใผใธๆบถๆถฒใใ 2ๆฌกใใกใผใธใฉใคใใฉใชใผใ 3ๆฌกใใกใผใธใฉใคใใฉใชใผใจ ใใใใใ ใใ 2ๆฌกใใณใใณใฐใ 3ๆฌกใใณใฆใณใฐใงใฏใใใญใใญใณใฐๆบถๆถฒใซใใใใใ 5 %ในใญใ ใใซใฏใ 0.5%BSAใ็จใใใ ย [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] 2ๆฌกใใณใใณใฐใใชใใณใซ 3ๆฌกใใณใฆใณใฐๅพใฎ SOBAGใใฌใผใใใไปปๆใซใฏใญใผใณใ ๆฝๅบใใ็ญ้ Fvๆไฝใใกใผใธใฏใญใผใณใ่ชฟ่ฃฝใใฆใ B7RP-1-FCใซๅฏพใใ็น็ฐๆงใฎ็ขบ ่ชใชใใณใซๆไฝ้บไผๅญใฎ้
ๅ่งฃๆใ่กใฃใใ ย [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] ใๅฎๆฝไพ 3 :ในใฏใชใผใใณใฐ B7RP-1 ELISA)) ย [0093] << Example 3: Screening B7RP-1 ELISA))
ๅ้ขใใๅ้ Fvๆไฝใใกใผใธใฎ ELISAใซใใ็ตๅ็น็ฐๆงใฎ็ขบ่ชใฏใไปฅไธใฎๆนๆณ ใง่กใฃใใ ELISAใใฌใผใ๏ผNunc)ใซ B7RP- 1- Fc (80ng/well)ใ 4ยฐCใงใผๆใณใผใใใ 0 .5%ใผใฉใใณ/ PBSใงใใญใใญใณใฐใ่กใฃใใ 0.1%Tween20/PBSใงๆดๆตๅพใๅ้ขใใ ๅ้ Fvใใกใผใธใฏใญใผใณ๏ผl X 1013pfo/ml) 40 1ใๅ ใใๅธธๆธฉใง 2ๆ้ๅๅฟใใใใ็ต ๅใใกใผใธใฎๆคๅบใฏใไธๆฌกๆไฝใจใใฆใใชใใณๅๆ M13ใใกใผใธๆไฝ๏ผPharmacia)ใ ใไบๆฌกๆไฝใจใใฆ APๆจ่ญใใในใใฌใใใขใใธใณใๅ ใๅๅฟใใใๅพใๅบ่ณชๆบถๆถฒ๏ผ10% 2,2-ใคใใใธใจใฟใใผใซใๅซใ lmg/ml PNP-ใชใณ้
ธใฎ PBSๆบถๆถฒ๏ผใใซๅฉใใ 405nmใงใฎๅธ ๅ
ๅบฆใใใใซใใใฌใผใใชใผใใชใผใใผ NJ-2001 (Nunc็คพ๏ผใซใฆๆธฌๅฎใใใใใฎ็ตๆใ ๆ็ต็ใซ่ฉไพกใใใฏใญใผใณใในใฆๅ B7RP-1ใซ็น็ฐ็ใงใใใใจใใๅใคใ๏ผๅณ 1)ใ 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 ยฐ C and blocked with 0.5% gelatin / PBS. After washing with 0.1% Tween20 / PBS, an isolated single-chain Fv phage clone (1 ร 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] ใๅฎๆฝไพ 4 :ใฏใญใผใณใฎ้
ๅๅๆใ ย [0094] << Example 4: Sequence analysis of clones >>
ๅ้ขใใใฏใญใผใณใฎ scFv้บไผๅญใฎ VHใใใณ VLใฎ DNAๅกฉๅบ้
ๅใ Dye terminate r cycle sequencing FS Ready Reaction kit (Applied Biosystems)ใ็จใ ๆฑบๅฎใ 7ใใ E LISAใใใณ้
ๅๅๆใฎ็ตๆใๅ้ขใใใฏใญใผใณใฏ 4็จฎใฎ scFvใซๅ้กใใใใ ย 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.
[0095] ใๅฎๆฝไพ 5 :ใใ็ฑๆฅๆใใ B7RP- IscFvใฎ็บ็พใจ็ฒพ่ฃฝใ ย [Example 5: Expression and purification of human-derived anti-human B7RP-IscFv]
ๅ่จๅฎๆฝไพ 2ใ 3ใงๅ้ขใใใใ B7RP-1ใซๅๅฟใใ scFvใฏใญใผใณๅใใใฉในใใ DN Aใๅๅใใฆใๅธธๆณใซๅพใฃใฆๅคง่
ธ่ HB2151ใๅฝข่ณช่ปขๆใใใ 2%ใฐใซใณใผในใใใณ 1
00 g/mLใฎใขใณใใทใชใณใๅซใ 2 XYTๅนๅฐใงใใใใฎๅคง่
ธ่ใไธๅคๅๅน้คๅพใใใซ ใณใผในใใชใผใฎ 2 ฮงฮฅฮคๅนๅฐใซไธ้จ็งปๆคใใ็ตๆฟๅบฆ ImM IPTGใ 100 g/mLใฎใขใณใ ใทใชใณใๅ ใใฆ 4ๆ้ๅน้คใใฆ scFvใฎ็บ็พ่ชๅฐใ่กใฃใใๅน้ค็ตไบๅพ่ไฝใ้ ๅฟๅ ๅใใ ImM EDTAใๅซใ PBSใซๆธๆฟใใฆๆฐทไธญใซ 30ๅ่ไฝใๆพ็ฝฎใใใๆฌกใใง 8,90 O X gใง 30ๅ้้ ๅฟใใไธๆธ
ใ 0.45 ฮผ mใใฃใซใฟใผๆฟพ้ใใใใฎใใใชใใฉใบใ ็ปๅใจ ใใ scFvใฎ็ฒพ่ฃฝๅบ็บๆๆใจใใใ 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 ฮงฮฅฮค 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 ฮผm filter to obtain a periplasm fraction, which was used as a starting material for scFv purification.
[0096] ใใฎใใใซใใฆ่ชฟ่ฃฝใใ็ฒพ่ฃฝใฎๅบ็บๆๆใใๆ E tagๆไฝใ็จใใใกใใฃใฆใใฃใผใฏใญ ใใใฐใฉใใฃไธใงๅธธๆณใซๅพใฃใฆ็ฒพ่ฃฝใใใ PBSใง้ๆๅพใใจใณใใใญใทใณ้คๅปใซใฉใ De toxi-gel (PIERCE็คพ๏ผใงๆทปไปใฎใใญใใณใซใซๅพ!ใใจใณใใใญใทใณใ้คๅปใใใๅๅญ้ๅ ใใ 10,000ใฎ Centricon (Amicon็คพ๏ผใงๆฟ็ธฎๅพใ 0.45 ฮผ mใใฃใซใฟใผๆฟพ้ใใฆ็ฒพ่ฃฝๆจๅ ใจใใใไฟๅญใฏ- 20ยฐCใง่กใฃใใ ย [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 ฮผm filter to obtain a purified sample. Storage was performed at -20 ยฐ C.
[0097] ใๅฎๆฝไพ 6๏ผ็ฒพ่ฃฝ scFvใฎใใ B7RP- 1ใจใฎ็ตๅๆงใ ย << Example 6: Binding of purified scFv to human B7RP-1 >>
็ฒพ่ฃฝใใๅ้ Fvๆไฝใฎ B7RP-1ใซๅฏพใใ็ตๅๆดปๆงใฎๆธฌๅฎใฏใไปฅไธใฎๆนๆณใง่กใฃใ ใ ELISAใใฌใผใ๏ผNunc)ใซ B7RP- 1- Fc (80ng/well)ใ 4ยฐCใงใผๆใณใผใใใ 0.5%ใผใฉ ใใณ/ PBSใงใใญใใญใณใฐใ่กใฃใใ 0.1%Tween20/PBSใงๆดๆตๅพใๅ้ขใใๅ้ Fv ๆไฝ g/ml)ใจๆ E- tagๆไฝใๆททๅใใใใฎใ 40 1ๅ ใใ 2ๆ้ๅๅฟใใใใๆคๅบ ใฏใไบๆฌกๆไฝใจใใฆ APๆจ่ญใใๆใใฆใน IgG Fe yๆไฝใๅ ใ 60ๅๅๅฟใใใๅพใๅบ ่ณชๆบถๆถฒใๅ ใใ 405nmใงใฎๅธๅ
ๅบฆใใใใซใใใฌใผใใชใผใใชใผใใผ NJ-2001ใซใฆๆธฌ ๅฎใใใใใฎ็ตๆใๆ็ต็ใซ่ฉไพกใใใฏใญใผใณใในใฆใใ B7RP-1ใซ็น็ฐ็ใงใใใใจ ใใใใฃใ๏ผๅณ 2)ใ ย 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 ยฐ 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] ใพใใใฏใญใผใณ 223ใซใคใใฆใๅ้ Fvๆไฝใจ B7RP-1ใจใฎ็ตๅ่งฃ้ขๅฎๆฐใ BIAcoreใ ็จใใฆๆค่จใใใใปใณใตใผใใใ CM5 (Biacore AB, Uppsala, Sweden)ไธใฎ 4ใคใฎใ ใญใผใปใซใซ 20ยฐCใง 10mM้
ข้
ธ็ทฉ่กๆถฒ๏ผpH4.0)ใงๅธ้ใใ B7RP- 1- Fcใ ICOS- Fcใ CT LA- 4- Fcใๆใใ Fc F(ab')ๆไฝ๏ผๅ 10 ฮผ g/mL)ใใใใใๆต้ 5 ฮผ L/ๅใงๅบๅฎๅใ ย [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 ยฐ C in 4 flow cells on sensor chip CM5 (Biacore AB, Uppsala, Sweden) , Anti-human Fc F (ab ') antibody (each 10 ฮผg / mL) was immobilized at a flow rate of 5 ฮผL / min.
2 ย 2
ใใ HBS็ทฉ่กๆถฒ๏ผlOOmM HEPES/5M NaCl/0.5M EDTA/0.005%Tween20 (Pharmaci a biotech) )ใซใใใปใณใตใผใใใใๆดๆตใใๅพใ HBS็ทฉ่กๆถฒ /0.01%BSAใงๅธ้ใใ B 7RP- 1- Fcใๅๅฟใใใๆใใ Fc F(ab')ๆไฝใซใญใคใใใคใผใใใใๅผใ็ถใใๅ scF ย 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 โฒ) antibody was captulated. Continue with each scF
2 ย 2
Vใตใณใใซ๏ผ100nMใซ่ชฟๆด๏ผใๆต้ 10 L/ๅใงๆณจๅ
ฅใใฆๅๅฟใใใใตใณใใซๆฏใฎ็ตๅ
่งฃ้ขๅฎๆฐใๆธฌๅฎใใใใพใใๅใตใณใใซใๅๅฟใใใๅทฅ็จใฎๅใซใ 200mM NaClใๅซ ใ 200mMใฐใชใทใณ- HC1็ทฉ่กๆถฒ (pH2.2)ใซใใ scFvใๆบถๅบใๆดๆตใใใใใผใฟ่งฃๆใซ ใฏ BIAevaluationใฝใใใไฝฟ็จใใใใใฎ็ตๆใใพใ B7RP- 1- Fcใฏ ICOS- Fcใจๆใใ Fc F(ab')ๆไฝใๅบๅฎใใใใใใซใฎใฟๅๅฟใ็คบใใใใฎ็ตๆใใใ B7RP-1ใจ ICOSใจใฎV 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 2
็ตๅ่งฃ้ขๅฎๆฐใฏ็ด 7nMใงใใใใจใๅๅใคใใๅผใ็ถใใ 233ใๅๅฟใใใใจใใใ B7 - RPl-Fcใๅบๅฎใใใใใใจใๆใใ Fc F(ab')ๆไฝใซ B7-RPl-Fcใใญใคใใใคใผใใ ย 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 ย 2
ใใใใใซใฎใฟ็ตๅๆงใ็คบใใ ICOSใจ็ตๅใใฆ ใใ B7-RPใ Fcใซใฏ็ตๅๆงใ็คบใใช ๅใคใใใจใใใ 233ใซใคใใฆใฏ ICOSใจใฎ็ตๅ้จไฝใซ็ตๅใใใใจใๆจๅฎใใใใ B7 RP-1ใจใฎ็ตๅ่งฃ้ขๅฎๆฐใฏ็ด 13ใ15nMใจ็ฎๅบใใใใใจใใใใฌใปใใฟใผใงใใ ICOS ใจใปใผๅ็ญใงใใใใจใ็ขบ่ชใใใ๏ผๅณ 3)ใ ย 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).
[0099] ใๅฎๆฝไพ 7 :ๆ B7RP-1ๅ้ Fvๆไฝใซใใ ICOS-Fcใจ B7RP-1-FC้ใฎ็ตๅ้ปๅฎณใ Example 7: Inhibition of binding between ICOS-Fc and B7RP-1-FC by anti-B7RP-1 single chain Fv antibody
96็ฉดใใฌใผใใซ B7RP- 1- Fc (40ng/well)ใ 4ยฐCใงใผๆใณใผใใใ 0.5%ใผใฉใใณ/ PBS ใงใใญใใฏใใๅพใ 0.1%Tween20 /PBSใงๆดๆตใใใ 125ฮทฮใ2 Mใฎๆ B7RP- 1ๅ้ Fvๆไฝๆบถๆถฒใจใใชใใณๆจ่ญใใ ICOS-Fcใๆททๅใใใใฎใๅ ใใ 90ๅๅๅฟใใใใ ๆดๆตๅพใ APๆจ่ญใใในใใฌใใใขใใธใณใๅ ใใ 30ๅๅๅฟใใใๅพใๅบ่ณชๆบถๆถฒใๅ ใ ใ 405nmใฎๅธๅ
ๅบฆใๆธฌๅฎใใใใใฎ็ตๆใใฏใญใผใณ 223ใ 323ใ 325ใซใคใใฆใฏใๅ ใ ใๅ้ Fvๆไฝใฎ็จ้ไพๅญ็ใซใ ICOSใจ B7RP-1้ใฎ็ตๅใ้ปๅฎณใใฆใใใใจใใใ ใคใ๏ผๅณ 4)ใ ย A 96-well plate was coated with B7RP-1-Fc (40 ng / well) at 4 ยฐ C, blocked with 0.5% gelatin / PBS, and washed with 0.1% Tween20 / PBS. A mixture of 125ฮทฮ 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).
[0100] ใๅฎๆฝไพ 8๏ผๅ้ Fvๆไฝใฎๆซๆขข่ก Bใชใณใ็ไธใฎ B7RP-1ใธใฎ็ตๅใฎใใญใผใตใคใใกไธ ใฟใซใใ่งฃๆใ ย Example 8: Analysis of single-chain Fv antibody binding to B7RP-1 on peripheral blood B lymphocytes by flow cytometry
ๅ้ขใใใใๆ B7RP-1ๅ้ Fvๆไฝใฎ็ดฐ่ไธใซ็บ็พใใใ B7RP-1ๅๅญใซๅฏพใใ็ต ๅๆดปๆงใ็ขบ่ชใใใใใซใใใๆซๆขข่กใชใณใ็ใ็จใใฆใ่ๅ
ใใญใผใตใคใใกใผใฟใไฝฟ ใคใฆ่งฃๆใใใใใใธใ ใชใณๆซๆขข่กใซใใใคใณใผใซใ็จใใๅฎๆณใซใใ PBMCใ็ฒพ่ฃฝใ ใใ PMA(5ng/ml)ใจ PHA(2 /z g/ml)ใง 41ๆ้ๅบๆฟใใๅพใใใ IgGๆไฝใใซๅฉใใใใจใง ใใ IgG Fe yใฌใปใใฟใผใฎใใญใใฏใ่กใใๅ้ Fvๆไฝใจๆ E tagๆไฝใฎๆททๅ็ฉใใซใญ ใ 90ๅๅฟใใใใ็ดฐ่ใๆดๆตๅพใ PEใฉใใซใคๅใใในใใฌใใใขใใธใณใชใใณใซใใใ B็ดฐ ่็น็ฐ็ใใผใซใผใจใใฆใ FITCใงใฉใใซใคๅใใๆ CD19ๆไฝใง 30ๅ้ๅๅฟใใใ่ ๅ
ใฉใใซๅใใใๆดๆตๅพใ Coulter EPICS XL (Coulter็คพ Miami, FL)ใใญใผใตใคใใก
ใผใฟใ็จใใฆใ PEใชใใณใซ FITCใใฃใณใใซใงใฎ 2ๆฌกๅ
ใใญใผใตใคใใกใใชใผ่งฃๆใ่กใค ใใใใฎ็ตๆใใฏใญใผใณๅใ 223ใ 323ใ 325ใใใ!ใฝใฆใๆ CD19ๆไฝใใใใฃใฆ FITCใฉ ใใซใคๅใใใ B็ดฐ่้ๅฃใฎ PEใซใใๆ่ฒใ็ขบ่ชใใใใใจใใใใใใใฎใฏใญใผใณใซ ใค!ใฝใฆใฏใ B็ดฐ่ไธใฎ B7RP-1ใซๅฏพใใ็ตๅๆดปๆงใๆใใใใจใ็คบใใใ๏ผๅณ 5)ใ ใๅฎๆฝไพ 9๏ผใใๆ B7RP-1ๅ้ Fvๆไฝใซใใ T็ดฐ่่ฃๅฉๅบๆฟใทใฐใใซใซๅฏพใใ T็ดฐ่ ๅขๆฎ้ปๅฎณๆดปๆงใ 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
ๆๅๆ็คบ็ดฐ่ไธใฎ B7RP-1ใจ T็ดฐ่ไธใฎ ICOS้ใฎ่ฃๅฉใทใฐใใซใฎไผ้ใซๅฏพใใใ ๅพใใใๅ้ Fvๆไฝใฎ้ปๅฎณๆดปๆงใ T็ดฐ่ๅขๆฎๆดปๆงใๆๆจใซ่งฃๆใใใ 96็ฉดใฎไธธๅบ ใใฌใผใใซใๆ CD3ๆไฝ๏ผ2 ฮผ g/ml)ใจๆใ HgG Fcใใฉใฐใกใณใ F(ab')ๆไฝ๏ผ2.4 /z g/ml) ย 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 ฮผg / ml) and anti-HgG Fc fragment F (ab ') antibody (2.4 / z g / ml) on 96-well round bottom plate
2 ย 2
ใฎๆททๅๆถฒใ 37ยฐCใง 90ๅ้ใณใผใใใใใใฌใผใใ PBSใงๆดๆตใใใฎใกใ B7RP-1- Fc (l ฮผ g/ml)ใๅ ใใฆ 37ยฐCใง 2ๆ้ๅๅฟใใใใๅๅบฆใใฌใผใใๆดๆตใใๅพใ RPMI1640ใง ่ชฟ่ฃฝใใ 1ฮทฮใ500ฮทฮใฎๅ้ Fvๆไฝใใใณๆ B7RP-1ๆไฝใใซๅฉใ 30ๅๅๅฟใใใใ ใฎๅพใใๆซๆขข่กๅใ่ชฟ่ฃฝใใๆซๆขข่กใชใณใ็๏ผ1 X 105็ดฐ่/ well)ใๅ ใใ 37ยฐCใงๅน้ค ใใใๅน้ค้ๅงๅใ 48ๆ้ๅพใซใใชใใฆใ ใใใธใณ (0.5 Ci/well)ใๅ ใใใใใซ 18ๆ ้ๅน้คใใใ็ดฐ่ใฎ DNAใใฌใฉในใใฃใซใฟใผใซๅธ็ใใใๅพใๆถฒไฝใทใณใใฌไธใฟใซๆบถ ใใใ DNAไธญใฎใใชใใฆใ ใใใธใณ้ใใทใณใใฌใผใทใจใณใซใฆใณใฟใผใงๆธฌๅฎใใใใในใฆ ใฎใฏใญใผใณใงใใ HCOS-Fcใๅ ใใๅ ดๅใจใปใผๅ็ญใฎๅ้ Fvๆไฝใซใใ็จ้ไพๅญ ็ใช T็ดฐ่ๅขๆฎ้ปๅฎณใ่ตทใใฃใฆใใใใใใใฎๅ้ Fvใฏใญใผใณ 223ใ 323ใ 325ใฎ IC OSใทใฐใใซใซๅฏพใใ้ปๅฎณๆดปๆงใ็คบใใใ๏ผๅณ 6)ใ
The mixture was coated at 37 ยฐ C for 90 minutes. After the plate was washed with PBS, B7RP-1-Fc (l ฮผg / ml) was added and reacted at 37 ยฐ C. for 2 hours. After washing the plate again, the 1ฮทฮ to 500ฮทฮ 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 ยฐ 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
[1] ใใ B7 Related Protein 1 (ไปฅไธใ B7RP- 1)ใซ็ตๅๆงใๆใใใใๆใใ B7RP- 1ๆไฝใ ย [1] A human anti-human B7RP-1 antibody having binding property to human B7 Related Protein 1 (hereinafter referred to as B7RP-1).
[2] H้ใฎ็ธ่ฃๆงๆฑบๅฎ้ ๅ (CDR)ใไปฅไธใฎ (a)ใพใใฏ (b)ใฎใขใใ้
ธ้
ๅใๆใใ L้ ใฎ็ธ่ฃๆงๆฑบๅฎ้ ๅ (CDR)ใไปฅไธใฎ๏ผc)ใพใใฏ (d)ใฎใขใใ้
ธ้
ๅใๆใใใ่ซๆฑ้
[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
1ใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝ๏ผ 1. Human anti-human B7RP-1 antibody according to 1:
(a) CDRlใจใใฆ้
ๅ็ชๅท 1ใ3ใฎใใใใไธใคใ CDR2ใจใใฆ้
ๅ็ชๅท 4ใ6ใฎใ ใใใไธใคใใใใณ CDR3ใจใใฆ้
ๅ็ชๅท 7ใ9ใฎใใใใไธใคใซใใใใใใ็คบใ ใใใขใใ้
ธ้
ๅ๏ผ ย (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) CDRlใ3ใฎใขใใ้
ธ้
ๅใจใใฆใใใใใใ้
ๅ็ชๅท 1ใ3ใ 4ใ6ใใพใใฏ 7ใ9 ใใพใใฏใใใใขใใ้
ธ้
ๅใซใใใฆ 1ใพใใฏใใไปฅไธใฎใขใใ้
ธใ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅ ใใใใณ/ใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใงใใฃใฆใใใ B7RP-1ใซๅฏพใใ H้ใฎ็ธ่ฃ ๆงๆฑบๅฎ้ ๅใจใชใใใใใฎ๏ผ ย (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) CDRlใจใใฆ้
ๅ็ชๅท 10ใ12ใฎใใใใไธใคใ CDR2ใจใใฆ้
ๅ็ชๅท 13ใ15 ใฎใใใใไธใคใ CDR3ใจใใฆ้
ๅ็ชๅท 16ใ18ใฎใใใใไธใคใซใใใใใใ็คบ ใใใใขใใ้
ธ้
ๅ๏ผ ย (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) CDRlใ3ใฎใขใใ้
ธ้
ๅใจใใฆใใใใใใ้
ๅ็ชๅท 10ใ12ใ 13ใ15ใใพใใฏ 16ใ18ใใพใใฏใใใใขใใ้
ธ้
ๅใซใใใฆ 1ใพใใฏใใไปฅไธใฎใขใใ้
ธใ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅใใใใณ Zใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใงใใฃใฆใใใ B7RP-1ใซๅฏพใใ L้ ใฎ็ธ่ฃๆงๆฑบๅฎ้ ๅใจใชใใใใใฎใ ย (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] H้ใฎ CDR1ใ3ใฎใขใใ้
ธ้
ๅๅ ้
ๅ็ชๅท 1ใ 4ใใใณ 7ใ้
ๅ็ชๅท 2ใ 5ใใใณย [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
8ใใพใใฏ้
ๅ็ชๅท 3ใ 6ใใใณ 9ใฎ็ตใฟๅใใๅ ้ธๆใใใใขใใ้
ธ้
ๅใงใใใ L ้ใฎ CDR1ใ3ใฎใขใใ้
ธ้
ๅๅ ้
ๅ็ชๅท 10ใ 13ใใใณ 16ใ้
ๅ็ชๅท 11ใ 14ใ ใใณ 17ใใพใใฏ้
ๅ็ชๅท 12ใ 15ใใใณ 18ใฎ็ตใฟๅใใๅใ้ธๆใใใใขใใ้
ธ้
ๅ ใงใใ่ซๆฑ้
1ใพใใฏ 2ใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใ 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] H้ใฎ CDR1ใ3ใจ L้ใฎ CDR1ใ3ใจใฎ็ตใฟๅใใใฎใขใใ้
ธ้
ๅใใ้
ๅ็ชๅท 1ย [4] The amino acid sequence of the combination of H chain CDR1-3 and L chain CDR1-3 is SEQ ID NO: 1.
ใ 4ใใใณ 7ใจ้
ๅ็ชๅท 10ใ 13ใใใณ 16ใ้
ๅ็ชๅท 2ใ 5ใใใณ 8ใจ้
ๅ็ชๅท 11ใ 14 ใใใณ 17ใใพใใฏ้
ๅ็ชๅท 3ใ 6ใใใณ 9ใจ้
ๅ็ชๅท 12ใ 15ใใใณ 18ใจใฎใใใใ ไธใคใฎ็ตใฟๅใใใงใใใ่ซๆฑ้
3ใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใ
, 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] H้ๅฏๅค้ ๅใไปฅไธใฎ๏ผe)ใพใใฏ (f)ใฎใขใใ้
ธ้
ๅใๆใใ L้ๅฏๅค้ ๅใไปฅไธ ใฎ๏ผg)ใพใใฏ (h)ใฎใขใใ้
ธ้
ๅใๆใใใ่ซๆฑ้
1ใใ 4ใฎใใใใใซ่จ่ผใฎใใๆใ ใ B7RP- 1ๆไฝ๏ผ [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:
(e)้
ๅ็ชๅท 19ใ21ใฎใใใใไธใคใฎใขใใ้
ธ้
ๅๅ ้ธๆใใใใขใใ้
ธ้
ๅ๏ผย (e) the amino acid sequence ability of any one of SEQ ID NOS: 19 to 21; selected amino acid sequence;
(f)้
ๅ็ชๅท 19ใ21ใซ็คบใใใใขใใ้
ธ้
ๅใซใใใฆ 1ใพใใฏใใไปฅไธใฎใขใใ้
ธใ ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅใใใใณ Zใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใงใใฃใฆใใใ B7RP-1ใซ ๅฏพใใ H้ๅฏๅค้ ๅใจใชใใใใใฎ๏ผ (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)้
ๅ็ชๅท 22ใ24ใฎ!ใใใใไธใคใฎใขใใ้
ธ้
ๅๅ ้ธๆใใใใขใใ้
ธ้
ๅ๏ผย (g) SEQ ID NOS: 22 to 24 !, any one amino acid sequence ability Selected amino acid sequence;
(h)้
ๅ็ชๅท 22ใ24ใซ็คบใใใใขใใ้
ธ้
ๅใซใใใฆ 1ใพใใฏใใไปฅไธใฎใขใใ้
ธใ ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅใใใใณ Zใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใงใใฃใฆใใใ B7RP-1ใซ ๅฏพใใ L้ๅฏๅค้ ๅใจใชใใใใใฎใ (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.
[6] H้ๅฏๅค้ ๅใจ L้ๅฏๅค้ ๅใจใฎ็ตใฟๅใใใฎใขใใ้
ธ้
ๅๅ ้
ๅ็ชๅท 19ใจ้
ๅ็ชๅท 22ใ้
ๅ็ชๅท 20ใจ้
ๅ็ชๅท 23ใใพใใฏ้
ๅ็ชๅท 21ใจ้
ๅ็ชๅท 24ใจใฎ็ตใฟ ๅใใใซใใ็คบใใใใขใใ้
ธ้
ๅใงใใใ่ซๆฑ้
1ใใ 5ใฎใใใใใซ่จ่ผใฎใใๆใ ใ B7RP- 1ๆไฝใ ย [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] ใใ B7RP-1ใซ็ตๅๆงใๆใใใใๆใใ B7RP-1ๆไฝใฎ H้ๅฏๅค้ ๅใใฉใฐใกใณใใ ย [7] A heavy chain variable region fragment of a human anti-human B7RP-1 antibody having binding ability to human B7RP-1.
[8] ็ธ่ฃๆงๆฑบๅฎ้ ๅ (CDR)ใไปฅไธใฎ๏ผa)ใพใใฏ (b)ใฎใขใใ้
ธ้
ๅใๆใใใ่ซๆฑ้
7 ใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ H้ๅฏๅค้ ๅใใฉใฐใกใณใ๏ผ [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):
(a) CDRlใจใใฆ้
ๅ็ชๅท 1ใ3ใฎใใใใไธใคใ CDR2ใจใใฆ้
ๅ็ชๅท 4ใ6ใฎใ ใใใไธใคใใใใณ CDR3ใจใใฆ้
ๅ็ชๅท 7ใ9ใฎใใใใไธใคใซใใใใใใ็คบใ ใใใขใใ้
ธ้
ๅ๏ผ ย (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) CDRlใ3ใฎใขใใ้
ธ้
ๅใจใใฆใใใใใใ้
ๅ็ชๅท 1ใ3ใ 4ใ6ใใพใใฏ 7ใ9 ใใพใใฏใใใใขใใ้
ธ้
ๅใซใใใฆ 1ใพใใฏใใไปฅไธใฎใขใใ้
ธใ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅ ใใใใณ/ใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใงใใฃใฆใใใ B7RP-1ใซๅฏพใใ H้ใฎ็ธ่ฃ ๆงๆฑบๅฎ้ ๅใจใชใใใใใฎใ ย (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.
[9] CDR1ใ3ใฎใขใใ้
ธ้
ๅๅ ้
ๅ็ชๅท 1ใ 4ใใใณ 7ใ้
ๅ็ชๅท 2ใ 5ใใใณ 8ใใพใ ใฏ้
ๅ็ชๅท 3ใ 6ใใใณ 9ใฎ็ตใฟๅใใๅ ้ธๆใใใใขใใ้
ธ้
ๅใงใใใ่ซๆฑ้
8 ใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ H้ๅฏๅค้ ๅใใฉใฐใกใณใใ
[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] ไปฅไธใฎ๏ผe)ใพใใฏ (f)ใฎใขใใ้
ธ้
ๅใๆใใใ่ซๆฑ้
7ใใ 9ใฎ ใใใใใซ่จ่ผใฎ ใใๆใใ B7RP-1ๆไฝใฎ H้ๅฏๅค้ ๅใใฉใฐใกใณใ๏ผ [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):
(e)้
ๅ็ชๅท 19ใ21ใฎใใใใไธใคใฎใขใใ้
ธ้
ๅๅ ้ธๆใใใใขใใ้
ธ้
ๅ๏ผย (e) the amino acid sequence ability of any one of SEQ ID NOS: 19 to 21; selected amino acid sequence;
(f)้
ๅ็ชๅท 19ใ21ใซ็คบใใใใขใใ้
ธ้
ๅใซใใใฆ 1ใพใใฏใใไปฅไธใฎใขใใ้
ธใ ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅใใใใณ Zใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใงใใฃใฆใใใ B7RP-1ใซ ๅฏพใใ H้ๅฏๅค้ ๅใจใชใใใใใฎใ (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.
[11] ใใ B7RP-1ใซ็ตๅๆงใๆใใใใๆใใ B7RP-1ๆไฝใฎ L้ๅฏๅค้ ๅใใฉใฐใกใณใใ ย [11] A light chain variable region fragment of a human anti-human B7RP-1 antibody capable of binding to human B7RP-1.
[12] ็ธ่ฃๆงๆฑบๅฎ้ ๅ (CDR)ใไปฅไธใฎ๏ผc)ใพใใฏ (d)ใฎใขใใ้
ธ้
ๅใๆใใใ่ซๆฑ้
1 1ใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ L้ๅฏๅค้ ๅใใฉใฐใกใณใ๏ผ [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):
(c) CDRlใจใใฆ้
ๅ็ชๅท 10ใ12ใฎใใใใไธใคใ CDR2ใจใใฆ้
ๅ็ชๅท 13ใ15 ใฎใใใใไธใคใใใใณ CDR3ใจใใฆ้
ๅ็ชๅท 16ใ18ใฎใใใใไธใคใซใใใใ ใใ็คบใใใใขใใ้
ธ้
ๅ๏ผ ย (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) CDRlใ3ใฎใขใใ้
ธ้
ๅใจใใฆใใใใใใ้
ๅ็ชๅท 10ใ18ใ 13ใ15ใฎใใ ใใไธใคใใพใใฏ 16ใ18ใใพใใฏใใใใขใใ้
ธ้
ๅใซใใใฆ 1ใพใใฏใใไปฅไธใฎใก ใใ้
ธใ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅใใใใณ/ใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใงใใฃใฆใใใๆใ ใ B7RP-1ๆไฝใฎ L้ใฎ็ธ่ฃๆงๆฑบๅฎ้ ๅใจใชใใใใใฎใ ย (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] CDR1ใ3ใฎใขใใ้
ธ้
ๅๅ ้
ๅ็ชๅท 10ใ 13ใใใณ 16ใ้
ๅ็ชๅท 11ใ 14ใใใณ 17ใใพใใฏ้
ๅ็ชๅท 12ใ 15ใใใณ 18ใฎ็ตใฟๅใใๅ ้ธๆใใใใขใใ้
ธ้
ๅใงใ ใใ่ซๆฑ้
12ใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ L้ๅฏๅค้ ๅใใฉใฐใกใณใใ ย [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] ไปฅไธใฎ (g)ใพใใฏ (h)ใฎใขใใ้
ธ้
ๅใๆใใใ่ซๆฑ้
11ใใ 13ใฎใใใใใซ่จ่ผ ใฎใใๆใใ B7RP-1ๆไฝใฎ L้ๅฏๅค้ ๅใใฉใฐใกใณใ๏ผ ย [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):
(g)้
ๅ็ชๅท 22ใ24ใฎ!ใใใใไธใคใฎใขใใ้
ธ้
ๅๅ ้ธๆใใใใขใใ้
ธ้
ๅ๏ผย (g) SEQ ID NOS: 22 to 24 !, any one amino acid sequence ability Selected amino acid sequence;
(h)้
ๅ็ชๅท 22ใ24ใซ็คบใใใใขใใ้
ธ้
ๅใซใใใฆ 1ใพใใฏใใไปฅไธใฎใขใใ้
ธใ ็ฝฎๆใๆฌ ๅคฑใๆฟๅ
ฅใใใใณ Zใพใใฏไปๅ ใใใใขใใ้
ธ้
ๅใงใใฃใฆใใใ B7RP-1ใซ ๅฏพใใ L้ๅฏๅค้ ๅใจใชใใใใใฎใ (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.
[15] ่ซๆฑ้
7ใใ 10ใฎใใใใใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ H้ๅฏๅค้ ๅใใฉใฐใก ใณใใจใ่ซๆฑ้
11ใใ 14ใฎใใใใใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ L้ๅฏๅค้ ๅ ใใฉใฐใกใณใใจใ้ฃ็ตใใฆใชใใใใ B7RP-1ใซๅฏพใใใใ็ฑๆฅใฎๆไฝใฎไธๆฌ้ๅฏๅค้ ๅ
ใใฉใฐใกใณใใ [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] ่ซๆฑ้
7ใใ 10ใฎใใใใใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ H้ๅฏๅค้ ๅใใฉใฐใก ใณใใใใใณ Zใพใใฏ่ซๆฑ้
11ใใ 14ใฎใใใใใซ่จ่ผใฎใใๆใใ B7RP-1ๆไฝใฎ L ้ๅฏๅค้ ๅใใฉใฐใกใณใใซใใใ็ฑๆฅใฎๆไฝๅฎๅธธ้ ๅใ้ฃ็ตใใฆใชใใใใ B7RP-1ใซๅฏพ ใใใใ็ฑๆฅใฎๆไฝใพใใฏใใฎๆไฝใใฉใฐใกใณใใ ย [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] ๅฝ่ฉฒๆไฝใใฉใฐใกใณใใใ Fab, Fab'ใ F(ab')ใ scAbใใพใใฏ scFv-Fcใงใใ่ซๆฑ้
1 ย [17] The antibody fragment is Fab, Fab โฒ, F (ab โฒ), scAb, or scFv-Fc.
2 ย 2
6ใซ่จ่ผใฎๆไฝใใฉใฐใกใณใใ ย The antibody fragment according to 6.
[18] ่ซๆฑ้
1ใใ 17ใฎใใใใใซ่จ่ผใฎๆไฝใพใใฏใใฎใใฉใฐใกใณใใจใใใใๆใใฏ ไปใฎใฟใณใใฏ่ณชใจใ่ๅใใใ่ๅๆไฝใพใใฏใใฎใใฉใฐใกใณใใ [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] ่ซๆฑ้
1ใใ 18ใฎใใใใใซ่จ่ผใฎๆไฝใใใใฏ่ๅๆไฝใพใใฏใใฎใใฉใฐใกใณใ ใซไฟฎ้ฃพๅคใ็ตๅใใใฆใชใไฟฎ้ฃพๆไฝใพใใฏใใฎใใฉใฐใกใณใใ [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] ่ซๆฑ้
1ใใ 19ใฎใใใใใซ่จ่ผใฎๆไฝใใใใฏ่ๅๆไฝใพใใฏใใฎใใฉใฐใกใณใ ใใณใผใใใ้บไผๅญใ [20] A gene encoding the antibody or fusion antibody or fragment thereof according to any one of claims 1 to 19.
[21] ่ซๆฑ้
20ใซ่จ่ผใฎ้บไผๅญใๅซใ็ตๆใ็บ็พใใฏใฟใผใ [21] A recombinant expression vector comprising the gene according to claim 20.
[22] ่ซๆฑ้
20ใซ่จ่ผใฎ้บไผๅญใๅฐๅ
ฅใใใๅฝข่ณช่ปขๆไฝใ [22] A transformant into which the gene according to claim 20 has been introduced.
[23] ่ซๆฑ้
20ใซ่จ่ผใฎ้บไผๅญใๅฎฟไธปใซ็บ็พใใใใใจใซใใฃใฆใใใๆ B7RP-1ๆไฝใพ ใใฏใใฎๆญ็ใ็็ฃใใๆนๆณใ ย [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] ่ซๆฑ้
1ใใ 17ใฎใใใใใซ่จ่ผใฎๆไฝใพใใฏใใฎใใฉใฐใกใณใใใพใใฏ่ซๆฑ้
18 ใซ่จ่ผใฎ่ๅๆไฝใพใใฏใใฎใใฉใฐใกใณใใใพใใฏ่ซๆฑ้
19ใซ่จ่ผใฎไฟฎ้ฃพๆไฝใพใ ใฏใใฎใใฉใฐใกใณใใ็จใใ B7RP-1ใฎๆคๅบ่ฉฆ่ฌใ [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] ่ซๆฑ้
20ใซ่จ่ผใฎ้บไผๅญใๅซใ้บไผๅญๆฒป็ๅคใ [25] A gene therapy agent comprising the gene according to claim 20.
[26] ่ซๆฑ้
1ใใ 17ใฎใใใใใซ่จ่ผใฎๆไฝใพใใฏใใฎใใฉใฐใกใณใใใพใใฏ่ซๆฑ้
18 ใซ่จ่ผใฎ่ๅๆไฝใพใใฏใใฎใใฉใฐใกใณใใใพใใฏ่ซๆฑ้
19ใซ่จ่ผใฎไฟฎ้ฃพๆไฝใพใ ใฏใใฎใใฉใฐใกใณใใ็จใใ B7RP-1ใจ ICOSใจใฎ็ตๅๆดปๆง้ปๅฎณๅคใ ย [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] ่ซๆฑ้
1ใใ 17ใฎใใใใใซ่จ่ผใฎๆไฝใพใใฏใใฎใใฉใฐใกใณใใใพใใฏ่ซๆฑ้
18 ใซ่จ่ผใฎ่ๅๆไฝใพใใฏใใฎใใฉใฐใกใณใใใพใใฏ่ซๆฑ้
19ใซ่จ่ผใฎไฟฎ้ฃพๆไฝใพใ ใฏใใฎใใฉใฐใกใณใใ่ช่ญใใ B7RP-1ไธใฎๆๅๆฑบๅฎ้ ๅใซๅบใฅใใฆๅๅญ่จญ่จใใใ ไฝๅๅญๅๅ็ฉใพใใฏใใฎ่ชๅฐไฝใ
่ซๆฑ้
27ใซ่จ่ผใฎไฝๅๅญๅๅ็ฉใพใใฏใใฎ่ชๅฐไฝใ็จใใ B7RP-1ใจ ICOSใจใฎ ็ตๅๆดปๆง้ปๅฎณๅคใ [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.
่ซๆฑ้
26ใพใใฏ่ซๆฑ้
28ใซ่จ่ผใฎ็ตๅๆดปๆง้ปๅฎณๅคใ็จใใ B7RP-1ใจ ICOSใจใฎ ็ธไบไฝ็จใซใใๆน่ตทใใใ็็ใใใณๅ
็ซ็ฐๅธธๆง็พๆฃใฎไบ้ฒใพใใฏๆฒป็่ฌใ
ย 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.
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| Application Number | Priority Date | Filing Date | Title |
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| JP2006528814A JPWO2006003999A1 (en) | 2004-07-05 | 2005-06-30 | Human anti-human B7RP-1 antibody and antibody fragment thereof |
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| JP2004198224 | 2004-07-05 | ||
| JP2004-198224 | 2004-07-05 |
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| JP2016524592A (en) * | 2013-04-23 | 2016-08-18 | ใถ ใฆใใใผใทใใฃ ใณใผใ ใชใ ใถ ใฆใใใผใทใใฃ ใชใ ใขใใใฃใผใณ | Isolation of therapeutic target-specific VNAR domains into ICOSL |
| US10421823B2 (en) | 2013-03-13 | 2019-09-24 | Amgen Inc. | Proteins specific for BAFF and B7RP1 and uses thereof |
| US10421824B2 (en) | 2013-03-13 | 2019-09-24 | Amgen Inc. | Proteins specific for BAFF and B7RP1 |
| CN111902423A (en) * | 2018-01-18 | 2020-11-06 | ็ฎๅ่็น้ฟ็ฑณ้ฟไผไผฝๅพท็ฝไธๆนๅคงๅญฆ | Antitumor therapeutic agents based on ligands of B7H receptors |
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| US11607451B2 (en) | 2005-06-14 | 2023-03-21 | Amgen Inc. | Self-buffering antibody formulations |
| US10072090B2 (en) | 2005-07-18 | 2018-09-11 | Amgen Inc. | Human anti-B7RP1 neutralizing antibodies |
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| US9266945B2 (en) | 2005-07-18 | 2016-02-23 | Amgen Inc. | Human anti-B7RP1 neutralizing antibodies |
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| US7868140B2 (en) | 2005-07-18 | 2011-01-11 | Amgen Inc. | Human anti-B7RP1 neutralizing antibodies |
| US11492417B2 (en) | 2013-03-13 | 2022-11-08 | Amgen Inc. | Proteins specific for BAFF and B7RP1 and uses thereof |
| US10421823B2 (en) | 2013-03-13 | 2019-09-24 | Amgen Inc. | Proteins specific for BAFF and B7RP1 and uses thereof |
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| JP2016524592A (en) * | 2013-04-23 | 2016-08-18 | ใถ ใฆใใใผใทใใฃ ใณใผใ ใชใ ใถ ใฆใใใผใทใใฃ ใชใ ใขใใใฃใผใณ | Isolation of therapeutic target-specific VNAR domains into ICOSL |
| US10472410B2 (en) | 2013-04-23 | 2019-11-12 | The University Court Of The University Of Aberdeen | Isolation of therapeutic target specific VNAR domains to ICOSL |
| US11491223B2 (en) | 2016-10-21 | 2022-11-08 | Amgen Inc. | Pharmaceutical formulations and methods of making the same |
| US12214013B2 (en) | 2016-10-21 | 2025-02-04 | Amgen Inc. | Methods of treatment of arthritis and/or psoriasis with pharmaceutical formulations of etanercept |
| CN111902423A (en) * | 2018-01-18 | 2020-11-06 | ็ฎๅ่็น้ฟ็ฑณ้ฟไผไผฝๅพท็ฝไธๆนๅคงๅญฆ | Antitumor therapeutic agents based on ligands of B7H receptors |
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| JPWO2006003999A1 (en) | 2008-07-31 |
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