WO2006003999A1 - Anticorps de b7rp-1 antihumain humain et son fragment d’anticorps - Google Patents

Anticorps de b7rp-1 antihumain humain et son fragment d’anticorps Download PDF

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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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antibody
seq
amino acid
human
b7rp
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PCT/JP2005/012094
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Japanese (ja)
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Kazuhisa Sugimura
Yuji Ito
Toshihiro Nakashima
Masaharu Torikai
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Juridical Foundation The Chemo-Sero-Therapeutic Research Institute
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    • 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 Pl”) 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 ′) 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 “mutation” mainly means a mutation artificially introduced by a known method for producing a mutant protein, but a similar mutant polypeptide existing in nature (eg, human) is isolated and purified. There may be.
  • 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).
  • 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 ( ⁇ ) —V ⁇ , VH ( ⁇ ) —V, VH () —V ⁇ , 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 ° C for 30 minutes, and then partly spread on a SOBAG plate.
  • the medium was changed to 30 ml of 2 XYTAG, and then cultured at 30 ° C. After centrifuging the culture solution at 2200 rpm ⁇ 10 minutes, the precipitated Escherichia coli was suspended in 3 ml of 2 XYTAG, and used as the primary pan-Jung coli library.
  • This TG1 solution was planted in 2 XYTAG medium, rescued using helper phage, and a phage library after screening was prepared.
  • 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 ⁇ 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 ⁇ 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 ⁇ m filter to obtain a purified sample. Storage was performed at -20 ° 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 ° 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 ° C, blocked with 0.5% gelatin / PBS, and washed with 0.1% Tween20 / PBS.
  • a mixture of 125 ⁇ 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 ° C for 90 minutes. After the plate was washed with PBS, B7RP-1-Fc (l ⁇ g / ml) was added and reacted at 37 ° C. for 2 hours. After washing the plate again, the 1 ⁇ to 500 ⁇ single-chain Fv antibody and anti-B7RP-1 antibody prepared with RPMI1640 were reacted for 30 minutes, and then peripheral blood lymphocytes (1 X 10 5 cells / well) were added and cultured at 37 ° C. Tritium thymidine (0.5 Ci / well) was added 48 hours later, and the culture was further cultured for 18 hours.
  • the cell DNA was adsorbed on a glass filter, dissolved in a liquid scintillator, and the amount of tritium thymidine in the DNA was measured with a scintillation counter.
  • 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

On propose un anticorps de protéine 1 apparentée à B7 (ci-après désignée par B7RP-1) antihumain humain, son fragment d’anticorps et un procédé pour les utiliser. En utilisant le procédé de phage anticorps, un anticorps de B7RP-1 antihumain humain ayant une affinité élevée pour la B7RP-1 humaine et son fragment d’anticorps sont obtenus. L’anticorps et son fragment d’anticorps ainsi obtenus sont prévus pour être aussi utiles que des remèdes pour l’inflammation et l’anomalie immunitaire.
PCT/JP2005/012094 2004-07-05 2005-06-30 Anticorps de b7rp-1 antihumain humain et son fragment d’anticorps WO2006003999A1 (fr)

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WO2007011941A3 (fr) * 2005-07-18 2007-05-03 Amgen Inc Anticorps neutralisants anti-b7rp1 humains
JP2016524592A (ja) * 2013-04-23 2016-08-18 ザ ユニバーシティ コート オブ ザ ユニバーシティ オブ アバディーン Ｉｃｏｓｌへの治療上の標的特異的ｖｎａｒドメインの単離
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CN111902423A (zh) * 2018-01-18 2020-11-06 皮埃蒙特阿米阿伏伽德罗东方大学 基于b7h受体的配体的抗肿瘤治疗剂
US11491223B2 (en) 2016-10-21 2022-11-08 Amgen Inc. Pharmaceutical formulations and methods of making the same
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JP6580791B2 (ja) * 2016-02-26 2019-09-25 エスシーエム ライフサイエンス カンパニー リミテッド 調節ｔ細胞媒介性疾患の予防または治療用薬学的組成物

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US8981071B2 (en) 2005-07-18 2015-03-17 Amgen, Inc. Human anti-B7RP1 neutralizing antibodies
EA021669B1 (ru) * 2005-07-18 2015-08-31 Амджен Инк. Нейтрализующие антитела человека против b7rp1
US9266945B2 (en) 2005-07-18 2016-02-23 Amgen Inc. Human anti-B7RP1 neutralizing antibodies
WO2007011941A3 (fr) * 2005-07-18 2007-05-03 Amgen Inc Anticorps neutralisants anti-b7rp1 humains
US7868140B2 (en) 2005-07-18 2011-01-11 Amgen Inc. Human anti-B7RP1 neutralizing antibodies
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
US11492417B2 (en) 2013-03-13 2022-11-08 Amgen Inc. Proteins specific for BAFF and B7RP1 and uses thereof
JP2016524592A (ja) * 2013-04-23 2016-08-18 ザ ユニバーシティ コート オブ ザ ユニバーシティ オブ アバディーン Ｉｃｏｓｌへの治療上の標的特異的ｖｎａｒドメインの単離
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
CN111902423A (zh) * 2018-01-18 2020-11-06 皮埃蒙特阿米阿伏伽德罗东方大学 基于b7h受体的配体的抗肿瘤治疗剂

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