WO2021020416A1 - 二重特異性抗体 - Google Patents

二重特異性抗体 Download PDF

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Publication number
WO2021020416A1
WO2021020416A1 PCT/JP2020/028972 JP2020028972W WO2021020416A1 WO 2021020416 A1 WO2021020416 A1 WO 2021020416A1 JP 2020028972 W JP2020028972 W JP 2020028972W WO 2021020416 A1 WO2021020416 A1 WO 2021020416A1
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seq
amino acid
acid sequence
antibody
arm
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PCT/JP2020/028972
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English (en)
French (fr)
Japanese (ja)
Inventor
史朗 柴山
拓也 新坊
智也 手塚
マーク スロスビー
クルイフ コルネリス アドリアン デ
ロー ピーテル フォッコ ヴァン
リンス クロースター
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Ono Pharmaceutical Co Ltd
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Ono Pharmaceutical Co Ltd
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Priority to KR1020227003026A priority Critical patent/KR20220039720A/ko
Priority to MX2022000971A priority patent/MX2022000971A/es
Priority to EP20846826.4A priority patent/EP4008348A4/en
Priority to BR112022001473A priority patent/BR112022001473A2/pt
Priority to CA3149309A priority patent/CA3149309A1/en
Priority to JP2020570992A priority patent/JP6856183B1/ja
Priority to US17/630,426 priority patent/US20220281977A1/en
Priority to AU2020322765A priority patent/AU2020322765A1/en
Application filed by Ono Pharmaceutical Co Ltd filed Critical Ono Pharmaceutical Co Ltd
Priority to PH1/2022/550190A priority patent/PH12022550190A1/en
Priority to CN202080054077.0A priority patent/CN114174343A/zh
Publication of WO2021020416A1 publication Critical patent/WO2021020416A1/ja
Priority to IL290050A priority patent/IL290050A/en
Priority to ZA2022/01247A priority patent/ZA202201247B/en
Anticipated expiration legal-status Critical
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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
    • C07K16/2809—Immunoglobulins [IG], 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 the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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
    • C07K16/2818—Immunoglobulins [IG], 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 CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention is a bispecific antibody capable of specifically binding to PD-1 and CD3 (hereinafter, may be abbreviated as "PD-1 / CD3 bispecific antibody”) or an antibody thereof.
  • the present invention relates to fragments (hereinafter, these may be collectively abbreviated as “PD-1 / CD3 bispecific antibody, etc.”), pharmaceutical compositions containing this as an active ingredient, and their pharmaceutical therapeutic uses.
  • PD-1 is an immunosuppressive receptor belonging to the immunoglobulin family, and is a molecule having a function of suppressing the immune activation signal of T cells activated by stimulation from an antigen receptor.
  • PD-1 signals are obtained from autoimmune dilated cardiomyopathy, lupus-like syndrome, autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease, type I diabetes and It is known to play an important role in suppressing autoimmune diseases such as rheumatoid arthritis. Therefore, it has been pointed out that substances that enhance PD-1 signals can be prophylactic or therapeutic agents for autoimmune diseases and the like.
  • bispecific antibodies that recognize PD-1 have been known as substances that enhance PD-1 signals (Patent Documents 1 to 3).
  • This bispecific antibody is a genetically engineered link between the antigen recognition site of an antibody that recognizes CD3, which is a member of the T cell receptor complex, and the antigen recognition site of an antibody that recognizes PD-1. It has the effect of enhancing the inhibitory signal of PD-1 to the T cell receptor complex by increasing the frequency with which PD-1 is located near the T cell receptor complex.
  • the patent document also describes that PD-1 bispecific antibody can be used for the prevention or treatment of autoimmune diseases and the like.
  • the stimulation of cytokine production after administration which is considered to be the cause of such side effects, is sufficiently reduced, and therefore the occurrence of side effects of concern is suppressed. It is expected to become a drug that has been used. No bispecific antibody having such characteristics has been reported to date.
  • An object of the present invention is to provide a new drug for prevention, suppression of symptom progression, suppression of recurrence or treatment of autoimmune diseases and the like.
  • the inventors of the present invention focused on the PD-1 / CD3 bispecific antibody of the present invention and the like as substances capable of solving such a problem, and they were infusion reaction or cytokine release.
  • the present invention has been completed by confirming that it can be a drug in which the occurrence of side effects called syndrome is reduced.
  • the inventors of the present invention confirmed that the PD-1 / CD3 bispecific antibody and the like have a characteristic of allowing interaction with PD-1 and its ligand PD-L1. It was found that such characteristics contribute to prevention of autoimmune diseases such as PD-1 / CD3 bispecific antibody, suppression of symptom progression, suppression of recurrence, or enhancement or persistence of therapeutic effect.
  • a bispecific antibody having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3 (hereinafter, also in the same manner as described above, "PD-1 / CD3”. (Sometimes abbreviated as “bispecific antibody”) or an antibody fragment thereof, the first arm that specifically binds to PD-1.
  • PD-1 / CD3 a bispecific antibody
  • an antibody fragment thereof the first arm that specifically binds to PD-1.
  • VH-CDR1 Complementarity determining regions 1 of heavy chain variable regions
  • VH-CDR2 Complementarity determining regions 2 of heavy chain variable regions consisting of the amino acid sequence of SEQ ID NO: 7
  • VH-CDR3 Complementarity determining regions 3 of heavy chain variable regions consisting of the amino acid sequence of SEQ ID NO: 8
  • VH-CDR3 Complementarity determining regions 3 of heavy chain variable regions
  • C (a) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 12
  • VH having (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 13 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 14.
  • D (a) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 15.
  • VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 17 and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 18 CDR1, It has any one VH selected from (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 20.
  • the second arm that specifically binds to CD3 (A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 37, The first has VH having (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 38 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 39, where it specifically binds to PD-1.
  • VH-CDRs of VH-CDR1, VH-CDR2 and VH-CDR3 in the arm each of its arbitrary 1-5 amino acid residues is another amino acid (preferably its conservative).
  • VH-CDR1 VH-CDR2 and VH-CDR3 in one or more VH-CDRs in the second arm that may be substituted with (amino acids) and / or specifically binds to CD3.
  • the first arm that specifically binds to PD-1 (A) (a) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 6.
  • VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 9.
  • C (a) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 12
  • D (a) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 15.
  • VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 17 and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 18 CDR1, It has any one VH selected from (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 20.
  • the second arm that specifically binds to CD3 (A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 37, PD-1 / CD3 bispecificity according to the preceding paragraph [1], which has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 38 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 39. An antibody or an antibody fragment thereof. [3] (i) The VH of the first arm that specifically binds to PD-1 is (A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 6.
  • VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7
  • VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8.
  • VH of the second arm that specifically binds to CD3 A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 37, PD-1 / CD3 bispecific according to the preceding paragraph [1] or [2], which has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 38 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 39. Sex antibody or antibody fragment thereof.
  • VH of the first arm that specifically binds to PD-1 is (A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 9. It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 10 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 11.
  • VH of the second arm that specifically binds to CD3 (A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 37, PD-1 / CD3 bispecific according to the preceding paragraph [1] or [2], which has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 38 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 39. Sex antibody or antibody fragment thereof. [5] (i) The VH of the first arm that specifically binds to PD-1 is (A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 12.
  • VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 13 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 14.
  • VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 14.
  • A VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 37, PD-1 / CD3 bispecific according to the preceding paragraph [1] or [2], which has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 38 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 39. Sex antibody or antibody fragment thereof.
  • VH of the first arm that specifically binds to PD-1 is (A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 15. It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 17.
  • VH of the second arm that specifically binds to CD3 (A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 37, PD-1 / CD3 bispecific according to the preceding paragraph [1] or [2], which has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 38 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 39. Sex antibody or antibody fragment thereof. [7] (i) The VH of the first arm that specifically binds to PD-1 is (A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 18.
  • VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 20.
  • VH of the second arm that specifically binds to CD3 A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 37, PD-1 / CD3 bispecific according to the preceding paragraph [1] or [2], which has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 38 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 39. Sex antibody or antibody fragment thereof.
  • a bispecific antibody or antibody fragment thereof having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3, and specifically binds to PD-1.
  • the first arm is (A) Amino acid sequence represented by HYJ 1 LH [In the sequence, J 1 represents G (glycine) or A (alanine), where J 1 represents or the other alphabet is one letter of each amino acid. Represents an abbreviation. ] VH-CDR1, consisting of (B) Amino acid sequence represented by WJ 2 NTTU 2 NPTX 2 AQGFTG [In the sequence, J 2 represents L (leucine) or I (isoleucine), and U 2 represents E (glutamic acid) or G (glycine).
  • X 2 represents F (phenylalanine) or Y (tyrosine), where J 2 , U 2 and X 2 represent, respectively, or the other alphabets each represent the same meanings as above.
  • GDJ 3 VVPTTIWNYYU 3 X 3 MZ 3 V amino acid sequence [in the sequence, J 3 represents M (methionine) or L (leucine), and U 3 represents.
  • H histidine
  • Y tyrosine
  • X 3 F (phenylalanine) or Y (tyrosine)
  • Z 3 represents D (aspartic acid) or E (glutamic acid)
  • J 3 , U 3 , X 3 and Z 3 respectively represent, or the other alphabets each represent the same meaning as above.
  • VH with VH-CDR3 consisting of The second arm that specifically binds to CD3
  • A VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 37, A PD-1 / CD3 bispecific antibody or antibody fragment thereof having VH having (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 38 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 39.
  • J 1 represents G (glycine)
  • J 2 represents L (leucine)
  • U 2 represents E (glutamic acid)
  • X 2 represents F (phenylalanine)
  • J 3 represents M.
  • U 3 represents Y (tyrosine), X 3 represents Y (tyrosine), and Z 3 represents D (aspartic acid), or (d) J 1 represents A (alanine).
  • J 2 stands for L (leucine)
  • U 2 stands for E (glutamic acid)
  • X 2 stands for F (phenylalanine)
  • J 3 stands for M (methionine)
  • U 3 stands for H (histidine), and so on.
  • the PD-1 / CD3 bispecific antibody or antibody fragment thereof according to the previous section [8], wherein X 3 represents F (phenylalanine) and Z 3 represents D (aspartic acid).
  • framework region of the first arm VH that specifically binds to PD-1
  • framework 1 hereinafter, “FR1”
  • FR2 The regions of Framework 2 (hereinafter, may be abbreviated as FR2) and Framework 3 (hereinafter, may be abbreviated as FR3) are the germline type V gene IGHV7-.
  • the PD-1 / CD3 bispecific antibody according to any one of the above items [1] to [9], which corresponds to the amino acid sequence encoded by the gene encoded by 4-1 or its somatic cell mutation. Or its antibody fragment.
  • the first arm VH framework 4 (hereinafter, “framework 4” may be abbreviated as FR4) region that specifically binds to PD-1 is the germline J gene JH6c.
  • the PD-1 / CD3 bispecific according to the previous section [10], which comprises the amino acid sequence encoded by the gene that has undergone the somatic mutation (excluding the amino acid sequence contained in the VH-CDR3 region). A sex antibody or an antibody fragment thereof.
  • the FR region of the first arm VH that specifically binds to PD-1 is encoded by the somatic mutation of the germline V gene IGHV7-4-1.
  • the somatic mutation replaces lysine at position 13 in the amino acid sequence of SEQ ID NO: 21 with glutamine, alanine at position 16 with valine, or lysine at position 19 with methionine, or any of them.
  • the PD-1 / CD3 bispecific antibody or antibody fragment thereof according to the preceding item [10] or [11], which comprises a FR1 region substituted or optionally substituted with a plurality of combinations. [13]
  • the FR region of the VH of the first arm that specifically binds to PD-1 is encoded by the somatic mutation of the germline V gene IGHV7-4-1.
  • the FR region of the VH of the first arm that specifically binds to PD-1 is encoded by the somatic mutation of the germline V gene IGHV7-4-1.
  • the somatic mutation replaces serine at position 77 in the amino acid sequence of SEQ ID NO: 21 with threonine, or cysteine at position 84 with serine or asparagine, respectively, or with any combination of them.
  • the PD-1 / CD3 bispecific antibody or antibody fragment thereof according to any one of the above items [10] to [13], which comprises the FR3 region which may be substituted or substituted.
  • the FR4 region of VH in the first arm that specifically binds to PD-1 may have undergone a somatic mutation in the germline J gene JH6c (however, the VH-CDR3 region).
  • the VH of the first arm that specifically binds to PD-1 is any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, or PD-1 / CD3 double according to any one of the above items [1] to [15], which comprises an amino acid sequence that is at least 80%, 90%, 95%, 98% or 99% identical to the amino acid sequence of the VH. Specific antibody or antibody fragment thereof.
  • the VH of the first arm that specifically binds to PD-1 consists of any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
  • PD-1 / CD3 bispecific antibody or antibody fragment thereof according to any one of the above items [1] and [3] to [7].
  • the amino acid sequence of the second arm that specifically binds to CD3 is at least 80%, 90%, 95%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence of the VH.
  • PD-1 / CD3 according to any one of the above items [1] and [3] to [18], wherein the VH of the second arm that specifically binds to CD3 comprises the amino acid sequence of SEQ ID NO: 36. Bispecific antibody or antibody fragment thereof.
  • the VH of the first arm that specifically binds to PD-1 consists of any one of the amino acid sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
  • PD-1 / CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1], wherein the VH of the second arm that specifically binds to CD3 comprises the amino acid sequence of SEQ ID NO: 36.
  • a bispecific antibody or antibody fragment thereof having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3.
  • the VH of the first arm that specifically binds to PD-1 is any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, or the VH of the said VH. It consists of an amino acid sequence that is at least 80%, 90%, 95%, 98% or 99% identical to the amino acid sequence.
  • the VH of the second arm that specifically binds to CD3 comprises the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence that is at least 80%, 90%, 95%, 98% or 99% identical to the amino acid sequence of the VH.
  • PD-1 / CD3 bispecific antibody or antibody fragment thereof is any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, or the VH of the said VH. It consists of an amino acid sequence that is at least 80%, 90%, 95%, 98% or 99% identical to the amino
  • the VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 1
  • the VH in the second arm that specifically binds to CD3 consists of the amino acid sequence of SEQ ID NO: 36. , PD-1 / CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [3].
  • the VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 2
  • the VH in the second arm that specifically binds to CD3 consists of the amino acid sequence of SEQ ID NO: 36. , PD-1 / CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [4].
  • the VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 3, and the VH in the second arm that specifically binds to CD3 consists of the amino acid sequence of SEQ ID NO: 36. , PD-1 / CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [5].
  • the VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 4, and the VH in the second arm that specifically binds to CD3 consists of the amino acid sequence of SEQ ID NO: 36. , PD-1 / CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [6].
  • VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 5
  • the VH in the second arm that specifically binds to CD3 consists of the amino acid sequence of SEQ ID NO: 36. , PD-1 / CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [7].
  • VL-CDR1 Complementarity determining regions 1 of the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 26
  • VL-CDR2 Complementarity determining regions 2 of the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 27
  • VL-CDR3 complementarity determining regions 3 of the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 28
  • VL-CDR3 complementarity determining regions 3 of the light chain variable region
  • CD3 bispecific antibodies or antibody fragments thereof [28]
  • the first arm that specifically binds to PD-1 and / or the second arm that specifically binds to CD3 each has a VL consisting of the amino acid sequence of SEQ ID NO: 25.
  • the PD-1 / CD3 bispecific antibody or antibody fragment thereof according to any one of the above.
  • a bispecific antibody or antibody fragment thereof having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3.
  • the second arm that specifically binds to CD3 has a VH consisting of the amino acid sequence of SEQ ID NO: 36 and a VL consisting of the amino acid sequence of SEQ ID NO: 25.
  • PD-1 / CD3 bispecific antibody or antibody fragment thereof bispecific antibody or antibody fragment thereof.
  • a bispecific antibody or antibody fragment thereof having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3, and specifically binds to the PD-1.
  • the first arm to be used (1) consists of VH consisting of any one of the amino acid sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and the amino acid sequence of SEQ ID NO: 25. Binding of the first arm that has VL and specifically binds to PD-1 to PD-1, or (2) PD-in the variable region of the monoclonal antibody that specifically binds to PD-1 consisting of the VH and VL.
  • a bispecific antibody or antibody fragment thereof having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3, and specifically binds to the PD-1. Binding to PD-1 by the first arm is (1) VH and sequence consisting of any one of the amino acid sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
  • the second arm that specifically binds to the CD3 (1) specifically binds to CD3 having a VH consisting of the amino acid sequence of SEQ ID NO: 36 and a VL consisting of the amino acid sequence of SEQ ID NO: 25.
  • the second arm that specifically binds to CD3 (A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 37, PD-1 / CD3 according to the preceding paragraph [30] or [31], which has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 38 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 39. Bispecific antibody or antibody fragment thereof.
  • the amino acid sequence of the second arm that specifically binds to CD3 is at least 80%, 90%, 95%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence of the VH.
  • the PD-1 / CD3 bispecific antibody or antibody fragment thereof according to the preceding item [30] or [31], wherein the VH of the second arm that specifically binds to CD3 comprises the amino acid sequence of SEQ ID NO: 36. ..
  • the second arm that specifically binds to CD3 (A) VL-CDR1, consisting of the amino acid sequence of SEQ ID NO: 26, (B) VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27 and (c) VL having VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28, the preceding paragraphs [30], [31] and [33] to [35]. ], PD-1 / CD3 bispecific antibody or antibody fragment thereof according to any one of the above. [37] The item according to any one of the preceding items [30], [31] and [33] to [35], wherein the second arm specifically binding to CD3 has a VL consisting of the amino acid sequence of SEQ ID NO: 25.
  • PD-1 / CD3 bispecific antibody or antibody fragment thereof [38] The PD-1 / CD3 bispecific antibody or an antibody fragment thereof according to any one of the above items [1] to [37], wherein the PD-1 / CD3 bispecific antibody is an IgG antibody. [39] The PD-1 / CD3 bispecific antibody or antibody fragment thereof according to the previous item [38], wherein the IgG antibody according to the previous item [38] is an IgG 1 antibody or an IgG 4 antibody. [40] The PD-1 / CD3 bispecific antibody or antibody fragment thereof according to the previous item [38], wherein the IgG antibody according to the previous item [38] is an IgG 1 antibody.
  • leucine at position 235 in the EU numbering system in the two heavy chain constant regions was replaced with glycine, and / or glycine at position 236 was replaced with arginine, respectively.
  • Leucine at position 351 is replaced with aspartic acid and leucine at position 368 is replaced with glutamic acid by the EU numbering system in the constant region in the heavy chain with VH of the first arm that specifically binds to PD-1.
  • leucine at position 351 and threonine at position 366 in the constant region were both replaced with lysine in the previous section [40], [42] or [43].
  • the IgG 1 antibody according to the preceding paragraph [48] or [49] has a feature that the half-life in blood is shortened as compared with the original antibody in which the amino acid is not substituted. 49] The PD-1 / CD3 bispecific antibody or antibody fragment thereof. [51] The IgG 1 antibody according to the preceding paragraph [48] or [49] has a half-life in blood of at least 50%, at least 60%, and at least 70% of that of the original antibody without the amino acid substitution. The PD-1 / CD3 bispecific antibody or antibody fragment thereof according to the preceding paragraph [48] or [49], which has a characteristic shortened by at least 80% or at least 90%.
  • the heavy chain having the VH of the first arm that specifically binds to PD-1 is SEQ ID NO: 23, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47.
  • the heavy chain with the VH of the second arm that specifically binds to CD3 is selected from SEQ ID NO: 24, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53.
  • a light chain having a VL of the first arm that specifically binds to PD-1 and / or a light chain having a VL of the second arm that specifically binds to CD3 comprises the amino acid sequence of SEQ ID NO: 29.
  • Any one of the heavy chains having the VH of the first arm that specifically binds to PD-1 is selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.
  • a heavy chain constant region consisting of VH consisting of an amino acid sequence and any one of the amino acid sequences selected from SEQ ID NO: 23, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47.
  • the light chain having the VL of the first arm that specifically binds to PD-1 contains the VL consisting of the amino acid sequence of SEQ ID NO: 25 and the light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29.
  • the heavy chain having the VH of the second arm that specifically binds to CD3 is the VH consisting of the amino acid sequence of SEQ ID NO: 36 and SEQ ID NO: 24, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51.
  • a PD-1 / CD3 bispecific antibody or antibody fragment thereof comprising a VL consisting of the amino acid sequence of No. 25 and a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29.
  • the PD-1 / CD3 bispecific antibody or antibody fragment thereof according to any one of the above items [1] to [56], which specifically binds to PD-1 and CD3 expressed on the target cell, respectively. ..
  • PD-1 / according to any one of the preceding paragraphs [1] to [57], wherein the first arm specifically bound to PD-1 allows interaction between PD-1 and PD-L1.
  • the first arm that specifically binds to PD-1 tolerates the interaction between PD-1 and PD-L1 and the cytokine production is sufficiently reduced, according to the preceding paragraphs [1] to [57].
  • PD-1 / CD3 bispecific antibody or antibody fragment thereof according to any one of the above.
  • PD-1 / CD3 bispecific antibody selected from any one of the above items [1] to [65] or an antibody fragment thereof as an active ingredient. Suppression, recurrence suppression and / or therapeutic agent.
  • Autoimmune diseases include Bechette's disease, systemic erythematosus, chronic discoid erythematosus, polyarteritis nodosa, systemic sclerosis, progressive systemic sclerosis, chondrosis, polyarteritis nodosa, and skin.
  • polyarteritis nodosa polyarteritis nodosa, microangiitis nodosa
  • aortitis syndrome hyperan arteritis
  • rheumatoid arthritis rheumatoid arthritis
  • juvenile idiopathic arteritis spondyloarthritis
  • mixed connective tissue Diseases Castleman's disease, Schegren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis, macroangiitis
  • ANCA-related vasculitis eg, polyarteritis nodosa) (Sickness and polyarteritis nodosa)
  • Cogan syndrome RS3PE syndrome
  • temporal arteritis polyarteritis nodosa
  • fibromyalgia antiphospholipid antibody syndrome
  • autoimmune myocardium Flames IgG
  • GVHD Graft-versus-host disease
  • Insulin preparations eg, human insulin, insulin glargine, insulin lispro, insulin detemil, insulin aspart, etc.
  • sulfonylureas eg, glibenclamide, gliclazide, glimepiride, etc.
  • fast-acting insulin secretagogues eg, glimepiride, etc.
  • biguanide preparations eg, metformin, etc.
  • insulin-resistant improving agents eg, pioglycazone, etc.
  • ⁇ -glucosidase inhibitors eg, acarbose, boglibose, etc.
  • diabetic neuropathy therapeutic agents eg, epalrestat, etc.
  • GLP-1 analogs eg, liraglutide, exenatide, lixisenatide, etc.
  • Steroid drugs eg, cortisone, cortisone acetate, hydrocortisone, sodium hydrocortisone phosphate, sodium hydrocortisone succinate, fludrocortisone acetate, prednisolone, prednisolone acetate, sodium prednisolone succinate, butylacetic acid Prednisolone, sodium prednisolone phosphate, halopredon acetate, methylprednisolone, methylprednisolone acetate, sodium methylpredonizolone succinate, triamsinolone, triamsinolone acetate, triamsinolone acetonide, dexamethasone, dexamethasone acetate, dexamethasone, dexamethasone Dexamethasone, dexamethasone palmitate, dexamethasone propionate
  • steroid drugs eg, the steroid drug described in the preceding paragraph [2-5]
  • immunosuppressive drugs eg, cyclosporine, tacrolimus, fingolimod, etc.
  • berimumab Prevention of systemic lupus erythematosus, which comprises PD-1 / CD3 bispecific antibody selected from any one of the above items [1] to [65] or an antibody fragment thereof as an active ingredient.
  • Steroids eg, the steroids described in [2-5] above
  • anti-rheumatic drugs eg, methotrexate, sulfasalazine, bushilamine, reflunomide, misolibin, tacrolimus, etc.
  • anti-cytocytosis drugs eg, infliximab, etc.
  • PD- selected from any one of the preceding paragraphs [1] to [65], characterized in that it is administered with any one or more agents selected from adalimumab, tocilizumab, etanercept, golimumab, sertrizumab) and abatacept.
  • An agent for preventing rheumatoid arthritis, suppressing symptom progression, suppressing recurrence, and / or treating rheumatoid arthritis which comprises a 1 / CD3 bispecific antibody or an antibody fragment thereof as an active ingredient.
  • a 1 / CD3 bispecific antibody or an antibody fragment thereof as an active ingredient.
  • a PD-1 / CD3 bispecific antibody or an antibody fragment thereof as an active ingredient which is a prophylactic, symptom progression inhibitory, recurrence inhibitory and / or therapeutic agent for autoimmune diseases.
  • [4-1] Prevention of autoimmune diseases which comprises administering to a patient an effective amount of the bispecific antibody or antibody fragment thereof selected from any one of the above items [1] to [65]. Symptom progression suppression, recurrence suppression and / or treatment method.
  • the PD-1 / CD3 bispecific antibody of the present invention has reduced cytokine production induction, it can be expected that the expression of infusion reaction or cytokine release syndrome after administration is suppressed.
  • the feature that allows the interaction between PD-1 and PD-L1 can be expected to contribute to its prevention, suppression of symptom progression, suppression of recurrence, and / or enhancement or persistence of therapeutic effect.
  • the amino acid sequences of the VL and constant regions of the common light chain are shown.
  • the amino acid sequence of each CDR of VL of the common light chain is shown.
  • the amino acid sequences encoded by the germline V genes IGHV7-4-1 and IGHV3-33 are shown, respectively.
  • Antibody that specifically binds to PD-1 (hereinafter, may be abbreviated as anti-PD-1 antibody) Shows the sequence alignment of the VH of each clone and the germline genes IGHV7-4-1 and JH6c. ..
  • amino acid sequence of each clone represents an amino acid that is the same as the amino acid sequence of the corresponding germline gene IGHV7-4-1 or JH6c, and the part where the abbreviation of the amino acid is described is , Represents an amino acid that differs from the amino acid sequence of the germline gene.
  • the amino acid sequence of VH of each clone of anti-PD-1 antibody is shown.
  • the amino acid sequence of each CDR in the VH of each anti-PD-1 antibody clone is shown.
  • An antibody that specifically binds to CD3 hereinafter, may be abbreviated as anti-CD3 antibody
  • the amino acid sequence of VH of clone CD3-2 is shown.
  • the amino acid sequence of each CDR in the VH of the anti-CD3 antibody clone CD3-2 is shown.
  • the amino acid sequence of each heavy chain constant region of the PD-1 / CD3 bispecific monoclonal antibody is shown.
  • the amino acid sequence of VH of the anti-CD3 antibody clone 15C3 described in WO2005 / 118635 is shown.
  • the underlined amino acid represents the 55th glycine converted to alanine in the preparation of clone CD3-1.
  • PD-1 / CD3 bispecific monoclonal antibody Biacore® measurement results are shown in which the binding activity of each clone to PD-1 and CD3 was confirmed.
  • PD-1 / CD3 bispecific monoclonal antibody Flow cytometry showing the simultaneous binding of each clone to PD-1 and CD3 is shown.
  • PD-1 / CD3 bispecific monoclonal antibody Flow cytometry showing the effect of each clone on PD-1 / PD-L1 interaction is shown.
  • the results of the action of each PD-1 / CD3 bispecific monoclonal antibody clone on the production of IFN- ⁇ from activated human T cells are shown.
  • “Ctrl" in the figure represents a control group.
  • the therapeutic effect of PD-1 / CD3 bispecific monoclonal antibody clones (PD1-1 (Bi), PD1-2 (Bi)) in an experimental allergic encephalomyelitis mouse model (EAE model) is shown.
  • the therapeutic effect of PD-1 / CD3 bispecific monoclonal antibody clones (PD1-3 (Bi), PD1-4 (Bi)) in a mouse model of experimental allergic encephalomyelitis is shown.
  • the therapeutic effect of PD-1 / CD3 bispecific monoclonal antibody clones (PD1-5 (Bi), PD1-6 (Bi)) in a mouse model of experimental allergic encephalomyelitis is shown.
  • PD-1 (P rogrammed Cell D eath -1) , in humans, is a membrane protein consisting of the amino acid sequence shown in GenBank accession number NP_005009. When referred to herein as "PD-1", unless otherwise specified, all isoforms thereof and the epitope of the "first arm specifically bound to PD-1" according to the invention are conserved. In addition, it may be used as including those variants. In the present invention, PD-1 is preferably human PD-1.
  • CD3 is a membrane-type protein that associates with T cell receptors to form a T cell receptor complex.
  • CD3 its subtypes ( ⁇ , ⁇ , ⁇ and ⁇ subtypes) and the “second arm specifically bound to CD3” according to the present invention. It may be used as a conserved epitope of, including those variants.
  • the CD3 is preferably CD3 ⁇ , human CD3, and more preferably human CD3 ⁇ .
  • isolated is defined as substantially a single pure substance by being identified, separated and / or purified from impurities containing multiple or innumerable components extracted from a host cell. It means that it becomes an ingredient.
  • the term "monoclonal antibody” means an antibody obtained from a substantially homogeneous population of antibodies that binds to the same specific antigen.
  • bispecific antibody means an antibody having binding specificity to two different antigen molecules or epitopes in one molecule, and the term “bispecific monoclonal antibody” further means substantially. Means a bispecific antibody obtained from an antibody population that is uniformly homogeneous.
  • the present invention relates to a bispecific antibody capable of specifically binding to PD-1 and CD3, respectively (in this specification, it may be abbreviated as PD-1 / CD3 bispecific antibody).
  • the PD-1 / CD3 bispecific antibody is preferably a PD-1 / CD3 bispecific monoclonal antibody, more preferably an isolated PD-1 / CD3 bispecific monoclonal antibody. Yes, more preferably an isolated human PD-1 / human CD3 bispecific monoclonal antibody.
  • the "isolated human PD-1 / human CD3 bispecific monoclonal antibody” means an isolated bispecific monoclonal antibody against human PD-1 and human CD3.
  • the morphology of the bispecific antibody includes, for example, diabody, bispecific sc (Fv) 2 , bispecific minibody, bispecific F (ab') 2 , bispecific.
  • Hybrid antibody covalently bound diabody (bispecific DART), bispecific (FvCys) 2 , bispecific F (ab'-zipper) 2 , bispecific (Fv-zipper) 2 , 2
  • bispecific mAb 2 Addbody® and Mirabody® and the like.
  • a diabody is a dimer of a single-stranded peptide in which VHs and VLs that recognize different antigens are linked by a peptide linker (Proc. Natl. Acad. Sci. USA (1993), Vol. 90, No. .14: See p.6444-6448).
  • Bispecific sc (Fv) 2 is a small molecule modified so that two sets of VH / VL of two antibodies that recognize different antigens are produced in a continuous single-chain form via a peptide linker. It is a peptide (see J. Biological Chemistry (1994), Vol. 269: p. 199-206).
  • Bispecific F (ab') 2 is a low molecular weight antibody in which Fab'fragments of an antibody that recognizes two different antigens are covalently bonded by a disulfide bond or the like.
  • the bispecific minibody is a low molecular weight antibody fragment in which a small molecule antibody fragment modified so that the constant region CH3 domain of an antibody is linked to scFv that recognizes different antigens is covalently bonded by a disulfide bond or the like on the CH3 domain. It is a molecularized antibody (see Biochemistry (1992), Vo.31, No.6, p.1579-1584).
  • a bispecific hybrid antibody is an intact antibody in which a heavy chain / light chain complex of an antibody that recognizes two different antigens is covalently bound by a disulfide bond or the like.
  • the preferred form of the bispecific antibody is a bispecific hybrid antibody.
  • the bispecific hybrid antibody can be produced, for example, from a hybridoma prepared by the hybrid hybridoma method (see US4474893).
  • a total of four types of cDNA encoding the heavy and light chains of antibodies that recognize different antigens can be co-expressed and secreted into mammalian cells for production.
  • the monoclonal antibody used in the present invention is a hybridoma method (for example, Kohler and Milstein et al., Natur (1975), Vol. 256, p.495-97, Hongo et al., Hybridoma (1995), Vol. 14, No. 3 , P.253-260, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press (1988), Vol.
  • an antibody or monoclonal antibody When administered to a human, it can be prepared in the form of a chimeric antibody, a humanized antibody, or a fully human antibody in order to reduce or eliminate its antigenicity.
  • the "chimeric antibody” means an antibody in which the variable region sequence and the constant region sequence are derived from mammals, for example, the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. ..
  • the chimeric antibody is a gene encoding an antibody variable region isolated by a known method from an antibody-producing hybridoma isolated by the above-mentioned hybridoma method, recombinant DNA method or phage display method, using a known method.
  • Human-derived antibody constant regions can be linked to genes encoding to make them (see, eg, US4816567 of Cabilly et al.).
  • Humanized antibody means an antibody in which a complementarity determining region (CDR) sequence derived from a germ cell type of another mammal such as a mouse is transplanted onto a human framework sequence.
  • CDR complementarity determining region
  • a gene encoding an antibody CDR region isolated by a known method from an antibody-producing hybridoma isolated by the above method can be obtained from a human-derived antibody framework region using a known method.
  • Can be made by linking to a gene encoding see, eg, US5225539 and US5530101 in Winter, US5585089 and US6180370 in Queen et al.).
  • Human antibody or “fully human antibody” means an antibody derived from a human germ cell type immunoglobulin sequence in both the variable region consisting of the framework region and the CDR region and the constant region.
  • the human antibodies used in the present invention are mice transformed to produce human antibodies, such as Humab mice (eg, US5545806, US5569825, US5625126, US5633425, US5789650, US5877397, US5661016, US5814318 by Lonberg and Kay et al.
  • mice see, eg, WO2002 / 43478 of Ishida et al.
  • Xeno mice see, eg, US5939598, US6075181, US6114598, US6150584 and US6162963
  • Tc mice eg, Tomizuka et al., Proc. Natl. It can be produced by the method using Acad. Sci. USA (2000), p.722-727. It can also be prepared using SCID mice in which human immune cells have been reconstituted (see, eg, US5476996 and US5698767 by Wilson et al.) So that immunization causes a human antibody response.
  • the human antibody used in the present invention can also be produced by the phage display method described above.
  • an "antibody fragment" of a PD-1 / CD3 bispecific antibody is an antibody that is part of a full-length antibody and has an antigen-binding portion to PD-1 and an antigen-binding portion to CD3.
  • F (ab') 2 an antigen-binding portion
  • the antigen-binding portion means the minimum unit that an antibody can bind to the antigen, and for example, the target antigen can be recognized by the combination of three CDRs each in VH and VL and those CDRs. It consists of a framework area where the CDR is placed.
  • the term "common light chain” means a light chain that is capable of associating with two or more different heavy chains and exhibiting binding ability to each antigen (De Wildt RM et al., J. Mol. Biol. (1999), Vol. 285, p.895-901, De Kruif et al., J. Mol. Biol. (2009), Vol. 387, p.548-58, WO2004 / 009618, WO2009 / 157771 and WO2014 / 051433).
  • a common light chain for example, a human ⁇ light chain IgV ⁇ 1-39 * 01 / IGJ ⁇ 1 * 01 (nominated by the IMGT database) a light chain encoded by a germline gene (hereinafter, IGVK1-39 /) It may be abbreviated as JK1 common light chain), and more preferably, for example, VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and the amino acid of SEQ ID NO: 28.
  • the light chain having a VL comprising the sequence VL-CDR3, more preferably, for example, the light chain having a VL consisting of the amino acid sequence of SEQ ID NO: 25.
  • a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29 is preferable.
  • the amino acid sequences of the VL and the constant region of the common light chain used in the present invention are shown in FIG. 1, and the amino acid sequence of each CDR of the variable region is shown in FIG.
  • isotype is used to refer to an antibody class (eg, IgM or IgG) encoded by a heavy chain constant region gene.
  • the preferred isotype of the PD-1 / CD3 bispecific antibody of the invention is IgG, more preferably IgG 1 or IgG 4 .
  • IgG 1 those in which the binding to the Fc receptor (for example, Fc ⁇ receptor) is lost or attenuated are preferable. Specifically, by substituting, deleting or inserting an arbitrary amino acid in the heavy chain constant region, an IgG 1 antibody in which the binding to the Fc receptor is lost or attenuated can be obtained.
  • an antibody according to the EU numbering system in which leucine at position 235 is replaced with glycine and / or glycine at position 236 is replaced with arginine.
  • an antibody lacking the C-terminal amino acid, for example, ricin at position 447 according to the EU numbering system is preferable.
  • those in which the binding to FcRn is lost or attenuated are particularly preferable.
  • the binding to the receptor can be eliminated or attenuated by substitution or deletion of an amino acid belonging to the FcRn binding site in the heavy chain constant region, but in the case of an IgG 1 antibody, such an antibody can be used.
  • an antibody can be used.
  • the 252nd methionine by the EU numbering system is to glutamic acid, proline, arginine or aspartic acid
  • the 434th aspartic acid by the EU numbering system is to leucine
  • / or (3) by the EU numbering system examples thereof include those in which the 438th glutamine is replaced with glutamic acid.
  • the bispecific antibody is IgG 4
  • a variant in which any amino acid in the heavy chain constant region is substituted, deleted or inserted so as to suppress swapping in the antibody molecule is more preferable.
  • an antibody in which serine at position 228 by the EU numbering system is replaced with proline, which is located in the hinge region is preferable.
  • the amino acid position assigned to the CDR of the variable region of an antibody and the framework may be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md.,). 1987) and (1991)).
  • the amino acids in the constant region are represented according to the EU numbering system according to the amino acid position of Kabat (see Sequences of proteins of immunological interest, NIH Publication No. 91-3242).
  • the Fc region of the PD-1 / CD3 bispecific antibody of the present invention may be substituted with any amino acid in that region so that two different heavy chains can easily associate with each other.
  • leucine at position 351 is replaced with lysine and threonine at position 366 by the EU numbering system of the constant region on the heavy chain having the VH of the first arm that specifically binds to PD-1.
  • Pad- 1 / CD3 bispecific antibody can be mentioned.
  • leucine at position 351 by the EU numbering system in the constant region in the heavy chain having VH of the first arm that specifically binds to PD-1 was replaced with aspartic acid, and leucine at position 368 was replaced with glutamic acid.
  • PD-1 / CD3 double in which leucine at position 351 and threonine at position 366 in the constant region in the heavy chain with VH of the second arm that specifically binds to CD3 was replaced with lysine.
  • Specific antibodies can also be mentioned.
  • first arm that specifically binds to PD-1
  • first arm is an antibody.
  • an antibody that specifically binds to PD-1 hereinafter, anti-PD-1 antibody
  • anti-PD-1 antibody regardless of whether it is contained in a part of the antibody fragment or is not a part of the antibody fragment and exists as a single substance.
  • anti-PD-1 antibody an antibody that specifically binds to PD-1
  • VH the VH of an anti-PD-1 antibody and said.
  • the first arm also contains the Fab portion of the antibody containing the VH and VL.
  • “specifically binding to PD-1” means having an affinity higher than at least 1x10 -5 M, preferably 1x10 -7 M, and more preferably 1x10 -9 M (dissociation constant (Kd value)). It can bind directly to PD-1 with its binding activity, and at least does not substantially bind to other receptor members belonging to the so-called CD28 family of receptors, such as CD28, CTLA-4 and ICOS. Used as a feature.
  • the "antibody” in the “antibody that specifically binds to PD-1" or “anti-PD-1 antibody” is a full-length antibody, that is, two heavy chains linked by a disulfide bond and two light chains. Means a full-length antibody, preferably a monoclonal antibody thereof.
  • X 2 represents F (phenylalanine) or Y (tyrosine), where J 2 , U 2 and X 2 represent, respectively, or the other alphabets each represent the same meanings as above.
  • VH-CDR2 and (c) GDJ 3 VVPTTIWNYYU 3 X 3 MZ 3 V amino acid sequence [in the sequence, J 3 represents M (methionine) or L (leucine), and U 3 represents Represents H (histidine) or Y (tyrosine), X 3 represents F (phenylalanine) or Y (tyrosine), Z 3 represents D (aspartic acid) or E (glutamic acid), where J 3 , U 3 , X 3 and Z 3 respectively represent, or the other alphabets each represent the same meaning as above.
  • VH having VH-CDR3.
  • J 1 in the HYJ 1 LH sequence, which is the VH-CDR 1 represents G (glycine)
  • J 2 in the WJ 2 NTTU 2 NPTX 2 AQGFTG sequence, which is the VH-CDR 2 represents L (leucine).
  • U 2 represents E (glutamic acid)
  • X 2 represents F (phenylalanine)
  • J 3 in the GDJ 3 VVPTTIWNYYU 3 X 3 MZ 3 V sequence, which is its VH-CDR3, represents M (methionine)
  • U 3 represents M (methionine).
  • H H (histidine), X 3 stands for F (phenylalanine), Z 3 stands for D (aspartic acid), each with VH-CDR.
  • J 1 in the HYJ 1 LH sequence, which is the VH-CDR 1 represents G (glycine), and J 2 in the WJ 2 NTTU 2 NPTX 2 AQGFTG sequence, which is the VH-CDR 2 , represents I (isoleucine).
  • U 2 represents G (glycine)
  • X 2 represents Y (tyrosine)
  • U 3 represents L (leucine).
  • VH each with a VH-CDR, representing H (histidine), X 3 representing Y (tyrosine), Z 3 representing E (glutamic acid), respectively.
  • J 1 in the HYJ 1 LH sequence, which is the VH-CDR 1 represents A (alanine)
  • J 2 in the VH-CDR 2 WJ 2 NTTU 2 NPTX 2 AQGFTG sequence represents L (leucine).
  • U 2 represents E (glutamic acid)
  • X 2 represents Y (tyrosine)
  • HYJ 1 LH sequence which represents Y (tyrosine)
  • X 3 represents Y (tyrosine)
  • Z 3 represents D (aspartic acid), each with VH-CDR, or (4a) its VH-CDR1.
  • J 1 represents A (alanine), and its VH-CDR2, WJ 2 NTTU 2 NPTX 2 AQGFTG sequence
  • J 2 is L (leucine)
  • U 2 is E (glutamic acid)
  • X 2 is F ( Phenylalanine)
  • VH-CDR3 GDJ 3 VVPTTIWNYYU 3 X 3 MZ 3 V sequence
  • J 3 is M (methionine)
  • U 3 is H (histidine)
  • X 3 is F (phenylalanine).
  • Z 3 represents D (aspartic acid), respectively, and those having VH each having VH-CDR can be mentioned.
  • VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 6, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7, and VH containing VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8.
  • VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 9, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 10 and VH containing VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 11.
  • VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 13 and VH containing VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 14.
  • VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 15, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and VH containing VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 17 and (5b) VH of SEQ ID NO: 18.
  • Examples thereof include those having any one VH selected from VH-CDR1 consisting of the amino acid sequence, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 20.
  • the "first arm that specifically binds to PD-1" includes each of the above (1a) to (4a) or (1b) to (5b) in any one of the VHs.
  • any 1-5 amino acid residues are replaced with other amino acids (preferably their conservative amino acids), and PD by the original first arm not replaced with the amino acid.
  • Those having substantially the same binding activity as the binding activity to -1 are also included.
  • one amino acid residue is replaced with another amino acid (preferably its conservative amino acid)
  • the amino acid residue of is replaced with another amino acid (preferably its conservative amino acid).
  • each amino acid different between each clone or any of them in each CDR of the anti-PD-1 antibody clone corresponding to the first arm that specifically binds to PD-1, each amino acid different between each clone or any of them.
  • the plurality of combinations can be replaced with each other among the clones.
  • substitution with conservative amino acids means the interchangeability of residues having similar side chains, for example, in the group of amino acids having aliphatic side chains, with glycine, alanine, valine, leucine and isoleucine.
  • Examples of preferred conservative amino acid substitutions include substitutions between valine, leucine and isoleucine, substitutions between phenylalanine and tyrosine, substitutions between lysine and arginine, substitutions between alanine and valine and between asparagine and glutamine. Replacement of. Further, here, the above-mentioned "having substantially the same binding activity to PD-1 by the original first arm not substituted with the amino acid" means that the first arm is substituted with the amino acid.
  • the binding activity of the arm to PD-1 is 95% or more, preferably 98% or more, and more preferably 99% or more of the binding activity of the original first arm not substituted with the amino acid. Means.
  • the "first arm that specifically binds to PD-1" in the present invention includes each VH-CDR having the above-mentioned specific amino acid sequence in the VH, and the amino acid sequence of the framework of the VH is included. Also included are those with a specific germline gene or a VH encoded by that gene that has undergone a somatic mutation thereof.
  • the VH indicated by any of the above (1a) to (4a) or (1b) to (5b) has a germline V gene of IGHV7-4-1 and is a germline type.
  • the J gene can be encoded by the VDJ recombinant gene, which is JH6c, or the gene that has undergone a somatic mutation thereof.
  • the amino acid sequences encoded by the germline V gene IGHV7-4-1 correspond to the amino acid sequence of SEQ ID NO: 21 (Fig. 3).
  • the first arm VH framework that specifically binds to PD-1 of the present invention may be encoded by the germline VDJ recombinant gene that has undergone somatic mutation.
  • FR1, FR2, FR3 of VH shown in any of the above (1a) to (4a) or (1b) to (5b) in which the germline type V gene is IGHV7-4-1 Since the amino acid position shown in FIG. 4 is different from the amino acid sequence encoded by the IGHV7-4-1 gene, it has undergone a somatic mutation at each position.
  • lysine at position 13 in the amino acid sequence of SEQ ID NO: 21 is replaced with glutamine, alanine at position 16 is replaced with valine, or lysine at position 19 is replaced with methionine, or any of them. It may be replaced by a plurality of combinations.
  • valine at position 37 in the amino acid sequence of SEQ ID NO: 21 may be replaced with leucine.
  • serine at position 77 in the amino acid sequence of SEQ ID NO: 21 may be replaced with threonine, cysteine at position 84 may be replaced with serine or asparagine, or any combination may be substituted.
  • the FR4 region of VH indicated by any of the above (1a) to (4a) or (1b) to (5b) is the FR4 region amino acid sequence (Trp-Gly-Lys) derived from the J gene JH6c.
  • -Lysine (Lys) in Gly-Thr-Thr * -Val-Thr-Val-Ser-Ser (SEQ ID NO: 41) is replaced with glutamine or asparagine, and / or threonine (Thr) marked with * is replaced with leucine.
  • FR1, FR2, FR3 and FR4 respectively, which have any of the above amino acid substitution combinations, also have no substantial effect on the function of the first arm that specifically binds to PD-1, and can be used as a framework. it can.
  • the "first arm that specifically binds to PD-1" in the present invention contains each CDR having the above-mentioned specific amino acid sequence, and the FR amino acid sequence of the VH is a specific germ cell. It also includes a lineage gene or one encoded by the gene that has undergone a somatic mutation thereof. For example, such a first arm may have a VH consisting of an amino acid sequence selected from SEQ ID NOs: 1-5.
  • first arm that specifically binds to PD-1 is, for example, at least 80% identical to any one amino acid sequence selected from SEQ ID NOs: 1-5, preferably at least. It has a VH consisting of an amino acid sequence that is 90% identical, more preferably at least 95% identical, even more preferably at least 98% identical, and even more preferably at least 99% identical, and the original first. It also includes those in which the difference from the amino acid sequence of VH of one arm does not substantially affect the binding activity to PD-1 (hereinafter, may be abbreviated as homologous first arm).
  • % identity as used in the comparison of identity with respect to an amino acid sequence is the amino acid sequence that aligns and references two sequences (where necessary to achieve the maximum percentage identity). Is defined as a percentage of the amino acid sequence that is identical to the reference amino acid sequence after the introduction of the gap). Further, here, “the difference from the amino acid sequence of VH of the original first arm does not substantially affect the binding activity to PD-1” means that the binding activity of the homologous first arm to PD-1 It means that it is 95% or more, preferably 98% or more, and more preferably 99% or more of the binding activity of the original first arm.
  • the "first arm that specifically binds to PD-1" in the present invention includes (1) any of the above (1a) to (4a) or (1b) to (5b). Binding to PD-1 of the first arm having VL of VH and common light chain consisting of VH represented by or amino acid sequence selected from SEQ ID NOs: 1 to 5 or (2) PD- consisting of the VH and VL.
  • the variable region of an anti-PD-1 antibody that cross-competites with the binding of the variable region of a monoclonal antibody that specifically binds to 1 to PD-1 (where the variable region comprises the VHs and VLs that compose it.
  • binding to PD-1 is (3) VH or SEQ ID NO: shown in any of the above (1a) to (4a) or (1b) to (5b).
  • Those having a variable region of the anti-PD-1 antibody to be used are also included.
  • cross-competition for binding to PD-1 means PD- of the first arm by binding to an epitope that is the same as or partially overlaps with that of the first arm exemplified herein.
  • Binding to PD-1 by an antibody that inhibits binding to 1 to any extent, or binds to an epitope that is the same as or partially overlaps with the exemplified first arm, is the said first arm. It means that it is inhibited regardless of its degree, and whether or not it cross-competites can be evaluated by a competitive binding assay. For example, it can be judged by using viacore analysis, ELISA assay, flow cytometry, enzyme-linked immunosorbent assay (ELISA), fluorescence energy transfer measurement method (FRET), or fluorescence trace measurement technology (FMAT (registered trademark)). ..
  • VH and VL of the common light chain represented by (preferably VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28.
  • First arm having VL further VH consisting of amino acid sequences selected from SEQ ID NOs: 1 to 4 and VL of common light chain (preferably VL consisting of amino acid sequence of SEQ ID NO: 25).
  • PD by the first arm having VH represented by any of the above (1b) to (4b) or VH consisting of the amino acid sequence selected from SEQ ID NOs: 1 to 4 and VL of the common light chain.
  • VH shown in (5b) above and VL of the common light chain preferably VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, amino acid sequence of SEQ ID NO: 27).
  • a first arm having a VL) consisting of the amino acid sequence of number 25 can be mentioned.
  • the first arm that specifically binds to PD-1 in the present invention, preferably, the first arm having VH indicated by any of the above (1b) to (5b) can be mentioned.
  • this preferred first arm in each CDR of the VH, any 1-5 amino acid residues thereof are replaced with other amino acids (preferably their conservative amino acids).
  • the amino acid substitution does not substantially affect the binding activity to PD-1, and as described above, the amino acid sequence of the VH framework is the germ cell lineage type V gene IGHV7. -4-1 or the J gene JH6c or those having VH encoded by the somatically mutated gene thereof are also included.
  • those having VH consisting of any one amino acid sequence selected from SEQ ID NOs: 1 to 5 can be mentioned.
  • the "first arm specifically bound to PD-1" in the present invention preferably contains the VL of the common light chain, and is preferably common to, for example, IGVK1-39 / JK1. It is a light chain, and more preferably, for example, VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and VL containing VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28. It is a light chain having VL, and more preferably, for example, a light chain having VL consisting of the amino acid sequence of SEQ ID NO: 25. Further, as the constant region of the common light chain, a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29 is preferable.
  • the "first arm that specifically binds to PD-1” allows interaction with PD-1 and PD-L1, interaction with PD-1 and PD-L2, or both.
  • the one is more preferable.
  • "allowing the interaction with PD-1 and PD-L1, the interaction with PD-1 and PD-L2, or both of them” means the PD-1 / CD3 dual of the present invention.
  • the interaction, or both of them, is maintained by 50% or more, preferably 70% or more, and more, as compared with the interaction in the absence of the PD-1 / CD3 bispecific antibody of the present invention. It means that it is preferably maintained at 80% or more.
  • the definition of "allowing interaction with PD-1 and PD-L1, interaction with PD-1 and PD-L2, or both of them” is defined as “interaction with PD-1 and PD-L1". It may be used synonymously with the meaning of "substantially not inhibiting interaction, interaction with PD-1 and PD-L2, or their interaction.”
  • FIG. 5 shows the correspondence between each clone of the anti-PD-1 monoclonal antibody obtained for constructing the PD-1 / CD3 bispecific antibody of the present invention, their VH amino acid sequences, and their SEQ ID NOs. , And the correspondence between the amino acid sequence of each CDR in the VH of each clone of the anti-PD-1 monoclonal antibody and its SEQ ID NO: is shown in FIG.
  • Second arm that specifically binds to CD3 means an antibody or antibody fragment.
  • An antibody that specifically binds to CD3 (hereinafter, may be abbreviated as an anti-CD3 antibody) regardless of whether or not it is contained as a part of, or not a part of, and exists as a single substance. )
  • the second arm also includes the Fab portion of the antibody containing the VH and VL.
  • “specifically binds to CD3” has at least an affinity (dissociation constant (Kd value)) higher than 1x10 -5 M, preferably 1x10 -7 M, and more preferably 1x10 -9 M. It can bind directly to CD3 with its binding activity and is used as a feature that does not substantially bind to other proteins.
  • the "antibody” in the “antibody that specifically binds to CD3” or “anti-CD3 antibody” is a full-length antibody, that is, a full-length antibody consisting of two heavy chains linked by a disulfide bond and two light chains. It means an antibody, preferably a monoclonal antibody thereof.
  • VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 37
  • VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 38
  • VH containing VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 39.
  • any 1 to 5 amino acid residues thereof are other amino acids (preferably) in each CDR in the VH described in (1c) above. Includes those that are substituted with the conservative amino acid) and have substantially the same binding activity to CD3 by the original second arm not substituted with the amino acid.
  • one amino acid residue is replaced with another amino acid (preferably its conservative amino acid)
  • 1 to 5 amino acid residues are each other. Amino acids (preferably its conservative amino acids) have been substituted.
  • each VH-CDR of the second arm includes, for example, the above-mentioned example of amino acid substitution in the first arm.
  • the "second arm that specifically binds to CD3" includes each CDR having the above-mentioned specific amino acid sequence in the VH, and the FR amino acid sequence of the VH is a specific germline. It also includes those having a VH encoded by the germline gene or the gene that has undergone a somatic mutation thereof.
  • the VH in (1c) above is encoded by a VDJ recombinant gene in which the germline V gene is IGHV3-33 or the gene that has undergone a somatic mutation thereof.
  • the amino acid sequence (SEQ ID NO: 22) encoded by the germline V gene IGHV3-33 is shown in FIG.
  • a second arm one having VH consisting of the amino acid sequence of SEQ ID NO: 36 can be mentioned.
  • a second arm is, for example, at least 80% identical, preferably at least 90% identical, more preferably at least 95% identical, and even more preferably at least the amino acid sequence of SEQ ID NO: 36. It has a VH consisting of an amino acid sequence that is 98% identical, and even more preferably at least 99% identical, and the difference from the original second arm VH amino acid sequence is substantially in the binding activity to CD3. Those that do not affect (hereinafter, may be abbreviated as homologous second arm) are also included.
  • the difference from the amino acid sequence of VH of the original second arm does not substantially affect the binding activity to CD3 means that the binding activity of the homologous second arm to CD3 is the original second arm. It means that it is 95% or more, preferably 98% or more, and more preferably 99% or more of the binding activity.
  • the "second arm that specifically binds to CD3" in the present invention includes (1) VH represented by (1c) above or VH consisting of the amino acid sequence of SEQ ID NO: 36 and a shared light chain.
  • the variable region also includes those having VH and VL constituting the variable region).
  • cross-competition in binding to CD3 means that the second arm binds to CD3 by binding to an epitope that is the same as or partially overlaps with the second arm exemplified herein. Means to inhibit regardless of the degree.
  • whether or not there is cross-competition can be similarly measured according to the method described in the description of "the first arm that specifically binds to PD-1".
  • the "second arm that specifically binds to CD3" in the present invention a second arm having VH shown in (1c) above is preferably mentioned, and further, the preferred second arm is described above.
  • any 1 to 5 amino acid residues are replaced with other amino acids (preferably the conservative amino acids), and the amino acid substitution is the binding activity to CD3.
  • the amino acid sequence of the VH framework encodes the germline gene IGHV3-33 or its somatic mutated gene. Those with VH to be used are also included.
  • the second arm having VH consisting of the amino acid sequence of SEQ ID NO: 36 can be mentioned.
  • FIG. 8 shows the correspondence between the amino acid sequence of each CDR in the VH of each clone of the anti-CD3 antibody and its SEQ ID NO:.
  • the "second arm that specifically binds to CD3" in the present invention preferably contains the VL of the common light chain, and as such a common light chain, for example, IGVK1-39 / JK1 common light chain. More preferably, for example, a light chain having VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28. Yes, more preferably, for example, a light chain having a VL consisting of the amino acid sequence of SEQ ID NO: 25. Further, as the constant region of the common light chain, a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29 is preferable.
  • the "second arm that specifically binds to CD3" in the present invention preferably includes one that specifically binds to CD3 ⁇ .
  • the preferred isotype of the PD-1 / CD3 bispecific antibody of the present invention is an IgG antibody, more preferably an IgG 1 or IgG 4 antibody, and even more preferably an IgG 1 antibody.
  • a preferred embodiment of the PD-1 / CD3 bispecific antibody of the present invention is, for example, a first arm that specifically binds to PD-1.
  • A Any one selected from VH-CDR1, VH-CDR2 and VH-CDR3 in the VH indicated by any of the above (1a) to (4a) or (1b) to (5b).
  • VH each of which any 1-5 amino acid residues may be replaced with another amino acid (preferably its conservative amino acid)
  • B the amino acid sequence of SEQ ID NO: 26.
  • VL has a common light chain VL comprising VL-CDR1 consisting of, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27 and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28, and The second arm that specifically binds to CD3 (C)
  • C In any one or more CDRs selected from VH-CDR1, VH-CDR2 and VH-CDR3 in VH shown in (1c) above, each of any 1 to 5 amino acid residues thereof.
  • VH may be substituted with another amino acid (preferably a conservative amino acid), and (D) VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27 and the sequence. Included are PD-1 / CD3 bispecific antibodies characterized by having a common light chain VL containing VL-CDR3 consisting of the amino acid sequence of number 28.
  • the first arm that specifically binds to PD-1 (A) Any one VH selected from (1a) to (4a) or (1b) to (5b) above, and (B) VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, the amino acid of SEQ ID NO: 27.
  • VL-CDR2 consisting of the sequence and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28, and The second arm that specifically binds to CD3 (C) VH shown in (1c) above, (D) VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and VL- consisting of the amino acid sequence of SEQ ID NO: 28.
  • PD-1 / CD3 bispecific antibodies characterized by having a common light chain VL containing CDR3.
  • a first arm that specifically binds to PD-1 is used.
  • VH consisting of any one amino acid sequence selected from SEQ ID NOs: 1 to 5, VH consisting of an amino acid sequence that is at least 80% identical to the amino acid sequence of the VH, and
  • B amino acid of SEQ ID NO: 25.
  • VH consisting of the amino acid sequence of SEQ ID NO: 36
  • VH consisting of the amino acid sequence of at least 80% identical to the amino acid sequence of the VH
  • D VL of the common light chain consisting of the amino acid sequence of SEQ ID NO: 25.
  • PD-1 / CD3 bispecific antibodies which are characterized by having.
  • a first arm that specifically binds to PD-1 It has (A) VH consisting of any one amino acid sequence selected from SEQ ID NOs: 1 to 5, and (B) VL of a common light chain consisting of the amino acid sequence of SEQ ID NO: 25, and The second arm that specifically binds to CD3 PD-1 / CD3 bispecific antibodies, characterized by having (C) a VH consisting of the amino acid sequence of SEQ ID NO: 36 and (D) a VL of a common light chain consisting of the amino acid sequence of SEQ ID NO: 25. Be done.
  • the 235th leucine according to the EU numbering system is located on each of the two heavy chain constant regions or hinge regions.
  • An IgG 1 antibody substituted with glycine and / or glycine at position 236 with arginine is preferred.
  • antibodies lacking the amino acid at the C-terminal of the heavy chain of these bispecific antibodies, for example, lysine at position 447 according to the EU numbering system are more preferable.
  • the PD-1 / CD3 bispecific antibody is an IgG 4 antibody is located in the hinge region, antibody was substituted with proline 228 serine by the EU numbering system are preferred.
  • PD-1 / CD3 bispecific antibodies are IgG 1 antibodies
  • a preferred embodiment is the EU in a constant region in a heavy chain with a first arm VH that specifically binds to PD-1.
  • leucine at position 351 is replaced with lysine
  • threonine at position 366 is replaced with lysine
  • Examples thereof include IgG 1 antibody in which aspartic acid is substituted and leucine at position 368 is substituted with glutamic acid.
  • leucine at position 351 was replaced with aspartic acid and leucine at position 368 was replaced with glutamic acid by the EU numbering system in the constant region in the heavy chain having VH of the first arm that specifically binds to PD-1.
  • An IgG 1 antibody in which leucine at position 351 and threonine at position 366 in the constant region in the heavy chain having VH of the second arm that specifically binds to CD3 is replaced with lysine is also preferable. ..
  • the preferred embodiment is that (1) the 252nd methionine by the EU numbering system in the constant region of each of the two heavy chains is glutamic acid.
  • Examples include IgG 1 antibody in which proline, arginine or aspartic acid is replaced by (2) aspartic acid at position 434 according to the EU numbering system and / or (3) glutamine at position 438 according to the EU numbering system is replaced with glutamic acid. .. Due to these amino acid substitutions, the half-life of the PD-1 / CD3 bispecific antibody in blood is at least 50%, preferably at least 60%, more preferably, as compared with the antibody without the amino acid substitution. It can be expected to be reduced by at least 70%, more preferably by at least 80%, and most preferably by at least 90%.
  • a preferred embodiment of the PD-1 / CD3 bispecific IgG 1 antibody incorporating the above amino acid substitutions in the heavy chain constant region is, for example, a heavy chain having a VH of the first arm that specifically binds to PD-1.
  • the heavy chain having the VH of the second arm specifically bound to is selected from SEQ ID NO: 24, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53.
  • Examples thereof include antibodies having a heavy chain constant region consisting of one amino acid sequence. Some of those amino acid sequences are shown in FIG.
  • the most preferable embodiment of the PD-1 / CD3 bispecific antibody of the present invention is clone PD1-1 (Bi), clone PD1-2 (Bi), and clone PD1 prepared in Example 8 of the present specification.
  • -3 (Bi), clone PD1-4 (Bi) and clone PD1-5 (Bi) and clone Mut1, clone Mut2, clone Mut3, clone Mut4, clone Mut5 and clone Mut6 prepared in Example 19.
  • Preferred features of the PD-1 / CD3 bispecific antibody of the present invention include (1) interaction with PD-1 and PD-L1, interaction with PD-1 and PD-L2, or both. Examples include those that allow interaction and / or (2) sufficiently reduced cytokine production.
  • tolerate interaction with PD-1 and PD-L1, interaction with PD-1 and PD-L2, or both of them means “specifically binds to PD-1.” It has the same meaning as the definition given in the description of "first arm”.
  • “cytokine production was sufficiently reduced” means that, for example, the PD-1 / CD3 bispecific antibody or the like of the present invention is intravenously administered by intravenous drip or the like, or within 24 hours after administration. For example, it means that the concentration of cytokines including IL-2, IFN- ⁇ and / or TNF- ⁇ in blood or tissue does not increase, or even if it increases, it can be suppressed by steroid administration.
  • the PD-1 / CD3 bispecific antibody of the present invention and an antibody fragment thereof are disclosed in WO2014 / 051433, WO2013 / 157953 or WO2013 / 157954. It can also be manufactured by the method.
  • a polynucleotide encoding a heavy chain having a VH of the first arm that specifically binds to PD-1 and (2) a weight having a VH of the second arm that specifically binds to CD3.
  • An expression vector in which a polynucleotide encoding a chain and (3) a polynucleotide encoding a common light chain are inserted is introduced into a mammalian cell and transformed to express both heavy chains and a common light chain. It can also be produced by secreting it.
  • the host cell expressing the PD-1 / CD3 bispecific antibody of the present invention may be any host cell capable of introducing an expression vector into the gene and expressing the introduced expression vector.
  • insect cells such as SF-9 and SF-21 cells, more preferably mouse cells containing CHO cells, BHK cells, SP2 / 0 cells and NS-0 myeloma cells, primates such as COS and Vero cells.
  • Examples include mammalian cells such as similar cells, MDCK cells, BRL3A cells, hybridomas, tumor cells, immortalized primary cells, embryonic retinal cells such as W138, HepG2, HeLa, HEK293, HT1080 or PER.C6.
  • an expression vector and host of mammalian cells may be used so that the antibody is appropriately glycosylated.
  • Human cell lines, preferably PER.C6, are advantageously used to obtain antibodies that match the glycosylation pattern in humans.
  • Protein production in host cells transformed by gene transfer of expression vectors is described, for example, in Current Protocols in Protein Science (1995), Coligan JE, Dunn BM, Ploegh HL, Speicher DW, Wingfield PT, ISBN 0-471-11184. -8, Bendig, 1988 can be referred to, and general guidelines, procedures and practical methods for maximizing host cell culture productivity can be found in Mammalian Cell Biotechnology: a Practical Approach. It can be carried out with reference to (M. Butler, ed., IRL Press, 1991). Expression of antibodies in host cells is described, for example, in publications such as EP0120694, EP0314161, EP0481790, EP0523949, US4816567 and WO2000 / 63403.
  • the culture conditions of the host cell can be optimized by a known method, and the amount of protein produced can be optimized.
  • the culture can be carried out, for example, in a culture dish, a roller bottle or a reaction vessel by batch culture, fed-batch culture, continuous culture, or culture with hollow yarn.
  • Antibodies expressed in host cells and recovered from the cells or cell culture medium by a known method can be purified by a known method. Purification methods include immunoprecipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography, hydrophobic interaction chromatography and the like. In addition, protein A or protein G affinity chromatography may be preferably used (see, eg, US4801687 and US5151504).
  • the PD-1 / CD3 bispecific antibody and the like of the present invention are useful for prevention, suppression of symptom progression, suppression of recurrence and / or treatment of autoimmune disease or graft-versus-host disease (GVHD).
  • GVHD graft-versus-host disease
  • treatment means, for example, curing or ameliorating a certain disease or a symptom thereof
  • prevention means preventing or delaying the onset of a certain disease or a symptom for a certain period of time. That is, “suppression of symptom progression” means suppressing the progression or worsening of symptoms and stopping the progression of the pathological condition. The meaning of “prevention” also includes suppression of recurrence. By “suppressing recurrence” is meant preventing or reducing the likelihood of recurrence of a disease or symptom.
  • the PD-1 / CD3 bispecific antibody and the like of the present invention are usually administered systemically or locally in a parenteral form.
  • Specific examples of such an administration method include injection administration, nasal administration, pulmonary administration, and transdermal administration.
  • Examples of the injection administration include intravenous injection, intramuscular injection, intraperitoneal injection and the like, and in the case of intravenous injection, administration by infusion is preferable.
  • the dose varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but is usually in the range of 0.1 ⁇ g / kg to 300 mg / kg per adult, particularly preferably.
  • the injection or infusion solution is an aqueous solution, suspension or emulsion.
  • it is formulated as a solid with a pharmaceutically acceptable carrier so that it can be dissolved, suspended or emulsified by adding a solvent at the time of use. May be good.
  • Solvents used in infusions for injections or infusions include, for example, distilled water for injection, saline, glucose solutions and isotonic solutions (eg, sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, etc. A solution of sodium chloride, propylene glycol, etc.) can be used.
  • examples of the pharmaceutically acceptable carrier include stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, preservatives, pH adjusters, antioxidants and the like.
  • Stabilizers include, for example, various amino acids, albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, polyethylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, Dibutylhydroxytoluene and the like can be used.
  • solubilizing agent examples include alcohols (eg, ethanol, etc.), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, polysorbate 20 (registered trademark), polysorbate 80 (registered trademark)). ), HCO-50, etc.) can be used.
  • alcohols eg, ethanol, etc.
  • polyalcohols eg, propylene glycol, polyethylene glycol, etc.
  • nonionic surfactants eg, polysorbate 20 (registered trademark), polysorbate 80 (registered trademark)).
  • the suspending agent for example, glycerin monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used.
  • emulsifier for example, gum arabic, sodium alginate, tragant and the like can be used.
  • the pain-relieving agent for example, benzyl alcohol, chlorobutanol, sorbitol and the like can be used.
  • the buffer for example, a phosphate buffer solution, an acetate buffer solution, a borate buffer solution, a carbonic acid buffer solution, a citrate buffer solution, a Tris buffer solution, a glutamate buffer solution, an epsilon aminocaproic acid buffer solution and the like can be used.
  • Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, and borax. Sand or the like can be used.
  • As the preservative for example, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
  • As the pH adjuster for example, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid and the like can be used.
  • antioxidants for example, (1) water-soluble antioxidants such as (1) ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, etc., (2) ascorbic palmitate, butylated hydroxyanisol, Use oil-soluble antioxidants such as butylated hydroxytoluene, lecithin, propyl gallate, ⁇ -tocopherol and (3) metal chelating agents such as citric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid, phosphoric acid and the like. Can be done.
  • the infusion solution for injection or infusion can be produced by sterilization in the final step or by aseptic technique, for example, filtering with a filter or the like to sterilize, and then filling in a sterile container.
  • Infusions for injections or infusions are vacuum-dried and lyophilized sterile powders (which may contain pharmaceutically acceptable carrier powders) dissolved in a suitable solvent before use. You can also do it.
  • the PD-1 / CD3 bispecific antibody of the present invention and the like are used in combination with other drugs used for prevention, suppression of symptom progression, suppression of recurrence and / or treatment of autoimmune diseases. You may.
  • the administration form when used in combination with other drugs (combination), the administration form may be a combination drug in which both components are mixed in one preparation, or may be administered as separate preparations. There may be.
  • the preventive effect of other drugs, suppression of symptom progression, suppression of recurrence and / or therapeutic effect can be complemented, and the dose or frequency of administration can be maintained or reduced.
  • the PD-1 / CD3 bispecific antibody or the like of the present invention and another drug are administered separately, they are co-administered for a certain period of time, and then only the PD-1 / CD3 bispecific antibody or the like or another drug is administered. Only the drug may be administered. Further, the PD-1 / CD3 bispecific antibody or the like of the present invention may be administered first and another drug may be administered after the administration is completed, or the other drug may be administered first and after the administration is completed. The PD-1 / CD3 bispecific antibody or the like of the present invention may be administered later, and the respective administration methods may be the same or different.
  • the dose of the other drug can be appropriately selected based on the clinically used dose.
  • other drugs may be administered in combination of any two or more at an appropriate ratio.
  • the other drugs include not only those found so far but also those found in the future.
  • insulin preparations for example, human insulin, insulin glalgin, insulin
  • Lispro insulin detemil, insulin aspart, etc.
  • sulfonylureas eg, glibenclamide, gliclazide, glimepiride, etc.
  • hasty insulin secretagogues eg, nateglunide, etc.
  • biguanide preparations eg, metformin, etc.
  • improvement of insulin resistance Drugs eg, pioglycazone, etc.
  • ⁇ -glucosidase inhibitors eg, acarbose, boglibose, etc.
  • therapeutic agents for diabetic neuropathy eg, epalrestat, mexiretin, imidapril, etc.
  • GLP-1 analog preparations eg, l
  • steroid drugs eg, cortisone, cortisone acetate
  • Interferon ⁇ -1a Interferon ⁇ -1b, Gratylama acetate, Mitoxamethasone, Azatiopurine, Cyclophosphamide, Cyclosporine, Metotrexate, Cladribine, Corticotropin, Misoribin, Tachlorimus, Fingolimod and Alemtuzumab, etc. It may be used in combination with any one or more agents selected from.
  • a steroid drug for example, the steroid drug described above
  • Immunosuppressants eg, cyclosporine, tacrolimus, fingolimod, etc.
  • belimumab may be used in combination with any one or more agents selected from the above.
  • a steroid drug for example, the steroid drug described above
  • anti One or more selected from rheumatoid drugs eg, methotrexate, sulfasalazine, bushilamine, leflunomide, misolivin, tacrolimus, etc.
  • anti-cytocytomous drugs eg, infliximab, adalimumab, tocilizumab, etanercept, golimumab and sertrizumab, etc.
  • infliximab, adalimumab, tocilizumab, etanercept, golimumab and sertrizumab, etc. It may be used in combination with a drug.
  • the PD-1 / CD3 bispecific antibody of the present invention When applied to the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of other autoimmune diseases, the PD-1 / CD3 bispecific antibody of the present invention or the like and any one of the other agents described above. It may be used in combination with the above.
  • Example 1 Immunization of MeMo® Mice Using Recombinant Human PD-1-Fc Fusion Protein As a method for obtaining a first arm that specifically binds to PD-1 of the present invention, MeMo (registered trademark) A method of immunizing recombinant human PD-1 protein in (trademark) mice (see WO2009 / 157771) was selected.
  • MeMo® mice are gene fragments containing the non-recombinant human heavy chain V gene region, D gene region and J gene region and the recombinant human kappa light chain IgV ⁇ 1-39 * 01 / IGJ ⁇ 1 * 01 germ cell lineage gene. Is a mouse genetically modified to be linked to a mouse constant region gene, and by directly immunizing the target protein of the antibody, an antibody consisting of a heavy chain having diversity and a common light chain can be produced. ..
  • Recombinant human PD-1-Fc fusion protein (R & D) emulsified with 12 MeMo® MS5B / MS9 mice aged 12 to 16 weeks using Gerbu-adjuvant MM (Gerbu Biotechnik, model number # 3001). Systems, model number 1086-PD) were each immunized at 14-day intervals.
  • the recombinant human PD-1-Fc fusion protein was subcutaneously administered on the 0th, 14th, and 28th days of immunization, and at the subsequent timing, the recombinant human PD-1-Fc fusion protein dissolved in PBS was administered. It was administered subcutaneously.
  • Serum antibody titers were evaluated by flow cytometry using a human PD-1 forced expression HEK293T cell line on days 21, 35, 56, 77 and 98 of immunization.
  • human PD-1 forced expression HEK293T cell line was stained with 1000-fold diluted serum, the lymphoid tissue of the mouse whose MFI value was increased by 3 times or more as compared with the control human PD-1 non-expressing HEK293T cell line was obtained. It was used to construct a phage display library.
  • mice that met the criteria for library construction were boost-immunized with the recombinant PD-1-Fc fusion protein for 3 days from the date of antibody titer evaluation, and the spleen and spleen lymph nodes were collected. Spleen and spleen lymph nodes were also recovered from mice with serum antibody titers of 1/100 or higher against human PD-1 and crab monkey PD-1 and whose antibody titers did not increase due to booster immunization. RNA was extracted from these lymphoid tissues, and then cDNA synthesis was performed.
  • Example 2 Construction of a phage display library for obtaining an anti-PD-1 antibody having a first arm that specifically binds to PD-1 (protein immunity)
  • a PCR reaction was carried out using primers specific for the immunoglobulin heavy chain variable region family.
  • the PCR product was cleaved with restriction enzymes SfiI and XhoI, and then cleaved with the restriction enzymes.
  • MV1473 Phagemid vector [Gene encoding common light chain (human ⁇ light chain IgV ⁇ 1-39 * 01 / IGJ ⁇ 1 * 01 germline) Includes type gene)] to construct the same library.
  • Example 3 Screening of an anti-PD-1 antibody having a first arm that specifically binds to PD-1, human PD-1-Fc fusion protein, human PD-1-His tag fusion protein, crab monkey PD-1-His A plate coated with a tag fusion protein or a mouse PD-1-His tag fusion protein was used to perform phage selection based on PD-1 binding.
  • Fc-reactive clones were absorbed by adding human IgG (SIGMA, model number I4506) during incubation with phage. Binding phage that bind to human PD-1, cynomolgus monkey PD-1 and mouse PD-1 were enriched.
  • Selection on the cynomolgus monkey PD-1 expression HEK293T cell line enriched the phage that bind to cynomolgus monkey PD-1.
  • a clone of Escherichia coli strain TG1 transformed with the phage obtained by the selection was obtained to prepare a master plate.
  • phage selection was performed from the cloned periplasmic extracts obtained from the above selection based on the binding to PD-1 on the plate on which the human PD-1-Fc fusion protein was adsorbed.
  • positive clones were those in which a signal of 3 times or more was obtained with respect to the signal (OD 450 value) obtained in the negative control well (PBS).
  • Example 4 DNA sequence of candidate clones of anti-PD-1 antibody having a first arm that specifically binds to PD-1 DNA sequence of heavy chain variable region gene of positive clone obtained by screening of Example 3 Carried out.
  • the analyzed DNA sequences were classified into superclusters (a group in which CDR3s have the same length and the amino acid sequences of CDR3s are 70% or more homologous) and clusters (a group in which the amino acid sequences of heavy chain CDR3s are the same). .. 924 clones were obtained and they were classified into 146 superclusters and 194 clusters.
  • Example 5 Screening by evaluation of binding to PD-1 expressing cells Anti-PD-1 monoclonal antibody clones satisfying the following conditions were selected and isolated from each classified supercluster. (1) Somatic mutations are frequently introduced into the CDR regions, It has (2) a germline gene for VH that is frequently used, and (3) a high signal was obtained in the binding screening for the human PD-1-Fc fusion protein.
  • the binding property to human PD-1-expressing CHO-S cell line and kanikuisaru PD-1-expressing CHO-S cell line was evaluated by detecting with anti-mouse IgG polyclonal antibody. did. Of the 117 clones evaluated (105 clusters), 22 clones including anti-PD-1 monoclonal antibody clones PD1-1, PD1-2, PD1-3 and PD1-4 expressed human PD-1 expression CHO. -Bindability to S cell line was observed.
  • Example 6 Preparation of amino acid substitutions for anti-PD-1 monoclonal antibody having a first arm that specifically binds to PD-1
  • Clones PD1-1 and PD1-4 are incorporated into framework 4 of their heavy chain variable regions. Includes deamidation motif (Asn-Gly). A mutant in which this deamidation motif was converted was prepared for the purpose of obtaining a first arm with a reduced risk of deamidation.
  • Clone PD1-5 was prepared and isolated so that asparagine (Asn) at position 119 by the EU numbering system of clone PD1-4 was modified to be glutamine by a known site-specific mutation method. The clone also had the same binding property to human PD-1-expressing CHO-S cells as clone PD1-4.
  • Example 7 Screening of an anti-CD3 monoclonal antibody having a second arm that specifically binds to CD3
  • An anti -CD3 antibody consisting of the VH and IGVK1-39 / JK1 common light chain of the anti-CD3 antibody clone 15C3 described in WO 2005/118635.
  • An anti-CD3 antibody having the "second arm that specifically binds to CD3" of the present invention by the following method in order to obtain a stable CD3 binding Fab with further reduced charge heterogeneity based on the clone. was acquired.
  • phages were extracted and screened for binding to cell surface-expressed CD3 by flow cytometry. Colony PCR was performed on all phages showing CD3 binding to amplify the VH-encoding cDNA and sequence their DNA.
  • the anti-CD3 antibody clone CD3-2 having VH consisting of the amino acid sequence of SEQ ID NO: 36 showed CD3 binding and charge homogeneity equivalent to that of clone CD3-1.
  • Example 8 Preparation of PD-1 / CD3 bispecific monoclonal antibody
  • the expression vector expressing each heavy chain of the first arm that specifically binds to PD-1 is the anti-PD selected in Example 5.
  • -1 Monoclonal antibody clones PD1-1 to PD1-5 and PD1-6 were prepared by ligating the DNA encoding each heavy chain variable region to the DNA encoding the IgG 1 heavy chain constant region.
  • the expression vector expressing the heavy chain of the second arm that specifically binds to CD3 is the DNA encoding the heavy chain variable region of CD3-2 in the anti-CD3 monoclonal antibody clone selected in Example 7, IgG. It was prepared by linking it to the DNA encoding the single heavy chain constant region.
  • the genes expressing those heavy chain constant regions include those expressing the Fc region having the L351D / L368E mutation (DE mutation) in the case of the first arm that specifically binds to PD-1.
  • the gene expressing the Fc region having the L351K / T366K mutation (KK mutation) was used.
  • These expression vectors were constructed to contain a gene encoding this so that the IGVK1-39 / JK1 common light chain was also expressed.
  • leucine at position 235 and glycine at position 236 of the heavy chain constant region are replaced with glycine and expressed.
  • PD-1 / CD3 bispecific monoclonal antibody clones are the anti-PD-1 monoclonal antibody clones PD1-1, PD1-2, PD1-3, PD1-4 and PD1-5 used in their preparation. It corresponds to each of these anti-PD-1 monoclonal antibody clones in that it has a first arm derived from PD1-6 that specifically binds to PD-1.
  • Example 9 Evaluation of binding of PD-1 / CD3 bispecific monoclonal antibody Human IgG1-Fc fusion human PD-1 extracellular region recombinant protein or 6 ⁇ His tag fusion crab quail PD-1 extracellular region recombinant protein
  • Biacore registered trademark
  • a Series S Sensor Chip CM5 sensor chip (GE Healthcare, model number 29-1049-88) was used to immobilize the recombinant protein.
  • the CD3 binding affinity of the second arm of the antibody was evaluated by Biacore® measurement using a human IgG1-Fc fusion CD3 ⁇ / CD3 ⁇ extracellular space recombinant protein.
  • the binding affinity (Kd value) of the first arm of each clone to PD-1 and the binding affinity of the second arm to CD3 ⁇ / CD3 ⁇ are shown in FIG.
  • Example 10 Confirmation of binding of PD-1 / CD3 bispecific monoclonal antibody
  • the PD-1 / CD3 bispecific monoclonal antibody obtained in Example 8 specifically binds to PD-1 and CD3 at the same time. It was confirmed.
  • clones PD1-1 (Bi) to PD1-6 (Bi) were added to each of the human PD-1-deficient Jurkat cell lines (human T cell lines) expressing human CD3, and 15 on ice. Incubated for minutes. After washing the cells, 3 times the amount of biotin-labeled soluble PD-1 recombinant protein (R & D systems, model number 1086-PD-050) was added and incubated on ice for 15 minutes. After washing the cells, 100 ⁇ L of Alexa Fluor 488-labeled streptavidin (BioLegend, model number 405235) 1.25 ⁇ g / mL was added and incubated on ice for 15 minutes. After washing the cells, the amount of soluble PD-1 recombinant protein bound was evaluated by flow cytometry. The results of the assay are shown in FIG.
  • Example 11 Evaluation of binding characteristics of the first arm of the PD-1 / CD3 bispecific monoclonal antibody PD-1 / PD of the first arm of the PD-1 / CD3 bispecific monoclonal antibody obtained in Example 8
  • a competitive binding assay was performed for the binding of the bispecific monoclonal antibody clone and the soluble PD-L1 recombinant protein to PD-1.
  • clones PD1-1 (Bi) to PD1-6 (Bi) were added to human PD-1-expressing CHO-S cell lines, respectively, and incubated on ice for 30 minutes.
  • the clones PD1-1 (Bi) to PD1-5 (Bi) are the soluble PD-L1 recombinant proteins for PD-1, despite the fact that they are present in 20-fold doses of the soluble PD-L1 recombinant proteins. Allowed binding.
  • PD1-6 (Bi) completely inhibited the binding of soluble PD-L1 recombinant protein to PD-1 under the same conditions.
  • Example 12 Activation of PD-1 / CD3 bispecific monoclonal antibody
  • CD4 T cells healthy human peripheral blood-derived CD4-positive T cells (LONZA, model number 2W-200) (human T cells)
  • LONZA human peripheral blood-derived CD4-positive T cells
  • Human T cells The inhibitory effect on IFN- ⁇ production from activated T cells was evaluated.
  • Human T cells are seeded on a cell culture plate on which an anti-human TCRV ⁇ 8 antibody (Thermo Scientific, model number TCR1750) is immobilized, and an anti-human CD28 antibody (BioLegend, model number 302923) is added and activated for 72 hours. It was.
  • Human T cells subjected to this activation treatment were then cultured overnight in fresh medium containing 100 Units / mL human IL-2 (R & D systems, model number 202-IL). Further, the recovered human T cells were seeded on another cell culture plate on which the anti-human TCRV ⁇ 8 antibody was immobilized, the anti-human CD28 antibody was added in the same manner, and the activation treatment was performed again. At that time, after adding clones PD1-1 (Bi) to PD1-6 (Bi), IFN- ⁇ contained in the culture supernatant 96 hours after the reactivation treatment was quantified by the ELISA method (pg / mL). The result is shown in FIG.
  • the IFN- ⁇ production inhibitory effect was confirmed for clones PD1-1 (Bi) to PD1-5 (Bi).
  • the inhibitory effect of clone PD1-6 (Bi) tended to be weaker than that of other clones.
  • As the control antibody an antibody obtained by immunizing tetanus toxoid was used as a non-specific antibody by using the same method as the method for obtaining the first arm.
  • Example 13 In vivo action in an experimental allergic encephalomyelitis mouse model (EAE model) of PD-1 / CD3 bispecific monoclonal antibody CD3 ⁇ and PD-1 genes were used for human CD3 ⁇ and human PD-1.
  • the in vivo action of the PD-1 / CD3 bispecific monoclonal antibody of the present invention was evaluated in an EAE model using human CD3 ⁇ / human PD-1 knock-in C57BL / 6 mice substituted with genes.
  • Dead tuberculosis H37Ra (BD Biosciences, model number 231141) and incomplete Freund's adjuvant (BD Biosciences, model number 263910) were mixed to prepare a complete Freund's adjuvant (CFA) containing 4 mg / mL killed tuberculosis H37Ra.
  • CFA complete Freund's adjuvant
  • An emulsion was prepared by mixing 1 mg / mL MOG peptide (ANASPEC, model number AS-60130) and an equal amount of CFA, and used as an inducer for the EAE model.
  • 200 ⁇ L of the inducer was subcutaneously administered to the ridge of the C57BL / 6 mouse, and 200 ⁇ L of 1 ⁇ g / mL pertussis toxin (SIGMA-ALDRICH, model number P7208) was intravenously administered on the day and the second day of the immune treatment. ..
  • clones PD1-1 (Bi) to PD1-6 (Bi) were added to the C57BL / 6 mice once daily at a dose of 2 mg / kg on the 6th and 7th days of immunotreatment. It was administered intraperitoneally. After the day of immunotreatment, neurological symptoms were evaluated according to Onuki et al.
  • Example 14 Evaluation of in vitro effect of PD-1 / CD3 bispecific monoclonal antibody on cytokine release from human peripheral blood mononuclear cells Analysis of cytokine release activity of PD-1 / CD3 bispecific monoclonal antibody
  • IL-2 contained in the culture supernatant 24 hours after culturing was quantified by a multiplex immunoassay (BIO-RAD, model number M50000007A). The result is shown in FIG.
  • Example 15 PD-1 / CD3 bispecific monoclonal antibody Evaluation of cross-competitiveness of PD1-5 (Bi) to binding of each clone to PD-1 PD-1 / CD3 double obtained in Example 8 Specific Monoclonal Antibodies
  • Competitive binding assays were performed to assess the cross-competitiveness of PD1-5 (Bi) to PD-1 binding of each clone PD1-1 (Bi) to PD1-5 (Bi).
  • clone PD1-5 (Bi) was added to a human PD-1-expressing CHO-S cell line and incubated on ice for 20 minutes. Further, 1/100 amount of biotin-labeled clones PD1-1 (Bi) to PD1-5 (Bi) were added to the added clone PD1-5 (Bi), and the clones were incubated on ice for 20 minutes. After washing the cells, PE-labeled streptavidin (BD Pharmagen, model number 554061) was added and incubated on ice for 20 minutes. After washing the cells, the amount of binding of biotin-labeled clones PD1-1 (Bi) to PD1-5 (Bi) was evaluated by flow cytometry. The result is shown in FIG.
  • PD1-5 inhibits the binding of clones PD1-1 (Bi) to PD1-4 (Bi) to PD-1, and the clones cross-compete for their binding to PD-1. It was shown to do.
  • Example 16 In vivo action of PD-1 / CD3 bispecific monoclonal antibody in human peripheral blood mononuclear cell transplanted hyperimmune deficient mouse Hyperimmune deficient mouse transplanted with human peripheral blood mononuclear cell (PBMC)
  • the PD-1 / CD3 bispecific monoclonal antibody of the present invention was administered to NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (hereinafter, NSG mouse), and its effect on T cell proliferation in vivo was evaluated.
  • an anti-CD4 antibody (Biolegend, model number 300518) fluorescently labeled with APC-Cy7 or an anti-CD8 antibody (Biolegend, model number 301006) fluorescently labeled with FITC was added, and a flow cytometer (BD Biosciences, The number of human CD4 positive cells and the number of human CD8 positive cells were measured using the model BD LSR Fortessa X-20) and the analysis software FlowJo (Biolegend, model number 300518). The evaluation result is shown in FIG.
  • the PD1-5 (Bi) -administered group had significantly lower numbers of human CD4 + and human CD8-positive cells in the spleen than the control antibody-administered group. Clone PD1-5 (Bi) showed an inhibitory effect on these cell number increases.
  • Example 17 In vivo action of PD-1 / CD3 bispecific monoclonal antibody in mouse antibody production model
  • the PD-1 of the present invention was applied to the human CD3 ⁇ / human PD-1 knock-in C57BL / 6 mouse prepared in Example 13.
  • a / CD3 bispecific monoclonal antibody was administered and the in vivo effect on antibody production was evaluated.
  • mice Dissolve Keyhole limpet hemocyanin (Thermo Scienctific, model number 77600, hereafter KLH) in DPBS to prepare a 200 ⁇ g / mL KLH solution, and mix with an equal volume of ALUM (Cosmo Bio, model number LG-6000) for 30 minutes at room temperature. Was used as an inducer.
  • the mice were immunotreated by intraperitoneally administering 200 ⁇ L of this inducer.
  • Control antibody or PD-1 / CD3 bispecific monoclonal antibody clone PD1-5 (Bi) was intraperitoneally administered once daily 7 and 8 days after immunization. From 7 to 374 days after immunization, 50 to 80 ⁇ L of blood was collected from the tail vein of mice to prepare serum.
  • the anti-KLH antibody (IgG 1 ) titer in serum was measured by the ELISA method. That is, an anti-KLH antibody in which KLH was immobilized and bound to KLH was detected using an HRP-labeled anti-mouse IgG 1 antibody (Bethyl Laboratories, model number A90-105P-38).
  • FIG. 21 shows the evaluation result.
  • PD1-5 (Bi) showed an inhibitory effect on the anti-KLH antibody production of the mouse. In addition, a significant inhibitory effect lasted up to 360 days after immunization.
  • Example 18 In vivo action of PD-1 / CD3 bispecific monoclonal antibody in spontaneous diabetes model mice PD-1 / CD3 of the present invention was applied to human CD3 ⁇ / human PD-1 knock-in NOD mice spontaneously developing diabetes. A bispecific monoclonal antibody was administered and the in vivo effect on changes in blood glucose level was evaluated.
  • the NOD mice were injured with a razor in the tail vein once a week from 16 weeks of age, 5 to 10 ⁇ L of blood was collected, and the glucose concentration in the blood was measured using Accucheck Aviva (registered trademark) (Roche). Mice showing a blood glucose concentration of 200 mg / dL or more (hyperglycemic region) twice in a row were considered to develop diabetes, and control antibody or PD-1 / CD3 bispecific monoclonal antibody was used in each of the 7 individuals who developed diabetes. Antibody clone PD1-5 (Bi) was intraperitoneally administered for 2 days. After the start of administration, blood was collected once a week and the blood glucose concentration was measured.
  • FIG. 22 shows the evaluation result.
  • the NOD mouse was prepared by mating the human CD3 ⁇ knock-in mouse, the human PD-1 knock-in mouse, and the NOD / ShiJcl mouse prepared in Example 13, respectively, by a known method.
  • the PD1-5 (Bi) -administered group had a significantly larger number of cases in the normoglycemic region than the control antibody-administered group, and PD1-5 (Bi) was after the onset of diabetes.
  • the administration showed a therapeutic effect.
  • Example 19 Preparation of PD-1 / CD3 bispecific monoclonal antibody in which a mutation was introduced into the amino acid at the human FcRn binding site and evaluation of FcRn binding PD-1 / CD3 bispecific monoclonal antibody clone PD1-5 (Bi) ) Antibody clones Mut1, Mut2, Mut3 and Mut4 in which the 252nd methionine in the EU numbering system in the heavy chain constant region was replaced with glutamic acid, proline, arginine and aspartic acid, respectively, to a known technique for amino acid substitution. It was prepared by the same method.
  • the antibody clone Mut5 in which asparagine at position 434 was replaced with leucine and the antibody clone Mut6 in which glutamine at position 438 was replaced with glutamic acid in the EU numbering system in the heavy chain constant region of the clone PD1-5 (Bi) were similarly prepared. Each was prepared by the method.
  • Mut1 to Mut6 for human FcRn is lower than that of PD1-5 (Bi) (dissociation constant (K D value): 2.75 ⁇ 10 -8 M), and Mut4 and Mut2 are particularly remarkable. It has decreased. Mut1 and the affinity for murine FcRn of Mut4 is PD1-5 (Bi) (K D value: 1.61 ⁇ 10 -8 M) lower than (K D value of Mut1: 4.67 ⁇ 10 -8 M) , in particular, Mut4 dropped significantly.
  • Example 20 Blood dynamics of PD-1 / CD3 bispecific monoclonal antibody clone Mut4 Using human CD3 ⁇ / human PD-1 knock-in C57BL / 6 mice prepared in Example 13, in vivo blood of clone Mut4 The kinetics was evaluated.
  • the tail vein of the mouse was partially injured with a shear blade, and about 40 ⁇ L.
  • Blood was collected in heparinized tubes. Blood was collected by centrifugation to prepare plasma, and the plasma concentration of the clone was measured using the electrochemical luminescence (ECL) method. The half-life in blood was calculated from the transition of plasma concentration.
  • ECL electrochemical luminescence
  • the half-life in blood of the clone was 8.0 hours, which was shorter than the half-life in blood of the original PD1-5 (Bi) of about 160 hours.
  • Example 21 In vivo action of PD-1 / CD3 bispecific monoclonal antibody clone Mut4 in EAE model In vivo action of clone Mut4 was evaluated in an EAE model prepared according to the method described in Example 13.
  • Each of the 8 EAE model mice was intraperitoneally administered with a control antibody or clone Mut4 once a day for 5 days from 6 to 10 days after immunization.
  • the neurological symptoms of the mice after the date of immunotreatment were evaluated by the method described in Example 13 according to the method of Onuki et al. (Onuki M, et al., Microsc Res Tech 2001; 52: 731-9.). Cumulative neurological symptom scores for the observation period from the day of immunization to 30 days after immunization were tabulated.
  • FIG. 26 shows the evaluation result.
  • the PD-1 / CD3 bispecific antibody or antibody fragment thereof of the present invention is useful for prevention of autoimmune diseases, suppression of symptom progression, suppression of recurrence and / or treatment.

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KR20250156121A (ko) 2023-03-16 2025-10-31 오노 야꾸힝 고교 가부시키가이샤 항체 제제

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WO2024190892A1 (ja) 2023-03-16 2024-09-19 小野薬品工業株式会社 抗体製剤
KR20250156121A (ko) 2023-03-16 2025-10-31 오노 야꾸힝 고교 가부시키가이샤 항체 제제
EP4681730A1 (en) 2023-03-16 2026-01-21 ONO Pharmaceutical Co., Ltd. Antibody preparation

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ZA202201247B (en) 2023-08-30
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US20220281977A1 (en) 2022-09-08
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