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WO2024144336A1 - Nouvelle protéine de fusion bispécifique et son utilisation - Google Patents

Nouvelle protéine de fusion bispécifique et son utilisation Download PDF

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Publication number
WO2024144336A1
WO2024144336A1 PCT/KR2023/021957 KR2023021957W WO2024144336A1 WO 2024144336 A1 WO2024144336 A1 WO 2024144336A1 KR 2023021957 W KR2023021957 W KR 2023021957W WO 2024144336 A1 WO2024144336 A1 WO 2024144336A1
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WO
WIPO (PCT)
Prior art keywords
fusion protein
protein
dual
antibody
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2023/021957
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English (en)
Korean (ko)
Inventor
진현탁
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Progen Co Ltd
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Progen Co Ltd
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Filing date
Publication date
Application filed by Progen Co Ltd filed Critical Progen Co Ltd
Priority claimed from KR1020230195667A external-priority patent/KR20240109202A/ko
Publication of WO2024144336A1 publication Critical patent/WO2024144336A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • immunosuppressants include tacrolimus, a calcineurin inhibitor, mycophenolate-mofetil, a gene synthesis inhibitor, and prednisone, an anti-inflammatory steroid.
  • tacrolimus a calcineurin inhibitor
  • mycophenolate-mofetil a gene synthesis inhibitor
  • prednisone an anti-inflammatory steroid.
  • these immunosuppressants can cause serious side effects because they are administered for a long period of time (Kalluri and Hardinger, World J. Translant . 2(4): 51-68, 2012; Tanabe et al ., Translantation , 93(7): 709 -719, 2012;Zaza et al ., Toxins 6(3): 869-891, 2014).
  • an anti-CD154 antibody was developed, and the antibody showed good effects in animal experiments, including monkeys.
  • anti-CD154 antibodies showed a serious side effect of thrombosis, and the development of antibody drugs targeting CD154 is currently halted (Kalunian et al ., Arthritis. Rheum. 46 (Kalunian et al., Arthritis. Rheum . 46) 12): 3251-3258, 2002; Arthritis Rheum 48(3): 719-727; Pilat et al., Curr. 17: 368-375, 2012).
  • IL-10 is known to have a dual characteristic of having the opposite effect of immunostimulatory activity.
  • IL-10 can stimulate B cell activation, prolong B cell survival, and contribute to class switching of B cells. It can also stimulate NK cell proliferation and cytokine production and act as a growth factor to promote proliferation of specific subpopulations of CD8 + T cells (Mosser & Yhang, Immunol. Rev. 226: 205-218, 2009; Cai et al ., J. Immunol. 29 : 2658-2665, 1999; J. Virol 74: 4729-4737 ; 160: 3188-3193 . ).
  • the present inventors aimed to solve various problems, including the above-mentioned problems, by providing a new dual-specific fusion protein that can reduce side effects compared to effectiveness by improving the administration ratio of anti-CD154:IL-10.
  • the purpose The purpose.
  • the scope of protection of the present invention is not limited to the above purpose.
  • a heavy chain variable region of an antibody that specifically binds to CD154 and e) a second fusion protein comprising a second modified Fc region that is mutated so as not to bind to the Fc gamma receptor, but does not include IL-10; and
  • a dual specificity fusion protein consisting of the amino acid sequence shown in SEQ ID NO: 1 including the anti-CD145 antibody heavy chain and IL-10 protein is provided.
  • a polynucleotide encoding the first fusion protein and/or the second fusion protein of the dual specificity fusion protein is provided.
  • a recombinant vector containing the polynucleotide is provided.
  • a transformed host cell transformed with the vector is provided.
  • a dual specificity fusion protein consisting of the amino acid sequence shown in SEQ ID NO: 1, an anti-CD145 antibody heavy chain protein containing the amino acid sequence shown in SEQ ID NO: 2, and an anti-CD145 antibody heavy chain protein consisting of the amino acid sequence shown in SEQ ID NO: 3.
  • Dual specificity fusion proteins comprising an anti-CD154 antibody light chain are provided.
  • a pharmaceutical composition for immunosuppression comprising the dual-specific fusion protein as an active ingredient.
  • a pharmaceutical composition for treating autoimmune diseases comprising the dual-specific fusion protein as an active ingredient is provided.
  • a method of suppressing the immune system of an individual comprising administering a therapeutically effective amount of the dual-specific fusion protein to an individual in need of immunosuppression.
  • Figure 3 shows the inflammatory response of the dual-specificity fusion protein (PG-405), the control group (NTIG), and various comparative examples (rhIL-10, PG-010-3, and PG-010) according to an embodiment of the present invention.
  • PG-405 dual-specificity fusion protein
  • NTIG control group
  • rhIL-10 various comparative examples
  • PG-010-3, and PG-010 various comparative examples
  • Figure 4 shows the dual specificity fusion protein (PG-405) according to an embodiment of the present invention and the dual specificity fusion protein (PG-410) of Comparative Example 1, which is a comparative group, and the anti-CD154 antibody (PG-400) of Comparative Example 5.
  • PG-405 the dual specificity fusion protein
  • PG-410 the dual specificity fusion protein
  • PG-400 the anti-CD154 antibody
  • Figure 5a is a schematic diagram schematically showing the operating mechanism of the HEK-Blue system, a reporter system used to analyze the CD40L-CD40 signaling pathway and the CD40L-CD40 signaling inhibition ability of the dual specificity fusion protein according to an embodiment of the present invention.
  • Figure 5b shows CD40L of the dual-specific fusion protein (PG-405) according to an embodiment of the present invention and PG-410 of Comparative Example 1 and PG-400 of Comparative Example 5 as a control using the HEK-Blue system.
  • PG-405 dual-specific fusion protein
  • PG-410 of Comparative Example 1
  • PG-400 Comparative Example 5
  • Figure 6 is an analysis of the immune response inhibition ability of human peripheral blood mononuclear cells of the dual specificity fusion protein (PG-405) and various comparison groups (NTIG, PG-400, C10, PG-410) according to an embodiment of the present invention. This is a graph showing the results.
  • Figure 7 shows the results of a pharmacokinetic analysis performed to measure the in vivo half-life of the dual-specific fusion protein (PG-405) according to an embodiment of the present invention and the dual-specific fusion protein (PG-410) of Comparative Example 1. It's a graph.
  • the IL-10 protein may be a wild-type or mutant IL-10 protein, and the mutant IL-10 protein may have its immune activation function removed, and the mutant IL-10 protein may be a wild-type IL-10 protein.
  • the protein may be a monomeric variant IL-10 protein.
  • the monomeric mutant IL-10 protein may be a mature form protein containing the 19th to 178th amino acid sequence from which the signal sequence has been removed from the amino acid sequence described in UniProtKB P22301, and the 87th amino acid of the mature protein.
  • the first modified Fc region and the second modified Fc region may form a heterodimer.
  • DD-KK used in this document refers to lysine (K), the 69th amino acid of CH3 of the first modified Fc region, as aspartic acid (D) (K409D), and lysine (K), the 52nd amino acid, as aspartic acid.
  • D was replaced with (K392D), aspartic acid (D), the 59th amino acid of CH3 in the second modified Fc region, was replaced with lysine (K) (D399K), and glutamic acid (E), the 16th amino acid, was replaced with lysine (K).
  • V (D399V)
  • T threonine
  • V405T hydrophobic/steric complementarity and long-range electrostatic interaction by substituting the 65th amino acid, phenylalanine (F), with threonine (T)
  • F405T threonine
  • EW-RVT SS used in this document is a structure in which cysteine is introduced into CH3 so that an intermolecular disulfide bond (intramolecular) can be formed in the EW-RVT structure, and CH3 in the first modified Fc region of the EW-RVT Tyrosine (Y), the 9th amino acid, was additionally substituted with cysteine (C) (Y349C), and serine (S), the 14th amino acid of CH3 of the second modified Fc region, was additionally substituted with cysteine (C) (S354C).
  • SEED refers to a chain between IgG and IgA by introducing 45 IgA-derived residues into CH3 of the first modified Fc region and 57 IgG1-derived residues into CH3 of the second modified Fc region. It refers to a technology that induces heterodimerization using hydrophobic/steric complementarity due to exchange (Davis et al ., Protein Eng, Des, Sel , 23(4):195-202, 2010) .
  • A107 refers to the 26th amino acid, lysine (K), of CH3 of the first modified Fc region, as glutamic acid (E) (K370E), and the 69th amino acid, lysine (K), as tryptophan (W).
  • the scFv has a light chain variable region (V L ) containing the amino acid sequence shown in SEQ ID NO: 16 and a heavy chain variable region (V H ) containing the amino acid sequence shown in SEQ ID NO: 17, which is a linker It may be connected by peptide.
  • the linker peptide may be any one of those described below, but is preferably a flexible linker peptide with a length of 10 to 20 aa.
  • mice experiment related to xenograft-versus-host disease The present inventors used a dual-specific fusion protein (PG-405) according to an embodiment of the present invention and various comparison proteins (PG-400, PG-400 + PG) The combination of -010 and PG-410) was investigated through experiments using an actual xenograft-versus-host disease model.
  • PG-405 dual-specific fusion protein
  • PG-400 + PG various comparison proteins
  • the fusion protein according to one embodiment of the present invention can be used to manufacture medicine, particularly pharmaceutical compositions for the treatment and prevention of diseases or symptoms requiring immunosuppression.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Mycology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne une nouvelle protéine de fusion bispécifique et son utilisation et, plus précisément, une protéine de fusion bispécifique, qui comprend : I) une première protéine de fusion comprenant séquentiellement a) une région variable de chaîne lourde d'un anticorps qui se lie spécifiquement à CD154, b) une première région Fc modifiée qui est modifiée de façon à ne pas se lier à un récepteur gamma Fc, et c) une protéine IL-10 ; une seconde protéine de fusion comprenant d) une région variable de chaîne lourde d'un anticorps qui se lie spécifiquement à CD154, et e) une seconde région Fc modifiée qui est modifiée de façon à ne pas se lier au récepteur gamma Fc, mais ne comprenant pas de protéine IL-10 ; et f) une chaîne légère d'anticorps anti-CD154 qui se lie à un premier fragment de liaison à l'antigène pour former le site de liaison à l'antigène d'un anticorps anti-CD154, ou qui comprend : II) une première protéine de fusion comprenant séquentiellement g) un analogue d'anticorps à base de chaîne unique qui se lie spécifiquement à CD154, h) une première région Fc modifiée qui est modifiée de façon à ne pas se lier au récepteur gamma Fc, et i) une protéine IL-10 ; et une seconde protéine de fusion comprenant j) un analogue d'anticorps à base de chaîne unique qui se lie spécifiquement à CD154, et k) une seconde région Fc modifiée qui est modifiée de façon à ne pas se lier au récepteur gamma Fc, mais ne comprenant pas de protéine IL-10.
PCT/KR2023/021957 2022-12-30 2023-12-28 Nouvelle protéine de fusion bispécifique et son utilisation Ceased WO2024144336A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20220190363 2022-12-30
KR10-2022-0190363 2022-12-30
KR1020230195667A KR20240109202A (ko) 2022-12-30 2023-12-28 신규 이중 특이성 융합단백질 및 그의 용도
KR10-2023-0195667 2023-12-28

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WO2024144336A1 true WO2024144336A1 (fr) 2024-07-04

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20150038012A (ko) * 2012-08-08 2015-04-08 로슈 글리카트 아게 인터루킨-10 융합 단백질 및 그의 용도
JP2019527200A (ja) * 2016-06-22 2019-09-26 アルカームス インコーポレーテッド Ｉｌ−１０の免疫刺激特性および抗炎症特性を調節するための組成物および方法
KR20210006302A (ko) * 2019-07-08 2021-01-18 주식회사 프로젠 신규 il-10 변이체 단백질 및 그의 용도
KR20220061644A (ko) * 2020-11-06 2022-05-13 주식회사 제넨바이오 Pd-l1 및 il-10의 융합단백질을 유효성분으로 하는 이식거부반응 치료용 약학적 조성물
US20220259278A1 (en) * 2019-07-08 2022-08-18 Progen Co., Ltd. Novel fusion protein and use of same

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20150038012A (ko) * 2012-08-08 2015-04-08 로슈 글리카트 아게 인터루킨-10 융합 단백질 및 그의 용도
JP2019527200A (ja) * 2016-06-22 2019-09-26 アルカームス インコーポレーテッド Ｉｌ−１０の免疫刺激特性および抗炎症特性を調節するための組成物および方法
KR20210006302A (ko) * 2019-07-08 2021-01-18 주식회사 프로젠 신규 il-10 변이체 단백질 및 그의 용도
US20220259278A1 (en) * 2019-07-08 2022-08-18 Progen Co., Ltd. Novel fusion protein and use of same
KR20220061644A (ko) * 2020-11-06 2022-05-13 주식회사 제넨바이오 Pd-l1 및 il-10의 융합단백질을 유효성분으로 하는 이식거부반응 치료용 약학적 조성물

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