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WO2024235270A1 - Inhibiteur du complément, son procédé de préparation et son utilisation - Google Patents

Inhibiteur du complément, son procédé de préparation et son utilisation Download PDF

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Publication number
WO2024235270A1
WO2024235270A1 PCT/CN2024/093492 CN2024093492W WO2024235270A1 WO 2024235270 A1 WO2024235270 A1 WO 2024235270A1 CN 2024093492 W CN2024093492 W CN 2024093492W WO 2024235270 A1 WO2024235270 A1 WO 2024235270A1
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omci
protein
acid
mutant
molecular weight
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冯军
上官雯雯
路建光
东圆珍
黄宗庆
汪燕丹
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Shanghai Duomirui Biotechnology Ltd
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Shanghai Duomirui Biotechnology Ltd
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    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43527—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from ticks
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the field of biomedicine, in particular to fatty acid side chain modified OmCI mutants, and also to methods for preparing these mutants and their use in treating diseases.
  • Complement is a group of globulins that are present in the blood, tissue fluid and cell membrane surface of humans and animals and have enzymatic activity after activation.
  • Complement is a complex protein network composed of more than 50 proteins, so it is also called the complement system (Ricklin D, Lambris JD. Complement therapeutics. Semin Immunol. 2016; 28(3): 205-207).
  • the components were named in the order of discovery of C1-C4, and then the order was corrected by the WHO in 1968 and named according to the activation order of C1-C9 (Kaufmann SH. Immunology's foundation: the 100-year anniversary of the Nobel Prize to Paul Ehrlich and Elie Metchnikoff. Nat Immunol. 2008; 9(7): 705-712.).
  • complement mainly exerts its immunological function by regulating antigens to stimulate phagocytes to remove foreign matter and damaged substances, attracting macrophages and neutrophils to cause inflammation, and activating cell-killing membrane attack complexes.
  • the complement-triggered immune function is achieved by a series of cascade reactions such as proteolysis to amplify the signal.
  • complement activation pathways There are currently three known complement activation pathways: a) the classical pathway, which requires antigen-antibody complexes to initiate C1 activation through specific immune responses; b) the alternative pathway, which does not require pathogen identification and can be initiated only by spontaneous hydrolysis or activation of C3; c) the lectin pathway, in which lectins in the blood can specifically recognize and bind to mannose on the surface of pathogenic microorganisms. This pathway can be activated by non-specific immune responses such as C3 hydrolysis or antigens in the absence of antibodies.
  • the three pathways of complement activation are shown in Figure 1.
  • C5 is cleaved into C5a and C5b (Rawal N, Pangburn MK. Structure/function of C5 convertases of complement. Int Immunopharmacol. 2001; 1(3): 415-422).
  • C5a is an anaphylatoxin and an important chemotactic protein that plays a key role in recruiting inflammatory cells.
  • C5b recruits the membrane attack complex (MAC) composed of C6, C7, C8 and C9.
  • MAC membrane attack complex
  • MAC As the end product of the complement cascade reaction, MAC has the function of dissolving cell membranes. It can bind to the cell membrane of pathogens to form a hole, namely a transmembrane channel, causing penetration of target cells, thereby killing or destroying pathogen cells (Serna M, Giles JL, Morgan BP, Bubeck D. Structural basis of complement membrane attack complex formation. Nat Commun. 2016; 7:10587).
  • complement activation has a positive impact on the body's self-protection and anti-infection effects, but the damage and dysregulation of complement response are considered to be important pathogenic factors for some autoimmune diseases and various inflammatory diseases, such as paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy, age-related macular degeneration (AMD) and many other diseases.
  • PNH paroxysmal nocturnal hemoglobinuria
  • aHUS atypical hemolytic uremic syndrome
  • AMD age-related macular degeneration
  • Complement C5 as a representative molecule of the membrane attack complex in the complement system, is a large protein of 188 kDa with a concentration of 75 ⁇ g/ml in serum. Targeting this protein can regulate the activation of all three different pathways of complement. Body signal.
  • a natural protein with a molecular weight of about 17 kDa was extracted from the salivary glands of the soft tick (Ornithodoros moubata). Studies have shown that this protein can specifically bind to complement C5, inhibit the activation pathway of complement downstream, and prevent the formation of MAC. In addition to targeting complement C5, studies have also shown that OmCI protein has the ability to inhibit the activity of leukotriene B4 (LTB4), providing additional anti-inflammatory function (Roversi P, Ryffel B, Togbe D, et al. Bifunctional lipocalin ameliorates murine immune complex-induced acute lung injury. J Biol Chem. 2013; 288(26): 18789-18802). Therefore, OmCI has a variety of clinical application potentials.
  • HSA Human serum albumin
  • Fatty acids reversibly bind to human serum albumin (HAS) in vivo through non-covalent bonds and participate in the circulation mediated by the neonatal receptor (FcRn), thereby achieving the long-acting effect of the drug.
  • FcRn neonatal receptor
  • Semaglutide developed by Novo Nordisk, is based on human GLP-1 and is modified with fatty acid side chains to extend its injection frequency to once a week (Lau J, Bloch P, L, et al. Discovery of the Once-Weekly Glucagon-Like Peptide-1 (GLP-1) Analogue Semaglutide. J Med Chem. 2015; 58(18):7370-7380).
  • the object of the present invention is to provide a method for extending the in vivo half-life of an OmCI protein, as well as an OmCI protein with an extended half-life and a pharmaceutical composition containing the same.
  • the present invention provides a modified protein, comprising a protein portion and a modification portion, wherein a cysteine residue is introduced into the protein portion by mutation, and the modification portion is connected to the protein portion via the cysteine residue introduced into the protein portion, and the protein is OmCI or an active fragment thereof.
  • the mutation is a substitution or an insertion; preferably a substitution.
  • the OmCI has the amino acid sequence shown in SEQ ID NO:1.
  • the OmCI has the amino acid sequence shown in SEQ ID NO:1, and T90, K95, T97, E126 or S156 is mutated to a cysteine residue.
  • the amino acid sequence of OmCI is as shown in any one of SEQ ID NO:4-8.
  • the amino acid sequence of OmCI is as shown in any one of SEQ ID NO: 4, 5, 7, and 8.
  • the modified moiety is capable of extending the in vivo half-life of the protein moiety.
  • the modified portion is capable of binding specifically or non-specifically to a protein in plasma.
  • the protein in the plasma has a long half-life; for example, the half-life of the protein in the plasma is 10 days or more, preferably 15 days or more, more preferably 19 days or more.
  • the protein in the plasma is human serum albumin.
  • K is lysine
  • R1 is a substituted or unsubstituted C1-20 acyl group
  • n is an integer from 0 to 5;
  • n is an integer from 0 to 5;
  • o is an integer of 1-3 (preferably 1 or 2);
  • X1 and X2 are independently selected from the following group: alanine (Ala), D-alanine (D-Ala), ⁇ -alanine ( ⁇ -Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), glutamic acid (Glu), D-glutamic acid (D-Glu), ⁇ -glutamic acid ( ⁇ -Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met),
  • X 3 is absent or is a linking moiety of the following formula:
  • R 3 is a substituted or unsubstituted C 1-3 acyl group
  • R 4 is a C 5-8 aryl or heteroaryl group
  • R 5 is a substituted or unsubstituted amino group
  • R2 is selected from halogen-substituted C1-6 acyl.
  • R 1 is selected from the group consisting of heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, nonanoyl, methylnonanoyl, decanoyl, methyldecanoyl, lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, 15-carboxypentadecanoyl, 13-carboxytridecanoyl, and 11-carboxyundecanoyl.
  • R 1 is selected from the group consisting of lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, and 19-carboxynonadecanoyl.
  • n is an integer of 1-3.
  • n is an integer of 1-3.
  • X1 and X2 are independently selected from the following group: 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), D-alanine (D-Ala), ⁇ -alanine ( ⁇ -Ala), aspartic acid (Asp), cysteine (Cys), glutamic acid (Glu), ⁇ -glutamic acid ( ⁇ -Glu), glycine (Gly), serine (Ser), tyrosine (Tyr), 8-amino-3,6-dioxaoctanoic acid (AEEA), lysine (Lys).
  • X1 and X2 are independently selected from the group consisting of glutamic acid (Glu), ⁇ -glutamic acid ( ⁇ -Glu), 8-amino-3,6-dioxaoctanoic acid (AEEA), and lysine (Lys).
  • Glu glutamic acid
  • ⁇ -Glu ⁇ -glutamic acid
  • AEEA 8-amino-3,6-dioxaoctanoic acid
  • Lys lysine
  • the amino acid is preferably an L-amino acid.
  • R 2 is selected from halogen-substituted C 1-3 acyl.
  • R 2 is selected from iodoacetyl or bromoacetyl; more preferably bromoacetyl.
  • modified protein is selected from the following group:
  • the present invention provides an OmCI mutant having an amino acid sequence as shown in SEQ ID NO: 1, and T90, K95, T97, E126 or S156 are mutated to cysteine residues.
  • the amino acid sequence of OmCI is as shown in any one of SEQ ID NO:4-8.
  • the amino acid sequence of OmCI is as shown in any one of SEQ ID NO: 4, 5, 7, and 8.
  • the present invention provides a pharmaceutical composition comprising the modified protein according to the first aspect or the OmCI mutant according to the second aspect and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is used as a complement inhibitor.
  • the pharmaceutical composition is suitable for use in autoimmune diseases, inflammation, leukotriene or hydroxyeicosanoid mediated diseases.
  • the pharmaceutical composition is suitable for paroxysmal nocturnal hemoglobinuria (PNH), atypical bullous pemphigoid, atopic keratoconjunctivitis, thrombotic microangiopathy (TMA), myasthenia gravis (gMG), atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy, age-related macular degeneration (AMD), respiratory viral infections, autoimmune bullous diseases, and eye diseases (e.g., proliferative retinal diseases).
  • PNH paroxysmal nocturnal hemoglobinuria
  • TMA thrombotic microangiopathy
  • gMG myasthenia gravis
  • aHUS atypical hemolytic uremic syndrome
  • C3 glomerulopathy C3 glomerulopathy
  • AMD age-related macular degeneration
  • respiratory viral infections e.g., proliferative retinal diseases.
  • the present invention provides use of the modified protein of the first aspect or the OmCI mutant of the second aspect in preparing a drug.
  • the drug is a complement inhibitor.
  • the medicament is suitable for paroxysmal nocturnal hemoglobinuria (PNH), atypical bullous pemphigoid, atopic keratoconjunctivitis, thrombotic microangiopathy (TMA), myasthenia gravis (gMG), atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy, age-related macular degeneration (AMD), respiratory viral infections, autoimmune blistering diseases, eye diseases (e.g., proliferative retinal diseases).
  • PNH paroxysmal nocturnal hemoglobinuria
  • TMA thrombotic microangiopathy
  • gMG myasthenia gravis
  • aHUS atypical hemolytic uremic syndrome
  • C3 glomerulopathy C3 glomerulopathy
  • respiratory viral infections e.g., autoimmune blistering diseases, eye diseases (e.g., proliferative retinal diseases).
  • the present invention provides an albumin binder, the structure of which is shown in the following formula:
  • K is lysine
  • R1 is a substituted or unsubstituted C1-20 acyl group
  • n is an integer from 0 to 5;
  • n is an integer from 0 to 5;
  • o is an integer of 1-3 (preferably 1 or 2);
  • X1 and X2 are independently selected from the following group: alanine (Ala), D-alanine (D-Ala), ⁇ -alanine ( ⁇ -Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asp), Asn, Cysteine (Cys), Glutamic acid (Glu), D-glutamic acid (D-Glu), ⁇ -glutamic acid ( ⁇ -Glu), Glutamine (Gln), Glycine (Gly), Histidine (His), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Proline (Pro), Phenylalanine (Phe), Serine (Ser), Tyrosine (Tyr), Threonine (Thr), Tryptophan (Trp), Valine (Val), Methionine (Met), Tra
  • X 3 is absent or is a linking moiety of the following formula:
  • R 3 is a substituted or unsubstituted C 1-3 acyl group
  • R 4 is a C 5-8 aryl or heteroaryl group
  • R 5 is a substituted or unsubstituted amino group
  • R2 is selected from halogen-substituted C1-6 acyl.
  • R 1 is selected from the group consisting of heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, nonanoyl, methylnonanoyl, decanoyl, methyldecanoyl, lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, 15-carboxypentadecanoyl, 13-carboxytridecanoyl, and 11-carboxyundecanoyl.
  • R 1 is selected from the group consisting of lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, and 19-carboxynonadecanoyl.
  • n is an integer of 1-3.
  • n is an integer of 1-3.
  • X1 and X2 are independently selected from the following group: 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), D-alanine (D-Ala), ⁇ -alanine ( ⁇ -Ala), aspartic acid (Asp), cysteine (Cys), glutamic acid (Glu), ⁇ -glutamic acid ( ⁇ -Glu), glycine (Gly), serine (Ser), tyrosine (Tyr), 8-amino-3,6-dioxaoctanoic acid (AEEA), lysine (Lys).
  • the amino acid is preferably an L-amino acid.
  • R 3 is formyl
  • R 4 is phenyl
  • R 5 is amino
  • R 2 is selected from halogen-substituted C 1-3 acyl.
  • R 2 is selected from iodoacetyl or bromoacetyl; more preferably bromoacetyl.
  • the albumin binder is selected from the group consisting of:
  • the present invention provides an expression vector comprising the isolated nucleic acid molecule of the sixth aspect.
  • the present invention provides a host cell, wherein the host cell comprises the expression vector described in the seventh aspect or the nucleic acid molecule described in the sixth aspect is integrated into the genome of the host cell.
  • the present invention provides use of the OmCI mutant described in the second aspect in preparing the modified protein described in the first aspect.
  • the present invention provides a therapeutic method, comprising the step of administering a therapeutically effective amount of the modified protein of the first aspect, the OmCI mutant of the second aspect, or the pharmaceutical composition of the third aspect to a subject in need thereof.
  • the treatment methods are applicable to autoimmune diseases, inflammation, leukotriene or hydroxyeicosanoid mediated diseases.
  • the treatment method is suitable for paroxysmal nocturnal hemoglobinuria (PNH), atypical bullous pemphigoid, atopic keratoconjunctivitis, thrombotic microangiopathy (TMA), myasthenia gravis (gMG), atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy, age-related macular degeneration (AMD), respiratory viral infections, autoimmune blistering diseases, and eye diseases (e.g., proliferative retinal diseases).
  • PNH paroxysmal nocturnal hemoglobinuria
  • TMA thrombotic microangiopathy
  • gMG myasthenia gravis
  • aHUS atypical hemolytic uremic syndrome
  • C3 glomerulopathy C3 glomerulopathy
  • AMD age-related macular degeneration
  • respiratory viral infections e.g., proliferative retinal diseases
  • autoimmune blistering diseases e.g., proliferative retinal diseases
  • Figure 1 is a schematic diagram of the three pathways of complement activation
  • Figure 2 shows the SDS-PAGE images of the purified recombinant OmCI protein and its four mutants described in Examples 1 and 2; wherein, “1" is OmCIT90C; “2” is OmCIK95C; “3” is OmCIT97C; “4" is OmCIS156C; “5" is OmCI; “M” is Marker; “6” is OmCIT90C+DTT; “7” is OmCIK95C+DTT; “8” is OmCIT97C+DTT; “9” is OmCIS156C+DTT; and “10” is OmCI+DTT;
  • FIG3 shows the inhibition of PNH-like erythrocyte hemolysis in vitro by the recombinant OmCI protein and two modified derivatives described in Example 8;
  • FIG. 4 shows the pharmacokinetic parameters of the recombinant OmCI protein and two modified derivatives described in Example 11 in mice.
  • the OmCI mutant modified derivative has good complement C5 inhibitory activity and excellent in vivo half-life, thereby laying a new material foundation for the development of related drugs and having great clinical development potential. On this basis, the present invention was completed.
  • isolated nucleic acid molecule refers to a nucleic acid molecule provided herein that is: 1) separated from at least about 50% of the proteins, lipids, carbohydrates or other materials that coexist with the nucleic acid when the nucleic acid is separated from the source cell, 2) not connected to all or part of a polynucleotide that is naturally connected to the "isolated nucleic acid molecule", 3) effectively connected to a polynucleotide that is not naturally connected to it, or 4) not naturally present as part of a larger polynucleotide sequence.
  • the isolated nucleic acid molecule is substantially free of any other contaminating nucleic acid molecules or other pollutants that may interfere with its use in polypeptide production or its treatment, diagnosis, prevention or research purposes that exist in its natural environment.
  • vector is used to refer to any molecule (eg, nucleic acid, plasmid or virus) used to transfer coding information to a host cell.
  • expression vector refers to a vector suitable for host cell transformation and containing a nucleic acid sequence that directs and/or controls the expression of an inserted heterologous nucleic acid sequence. Expression includes, but is not limited to, transcription, translation, and RNA splicing (if introns are present). process.
  • flanking sequence that is operably linked to a coding sequence may be capable of effecting replication, transcription, and/or translation of the coding sequence.
  • a coding sequence is operably linked to a promoter when the promoter is capable of directing transcription of the coding sequence.
  • a flanking sequence need not be contiguous with a coding sequence, as long as it functions properly.
  • an intervening non-translated but transcribed sequence may be present between a promoter sequence and a coding sequence, and the promoter sequence may still be considered to be "operably linked" to a coding sequence.
  • host cell is used to refer to a cell that is transformed or capable of being transformed by a nucleic acid sequence (e.g., a nucleic acid provided herein) and then capable of expressing a selected target gene.
  • a nucleic acid sequence e.g., a nucleic acid provided herein
  • the term includes progeny of a parent cell, whether or not the progeny is identical to the original parent in morphology or genetic composition, as long as the selected gene is present.
  • sequence identity is determined by aligning the sequence of a reference DNA with another DNA sequence, thereby maximizing the overlap between the two sequences and simultaneously minimizing sequence gaps, wherein any protruding sequences between the two sequences are ignored. For any sequence identity described herein, at least 80% is preferred, 85% is more preferred, 90% is more preferred, 95% sequence identity is still more preferred, and 96%, 97%, 98% and 99% sequence identity are most preferred.
  • amino acid residues in proteins are as follows: Phe or F for phenylalanine; Leu or L for leucine; Ile or I for isoleucine; Met or M for methionine; Val or V for valine; Ser or S for serine; Pro or P for proline; Thr or T for threonine; Ala or A for alanine; Tyrosine is Tyr or Y; His or H for histidine; Gln or Q for glutamine; Asn or N for asparagine; Lys or Lys; Aspartic acid is Asp or D; Glu or E for glutamate; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; Glycine is Gly or G.
  • an ethyl group is "optionally” substituted with a halogen, which means that the ethyl group may be unsubstituted (CH 2 CH 3 ), monosubstituted (such as CH 2 CH 2 F), polysubstituted (such as CHFCH 2 F, CH 2 CHF 2, etc.) or fully substituted (CF 2 CF 3 ). It will be understood by those skilled in the art that for any group containing one or more substituents, no substitution or substitution pattern that is sterically impossible and/or cannot be synthesized will be introduced.
  • Cm-n means that there are m-n carbon atoms in the moiety.
  • C0-6 alkylene means that the alkylene has 0-6 carbon atoms, and when the alkylene has 0 carbon atoms, the group is a bond.
  • C1-6 means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.
  • any variable e.g., R
  • its definition at each occurrence is independent.
  • each R has an independent choice.
  • substituted means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, as long as the particular atom The valence state is normal and the substituted compound is stable.
  • it means that two hydrogen atoms are replaced, and oxo will not occur on the aromatic group.
  • derivative refers to a modified protein, i.e. a protein to which a moiety is bonded in order to modify the properties of the protein; as a verb, the term refers to the process of bonding a moiety to a protein to modify the properties of the protein.
  • OmCI means a recombinant C5 complement inhibitor derived from ticks, which is a natural protein with a molecular weight of about 17 kDa extracted from the salivary glands of soft ticks (Ornithodoros moubata). Studies have found that the protein can specifically bind to complement C5, inhibit the activation pathway downstream of complement, and prevent the formation of MAC.
  • CN1798841B discloses the recombinant sequence of the complement inhibitor.
  • the inhibitor sequence consists of amino acids 1 to 168, wherein the first 18 amino acids of the protein sequence form a signal sequence, and its mature form is a protein composed of amino acid sequences 19 to 168 shown in CN1798841B, and it is named "OmCI protein", "Coversin” or "Nomacopan”.
  • the OmCI protein structure belongs to the lipocalin family. It has a compact folded structure, including a central eight-stranded antiparallel ⁇ barrel with three pairs of disulfide bonds at positions 24 and 146, 118 and 147, and 56 and 168, respectively (Roversi P, Lissina O, Johnson S, et al. The structure of OMCI, a novel lipocalin inhibitor of the complement system.
  • OmCI protein has the ability to inhibit the activity of leukotriene B4 (LTB4), providing additional anti-inflammatory function (Roversi P, Ryffel B, Togbe D, et al. Bifunctional lipocalin ameliorates murine immune complex-induced acute lung injury. J Biol Chem. 2013; 288(26): 18789-18802).
  • OmCI has a variety of clinical application potentials.
  • CN102066412A discloses its ability to bind to the eicosanoids of LTB4 and its use in treating diseases mediated by leukotrienes or hydroxy eicosanoids
  • CN101340926A discloses its effect in treating myasthenia gravis
  • CN102762223B discloses its effect in preventing respiratory viral infections and inflammation
  • CN106659767B discloses that it can treat or prevent subjects with C5 polymorphism and complement-mediated disorders
  • CN110896606A discloses that it can be used as an effective method for treating and preventing autoimmune bullous disease (AIBD);
  • CN110831617A discloses that it can be used to treat eye diseases;
  • CN114072206A discloses that it can treat or prevent proliferative retinal diseases.
  • mutant as used herein has the meaning conventionally understood by those skilled in the art.
  • a mutant is understood to be a compound obtained by the following steps: replacing one or more amino acid residues in the OmCI sequence with another natural or non-natural amino acid; and/or adding one or more natural or non-natural amino acids to the OmCI sequence; and/or deleting one or more amino acid residues from the OmCI sequence, wherein any of these steps may be optionally followed by further derivatization of one or more amino acid residues.
  • substitutions are conservative.
  • Amino acids can be suitably divided into the following groups based on their properties: basic amino acids (e.g., arginine, lysine, histidine), acidic amino acids (e.g., glutamic acid and aspartic acid), polar amino acids (e.g., glutamine, cysteine and asparagine), hydrophobic amino acids (e.g., leucine, isoleucine, proline, methionine and valine), aromatic amino acids (e.g., phenylalanine, tryptophan, tyrosine) and small amino acids (e.g., glycine, alanine, serine and threonine).
  • the OmCI mutant has at least 80%, such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the native OmCI from ticks.
  • OmCI fusion protein refers to the fusion of one or more amino acid residues (eg, a heterologous protein or peptide) to the N-terminus or C-terminus of any of the OmCI mutants described herein.
  • Heterologous peptides and polypeptides include, but are not limited to, epitopes for detecting and/or isolating OmCI protein mutants: transmembrane receptor proteins or portions thereof, such as extracellular domains or transmembrane and intracellular domains; ligands or portions thereof that bind to transmembrane receptor proteins; enzymes or catalytically active portions thereof; polypeptides or peptides that promote oligomerization, such as leucine zipper domains; polypeptides or peptides that improve stability, such as immunoglobulin constant regions (e.g., domains); half-life extension sequences comprising a combination of two or more (e.g., 2, 5, 10, 15, 20, 25, etc.) naturally occurring or non-naturally occurring, charged and/or uncharged amino acids (e.g., serine, glycine, glutamic acid, or aspartic acid) designed to form a fusion partner of a predominantly hydrophilic or predominantly hydrophobic mutant; functional or
  • OmCI fusion proteins can be prepared by fusing heterologous sequences to the N-terminus or C-terminus of OmCI polypeptide mutants.
  • the heterologous sequence described herein can be an amino acid sequence or a polymer containing non-amino acids.
  • the heterologous sequence can be directly fused to the OmCI mutant or fused through a joint or an adapter molecule.
  • the joint or adapter molecule can be one or more amino acid residues (or amino acid polymers), such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 residues (or amino acid polymers), preferably 10-50 amino acid residues (or amino acid polymers), such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 residues (or amino acid polymers), more preferably 15-35 amino acid residues (or amino acid polymers).
  • the joint or adapter molecule can also be designed to have a cleavage site for DNA restriction endonucleases or proteases for separating the fusion part.
  • OmCI analogs or OmCI derivatives refer to proteins that are or can be derived from natural OmCI, in particular, from or can be derived from SEQ ID NO: 1, i.e., through the modification of its amino acid sequence.
  • modification, amendment or change may include substitution, deletion and/or addition of one or more amino acids.
  • amino acids may be added and/or deleted at the C-terminus, N-terminus or inside the amino acid sequence.
  • amino acids are added and/or deleted at the C-terminus and/or N-terminus, more preferably at the N-terminus.
  • An amino acid sequence with amino acids deleted at the C-terminus or N-terminus may also be referred to as a truncated sequence, which is known in the art.
  • amino acids added inside the sequence may be referred to as insertions.
  • the albumin binder is a fatty acid.
  • the structure of the albumin binder is shown in the following formula:
  • K, R 1 , m, n, o, X 1 , X 2 and X 3 are as defined above.
  • OmCI and its mutants are prepared as follows:
  • OmCI and its mutants can be expressed in bacteria, such as E. coli, mammals, yeast, such as Pichia pastoris, and plant expression systems. Expression can be performed by exogenous expression (when the host cell naturally contains the desired genetic code) or by endogenous expression. In some embodiments, OmCI and its mutants are expressed in E. coli.
  • recombinant-based methods for preparing proteins typically involve constructing a nucleic acid encoding a desired polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming a host cell, and expressing the nucleic acid to produce the desired polypeptide or fragment.
  • Methods for producing and expressing recombinant polypeptides in vitro and in prokaryotic host cells are known to those of ordinary skill in the art.
  • a nucleotide sequence encoding an acid-sensitive tag in order to obtain an OmCI analog of the correct sequence, can be inserted or added to the encoded sequence in a frame-matched manner, thereby producing a fusion protein comprising a desired polypeptide and a polypeptide containing an acid-sensitive tag.
  • a method using genetic engineering is provided to artificially synthesize and express a fusion protein (precursor protein) comprising a desired polypeptide and a tag polypeptide, insert the recombinant gene sequence into an expression vector, transform host cells, and prepare OmCI and its mutants by fermentation, high-pressure cell disruption, enzyme digestion, purification, etc.
  • the purity of OmCI and its mutants can be determined by any of a variety of known analytical methods, including gel electrophoresis, high-performance liquid chromatography, etc., and its molecular weight can be identified by mass spectrometry.
  • OmCI cysteine mutants were prepared as follows:
  • rationally designed OmCI mutants are provided, in particular, mutants in which certain amino acids of OmCI are mutated to cysteine. Including, cysteine mutations were performed on T90, K95, T97, E126 and S156 based on the sequence of SEQ NO:1, respectively, to obtain five OmCI cysteine mutants with sequences of T90C (SEQ NO:4), K95C (SEQ NO:5), T97C (SEQ NO:6), E126C (SEQ NO:7) and S158C (SEQ NO:8).
  • OmCI cysteine mutants including T90C (SEQ NO:4), K95C (SEQ NO:5), T97C (SEQ NO:7) and S158C (SEQ NO:8) were successfully prepared and their activities were determined by the classical pathway hemolytic test.
  • compounds linked by disulfide bonds including but not limited to OmCI-Cys, OmCI-reduced glutathione, OmCI-cysteamine and the like obtained by reacting with free Cys of the OmCI mutant are also included in the OmCI mutant.
  • albumin binder of the present invention is prepared as follows:
  • the solid phase synthesis of the albumin binding fragment can be carried out according to conventional solid phase synthesis methods.
  • the albumin binding fragment can be purified by various known methods, and the purity and molecular weight of the albumin binding fragment can be verified by high performance liquid chromatography and mass spectrometry analysis.
  • the derivatives generally comprise an albumin binding fragment, the tag being non-toxic, non-naturally occurring and biocompatible, typically the albumin binding fragment having a binding affinity for human serum albumin of less than about 10 ⁇ M or even less than about 1 ⁇ M.
  • the OmCI mutant modified derivatives can be prepared as follows:
  • OmCI mutant derivatives with albumin binder modifications can be prepared as follows:
  • the albumin binding fragment reacts with free cysteine on the OmCI mutant through a halogenation reaction, ie, the fatty acid is modified to obtain an OmCI derivative.
  • the term "pharmaceutically acceptable salt” means a salt that is harmless to the patient.
  • the salt includes pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium salts and alkylated ammonium salts.
  • Acid addition salts include salts of inorganic acids and organic acids. Representative examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfuric acid, nitric acid, etc.
  • suitable organic acids include formic acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, propionic acid, benzoic acid, cinnamic acid, citric acid, fumaric acid, glycolic acid, lactic acid, maleic acid, malic acid, malonic acid, mandelic acid, oxalic acid, picric acid, pyruvic acid, salicylic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, tartaric acid, ascorbic acid, pamoic acid, dimethoxy salicylic acid, ethanedisulfonic acid, gluconic acid, citric acid, aspartic acid, stearic acid, palmitic acid, EDTA, glycolic acid, p-aminobenzoic acid, glutamic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.
  • metal salts include lithium salts, sodium salts, potassium salts, magnesium salts, etc.
  • Ammonium salts and alkylated ammonium salts include ammonium salts, methylammonium salts, dimethylammonium salts, trimethylammonium salts, ethylammonium salts, hydroxyethylammonium salts, diethylammonium salts, butylammonium salts, tetramethylammonium salts, etc.
  • the present application also includes isotope-labeled compounds of the present application that are identical to those described herein, but one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number commonly found in nature.
  • isotopes that can be incorporated into the compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 31P, 32P, 35S, 18F, 123I, 125I, and 36Cl, etc., respectively.
  • Certain isotopically labeled compounds of the present invention can be used in compound and/or substrate tissue distribution analysis. Tritiated (i.e., 3H) and carbon-14 (i.e., 14C) isotopes are particularly preferred for their ease of preparation and detectability.
  • substitution with heavier isotopes such as deuterium (i.e., 2H)
  • can provide certain therapeutic advantages e.g., increased in vivo half-life or reduced dosage requirements
  • Positron emitting isotopes such as 15O, 13N, 11C, and 18F can be used in positron emission tomography (PET) studies to determine substrate occupancy.
  • Isotopically labeled compounds of the present invention can generally be prepared by replacing an isotopically labeled reagent with an isotopically labeled reagent by the following procedures similar to those disclosed in the schemes and/or embodiments below.
  • OmCI analogs can be used to treat, diagnose, ameliorate or prevent a variety of diseases, disorders or conditions, including but not limited to immunotherapy. Pemphigus, atopic keratoconjunctivitis, thrombotic microangiopathy (TMA), myasthenia gravis (gMG), atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy, age-related macular degeneration (AMD) and many other diseases.
  • TMA thrombotic microangiopathy
  • gMG myasthenia gravis
  • aHUS atypical hemolytic uremic syndrome
  • AMD age-related macular degeneration
  • the OmCI analogs described herein can be administered to patients in need of the treatment of PNH or aHUS and other diseases or conditions. Administration can be as described herein, for example, by intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, or oral administration in the form of tablets or liquid preparations. In most cases, the required dose can be determined by clinical staff as described herein, and can represent a therapeutically effective dose of OmCI protein analogs. It is obvious to those skilled in the art that the therapeutically effective dose of OmCI analogs will depend on, among other things, the dosing regimen, the unit dose of the substance administered (whether or not the nucleic acid molecule or polypeptide is administered in combination with other therapeutic agents), the immune status and the health status of the recipient.
  • terapéuticaally effective dose means the amount of OmCI analogs that elicits a biological or drug response in a tissue system, animal or human that is sought by a researcher or other clinician, including the alleviation of the symptoms of the disease or condition to be treated.
  • the present invention provides a new modified protein
  • the modified protein of the present invention retains the original activity of the protein, and its half-life in vivo is also significantly improved;
  • the preparation process of the modified protein of the present invention is simple and can be easily obtained by biotechnology, chemical synthesis technology and other technical means;
  • the modified protein of the present invention lays a new material foundation for the development of therapeutic drugs for various diseases such as paroxysmal nocturnal hemoglobinuria (PNH), atypical bullous pemphigoid, atopic keratoconjunctivitis, thrombotic microangiopathy (TMA), myasthenia gravis (gMG), atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy, age-related macular degeneration (AMD), etc.
  • PNH paroxysmal nocturnal hemoglobinuria
  • TMA thrombotic microangiopathy
  • gMG myasthenia gravis
  • aHUS atypical hemolytic uremic syndrome
  • C3 glomerulopathy age-related macular degeneration (AMD), etc.
  • the protein concentration was determined by using a BCA kit.
  • Example 1 Preparation of recombinant OmCI protein
  • OmCI is naturally derived from the salivary glands of ticks, and its sequence is available on Uniport (ID: Q5YD09), such as SEQ ID NO: 1.
  • the natural OmCI protein consists of amino acids 1 to 168, of which the first 18 amino acids of the protein sequence form a signal sequence that is not essential for C5 binding activity or for LTB4 binding activity.
  • the mature form of the amino acid sequence 19 to 168 constitutes the protein, so the amino acid sequence of the mature protein can be used for the efficiency of recombinant protein production.
  • an extracellular expression system of recombinant OmCI protein was established in the form of fusion protein.
  • An expression cassette containing an N-terminal enterokinase recognition site (GSGDEGD) and a periplasmic binding protein (ArgT; UniProt ID P09551) for OmCI expression was constructed and inserted into the pET30 plasmid. Then it was transformed into competent E. coli BL21 (DE3) bacteria, and an engineered strain for expressing the ArgT-GSGDEGD-OmCI (SEQ ID NO: 2) fusion protein was obtained by screening. After high-density fermentation, the engineered bacteria expressed the ArgT-GSGDEGD-OmCI fusion protein in a soluble form outside the cell.
  • Soluble fusion protein ArgT-GSGDEGD-OmCI (SEQ ID NO: 2), after enzyme cleavage, purified to obtain soluble target OmCI recombinant protein.
  • the pH of the fermentation broth was adjusted to 4.0, and the broth was clarified after centrifugation. The pH was then adjusted to 8.0, and recombinant bovine enterokinase light chain (which can specifically cleave the GSGDEGD site) was added at 37°C for enzyme cleavage reaction.
  • the pH of the treated enzyme cleavage solution was adjusted to 6.5, and anion exchange chromatography (HiTrap Q HP (5 ml, purchased from Cytiva) was used to maintain the chromatography pH at 6.5.
  • the elution was performed with a linear salt gradient of 0-500 mM NaCl. After SDS-PAGE analysis and RP-HPLC analysis, the soluble target OmCI protein with high purity was obtained. Finally, the OmCI protein was concentrated by ultrafiltration using a MWCO 3kDa filter membrane and replaced in PBS.
  • the measured molecular weight of OmCI prepared according to the above method was 16779.85 Da, which was consistent with the theoretical molecular weight (16779.59 Da).
  • the position of the cysteine mutation should not affect the function and structure of OmCI itself and should be easy to modify the subsequent albumin binding fragment, that is, it should meet the following three points: (1) conservative mutations should be selected to avoid affecting the binding of OmCI to its receptor, that is, the amino acids involved in the interaction should not be mutated; (2) the formation of three pairs of disulfide bonds should not be affected; (3) the position should be solvent accessible on the protein surface.
  • T90 (SEQ ID NO: 4), K95 (SEQ ID NO: 5), T97 (SEQ ID NO: 6), E126 (SEQ ID NO: 7) and S156 (SEQ ID NO: 8) of OmCI were selected for cysteine mutation.
  • These OmCI cysteine mutants were engineered, fermented and purified as described in Example 2. Except for the OmCIE126C mutant, which was difficult to express and purify, the remaining mutants were successfully prepared.
  • OmCIT90C, OmCIK95C, OmCIT97C, OmCIE126C, and OmCIS156C were expressed in the form of tag fusion proteins.
  • OmCIT90C an expression cassette containing an N-terminal 6-histidine (His 6 ) tag and a ubiquitin-like modifier protein (SUMO) tag for the expression of OmCI mutants was constructed and inserted into the pET30 plasmid. Then it was transformed into competent E. coli BL21 (DE3) bacteria, and an engineered strain for expressing His 6 -SUMO-OmCIT90C (SEQ ID NO: 3) fusion protein was obtained by screening. The engineered bacteria were fermented at high density to express the His 6 -SUMO-OmCIT90C fusion protein in a soluble form in the periplasmic space.
  • His 6 N-terminal 6-histidine
  • SUMO ubiquitin-like modifier protein
  • the fusion protein was enriched by metal ion chelation chromatography (IMAC) HisTrap Ni excel (5 ml, purchased from Cytiva), and the fusion protein containing the His 6 tag was eluted by 0.3 M imidazole.
  • the eluted peak was collected and subjected to anion exchange chromatography (HiTrap Q HP (5 ml, purchased from Cytiva), the chromatography pH was maintained at 7.4, and eluted with a linear salt gradient of 0-500 mM NaCl, and then ULP1 enzyme (which can specifically cut the SUMO tag) was added to the eluted peak for enzyme cleavage at 32°C, and the cleaved solution was subjected to metal ion chelation chromatography (IMAC) to remove the tag protein (His 6 -SUMO), and the effluent was collected and combined after SDS-PAGE analysis and RP-HPLC analysis to obtain the target OmCIT90C (SEQ ID NO:4) mutant, which
  • the measured molecular weight of the monomer OmCIT90C without blocked cysteine prepared according to the above method was 16781.2Da, which was consistent with the theoretical molecular weight (16781.63Da).
  • the measured molecular weight of the cysteine-blocked OmCIT90C-Cys mutant was 16900.80Da, which was consistent with the theoretical molecular weight (16900.79Da).
  • OmCI mutant proteins were expressed in a bacterial expression system such as E. coli BL21 (DE3). Unless otherwise specified, the expression and purification were performed according to the method described in this example. The results are as follows:
  • Theoretical molecular weight 966.96Da; measured molecular weight: 967.43Da.
  • Theoretical molecular weight 995.02Da; measured molecular weight: 995.55Da.
  • Theoretical molecular weight 1023.07Da; measured molecular weight: 1023.60Da.
  • Theoretical molecular weight 954.95Da; measured molecular weight: 955.44Da.
  • Theoretical molecular weight 983.01 Da; measured molecular weight: 983.49 Da.
  • Theoretical molecular weight 953.02Da; measured molecular weight: 953.30Da.
  • Theoretical molecular weight 1011.06Da; measured molecular weight: 1011.51Da.
  • Theoretical molecular weight 949.04Da; measured molecular weight: 949.53Da.
  • Theoretical molecular weight 977.09 Da; measured molecular weight: 977.59 Da.
  • OmCI-Cys 90 [S-CH 2 CO-Lys- ⁇ Glu- ⁇ Glu-AEEA-CO-(CH 2 ) 16 -CO 2 H]
  • the compound is an OmCIT90C mutant derivative (OmCIT90C-CM03) modified by the albumin binder (CM03) as described in Example 3.1, such as the OmCIT90C mutant of SEQ ID NO:4 (Example 2).
  • the specific preparation process and identification are as follows.
  • the cysteine-blocked OmCIT90C-Cys mutant was ultrafiltered into PBS buffer (pH 7.4) with a final concentration of 1 mg/mL.
  • PBS buffer pH 7.4
  • 0.5 mM TCEP tris(2-carboxyethyl)phosphine, dissolved in PBS buffer, adjusted to pH 7.4
  • the albumin binder as described in Example 3.1 was dissolved in a saturated ammonium bicarbonate solution, and the dissolved albumin binding fragment solution was added to the OmCI mutant at a molar ratio of 1:10 for OmCI mutant:albumin binding fragment, and reacted overnight under stirring.
  • the albumin binder-modified OmCI mutant derivative was purified by anion exchange chromatography 30Q column (purchased from Suzhou Saifen). The chromatography pH was maintained at 7.4, and eluted with a linear salt gradient of 0–500 mM NaCl. The target peaks were merged after RP-HPLC analysis and SDS-PAGE analysis to obtain the OmCIT90C mutant of SEQ ID NO: 4 with higher purity (Example 2), which was an OmCI mutant derivative modified with the albumin binding fragment (CM03) as described in Example 3.1.
  • Theoretical molecular weight 17667.68Da; measured molecular weight: 17667.12Da.
  • OmCIT90C-CM05 OmCI-Cys 90 -[S-CH 2 CO-Lys- ⁇ Glu- ⁇ Glu-AEEA-CO-(CH 2 ) 18 -CO 2 H]
  • the compound is an OmCI mutant derivative modified by the OmCIT90C mutant of SEQ ID NO:4 (Example 2) and the albumin binding fragment (CM05) as described in Example 3.3.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17695.68Da; measured molecular weight: 17694.83Da.
  • OmCIT90C-CM06 OmCI-Cys 90 -[S-CH 2 CO-Lys- ⁇ Glu- ⁇ Glu-AEEA-CO-(CH 2 ) 20 -CO 2 H]
  • the compound is an OmCI mutant derivative modified by the OmCIT90C mutant of SEQ ID NO:4 (Example 2) and the albumin binding fragment (CM06) as described in Example 3.4.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17725.68Da; measured molecular weight: 17724.12Da.
  • the compound is an OmCI mutant derivative obtained by modifying the OmCIT90C mutant of SEQ ID NO: 4 (Example 2) with an albumin binder (CM10) as described in Example 3.8.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17711.78Da; measured molecular weight: 17711.29Da.
  • the compound is an OmCI mutant derivative modified by the OmCIT90C mutant of SEQ ID NO:4 (Example 2) and the albumin binder (CM14) as described in Example 3.11.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17677.81Da; measured molecular weight: 17677.10Da.
  • the compound is an OmCI mutant derivative modified with an albumin binder (CM03) as described in Example 3.1 by the OmCIT97C mutant of SEQ ID NO:6 (Example 2), and the preparation process and identification are as follows.
  • Theoretical molecular weight 17667.68Da; measured molecular weight: 17668.24Da.
  • OmCIT97C-CM05 OmCI-Cys 97 -[S-CH 2 CO-Lys- ⁇ Glu- ⁇ Glu-AEEA-CO-(CH 2 ) 18 -CO 2 H]
  • the compound is the OmCIT97C mutant of SEQ ID NO: 6 (Example 2) obtained by mixing the white
  • the preparation process and identification of the OmCI mutant derivative modified with the protein binding fragment (CM05) are as follows.
  • Theoretical molecular weight 17695.68Da; measured molecular weight: 17695.25Da.
  • OmCIT97C-CM06 OmCI-Cys 97 -[S-CH 2 CO-Lys- ⁇ Glu- ⁇ Glu-AEEA-CO-(CH 2 ) 20 -CO 2 H]
  • the compound is an OmCI mutant derivative modified by the OmCIT97C mutant of SEQ ID NO:6 (Example 2) and the albumin binding fragment (CM06) as described in Example 3.4.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17653.74Da; measured molecular weight: 17651.22Da.
  • the compound is an OmCI mutant derivative modified by the OmCIT97C mutant of SEQ ID NO:6 (Example 2) and the albumin binder (CM10) as described in Example 3.8.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17711.78Da; measured molecular weight: 17711.26Da.
  • the compound is an OmCI mutant derivative modified by the OmCIT97C mutant of SEQ ID NO:6 (Example 2) and the albumin binder (CM14) as described in Example 3.11.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17677.81Da; measured molecular weight: 17677.28Da.
  • OmCIS156C-CM03 OmCI-Cys 156 -[S-CH 2 CO-Lys- ⁇ Glu- ⁇ Glu-AEEA-CO-(CH 2 ) 16 -CO 2 H]
  • the compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binder (CM03) as described in Example 3.1.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17681.70Da; measured molecular weight: 17681.64Da.
  • OmCIS156C-CM05 OmCI-Cys 156 -[S-CH 2 CO-Lys- ⁇ Glu- ⁇ Glu-AEEA-CO-(CH 2 ) 18 -CO 2 H]
  • the compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binding fragment (CM05) as described in Example 3.3.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17709.70Da; measured molecular weight: 17709.74Da.
  • OmCIS156C-CM06 OmCI-Cys 156 -[S-CH 2 CO-Lys- ⁇ Glu- ⁇ Glu-AEEA-CO-(CH 2 ) 20 -CO 2 H]
  • the compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binding fragment (CM06) as described in Example 3.4.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17737.70Da; measured molecular weight: 17737.80Da.
  • OmCIS156C-CM07 OmCI-Cys 156 -[S-CH 2 -CO-Lys-AEEA-AEEA- ⁇ Glu-CO-(CH 2 ) 14 -CO 2 H]
  • the compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binding fragment (CM07) as described in Example 3.5.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17669.69 Da; measured molecular weight: 17669.28 Da.
  • OmCIS156C-CM08 OmCI-Cys 156 -[S-CH 2 -CO-Lys-AEEA-AEEA- ⁇ Glu-CO-(CH 2 ) 16 -CO 2 H]
  • the compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binder (CM08) as described in Example 3.6.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17697.75Da; measured molecular weight: 17697.34Da.
  • OmCIS156C-CM09 OmCI-Cys 156 -[S-CH 2 -CO-Lys-AEEA-AEEA- ⁇ Glu-CO-(CH 2 ) 16 -CH 3 ]
  • the compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binder (CM09) as described in Example 3.7.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17667.76Da; measured molecular weight: 17667.39Da.
  • OmCIS156C-CM10 OmCI-Cys 156 -[S-CH 2 -CO-Lys-AEEA-AEEA- ⁇ Glu-CO-(CH 2 ) 18 -CO 2 H]
  • the compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binder (CM10) as described in Example 3.8.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17725.80Da; measured molecular weight: 17725.24Da.
  • OmCIS156C-CM11 OmCI-Cys 156 -[S-CH 2 -CO-Lys- ⁇ Lys- ⁇ Lys- ⁇ Glu-CO-(CH 2 ) 14 -CO 2 H]
  • the compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binder (CM11) as described in Example 3.9.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17635.73Da; measured molecular weight: 17636.43Da.
  • the compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binder (CM12) as described in Example 3.10.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17663.78Da; measured molecular weight: 17662.74Da.
  • the compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binder (CM14) as described in Example 3.11.
  • the preparation process and identification are as follows.
  • Theoretical molecular weight 17691.83Da; measured molecular weight: 17691.31Da.
  • Theoretical molecular weight 18702.74Da; measured molecular weight: 18702.19Da.
  • CH50 classical pathway hemolysis inhibition assay
  • 2 mL of fresh sterile sheep defibrinated blood in Alsever's (1:1 v/v) was washed once with 20 mL of GVBE (0.1% gelatin, 5 mM barbital, 145 mM NaCl, 10 mM EDTA; pH 7.4) buffer, centrifuged at 2500 x g for 10 min, the supernatant was discarded, and then washed three times with 20 mL of GVB++ (0.1% gelatin, 5 mM barbital, 145 mM NaCl, 0.15 mM CaCl 2 , 0.5 mM MgCl 2 ; pH 7.4) buffer.
  • the precipitated red blood cells were diluted in GVB++ buffer in proportion to prepare 4 ⁇ 10 8 cell/mL of sheep erythrocytes were reacted with 2 units (1:4000) of rabbit hemolysin (purchased from Shanghai Yuanye) in a 1:1 ratio and incubated in a 37°C water bath for 30 min to prepare 2x10 8 cell/mL sensitized sheep erythrocytes (EA) (antigen-antibody immune complex).
  • EA antigen-antibody immune complex
  • the alternative pathway hemolysis inhibition assay was similar to the CH50 and tested according to the method described in CN1798841B. 2 mL of sterile rabbit defibrinated blood in Alsever's was washed three times with 20 mL of GVBMg-EGTA buffer (0.1% gelatin, 5 mM barbital, 145 mM NaCl, 2.5 mM MgCl 2 , 8 mM EGTA; pH 7.4). The precipitated red blood cells were diluted in GVBMg-EGTA buffer in proportion to prepare 2 ⁇ 10 8 cell/mL of rabbit red blood cells.
  • Inhibition rate % 1 - hemolysis rate % ((A 412nm sample - A 412nm positive) / (A 412nm negative - A 412nm positive)
  • Example 7 In vitro anti-complement classical/alternative pathway activity (CH50/AH50) of OmCI recombinant protein, OmCI mutants and their modified derivatives
  • Example 8 Evaluation of complement hemolysis inhibition activity (PNH50) using PNH-like erythrocytes
  • the concentration of erythrocytes in the erythrocyte stock solution was adjusted with PBS so that the absorbance at 405 nm was 1.5-2.0 (indicating hemoglobin release) when 10 ⁇ L of the erythrocyte stock solution was diluted with 140 ⁇ L of water.
  • Normal human serum was acidified to pH 6.4 with 0.2M HCl and supplemented with MgCl 2 and EGTA to a final concentration of 2.5mM and 8mM, respectively.
  • 60 ⁇ L normal serum NHS final concentration of about 53% v/v
  • PBS-Mg set to 100% hemolysis as a negative control
  • PBS-EDTA set hemolysis rate to 0% as a positive control
  • samples of different concentrations 0.6nM-1500nM
  • Inhibition rate % 1 - hemolysis rate % ((A 405nm sample - A 405nm positive) / (A 405nm negative - A 405nm positive)
  • the recombinant OmCI protein (Example 1), the OmCI modified derivative OmCIT90C-CM05 (Example 4.2) and OmCIT90C-CM14 (Example 4.5) was evaluated for complement inhibition activity using PNH-like erythrocytes. The results are shown in Table 4, and the successfully expressed recombinant OmCI protein and its modified derivatives have considerable in vitro complement hemolytic inhibition activity.
  • the affinity between the recombinant OmCI protein (Example 1), the mutant OmCIT90C (Example 2), the OmCI modified derivative OmCIT90C-CM05 (Example 4.2) and OmCIT90C-CM14 (Example 4.5) and the receptor C5 was determined using surface plasmon resonance (SPR) technology. Real-time affinity measurements were performed at 25°C on a Biacore 8000 system, using PBST-0.02 buffer as a running buffer and a blank control, and the samples to be tested were gradiently diluted using the running buffer to a concentration of 300nM-3nM.
  • SPR surface plasmon resonance
  • the receptor protein (human complement C5 protein) was diluted to a concentration of 10 ⁇ g/mL with a 10mM sodium acetate (pH 5.0) solution, and the human complement C5 protein was fixed on a CM5 sensor chip using an amino coupling method in PBS buffer at a continuous flow rate of 10 ⁇ L/min and 200s, and its ligand density was approximately 1900-2400 resonance units (RU).
  • the experiment was carried out in PBST-0.02 running buffer with different concentrations of the sample dilution and blank control, and flowed through the control channel and the experimental channel at a flow rate of 30 ⁇ L/min, with a binding time of 60 s and a dissociation time of 200 s.
  • Real-time data signals were collected using BiaControl Software 2.0. 2M MgCl 2 solution was used for regeneration at a flow rate of 30 ⁇ L/min for 30 s to interrupt the strong electrostatic interaction between OmCI protein and C5.
  • the affinity between recombinant OmCI protein (Example 1), mutant OmCIT90C (Example 2), OmCI modified derivatives OmCIT90C-CM05 (Example 4.2) and OmCIT90C-CM14 (Example 4.5) and human serum albumin (HSA) was determined using surface plasmon resonance (SPR) technology. Real-time affinity measurements were performed at 25°C on a Biacore 8000 system (GE Healthcare). 0.02% PBST buffer was used as the running buffer and blank control, and the samples to be tested were gradiently diluted with the running buffer to a concentration of 3000nM-40nM.
  • HSA Human serum albumin
  • PBS buffer pH 4.5
  • a ligand density approximately 3500 resonance units (RU).
  • the experiment was carried out in the running buffer with different concentrations of the sample dilution and blank control, flowing through the control channel and the experimental channel at a flow rate of 30 ⁇ L/min, with a binding time of 60s and a dissociation time of 200s.
  • Real-time data signals were collected using BiaControl Software 2.0. 10mM Gly-HCl solution (pH 2.5) was used for regeneration at a flow rate of 30 ⁇ L/min for 30s to interrupt the strong electrostatic interaction between fatty acids and albumin.
  • mice Female C57BL/6J mice aged 6-8 weeks were housed under standard conditions. Mice (approximately 20 g) were divided into three groups and given a single dose of OmCI (s.c. 15 mg/kg); OmCI-T90C-CM05 (s.c. 15 mg/kg); OmCIT90C-CM14 (s.c. 15 mg/kg) in PBS by subcutaneous injection in the neck. Blood samples were collected through the tail vein at 0.1, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 72 h after administration (three animals per group at each time point). The blood was drawn into EDTA ⁇ 2K-coated tubes, placed on ice, and centrifuged at 1200 ⁇ g for 10 min at 4°C. The plasma was then transferred to Micronic tubes and stored at -20°C. The target protein concentration in plasma was determined by sandwich ELISA.
  • a transparent 96-well plate was supplemented with 50 ⁇ L of human complement C5 protein (purchased from Complement Technology) dissolved in PBS at 10 ⁇ g/mL and coated overnight at 4°C. After washing three times with PBS containing 0.05% v/v Tween 20 (PBST-0.05), 200 ⁇ L Blocker Casein (purchased from Thermo Fisher Scientific) was used at room temperature in the blocking Block for 1 hour and wash again. The plasma samples were diluted 1:5 in PBS to a constant content of up to 20% v/v mouse plasma in the sample matrix and incubated at 37°C for 1 hour.
  • a separate standard curve was constructed to determine the plasma level of each protein, which was obtained from a dilution series of purified recombinant OmCI protein with a determined concentration in PBS, also supplemented with up to 20% v/v mouse plasma from untreated animals.
  • the plasma concentration ( ⁇ g/mL) of each sample was calculated using the parameters derived from the standard curve (log-log regression analysis).
  • the pharmacokinetic parameters were calculated using a two-compartment model and drug statistics software (DAS, version 2.0; Chinese Mathematical Pharmacology Committee). The results are listed in Table 7.

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Abstract

La présente invention concerne une protéine modifiée. La protéine modifiée comprend une fraction protéique et une fraction modifiée ; la fraction protéique est un mutant OmCI dans lequel un résidu cystéine est introduit par mutation ; et la fraction modifiée est liée à la fraction protéique au moyen du résidu cystéine introduit dans la fraction protéique. La protéine modifiée selon la présente invention conserve l'activité d'origine de la protéine, et la période de demi-vie in vivo de la protéine est significativement améliorée, ce qui permet de poser une nouvelle fondation de matériau pour le développement de médicaments destinés au traitement de plusieurs maladies.
PCT/CN2024/093492 2023-05-15 2024-05-15 Inhibiteur du complément, son procédé de préparation et son utilisation Pending WO2024235270A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1798841A (zh) * 2003-06-02 2006-07-05 发展技术有限公司 来源于蜱的补体抑制剂
CN102341407A (zh) * 2009-02-05 2012-02-01 自然环境研究会 作为补体抑制剂的经修饰的omci
CN103002918A (zh) * 2010-01-22 2013-03-27 诺沃—诺迪斯克保健股份有限公司 体内功效延长的生长激素

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1798841A (zh) * 2003-06-02 2006-07-05 发展技术有限公司 来源于蜱的补体抑制剂
CN102341407A (zh) * 2009-02-05 2012-02-01 自然环境研究会 作为补体抑制剂的经修饰的omci
CN103002918A (zh) * 2010-01-22 2013-03-27 诺沃—诺迪斯克保健股份有限公司 体内功效延长的生长激素

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JUSTIN M. CHALKER ET AL.: "Chemical Modification of Proteins at Cysteine: Opportunities in Chemistry and Biology", CHEMISTRY – AN ASIAN JOURNAL, vol. 4, 23 February 2009 (2009-02-23), XP072419569, DOI: 10.1002/asia.200800427 *
RAMÍREZ-ANDERSEN HENRIK S., BEHRENS CARSTEN, BUCHARDT JENS, FELS JOHANNES J., FOLKESSON CHARLOTTA G., JIANHE CHEN, NØRSKOV-LAURITS: "Long-Acting Human Growth Hormone Analogue by Noncovalent Albumin Binding", BIOCONJUGATE CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 29, no. 9, 19 September 2018 (2018-09-19), US , pages 3129 - 3143, XP093090893, ISSN: 1043-1802, DOI: 10.1021/acs.bioconjchem.8b00463 *
SHANGGUAN WENWEN, LI XIAOWAN, WANG YANDAN, HUANG ZONGQING, DONG YUANZHEN, FENG MEIQING, FENG JUN: "Design and Biological Evaluation of the Long-Acting C5-Inhibited Ornithodoros moubata Complement Inhibitor (OmCI) Modified with Fatty Acid", BIOCONJUGATE CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 35, no. 5, 15 May 2024 (2024-05-15), US , pages 653 - 664, XP093239322, ISSN: 1043-1802, DOI: 10.1021/acs.bioconjchem.4c00126 *

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