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WO2024174819A1 - Acide aminé non naturel contenant un hétéroaryle et son utilisation - Google Patents

Acide aminé non naturel contenant un hétéroaryle et son utilisation Download PDF

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Publication number
WO2024174819A1
WO2024174819A1 PCT/CN2024/074706 CN2024074706W WO2024174819A1 WO 2024174819 A1 WO2024174819 A1 WO 2024174819A1 CN 2024074706 W CN2024074706 W CN 2024074706W WO 2024174819 A1 WO2024174819 A1 WO 2024174819A1
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Prior art keywords
amino acid
natural amino
alkyl
group
recombinant human
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Ceased
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English (en)
Chinese (zh)
Inventor
王谦
霍鹏超
叶诚浩
杨金纬
刘利
丁文
李静
焦琳
张凤霞
曹锴
靳婷
林欣
应跃斌
祝静静
夏钢
梁学军
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Novocodex Biopharmaceuticals Co Ltd
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Novocodex Biopharmaceuticals Co Ltd
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Publication of WO2024174819A1 publication Critical patent/WO2024174819A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/55—Acids; Esters
    • 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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • 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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/44—Radicals substituted by doubly-bound oxygen, sulfur, or nitrogen atoms, or by two such atoms singly-bound to the same carbon atom
    • C07D213/46—Oxygen atoms
    • C07D213/50—Ketonic radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/56—Amides
    • 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]
    • C07K14/55—IL-2
    • 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
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers

Definitions

  • the present invention relates to the field of biopharmaceuticals, and in particular to a non-natural amino acid containing a heteroaryl group and uses of the non-natural amino acid.
  • non-natural amino acids containing special groups By introducing non-natural amino acids containing special groups into proteins, a variety of scientific research and product development applications can be achieved.
  • photosensitive non-natural amino acids can be introduced into proteins, or non-natural amino acids can be specially labeled to facilitate the study of protein-protein interactions; for example, non-natural amino acids can be used to modify enzymes in a targeted manner to improve enzyme activity and enzyme stability, or to facilitate efficient enzyme immobilization; for example, the characteristics of non-natural amino acid insertion that cannot be achieved in conventional hosts can be used to prepare safe live bacteria or live virus vaccines.
  • Chinese patent ZL 202111019771.1 discloses a non-natural amino acid derived from lysine, which introduces a carbonyl group as an active reactive group at its terminal, and the structure also contains an aromatic group connected to the terminal carbonyl group.
  • non-natural amino acids with terminal azido groups (e.g., Lys-azido)
  • such non-natural amino acids are easier to prepare, safer, less likely to be inactivated when inserted into proteins, have a higher binding rate with the coupling part, and the resulting conjugates are more stable, so they can be used to prepare recombinant proteins, recombinant protein conjugates, etc.
  • Para-acetylphenylalanine is also one of the most common non-natural amino acids (for example, Chinese patent application CN 111212661A). pAF can also be easily inserted into proteins and coupled with other substances (such as polymers, drugs, etc.).
  • One object of the present invention is to provide a non-natural amino acid containing a heteroaryl group and its use.
  • the non-natural amino acid structure introduces a heteroaryl group containing a nitrogen atom, which can increase the protein expression when inserted into a recombinant protein, and when a conjugate is formed, The synthesis efficiency is also higher, so its applicability is greatly improved.
  • Another object of the present invention is to provide a recombinant human interleukin 2-polyethylene glycol conjugate and its use.
  • the non-natural amino acid containing a heteroaryl group provided by the present invention is characterized in that it is a compound having a structure as shown in formula (I) or an enantiomer thereof,
  • ring A represents a substituted or unsubstituted 5- to 10-membered heteroaryl group containing 1, 2 or 3 nitrogen atoms;
  • L represents a substituted or unsubstituted C1-C20 straight or branched alkylene group, wherein one or more -CH 2 - is optionally replaced by one or more of -O-, -NH-, -C(O)-, -S-, -S(O)-;
  • R represents a substituted or unsubstituted C0-C10 straight or branched alkylene group, wherein one or more -CH 2 - is optionally replaced by one or more of -O-, -NH-, and -C(O)-;
  • the substituent is selected from one or more of C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, hydroxyl, mercapto, halogen, nitro, cyano, amino, -NH(C1-C6 alkyl), -N(C1-C6 alkyl) 2 , -CO(C1-C6 alkyl), -COO(C1-C6 alkyl), -CONH 2 , -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl) 2 , C3-C10 cycloalkyl, C3-C10 heterocyclic group, and C6-C20 aryl.
  • C0-Cn includes C0-C1, C0-C2, ... C0-Cn.
  • C0 when C0 is represented, it means that the group does not exist, and the C atoms at both ends are directly connected to form a bond.
  • the "C1-C20” group means that the part has 1 to 20 carbon atoms
  • the "C0-C10” group means that the part has 0 to 10 carbon atoms.
  • the "C0-C6” group means that the part has 0 to 6 carbon atoms, that is, the group does not exist, contains 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms. And so on.
  • non-natural amino acids provided by the present invention include optically pure enantiomers and racemates.
  • the non-natural amino acid is a compound having a structure as shown in formula (I-1) or formula (I-2) or an enantiomer thereof,
  • rings A and R are as defined in any of the above technical solutions
  • X represents a substituted or unsubstituted C0-C10 straight or branched alkylene group, wherein one or more -CH 2 - is optionally replaced by one or more of -O-, -NH-, and -C(O)-;
  • the substituent is selected from one or more of C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, hydroxyl, mercapto, halogen, nitro, cyano, amino, -NH(C1-C6 alkyl), -N(C1-C6 alkyl) 2 , -CO(C1-C6 alkyl), -COO(C1-C6 alkyl), -CONH 2 , -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl) 2 , C3-C10 cycloalkyl, C3-C10 heterocyclic, and C6-C20 aryl.
  • the ring A represents a 5- to 6-membered heteroaryl group containing 1 or 2 nitrogen atoms, including but not limited to pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl and the like.
  • the ring A represents a pyridyl group.
  • the R and X each independently represent a C0-C6 straight chain or branched alkylene group, wherein one or more -CH 2 - groups are optionally replaced by -O-.
  • the non-natural amino acid is a compound having a structure as shown in formula (I-3) or formula (I-4) or an enantiomer thereof,
  • rings A and X are defined as in any one of the aforementioned technical solutions.
  • the substituent is selected from one or more of halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, including but not limited to F, Cl, Br, I, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy, trifluoromethyl, and the like.
  • the non-natural amino acid is a non-natural amino acid having the formula (I- 5), a compound represented by the structure of formula (I-6) or formula (I-7) or an enantiomer thereof,
  • Y represents a C0-C6 straight chain or branched alkylene group
  • R' represents one or more of hydrogen, halogen, C1-C4 alkyl, C1-C4 alkoxy, and C1-C4 haloalkyl;
  • n 0, 1, 2 or 3.
  • Y represents a C0-C4 straight-chain alkylene group.
  • the non-natural amino acid is a compound having one of the following structures:
  • the present invention also provides the use of the non-natural amino acid described in any one of the above technical solutions in preparing a recombinant protein or a recombinant protein conjugate.
  • the recombinant protein can be a recombinant protein obtained by inserting the non-natural amino acid described in any of the above technical solutions into any type of protein commonly used in the art at any site and in any number.
  • the recombinant protein conjugate can be a conjugate obtained by coupling any recombinant protein obtained with a coupling part commonly used in the art, wherein the coupling part includes but is not limited to polymers (e.g., polyethylene glycol of any molecular weight), proteins, polypeptides, small molecule drugs, etc.
  • the recombinant protein may be recombinant human interleukin 2, and/or the recombinant protein conjugate may be a recombinant human interleukin 2-polyethylene glycol conjugate.
  • the recombinant protein may be recombinant human growth hormone, and/or the recombinant protein conjugate may be a recombinant human growth hormone-polyethylene glycol conjugate.
  • the present invention also provides a recombinant protein, wherein at least one site in the amino acid sequence of the recombinant protein is a non-natural amino acid as described in any one of the above technical solutions.
  • the recombinant protein may have a structure as shown in formula (II),
  • D represents the residue of the non-natural amino acid described in any one of the above technical solutions minus the aminocarboxylic acid part
  • P1 and P2 represent the connecting part of the amino group and carboxyl group of the non-natural amino acid in the amino acid sequence, respectively.
  • the recombinant protein provided by the present invention can be prepared using common preparation methods in the art, including using gene codon expansion technology to achieve cloning and expression of recombinant proteins containing non-natural amino acids.
  • the recombinant protein provided by the present invention can be a recombinant protein obtained by inserting the non-natural amino acid described in any of the above technical solutions into any type of protein commonly used in the art at any site and in any number, such as recombinant human growth hormone, recombinant human interleukin 2, etc.
  • the present invention also provides a recombinant protein conjugate, wherein the conjugate is formed by forming an oxime bond between the terminal carbonyl group of the non-natural amino acid in the recombinant protein described in any one of the above technical solutions and the coupling part containing the "NH 2 -O-" terminal group.
  • the recombinant protein conjugate may have a structure as shown in formula (III),
  • D' represents the residue of the recombinant protein as described in any of the above technical solutions minus the terminal carbonyl part of the non-natural amino acid
  • D" represents the residue of the coupling part minus the " NH2 -O-" terminal group.
  • the coupling part may include a polymer (e.g., polyethylene glycol of any molecular weight), a protein, a polypeptide, a small molecule drug, etc.
  • a polymer e.g., polyethylene glycol of any molecular weight
  • Different types of coupling parts may be coupled to the recombinant protein alone, or different types of coupling parts may be connected first and then coupled to the recombinant protein.
  • the recombinant protein may be recombinant human growth hormone, recombinant human interleukin 2, etc.
  • the coupling part containing an "NH 2 -O-" terminal group may be polyethylene glycol containing an "NH 2 -O-" terminal group.
  • the polyethylene glycol containing "NH 2 -O-" terminal groups has the following structural formula:
  • the molecular weight of the polyethylene glycol containing " NH2 -O-" terminal groups may be 10-100KD, including but not limited to about 10KD, 20KD, 30KD, 40KD, 50KD, 60KD, 70KD, 80KD, 90KD, 100KD, etc. or any combination of molecular weight intervals.
  • the molecular weight of the polyethylene glycol containing " NH2 -O-" terminal groups may be 20-50KD.
  • the present invention also provides a recombinant human interleukin 2-polyethylene glycol conjugate, which comprises recombinant human interleukin 2 containing at least one non-natural amino acid and PEG coupled to the at least one non-natural amino acid, wherein the non-natural amino acid is the non-natural amino acid as described in any of the aforementioned technical solutions, and the PEG is coupled to the at least one non-natural amino acid by forming an oxime bond between the terminal carbonyl group of the non-natural amino acid and the PEG containing an " NH2 -O-" terminal group.
  • the recombinant human interleukin 2 is the protein shown in SEQ ID NO: 3 or a functionally active fragment thereof.
  • the position of at least one non-natural amino acid is selected from one or more sites corresponding to P34, K35, T37, R38, L40, T41, F42, K43, F44, Y45, E61, E62, K64, P65, E67, E68, N71, L72 and Y107 of SEQ ID NO:2.
  • the molecular weight of the PEG containing an "NH 2 -O-" terminal group is 20 to 50 KD.
  • the preparation and purification methods of the recombinant human interleukin 2-polyethylene glycol conjugate provided by the present invention can refer to Chinese patent The method recorded in ZL 202111019771.1.
  • the present invention also provides the use of the recombinant human interleukin-2-polyethylene glycol conjugate as described in any one of the aforementioned technical solutions as a medicine (i.e., for treatment).
  • the present invention also provides use of the recombinant human interleukin-2-polyethylene glycol conjugate as described in any one of the aforementioned technical solutions in the preparation of a drug for promoting the immune function of interleukin-2.
  • the present invention also provides use of the recombinant human interleukin 2-polyethylene glycol conjugate as described in any one of the aforementioned technical solutions in the preparation of a drug for treating cancer.
  • the cancer includes but is not limited to renal cell carcinoma, liver cancer, colorectal cancer, lymphoma, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, etc.
  • the present invention also provides use of the recombinant human interleukin 2-polyethylene glycol conjugate as described in any one of the aforementioned technical solutions in the preparation of a drug for expanding CD8 + T cells.
  • the present invention also provides a method for promoting the immune function of interleukin 2, comprising: administering an effective amount of the recombinant human interleukin 2-polyethylene glycol conjugate as described in any of the aforementioned technical solutions to an individual in need.
  • the present invention also provides a method for treating cancer, comprising: administering an effective amount of the recombinant human interleukin-2-polyethylene glycol conjugate as described in any of the aforementioned technical solutions to an individual in need.
  • the cancer includes but is not limited to renal cell carcinoma, liver cancer, colorectal cancer, lymphoma, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, etc.
  • the present invention also provides a method for expanding CD8 + T cells, comprising: administering an effective amount of the recombinant human interleukin 2-polyethylene glycol conjugate as described in any of the aforementioned technical solutions to an individual in need.
  • the non-natural amino acids provided by the present invention introduce terminal carbonyl groups and nitrogen-containing heteroaryl groups connected thereto into their structures.
  • the terminal carbonyl groups can easily form conjugates, and the heteroaryl groups can further enhance the stability of the resulting conjugates.
  • the non-natural amino acids provided by the present invention have higher protein expression levels when preparing recombinant proteins and higher synthesis efficiency when forming conjugates.
  • the non-natural amino acids provided by the present invention also have higher solubility and are easier to operate during fermentation preparation and feeding. Therefore, the non-natural amino acids provided by the present invention have better applicability.
  • the non-natural amino acids provided by the present invention expand the types of amino acids, so that they can be used as amino acid derivatives in many fields, especially in the preparation of recombinant proteins or recombinant protein conjugates, and also expand the types and application scope of recombinant proteins or recombinant protein conjugates.
  • the recombinant human interleukin 2-polyethylene glycol conjugate provided by the present invention achieves precise site-specific coupling of PEG and IL-2 by using the non-natural amino acid containing heteroaryl groups of the present invention, thereby achieving an extension of the half-life of IL-2 in vivo.
  • the recombinant human interleukin 2-polyethylene glycol conjugate provided by the present invention can reduce the binding activity of IL-2R ⁇ and keep the binding activity of IL-2R ⁇ and IL-2R ⁇ relatively unchanged, so it can specifically promote the proliferation of CD8 + T cells in the tumor microenvironment, but has no obvious effect on the proliferation of CD4 + T cells, thereby facilitating the immunotherapy of tumors.
  • FIG. 1 is a schematic diagram of the expression plasmid pET21-rhIL2T41 in Example 4.
  • FIG. 2 is an SDS-PAGE electrophoresis diagram of the fermentation products obtained by adding different types of unnatural amino acids to the rhIL-2 expression strain in Example 5.
  • FIG. 3A is the RP-HPLC spectrum of PEG-rhIL2-pAF5 and PEG-rhIL2-pAF5 in Example 6.
  • FIG. 3B is the RP-HPLC spectrum of PEG-rhIL2-NBOK and PEG-rhIL2-NBOK3 in Example 6.
  • FIG. 3C is the RP-HPLC spectrum of PEG-rhIL2-NPAK and PEG-rhIL2-NPAK2 in Example 6.
  • the reagents or raw materials used in the examples of the present invention are all commercially available products; the experimental methods used are all conventional methods in the art unless otherwise specified.
  • non-natural amino acids NBOK and NPAK used in the embodiments of the present invention have the following structural formulas, respectively, and are prepared with reference to Chinese Patent ZL 202110863816.7.
  • NPAK2 The structural formula of NPAK2 is shown below:
  • auxiliary plasmids The auxiliary plasmids pEVOL-pAcFRS.2.t1 (Cat. No. 73544) and pSupAR-MbPylRS (Cat. No. 91705) are from the plasmid collection organization Addgene.
  • pEVOL-pAcFRS.2.t1 can express tRNA and tRNA synthetase that specifically recognize p-Acetylphenylalanine (pAF) and pAF5 in Escherichia coli;
  • pSupAR-MbPylRS can express tRNA and tRNA synthetase that specifically recognize NPAK, NBOK, NPAK2 and NBOK3 in Escherichia coli.
  • the auxiliary plasmids were extracted after shaking flask culture in LB medium supplemented with 37.5 mg/L chloramphenicol.
  • the cysteine codon at position 125 was mutated to a serine codon (the cysteine at position 125 does not participate in the formation of disulfide bonds, but interferes with the normal formation of disulfide bonds during the renaturation of the protein inclusion body of recombinant human IL-2.
  • the mutation can improve the efficiency of renaturation without significantly affecting its activity).
  • methionine Met needs to be added to the N-terminus of the protein sequence to start the translation of the protein, so that a mature recombinant protein is obtained.
  • the protein sequence of human IL-2 is SEQ ID NO:3.
  • the codon for Threonine at position 41 of SEQ ID NO:3 was changed to an amber codon (TAG) for inserting non-natural amino acids, and finally the amino acid sequence of recombinant human IL-2 was obtained (X represents non-natural amino acids, SEQ ID NO:4).
  • TAG amber codon
  • X represents non-natural amino acids, SEQ ID NO:4
  • the complete DNA sequence was then synthesized by total gene synthesis to obtain the gene sequence of recombinant human IL-2 (SEQ ID NO:5).
  • the gene sequence of recombinant human IL-2 (SEQ ID NO: 5) was cloned between the two restriction sites of Nde I and Xho I of pET21b (Novagen, catalog number #69741-3) to obtain the expression plasmid pET21-rhIL2T41, which was verified by sequencing to be consistent with the expected sequence.
  • pET21-rhIL2T41 can be used to express recombinant human IL-2 in which the 41st amino acid codon is replaced with an amber codon.
  • the map of the recombinant human IL-2 expression plasmid pET21-rhIL2T41 is shown in Figure 1.
  • auxiliary plasmid pSupAR-MbPylRS and expression plasmid pET21-rhIL2T41 were co-transformed into Escherichia coli OrigamiB (DE3) (Novagen, catalog number #70911-3) competent cells, and LB medium containing 100 mg/L ampicillin and 37.5 mg/L chloramphenicol was used to screen to obtain a double-resistant strain, which is the expression strain of recombinant human IL2, named "IL2 (NPAK/NBOK/NPAK-2/NBOK-3)-OB".
  • pAF and NPAK were dissolved in 2 ⁇ YT medium to prepare a mother solution with a final concentration of 80 mM (due to solubility, it is difficult to reach a higher concentration), and sterilized at high temperature for use;
  • NBOK was dissolved in 2 ⁇ YT medium after heating to prepare a mother solution with a final concentration of 10 mM (due to solubility, it is difficult to reach a higher concentration), and sterilized at high temperature for use.
  • the IL2(pAF/pAF5)-OB expression strain obtained in Example 4 was inoculated into three 2 ⁇ YT medium (10 g/L yeast extract, 16 g/L tryptone, 5 g/L NaCl, containing 100 mg/L ampicillin and 37.5 mg/L chloramphenicol), and cultured at 37°C until the bacterial solution OD 600 was 2.0 ⁇ 0.2, and IPTG (final concentration of 1 mM) and arabinose (final concentration of 0.2%) were added to the three bacterial solutions respectively.
  • the corresponding unnatural amino acids pAF and pAF5 final concentrations of both were 1 mM were added to two of them, and the remaining IL2-OB bacterial solution was used as a negative control without the addition of unnatural amino acids.
  • Example 3 The IL2(NPAK/NBOK/NPAK2/NBOK3)-OB expression strain obtained in Example 4 was inoculated into 5 portions of 2 ⁇ YT medium (10 g/L yeast extract, 16 g/L tryptone, 5 g/L NaCl, containing 100 mg/L ampicillin and 37.5 mg/L chloramphenicol), and cultured at 37°C until the bacterial solution OD 600 was 2.0 ⁇ 0.2, and then IPTG (final concentration of 1 mM) and arabinose (final concentration of 0.2%) were added to the 5 bacterial solutions respectively.
  • 2 ⁇ YT medium 10 g/L yeast extract, 16 g/L tryptone, 5 g/L NaCl, containing 100 mg/L ampicillin and 37.5 mg/L chloramphenicol
  • IL2-OB bacterial solution was used as a negative control without the addition of non-natural amino acids, and the other 4 portions were added with the corresponding non-natural amino acids NPAK, NBOK, NPAK2 and NBOK3 (final concentrations of 1 mM each).
  • the results in Figure 2 show that the expression strain can express the target protein when 6 non-natural amino acids are added respectively, among which the expression level of IL-2 containing pAF5 and NPAK2 is about 3g/L, and the expression level of IL-2 containing NBOK3 is about 2.5g/L; correspondingly, the expression level of IL-2 containing pAF and NPAK is about 1g/L, and the expression level of IL-2 containing NBOK is about 1.5g/L. It can be seen that the expression levels of IL-2 containing non-natural amino acids of pyridine ring are all The expression level is significantly greater than that of IL-2 containing a non-natural amino acid with a benzene ring.
  • the collected cells were resuspended in buffer (25mM Tris, 6mM EDTA, 1mM DTT, pH8.0), 1% DNA enzyme (1mg/mL) was added, mixed evenly, and homogenized 3 times with an ultra-high pressure homogenizer at a pressure of 50-80MPa.
  • the homogenate was centrifuged at 10000rpm for 20min, and the lower layer of inclusion bodies was collected.
  • the obtained inclusion bodies were washed twice with washing buffer (20mM Tris-HCl, 100mM NaCl, 2% TritonX-100, pH8.0), and then washed once with ultrapure water to obtain purified inclusion bodies.
  • the purified inclusion bodies were dissolved with denaturing buffer (20mM Tris-HCl, 100mM NaCl, 6M guanidine hydrochloride, 1mM DTT, pH8.0), centrifuged at 10000rpm after 30min, and the supernatant was collected as the denatured protein solution.
  • denaturing buffer (20mM Tris-HCl, 100mM NaCl, 6M guanidine hydrochloride, 1mM DTT, pH8.0
  • refolding buffer 20mM Tris-HCl, 100mM NaCl, pH8.0
  • the site-directed coupling of PEG to rhIL-2 with site-directed mutations inserted into pAF5, NBOK3, and NPAK2 is shown in synthetic routes AC (wherein the direction from P1 to P2 is the direction from the N-terminus to the C-terminus of the amino acid sequence).
  • the site-directed coupling of PEG to rhIL-2 with site-directed mutations inserted into pAF, NBOK, and NPAK is shown in synthetic routes DF (wherein the direction from P1 to P2 is the direction from the N-terminus to the C-terminus of the amino acid sequence).
  • the coupling reaction operation is as follows: before the coupling reaction, the target protein obtained above is adjusted to 2.0 mg/ml with a citric acid buffer of pH 4.0 and 20 mM, and 30KD aminooxy PEG solid (purchased from Beijing Jiankai Technology Co., Ltd.) is added according to 1:15 (molar ratio, protein: aminooxy PEG), and fully shaken to dissolve to obtain a clear and transparent solution, and then the reaction solution is sealed and shaken in a constant temperature shaker (25°C, 100 rpm) for reaction. After 48 hours, the coupling situation is analyzed by RP-HPLC, as shown in Figures 3A-3C.
  • the target protein components were collected to obtain a target protein sample with a purity of approximately 95%.
  • This method uses two cell lines, mouse CTLL-2 cells are cell lines containing IL-2Ra ⁇ , and human YT cells are cell lines containing IL-2R ⁇ .
  • PEG-rhIL2 activates the JAK-STAT signaling pathway by binding to IL-2R on the cell surface.
  • mice CTLL-2 cells purchased from American Type Culture Collection
  • human YT cells were cultured with their respective culture media
  • CTLL-2 cell culture medium RPMI 1640 + 10% FBS + 400IU / mL rhIL-2, 2mM L-glutamine, 1mM sodium pyruvate
  • YT cell culture medium RPMI 1 640 + 10% FBS + 1mM Non-Essential Amino Acids Solution (purchased from Gibco, product catalog number 11140050)) at 37 ° C, 5% carbon dioxide to a sufficient amount, starved for 4 hours before detection, and then adjusted to a cell density of 1 ⁇ 10 6 cells / mL for use.
  • the calculation formula is: [EC50 of YT cells of 30KD PEG-rhIL2 sample/EC50 of CTLL-2 cells of 30KD PEG-rhIL-2 sample]/[EC50 of YT cells of reference product/EC50 of CTLL-2 cells of reference product] ⁇ 100%

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Abstract

L'invention concerne un acide aminé non naturel contenant un hétéroaryle, qui est un composé ayant une structure telle que représentée par la formule (I), ou un énantiomère de celui-ci. Le niveau d'expression de protéine est plus élevé lorsque l'acide aminé non naturel contenant un hétéroaryle est utilisé pour préparer une protéine recombinante, et l'efficacité de synthèse est également plus élevée lorsque l'acide aminé non naturel contenant un hétéroaryle est utilisé pour former un conjugué. Par conséquent, l'acide aminé non naturel contenant un hétéroaryle a une bonne applicabilité, et a un procédé de préparation simple et pratique, ne nécessite pas de coûts élevés, et est ainsi approprié pour une production à grande échelle. Le conjugué d'interleukine 2-polyéthylène glycol humain recombinant selon l'invention réalise un couplage précis orienté sur un site de PEG avec l'IL-2 au moyen de l'acide aminé non naturel, ce qui permet de prolonger la demi-vie d'IL-2 in vivo et de faciliter l'immunothérapie de tumeurs.
PCT/CN2024/074706 2023-02-20 2024-01-30 Acide aminé non naturel contenant un hétéroaryle et son utilisation Ceased WO2024174819A1 (fr)

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