WO2024046280A1 - Dérivé d'il-21 modifié par du polyéthylène glycol et son utilisation - Google Patents

Dérivé d'il-21 modifié par du polyéthylène glycol et son utilisation Download PDF

Info

Publication number
WO2024046280A1
WO2024046280A1 PCT/CN2023/115300 CN2023115300W WO2024046280A1 WO 2024046280 A1 WO2024046280 A1 WO 2024046280A1 CN 2023115300 W CN2023115300 W CN 2023115300W WO 2024046280 A1 WO2024046280 A1 WO 2024046280A1
Authority
WO
WIPO (PCT)
Prior art keywords
mutant
amino acid
polyethylene glycol
peg
acid sequence
Prior art date
Application number
PCT/CN2023/115300
Other languages
English (en)
Chinese (zh)
Inventor
周家杰
张琪
王礼翠
张天赋
赵艳宇
谭伟
王国永
王雅媛
张秋磊
张建军
刘懿
金秀丽
黄英姿
高翔
张维
Original Assignee
北京志道生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京志道生物科技有限公司 filed Critical 北京志道生物科技有限公司
Publication of WO2024046280A1 publication Critical patent/WO2024046280A1/fr

Links

Classifications

    • 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]
    • 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]
    • 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
    • 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
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of protein engineering, and specifically relates to a polyethylene glycol-modified IL-21 derivative and its application.
  • Interleukin-21 is a member of the IL-2 family. It is a multifunctional cytokine that has a variety of biological effects on lymphocytes and bone marrow cells. It plays a role in natural immunity and acquired immunity. play an important role in immunity. IL-21 is mainly secreted by activated CD4+T cells, NK cells, Tfh cells, and Th17 cells, and can improve the antigen-specific response of immune cells. IL-21 has multiple effects on T cells, including promoting cell proliferation and enhancing cytotoxicity. IL-21 can induce the proliferation and maturation of CD8+ T cells and NK cells and enhance their cytotoxicity, thereby enhancing the body's immune response. IL-21 carries out immune responses by regulating the proliferation and differentiation of B cells into plasma cells 1 . IL-21 can also promote memory CD8+ T cell differentiation and suppress Treg cells2 .
  • Human IL-21 contains 134 amino acids and forms four ⁇ -helical bundles in structure. There are four cysteine residues (C42, C49, C93 and C96) in IL-21, of which cysteine at positions 42 and 93 forms a disulfide bond, and cysteine at positions 49 and 96 Acids form disulfide bonds 3 .
  • IL-21 receptor is a heterodimer composed of IL-21's unique subunit IL-21R and ⁇ chain, where IL-21R is the ligand recognition and binding site and ⁇ chain is the signal transduction unit. IL-21R is widely expressed on the surface of T cells, B cells, NK cells, dendritic cells, and monocytes/macrophages. IL-21 has a high affinity to IL-21R, up to 70pM.
  • IL-21 After IL-21 binds to its heterodimeric receptor, it mainly conducts signal transduction through the protein tyrosine kinase JAK/signal transduction and transcription agonist SATA pathway, activates JAK (JAK1 and JAK3), and subsequently phosphorylates STAT1, STAT3, STAT4 and STAT5 finally enter the cell nucleus to regulate the expression of corresponding genes 4 .
  • the gamma receptor is a universal receptor for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21.
  • IL-21 has a low affinity for gamma receptors (160 ⁇ M), However, the interaction between IL-21 and ⁇ receptors is necessary for signal transduction 5 .
  • IL-2 interleukin proteins
  • High- dose IL-2 has shown good results in the treatment of melanoma and renal cell carcinoma6,7.
  • high doses of IL-2 can cause strong side effects, such as vascular leak syndrome (VLS), which causes water accumulation in human organs, such as pulmonary edema and liver cell damage.
  • VLS vascular leak syndrome
  • studies have found that low-dose IL-2 can induce regulatory T cells Tregs, suppress immune responses, and promote tumor escape8 . Therefore, low-dose IL-2 cannot be used to treat tumors, and the clinical application prospects of IL-2 are greatly limited by dosage and toxicity issues.
  • IL-21 as an emerging anti-tumor cytokine, has attracted more and more attention because of its significantly lower toxicity than IL-2. At the same time, low-dose IL-21 does not induce regulatory T cells Tregs and still maintains the activation effect on immune cells. Recombinant human IL-21 has also shown good anti-tumor effects in clinical trials of melanoma and renal cell carcinoma9. In mouse models of colon cancer and breast cancer, low doses of IL-21 can induce good anti-tumor effects10,11 . Although IL-21 has low toxic and side effects on the body, clinical experiments show that the maximum tolerated dose of human IL-21 is 30 ⁇ g/kg. As the dosage of IL-21 increases, toxic side effects begin to appear. The most common adverse reactions of IL-21 include headache, fatigue, fever, nausea, myalgia/arthralgia, rash, diarrhea, thrombocytopenia, etc.12,13 .
  • the short half-life and poor stability of IL-21 also greatly limit the development of IL-21 as an anti-tumor drug 8 .
  • the plasma half-life of wild-type IL-21 is short, only 0.2 hours in mice, 0.4-0.8 hours in cynomolgus monkeys, and approximately 2 hours in humans 10 . Therefore, it is of great significance to design a reliable, long-acting and stable IL-21 for the development of anti-tumor drugs.
  • the present invention analyzes the structure of IL-21 and mutates specific sites in IL-21 into cysteine without affecting the overall structure of IL-21. acid, and then connect the appropriate PEG molecule to the mutated cysteine on IL-21 through sulfhydryl coupling.
  • Plasma half-life of IL-21 after PEG modification The treatment period is prolonged and has excellent dose-dependent anti-tumor efficacy.
  • PEG-modified IL-21 has better water solubility, which is beneficial to drug formulations.
  • the IL-21 skeleton protein used for PEG modification in the present invention is expressed in E. coli and obtained through renaturation and purification, and has low Industrialization advantages of low cost and high productivity.
  • the invention provides an IL-21 mutant, the IL-21 mutant is selected from any one of the following:
  • the IL-21 mutant has 95%, preferably 98%, more preferably 99% sequence identity with the amino acid sequence described in (a), and has the function of the protein described in (a), wherein corresponds to The amino residues at positions N25, K52, K56, N59, G61, N82, and S98 of the amino acid sequence shown in SEQ ID NO:1 are the same as those in the amino acid sequence described in (a);
  • the IL-21 mutant consists of adding or deleting 1-30 elements at the C-terminus and/or N-terminus of the amino acid sequence described in (a), more preferably 1-10 elements, and still more preferably 1-6 elements , most preferably formed of 1-3 amino acid residues, and having the function of the IL-21 mutant described in (a), wherein N25, K52, K56, N59, G61 corresponding to the amino acid sequence shown in SEQ ID NO:1 , N82 and S98 are the same as those in the amino acid sequence described in (a).
  • amino acid sequence of the IL-21 mutant (a) is mutated from the sequence shown in SEQ ID NO: 1, and an amino acid residue selected from the following group is mutated to cysteine Acid C: N25, K56, N59, G61, N82, S98.
  • amino acid sequence of the IL-21 mutant (a) is mutated from the sequence shown in SEQ ID NO: 1, and an amino acid residue selected from the following group is mutated to cysteine Acid C: N59, G61, N82, S98.
  • amino acid sequence of the IL-21 mutant (a) is mutated from the sequence shown in SEQ ID NO: 1, and an amino acid residue selected from the following group is mutated to cysteine Acid C: N59, N82, S98.
  • the invention provides a nucleotide sequence encoding the IL-21 mutant of the first aspect of the invention.
  • the present invention provides an expression vector comprising the nucleoside described in the second aspect of the present invention. acid sequence.
  • the present invention provides a host cell, which contains the expression vector of the third aspect of the present invention or the nucleotide sequence of the second aspect of the present invention integrated into its genome.
  • the host cell is a bacterium; more preferably, the host cell is E. coli, Corynebacterium glutamicum; most preferably, the host cell is Escherichia coli (E.coli).
  • the present invention provides a polyethylene glycol derivative of the IL-21 mutant described in the first aspect, wherein the polyethylene glycol modifier is connected to the cysteine at the mutation site of the IL-21 mutant. On the amino acid.
  • the polyethylene glycol modifier is a mercapto polyethylene glycol modifier, which is a polyol derivative having a functional group selected from vinyl sulfone, maleimide or iodoacetamide.
  • the molecular weight of the polyethylene glycol modifier is 20 kDa.
  • the present invention provides a method for preparing a polyethylene glycol derivative of the IL-21 mutant described in the fifth aspect, comprising the following steps: using a thiol-reactive polyol agent and the polyethylene glycol derivative described in the first aspect. Reactions to IL-21 mutants having a single free cysteine residue, the polyol reagent and the single free cysteine residue specificity of the IL-21 mutant Covalently combine to form a covalent thioether bond to obtain a polyol-IL-21 mutant conjugate, and then recover and purify the target polyol-IL-21 mutant conjugate to obtain.
  • the polyethylene glycol portion of the polyethylene glycol derivative of the IL-21 mutant has a molecular weight between 10 and 40 kDa.
  • the polyethylene glycol portion of the polyethylene glycol derivative of the IL-21 mutant has a molecular weight of 20 kDa.
  • the present invention provides a pharmaceutical composition comprising the IL-21 mutant described in the first aspect, or a polyethylene glycol derivative of the IL-21 mutant described in the fifth aspect. , and pharmaceutically acceptable carriers or excipients.
  • the present invention provides the IL-21 mutant described in the first aspect, or the polyethylene glycol derivative of the IL-21 mutant described in the fifth aspect, or the pharmaceutical composition described in the seventh aspect.
  • the present invention provides the IL-21 mutant described in the first aspect, or the polyethylene glycol derivative of the IL-21 mutant described in the fifth aspect, or the pharmaceutical composition described in the seventh aspect. Application in preparing preparations that promote B cell differentiation and proliferation, T cell differentiation and proliferation, and NK cell differentiation and proliferation.
  • the present invention provides the IL-21 mutant described in the first aspect, or the polyethylene glycol derivative of the IL-21 mutant described in the fifth aspect, or the pharmaceutical composition described in the seventh aspect. Use in the preparation of drugs for the prevention and/or treatment of tumors.
  • All amino acid sequence numbers in this article are based on the natural IL-21 (SEQ ID NO: 1) sequence. The first amino acid number is 1, and so on. Since all proteins in the present invention are expressed using Escherichia coli, all proteins in the present invention contain the starting amino acid methionine at the -1 position, which does not affect the activity of IL-21, among which only the -1 position of natural IL-21 IL-21 with the starting amino acid methionine added is defined herein as wild-type IL-21 (wt-IL-21).
  • the present invention successfully found seven sites and mutated them into cysteine. These seven sites are far away from the interaction interface between IL-21 and IL-21R. The mutation will not significantly change the structure of IL-21, nor will it affect the interaction between IL-21 and IL-21R. PEG modification was further performed on the cysteine introduced by point mutation through chemical coupling. PEG-modified IL-21 mutants reduce glomerular permeability as their molecular weight increases, thereby prolonging plasma half-life. On the other hand, PEG molecules will prevent proteases from approaching the IL-21 mutant, preventing the IL-21 mutant from being hydrolyzed by proteases and thus prolonging the plasma half-life of IL-21.
  • PEG-modified IL-21 mutants have better water solubility, which is beneficial to drug formulations.
  • the IL-21 mutant skeleton protein used for PEG modification in the present invention is expressed in E. coli and purified through denaturation and renaturation. It has the industrialization advantages of low cost and high yield.
  • the PEG-modified IL-21 mutant molecule has mild efficacy and maintains low toxicity. It still does not show obvious toxic and side effects at a dosage of up to 18 mg/kg, and has high drug safety.
  • Interleukin-21 refers to any natural IL-21 from any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats).
  • the term encompasses unprocessed IL-21 as well as any form of IL-21 derived from processing in the cell.
  • the term also encompasses naturally occurring IL-21 variants, such as splice variants or allelic variants.
  • the amino acid sequence of an exemplary natural human IL-21 is shown in SEQ ID NO: 1.
  • IL-21 mutant As used herein, the terms "IL-21 mutant”, “IL-21 derivative”, “IL-21 derivative protein” and “modified IL-21 molecule” are used interchangeably and refer to the first A kind of IL-21 mutant described in aspect, compared with natural IL-21SEQ ID NO:1, the IL-21 mutant is at the 25th and 52nd positions of the amino acid sequence shown in SEQ ID NO:1 , one of the amino acids at positions 56, 59, 61, 82, and 98 is mutated to cysteine.
  • “Derivatives” are intended to be construed broadly to include any IL-21 related product. Including but not limited to human and non-human IL-21 homologs, fragments or truncations, fusion proteins (such as fusion with signal peptide or other active or inactive components, active components such as antibodies or antigen-binding fragments thereof), modifications forms (e.g. PEGylated, glycosylated, albumin Conjugation/fusion, Fc conjugation/fusion, hydroxyethylation, etc.) and conservatively modified proteins, etc.
  • fusion proteins such as fusion with signal peptide or other active or inactive components, active components such as antibodies or antigen-binding fragments thereof
  • modifications forms e.g. PEGylated, glycosylated, albumin Conjugation/fusion, Fc conjugation/fusion, hydroxyethylation, etc.
  • amino acid mutation is meant to encompass amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions, and modifications can be made to achieve the final construct, so long as the final construct possesses the desired properties.
  • Preferred amino acid mutations are amino acid substitutions, ie the replacement of one amino acid with another amino acid having different structural and/or chemical properties.
  • Preferred amino acid substitutions include hydrophilic charged or uncharged amino acids, such as Ser, Thr, Ala, Gly, Glu, Arg, His and Lys.
  • Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc.
  • reference to an amino acid substitution such as "N82C” refers to the position number (82) of the original residue asparagine (N), followed by the substituted residue cysteine (C ).
  • the molecular weight of polyethylene glycol was not included when calculating the concentration and mass of the polyethylene glycol derivative of the IL-21 mutant. Only the molecular weight of the protein part of the IL-21 mutant was calculated.
  • preparation of IL-21 mutants of the invention can be accomplished by the procedures disclosed herein and by recognized recombinant DNA techniques, including, for example, polymerase chain reaction (PCR), preparation of plasmid DNA, Cleavage of DNA with restriction enzymes, preparation of oligonucleotides, ligation of DNA, isolation of mRNA, introduction of DNA into appropriate cells, transformation or transfection of hosts, and culture of hosts.
  • fusion molecules can be isolated and purified using chaotropes and well-known electrophoresis, centrifugation and chromatography methods. Disclosures regarding these methods are generally found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. (1989); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989)).
  • Genes encoding variant proteins of the invention include restriction enzyme digestion and ligation as the basic steps for generating DNA encoding the desired fusion.
  • the ends of the DNA fragments may need to be modified before ligation, and this can be accomplished by filling in overhangs, deleting the end portions of the fragments with nucleases (eg, ExoIII), site-directed mutagenesis, or adding new base pairs by PCR.
  • nucleases eg, ExoIII
  • Expression constructs are typically assembled in stages using several rounds of restriction enzyme digestion, ligation, and E. coli transformation.
  • Many cloning vectors suitable for construction of expression constructs are known in the art (lambda.ZAP, Agilent; pET, EMD Millipore) and the specific choice is not critical to the invention.
  • the choice of cloning vector will be influenced by the gene transfer system chosen to introduce the expression construct into the host cell. At the end of each stage, all results can be analyzed by restriction enzyme digestion, DNA sequencing, hybridization, and PCR analysis. obtained construct.
  • Site-directed mutagenesis is generally used to introduce specific mutations into the gene encoding the IL-21 mutants of the invention by methods known in the art. See, e.g., U.S. Patent Application Publication 2004/0171154; Storici et al., 2001, Nature Biotechnology 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet .Newslett.43:15-16. Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available for preparing variants of the invention.
  • promoters transcription initiation regulatory regions
  • the selection of a suitable promoter depends on the proposed expression host. Promoters from heterologous sources can be used as long as they are functional in the host of choice.
  • IL-21 mutants can be expressed in E. coli without a signal peptide sequence, and the protein is recovered from inclusion bodies and refolded into an active form.
  • vector or "expression vector” is synonymous with "expression construct” and refers to a DNA molecule used to introduce a specific gene in operative association with it and direct its expression in a target cell.
  • the term includes vectors that are autonomously replicating nucleic acid structures as well as vectors that are incorporated into the genome of the host cell into which they are introduced.
  • the expression vector of the present invention contains an expression cassette.
  • the expression cassette allows for the transcription of large amounts of stable mRNA. Once the expression vector is in the target cell, the gene-encoded ribonucleic acid molecule or protein is produced by the cell's transcription and/or translation systems.
  • the expression vector of the invention comprises an expression cassette comprising a polynucleotide sequence encoding an IL-21 derivative of the invention.
  • transformation herein has the meaning generally understood by those skilled in the art, that is, the process of introducing exogenous DNA into a host.
  • the transformation method includes any method of introducing nucleic acid into cells, including but not limited to electroporation, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol alcohol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which includes the initially transformed cell and progeny derived therefrom (regardless of passage number).
  • the offspring may not be identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected in the original transformed cell are included herein.
  • the host cells herein can be cultured according to conventional methods in the field, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture, fed-batch culture, etc., and each method can be appropriately adjusted according to the actual situation.
  • Various culture conditions such as temperature, time and pH value of the culture medium.
  • IL-21 receptor means that the IL-21 receptor is a subunit unique to IL-21 IL- A heterodimer composed of 21R and ⁇ chain, in which IL-21R is the ligand recognition and binding site and ⁇ chain is the signal transduction unit.
  • Binding affinity refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, a receptor) and its binding partner (eg, a ligand). Unless otherwise indicated, as used herein, "binding affinity” refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, a receptor and a ligand). The affinity of a molecule Thus, equal affinities may involve different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by established methods known in the art, including those described herein.
  • Mutated or wild-type IL-21 can be determined by surface plasmon resonance (SPR) according to the methods set forth in the examples, using standard instruments such as Biacore instruments (GE Healthcare) and receptor subunits (as can be obtained by recombinant expression) Affinity for various forms of IL-21 receptor.
  • SPR surface plasmon resonance
  • receptor subunits as can be obtained by recombinant expression
  • Affinity for various forms of IL-21 receptor can be assessed using cell lines known to express one or another form of such receptors. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.
  • an “effective amount” of an agent is that amount necessary to induce physiological changes in the cells or tissues to which it is administered.
  • a “therapeutically effective amount” of an agent, such as a pharmaceutical composition is that amount effective in the dosage and time period necessary to achieve the desired therapeutic or preventive result.
  • a therapeutically effective amount of an agent for example, eliminates, reduces, delays, minimizes or prevents the adverse effects of a disease.
  • composition refers to a preparation in a form such that the biological activity of the active ingredients contained therein is effective and does not contain other ingredients that would be unacceptable toxicity to a subject to whom the composition is administered.
  • “Pharmaceutically acceptable carrier” refers to ingredients in a pharmaceutical composition other than the active ingredients that are non-toxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • Coupling PEG to proteins through sulfhydryl coupling is a commonly used method for long-lasting proteins.
  • Natural IL-21 There are 4 cysteines in it, and exactly two pairs of disulfide bonds are formed. Therefore, if you want to PEG-modify IL-21 through sulfhydryl coupling method, you need to mutate other amino acids in IL-21 to separate of cysteine.
  • cysteine thiol coupling has the advantages of convenience, easy operation and strong specificity.
  • the present invention analyzes and calculates the structure of natural IL-21 to search for amino acids that can be mutated into cysteine without affecting the function of IL-21.
  • the mutation site excludes amino acids involved in the formation of secondary structure (such as ⁇ -helix) in IL-21.
  • the mutation site needs to be away from the key amino acids (such as Q12) in the interaction interface between IL-21 and IL-21R.
  • the present invention also fully considers that the mutation site may affect the formation of the original disulfide bond in IL-21, so it is also as far away from the original disulfide bond as possible (in principle, it is larger than ).
  • natural IL-21 is mutated, and N at position 25, K at position 52, K at position 56, N at position 59, G at position 61, N at position 82 and The S at position 98 was mutated to C respectively, resulting in seven different mutants, named 1327(N25C), 1328(K52C), 1329(K56C), 1330(N59C), 1331(G61C), 1332(N82C). and 1333(S98C).
  • all wild-type and mutant plasmids are transformed into Escherichia coli host engineering bacteria for expression, and expression produces insoluble inclusion bodies.
  • the bacterial cells are disrupted by high pressure and washed, and the inclusion bodies are recovered to prepare for later protein refolding.
  • the inclusion bodies of the above wt-IL-21 and IL-21 mutants were dissolved in 20 times (v/w) volume of denaturing solution (6M Guanidine Hydrochloride, 50mM Tris, pH 9.0) at room temperature for 30 minutes. Add DTT and continue reduction for 30 minutes. Drop the denaturing solution into 30 times the volume of the refolding solution (20mM Tris, 0.5M Arginine, 2.5mM Cysteine, pH 8.0), stir at room temperature overnight, and detect the next day Refolding effect.
  • denaturing solution 6M Guanidine Hydrochloride, 50mM Tris, pH 9.0
  • the above renatured proteins were adjusted to pH 5.3 with sodium acetate, and the conductivity was diluted to less than 20 ms/cm with deionized water. After centrifugation, the supernatant was directly flowed through GE's SP BB column for capture.
  • the operation is as follows: Before purification, balance the SP BB column with 5 times the column volume of equilibrium solution (20mM sodium acetate, pH 5.3); flow the refolding solution through the column, and then wash the column with 5 times the column volume of the equilibrium solution to remove non-specific binding. Protein; use gradient elution with 15 times column volume of elution buffer (20mM sodium acetate, 1M NaCl, pH 5.3), and collect the eluate containing the target protein.
  • Polyethylene glycol is an inert, non-carcinogenic polymer.
  • Polyethylene glycol modification also known as PEGylation of molecules, can enhance the solubility of hydrophobic drugs, proteins, liposomes, and nucleic acids, improve their stability, and extend their half-life. It can also reduce the immunogenicity of the modified products. properties, improve the clinical application scope of modified substances, etc.
  • IL-21 molecules containing free cysteine 1327, 1328, 1329, 1330, 1331, 1332 and 13373 were PEG modified through sulfhydryl coupling.
  • the specific method is as follows:
  • IL-21 mutants containing free cysteine was controlled between 0.5-2.0 mg/mL.
  • MethoxyPEG maleimide (mPEG-MAL, synthesized by Xiamen Sanobange Biotechnology Co., Ltd.) was dissolved in 50mM disodium hydrogen phosphate solution with a pH value of 7.0 and a final concentration of 100mg/mL.
  • Example 4 In vitro affinity detection of wt-IL-21 and IL-21 mutants with IL-21 receptor (IL-21R)
  • the modified IL-21 mutant of the present invention introduces cysteine mutations at different positions, so it is necessary to detect whether the mutation sites will affect the binding of IL-21 to its receptor IL-21R.
  • a further 20 kDa PEG was introduced. It is also necessary to detect whether the PEG modification will affect the binding of IL-21 and IL-21R.
  • affinity testing is to measure the dissociation constant (KD) of IL-21 and IL-21R through the BiacoreSPR 8K instrument. The specific method is as follows:
  • Protein-A chip (Cytiva) to immobilize IL-21R with Fc tag on the chip surface.
  • the mobile phase is PBS buffer containing 0.05% Tween20.
  • IL-21 and its mutants or derivatives at different concentrations (50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.56nM, 0.78nM, 0nM) as analytes to detect their response to IL-21R value.
  • concentrations 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.56nM, 0.78nM, 0nM
  • the molecular weight of polyethylene glycol was not included in the calculation of the concentration and mass of the PEG-modified IL-21 derivative, and only the molecular weight of the IL-21 mutant protein was calculated.
  • SPR detects the affinity of IL-21 and IL-21R after point mutation
  • the present invention initially screened out four molecules, 1330, 1331, 1332 and 1333, for subsequent cell experiments to test the ability of the PEG-modified protein to promote immune cell proliferation and activate immune cells. ability.
  • Table 4 Affinity values of wt-IL-21 and IL-21 mutants and their derivatives with IL-21R
  • Example 5 Activation experiment of immune cells in mouse spleen by PEG-modified IL-21 mutants
  • mice spleen cells In order to further verify and screen the biological activity of the IL-21 mutants designed in the present invention, we selected mouse spleen cells and used 1330-PEG, 1331-PEG, 1332-PEG, 1333-PEG and wt-IL- 21 acts on mouse spleen cells to detect the ability of these molecules to promote proliferation and activation of immune cells.
  • the specific operation is as follows: Dilute an appropriate amount of mouse anti-CD3e with PBS to 0.25 ⁇ g/mL, add 1 mL/well to a 24-well plate, and incubate the plate at 37°C for 2 hours. In the incubated 24-well plate, discard the coating solution, add 1 mL PBS to each well, wash twice and set aside. Take the spleen of a C57 mouse, grind it and separate single cells through a 40 ⁇ m cell sieve. Add 40mL PBS to the single cells and centrifuge at 300g for 5 minutes.
  • both wt-IL-21 and PEG-modified IL-21 mutants When wt-IL-21 and PEG-modified IL-21 mutants were administered alone, they had no stimulatory effect on immune cells in the mouse spleen. After combined use with CD3 antibodies, both wt-IL-21 and PEG-modified IL-21 mutants can promote the proliferation of CD4-CD8+T cells and NKT cells, and can activate CD4-CD8+T cells, CD4+CD8-T cells and NKT cells. The difference is that the stimulatory effect mediated by wt-IL-21 is dose-dependent, while the stimulatory effect mediated by the PEG-modified IL-21 mutant increases at low concentrations and decreases at high concentrations.
  • IL-21 molecule has significantly lower cytotoxicity than the widely studied IL-2 molecule.
  • the highest dosage of wt-IL-21 reaches 200 ⁇ g/mouse, and it does not show abnormal toxic and side effects.
  • the present invention selected 1330-PEG and 1332-PEG as research subjects, further increased the dosage to 18 mg/kg, and conducted experiments in mice.
  • mice Female C57BL/6 mice aged 6-8 weeks were randomly divided into 3 groups (Vehicle, 1330-PEG, 1332-PEG), with 2 mice in each group. The administration cycle was twice a week, and the administration route was repeated through the tail vein. The test drug was injected for a total of 3 weeks. Animals were monitored individually. Animal body weights were measured twice weekly. Daily health observations included animal mortality, appearance, spontaneous activity, body posture, food and water intake. Any other lesions and adverse reactions were recorded. At the end of the experiment, dissection was performed and the organs were inspected for visible lesions.
  • mice in both the experimental group and the control group were in good condition. As shown in Figure 5, the weight changes of the six mice were all within a reasonable range without significant reduction. After the mice were euthanized, no obvious lesions were found in the main organs of the mice. It can be seen that the PEG-modified IL-21 molecule has very low toxic and side effects. It still does not show obvious toxic and side effects at doses as high as 18 mg/kg, and has high drug safety.
  • the plasma half-life of human IL-21 is short, only 0.2 hours in mice, 0.4-0.8 hours in cynomolgus monkeys, and about 2 hours in humans.
  • the short plasma half-life greatly limits the anti-tumor efficacy of IL-21.
  • PEG modification is a commonly used and efficient way to make proteins long-lasting. In this example, the plasma half-life of the PEG-modified IL-21 mutant was detected.
  • mice 6-8 week old female C57BL/6 mice were randomly divided into 4 groups (wt-IL-21, 1330-PEG, 1332-PEG, 1333-PEG), with 6 mice in each group, and the dosage was 0.15 mg/kg.
  • the dose of the test drug was injected through the tail vein, and the concentration of the test drug in the peripheral blood of the mice was measured at 0, 0.5, 2, 6, 24, 48, 72, and 96 hours respectively.
  • the 1332-PEG molecule Due to the limitations of the detection method, the 1332-PEG molecule is insensitive to the detection method and its concentration cannot be detected, while wt-IL-21, 1330-PEG and 1333-PEG are not affected.
  • the results are shown in Figure 6. After wt-IL-21 enters the mouse plasma, it is rapidly metabolized and eliminated, and the half-life is basically consistent with the 0.2h reported in the literature. After PEG modification, the half-life of 1330-PEG is about 22 hours, and the half-life of 1333-PEG is about 18 hours. It can be seen that after PEG modification, the plasma half-life of IL-21 is significantly extended.
  • IL-21 can promote immune cell proliferation and inhibit tumor growth.
  • the short half-life of wild-type IL-21 limits the ability of IL-21 to inhibit tumors.
  • PEG-modified IL-21 has a half-life that is significantly longer than that of the wild type, so in this example, the effect of extended half-life on the anti-tumor efficacy of IL-21 was further tested.
  • MC38 colon cancer model mice and EMT-6 breast cancer model mice were used for experiments, and the effects of long-acting IL-21 (1330-PEG, 1332-PEG, 1333-PEG) were detected in the mice. Anti-tumor efficacy.
  • the experimental method is as follows: resuscitate MC38 or EMT-6 cells and culture them in vitro to obtain 1.7 ⁇ 10 8 cells.
  • Female C57BL/6 mice aged 7-8 weeks were selected and adaptively raised for one week. 1 ⁇ 10 6 cells were taken and inoculated subcutaneously in the experimental animals. After the tumor volume is 50-100mm, the drug is divided into groups, with a total of 5 groups, 6 mice in each group. The drug is administered intraperitoneally twice a week for a total of three weeks. The body weight and tumor volume of the mice are measured regularly. Dosing information and grouping information are shown in Table 5.
  • the results of the MC38 colon cancer tumor model are shown in Figure 7.
  • wt-IL-21 had no inhibitory effect on tumor growth.
  • the long-acting IL-21 (1330-PEG, 1332-PEG, 1333-PEG) showed significant efficacy in inhibiting tumor growth compared with the control group.
  • 1330-PEG has the best anti-tumor effect, followed by 1333-PEG.
  • 1332-PEG has the weakest anti-tumor effect, but 1332-PEG still reduced the tumor volume by nearly 40% compared to the control group. .
  • the results of the EMT-6 breast cancer tumor model are shown in Figure 8.
  • the wt-IL-21 group had a weaker tumor growth inhibitory effect than the control group.
  • the tumor growth rate in mice was significantly inhibited. Comparing the efficacy of the three molecules in the EMT-6 tumor model, 1332-PEG has the best efficacy, followed by 1333-PEG, and 1330-PEG is the weakest, but the difference is small and basically at a similar level.
  • Example 9 The anti-tumor efficacy of 1332-PEG is dose-dependent
  • Example 8 shows that a low dose of PEG-modified IL-21 can induce significant anti-tumor effects.
  • 1332-PEG was selected as the research object, and different dosages were set in the MC38 colon cancer mouse model to conduct anti-tumor efficacy experiments.
  • the experimental method is basically consistent with Example 8. Dosing information and grouping information are shown in Table 6.

Landscapes

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

Abstract

L'invention concerne un dérivé d'IL-21 modifié par du polyéthylène glycol et son utilisation. Par analyse de la structure d'IL-21, à condition que la structure globale de l'IL-21 n'est pas affectée, des sites spécifiques dans l'IL-21 sont mutés en cystéine, puis les molécules PEG appropriées sont reliées à la cystéine mutée sur IL-21 au moyen d'un couplage par l'intermédiaire de sulfydryle. La demi-vie plasmatique de l'IL-21 modifiée par PEG est prolongée, et l'IL-21 modifiée par PEG présente un excellent effet de médicament antitumoral dépendant de la dose. De plus, l'IL-21 modifiée par PEG présente une meilleure solubilité dans l'eau, ce qui est bénéfique pour la formule de préparation d'un médicament. Par comparaison avec d'autres protéines de fusion IL-21 à action prolongée, qui doivent être exprimées dans des cellules de mammifère, la protéine de squelette d'IL-21 utilisée par modification de PEG est obtenue au moyen de l'expression dans Escherichia coli, la dénaturation et la renaturation et la purification, ce qui présente les avantages d'industrialisation à faible coût et de rendement élevé.
PCT/CN2023/115300 2022-09-02 2023-08-28 Dérivé d'il-21 modifié par du polyéthylène glycol et son utilisation WO2024046280A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211074184.7A CN117645661A (zh) 2022-09-02 2022-09-02 一种聚乙二醇修饰的il-21衍生物及其应用
CN202211074184.7 2022-09-02

Publications (1)

Publication Number Publication Date
WO2024046280A1 true WO2024046280A1 (fr) 2024-03-07

Family

ID=90043837

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/115300 WO2024046280A1 (fr) 2022-09-02 2023-08-28 Dérivé d'il-21 modifié par du polyéthylène glycol et son utilisation

Country Status (2)

Country Link
CN (1) CN117645661A (fr)
WO (1) WO2024046280A1 (fr)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060228331A1 (en) * 2003-10-10 2006-10-12 Novo Nordisk A/S IL-21 Derivatives and variants
CN101189024A (zh) * 2005-06-06 2008-05-28 诺沃-诺迪斯克有限公司 稳定的il-21组合物
WO2008074863A1 (fr) * 2006-12-21 2008-06-26 Novo Nordisk A/S Variants de l'interleukine-21 avec une liaison modifiée au récepteur de il-21
WO2010076339A1 (fr) * 2009-01-05 2010-07-08 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Moyens et procédé de traitement de maladies de déficience en anticorps sur la base de il-21 et de variants de il-21
CN102516386A (zh) * 2003-10-10 2012-06-27 诺沃挪第克公司 Il-21衍生物
CN107698682A (zh) * 2010-05-03 2018-02-16 百时美施贵宝公司 血清白蛋白结合分子
CN111164100A (zh) * 2017-08-03 2020-05-15 美国安进公司 白介素-21突变蛋白和治疗方法
WO2022135469A1 (fr) * 2020-12-23 2022-06-30 信达生物制药(苏州)有限公司 Mutant de l'interleukine-21 et son utilisation

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060228331A1 (en) * 2003-10-10 2006-10-12 Novo Nordisk A/S IL-21 Derivatives and variants
CN102516386A (zh) * 2003-10-10 2012-06-27 诺沃挪第克公司 Il-21衍生物
CN101189024A (zh) * 2005-06-06 2008-05-28 诺沃-诺迪斯克有限公司 稳定的il-21组合物
WO2008074863A1 (fr) * 2006-12-21 2008-06-26 Novo Nordisk A/S Variants de l'interleukine-21 avec une liaison modifiée au récepteur de il-21
WO2010076339A1 (fr) * 2009-01-05 2010-07-08 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Moyens et procédé de traitement de maladies de déficience en anticorps sur la base de il-21 et de variants de il-21
CN107698682A (zh) * 2010-05-03 2018-02-16 百时美施贵宝公司 血清白蛋白结合分子
CN111164100A (zh) * 2017-08-03 2020-05-15 美国安进公司 白介素-21突变蛋白和治疗方法
WO2022135469A1 (fr) * 2020-12-23 2022-06-30 信达生物制药(苏州)有限公司 Mutant de l'interleukine-21 et son utilisation

Also Published As

Publication number Publication date
CN117645661A (zh) 2024-03-05

Similar Documents

Publication Publication Date Title
US10898545B2 (en) Tear lipocalin muteins binding IL-4 r alpha
JP6941664B2 (ja) 自己免疫疾患の処置のために調節性ｔ細胞を選択的に活性化させる改変ｉｌ−２変異体
EP3733693A1 (fr) Variant d'il-2
US10174302B1 (en) Modified L-asparaginase
KR101149607B1 (ko) 인터페론 알파 돌연변이체 및 이의 폴리에틸렌 글리콜 유도체
WO2015103928A1 (fr) Protéine dimérique hétérogène il-15 et ses utilisations
US20180360977A1 (en) Interleukin-15 Compositions and Uses Thereof
CN112724259B (zh) 人血清白蛋白与白介素2的融合蛋白及其用途
CN104245722A (zh) 白细胞介素-10多肽结合物和其用途
WO1996010089A1 (fr) Modification d'un peptide et d'une proteine
AU2002358467A1 (en) Adiponectin fragments and conjugates
TWI827593B (zh) 人類犬尿胺酸酶及其用途
JP2021535140A (ja) 抗ｃｄ３抗体葉酸生物複合体およびその使用
JP2013515474A (ja) 組換え体ｈ因子ならびにそのバリアントおよびコンジュゲート
CN111944036B (zh) 一种增殖免疫细胞的突变体蛋白
JP4092081B2 (ja) 顆粒球形成活性を有するｇ−ｃｓｆ変異体相当核酸およびタンパク質
JPH0463595A (ja) ヒトインターロイキン３誘導体
WO2024046280A1 (fr) Dérivé d'il-21 modifié par du polyéthylène glycol et son utilisation
CN117362411A (zh) 白细胞介素15蛋白突变体及其应用
CN117384273A (zh) 一种聚乙二醇修饰的il-2衍生物及其应用
WO2023155872A1 (fr) Procédé pour réaliser une libération contrôlée et une libération prolongée d'activité de molécules bioactives et utilisation pour des médicaments
US20220324932A1 (en) Modified il-2 proteins, peg conjugates, and uses thereof
WO2011147138A1 (fr) Protéine de fusion de l'interleukine de ciblage et son procédé de préparation et son utilisation
KR20220077590A (ko) Candida Utilis 유래 요산산화효소-알부민 복합체 및 이의 제조 방법
CN115947820A (zh) 一种水溶性提高的il-2衍生物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23859310

Country of ref document: EP

Kind code of ref document: A1