WO2024162772A1 - Conjugués de substances bioactives à persistance in vivo améliorée - Google Patents

Conjugués de substances bioactives à persistance in vivo améliorée Download PDF

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WO2024162772A1
WO2024162772A1 PCT/KR2024/001489 KR2024001489W WO2024162772A1 WO 2024162772 A1 WO2024162772 A1 WO 2024162772A1 KR 2024001489 W KR2024001489 W KR 2024001489W WO 2024162772 A1 WO2024162772 A1 WO 2024162772A1
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seq
amino acid
acid sequence
heavy chain
light chain
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PCT/KR2024/001489
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Korean (ko)
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장성수
이학섭
최두진
이진혁
정유진
이원경
지황우
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주식회사 에이티지씨
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Publication of WO2024162772A1 publication Critical patent/WO2024162772A1/fr

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    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment

Definitions

  • the present invention relates to a conjugate of a physiologically active substance with increased in vivo persistence, and more specifically, to a conjugate in which a physiologically active substance is bound to a carrier protein comprising a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 2 or 6; and a light chain comprising an amino acid sequence represented by SEQ ID NO: 10 or 14, and a method for enhancing the in vivo stability of a physiologically active substance using the same.
  • a physiologically active material is a substance that has a significant effect on the function of the body in small amounts, and refers to vitamins, hormones, enzymes, neurotransmitters, etc.
  • Most physiologically active substances are composed of peptides or polypeptides, so they are generally unstable and easily denatured and decomposed by proteolytic enzymes in the body, or are easily eliminated through the kidneys due to their relatively small size. Therefore, in order to maintain physiological activity in the body for a long time as a pharmacological ingredient, the blood concentration and potency of the physiologically active substance must be maintained, so the drug must be administered frequently to the patient. In particular, when administered to the patient in the form of an injection, frequent administration not only causes great pain to the patient, but also causes high treatment costs.
  • the present invention has been devised to solve the problems in the prior art as described above, and comprises a combination having the following chemical formula 1:
  • G is a physiologically active substance
  • I is a carrier protein capable of increasing the in vivo half-life of the physiologically active substance
  • I is a carrier protein consisting of a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 2 or 6; and a light chain comprising an amino acid sequence represented by SEQ ID NO: 10 or 14, and a method for increasing the stability of a physiologically active substance in the body, including a step of binding the carrier protein to the physiologically active substance.
  • the present invention provides a complex having the following chemical formula 1.
  • G is a physiologically active substance
  • I is a carrier protein capable of increasing the in vivo half-life of the physiologically active substance, the carrier protein being composed of a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 2 or 6; and a light chain comprising an amino acid sequence represented by SEQ ID NO: 10 or 14.
  • the carrier protein is in the form of immunoglobulin G composed of a light chain and a heavy chain, and the light chain is most preferably composed of the amino acid sequence of SEQ ID NO: 10 or 14, but may preferably include an amino acid sequence having 80% or more, more preferably 90% or more, and even more preferably 95% or more sequence homology with the amino acid sequence of SEQ ID NO: 10 or 14.
  • the “% of sequence homology” in the present invention is confirmed by comparing the optimally arranged sequence with the comparison region, and a part of the amino acid sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence for the optimal arrangement of the sequence (which does not include additions or deletions).
  • the heavy chain most preferably consists of the amino acid sequence of SEQ ID NO: 2 or 6, but may preferably include an amino acid sequence having a sequence identity of at least 80%, more preferably at least 90%, and even more preferably at least 95% with the amino acid sequence of SEQ ID NO: 2 or 6.
  • the physiologically active substance and the carrier protein may be connected by a linker, and the linker may be a glycine-serine linker, preferably [Gly-Gly-Gly-Gly-Ser]n, where n is 1, 2, 3, 4, or 5, but there is no limitation as long as it is a linker that can connect the physiologically active substance and the carrier protein.
  • the linker may be a glycine-serine linker, preferably [Gly-Gly-Gly-Gly-Gly-Ser]n, where n is 1, 2, 3, 4, or 5, but there is no limitation as long as it is a linker that can connect the physiologically active substance and the carrier protein.
  • the I may be a carrier protein consisting of a heavy chain comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8; and a light chain comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16.
  • the light chain is most preferably composed of the amino acid sequence of SEQ ID NO: 10, 12, 14 or 16, but may preferably include an amino acid sequence having 80% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 10, 12, 14 or 16.
  • the heavy chain most preferably consists of an amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, but may preferably include an amino acid sequence having a sequence identity of at least 80%, more preferably at least 90%, and even more preferably at least 95% with the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8.
  • the I is preferably a carrier protein comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 4 and a light chain comprising an amino acid sequence of SEQ ID NO: 12, a carrier protein comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 2 and a light chain comprising an amino acid sequence of SEQ ID NO: 16, a carrier protein comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 4 and a light chain comprising an amino acid sequence of SEQ ID NO: 14, a carrier protein comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 8 and a light chain comprising an amino acid sequence of SEQ ID NO: 10, a carrier protein comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 2 and a light chain comprising an amino acid sequence of SEQ ID NO: 14, a carrier protein comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 8 and a light chain comprising an amino acid sequence of SEQ ID NO: 10.
  • the physiologically active substance is a concept that collectively refers to all substances having a physiological action in a living body, and since the half-life in a living body is increased by being linked to the carrier protein of the present invention, there is no limitation on the type of the physiologically active substance, but preferably, it is a physiologically active peptide and polypeptide, and examples of the physiologically active peptide and polypeptide may include an antibody, an antibody fragment, a structural protein, a regulatory protein, a transcription factor, a toxic protein, a hormone, a hormone analog, a cytokine, an enzyme, an enzyme fragment, an enzyme inhibitor, a transport protein, a receptor, a receptor fragment, a biological defense inducer, a storage protein, a movement protein, an exploitative protein, a reporter protein, and the like, and further examples include a toxin; a GLP-1 (Glucagon like peptide-1) receptor agonist; a glucagon receptor agonist; a GIP (G)
  • Another example may include a naturally occurring exendin-3 or an analogue thereof, a naturally occurring exendin-4 or an analogue thereof, a naturally occurring insulin or an analogue thereof, a naturally occurring GLP-1 or an analogue thereof, a naturally occurring GLP-2 or an analogue thereof, a naturally occurring oxyntomodulin or an analogue thereof, a naturally occurring glucagon or an analogue thereof, a naturally occurring fibroblast growth factor or an analogue thereof, a naturally occurring ghrelin or an analogue thereof, a naturally occurring calcitonin or an analogue thereof, a naturally occurring granulocyte colony-stimulating factor or an analogue thereof, or a substance that binds to two or more receptors among a GLP receptor, a glucagon receptor, and a GIP receptor.
  • the analogue is a concept encompassing all of derivatives, fragments, and variants.
  • the present invention provides a method for improving the stability of a physiologically active substance in the body, comprising a step of binding the carrier protein to the physiologically active substance.
  • the present invention provides a pharmaceutical composition for preventing or treating a disease, comprising the above complex as an effective ingredient.
  • the present invention also provides a method for treating a disease, comprising a step of administering to a subject a composition comprising the above complex as an active ingredient.
  • the present invention also provides a composition containing the above complex as an effective ingredient for the prevention or treatment of diseases.
  • the present invention also provides a use of the above combination for producing a medicament for preventing or treating a disease.
  • the disease there is no limitation on the disease as long as it is a disease that can be treated by a physiologically active substance included in the combination.
  • the bioactive substance conjugate with increased in vivo persistence can increase the in vivo activity, stability, and persistence of various bioactive substances by conjugating a carrier protein capable of increasing the in vivo half-life of various bioactive substances, and therefore can be effectively used in the treatment of all diseases in which bioactive substances are used for treatment.
  • Figure 1 is a schematic diagram showing the form of a bioactive substance conjugate according to one embodiment of the present invention.
  • FIG. 2 is a drawing showing the results of confirming the physiological activity of a physiologically active substance conjugate according to one embodiment of the present invention by measuring the amount of insulin secretion and the amount of cAMP change.
  • the present inventors have conducted extensive research into a method for preventing frequent administration of a bioactive substance therapeutic agent by increasing the stability of the bioactive substance in the body, i.e., persistence in the body, and as a result, they have confirmed that the persistence, stability and bioactivity of the bioactive substance in the body are significantly increased by binding the bioactive substance to a carrier protein having an immunoglobulin structure, which is composed of a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 2 or 6; and a light chain comprising an amino acid sequence represented by SEQ ID NO: 10 or 14, thereby completing the present invention.
  • bioactive substance active ingredient
  • active drug active drug
  • active preparation active preparation
  • drug therapeutic preparation
  • step of ⁇ or “step of ⁇ ” do not mean “step for ⁇ .”
  • the term “combination of these” included in the expression in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the expression in the Makushi format, and means including one or more selected from the group consisting of said components.
  • the term “recombinant vector” refers to a vector capable of expressing a peptide or protein encoded by a heterologous nucleic acid inserted into the vector, and preferably means a vector manufactured to include a gene encoding the complex of the present invention.
  • the “vector” refers to any medium for the introduction and/or transfer of a base into a host cell in vitro, ex vivo or in vivo, and may be a replicating unit (replicon) to which another DNA fragment can be bound and replication of the bound fragment can be brought about, and the “replication unit” refers to any genetic unit (e.g., plasmid, phage, cosmid, chromosome, virus, etc.) that functions as an autonomous unit of DNA replication in vivo, i.e., is capable of replicating by its own regulation.
  • the recombinant expression vector of the present invention may preferably include a promoter, which is a transcription initiation factor to which RNA polymerase binds, an arbitrary operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site and a sequence for regulating the termination of transcription and translation, a terminator, a signal peptide, etc., and more preferably, may further include a 5' UTR region gene of M17 for increasing the amount of protein synthesis, an HDEL gene for minimizing protein degradation so that the protein can be stably maintained in the endoplasmic reticulum, and more preferably, may further include a tag gene for increasing the production amount of the recombinant protein, a tag gene for maintaining the structural stability of the recombinant protein, a tag gene for easily isolating the recombinant protein, a selection marker gene such as an antibiotic resistance gene for selecting a transformant, and the like.
  • a promoter which is a transcription initiation factor to
  • prevention means any act of suppressing or delaying the onset of a disease by administering a combination according to the present invention.
  • treatment means any action by which the symptoms of a disease are improved or beneficially changed by administration of a combination according to the present invention.
  • subject refers to a subject to which the combination of the present invention can be administered, and there is no limitation on the subject.
  • the “pharmaceutical composition” refers to a composition including a combination according to the present invention, and may be characterized in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be characterized in that it is for human use.
  • the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc. for oral administration, and may include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc.
  • the formulation of the pharmaceutical composition of the present invention may be variously prepared by mixing with the pharmaceutically acceptable carrier as described above.
  • the pharmaceutically acceptable carrier for example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit dose ampoules or multiple doses.
  • it can be formulated and used as sugar-coated tablets, gels, pills, powders, granules, suppositories, external preparations, solutions, suspensions, sustained-release preparations, slurries, etc.
  • examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc.
  • fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. can be additionally included.
  • the route of administration of the pharmaceutical composition according to the present invention is not limited to these, but oral or parenteral administration is preferable, and includes, for example, oral, intravenous, intramuscular, intraarticular, intrasynovial, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, intradermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, rectal, intrasternal, intralesional, intracranial, etc.
  • the dosage of the pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination, and the severity of the specific disease to be prevented or treated, and may be appropriately selected by those skilled in the art depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, and may be administered at 0.0001 to 500 mg/kg or 0.001 to 500 mg/kg per day. Administration may be done once a day or divided into several times. The above dosage does not limit the scope of the present invention in any way.
  • Example 1 Preparation of a bioactive substance conjugate with increased in vivo persistence
  • a hybrid immunoglobulin G (IgG) structure was used. More specifically, CH1 constituting the heavy chain of IgG was manufactured to include the amino acid sequence of IgG3 or IgG4, and CH2 and CH3 were manufactured to include the amino acid sequence of IgG4. In the case of the light chain, it was manufactured to include the amino acid sequence of kappa or lambda2. In addition, the hinge connecting CH1 and CH2 of the heavy chain was manufactured to include the amino acid sequence of IgG3.
  • PCR was performed using pFUSE-CHIg-hG3 and pFUSE-CHIg-hG4 (Invivogen) vectors as templates to obtain DNA corresponding to CH1 and hinge of IgG3, and CH1, CH2, and CH3 of IgG4, respectively.
  • kappa DNA was obtained using pFUSE2-CLIg-hK (InvivoGen)
  • lambda2 DNA was obtained using pFUSE2-CLIg-hl2 (InvivoGen).
  • the obtained DNAs were constructed into two recombinant vectors, one for the light chain and one for the heavy chain.
  • each of the heavy chain including a sequence in which CH1 and CH2-CH3 are composed of IgG4 and the hinge is composed of IgG3
  • the heavy chain including a sequence in which CH1 and the hinge are composed of IgG3 and CH2-CH3 is composed of IgG4
  • the light chain including a sequence in which kappa is composed
  • the light chain hereinafter referred to as “L ⁇ 2”
  • a bioactive substance conjugate was manufactured using Glucagon-like peptide-1 (GLP-1), a GLP-1 mutant, and Exendin-4 as examples of bioactive substances.
  • GLP-1 (hereinafter referred to as “G0”), GLP-1 mutant (hereinafter referred to as “G1”), or Exendin-4 (hereinafter referred to as “E0”) DNA was obtained by performing PCR using GLP-1 ORF clone (Origene TM ) and exendin-4 ORF clone (Cosmogenetech Inc.) as templates, respectively, and light chain DNA and heavy chain DNA were obtained by performing PCR using the recombinant vector manufactured by the method of Example 1.1 as a template.
  • GLP-1 ORF clone Origene TM
  • exendin-4 ORF clone Cosmogenetech Inc.
  • the obtained DNAs were respectively linked to the physiologically active substance and the light chain, and the physiologically active substance and the heavy chain using PCR, and some of them were linked to the physiologically active substance and the heavy chain or the physiologically active substance and the light chain using a “(G4S)3” linker. Then, the linked DNAs were each inserted into the pcDNA3.4 TOPO expression vector (Thermo Fisher Scientific), transformed into E. coli DH5 ⁇ , and stored.
  • the sequences of the conjugates linking the light chain or the heavy chain with G0, G1, or E0, respectively, are shown in Table 2 below.
  • Example 2 Expression and purification of a bioactive substance conjugate
  • ExpiCHO-STM cells were used. More specifically, ExpiCHO-STM cells were inoculated into ExpiCHOTM Expression Medium at a concentration of 3x106 cells/mL, and then cultured in a 125 mL Erlenmeyer flask under the conditions of 8% CO2 , 37°C, and 125 rpm for 1 day.
  • the cultured cells were diluted to a concentration of 6x106 cells/mL, and 12.5 ⁇ g of a recombinant vector containing a bioactive substance and a light chain sequence and 12.5 ⁇ g of a recombinant vector containing a bioactive substance and a heavy chain sequence were mixed, and co-transfection was performed using an ExpiCHOTM Expression System kit (Thermo Fisher Scientific), and cultured for 1 day. Thereafter, the enhancer and feed included in the kit were added to the cells, and the cells were cultured for 2 additional days, and then the cells were removed by centrifugation at 4,500 rpm for 30 minutes.
  • cAMP is an important regulatory factor that acts in the insulin secretion process, and when the cAMP concentration increases, cAMP-dependent protein kinase is activated and cAMP-response element binding protein (CREB) is phosphorylated, through which the transcription of target genes in ⁇ -cells is regulated, thereby causing insulin secretion.
  • CREB cAMP-response element binding protein
  • RIN-m5F cell line was seeded in a 24-well plate at a density of 1x106 cells/well in RPMI-1640 medium supplemented with 2 mM L-glutamine, 10 mM HEPES, 1 mM Sodium pyruvate, 1 % penicillin-streptomycin, 2500 mg/L Glucose solution, and 10% FBS, cultured for 48 hours, and the cultured cells were washed three times with 1 mL of HBSS.
  • HBSS containing 2.8 mM Glucose was added to the prepared cells, and the cells were cultured for 1 hour in a 37°C, 5% CO2 incubator. Then, the cells were treated again with the bioactive substance conjugate added to HBSS containing 16.8 mM Glucose, and reacted for 1 hour in a 37°C, 5% CO2 incubator. After 1 hour, the supernatant was centrifuged at 2,000 rpm, 4°C for 5 minutes, and the amount of insulin contained in the supernatant was measured using an insulin ELISA kit (ALPCO®).
  • ALPCO® insulin ELISA kit
  • HBSS containing 2.5 mM Glucose was added to the prepared cells, and cultured for 1 hour in a 37°C, 5% CO2 incubator. Then, 20 mM Glucose, 500 M IBMX (3-isobutyl-1-methylxanthine), and a bioactive substance conjugate were added to HBSS, and the cells were treated again and reacted at room temperature for 20 minutes. After the solution was completely removed, 0.1 M HCl was added to completely lyse the cells, and the amount of cAMP contained in the lysed cells was measured using a cAMP ELISA kit (Enzo life science, Inc.). Sample No.
  • Sample No. 12 is a bioactive substance conjugate composed of a heavy chain of SEQ ID NO. 56 and a light chain of SEQ ID NO. 20
  • Sample No. 14 is a bioactive substance conjugate composed of a heavy chain of SEQ ID NO. 18 and a light chain of SEQ ID NO. 22
  • Sample No. 15 is a bioactive substance conjugate composed of a heavy chain of SEQ ID NO. 56 and a light chain of SEQ ID NO. 58
  • Sample No. 19 is a bioactive substance conjugate composed of a heavy chain of SEQ ID NO. 60 and a light chain of SEQ ID NO. 62
  • Sample No. 37 is a bioactive substance conjugate composed of a heavy chain of SEQ ID NO. 24 and a light chain of SEQ ID NO. 36
  • sample No. 38 is a bioactive substance conjugate composed of a heavy chain of SEQ ID NO. 24 and a light chain of SEQ ID NO. 38
  • Sample No. 39 is a bioactive substance conjugate composed of a heavy chain of SEQ ID NO. 26 and a light chain of SEQ ID NO. 36
  • Sample No. 44 is a bioactive substance conjugate composed of a heavy chain of SEQ ID NO. 30 and a light chain of SEQ ID NO.
  • sample no. 45 is a bioactive substance conjugate composed of a heavy chain of sequence number 28 and a light chain of sequence number 36, sample no.
  • sample no. 47 is a bioactive substance conjugate composed of a heavy chain of sequence number 30 and a light chain of sequence number 36
  • sample no. 52 is a bioactive substance conjugate composed of a heavy chain of sequence number 64 and a light chain of sequence number 48
  • sample no. 57 is a bioactive substance conjugate composed of a heavy chain of sequence number 40 and a light chain of sequence number 46
  • sample no. 58 is a bioactive substance conjugate composed of a heavy chain of sequence number 40 and a light chain of sequence number 48
  • sample no. 59 is a bioactive substance conjugate composed of a heavy chain of sequence number 42 and a light chain of sequence number 46, sample no.
  • Sample No. 70 is a bioactive substance conjugate composed of a heavy chain of SEQ ID NO. 54 and a light chain of SEQ ID NO. 52
  • Sample No. 71 is a bioactive substance conjugate composed of a heavy chain of SEQ ID NO. 44 and a light chain of SEQ ID NO. 50
  • Sample No. 72 is a bioactive substance conjugate composed of a heavy chain of SEQ ID NO. 44 and a light chain of SEQ ID NO. 52. All experiments were repeated at least three times, and statistical significance was confirmed by Student's t-test. If P ⁇ 0.05, it was determined that there was statistical significance. The results are shown in Fig. 2.
  • Example 3 it was confirmed that the physiological activity of the physiologically active substances was normally maintained even when various physiologically active substances were bound to the carrier protein of the present invention.
  • the binding affinity with neonatal fragment crystallizable receptor (FcRn) was confirmed by Bio Layer Interferometry (BLI).
  • FcRn neonatal fragment crystallizable receptor
  • BLI was performed using an Octet (model# R8) device.
  • the complex of the present invention to be analyzed was treated with the SA biosensor to which FcRn was bound, and the association step and dissociation step were confirmed at pH 6.0 and pH 7.4, respectively, and the KD value and Kdis value were measured.
  • KD is a value for binding affinity and means molar concentration. A smaller value means a higher binding affinity, and Kdis is dissociation, that is, the time it takes for the complex to detach from the biosensor. A smaller value means a longer time is needed for dissociation, and a smaller value means a higher binding affinity.
  • the KD and Kdis values at pH 6.0 are shown in Table 3 below.
  • the carrier protein for increasing the in vivo persistence and stability of the bioactive substance of the present invention can be effectively used in the development of therapeutic agents for various diseases because it can increase the in vivo activity, stability, and persistence of bioactive substances by binding various bioactive substances.

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Abstract

La présente invention concerne des conjugués de substances bioactives ayant une persistance in vivo accrue et, spécifiquement, un conjugué où une substance bioactive est liée à une protéine porteuse composée d'une chaîne lourde comprenant la séquence d'acides aminés représentée par SEQ ID NO : 2 ou 6, et une chaîne légère comprenant la séquence d'acides aminés représentée par SEQ ID NO : 10 ou 14, ainsi qu'une méthode d'amélioration de la stabilité in vivo de substances bioactives les utilisant.
PCT/KR2024/001489 2023-02-01 2024-01-31 Conjugués de substances bioactives à persistance in vivo améliorée WO2024162772A1 (fr)

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