WO2020032366A1 - Support immunoréactif - Google Patents

Support immunoréactif Download PDF

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Publication number
WO2020032366A1
WO2020032366A1 PCT/KR2019/006682 KR2019006682W WO2020032366A1 WO 2020032366 A1 WO2020032366 A1 WO 2020032366A1 KR 2019006682 W KR2019006682 W KR 2019006682W WO 2020032366 A1 WO2020032366 A1 WO 2020032366A1
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Prior art keywords
silica particles
porous silica
antibody
hours
cytokine
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PCT/KR2019/006682
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English (en)
Korean (ko)
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WO2020032366A9 (fr
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원철희
민달희
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주식회사 레모넥스
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Application filed by 주식회사 레모넥스 filed Critical 주식회사 레모넥스
Priority to US17/265,692 priority Critical patent/US20210177988A1/en
Priority to EP19846510.6A priority patent/EP3834820A4/fr
Priority to JP2021506399A priority patent/JP2021532163A/ja
Priority to SG11202101258VA priority patent/SG11202101258VA/en
Priority to CN201980052469.0A priority patent/CN112543631B/zh
Priority to CA3108251A priority patent/CA3108251C/fr
Priority to AU2019317939A priority patent/AU2019317939A1/en
Priority claimed from KR1020190065620A external-priority patent/KR102316722B1/ko
Publication of WO2020032366A1 publication Critical patent/WO2020032366A1/fr
Publication of WO2020032366A9 publication Critical patent/WO2020032366A9/fr
Priority to AU2023208105A priority patent/AU2023208105A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • 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
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • 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

Definitions

  • the present invention relates to an immunoreactive substance carrier having an excellent immunotherapeutic effect by delivering an immunoreactive substance stably to a target site.
  • Drug delivery system refers to a medical technology that can efficiently deliver the required amount of drugs, such as proteins, nucleic acids, or other small molecules by minimizing the side effects and maximizing the efficacy and effects of existing drugs.
  • This technology which saves the cost and time required for the development of new drugs, has recently become one of the cutting-edge technologies that create new added value in the pharmaceutical industry, in combination with nanotechnology.
  • the company has been focusing on the development of drug delivery systems along with the development of new drugs, especially companies and companies.
  • Efficient delivery systems are needed for the study of the function of bioactive substances in cells or for intracellular delivery.
  • a universal delivery system capable of delivering a wide range of bioactive substances, a system capable of accommodating and delivering a large amount of drugs, and a system for releasing drugs in a sustained manner is far from under development.
  • An object of the present invention is to stably deliver various immunoreactive substances including antibodies and cytokines to a target site, thereby providing an immunoreactive substance carrier having excellent immunotherapeutic effects.
  • the porous silica particles react with silica particles having pores less than 5 nm in diameter at 120 ° C. to 180 ° C. for 24 to 96 hours to expand the pores less than 5 nm in diameter; And calcining the pores of expanded silica particles at a temperature of 400 ° C. or higher for at least 3 hours.
  • the average diameter of the porous silica particles is 150 nm to 1000 nm, the BET surface area is 200m 2 / g to 700m 2 / g, the volume per g is 0.7ml to 2.2ml,
  • the porous silica particles have an immunoreactive substance carrier in which t is 24 or more such that the ratio of absorbance of Equation 1 is 1/2:
  • a 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles into a cylindrical permeable membrane having pores having a diameter of 50 kDa,
  • the pH of the suspension is 7.4,
  • a t is the absorbance of the porous silica particles measured after t hours have elapsed since the measurement of A 0 ).
  • the antibody of 1 above wherein the antibody is an IgG; PD-1, CTLA-4, TIM-3, BTLA, VISTA, LAG-3, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD27, CD137, HVEM, GITR, VEGFR, VEGF,
  • the antibody is an antibody that specifically binds to at least one protein selected from the group consisting of PD-1, PD-L1, and CTLA-4.
  • the cytokine is IL-7, IL-10, IL-12, IL-13, IL-15, IL-21, IL-23, IL-24, IL-27, G-CSF , GM-CSF, HGF, EGF, VEGF, LTF, TGF- ⁇ , and at least one carrier selected from the group consisting of IL-2.
  • cytokine is at least one selected from the group consisting of IL-2, IL-12, IL-15, IL-21, IL-24, and IL-13.
  • porous silica particles have hydrophilic or hydrophobic functional groups on the outer surface or inside the pores.
  • composition for immunotherapy comprising the carrier of any one of the above 1 to 10.
  • a pharmaceutical composition for preventing or treating cancer or immune disease comprising the carrier of any one of 1 to 10 above.
  • the immune-responsive substance carrier of the present invention stably delivers various immune-responsive substances, including antibodies and cytokines, to the target site, and has an excellent immunotherapeutic effect.
  • FIG. 1 is a micrograph of porous silica particles according to an embodiment of the present invention.
  • FIG. 2 is a micrograph of porous silica particles according to one embodiment of the present invention.
  • Figure 3 is a micrograph of the small pore particles during the manufacturing process of the porous silica particles according to an embodiment of the present invention.
  • Figure 4 is a micrograph of the small pore particles according to an embodiment of the present invention.
  • Figure 5 is a micrograph of the pore diameter of the porous silica particles according to an embodiment of the present invention.
  • DDV Delivery Vehicle
  • the number in parenthesis means the diameter of the particle
  • the number of subscripts means the pore diameter.
  • DDV (200) 10 means the particles of the embodiment having a particle diameter of 200 nm
  • the pore diameter is 10 nm.
  • Figure 6 is a micrograph to confirm the biodegradability of the porous silica particles according to an embodiment of the present invention.
  • FIG. 7 is a tube having a cylindrical permeable membrane according to one example.
  • Figure 11 is a result of reducing the absorbance for each pH of the environment over time of the porous silica particles according to an embodiment of the present invention.
  • 13 to 15 show cumulative amounts released after supporting IgG, PD-1, and PD-L1 antibodies on porous silica particles, respectively.
  • FIG. 16 shows TEM images of porous silica particles used to support IL-2 cytokines.
  • 17 to 21 show the cumulative amount released after supporting IL-10, IL-15, HGF, EGF, IL-2 on porous silica particles.
  • an immunoreactive substance transporter comprising; porous silica particles carrying an antibody or cytokine.
  • the cytokine may be specifically interleukins or interferons.
  • the cytokines are IL-7, IL-10, IL-12, IL-13, IL-15, IL-21, IL-23, IL-24, IL-27, G-CSF, GM-CSF, HGF, EGF, VEGF, LTF, TGF- ⁇ , IL-2, A2M, ABI3BP, acidic fibroblast growth factor, ACVR1B, ADAM17, ADAMTS6, ADMLX, aFGF, AGPAT1, AGPAT2, AIF1, AIMP1, AKR1C1, AKT1S1, allograft-inflammatory factor- 1, amac-1, AMH, ANGPTL2, ANGPTL3, ANKFN1, ANKRD1, ankyrin repeat and SOCS box-containing protein 3 isoform a variant, ankyrin repeat domain-containing SOCS box protein ASB11, ankyrin repeat domain-containing SOCS box protein Asb-13 , ankyrin repeat domain-containing SOCS box protein Asb-14, ankyrin repeat domain-containing
  • the antibody may be an antibody that specifically binds to an interleukins or interferons protein.
  • the antibody is an IgG; Or PD-1, CTLA-4, TIM-3, BTLA, VISTA, LAG-3, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD27, CD137, HVEM, GITR, VEGFR, VEGF Antibodies that specifically bind to at least one protein selected from the group consisting of EGFR, EGF, IL-1, IL-6, IL-23, TGF-beta, CTGF, TSLP, TNF-alpha, Notch, and OX40 But, if it is recognized as a conventional antibody from a biological point of view is not necessarily limited thereto.
  • the porous silica particles are particles of silica (SiO 2 ) material and have a particle size of nano size.
  • Porous silica nanoparticles of the present invention is a porous particle, having nano-sized pores, and can carry an antibody or cytokine on its surface and / or inside the pores.
  • Porous silica particles of the present invention can carry an antibody or cytokine and release the antibody or cytokine while being biodegraded in the body when administered to the body. Porous silica particles of the present invention can be slowly degraded in the body to allow the sustained release of the supported antibody (cytokine) or cytokine (cytokine).
  • t which is the ratio of the absorbance of the following formula 1 to 1/2, is 24 or more:
  • a 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles into a cylindrical permeable membrane having pores having a diameter of 50 kDa,
  • the pH of the suspension is 7.4,
  • a t is the absorbance of the porous silica particles measured after t hours have elapsed since the measurement of A 0 ).
  • Equation 1 means that the rate at which the porous silica particles are degraded in an environment similar to the body.
  • Absorbance A 0 , A t in Equation 1 may be measured by putting porous silica particles and a suspension in a cylindrical permeable membrane and putting the same suspension outside the permeable membrane, as illustrated in FIG. 34, for example.
  • the porous silica particles of the present invention are biodegradable, and can be slowly decomposed in suspension, 50 kDa in diameter corresponds to about 5 nm, and biodegradable porous silica particles can pass through a permeable membrane of 50 kDa in diameter, and a cylindrical permeable membrane is 60 rpm horizontal. Under stirring, the suspension can be mixed evenly and the degraded porous silica particles can come out of the permeable membrane.
  • the absorbance in Equation 1 may be measured, for example, under an environment in which the suspension outside the permeable membrane is replaced with a new suspension.
  • the suspension can be one that is constantly replaced, one that can be replaced every period, and the period can be periodic or irregular. For example, within the range of 1 hour to 1 week, 1 hour interval, 2 hours interval, 3 hours interval, 6 hours interval, 12 hours interval, 24 hours interval, 2 days interval, 3 days interval, 4 days interval, 7 It may be replaced at day intervals, but is not limited thereto.
  • the ratio of the absorbance to 1/2 means that the absorbance is half of the initial absorbance after t hours, which means that approximately half of the porous silica particles are decomposed.
  • the suspension may be a buffer solution, for example, at least one selected from the group consisting of phosphate buffered saline (PBS) and simulated body fluid (SBF), and more specifically, PBS.
  • PBS phosphate buffered saline
  • SBF simulated body fluid
  • T of the absorbance ratio of Equation 1 of the present invention is 1/2 or more, for example, t may be 24 to 120, for example, 24 to 96, 24 to 72, 30 within the above range To 70, 40 to 70, 50 to 65 and the like, but is not limited thereto.
  • t for example, the absorbance ratio of Equation 1 is 1/5 may be, for example, 70 to 140, for example, 80 to 140, 80 to 120, and 80 to 110 within the above range. , 70 to 140, 70 to 120, 70 to 110, and the like, but is not limited thereto.
  • t may be 130 to 220, for example, wherein the ratio of absorbance of Equation 1 is 1/20, for example, 130 to 200, 140 to 200, 140 to 180 within the above range. , 150 to 180, and the like, but is not limited thereto.
  • the porous silica particles of the present invention may have a measured absorbance of 0.01 or less, for example, 250 or more, for example, 300 or more, 350 or more, 400 or more, 500 or more, 1000 or more, and the upper limit thereof is 2000 days. May be, but is not limited thereto.
  • the ratio of the absorbance of Formula 1 and t have a high positive correlation.
  • the Pearson correlation coefficient may be 0.8 or more, for example, 0.9 or more and 0.95 or more. .
  • T in Equation 1 means how fast the porous silica particles decompose in an environment similar to the body, for example, the surface area, particle diameter, pore diameter, surface and / or inside the pores of the porous silica particles. It can be controlled by controlling the substituent, the degree of compactness of the surface, and the like.
  • the surface area of the particles can be increased to reduce t, or the surface area can be reduced to increase t.
  • the surface area can be adjusted by adjusting the diameter of the particles and the diameter of the pores.
  • substituents on the surface and / or within the pores, it is possible to increase t by reducing the direct exposure of porous silica particles to the environment (such as solvents).
  • the porous silica silica particles are directly exposed to the environment.
  • the surface may be made more densely at the time of preparation of the particles to increase t.
  • Porous silica particles of the present invention may be, for example, spherical particles, but is not limited thereto.
  • the porous silica particles of the present invention may have an average diameter of, for example, 150 nm to 1000 nm, for example, within the above range, for example, 150 nm to 800 nm, 150 nm to 500 nm, 150 nm to 400 nm, 150 nm to 300 nm, and 150 nm to 200 nm. May be, but is not limited thereto.
  • the porous silica particles of the present invention may have an average pore diameter of, for example, 1 nm to 100 nm, for example, within the above range, for example, 5 nm to 100 nm, 7 nm to 100 nm, 7 nm to 50 nm, 10 nm to 50 nm, 10 nm to 30 nm. , 7 nm to 30 nm, but is not limited thereto. Having such a large diameter can carry a large amount of antibody (cytokine) or cytokine (cytokine), it is also possible to carry a large antibody (cytokine) or cytokine (cytokine).
  • the porous silica particles of the present invention may have a BET surface area of, for example, 200 m 2 / g to 700 m 2 / g.
  • a BET surface area of, for example, 200 m 2 / g to 700 m 2 / g, 200 m 2 / g to 650 m 2 / g, 250 m 2 / g to 650 m 2 / g, 300 m 2 / g to 700 m 2 / g, 300 m 2 / g to 650m 2 / g, 300m 2 / g to 600m 2 / g, 300m 2 / g to 550m 2 / g, 300m 2 / g to 500m 2 / g, 300m 2 / g to 450m 2 / g, etc. It is not limited to this.
  • the porous silica nanoparticles of the present invention may have a volume per g, for example, 0.7 ml to 2.2 ml.
  • a volume per g for example, 0.7 ml to 2.2 ml.
  • within the above range may be 0.7ml to 2.0ml, 0.8ml to 2.2ml, 0,8ml to 2.0ml, 0.9ml to 2.0ml, 1.0ml to 2.0ml and the like, but is not limited thereto. If the volume per gram is too small, the rate of decomposition may be too high, and excessively large particles may be difficult to manufacture or may not have an intact shape.
  • the porous silica particles of the present invention may have hydrophilic substituents and / or hydrophobic substituents on the outer surface and / or inside the pores.
  • hydrophilic substituents may exist on both the surface and inside of the pores, or only hydrophobic substituents may exist, hydrophilic substituents may exist on the surface or inside of the pores, hydrophobic substituents may exist on the surface, hydrophilic substituents on the surface, and hydrophobic substituents inside the pores. It may be present and vice versa.
  • the release of an antibody or cytokine supported on the porous silica particles of the present invention is mainly carried out by decomposition of the nanoparticles, and the antibody or cytokine is controlled by the control of the substituent.
  • the interaction of the porous silica particles with respect to the release environment of is controlled to control the rate of degradation of the nanoparticles themselves, thereby controlling the release rate of the antibody or cytokine, and also the antibody or cytokine.
  • the cytokine may be diffused from the nanoparticles to be released, and the binding force of the antibody or cytokine to the nanoparticles is controlled by the control of the substituents, thereby controlling the antibody or cytokine. Release can be controlled.
  • hydrophobic substituents are present inside the pores to enhance binding to poorly soluble (hydrophobic) antibodies, cytokines, or substances, and the surface of the particles is characterized by hydrophilic substituents in terms of ease of use and formulation. May be present.
  • Hydrophilic substituents are, for example, hydroxyl groups, carboxy groups, amino groups, carbonyl groups, sulfhydryl groups, phosphate groups, thiol groups, ammonium groups, ester groups, imide groups, thiimide groups, keto groups, ether groups, indene groups, sulfonyl groups, polyethylene Glycol groups and the like
  • the hydrophobic substituent is, for example, substituted or unsubstituted C1 to C30 alkyl group, substituted or unsubstituted C3 to C30 cycloalkyl group, substituted or unsubstituted C6 to C30 aryl group, substituted Or an unsubstituted C2 to C30 heteroaryl group, a halogen group, a C1 to C30 ester group, a halogen-containing group, and the like.
  • the porous silica particles of the present invention may be one in which the outer surface and / or the inside of the pores are positively and / or negatively charged.
  • both the surface and the inside of the pore may be positively charged, or may be negatively charged, only the surface or the inside of the pore may be positively charged, or may be negatively charged, the surface may be positively charged, and the interior of the pore may be negatively charged. The opposite is also true.
  • the charging may be, for example, by the presence of a cationic substituent or an anionic substituent.
  • the cationic substituent may be, for example, an amino group, other nitrogen-containing groups, etc. as the basic group, and the anionic substituent may be, for example, a carboxy group (-COOH), a sulfonic acid group (-SO 3 H), a thiol group as an acidic group, and the like. (-SH) and the like, but is not limited thereto.
  • the interaction of the porous silica particles with respect to the release environment of the antibody or cytokine is controlled by the control of the substituent, thereby controlling the rate of decomposition of the nanoparticles themselves, thereby controlling the antibody or cytokine.
  • the release rate of the cytokine may be controlled, and the antibody or the cytokine may be released from the nanoparticles by being diffused, and by controlling the substituent, the antibody or the cytokine may be released.
  • the binding force to the nanoparticles of the can be controlled to control the release of antibody or cytokine (cytokine).
  • porous silica particles of the present invention may carry an antibody or cytokine in addition to the above, inside the surface and / or the pores, transfer the antibody or cytokine to target cells, and the like.
  • Substituents for supporting other materials or binding of other additional substituents may be present, and may further include antibodies, ligands, cell permeable peptides, or aptamers bound thereto.
  • Substituents, charges, binders and the like within the aforementioned surfaces and / or pores may be added, for example, by surface modification.
  • Surface modification can be carried out, for example, by reacting a compound having a substituent to be introduced with the particles, which may be, for example, an alkoxysilane having a C1 to C10 alkoxy group, but is not limited thereto.
  • the alkoxysilane has one or more alkoxy groups, and may have, for example, 1 to 3, and there may be a substituent to be introduced into a site where the alkoxy group is not bonded or a substituent substituted therewith.
  • the porous silica particles of the present invention may be manufactured through a small pore particle preparation and a pore expansion process, and may be manufactured through a calcination process, a surface modification process, and the like, as necessary. If both the calcination and the surface modification process has gone through may be surface modified after calcination.
  • the small pore particles may be, for example, particles having an average pore diameter of 1 nm to 5 nm.
  • the small pore particles can be obtained by adding a surfactant and a silica precursor in a solvent, stirring and homogenizing.
  • the solvent may be water and / or an organic solvent
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone, ⁇ -butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.
  • ethers such as 1,4-dioxane (particularly cyclic ethers)
  • Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachlor
  • Ketones Carbon-based aromatics such as benzene, toluene, xylene and tetramethylbenzene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether Glycol ethers (cellosolve) such as dipropylene glycol diethyl ether and triethylene glycol monoethyl ether; Dimethylacetamide (DMAc), N, N-diethylacetamide,
  • the ratio may be, for example, water and the organic solvent in a volume ratio of 1: 0.7 to 1.5, for example, 1: 1: 0.8 to 1.3, but is not limited thereto.
  • the surfactant may be, for example, cetyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium bromide (TMABr), hexadecyltrimethylpyridinium chloride (TMPrCl), tetramethylammonium chloride (TMACl), and the like, and specifically, CTAB may be used.
  • CTAB cetyltrimethylammonium bromide
  • TMABr hexadecyltrimethylammonium bromide
  • TMPrCl hexadecyltrimethylpyridinium chloride
  • TMACl tetramethylammonium chloride
  • the surfactant may be added, for example, in an amount of 1 g to 10 g, for example, 1 g to 8 g, 2 g to 8 g, 3 g to 8 g, etc., per liter of solvent, but is not limited thereto.
  • the silica precursor may be added after stirring with the addition of a surfactant to the solvent.
  • the silica precursor may be, for example, tetramethyl orthosilicate (TMOS), but is not limited thereto.
  • the stirring may be performed, for example, for 10 minutes to 30 minutes, but is not limited thereto.
  • the silica precursor may be added, for example, 0.5 ml to 5 ml per liter of solvent, for example, 0.5 ml to 4 ml, 0.5 ml to 3 ml, 0.5 ml to 2 ml, 1 ml to 2 ml, etc. within the above range, but is not limited thereto. It doesn't happen.
  • sodium hydroxide may further be used as a catalyst, which may be added with stirring after adding the surfactant to the solvent and before adding the silica precursor.
  • the sodium hydroxide may be, for example, 0.5 ml to 8 ml per liter of solvent, for example, 0.5 ml to 5 ml, 0.5 ml to 4 ml, 1 ml to 4 ml, 1 ml to 3 ml, 2 ml to 3 ml, etc., based on 1 M aqueous sodium hydroxide solution. However, it is not limited thereto.
  • the solution can be reacted with stirring.
  • the stirring may be performed for example, for 2 hours to 15 hours, for example, within the above range, for example, 3 hours to 15 hours, 4 hours to 15 hours, 4 hours to 13 hours, 5 hours to 12 hours, 6 hours to 12 hours , 6 hours to 10 hours, and the like, but is not limited thereto. If the stirring time (reaction time) is too short, nucleation may be insufficient.
  • the solution may be aged. Aging may be performed for example, from 8 hours to 24 hours, for example, within the range of 8 hours to 20 hours, 8 hours to 18 hours, 8 hours to 16 hours, 8 hours to 14 hours, 10 hours to 16 hours. , 10 hours to 14 hours, and the like, but is not limited thereto.
  • reaction product may be washed and dried to obtain porous silica particles, and if necessary, separation of unreacted material may be preceded before washing.
  • Separation of the unreacted material may be carried out by separating the supernatant, for example by centrifugation, centrifugation may be carried out, for example at 6,000 to 10,000 rpm, the time is for example 3 minutes to 60 minutes, For example, it may be performed within 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes to 30 minutes, and the like, but is not limited thereto.
  • the washing may be performed with water and / or an organic solvent, and in particular, since a substance that can be dissolved in each solvent may be different, water and an organic solvent may be used once or several times, or once or even with water or an organic solvent alone. Can be washed several times.
  • the number of times may be, for example, two or more, ten or less, for example, three or more and ten or less, four or more and eight or less, four or more and six or less.
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone, ⁇ -butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.
  • ethers such as 1,4-dioxane (particularly cyclic ethers)
  • Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichlor
  • Ketones Carbon-based aromatics such as benzene, toluene, xylene and tetramethylbenzene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether Glycol ethers (cellosolve) such as dipropylene glycol diethyl ether and triethylene glycol monoethyl ether; Dimethylacetamide (DMAc), N, N-diethylacetamide,
  • the washing may be carried out under centrifugation, for example at 6,000 to 10,000 rpm, the time being for example 3 to 60 minutes, for example 3 to 30 minutes, 3 within the above range. It may be performed in minutes to 30 minutes, 5 minutes to 30 minutes and the like, but is not limited thereto.
  • the washing may be performed by filtering out particles with a filter without centrifugation.
  • the filter may have pores less than or equal to the diameter of the porous silica particles. Filtering the reaction liquid with such a filter leaves only particles on the filter, which can be washed by pouring water and / or an organic solvent on the filter.
  • water and an organic solvent may be used alternately once or several times, and may be washed once or several times even with water or an organic solvent alone.
  • the number of times may be, for example, two or more, ten or less, for example, three or more and ten or less, four or more and eight or less, four or more and six or less.
  • the drying may be performed at 20 ° C. to 100 ° C., but is not limited thereto, and may be performed in a vacuum state.
  • the pores of the obtained porous silica particles are expanded, and the pore expansion may be performed using a pore swelling agent.
  • the pore swelling agent may be trimethylbenzene, triethylbenzene, tripropylbenzene, tributylbenzene, tripentylbenzene, trihexylbenzene, toluene, benzene, and the like, and specifically, trimethylbenzene may be used. It is not limited.
  • the pore swelling agent may use, for example, N, N-dimethylhexadecylamine (N, N-dimethylhexadecylamine, DMHA), but is not limited thereto.
  • the pore expansion can be carried out, for example, by mixing porous silica particles in a solvent with a pore swelling agent and heating to react.
  • the solvent may be, for example, water and / or an organic solvent
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone and cyclohexanone; Carbon-based aromatics such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; And the like
  • the porous silica particles are, for example, 10 g to 200 g per liter of solvent, for example, 10 g to 150 g, 10 g to 100 g, 30 g to 100 g, 40 g to 100 g, 50 g to 100 g, 50 g to 80 g, 60 g to 80 g, etc., within the above range. It may be added in a ratio of, but is not limited thereto.
  • the porous silica particles may be evenly dispersed in a solvent, for example, the porous silica particles may be added to the solvent and ultrasonically dispersed.
  • the second solvent may be added after the porous silica particles are dispersed in the first solvent.
  • the pore swelling agent is for example 10 to 200 parts by volume, 100 to 150 parts by volume, 10 to 100 parts by volume, 10 to 80 parts by volume, 30 to 80 parts by volume, 30 to 80 parts by volume based on 100 parts by volume of solvent. 70 parts by volume may be added, but is not limited thereto.
  • the reaction can be carried out, for example, at 120 ° C to 190 ° C.
  • 120 ° C to 190 ° C For example, within the range of 120 °C to 190 °C, 120 °C to 180 °C, 120 °C to 170 °C, 130 °C to 170 °C, 130 °C to 160 °C, 130 °C to 150 °C, 130 °C to 140 °C It may be performed, but is not limited thereto.
  • the reaction may be performed, for example, for 6 hours to 96 hours.
  • 6 hours to 96 hours within the range of 30 hours to 96 hours, 30 hours to 96 hours, 30 hours to 80 hours, 30 hours to 72 hours, 24 hours to 80 hours, 24 hours to 72 hours, 36 hours to 96 hours, 36 36 hours to 80 hours, 36 hours to 72 hours, 36 hours to 66 hours, 36 hours to 60 hours, 48 hours to 96 hours, 48 hours to 88 hours, 48 hours to 80 hours, 48 hours to 72 hours, 6 hours to 96 hours, 7 hours to 96 hours, 8 hours to 80 hours, 9 hours to 72 hours, 9 hours to 80 hours, 6 hours to 72 hours, 9 hours to 96 hours, 10 hours to 80 hours, 10 hours to 72 hours , 12 hours to 66 hours, 13 hours to 60 hours, 14 hours to 96 hours, 15 hours to 88 hours, 16 hours to 80 hours, 17 hours to 72 hours, and the like, but is not limited thereto.
  • the time and temperature can be adjusted within the ranges exemplified above so that the reaction can be carried out sufficiently without excess. For example, when the reaction temperature is lowered, the reaction time may be increased, or when the reaction temperature is lowered, the reaction time may be shortened. If the reaction is not sufficient, the expansion of the pores may not be sufficient, and if the reaction proceeds excessively, the particles may collapse due to the expansion of the pores.
  • the reaction can be carried out, for example, by gradually raising the temperature. Specifically, it may be carried out by gradually raising the temperature at a rate of 0.5 °C / min to 15 °C / min from the room temperature to the above temperature, for example, 1 °C / min to 15 °C / min, 3 °C / min within the above range To 15 ° C./minute, 3 ° C./minute to 12 ° C./minute, 3 ° C./minute to 10 ° C./minute, and the like, but are not limited thereto.
  • the reaction can be carried out under stirring. For example, it may be stirred at a speed of 100 rpm or more, and specifically, may be performed at a speed of 100 rpm to 1000 rpm, but is not limited thereto.
  • the reaction solution may be cooled slowly, for example, it may be cooled by gradually reducing the temperature. Specifically, it may be carried out by gradually decreasing the temperature at a rate of 0.5 °C / min to 20 °C / min from the temperature to room temperature, for example, 1 °C / min to 20 °C / min, 3 °C / min to within the above range 20 ° C./minute, 3 ° C./minute to 12 ° C./minute, 3 ° C./minute to 10 ° C./minute, and the like, but is not limited thereto.
  • reaction product After cooling, the reaction product may be washed and dried to obtain porous silica particles having expanded pores, and if necessary, separation of unreacted material may be preceded before washing.
  • Separation of the unreacted material may be carried out by separating the supernatant, for example by centrifugation, centrifugation may be carried out, for example at 6,000 to 10,000 rpm, the time is for example 3 minutes to 60 minutes, For example, it may be performed within 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes to 30 minutes, and the like, but is not limited thereto.
  • the washing may be performed with water and / or an organic solvent, and in particular, since a substance that can be dissolved in each solvent may be different, water and an organic solvent may be used once or several times, or once or even with water or an organic solvent alone. Can be washed several times.
  • the number of times may be, for example, two or more times, ten times or less, for example, three times, four times, five times, six times, seven times, eight times, and the like.
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone and cyclohexanone; Carbon-based aromatics such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; And the like, and specifically, alcohol, more specifically ethanol may be used, but is not limited
  • the washing may be carried out under centrifugation, for example at 6,000 to 10,000 rpm, the time being for example 3 to 60 minutes, for example 3 to 30 minutes, 3 within the above range. It may be performed in minutes to 30 minutes, 5 minutes to 30 minutes and the like, but is not limited thereto.
  • the washing may be performed by filtering out particles with a filter without centrifugation.
  • the filter may have pores less than or equal to the diameter of the porous silica particles. Filtering the reaction liquid with such a filter leaves only particles on the filter, which can be washed by pouring water and / or an organic solvent on the filter.
  • water and an organic solvent may be used alternately once or several times, and may be washed once or several times even with water or an organic solvent alone.
  • the number of times may be, for example, two or more, ten or less, for example, three or more and ten or less, four or more and eight or less, four or more and six or less.
  • the drying may be performed at 20 ° C. to 100 ° C., but is not limited thereto, and may be performed in a vacuum state.
  • the obtained particles may be calcined, which is a process of heating the particles to remove silanol groups on the surface and inside thereof to lower the reactivity of the particles, to have a more compact structure, and to remove organic substances filling the pores.
  • it may be heated to a temperature of 400 °C or more.
  • the upper limit thereof is not particularly limited, and may be, for example, 1000 ° C, 900 ° C, 800 ° C, 700 ° C, or the like. Heating can be carried out for example for 3 hours or more.
  • the upper limit is not particularly limited and may be, for example, 24 hours, 12 hours, 10 hours, 8 hours, 6 hours, or the like. More specifically, it may be performed for 3 hours to 8 hours at 400 ° C to 700 ° C, specifically 4 hours to 5 hours at 500 ° C to 600 ° C, but is not limited thereto.
  • the porous silica particles obtained can then be surface modified, and the surface modification can be carried out on the surface and / or inside the pores.
  • the particle surface and the inside of the pore may be surface-modified identically or may be surface-modified differently.
  • the surface modification can cause the particles to charge or to have hydrophilic and / or hydrophobic properties.
  • At least one selected from the group consisting of an amino group, an aminoalkyl group, an alkylamino group, a heterocyclic aromatic compound group containing a nitrogen atom, a cyan group and a guanidine group for effective loading of an antibody or cytokine By having a substituent of, the surface modification of the porous silica particles can be carried out.
  • Surface modification can be carried out, for example, by reacting a compound having substituents such as hydrophilic, hydrophobic, cationic, anionic and the like to be introduced with the particles, and the compound can be, for example, an alkoxysilane having a C1 to C10 alkoxy group. However, it is not limited thereto.
  • the alkoxysilane has one or more alkoxy groups, and may have, for example, 1 to 3, and there may be a substituent to be introduced into a site where the alkoxy group is not bonded or a substituent substituted therewith.
  • the alkoxysilane reacts with the porous silicon particles, a covalent bond is formed between the silicon atom and the oxygen atom so that the alkoxysilane may be bonded to the surface and / or the inside of the pores of the porous silicon particle, and the alkoxysilane has a substituent to be introduced.
  • the corresponding substituents may be introduced into the surface of the porous silicon particles and / or within the pores.
  • the reaction may be carried out by reacting porous silica particles dispersed in a solvent with an alkoxysilane.
  • the solvent may be water and / or an organic solvent
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone, ⁇ -butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.
  • ethers such as 1,4-dioxane (particularly cyclic ethers)
  • Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachlor
  • Ketones Carbon-based aromatics such as benzene, toluene, xylene and tetramethylbenzene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether Glycol ethers (cellosolve) such as dipropylene glycol diethyl ether and triethylene glycol monoethyl ether; Dimethylacetamide (DMAc), N, N-diethylacetamide,
  • the charge to the positive charge can be carried out by reacting with an alkoxysilane having a basic group such as a nitrogen-containing group such as an amino group, an aminoalkyl group, for example.
  • an alkoxysilane having a basic group such as a nitrogen-containing group such as an amino group, an aminoalkyl group, for example.
  • Charging to the negative charge may be carried out by reacting with an alkoxysilane having an acidic group such as, for example, a carboxyl group, a sulfonic acid group, a thiol group, and the like.
  • an alkoxysilane having an acidic group such as, for example, a carboxyl group, a sulfonic acid group, a thiol group, and the like.
  • 3-Mercaptopropyl) trimethoxysilane may be used, but is not limited thereto.
  • the hydrophilic property is a hydrophilic group such as hydroxy group, carboxy group, amino group, carbonyl group, sulfhydryl group, phosphate group, thiol group, ammonium group, ester group, imide group, thiimide group, keto group, ether group, indene group, sulfo It may be made to react with the alkoxysilane which has a silyl group, a polyethyleneglycol group, etc.
  • the hydrophobic nature may include hydrophobic substituents such as substituted or unsubstituted C1 to C30 alkyl groups, substituted or unsubstituted C3 to C30 cycloalkyl groups, substituted or unsubstituted C6 to C30 aryl groups, substituted or unsubstituted It can be made to react with the alkoxysilane which has a C2-C30 heteroaryl group, a halogen group, C1-C30 ester group, a halogen containing group, etc.
  • Trimethoxy (octadecyl) silane, Trimethoxy-n-octylsilane, Trimethoxy (propyl) silane, Isobutyl (trimethoxy) silane, Trimethoxy (7-octen-1-yl) silane, Trimethoxy (3,3,3-trifluoropropyl) Silane, Trimethoxy (2-phenylethyl) silane, Vinyltrimethoxysilane, Cyanomethyl, 3- (trimethoxysilyl) propyl] trithiocarbonate, (3-Bromopropyl) trimethoxysilane, etc. may be used, but is not limited thereto.
  • hydrophobic substituents are present in the pores to enhance binding to poorly soluble (hydrophobic) antibodies, cytokines or substances through the surface modification, and particles in terms of ease of use and formulation.
  • the surface of may also be treated such that a hydrophilic substituent is present, and a substituent may be present on the surface to bind other antibodies, cytokines, or substances.
  • the surface modification may be carried out in combination.
  • two or more surface modifications may be performed on the outer surface or inside the pores.
  • a compound including a carboxyl group may be bonded to silica particles into which amino groups are introduced by amide bonds to change the positively charged particles to have different surface properties, but is not limited thereto.
  • the reaction of the porous silica particles with the alkoxysilane can be carried out, for example, under heating, and the heating is for example from 80 ° C. to 180 ° C., for example from 80 ° C. to 160 ° C., from 80 ° C. to 150 ° C. within the above range. , 100 ° C. to 160 ° C., 100 ° C. to 150 ° C., 110 ° C. to 150 ° C., etc., but is not limited thereto.
  • the reaction of the porous silica particles with the alkoxysilane is, for example, 4 hours to 20 hours, for example, 4 hours to 18 hours, 4 hours to 16 hours, 6 hours to 18 hours, 6 hours to 16 hours within the above range. , 8 hours to 18 hours, 8 hours to 16 hours, 8 hours to 14 hours, 10 hours to 14 hours, etc., but is not limited thereto.
  • the reaction temperature, time, and the amount of the compound used for surface modification may be selected according to the degree to which the surface is to be modified, and the degree of hydrophilicity, hydrophobicity, and charge of an antibody, cytokine, or materials may be selected. Accordingly, by controlling the hydrophilicity, hydrophobicity, and charge of the porous silica particles by varying the reaction conditions, the release rate of the antibody, cytokine, or substances can be controlled. For example, if antibodies, cytokines or substances have a strong negative charge at neutral pH, the reaction temperature may be increased or the reaction time may be increased in order for the porous silica particles to have a strong positive charge. It is possible to increase the compound throughput, but is not limited thereto.
  • porous silica particles of the present invention may be produced through, for example, the preparation of small pores, pore expansion, surface modification, and internal pore modification.
  • the small pore particle production and pore expansion process may be based on the above-described process, and the washing and drying process may be performed after the small pore particle production and after the pore expansion process.
  • separation of the unreacted material may be preceded before washing, and separation of the unreacted material may be performed by separating the supernatant, for example, by centrifugation.
  • the centrifugation may be performed, for example, at 6,000 to 10,000 rpm, and the time may be, for example, 3 to 60 minutes, specifically, 3 to 30 minutes, 3 to 30 minutes, and 5 minutes within the above range. To 30 minutes, etc., but is not limited thereto.
  • the washing after the preparation of the particles of the small pores may be performed by a method / condition within the above-described range, but is not limited thereto.
  • the washing after the pore expansion may be performed under more relaxed conditions than the above example.
  • washing may be performed within three times, but is not limited thereto.
  • the surface modification and internal pore modification may be by the processes described above, respectively, the process may be performed in the order of surface modification and internal pore modification, and the washing process of the particles may be further performed between the two processes. Can be.
  • the reaction solution such as a surfactant used for particle production and pore expansion is filled in the pores so that the inside of the pores is not modified during surface modification. Only the surface can be modified. Then, washing the particles may remove the reaction solution in the pores.
  • Particle washing between the surface modification and the internal pore reforming process may be water and / or an organic solvent, and in particular, water and an organic solvent may be alternately used once or several times because different materials may be dissolved for each solvent.
  • Water or organic solvents alone may be washed once or several times. The number of times may be, for example, two or more, ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less.
  • the washing may be carried out under centrifugation, for example at 6,000 to 10,000 rpm, the time being for example 3 to 60 minutes, specifically 3 to 30 minutes, 3 within the above range. It may be performed in minutes to 30 minutes, 5 minutes to 30 minutes and the like, but is not limited thereto.
  • the washing may be performed by filtering out particles with a filter without centrifugation.
  • the filter may have pores less than or equal to the diameter of the porous silica particles. Filtering the reaction liquid with such a filter leaves only particles on the filter, which can be washed by pouring water and / or an organic solvent on the filter.
  • water and an organic solvent may be used alternately once or several times, and may be washed once or several times even with water or an organic solvent alone.
  • the number of times may be, for example, two or more, ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less.
  • the drying may be performed at 20 ° C. to 100 ° C., but is not limited thereto, and may be performed in a vacuum state.
  • the antibody or cytokine may be supported on the surface and / or inside the pores of the porous silica particles, and the support may be mixed with, for example, the porous silica particles and the antibody or cytokine in a solvent. Can be performed.
  • the solvent may be water and / or an organic solvent
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone and cyclohexanone; Carbon-based aromatics such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Etc. can be used.
  • PBS phosphate buffered saline solution
  • SBF Simulated Body Fluid
  • Borate-buffered saline Borate-buffered saline
  • Tris-buffered saline may be used as the solvent.
  • the ratio of the porous silica particles and the oligonucleotide of the present invention is not particularly limited, for example, the weight ratio is 1: 0.05 to 0.8, for example within the above range 1: 0.05 to 0.7, 1: 0.05 to 0.6, 1: 0.1 to 0.8, 1: 0.1 to 0.6, 1: 0.2 to 0.8, 1: 0.2 to 0.6, and the like.
  • Antibodies or cytokines supported on the porous silica particles may be gradually released over an extended time. Such slow release may be continuous or discontinuous, linear or nonlinear, and may vary due to the characteristics of the porous silica particles and / or their interaction with antibodies or cytokines.
  • Antibodies or cytokines supported on the porous silica particles are released as the porous silica particles are biodegraded, and the porous silica particles according to the present invention are gradually decomposed and supported on an antibody or cytokine. This can be released slowly. This may be controlled by, for example, adjusting the surface area, particle diameter, pore diameter, substituents on the surface and / or pores, degree of compactness of the porous silica particles, and the like, but are not limited thereto.
  • the antibody or cytokine supported on the porous silica particles may be released while being diffused from the porous silica particles, which may be released. It is affected by the relationship with the release environment of the antibody or cytokine (cytokine), it can be controlled by controlling the release of the antibody (cytokine) or cytokine (cytokine). For example, it can be controlled by strengthening or weakening the binding strength of the porous silica particles to the antibody or cytokine by surface modification.
  • the surface of the particles and / or the inside of the pores may have a hydrophobic substituent, so that the porous silica particles and the antibody
  • the cytokine (cytokine) or the binding force with the substance may be increased, whereby the antibody (cytokine) or the substance (cytokine) or the substance may be released in a sustained manner.
  • This may be, for example, the surface-modified porous silica particles with an alkoxysilane having a hydrophobic substituent.
  • “poorly soluble” means to be insoluble (practically insoluble) or only slightly soluble (with respect to water), which means “Pharmaceutical Science” 18 th Edition ( USP, Remington, Mack Publishing Company).
  • the poorly water-soluble material may be, for example, water solubility of less than 10 g / L, specifically less than 5 g / L, more specifically less than 1 g / L at 1 atmosphere and 25 ° C., but is not limited thereto.
  • the surface and / or the inside of the pores have a hydrophilic substituent which has a porous silica particle and an antibody, cytokine or The binding force with the substance may be increased, whereby an antibody, cytokine or substance may be released in a sustained manner.
  • This may be, for example, the surface of the porous silica particles modified with an alkoxysilane having a hydrophilic substituent.
  • the water-soluble substance may have a water solubility of 10 g / L or more at 1 atmosphere and 25 ° C., but is not limited thereto.
  • the surface of the particles and / or the interior of the pores are charged with opposite charges so that the porous silica particles, antibodies, and cytokines ), Or the binding force with the substance may be increased, whereby an antibody, cytokine or substance may be released in a sustained manner.
  • This may be, for example, the surface-modified porous silica particles with an alkoxysilane having an acidic group or a basic group.
  • porous silica particles may be surface-modified with an alkoxysilane having an acidic group such as a carboxyl group (-COOH) and a sulfonic acid group (-SO 3 H).
  • the surface of the particles and / or the inside of the pores may be positively charged, whereby the porous silica particles and the antibody (antibody) ), Cytokines (cytokine) or the binding force with the substance is increased, the antibody (cytokine) or the substance can be released in a sustained release.
  • the porous silica particles may be surface-modified with an alkoxysilane having a basic group such as an amino group or another nitrogen-containing group.
  • Antibodies, cytokines or substances may be released for a period of, for example, 7 days to 1 year or more, depending on the type of treatment required, the release environment, and the porous silica particles used.
  • porous silica particles of the present invention are 100% degradable as biodegradable, the antibody, cytokine or substance supported thereon may be released 100%.
  • the present invention provides a composition for immunotherapy comprising the carrier described above.
  • Immunotherapy refers to the planning of patient treatment by immunological methods, which include specific immunotherapy involving only the immune response to specific antigens and nonspecific immunotherapy that is effective for the entire immune system but not limited to specific antigens.
  • the method of administering an antibody as said specific therapy is the typical example of the method of administering cytokines as said non-specific method.
  • the immunotherapeutic composition of the present invention comprises porous silica particles carrying an immunoreactive substance containing the above-described antibody or cytokine, and delivers the supported immunoreactive substance to the body stably and sustainably, and immunoreactive substance.
  • porous silica particles carrying an immunoreactive substance containing the above-described antibody or cytokine and delivers the supported immunoreactive substance to the body stably and sustainably, and immunoreactive substance.
  • it has an effective superiority to reduce the inherent side effects, it can be used in immunotherapy to exert an excellent effect in the prevention or treatment of various cancers or immune diseases.
  • the cancer includes carcinoma including skin cancer including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, cervical cancer, thyroid cancer and squamous cell carcinoma; Lymphoid hematopoietic tumors including leukemia, acute lymphocytic leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Bucket lymphoma; Myeloid hematopoietic tumors, including acute and chronic myeloid leukemia and promyelocytic leukemia; Mesenchymal-derived tumors including fibrosarcoma and rhabdomyosarcoma; Other tumors including melanoma, normal carcinoma, teratocarcinoma, neuroblastoma and glioma; Tumors of the central and peripheral nervous system,
  • the immune disease refers to a disease in which components of the mammalian immune system cause, mediate or otherwise contribute to a mammalian pathology, and all diseases in which stimulation or interruption of an immune response has a compensatory effect on the progression of the disease. And may include all autoimmune diseases, infectious diseases, inflammatory diseases or transplant rejection diseases of cells, tissues or organs, and the like, specifically, Behcet's disease, multiple myositis / skin myositis, autologous diseases Immunocytopenia, Autoimmune myocarditis, Atopic dermatitis, Asthma, Primary cirrhosis, Dermatitis, Goodfiction syndrome, Autoimmune meningitis, Obesity, Sjogren's syndrome, Ankylosing myelitis, Systemic lupus erythematosus, Addison's disease, Alopecia areata, Autoimmunity Hepatitis, autoimmune mumps, Crohn's disease, insulin dependent diabetes mellitus, dystrophic
  • Autoimmune diseases as one type of the immune disease is not limited to the type, Crohn's disease, erythema, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Edison's disease, type 1 diabetes, Lupus, chronic fatigue syndrome, fiber Myalgia, hypothyroidism and hyperplasia, scleroderma, Behcet's disease, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's syndrome, Guilian-Barre syndrome, Sjogren's syndrome, vitiligo Endometriosis, psoriasis, systemic scleroderma, asthma or ulcerative colitis.
  • the infectious disease may be an infectious disease caused by bacteria, parasites, fungi, viruses, viroids and prions.
  • the virus may be enterovirus, rotorvirus, adenovirus and hepatitis virus, and in addition to the virus, the retroviral family (e.g., HIV-I (also HTLV-III, LAV or HTLV-III / LAV, or HIV-III) Human immunodeficiency virus such as; and other isolates such as HIV-LP; Picornaviridae (eg, polio virus, hepatitis A virus, enterovirus, human Coxsackie virus, rhinovirus, echovirus); Calciviridae (eg, species responsible for gastroenteritis); Togaviridae (eg, equine encephalitis virus, rubella virus); Flaviviridae (eg, dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (eg, coronavirus); Rhabdoviridae (eg, vesicular stomatitis virus, rabies virus); Phyllovirus family (eg,
  • Pox virus and Iridoviridae (e.g. African swine fever virus); and unclassified Viruses (for example delta hepatitis (presumed to be a satellite deficient in hepatitis B), hepatitis C); Norwalk and related viruses, and astroviruses ( preparations of astro virus), but are not limited thereto.
  • Iridoviridae e.g. African swine fever virus
  • unclassified Viruses for example delta hepatitis (presumed to be a satellite deficient in hepatitis B), hepatitis C); Norwalk and related viruses, and astroviruses ( preparations of astro virus), but are not limited thereto.
  • the present invention also provides a pharmaceutical composition for the prophylaxis or treatment of cancer or immune disease, including the above-mentioned carrier.
  • composition of the present invention has a prophylactic or therapeutic effect of cancer or immune disease, which stably delivers the supported antibody or cytokine to the body and releases it to the target in a slow manner, thereby inhibiting the growth of cancer (or tumor). And the effect achieved by suppressing the transition.
  • composition of the present invention may further comprise a pharmaceutically acceptable carrier, and may be formulated with the carrier.
  • pharmaceutically acceptable carrier refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound.
  • Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and biocompatible, which include saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary.
  • Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • composition of the present invention is applicable to any formulation containing the carrier of the present invention as an active ingredient, and can be prepared in oral or parenteral formulations.
  • Pharmaceutical formulations of the present invention may be oral, rectal, nasal, topical (including the cheek and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous). And forms suitable for administration by inhalation or insufflation.
  • composition of the present invention is administered in a pharmaceutically effective amount. Effective dose levels depend on the type of disease, severity, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent medications, and other factors well known in the medical field. Can be determined.
  • the pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.
  • the dosage of the composition of the present invention varies widely depending on the weight, age, sex, health condition, diet, time of administration, administration method, excretion rate and severity of the disease, and the appropriate dosage is, for example, Depending on the amount of drug accumulated in the body and / or the specific efficacy of the carrier of the invention used. It can be calculated on the basis of EC50, which is generally determined to be effective in in vivo animal models and in vitro, for example from 0.01 ⁇ g to 1 g per kg of body weight, in unit periods of daily, weekly, monthly or yearly It may be administered once or several times per unit period, or may be continuously administered for a long time using an infusion pump. The number of repeated doses is determined in consideration of the time the drug stays in the body, the drug concentration in the body, and the like. Even after treatment according to the course of the disease treatment, the composition can be administered for relapse.
  • composition of the present invention may further contain a compound which maintains / increases the solubility and / or absorption of one or more of the active ingredients or the active ingredients exhibiting the same or similar function in the treatment of cancer. It may also optionally further comprise chemotherapeutic agents, anti-inflammatory agents, antiviral agents and / or immunomodulators and the like.
  • compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
  • the formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.
  • porous silica particles of the present invention may be abbreviated as 'DegradaBALL or DDV'
  • the DegradaBALL loaded with IL-2 may be abbreviated as 'BALLkine-2'.
  • peri-tumoral injection may be abbreviated as pt, intra-peritoreal injection as ip, subcutaneous injection as sc, and intravenous injection as iv. .
  • Example 1 Porous Silica Particles (DDV or DegradaBALL)
  • reaction solution was then centrifuged at 8000 rpm for 10 minutes at 25 ° C. to remove the supernatant, centrifuged at 8000 rpm for 10 minutes at 25 ° C., and washed five times with alternating ethanol and distilled water.
  • the reaction was carried out starting at 25 ° C. and warming up at a rate of 10 ° C./min, then slowly cooling at a rate of 1-10 ° C./min in the autoclave.
  • the cooled reaction solution was centrifuged at 8000 rpm for 10 minutes at 25 ° C. to remove the supernatant, and centrifuged at 8000 rpm for 10 minutes at 25 ° C. and washed five times with ethanol and distilled water.
  • porous silica particles prepared in 2) were placed in a glass vial, heated at 550 ° C. for 5 hours, and cooled to room temperature after completion of the reaction to prepare particles.
  • Porous silica particles were prepared in the same manner as in 1-1- (1), except that the reaction conditions at the time of pore expansion were changed to 140 ° C. and 72 hours.
  • Porous silica particles were prepared in the same manner as in Example 1-1- (1), except that a 5-fold large container was used, and each material was used in 5-fold volume.
  • Porous silica particles were prepared in the same manner as in 1-1- (1), except that 920 ml of distilled water and 850 ml of methanol were used to prepare the small pore particles.
  • Porous silica particles were prepared in the same manner as in 1-1- (1), except that 800 ml of distilled water, 1010 ml of methanol, and 10.6 g of CTAB were used to prepare the small pore particles.
  • Porous silica particles were prepared in the same manner as in 1-1- (1), except that 620 ml of distilled water, 1380 ml of methanol, and 7.88 g of CTAB were used to prepare the small pore particles.
  • Porous silica particles were prepared in the same manner as in 1-1- (1), except that 2.5 mL of TMB was used for pore expansion.
  • Porous silica particles were prepared in the same manner as in 1-1- (1), except that 4.5 mL of TMB was used for pore expansion.
  • Porous silica particles were prepared in the same manner as in 1-1- (1), except that 11 mL of TMB was used for pore expansion.
  • Porous silica particles were prepared in the same manner as in 1-1- (1), except that 12.5 mL of TMB was used for pore expansion.
  • Example 1-1- (1) -2 In the same manner as in Example 1-1- (1) -2), the small pore particles were reacted with TMB, cooled, and centrifuged to remove the supernatant. Thereafter, centrifuged under the same conditions as in Example 1-1- (1) -2), washed three times with alternating ethanol and distilled water, and then dried under the same conditions as in Example 1-1- (1) -2). Powdery porous silica particles (pore diameter 10-15 nm, particle diameter 200 nm) were obtained.
  • N-Hydroxysuccinimide 200 mg was dispersed in 30 mL of PBS and allowed to react for 12 hours while stirring at room temperature. The product is then washed and dried.
  • reaction solution of the previous step remains inside the pore, so that the inside of the pore is not modified.
  • the cooled reaction solution was centrifuged at 8000 rpm for 10 minutes to remove the supernatant, centrifuged at 8000 rpm for 10 minutes at 25 ° C, and washed five times with alternating ethanol and distilled water.
  • Example 1-1- (4) The porous silica particles of Example 1-1- (4) were reacted with (3-Aminopropyl) triethoxysilane (APTES) to charge with a positive charge.
  • APTES (3-Aminopropyl) triethoxysilane
  • porous silica particles in a 100 mL round bottom flask was dispersed in 10 mL of toluene with a bath sonicator. Then 1 mL of APTES was added and stirred at 400 rpm and stirred at 130 ° C. for 12 hours.
  • Example 1-1- (1) The porous silica particles of Example 1-1- (1) were charged with positive charge by reacting with (3-Aminopropyl) triethoxysilane (APTES), except that 0.4 ml of APTES was added and the reaction time was 3 hours.
  • APTES (3-Aminopropyl) triethoxysilane
  • Example 1-1- (9) The porous silica particles of Example 1-1- (9) were charged with positive charge by reacting with (3-Aminopropyl) triethoxysilane (APTES), and the other method was the method of 9-2- (1) -1). Modified in the same manner as
  • Example 1-1- (10) were charged with positive charge by reacting with (3-Aminopropyl) triethoxysilane (APTES), and were modified in the same manner as in the method of 9-2- (1) -1). It was.
  • APTES (3-Aminopropyl) triethoxysilane
  • Example 1-1- (1) The porous silica particles of Example 1-1- (1) were reacted with Trimethoxy (propyl) silane to introduce a propyl group into the surface and the pores, and 0.35ml of Trimethoxy (propyl) silane was added instead of APTES, followed by reaction for 12 hours. Modification was carried out in the same manner as in Example 1-2- (1) except for the above.
  • Example 1-1- (1) The porous silica particles of Example 1-1- (1) were reacted with Trimethoxy-n-octylsilane to introduce propyl groups into the surface and the pores, and 0.5 ml of Trimethoxy-n-octylsilane was added instead of APTES, followed by 12 hours of reaction. Modification was carried out in the same manner as in Example 1-2- (1) except for the above.
  • the porous silica particles of Example 1-1- (1) were charged with negative charge by reacting with succinic anhydride,
  • DMSO Dimethyl sulfoxide
  • 80 mg of succinic anhydride was added instead of APTES, and reacted at room temperature for 24 hours, except that DMSO was used instead of distilled water.
  • the modification was carried out in the same manner as in the method of -2- (1) -1).
  • Example 1-2- (1) -1) It was modified in the same manner as in Example 1-2- (1) -1) except that 1.1 mL of MPTES was used instead of APTES.
  • Example 1-2- (3) -2 100 mg of the porous silica nanoparticles of Example 1-2- (3) -2) were dispersed in 1 mL of 1 M aqueous sulfuric acid solution and 20 mL of 30% hydrogen peroxide solution, and stirred at room temperature to induce an oxidation reaction. Oxidized to a group. After the same washing and drying in the same manner as in Example 1-2- (1) -1).
  • FIG. 1 is a photograph of porous silica particles of Example 1-1- (1)
  • FIG. 2 is a photograph of porous silica particles of Example 1-1- (2). You can see that it was created
  • Figure 3 is a photograph of the small pore particles of Example 1-1- (1)
  • Figure 4 is a comparison photograph of the small pore particles of Example 1-1- (1) and 1-1- (3), spherical It can be seen that the small pore particles of evenly generated.
  • the surface area and pore volume of the small pore particles of Example 1-1- (1) and the porous silica particles of Examples 1-1- (1), (7), (8) and (10) were calculated.
  • the surface area was calculated by Brunauer-Emmett-Teller (BET) method, and the pore size distribution was calculated by Barrett-Joyner-Halenda (BJH) method.
  • Example 1-1- (1) 2.1 1337 0.69
  • Example 1-1- (7) 4.3 630 0.72
  • Example 1-1- (8) 6.9 521 0.79
  • Example 1-1- (1) 10.4 486 0.82
  • Example 1-1- (10) 23 395 0.97
  • porous silica particles are biodegraded and nearly decomposed after 360 hours.
  • a 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles into a cylindrical permeable membrane having pores having a diameter of 50 kDa,
  • a t is the absorbance of the porous silica particles measured after t hours have elapsed since the measurement of A 0 ).
  • porous silica particle powder was dissolved in 5 ml of SBF (pH 7.4). Thereafter, 5 ml of the porous silica particle solution was placed in a permeable membrane having pores having a diameter of 50 kDa shown in FIG. 7. 15 ml of SBF was added to the outer membrane, and the SBF of the outer membrane was replaced every 12 hours. Decomposition of the porous silica particles was performed at 37 ° C. with 60 rpm horizontal stirring.
  • porous silica particles of the example have a significantly larger t than the control.
  • t which is a ratio of absorbance 1/2
  • t which has a ratio of absorbance 1/2 of the positively charged particles, was 24 or more.
  • IgG is released in both SBF and PBS in a sustained manner, and it can be seen that IgG, PD-1, and PD-L1 are released to almost 100% over 250 hours or more.
  • Porous silica particles bearing FITC-bound cytokines were resuspended in SBF (pH 7.4) at 37 ° C. and then observed for a predetermined period (1, 2, 3, 4, 5, 6, 7 days). The solution was centrifuged at 8,000 rpm for 10 minutes and the release of cytokines was confirmed by fluorescence intensity spectra of FITC.
  • IL-10, IL-15, HGF, EGF, IL-2 are all released slowly, it can be seen that the release to almost 100% over 7 hours or more.
  • porous silica particles of the present invention 31.25, 62.5, 125, and 250 mg / kg of porous silica particles (DegradaBALL) were respectively administered to all mice (four per group), and 5 days after the administration. Mice were sacrificed to separate major organs (liver, spleen, lung, heart and kidney).
  • porous silica particles of the present invention 0.5, 5, 50 mg / kg of porous silica particles (DegradaBALL) were added up to 9 times every 3 or 4 days to all mice (8 per group). Subcutaneously, 48 hours after the last administration, all mice were sacrificed to isolate the major organs (liver, spleen, lung, heart and kidney).
  • the porous silica particles of the present invention are organs (liver, spleen, kidney, thymus, heart, Lung and lymph nodes) did not induce significant weight loss (data is expressed as mean ⁇ standard deviation; significant difference from normal group ( * p ⁇ 0.05)) and did not affect the various parameters obtained by whole blood analysis and serum biochemical analysis.
  • ALT Alanine transaminase
  • AST aspartate transaminase
  • ALP alkaline phosphatase
  • GGT gamma-glutamyltransferase
  • MUSST myeloid U937 skin sensitivity test
  • the fluorescence intensity of PD-L1 antibody and DDV can be measured. It can be seen that DDV and anti-PD-L1 antibody remain at the administration site for a long time, which is the anti-PD- loaded carrier of the present invention.
  • the L1 antibody can be delivered stably, and it can be long-lived in the tumor and at the site of administration, demonstrating that it can have long-term efficacy.
  • IL-2 and DDV after labeling Cy7 and Cy5 to IL-2 and DDV, and subcutaneously administering DDV (BALLkine-2) carrying IL-2 (SC injection), IL-2 and DDV at each hour. It can be confirmed that the result of measuring the fluorescence intensity of, DDV and BALLkine-2 can be confirmed that it remains at the administration site for a long time, which proves that the present invention can be delivered stably delivered with the IL-2.
  • FITC fluorescence was added to IL-2, TAMRA fluorescence was applied to DDV, and subcutaneously administered. After 24 hours, lectin labeled with Cy5 fluorescence was injected into the tail vein. After staining the blood vessels, the tumor was extracted to confirm the results of the fluorescence image. It can be seen that DDV and BALLkine-2 are specifically distributed in the tumor tissue, which indicates IL-2 carrying the carrier of the present invention. To demonstrate tumor-specific targeting and delivery.
  • FIG. 52 shows histological cross-sectional images of tumor tissue showing tumor cells (DAPI), vehicle (TAMRA), IL-2 (FITC) and dylight 649 conjugated lectin
  • FIG. 53 shows BALLkine-2 (pt) ) And IL-2 (ip or pt) intratumoral pharmacokinetics (graphs represent mean ⁇ standard deviation of 3 mice per measurement time), followed by subcutaneous injection to determine the amount of IL-2 in the tumor
  • DAPI tumor cells
  • TAMRA vehicle
  • IL-2 FITC
  • FIG. 53 shows BALLkine-2 (pt) ) And IL-2 (ip or pt) intratumoral pharmacokinetics (graphs represent mean ⁇ standard deviation of 3 mice per measurement time), followed by subcutaneous injection to determine the amount of IL-2 in the tumor
  • the porous silica particles of the present invention can keep IL-2 in the tumor and the administration site for a long time, and stably deliver IL-2.
  • Figures 54 and 55 show subcutaneous infusion of 1xPBS buffer (100 ⁇ L) and Cy5 conjugated DegradaBALL (2.5 mg / kg) and sacrifice of mice at 1, 3 and 5 days after administration to organs (skin, thymus, spleen and lymph nodes). After separation), the results of photographing the fluorescence image of the organ by the FOBI imaging system can be confirmed, which can be confirmed through the fluorescence image to be distributed to the immune organs on day 1, 3, 5, the porous silica of the present invention It is to prove that the particles can carry a variety of immunoreactive substances to the immune system stably.
  • mice normal and melanoma tumor mice were injected subcutaneously or around tumors, respectively.
  • FIG. 57 all mice were injected subcutaneously with DegradaBALL with TAMRA at doses of 2.5, 10, and 50 mg / kg, respectively, and skin tissues were extracted at 1, 3, 5, and 7 days later to fluoresce.
  • the porous silica particles of the present invention have a high in vivo stability, and thus can carry the sustained sustained release of the immunoreactive substance stably for a long time. Imply that there is.
  • Dosing Group Dosing Protocol Total capacity Buffer i.p 7 injections every 3 days (0,3,6,9,12,15,18 days)
  • aPD-1 antibody 70 mg / kg IL-2: 10 mg / kg aPD-1 antibody (i.p) + IL-2
  • aPD-1 antibody 7 intraperitoneal injections every 3 days (0,3,6,9,12,15,18 days) IL-2: 4 mass peripher
  • TIL assays were isolated from mice on the target day (1,3,5,7 days after BALLkine-2 administration) to activate immune cell populations ((b) CD8 + T cells, (c) CD4 + T cells, (d) activation).
  • NK cells, (e) Treg cells, (f) CD8 + / Treg cells) were analyzed by flow cytometry (dots represent individual tumor tissues, lines represent the average of tumor tissues of 8 mice), 3, 5 days Analyzes were performed by staining the immune tissues of CD8 + T cells at the time point. Referring to the representative image of the BALLkine-2 administration group (FIG. 70), the number of CD8 + T cells increased dramatically and it was confirmed that they were distributed among a wide range of tumor cells. Can be.
  • spleen and drainage lymph nodes (iliac and inguinal lymph nodes) were isolated on days 1 and 3 and 5 after BALLkine-2 was injected into the right flank of mice with B16F10 melanoma tumors.
  • CD8 + T cells and NK cells were isolated to evaluate immune cell populations (data is expressed as mean ⁇ standard deviation of three mice), indicating that the number of immune cells compared to control group was significantly increased in BALLkine-2 administration group.
  • kidney size of BALLkine-2 was significantly smaller than that of other groups, and H & E staining of the kidney tissue of FIG. It can be seen that the degree is significantly higher.
  • wet lungs were harvested and weighed, and then dried in a 60 ° C. oven for 3 days and then weighed (FIG. 81, data are mean ⁇ SD, 3 mice per group, # #p ⁇ 0.01: significantly different than control group (Buffer group), ** p ⁇ 0.01: significantly different group than IL-2 group), the weight ratio of wet and dry lungs compared to IL-2 group in BALLkine-2 group It was found to be significantly lower.
  • mice results of analysis of leukocytes, neutrophils and lymphocytes in the blood of mice (FIG. 82, data are mean ⁇ SD, 3 mice per group, #p ⁇ 0.05: significantly different from the control group (Buffer administration group), * p ⁇ 0.05: Significantly different from IL-2 administration group), the number of BALLkine-2 treatment group showed significantly higher level than the other groups, and thus the remarkable excellence in the immune response promoting effect was confirmed.
  • the experiment was carried out according to the administration schedule of FIG. 83 and the CLS analysis protocol. Specifically, three days after the administration of IL-2 (15 ⁇ g) and BALLkine-2 (15 ⁇ g) to C57BL / 6 mice, the analysis was performed on the fourth day. It was. Referring to a representative H & E staining image of mouse lung tissue administered buffer (iv or sc), DegradaBALL (iv or sc), IL-2 (iv or sc) or BALLkine-2 (iv or sc) (FIG.
  • the number of inflammatory cells in and around the blood vessels was reduced compared to the IL-2 (iv or sc) administration group (the rod indicates 200 ⁇ m).
  • wet lungs were separated and weighed to evaluate pulmonary edema, and lungs were dried and weighed for 3 days in an oven at 60 ° C. (FIG. 85), IL-2 (iv or sc) in the BALLkine-2 administration group. It was confirmed that the weight ratio (wet lung weight / dry weight) of the dry lung to the wet lung was significantly lower.
  • mice were injected 2 hours before sacrificing Evans blue dye through the tail vein, and after sacrifice, the liver (Fig.
  • Vascular Leakage Syndrome (VLS) analysis was performed in a tumor bearing mouse model according to the protocol of FIG. 89 to determine if vascular leakage syndrome could be reduced even in actual cancer immunotherapy.
  • VLS Vascular Leakage Syndrome
  • wet lungs were separated and weighed, and lungs were dried and weighed for 3 days in an oven at 60 ° C. (FIG. 90), compared to IL-2 (iv or sc) -administered groups in the BALLkine-2 administration group. The weight ratio was significantly lower and showed similar values to the normal group.
  • mice injected 2 hours before sacrificing Evans blue dye through the tail vein, and then separated the liver ( Figure 91) and lung ( Figure 92) and measured VLS through extravasation of Evans Blue It was confirmed that the BALLkine-2 administration group was significantly lower than the IL-2 (iv or sc) administration group.

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Abstract

Un support immunoréactif de la présente invention a un excellent effet immunothérapeutique en administrant de manière stable divers immunoréactifs comprenant des anticorps et des cytokines à un site cible. Par conséquent, une composition comprenant un support de la présente invention peut être utilisée pour l'immunothérapie, ayant ainsi d'excellents effets dans la prévention ou le traitement du cancer ou de diverses maladies immunitaires.
PCT/KR2019/006682 2018-08-06 2019-06-03 Support immunoréactif WO2020032366A1 (fr)

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US17/265,692 US20210177988A1 (en) 2018-08-06 2019-06-03 Immunoreactant carrier
EP19846510.6A EP3834820A4 (fr) 2018-08-06 2019-06-03 Support immunoréactif
JP2021506399A JP2021532163A (ja) 2018-08-06 2019-06-03 免疫反応物質送達体
SG11202101258VA SG11202101258VA (en) 2018-08-06 2019-06-03 Immunoreactant carrier
CN201980052469.0A CN112543631B (zh) 2018-08-06 2019-06-03 免疫反应性物质运载体
CA3108251A CA3108251C (fr) 2018-08-06 2019-06-03 Support immunoreactif
AU2019317939A AU2019317939A1 (en) 2018-08-06 2019-06-03 Immunoreactant carrier
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KR20160011565A (ko) * 2014-07-22 2016-02-01 주식회사 레모넥스 생리활성 물질 또는 단백질 전달용 조성물 및 이의 용도
KR20180091768A (ko) * 2017-02-06 2018-08-16 주식회사 레모넥스 생리활성물질 전달체

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