Patent Application
20250188142
The present invention belongs to the technical field of biopharmaceuticals and relates to a polyethylene glycol derivative modified interleukin-12, a preparation method thereof and an application thereof.
Interleukin-12 (IL-12), as a cytokine with broad biological activities, plays an indispensable role in immune regulation. IL-12 can promote the differentiation of Th0 cells into Th1 cells, prevent the development of Th0 cells into Th2 cells, induce T cells and NK cells to produce several cytokines: IFN-γ, IFN-α, GM-CSF, M-CSF, IL-13, IL-8, and IL-2, act through these mediators to inhibit the secretion of IL-4 by Th2 cells, etc. IL-12 can also induce enhanced cellular toxicity, which is weaker than that induced by IL-2 but comparable to that induced by IFN-γ. When IL-12 works in conjunction with IL-2, the most significant enhancement of NK cell-mediated cytotoxicity can be observed, which is an additive effect rather than a synergistic effect between the two factors. IL-12 plays an important regulatory role in both innate immunity and adaptive immunity. Therefore, IL-12 has obvious application value in the body's resistance to infection by various bacteria, viruses, fungi, and parasites, as well as in anti-tumor aspect. In addition, IL-12 also has a regulatory effect on hematopoietic cells. IL-12 works synergistically with iron factor (SF), IL-13 and other hematopoietic cell growth factors to increase the survival rate and proliferation of hematopoietic stem cells in the broth, and to increase the number and size of colonies of early pluripotent hematopoietic stem cells (including cells with bipotential for bone marrow B-lymphoid systems). Therefore, IL-12 also has great positive significance in the hemogram recovery and can be used to improve the side effects of abnormal hemogram caused by radiotherapy and chemotherapy, as well as for the prevention and treatment of acute radiation syndrome caused by nuclear leakage accidents and nuclear warfare.
Interleukin-12 itself, as a protein, also has many defects in its application as a drug. The half-life of interleukin-12 in the body is short, only 5.3 to 9.6 hours. In addition, systemic administration of interleukin-12 would also cause many side effects, such as: fever/chills, fatigue, nausea, vomiting, headache, especially when the dose of IL-12 is increased, the side effects increase significantly. In previous clinical trials, continuous administration of 500 ng/Kg/day of IL-12 to patients would cause serious toxic side effects (Effects of Single-Dose Interleukin-12 Exposure on Interleukin-12-Associated Toxicity and Interferon-g Production). The toxic side effects of IL-12 are primarily related to the excess production of interferon-γ by T cells or NK cells stimulated by IL-12. In order to cope with the increase in toxic side effects, the dose of IL-12 in general treatment is low. However, the use of IL-12 at lower doses requires frequent injections to maintain blood concentration, due to IL-12's short half-life. Although frequent injections of IL-12 can maintain the blood concentration, it poses significant inconvenience to the patient. Moreover, the frequently violent fluctuations in IL-12 levels in the blood are not conducive to treatment. Too long an interval between injections results in discontinuous treatment and poorer treatment effectiveness, while too short an interval between injections can lead to cumulative time toxicity.
Polyethylene glycol (PEG), also known as polyoxyethylene alcohol or polyoxyethylene glycol, is synthesized by the polymerization of ethylene oxide with ethylene glycol (or diethylene glycol) under the catalysis of a basic catalyst. Due to its stable chemical properties and good biological safety, it has been used in the medical field for a long time. Polyethylene glycol-modified proteins or polypeptide drugs prolong the drug's blood half-life in vivo, stabilize the drug, and suppress immunogenicity in various ways.
CN 1186121 A discloses a construction of a eukaryotic expression vector, with the IL-12 gene carried by PEG. This method involves using PEG to carry the IL-12 gene and transfecting it into eukaryotic cells to translate and express the synthetic IL-12 protein, and secreting the protein out of the cells to exert its efficacy. However, the physical carrying is not as strong and stable as the chemical bond and needs to go through more steps, the drug carried is slow to take effect, and the final expression level of IL-12 is also difficult to control. If the expression level is too low, the drug effect would unclear. If the expression level is too high, drug side effects increase. In addition, IL-12 itself is a non-monodisperse macromolecule containing multiple amino acid chains and has a short metabolic half-life in the body. Even if IL-12 is expressed, IL-12 protein alone also has the defects of small therapeutic window and great toxic side effects in therapy as a drug.
CN 109438568 A discloses a method for preparing monodisperse polyethylene glycol monomethyl ether-modified interleukin IL-12 prodrug. Specifically, in this method, interleukin-12, an alkali, and a solvent are added to monodisperse polyethylene glycol monomethyl ether lysine active ester, and the mixture is reacted at 50° C. to obtain monodisperse polyethylene glycol monomethyl ether lysine interleukin-12 prodrug; In this method, although the molecular weight of the product is increased by connecting interleukin-12 with polyethylene glycol through chemical bonds, the molecular weight of the polyethylene glycol used is small. In order to achieve a good effect of prolonging the half-life, this method uses polyethylene glycol to modify IL-12 randomly at multiple sites. Random modifications at multiple sites tend to mask more surface of IL-12 and reduce the activity of IL-12 further. Moreover, the reaction is carried out in an organic solvent in an alkaline environment at 50° C., which is extremely unfriendly to the protein IL-12, easily cause protein denaturation and inactivation, and leads to further decline in IL-12 activity. Further, random modification leads to complex products, posing greater potential risks when used as drugs.
Based on the defects existing in the prior art, the first purpose of the present invention is to provide a polyethylene glycol derivative modified interleukin-12. By improving the structure of interleukin-12, its side effects in clinical application are reduced. The second purpose of the present invention is to provide a method for preparing the polyethylene glycol derivative modified interleukin-12. By controlling the reaction conditions and preparation conditions, an interleukin-12 site-specifically modified at single site with polyethylene glycol derivative or an interleukin-12 reversibly modified at multiple sites can be obtained. The third purpose of the present invention is to provide the application of the polyethylene glycol derivative modified interleukin-12 in the preparation of an immunity-improving drug and an anti-infective drug; The fourth purpose of the present invention is to provide the application of the polyethylene glycol derivative modified interleukin-12 in the preparation of a medicament for preventing or treating an disease with abnormal hemogram; The fifth purpose of the present invention is to provide the application of the polyethylene glycol derivative modified interleukin-12 in the preparation of an anti-tumor drug for preventing or treating a tumor; The sixth purpose of the present invention is to provide a pharmaceutical preparation containing the polyethylene glycol derivative modified interleukin-12.
In an aspect, the present invention provides a polyethylene glycol derivative modified interleukin-12, which is obtained by performing a modification reaction on a polyethylene glycol derivative and interleukin-12; wherein, an active group of the polyethylene glycol derivative and an active group of the interleukin-12 participate in the modification reaction. The active group of the polyethylene glycol derivative comprises one or more among a aldehyde group, a ketone group, a maleimide group, a vinyl sulfone group, a terminal alkyne group, a succinimide group, a p-nitrophenyl group, an isobutyl chloroformate group, a halo group (haloalkyl group), a thio group and a carboxyl group, but not limited to these. The active group of the interleukin-12 comprises one or more among an N-terminal α-amino group, an ε-amino group of a lysine side chain and a free sulfhydryl group of a cysteine, but not limited to these.
In the present invention, the polyethylene glycol derivative and IL-12 are coupled through covalent bonds to obtain the interleukin-12 with polyethylene glycol derivative modification introduced, so that the interleukin-12 has more advantages of becoming a potential drug product while maintaining the original biological activity of the interleukin-12. The advantages comprise, but are not limited to, increased molecular weight and hydration radius, improved in vivo stability, prolonged in vivo half-life, reduced immunogenicity, improved storage stability, storage capacity, and safe use as a liquid preparation, a frozen formulation, and a dried lyophilized formulation after several days. Polyethylene glycol derivative modified interleukin-12 can not only prolong the half-life, but also balance the drug activity and drug toxicity of IL-12. The toxic side effects of IL-12 are mainly caused by excessive interferon γ produced by T cells or NK cells stimulated by IL-12. A little excessive IL-12 would bring great toxic side effects, which is very troublesome for treatment, and the dose is difficult to control. When natural IL-12 is used for treatment, the toxic side effects can only be reduced by reducing the dose of a single injection. However, the lower dose makes it difficult to maintain the therapeutic effect of the drug, and frequent low-dose injections are needed to maintain blood concentration. Polyethylene glycol derivative modified interleukin-12 can reduce the toxic side effects of IL-12 and balance the drug activity and drug toxicity, and a larger dose may be used in a single dose. In addition, the IL-12 may maintain an effective blood concentration in the body for a longer period of time without causing serous toxic side effects, which is beneficial for the continuity of treatment and improves the treatment effects of diseases.
Interleukin-12 in the present invention may further comprise mutants, monomers, polymers, bioactive peptides, functional fragments, functional derivatives and fusion proteins of interleukin-12, subunits P40 and P35 that constitute interleukin-12, mutants, monomers, polymers, bioactive peptides, functional fragments, functional derivatives and fusion proteins of subunits P40 and P35 of interleukin-12, etc.
In the above-mentioned polyethylene glycol derivative modified interleukin-12, preferably, the active group of the polyethylene glycol derivative is an aldehyde group, a ketone group, a succinimide group, an isobutyl chloroformate group, a p-nitrophenyl group or a carboxyl group, the active group of the interleukin-12 that undergoes the modification reaction is an N-terminal α-amino group (any one or both of the N-terminal α-amino groups); or
the active group of the polyethylene glycol derivative is a maleimide group, a vinyl sulfone group, a terminal alkyne group, a halo group or a thio group, and the active group of the interleukin-12 that undergoes the modification reaction is a free sulfhydryl group of a cysteine; preferably a free sulfhydryl group of a cysteine at the position C252 of the P40 subunit; or
The active group of the polyethylene glycol derivative is a succinimide group, an isobutyl chloroformate group, a p-nitrophenyl group or a carboxyl group, and the active group of the interleukin-12 that undergoes the modification reaction is an ε-amino group of a lysine side chain (any one or more of the ε-amino groups of multiple lysine side chains).
In the above-mentioned polyethylene glycol derivative modified interleukin-12, preferably, the molecular weight of the polyethylene glycol derivative is 5-40 KD; preferably 10-30 KD; and more preferably, 20 KD.
Among the above-mentioned polyethylene glycol derivative modified interleukin-12, preferably, the polyethylene glycol derivative comprises one or more of a polyethylene glycol aldehyde derivative, a polyethylene glycol ketone derivative, a polyethylene glycol active ester derivative, a polyethylene glycol carboxyl derivative, a polyethylene glycol unsaturated bond-containing derivative, a polyethylene glycol haloalkane derivative, a polyethylene glycol sulfur activity-containing derivative; but not limited to these. Preferably, the polyethylene glycol aldehyde derivative comprises one or more of a polyethylene glycol acetaldehyde derivative, a polyethylene glycol propionaldehyde derivative and a polyethylene glycol butyraldehyde derivative; but not limited to these. The polyethylene glycol propionaldehyde derivative comprises monomethoxy polyethylene glycol propionaldehyde and/or two-arm polyethylene glycol propionaldehyde; but not limited to these. The polyethylene glycol active ester derivative comprises one or more of a polyethylene glycol succinimide active ester derivative, a polyethylene glycol p-nitrobenzene active ester derivative and a polyethylene glycol isobutyl chloroformate active ester derivative; but not limited to these. The polyethylene glycol succinimide active ester derivative comprises monomethoxy polyethylene glycol succinimide succinate and/or monomethoxy polyethylene glycol succinimide carbonate; but not limited to these. The polyethylene glycol carboxyl derivative comprises one or more of a polyethylene glycol acetic acid derivative, a polyethylene glycol propionic acid derivative, a polyethylene glycol butyric acid derivative, a polyethylene glycol valeric acid derivative, a polyethylene glycol hexanoic acid derivative, a polyethylene glycol oxalic acid derivative, a polyethylene glycol malonic acid derivative, a polyethylene glycol succinic acid derivative, a polyethylene glycol glutaric acid derivative and a polyethylene glycol hexanedioic acid derivative; but not limited to these. The polyethylene glycol carboxyl derivative comprises monomethoxy polyethylene glycol acetic acid. The polyethylene glycol unsaturated bond-containing derivative comprises one or more of polyethylene glycol vinyl sulfones, polyethylene glycol maleimides, and polyethylene glycol terminal acetylenes; but not limited to these. the polyethylene glycol unsaturated bond-containing derivative comprises one or more of a monomethoxy polyethylene glycol vinyl sulfone, a monomethoxy polyethylene glycol maleimide and a two-arm polyethylene glycol maleimide; but not limited to these. the polyethylene glycol haloalkane derivative comprises one or more of a polyethylene glycol iodopropionamide derivative, a polyethylene glycol iodoacetamide derivative, a polyethylene glycol chloropropionamide derivative, a polyethylene glycol chloroacetamide derivative, a polyethylene glycol bromopropionamide derivative, a polyethylene glycol bromoacetamide derivative, a polyethylene glycol fluoropropionamide derivative and a polyethylene glycol fluoroacetamide derivative; but not limited to these. The polyethylene glycol haloalkane derivative comprises monomethoxy polyethylene glycol iodoacetamide. The polyethylene glycol sulfur activity-containing derivative comprises one or more of polyethylene glycol dithioisopyridines, polyethylene glycol thiosulfonates, and polyethylene glycol thiols; but not limited to these. The polyethylene glycol sulfur activity-containing derivative comprises one or more of monomethoxy polyethylene glycol dithioisopyridine, monomethoxy polyethylene glycol thiomethanesulfonate and monomethoxy polyethylene glycol thiobenzenesulfonate; but not limited to these.
According to a specific embodiment of the present invention, the polyethylene glycol derivative modified interleukin-12 of the present invention comprises one or more of the following substances: monomethoxy polyethylene glycol propionaldehyde-modified interleukin-12, two-arm polyethylene glycol propionaldehyde-modified interleukin-12, monomethoxy polyethylene glycol succinimide succinate-modified interleukin-12, monomethoxy polyethylene glycol maleimide-modified interleukin-12, branched monomethoxy polyethylene glycol maleimide-modified interleukin-12, monomethoxy polyethylene glycol succinimide carbonate-modified interleukin-12, monomethoxy polyethylene glycol succinimide carbonate-modified interleukin-12, monomethoxy polyethylene glycol vinyl sulfone-modified interleukin-12, monomethoxy polyethylene glycol iodoacetamide-modified interleukin-12, monomethoxy polyethylene glycol dithioisopyridine-modified interleukin-12, monomethoxy polyethylene glycol acetic acid-modified interleukin-12.
In another aspect, the present invention further provides a method for preparing the above-mentioned polyethylene glycol derivative modified interleukin-12, and the method comprises:
In the preparation method of the present invention, an IL-12 site-specifically modified at single site with polyethylene glycol derivative or an IL-12 reversibly modified at multiple sites can be obtained by controlling the reaction conditions. Modification at single site occupies less surface area of the protein than modification at multiple sites, offering a more distinct modification site. At the same time, a single polyethylene glycol derivative with a larger molecular weight is used, the biological activity of IL-12 can be better retained when the molecular weight of the product increases significantly and appropriately reduced activity also makes it easier to combine with other drugs. The IL-12 reversibly modified at multiple sites has a larger molecular weight and longer blood drug half-life when used. PEG can be dissociated again to restore the activity of IL-12. At the same time, the reaction of the present invention is carried out at low temperature or normal temperature, which can better retain the biological activity of IL-12. This step is performed in particular as follows: when a polyethylene glycol aldehyde derivative is used as the polyethylene glycol derivative, IL-12 only has two subunits, but it also has 39 lysine side chain amino groups, and by controlling the reaction conditions, it is able to achieve selective site-specific coupling of the polyethylene glycol aldehyde derivative to the terminal α-amino group of interleukin-12, rather than random modification of the terminal α-amino group or the ε-amino group of the lysine side chains of interleukin-12. when a polyethylene glycol active ester derivative and a polyethylene glycol carboxyl derivative are used as polyethylene glycol derivatives, by controlling the reaction conditions, it is able to achieve selective site-specific coupling of the polyethylene glycol active ester derivative or the polyethylene glycol carboxyl derivative to the terminal α-amino group or the ε-amino group of the lysine side chains of interleukin-12; this type of ester bond-containing linker group can allow the modified interleukin-12 to have the characteristic of reversible modification, and during use, it enables polyethylene glycol to separate from IL-12 again through a hydrolysis reaction, thereby allowing IL-12 to retain more complete biological activity. when a polyethylene glycol unsaturated bond-containing derivative, a polyethylene glycol haloalkane derivative, or a polyethylene glycol sulfur activity-containing derivative is used as the polyethylene glycol derivative, it is able to achieve selective site-specific coupling of the polyethylene glycol unsaturated bond-containing derivative, the polyethylene glycol haloalkane derivative, or the polyethylene glycol sulfur activity-containing derivative to the free sulfhydryl group of cysteine at the position C252 of the P40 subunit of interleukin-12 by controlling the reaction conditions.
Preferably, the chromatography comprises ion exchange chromatography, size exclusion chromatography, hydrophobic chromatography or reversed phase chromatography; but not limited to these.
Preferably, the molar ratio of the polyethylene glycol derivative to the interleukin-12 is (5.5-20):1.
Preferably, the polyethylene glycol derivative is a polyethylene glycol aldehyde derivative or a polyethylene glycol ketone derivative, and the solvent comprises an acidic buffer solution with a pH value of 4 to 6.5, preferably an acidic buffer solution with a pH value of 5, so as to control the reaction conditions to be an acidic environment; the temperature of the reaction under stirring is 0-40° C., preferably 10° C.; a reducing agent further needs to be added during the reaction.
Preferably, the acidic buffer solution comprises one or more of a MES buffer, a Bis-Tris buffer, a glycine-hydrochloric acid buffer, a disodium hydrogen phosphate-citric acid buffer, a citric acid-sodium citrate buffer, a citric acid-sodium hydroxide-hydrochloric acid buffer, a acetic acid-sodium acetate buffer, a potassium hydrogen phthalate-sodium hydroxide buffer, a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer and a potassium dihydrogen phosphate-sodium hydroxide buffer, but not limited to these.
Preferably, the reducing agent comprises one or more of sodium cyanoborohydride, sodium borohydride, sodium triacetylborohydride, borane and lutidine borane, but not limited to these; preferably, the reducing agent is sodium cyanoborohydride.
Preferably, the molar ratio of the reducing agent to the interleukin-12 is (30-500):1.
Preferably, when the polyethylene glycol derivative is a polyethylene glycol carboxyl derivative, the polyethylene glycol carboxyl derivative needs to be activated with an active condensing agent before reacting with interleukin-12.
Preferably, the active condensing agent comprises one or more of a carbodiimide condensing agent, an onium salt condensing agent and an organophosphorus condensing agent; but not limited to these.
Preferably, the carbodiimide condensing agent comprises one or more of dicyclohexylcarbodiimide, diisopropylcarbodiimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; but not limited to these.
Preferably, the onium salt condensing agent comprises one or more of 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate, benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate, 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate, O—(N-succinimide)-1,1,3,3-tetramethylurea tetrafluoroborate, 2-(endo-5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethylurea tetrafluoroborate, O-(7-azabenzotriazol-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate and O-(benzotriazol-1-yl)-N,N,N′,N′-dipyrrolylurea hexafluorophosphate; but not limited to these.
Preferably, the organophosphorus condensing agent comprises one or more of diethyl cyanophosphate, diphenyl azidophosphate, diphenyl chlorophosphate, dimethylthiophosphoryl azide, benzotriazole-1-bis(trimethylamino)phosphonium-hexafluorophosphate, 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate, triphenylphosphine-polyhalomethane, triphenylphosphine-hexachloroacetone and triphenylphosphine-NBS; but not limited to these.
Preferably, the polyethylene glycol derivative is a polyethylene glycol active ester derivative, and the solvent comprises an alkaline buffer solution with a pH value of 7.5 to 10, preferably an alkaline buffer solution with a pH value of 8, so as to control the reaction conditions to be an alkaline environment; the temperature of the reaction under stirring is 0-40° C., preferably 10° C.
Preferably, the alkaline buffer solution comprises one or more of a MES buffer, a Bis-Tris buffer, a HEPES buffer, a TEA buffer, a disodium hydrogen phosphate-citric acid buffer, a citric acid-sodium citrate buffer, a citric acid-sodium hydroxide-hydrochloric acid buffer, a acetic acid-sodium acetate buffer, a potassium hydrogen phthalate-sodium hydroxide buffer, a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, a potassium dihydrogen phosphate-sodium hydroxide buffer, a sodium barbiturate-hydrochloric acid buffer and a Tris-hydrochloric acid buffer, but not limited these.
Preferably, the polyethylene glycol derivative is a polyethylene glycol carboxyl derivative, a polyethylene glycol unsaturated bond-containing derivative, a polyethylene glycol haloalkane derivative or a polyethylene glycol sulfur activity-containing derivative, and the solvent comprises a neutral or near-neutral buffer solution with a pH value of 6 to 8, preferably a neutral buffer solution with a pH value of 7, so as to control the reaction conditions to be a neutral or near-neutral environment. the temperature of the reaction under stirring is 0-40° C., preferably 10° C.
Preferably, the neutral or near-neutral buffer solution comprises one or more of a MES buffer, a Bis-Tris buffer, a HEPES buffer, a TEA buffer, a disodium hydrogen phosphate-citric acid buffer, a citric acid-sodium citrate buffer, a citric acid-sodium hydroxide-hydrochloric acid buffer, a acetic acid-sodium acetate buffer, a potassium hydrogen phthalate-sodium hydroxide buffer, a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, a potassium dihydrogen phosphate-sodium hydroxide buffer, a sodium barbiturate-hydrochloric acid buffer and a Tris-hydrochloric acid buffer; but not limited to these.
In another aspect, the present invention further provides the application of the above-mentioned polyethylene glycol derivative modified interleukin-12 in the preparation of an immunity-improving drug and an anti-infective drug;
Preferably, the resistance to infection comprises resistance to one or more of viral infection diseases, bacterial infection diseases and parasitic infection diseases, but not limited to these; the resistance to viral infection diseases comprise resistance to one or more of HSV, HIV, hepatitis B and hepatitis C, but not limited to these; the resistance to bacterial infection diseases comprise resistance to one or more of pulmonary tuberculosis, salmonellosis and listeriosis, but not limited to these; the resistance to parasitic infection diseases comprise resistance to one or more of malaria, leishmaniasis and schistosomiasis, but not limited to these.
In another aspect, the present invention further provides the application of the above-mentioned polyethylene glycol derivative modified interleukin-12 in the preparation of a medicament with one or more of the following efficacies: improving immunity; resistance to infection; preventing and/or treating a disease with abnormal hemogram; and preventing and/or treating a tumor. Accordingly, the present invention provides a corresponding method for treating one or more of the following diseases, which method comprises administering an effective amount of the polyethylene glycol derivative modified interleukin-12 of the present invention to a subject in need: improving immunity; resistance to infection; preventing and/or treating a disease with abnormal hemogram; and preventing and/or treating a tumor.
Preferably, the disease with abnormal hemogram comprises one or more of a disease with abnormal hemogram caused by a hematopoietic malignant tumor, a disease with abnormal hemogram caused by radiotherapy, chemotherapy, or myeloablative therapy, and a disease with abnormal hemogram caused by acute radiation syndrome; but not limited to these.
Preferably, the disease with abnormal hemogram caused by a hematopoietic malignant tumor comprises one or more of chronic myeloid leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, low-grade non-Hodgkin lymphoma, acute myeloid leukemia, moderate lymphoma, multiple myeloma, myelodysplastic syndrome, and Hodgkin's disease; but not limited to these.
Preferably, the tumor comprises various types of solid tumors and/or non-solid tumors.
In another aspect, the present invention further provides a pharmaceutical preparation, which comprises an effective amount of the above-mentioned polyethylene glycol derivative modified interleukin-12. A pharmaceutically acceptable excipient may further be comprised.
Preferably, the dosage form of the pharmaceutical preparation comprises a liquid preparation and/or a lyophilized preparation; but not limited to these.
Preferably, the administration route of the pharmaceutical preparation comprises injection, oral administration, external application or administration via a combination of multiple routes; but not limited to these.
Preferably, a treatment method using the pharmaceutical preparation comprises one of or a combination of a radiotherapy, a chemotherapy, a myeloablative therapy, a CAR-T therapy, a CAR-NK therapy, an antibody-targeted therapy, an oncolytic virus therapy and a gene vaccine therapy; but not limited to these.
In the present invention, the polyethylene glycol derivative and IL-12 are coupled through covalent bonds to obtain the interleukin-12 with polyethylene glycol derivative modification introduced, so that the interleukin-12 has more advantages of becoming a potential drug product while maintaining the original biological activity of the interleukin-12. The advantages comprise, but are not limited to, increased molecular weight and hydration radius, improved in vivo stability, prolonged in vivo half-life, reduced immunogenicity, improved storage stability, storage capacity, and safe use as a liquid preparation, a frozen formulation, and a dried lyophilized formulation after several days. Polyethylene glycol derivative modified interleukin-12 is able to balance the drug activity and drug toxicity of IL-12. Compared to natural IL-12, the polyethylene glycol derivative modified interleukin-12 can be administered in larger single doses and maintain effective blood concentration for a longer period of time without causing serous toxic side effects, which is beneficial for the continuity of treatment and improves the treatment effects of diseases. Therefore, polyethylene glycol-modified interleukin-12 has greater advantages over natural IL-12 for use in treatments such as anti-tumor, resistance to infection, and hemogram recovery. Compared to PEG-G-CSF and PEG-IL-2, polyethylene glycol derivative modified interleukin-12 has a larger molecular weight, which translates to a longer half-life. This allows for the maintenance of blood concentration for a longer period of time with each administration, thereby increasing the intervals between administrations, reducing the patient's discomfort, and saving time costs. Additionally, unlike PEG-G-CSF, which primarily functions to improve the hemogram recovery, and unlike PEG-IL-2, which primarily stimulates T cell proliferation and induces cytotoxic effects for anti-tumor purposes, the polyethylene glycol derivative modified interleukin-12 has a more extensive range of applications as it not only can promote the hemogram recovery but also can stimulate T cells to differentiate towards Th1, stimulate NK cells to exert more natural killer toxicity, and stimulate cells to express a series of cytokines. This overall enhances immunity, presenting significant potential in applications such as resistance to infection, anti-tumor, and hemogram recovery. The polyethylene glycol derivative modified interleukin-12 of the present invention can be widely used in one of or a combination of radiotherapy, chemotherapy, myeloablative therapy, CAR-T therapy, CAR-NK therapy, antibody-targeted therapy, oncolytic virus therapy and gene vaccine therapy, and has effects such as improving immunity, resistance to infection, anti-tumor and hemogram recovery.
In order to understand the technical features, object and beneficial effects of the present invention more clearly, then the technical solution of the present invention is described in detail as below, but it cannot be construed as a limitation for the implementable scope of the present invention. Unless otherwise specified, the raw material components in the following examples are all commercially available in the art.
This example provides a synthesis of monodisperse 20 KD monomethoxy polyethylene glycol propionaldehyde (mPEG-pALD-20K)-modified interleukin-12. The specific preparation method is as follows:
It can be seen from Table 1 and
The specific reaction flow for the preparation method of this example is as follows:
The value of n was approximately 454.
In this example, selective site-specific coupling of mPEG-pALD-20KD on the N-terminal α-amino group of interleukin-12 was achieved by controlling the above reaction conditions.
The HLC-8420GPC&LenS3MALS detector was used to detect the molecular weight of the synthesized mPEG-pALD-20KD-IL-12. The experimental results are shown in
SDS-PAGE was used to detect the purity of purified mPEG-pALD-20KD-IL-12. The electrophoresis image is shown in
This example provides the synthesis of monodisperse 20 KD two-arm polyethylene glycol propionaldehyde (2-arm PEG-CHO-20K)-modified interleukin-12. The specific preparation method is as follows:
It can be seen from Table 2 and
The specific reaction flow for the preparation method of this example is as follows:
The n or m value was approximately 227.
In this example, selective site-specific coupling of 2-arm PEG-CHO-20K on the N-terminal α-amino group of interleukin-12 was achieved by controlling the above reaction conditions.
It can be seen from the UPLC chromatogram that the target products of Example 1 and Example 2 have the same theoretical molecular weight, but the retention times are different. The retention time of Example 1 is 14.360 min, and the retention time of Example 2 is 12.409 min. The retention time of the two relative to IL-12 is also different, and the relative retention time of Example 2 is smaller. This is because the product molecule in Example 2 is two-armed and has a larger apparent molecular weight, and the packing of the chromatographic column used has a pore size of 450 A and has certain characteristics of size exclusion chromatography, so the product of Example 2 with a larger apparent molecular weight has a shorter retention time in the column than Example 1 and comes out earlier.
Therefore, under the same molecular weight, multi-arm PEG modification brings better modification effect than single-arm PEG modification. This is mainly because multi-arm PEG has a larger apparent molecular weight, a greater steric hindrance, and at the same time covers a smaller area of IL-12 and can retain more IL-12 activity.
This example provides a synthesis of monodisperse 20 KD monomethoxy polyethylene glycol succinimide succinate (mPEG-SS-20KD)-modified interleukin-12. The specific preparation method is as follows:
It can be seen from Table 3 and
The specific reaction flow for the preparation method of this example is as follows:
The value of n was approximately 454.
In this example, selective site-specific coupling of mPEG-SS-20KD on the N-terminal α-amino group or the ε-amino group of the lysine side chains of interleukin-12 was achieved by controlling the above reaction conditions. The polyethylene glycol active ester derivative modified interleukin-12 of this example has the characteristic of reversible modification, and during use, it enables polyethylene glycol to separate from IL-12 again through a hydrolysis reaction, thereby allowing IL-12 to retain more complete biological activity.
The HLC-8420GPC&LenS3MALS detector was used to detect the molecular weight of the synthesized mPEG-SS-20KD-IL-12. The experimental results are shown in
This example provides a synthesis of monodisperse 20 KD monomethoxy polyethylene glycol maleimide (mPEG-MAL-20KD)-modified interleukin-12. The specific preparation method is as follows:
It can be seen from Table 4 and
The specific reaction flow for the preparation method of this example is as follows:
The value of n was approximately 454.
In this example, selective site-specific coupling of mPEG-MAL-20KD on the free sulfhydryl group of cysteine at the position C252 of the P40 subunit of interleukin-12 was achieved by controlling the above reaction conditions.
The HLC-8420GPC&LenS3MALS detector was used to detect the molecular weight of the synthesized mPEG-MAL-20KD-IL-12. The experimental results are shown in
IL-12, mPEG-pALD-20KD-IL-12 prepared in Example 1 and mPEG-MAL-20KD-IL-12 prepared in Example 4 were subjected to UPLC examination after reduction with mercaptoethanol. The UPLC chromatogram is shown in
This example provides a synthesis of monodisperse 20 KD branched monomethoxy polyethylene glycol maleimide (2-arm-PEG-MAL-20KD)-modified interleukin-12. The specific preparation method is as follows:
It can be seen from Table 5 and
The specific reaction flow for the preparation method of this example is as follows:
The value of n or m was approximately 227.
In this example, selective site-specific coupling of 2-arm-PEG-MAL-20KD on the free sulfhydryl group of cysteine at the position C252 of the P40 subunit of interleukin-12 was achieved by controlling the above reaction conditions.
It can be seen from the UPLC chromatogram that the target products of Example 4 and Example 5 have the same theoretical molecular weight, but the retention times are different. The retention time of Example 4 is 14.245 min, and the retention time of Example 5 is 12.273 min. The retention time of the two relative to IL-12 is also different, and the relative retention time of Example 5 is smaller. This is because the product molecule in Example 5 is two-armed and has a larger apparent molecular weight, and the packing of the chromatographic column used has a pore size of 450 A and has certain characteristics of size exclusion chromatography, so the product of Example 5 with a larger apparent molecular weight has a shorter retention time in the column than Example 4 and comes out earlier.
Therefore, under the same molecular weight, multi-arm PEG brings better modification effect than single-arm PEG. This is mainly because multi-arm PEG has a larger apparent molecular weight, a greater steric hindrance, and at the same time covers a smaller area of IL-12 and can retain more IL-12 activity.
This example provides a synthesis of monodisperse 20 KD monomethoxy polyethylene glycol succinimide carbonate (mPEG-SC-20KD)-modified interleukin-12. The specific preparation method is as follows:
It can be seen from Table 6 and
The specific reaction flow for the preparation method of this example is as follows:
The value of n was approximately 454.
In this example, the saturated and reversible modification of interleukin-12 by mPEG-SC-20KD was achieved by controlling the above reaction conditions. Selective site-specific coupling modification on the terminal α-amino group or the ε-amino group of the lysine side chains of interleukin-12 was achieved. The polyethylene glycol active ester derivative modified interleukin-12 of this example has the characteristic of reversible modification, and during use, it enables polyethylene glycol to separate from IL-12 again through a hydrolysis reaction, thereby allowing IL-12 to retain more complete biological activity.
This example provides a synthesis of monodisperse 5 KD monomethoxy polyethylene glycol succinimide carbonate (mPEG-SC-5KD)-modified interleukin-12. The specific preparation method is as follows:
It can be seen from Table 7 and
The specific reaction flow for the preparation method of this example is as follows:
The value of n was approximately 113.
In this example, selective site-specific coupling of mPEG-SC-5KD on the terminal α-amino group or the ε-amino group of the lysine side chains of interleukin-12 was achieved by controlling the above reaction conditions. The polyethylene glycol active ester derivative modified interleukin-12 of this example has the characteristic of reversible modification, and during use, it enables polyethylene glycol to separate from IL-12 again through a hydrolysis reaction, thereby allowing IL-12 to retain more complete biological activity.
This example provides a synthesis of monodisperse 20 KD monomethoxy polyethylene glycol vinyl sulfone (mPEG-VS-20KD)-modified interleukin-12. The specific preparation method is as follows:
The specific reaction flow for the preparation method of this example is as follows:
The value of n was approximately 454.
This example provides a synthesis of monodisperse 20 KD monomethoxy polyethylene glycol iodoacetamide (mPEG-IA-20KD)-modified interleukin-12. The specific preparation method is as follows:
The specific reaction flow for the preparation method of this example is as follows:
The value of n was approximately 454.
This example provides a synthesis of monodisperse 20 KD monomethoxy polyethylene glycol dithioisopyridine (mPEG-OPSS-20KD)-modified interleukin-12. The specific preparation method is as follows:
The specific reaction flow diagram for the preparation method of this example is as follows:
The value of n was approximately 454.
This example provides a synthesis of monodisperse 20 KD monomethoxy polyethylene glycol acetic acid (mPEG-AA-20KD)-modified interleukin-12. The specific preparation method is as follows:
The specific reaction flow diagram for the preparation method of this example is as follows:
The value of n was approximately 454.
Through comparative experiment of IFN-γ production of NK-92 cells stimulated by mPEG-pALD-20KD-IL-12 (Example 1), mPEG-MAL-20KD-IL-12 (Example 4), mPEG-SS-20KD-IL-12 (Example 3) and IL-12 using IFN-γ detection ELISA kit, the difference in activity and the degree of toxicity reduction between mPEG-pALD-20KD-IL-12, mPEG-MAL-20KD-IL-12, mPEG-SS-20KD-IL-12 and IL-12 were evaluated. The specific method was as follows:
For result processing, GraphPad Prism6 software was used to process the data. The OD value was used as the ordinate and the dilution factor was used as the abscissa. Four parameters were used for fitting to obtain the activity curve, Top value, Bottom value and R2 value, the test standard required R2 value≥0.95. The activity of the test product was calculated according to the following formula:
Among them, Pr is the biological activity of the standard product, that is, the biological activity of the WHO IL-12 standard product=1×104 U/ml; Ds is the pre-dilution factor of the test product; Dr is the pre-dilution factor of the standard product; Es is the dilution factor of the test product equivalent to the median effective dose of the standard product; Er is the dilution factor of the median effective dose of the standard product. Specific activity of the test product (U/mg)=titer of the test product (U/ml)/concentration of the test product (mg/ml).
The experimental results are shown in Table 8:
It can be seen from the experimental results in Table 8 above that: Compared with the IL-12 standard, the activity of the product mPEG-pALD-20KD-IL-12 obtained in Example 1 decreased by 8.0-fold, and the activity of the product mPEG-MAL-20KD-IL-12 obtained in Example 4 decreased by 20.3-fold, and the activity of the product mPEG-SS-20KD-IL-12 with single modification obtained in Example 3 decreased by 11.70-fold. The decrease of activity means that the dose of polyethylene glycol-modified interleukin-12 was larger than that of IL-12 in order to produce the same amount of IFN-γ. On the other hand, polyethylene glycol-modified interleukin-12 exhibited lower side effects at the same dose, which facilitates the administration of a larger dose in a single time and maintains stable drug concentration in the blood for a longer period of time without causing major side effects. The dose of polyethylene glycol-modified interleukin-12 used in subsequent pharmacodynamic tests was determined with reference to the experimental results obtained in Example 12.
This example provided an analysis experiment on the effect of the drug action duration of mPEG-pALD-20KD-IL-12 prepared in Example 1 in normal rats. Through the comparative experiment of the action duration of mPEG-pALD-20KD-IL-12 prepared in Example 1 and IL-12 in normal rats, it was confirmed that PEG modification of long-acting interleukin-12 could prolong the drug action duration.
Eight-week-old normal rats were divided into a control group (vehicle), a group administered with mPEG-pALD-20KD-IL-12, and a group administered with interleukin-12. For 3 rats in each group, test substances were administered to normal rats subcutaneously once, and whole blood samples were collected from their tail veins at 1, 4, 8, 24, 48, and 72 hours respectively. The whole blood samples were added into each 1.5 mL microtube, and the microtubes were centrifuged at 5000 rpm for 10 min at room temperature. Each serum was separated and stored at −20° C., respectively. The polyethylene glycol-modified interleukin-12 or simple interleukin-12 contained in the serum of each group was quantified by ELISA. The ELISA assay was performed in the following manner: the serum collected each time and anti-human interleukin-12-HRP were added to a plate coated with interleukin-12 monoclonal antibody at the same time. The reaction was carried out at room temperature for 1 h. TMB reagent was used to develop the color. The absorbance was measured at a wavelength of 450 nm, respectively.
The results showed that: the group administered long-acting interleukin-12 containing a 20 kDa PEG linker showed an increase in AUC compared to the group administered interleukin-12.
This example provided an experiment on the resistance effect to infection of polyethylene glycol derivative modified interleukin-12; The unique biological activity of IL-12 enables it to promote the differentiation and maturation of Th1 cells, and can stimulate T cells and NK cells to secrete a large number of IFN-γ, TNF-α and GM-CSF, and activate the killing effect of T cells and NK cells. IL-12 plays an extremely important role in the body's early non-specific immunity and antigen-specific adaptive immunity. Therefore, IL-12 plays a significant role in resistance to bacterial infection, resistance to viral infection, resistance to parasitic infection, etc. Polyethylene glycol derivative modified interleukin-12 was used in the experiment on resistance effect to infection. The unique biological activity of IL-12 enables it to promote the differentiation and maturation of Th1 cells, stimulate human peripheral blood mononuclear cells, and kill target cells infected with herpes simplex virus type I to evaluate the resistance effect to infection of PEG modified interleukin-12. The detailed experimental steps are as follows:
This example provided an experiment on the effect of polyethylene glycol derivative modified interleukin-12 on hemogram recovery; Myeloablative therapy is often used in the treatment of hematological cancers. As the therapy progresses, various blood cells undergo a process of decline and then gradual recovery. In the treatment of cancers, conventional chemotherapy and radiotherapy can also cause abnormal decreases in various cells in the blood, including decrease of neutrophils, decrease of platelets and decrease of lymphocytes, and are easy to bring serious side effects, including spontaneous bleeding caused by decrease of platelets, usually accompanied by fatigue and sometimes depression. The decrease in white blood cells, neutrophils, and lymphocytes can also lead to a decrease in the body's own immunity, making it susceptible to infection and other complications. Acute radiation syndrome, also known as radiation poisoning or radiation sickness (ARS), has an onset and type of symptoms that depend on the patient's radiation exposure. A low dose of radiation can cause effects on the digestive system, such as nausea, vomiting, and symptoms associated with a decrease in blood indices such as infection and bleeding. A high dose of radiation can cause symptoms of neurological damage and rapid death. In the hematopoietic subsyndrome of ARS (HSARS), rapid bone marrow clearance caused by large amounts of harmful rays reduces the number of various blood cells. Depending on the dose of radiation exposed, symptoms such as infection and bleeding generally occur within 2 weeks to 2 months, and eventually leading to death. The general treatment for acute radiation syndrome is blood transfusion and antibiotics.
IL-12 can stimulate hematopoietic function and promote hemogram recovery. IL-12 induces the production of IFN-γ through the Stat4 pathway, and then IFN-γ acts on hematopoietic stem cells. However, studies have shown that IFN-γ plays a dual role (stimulatory/inhibitory role) in the hematopoietic process. IL-12 toxicity is associated with overproduction of IFN-γ, suggesting that the effects of IL-12 on blood recovery may be dose-related: low-dose IL-12 can promote hematopoiesis, while high-dose IL-12 can inhibit hematopoiesis by inducing excessive IFN-γ production and toxic reactions.
In this example, PEG-modified IL-12 is used to balance the drug activity and drug toxicity of IL-12, which can not only stimulate cells to produce an appropriate amount of IFN-γ, but also make IL-12 suitable for hemogram recovery. CD34+ cells are hematopoietic stem cells. We evaluated the effect of polyethylene glycol-modified IL-12 on the hemogram recovery by comparing the proliferation and differentiation of CD34+ cells stimulated in vitro with mPEG-MAL-20K-IL-12, mPEG-pALD-20K-IL-12 and IL-12. The specific experimental steps are as follows:
This example provided an IL-12 stimulation experiment on cynomolgus monkeys after irradiation with radioactive radiation to evaluate the effect of polyethylene glycol derivative modified interleukin-12 on hemogram recovery. The specific experimental steps are as follows:
In this experiment, cynomolgus monkeys were randomly divided into three groups and irradiated with 500 Rad radiation simultaneously. In order to distribute the dose evenly, the first half of the dose was irradiated from front to back and the second half of the dose was irradiated from back to front. 24 h before receiving radiation or 30 min after receiving radiation, the experimental group was injected with mPEG-MAL-20K-IL-12 (2 ug/Kg) and IL-12 (100 ng/Kg) respectively, and the control group was injected with PBS buffer. Clinical signs were recorded twice daily. Decreased appetite (based on food intake) and physical activity were recorded daily and scored as follows: 1=mild; 2=moderate; and 3=severe. Detailed physical examinations were performed before administration and twice weekly after administration. Body temperature (ear) was obtained before irradiation and on days 3, 10, 12, 14, 16, 18, 30, 45 and 60. Blood sampling (0.5 mL) for peripheral blood counting was performed before irradiation and on days 5, 10, 12, 14, 16, 18, 30, 45, and 60 to observe the dynamic changes of red blood cells, white blood cells, and platelets. In the early stage, the decline in the counts of various types of peripheral blood cells in the experimental group was less than that observed in the control group. The early recovery of platelets, red blood cells, and monocytes was significantly improved, and lymphocytes and neutrophils also recovered well. In the later stage, the experimental group that received injections of mPEG-MAL-20KD-IL-12 prepared in Example 4 exhibited a faster recovery rate of platelets, red blood cells, and monocytes compared to the experimental group that received injections of IL-12. This may be because PEGylated IL-12 had a longer half-life in vivo, so it could exert its effects over an extended period. Experimental results proved that IL-12 was useful in the prevention and treatment of hemogram abnormalities caused by myeloablative therapy, radiotherapy, chemotherapy, and acute radiation syndrome. mPEG-MAL-20KD-IL-12 had a longer half-life in vivo and a longer therapeutic effect due to the modification of IL-12 by PEG.
This example provided an evaluation of the anti-tumor effect of polyethylene glycol derivative modified interleukin-12 via stimulating T cells to kill tumor cells by the polyethylene glycol derivative modified interleukin-12. The specific experimental steps are as follows:
This example used a mouse tumor model to evaluate the in vivo anti-tumor effect of mPEG-MAL-20KD-IL-12 prepared in Example 4. Human hematopoietic stem cells (HSC) were transplanted into irradiated myeloablative M-NSG mice to construct CD34+ humanized mice, and pp65-MCF-7 cancer cells were transplanted into mice to create cancer models for evaluation of the in vivo anti-tumor activity of PEG-modified IL-12. 10 mice/group were given the specified dose of the test article every week (IP), and the experimental groups were injected with mPEG-MAL-20KD-IL-12 prepared in Example 4 (2 ug/Kg) and IL-12 (100 ng/Kg) respectively, and the control group was injected with PBS buffer. Tumor volume was measured before injection and on days 5, 10, 12, 14, 16, 18, and 30 after injection, and blood was collected to analyze lymphocyte activation/proliferation and blood IFN-γ content. The differences in anti-tumor ability and side effects of mPEG-MAL-20K-IL-12 and IL-12 in vivo were statistically analyzed.
This example provided long-term stimulation of NK-92 cells by mPEG-pALD-20KD-IL-12 (Example 1), mPEG-MAL-20KD-IL-12 (Example 4), and IL-12, and the long-term effect in vitro of polyethylene glycol-modified interleukin-12 was evaluated by cell counting and detection of IFN-γ release amount.
In the experiment, NK92 cells were divided into four groups, with two T25 bottles in each group, and 6 mL of NK92 cells was added into each bottle. The cell density was 1×10{circumflex over ( )}5/mL, and 100 pg/mL of IL-12 or mPEG-MAL-20KD-IL-12 or mPEG-pALD-20KD-IL-12 was added to each group, respectively, or no drug was added as the control group. Then, the cells were subjected to stationary culture in a 5% CO2 incubator at 37° C., and 120 μL was sampled on days 1, 2, 3, 5, 7, 9, 11, 13, and 15 respectively to detect the release amount of IFN-γ, and 20 μL was sampled on days 3, 7, 11, and 15 respectively to detect the changes of number and viability of the cells.
The results are as shown below. The bar graph in
IL-12 could stimulate T cells to differentiate towards Th1, and could stimulate T cells or NK cells to produce pro-inflammatory cytokines, thereby improving the body's immunity and enhancing the body's resistance ability to viral infection, bacterial infection, parasitic infection and tumors. This example provided an evaluation of the effect on improving immunity, resistance to infection and anti-tumor of polyethylene glycol derivative modified interleukin-12 via stimulating NK cells to kill tumor cells by the polyethylene glycol derivative modified interleukin-12. The specific experimental steps are as follows:
The stability of the sample was evaluated by analyzing the particle size distribution of protein from multiple perspectives, that is, detecting natural IL-12(S1), mPEG-MAL-20K-IL-12 (S2) and mPEG-pALD-20K-IL-12 (S3) using high-throughput protein stability analysis system (Uncle), and analyzing the result parameters Tm (protein melting temperature), Tagg (protein aggregation temperature), Z-Ave.Dia (average protein particle size), PDI (particle size polydispersity), Pk1. Mode Dia, Pk1 Mass % and other parameters.
Table 9 is a summary of the results. It can be seen that polyethylene glycol modification could increase the Tm value and Tagg value of IL-12, and could also increase the particle size of the protein while maintaining a relatively uniform dispersity.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111166180.7 | Sep 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/123400 | 9/30/2022 | WO |
