Deflective mutant of interleukin-12 and production method and application thereof

Abstract

The invention belongs to the technical field of biological pharmacy, and relates to a biased mutant of interleukin-12 as well as a production method and application of the biased mutant. According to the application, the subunit of the wild type interleukin-12 is modified, so that an interleukin-12 biased mutant with specific mutation is obtained, and the affinity of the interleukin-12 biased mutant with a receptor is reduced compared with that of the wild type interleukin-12.

Description

Technical Field

[0001] The present application belongs to the field of biopharmaceutical technology and relates to a biased mutant of interleukin-12 and a production method and application thereof. Background Art

[0002] Interleukin-12 (IL-12), discovered in 1988, is a cytokine with a wide range of biological activities, primarily synthesized and secreted by activated inflammatory cells (monocytes, macrophages, dendritic cells, and other antigen-presenting cells). Human IL-12 is a heterodimer composed of two subunits, p35 (IL-12α) and p40 (IL-12β), linked by a disulfide bond. p40 consists of 306 amino acids, has a molecular weight of approximately 40 kDa, and contains 10 cysteine ​​residues and four potential glycosylation sites. p35 consists of 197 amino acids, has a molecular weight of approximately 35 kDa, and contains seven cysteine ​​residues and three potential glycosylation sites.

[0003] As research deepened, researchers discovered that IL-12's immunomodulatory and anti-angiogenic properties give it potential as an anti-tumor agent. Over a decade ago, clinical trials used IL-12 to treat tumors, including T-cell lymphoma, non-Hodgkin's lymphoma, melanoma, ovarian cancer, Kaposi's sarcoma, and renal cancer. Systemic IL-12 has been shown to inhibit tumor growth, metastasis, and angiogenesis. However, IL-12 should not be used in high doses. In early studies, a single injection of 500 ng / kg / day of IL-12 caused severe toxicity (Effects of Single-Dose 25 Interleukin-12 Exposure on Interleukin-12-Associated Toxicity and Interferon-g Production). Common side effects include fever / chills, fatigue, nausea, vomiting, and headache. Anemia, neutropenia, lymphopenia, and hyperglycemia have also been observed in clinical studies. Thrombocytopenia and hypoalbuminemia have also been observed. Furthermore, these side effects can accumulate over time. Most research reports believe that the toxic side effects of IL-12 are caused by IL-12 stimulating NK or T cells to produce excessive IFN-γ. Some literature also believes that it is caused by IL-12 stimulating macrophages, causing them to proliferate and then express excessive TNF-α (Abrogation of TNF-aProduction during Cancer Immunotherapy Is Crucial for Suppressing Side Effects Due to the Systemic Expression of IL-12).

[0004] To overcome the toxic side effects of IL-12, researchers have developed a variety of methods:

[0005] 1. Improvements in drug delivery methods and procedures, including localized administration at the tumor site and pre-administration of a small dose followed by a therapeutic dose, still have drawbacks. Localized administration is not effective for systemic tumors, and while pre-administration can reduce the incidence of adverse reactions in patients, it cannot completely eliminate them.

[0006] 2. Intratumoral injection of DNA and RNA encoding IL-12 has the potential to localize and sustain IL-12 production within the tumor microenvironment. Compared to recombinant cytokines, nucleic acids are easier to produce, purify, and manipulate. However, mammalian host cells are difficult to transfect, typically requiring chemical, physical, or electrical assistance to achieve reasonable transfection rates, or treatment with IL-12 packaged with lentivirus or oncolytic viruses.

[0007] 3. At the formulation level, adding IL-12 to polymer microspheres, liposomes, or gels can achieve a sustained release effect. Although this is unlikely to cause toxic side effects as frequently administered as in early studies, it still does not fundamentally address the toxic side effects of IL-12, and high doses can still cause toxic side effects.

[0008] 4. Design IL-12 and antibodies into fusion proteins, or connect the peptide to the receptor to make a prodrug fusion protein. The peptide can be degraded by metalloproteinases highly expressed in the tumor microenvironment, making it targeted. The treatment has better targeting and can also reduce some toxic side effects. However, the dosage of antibody drugs is about 100-200 mg, while the dosage of IL-12 is only 5 μg. The difference is too large and difficult to match.

[0009] In view of this, this application is hereby filed. Summary of the Invention

[0010] Based on this, one or more embodiments of the present application provide a biased mutant of interleukin-12 and a production method and application thereof.

[0011] One or more embodiments of the present application provide a biased mutant of interleukin-12, which has a p35 subunit biased mutant and / or a p40 subunit biased mutant;

[0012] Relative to the p35 subunit of wild-type interleukin-12 or a conservative mutant thereof, the p35 subunit-biased mutant has one or more of the following active site amino acid mutations: F39, Y40, I47, D126, K128, R129, Q130, F166, Y167, K168, and K170;

[0013] Compared to the wild-type interleukin-12 p40 subunit or a conservative mutant thereof, the p40 subunit-biased mutant has one or more of the following active site amino acid mutations: D18, E45, K58, E86, K195, and K197;

[0014] Compared with wild-type interleukin-12, the interleukin-12 biased mutant has a decreased affinity for the receptor.

[0015] In some embodiments of the present application, the amino acid mutation includes an alanine mutation.

[0016] In some embodiments of the present application, the p35 subunit-biased mutant has the following amino acid mutations: D126A, K128A, Y167A, and K168A.

[0017] In some embodiments of the present application, the amino acid sequence of the p35 subunit-biased mutant is shown in SEQ ID NO: 3.

[0018] In some embodiments of the present application, the p40 subunit-biased mutant has the following amino acid mutations: D18A, E45A, K58A, E86A, K195A, and K197A.

[0019] In some embodiments of the present application, the amino acid sequence of the p40 subunit biased mutant is shown in SEQ ID NO: 6.

[0020] In some embodiments of the present application, the amino acid mutation comprises an approximate amino acid substitution mutation.

[0021] In some embodiments of the present application, the p35 subunit-biased mutant has the following amino acid mutations: D126N, K128R, Y167F, and K168R.

[0022] In some embodiments of the present application, the amino acid sequence of the p35 subunit-biased mutant is shown in SEQ ID NO: 4.

[0023] In some embodiments of the present application, the p40 subunit-biased mutant has the following amino acid mutations: D18N, E45Q, K58R, E86Q, K195R, and K197N.

[0024] In some embodiments of the present application, the amino acid sequence of the p40 subunit biased mutant is shown in SEQ ID NO: 7.

[0025] In some embodiments of the present application, the amino acid mutation includes a transposition mutation.

[0026] In some embodiments of the present application, the p35 subunit-biased mutant has the following amino acid mutations: F39Y, Y40F, I47K, D126N, R129Q, Q130R, F166H, Y167F, K168E and K170R.

[0027] In some embodiments of the present application, the amino acid sequence of the p35 subunit-biased mutant is shown in SEQ ID NO: 5.

[0028] In some embodiments of the present application, the interleukin-12 biased mutant is:

[0029] A heterodimer of the polypeptides represented by SEQ ID NO: 3 and SEQ ID NO: 2, or

[0030] A heterodimer of the polypeptides represented by SEQ ID NO: 4 and SEQ ID NO: 2, or

[0031] A heterodimer of the polypeptides represented by SEQ ID NO: 5 and SEQ ID NO: 2, or

[0032] A heterodimer of the polypeptides represented by SEQ ID NO: 1 and SEQ ID NO: 6, or

[0033] A heterodimer of the polypeptides represented by SEQ ID NO: 1 and SEQ ID NO: 7.

[0034] One or more embodiments of the present application further provide the p35 subunit-biased mutant or the p40 subunit-biased mutant defined above.

[0035] One or more embodiments of the present application further provide a nucleic acid molecule encoding the biased mutant of interleukin-12, or,

[0036] It encodes a p35 subunit-biased mutant or a p40 subunit-biased mutant as defined above.

[0037] In some embodiments of the present application, the nucleic acid molecule further includes a nucleic acid fragment encoding a signal peptide or a leader peptide.

[0038] One or more embodiments of the present application also provide a vector comprising the nucleic acid molecule.

[0039] In some embodiments of the present application, the vector comprises a co-expression vector of the p35 subunit biased mutant and the p40 subunit biased mutant.

[0040] One or more embodiments of the present application further provide a cell expressing the interleukin-12 biased mutant, or,

[0041] It expresses the p35 subunit-biased mutant or the p40 subunit-biased mutant defined above.

[0042] In some embodiments of the present application, the cell is a eukaryotic cell.

[0043] One or more embodiments of the present application also provide a method for constructing the cell, which includes the step of transferring the vector into a host to construct the cell.

[0044] One or more embodiments of the present application also provide a method for producing the interleukin-12 biased mutant, the p35 subunit biased mutant or the p40 subunit biased mutant defined above, which comprises the steps of culturing the cells and isolating the interleukin-12 biased mutant, the p35 subunit biased mutant or the p40 subunit biased mutant from the obtained culture.

[0045] One or more embodiments of the present application further provide a drug comprising:

[0046] The interleukin-12 biased mutant, the p35 subunit biased mutant or the p40 subunit biased mutant defined above, the nucleic acid molecule, the vector, and the cell;

[0047] and a pharmaceutically acceptable carrier.

[0048] One or more embodiments of the present application also provide the use of the interleukin-12 biased mutant, the p35 subunit biased mutant or the p40 subunit biased mutant defined above, the nucleic acid molecule, the vector, and the cell in the preparation of a drug for treating interleukin-12-mediated diseases.

[0049] In some embodiments of the present application, the interleukin-12-mediated disease includes cancer or autoimmune disease.

[0050] In some embodiments of the present application, the cancer comprises acute myeloid leukemia, anaplastic lymphoma, astrocytic lymphoma, B cell cancer, breast cancer, colon cancer, ependymoma, esophageal cancer, glioblastoma, glioma, leiomyosarcoma, liposarcoma, liver cancer, lung cancer, mantle cell lymphoma, melanoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, ovarian cancer, pancreatic cancer, peripheral T cell lymphoma, renal cancer, sarcoma, gastric cancer, carcinoma, mesothelioma or sarcoma.

[0051] In some embodiments of the present application, the autoimmune disease comprises rheumatoid arthritis, insulin-dependent diabetes mellitus, hemolytic anemia, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, alopecia areata, psoriasis, vitiligo, dystrophic epidermolysis bullosa, systemic lupus erythematosus, moderate to severe plaque psoriasis, psoriatic arthritis, Crohn's disease, ulcerative colitis or graft-versus-host disease.

[0052] The details of one or more embodiments of the present application are set forth in the description below, and other features, objects, and advantages of the application will become apparent from the description and from the claims thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0054] Figure 1 This is the DOT result of transiently expressing biased interleukin-12 in Example 1;

[0055] Figure 2 is an SDS-PAGE image of the purified sample in Example 2;

[0056] Figure 3 The affinity test results of the biased interleukin-12 and the corresponding receptor in Example 3;

[0057] Figure 4 The results of flow cytometry analysis of the phosphorylation levels of STAT4 in NK cells and T cells stimulated by interleukin-12 in Example 4 were compared with those of wild-type IL-12;

[0058] Figure 5 This is a graph showing the results of cytokine detection by flow cytometry in Example 5;

[0059] Figure 6 The killing rate of NK92 cells against Jurkat cells stimulated by interleukin-12 is shown. DETAILED DESCRIPTION

[0060] Below in conjunction with accompanying drawing, embodiment and example, the application is described in further detail.It should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms and is not limited to the embodiment and example described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large amount of specific details are given in order to provide a more complete understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing embodiments and examples only and are not intended to limit this application.

[0062] the term

[0063] Unless otherwise specified or incompatible therewith, terms and phrases used in this application shall have the following meanings:

[0064] The terms "and / or", "or / and", and "and / or" used in this application include any one of two or more related listed items, and also include any and all combinations of the related listed items, and the said any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0065] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0066] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0067] In this application, the "suitable" mentioned in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0068] In this application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute a limitation on the scope of protection of this application.

[0069] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0070] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0071] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Furthermore, "first," "second," "third," "fourth," etc. serve only as non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0072] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0073] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0074] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0075] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0076] “IL-12,” “IL-12 protein,” and “IL-12 protein” may be used as concepts including “IL-12 protein and fragments thereof.” Unless otherwise specified, the terms “protein,” “polypeptide,” and “peptide” may be used as interchangeable concepts.

[0077] A "subunit" is a part of a protein's quaternary structure. In proteins, each amino acid chain is a subunit. IL-12 is composed of two subunits: p35 and p40.

[0078] "Nucleic acid molecule" or "nucleic acid fragment" refers to a deoxyribonucleotide or ribonucleotide polymer in single- or double-stranded form and, unless otherwise limited, includes known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid fragment includes its complementary fragment.

[0079] "Amino acid sequence" refers to the sequence of amino acid residues from the amino terminus to the carboxyl terminus on a peptide chain. There is a corresponding relationship between the amino acid sequence and the nucleic acid sequence. A nucleic acid molecule with a specific nucleic acid sequence can be translated and synthesized into a peptide chain with the corresponding amino acid sequence.

[0080] "Mutation" refers to a change in the nucleotide sequence of the genome of an organism, virus or extrachromosomal DNA, including changes in the sequence caused by base substitutions, insertions, deletions or duplications. "Missense mutation" refers to a change in a DNA base pair that causes the encoded amino acid to change from one type to another. Site-directed mutations of amino acids are mainly achieved through missense mutations in nucleic acid sequences. "Nonsense mutation" is similar to missense mutation in that it also causes the encoded amino acid to change from one type to another due to a change in a base pair, but the difference is that this change causes the cell to translate the peptide chain prematurely. "Frameshift mutation" generally means that the code for (amino acid) is misplaced, thereby changing the type of amino acid encoded for synthesis, and ultimately leading to changes in the structure and function of the protein. Among them, insertions, deletions and duplications can all be frameshift mutations. The knockout and increase of amino acids are achieved through frameshift mutations in the nucleic acid sequence that correspond to a reduction or increase of three times.

[0081] "Affinity" or "binding capacity" refers to the ability of a receptor to specifically form an intermediate complex with its ligand.

[0082] In order to overcome the problem of IL-12 producing toxic side effects during application, the embodiment of the present application uses the difference in expression of IL-12 receptors on NK cells and T cells to change the binding ability of IL-12 to the receptor through mutation, thereby weakening the ability of IL-12 to activate NK cells while maintaining or less weakening the ability to activate T cells. On the one hand, the ability of IL-12 to activate NK cells and T cells is balanced. While producing an appropriate amount of IFN-γ, both NK cells and T cells are activated. On the other hand, the activity of IL-12 is reduced as a whole, so that its dosage can be increased to a level equivalent to that of the antibody dosage, thereby improving the safety and convenience of interleukin-12 administration, and allowing it to be used in combination with antibody anti-tumor drugs or with cell therapy gene therapy.

[0083] The technical solutions provided in the embodiments of this application include:

[0084] The first aspect of the embodiments of the present application

[0085] The present embodiment provides a biased mutant of interleukin-12, which has a p35 subunit biased mutant and / or a p40 subunit biased mutant;

[0086] Relative to the p35 subunit of wild-type interleukin-12 or a conservative mutant thereof, the p35 subunit-biased mutant has one or more of the following active site amino acid mutations: F39, Y40, I47, D126, K128, R129, Q130, F166, Y167, K168, and K170;

[0087] Compared to the wild-type interleukin-12 p40 subunit or a conservative mutant thereof, the p40 subunit-biased mutant has one or more of the following active site amino acid mutations: D18, E45, K58, E86, K195, and K197;

[0088] Compared with wild-type interleukin-12, the interleukin-12 biased mutant has a decreased affinity for the receptor.

[0089] "Interleukin-12-biased mutants" are synonymous with "biased interleukin-12," meaning they have been modified to differ from native interleukin-12 (or wild-type interleukin-12) in terms of receptor affinity, downstream signaling pathway activation, cell activation, and, consequently, tumor-killing ability. "Signaling pathways" refer to the process by which a cell's signal, transmitted from outside to inside, triggers a response. The cell then responds accordingly.

[0090] A "conservative mutant" refers to a mutant modified on the basis of wild-type interleukin-12, wherein the activity or function (including affinity for the receptor) of the resulting mutant remains consistent with that of the wild-type interleukin-12.

[0091] For example, a conservative variant of the p35 subunit, compared to the p35 subunit of wild-type interleukin-12, or a conservative variant of the p40 subunit, compared to the p40 subunit of wild-type interleukin-12, has up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids replaced with amino acids having similar or similar properties. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table 1 below.

[0092] Table 1

[0093] Initial residue Representative replacement Preferred substitutions Ala Val; Leu; Ile Val Arg Lys; Gln; Asn Lys Asn Gln; His; Lys; Arg Gln Asp Glu Glu Cys Ser Ser Gln Asn Asn Glu Asp Asp Gly Pro; Ala Ala His Asn; Gln; Lys; Arg Arg Ile Leu; Val; Met; Ala; Phe Leu Leu Ile; Val; Met; Ala; Phe Ile Lys Arg; Gln; Asn Arg Met Leu; Phe; Ile Leu Phe Leu; Val; Ile; Ala; Tyr Leu Pro Ala Ala Ser Thr Thr Thr Ser Ser Trp Tyr; Phe Tyr Tyr Trp; Phe; Thr; Ser Phe Val Ile;Leu;Met;Phe;Ala Leu

[0094] For example, a conservative mutant of the p35 subunit is prepared by replacing, deleting or adding at least one amino acid in the p35 subunit of wild-type interleukin-12 without changing the function of IL-12, or a conservative mutant of the p40 subunit is prepared by replacing, deleting or adding at least one amino acid in the p40 subunit of wild-type interleukin-12 without changing the function of IL-12, and these conservative mutants have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with their corresponding wild types.

[0095] Traditionally, wild-type amino acid residues are substituted with alanine, but this substitution can be a conservative amino acid substitution that has no or weak effect on the overall protein charge, i.e., polarity or hydrophobicity. For conservative amino acid substitutions, reference can be made to Table 2 below.

[0096] Table 2

[0097]

[0098] For each amino acid, additional conservative substitutions include "homologs" of the amino acid. Specifically, "homologs" refer to amino acids obtained by inserting a methylene group (CH2) into the β-side chain of the amino acid side chain. Examples of "homologs" include, but are not limited to, homophenylalanine, homoarginine, and homoserine.

[0099] In some examples, the amino acid mutation includes alanine mutation, i.e., other amino acids at appropriate sites are mutated to alanine.

[0100] Optionally, the p35 subunit-biased mutant has the following amino acid mutations: D126A, K128A, Y167A, and K168A. Further optionally, the amino acid sequence of the p35 subunit-biased mutant is shown in SEQ ID NO: 3.

[0101] Optionally, the p40 subunit-biased mutant has D18A, E45A, K58A, E86A, K195A and K197A.

[0102] Further optionally, the amino acid sequence of the p40 subunit biased mutant is shown in SEQ ID NO: 6.

[0103] In some examples, the amino acid mutation includes an approximate amino acid substitution mutation, which refers to a substitution of an amino acid at a suitable site by an amino acid with similar or similar properties.

[0104] Optionally, the p35 subunit-biased mutant has the following amino acid mutations: D126N, K128R, Y167F, and K168R. Further optionally, the amino acid sequence of the p35 subunit-biased mutant is shown in SEQ ID NO:4.

[0105] Optionally, the p40 subunit-biased mutant has the following amino acid mutations: D18N, E45Q, K58R, E86Q, K195R, and K197N. Further optionally, the amino acid sequence of the p40 subunit-biased mutant is shown in SEQ ID NO: 7.

[0106] In some examples, the amino acid mutation comprises a transposition mutation.

[0107] Optionally, the p35 subunit-biased mutant has the following amino acid mutations: F39Y, Y40F, I47K, D126N, R129Q, Q130R, F166H, Y167F, K168E, and K170R. Further optionally, the amino acid sequence of the p35 subunit-biased mutant is shown in SEQ ID NO: 5.

[0108] Furthermore, the biased mutant of interleukin-12 is:

[0109] A heterodimer of the polypeptides represented by SEQ ID NO: 3 and SEQ ID NO: 2, or

[0110] A heterodimer of the polypeptides represented by SEQ ID NO: 4 and SEQ ID NO: 2, or

[0111] A heterodimer of the polypeptides represented by SEQ ID NO: 5 and SEQ ID NO: 2, or

[0112] A heterodimer of the polypeptides represented by SEQ ID NO: 1 and SEQ ID NO: 6, or

[0113] A heterodimer of the polypeptides represented by SEQ ID NO: 1 and SEQ ID NO: 7.

[0114] The second aspect of the embodiment of the present application

[0115] The embodiments of the present application provide the p35 subunit-biased mutant or the p40 subunit-biased mutant defined in the first aspect.

[0116] The third aspect of the embodiments of the present application

[0117] The embodiments of the present application provide a nucleic acid molecule encoding the interleukin-12 biased mutant described in the first aspect, or encoding the p35 subunit biased mutant or the p40 subunit biased mutant defined in the first aspect.

[0118] The nucleic acid molecule may further include a nucleic acid fragment encoding a signal peptide or a nucleic acid fragment encoding a leader peptide.

[0119] As used herein, the term "signal peptide" refers to a fragment that guides the secretion of bioactive molecule drugs and fusion proteins, and is cut off after translation in the host cell. The "nucleic acid fragment encoding a signal peptide" of the present application is a polynucleotide that encodes an amino acid sequence that starts the protein movement through the endoplasmic reticulum (ER) membrane. The signal peptides in the present application embodiment include: a signal peptide of a human interleukin 12-p35 subunit, a signal peptide of a human interleukin-12p40 subunit, a signal peptide of a mouse interleukin-12p35 subunit, and a signal peptide of a mouse interleukin-12p40 subunit. The characteristics of signal peptides are well known in the art, and signal peptides generally have 16 to 30 amino acids, but they can contain more or less number of amino acid residues. Conventional signal peptides are composed of three regions: an alkaline N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region includes 4 to 12 hydrophobic residues, which fix the signal peptide by the membrane lipid bilayer during the translocation of the immature polypeptide. After activation, the signal peptide is cut off in the lumen of the ER by a cellular enzyme commonly referred to as a signal peptidase. Specifically, the signal peptide can be a secretion signal peptide of tissue plasminogen activator (tPa), a signal peptide of herpes simplex virus glycoprotein D (HSV gDs), or growth hormone. Alternatively, a secretion signal peptide used in higher eukaryotic cells including mammals can be used. In addition, as a secretion signal peptide, a signal peptide contained in wild-type IL-7 can be used, or it can be used after replacing a codon with a high expression frequency in the host cell.

[0120] The fourth aspect of the embodiments of the present application

[0121] An embodiment of the present application provides a vector comprising the nucleic acid molecule described in the third aspect.

[0122] An "expression system" or "expression vector (or vector)" refers to a nucleic acid sequence containing a desired coding sequence and control sequences in operable association so that a host transformed with these sequences can produce the encoded protein. To achieve transformation, the expression system can be contained on a vector; however, the relevant nucleic acid molecule can also subsequently be integrated into the host chromosome.

[0123] In some examples, the vector comprises a co-expression vector of the p35 subunit-biased mutant and the p40 subunit-biased mutant.

[0124] The fifth aspect of the embodiment of the present application

[0125] The present application provides a cell that expresses the interleukin-12 biased mutant described in the first aspect, or,

[0126] It expresses the p35 subunit-biased mutant or the p40 subunit-biased mutant defined in the first aspect.

[0127] In some examples, the cell comprises a eukaryotic cell. In some examples, in some embodiments, the cell is an animal cell. In some examples, the animal cell is a mammalian cell. In some embodiments, the animal cell is a human cell.

[0128] The sixth aspect of the embodiments of the present application

[0129] The embodiment of the present application provides a method for constructing the cell described in the fifth aspect, which includes the step of transferring the vector described in the fourth aspect into a host to construct the cell.

[0130] "Transfection," "stable transfection," or "transient transfection" refers to the uptake of an expression vector by a host cell, regardless of whether any coding sequence is actually expressed. A variety of transfection methods are known to those skilled in the art. For example, transfection can be accomplished in the presence of an expression vector and a high concentration of calcium phosphate, by electroporation, by using a bacteriophage or viral expression vector for insertion into a host cell, by mechanical insertion of nucleic acid, or even by culturing host cells in the presence of unpackaged nucleic acid fragments. Successful transfection is typically confirmed when any indication of manipulation of the vector of interest appears in the host cell.

[0131] The seventh aspect of the embodiment of the present application

[0132] The embodiments of the present application provide a method for producing the interleukin-12 biased mutant described in the first aspect, the p35 subunit biased mutant, or the p40 subunit biased mutant defined in the first aspect, which comprises the steps of culturing the cells described in the fifth aspect and isolating the interleukin-12 biased mutant, the p35 subunit biased mutant, or the p40 subunit biased mutant from the obtained culture.

[0133] The eighth aspect of the embodiments of the present application

[0134] The present invention provides a drug comprising:

[0135] The interleukin-12 biased mutant described in the first aspect, the p35 subunit biased mutant or the p40 subunit biased mutant defined in the first aspect, the nucleic acid molecule described in the third aspect, the vector described in the fourth aspect, the cell described in the fifth aspect; and a pharmaceutically acceptable carrier.

[0136] The medicine that is suitable for injection includes sterile aqueous solution (water-soluble) or dispersion, and the sterile powder for the temporary preparation of sterile injectable solution or dispersion.For intravenous administration, suitable carrier includes physiological saline, antibacterial water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).In all cases, medicine should be sterile and should be the fluid of the degree that is easy to inject.It should be stable and must resist the contamination of microorganisms (such as bacteria and fungi) and preserve under manufacturing and storage conditions.Carrier can be solvent or dispersion medium, and described solvent or dispersion medium contain such as water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol etc.) and its suitable mixture.For example, can by using coating such as lecithin, by maintaining required particle size and by using surfactant (for example, sodium lauryl sulfate), maintain suitable fluidity.Preventing the effect of microorganism can be realized by various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal etc.). In many cases, isotonic agents, for example, sugars, polyalcohols (such as mannitol, sorbitol), and / or sodium chloride will generally be included in the composition. Prolonged absorption of the injectable compositions can be achieved by including in the composition an agent that delays absorption (for example, aluminum monostearate and gelatin).

[0137] Sterile injectable solutions can be prepared by incorporating the active ingredient in the desired amount into a suitable solvent optionally with one or a combination of the ingredients listed above, followed by sterilization by filtration. Typically, dispersions are prepared by incorporating the active ingredient into a sterile vehicle containing a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution.

[0138] In some cases, the medicine of the present application is prepared from a carrier that protects active ingredients from being rapidly eliminated from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Such formulations can be prepared using standard techniques. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies directed against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods well known to those skilled in the art.

[0139] A ninth aspect of the embodiments of the present application

[0140] The embodiments of the present application provide the use of the interleukin-12 biased mutant described in the first aspect, the p35 subunit biased mutant or the p40 subunit biased mutant defined in the first aspect, the nucleic acid molecule described in the third aspect, the vector described in the fourth aspect, and the cell described in the fifth aspect in the preparation of a drug for treating interleukin-12-mediated diseases.

[0141] The interleukin-12 mediated disease is, for example, cancer or autoimmune disease.

[0142] The term "cancer" generally refers to the presence of cells with typical characteristics of cancer cells (such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features). Cancer cells are usually observed to aggregate into tumors, but such cells can exist alone in animal subjects, or can be non-tumorigenic cancer cells, such as leukemia cells. Therefore, the term "cancer" can encompass reference to solid tumors, soft tissue tumors, or metastatic lesions. As used herein, the term "cancer" includes precancerous and malignant cancers. In some embodiments, the cancer is a solid tumor, soft tissue tumor, or metastatic lesion. In some embodiments, provided herein are methods for treating a disorder in a subject in need thereof, wherein the disorder is a cancer selected from the group consisting of acute myeloid leukemia, anaplastic lymphoma, astrocytic lymphoma, B-cell cancer, breast cancer, colon cancer, ependymoma, esophageal cancer, glioblastoma, glioma, leiomyosarcoma, liposarcoma, liver cancer, lung cancer, mantle cell lymphoma, melanoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, ovarian cancer, pancreatic cancer, peripheral T-cell lymphoma, renal cancer, sarcoma, gastric cancer, carcinoma, mesothelioma, and sarcoma.

[0143] In some embodiments, the immune disease is an autoimmune disease. In some embodiments, the autoimmune disease is selected from rheumatoid arthritis, insulin-dependent diabetes mellitus, hemolytic anemia, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, alopecia areata, psoriasis, vitiligo, dystrophic epidermolysis bullosa, systemic lupus erythematosus, moderate to severe plaque psoriasis, psoriatic arthritis, Crohn's disease, ulcerative colitis, and graft-versus-host disease.

[0144] The tenth aspect of the embodiment of the present application

[0145] An embodiment of the present application provides a method for alleviating interleukin-12-mediated diseases, comprising administering to the subject the interleukin-12 biased mutant described in the first aspect, the p35 subunit biased mutant or the p40 subunit biased mutant described in the second aspect, the nucleic acid molecule described in the third aspect, the vector described in the fourth aspect, the cell described in the fifth aspect, or the drug described in the eighth aspect.

[0146] In this application, "alleviate" and "prevent and / or treat" have the same meaning and can be used interchangeably. In this application, "alleviate" includes aspects such as prevention, treatment, and adjuvant therapy. As used herein, "alleviate" means to reduce, slow the progression, attenuate, prevent, or maintain an existing disease or condition. "Alleviate" also includes curing, preventing the development of, or alleviating to a certain extent one or more symptoms of a disease or condition.

[0147] In the present application, the subject is a mammal. In some examples, the mammal is a human.

[0148] The definition of "interleukin-12-mediated disorder" refers to the definition in the ninth aspect.

[0149] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0150] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0151] In the examples of this application, the following are firstly analyzed: the amino acid sites that affect the affinity of interleukin-12 receptor binding are identified by structural analysis. Secondly, the corresponding nucleic acid fragments are obtained through site-directed amino acid mutagenesis or whole gene synthesis, and then constructed into a plasmid vector for transient or stable transfection and purification to obtain the biased interleukin-12.

[0152] This application mutates the nucleic acid fragment of natural interleukin-12, expresses the corresponding protein by transient transfection, and performs affinity detection on the modified biased interleukin-12 and the interleukin-12 receptors RB1 and RB2 to observe the affinity changes. Stimulate NK cells and T cells with biased interleukin-12, detect the activation of the main downstream signaling pathways, detect the production of cell activity-related cytokines, and evaluate the differences in cell activation levels. Use biased interleukin-12 to activate NK92 cells to kill tumor cells and evaluate the potential of biased interleukin-12 for tumor killing.

[0153] Example 1: Biased modification of IL-12

[0154] IL-12 has two subunits, p35 and p40. In the present embodiment, the amino acids that affect the activity are mutated by gene mutation.

[0155] The sequence of the biased mutant obtained by alanine mutation of the active site in the p35 subunit of human IL-12 is: SEQ ID NO: 3;

[0156] The sequence of the biased mutant obtained by performing a near substitution mutation on the active site of the p35 subunit of human IL-12 is: SEQ ID NO: 4;

[0157] The sequence of the biased mutant obtained by performing substitution mutation on the active site of the p35 subunit of human IL-12 is: SEQ ID NO: 5;

[0158] The sequence of the biased mutant obtained by alanine mutation of the active site in the p40 subunit of human IL-12 is: SEQ ID NO: 6;

[0159] The sequence of the biased mutant obtained by performing an approximate substitution mutation on the active site of the p40 subunit of human IL-12 is: SEQ ID NO: 7.

[0160] Nucleic acid fragments corresponding to the amino acid fragments were synthesized using whole-gene synthesis technology. Nucleic acid fragments encoding the p40 and p35 subunits were constructed on the co-expression plasmid pCGS3. Nucleic acid fragments were optimized for 293F cells prior to synthesis, avoiding restriction enzyme cleavage sites such as HindIII, EcoRI, SalI, NotI, PvuI, BamHI, and ScaI. Following optimization, a Kozak fragment (GCCACC) and a signal peptide were introduced at the N-terminus, and a TAA stop codon was added to the C-terminus. Of note, six His residues were added to the C-terminus of the weakly expressed p35 subunit as a purification tag to reduce the number of purification steps and complexity.

[0161] The synthesized vector containing the target nucleic acid fragment was transformed and shaken, and the endotoxin removal plasmid was extracted and sequenced. Subsequently, according to the combination in the table below, the expression vector containing the nucleic acid fragment encoding the P35 subunit and the nucleic acid fragment encoding the P40 subunit was transiently transfected into 293F cells for transient expression. The concentration of the expression vector was 0.8 μg / mL, the transfection reagent was 1.2 μL / mL, the transfection system was Opti-MEM, and the transfection reagent was PEI4W. The day before transfection, 1×10 6Prepare 1000 mL of cell suspension at 100 cells / mL. Incubate cells overnight at 37°C, 125 rpm, and 5% CO2. For transfection, add the corresponding volume of PEI4W to a 50 mL empty tube. In a second 50 mL tube, dilute the desired concentration of DNA plasmid in serum-free medium to a final volume of 10% of the total volume. Gently mix thoroughly. Pour the diluted DNA into the pure PEI4W reagent all at once, immediately mix the solution, and incubate at room temperature for 10 minutes. Pour the PEI4W / DNA transfection mixture into the cells and mix thoroughly with the cell suspension. Continue incubating until the viability falls below 80%. Harvest the cell supernatant and assess protein expression by DOT. Purify the target protein using a Ni column and analyze its purity by SDS-PAGE.

[0162] Table 3

[0163]

[0164]

[0165] Figure 1 The DOT results of transient expression of biased interleukin-12, where the first number represents the combination number and the second number represents the type of culture medium used (1 represents Expi293 TM Expression Medium, 2 represents OMP-CD03). The results shown in the figure show that all five IL-12 tropisms are expressed, with combinations 2 and 3 showing higher expression levels, and combinations 4, 5, and 6 showing lower expression levels.

[0166] Figure 2 This is the SDS-PAGE image of the purified samples. Lane 1 is wild-type human interleukin-12, and lanes 2 to 6 correspond to the biased interleukin-12 of combination numbers 2 to 6 in the table, respectively. The purity is above 90% and can be used for subsequent experiments.

[0167] Wild-type interleukin-12p35 subunit (SEQ ID NO: 1):

[0168] RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS.

[0169] Wild-type interleukin-12 p40 subunit (SEQ ID NO: 2):

[0170] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS.

[0171] Bias interleukin-12 p35A subunit (SEQ ID NO: 3):

[0172] RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMAPARQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFAATKIKLCILLHAFRIRAVTIDRVMSYLNAS.

[0173] Bias interleukin-12 P35R subunit (SEQ ID NO: 4):

[0174] RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMNPRRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFFRTKIKLCILLHAFRIRAVTIDRVMSYLNAS.

[0175] Bias interleukin-12 p35Q subunit (SEQ ID NO: 5):

[0176] RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEYFPCTSEEKDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMNPKQRIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDHFETRIKLCILLHAFRIRAVTIDRVMSYLNAS。

[0177] Biased interleukin-12 p40 A subunit (SEQ ID NO: 6):

[0178] IWELKKDVYVVELDWYPAAPGEMVVLTCDTPEEDGITWTLDQSSAVLGSGKTLTIQVAEFGDAGQYTCHKGGEVLSHSLLLLHKKADGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHALAYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS。

[0179] Biased interleukin-12 p40 R subunit (SEQ ID NO: 7):

[0180] IWELKKDVYVVELDWYPNAPGEMVVLTCDTPEEDGITWTLDQSSQVLGSGKTLTIQVREFGDAGQYTCHKGGEVLSHSLLLLHKKQDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHRLNYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS。

[0181] Example 2

[0182] In this example, the IFN-γ ELISA kit was used to detect the production of IFN-γ by NK-92 cells stimulated by biased interleukin-12, and to evaluate the difference in activity between biased interleukin-12 and wild-type IL-12. The specific method is as follows:

[0183] (1) Take the cultured NK-92 cells and record the passage number. Observe the cell status under a microscope. If the cells are in good condition, collect the cells in a 15 mL or 50 mL sterile centrifuge tube, centrifuge at 600 rpm for 5 minutes, discard the supernatant, and resuspend the cells in 5 mL of fresh complete culture medium and count them.

[0184] (2) Based on the cell count results, dilute the cell suspension to 2×10 5 cells / mL, and then add the cell suspension to a 96-well plate, with 100 μL per well.

[0185] (3) The sample was sterilized by filtration and graded diluted with NK-92 complete medium to a final concentration of 1000 to 0.46 U / mL. The diluted sample was added to a 96-well plate containing 100 μL of cell suspension. The final culture volume was 200 μL and the final cell density was 1×10 5 cells / mL, and stimulate cells for 24 ± 2 hours.

[0186] (4) After stimulation, centrifuge the 96-well plate at 1000 rpm for 5 minutes and dilute 60 times with the standard diluent (1X Dilution buffer R) in the ELISA kit. Use a 10 μL 8-channel pipette to draw 5 μL of the supernatant and add it to 295 μL of the standard diluent. Use a plate washer to shake at high speed for 5 minutes. Then take 100 μL of the diluted supernatant and add it to the ELISA plate well of the IFN-γ detection kit. Add 50 μL of biotinylated antibody, mix well, cover with a sealing film, and incubate at room temperature (18-25°C) for 120 minutes. After incubation, wash the plate four times with washing solution and pat dry.

[0187] (5) Then add 100 μL of streptavidin-HRP and incubate at room temperature (18-25°C) for 20 minutes. After incubation, wash the plate four times with washing solution and pat dry.

[0188] (6) Add 100 μL of TMB to each well and incubate at room temperature (18-25°C) in the dark for 5-30 minutes. The liquid in the well gradually turns blue. After incubation at room temperature, add 100 μL of Stop solution to each well to terminate the reaction. Detect the OD value at 450 nm using a SpectraMax M2 microplate reader.

[0189] Result processing: GraphPad Prism 6 software was used to process the data. OD value was used as the vertical axis and dilution factor as the horizontal axis. A four-parameter fitting was used to obtain the activity curve, top and bottom values, and R2 value. The test standard requires an R2 value of ≥ 0.95. The activity of the test sample was calculated according to the following formula:

[0190]

[0191] Where: Pr is the biological activity of the standard, that is, the biological activity of the WHO IL-12 standard = 1×10 4 U / mL; Ds is the pre-dilution multiple of the test sample; Dr is the pre-dilution multiple of the standard; Es is the dilution multiple of the test sample equivalent to the half-effective dose of the standard; Er is the dilution multiple of the standard equivalent to the half-effective dose.

[0192] Specific activity of the test article (U / mg) = test article potency (U / mL) / test article concentration (mg / mL).

[0193] The experimental results are shown in the following table:

[0194] Table 4

[0195] Combination number Group Name Protein content mg / mL <![CDATA[Specific activity of sample 10 6 U / mg]]> Activity reduction factor 1 hIL-12 1.0 12.58 2 pIL-12-2 0.67 0.0033 3784.3 3 pIL-12-3 0.57 0.25 51.6 4 pIL-12-4 0.5 0.0062 2056.8 5 pIL-12-5 0.61 0.02 619.9 6 pIL-12-6 0.65 0.23 52.9

[0196] As can be seen from the above table, compared with wild-type IL-12, the activity of interleukin-12 decreased to varying degrees, among which pIL-12-2 and pIL-12-4 decreased the most, by 3784 times and 2056 times, respectively, while pIL-12-3, pIL-12-5 and pIL-12-6 decreased less.

[0197] Example 3

[0198] In this example, the binding ability of biased interleukin-12 to receptors was studied by ELISA method. The specific method is as follows:

[0199] (1) Interleukin-12 receptors RB1 and RB2 were diluted to 0.1 μg / mL with 0.01 M PBS, added to a blank 96-well ELISA plate, 100 μL per well, and coated at 4°C overnight.

[0200] (2) Discard the incubated receptors and add 5 wt% skim milk powder as blocking solution, 250 μL per well, at 37°C for 3 hours;

[0201] (3) Discard the blocking solution, add 200 μL PBST, wash three times, and pat dry;

[0202] (4) The obtained biased interleukin-12 was diluted from a starting concentration of 40 μg / mL to 0.005 μg / mL, added to the above-mentioned ELISA plate, 100 μL per well, and incubated at 37°C for 1 hour. After the incubation, 200 μL PBST was added, washed three times, and patted dry;

[0203] (5) Add anti-His antibody diluted 1000-fold with 0.01 M PBST and incubate at 37°C for 1 hour. After incubation, add 200 μL PBST, wash three times, and pat dry.

[0204] (6) Add goat anti-mouse secondary antibody-HRP diluted 3000 times with 0.01 M PBST and incubate at 37°C for 1 hour. After incubation, add 200 μL PBST, wash three times, and pat dry.

[0205] (7) Add 50 μL of TMB colorimetric solution to each well, place in the dark, incubate for 5 minutes, and add 50 μL of stop solution (0.2 M sulfuric acid) to terminate the reaction;

[0206] The results were read at 450 nm within 30 minutes using a microplate reader and processed using Graphpad. Figure 3 shown.

[0207] from Figure 3 It can be seen that the affinity of the modified subunit of biased interleukin-12 to the corresponding receptor is significantly reduced, indicating that it has bias at the receptor binding level.

[0208] Example 4

[0209] In this example, NK cells and T cells were stimulated by biased interleukin-12, and the phosphorylation level of STAT4, the main signaling pathway of interleukin-12-stimulated cells, was detected by flow cytometry to evaluate the biased differences in activating the main signaling pathways by biased interleukin-12. The specific implementation method is as follows:

[0210] (1) NK cells and T cells were starved overnight in reduced serum medium.

[0211] (2) Dilute the 5× fixative with deionized water and use it immediately. Preheat the 1× fixative at 37°C for 5-10 minutes. Precool the membrane permeabilization solution at -20°C for 10 minutes.

[0212] (3) Preparation: Sterile filter the interleukin-12 and prepare it at a 2× concentration, starting at a concentration of 1400 ng / mL and diluting it 10-fold.

[0213] Set up control wells without drug addition

[0214] (4) Cell treatment: Centrifuge at 1300 r for 5 minutes, adjust the cell density to 4E6 / mL, plate into a 96-well plate, add 50 μL per well, i.e., 2E5 / well, add 50 μL / well of drug, and stimulate at 37°C for 20 minutes.

[0215] (5) Fixation: After stimulation, quickly add the same volume of preheated cell fixative (100 μL), mix the cells, and incubate at 37°C for 10 minutes.

[0216] (6) Wash cells: Centrifuge at 1300 r for 5 minutes, discard the supernatant, and then wash once with PBS.

[0217] (7) Membrane permeabilization: 100-200 μL / well permeabilization solution, permeabilize the membrane on ice for at least 30 minutes (for 1E6-1E7 cells, add at least 1 ml of permeabilization solution, and add slowly).

[0218] (8) Wash cells: Wash twice with Intracellular Staining Perm Wash Buffer (Biolegend).

[0219] (9) Incubation with primary antibody: The primary antibody was prepared in Intracellular Staining Perm Wash Buffer (Biolegend) and incubated at room temperature in the dark for 60 minutes. Antibody dosage: 1E6 cells, 100 μl, is counted as one test, and 20 μl of antibody is added to each test. For a cell volume of 3E5 / well, 3 μL of antibody is actually added to each well.

[0220] (10) Wash cells: Wash twice with Intracellular Staining Perm Wash Buffer (Biolegend), resuspend in 100-200 μL / well FACS buffer and analyze.

[0221] Test results such as Figure 4 shown. Figure 4 Flow cytometry was used to detect the phosphorylation levels of STAT4 in NK cells and T cells stimulated by biased interleukin-12 and compared with wild-type IL-12. As can be seen from the figure, pIL-12-2, pIL-12-4, and pIL-12-5 have obvious bias, and pIL-12-5 has bias in a larger concentration range, while pIL-12-2 and pIL-12-4 have bias only at low concentrations.

[0222] Example 5

[0223] In this example, NK cells and T cells were stimulated by biased interleukin-12, and the amount of activity-related cytokines expressed by the cells was detected by flow cytometry to evaluate the biased difference in activating cell activity by biased interleukin-12. The specific implementation method is as follows:

[0224] (1) NK cells and T cells were starved overnight in reduced serum medium.

[0225] (2) Dilute the 5× fixative with deionized water and use immediately. Preheat the 1× fixative at 37°C for 5-10 minutes. Precool the permeabilization solution at -20°C for 10 minutes.

[0226] (3) Drug preparation: Prepare drugs at a 2X concentration, with a final concentration of 0.1 nM. The wells should contain Golgistop (BD) and be sterilized by filtration. Set up control wells without drug addition, and set up positive wells containing 1 mg / mL ionomycin (Sigma), 50 ng / mL phorbol 12-myristate 13-acetate (PMA, Sigma), and Golgistop (BD).

[0227] (4) Cell treatment: Centrifuge at 1300 r for 5 minutes, adjust the density of CD8+ T cells and NK cells to 4E6 / mL, plate into a 96-well plate, add 50 μL per well, i.e., 2E5 / well, add 50 μL / well of drug, and stimulate at 37°C for 4 hours.

[0228] (5) Fixation: After stimulation, quickly add the same volume of preheated cell fixative (100 μL), mix the cells, and incubate at 37°C for 10 minutes.

[0229] (6) Wash cells: Centrifuge at 1300 r for 5 minutes, discard the supernatant, and then wash once with PBS.

[0230] (7) Permeabilization: Add 100-200 μL / well of permeabilization buffer on ice for at least 30 minutes. (For 1E6-1E7 cells, add at least 1 mL of permeabilization buffer, and add slowly.)

[0231] (8) Wash cells: Wash twice with Intracellular Staining Perm Wash Buffer (Biolegend).

[0232] (9) Incubation with primary antibody: Dilute the primary antibody with Intracellular Staining Perm Wash Buffer (Biolegend) and incubate at room temperature in the dark for 60 minutes. Antibody dosage: 1E6 cells are counted as one test, and 5 μL of antibody is added to each test. For 3E5 cells / well, 1.5 μL of antibody is actually added to each well.

[0233] (10) Wash cells: Wash twice with Intracellular Staining Perm Wash Buffer (Biolegend), resuspend in 100-200 μL / well FACS buffer and analyze.

[0234] Figure 5 This is the result of cytokine detection by flow cytometry. It can be seen from the figure that the cytokines released after biased interleukin-12 stimulates CD8+T cells are not significantly different from those released by wild-type interleukin-12, but the amount of cytokines produced by biased interleukin 12 when stimulating NK cells is significantly lower than that of wild-type interleukin 12. Among them, the combinations of biased interleukin 12 No. 3, No. 5, and No. 6 have significant differences, and the significance of combination No. 3 is stronger.

[0235] Example 6

[0236] In this example, the biased interleukin-12 prepared in Example 1 was used to stimulate NK92 cells in vitro, and the stimulated NK92 cells were used for co-culture with tumor cells to examine the bias of the tumor killing ability of the biased interleukin-12.

[0237] The density of starved NK92 cells was adjusted to 1×10 5 / mL, divided into six parts, and plated into 6-well plates, 1.5mL per well, and pIL-12-2, pIL-12-4, pIL-12-5 and natural IL-12 were prepared to 50ng / mL. They were added to the above 6-well plates, 1.5mL per well, 37℃, 5% CO2, and stimulated for 48 hours. A group without drug was set as a control group. The stimulated NK92 cells were then harvested, centrifuged at 1300r / min for 5min, and resuspended in culture medium to 1×10 6 / mL. The cell density of Jurkat tumor cells was adjusted to 2×10 5 / mL and plated into 96-well plates, 50 μL per well. Stimulated NK92 cells were then added for co-culture at NK92:Jurkat ratios of 1:2.5, 1:1, 2.5:1, 5:1, 7.5:1, and 10:1. A group of NK92 cells with natural death and a control group with blank culture medium were set up. After 24 hours of co-culture, tumor cytotoxicity was analyzed using a CCK8 assay, and IFN-γ levels in the cell supernatant were measured using an ELISA kit.

[0238] from Figure 6 It can be seen that the tumor killing ability of NK92 cells stimulated by biased interleukin 12 is slightly higher than that of the blank control group, but lower than that of the natural interleukin 12 group.

[0239] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0240] The above-described embodiments only express several implementation methods of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that, for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of the present application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A biased mutant of interleukin-12, which has a p35 subunit biased mutant and / or a p40 subunit biased mutant; Compared with the p35 subunit of wild-type interleukin-12 or its conservative mutant, the p35 subunit biased mutant has amino acid mutations at one or more of the following active sites: F39, Y40, I47, D126, K128, R129, Q130, F166, Y167, K168 and K170; Compared with the p40 subunit of wild-type interleukin-12 or its conservative mutant, the p40 subunit biased mutant has amino acid mutations at one or more of the following active sites: D18, E45, K58, E86, K195 and K197; Compared with wild-type interleukin-12, the biased mutant of interleukin-12 has a decreased affinity for the receptor.

2. The biased mutant of interleukin-12 according to claim 1, which satisfies one of the conditions shown in the following (1) to (3): (1) The amino acid mutations include alanine mutations; Optionally, the p35 subunit biased mutant has the following amino acid mutations: D126A, K128A, Y167A and K168A; Further optionally, the amino acid sequence of the p35 subunit biased mutant is as shown in SEQ ID NO: 3; Optionally, the p40 subunit biased mutant has the following amino acid mutations: D18A, E45A, K58A, E86A, K195A and K197A; Further optionally, the amino acid sequence of the p40 subunit biased mutant is as shown in SEQ ID NO: 6; (2) The amino acid mutations include approximate amino acid substitution mutations; Optionally, the p35 subunit biased mutant has the following amino acid mutations: D126N, K128R, Y167F and K168R; Further optionally, the amino acid sequence of the p35 subunit biased mutant is as shown in SEQ ID NO: 4; Optionally, the p40 subunit biased mutant has the following amino acid mutations: D18N, E45Q, K58R, E86Q, K195R and K197N; Further optionally, the amino acid sequence of the p40 subunit biased mutant is as shown in SEQ ID NO: 7; (3) The amino acid mutations include transposition mutations; Optionally, the p35 subunit biased mutant has the following amino acid mutations: F39Y, Y40F, I47K, D126N, R129Q, Q130R, F166H, Y167F, K168E and K170R; Further optionally, the amino acid sequence of the p35 subunit biased mutant is as shown in SEQ ID NO: 5; Even more optionally, the biased mutant of interleukin-12 is: A heterodimer of the polypeptides shown in SEQ ID NO: 3 and SEQ ID NO: 2, or, A heterodimer of the polypeptides shown in SEQ ID NO: 4 and SEQ ID NO: 2, or, A heterodimer of the polypeptides shown in SEQ ID NO: 5 and SEQ ID NO: 2, or, A heterodimer of the polypeptides shown in SEQ ID NO: 1 and SEQ ID NO: 6, or, A heterodimer of the polypeptides shown in SEQ ID NO: 1 and SEQ ID NO:

7.

3. The p35 subunit-biased mutant or p40 subunit-biased mutant as defined in any one of claims 1 to 2.

4. A nucleic acid molecule encoding the biased mutant of interleukin-12 as described in any one of claims 1 to 2, or, Encoding the p35 subunit-biased mutant or p40 subunit-biased mutant as defined in any one of claims 1 to 2; Optionally, the nucleic acid molecule further comprises a nucleic acid fragment encoding a signal peptide or a leader peptide.

5. A vector comprising the nucleic acid molecule of claim 4; Optionally, the vector comprises a co-expression vector of the p35 subunit-biased mutant and the p40 subunit-biased mutant.

6. A cell expressing the biased mutant of interleukin-12 as described in any one of claims 1 to 2, or, Expressing the p35 subunit-biased mutant or p40 subunit-biased mutant as defined in any one of claims 1 to 2; Optionally, the cell is a eukaryotic cell.

7. A method for constructing the cell of claim 6, which comprises the step of transferring the vector of claim 5 into a host to construct the cell.

8. A method for producing the biased mutant of interleukin-12 as described in any one of claims 1 to 2, the p35 subunit-biased mutant or p40 subunit-biased mutant as defined in any one of claims 1 to 2, which comprises culturing the cell of claim 6 and isolating the biased mutant of interleukin-12, the p35 subunit-biased mutant or the p40 subunit-biased mutant from the resulting culture.

9. A drug, which comprises: The biased mutant of interleukin-12 as described in any one of claims 1 to 2, the p35 subunit-biased mutant or p40 subunit-biased mutant as defined in any one of claims 1 to 2, the nucleic acid molecule of claim 4, the vector of claim 5, the cell of claim 6; And a pharmaceutically acceptable carrier.

10. Use of the biased mutant of interleukin-12 as described in any one of claims 1 to 2, the p35 subunit-biased mutant or p40 subunit-biased mutant as defined in any one of claims 1 to 2, the nucleic acid molecule of claim 4, the vector of claim 5, the cell of claim 6 in the preparation of a drug for treating interleukin-12-mediated diseases; Optionally, the interleukin-12-mediated diseases include cancer or autoimmune diseases; Further optionally, the cancer includes acute myeloid leukemia, anaplastic lymphoma, astrocytic lymphoma, B-cell carcinoma, breast cancer, colon cancer, ependymoma, esophageal cancer, glioblastoma, glioma, leiomyosarcoma, liposarcoma, liver cancer, lung cancer, mantle cell lymphoma, melanoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, ovarian cancer, pancreatic cancer, peripheral T-cell lymphoma, kidney cancer, sarcoma, stomach cancer, carcinoma, mesothelioma or sarcoma; Further optionally, the autoimmune disease includes rheumatoid arthritis, insulin-dependent diabetes, hemolytic anemia, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, alopecia areata, psoriasis, vitiligo, dystrophic epidermolysis bullosa, systemic lupus erythematosus, moderate to severe plaque psoriasis, psoriatic arthritis, Crohn's disease, ulcerative colitis or graft-versus-host disease.

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