Interleukin 12 mutant and use thereof

Abstract

Disclosed are an interleukin 12 mutant and the use thereof, belonging to the technical field of genetic engineering. The IL-12 mutant protein comprises a mutated P40 subunit and / or a mutated P35 subunit, wherein the mutated P40 subunit is generated by means of mutating a wild-type P40 subunit at the following positions: positions 37, 38, 39, 40, 81, 82, 106, 108, 114, 115 or / and 219, and the mutation eliminates or reduces the binding affinity of the wild-type P40 subunit for IL-12Rβ1; and the mutated P35 subunit is generated by means of mutating a wild-type P35 subunit at the following positions: positions 72, 151 or / and 190, and the mutation eliminates or reduces the binding affinity for IL-12Rβ2. The IL12 mutant protein and fusion protein can significantly improve a therapeutic effect by means of reducing toxicity and enhancing tumor targeting.

Description

Interleukin 12 mutants and uses thereof TECHNICAL FIELD

[0001] The present invention relates to interleukin 12 mutants and uses thereof. BACKGROUND

[0002] Interleukin 12 (IL-12) is an important cytokine, mainly produced by activated antigen-presenting cells, is a natural interleukin produced by dendritic cells, macrophages, neutrophils and human B lymphoblasts (NC-37) in response to antigen stimulation. IL-12 is composed of 4 a-helices, a heterodimer formed by two subunits IL-12A (p35) and IL-12B (p40) through disulfide bond, and IL12 receptor is composed of two subunits IL12Rβ1 and IL12Rβ2, mainly expressed on T cells and NK cells.

[0003] IL-12 belongs to the interleukin 12 family, induces NK cell, NKT cell and T cell proliferation, enhances cytotoxicity and cytotoxicity mediator expression and cytokine production, especially interferon-γ (IFN-γ), and is conducive to differentiation into 1 type cytokine-producing cells (TH1, TC1 and NK1 cells); for B cells, IL-12 enhances the activation and production of TH1-related immunoglobulin (e.g. mouse IgG2a) directly or through the action of 1 type cytokine (such as IFN-γ), which plays an important role in the regulation of the immune system and anti-viral, anti-tumor and other aspects.

[0004] IL-12 was first discovered in the early 1990s, and scientists found that it played a key role in regulating the activities of immune cells such as T cells and natural killer cells (NK cells). With the deepening of the study of IL-12, it was found that IL-12 can enhance the body's resistance by activating immune cells, and can activate immune cells to attack tumor cells, so IL-12 is widely studied for use in the field of immunotherapy and the field of tumor treatment.

[0005] Years of clinical experience found that although IL-12 has good anti-tumor effect, it also causes serious drug-related side effects, studies have shown that these toxicities are likely to be due to over-activation of IL-12 on lymphocytes, stimulating the release of inflammatory factors. In addition, due to the half-life of IL-12 in the human body is only about 5-10 hours. In order to achieve a high enough human exposure, multiple doses are often used in clinical practice. However, frequent dosing not only brings a heavy burden to patients, but more importantly, it can cause high peak blood drug concentration (Cmax), which is another key factor causing drug toxicity.

[0006] In the production aspect, the natural IL-12 molecule is difficult to express in mammalian cells (CHO or HEK293) due to the characteristics of the amino acid sequence, and the molecule has poor stability, so there is a need in the art to improve the expression of IL-12 molecules and IL-12-Fc molecules in mammalian cells.

[0007] Some modification schemes of IL-12 molecules have been proposed in the art. For example, to extend the half-life, Fc fusion or fusion of HAS protein is performed. In terms of activity, Dragonfly chooses not to reduce the activity of IL-12, while Xencor chooses to reduce the activity of IL-12, reduce the affinity, reduce the activity, compensate the efficacy by extending the half-life, and improve the safety window. However, at present, there is no optimized IL-12 molecule on the market, and the fastest one is still in the clinical research and development stage.

[0008] In view of the above problems related to IL-12 immunotherapy and production, there is a need in the art to further develop new IL-12 molecules with optimized properties, to exhibit IL-12 molecules that are beneficial to production, purification, and have improved pharmacokinetic and pharmacodynamic properties.

[0009] SUMMARY

[0010] The main problem to be solved by the present application is to obtain an IL-12 molecule with improved pharmacokinetic and pharmacodynamic properties.

[0011] In order to solve the above problems, the present application provides an IL-12 mutant protein.

[0012] The IL-12 mutant protein provided by the present application is a protein obtained by mutating the amino acids of wild-type IL-12, wherein the wild-type IL-12 comprises a wild-type P40 subunit and a wild-type P35 subunit, and the IL-12 mutant protein comprises a mutated P40 subunit and / or a mutated P35 subunit, the mutated P40 subunit is a subunit obtained by mutating at least one of the following positions of the wild-type P40 subunit: positions 36, 37, 38, 40, 81, 82, 106, 108, 114, 115, or / and 219, the mutation has a mutation that eliminates or reduces the binding affinity of the wild-type P40 subunit to IL-12Rβ1; the mutated P35 subunit is a subunit obtained by mutating at least one of the following positions of the wild-type P35 subunit: positions 72, 151, or / and 190, the mutation eliminates or reduces the binding affinity to IL-12Rβ2;

[0013] The positions 36, 37, 38, 40, 81, 82, 106, 108, 114, 115, and 219 correspond to positions 36, 37, 38, 40, 81, 82, 106, 108, 114, 115, and 219 of SEQ ID No. 3, respectively.

[0014] the 72nd, 151st or / and 190th position of SEQ ID No. 4, respectively.

[0015] The wild type IL-12 can be IL-12 derived from a mammal, such as human IL-12.

[0016] Further, the P40 subunit binding interface with IL-12Rβ1 comprises one or more mutations selected from the group consisting of: W37E; W37K; W37A; W37N; W37T; D36K; T38K; D40P; E81A; E81K; F82A; K106S; K106E; K106Q; E108A; T114A; D115K; D115P; K219E;

[0017] Preferably, the IL-12 binding interface mutation with IL-12Rβ1 comprises:

[0018] selected from the group consisting of: W37A+F82A; W37A+K219E; E81K+K106E; E81A+K106E; W37K+E81A+K106S; W37A+F82A+E108A; W37E+E81A+F82A; D40P+K106E+K219E; D36K+W37K+T38K; W37K+K106S+K219E; E81K+D115K+K219E; E81A+F82A+K219E; W37N+T114A+D115P; W37T+T114A+D115P.

[0019] Further, the P35 subunit binding interface with IL-12Rβ2 comprises one or more mutations selected from the group consisting of: E72S; R151E; K190E.

[0020] Further, the IL-12 mutein binding interface with IL-12R comprises IL-12 binding interface mutation with IL-12Rβ1 and / or IL-12 binding interface mutation with IL-12Rβ2: the mutation comprises:

[0021] 1) the IL-12 binding interface mutation with IL-12Rβ2 is: E72S; R151E; K190E; and / or

[0022] 2) the IL-12 binding interface mutation with IL-12Rβ1 is: W37K+K219E.

[0023] Further, the mutant protein has the following amino acid mutations relative to the natural IL-12: introducing mutations C199S and / or C274S at positions 199 and / or 274 of the P40 subunit of IL-12, and introducing mutation C96S at position 96 of the P35 subunit.

[0024] In the present application, 1) the P40 subunit of the mutant protein has an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, or 98% identity to the amino acid sequence selected from SEQ ID No. 3;

[0025] and / or,

[0026] 2) the P35 subunit of the mutant protein has an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, or 98% identity to the amino acid sequence selected from SEQ ID No. 4.

[0027] In specific embodiments, 1) the amino acid sequence of the P40 subunit of the mutant protein is SEQ ID No. 22, SEQ ID No. 26, SEQ ID No. 16, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 23, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21;

[0028] 2) the amino acid sequence of the P35 subunit of the mutant protein is SEQ ID No. 4, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13.

[0029] In the present application, the mutant protein has reduced binding affinity to IL-12R compared to before weakening of the introduced mutations, and the mutant protein has at least one or more of the following properties:

[0030] 1) reduced activation of T cells (such as CD4 + or / and CD8 + T cells) compared to before weakening;

[0031] 2) reduced activation of NK compared to before weakening;

[0032] 3) reduced activation of IL-12 pSTAT4 phosphorylation signaling pathway compared to before attenuation;

[0033] 4) reduced activation of IL-12 reporter gene signaling compared to before attenuation;

[0034] 5) increased safety in animals compared to before attenuation.

[0035] The present application also provides IL-12 mutein fusion proteins, comprising any of the IL-12 muteins described herein.

[0036] In the present application, the fusion protein is fused from an IL-12 mutein and an Fc antibody fragment.

[0037] Preferably, the IL-12 mutein is fused to the Fc via a linker, preferably (G4S)n, where n can be 0, 1, 2, 3, 4, 5, etc.

[0038] Preferably, the Fc fragment comprises mutations that reduce or eliminate Fc binding to FcyR, such as L234A+L235A,

[0039] Preferably, the Fc fragment comprises an amino acid sequence that is at least 85%, at least 95%, at least 96%, or 100% identical to SEQ ID NO. 5.

[0040] Further, the Fc fragment comprises the following mutations:

[0041] 1) comprises Knob mutations, such as mutations T366W and S354C;

[0042] 2) comprises Hole mutations, such as mutations Y349C, T366S, L368A, Y407V.

[0043] The present application also provides IL12-Fc dimeric proteins, comprising the IL-12 mutein fusion proteins described herein, preferably, the dimeric proteins have one or more of the following properties compared to the corresponding dimeric protein of wild-type IL12-Fc fusion protein:

[0044] 1) reduced or avoided lymphocyte over-activation and / or inflammatory cytokine release due to IL-12;

[0045] 2) lower toxicity when used in vivo;

[0046] 3) increased expression and / or purity when expressed in mammalian cells (e.g., CHO or HEK293 cells);

[0047] Further, the IL12-Fc dimeric protein is a heterodimer, comprising:

[0048] a first monomer, wherein the first monomer comprises from N-terminus to C-terminus: 1) an IL-12 P40 subunit or a mutein thereof; 2) a linker; 3) a first Fc fragment (also referred to as Fc1); the linker is preferably (G4S)n, n can be 0, 1, 2, 3, 4, 5, etc. natural numbers; preferably, n = 0; a second monomer, wherein the second monomer comprises from N-terminus to C-terminus: 1) an IL-12 P35 subunit or a mutein thereof; 2) a linker; 3) a second Fc fragment (also referred to as Fc2); the linker is preferably (G4S)n, n can be 0, 1, 2, 3, 4, 5, etc. natural numbers; preferably, n = 2;

[0049] Further, the IL12-Fc dimeric protein is a heterodimer, wherein comprises:

[0050] a first monomer, wherein the first monomer comprises from N-terminus to C-terminus: 1) an IL-12 P40 subunit or a mutein thereof; 2) a linker; 3) an IL-12 P35 subunit or a mutein thereof; 4) a linker; 5) a first Fc fragment, the 1) and 2) can be replaced in position; the linker is preferably (G4S)n, n can be 0, 1, 2, 3, 4, 5, etc. natural numbers; preferably, n = 3;

[0051] a second monomer, wherein the second monomer comprises a second Fc fragment and optionally an IgG1 hinge region.

[0052] In the above IL12-Fc dimeric protein, wherein the first Fc fragment and the second Fc fragment respectively comprise a first and a second heterodimerization mutation facilitating the formation of a heterodimer between the first monomer and the second monomer, the IL12-Fc dimeric protein is any one of the following:

[0053] 1) the first and the second heterodimerization mutation comprise a Knob-into-Hole mutation combination, for example, T366W, S354C and Y349C, T366S, L368A, Y407V;

[0054] 2) the first heterodimerization mutation on the first Fc fragment comprises a Knob mutation, and the second heterodimerization mutation on the second Fc fragment comprises a Hole mutation;

[0055] 3) the first heterodimerization mutation on the first Fc fragment comprises a Hole mutation, and the second heterodimerization mutation on the second Fc fragment comprises a Knob mutation.

[0056] The present application also provides immunoconjugates comprising the IL-12 mutein described supra and an antigen binding molecule, preferably the antigen binding molecule is an immunoglobulin molecule, in particular an IgG molecule, or an antibody, or an antibody fragment, in particular a Fab molecule and a scFv molecule.

[0057] The present application also provides polynucleotides encoding the IL-12 mutein described supra, the fusion protein described supra, or the IL12-Fc dimer fusion protein described supra, or the immunoconjugate described supra.

[0058] The present application also provides expression vectors comprising the polynucleotides described supra.

[0059] The present application also provides host cells comprising the polynucleotides described supra, or the vectors described supra, preferably the host cells are mammalian cells, in particular HEK293 cells or CHO cells, and yeast.

[0060] The present application also provides pharmaceutical compositions comprising the IL-12 mutein described supra, the fusion protein described supra, or the IL12-Fc dimer fusion protein described supra, or the immunoconjugate described supra and a pharmaceutically acceptable carrier.

[0061] The present application also provides the use of the IL-12 mutein described supra, the fusion protein described supra, or the IL12-Fc dimer fusion protein described supra, or the immunoconjugate described supra for the preparation of a medicament for stimulating the immune system of a subject.

[0062] The present application also provides a method of treating a disease in a subject, the method comprising administering to the subject the IL-12 mutein described supra, the fusion protein described supra, or the IL12-Fc dimer fusion protein described supra, or the immunoconjugate described supra or the pharmaceutical composition described supra.

[0063] The present application also provides a method of stimulating the immune system of a subject, the method comprising administering to the subject an effective amount of the IL-12 mutein described supra, the fusion protein described supra, or the IL12-Fc dimer fusion protein described supra, or the immunoconjugate described supra or the pharmaceutical composition described supra. Advantageous effects

[0064] The IL-12 mutein provided by the application can reduce the activity of IL12 in stimulating immune cells, reduce the ability of IL12 in mediating T cells and NK cells to secrete IFN-γ, avoid excessive activation of T cells, balance the toxicity of drug efficacy, maintain good anti-tumor effect, avoid toxic side effects caused by excessive activity in clinical application, widen the clinical treatment window, and achieve better therapeutic effect; the fusion protein of the IL-12 mutein provided by the application can prolong the half-life, reduce the frequency of administration, improve the production yield, and show good drug properties on the basis of retaining the optimized properties of the IL-12 mutein. BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1 is a contact interface of a subunit of IL-12 and an IL-12Rβ1 complex or an IL-12Rβ2 complex. Wherein A is the crystal structure of the P40 subunit of IL-12 and the IL-12Rβ1 complex (PDB: 6WDQ) and the contact interface thereof; B is the contact interface of the P35 subunit of IL-12 and the IL-12Rβ2 complex.

[0066] Figure 2 is a molecular mode diagram of IL-12-Fc dimer. Wherein Format 1 represents: one IL12-Fc heterodimer protein structure, linker is (G4S)2; Format 2 represents: another IL12-Fc heterodimer protein structure; linker is (G4S)3.

[0067] Figure 3 is an SDS-PAGE electrophoretogram of IL12 mutant -Fc dimer protein. Wherein M is a standard control protein with known molecular weight (also known as Marker); R is the molecular weight of reduced protein; NR is the molecular weight of non-reduced protein.

[0068] Figure 4 is the activation signal curve (A), (B), (C) and (D) of the weakened IL12 mutant -Fc dimer protein on IL-12 reporter cell lines. Wherein IL12 WT is wild-type IL-12; IL12 WT -Fc (Format 1) is a wild-type IL12-Fc heterodimer fusion protein with Format 1 protein structure; IL12 WT-Fc (Format 2) is a wild-type IL12-Fc heterodimer fusion protein with the protein structure Format 2; HK54029, HK54032, HK54033, HK54037, HK54040, HK54042, HK54043, HK54044, HK54045, HK54046, HK54051, HK54052, HK54053, HK54054, HK54064, HK54065, HK54071, HK54073, and HK54074 are mutant fusion proteins (collectively referred to as IL12). mutant -Fc).

[0069] Figure 5 shows the weakened IL12. mutant -Fc dimer protein activates the secretion level of IFN-γ in cells. (A) represents attenuated IL12. mutant -Fc dimer protein activates normal CD8 + T cells, where the protein concentrations were examined at 20 nM, 4 nM, 0.2 nM, 0.01 nM, 0.00025 nM, and 0.00000625 nM respectively; (B) and (C) show the IFN-γ secretion levels of PBMC cells after activation by the IL-12 mutant fusion protein. (B) shows wild-type IL-12 (IL12... WT The protein concentrations examined in (C) were 350 nM, 17 nM, and 0.03 nM, respectively; the protein concentrations examined in (C) were 350 nM, 90 nM, 9 nM, 1.8 nM, and 0.36 nM, respectively; and the protein concentrations examined in (C) were 2000 nM, 500 nM, 50 nM, 5 nM, and 0.1 nM, respectively.

[0070] Figure 6 shows the weakened IL12. mutant -Fc dimer protein activates NK cells and increases IFN-γ secretion levels. Among these, wild-type IL-12 (IL12...) WT The protein concentrations examined were 170 nM, 17 nM, and 0.09 nM, respectively, and the concentrations examined for mutant fusion proteins were 4500 nM, 2250 nM, and 450 nM, respectively.

[0071] Figure 7 shows the weakened IL12. mutant -Fc dimer protein activates pSTAT4 signaling curve in normal CD8+ T cells, including wild-type IL-12 (IL12) WTThe concentrations of the proteins of interest were 15 nM, 3 nM, 0.6 nM, 0.12 nM, 0.024 nM, 0.0048 nM, 0.00096 nM, and 0.000048 nM, and the concentrations of the mutant fusion proteins of interest were 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, and 0.064 nM, respectively.

[0072] Figure 8 shows the effect of the weakened IL12 mutant Figure 9 shows the effect of the weakened IL12

[0073] Figure 9 shows the effect of the weakened IL12 mutant Figure 9 shows the effect of the weakened IL12 Embodiments of the present application

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. For purposes of the present application, the following terms are defined below.

[0075] In this context, wild-type "interleukin-12" or "IL-12" or "IL12 WT"parent IL-12 protein" refers to a naturally occurring IL-12 protein, including unprocessed (e.g., signal peptide not removed) and processed (e.g., signal peptide removed) forms. A sequence of a full-length P40 subunit of a naturally occurring IL-12 protein including signal peptide is shown in SEQ ID No. 1, and a sequence of a full-length P35 subunit of a naturally occurring IL-12 protein including signal peptide is shown in SEQ ID No. 2. In addition, the term also includes variants of naturally occurring IL-12, e.g., the variants can have at least 95-99% or more identity to naturally occurring IL-12 or have no more than 1-10 or 1-5 amino acid mutations (e.g., conservative substitutions). Thus, in some embodiments, wild-type IL-12 can contain amino acid mutations that do not affect its binding to IL-12 receptor and contain 6xHis at the C-terminus compared to naturally occurring IL-12 protein. The P40 subunit of wild-type IL-12 is wild-type P40 of the present application, and its sequence is shown in SEQ ID No. 3, and the P35 subunit of wild-type IL-12 is wild-type P35 of the present application, and its sequence is shown in SEQ ID No. 4. In some embodiments, the P40 subunit and P35 subunit of wild-type IL-12 can have at least 85%, 95%, or even more amino acid sequence identity to the amino acid sequence of SEQ ID No. 1 or 2, respectively. In some embodiments, wild-type P40 and wild-type P35 can have at least 85%, 95%, or even more amino acid sequence identity to the amino acid sequence of SEQ ID No. 3 or 4, respectively.

[0076] In this context, an amino acid mutation can be an amino acid substitution, deletion, insertion, and addition.

[0077] In the present application, when referring to the position of an amino acid in an IL-12 protein or IL-12 sequence, it is determined by reference to the amino acid sequence of wild-type human IL-12 protein (also referred to as IL12 WT ) wherein the sequence of the P40 subunit of wild-type IL-12 is shown in SEQ ID No. 3, and the sequence of the P35 subunit of wild-type IL-12 is shown in SEQ ID No. 4. For example, when referring to P40 "W37", it refers to the 37th tyrosine residue W of SEQ ID No. 3.

[0078] In the present application, the antibody Fc fragment refers to the C-terminal region of the immunoglobulin heavy chain containing at least part of the constant region, which can be the Fc region of various Ig subtypes and allotypes, and can also include some mutated Fc. Preferably, in some embodiments, the human IgG1 heavy chain Fc fragment is selected, which contains mutations such as L234A / L235A mutations (also known as LALA mutations), and the amino acid sequence is as shown in SEQ ID No. 5. In some embodiments, the Fc fragment contains Knob mutations, such as mutations T366W and S354C; or the Fc fragment contains Hole mutations, such as mutations Y349C, T366S, L368A, Y407V.

[0079] In the present application, the antibody Fc fragment refers to the C-terminal region of the immunoglobulin heavy chain containing at least part of the constant region, which can be the Fc region of various Ig subtypes and allotypes, and can also include some mutated Fc. Preferably, in some embodiments, the human IgG1 heavy chain Fc fragment is selected, which contains mutations such as L234A / L235A mutations (also known as LALA mutations), and the amino acid sequence is as shown in SEQ ID No. 5. In some embodiments, the Fc fragment contains Knob mutations, such as mutations T366W and S354C; or the Fc fragment contains Hole mutations, such as mutations Y349C, T366S, L368A, Y407V.

[0080] In the present application, the antibody Fc fragment refers to the C-terminal region of the immunoglobulin heavy chain containing at least part of the constant region, which can be the Fc region of various Ig subtypes and allotypes, and can also include some mutated Fc. Preferably, in some embodiments, the human IgG1 heavy chain Fc fragment is selected, which contains mutations such as L234A / L235A mutations (also known as LALA mutations), and the amino acid sequence is as shown in SEQ ID No. 5. In some embodiments, the Fc fragment contains Knob mutations, such as mutations T366W and S354C; or the Fc fragment contains Hole mutations, such as mutations Y349C, T366S, L368A, Y407V.

[0081] 1、the IL-12 mutein of the present application

[0082] The IL-12 mutein of the present application has the following advantageous biological properties: through long-term research, the following molecular mutations and modifications can be combined to simultaneously improve the efficacy of IL-12, reduce the side effects of IL-12, and achieve good production performance.

[0083] 1) Introducing specific amino acid mutations at the binding interface of IL-12 and IL-12R, weakening the binding of the P40 subunit of IL-12 to IL-12Rβ1 or / and weakening the binding of the P35 subunit of IL-12 to IL-12Rβ2, and down-regulating the activity of IL-12 to a certain extent. The IL-12 mutein of the present application can activate lymphocytes to kill tumor cells while avoiding the release of a large amount of inflammatory factors caused by the overactivation of lymphocytes and the drug-related toxicity caused thereby.

[0084] 2) The mutant IL-12 protein of the present application is constructed into an IL12-Fc dimer. The formation of the dimer can be recycled in vivo through FcRn-mediated circulation, prolonging the half-life of the IL12-Fc fusion protein. Thus, the problem of high peak blood concentration caused by the short half-life of IL-12 and high-frequency dose administration is overcome.

[0085] 3) The mutant IL-12 protein of the present application constructed into an IL12-Fc dimer has improved drug properties. For example, when expressed in mammalian HEK293 or CHO cells, etc., especially when expressed as an Fc fusion protein, it has one or more properties selected from the following: (a) better expression than wild-type IL-12 protein; (b) easy to purify to a higher protein purity.

[0086] Therefore, through modification, the present application provides an IL-12 mutein with improved drug properties, an IL12-Fc series molecule comprising the IL-12 protein of the present application, which on the one hand weakens the affinity with its receptor, achieves a comparable or even better efficacy than the natural IL-12 molecule, and on the other hand significantly improves the drug properties such as protein expression amount and purity.

[0087] In some embodiments of the present application, the IL-12 mutein of the present application exhibits an increase in expression level compared to wild-type IL-12, with an increase in expression amount in mammalian cells of at least 1.1 times, or at least 1.5 times, or at least 2 times, 3 times, or at least 3 times or more.

[0088] In some embodiments, the IL-12 muteins of the present application, or the IL12-Fc fusion proteins of the IL-12 muteins, exhibit higher purity relative to the wild-type IL-12 protein, as shown by determining the purity of the purified protein after protein A affinity chromatography. The IL-12 muteins of the present application, or the IL12-Fc fusion proteins of the IL-12 muteins, can achieve a purity of 70%, or 80%, or 90% or more, preferably 92%, 93%, 94%, 95%, 98% or 99% or more after one-step protein A affinity chromatography purification.

[0089] In some embodiments, the IL-12 muteins of the present application, or the IL12-Fc fusion proteins of the IL-12 muteins, have attenuated IL-12Rβ1 or / and attenuated IL-12Rβ2 binding affinity relative to the corresponding protein prior to the introduction of the mutation at the binding interface of IL-12 and IL-12R, for example, 1-100 fold or more reduction.

[0090] In some embodiments, the IL-12 muteins of the present application, or the IL12-Fc fusion proteins of the IL-12 muteins, have attenuated IL-12 activity relative to the IL-12 activity prior to the introduction of the mutation for attenuating, for example, at least one of the IL-12 activities selected from:

[0091] a. reduced activation of T cells (such as CD4+ or / and CD8+ T cells) relative to the IL-12 activity prior to the introduction of the mutation for attenuating;

[0092] b. reduced activation of NK cells relative to the IL-12 activity prior to the introduction of the mutation for attenuating;

[0093] c. reduced activation of IL-12 pSTAT4 phosphorylation signaling pathway relative to the IL-12 activity prior to the introduction of the mutation for attenuating;

[0094] d. reduced activation of IL-12 reporter gene signaling relative to the IL-12 activity prior to the introduction of the mutation for attenuating.

[0095] In some embodiments, the IL-12 muteins of the present application result in reduced activation of lymphocytes (such as T cells and / or NK cells) mediated by IL-12 relative to the IL-12 activity prior to the introduction of the mutation for attenuating. In some embodiments, the lymphocytes are PBMC total or isolated CD8 + T cells or isolated CD3 +T cells. In some embodiments, the ability of the IL-12 mutein to activate lymphocytes is identified by detecting the level of IFN-g secretion upon activation of lymphocytes, such as T cells or NK cells. In some embodiments, the ability of the IL-12 mutein to activate lymphocytes is identified by detecting the activation of the pSTAT4 phosphorylation signaling pathway in lymphocytes, such as T cells. In some embodiments, the ability of the IL-12 mutein to activate lymphocytes is identified by detecting the activation of the JAK1 and TYK2 phosphorylation pathway in IL-12 reporter cell lines. In embodiments of the application, the T cell activation activity of the IL-12 mutein of the application is reduced by, for example, 2-fold or more, or 5-fold or more, or 10-fold or more, or 50-fold or more, or 100-fold or more, or even 1000-fold or more, as compared to before attenuation.

[0096] In some embodiments, the IL-12 mutein of the application has reduced toxic side effects mediated by IL-12 and its receptors, as compared to wild-type IL-12.

[0097] The muteins of the application:

[0098] In one aspect, the application provides an IL-12 mutein, wherein the mutein comprises a mutation, as compared to wild-type IL-12 (preferably human IL-12):

[0099] 1) a mutation at the IL-12 P40 subunit binding interface with IL-12Rb1, particularly at the P40 subunit selected from at least one of positions 36, 37, 38, 40, 81, 82, 106, 114, 115, and 219, wherein the amino acid positions are according to SEQ ID No. 3, which abrogates or reduces the binding affinity to IL-12Rb1.

[0100] 2) a mutation at the IL-12 P35 subunit binding interface with IL-12Rb2, particularly at the P35 subunit selected from at least one of positions 72, 151, 190, wherein the amino acid positions are according to SEQ ID No. 4, which abrogates or reduces the binding affinity to IL-12Rb2.

[0101] In some embodiments, the IL-12 binding interface with IL-12Rb1 mutation comprises a mutation selected from one or more of the following:

[0102] W37E; W37K; W37N; W37T; W37A; D36K; T38K; D40P; E81A; E81K; F82A; K106S; K106E; K106Q; E108A; T114A; D115K; D115P; K219E.

[0103] Preferably, the IL-12 and IL-12Rβ1 binding interface mutations comprise:

[0104] selected from the following exemplary combinations of mutations: W37A + F82A; W37A + K219E; E81K + K106E; E81A + K106E; W37K + E81A + K106S; W37A + F82A + E108A; W37E + E81A + F82A; D40P + K106E + K219E; D36K + W37K + T38K; W37K + K106S + K219E; E81K + D115K + K219E; E81A + F82A + K219E; W37N + T114A + D115P; W37T + T114A + D115P.

[0105] In some embodiments, the IL-12 and IL-12Rβ2 binding interface mutations comprise mutations selected from one or more of the following:

[0106] E72S; R151E; K190E.

[0107] In some embodiments, the IL-12 and IL-12R binding interface mutations comprise IL-12 and IL-12Rβ1 binding interface mutations and / or IL-12 and IL-12Rβ2 binding interface mutations: Preferably, the mutations comprise:

[0108] selected from the following IL-12 and IL-12Rβ2 binding interface mutations: E72S; R151E; K190E; and / or

[0109] selected from the following IL-12 and IL-12Rβ1 binding interface mutations: W37K + K219E.

[0110] In some embodiments, the present application provides IL-12 muteins, wherein the muteins comprise, relative to wild-type IL-12:

[0111] Additional mutations:

[0112] In addition to the above "IL-12 and IL-12R binding interface" mutations, the IL-12 muteins of the present application can have one or more mutations in other regions or positions, so long as they retain one or more of the above beneficial properties of the IL-12 muteins of the present application. For example, the IL-12 muteins of the present application can further comprise mutations C199S and / or C274S at position 199 and / or 274 of the IL-12 P40 subunit and a mutation C96S at position 96 of the P35 subunit to provide additional advantages, such as improved expression or homogeneity or stability. Those skilled in the art will know how to determine additional mutations that can be incorporated into the IL-12 muteins of the present application.

[0113] The sequence difference between the IL-12 mutein and the wild-type protein can be expressed in terms of sequence identity, or in terms of the number of amino acids that differ between the two. In some embodiments, the IL-12 mutein has at least 85%, 86%, 87%, 88%, 89% identity, preferably 90% or more identity, preferably 95% identity with the wild-type protein. In some embodiments, the IL-12 mutein can have no more than 15, for example 1-10, or 1-5 mutations, for example 0, 1, 2, 3, 4 mutations, in addition to the mutations described above in the present application. In some embodiments, the remaining mutations can be conservative substitutions, meaning substitutions of an amino acid residue with an amino acid residue having a similar side chain, i.e. an amino acid substitution that does not adversely affect or alter the biological function of the protein comprising the amino acid sequence.

[0114] As used herein, the term "conservative substitution" is for example a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue, e.g. has similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc. Families of amino acid residues having similar side chains have been defined in the art, see Table 1.

[0115] Table 1, conservative amino acid substitutions

[0116] Thus, it is preferred that the corresponding amino acid residue is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997)).

[0117] 2. Fusion proteins and IL12-Fc dimeric proteins

[0118] In one aspect, the present application also provides fusion proteins comprising the IL-12 muteins of the present application. In preferred embodiments, the IL-12 muteins of the present application are fused to another polypeptide that can improve the pharmacokinetic properties, such as serum albumin, preferably an antibody Fc fragment.

[0119] In some embodiments, the present application provides IL-12 mutein fusion proteins comprising an IL-12 mutein of the present application fused to an antibody Fc fragment. The Fc fragment used in the present application can comprise mutations that reduce or eliminate effector function, for example, in some embodiments, the Fc fragment used in the present application has L234A / L235A mutations (also known as LALA mutations) that reduce binding to Fcy receptors. The Fc fragment fused to the IL-12 mutein is a human IgG Fc, for example, a human IgG1 Fc, a human IgG2 Fc, a human IgG4 Fc. In some embodiments, the Fc fragment comprises the amino acid sequence of SEQ ID No. 5 or has at least 90% identity, for example, 95%, 96%, 97%, 99% or more, thereto. In some embodiments, the IL-12 mutein is fused to the Fc via a linker, which is (G4S) n , preferably (G4S)2.

[0120] In yet another aspect, the present application also provides dimeric molecules comprising an IL-12 mutein of the present application fused to an Fc fragment. The molecules are recycled in vivo by FcRn-mediated recycling, which can prolong the half-life of the IL12-Fc fusion protein. Compared to the corresponding dimeric molecules comprising wild-type IL12-Fc fusion proteins, the dimeric molecules of the present application have one or more of the following properties:

[0121] a) reduced or avoided lymphocyte over-activation and / or inflammatory cytokine release caused by IL-12;

[0122] b) improved pharmacokinetic properties, such as prolonged half-life in vivo;

[0123] c) increased expression level and / or purity when expressed in mammalian cells (e.g., CHO or HEK293 cells);

[0124] Preferably, the dimeric molecules of the present application exhibit good anti-tumor efficacy and tolerability when administered to animals. Anti-tumor efficacy can be determined by measuring tumor size or tumor inhibition rate in the animal tumor-bearing experiments described in the Examples. Tolerability can be determined by measuring the body weight of the animal model and its body weight change after administration as described in the Examples.

[0125] In some embodiments, the present application provides IL12-Fc proteins, which are heterodimers, for example, Format 1, comprising:

[0126] a) a first monomer, wherein the first monomer comprises, from N-terminus to C-terminus: 1) an IL-12 P40 subunit or a mutein thereof; 2) a linker; 3) a first Fc fragment (also referred to as Fc1); the linker is preferably (G4S)n, n can be 0, 1, 2, 3, 4, 5, etc. natural numbers; preferably, n = 0;

[0127] b) a second monomer, wherein the second monomer comprises, from N-terminus to C- terminus: 1) an IL-12 P35 subunit or mutein thereof; 2) a linker; 3) a second Fc fragment (also referred to as Fc2); the linker is preferably (G4S)n, n can be 0, 1, 2, 3, 4, 5, etc. natural number; preferably, n = 2.

[0128] In some embodiments, the present application provides IL12-Fc proteins, which are heterodimers, e.g., Format 2, comprising:

[0129] a) a first monomer, wherein the first monomer comprises, from N-terminus to C- terminus: 1) an IL-12 P40 subunit or mutein thereof; 2) a linker; 3) an IL-12 P35 subunit or mutein thereof; 4) a linker; 5) a first Fc fragment. The 1) and 3) can be replaced in position; the linker is preferably (G4S)n, n can be 0, 1, 2, 3, 4, 5, etc. natural number; preferably, n = 3;

[0130] b) a second monomer, wherein the second monomer comprises a second Fc fragment and optionally an IgG hinge region.

[0131] The above heterodimer formats, in some embodiments, the first Fc fragment and the second Fc fragment respectively comprise first and second heterodimer mutations that promote the first monomer and the second monomer to form a heterodimer. In some preferred embodiments, the first and second heterodimer mutations comprise a Knob-into-Hole mutation combination, e.g., T366W / S354C: Y349C / T366S / L368A / Y407V.

[0132] In some preferred embodiments, the first heterodimer mutation on the first Fc fragment comprises a Knob mutation, and the second heterodimer mutation of the second Fc fragment comprises a Hole mutation; or, the first heterodimer mutation on the first Fc fragment comprises a Hole mutation, and the second heterodimer mutation of the second Fc fragment comprises a Knob mutation.

[0133] As understood by those skilled in the art, the Fc fragment suitable for use in the fusion proteins and dimeric molecules of the present application can be any mutations known in the art that promote heterodimer formation.

[0134] As understood by those skilled in the art, the linker suitable for use in the fusion proteins and dimeric molecules of the present application to link the IL-12 mutein and the Fc fragment can be any linker known in the art.

[0135] 3. Polynucleotides, vectors, and hosts

[0136] The present application provides nucleic acids encoding any of the above IL-12 muteins or fusion proteins or dimer molecules. The polynucleotide sequences encoding the muteins of the present application can be generated by de novo DNA synthesis or by PCR mutagenesis of an existing sequence encoding wild-type IL-12 using methods well known in the art. In addition, the polynucleotides and nucleic acids of the present application can comprise a segment encoding a secretion signal peptide operably linked to a segment encoding a mutein of the present application, thereby directing the secretory expression of the mutein of the present application.

[0137] The present application also provides vectors comprising the nucleic acids of the present application. In some embodiments, the vectors are expression vectors, such as eukaryotic expression vectors. The vectors include, but are not limited to, viruses, plasmids, phages. In preferred embodiments, the expression vectors of the present application are pcDNA3.4.

[0138] The present application also provides host cells comprising the nucleic acids or vectors described. Host cells suitable for replicating and supporting expression of the mutant IL-12 proteins or fusion proteins or dimers are well known in the art. Such cells can be transfected or transduced with the particular expression vectors, and large quantities of vector-containing cells can be grown for seeding large scale fermentors to obtain sufficient quantities of the IL-12 mutants or fusion proteins or dimers for clinical applications. In some embodiments, the host cells are eukaryotic, and the host cells are selected from the group consisting of yeast cells, mammalian cells (e.g., CHO cells or 293 cells).

[0139] 4. Methods of manufacture

[0140] The present application provides methods of making the IL-12 muteins or fusion proteins or dimers of the present application, wherein the methods comprise culturing host cells comprising nucleic acids encoding the proteins or fusion proteins or dimers under conditions suitable for expression of the IL-12 muteins or fusion proteins or dimers, as described above, and optionally recovering the proteins from the host cells.

[0141] 5. Methods of detection

[0142] The IL-12 muteins of the present application can be identified, screened, or characterized for their physical / chemical properties and biological activities by a variety of assays known in the art.

[0143] In one aspect, the IL-12 muteins of the present application can be tested for their binding activity to IL-12 receptors. For example, the binding of IL-12 to IL-12Rβl or IL-12Rβ2 can be determined by methods known in the art, such as ELISA, Western blotting, and the like, or the exemplary methods disclosed in the Examples herein.

[0144] In yet another aspect, the ability of an IL-12 mutein to bind to the IL-12 receptor can be measured indirectly by assaying signal transduction and / or immune activation effects that occur downstream of receptor binding.

[0145] Thus, in some embodiments, assays are provided for identifying IL-12 muteins or dimer molecules having biological activities. Biological activities can include, for example, the ability to induce T cell and / or NK cell signal transduction in cells having the IL-12 receptor, the ability to induce T cell and / or NK cell secretion of IFN-γ in cells having the IL-12 receptor, the level of STAT4 phosphorylation downstream of JAK and TYK2 upon IL-12 interaction with the receptor, the ability to induce tumor regression and / or improved survival, and reduced in vivo toxicity properties.

[0146] 6. Methods of IL-12 protein engineering

[0147] In one aspect, the present application provides methods for obtaining IL-12 muteins having improved properties and IL-12 muteins obtained by such methods.

[0148] In some embodiments, the methods of the present application comprise the following steps:

[0149] a) optionally introducing one or more mutations at the IL-12 / IL-12R binding interface.

[0150] b) expressing the IL-12 mutein, e.g., as an Fc fusion protein, in mammalian cells, such as HEK293 or CHO cells.

[0151] In the above embodiments, preferably, the IL12 / IL12Rβ1 binding interface mutations comprise the mutations described above;

[0152] Preferably, the muteins have improved properties including (1) improved expression levels and / or protein purity; and optionally (2) attenuated IL12β1 binding.

[0153] 7. Therapeutic methods and uses

[0154] In the present context, the terms "individual" or "subject" are used interchangeably and refer to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0155] In one aspect, the present application provides a method of stimulating the immune system of a subject, the method comprising administering to the subject an effective amount of an IL-12 mutein or fusion protein or dimer molecule of the present application.

[0156] In one aspect, the present application relates to a method of treating a disease, such as cancer, in a subject, the method comprising administering to the subject an effective amount of an IL-12 mutein or fusion protein or dimer molecule of the present application. The cancer can be in early, intermediate, or advanced metastatic cancer. In some embodiments, the cancer can be, for example, a gastrointestinal cancer.

[0157] The present application will be further described in the following detailed description of specific embodiments with reference to the attached drawings, of which the embodiments are presented by way of illustration and not of limitation. The following examples are provided as guidance to one of ordinary skill in the art and are not intended to limit the scope of the application.

[0158] The experimental methods in the following examples are routine methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0159] The quantitative experiments in the following examples are performed in triplicate, unless otherwise specified.

[0160] The specific information of some proteins and subunits in the present application is shown in Table 2 below.

[0161] Table 2, specific description information of some proteins and subunits in the present application

[0162] Example 1, design of IL-12 mutants

[0163] According to the binding interface of interleukin 12 (IL-12) and its receptor, including the crystal structure of IL-12 and IL-12Rβ1 complex (PDB: 6WDQ) (Figure 1A) and the computer simulation of the binding interface of IL-12 and IL-12Rβ2 (Figure 1B), the interaction sites are listed. The wild-type IL-12 is used as a mutation template (wild-type IL-12, also referred to as IL12 WT in this paper) to design and obtain IL-12 mutants. The wild-type P40 subunit has an amino acid sequence of SEQ ID No. 3, and a C274S mutation is introduced at position 274 of SEQ ID No. 3 to avoid disulfide bridged IL-12 dimers; the wild-type P35 subunit has an amino acid sequence of SEQ ID No. 4, and a C96S mutation is introduced at position 96 of SEQ ID No. 4 to avoid disulfide bridged IL-12 dimers.

[0164] Example 2, expression and purification of IL12 mutant fusion proteins

[0165] 1. Construction of IL12 mutant fusion protein expression vector

[0166] This example uses Fc to construct IL12 mutant fusion protein, also known as IL12 mutant -Fc, the Fc used refers to the Fc of human IgG1 with mutations L234A and L235A, the corresponding Fc sequence is shown in SEQ ID No. 6 (Fc1 sequence) and SEQ ID No. 7 (Fc2 sequence). IL12 mutant -Fc fusion protein has two heterodimeric forms, Format 1 and Format 2, as shown in Figure 2.

[0167] Construct wild type IL12 (IL12 WT ) as a control, the C-terminal of the P40 sequence of wild type IL12 is connected with 6 histidine tags (Hisx6), the corresponding nucleotide sequence is synthesized by full gene synthesis, as shown in SEQ ID No. 35, and is cloned into the XbaI and HindIII sites of pcDNA3.4 vector pcDNA TM 3.4TOPO TM vector (Invitrogen, Cat: A14697, hereinafter referred to as pcDNA3.4). The nucleotide sequence encoding the p35 subunit of wild type IL12 is synthesized by full gene synthesis, as shown in SEQ ID No. 36, and is cloned into the XbaI and HindIII sites of pcDNA3.4 vector to obtain pcDNA3.4-p35 vector.

[0168] Construct Format 1 wild type IL12-Fc fusion protein (IL12 WT -Fc Format 1) as a control, the first monomer connection mode is P40-Fc1, the second monomer connection mode is P35-(G4S)2-Fc2, the construction method is as follows: the synthesized gene sequence of the p40 subunit of wild type IL12 is connected to Fc1 and cloned into pcDNA3.4 vector to obtain pcDNA3.4-p40-Fc1 vector; the synthesized gene sequence of the p35 subunit of wild type IL12 is connected to Fc2 through (G4S)2 and cloned into pcDNA3.4 vector to obtain pcDNA3.4-p35-Fc2 vector.

[0169] Construct Format 2 wild type IL12-Fc fusion protein (IL12 WT- Fc Format 2) as a control, the first monomer connection mode is P40-(G4S)3-P35-(G4S)3-Fc1, the second monomer Fc2, the construction method is as follows: the gene sequence of the synthesized wild type IL12 p40 subunit is connected to the gene sequence of the P35 subunit through (G4S)3, and then connected to Fc1 through (G4S)3, cloned into the pcDNA3.4 vector to obtain the pcDNA3.4-IL12-Fc1 vector; the Fc2 gene sequence is directly constructed into the pcDNA TM 3.4TOPO TM vector to obtain the recombinant vector pcDNA3.4-Fc2.

[0170] IL12 mutant The vector construction of the IL12

[0171] A, IL12 mutant -Fc fusion protein HK54046 as an example, HK54046 is an IL12 mutant -Fc fusion protein, the first monomer connection mode is mutant P40-Fc1, and the second monomer connection mode is mutant P35-(G4S)2-Fc2. The fusion protein HK54046 is constructed in the following manner:

[0172] (1) Design primers at the nucleotide sequences corresponding to the 81st and 106th amino acids of the p40 subunit of wild type IL12, mutate E at the 81st position of SEQ ID No. 3 to A and K at the 106th position to E, and then connect the p40 nucleotide subunit sequence of the IL12 mutant to Fc1 to obtain a DNA sequence with a nucleotide of SEQ ID No. 37, wherein the 1st-984th of SEQ ID No. 37 is the nucleotide sequence of the mutant subunit P40 mutant， , and the 985th-1680th is the nucleotide sequence of Fc1. Clone the nucleotide sequence into the XbaI and HindIII recognition sites of the expression vector pcDNA TM 3.4TOPO TM vector to obtain the recombinant vector pcDNA3.4-IL12(P40) mutant -Fc1-HK54046;

[0173] (2) Connect the P35 subunit nucleotide sequence of the IL12 mutant to Fc2 through the linker (G4S)2 to obtain a DNA sequence with a nucleotide of SEQ ID No. 38, wherein the 1st-657th of SEQ ID No. 38 is the nucleotide sequence of the mutant subunit P35 mutant，nucleotide sequence of Fc2. The nucleotide sequence was constructed into pcDNA3.4-IL12(P35) by using Xbal and Hindlll restriction sites of the vector. TM 3.4 TOPO TM The recombinant vector pcDNA3.4-IL12(P35) was obtained between Xbal and Hindlll recognition sites of the vector, corresponding to P35 of IL12. mutant -Fc2-HK54046.

[0174] IL12 mutant The IL12-Fc fusion proteins HK54029, HK54030, HK54037, HK54040, HK54042, HK54043, HK54044, HK54045, HK54046, HK54051, HK54052, HK54053, HK54064, HK54066, HK54067, HK54068, HK54071, HK54073, HK54074, HK54075, HK54077, and HK54078 were constructed according to the above construction method, and the corresponding mutation sites and mutant protein subunit amino acid sequences are shown in Table 3.

[0175] Table 3, list of IL12 mutant fusion proteins and their corresponding IL12 mutant mutation sites

[0176] Note: The "mutation note" column in the table describes the mutation information of the IL-12 mutant at the corresponding site of the P40 subunit and the P35 subunit amino acid sequence by "SEQ ID No. 3 and "mutation site", or "SEQ ID No. 4" and "mutation site".

[0177] B, IL12 mutant For example, the IL12-Fc fusion protein HK54065 is in the format 2 structure form. mutant The first monomer of the IL12-Fc fusion protein is mutated P40-(G4S)3-P35-(G4S)3-Fc1, and the second monomer is Fc2. The construction method of the fusion protein HK54065 is as follows:

[0178] (1) Design primers at the nucleotide sequences corresponding to the 37th and 219th amino acids of the p40 subunit of wild-type IL12, respectively, mutate W at the 37th position of SEQ ID No. 3 to K and K at the 219th position to E, then connect the p40 subunit sequence of the IL12 mutant to the P35 subunit nucleotide sequence through a linker (G4S)3, and then connect it to Fc1 through a linker (G4S)3 to obtain a DNA sequence with the nucleotide of SEQ ID No. 39, wherein the 1st-984th of SEQ ID No. 39 is the nucleotide sequence of the mutant subunit P40 mutant , the 985th-1029th is a connecting sequence, the 1030th-1620th is the nucleotide sequence of the mutant subunit P35 mutant , the 1621st-1665th is a connecting sequence, and the 1666th-2361st is the nucleotide sequence of Fc1, construct SEQ ID No. 39 into the XbaI and HindIII recognition sites of pcDNA TM 3.4TOPO TM vector, and obtain the recombinant vector pcDNA3.4-IL12 mutant -Fc1-HK54065.

[0179] (2) Add a signal peptide sequence MEFGLSWVFLVAILKGVQC (SEQ ID No. 46) to the N terminus of Fc2, and synthetically obtain the corresponding nucleotide sequence to obtain a DNA sequence with the nucleotide of SEQ ID No. 40, and directly construct it into pcDNA TM 3.4TOPO TM vector, and obtain the recombinant vector pcDNA3.4-Fc2-HK54065.

[0180] IL12 mutant -Fc fusion protein HK54032, HK54033 are constructed according to the above construction method, and the corresponding mutation sites and amino acid sequences are shown in Table 3.

[0181] Two IL12 mutant -Fc fusion protein structure forms are designed, which are shown as Format 1 and Format 2 in FIG. 2, respectively, wherein Format 1 and Format 2 are heterodimers.

[0182] 2、IL12 mutant -Fc fusion protein expression and purification

[0183] The recombinant vectors containing the genes encoding the P40 and P35 subunit of the fusion protein (HK54046 fusion protein vector in Format 1 form is pcDNA3.4-IL12(P40) mutant -Fc1 and pcDNA3.4-IL12(P35) mutant -Fc2; HK54065 fusion protein vector in Format 2 form is pcDNA3.4-IL12 mutant -Fc1 and pcDNA3.4-Fc2) into ExpiCHO-S cells (Gibco, item number A29127) at a mass ratio of 1:1, and cultured at 37°C in 6% CO2 for 7 days. The antibody protein was purified from the culture supernatant using a Protein A affinity chromatography column. The Protein A column (GE) was first equilibrated with PBS, and then the culture supernatant was passed through the column. Five column volumes of A solution (formula: solvent is water, solute and concentration: 20mM sodium phosphate, 500mM NaCl, pH 5.0) were used for pre-elution, and five column volumes of B solution (formula: solvent is water, solute and concentration: 20mM sodium acetate, 150mM NaCl, pH 3.5) were used for elution. The affinity elution peak was collected, and further fine purification was performed by gel filtration chromatography. After the gel filtration chromatography column (GE, SUPERDEX 200Pg) was equilibrated with PBS, the affinity collection liquid was loaded onto the column. When the target protein peak appeared, the gel filtration elution peak was collected, and then the above IL12 mutant -Fc fusion protein molecules were obtained by concentrating in a 30KDa concentration centrifuge tube. The protein electrophoretic bands identified by SDS-PAGE are shown in Figure 3.

[0184] As can be seen from Figure 3, both the Format 1 and Format 2 forms of IL12 mutant -Fc fusion proteins can be correctly expressed and produced. The reduced and non-reduced molecular weights in the SDS-PAGE electrophoretic results are consistent with the theoretical size, and the non-reduced electrophoretic results show that the purity of the fusion protein is >90%, which meets the subsequent detection requirements, i.e., the IL12 fusion protein designed in the present application has good producibility.

[0185] Example 3, Reporter gene functional activity of IL12 mutant -Fc fusion protein

[0186] IL12 Reporter 293 cell (Gibco, item number GM-C19224) was used to detect IL12 mutant- Fc fusion protein reporter gene activity. This reporter cell line is a luciferase reporter cell line based on the JAK-STAT4 signal transduction pathway. When IL12 binds to the receptor to form a complex, the receptor complex is phosphorylated by JAK1 and Tyk2, which ultimately leads to the phosphorylation and dimerization of STATs, thereby activating the expression of luciferase. The reading of luciferase represents the activation of the signal pathway, which can evaluate the signal pathway activation activity of IL12.

[0187] IL12 Reporter 293 cells (1.5 x 10 4 cells / well) were inoculated into a 96-well cell culture plate, and then gradient-diluted test samples HK54029, HK54030, HK54032, HK54033, HK54037, HK54040, HK54042, HK54043, HK54044, HK54045, HK54046, HK54051, HK54052, HK54053, HK54054, HK54064, HK54065, HK54071, HK54073, HK54074 were added, and wild-type IL12 was used as a control. After overnight incubation in a 37°C CO2 incubator, color developing agent ONE-Glo TM Luciferase Assay System reagent (Meilunbio, Catalog No. MA0519-2) was added and incubated for 10 min, and the chemiluminescence value was detected.

[0188] The results are shown in Figures 4 (A) and (B): wild-type IL12 (abbreviated as IL12 WT ) and wild-type IL12-Fc fusion protein (abbreviated as IL12 WT -Fc) have significant activation activity in the in vitro reporter gene system. The addition of Fc fragments does not reduce the activity of IL12. There is no difference in the activity of IL12-Fc fusion proteins IL12 WT -Fc (Format 1) and IL12 WT -Fc (Format 2) in the heterodimer Format 1 and Format 2, so wild-type IL12 or one structure of wild-type IL12-Fc fusion protein is selected as a control in subsequent experiments.

[0189] Experiments have proved that IL12 mutant -Fc fusion protein and the control wild-type IL12 can activate the expression signal of the reporter gene, and IL12 mutantThe Fc fusion proteins reduced the binding ability of IL12 to IL12R and the activation ability, and the activation activity of different mutations was reduced by 1-100 times or more compared with wild-type IL12, as shown in Figures 4(A), (B), (C), and (D).

[0190] Example 4, IL12 mutant In vitro lymphocyte activation function verification of Fc fusion protein

[0191] Test sample: IL12 mutant -Fc fusion proteins HK54037, HK54046, HK54052, HK54053, HK54053, HK54064, HK54065, HK54066, HK54067, HK54068, HK54075, HK54077, HK54078.

[0192] Anti-CD3 antibody (Bing Biological, Catalog No. B6928) was diluted to 0.5 μg / ml with PBS, added to a 96-well plate (Corning), 60 μl / well, and the 96-well plate was incubated at 37°C for 1 hour.

[0193] 1. Human CD8 + T cell activation

[0194] CD8 + T cells were isolated from PBMC (Shanghai Heyuosheng Biological Technology Co., Ltd., Catalog No. HPB050C) using IIMagTM Human CD8 T Lymphocyte Enrichment Set-DM magnetic beads sorting (BD Biosciences, Catalog No. 557941) according to the instructions. + T cells were isolated and purified according to the instructions.

[0195] In a 96-well cell culture plate, 0.5ug / ml anti-CD3 was coated for 2h, then 100 μl of human CD8 + T cells were added to each well at 50000 cells / well, and the test samples HK54037, HK54053, and HK54064 were diluted to the target concentration with culture medium and added to the 96-well plate at 100 μl / well, with 3 replicate wells for each concentration. The 96-well plate was incubated in a cell incubator for 3 days, and the secretion of cytokine IFN-γ in the cell culture supernatant was detected by ELISA, as shown in Figure 5(A).

[0196] Experiments showed that IL12 mutant -Fc fusion proteins and control wild-type IL12 fusion proteins can activate CD8 +T cells, induce CD8 + T cells secrete IFN-γ. Compared with wild type IL12 fusion protein, IL12 mutant -Fc fusion protein increases the EC50 (half effective concentration) of CD8+ T cells secreting IFN-γ, and reduces the plateau on the curve, indicating that IL12 mutant -Fc fusion protein reduces the ability of wild type IL12 fusion protein to activate CD8 + T cells, the mutations of HK54037, HK54053 and HK54064 are different, and the degree of reduction of activation activity is also different, which is reduced by 1-100 times or more, reaching the expected goal of mutation.

[0197] 2, human PBMC cell activation

[0198] In a 96-well cell culture plate, 0.5 ug / ml anti-CD3 was coated respectively, and after 2 hours, the plate was washed with PBS, and then 100 μl of human PBMC (Shanghai Heyousheng Biological Technology Co., Ltd.) was plated, 20000 cells / well; then add dilution sample to be tested, 100ul per well, set 3 replicate wells for each concentration. Put the 96-well plate into the cell incubator for 3 days, detect the secretion amount of cytokine IFN-γ in cell culture supernatant by ELISA method, as shown in (B) and (C) of Figure 5.

[0199] Experiments prove that: IL12 mutant -Fc fusion protein and the control wild type IL12 can activate PBMC cells and induce PBMC cells to secrete IFN-γ. Compared with wild type IL12, IL12 mutant -Fc fusion protein induces PBMC to secrete IFN-γ at a higher concentration of sample, and the maximum value of PBMC secreting IFN-γ is also reduced, indicating that compared with wild type IL12, IL12 mutant -Fc fusion protein reduces the ability of wild type IL12 fusion protein to activate PBMC cells, and the degree of reduction of activation activity of different mutations is different, which is reduced by 1-100 times or more, reaching the expected goal of mutation.

[0200] 3, NK cell activation

[0201] NK cells were isolated from PBMC using Human NK Cell Enrichment Set-DM magnetic beads sorting (BD Biosciences, Cat#2257429), resuspended with culture medium with final concentration of 0.5 ng / mL Recombinant Human IL-2 (Peprotech, Cat#200-02-100), then cells were added into 384-well plate, 10000 cells / well, 40ul / well, 40ul / well for test sample, 384-well plate was put into cell incubator for 2 days, then the secretion of IFN-gamma was detected by ELISA method, as shown in Figure 6.

[0202] Experiments show that, compared with wild type IL12, IL12 mutant The level of Fc fusion protein activating NK cells to secrete IFN-gamma is significantly reduced, and a higher concentration than wild type IL12 is needed to reach the level of wild type IL12 activating NK cells. Because the drug toxicity side effect of activating peripheral blood NK cells is large, reducing the level of IL12 mutant activating NK cells is a strategy to improve its safety. IL12 mutant The Fc fusion proteins HK54037, HK54051, HK54052, HK54053, HK54046, HK54064 all reduce the activation ability of NK cells, and are safer than wild type IL12.

[0203] Example 5, IL12 mutant STAT4 phosphorylation activity of Fc fusion protein

[0204] Test sample: IL12 mutant Fc fusion proteins K54037, HK54046, HK54052, HK54053, HK54064.

[0205] PBMC (Shanghai Heyousheng Biological Technology Co., Ltd., Cat# HPB050C) was stimulated with Phytohemagglutinin-L (PHA-L) Solution (500x) (Invitrogen, 2514949) for 24 hours, then washed and stimulated with 1000 IU Recombinant Human IL-2 (PEPROTECH, Cat#200-02-100) for 1 day, and then rested overnight; diluted test sample was added to PBMC (3x10 5The concentration of the sample to be tested HK54037, HK54046, HK54052, HK54053, HK54064 added in turn: 1000, 200, 40, 8, 1.6, 0.32, 0.064 nM, and the concentration of the control wild-type IL12 added in turn: 3, 0.6, 0.12, 0.024, 0.0048, 0.00096, 0.000048 nM, 37°C for 30 min, centrifugation to take the supernatant, then add 100ul Phosflow TM Fix Buffer I (BD Biosciences, item number 557870) fixation buffer, 37°C for 15 min, wash twice with staining buffer (PBS+3%FBS), then add-20°C pre-cooled permeation buffer Phosflow Perm Buffer III (BD Biosciences, item number 558050) to resuspend the cells, incubate on ice for 30-45 min, wash twice with PBS+3%FBS, then add FITC Mouse Anti-Human CD8 (BD), PE Mouse Anti Human PSTAT4 (PY693) (BD Biosciences, item number 558249), incubate at room temperature for 1 h, wash twice with PBS and then detect on the machine.

[0206] Experiments show that: IL12 mutant -Fc fusion protein and wild-type IL12 can activate the Jak-STAT signal pathway downstream of T cell IL12R to activate pSTAT4, compared with wild-type IL12 fusion protein, IL12 mutant -Fc fusion protein activates T cell-mediated pSTAT4 fluorescence curve EC50 (half effective concentration) increases, the curve platform decreases, that is, IL12 mutant -Fc fusion protein compared with wild-type IL12 fusion protein reduces the ability to activate T cells, HK54037, HK54046, HK54052, HK54053, HK54064 mutations are different, the degree of reduced activation activity is also different, reduced by 1-100 times or more.

[0207] Example 6, IL12 mutant -Fc fusion protein in vivo anti-tumor activity

[0208] Test sample: IL12 mutant -Fc fusion protein mHK54037, mHK54046, mHK54052, mHK54053, mHK54064.

[0209] The mouse IL12 is used to construct a mutant molecule to replace human IL12, and the wild-type mouse IL12 is used as a control. The amino acid sequence of the P40 subunit of the wild-type mouse IL12 is SEQ ID No. 33, and the amino acid sequence of the P35 subunit of the wild-type mouse IL-12 is SEQ ID No. 34. The correspondence and sequence details of the mouse IL12 mutant fusion protein and the human IL12 mutant fusion protein are shown in Table 4.

[0210] Table 4, correspondence table of human and mouse IL12 mutant fusion proteins and their sequences

[0211] 1, IL12 mutant -Fc fusion protein resistance to mouse colon cancer cells MC38

[0212] The mouse colon cancer cell line MC38 (Nanjing Kebai Biotechnology Co., Ltd., Catalog No. CBP60825) was inoculated into C57BL / 6 mice (Beijing VitoLihua Experimental Animal Technology Co., Ltd., Catalog No. 213), and the CT26 cell line (Nanjing Kebai Biotechnology Co., Ltd., Catalog No. CBP61189) was inoculated into Balb / c mice (Beijing VitoLihua Experimental Animal Technology Co., Ltd., Catalog No. 211) to evaluate the anti-tumor activity of the mutant molecule.

[0213] The MC38 cells were cultured to 80% fullness, trypsinized, centrifuged at 1000 rpm for 5 min, the cells were collected, washed, centrifuged and resuspended to obtain a cell suspension, and the cell viability was ensured to be >95%, ready for use. The MC38 cell line was inoculated subcutaneously on the back (shaved) of the test mice (5x10 5 cells per mouse). When the average tumor volume of the tumor-bearing mice reached 100 mm 3 around, the mice were randomly divided into groups according to the experimental design, with 5 mice in each group. The saline group (Vehicle) was set as the negative control group without drug administration. Wild-type IL12 and mHK54037, mHK54052, mHK54053 were diluted with saline according to the required concentration, and administered by intraperitoneal injection, twice a week. After tumor inoculation, the survival and activity of the animals were checked twice a week. This included: tumor growth, activity, diet, body weight and other abnormal behaviors.

[0214] The specific results are shown in Figure 8. As shown in Figure 8(A), compared with the saline group (referred to as Vehicle), mHK54037, mHK54052 and mHK54053 can significantly inhibit tumor growth. As shown in Figure 8(B), wild-type IL12 can cause a significant decrease in mouse body weight (decrease of more than 20%) and even cause mouse death, while mIL12 mutant -Fc fusion protein administration group and the saline control group, indicating that mIL12mutant -Fc fusion proteins can inhibit the growth of MC38 tumors, and do not affect the activity and body weight of mice, and are safer than wild-type IL12.

[0215] 2、IL12 mutant -Fc fusion proteins against CT26 colon cancer cells in mice

[0216] Mouse colon cancer cells (CT26 cells, ATCC) were cultured to 80% fullness, trypsinized, centrifuged at 1000 rpm for 5 min, and the cells were collected, washed, centrifuged and resuspended to obtain a cell suspension with a cell viability of >95%, ready for use. The CT26 cell line was inoculated subcutaneously on the back (shaved) of the test mice (2x10 5 cells per mouse). When the average tumor volume of the tumor-bearing mice reached 100mm 3 around, the mice were randomly divided into groups according to the experimental design, with 5 mice in each group. The saline group (Vehicle) was set as the negative control group without drug administration. Wild-type mIL12 and mHK54037, mHK54046, mHK54052, mHK54064 were diluted with saline according to the required concentration, and administered intraperitoneally, once a week. After tumor inoculation, the survival and activity of the animals were checked twice a week. This included: tumor growth, activity, diet, body weight and other abnormal behaviors.

[0217] The specific results are shown in Figure 9: compared with the saline group (Vehicle), mIL12 mutant -Fc fusion proteins mHK54037, mHK54046, mHK54052, mHK54064 can significantly inhibit the growth of CT26 tumors, and mHK064 can achieve the same efficacy as mHK54037, mHK54046, mHK54052 when the dose is increased.

[0218] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range of equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, this application intends to include any changes, uses or improvements of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application. Industrial applicability

[0219] The present application provides a novel interleukin 12 (IL-12, IL12) mutant protein with improved binding properties to IL12 receptor and drugability compared to wild-type IL12. The IL12 mutant protein can be applied in the field of anti-tumor therapy. By reducing the binding ability to IL12 receptor, excessive lymphocyte activation and inflammatory factor release can be avoided, the systemic inflammatory response induced by IL12 can be reduced, the adverse reactions of patients during treatment can be reduced, and the tolerance and safety of treatment can be improved. The present application prolongs the half-life of IL12 in vivo and enhances the stability of the molecule in the form of a fusion protein, so that the IL12 fusion protein can continuously exert an anti-tumor effect at a lower dose, reduce the frequency of administration, and further reduce the systemic inflammatory response during treatment and improve patient compliance. The design of the fusion protein not only helps the stability of the protein, but also improves the efficiency of expression and purification, making IL12 suitable for large-scale production and commercialization.

[0220] The weakened IL12 molecule can be used alone for anti-tumor therapy, or can be used in combination with other immunotherapy (such as immune checkpoint inhibitors, CAR-T cell therapy) to enhance anti-tumor efficacy. The weakened IL12 molecule can also be used for gene therapy, by introducing the IL-12 gene into the tumor or specific immune cells, sustained and localized IL12 expression can be achieved, anti-tumor immune response can be enhanced, and systemic toxicity can be reduced.

[0221] Overall, the IL12 mutant protein and fusion protein provided by the present application provide a powerful tool for tumor immunotherapy, which can significantly improve the therapeutic effect, improve the prognosis of patients, and also can be personalized according to the tumor type and individual differences of patients, providing more choices for clinical treatment.

[0222] SEQUENCE LISTING

Claims

1. An IL-12 mutein characterized in that, The IL-12 mutein is a protein obtained by mutating the amino acids of wild type IL-12, which comprises wild type P40 subunit and wild type P35 subunit, the IL-12 mutein comprises mutated P40 subunit and / or mutated P35 subunit, the mutated P40 subunit is a subunit obtained by mutating at least one of the following positions of the wild type P40 subunit: 37, 38, 39, 40, 81, 82, 106, 108, 114, 115 or / and 219, the mutation is a mutation that eliminates or reduces the binding affinity of the wild type P40 subunit to IL-12Rβ1; the mutated P35 subunit is a subunit obtained by mutating at least one of the following positions of the wild type P35 subunit: 72, 151 or / and 190, the mutation eliminates or reduces the binding affinity to IL-12Rβ2. The wild type IL-12 can be IL-12 derived from mammals, such as human IL-12.

2. The mutein according to claim 1, characterized in that, The P40 subunit binding interface with IL-12Rβ1 comprises one or more mutations selected from the following: W37K; W37A; D36K, T38K; D40P; E81A; E81K; F82A; K106S; K106E; K106Q; E108A; T114A; D115K; K219E; K219S.

3. The mutein according to claim 1 or 2, characterized in that, The P35 subunit binding interface with IL-12Rβ2 comprises one or more mutations selected from the following: E72S; R151E; K190E.

4. The mutein according to any one of claims 1 - 3, characterized in that, The IL-12 mutein binding interface with IL-12R comprises IL-12 binding interface mutation with IL-12Rβ1 and / or IL-12 binding interface mutation with IL-12Rβ2: the mutation comprises: 1) the IL-12 binding interface mutation with IL-12Rβ2 is: E72S; R151E; K190E; and / or 2) the IL-12 binding interface mutation with IL-12Rβ1 is: W37K+K219E.

5. The mutein according to any one of claims 1 -4, characterized in that, The mutein, relative to native IL-12, has the following amino acid mutations: introducing mutation C199S and / or C274S at position 199 and / or 274 of IL-12 P40 subunit, and introducing mutation C96S at position 96 of P35 subunit.

6. The mutein of claims 1-5, characterized in that, 1) the P40 subunit of the mutein has an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, or 98% identity to the amino acid sequence selected from SEQ ID No. 3; and / or, 2) the P35 subunit of the mutein has an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, or 98% identity to the amino acid sequence selected from SEQ ID No.

4.

7. The mutein according to claims 1-6, characterized in that, 1) the amino acid sequence of the P40 subunit of the mutein is SEQ ID No. 22, SEQ ID No. 26, SEQ ID No. 16, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 23, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21; 2) the amino acid sequence of the P35 subunit of the mutein is SEQ ID No. 4, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No.

13.

8. The mutein according to any one of claims 1-7, characterized in that, compared to before the weakening, and the protein has at least one or more of the following properties: 1) reduced activation of T cells (such as CD4+ or / and CD8+ T cells) compared to before the weakening; 2) reduced activation of NK compared to before the weakening; 3) reduced activation of IL-12 pSTAT4 phosphorylation signaling pathway compared to before the weakening; 4) reduced IL-12 reporter gene signal activation compared to before the weakening; 5) increased safety in animals compared to before the weakening.

9. An IL-12 mutein fusion protein characterized in that, The fusion protein comprises the IL-12 mutein according to any one of claims 1-8.

10. The fusion protein of claim 9, characterized in that, The fusion protein is formed by fusing the IL-12 mutein with an Fc antibody fragment.

11. The fusion protein of claim 8, characterized in that, The Fc fragment comprises the following mutations: 1) comprises Knob mutations, such as mutations T366W and S354C; 2) comprises Hole mutations, such as mutations Y349C, T366S, L368A, Y407V.

12. An IL12-Fc dimeric protein characterized in that, The IL-12 mutein fusion protein according to claim 10 or 11, preferably the dimeric protein, has one or more of the following properties compared to the corresponding dimeric protein of wild-type IL12-Fc fusion protein: 1) reduced or avoided lymphocyte over-activation and / or inflammatory factor release caused by IL-12; 2) lower toxicity when used in vivo; 3) increased expression amount and / or purity when expressed in mammalian cells (such as CHO or HEK293 cells).

13. The IL12-Fc dimeric protein of claim 12, which is a heterodimer, wherein comprises: a first monomer, wherein the first monomer comprises, from N-terminus to C-terminus: 1) an IL-12 P40 subunit or a mutein thereof; 2) a linker; 3) a first Fc fragment; a second monomer, wherein the second monomer comprises, from N-terminus to C-terminus: 1) an IL-12 P35 subunit or a mutein thereof; 2) a linker; 3) a second Fc fragment.

14. The IL12-Fc dimeric protein of claim 12, which is a heterodimer, wherein comprises: a first monomer, wherein the first monomer comprises, from N-terminus to C-terminus: 1) an IL-12 P35 subunit or mutein thereof; 2) a linker; 3) an IL-12 P40 subunit or mutein thereof; 4) a linker; 5) a first Fc fragment, the 1) and 2) can be replaced in position; a second monomer, wherein the second monomer comprises a second Fc fragment and optionally an IgGl hinge region.

15. The IL12-Fc dimeric protein of claim 13 or 14, wherein the first Fc fragment and the second Fc fragment each comprises a first and a second heterodimerization mutation, respectively, that promotes the formation of a heterodimer between the first monomer and the second monomer, the IL12-Fc dimeric protein being any one of: 1) the first and the second heterodimerization mutations comprise a Knob-into-Hole mutation combination; 2) the first heterodimerization mutation on the first Fc fragment comprises a Knob mutation and the second heterodimerization mutation of the second Fc fragment comprises a Hole mutation; 3) the first heterodimerization mutation on the first Fc fragment comprises a Hole mutation and the second heterodimerization mutation of the second Fc fragment comprises a Knob mutation.

16. An immunoconjugate comprising the IL-12 mutein of claims 1-7 and an antigen binding molecule, preferably the antigen binding molecule is an immunoglobulin molecule, in particular an IgG molecule, or an antibody, or an antibody fragment, in particular a Fab molecule and a scFv molecule.

17. A polynucleotide encoding the IL-12 mutein of claims 1-7, or the fusion protein of claims 9-11, or the IL12-Fc dimeric fusion protein of claims 12-15, or the immunoconjugate of claim 16.

18. An expression vector comprising the polynucleotide of claim 17.

19. A host cell comprising the polynucleotide of claim 17, or the vector of claim 18, preferably the host cell is a mammalian cell, in particular a HEK293 cell or a CHO cell, and a yeast.

20. The host cell of claim 19, wherein the host cell is a mammalian cell or a yeast cell.

21. The host cell of claim 19, wherein the host cell is a HEK293 cell or a CHOS cell.

22. A pharmaceutical composition comprising the IL-12 mutein of claims 1-8 or the fusion protein of claims 9-11 or the IL12-Fc dimeric fusion protein of claims 12-15 or the immunoconjugate of claim 16 and a pharmaceutically acceptable carrier.

23. Use of the IL-12 mutein of claims 1-8 or the fusion protein of claims 9-11 or the IL12-Fc dimeric fusion protein of claims 12-15 or the immunoconjugate of claim 16 for the manufacture of a medicament for stimulating the immune system of a subject.

24. A method of treating a disease in a subject, the method comprising administering to the subject the IL-12 mutein of claims 1-8 or the fusion protein of claims 9-11 or the IL12-Fc dimer fusion protein of claims 12-15 or the immunoconjugate of claim 16 or the pharmaceutical composition of claim 22.

25. A method of stimulating the immune system of a subject, the method comprising administering to the subject an effective amount of the IL-12 mutein of claims 1-8 or the fusion protein of claims 9-11 or the IL12-Fc dimer fusion protein of claims 12-15 or the immunoconjugate of claim 16 or the pharmaceutical composition of claim 22.

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