Interleukin 12 mutant and application thereof

Abstract

The invention discloses an interleukin 12 mutant and application thereof, and belongs to the technical field of gene engineering. An IL-12 mutant protein comprises a mutated P40 subunit and / or a mutated P35 subunit wherein the mutated P40 subunit is obtained by mutating a wild-type P40 subunit at the following positions: position 37, 38, 39, 40, 81, 82, 106, 108, 114, 115 or / and 219, said mutation having a mutation that eliminates or reduces the binding affinity of the wild-type P40 subunit to IL-12R [beta] 1; wherein the mutated P35 subunit is obtained by mutating the wild-type P35 subunit at the following positions: the 72,151st or / and 190th position, and the mutation eliminates or reduces the mutation of binding affinity to IL-12R beta 2. The IL12 mutant protein and the fusion protein can significantly improve the treatment effect by reducing toxicity and enhancing tumor targeting.

Description

Technical Field

[0001] The present invention relates to an interleukin-12 mutant and uses thereof. Background Art

[0002] Interleukin-12 (IL-12) is an important cytokine primarily produced by activated antigen-presenting cells. It is naturally produced by dendritic cells, macrophages, neutrophils, and human B lymphoblastoid cells (NC-37) in response to antigen stimulation. IL-12 is composed of four α-helices and is a heterodimer formed by two subunits, IL-12A (p35) and IL-12B (p40), bonded by disulfide bonds. The IL12 receptor, composed of two subunits, IL12Rβ1 and IL12Rβ2, is primarily expressed on T cells and NK cells.

[0003] IL-12 belongs to the interleukin 12 family, which induces the proliferation of NK cells, NKT cells and T cells, enhances the expression of cytotoxicity and cytotoxic mediators, and produces cytokines, especially interferon-γ (IFN-γ), and is conducive to differentiation into cells that produce type 1 cytokines (TH1, TC1 and NK1 cells); for B cells, IL-12 directly or through the action of type 1 cytokines (such as IFN-γ) enhances the activation and production of TH1-related immunoglobulins (such as mouse IgG2a), and plays an important role in the regulation of the immune system and antiviral and anti-tumor effects.

[0004] IL-12 was first discovered in the early 1990s, and scientists have shown that it plays a key role in regulating the activity of immune cells such as T cells and natural killer cells (NK cells). As research on IL-12 continues to deepen, it has been found that IL-12 can enhance the body's resistance by activating immune cells and can activate immune cells to attack tumor cells. Therefore, IL-12 has been widely studied in the fields of immunotherapy and tumor treatment.

[0005] Years of clinical experience have revealed that while IL-12 has promising anti-tumor effects, it can also cause serious drug-related side effects. Studies have shown that these toxicities are likely due to IL-12's overactivation of lymphocytes, stimulating the release of inflammatory factors. Furthermore, because IL-12 has a half-life of only approximately 5-10 hours in the human body, daily dosing is often used clinically to achieve sufficiently high human exposure. However, frequent dosing not only places a heavy burden on patients but, more importantly, results in peak blood drug concentrations (Cmax), another key factor in drug toxicity.

[0006] In terms of production, natural IL-12 molecules are difficult to express in mammalian cells (CHO or HEK293) due to the characteristics of their amino acid sequences and have poor molecular stability. Therefore, there is a need in the art to improve the expression of IL-12 molecules and IL-12-Fc molecules in mammalian cells.

[0007] Several IL-12 molecule modification schemes have been proposed in the field. For example, extending half-life involves Fc fusion or HAS protein fusion. Regarding activity, Dragonfly chose not to reduce IL-12 activity, while Xencor opted to reduce IL-12 activity, lowering affinity and activity, thereby compensating for efficacy and increasing the safety window by extending half-life. However, no optimized IL-12 molecules have yet reached market, and the earliest is still in clinical development.

[0008] In view of the above-mentioned IL-12 immunotherapy and production-related problems, there is a need in the art to further develop new IL-12 molecules with optimized properties, showing IL-12 molecules that are easy to produce and purify and have improved pharmacokinetic and pharmacodynamic properties. SUMMARY OF THE INVENTION

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

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

[0012] The IL-12 mutant protein provided by the present invention is a protein obtained by subjecting wild-type IL-12 to amino acid mutations, wherein the wild-type IL-12 includes a wild-type P40 subunit and a wild-type P35 subunit. The IL-12 mutant protein comprises a mutated P40 subunit and / or a mutated P35 subunit, wherein 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, and / or 219, wherein the mutation 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, and / or 190, wherein the mutation eliminates or reduces the binding affinity to IL-12Rβ2;

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

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

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

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

[0017] Preferably, the IL-12 and IL-12Rβ1 binding interface mutations include:

[0018] Selected from the following exemplary mutation combinations: 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] Furthermore, the mutations at the binding interface between the P35 subunit and IL-12Rβ2 include one or more mutations selected from the following: E72S; R151E; K190E.

[0020] Furthermore, the mutations in the IL-12 mutant protein and IL-12R binding interface include IL-12 and IL-12Rβ1 binding interface mutations and / or IL-12 and IL-12Rβ2 binding interface mutations: the mutations include:

[0021] 1) The mutations in the IL-12 and IL-12Rβ2 binding interface are: E72S; R151E; K190E; and / or

[0022] 2) The mutations at the binding interface between IL-12 and IL-12Rβ1 are: W37K+K219E.

[0023] Furthermore, the mutant protein has the following amino acid mutations relative to native IL-12: C199S and / or C274S mutations are introduced at position 199 and / or 274 of the IL-12P40 subunit, and C96S mutations are introduced at position 96 of the P35 subunit.

[0024] In the present invention, 1) the P40 subunit of the mutant protein has an amino acid sequence that is at least 90%, 92%, 94%, 95%, 96%, 97%, or 98% identical 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 that is at least 90%, 92%, 94%, 95%, 96%, 97%, or 98% identical to the amino acid sequence selected from SEQ ID No. 4.

[0027] In a specific embodiment, 1) the amino acid sequence of the mutant protein P40 subunit 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 mutant protein P35 subunit is SEQ ID No.4, SEQ ID No.11, SEQ ID No.12, and SEQ ID No.13.

[0029] In the present invention, compared with the attenuation before the introduction of the mutation, the binding affinity for IL-12R is reduced, and the mutant protein has at least one or more properties selected from the following:

[0030] 1) Compared with before weakening, the T cells (such as CD4 + or / and CD8 + T cell) activation;

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

[0032] 3) Compared with before attenuation, the activation of IL-12pSTAT4 phosphorylation signaling pathway was reduced;

[0033] 4) Reduced IL-12 reporter gene signal activation compared to before attenuation;

[0034] 5) Compared with before weakening, the safety in animals is increased.

[0035] The present invention also provides an IL-12 mutant protein fusion protein, wherein the fusion protein comprises any one of the above-mentioned IL-12 mutant proteins.

[0036] In the present invention, the fusion protein is formed by fusing IL-12 mutant protein with Fc antibody fragment.

[0037] Preferably, the IL-12 mutant protein is fused to Fc via a linker, and the linker is preferably (G4S)n, where n can be a natural number such as 0, 1, 2, 3, 4, or 5;

[0038] Preferably, the Fc fragment comprises a mutation that reduces or eliminates the binding of Fc to FcγR, such as L234A+L235A,

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

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

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

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

[0043] The present invention also provides an IL12-Fc dimer protein comprising the aforementioned IL-12 mutant fusion protein. Preferably, the dimer protein has one or more of the following properties compared to the corresponding dimer protein of the wild-type IL12-Fc fusion protein:

[0044] 1) Reduce or avoid excessive lymphocyte activation and / or inflammatory factor release caused by 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] Furthermore, the IL12-Fc dimer protein is a heterodimer, comprising:

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

[0049] Furthermore, the IL12-Fc dimer protein is a heterodimer, comprising:

[0050] A first monomer, wherein the first monomer comprises, from N-terminus to C-terminus: 1) an IL-12P40 subunit or a mutant protein thereof; 2) a linker; 3) an IL-12P35 subunit or a mutant protein thereof; 4) a linker; 5) a first Fc fragment, wherein 1) and 2) can be interchangeable; the linker is preferably (G4S)n, where n can be a natural number such as 0, 1, 2, 3, 4, or 5; 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-mentioned IL12-Fc dimer protein, the first Fc fragment and the second Fc fragment respectively comprise first and second heterodimer mutations that promote the formation of a heterodimer between the first monomer and the second monomer, and the IL12-Fc dimer protein is any one of the following:

[0053] 1) The first and second heterodimer mutations comprise a combination of Knob-into-Hole mutations, such as T366W, S354C and Y349C, T366S, L368A, Y407V;

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

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

[0056] The present invention also provides an immunoconjugate comprising the IL-12 mutant protein described above and an antigen-binding molecule. Preferably, the antigen-binding molecule is an immunoglobulin molecule, particularly an IgG molecule, or an antibody, or an antibody fragment, particularly a Fab molecule and a scFv molecule.

[0057] The present invention also provides a polynucleotide encoding the aforementioned IL-12 mutant protein, the aforementioned fusion protein, the aforementioned IL12-Fc dimer fusion protein, or the aforementioned immunoconjugate.

[0058] The present invention also provides an expression vector comprising the polynucleotide described above.

[0059] The present invention also provides a host cell comprising the polynucleotide or the vector described above. Preferably, the host cell is a mammalian cell, in particular a HEK293 cell or a CHO cell, and a yeast.

[0060] The present invention also provides a pharmaceutical composition comprising the aforementioned IL-12 mutant protein, the aforementioned fusion protein, or the aforementioned IL12-Fc dimer fusion protein, or the aforementioned immunoconjugate and a pharmaceutically acceptable carrier.

[0061] The present invention also provides use of the aforementioned IL-12 mutant protein, the aforementioned fusion protein, or the aforementioned IL12-Fc dimer fusion protein, or the aforementioned immunoconjugate in the preparation of a medicament for stimulating the immune system of a subject.

[0062] The present invention also provides a method for treating a disease in a subject, comprising administering to the subject the aforementioned IL-12 mutant protein, the aforementioned fusion protein, or the aforementioned IL12-Fc dimer fusion protein, or the aforementioned immunoconjugate or pharmaceutical composition.

[0063] The present invention also provides a method for stimulating a subject's immune system, comprising administering to the subject an effective amount of the aforementioned IL-12 mutant protein, the aforementioned fusion protein, or the aforementioned IL12-Fc dimer fusion protein, or the aforementioned immunoconjugate or pharmaceutical composition.

[0064] Beneficial effects

[0065] The IL-12 mutant protein provided by the present invention can reduce the activity of IL12 in stimulating immune cells by reducing its binding activity to IL-12R, reduce the ability of IL12 to mediate the secretion of IFN-γ by T cells and NK cells, avoid excessive activation of T cells, balance the toxicity of the drug efficacy, maintain a good anti-tumor effect, and avoid the toxic and side effects caused by excessive activity in clinical applications, broaden the clinical treatment window, and achieve better therapeutic effects; the fusion protein of the IL-12 mutant protein provided by the present invention can prolong the half-life, reduce the frequency of administration, and increase the production yield on the basis of retaining the optimized characteristics of the IL-12 mutant protein, and exhibit good drug-forming properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 The contact interfaces between the IL-12 subunits and the IL-12Rβ1 or IL-12Rβ2 complexes. A represents the crystal structure of the IL-12 P40 subunit complexed with IL-12Rβ1 (PDB: 6WDQ) and their interface; B represents the contact interface between the IL-12 P35 subunit complexed with IL-12Rβ2.

[0067] Figure 2 The molecular model diagram of IL-12-Fc dimer. Format 1 represents an IL12-Fc heterodimer protein structure with a linker of (G4S)2; Format 2 represents another IL12-Fc heterodimer protein structure with a linker of (G4S)3.

[0068] Figure 3 IL12 mutant - SDS-PAGE electrophoresis of Fc dimer protein. M is a standard control protein (also called a marker) with a known molecular weight; R is the molecular weight of the reduced protein; and NR is the molecular weight of the non-reduced protein.

[0069] Figure 4 For weakened IL12 mutant Activation signal curves of -Fc dimer protein on IL-12 reporter gene cell line (A), (B), (C) and (D). WT is wild-type IL-12; IL12 WT -Fc (Format1) is a wild-type IL12-Fc heterodimer fusion protein with the protein structure of Format1; IL12 WT-Fc (Format 2) is a wild-type IL12-Fc heterodimer fusion protein with a protein structure of Format 2; HK54029, HK54032, HK54033, HK54037, HK54040, HK54042, HK54043, HK54044, HK54045, HK54046, HK54051, HK54052, HK54053, HK54054, HK54064, HK54065, HK54071, HK54073, HK54074, are mutant fusion proteins (collectively referred to as IL12 mutant -Fc).

[0070] Figure 5 For weakened IL12 mutant - The secretion level of IFN-γ in cells activated by Fc dimer protein. (A) is the weakened IL12 mutant -Fc dimer protein activates normal CD8 + T cells, where the protein concentrations tested were 20nM, 4nM, 0.2nM, 0.01nM, 0.00025nM, and 0.00000625nM; (B) and (C) are the levels of IFN-γ secretion after PBMC cells were activated by IL-12 mutant fusion proteins. (B) Wild-type IL-12 (IL12 WT ) were 350nM, 17nM, and 0.03nM, respectively; the mutant fusion protein concentrations were 350nM, 90nM, 9nM, 1.8nM, and 0.36nM, respectively; the mutant fusion protein concentrations in (C) were 2000nM, 500nM, 50nM, 5nM, and 0.1nM, respectively.

[0071] Figure 6 For weakened IL12 mutant -Fc dimer protein activates NK cells to secrete IFN-γ. WT The concentrations of the proteins tested were 170 nM, 17 nM, and 0.09 nM, respectively, and the concentrations of the mutant fusion proteins tested were 4500 nM, 2250 nM, and 450 nM, respectively.

[0072] Figure 7 For weakened IL12 mutant -Fc dimer protein activated normal CD8+ T cells pSTAT4 signal curve, in which wild-type IL-12 (IL12 WT) protein inspection concentrations were 15nM, 3nM, 0.6nM, 0.12nM, 0.024nM, 0.0048nM, 0.00096nM, and 0.000048nM, and the mutant fusion protein inspection concentrations were 1000nM, 200nM, 40nM, 8nM, 1.6nM, 0.32nM, and 0.064nM, respectively.

[0073] Figure 8 For weakened IL12 mutant Effects of the mouse counterpart of the -Fc dimer protein on tumor growth (A) and animal body weight (B) in tumor-bearing C57 mice.

[0074] Figure 9 For weakened IL12 mutant -The effect of the mouse molecule corresponding to the Fc dimer protein on tumors in tumor-bearing Balb / c mice.

[0075] Modes for Carrying Out the Invention

[0076] 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. For the purposes of the present invention, the following terms are defined below.

[0077] In this article, wild-type "interleukin-12" or "IL-12" or "IL12 WT" refers to the parent IL-12 protein used as a template for introducing the mutations or combinations of mutations of the present invention, preferably a naturally occurring IL-12 protein, including unprocessed (e.g., signal peptide not removed) and processed (e.g., signal peptide removed) forms. The sequence of a full-length natural IL-12 P40 subunit including the signal peptide is shown in SEQ ID No. 1, and the sequence of a full-length natural IL-12 P35 subunit including the signal peptide is shown in SEQ ID No. 2. In addition, the expression also includes variants of natural IL-12, for example, the variant may have at least 95%-99% or higher identity with natural IL-12 or have no more than 1-10 or 1-5 amino acid mutations (e.g., conservative substitutions). Therefore, in some embodiments, wild-type IL-12 may contain amino acid mutations that do not affect its binding to the IL-12 receptor compared to the natural IL-12 protein and contain 6×His at the C-terminus. The P40 subunit of wild-type IL-12 is the wild-type P40 of the present invention, and its sequence is shown in SEQ ID In No. 3, the wild-type IL-12 P35 subunit is the wild-type P35 of the present invention, and its sequence is shown in SEQ ID No. 4. In some embodiments, the wild-type IL-12 P40 subunit and P35 subunit may have at least 85%, 95%, or even higher amino acid sequence identity with the amino acid sequence of SEQ ID No. 1 or 2, respectively. In some embodiments, the wild-type P40 and wild-type P35 may have at least 85%, 95%, or even higher amino acid sequence identity with the corresponding amino acid sequences of SEQ ID No. 3 and SEQ ID No. 4, respectively.

[0078] Herein, the amino acid mutation may be amino acid substitution, deletion, insertion and addition.

[0079] In the present invention, when referring to the position of amino acids in the IL-12 protein or IL-12 sequence, reference is made to the wild-type human IL-12 protein (also known as IL12 WT ), wherein the amino acid sequence of the wild-type IL-12 P40 subunit is shown in SEQ ID No. 3, and the sequence of the wild-type IL-12 P35 subunit is shown in SEQ ID No. 4. For example, when referring to P40 "W37", it refers to the tyrosine residue W at position 37 of SEQ ID No. 3.

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

[0081] In this article, mutations in the "binding interface between IL-12 and IL-12R" include: 1) the binding interface between the IL-12P40 subunit and IL-12Rβ1; 2) or / and the binding interface between the IL-12P35 subunit and IL-12Rβ2. The "binding interface between the IL-12P40 subunit and IL-12Rβ1" mutation refers to a mutation that occurs at the amino acid site where IL-12, especially the P40 subunit, interacts with IL-12Rβ1. These interacting amino acid sites can be determined by crystal structure analysis of the complex of IL-12 and its receptor (such as PDB: 6WDQ). In some embodiments, the mutation refers in particular to a mutation at the following amino acid sites of the P40 subunit: 36, 37, 38, 40, 81, 82, 106, 108, 114, 115, 219. Preferably, the IL-12 protein comprising the mutation has weakened IL-12Rβ1 binding compared to the corresponding protein before the mutation. The term "IL-12 p35 subunit and IL-12Rβ2" refers to mutations occurring at amino acid sites in IL-12, particularly the p35 subunit, that interact with IL-12Rβ2. In some embodiments, the mutations specifically refer to mutations at the following amino acid sites in the p35 subunit: 72, 151, and 190. Preferably, the IL-12 protein comprising the mutations exhibits reduced IL-12Rβ2 binding compared to the corresponding protein before the mutations.

[0082] Herein, "weakened" IL-12 protein molecules with respect to IL-12R binding refer to the introduction of mutations into the IL-12R binding interface, wherein the mutations result in reduced binding to IL-12Rβ1 and / or IL-12Rβ2 relative to the corresponding IL-12 protein before the introduction of the mutations. Further preferably, the weakened molecules have reduced T cell (such as PBMC, CD8+ T cell) and / or NK cell activation activity relative to the corresponding proteins.

[0083] 1. IL-12 mutant protein of the present invention

[0084] The IL-12 mutant protein of the present invention has favorable biological properties: After long-term research, the following molecular mutations and modifications can be combined to simultaneously improve the efficacy of IL-12, reduce the toxic side effects of IL-12, and achieve good production performance.

[0085] 1) Introducing specific amino acid mutations at the binding interface between IL-12 and IL-12R weakens the binding of the IL-12 P40 subunit to IL-12Rβ1 and / or weakens the binding of the IL-12 P35 subunit to IL-12Rβ2, thereby downregulating IL-12 activity to a certain extent. The IL-12 mutein of the present invention can activate lymphocytes to kill tumor cells while avoiding the release of large amounts of inflammatory factors caused by excessive lymphocyte activation and the resulting drug-related toxicity.

[0086] 2) The mutant IL-12 protein of the present invention is constructed into an IL12-Fc dimer. This dimer formation allows for FcRn-mediated recycling in vivo, extending the half-life of the IL12-Fc fusion protein. This overcomes the high peak plasma concentrations associated with IL-12's short half-life and high-frequency dosing.

[0087] 3) The mutant IL-12 proteins of the present invention constructed as IL12-Fc dimers have improved druggability. For example, when expressed in mammalian cells such as HEK293 or CHO cells, particularly when expressed as Fc fusion proteins, they exhibit one or more of the following properties: (a) superior expression compared to wild-type IL-12 protein; and (b) ease of purification to a higher protein purity.

[0088] Therefore, through modification, the present invention provides an IL-12 mutant protein with improved drugability. The IL12-Fc series molecules comprising the IL-12 protein of the present invention, on the one hand, weaken the affinity with its receptor, achieving a drug efficacy equivalent to or even better than that of the natural IL-12 molecule, while achieving higher safety. On the other hand, the drugability, such as protein expression and purity, is significantly improved.

[0089] In some embodiments of the present invention, the IL-12 mutant protein of the present invention exhibits increased expression levels compared to wild-type IL-12, with the expression level in mammalian cells increased by at least 1.1-fold, or at least 1.5-fold, or at least 2-fold, 3-fold, or at least 3-fold or more.

[0090] In some embodiments, the IL-12 muteins and IL12-Fc fusion proteins of the present invention exhibit higher purity relative to wild-type IL-12 protein, as determined by protein A affinity chromatography purification. After one-step protein A affinity chromatography purification, the purity of the IL-12 muteins and IL12-Fc fusion proteins of the present invention can reach 70%, 80%, or 90% or greater, preferably 92%, 93%, 94%, 95%, 98%, or 99% or greater.

[0091] Mutations are introduced into the binding interface between IL-12 and IL-12R. In some embodiments, the IL-12 mutant protein of the present invention, or the IL12-Fc fusion protein of the IL12 mutant protein, has a weakened IL-12Rβ1 and / or weakened IL-12Rβ2 binding affinity relative to the corresponding protein before the mutation, for example, a decrease of 1-100 times or more.

[0092] In some embodiments, the IL-12 mutant protein or IL12-Fc fusion protein of the present invention has a weakened IL-12 activity relative to that before the introduction of the mutation, for example, an IL-12 activity selected from at least one of the following:

[0093] a. Reduced activation of T cells (such as CD4+ and / or CD8+ T cells) compared to before weakening;

[0094] b. Reduced NK activation compared to before weakening;

[0095] c. Compared with before attenuation, the activation of IL-12pSTAT4 phosphorylation signaling pathway was reduced;

[0096] d. Reduced IL-12 reporter gene signal activation compared to before attenuation.

[0097] In some embodiments, the IL-12 mutant protein of the present invention results in reduced IL-12-mediated activation of lymphocytes (such as T cells and / or NK cells) compared to before attenuation. In some embodiments, the lymphocytes are PBMC total or isolated CD8 + T cells or isolated CD3 +T cells. In some embodiments, the ability of IL-12 mutant proteins to activate lymphocytes is identified by detecting the level of IFN-γ secretion after activation of lymphocytes, such as T cells or NK cells. In some embodiments, the ability of IL-12 mutant proteins 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 IL-12 mutant proteins to activate lymphocytes is identified by detecting the activation of JAK1 and TYK2 phosphorylation pathways using an IL-12 reporter gene cell line. In an embodiment of the present application, the T cell activation activity of the IL-12 mutant protein of the present invention can be reduced by, for example, more than 2 times, or more than 5 times, or more than 10 times, or more than 50 times, or more than 100 times, or even more than 1000 times compared to before attenuation.

[0098] In some embodiments, the IL-12 muteins of the present invention have reduced toxic side effects mediated by IL-12 and its receptor relative to wild-type IL-12.

[0099] The mutant protein of the present invention:

[0100] In one aspect, the present invention provides an IL-12 mutant protein, wherein the mutant protein, compared to wild-type IL-12 (preferably human IL-12), comprises the mutation:

[0101] 1) a mutation that eliminates or reduces the binding affinity to IL-12Rβ1 at the binding interface between the IL-12P40 subunit and IL-12Rβ1, particularly at at least one position selected from positions 36, 37, 38, 40, 81, 82, 106, 114, 115 and 219 of the P40 subunit, wherein the amino acid positions are according to SEQ ID No. 3.

[0102] 2) A mutation at the binding interface between the IL-12P35 subunit and IL-12Rβ2, particularly at at least one position selected from positions 72, 151, and 190 of the P35 subunit, that eliminates or reduces the binding affinity to IL-12Rβ2, wherein the amino acid positions are according to SEQ ID No. 4.

[0103] In some embodiments, the IL-12 and IL-12Rβ1 binding interface mutations comprise one or more mutations selected from the group consisting of:

[0104] W37E; W37K; W37N; W37T; W37A; D36K; T38K; D40P; E81A; E81K; F82A; K106S; K106E; K106Q; E108A; T114A; D115K; D115P; K219E.

[0105] Preferably, the IL-12 and IL-12Rβ1 binding interface mutations include:

[0106] Selected from the following exemplary mutation combinations: 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.

[0107] In some embodiments, the IL-12 and IL-12Rβ2 binding interface mutations comprise one or more mutations selected from the group consisting of:

[0108] E72S;R151E;K190E.

[0109] In some embodiments, the mutations in the IL-12 and IL-12R binding interface comprise mutations in the IL-12 and IL-12Rβ1 binding interface and / or mutations in the IL-12 and IL-12Rβ2 binding interface. Preferably, the mutations comprise:

[0110] selected from the following mutations in the IL-12 binding interface to IL-12Rβ2: E72S; R151E; K190E; and / or

[0111] The mutations selected from the following IL-12 and IL-12Rβ1 binding interface: W37K+K219E.

[0112] In some embodiments, the present invention provides IL-12 mutant proteins, wherein the mutant proteins, relative to wild-type IL-12, comprise:

[0113] Other mutations:

[0114] In addition to the aforementioned mutations in the "IL-12 and IL-12R binding interface," the IL-12 muteins of the present invention may also have one or more mutations in other regions or positions, as long as they retain one or more of the aforementioned beneficial properties of the IL-12 muteins of the present invention. For example, the IL-12 muteins of the present invention may also include the introduction of C199S and / or C274S mutations at positions 199 and / or 274 of the IL-12 P40 subunit, and the introduction of C96S mutations at position 96 of the P35 subunit, to provide additional advantages, such as improved expression, homology, or stability. Those skilled in the art will appreciate how to identify additional mutations that can be incorporated into the IL-12 muteins of the present invention.

[0115] The sequence differences between the IL-12 mutant protein and the wild-type protein can be expressed as sequence identity or as the number of amino acid differences between the two. In some embodiments, the IL-12 mutant protein has at least 85%, 86%, 87%, 88%, 89% identity with the wild-type protein, preferably more than 90% identity, preferably 95%. In some embodiments, in addition to the above-mentioned mutations of the present invention, the IL-12 mutant protein and the wild-type protein may also have no more than 15, such as 1-10, or 1-5 mutations, such as 0, 1, 2, 3, 4 mutations. In some embodiments, the remaining mutations may be conservative substitutions, meaning replacement of amino acid residues with amino acid residues having similar side chains, i.e., amino acid substitutions that do not adversely affect or change the biological function of the protein comprising the amino acid sequence.

[0116] As used herein, the term "conservative substitution" refers to a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art and are described in Table 1.

[0117] Table 1. Conservative amino acid substitutions

[0118] Sidechain amino acids Contains non-polar side chains A, I, L, M, F, P, W, Y, V Contains aliphatic side chains G, A, V, L, T, P Hydroxyl side chain S, T, Y Contains carboxylic acid and amide side chains D, N, E, Q Containing basic side chains R, K, H Contains aromatic side chains H, F, Y, W

[0119] Therefore, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative replacements of amino acids 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)).

[0120] 2. Fusion protein and IL12-Fc dimer protein

[0121] In one aspect, the present invention further provides a fusion protein comprising an IL-12 mutein of the present invention. In a preferred embodiment, the IL-12 mutein of the present invention is fused to another polypeptide that can improve pharmacokinetic properties, such as serum albumin, preferably an antibody Fc fragment.

[0122] In some embodiments, the present invention provides an IL-12 mutant protein fusion protein, which comprises an IL-12 mutant protein of the present invention fused to an antibody Fc fragment. The Fc fragment used in the present invention may comprise a mutation that reduces or removes effector function. For example, in some embodiments, the Fc fragment used in the present invention has an L234A / L235A mutation (also known as a LALA mutation) that reduces binding to Fcγ receptors. The Fc fragment fused to the IL-12 mutant protein is a human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG4 Fc. In some embodiments, the Fc fragment comprises the amino acid sequence SEQ ID No. 5 or has at least 90% identity thereto, for example, 95%, 96%, 97%, 99% or higher identity. In some embodiments, the IL-12 mutant protein is fused to the Fc via a linker, and the linker is (G4S) n , preferably (G4S)2.

[0123] In another aspect, the present invention further provides a dimeric molecule comprising an IL-12 mutein of the present invention fused to an Fc fragment. This molecule is recycled through FcRn-mediated in vivo circulation, extending the half-life of the IL12-Fc fusion protein. Compared to a corresponding dimeric molecule comprising a wild-type IL12-Fc fusion protein, the dimeric molecule exhibits one or more of the following properties:

[0124] a) Reduce or avoid excessive lymphocyte activation and / or inflammatory factor release caused by IL-12;

[0125] b) Better pharmacokinetic properties, such as extended half-life in vivo;

[0126] c) increased expression level and / or purity when expressed in mammalian cells (e.g., CHO or HEK293 cells);

[0127] Preferably, the IL-12 mutein dimer of the present invention exhibits 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 tumor-bearing animals as described in the Examples. Tolerability can be determined by measuring body weight and weight change in animal models after administration as described in the Examples.

[0128] In some embodiments, the present invention provides an IL12-Fc protein that is a heterodimer, such as Format 1, comprising:

[0129] a) a first monomer, wherein the first monomer comprises, from N-terminus to C-terminus: 1) an IL-12P40 subunit or a mutant protein thereof; 2) a linker; 3) a first Fc fragment (also referred to as Fc1); the linker is preferably (G4S)n, where n can be a natural number such as 0, 1, 2, 3, 4, or 5; preferably, n=0;

[0130] b) a second monomer, wherein the second monomer comprises, from N-terminus to C-terminus: 1) an IL-12P35 subunit or a mutant protein thereof; 2) a linker; 3) a second Fc fragment (also referred to as Fc2); the linker is preferably (G4S)n, where n can be a natural number such as 0, 1, 2, 3, 4, or 5; preferably, n=2.

[0131] In some embodiments, the present invention provides an IL12-Fc protein that is a heterodimer, such as Format 2, comprising:

[0132] a) a first monomer, wherein the first monomer comprises, from N-terminus to C-terminus: 1) an IL-12P40 subunit or a mutant thereof; 2) a linker; 3) an IL-12P35 subunit or a mutant thereof; 4) a linker; and 5) a first Fc fragment. 1) and 3) may be interchangeable; the linker is preferably (G4S)n, where n can be a natural number such as 0, 1, 2, 3, 4, or 5; preferably, n=3;

[0133] b) a second monomer, wherein the second monomer comprises a second Fc fragment and optionally an IgG hinge region.

[0134] In the aforementioned heterodimer form, in some embodiments, the first Fc fragment and the second Fc fragment respectively comprise first and second heterodimer mutations that promote heterodimer formation between the first monomer and the second monomer. In some preferred embodiments, the first and second heterodimer mutations comprise a combination of knob-into-hole mutations, such as T366W / S354C:Y349C / T366S / L368A / Y407V.

[0135] In some preferred embodiments, the first heterodimer mutation on the first Fc fragment comprises a Knob mutation, and the second heterodimer mutation on 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 on the second Fc fragment comprises a Knob mutation.

[0136] As will be appreciated by those skilled in the art, the Fc fragments suitable for use in the fusion proteins and dimer molecules of the present invention may be mutations known in the art that promote heterodimer formation.

[0137] As will be appreciated by those skilled in the art, the linker suitable for linking the IL-12 mutant protein and the Fc fragment in the fusion protein and dimer molecule of the present invention can be any linker known in the art.

[0138] 3. Polynucleotides, vectors, and hosts

[0139] The present invention provides nucleic acids encoding any of the above-described IL-12 muteins, fusion proteins, or dimer molecules. Polynucleotide sequences encoding the muteins of the present invention can be generated by de novo DNA synthesis or by PCR mutagenesis of existing sequences encoding wild-type IL-12 using methods well known in the art. Furthermore, the polynucleotides and nucleic acids of the present invention may include a segment encoding a secretory signal peptide operably linked to the segment encoding the mutein of the present invention, thereby directing secretory expression of the mutein of the present invention.

[0140] The present invention also provides vectors comprising the nucleic acids of the present invention. In some embodiments, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, and phages. In a preferred embodiment, the expression vector of the present invention is pcDNA3.4.

[0141] The present invention also provides host cells comprising the nucleic acid or the vector. Host cells suitable for replicating and supporting the expression of mutant IL-12 proteins, fusion proteins, or dimers are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and large quantities of vector-containing cells can be grown for inoculating large-scale fermenters, thereby obtaining sufficient quantities of IL-12 mutants, fusion proteins, or dimers for clinical applications. In some embodiments, the host cell is eukaryotic and is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells).

[0142] 4. Preparation method

[0143] The present invention provides a method for preparing an IL-12 mutant or fusion protein or dimer of the present invention, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the protein or fusion protein or dimer under conditions suitable for expression of the IL-12 mutant or fusion protein or dimer, as described above, and optionally recovering the protein from the host cell.

[0144] 5. Detection method

[0145] The IL12 muteins of the present invention can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0146] In one aspect, the IL-12 muteins of the present invention can be tested for their binding activity to the IL-12 receptor. For example, the binding of IL-12 to IL-12Rβ1 or IL-12Rβ2 can be determined by methods known in the art, such as ELISA, Western blotting, or the exemplary methods disclosed in the Examples herein.

[0147] In yet another aspect, the ability of an IL-12 mutein to bind to the IL-12 receptor can be measured indirectly by measuring the signaling and / or immune activation effects that occur downstream of receptor binding.

[0148] Therefore, in some embodiments, an assay method for identifying an IL-12 mutant protein or dimer molecule having biological activity is provided. Biological activity may include, for example, the ability to induce signal transduction of T cells and / or NK cells having an IL-12 receptor, the ability to induce IFN-γ secretion by T cells and / or NK cells having an IL-12 receptor, the level of STAT4 phosphorylation downstream of JAK and TYK2 caused by interaction of IL-12 with the receptor, the ability to induce tumor regression and / or improve survival, and reduced in vivo toxicity properties.

[0149] 6. IL-12 protein modification method

[0150] In one aspect, the present invention provides a method for obtaining IL-12 muteins with improved properties and the IL-12 muteins obtained by the method.

[0151] In some embodiments, the method of the present invention comprises the steps of:

[0152] a) optionally introducing one or more mutations in the IL-12 and IL-12R binding interface.

[0153] b) expressing the IL-12 mutein in mammalian cells (such as HEK293 or CHO cells), for example as an Fc fusion protein.

[0154] In the above embodiments, preferably, the IL12 and IL12Rβ1 binding interface mutations include the mutations described above;

[0155] Preferably, the mutant protein has the following improved properties: (1) improved expression level and / or protein purity; and optionally (2) weakened IL12β1 binding.

[0156] 7. Treatment methods and uses

[0157] As used herein, the terms "individual" or "subject" are used interchangeably to refer to mammals. 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).

[0158] In one aspect, the present invention provides a method for stimulating the immune system of a subject, comprising administering to the subject an effective amount of a molecule comprising an IL-12 mutein, fusion protein, or dimer of the present invention.

[0159] In one aspect, the present invention relates to a method for treating a disease, such as cancer, in a subject, comprising administering to the subject an effective amount of a molecule comprising an IL-12 mutein, fusion protein, or dimer of the present invention. The cancer can be in the early, middle, or late stages of metastatic cancer. In some embodiments, the cancer can be, for example, a gastrointestinal cancer.

[0160] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0161] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0162] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0163] Detailed information of some proteins and subunits in the present invention is shown in Table 2 below.

[0164] Table 2. Detailed description of some proteins and subunits of the present invention

[0165]

[0166] Example 1. Design of IL-12 mutants

[0167] 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 1 Middle A) and computer simulation of the binding interface between IL-12 and IL-12Rβ2 ( Figure 1 The interaction sites are listed in Figure B. Wild-type IL-12 was used as a mutation template (wild-type IL-12, also referred to as IL12 in this article). WT ) designed and obtained IL-12 mutants. Wild-type IL-12 has two subunits, P40 and P35. The amino acid sequence of the wild-type P40 subunit is SEQ ID No. 3, with a C274S mutation introduced at position 274 of SEQ ID No. 3 to prevent the formation of disulfide-bridged IL-12 dimers. The amino acid sequence of the wild-type P35 subunit is SEQ ID No. 4, with a C96S mutation introduced at position 96 of SEQ ID No. 4 to prevent the formation of disulfide-bridged IL-12 dimers.

[0168] Example 2. Expression and purification of IL12 mutant fusion protein

[0169] 1. Construction of IL12 mutant fusion protein expression vector

[0170] In this example, Fc was used 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, and the corresponding Fc sequences are 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, such as Figure 2 shown.

[0171] Constructing wild-type IL12 (IL12 WT ) was used as a control, and the C-terminus of the wild-type IL12 P40 sequence was connected to a 6-histidine tag (His×6). The corresponding nucleotide sequence was synthesized, as shown in SEQ ID No. 35, and cloned into the pcDNA3.4 vector pcDNA TM 3.4TOPO TM The pcDNA3.4-p40-His vector was generated between the XbaI and HindIII sites of the vector (Invitrogen, Cat: A14697, hereinafter referred to as pcDNA3.4). A nucleotide sequence encoding the p35 subunit of wild-type IL-12, as shown in SEQ ID No. 36, was synthesized and cloned into the pcDNA3.4 vector between the XbaI and HindIII sites to generate the pcDNA3.4-p35 vector.

[0172] Construction of Format 1 wild-type IL12-Fc fusion protein (IL12 WT -Fc Format 1) was used as a control, in which the first monomer connection mode was P40-Fc1 and the second monomer connection mode was P35-(G4S)2-Fc2. The construction method was as follows: the gene sequence of the synthetic wild-type IL12 p40 subunit was connected to Fc1 and cloned into the pcDNA3.4 vector to obtain the pcDNA3.4-p40-Fc1 vector; the gene sequence of the synthetic wild-type IL12 p35 subunit was connected to Fc2 via (G4S)2 and cloned into the pcDNA3.4 vector to obtain the pcDNA3.4-p35-Fc2 vector.

[0173] Construction of Format 2 wild-type IL12-Fc fusion protein (IL12 WT-Fc Format 2) was used as a control. The first monomer connection mode was P40-(G4S)3-P35-(G4S)3-Fc1, and the second monomer Fc2 was constructed as follows: the gene sequence of the synthetic wild-type IL12 p40 subunit was connected to the gene sequence of the p35 subunit via (G4S)3, and then connected to Fc1 via (G4S)3, and cloned into the pcDNA3.4 vector to obtain the pcDNA3.4-IL12-Fc1 vector; the Fc2 gene sequence was directly constructed into the pcDNA TM 3.4TOPO TM The recombinant vector pcDNA3.4-Fc2 was obtained.

[0174] IL12 mutant -Fc fusion protein vector is constructed as follows:

[0175] A. IL12 mutant -Fc fusion protein HK54046 is taken as an example, HK54046 is IL12 in Format 1 structure mutant -Fc fusion protein, wherein the first monomer connection mode is mutant P40-Fc1, and the second monomer connection mode is mutant P35-(G4S)2-Fc2. The specific construction method of fusion protein HK54046 is as follows:

[0176] (1) Primers were designed at the nucleotide sequences corresponding to amino acids 81 and 106 of the p40 subunit of wild-type IL12, respectively, to mutate E at position 81 to A and K at position 106 to E in SEQ ID No. 3, and then the p40 nucleotide subunit sequence of the IL12 mutant was ligated with Fc1 to obtain a DNA sequence of SEQ ID No. 37, wherein positions 1-984 of SEQ ID No. 37 are the nucleotide sequence of the mutant subunit p40. mutant， The nucleotide sequence of Fc1 is shown in Figure 1. The nucleotide sequence is cloned into the expression vector pcDNA3. TM 3.4TOPO TM The recombinant vector pcDNA3.4-IL12 (P40) corresponding to P40 was obtained between the XbaI and HindIII recognition sites of the vector. mutant -Fc1-HK54046;

[0177] (2) The nucleotide sequence of the P35 subunit of the IL12 mutant was connected to Fc2 via a linker (G4S)2 to obtain a DNA sequence of SEQ ID No. 38, wherein positions 1-657 of SEQ ID No. 38 are the mutant subunit P35 mutant，The nucleotide sequence of the nucleotide sequence of Fc2 is constructed into pcDNA3. TM 3.4TOPO TM The recombinant vector pcDNA3.4-IL12 (P35) corresponding to P35 was obtained between the XbaI and HindIII recognition sites of the vector. mutant -Fc2-HK54046.

[0178] IL12 mutant -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.

[0179] Table 3. List of IL12 mutant fusion proteins and their corresponding IL12 mutant mutation sites

[0180]

[0181] Note: The "Mutation Annotation" column in the table describes the mutation information of the corresponding sites in the amino acid sequences of the P40 subunit and P35 subunit of the mutant IL-12 through "SEQ ID No.3 and "Mutation Site", or "SEQ ID No.4 and "Mutation Site".

[0182] B.IL12 mutant -Fc fusion protein HK54065 is taken as an example, HK54065 is Format 2 structure of IL12 mutant -Fc fusion protein, wherein the first monomer is connected in a manner of mutant P40-(G4S)3-P35-(G4S)3-Fc1, and the second monomer is Fc2. The specific method for constructing the fusion protein HK54065 is as follows:

[0183] (1) Primers were designed at the nucleotide sequences corresponding to amino acids 37 and 219 of the p40 subunit of wild-type IL12, respectively, to mutate W at position 37 of SEQ ID No. 3 to K and K at position 219 to E. The p40 subunit sequence of the IL12 mutant was then linked to the p35 subunit nucleotide sequence via a linker (G4S) 3, and then to Fc1 via a linker (G4S) 3, to obtain a DNA sequence of SEQ ID No. 39, wherein positions 1-984 of SEQ ID No. 39 are the mutant subunit p40. mutant The nucleotide sequence of the ... mutant The nucleotide sequence of SEQ ID No. 39 is constructed into pcDNA TM 3.4TOPO TM The recombinant vector pcDNA3.4-IL12 was obtained between the XbaI and HindIII recognition sites of the vector. mutant -Fc1-HK54065;

[0184] (2) The signal peptide sequence MEFGLSWVFLVAILKGVQC (SEQ ID No. 46) was added to the N-terminus of Fc2, and the corresponding nucleotide sequence was synthesized to obtain a DNA sequence with SEQ ID No. 40, which was directly constructed into pcDNA TM 3.4TOPO TM The recombinant vector pcDNA3.4-Fc2-HK54065 was obtained between the XbaI and HindIII recognition sites of the vector.

[0185] IL12 mutant -Fc fusion proteins HK54032 and HK54033 were constructed according to the above construction method, and the corresponding mutation sites and amino acid sequences are shown in Table 3.

[0186] Two IL12s were designed mutant -Fc fusion protein structures, such as Figure 2 As shown in Format 1 and Format 2, Format 1 and Format 2 are heterodimers.

[0187] 2. IL12 mutant Expression and purification of α-Fc fusion proteins

[0188] The recombinant vector containing the P40 and P35 subunit genes encoding the fusion protein (e.g., the HK54046 fusion protein vector in Format 1 is pcDNA3.4-IL12(P40)) was transfected into the IL12 cell line using chemical transfection. mutant -Fc1 and pcDNA3.4-IL12(P35) mutant -Fc2; Format 2 HK54065 fusion protein vector is pcDNA3.4-IL12 mutant pcDNA3.4-Fc1 and pcDNA3.4-Fc2) were transferred into ExpiCHO-S cells (Gibco, Catalog No. A29127) at a 1:1 mass ratio and cultured at 37°C, 6% CO2 for 7 days. The antibody protein was purified from the culture supernatant using a Protein A affinity chromatography column. First, PBS was used to balance the Protein A column (GE), and then the culture supernatant was passed through the column. First, 5 column volumes of solution A (formula: solvent is water, solute and concentration are: 20mM sodium phosphate, 500mM NaCl, pH5.0) were used for pre-elution, and then 5 column volumes of solution B (formula: solvent is water, solute and concentration are: 20mM sodium acetate, 150mM NaCl, pH3.5) were used for elution. The affinity elution peak was collected and further purified by gel filtration chromatography. After the gel filtration chromatography column (GE, SUPERDEX 200Pg) was balanced with PBS, the affinity collection solution was loaded onto the column. After the target protein peak appeared, the gel filtration elution peak was collected, and then concentrated in a 30KDa concentrating centrifuge tube to obtain the above-mentioned IL12. mutant -Fc fusion protein molecules, SDS-PAGE identification protein electrophoresis bands such as Figure 3 shown.

[0189] Depend on Figure 3 It can be seen that there are two forms of IL12, Format 1 and Format 2 mutant -Fc fusion proteins can be correctly expressed and produced. The SDS-PAGE electrophoresis results show that both the reduced and non-reduced molecular weights are consistent with the theoretical size. In addition, the non-reduced electrophoresis results show that the purity of the fusion protein is greater than 90%, which meets the requirements of subsequent testing. That is, the IL12 fusion protein designed by the present invention has good manufacturability.

[0190] Example 3, IL12 mutant -Fc fusion protein reporter gene functional activity

[0191] IL12 was detected using IL12 Reporter 293 cell (Jiman Bio, Cat. No. GM-C19224). mutant-Fc fusion protein reporter gene activity. This reporter gene cell line is a luciferase reporter gene cell line constructed 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, ultimately leading to the phosphorylation and dimerization of STATs, thereby activating the expression of luciferase. The luciferase reading represents the activation of the signaling pathway and can be used to evaluate the signaling pathway activation activity of IL12.

[0192] IL12 Reporter 293 cells (1.5×10 4 Cells were seeded into 96-well cell culture plates (100 cells / well) and serially diluted samples of HK54029, HK54030, HK54032, HK54033, HK54037, HK54040, HK54042, HK54043, HK54044, HK54045, HK54046, HK54051, HK54052, HK54053, HK54054, HK54064, HK54065, HK54071, HK54073, and HK54074 were added. Wild-type IL12 was used as a control. After incubation at 37°C in a CO2 incubator overnight, the colorimetric ONE-Glo™ Luciferase Assay System reagent (Meilunbio, Catalog No. MA0519-2) was added and incubated for 10 min. The chemiluminescence value was detected.

[0193] The results are as follows Figure 4 (A) and (B) show: 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. Adding Fc fragments will not reduce the activity of IL12. The two forms of IL12-Fc fusion protein IL12 in the heterodimer Format 1 and Format 2 WT -Fc (Format 1) and IL12 WT There was no difference in the activity of -Fc (Format 2), so wild-type IL12 or a wild-type IL12-Fc fusion protein of a certain structure was selected as a control in subsequent experiments.

[0194] Experimental results show that IL12 mutant -Fc fusion protein and wild-type IL12 control can activate reporter gene expression signal. mutant-Fc fusion protein reduces the binding ability of IL12 to IL12R, and its activation ability is also reduced. Compared with wild-type IL12, the degree of reduction in activation activity of different mutations varies, ranging from 1 to 100 times or more. Figure 4 As shown in (A), (B), (C) and (D).

[0195] Example 4, IL12 mutant Verification of the lymphocyte activation function of -Fc fusion protein in vitro

[0196] Sample to be tested: IL12 mutant -Fc fusion proteins HK54037, HK54046, HK54052, HK54053, HK54053, HK54064, HK54065, HK54066, HK54067, HK54068, HK54075, HK54077, HK54078.

[0197] Anti-CD3 antibody (Bio-Ying Biotechnology, catalog number B6928) was diluted to 0.5 μg / ml with PBS and added to a 96-well plate (Corning) at 60 μl / well. The 96-well plate was incubated at 37° C. for 1 hour.

[0198] 1. Human CD8 + T cell activation

[0199] CD8 was isolated from PBMC (Shanghai Heyousheng Biotechnology Co., Ltd., catalog number HPB050C). + T cells were isolated and purified using IIMag™ Human CD8 T Lymphocyte Enrichment Set-DM magnetic beads (BD Biosciences, Cat. No. 557941) according to the instructions. + T cells, spare.

[0200] In a 96-well cell culture plate, 0.5ug / ml anti-CD3 was coated for 2h, and then 100μl human CD8 + T cells, 50,000 cells / well, and then the test samples HK54037, HK54053, and HK54064 were diluted with culture medium to the target concentration and added to a 96-well plate, 100 μl / well, with 3 replicates for each concentration. The 96-well plate was placed in a cell culture incubator and incubated for 3 days. The secretion of cytokine IFN-γ in the cell culture supernatant was detected by ELISA. Figure 5 As shown in (A).

[0201] Experimental results show that IL12 mutant-Fc fusion protein and wild-type IL12 fusion protein can activate CD8 + T cells, induced CD8 + T cells secrete IFN-γ. Compared with wild-type IL12 fusion protein, IL12 mutant -Fc fusion protein induced CD8+ T cells to secrete IFN-γ curve EC50 (half effective concentration) increased, the platform on the curve decreased, indicating that IL12 mutant -Fc fusion protein reduces activation of CD8 compared to wild-type IL12 fusion protein + The ability of T cells, HK54037, HK54053, and HK54064 have different mutations, and the degree of reduction in activation activity is also different, with a reduction of 1-100 times or more, achieving the expected goal of the mutation.

[0202] 2. Human PBMC cell activation

[0203] In a 96-well cell culture plate, the plate was pre-coated with 0.5ug / ml anti-CD3, washed with PBS after 2 hours, and then 100μl human PBMC (Shanghai Heyousheng Biotechnology Co., Ltd.) was plated, 20,000 cells / well; then the diluted sample to be tested was added, 100ul per well, and 3 replicates were set for each concentration. The 96-well plate was placed in a cell culture incubator and incubated for 3 days. The secretion of cytokine IFN-γ in the cell culture supernatant was detected by ELISA, as shown in the following figure. Figure 5 As shown in (B) and (C).

[0204] Experimental results show that IL12 mutant -Fc fusion protein and wild-type IL12 can activate PBMC cells and induce PBMC cells to secrete IFN-γ. mutant -Fc fusion protein induction requires a higher concentration of sample to induce PBMC to secrete IFN-γ, and the maximum value of PBMC secretion of IFN-γ is also reduced, indicating that compared with wild-type IL12, IL12 mutant -Fc fusion protein reduced the ability to activate PBMC cells. The degree of reduction in activation activity varied among different mutations, ranging from 1-100 times or more, achieving the expected mutation target.

[0205] 3. NK cell activation

[0206] NK cells were isolated from PBMC using Human NK Cell Enrichment Set-DM magnetic bead sorting (BD Biosciences, Catalog No. 2257429) and resuspended in culture medium with a final concentration of 0.5 ng / mL Recombinant Human IL-2 (Peprotech, Catalog No. 200-02-100). The cells were then added to a 384-well plate at 10,000 cells / well, 40 μl per well. The test sample was added at 40 μl per well, and the 384-well plate was placed in a cell culture incubator and incubated for 2 days. The secretion of the cytokine IFN-γ in the cell culture supernatant was detected by ELISA, as shown in the following example. Figure 6 shown.

[0207] Experiments have shown that compared with wild-type IL12, IL12 mutant -Fc fusion protein significantly reduces the level of IFN-γ secreted by NK cells when activated, and a higher concentration than wild-type IL12 is required to reach the level of wild-type IL12 activating NK cells. Because the drug toxicity side effects caused by activating peripheral blood NK cells are large, reducing the level of IL12 mutants activating NK cells is a strategy to improve its safety. mutant -Fc fusion proteins HK54037, HK54051, HK54052, HK54053, HK54046, and HK54064 all reduce the activation ability of NK cells and are safer than wild-type IL12.

[0208] Example 5, IL12 mutant Study on STAT4 phosphorylation activity of -Fc fusion protein

[0209] Sample to be tested: IL12 mutant -Fc fusion proteins K54037, HK54046, HK54052, HK54053, HK54064.

[0210] PBMC (Shanghai Heyousheng Biotechnology Co., Ltd., Catalog No. HPB050C) were stimulated with Phytohemagglutinin-L (PHA-L) Solution (500×) (Invitrogen, 2514949) for 24 h. After washing, PBMC were stimulated with 1000 IU Recombinant Human IL-2 (PEPROTECH, Catalog No. 200-02-100) for 1 day and rested overnight. The diluted test samples were added to PBMC (3×10 5The concentrations of the test samples HK54037, HK54046, HK54052, HK54053, and HK54064 were 1000, 200, 40, 8, 1.6, 0.32, and 0.064 nM, respectively. The concentrations of the control wild-type IL12 were 3, 0.6, 0.12, 0.024, 0.0048, 0.00096, and 0.000048 nM, respectively. The cells were incubated at 37°C for 30 min, the supernatant was collected by centrifugation, and 100 μl of Phosflow TM Fix Buffer I (BD Biosciences, Catalog No. 557870) and fix the cells at 37°C for 15 minutes. Wash twice with staining buffer (PBS + 3% FBS) and resuspend the cells in -20°C pre-cooled permeabilization buffer Phosflow PermBuffer III (BD Biosciences, Catalog No. 558050). Incubate on ice for 30-45 minutes and wash twice with PBS + 3% FBS. Then, add FITC Mouse Anti-Human CD8 (BD) and PE Mouse Anti-Human PSTAT4 (PY693) (BD Biosciences, Catalog No. 558249). Incubate at room temperature for 1 hour and wash twice with PBS before detection.

[0211] Experimental results show that IL12 mutant -Fc fusion protein and wild-type IL12 can activate the Jak-STAT signaling pathway downstream of T cell IL12R to activate pSTAT4. Compared with wild-type IL12 fusion protein, IL12 mutant -Fc fusion protein activated T cells mediated by pSTAT4 fluorescence curve EC50 (half effective concentration) increased, the curve platform decreased, which means that IL12 mutant Compared with wild-type IL12 fusion protein, -Fc fusion protein has reduced ability to activate T cells. HK54037, HK54046, HK54052, HK54053, and HK54064 have different mutations, and the degree of reduced activation activity is also different, ranging from 1-100 times or more.

[0212] Example 6, IL12 mutant Study on the anti-tumor activity of -Fc fusion protein in vivo

[0213] Sample to be tested: IL12 mutant -Fc fusion proteins mHK54037, mHK54046, mHK54052, mHK54053, and mHK54064.

[0214] Mouse IL12 was used to construct a mutant molecule to replace human IL12, with wild-type mouse IL12 as a control. The amino acid sequence of the P40 subunit of wild-type mouse IL12 is SEQ ID No. 33, and the amino acid sequence of the P35 subunit of 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.

[0215] Table 4. Corresponding list of human and mouse IL12 mutant fusion proteins and their sequences

[0216] Name of human IL12 mutant Name of mouse IL12 mutant Mouse P40 subunit SEQ ID NO: Mouse P35 subunit SEQ ID NO: HK54037 mHK54037 41 34 HK54052 mHK54052 43 34 HK54053 mHK54053 44 34 HK54046 mHK54046 42 34 HK54064 mHK54064 45 34

[0217] 1. IL12 mutant Anti-tumor effect of -Fc fusion protein on mouse colon cancer cell MC38

[0218] Mouse colon cancer cell MC38 cells (Nanjing Kebai Biotechnology Co., Ltd., Catalog No. CBP60825) were inoculated into C57BL / 6 mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., Catalog No. 213), and CT26 cells (Nanjing Kebai Biotechnology Co., Ltd., Catalog No. CBP61189) were inoculated into Balb / c mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., Catalog No. 211) to evaluate the anti-tumor activity of the mutant molecules.

[0219] MC38 cells were cultured to 80% confluence, digested with trypsin, and centrifuged at 1000 rpm for 5 min. The cells were collected, washed, centrifuged, and resuspended to obtain a cell suspension with a cell viability of >95% for later use. MC38 cells were subcutaneously inoculated on one side of the back (shaved) of the test mice (5×10 cells per mouse). 5 When the average tumor volume of tumor-bearing mice reached 100 mm 3 Around 6:00 p.m., mice were randomly divided into groups of 5 according to the experimental design. A saline group (Vehicle) served as a negative control group, receiving no medication. Wild-type IL12 and mHK54037, mHK54052, and mHK54053 were diluted in saline at the desired concentration and administered intraperitoneally twice weekly. Following tumor inoculation, animal survival and activity were monitored twice weekly, including tumor growth, activity, diet, body weight, and any abnormal behaviors.

[0220] Specific results can be found in Figure 8 Shown: By Figure 8 As shown in (A), compared with the saline group (referred to as Vehicle), mHK54037, mHK54052, and mHK54053 can significantly inhibit tumor growth. Figure 8As shown in (B), wild-type IL12 can cause a significant decrease in the body weight of mice (a decrease of more than 20%), and even cause the death of mice, while mIL12 mutant The body weights of mice in the -Fc fusion protein administration group and the normal saline control group were the same, indicating that mIL12 mutant -Fc fusion proteins can inhibit the growth of MC38 tumors without affecting the activity and body weight of mice, and are safer than wild-type IL12.

[0221] 2. IL12 mutant Anti-tumor effect of -Fc fusion protein on mouse colon cancer cell CT26

[0222] Mouse colon cancer cells (CT26 cells, ATCC) were cultured to 80% confluence, digested with trypsin, and centrifuged at 1000 rpm for 5 min. The cells were collected, washed, centrifuged, and resuspended to obtain a cell suspension with a cell viability of >95% for later use. CT26 cells were subcutaneously inoculated on one side of the back (shaved) of the test mice (2×10 cells per mouse). 5 When the average tumor volume of tumor-bearing mice reached 100 mm 3 Around 60 days, mice were randomly divided into groups of 5 according to the experimental design. A saline group (Vehicle) served as a negative control group without medication. Wild-type mIL12 and mHK54037, mHK54046, mHK54052, and mHK54064 were diluted in saline according to the required concentration and administered intraperitoneally once a week. After tumor inoculation, animal survival and activity were monitored twice a week, including tumor growth, activity, diet, body weight, and other abnormal behaviors.

[0223] Specific results such as Figure 9 Compared with the saline group (Vehicle), mIL12 mutant -Fc fusion proteins mHK54037, mHK54046, mHK54052, and mHK54064 can all significantly inhibit the growth of CT26 tumors. At the same time, increasing the dosage of mHK064 can achieve the same efficacy results as mHK54037, mHK54046, and mHK54052.

[0224] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

[0225] Industrial Applicability

[0226] The present invention provides a novel interleukin 12 (IL-12, IL12) mutant protein, which has improved IL12 receptor binding properties and drug-forming IL12 mutant proteins compared to wild-type IL12, and can be applied to the field of anti-tumor treatment. By weakening the binding ability to the IL12 receptor, excessive lymphocyte activation and inflammatory factor release can be avoided, the systemic inflammatory response caused by IL12 can be reduced, and the adverse reactions of patients during treatment can be reduced, thereby improving the tolerance and safety of treatment. The present invention prolongs the half-life of IL12 in the body and enhances the stability of the molecule in the form of a fusion protein, so that the IL12 fusion protein can continue to exert an anti-tumor effect at a lower dose, reducing the frequency of administration, thereby further reducing the systemic inflammatory response during treatment and improving patient compliance. The design of the fusion protein not only contributes to the stability of the protein, but also improves the efficiency of expression and purification, making IL12 suitable for large-scale production and commercialization.

[0227] Weakened IL12 molecules can be used alone for anti-tumor treatment or in combination with other immunotherapies (such as immune checkpoint inhibitors and CAR-T cell therapy) to enhance anti-tumor efficacy. Weakened IL12 molecules can also be used in gene therapy. By introducing the IL-12 gene into local tumors or specific immune cells, sustained and localized IL12 expression can be achieved, enhancing anti-tumor immune responses while reducing systemic toxicity.

[0228] In general, the IL12 mutant protein and fusion protein provided by the present invention provide a powerful tool for tumor immunotherapy. By reducing toxicity and enhancing tumor targeting, they can significantly enhance therapeutic efficacy and improve patient prognosis. They can also be personalized according to the patient's tumor type and individual differences, providing more options for clinical treatment.

[0229] Sequence Listing

[0230] SEQ ID No.1

[0231] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0232] SEQ ID No.2

[0233] MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPPDFYKTKIKLCILLHAFRIVTIDRVMSYLNAS

[0234] SEQ ID No. 3

[0235] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0236] SEQ ID No.4

[0237] MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESSLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPPDFYKTKIKLCILLHAFRIVTIDRVMSYLNAS

[0238] SEQ ID No.5

[0239] EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0240] SEQ ID No. 6

[0241] EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0242] SEQ ID No.7

[0243] EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0244] SEQ ID No.8

[0245] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFT DKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0246] SEQ ID No. 9

[0247] RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESSLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSEPKSSDKTHTCP PCPAPEAAGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK

[0248] SEQ ID No.10

[0249] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATAVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSGNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVE ACLPLELTKNESSLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSEPKSSDKTHTCPPPAPEAAGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0250] SEQ ID No.11

[0251] RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESSLNSRETSFITNGSCLASRKTSFMMACLSSIYEDLKMYQVEFKTMNAKLLMDPKEQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

[0252] SEQ ID No.12

[0253] RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESSLNSRETSFITNGSCLASRKTSFMMACLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYETKIKLCILLHAFRIRAVTIDRVMSYLNAS

[0254] SEQ ID No.13

[0255] RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHSDITKDKTSTVEACLPLELTKNESSLNSRETSFITNGSCLASRKTSFMMACLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

[0256] SEQ ID No.14

[0257] IWELKKDVYVVELDAYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEAGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0258] SEQ ID No.15

[0259] IWELKKDVYVVELDAYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEAGDAGQYTCHKGGEVLSHSLLLLHKKADGIWSTILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0260] SEQ ID No.16

[0261] IWELKKDVYVVELDKYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0262] SEQ ID No.17

[0263] IWELKKDVYVVELDEYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKAAGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0264] SEQ ID No.18

[0265] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKKFGDAGQYTCHKGGEVLSHSLLLLHSKEDGIWSTILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0266] SEQ ID No.19

[0267] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKKFGDAGQYTCHKGGEVLSHSLLLLHNKEDGIWSTILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0268] SEQ ID No.20

[0269] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKKFGDAGQYTCHKGGEVLSHSLLLLHEKEDGIWSTILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0270] SEQ ID No.21

[0271] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKKFGDAGQYTCHKGGEVLSHSLLLLHQKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0272] SEQ ID No.22

[0273] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKAFGDAGQYTCHKGGEVLSHSLLLLHEKEDGIWSTILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0274] SEQ ID No.23

[0275] IWELKKDVYVVELDWYPPAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHEKEDGIWSTILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLEYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0276] SEQ ID No.24

[0277] IWELKKDVYVVELDKYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKAFGDAGQYTCHKGGEVLSHSLLLLHSKEDGIWSTILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0278] SEQ ID No.25

[0279] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKAAGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLEYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0280] SEQ ID No.26

[0281] IWELKKDVYVVELDKYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLEYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0282] SEQ ID No.27

[0283] IWELKKDVYVVELDKYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKAFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLEYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0284] SEQ ID No.28

[0285] IWELKKDVYVVELKKKPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0286] SEQ ID No.29

[0287] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKKFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTKILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLEYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0288] SEQ ID No.30

[0289] IWELKKDVYVVELDKYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHSKEDGIWSTILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLEYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0290] SEQ ID No.31

[0291] IWELKKDVYVVELDNYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSAPILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0292] SEQ ID No.32

[0293] IWELKKDVYVVELDTYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSAPILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0294] SEQ ID No.33

[0295] MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTSPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS

[0296] SEQ ID No.34

[0297] RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESSLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA

[0298] SEQ ID No.35

[0299]

[0300] SEQ ID No.36

[0301] ATGTGCCCAGCCAGATCTCTGCTGCTCGTGGCTACACTGGTGCTGCTGGATCACCTGAGCCTGGCCAGAAATCTGCCAGTGGCTACCCCAGACCCAGGAATGTTCCCTTGCCTGCACCACAGCCAGAACCTGCTGAGAGCCGTGTCCAACATGCTGCAGAAGGCCAGACAGACCCTGGAGTTCTACCCTTGCACCAGCGAGGAGATCGACCACGAGGACATCACCAAGGACAAGACCTCTACCGTTGAGGCTTGCCTGCCTCTGGAGCTGACCAAGAACGAGTCTAGCCTGAACAGCCGGGAGACCAGCTTCATCACCAACGGCTCTTGCCTGGCCAGCAGGAAGACCAGCTTCATGATGGCCCTGTGTCTGAGCAGCATCTACGAGGACCTGAAGATGTACCAGGTGGAGTTCAAGACCATGAACGCCAAGCTGCTGATGGACCCCAAGCGGCAGATCTTCCTGGACCAGAACATGCTGGCCGTGATCGACGAACTGATGCAGGCTCTGAACTTCAACAGCGAGACCGTTCCTCAGAAAAGCAGCCTGGAGGAGCCCGACTTCTACAAGACCAAGATCAAGCTCTGCATCCTGCTGCACGCCTTCAGGATCAGAGCCGTGACCATCGACCGCGTGATGAGCTATCTGAACGCCAGCTGA

[0302] SEQ ID No.37

[0303]

[0304] SEQ ID No.38

[0305]

[0306] SEQ ID No.39

[0307]

[0308] SEQ ID No.40

[0309] ATGGAGTTCGGCCTGTCTTGGGTGTTCCTGGTGGCTATCCTGAAGGGTGTACAGTGCGAACCTAAGAGCAGCGACAAGACCCACACTTGTCCTCCATGTCCAGCCCCTGAAGCAGCAGGAGGGCCCAGCGTGTTCCTGTTTCCACCCAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCAGAGGTGACATGCGTGGTGGTGGACGTGTCTCACGAGGACCCCGAGGTGAAGTTTAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACAGCACCTATAGAGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCAAGGTGAGCAATAAGGCCCTGCCCGCCCCTATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAGCCACAGGTATGCACACTGCCTCCAAGCCGCGACGAGCTGACCAAGAACCAGGTGTCCCTGAGCTGTGCCGTGAAGGGCTTCTATCCTTCCGATATCGCCGTGGAGTGGGAGTCTAATGGCCAGCCAGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTGATGGCAGCTTCTTTCTGGTGTCTAAGCTGACCGTGGATAAGAGCAGGTGGCAGCAGGGCAACGTGTTTTCCTGCTCTGTGATGCACGAGGCCCTGCACAATCACTATACACAGAAGAGCCTGTCCCTGTCTCCCGGCAAGTGA

[0310] SEQ ID No.41

[0311] MWELEKDVYVVEVDKTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTSPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRRYYNSSCSKWACVPCRVRS

[0312] SEQ ID No.42

[0313] MWELEKDVYVVEVDWTDPAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKAFLDAGQYTCHKGGETLSHSHLLLHEKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTSPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRRYYNSSCSKWACVPCRVRS

[0314] SEQ ID No.43

[0315] MWELEKDVYVVEVDKTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKAFLDAGQYTCHKGGETLSHSHLLLHSKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTSPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRRYYNSSCSKWACVPCRVRS

[0316] SEQ ID No.44

[0317] MWELEKDVYVVEVDWTDPAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKAALDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTSPTAEETLPIELALEARQQNEYENYSTSFFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRRYYNSSCSKWACVPCRVRSEPKSSDKTHTCPPCPAPEAAGGP

[0318] SEQ ID No.45

[0319] MWELEKDVYVVEVDKTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTSPTAEETLPIELALEARQQNEYENYSTSFFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRRYYNSSCSKWACVPCRVRS

[0320] SEQ ID No.46

[0321] MEFGLSWVFLVAILKGVQC

Claims

1. An IL-12 mutant protein, characterized in that The IL-12 mutant protein is a protein obtained by subjecting wild-type IL-12 to amino acid mutations, wherein the wild-type IL-12 includes 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, wherein the mutated P40 subunit is a subunit obtained by subjecting the wild-type P40 subunit to a mutation at least one of the following positions: positions 37, 38, 39, 40, 81, 82, 106, 108, 114, 115, and / or 219, wherein 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 subjecting the wild-type P35 subunit to a mutation at least one of the following positions: positions 72, 151, and / or 190, wherein the mutation eliminates or reduces the binding affinity to IL-12Rβ2; The wild-type IL-12 may be IL-12 derived from a mammal, such as human IL-12.

2. The mutant protein according to claim 1, characterized in that The binding interface between the P40 subunit and 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 mutant protein according to claim 1 or 2, characterized in that The mutations in the binding interface between the P35 subunit and IL-12Rβ2 include one or more mutations selected from the following: E72S; R151E; K190E.

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

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

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

4.

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

13.

8. The mutant protein according to any one of claims 1 to 7, characterized in that Compared with before the attenuation by the introduction of mutations, the binding affinity to IL-12R is reduced, and the protein has at least one or more properties selected from the following: 1) Compared with before weakening, the activation of T cells (such as CD4+ and / or CD8+ T cells) is reduced; 2) Reduced activation of NK cells compared to before weakening; 3) Compared with before attenuation, the activation of IL-12pSTAT4 phosphorylation signaling pathway was reduced; 4) Reduced IL-12 reporter gene signal activation compared to before attenuation; 5) Compared with before weakening, the safety in animals is increased.

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

10. The fusion protein according to claim 9, characterized in that The fusion protein is formed by fusing the IL-12 mutant protein with the Fc antibody fragment.

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

12. An IL12-Fc dimer protein, characterized in that The IL-12 mutant fusion protein according to claim 10 or 11, preferably, the dimer protein has one or more of the following properties compared to the corresponding dimer protein of the wild-type IL12-Fc fusion protein: 1) Reduce or avoid excessive lymphocyte activation and / or release of inflammatory factors caused by IL-12; 2) Lower toxicity when used in vivo; 3) When expressed in mammalian cells (e.g., CHO or HEK293 cells), the expression level and / or purity is increased.

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

14. The IL12-Fc dimer protein of claim 12, which is a heterodimer, comprising: A first monomer, wherein the first monomer comprises from N-terminus to C-terminus: 1) IL-12P35 subunit or a mutant protein thereof; 2) a linker; 3) an IL-12P40 subunit or a mutant protein thereof; 4) a linker; 5) a first Fc fragment, wherein 1) and 2) can be replaced; A second monomer, wherein the second monomer comprises a second Fc fragment and optionally an IgG1 hinge region.

15. The IL12-Fc dimer protein according to claim 13 or 14, wherein the first Fc fragment and the second Fc fragment respectively comprise a first and a second heterodimer mutation that promotes the first monomer to form a heterodimer with the second monomer, and the IL12-Fc dimer protein is any one of the following: 1) The first and second heterodimer mutations comprise a Knob-into-Hole mutation combination; 2) the first heterodimer mutation on the first Fc fragment comprises a Knob mutation, and the second heterodimer mutation on the second Fc fragment comprises a Hole mutation; 3) The first heterodimer mutation on the first Fc fragment comprises a Hole mutation, and the second heterodimer mutation on the second Fc fragment comprises a Knob mutation.

16. An immunoconjugate comprising the IL-12 mutant protein of claims 1 to 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 mutant protein of claims 1 to 7, or the fusion protein of claims 9 to 11, or the IL12-Fc dimer fusion protein of claims 12 to 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 according to claim 19, wherein the host cell is a HEK293 cell or a CHOS cell.

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

23. Use of the IL-12 mutant protein 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 in the preparation of a medicament for stimulating the immune system of a subject.

24. A method for treating a disease in a subject, the method comprising administering to the subject the IL-12 mutant protein 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 for stimulating the immune system of a subject, the method comprising administering to the subject an effective amount of the IL-12 mutant protein 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.

Images