Il12 mutant fusion proteins and uses thereof

Abstract

The application discloses an IL12 mutant fusion protein and application thereof. Specifically disclosed are a fusion protein comprising an antibody binding to human PD1 and an IL12 mutant protein and a preparation method and therapeutic use thereof, wherein the IL12 mutant protein is obtained by mutation and modification of a wild-type P40 subunit. The fusion protein of the application has reduced binding capacity to an IL12 receptor, reduced drug toxicity of IL12, and obviously improved drugability of a cytokine. Meanwhile, through the specificity of the antibody, the fusion protein effectively enhances the targeting of the cytokine, weakens the potential toxicity of the cytokine, can exert the dual functions of the PD1 antibody and the IL12 cytokine, and thus has better anti-tumor potential. The fusion protein of the application can obviously inhibit tumor growth, has high anti-tumor effect, can prolong the half-life, reduce the administration frequency, has high safety, and has good clinical transformation value and broad application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to IL12 mutant fusion protein and its applications. Background Technology

[0002] Interleukin-12 (IL12) is an important cytokine, primarily produced by activated antigen-presenting cells. It is a naturally occurring interleukin produced by dendritic cells, macrophages, neutrophils, and human B lymphoblasts (NC-37) in response to antigen stimulation. IL12 belongs to the interleukin-12 family and induces the proliferation of NK cells, NKT cells, and T cells, enhances cytotoxicity and the expression of cytotoxic mediators, and produces cytokines. For B cells, IL12 enhances the activation and production of TH1-associated immunoglobulins directly or through the action of type 1 cytokines (such as IFN-γ). It plays a crucial role in the regulation of the immune system and in antiviral and antitumor activities; therefore, IL12 is widely studied for its application in immunotherapy and cancer treatment.

[0003] Clinical experience has shown that although IL12 has good anti-tumor effects, excessive stimulation of inflammatory factors by IL12 can cause drug-related side effects. Furthermore, because IL12 has a half-life of only about 5-10 hours in the human body, increasing human exposure through daily dosing can lead to peak plasma concentrations (Cmax), resulting in drug toxicity. Several modifications to the IL12 molecule have been proposed in this field, but currently, no optimized IL12 molecules are on the market; the most advanced are still in clinical development.

[0004] Programmed cell death 1 (PD1, also known as CD279) is a type I transmembrane receptor of approximately 55 kDa, belonging to the CD28 gene family. It is primarily expressed on the surface of immune cells such as T cells, B cells, and myeloid cells. PD1 has two ligands: PDL1 (also known as B7-H1) and PDL2 (also known as B7-DC). PD1 / PDL1 is an important specific immune checkpoint. In the tumor microenvironment, the expression of PD1 molecules on the surface of tumor-specific T cells is upregulated. After binding to PDL1 on the tumor surface, it transmits an inhibitory signal, downregulating T cell activity. Blocking this signaling pathway can activate suppressed T cells, thereby killing tumor cells. Antagonists such as antibodies against PD1 or PDL1 can block the binding of PD-1 on the surface of immune cells to PDL-1 or PDL-2, effectively preventing T cell inhibitory signals, activating immune cells, and enhancing anti-tumor immunity.

[0005] In view of the aforementioned issues related to IL12 immunotherapy, there is a need in the field to further develop new IL12 molecules with optimized properties, especially immunoconjugates with PD1 antibodies, which exhibit improved pharmacodynamic properties and provide patients with more precise, efficient and safe immunotherapy strategies. Summary of the Invention

[0006] The purpose of this invention is to provide an IL12 mutant fusion protein and its use in targeted cancer therapy. The technical problem to be solved is not limited to the described technical subject matter; other technical subjects not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0007] To achieve the above objectives, the present invention first provides a fusion protein comprising an antibody against an immune checkpoint molecule and an IL12 mutant protein.

[0008] The immune checkpoint molecules include, but are not limited to, PD1, PD-L1, B7-H3, TIM-3, CTLA-4, and LAG3.

[0009] Furthermore, the antibodies against immune checkpoint molecules include antibodies that bind to human PD1 (hereinafter referred to as PD1 antibodies).

[0010] The antibodies that bind to human PD1 include anti-PD1 single-domain antibodies and / or anti-PD1 single-chain antibodies.

[0011] Furthermore, the fusion protein of the present invention comprises an antibody that binds to human PD1 and an IL12 mutant protein, wherein the IL12 mutant protein comprises a mutated P40 subunit and a wild-type P35 subunit; the mutated P40 subunit contains a mutation at at least one of the following positions relative to the amino acid sequence of the wild-type P40 subunit: positions 37, 81, 106, and 219.

[0012] The amino acid sequence of the wild-type P40 subunit includes the sequence shown in A1) or A2):

[0013] A1) The first 328th bits of SEQ ID NO:3;

[0014] A2) A sequence obtained by substituting, deleting and / or adding amino acid residues to the amino acid sequence shown in A1), which has more than 80% identity with the sequence and retains the same function; wherein the substituents, deletions and / or additions are not at positions 37, 81, 106 and 219 of the amino acid sequence shown in A1).

[0015] The amino acid sequence of the wild-type P35 subunit includes the sequence shown in B1) or B2):

[0016] B1) SEQ ID NO:4 or bits 23-219 of SEQ ID NO:4;

[0017] B2) A sequence obtained by substituting, deleting and / or adding amino acid residues to the amino acid sequence shown in B1), which has more than 80% identity with the sequence and retains the same function.

[0018] Furthermore, mutations at positions 37, 81, 106, and 219 could be mutations that eliminate or reduce the binding affinity of the wild-type P40 subunit to IL-12Rβ1.

[0019] Furthermore, the mutated P40 subunit contains one or more mutations selected from the following: W37K, E81K, K106E, K219E.

[0020] Furthermore, the mutated P40 subunit contains any one of the following mutations (C1)-C3):

[0021] C1)W37K and K219E;

[0022] C2)W37K;

[0023] C3)E81K and K106E.

[0024] Furthermore, the amino acid sequence of the mutated P40 subunit includes the sequence shown in D1) or D2):

[0025] D1) SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14;

[0026] D2) A sequence obtained by substituting, deleting and / or adding amino acid residues to the amino acid sequence shown in D1), which has more than 80% identity with the sequence and retains the same function; wherein the substituents, deletions and / or additions are not at positions 37, 81, 106 and 219 of the amino acid sequence shown in D1).

[0027] In the IL12 mutant protein, the mutated P40 subunit may be located at the N-terminus or C-terminus of the wild-type P35 subunit. Specifically, the mutated P40 subunit may be located at the N-terminus of the wild-type P35 subunit.

[0028] In the IL12 mutant protein, the mutated P40 subunit and the wild-type P35 subunit can be connected via a linker.

[0029] The linker may be a flexible peptide linker, such as a peptide linker comprising glycine, serine, proline, and / or lysine residues. The peptide linker may consist of 1-40 amino acids.

[0030] The connectors include, but are not limited to: SGGGGS, (GxS)n, (GSSGG)n, (GGSGG)n, (GSGGSG)n, (GSGSGS)n, and (EAAAK)n, and various combinations thereof. Wherein: n can be any integer between 1 and 10; x can be any integer between 1 and 6. Preferably, the connector is (G4S)n. In one or more embodiments of the invention, the connector (connector 1) is (G4S)3.

[0031] The IL12 mutant protein, from N-terminus to C-terminus, can be the mutated P40 subunit, linker 1, and the wild-type P35 subunit, respectively.

[0032] Furthermore, the antibody binding to human PD1 includes a variable region and a constant region. The variable region includes PD1 single-domain antibody 1 (also known as VHH1), PD1 single-domain antibody 2 (also known as VHH2), or PD1 single-chain antibody, or a combination thereof. Both PD1 single-domain antibody 1 and PD1 single-domain antibody 2 bind to different or non-overlapping or partially overlapping epitopes of PD1 with the PD1 single-chain antibody, and / or do not compete for binding to PD1.

[0033] The PD1 single-domain antibody and the PD1 single-chain antibody bind to epitopes that do not completely overlap with the PD1 antigen, meaning that they bind to the PD1 antigen in a non-competitive manner, which can increase the affinity and specificity with PD1.

[0034] Furthermore, the amino acid sequence of the PD1 single-domain antibody 1 (VHH1) comprises SEQ ID NO:9, or an amino acid sequence having more than 80% identity with SEQ ID NO:9;

[0035] The amino acid sequence of the PD1 single-domain antibody 2 (VHH2) contains SEQ ID NO:10, or has more than 80% identity with SEQ ID NO:10.

[0036] The amino acid sequence of the PD1 single-chain antibody contains SEQ ID NO:11, or has more than 80% identity with SEQ ID NO:11.

[0037] The constant region may be selected from the heavy chain constant region of IgG, IgA, IgM, IgD or IgE or its variants, the Kappa(κ) or lambda(λ) type light chain constant region or its variants, and may also be selected from the CH1, Fc and CH3 domains or their variants, but is not limited thereto. Further, the constant region may be selected from the heavy chain constant region, CH1, Fc and CH3 domains or their variants of human IgG subclasses such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2; or may be selected from the heavy chain constant region, CH1, Fc and CH3 domains or their variants of mouse IgG subclasses such as IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgG5 and IgG6.

[0038] Further, the constant region may be an Fc fragment. The Fc fragment may refer to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region, and may be the Fc region of various Ig subtypes and their allotypes, or may contain some mutated Fc. Preferably, the constant region may be selected from the human IgG1 heavy chain Fc fragment, or an Fc variant containing mutations that reduce or eliminate Fc binding to FcγR (e.g., L234A+L235A+P329G). In some embodiments, the Fc fragment may also contain a Knob-into-Hole mutation combination, such as T366W, S354C and Y349C, T366S, L368A, Y407V.

[0039] Further, the constant region includes Fc1 and Fc2. Fc1 is located in the first polypeptide chain of the fusion protein of the present invention and contains L234A+L235A+P329G mutation and Knob mutation (T366W and S354C), and the sequence may be as shown in SEQ ID NO:7; Fc2 is located in the second polypeptide chain of the fusion protein of the present invention and contains L234A+L235A+P329G mutation and Hole mutation (Y349C+T366S+L368A+Y407V), and the sequence may be as shown in SEQ ID NO:8.

[0040] Furthermore, the constant region includes Fc1 and Fc2, wherein the amino acid sequence of Fc1 contains SEQ ID NO:7 and the amino acid sequence of Fc2 contains SEQ ID NO:8.

[0041] The antibody binding to human PD1 comprises a first chain containing Fc1 (referred to as the Fc1 chain) and a second chain containing Fc2 (referred to as the Fc2 chain). The structure of the antibody binding to human PD1 may be any of the following:

[0042] (1) The Fc1 chain, from the N-terminus to the C-terminus, can be the PD1 single-domain antibody 1 or the PD1 single-domain antibody 2, the adapter, the PD1 single-chain antibody, and the Fc1 in sequence; the Fc2 chain, from the N-terminus to the C-terminus, can be the PD1 single-domain antibody 1 or the PD1 single-domain antibody 2, and the Fc2 in sequence;

[0043] (2) The Fc1 chain, from the N-terminus to the C-terminus, can be either the PD1 single-domain antibody 1 or the PD1 single-domain antibody 2, or the Fc1 chain; the Fc2 chain, from the N-terminus to the C-terminus, can be either the PD1 single-chain antibody or the Fc2 chain.

[0044] (3) The Fc1 chain can be the PD1 single-domain antibody 1 or the PD1 single-domain antibody 2 and the Fc1 in sequence from the N end to the C end; the Fc2 chain can be the PD1 single-domain antibody 1 or the PD1 single-domain antibody 2 and the Fc2 in sequence from the N end to the C end.

[0045] (4) The Fc1 chain, from the N-terminus to the C-terminus, may be the PD1 single-domain antibody 1 or the PD1 single-domain antibody 2, a linker, the PD1 single-chain antibody, and the Fc1; the Fc2 chain may be the Fc2.

[0046] (5) The Fc1 chain can be the PD1 single-domain antibody 1 or the PD1 single-domain antibody 2, or the Fc1 chain from the N-terminus to the C-terminus; the Fc2 chain can be the Fc2 chain.

[0047] In the structure of the antibody that binds to human PD1, the linker is not limited. Specifically, the linker (linker 2) may be SGGGGS.

[0048] The Fc1 and Fc2 chains can form a heterodimer through disulfide bonds and a Knob-into-Hole structure. This pairing method is well known to those skilled in the art.

[0049] The IL12 mutant protein can be fused to the C-terminus of the Fc1 or Fc1 chain, or to the N-terminus of the Fc2 or Fc2 chain.

[0050] Furthermore, the IL12 mutant protein can fuse with the PD1 antibody via a linker. The linker is not limiting. Specifically, the linker (linker 3) can be (G4S)2.

[0051] Furthermore, the fusion protein comprises a first polypeptide chain and a second polypeptide chain selected from any one of the following (E1)-E5):

[0052] E1) The first polypeptide chain, from N-terminus to C-terminus, sequentially includes the PD1 single-domain antibody 1 or PD1 single-domain antibody 2, a linker (as described herein as linker 2), the PD1 single-chain antibody, Fc1, a linker (as described herein as linker 3), and any of the IL12 mutant proteins described herein; the second polypeptide chain, from N-terminus to C-terminus, sequentially includes the PD1 single-domain antibody 1 or PD1 single-domain antibody 2 and the Fc2;

[0053] E2) The first polypeptide chain, from the N-terminus to the C-terminus, sequentially includes the PD1 single-domain antibody 1 or PD1 single-domain antibody 2, the Fc1, a linker (as described herein as linker 3), and any of the IL12 mutant proteins described herein; the second polypeptide chain, from the N-terminus to the C-terminus, sequentially includes the PD1 single-chain antibody and the Fc2.

[0054] E3) The first polypeptide chain, from the N-terminus to the C-terminus, sequentially includes the PD1 single-domain antibody 1 or PD1 single-domain antibody 2, the Fc1, a linker (as described herein as linker 3), and any of the IL12 mutant proteins described herein; the second polypeptide chain, from the N-terminus to the C-terminus, sequentially includes the PD1 single-domain antibody 1 or PD1 single-domain antibody 2, and the Fc2.

[0055] E4) The first polypeptide chain, from N-terminus to C-terminus, sequentially includes the PD1 single-domain antibody 1 or PD1 single-domain antibody 2, a linker (as described herein as linker 2), the PD1 single-chain antibody, and the Fc1; the second polypeptide chain, from N-terminus to C-terminus, sequentially includes any of the IL12 mutant proteins described herein, a linker (as described herein as linker 3), and the Fc2.

[0056] E5) The first polypeptide chain, from the N-terminus to the C-terminus, sequentially includes the PD1 single-domain antibody 1 or PD1 single-domain antibody 2 and the Fc1; the second polypeptide chain, from the N-terminus to the C-terminus, sequentially includes any of the IL12 mutant proteins described herein, a linker (such as linker 3 described herein), and the Fc2.

[0057] Furthermore, the fusion protein comprises a first polypeptide chain and a second polypeptide chain selected from any one of the following F1)-F4):

[0058] F1) The amino acid sequence comprises a first polypeptide chain as shown in or having more than 80% identity with the amino acid sequence shown in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, and the amino acid sequence comprises a second polypeptide chain as shown in or having more than 80% identity with the amino acid sequence shown in SEQ ID NO:18.

[0059] F2) The amino acid sequence comprises a first polypeptide chain as shown in SEQ ID NO:15, SEQ ID NO:25 or SEQ ID NO:26 or having more than 80% identity with it, and the amino acid sequence comprises a second polypeptide chain as shown in SEQ ID NO:27 or having more than 80% identity with it.

[0060] F3) The amino acid sequence comprises a first polypeptide chain as shown in SEQ ID NO:22, SEQ ID NO:28 or SEQ ID NO:29 or having more than 80% identity with it, and the amino acid sequence comprises a second polypeptide chain as shown in SEQ ID NO:30 or having more than 80% identity with it.

[0061] F4) The amino acid sequence comprises a first polypeptide chain as shown in or having more than 80% identity with the amino acid sequence shown in SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:34, and a second polypeptide chain as shown in or having more than 80% identity with the amino acid sequence shown in or having more than 80% identity with the amino acid sequence shown in SEQ ID NO:33.

[0062] The first and second polypeptide chains can form a heterodimer through disulfide bonds and a Knob-into-Hole structure. This pairing method is well known to those skilled in the art.

[0063] The substitutions described in this article can be conservative substitutions.

[0064] The fusion protein described in this article can specifically bind to human PD1, reduce the binding affinity for IL-12R, and has at least one of the following characteristics: (1) it has PD1 / PDL1 blocking activity; (2) compared with wild-type IL12, it reduces the activation of general T cells, specifically in the secretion of cytokine INF-γ; (3) compared with wild-type IL12, it reduces the activation of IL-12 reporter gene signaling; (4) it has synergistic activation activity of PD1 and IL12; (5) it has significant anti-tumor activity; and (6) compared with wild-type IL12, it increases safety in animals.

[0065] The present invention also provides a biomaterial, which may be any of the following:

[0066] G1) is a nucleic acid molecule that encodes any of the fusion proteins described herein;

[0067] G2) contains an expression cassette containing the nucleic acid molecules described in G1);

[0068] G3) is a recombinant vector containing the nucleic acid molecules described in G1;

[0069] G4) Recombinant microorganisms containing the nucleic acid molecules described in G1);

[0070] G5) is a recombinant host cell containing the nucleic acid molecules described in G1).

[0071] In the aforementioned biological materials, the recombinant vector can be either a cloning vector or an expression vector.

[0072] Furthermore, the recombinant vector may be a recombinant expression vector obtained by cloning a nucleic acid molecule encoding any of the fusion proteins described herein into an expression vector (such as a prokaryotic expression vector, a eukaryotic expression vector, and a viral expression vector). Although the expression vector used in the embodiments provided by the present invention is the pcDNA3.4 vector, the present invention is not limited to this specific vector.

[0073] In the above-mentioned biological materials, the nucleic acid molecule may be any of the following:

[0074] H1) The coding sequence contains a DNA molecule as shown in SEQ ID NO:48, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or SEQ ID NO:57, and the coding sequence contains a DNA molecule as shown in SEQ ID NO:45;

[0075] H2) The coding sequence contains a DNA molecule as shown in SEQ ID NO:48, SEQ ID NO:46 or SEQ ID NO:49, and the coding sequence contains a DNA molecule as shown in SEQ ID NO:47;

[0076] H3) The coding sequence contains a DNA molecule as shown in SEQ ID NO:55, SEQ ID NO:50 or SEQ ID NO:51, and the coding sequence contains a DNA molecule as shown in SEQ ID NO:52;

[0077] H4) The coding sequence contains a DNA molecule as shown in SEQ ID NO:60, SEQ ID NO:58, SEQ ID NO:59 or SEQ ID NO:61, and the coding sequence contains a DNA molecule as shown in SEQ ID NO:62.

[0078] Those skilled in the art can readily employ known methods, such as site-directed mutagenesis (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis, and cassette mutagenesis) or directed evolution (including error-prone PCR, DNA shuffling, and in vitro random recombination), to mutate the nucleotide sequence encoding any of the fusion proteins described herein (the mutations do not include the four mutations W37K, K219E, E81K, and K106E described herein). Artificially modified nucleotide sequences that possess more than 75% identity with the nucleotide sequence encoding any of the fusion proteins described herein, provided they encode any of the fusion proteins described herein and have the same function as any of the fusion proteins described herein, are nucleotide sequences derived from and equivalent to those of this invention.

[0079] This invention also provides for the use of any of the fusion proteins described herein, or the biomaterials described herein, in any of the following:

[0080] K1) in the preparation of products for the prevention and / or treatment of tumors;

[0081] The use of K2 in the preparation of products for the prevention and / or treatment of PD1 target-related diseases;

[0082] The application of K3 in the preparation of products for inhibiting the proliferation of PD1-positive or PDL1-positive tumor cells;

[0083] The application of K4 in the preparation of products for inhibiting the growth of PD1-positive or PDL1-positive tumors;

[0084] Application of K5 in the preparation of products for stimulating the immune system of subjects.

[0085] The stimulation of the subject's immune system may include, but is not limited to, promoting the activation and proliferation of T cells, regulating the secretion of cytokines, and / or stimulating immune cells to produce a stronger immune response.

[0086] The tumors or PD1 target-related diseases mentioned in this article include, but are not limited to, colorectal cancer, pancreatic cancer, breast cancer, lung cancer, head and neck squamous cell carcinoma, gastric cancer, esophageal squamous cell carcinoma, urothelial carcinoma, melanoma, liver cancer, lymphoma, kidney cancer, esophageal cancer, cervical cancer, and ovarian cancer.

[0087] The tumors described in K1) or the PD1 target-related diseases described in K2) can be PD1-positive or PDL1-positive tumors.

[0088] The cancers mentioned also include those resistant to PD1 antibody therapy.

[0089] The products described herein include, but are not limited to, reagents, kits (such as therapeutic kits), formulations, drugs, or drug compositions.

[0090] In the above applications, the product can be used alone as a cancer treatment drug, or in combination with other anticancer drugs.

[0091] The present invention also provides pharmaceutical compositions comprising any of the fusion proteins described herein, and one or more pharmaceutically acceptable carriers.

[0092] Furthermore, the pharmaceutical composition may have at least one of the following uses: (1) for the prevention and / or treatment of tumors; (2) for the prevention and / or treatment of PD1 target-related diseases; (3) for the inhibition of the proliferation of PD1-positive or PDL1-positive tumor cells; (4) for the inhibition of the growth of PD1-positive or PDL1-positive tumors; and (5) for the stimulation of the immune system of a subject.

[0093] The active ingredient of the pharmaceutical composition may be any of the fusion proteins described herein.

[0094] The pharmaceutical composition may further include a second therapeutic agent, which may be an immune checkpoint inhibitor (such as a PD-1 inhibitor or a CTLA-4 inhibitor), an immunomodulatory drug (IMiDs) (such as lenalidomide and pomalidomide), an immunosuppressant (such as methotrexate, cyclosporine, DMF, and azathioprine), a cytokine (such as recombinant human IL-2), an antitumor drug, an anti-inflammatory drug, or other therapeutically active drugs.

[0095] The present invention also provides a method for preparing a fusion protein, the method comprising expressing any of the fusion proteins described herein in a host cell and recovering or separating the fusion protein.

[0096] The host cells include yeast cells or mammalian cells, such as HEK293 cells or CHO cells.

[0097] In one or more embodiments of the present invention, the host cell is an ExpiCHO-S cell.

[0098] In one or more embodiments of the present invention, the introduction is a chemical transfection method.

[0099] This invention also provides any of the fusion proteins described herein for use as pharmaceuticals.

[0100] Further, the fusion protein may be any of the following: (1) a fusion protein for the prevention and / or treatment of tumors; (2) a fusion protein for the prevention and / or treatment of PD1 target-related diseases; (3) a fusion protein for inhibiting the proliferation of PD1-positive or PDL1-positive tumor cells; (4) a fusion protein for inhibiting the growth of PD1-positive or PDL1-positive tumors; (5) a fusion protein for stimulating the immune system of the subject.

[0101] The present invention also provides a method for preventing or treating tumor diseases, the method comprising administering to a subject suffering from a tumor disease any of the fusion proteins described herein, or the pharmaceutical composition thereof.

[0102] The present invention also provides a method for preventing or treating PD1 target-related diseases, the method comprising administering to a subject suffering from PD1 target-related diseases any of the fusion proteins described herein, or the pharmaceutical compositions thereof.

[0103] The PD1 target-related diseases described in this article can be PD1-positive or PDL1-positive tumors.

[0104] The tumors or PD1 target-related diseases mentioned in this article include colorectal cancer, pancreatic cancer, breast cancer, lung cancer, head and neck squamous cell carcinoma, gastric cancer, esophageal squamous cell carcinoma, urothelial carcinoma, melanoma, liver cancer, lymphoma, kidney cancer, esophageal cancer, cervical cancer, and ovarian cancer.

[0105] The fusion protein described in this invention can also be called an IL12 mutant fusion protein, and may be named PD1-IL12v. In the IL12 mutant fusion protein, the IL12 mutant protein portion is a molecule obtained by amino acid mutation of wild-type IL12. At the binding interface between wild-type IL12 and IL12R, a specific amino acid mutation is introduced to weaken the binding of the IL12 P40 subunit to IL12Rβ1, thereby downregulating IL-12 activity to a certain extent. The IL12 mutant fusion protein described in this 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. The wild-type IL12 may be derived from mammalian IL12, such as human IL12. The wild-type IL12 includes the wild-type P40 subunit (sequence shown as positions 1-328 of SEQ ID NO:3) and the wild-type P35 subunit (sequence shown as positions 23-219 of SEQ ID NO:4 or SEQ ID NO:4).

[0106] In this invention, when referring to the position of an amino acid in the IL12 sequence, it is determined by referring to the amino acid sequence of the wild-type human IL-12 protein. When referring to the P40 subunit protein or the position of an amino acid in the P40 sequence, it is determined by referring to positions 1-328 of the amino acid sequence of wild-type P40 (SEQ ID NO:3). When referring to the P35 subunit protein or the position of an amino acid in the P35 sequence, it is determined by referring to the amino acid sequence of wild-type P35 (SEQ ID NO:4). For example, when referring to P40 "W37", it refers to the tryptophan residue W at position 37 in positions 1-328 of SEQ ID NO:3.

[0107] The inventors of this application, through extensive and in-depth research, mutated the P40 subunit of wild-type IL12 to eliminate or reduce the binding affinity of the wild-type P40 subunit to IL-12Rβ1, obtaining an IL12 mutant. Based on this, they designed and developed a fusion protein (IL12 mutant fusion protein PD1-IL12v) comprising a PD1 antibody and the IL12 mutant, with the structure as shown below. Figure 1 As shown. Experiments show that, compared with the prior art, the fusion protein of the present invention has the following beneficial effects:

[0108] (1) It has good manufacturability, can effectively bind to recombinant human PD-1 antigen, and has a reduced binding ability to IL12 receptor (IL12R) compared to the original, as well as a reduced activation activity of downstream IL12 signaling pathways.

[0109] (2) PD-1 / PDL-1 blocking experiments show that the fusion protein of the present invention can effectively block the PD-1 / PDL-1 signaling pathway, thereby exerting the immune function of PD1 antibody.

[0110] (3) The fusion protein of the present invention has IL12 activation activity, and its IL12 mutant protein has reduced activation ability compared with wild-type IL12. After binding to PD1, the activation ability is significantly restored. The fusion protein of the present invention can bind to PD1 and IL12R simultaneously, enhance the activation activity of IL12, and exert a synergistic effect.

[0111] (4) The fusion protein of the present invention can activate PBMC cells and T cells, induce them to secrete IFN-γ, and has a reduced ability to activate PBMC cells and T cells compared with the original protein.

[0112] (5) In vivo experiments showed that the fusion protein of the present invention can significantly inhibit tumor growth, and its anti-tumor effect is significantly better than that of Keytrud. Furthermore, the efficacy of the fusion protein of the present invention is enhanced with increasing the dosage, and it can achieve a highly efficient and safe anti-tumor effect by increasing the dosage, which is safer than wild-type IL12. The results of in vivo model evaluation experiments also showed that the safety of the fusion protein of the present invention is significantly better than that of wild-type IL12.

[0113] In summary, the IL12 mutant fusion protein provided by this invention, on the one hand, reduces the binding activity with IL12R, thereby decreasing the activity of IL12 in stimulating immune cells and avoiding excessive activation of T cells and NK cells, thus balancing the toxicity of the drug. On the other hand, it precisely targets PD1-highly expressed T cells in tumors with a PD1 antibody, exerting a dual mechanism of action of PD1 antibody and IL12, resulting in highly effective anti-tumor activity. This avoids the toxic side effects caused by excessive activity in clinical applications, broadens the clinical treatment window, and achieves better therapeutic effects. In addition to possessing the optimized characteristics of IL12 mutant proteins, the IL12 mutant fusion protein provided by this invention can also prolong the half-life, reduce the dosing frequency, and increase production yield, exhibiting good drug-like properties and possessing good clinical translational value and broad application prospects.

[0114] Terminology Definition

[0115] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0116] In this article, the term "amino acid" refers to one of 20 naturally occurring amino acids or any non-natural analogues that can be found at a specific, defined location. Natural amino acids can be abbreviated using either a three-letter code or a single-letter code. For example, alanine can be represented by A or Ala.

[0117] The term "conservative substitution" generally refers to the replacement of one amino acid residue with another amino acid residue in a side chain that has similar physicochemical properties. For example, 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 or hydrogen bonds). It is known in the art that conservative substitutions generally do not cause significant changes in protein conformation and structure, and essentially do not alter the protein's biological activity. Conservative substitutions in the protein sequence that are expected to have only a minimal or no effect on protein structure or function can be readily designed by those skilled in the art.

[0118] In this paper, amino acid mutations can be amino acid substitutions, deletions, insertions, and / or additions.

[0119] In this document, wild-type “interleukin-12” or “IL12” or “IL12-WT” refers to the parental IL12 protein that serves as a template for introducing the mutations or combinations of mutations of the present invention, preferably naturally occurring IL12 protein, including unprocessed (e.g., without removal of the signal peptide) and processed (e.g., with removal of the signal peptide) forms. The p40 subunit sequence of full-length natural IL12 containing the signal peptide is shown in SEQ ID NO:1, and the p35 subunit sequence of full-length natural IL-12 containing the signal peptide is shown in SEQ ID NO:2. Furthermore, this description also includes variants of natural IL12 that 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., conserved substitutions). Thus, in some embodiments, wild-type IL12 may contain amino acid mutations that do not affect its binding to the IL12 receptor and / or contain 6×His at the C-terminus compared to the natural IL12 protein. An exemplary wild-type IL12 P40 subunit sequence is shown in positions 1-328 of SEQ ID NO:3, and an exemplary wild-type IL12 P35 subunit sequence is shown in positions 23-219 of SEQ ID NO:4 or SEQ ID NO:4. In some embodiments, the P40 and P35 subunits of wild-type IL12 may have at least 85%, 95%, or even higher amino acid sequence identity with the amino acid sequences of SEQ ID NO:1 or 2, respectively. In some embodiments, 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 positions 1-328 of SEQ ID NO:3, positions 23-219 of SEQ ID NO:4, respectively.

[0120] In this invention, when referring to the position of amino acids in the IL12 protein or IL12 sequence, it is determined by referring to the amino acid sequence of wild-type human IL12 protein (also known as IL12-WT), wherein the P40 subunit sequence of wild-type IL12 is shown in positions 1-328 of SEQ ID NO:3, and the P35 subunit sequence of wild-type IL12 is shown in positions 23-219 of SEQ ID NO:4 or SEQ ID NO:4. For example, when referring to P40 "W37", it refers to the tryptophan residue W at position 37 in positions 1-328 of SEQ ID NO:3.

[0121] As used herein, the term "antibody" is not limited to any particular method of antibody production. The term "single-domain antibody," also known as a "nanobody," refers to an antibody fragment containing only a single heavy chain variable region (VHH) of the nanobody. It is a VHH single-domain antibody derived from alpaca heavy chain antibodies. Compared to traditional antibody heavy chains, nanobodies have a longer CDR3, which to some extent compensates for the decreased antigen-binding power caused by the absence of light chains. The term "single-chain antibody (ScFv)" generally refers to an antibody that expresses a single polypeptide chain by linking the light chain variable region and heavy chain variable region genes with an appropriate linker.

[0122] In this invention, the antibody Fc fragment can refer 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 their allotypes, and may also contain some mutated Fc fragments. Preferably, in some embodiments, a human IgG1 heavy chain Fc fragment containing mutations such as L234A / L235A / P329G is selected. In some embodiments, the Fc fragment contains Knob mutations, such as mutations T366W and S354C; or the Fc fragment contains Hole mutations, such as mutations Y349C, T366S, L368A, and Y407V, the amino acid sequences of which may be as shown in SEQ ID NO:7 and SEQ ID NO:8.

[0123] In this paper, IL12 mutants refer to mutant amino acids at the "IL12 P40 subunit-IL12Rβ1 binding interface" that eliminate or weaken the binding of IL12 to IL12R. Mutations at the "IL12 P40 subunit-IL12Rβ1 binding interface" refer to mutations occurring at amino acid sites in IL12, particularly the P40 subunit, that interact with IL-12Rβ1. These interacting amino acid sites can be identified through crystal structure analysis of the IL-12-receptor complex (e.g., PDB: 6WDQ).

[0124] In this article, "IL12 mutant fusion protein" can refer to a fusion protein containing an antibody that binds to PD1 and an IL12 mutant, and can be summarized as "PD1-IL12v".

[0125] The term "expression cassette" generally refers to a nucleic acid construct that contains enough nucleic acid elements to express the target gene.

[0126] The term "vector" generally refers to a vector that can carry foreign DNA or a target gene into a host cell for amplification and / or expression. The vector can be a cloning vector or an expression vector.

[0127] The term "host cell," also known as recipient cell, generally refers to any type of cell that can be used to introduce a vector, such as plant cells and animal cells.

[0128] The term "linker," also known as a linker peptide, peptide linker, or connector, is used to fuse, couple, link, or join two proteins or polypeptides to prevent steric hindrance. It should be understood that the presence of a linker is optional, and the length of a flexible linker can be adjusted to allow for proper folding of the fusion protein or to achieve optimal biological activity.

[0129] The term "identity" generally refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is usually expressed as a percentage. The identity described herein can refer to the identity of an amino acid sequence or a nucleotide sequence. Two copies having completely identical sequences have 100% identity. The 75% or greater identity described herein can be at least 75%, 80%, 85%, 90%, or 95% or greater. The 80% or greater identity described herein can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater.

[0130] The term “PDL1 positive tumor or PDL1 positive tumor cell” usually refers to a tumor or tumor cell that expresses the PDL1 protein.

[0131] The term "PD1 positive tumor or PD1 positive tumor cell" usually refers to a tumor or tumor cell that expresses the PD1 protein.

[0132] The term "prevention" generally refers to methods implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject.

[0133] The term "treatment" generally refers to a method implemented to achieve a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, narrowing of disease extent, stabilization of disease (i.e., cessation of disease progression), delay or slowing of disease progression, improvement or relief of disease status, and remission (whether partial or complete), whether detectable or undetectable. Furthermore, treatment can also refer to an extension of survival compared to the expected survival of a subject without treatment. Attached Figure Description

[0134] Figure 1 This is a molecular schematic diagram of the IL12 mutant fusion protein (PD1-IL12v).

[0135] Figure 2This is an SDS-PAGE electrophoresis image of the IL12 mutant fusion protein (PD1-IL12v). M represents a standard control protein with a known molecular weight (also known as a marker); R represents the molecular weight of the reduced protein; and NR represents the molecular weight of the non-reduced protein.

[0136] Figure 3 The binding activity of the IL12 mutant fusion protein (PD1-IL12v) to recombinant human PD-1 protein was detected by ELISA.

[0137] Figure 4 To detect the non-competitive binding activity of PD1 single-domain antibody and PD1 single-chain antibody in the IL12 mutant fusion protein (PD1-IL12v) using ELISA.

[0138] Figure 5 The binding activity of the IL12 mutant fusion protein (PD1-IL12v) to the IL12 receptor protein was detected by FACS.

[0139] Figure 6 To detect the activity of the IL12 mutant fusion protein (PD1-IL12v) in blocking the PD-1 / PDL-1 signaling pathway using a reporter gene assay.

[0140] Figure 7 To detect the activity of the IL12 mutant fusion protein (PD1-IL12v) in activating downstream signaling pathways of IL12 using a reporter gene assay.

[0141] Figure 8 To detect the activity of the IL12 downstream signaling pathway activated by the IL12 mutant fusion protein (PD1-IL12v) after binding to PD1 using the reporter gene assay.

[0142] Figure 9 The IL12 mutant fusion protein (PD1-IL12v) was used to activate the secretion level of IFN-γ in human peripheral blood PBMCs.

[0143] Figure 10 The IL12 mutant fusion protein (PD1-IL12v) was used to activate the secretion level of IFN-γ in CD3+ T cells in the mixed lymphocyte response system.

[0144] Figure 11 The effect of the murine molecule corresponding to the IL12 mutant fusion protein (PD1-IL12v) on tumor growth in tumor-bearing C57 mice.

[0145] Figure 12 The effect of the IL12 mutant fusion protein (PD1-IL12v) on tumor growth in PBMC immune reconstitution mice.

[0146] Figure 13The effect of IL12 mutant fusion protein (PD1-IL12v) on body weight in PBMC-immunized reconstituted mice ( Figure 13 (A) Liver Figure 13 (B) Spleen ( Figure 13 The impact of C). Detailed Implementation

[0147] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0148] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0149] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0150] The following examples do not include a detailed description of conventional methods.

[0151] The natural IL12 protein described herein includes both unprocessed (e.g., without removal of the signal peptide) and processed (e.g., with removal of the signal peptide) forms. The p40 subunit sequence of a full-length natural IL12 containing the signal peptide is shown in SEQ ID NO:1, and the p35 subunit sequence of a full-length natural IL12 containing the signal peptide is shown in SEQ ID NO:2. The wild-type IL12 (also referred to as IL12-WT) described in the examples refers to the parental IL12 protein that serves as a template for introducing the mutations or combinations of mutations of this invention, and may contain amino acid mutations compared to the natural IL12 protein that do not affect its binding to the IL12 receptor. Specifically, to avoid the formation of disulfide-bridged IL-12 dimers and without affecting the binding activity of IL12 to its receptor, compared to native IL12, the P40 subunit of wild-type IL12 containing the signal peptide has C199S and C274S mutations introduced at positions 199 and 274 of the P40 subunit of native IL12 (SEQ ID NO:1), as shown in positions 1-328 of SEQ ID NO:3; the P35 subunit of wild-type IL12 containing the signal peptide has a C96S mutation introduced at position 96 of the P40 subunit of native IL12 (SEQ ID NO:2), as shown in SEQ ID NO:4.

[0152] The sequence obtained by introducing C199S and C274S mutations into SEQ ID NO:1 (positions 1-328 of SEQ ID NO:3) is defined as the amino acid sequence of the P40 subunit of wild-type IL12 (also referred to as the wild-type P40 subunit in this article).

[0153] The sequence obtained by introducing the C96S mutation into SEQ ID NO:2 (SEQ ID NO:4), including the amino acid sequence of SEQ ID NO:4 (positions 23-219 of SEQ ID NO:4) with the signal peptide removed, is defined as the P35 subunit of wild-type IL12 (also referred to as wild-type P35 subunit in this article).

[0154] Example 1: Design and Construction of IL12 Mutant Fusion Protein

[0155] Based on the interface between interleukin-12 (IL12) and its receptor, including the crystal structure of the IL-12 and IL-12Rβ1 complex (PDB: 6WDQ), the interaction sites are listed using wild-type IL12 as a mutation template (wild-type IL12, also referred to as IL12-WT in this paper). The amino acid sequence of the wild-type P40 subunit is positions 1-328 of SEQ ID NO:3, and the amino acid sequence of the wild-type P35 subunit is positions 23-219 of SEQ ID NO:4 or SEQ ID NO:4. Mutations are made at at least at positions 37, 81, 106, and 219 of the wild-type P40 subunit (positions 1-328 of SEQ ID NO:3) to obtain an IL12 mutant, eliminating or reducing the affinity of the wild-type P40 subunit for the IL12 receptor (IL12 Rβ1).

[0156] Wild-type IL12 (IL12-WT) was constructed as a control. Six histidine tags (His×6) were appended to the C-terminus of the P40 sequence of wild-type IL12 (positions 1-328 of SEQ ID NO:3) to obtain SEQ ID NO:3. The nucleotide sequence corresponding to SEQ ID NO:3 was synthesized from the whole genome, as shown in SEQ ID NO:43, and cloned into pcDNA. TM 3.4 TOPO TM The pcDNA3.4-p40-His vector was obtained by cloning the nucleotide sequence encoding the p35 subunit (SEQ ID NO:4) of wild-type IL12 between the XbaI and HindIII sites on the pcDNA3.4 vector. The nucleotide sequence of the whole genome encoding the p35 subunit of wild-type IL12 (SEQ ID NO:44), as shown in SEQ ID NO:44, was synthesized and cloned into the pcDNA3.4 vector between the XbaI and HindIII sites to obtain the pcDNA3.4-p35 vector.

[0157] This invention designs five structural forms of IL12 mutant fusion protein (PD1-IL12v), namely Format 1, Format 2, Format 3, Format 4, and Format 5, as follows: Figure 1 As shown in the figure. All five structures are heterodimers. The Fc used in the examples refers to the Fc of human IgG1 with the mutation L234A+L235A+P329G. The amino acid residues in the Fc region are numbered according to the EU code. Specifically, the corresponding Fc contains only CH2 and CH3, and the sequences are shown in SEQ ID NO:7 (Fc1 sequence) and SEQ ID NO:8 (Fc2 sequence).

[0158] The vector construction of the PD1-IL12v fusion protein is as follows:

[0159] Taking the construction of the IL12 mutant fusion protein CyF18a02701 as an example, CyF18a02701 is a PD1-IL12v fusion protein with a Format 1 structure. Its first polypeptide chain is linked from the N-terminus to the C-terminus as a PD1 single-domain antibody PD1 VHH1-SGGGGS-PD1 scFv-Fc1-(G4S)2-IL12 mutant, and its amino acid sequence is shown in SEQ ID NO:25. The second polypeptide chain is linked from the N-terminus to the C-terminus as a PD1 single-domain antibody PD1 VHH1-Fc2, and its amino acid sequence is shown in SEQ ID NO:27.

[0160] Taking the construction of the IL12 mutant fusion protein CyF17a02701 as an example, CyF17a02701 is a PD1-IL12v fusion protein with a Format 2 structure. Its first polypeptide chain is linked from the N-terminus to the C-terminus as a PD1 single-domain antibody PD1 VHH1-Fc1-(G4S)2-IL12 mutant, and its amino acid sequence is shown in SEQ ID NO:15. The second polypeptide chain is linked from the N-terminus to the C-terminus as a PD1 single-chain antibody PD1 scFv-Fc2, and its amino acid sequence is shown in SEQ ID NO:18.

[0161] Taking the construction of the IL12 mutant fusion protein CyF29a02701 as an example, CyF29a02701 is a PD1-IL12v fusion protein with a Format 3 structure. Its first polypeptide chain is linked from the N-terminus to the C-terminus as a PD1 single-domain antibody PD1 VHH1-Fc1-(G4S)2-IL12 mutant, and its amino acid sequence is shown in SEQ ID NO:15. The second polypeptide chain is linked from the N-terminus to the C-terminus as a PD1 single-chain antibody PD1 VHH1-Fc2, and its amino acid sequence is shown in SEQ ID NO:27.

[0162] Taking the fusion protein CyF30a02701 as an example, CyF30a02701 is a PD1-IL12v fusion protein with a Format 4 structure. The first polypeptide chain is linked from the N-terminus to the C-terminus in the form of PD1 single-domain antibody PD1 VHH1-SGGGGS-PD1 scFv-Fc1, and its amino acid sequence is shown in SEQ ID NO:31. The second polypeptide chain is linked from the N-terminus to the C-terminus in the form of IL12 mutant-Fc2, and its amino acid sequence is shown in SEQ ID NO:33. Taking the fusion protein CyF14a02701 as an example, CyF14a02701 is a PD1-IL12v fusion protein with a Format 5 structure. Its first polypeptide chain is linked from the N-terminus to the C-terminus as PD1 single-domain antibody PD1 VHH1-Fc1, and its amino acid sequence is shown in SEQ ID NO:21. The second polypeptide chain is linked from the N-terminus to the C-terminus as IL12 mutant-Fc2, and its amino acid sequence is shown in SEQ ID NO:33.

[0163] The construction method is as follows: the corresponding nucleotide sequence of the whole gene is synthesized, and the corresponding nucleotide sequence is cloned into the XbaI and HindIII sites of the pcDNA3.4 vector to obtain the recombinant vectors corresponding to the first polypeptide chain and the second polypeptide chain of the corresponding structure, respectively.

[0164] The PD1-IL12 fusion proteins CyF18a02702, CyF18a90201, CyF18a90202, CyF17a02702, CyF17a02703, CyF17a90201, CyF17a90202, CyF17a90203, CyF29a90201, CyF30a90201, CyF14a90201, and CyF17a02701 correspond to the IL12-terminal parent molecule (Parental IL12-F17a02701), CyF17a02702 corresponds to the IL12-terminal parent molecule (Parental IL12-F17a02702), and the PD1-terminal parent molecule corresponding to the Format 2 structure (Parental IL12-F17a02702). PD1-F17a027 was also constructed according to the above method, and the corresponding IL12 mutant and the amino acid sequences of the first and second polypeptide chains are shown in Table 1. In the above molecular construction process, a signal peptide was added to the N-terminus of the corresponding polypeptide chain amino acid sequence. The signal peptide sequence used was MEFGLSWVFLVAILKGVQC (SEQ ID NO:6).

[0165] Table 1. Information on the amino acid and nucleotide sequences of the IL12 mutant fusion protein and its subunits.

[0166]

[0167] Note: When constructing the IL12 mutant protein, the amino acid sequence of the wild-type P35 subunit is the amino acid sequence after removing the signal peptide in SEQ ID NO:4.

[0168] like Figure 1 As shown, the fusion protein designed in this invention includes an antibody that binds to human PD1 (hereinafter referred to as PD1 antibody) and an IL12 mutant protein, wherein the IL12 mutant protein includes a mutated P40 subunit and a wild-type P35 subunit (SEQ ID NO:4 of the de-signal peptide), and the IL12 mutant protein is fused to the N-terminus or C-terminus of the PD1 antibody through a linker.

[0169] This invention designed 16 fusion proteins with 5 different structures. The mutation types of the IL12 mutant protein portion contained therein are shown in Table 1. Among them, the amino acid sequence of the mutated P40 subunit is shown in SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14.

[0170] SEQ ID NO:12 is a mutant P40 subunit obtained by performing W37K and K219E mutations on the amino acid sequence of the wild-type P40 subunit (positions 1-328 of SEQ ID NO:3) and removing the signal peptide (positions 1-22 of SEQ ID NO:3).

[0171] SEQ ID NO:13 is a mutant P40 subunit obtained by performing a W37K mutation on the amino acid sequence of the wild-type P40 subunit (positions 1-328 of SEQ ID NO:3) and removing the signal peptide (positions 1-22 of SEQ ID NO:3).

[0172] SEQ ID NO:14 is a mutant P40 subunit obtained by performing E81K and K106E mutations on the amino acid sequence of the wild-type P40 subunit (positions 1-328 of SEQ ID NO:3) and removing the signal peptide (positions 1-22 of SEQ ID NO:3).

[0173] The PD1 antibody moiety contained in the five fusion proteins designed in this invention can be as follows: Figure 1As shown, the PD1 antibody portion includes a PD1 single-domain antibody (PD1 VHH) and / or a PD1 single-chain antibody (PD1 scFv), as well as a constant region Fc. The constant region in the PD1 antibody is the Fc of human IgG1 with the mutation L234A+L235A+P329G. Two Fc types were selected as the constant region: Fc1 (SEQ ID NO:7) carries the mutation L234A+L235A+P923G and the Knob (S354C+T366W) mutation; Fc2 (SEQ ID NO:8) carries the mutation L234A+L235A+P923G and the Hole (Y349C+T366S+L368A+Y407V) mutation. The amino acid residues in the Fc are numbered according to the EU numbering system. In this example, the Fc contains only CH2 and CH3.

[0174] The amino acid sequence of the PD1 single-chain antibody (PD1 scFv) is SEQ ID NO:11.

[0175] The amino acid sequence of the PD1 single-domain antibody (PD1 VHH) is SEQ ID NO:9 or SEQ ID NO:10. Specifically, the amino acid sequence of the PD1 single-domain antibody (PD1 VHH) contained in the CyF17a02701, CyF17a02702, CyF17a02703, CyF18a02701, CyF18a02702, CyF29a02701, CyF30a02701, and CyF14a02701 fusion proteins is SEQ ID NO:9; the PD1 single-domain antibody (PD1 VHH) contained in the CyF17a90201, CyF17a90202, CyF17a90203, CyF18a90201, CyF18a90202, CyF29a90201, CyF30a90201, and CyF14a90201 fusion proteins is SEQ ID NO:9; The amino acid sequence of VHH is SEQ ID NO:10.

[0176] Among them, CyF18a02701, CyF18a02702, and CyF29a02701 each contain two identical single-domain antibodies (i.e., the single-domain antibody shown in SEQ ID NO:9).

[0177] The three fusion proteins CyF18a90201, CyF18a90202, and CyF29a90201 each contain two identical single-domain antibodies (i.e., the single-domain antibody shown in SEQ ID NO:10).

[0178] Example 2: Expression and purification of IL12 mutant fusion protein

[0179] The recombinant vectors (e.g., CyF17a02701 fusion protein vectors pcDNA3.4-PD1 VHH-17a01-Fc1 and pcDNA3.4-PD1 scFv-Fc2, respectively) containing the first and second polypeptide chains encoding the IL12 mutant fusion protein (PD1-IL12v) constructed in Example 1 were transfected into ExpiCHO-S cells (Gibco, catalog number A29127) at a 1:1 mass ratio using chemical transfection and cultured at 37°C and 6% CO2 for 7 days. The antibody protein was purified from the culture supernatant using a Protein A affinity chromatography column. First, equilibrate the Protein A column (GE) with PBS. Then, pass the culture supernatant through the column. Pre-elute 5 column volumes with solution A (solvent: water; solute and concentration: 20 mM sodium phosphate, 500 mM NaCl, pH 5.0). Then elute 5 column volumes with solution B (solvent: water; solute and concentration: 20 mM sodium acetate, 150 mM NaCl, pH 3.5). Collect the affinity elution peak. Further refine the purification using gel filtration chromatography. After equilibration with PBS on the gel filtration chromatography column (GE, SUPERDEX 200 Pg), load the affinity collection solution onto the column. Once the target protein peak appears, collect the gel filtration elution peak. Then, concentrate the protein using a 30 kDa centrifuge tube to obtain the IL12 mutant fusion protein. Taking CyF17a02702 as an example, its SDS-PAGE protein electrophoresis bands are shown below. Figure 2 As shown.

[0180] Depend on Figure 2 It can be seen that the five forms of IL12 mutant fusion protein (PD1-IL12v), namely Format 1 (CyF18a02701), Format 2 (CyF17a02701), Format 3 (CyF29a02701), Format 4 (CyF30a02701), and Format 5 (CyF14a02701), can all be correctly expressed and produced. In their SDS-PAGE electrophoresis results, M is the standard control protein (also known as the 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. Figure 2 It can be seen that the molecular weights of both reduced and non-reduced proteins are consistent with the theoretical size, and the non-reduced electrophoresis results show that the purity of the fusion protein is >90%, which meets the requirements for subsequent testing. That is, the IL12 mutant fusion protein designed in this invention has good producibility.

[0181] Sixteen IL12 mutant fusion proteins and parental control molecules were obtained through expression and purification (as shown in Table 1).

[0182] Example 3: ELISA detection of the binding activity of IL12 mutant fusion protein to recombinant human PD-1 antigen.

[0183] The preparation method of recombinant human PD1-His antigen is as follows: Six histidine tags (His×6) are appended to the C-terminus of the extracellular amino acid sequence of human PD1-His antigen (positions 1-170 of SEQ ID NO:5). The corresponding nucleotide sequence is synthesized from the whole gene (NCBI No.: MW051356) and cloned into the XbaI and HindIII sites of the pcDNA3.4 vector to obtain the recombinant vector. This recombinant vector is then transfected into Expi293F using Lipofectamine 3000 transfection reagent (Invitrogen). TM The cells (Thermo Fisher, catalog number A14527) were used to express the recombinant human PD1-His antigen, which was then purified to obtain the recombinant human PD1-His antigen.

[0184] Dilute recombinant human PD1-His antigen (recombinant human PD-1 antigen) to 1 μg / ml with NaHCO3, add 100 μl to each well of the ELISA plate, incubate overnight at 4°C, and wash 3 times with PBST (PBS + 0.1% Tween 20). Block the plate with PBST containing 5% milk at 37°C for 2 hours, then wash 3 times with PBST. Then, serially dilute the test samples with PBST containing 1% milk, add 100 μl to each well of the ELISA plate, and incubate at room temperature for 1 hour. After washing 3 times with PBST, dilute goat anti-human-HRP (Jackson Immuno Research, catalog number 109-035-088) with 1% milk PBST, add 100 μl to each well of the ELISA plate, and incubate at room temperature for 0.5 hours. Add TMB to each well and develop the color at room temperature in the dark. Stop the reaction by adding H2SO4. Analyze using SeptraMax. The absorbance (OD value) was measured at 450 nm using a Versa microplate reader, and the results were statistically analyzed and the EC50 value was calculated using a GraphPad Prism.

[0185] The samples to be tested were the IL12 mutant fusion proteins CyF17a02701, CyF18a02702, CyF18a90201, CyF18a90202, CyF14a02701, CyF29a02701 and CyF30a02701 prepared by the method in Example 2. The commercially available anti-human PD1 antibody Pembrolizumab (MSD, catalog number: X007819) was used as a positive control, and the parental IL12 mutant fusion protein of CyF17a02701 fusion protein (Parental IL12-F17a02701) was used as a parental control.

[0186] The results are as follows Figure 3As shown in Figure A, both the IL12 mutant fusion protein CyF17a02701 and the positive control Pembrolizumab can effectively bind to the recombinant human PD-1 antigen. However, the parent IL12 mutant molecule of the CyF17a02701 fusion protein (ParentalIL12-F17a02701) does not contain the PD1 binding antibody portion and therefore does not bind nonspecifically to the PD1 antigen.

[0187] like Figure 3 As shown in Figure B, the L12 mutant fusion proteins CyF18a02702, CyF18a90201, CyF18a90202, CyF14a02701, CyF29a02701, and CyF30a02701 can all effectively bind to the recombinant human PD-1 antigen. Due to their different structures, their corresponding affinities vary to some extent.

[0188] Example 4: ELISA detection of the competitive activity of PD1 single-domain antibody and PD1 single-chain antibody antigen-binding epitope against IL12 mutant fusion protein.

[0189] The relationship between the binding of PD1 single-domain antibodies and PD1 single-chain antibodies to PD1 epitopes of the IL12 mutant fusion protein was detected using a competitive ELISA method. The amino acid sequences of PD1 single-domain antibodies (VHH1 and VHH2) and PD1 single-chain antibody (scFv) were ligated to the N-terminus of the corresponding Fc of IgG1 (containing only CH2 and CH3 domains). Vectors were constructed according to the method described in Example 2, and VHH1-Fc, VHH2-Fc, and scFv-Fc proteins were expressed and purified. Recombinant human PD1-His antigen (recombinant human PD-1 antigen) was diluted with NaHCO3 to 1 μg / ml, and 100 μl was added to each well of the ELISA plate. The plate was incubated overnight at 4°C. PBST (PBS + 0.1% Tween) was then applied. 20) Wash the plate 3 times; after blocking with 5% milk PBST at 37°C for 2 hours, wash the plate 3 times with PBST; label scFv-Fc with biotin (Thermo, catalog number: A39259) as Bio-scFv-Fc, set as the competing sample, dilute with 1% milk PBST to a concentration of 2 nM, then add 1% milk PBST serially diluted VHH1-Fc, VHH2-Fc and scFv-Fc proteins at a 1:1 volume ratio, mix well, add 100 μl to each well of the microplate, and incubate at room temperature for 1 hour; after washing the plate 3 times with PBST, dilute SA-HRP (Thermo, catalog number 221130) with 1% milk PBST, add 100 μl to each well of the microplate, and incubate at room temperature for 0.5 hours; add TMB to each well, and develop color at room temperature in the dark; stop by adding H2SO4; use SeptraMax The absorbance (OD value) was measured at 450 nm using a Versa microplate reader, and the results were statistically analyzed and the EC50 value was calculated using a GraphPad Prism.

[0190] The results are as follows Figure 4 As shown, the PD1 single-domain antibodies (VHH1 and VHH2) in the IL12 mutant fusion protein do not compete with the PD1 single-chain antibody for binding to PD1. That is, the PD1 single-domain antibodies (VHH1 and VHH2) bind to the PD1 single-chain antibody at epitopes that do not completely overlap with PD1.

[0191] Example 5: FACS detection of the binding activity of the IL12 mutant fusion protein to the human IL12 receptor (IL12R).

[0192] The binding activity of the IL12 mutant fusion protein to the human IL12 receptor (IL12R) was detected using IL12 Reporter HEK-293 cells (Jimon Biotech, catalog number: GM-C19224). IL12 Reporter HEK-293 cells, derived from Jimon Biotech, are a Luciferase reporter gene cell line constructed based on the JAK-STAT signaling pathway, expressing the IL12 receptor on their cell surface. Cells were cultured in T75 flasks to 80% confluence, trypsinized, centrifuged at 1000 rpm for 5 min, and collected (number of cells per flask was approximately 10). 6 (Approximately 1000 cells), resuspend and wash with about 1 ml of PBS buffer, centrifuge, and aliquot into centrifuge tubes, 7.5 × 10⁻⁶ per tube. 5 Cells. Prepare 100 μl of the test samples (CyF17a02701, CyF17a02702, CyF17a02703, CyF18a02701 prepared in Example 2) at various dilution gradients, add them to centrifuge tubes and incubate with cells. Use the wild-type IL12 Fc fusion protein (IL12 WT-Fc, whose first polypeptide chain is linked from the N-terminus to the C-terminus of wild-type IL12 P40 subunit-Fc1, amino acid sequence SEQ ID NO:41; whose second polypeptide chain is linked from the N-terminus to the C-terminus of wild-type IL12 P35 subunit-(G4S)2-Fc2, amino acid sequence SEQ ID NO:42) as a positive control, and centrifuge tubes with PBS dilution as a blank control. After incubating at 4°C for 1 hour, wash twice with 1 ml of PBS buffer, add goat anti-human APC secondary antibody (Jackson, catalog number: 109-605-088), resuspend by pipetting, and incubate in the dark for 30 min. Wash twice again with 1 ml of PBS buffer, then add 400 μl of PBS buffer to each tube for resuspending before analysis.

[0193] The results are as follows Figure 5 As shown, the fusion protein PD1-IL12v and the control IL12 WT-Fc can both bind to IL12R. Due to different mutation schemes corresponding to IL12, the binding ability of CyF17a02701, CyF17a02702, CyF17a02703, and CyF18a02701 to IL12R is reduced to different degrees compared with wild-type IL12, which is in line with the expected design. Therefore, the fusion protein PD1-IL12v corresponding to the same IL12 mutant can bind to IL12R, and the binding ability has a similar degree of weakening.

[0194] Example 6: Detection of the blocking activity of IL12 mutant fusion protein against PD-1 / PDL-1 using reporter gene assay.

[0195] Methods: The reporter gene activity of the analyte molecules was detected using the PD1 / NFAT / Jurkat system. PD1 / NFAT / Jurkat cells (BPS Bioscience, catalog number: 60535) stably expressed human PD-1 protein and NFAT-induced luciferase, while CHO-K1 / PD-L1 / TCR cells (BPS Bioscience, catalog number: 60536) stably expressed human PDL-1 and a cell surface protein that can activate homologous TCRs in an antigen-independent manner. When the two cell types were co-cultured, the PD-1 / PDL-1 interaction inhibited TCR signaling and NFAT-mediated luciferase activity. The addition of an antibody against PDL-1 relieved the inhibition, thereby activating the TCR signaling pathway and enhancing NFAT-mediated luciferase activity. Fluorescence signals could be detected after the addition of the corresponding chromogenic reagents.

[0196] PD1 / NFAT / Jurkat(4.5×10 4 (pieces / well) and CHO-K1 / PDL1 / TCR (2.25×10) 4 (Number of cells / well) were seeded into 96-well cell culture plates, and then serially diluted test samples were added. After incubation overnight in a CO2 incubator at 37°C, the chromogenic reagent ONE-Glo™ Luciferase Assay System (Meilunbio, catalog number MA0519-2) was added and incubated for 10 min. The chemiluminescence value was then detected.

[0197] The samples to be tested were the IL12 mutant fusion proteins CyF17a02701, CyF17a90201, CyF18a02701, CyF18a90201, CyF29a0270 and the control antibody Pembrolizumab prepared in Example 2.

[0198] The results are as follows Figure 6 As shown, CyF17a02701, CyF17a90201, CyF18a02701, CyF18a90201, CyF29a0270, and Pembrolizumab can all effectively block the PD-1 / PDL-1 signaling pathway, which is in line with the expected design. Therefore, the fusion protein PD1-IL12v provided by this invention can effectively block the PD-1 / PDL-1 signaling pathway, thereby exerting the immune function of PD1 antibody.

[0199] Example 7: Detection of the activation activity of IL12 mutant fusion protein on downstream signaling pathways of IL12 using reporter gene assay.

[0200] The activation activity of the IL12 mutant fusion protein (PD1-IL12v) on downstream signaling pathways of IL12 was detected using the reporter gene assay in HEK-293 cells (Jiman Biotechnology, catalog number: GM-C19224). This reporter gene cell line is a luciferase reporter gene cell line constructed based on the JAK-STAT4 signaling pathway. When IL12 binds to its receptor to form a complex, the receptor complex is phosphorylated through JAK1 and Tyk2, ultimately leading to 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 IL12 signaling pathway activation activity.

[0201] IL12 Reporter HEK-293 cells (1.5×10⁻⁶) 4 Cells were seeded per well into 96-well cell culture plates, and then serially diluted test samples were added. Wild-type IL12 (IL12-WT) was used as a control. After incubation overnight in a CO2 incubator at 37°C, ONE-Glo™ Luciferase Assay System reagent (Meilunbio, catalog number MA0519-2) was added and incubated for 10 min. The chemiluminescence value was then detected.

[0202] The results are as follows Figure 7 As shown, PD1-IL12v (CyF17a02701, CyF17a02702, CyF17a02703, CyF18a02701, CyF29a02701, CyF30a02701, CyF17a90201, CyF17a90202, CyF17a90203, CyF29a90201, CyF14a90201) and IL12-WT both exhibit activation activity in the in vitro reporter gene system. Due to different mutation schemes corresponding to IL12, the binding ability with IL12R is weakened, and the ability to activate downstream signaling pathways of IL12R is also reduced. Compared with wild-type IL12, the activation activity intensity of fusion proteins from different mutants varies.

[0203] Example 8: Reporter gene assay to detect the effect of IL12 mutant fusion protein binding to PD1 on IL12 activation activity.

[0204] The activation activity of the IL12 mutant fusion protein (PD1-IL12v) on downstream IL12 signaling pathways after binding to PD1 was detected in IL12 / PD1 Reporter HEK-293 cells using a reporter gene assay. This reporter gene cell line was developed by inserting the full-length human PD-1 target sequence (amino acid sequence as shown in SEQ ID NO:5, corresponding GenBank number UMM61402.1) into the pCDNA3.4 vector between the XbaⅠ (5'-TCTAGA-3') and HindⅢ (5'-AAGCTT-3') sites, following conventional methods in the art, into IL12Reporter HEK-293 cells after the recombinant plasmid was verified by sequencing. The resulting recombinant plasmid was then transfected into IL12Reporter HEK-293 cells using Lipofectamine 3000 transfection reagent (Invitrogen), resulting in a cell line highly expressing human PD-1, IL12 / PD1 Reporter. HEK-293 cells also express IL12R. When IL12 binds to its receptor to form a complex, the receptor complex is phosphorylated through JAK1 and Tyk2, which eventually leads 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 IL12 signaling pathway activation activity.

[0205] IL12 / PD1 Reporter HEK-293 cells (1.5×10⁻⁶) 4 Cells (number per well) were seeded into 96-well cell culture plates, and then serially diluted test samples were added. Taking CyF17a02702 as an example, its PD1-terminal parental molecule Parental PD1-F17a027, its IL12 mutant-terminal parental molecule Parental IL12-F17a02702, and IL12-WT were used as controls. After overnight incubation in a 37°C CO2 incubator, the chromogenic reagent ONE-Glo™ Luciferase Assay System (Meilunbio, catalog number MA0519-2) was added and incubated for 10 min, and the chemiluminescence value was detected. The PD1 and IL12 dual-target activation activities of CyF18a02702, CyF18a90202, CyF17a90202, CyF17a02702, and CyF17a02701 were compared using the same method.

[0206] The results are as follows Figure 8As shown in Figure A: PD1-IL12v (e.g., CyF17a02702), along with its corresponding IL12 mutant parental molecules, Parental IL12v-F17a02 and IL12-WT, all exhibit IL12 activation activity in in vitro reporter gene systems. Because the IL12 mutant weakens the binding affinity of IL12 to IL12R, its activation capacity is reduced compared to wild-type IL12. However, after fusion with a PD1 antibody, the activation capacity of CyF17a02702 is significantly restored compared to its parental molecule, ParentalIL12-F17a02702, due to the simultaneous binding of PD1. Therefore, the simultaneous binding of PD1 and IL12 can exert a synergistic effect, enhancing IL12 activation activity. Figure 8 As shown in Figure B: fusion proteins with different structures, namely CyF18a02702, CyF18a90202, CyF17a90202, CyF17a02702, and CyF17a02701, exhibit different degrees of synergistic effects between PD1 and IL12 due to differences in their binding ability to PD1 and IL12R, and ultimately show different IL12 activation activities.

[0207] Example 9: Validation of the in vitro lymphocyte activation function of the IL12 mutant fusion protein

[0208] 1) Activation activity of human PBMCs

[0209] Anti-CD3 antibody (Baiying Biotechnology, catalog number B6928) was diluted to 0.5 μg / ml with PBS and added to a 384-well plate (Corning), 40 μl / well. The 384-well plate was incubated at 37°C for 2 hours, washed with PBS, and then 40 μl of human PBMCs (Shanghai Heyousheng Biotechnology Co., Ltd.), 20,000 cells / well, was added. Then, the diluted test sample was added, 40 μl per well, with 3 replicates for each concentration. The 384-well plate was incubated in a cell culture incubator for 2 days, and the secretion of cytokine IFN-γ in the cell culture supernatant was detected by ELISA. The test samples were the IL12 mutant fusion proteins CyF17a02701, CyF17a02702, and CyF17a02703 prepared in Example 2, as well as the PD1-terminal parental molecules Parental PD1-F17a027 and IL12-WT. The results are as follows: Figure 9 As shown in Figure A. Using the same method, the PBMC activation activities of CyF17a90201, CyF17a90202, CyF17a90203, CyF18a90202, CyF14a02701, CyF30a02701, and CyF29a02701 were compared, and the results are as follows. Figure 9 As shown in B.

[0210] The results showed that both the IL12 mutant fusion protein (PD1-IL12v) and wild-type IL12 could activate PBMCs and induce IFN-γ secretion. Compared with wild-type IL12, PD1-IL12v required a higher concentration of sample to induce IFN-γ secretion from PBMCs, and the maximum value of IFN-γ secretion by PBMCs was also reduced. This indicates that compared with wild-type IL12, PD1-IL12v reduced the ability to activate PBMCs. The degree of reduction in activation activity varied among different mutations, achieving the expected target of the mutation. In the same experimental system, the parental molecule Parental PD1, lacking the effect of IL12, exhibited very weak activation activity.

[0211] 2) Detection of activation activity using a mixed lymphatic reaction system

[0212] CD14+T cells were isolated from PBMCs (Shanghai Heyousheng Biotechnology Co., Ltd.) using Anti-Human CD14 Magnetic Particles-DM (BD) magnetic beads. The cells were cultured in 1640 medium (gibco, catalog number: C11875500BT) containing 500 U / ml IL-4 and 1000 U / ml GM-CSF. After 5 days, the medium was replaced with 1640 medium containing 10 ng / ml IL-6, 10 ng / mL IL-1β, 10 ng / mL TNF-α and 1 μg / mL PGE2. After 2 days of culture, the cells were activated into mature DCs. CD3+ T cells were isolated from PBMCs using Anti-Human CD3 T Magnetic Particles-DM Clone: ​​H1T3a magnetic bead sorting (BD, 552593). CD3+ T cells and DC cells were adjusted to target concentrations using 1640 medium, with a CD3+ T / DC ratio of 10:1 (number ratio, CD3+ T = 60,000 cells / well). The cells were added to 384-well cell culture plates, with 30 μL of each cell type added to each well. The test sample was diluted with culture medium, and 30 μL was added to each well to achieve the desired antibody drug concentration. Figure 10 The concentrations shown are as follows; the 384-well plates were incubated in a cell culture incubator for 5 days, and the secretion of cytokine INF-γ in the cell culture supernatant was detected by ELISA. The test samples were the IL12 mutant fusion proteins CyF17a02701, CyF17a02702, and CyF17a02703 prepared in Example 2, as well as the PD1 antibody Pembrolizumab and IL12-WT. The results are as follows. Figure 10As shown in Figure A. Using the same method, the PBMC activation activities of CyF17a90201, CyF18a90201, CyF18a02701, CyF17a02701, CyF14a02701, CyF30a02701, and CyF29a02701 were compared, and the results are as follows. Figure 10 As shown in B.

[0213] The results showed that both the IL12 mutant fusion protein (PD1-IL12v) and wild-type IL12 could significantly activate T cells and secrete IFN-γ. The activation activities of different PD1-IL12v molecules differed, but the activation activity of PD1-IL12v was significantly better than that of Pembrolizumab. Compared with wild-type IL12, PD1-IL12v required a higher concentration of sample to induce PBMC secretion of IFN-γ.

[0214] Example 10: In vivo efficacy detection of the antitumor activity of the IL12 mutant fusion protein

[0215] 1. Antitumor activity of PD1-IL12v against MC38 colon cancer in mice

[0216] A mutant molecule that replaces human IL12 was constructed using mouse 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:35, and the amino acid sequence of the P35 subunit of wild-type mouse IL12 is SEQ ID NO:36. The correspondence and sequence details between the mouse IL12 mutant fusion protein and the human IL12 mutant fusion protein are shown in Table 2.

[0217] Table 2. List of Human and Mouse IL12 Mutant Fusion Proteins and Their Sequences

[0218] Human fusion protein name Mouse fusion protein name Mouse fusion protein first polypeptide chain second polypeptide chain of mouse fusion protein CyF17a02701 mCyF17a02701 SEQ ID NO:37 SEQ ID NO:18 CyF17a02702 mCyF17a02702 SEQ ID NO:38 SEQ ID NO:18 CyF14a02701 mCyF14a02701 SEQ ID NO:21 SEQ ID NO:39 CyF18a02701 mCyF18a02701 SEQ ID NO:40 SEQ ID NO:27

[0219] MC38 cells (Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60825) were cultured in vitro, digested with trypsin, and then added at a rate of 5 × 10⁻⁶. 5 / Subcutaneously inoculate 6-8 week old PD1 humanized mice (B6-hPD1 mice, Biocytogen Biotechnology Co., Ltd.) when the average tumor volume of the tumor-bearing mice reaches 100 mm 3 At approximately 10:00 AM, mice were randomly divided into groups of 5 mice each according to the experimental design. A saline group (Vehicle) was set up as a negative control group without drug administration. Wild-type IL12 (mIL12-WT) and mCyF17a02701, mCyF17a02702, mCyF14a02701, mCyF18a02701, and Pembrolizumab were diluted with saline according to their concentration requirements. Specific dosage concentrations are as follows: Figure 11 As shown, mIL12-WT was administered via intraperitoneal injection once daily for a total of three doses, while other groups received the drug twice weekly for a total of three doses. Following tumor inoculation, animal survival and activity were assessed twice weekly, including tumor growth, activity level, diet, weight, and any abnormal behaviors.

[0220] For details, please see [link / details]. Figure 11 As shown: by Figure 11 The results showed that, compared with the saline group (Vehicle), mCyF14a02701, mCyF18a02701, mCyF17a02701, and mCyF17a02702 significantly inhibited tumor growth, and their anti-tumor effects were significantly better than Pembrolizumab. Increasing the dosage of mCyF17a02701 enhanced its efficacy, achieving anti-tumor effects consistent with mIL12-WT, and no abnormalities were observed in the mice during administration. In contrast, the mIL12-WT group, due to its short half-life, required daily administration, and subsequent re-administration resulted in weight loss in the mice, posing a certain safety risk. Therefore, compared to mIL12-WT, PD1-IL12v can achieve highly effective and safe anti-tumor effects by increasing the dosage, and is safer than wild-type IL12.

[0221] 2. Antitumor activity of PD1-IL12v against human breast cancer MDA-MB-231

[0222] The antitumor activity of D1-IL12v against human breast cancer MDA-MB-231 was evaluated using a PBMC immune reconstitution mouse model. The specific procedures were as follows: MDA-MB-231 cells (Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60382) were cultured in vitro, digested with trypsin, and then injected at a rate of 4 × 10⁻⁶ cells / mL. 6 / 6-8 week old immunodeficient mice (B-NDG MHCI / IIDKO mice plus, Biocytogen Biotechnology Co., Ltd.) were subcutaneously inoculated with tumor cells, followed by administration of 6×10 via the tail vein. 6 The immune system of mice was rebuilt by inoculating them with human PBMCs (Miaoshun (Shanghai) Biotechnology Co., Ltd.). When the average tumor volume of the tumor-bearing mice reached 80 mm... 3 At approximately [time missing], mice were randomly divided into groups of 5 mice each according to the experimental design. A saline group (Vehicle) was set up as a negative control group without drug administration. CyF17a02701, CyF17a02702, CyF17a02703, CyF17a90202, CyF30a02701, CyF18a02701, and Pembrolizumab were diluted with saline according to their concentration requirements, with the same molar concentration. The specific dosage concentrations were calculated based on the molecular weight as follows: Figure 12As shown, the medication was administered via intraperitoneal injection twice a week for a total of six times. Following tumor inoculation, the animals' survival and activity levels were monitored twice weekly, including tumor growth, activity level, diet, weight, and any abnormal behaviors.

[0223] Experimental results are as follows Figure 12 As shown, compared with the saline group (referred to as Vehicle), CyF17a02701, CyF17a02702, CyF17a02703, CyF17a90202, CyF30a02701, CyF18a02701 and Pembrolizumab can significantly inhibit tumor growth, and the fusion protein has a better ability to inhibit the growth of MDA-MB-231 tumors than Pembrolizumab.

[0224] Example 11: In vivo model assessment of the safety of the IL12 mutant fusion protein

[0225] The safety of PD1-IL12v in tumor-bearing mice was evaluated using a PBMC immune reconstitution mouse model. The specific procedure was as follows: 6-8 week old immunodeficient mice (B-NDG MHC I / IIDKO mice plus, Biocytogen Biotechnology Co., Ltd.) were selected, and PD1-IL12v was administered via tail vein at a dose of 1×10⁻⁶ mg / L. 7 The immune system of mice was reconstructed by inoculating them with only human PBMCs (Miaoshun (Shanghai) Biotechnology Co., Ltd.). Two days later, human pancreatic cancer BxPc-3 cells (Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60542) cultured in vitro were added at a rate of 5 × 10⁻⁶ cells / year. 6 / subcutaneously injected into immune-reconstituted mice. When the average tumor volume of tumor-bearing mice reached 60 mm. 3 Around 10:00 AM, mice were randomly divided into groups of five according to the experimental design. A saline group (Vehicle) served as a negative control group without drug administration. CyF17a02701 and IL12-WT were diluted with saline according to their concentration requirements. The molar concentration was calculated based on molecular weight, and the final molar concentration of CyF17a02701 was four times that of IL12-WT. Administered via intraperitoneal injection, IL12-WT was administered once daily for three days, and CyF17a02701 was administered every three days for three days. During the administration process, the animals' survival and activity levels were monitored, including tumor growth, activity level, diet, weight, and any abnormal behaviors. One week later, the mice were euthanized, dissected, and their livers and spleens were weighed.

[0226] Experimental results are as follows Figure 13As shown in Figure A, during the CyF17a02701 administration period, mice did not exhibit any abnormalities such as weight loss or inactivity. However, in the wild-type IL12 administration group, mice showed a significant decrease in weight, inactivity, and exhibited arched backs and ruffled fur. Dissection revealed the liver and spleen of the mice, and their weights were measured. Figure 13 As shown in Figures B and C, compared to the saline group (Vehicle), the spleen and liver of mice in the CyF17a02701 treatment group showed no abnormalities, while the spleen and liver of mice in the wild-type IL12 treatment group were significantly enlarged and heavier. In the experimental design, the molar concentration of CyF17a02701 was four times the molar concentration of IL12-WT; therefore, the safety profile of CyF17a02701 is significantly superior to that of wild-type IL12.

[0227] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises an antibody that binds to human PD1 and an IL12 mutant protein, the IL12 mutant protein comprising a mutated P40 subunit and a wild-type P35 subunit; the mutated P40 subunit contains a mutation at at least one of the following positions relative to the amino acid sequence of the wild-type P40 subunit: positions 37, 81, 106, and 219. The amino acid sequence of the wild-type P40 subunit includes the sequence shown in A1) or A2): A1) The first 328th bits of SEQ ID NO:3; A2) A sequence obtained by substituting, deleting and / or adding amino acid residues to the amino acid sequence shown in A1), which has more than 80% identity with the sequence and retains the same function; wherein the substituents, deletions and / or additions are not at positions 37, 81, 106 and 219 of the amino acid sequence shown in A1). The amino acid sequence of the wild-type P35 subunit includes the sequence shown in B1) or B2): B1) SEQ ID NO:4 or bits 23-219 of SEQ ID NO:4; B2) A sequence obtained by substituting, deleting and / or adding amino acid residues to the amino acid sequence shown in B1), which has more than 80% identity with the sequence and retains the same function.

2. The fusion protein according to claim 1, characterized in that, The mutated P40 subunit contains any one of the following mutations (C1)-C3): C1)W37K and K219E; C2)W37K; C3)E81K and K106E.

3. The fusion protein according to claim 1 or 2, characterized in that, The amino acid sequence of the mutated P40 subunit includes the sequence shown in D1) or D2): D1) SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14; D2) A sequence obtained by substituting, deleting and / or adding amino acid residues to the amino acid sequence shown in D1), which has more than 80% identity with the sequence and retains the same function; wherein the substituents, deletions and / or additions are not at positions 37, 81, 106 and 219 of the amino acid sequence shown in D1).

4. The fusion protein according to any one of claims 1-3, characterized in that, The antibody that binds to human PD1 includes a variable region and a constant region, wherein the variable region includes PD1 single-domain antibody 1, PD1 single-domain antibody 2 or PD1 single-chain antibody, or a combination thereof; Both the PD1 single-domain antibody 1 and the PD1 single-domain antibody 2 bind to different or non-overlapping or partially overlapping epitopes of PD1 with the PD1 single-chain antibody, and / or do not compete for binding to PD1.

5. The fusion protein according to claim 4, characterized in that, The amino acid sequence of the PD1 single-domain antibody 1 contains SEQ ID NO:9, or an amino acid sequence that has more than 80% identity with SEQ ID NO:9; The amino acid sequence of the PD1 single-domain antibody 2 contains SEQ ID NO:10, or an amino acid sequence that has more than 80% identity with SEQ ID NO:

10. The amino acid sequence of the PD1 single-chain antibody contains SEQ ID NO:11, or has more than 80% identity with SEQ ID NO:

11.

6. The fusion protein according to claim 4 or 5, characterized in that, The constant region includes Fc1 and Fc2, wherein the amino acid sequence of Fc1 contains SEQ ID NO:7 and the amino acid sequence of Fc2 contains SEQ ID NO:

8.

7. The fusion protein according to any one of claims 1-6, characterized in that, The fusion protein comprises a first polypeptide chain and a second polypeptide chain selected from any one of the following (E1)-E5): E1) The first polypeptide chain, from N-terminus to C-terminus, sequentially comprises PD1 single-domain antibody 1 or PD1 single-domain antibody 2 as described in claim 4 or 5, a linker, PD1 single-chain antibody as described in claim 4 or 5, Fc1 as described in claim 6, a linker, and IL12 mutant protein as described in any one of claims 1-3; the second polypeptide chain, from N-terminus to C-terminus, sequentially comprises PD1 single-domain antibody 1 or PD1 single-domain antibody 2 as described in claim 4 or 5, and Fc2 as described in claim 6; E2) The first polypeptide chain, from the N-terminus to the C-terminus, sequentially comprises the PD1 single-domain antibody 1 or PD1 single-domain antibody 2 as described in claim 4 or 5, the Fc1 as described in claim 6, a linker, and the IL12 mutant protein as described in any one of claims 1-3; the second polypeptide chain, from the N-terminus to the C-terminus, sequentially comprises the PD1 single-chain antibody as described in claim 4 or 5 and the Fc2 as described in claim 6. E3) The first polypeptide chain, from the N-terminus to the C-terminus, sequentially comprises PD1 single-domain antibody 1 or PD1 single-domain antibody 2 as described in claim 4 or 5, Fc1 as described in claim 6, a linker, and IL12 mutant protein as described in any one of claims 1-3; the second polypeptide chain, from the N-terminus to the C-terminus, sequentially comprises PD1 single-domain antibody 1 or PD1 single-domain antibody 2 as described in claim 4 or 5, and Fc2 as described in claim 6. E4) The first polypeptide chain, from the N-terminus to the C-terminus, sequentially comprises the PD1 single-domain antibody 1 or PD1 single-domain antibody 2 as described in claim 4 or 5, a linker, the PD1 single-chain antibody as described in claim 4 or 5, and Fc1 as described in claim 6; the second polypeptide chain, from the N-terminus to the C-terminus, sequentially comprises the IL12 mutant protein as described in any one of claims 1-3, a linker, and Fc2 as described in claim 6. E5) The first polypeptide chain, from the N-terminus to the C-terminus, sequentially includes PD1 single-domain antibody 1 or PD1 single-domain antibody 2 as described in claim 4 or 5, and Fc1 as described in claim 6; the second polypeptide chain, from the N-terminus to the C-terminus, sequentially includes IL12 mutant protein as described in any one of claims 1-3, a linker, and Fc2 as described in claim 6.

8. The fusion protein according to any one of claims 1-7, characterized in that, The fusion protein comprises a first polypeptide chain and a second polypeptide chain selected from any one of the following F1)-F4): F1) The amino acid sequence comprises a first polypeptide chain as shown in or having more than 80% identity with the amino acid sequence shown in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, and the amino acid sequence comprises a second polypeptide chain as shown in or having more than 80% identity with the amino acid sequence shown in SEQ ID NO:

18. F2) The amino acid sequence comprises a first polypeptide chain as shown in SEQ ID NO:15, SEQ ID NO:25 or SEQ ID NO:26 or having more than 80% identity with it, and the amino acid sequence comprises a second polypeptide chain as shown in SEQ ID NO:27 or having more than 80% identity with it. F3) The amino acid sequence comprises a first polypeptide chain as shown in SEQ ID NO:22, SEQ ID NO:28 or SEQ ID NO:29 or having more than 80% identity with it, and the amino acid sequence comprises a second polypeptide chain as shown in SEQ ID NO:30 or having more than 80% identity with it. F4) The amino acid sequence comprises a first polypeptide chain as shown in or having more than 80% identity with the amino acid sequence shown in SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:34, and a second polypeptide chain as shown in or having more than 80% identity with the amino acid sequence shown in or having more than 80% identity with the amino acid sequence shown in SEQ ID NO:

33.

9. A biomaterial, characterized in that, The biomaterial is any one of the following: G1) is a nucleic acid molecule encoding any of the fusion proteins described in claims 1-8; G2) contains an expression cassette containing the nucleic acid molecules described in G1); G3) is a recombinant vector containing the nucleic acid molecules described in G1; G4) Recombinant microorganisms containing the nucleic acid molecules described in G1); G5) is a recombinant host cell containing the nucleic acid molecules described in G1).

10. The use of the fusion protein according to any one of claims 1-8 or the biomaterial according to claim 9 in any of the following: K1) in the preparation of products for the prevention and / or treatment of tumors; The use of K2 in the preparation of products for the prevention and / or treatment of PD1 target-related diseases; The application of K3 in the preparation of products for inhibiting the proliferation of PD1-positive or PDL1-positive tumor cells; The application of K4 in the preparation of products for inhibiting the growth of PD1-positive or PDL1-positive tumors; Application of K5 in the preparation of products for stimulating the immune system of subjects.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the fusion protein of any one of claims 1-8, and one or more pharmaceutically acceptable carriers.

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