Bifunctional fusion protein of anti-PD-1 antibody and IL-2 mutant

Abstract

The present invention provides a bifunctional fusion protein of an anti-PD-1 antibody and an IL-2 mutant. The bifunctional fusion protein of the present invention comprises a PD-1 antibody and an IL-2 mutant connected by a mutated FC. The bifunctional fusion protein of the present invention has the functions of activating and amplifying PD-1 positive lymphocytes and regulating the activity of Treg cells, and can activate tumor immunity to treat cancer. The present invention also provides the application of the bifunctional fusion protein in the field of tumor treatment. The present invention also provides a combination therapy for treating tumors by combining the bifunctional fusion protein with atezolizumab.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to a bifunctional fusion protein of an anti-PD-1 antibody and an IL-2 mutant. Background Art

[0002] Cancer immunotherapy has always been a hot topic in cancer research, aiming to activate or stimulate the immune system to recognize, attack, and eliminate cancer cells through natural mechanisms, thereby achieving the effect of treating cancer. Cancer immunotherapy mainly includes cytokine therapy, immune checkpoint inhibitors, and adoptive immunocyte therapy. Among them, cytokine therapy is the earliest cancer immunotherapy and is of great significance in cancer treatment.

[0003] Immune checkpoint inhibitors are represented by PD-1 (Programmed Death-1) inhibitors. PD-1 is an immune negative regulatory factor that binds to its ligands PD-L1 or PD-L2, downregulates the activity of T cells, mediates immune tolerance, and mediates the immune escape of cancer cells in the tumor microenvironment. PD-1 inhibitors can block the binding of PD-1 to PD-L1 and restore the recognition and killing of tumor cells by T cells. Currently, marketed PD-1 inhibitors include Nivolumab, Pembrolizumab, etc., which are used for the first- and second-line treatment of different tumor indications. However, some patients do not respond to PD-1 inhibitors, and this problem can be solved by cytokine-stimulated effector T cells.

[0004] Cytokine therapy is represented by interleukin 2 (IL-2). IL-2 is a 15 kDa pleiotropic secreted protein mainly secreted by activated CD4+ T cells. It binds to the IL-2 receptor (IL-2R) and activates downstream JAK1-JAK3-STAT5, PI3K-mTOR1, and MAPK signaling pathways through CD122 and CD132 to regulate the immune response. When the immune environment is stable, CD4+ T cells secrete low concentrations of IL-2. After immune activation, CD4+ and CD8+ T cells, NK cells, dendritic cells (DCs), and mast cells act on effector immune cells expressing the IL-2 receptor (IL-2R+) through autocrine and paracrine IL-2. IL-2 exerts its immunomodulatory effect through the trimer / dimer formed by the receptor IL-2α / β / γ. The trimeric IL-2R consists of an α chain (IL-2Rα; CD25), a β chain (IL-2Rβ; CD122), and a common chain (γc / CD132, shared with IL-4, etc.), has high affinity, and is mainly expressed on Treg cells, newly activated CD4+ and CD8+ T cells, and some NK cells. The dimer composed of the IL-2Rβ and γ chains shows medium affinity and is mainly expressed on CD8+ memory T cells and NK cells. At low doses, IL-2 tends to bind to high-affinity receptors, and at high doses, IL-2 can be bound by medium-affinity receptors.

[0005] The combination therapy of IL-2 and PD-1 inhibitors is one of the new strategies for cancer immunotherapy. However, although IL-2 has good efficacy, due to its short half-life, high-dose administration is required, which easily leads to serious adverse reactions such as cytokine release syndrome and vascular leakage syndrome, limiting the clinical application of IL-2.

[0006] Therefore, there is a need in the art to develop a combined therapeutic drug of IL-2 / PD-1 inhibitors with high efficacy and low side effects. Summary of the Invention

[0007] The object of the present invention is to provide a combined therapeutic drug of IL-2 / PD-1 inhibitors with high efficacy and low side effects.

[0008] In the first aspect of the present invention, an IL-2 mutant protein is provided, and the mutant protein has the following mutations relative to the wild-type IL-2 protein shown in SEQ ID NO:7:

[0009] The P at position 65 is mutated to K, R, H, D, E, N, Q, Y, W, or F;

[0010] The C at position 125 is mutated to S, A, G, T, or V.

[0011] In another preferred embodiment, the ability of the IL-2 mutant protein to activate Treg cells is 20% (preferably 10%, more preferably 1%, even more preferably 0.1%) or less of that of wild-type IL-2.

[0012] In another preferred embodiment, the ability of the IL-2 mutant protein to bind to IL-2Rβγ is 80% (preferably 90%, more preferably 95%, even more preferably 99%) or more of that of wild-type IL-2.

[0013] In another preferred embodiment, except for the mutations (such as the amino acids at positions 65 and 125), the remaining amino acid sequence of the IL-2 mutant is the same as or substantially the same as the sequence shown in SEQ ID NO.7.

[0014] In another preferred embodiment, the amino acid sequence of the IL-2 mutant protein is as shown in SEQ ID NO:6, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

[0015] In a second aspect of the present invention, there is provided a fusion protein, which comprises the IL-2 mutant protein as described in the first aspect of the present invention.

[0016] In another preferred embodiment, the fusion protein comprises the following protein elements:

[0017] (i) an anti-PD-1 antibody;

[0018] (ii) an Fc fragment;

[0019] (iii) the IL-2 mutant protein as described in the first aspect of the present invention.

[0020] In another preferred embodiment, the fusion protein has the structure shown in Formula I from the N-terminus to the C-terminus:

[0021] P-F-I(I)

[0022] In the formula, each "-" is independently a linker peptide or a peptide bond;

[0023] P is an anti-PD-1 antibody;

[0024] F is an Fc fragment;

[0025] I is the IL-2 mutant protein.

[0026] In another preferred embodiment, the PD-1 is human PD-1.

[0027] In another preferred embodiment, the anti-PD-1 antibody is selected from the group consisting of: Fab fragment, single-chain antibody, single-domain antibody, or a combination thereof.

[0028] In another preferred embodiment, the anti-PD-1 antibody has a light chain variable region and a heavy chain variable region.

[0029] In another preferred embodiment, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9.

[0030] In another preferred embodiment, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:10.

[0031] In another preferred embodiment, the anti-PD-1 antibody has a structure represented by the following formula IIa or IIb from the N-terminus to the C-terminus:

[0032] VH-L-VL (IIa);

[0033] VL-L-VH (IIb);

[0034] Wherein,

[0035] VL is the light chain variable region;

[0036] VH is the heavy chain variable region;

[0037] L is a linker peptide or a peptide bond.

[0038] In another preferred embodiment, the L is a flexible linker or a rigid linker.

[0039] In another preferred embodiment, the flexible linker is a sequence of 1-6 (preferably 3-5) consecutive G4S.

[0040] In another preferred embodiment, the amino acid sequence of the anti-PD-1 antibody is as shown in SEQ ID NO:8, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

[0041] In another preferred embodiment, the Fc fragment is a wild-type IgG1-derived Fc fragment or a mutant Fc fragment.

[0042] In another preferred embodiment, the mutant Fc fragment has the following mutations relative to the wild-type IgG1 Fc fragment shown in SEQ ID NO:12:

[0043] Leu at position 234 is mutated to Ala, Leu at position 235 is mutated to Ala, and Pro at position 329 is mutated to Gly.

[0044] In another preferred embodiment, the amino acid sequence of the Fc fragment is as shown in SEQ ID NO:11.

[0045] In another preferred embodiment, the amino acid sequence of the IL-2 mutant protein is as shown in SEQ ID NO:6.

[0046] In another preferred embodiment, the anti-PD-1 antibody and the Fc fragment are linked by a linker peptide, preferably a flexible linker peptide, more preferably a sequence shown by GGS.

[0047] In another preferred embodiment, the Fc fragment and the IL-2 mutant protein are linked by a linker peptide, preferably a flexible linker peptide, more preferably a sequence shown by GGGS (SEQ ID NO:13).

[0048] In another preferred embodiment, the fusion protein has the amino acid sequence as shown in SEQ ID NO:1, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

[0049] In the third aspect of the present invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

[0050] (1) the IL-2 mutant protein as described in the first aspect of the present invention; or

[0051] (2) the fusion protein as described in the second aspect of the present invention.

[0052] In the fourth aspect of the present invention, there is provided a vector containing the polynucleotide as described in the third aspect of the present invention.

[0053] In another preferred embodiment, the vector is selected from the group consisting of: DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.

[0054] In the fifth aspect of the present invention, there is provided an engineered host cell containing the vector as described in the fourth aspect of the present invention or having the polynucleotide as described in the third aspect of the present invention integrated into its genome.

[0055] In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell, a plant cell or a mammalian cell (including human and non-human mammals).

[0056] In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

[0057] In another preferred embodiment, the host cell is a 293 cell or a CHO cell.

[0058] In a sixth aspect of the present invention, there is provided a method for preparing the IL-2 mutant protein as described in the first aspect of the present invention or the fusion protein as described in the second aspect of the present invention, comprising the steps of:

[0059] (i) culturing the host cell as described in the fifth aspect of the present invention under suitable conditions to obtain a mixture containing the fusion protein as described in the first aspect of the present invention; and

[0060] (ii) purifying and / or isolating the mixture obtained in step (i) to obtain the IL-2 mutant protein as described in the first aspect of the present invention or the fusion protein as described in the second aspect of the present invention.

[0061] In a seventh aspect of the present invention, there is provided an immunoconjugate, which contains:

[0062] (a) the fusion protein as described in the second aspect of the present invention; and

[0063] (b) a conjugate moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0064] In another preferred embodiment, the conjugate moiety is selected from: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme, a radionuclide, a biological toxin, a cytokine, an antibody, an antibody Fc fragment, an antibody scFv fragment, gold nanoparticles / nanorods, virus particles, liposomes, magnetic nanoparticles, a prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), a chemotherapeutic agent (e.g., cisplatin), or any form of nanoparticles, etc.

[0065] In an eighth aspect of the present invention, there is provided a pharmaceutical composition, which contains:

[0066] (a) a first active ingredient selected from the group consisting of: the IL-2 mutant protein as described in the first aspect of the present invention, the fusion protein as described in the second aspect of the present invention, the host cell as described in the fifth aspect of the present invention, the immunoconjugate as described in the seventh aspect of the present invention, or a combination thereof; and

[0067] (b) a pharmaceutically acceptable carrier.

[0068] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0069] In another preferred embodiment, the pharmaceutical composition is an injection.

[0070] In another preferred embodiment, the pharmaceutical composition further contains a second active ingredient, and the second active ingredient is an anti-tumor drug.

[0071] In another preferred embodiment, the second active ingredient is selected from the group consisting of: chemotherapeutic drugs, targeted drugs, immune stimulants, antibody-drug conjugates, polypeptide drugs, nucleic acid drugs.

[0072] In another preferred embodiment, the second active ingredient is a PD-1 antibody or a PD-L1 antibody.

[0073] In another preferred embodiment, the second active ingredient is atezolizumab.

[0074] In another preferred embodiment, the pharmaceutical composition is used for treating a disease.

[0075] In another preferred embodiment, the disease is a tumor with high expression of PD-L1.

[0076] In a ninth aspect of the present invention, there is provided the use of the IL-2 mutant protein as described in the first aspect of the present invention, the fusion protein as described in the second aspect of the present invention, the host cell as described in the fifth aspect of the present invention, the immunoconjugate as described in the sixth aspect of the present invention, or the pharmaceutical composition as described in the eighth aspect of the present invention in the preparation of a drug for treating tumors.

[0077] In another preferred embodiment, the tumor is a tumor with high expression of PD-L1.

[0078] In another preferred embodiment, the tumor is selected from the group consisting of: hematological tumors, solid tumors, or a combination thereof.

[0079] In another preferred embodiment, the hematological tumor is selected from the group consisting of: acute myeloid leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute granulocytic leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, chronic granulocytic leukemia, chronic myeloid leukemia, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma (MM), myelodysplastic syndrome, or a combination thereof.

[0080] In another preferred embodiment, the solid tumor is selected from the group consisting of: prostate cancer, liver cancer, head and neck cancer, melanoma, non-Hodgkin lymphoma, bladder cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, kidney cancer, mesothelioma, osteosarcoma, cholangiocarcinoma, ovarian cancer, gastric cancer, bladder cancer, meningioma, pancreatic cancer, multiple squamous cell carcinoma, esophageal cancer, small cell lung cancer, colorectal cancer, breast cancer, medulloblastoma, breast cancer, nasopharyngeal cancer, thymic cancer, or a combination thereof.

[0081] In another preferred embodiment, the tumor is selected from the group consisting of: colon cancer, kidney cancer, or a combination thereof.

[0082] In another preferred embodiment, the drug further contains other anti-tumor drugs.

[0083] In another preferred embodiment, the anti-tumor drug is selected from the group consisting of: chemotherapeutic drugs, targeted drugs, immune stimulants, antibody-drug conjugates, polypeptide drugs, nucleic acid drugs, or combinations thereof.

[0084] In another preferred embodiment, the anti-tumor drug is a PD-1 antibody or a PD-L1 antibody.

[0085] In another preferred embodiment, the anti-tumor drug is atezolizumab.

[0086] In a tenth aspect of the present invention, a method for treating a tumor is provided, the method comprising administering to a subject in need thereof an effective amount of the IL-2 mutant protein as described in the first aspect of the present invention, the fusion protein as described in the second aspect of the present invention, the host cell as described in the fifth aspect of the present invention, the immunoconjugate as described in the seventh aspect of the present invention, or the pharmaceutical composition as described in the eighth aspect of the present invention, or combinations thereof.

[0087] In another preferred embodiment, the tumor is a tumor with high expression of PD-L1.

[0088] In another preferred embodiment, the method further comprises treating the subject with another disease treatment method.

[0089] In another preferred embodiment, the other disease treatment method is selected from the group consisting of: surgery, radiotherapy, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, adjuvant therapy, immunotherapy, or combinations thereof.

[0090] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] The following drawings are used to illustrate the specific embodiments of the present invention, and are not used to limit the scope of the present invention defined by the claims.

[0092] Figure 1 Shows a schematic diagram of the fusion protein structure.

[0093] Figure 2 Shows the binding activity of the fusion protein to human PD-1 protein.

[0094] Figure 3Shows the binding differences of the fusion protein with human IL-2Rα (A), IL-2Rβγ (B), and IL-2Rαβγ (C).

[0095] Figure 4 Shows the functional study of KY-0118 in activating immune cells in vitro.

[0096] Figure 5 Shows the evaluation of the in vitro amplification activity of KY-0118 on PBMC.

[0097] Figure 6 Shows the evaluation of the ADCC effect of KY-0118.

[0098] Figure 7 Shows that KY-0118 induces PBMC to release IFN-γ.

[0099] Figure 8A and 8B Shows that KY-0118 induces PBMC-cell tumor co-cultures to release IFN-γ.

[0100] Figure 9 Shows the changes in body weight and tumor volume of tumor-bearing hPD-1 mice.

[0101] Figure 10 Shows the changes in the volume of 786-O xenografts in the PBMC humanized mouse model.

[0102] Figure 11 Shows the changes in body weight and tumor volume of the C57-hPD-1 mouse model.

[0103] Figure 12 Shows the expression level of PD-L1 on tumor cells.

[0104] Figure 13 Shows the detection results of IFN-γ, TNF-α, and IL-10.

[0105] Figure 14 Shows the detection result of IL-6.

[0106] Figure 15 Shows the changes in body weight and tumor volume of the C57-hPD-1-hPD-L1 mouse model.

[0107] Figure 16 Shows the changes in the volume of 786-O xenografts in the huPBMC-NCG-dko mouse model. Detailed implementation manners

[0108] After extensive and in-depth research, the inventor has developed a bifunctional fusion protein of an anti-PD-1 antibody and an IL-2 mutant for the first time. The bifunctional fusion protein of the present invention comprises a PD-1 antibody and an IL-2 mutant linked by a mutated FC. The bifunctional fusion protein of the present invention has the effects of activating and amplifying PD-1 positive lymphocytes and regulating the activity of Treg cells, and can activate tumor immunity to treat cancer. On this basis, the present invention has been completed.

[0109] Term

[0110] To facilitate a better understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0111] As used herein, the terms "comprising", "including", "containing" are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the said terms include "consisting of" and "consisting essentially of".

[0112] As used herein, the components of the term "pharmaceutically acceptable carrier" refer to substances that are suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit / risk ratio.

[0113] As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. Those of ordinary skill in the art should understand that the said "therapeutically effective amount" may vary depending on factors such as the form of the pharmaceutical composition, the route of administration, the excipients used in the drug, the severity of the disease, and the combination with other drugs.

[0114] The bifunctional fusion protein of the present invention

[0115] As used herein, the terms "fusion protein of the present invention", "bifunctional fusion protein of the present invention", "PD-1 antibody / IL-2 fusion protein of the present invention", "KY-0118" are used interchangeably and all refer to the fusion protein described in the second aspect of the present invention.

[0116] The fusion protein of the present invention comprises the following protein elements:

[0117] (i) An anti-human PD-1 antibody;

[0118] (ii) Fc fragment;

[0119] (iii) IL-2 mutant protein.

[0120] In the fusion protein of the present invention, the anti-PD-1 antibody can be any antibody or its antigen-binding fragment that has specific binding affinity for PD-1.

[0121] As used herein, "antigen-binding fragment" refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, or single Fv fragment that has antigen-binding activity. An Fv antibody contains the variable region of the heavy chain and the variable region of the light chain of the antibody, but no constant region, and is the smallest antibody fragment with all antigen-binding sites. Generally, an Fv antibody also contains a polypeptide linker between the VH and VL domains and can form the structure required for antigen binding. In one embodiment, the anti-PD-1 antibody is a single-chain antibody (scFv).

[0122] In the present invention, the scFv of the present invention also includes its conservative variants, which refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or close properties compared to the amino acid sequence of the scFv of the present invention.

[0123] In a preferred embodiment, the amino acid sequence of the anti-PD-1 antibody is as shown in SEQ ID NO:8.

[0124] As used herein, unless otherwise specified, Fc refers to the Fc fragment of human immunoglobulin. The term "immunoglobulin Fc region" refers to the constant region of the immunoglobulin chain, especially the carboxyl terminus of the constant region of the immunoglobulin heavy chain or a part thereof. For example, the immunoglobulin Fc region may include a combination of two or more domains of the heavy chain CH1, CH2, CH3 and the immunoglobulin hinge region. In a preferred example, the Fc region of the immunoglobulin used includes at least one immunoglobulin hinge region, one CH2 domain, and one CH3 domain, and preferably lacks the CH1 domain.

[0125] In a preferred embodiment, the Fc fragment in the fusion protein of the present invention is a mutant of the human IgG1 immunoglobulin Fc fragment. In one embodiment, the Fc fragment of the present invention has the following mutations relative to the wild-type IgG1 Fc fragment: L234A, L235A, P329G, but is not limited thereto. The Fc fragment of the present invention may also include any other mutations as long as the resulting fusion protein has reduced ADCC and CDC activities compared to the fusion protein having a wild-type Fc fragment.

[0126] In a preferred embodiment, the amino acid sequence of the Fc fragment is as shown in SEQ ID NO:11.

[0127] As used herein, the terms "IL-2 mutant" and "IL-2 mutant protein" both refer to the IL-2 mutant protein described in the first aspect of the present invention. Most traditional IL-2 drugs lead to pipeline termination due to excessive peripheral blood toxicity. The present invention designs mutations in IL-2 to make the mutant IL-2 preferentially bind to the IL-2Rβγ receptor, thereby improving the safety of the drug.

[0128] In one embodiment, the IL-2 mutant protein of the present invention has the following mutations relative to the wild-type IL-2 protein: P65K and C125S, but not limited thereto. The IL-2 mutant protein of the present invention (or the fusion protein of the present invention containing the IL-2 mutant protein) may also include any other mutations, as long as the resulting mutant has a reduced binding ability to IL-2Rα and an activation ability of Treg cells relative to the wild-type IL-2 protein, and has the same or substantially the same binding ability to IL-2Rβγ.

[0129] In a preferred embodiment, the amino acid sequence of the IL-2 mutant protein is as shown in SEQ ID NO:6.

[0130] In one embodiment, the structure of the fusion protein of the present invention is as Figure 1 shown in KY-0118, and the amino acid sequence is as shown in SEQ ID NO:1.

[0131] The fusion protein of the present invention also includes variant forms of the above fusion protein. These variant forms include (but are not limited to): deletion, insertion and / or substitution of 1-5 (usually 1-3, more preferably 1) amino acids, addition or deletion of one or several (usually within 5, preferably within 3, more preferably within 1) amino acids at the C-terminus and / or N-terminus, or addition of an amino acid fragment with a smaller amino acid side chain as a linker (such as glycine, serine, etc.) at the N-terminus or C-terminus of the protein. For example, in the art, when substituting amino acids with similar or similar properties, the function of the protein is usually not changed. Also, for example, adding or deleting one or several amino acids at the C-terminus and / or N-terminus usually does not change the structure and function of the protein. In addition, the term also includes monomeric and polymeric forms of the polypeptides of the present invention. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0132] The present invention also includes active fragments, derivatives and analogs of the above fusion protein. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the function or activity of the fusion protein of the present invention.

[0133] The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) polypeptides having substituent groups in one or more amino acid residues, or (iii) polypeptides formed by fusion of a polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (a fusion protein formed by fusion with a leader sequence, a secretion sequence or a tag sequence such as 6His). According to the teachings herein, these fragments, derivatives and analogs are within the scope well known to those skilled in the art.

[0134] A preferred class of active derivatives refers to polypeptides formed by replacing, compared with the amino acid sequence of the present invention, at most 5, preferably at most 3, more preferably at most 1 amino acid with an amino acid having similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0135] Table A

[0136]

[0137]

[0138] The present invention also provides analogs of the fusion proteins of the present invention. These analogs may differ from the polypeptides of the present invention either in amino acid sequence, or in modified forms that do not affect the sequence, or both. Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0139] In addition, the fusion proteins of the present invention can also be modified. The modification (usually without changing the primary structure) forms include: chemically derivatized forms of the polypeptide in vivo or in vitro such as acetylation or carboxylation. The modification also includes glycosylation, such as those polypeptides produced by glycosylation modification during the synthesis and processing of the polypeptide or in further processing steps. This modification can be accomplished by exposing the polypeptide to enzymes that carry out glycosylation (such as mammalian glycosylation enzymes or deglycosylation enzymes). The modification forms also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes polypeptides that are modified to improve their anti-proteolytic properties or optimize their solubility properties.

[0140] The term "polynucleotide of the present invention" may be a polynucleotide encoding the fusion protein of the present invention, or may also be a polynucleotide further including additional coding and / or non-coding sequences.

[0141] The present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or fusion proteins having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, allelic variants are alternative forms of a polynucleotide, which may be substitutions, deletions or insertions of one or more nucleotides, but do not substantially change the function of the encoded fusion protein.

[0142] The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions (or high stringency conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and washing at a lower ionic strength and a higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90% or more, preferably 95% or more.

[0143] The fusion proteins and polynucleotides of the present invention are preferably provided in isolated form, and more preferably, are purified to homogeneity.

[0144] The full-length sequence of the polynucleotide of the present invention can generally be obtained by PCR amplification, recombination or artificial synthesis methods. For PCR amplification, primers can be designed according to the nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template for amplification to obtain the relevant sequences. When the sequence is relatively long, it is often necessary to perform PCR amplification twice or more times, and then splice the fragments amplified each time together in the correct order.

[0145] Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination methods. This is usually to clone it into a vector, then transfer it into cells, and then isolate the relevant sequence from the proliferated host cells by conventional methods.

[0146] In addition, the relevant sequence can also be synthesized by artificial synthesis methods, especially when the fragment length is relatively short. Usually, a very long fragment can be obtained by first synthesizing multiple small fragments and then ligating them.

[0147] Currently, it is already possible to completely obtain the DNA sequence encoding the protein (or its fragment, or its derivative) of the present invention by chemical synthesis. Then this DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.

[0148] The method of amplifying DNA / RNA using PCR technology is preferably used to obtain the polynucleotides of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE method (rapid amplification of cDNA ends) can be preferably used. The primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.

[0149] Expression vector

[0150] The present invention also relates to a vector containing the polynucleotides of the present invention, a host cell genetically engineered with the vector of the present invention or the fusion protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

[0151] In the present invention, the polynucleotide sequence encoding the fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmid and vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene and translation control elements.

[0152] In the method for preparing the fusion protein of the present invention, any suitable vector can be used, which can be selected from one of pET, pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR. The expression vector includes a fusion DNA sequence ligated with appropriate transcriptional and translational regulatory sequences.

[0153] Both eukaryotic / prokaryotic host cells can be used for the expression of the fusion protein of the present invention. Eukaryotic host cells are preferably mammalian or insect host cell culture systems, preferably cells such as COS, CHO, NS0, sf9 and sf21; prokaryotic host cells are preferably one of DH5a, BL21(DE3), TG1.

[0154] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the fusion protein of the present invention and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The said DNA sequence can be effectively ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of such promoters are: the lac or trp promoter of Escherichia coli; the λ phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTRs of retroviruses, and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0155] In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0156] Vectors containing the appropriate DNA sequence as described above and appropriate promoters or control sequences can be used to transform appropriate host cells to enable them to express proteins.

[0157] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, plant cells (such as ginseng cells).

[0158] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs in length, which act on the promoter to enhance gene transcription. Examples include the 100 to 270 base pair SV40 enhancer on the late side of the replication origin, the polyomavirus enhancer on the late side of the replication origin, and the adenovirus enhancer, etc.

[0159] Those of ordinary skill in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0160] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well-known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps of which are well-known in the art. Another method is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0161] The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. The culture is carried out under conditions suitable for the growth of the host cell. After the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0162] The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well-known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0163] The fusion proteins disclosed in the present invention can be separated and purified by affinity chromatography. Depending on the characteristics of the affinity column used, conventional methods such as high salt buffer, pH change, etc. can be used to elute the fusion proteins bound to the affinity column.

[0164] Using the above methods, the fusion protein can be purified into a substantially homogeneous substance, such as a single band on SDS-PAGE electrophoresis.

[0165] Pharmaceutical composition

[0166] In the present invention, there is also provided a pharmaceutical composition containing the fusion protein or its immunoconjugate of the present invention.

[0167] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001 - 99 wt%, preferably 0.01 - 90 wt%, more preferably 0.1 - 80 wt%) of the fusion protein (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present invention can be made into an injection form, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms per kilogram of body weight per day - about 50 milligrams per kilogram of body weight. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents. The said fusion protein or its immunoconjugate can form a pharmaceutical preparation together with pharmaceutically acceptable excipients to exert its efficacy more stably. These preparations can ensure the structural integrity of the amino acid core sequence of the fusion protein of the present invention, and at the same time protect the multiple functional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation). The said preparations can be in various forms. Generally, for liquid preparations, they can be stored stably at least for one year at 2°C - 8°C, and for lyophilized preparations, they can remain stable at 30°C for at least six months. Here, the preparations can be suspensions, aqueous injections, lyophilized preparations, etc. commonly used in the pharmaceutical field, and aqueous injections or lyophilized preparations are preferred.

[0168] For the pharmaceutical composition of the present invention (such as aqueous injection or lyophilized preparation), the pharmaceutically acceptable excipients therein include one or a combination of a surfactant, a solution stabilizer, an isotonicity regulator, and a buffer solution. The surfactant includes non-ionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; myristyl, linoleyl or octadecyl sarcosine; Pluronics; MONAQUATTM, etc. The addition amount should minimize the tendency of the protein to granulate. The solution stabilizer can be sugars, including reducing sugars and non-reducing sugars, amino acids including monosodium glutamate or histidine, alcohols including trihydric alcohols, higher polyhydric alcohols, propylene glycol, polyethylene glycol, or a combination of them. The addition amount of the solution stabilizer should make the finally formed preparation maintain a stable state within a stable time considered by those skilled in the art. The isotonicity regulator can be one of sodium chloride and mannitol, and the buffer solution can be one of TRIS, histidine buffer solution, and phosphate buffer solution.

[0169] When using the pharmaceutical composition, a safe and effective amount of the fusion protein or its immunoconjugate of the present invention is administered to a mammal, wherein the safe and effective amount is usually at least about 50 micrograms per kilogram of body weight, and in most cases does not exceed about 100 milligrams per kilogram of body weight. Preferably, the dose is about 100 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight. Of course, the specific dose should also consider factors such as the administration route and the patient's health condition, which are all within the scope of the skills of a skilled physician. Typically, usually, the total administered amount cannot exceed a certain range. For example, the dose for intravenous injection is 10 to 3000 mg / day / 50 kg, preferably 100 to 1000 mg / day / 50 kg.

[0170] The fusion protein of the present invention and the pharmaceutical preparation containing the same can be used as an anti-tumor drug for tumor treatment. The anti-tumor drug referred to in the present invention means a drug having the effect of inhibiting and / or treating tumors, which may include the delay in the development of symptoms associated with tumor growth and / or the reduction in the severity of these symptoms. It further includes the alleviation of the symptoms already present in tumor growth and the prevention of the appearance of other symptoms, and also reduces or prevents metastasis.

[0171] The above-mentioned fusion protein and its pharmaceutical preparation can also be administered in combination with other anti-tumor drugs for the treatment of tumors. These anti-tumor drugs for combined administration include but are not limited to: 1. Cytotoxic drugs (1) Drugs that act on the chemical structure of DNA: alkylating agents such as nitrogen mustards, nitrosoureas, and methanesulfonates; platinum compounds such as cisplatin, carboplatin, and oxaliplatin; mitomycin (MMC); (2) Drugs that affect nucleic acid synthesis: dihydrofolate reductase inhibitors such as methotrexate (MTX) and Alimta; thymidine synthase inhibitors such as fluorouracils (5FU, FT-207, capecitabine, etc.; purine nucleoside synthetase inhibitors such as 6-mercaptopurine (6-MP) and 6-TG, etc.; ribonucleotide reductase inhibitors such as hydroxyurea (HU), etc.; DNA polymerase inhibitors such as cytarabine (Ara-C) and gemcitabine (Gemz), etc.; (3) Drugs that act on nucleic acid transcription: drugs that selectively act on DNA templates, inhibit DNA-dependent RNA polymerase, and thus inhibit RNA synthesis, such as actinomycin D, daunorubicin, doxorubicin, epirubicin, aclarubicin, mithramycin, etc.; (4) Drugs that mainly act on microtubules Drugs that inhibit protein synthesis: paclitaxel, taxotere, vinblastine, vinorelbine, podophyllotoxin, homoharringtonine; (5) other cytotoxic drugs: asparaginase mainly inhibits protein synthesis; 2. Hormonal antiestrogens: tamoxifen, droloxifene, exemestane, etc.; aromatase inhibitors: aminoglutethimide, lantron, letrozole, arimide, etc.; anti-androgen: flutamide RH-LH agonist / antagonist: nivolumab, enaton, etc.; 3. Biological response modifiers: interferon that mainly inhibits tumors through the body's immune function; other interleukins except IL-2; chest Adenosines; 4. Monoclonal antibodies: MabThera; Cetuximab (C225); Herceptin (Trastuzumab); Bevacizumab (Avastin); Yervoy (Ipilimumab); Pembrolizumab (Keytruda); Atezolizumab (Tecentriq); 5. Others, including some drugs whose mechanisms are currently unknown and require further research; cell differentiation inducers such as retinoids; apoptosis inducers.

[0172] The present invention also includes a pharmaceutical composition and a treatment method for treating tumors by combining the fusion protein of the present invention with atezolizumab.

[0173] The main advantages of the present invention include:

[0174] 1) In the bifunctional fusion protein of the present invention, the IL-2 mutant has been specially modified and designed (P65K and C125S mutations). Compared with wild-type IL-2, the bifunctional fusion protein of the present invention has substantially eliminated the affinity for human IL-2Rα and retained a high IL-2Rβγ dimer binding activity. The bifunctional fusion protein of the present invention preferentially activates lymphocytes expressing IL-2Rβγ, does not stimulate or stimulates Treg cells at a low level, and can significantly reduce the systemic toxicity caused by the IL-2 signaling pathway.

[0175] 2) The bifunctional fusion protein of the present invention can induce the release of a higher concentration of IFN-γ from PBMC. Compared with anti-PD-1 / PD-L1 monoclonal antibodies, the bifunctional fusion protein of the present invention can promote the release of a large amount of effector cytokines from PBMC, such as IFN-γ, and at the same time promote the efficient expansion of T cells. The bifunctional fusion protein of the present invention has a more efficient tumor cell killing ability.

[0176] 3) The bifunctional fusion protein of the present invention has a stronger tumor suppression effect at the animal experiment level, and its tumor suppression effect is significantly better than that of Nivolumab and commercial IL-2 in various tumor models.

[0177] 4) The present invention also provides a treatment method of combining the bifunctional fusion protein of the present invention with atezolizumab, which has high therapeutic efficacy and safety. Compared with the fusion protein alone and atezolizumab alone, the combined therapy significantly increases the release amount of IFN-γ, and at the same time, the release amounts of IL-6, IL-10, and TNF-α are not significantly increased, and the safety is good.

[0178] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0179] Example 1. Structural design, expression and preparation of the fusion protein

[0180] The structure of the fusion protein involved in the present invention is shown in Figure 1The basic information of each fusion protein is shown in Table 1, and the amino acid sequence information of the fusion protein is shown in Table 8. The structure of KY-0118 is VL-VH-Fc-IL-2v, the amino acid sequence of which is shown in SEQ ID NO: 1, the amino acid sequence of aPD-1-Fc-IL-2 is shown in SEQ ID NO: 2, the amino acid sequence of aPD-1-Fc is shown in SEQ ID NO: 3, the amino acid sequence of Fc-IL-2v is shown in SEQ ID NO: 4, the amino acid sequence of Fc-IL-2 is shown in SEQ ID NO: 5, the amino acid sequence of IL-2v is shown in SEQ ID NO: 6, and the amino acid sequence of IL-2 is shown in SEQ ID NO: 7.

[0181] The plasmid of the above fusion protein is transfected into 293 cells or CHO cells. After culture, the cells are lysed and the fusion protein is purified. The eluate is collected and concentrated, and the purified fusion protein is expressed for subsequent experiments.

[0182] Table 1 Summary of basic information of fusion proteins

[0183] SEQ ID NO: Fusion protein Number of amino acids Theoretical isoelectric point 1 KY-0118 600 8.36 2 aPD-1-Fc-IL-2 600 8.17 3 aPD-1-Fc 463 8.3 4 Fc-IL-2v 354 7.76 5 Fc-IL-2 354 7.25 6 IL-2v 133 7.99 7 IL-2 133 7.05

[0184] Example 2. Fusion protein can bind to human PD-1 protein

[0185] The experimental steps are as follows:

[0186] 1) Dilute the target protein PD-1 (His tag) to 0.5 μg / mL in PBS and add 100 μl per well of the ELISA plate for overnight coating.

[0187] 2) Remove the ELISA plate and wash it once by adding 300 μL PBST to each well;

[0188] 3) Prepare 3% BSA blocking solution with PBS buffer, add 300 μL of blocking solution to each well, cover the plate, and incubate at 37°C for 2 hours.

[0189] 4) Remove the ELISA plate and wash twice by adding 300 μL PBST to each well.

[0190] 5) Dilute the test samples (KY-0118, aPD-1-Fc-IL-2, Fc-IL-2v, Fc-IL-2, aPD-1-Fc), positive controls (Pembrolizumab, Nivolumab), and negative controls (Isotype) to 20 nM, and dilute them in a 5-fold to 8-fold series. Add 100 μl to each well, cover the plate, and incubate at 37°C for 1 hour.

[0191] 6) Remove the ELISA plate and wash the plate three times by adding 300 μL PBST to each well.

[0192] 7) After diluting the enzyme-labeled secondary antibody (Goat anti human-IgG Fc-HRP) at a ratio of 1:10000, add 100 μL to each well in the enzyme-labeled plate, cover the enzyme-labeled plate lid, and incubate statically at 37 °C for 0.5 hour;

[0193] 8) Take out the enzyme-labeled plate and wash the plate four times with 300 μL of PBST per well;

[0194] 9) Add 100 μL of TMB chromogenic solution to each well, develop color at room temperature for 5 min, add 50 μl of stop solution to terminate, and then perform detection on the machine;

[0195] The experimental results are as Figure 2 shown. KY-0118, aPD-1-Fc-IL-2, aPD-1-Fc and PD-1 (His tag) proteins all have good binding activities.

[0196] Example 3. Binding differences between the fusion protein and human IL-2Rα, IL-2Rβγ, IL-2Rαβγ proteins

[0197] The experimental steps are as follows:

[0198] 1) Dilute the target proteins IL-2Rα (His tag) and IL-2Rαβγ (His tag) to 1 μg / mL using PBS, and dilute the target protein IL-2Rβγ (His tag) to 2 μg / mL using PBS. Add 100 μl to each well of the enzyme-labeled plate and coat overnight;

[0199] 2) Take out the enzyme-labeled plate and wash the plate once with 300 μL of PBST per well;

[0200] 3) Prepare 3% BSA blocking solution using PBS buffer, add 300 μL of the blocking solution to each well, cover the enzyme-labeled plate lid, and incubate statically at 37 °C for 2 hours;

[0201] 4) Take out the enzyme-labeled plate and wash the plate twice with 300 μL of PBST per well;

[0202] 5) Dilute the test samples (KY-0118, aPD-1-Fc-IL-2, Fc-IL-2v, Fc-IL-2, aPD-1-Fc) and the negative control (Isotype) starting from 100 nM and 20 nM respectively, and perform serial dilutions in 8 gradients at a 5-fold ratio. Among them, the samples starting from 20 nM are added to the wells of the ELISA plate coated with IL-2Rα (His tag) and IL-2Rαβγ (His tag) at 100 μl per well, and the samples starting from 100 nM are added to the wells of the ELISA plate coated with IL-2Rβγ (His tag) at 100 μl per well. Cover the ELISA plate and incubate statically at 37 °C for 1 hour;

[0203] 6) Take out the ELISA plate and wash each well three times with 300 μL of PBST;

[0204] 7) Dilute the enzyme-labeled secondary antibody (Goat anti human-IgG Fc-HRP) at 1:10000, then add 100 μL to each well of the ELISA plate. Cover the ELISA plate and incubate statically at 37 °C for 0.5 hour;

[0205] 8) Take out the ELISA plate and wash each well four times with 300 μL of PBST;

[0206] 9) Add 100 μL of TMB chromogenic solution to each well, develop color at room temperature for 5 min, add 50 μl of stop solution to terminate the reaction, and then perform detection on the machine;

[0207] The experimental results are as Figure 3 shown, Figure 3 A, 3B indicate that aPD-1-Fc-IL-2 and Fc-IL-2 with an unmutated IL-2 segment have high binding activities to both IL-2Rα and IL-2Rαβγ proteins; KY-0118 and Fc-IL-2v with a mutated IL-2 segment do not show obvious binding activities to IL-2Rα and IL-2Rαβγ proteins; Figure 3 C indicates that the molecules before and after the mutation of the IL-2 segment maintain basically the same IL-2Rβγ binding activity.

[0208] IL-2 can efficiently activate immune cells, is the most potential tumor treatment target, and is also a target with relatively high R & D risks. IL-2 can bind to IL-2Rα, IL-2Rβγ, and IL-2Rαβγ, and activate T cells, Treg cells, and NK cells without discrimination. Therefore, in clinical research, the greatest R & D risk of IL-2-related drugs is excessive toxicity. KY-0118 preferentially activates lymphocytes expressing IL-2Rβγ, does not stimulate or stimulates Treg cells at a low level, and can significantly reduce the systemic toxicity caused by the IL-2 signaling pathway.

[0209] Example 4. Affinity of KY-0118 with FcγRs, FcRn, and C1q proteins

[0210] The experimental steps are as follows:

[0211] Detection of the affinity of KY-0118 with FcγRs:

[0212] 1) Chip preparation: Dilute the mouse anti-His antibody to 50 μg / mL with the immobilization reagent (10 mM sodium acetate, pH 4.5). Add 950 μL of the immobilization reagent to 50 μL of the mouse anti-His antibody for immobilizing eight channels. The surface of the CM5 chip was activated with 400 mM EDC and 100 mM NHS at a flow rate of 10 μL / min for 420 s. Subsequently, the 50 μg / mL antibody was injected into the channels (channel 1-8, Fc1,2) at a flow rate of 10 μL / min for about 420 s, and the immobilization amount was about 7000 to 14000 RU. After immobilization, the chip was blocked with 1 M ethanolamine at a flow rate of 10 μL / min for 420 s.

[0213] 2) Protein reconstruction: Reconstitute the protein lyophilized powder according to the product COA, and aliquot it in specifications of more than 10 μg per tube to avoid repeated freezing and thawing.

[0214] 3) Buffer replacement: Use a desalting column and running reagent 1 to replace the buffer of KY-0118, and measure the concentration of the replaced sample with SPECTROstarNano.

[0215] 4) Capture ligand: Dilute three Fcγ receptors to 0.25 μg / mL with running reagent 1 and inject them into the His capture chip experimental channel (Fc2) at a flow rate of 10 μL / min in sequence for about 100 RU. The reference channel (Fc1) does not require ligand capture.

[0216] 5) Multicycle analysis of the analyte: Dilute KY-0118 with running reagent 1. Inject the diluted KY-0118 into the experimental channel and the reference channel at a flow rate of 30 μL / min in sequence for the corresponding binding and dissociation times. The binding and dissociation steps are both carried out in running reagent 1. After each concentration analysis, the chip needs to be regenerated with 10 mM glycine hydrochloride at pH 1.5 at a flow rate of 30 μL / min for 60 s to wash away the ligand and the undissociated analyte. When performing the next concentration analysis, the experimental channel needs to recapture the same amount of ligand.

[0217] Detection of the affinity of KY-0118 with FcRn:

[0218] 1) Chip preparation: Dilute the mouse anti-His antibody with a fixation reagent (10 mM sodium acetate, pH 4.5) to 50 μg / mL. Add 950 μL of the fixation reagent to 50 μL of the mouse anti-His antibody to fix eight channels. First, activate the surface of the CM5 chip with 400 mM EDC and 100 mM NHS at a flow rate of 10 μL / min for 420 s. Second, inject the 50 μg / mL mouse anti-His antibody into the channels (channel1-8, Fc1, 2) at a flow rate of 10 μL / min for about 420 s, and the immobilization amount is about 7000 to 15000 RU. Finally, block the chip with 1 M ethanolamine at a flow rate of 10 μL / min for 420 s.

[0219] 2) Protein refolding: Refold the protein lyophilized powder according to the product COA, aliquot it in specifications of more than 10 μg per tube, and avoid repeated freezing and thawing.

[0220] 3) Buffer replacement: Use a desalting column and running reagent 2 to replace the buffer of KY-0118, and measure the concentration of the replaced sample with SPECTROstar Nano.

[0221] 4) Capture ligand: Dilute the FcRn protein with running reagent 2 to 0.25 μg / mL and inject it into the His capture chip experimental channel (Fc2) at a flow rate of 10 μL / min for about 100 RU. The reference channel (Fc1) does not require ligand capture.

[0222] 5) Multi-cycle analysis of analytes: Dilute KY-0118 with running reagent 2 by a factor of 2 in a serial dilution. Inject the three diluted antibodies into the experimental channel and the reference channel at a flow rate of 30 μL / min for the corresponding binding and dissociation times. The binding and dissociation steps are both carried out in running reagent 2. After each concentration analysis, the chip needs to be regenerated with glycine hydrochloride at pH 1.5 at a flow rate of 30 μL / min for 60 s to wash off the ligand and the undissociated analytes. When performing the next concentration analysis, the experimental channel needs to recapture the same amount of ligand.

[0223] Affinity detection of KY-0118 and C1q:

[0224] 1) Protein refolding: Refold the protein lyophilized powder according to the product COA, aliquot it in specifications of more than 10 μg per tube, and avoid repeated freezing and thawing.

[0225] 2) Buffer replacement: Use a desalting column and running reagent to replace the buffer of KY-0118 and C1q, and measure the concentration of the replaced sample with SPECTROstar Nano.

[0226] 3) Chip preparation: Dilute KY-0118 with a fixing reagent (10 mM sodium acetate, pH 4.5) to 10 μg / mL. Activate the surface of the CM5 chip with 400 mM EDC and 100 mM NHS at a flow rate of 10 μL / min for 420 s. Secondly, inject 10 μg / mL of KY-0118 into the experimental channel (Fc2) at a flow rate of 10 μL / min to immobilize approximately 1000 RU. Finally, block the chip with 1 M ethanolamine at a flow rate of 10 μL / min for 420 s. The reference channel (Fc1) is not injected with KY-0118, and other operations are the same as those of the experimental channel (Fc2).

[0227] 4) Multicycle analysis of analytes: Dilute C1q two-fold serially with running reagent 1. Inject the diluted C1q into the experimental channel and the reference channel at a flow rate of 30 μL / min in sequence, and record the corresponding binding and dissociation times. The binding and dissociation steps are both carried out in the running reagent. After analyzing each concentration, the chip needs to be regenerated with the running reagent at a flow rate of 30 μL / min for 60 s to wash away the undissociated analytes.

[0228] The experimental results are shown in Table 2. KY-0118 has a strong affinity for human FcRn, weak binding to CD64 and CD32a, and no binding to CD16a and C1q.

[0229] Table 2 Affinity test results of KY-0118 with human FcγRs, FcRn, and C1q proteins

[0230]

[0231] Example 5. Effect of KY-0118 on immune cell activation

[0232] The experimental procedure is as follows:

[0233] 1) Dilute the KY-0118 and wild-type IL-2 samples in 12 gradients, from 0.00001 to 1000000 pM.

[0234] 2) Wash the PBMC cells and resuspend them with PBS; centrifuge at 400 g for 8 min to remove the supernatant.

[0235] 3) Resuspend the PBMC cells with 1640 basal medium, seed them in 96-well plates at 50 μL / well, add the drugs (50 μL drug + 50 μL PBMC), and incubate at 37 °C for 15 min.

[0236] 4) Add 1 mL of BD Cytofix Buffer, vortex thoroughly, fix at 37 °C for 10 min, centrifuge at 2000 rpm for 5 min to remove the supernatant.

[0237] 5) Add 1 mL of Stain buffer (FBS) to each tube, resuspend and wash once, centrifuge at 2000 rpm for 5 min, and discard the supernatant.

[0238] 6) Add 0.5 mL of Perm Buffer III pre-cooled at -20°C to each tube, vortex to mix well, and incubate at 4°C for 30 min for membrane permeabilization (or incubate on ice for 30 min).

[0239] 7) Centrifuge at 2000 rpm for 5 min to discard the supernatant, add 1 mL of Stain buffer (FBS), add Fc-blocker to each 100 μL reaction system, and incubate at room temperature in the dark for 10 min; after the reaction is completed, add 1 mL of Stain buffer (FBS), wash the cells once, and centrifuge at 2000 rpm for 5 min to discard the supernatant.

[0240] 8) Prepare a Mix of CD3 (FITC), CD8 (PerCP), CD56 (PE), and STAT5 (APC) using Stain buffer (FBS) at 100 μL / tube, and incubate at room temperature in the dark for 40 min. Alexa Prepare a Mix of the Alexa 488 anti-human FOXP3 / CD25 PE / CD4 PerCP antibody mixture and STAT5 (APC) in the Treg detection kit. The staining method refers to the detection steps of the BD STAT5 antibody, and incubate at room temperature for 40 min.

[0241] 9) Add 1 mL of Stain buffer (FBS), centrifuge at 2000 rpm for 5 min to discard the supernatant; resuspend the cells with 200 μL of Stain buffer (FBS) and perform flow cytometry detection.

[0242] As Figure 4 The results showed that the activation ability of KY-0118 on CD8+ T cells and NK cells was similar to that of wild-type IL-2. The activation ability of KY-0118 on CD4+ T cells was weaker than that of wild-type IL-2. The activation ability of KY-0118 on Treg cells was significantly weaker than that of wild-type IL-2, indicating that the mutation at the IL-2 site in KY-0118 effectively reduced the binding to IL-2Rα and weakened the activation of Treg cells. The EC50 value of KY-0118 for the activation of Treg cells was 190.6 pM, and the EC50 value of IL-2 for the activation ability of Treg cells was <0.0001 pM.

[0243] Example 6. Effect of KY-0118 on the in vitro expansion activity of immune cells

[0244] The experimental procedure is as follows:

[0245] 1) Centrifuge PBMC cells (4×10⁶ cells), and wash them once with PBS.

[0246] 2) Dilute the 2 mM CFSE stock solution (100 μg dissolved in 90 μL DMSO) with PBS at a ratio of 1:20 to a final concentration of 0.1 nM.

[0247] 3) Resuspend the PBMC in pre-warmed PBS (10 mL), add 10 μL of the CFSE dilution, and incubate at 37 °C for 15 min.

[0248] 4) Add 10 mL of pre-warmed complete medium (1640 + 10% FBS + 1% triple antibody) to terminate the reaction, centrifuge at 400 g for 10 min, discard the supernatant, resuspend the cells in 20 mL of fresh medium, and incubate at 37 °C for 30 min.

[0249] 5) Centrifuge the cells at 400 g for 10 min, discard the supernatant, and resuspend them in 2.5 mL of fresh medium. The cell density is approximately 1×10⁶ - 1.5×10⁶ cells / mL.

[0250] 6) Seed the cells at 1×10⁵ - 1.5×10⁵ cells / well into a 96-well plate, 100 μL per well, and add 100 μL of the drug at the corresponding concentration.

[0251] 7) Incubate for 5 days. Wash the cells once with PBS containing FBS.

[0252] 8) Add the flow antibody mixture for staining. The antibody mixture: CD3 (PerCP), CD4 (APC), CD56 (PE).

[0253] 9) Incubate at room temperature in the dark for 40 min.

[0254] 10) Wash twice with PBS containing FBS, centrifuge at 400 g for 5 min, and perform flow cytometry analysis (cell population: CD8⁺ T cells are defined as CD3⁺CD4⁻, CD4⁺ T cells are defined as CD3⁺CD4⁺, and NK cells are defined as CD3⁻CD56⁺).

[0255] As Figure 5 The experimental results showed that KY-0118 could promote the expansion of CD4⁺ T cells and CD8⁺ T cells. Based on the P65K and C125S mutations of IL-2V in KY-0118, the expansion activity of KY-0118 was weaker than that of IL-2, and the results were in line with the design expectations.

[0256] Example 7. Evaluation of the ADCC effect of KY-0118

[0257] The experimental steps are as follows:

[0258] 1) Dilute the sample protein with medium containing 10% FBS to 200, 40, 10, 2.5, 0.625, 0.1562, 0.0390, and 0.0040 nM, transfer to a cell plate, 50 μL per well.

[0259] 2) Transfer PD-1-CHO cells to a centrifuge tube, centrifuge to remove the supernatant, resuspend with RPMI1640 medium containing 10% FBS, count, and adjust the cell density to 4×10 5 cells / mL.

[0260] 3) Centrifuge CD16-NF-AT-jurkat cells to remove the supernatant, resuspend with RPMI1640 medium containing 10% FBS, count, and adjust the cell density to 2×10 6 cells / mL.

[0261] 4) Mix PD-1-CHO cells and CD16-NF-AT-jurkat cells thoroughly, transfer to a 96-well plate containing the sample dilution, 50 μL per well, and culture in an incubator at 37 °C and 5% CO2 for 6 hours.

[0262] 5) Take out the cell plate before detection, equilibrate at room temperature for 10 minutes, add Bright-Lite detection solution equilibrated to room temperature, 50 μL per well, and perform flow cytometry detection.

[0263] As Figure 6 The experimental results showed that Anti-hPD-1-Ni-hIgG1 was the ADCC positive control antibody, and Anti-βGal-hIgG1 was the ADCC negative control antibody. Based on the LALA PG mutation in the Fc part of KY-0118, KY-0118 did not have an ADCC effect, which was consistent with the weak binding phenomenon of CD32a and CD64 shown in Example 4.

[0264] Example 8. KY-0118 induces PBMC to release IFN-γ

[0265] The experimental steps are as follows:

[0266] 1) Activation of hPBMC: Fresh hPBMC, after balancing, use a centrifuge at 400 G for 5 min, pour out the supernatant, tilt the centrifuge tube for about 2 min, and use a 200 μL pipette to aspirate the remaining liquid at the tube mouth. Resuspend with medium (X-Vivo15 Medium + 10% inactivated FBS + 1% P / S), and adjust the cell density to 2×10 6 cells / mL. Transfer to a T175 cell culture flask, add 10 nM Anti-CD3 mAb and 2 nM Anti-CD28 mAb, and incubate in a cell culture incubator for 24 h.

[0267] 2) After 24 h, wash the hPBMC three times with PBS solution. Resuspend with medium (RPMI 1640 + 10% inactivated FBS + 1% P / S + 55 μM 2-Mercaptoethanol) for the last time, and adjust the cell density to 2×10 6 cells / mL, and seed in 96-well plates at a density of 100 μL, 2×10 5 cells / well.

[0268] 3) Dissolve the sample (KY-0118) in water for injection to 10 mg / mL. Dilute the Isotype and KY-0118 samples with RPMI 1640 + 10% inactivated FBS + 1% P / S + 55 μM 2-Mercaptoethanol. There are two concentrations of Isotype: 100 nM and 10 nM. There are 9 concentrations of KY-0118, with the starting concentration of 2000 nM, diluted in a 4-fold gradient. Set up a negative control (Medium control). Add 100 μL of the sample and the control group to the wells, and incubate in a cell culture incubator for 72 h.

[0269] 4) After 72 h, centrifuge at 350 g for 5 minutes, and collect 150 μL of cell supernatant. Detect the IFN-γ content in the cell supernatant using a human IFN-γ ELISA detection kit.

[0270] The experimental results are as Figure 7 shown, KY-0118 can dose-dependently induce hPBMC cells to release IFN-γ factor.

[0271] Example 9. KY-0118 can promote the release of IFN-γ from T cell-tumor co-cultures

[0272] The experimental steps are as follows:

[0273] 1) Activation of hPBMC: For fresh hPBMC, after balancing, centrifuge at 400 G for 5 min using a centrifuge, pour out the supernatant, tilt the centrifuge tube for about 2 min, and aspirate the liquid remaining at the tube mouth using a 200 μL pipette. Resuspend with medium (X-Vivo15 Medium + 10% inactivated FBS + 1% P / S), and adjust the cell density to 2×10 6 cells / mL. Transfer to a T175 cell culture flask, add T cell Transact reagent or 10 nM Anti-CD3 mAb and 2 nM Anti-CD28 mAb, and incubate in a cell culture incubator for 24 h.

[0274] 2) After 24 h, wash the hPBMC three times with PBS solution. Resuspend with medium (RPMI 1640 + 10% inactivated FBS + 1% P / S + 55 μM 2-Mercaptoethanol) for the last time, and adjust the density to 1×10 6 cells / mL, and seed in 96-well plates at a density of 100 μL, 1×10 5 cells / well.

[0275] 3) Treat the tumor cells in the logarithmic growth phase with trypsin at room temperature for 3 - 10 min to detach the adherent cells from the bottom of the culture flask. Add 4 - 6 times the volume of trypsin of 1640 complete medium or an equal volume of FBS for termination treatment. Centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend with medium (RPMI 1640 + 10% inactivated FBS + 1% P / S + 55 μM 2-Mercaptoethanol). Seed the tumor cells in the hPBMC 96-well plates at 50 μL cells per well according to the ratios of hPBMC:Tumor cell = 2:1 and 4:1 respectively.

[0276] 4) Dissolve the sample (KY-0118) in water for injection to 10 mg / mL. Dilute the KY-0118 sample and Nivolumab with RPMI 1640 + 10% inactivated FBS + 1% P / S + 55 μM 2-Mercaptoethanol. Set up a negative control (Medium control). Add 50 μL of the sample and the control group to the wells and incubate in a cell culture incubator for 72 h.

[0277] 5) After 72 h, centrifuge at 350 g for 5 minutes and collect 150 μL of cell supernatant. Detect the IFN-γ content in the cell supernatant using the human IFN-γ ELISA detection kit.

[0278] Results: Figure 8A This is the effect of KY-0118 and Nivolumab on the release of IFN-γ under the co-incubation condition of PBMC and 4 kinds of tumor cells (H1993, A498, U-87MG, HCT116) at an effector-to-target ratio of 2:1.

[0279] Figure 8A The results showed that 31.25 nM and 0.488 nM of KY-0118 could significantly increase the release of IFN-γ in the co-incubation system of PBMC and tumor cells, showing significant differences compared with NC. In this experimental system, Nivolumab weakly promoted the release of IFN-γ, and the release of IFN-γ in the 31.25 nM KY-0118 experimental group was significantly better than that in the 33 nM Nivolumab experimental group.

[0280] Figure 8B The effects of KY-0118 and Nivolumab on the release of IFN-γ under the co-incubation condition of PBMC with 4 kinds of tumor cells (H1993, A498, U-87MG, HCT116) at an effector-to-target ratio of 4:1.

[0281] Figure 8B The results showed that KY-0118 at 31.25 nM and 0.488 nM could significantly increase the release of IFN-γ in the co-incubation system of PBMC and tumor cells, with significant differences compared to the NC group. In this experimental system, Nivolumab weakly promoted the release of IFN-γ, and the release of IFN-γ in the 31.25 nM KY-0118 experimental group was significantly better than that in the 33 nM Nivolumab experimental group.

[0282] Figure 8A and Figure 8B The results showed that, under the condition that other experimental conditions remained the same, the release of IFN-γ in the effector-to-target ratio of 4:1 experimental group was lower than that in the effector-to-target ratio of 2:1 experimental group.

[0283] Example 10. The experimental steps for KY-0118 to significantly inhibit the growth of MC38 transplanted tumors in hPD-1 mice at low doses are as follows:

[0284] 1) Inoculate MC38 cells subcutaneously in the right anterior hypochondrium of female C57-hPD-1 mice (8 - 10 weeks old, 50 mice);

[0285] 2) When the tumors grow to about 80 - 120 mm 3 divide them into groups, with 6 mice in each group, a total of 7 groups. The groupings and drug administrations are shown in Table 3:

[0286] Table 3 Information table of mouse grouping and drug administration

[0287]

[0288] 3) Administer by tail vein injection to the vehicle control group, KY-0118 0.05 mg / kg group, KY-0118 0.15 mg / kg group, KY-0118 0.5 mg / kg group, and Fc-IL-2v group at 0.29 mg / kg, once a week for 3 weeks; administer by intraperitoneal injection to the Nivolumab 3 mg / kg group, once a week for 3 weeks; administer by subcutaneous injection to the IL-2 group, once a day, 5 times a week for 3 weeks;

[0289] 4) Measure the tumor volume and body weight twice a week, and record the relationship between the changes in the body weight and tumor volume of tumor-bearing mice and the drug administration time;

[0290] 5) Calculate the tumor volume and tumor growth inhibition rate:

[0291] The tumor volume (V) is calculated as: (length × width 2 ) / 2

[0292] The tumor growth inhibition rate (TGI%) is calculated using the following formula:

[0293] Tumor growth inhibition rate = (1 - change in tumor volume in the drug - treated group / change in tumor volume in the control group) × 100%

[0294] The experimental results are as Figure 9 shown. In the MC38 tumor cell C57 - hPD - 1 mouse model, KY - 0118 at doses of 0.05 mg / kg, 0.15 mg / kg, and 0.5 mg / kg could effectively inhibit the growth and proliferation of tumor cells MC38 in mice. The tumor inhibition rates on Day 20 were 81.8%, 92.3%, and 99.2% respectively, showing a dose - dependent relationship; at the dose of Fc - IL - 2v 0.29 mg / kg, the tumor inhibition rate on Day 20 was - 26.4%, showing no tumor - inhibiting effect; at the dose of Nivolumab 3 mg / kg, the tumor inhibition rate on Day 20 was 95.2%, showing a significant tumor - inhibiting effect; after multiple administrations of IL - 2 (200,000 IU / time, QD×5 / week×3), the tumor inhibition rate on Day 20 was 7.6%, showing almost no tumor - inhibiting effect; the above results indicate that KY - 0118 at doses of 0.05 mg / kg and above can effectively inhibit the growth and proliferation of mouse colon cancer MC38 cells in mice, and shows a dose - dependent relationship; the tumor - inhibiting effect of KY - 0118 is better than that of non - targeted Fc - IL - 2v at equimolar doses; the tumor - inhibiting effect of KY - 0118 is better than that of high - dose IL - 2; at approximately the same tumor - inhibiting effect, the dose of KY - 0118 used is much lower than the dose of Nivolumab.

[0295] Example 11. The experimental steps for KY - 0118 to effectively inhibit the growth of 786 - O xenografts in PBMC humanized mice are as follows:

[0296] 1) Subcutaneously inoculate human renal cancer 786 - O cells on the right anterior hypochondrium of 80 female PBMC humanized B2M mice.

[0297] 2) When the tumor grows to about 100 - 150 mm 3 in size, randomly divide the mice into 6 groups, with 10 mice in each group. The grouping situation is shown in Table 4:

[0298] Table 4 Information table of mouse grouping and drug administration

[0299]

[0300] 3) The vehicle control group, KY-0118 0.08 mg / kg group, KY-0118 0.2 mg / kg group, and KY-0118 0.5 mg / kg group were given an injection via the tail vein once a week for 4 weeks; the Nivolumab 3 mg / kg group was given an injection via the abdominal cavity once a week for 4 weeks; the IL-2 group was given a subcutaneous injection once a day, 5 times a week for 4 weeks.

[0301] 4) The tumor volume and body weight were measured twice a week, and the relationship between the body weight and tumor volume of the tumor-bearing mice and the administration time was recorded.

[0302] The experimental results showed ( Figure 10 ), in the 786-O tumor cell PBMC humanized B2M mouse model, the doses of KY-0118 at 0.08 mg / kg, 0.2 mg / kg, and 0.5 mg / kg could effectively inhibit the growth and proliferation of tumor cells 786-O in mice. The tumor inhibition rates on Day 28 were 45.75%, 62.93%, and 73.78% respectively, showing a dose-dependent relationship; the tumor inhibition rate of the Nivolumab 3 mg / kg dose on Day 28 was 6.41%, showing no tumor inhibition effect; the tumor inhibition rate of IL-2 after multiple administrations (200,000 IU / time, QD×5 / week×4) on Day 28 was 64.95%, showing a tumor inhibition effect; the above results indicated that KY-0118 at a dose of 0.08 mg / kg and above could effectively inhibit the growth and proliferation of human renal cancer 786-O cells in mice, and showed a dose-dependent relationship; the Nivolumab 3 mg / kg group with a dose higher than that of KY-0118 also showed no tumor inhibition effect; the tumor inhibition effect of KY-0118 was better than that of high-dose IL-2; at an approximate tumor inhibition effect (64.95% vs 62.93%), the dosage of IL-2 (200,000 IU / time, QD×5 / week×4) was much higher than that of KY-0118 (0.2 mg / kg, QW×4).

[0303] Example 12. KY-0118 can significantly inhibit the growth of Panc02-CDX xenografts in hPD-1 mice

[0304] The experimental steps are as follows:

[0305] 1) Prepare 60 female C57-hPD-1 mice (8-10 weeks old) and 20 female C57 mice (8-10 weeks old).

[0306] 2) Inoculate Panc02 cells subcutaneously into the right anterior costal region of the above mice.

[0307] 3) When the tumor grows to about 80 - 120 mm3, group the mice. There are 8 C57-hPD-1 mice in each of the 6 groups; there are 8 C57 mice in each of the 2 groups. The groupings and drug administrations are shown in Table 5:

[0308] Table 5 Information Table of Mouse Grouping and Drug Administration

[0309]

[0310] 4) Administer by tail vein injection to the C57-hPD-1 vehicle control group, the C57-hPD-1 KY-0118 0.1 mg / kg group, the C57-hPD-1 KY-0118 0.3 mg / kg group, the C57-hPD-1 KY-0118 1 mg / kg group, as well as the C57 vehicle control group and the C57 KY-0118 1 mg / kg group, once a week for 4 weeks; administer by intraperitoneal injection to the C57-hPD-1 Nivolumab 3 mg / kg group, once a week for 4 weeks; administer by subcutaneous injection to the C57-hPD-1 IL-2 group, once a day, 5 days a week for 4 weeks;

[0311] 5) Measure the tumor volume and body weight twice a week, and record the relationship between the changes in the body weight and tumor volume of the tumor-bearing mice and the drug administration time;

[0312] 6) Calculate the tumor volume and tumor growth inhibition rate:

[0313] The tumor volume (V) is calculated as: (length × width 2 ) / 2

[0314] The tumor growth inhibition rate (TGI%) is calculated using the following formula:

[0315] Tumor growth inhibition rate = (1 - change in tumor volume of the drug treatment group / change in tumor volume of the control group) × 100%

[0316] The experimental results show ( Figure 11) In the Panc02 tumor cell C57-hPD-1 mouse model, doses of KY-0118 at 0.1 mg / kg, 0.3 mg / kg, and 1 mg / kg could effectively inhibit the growth and proliferation of tumor cells Panc02 in mice. The tumor inhibition rates on Day 27 were 78.1%, 96.2%, and 100% respectively, showing a dose-dependent relationship. Moreover, the tumor inhibition rate of KY-0118 at 1 mg / kg reached 100% on Day 17; the tumor inhibition rate of Nivolumab at 3 mg / kg on Day 27 was 49.9%, showing a tumor inhibition effect; after multiple administrations of IL-2 (200,000 IU / time, QD×5 / week×4), the tumor inhibition rate on Day 27 was 13.0%, showing almost no tumor inhibition effect; in the Panc02 tumor cell C57 mouse model, the tumor inhibition rate of KY-0118 at 1 mg / kg on Day 17 was 30.2%.

[0317] The above results indicate that KY-0118 at a dose of 0.1 mg / kg and above can effectively inhibit the growth and proliferation of mouse pancreatic cancer Panc02 cells in mice, and shows a dose-dependent relationship; the tumor inhibition effect of equal-dose KY-0118 in the targetable model is better than that in the non-targetable model; the tumor inhibition effect of 0.1 mg / kg KY-0118 is better than that of high-dose IL-2 at 200,000 IU / time (molar amount of KY-0118: molar amount of IL-2 = 1:187); with a better tumor inhibition effect, the dosage of 0.1 mg / kg KY-0118 is lower than the dosage of 3 mg / kg Nivolumab (molar amount of KY-0118: molar amount of Nivolumab = 1:14). KY-0118 shows a significant synergistic effect.

[0318] The results of Examples 10 - 12 show that compared with Nivolumab and commercial IL-2, KY-0118 has a stronger tumor inhibition effect and shows a dose-dependent trend.

[0319] Example 13. Screening of tumor cell lines with high expression of PD-L1 antigen

[0320] The experimental steps are as follows:

[0321] 1) Take out the cryopreserved tumor cells from liquid nitrogen, resuscitate them in a 37°C water bath, transfer them to a centrifuge tube containing complete medium, centrifuge and discard the supernatant, resuspend with 1×PBS, and centrifuge and wash once to remove DMSO in the cryopreservation solution.

[0322] 2) Resuspend each tumor cell with the corresponding medium, transfer it to a culture flask, and change the medium after culturing for 24 h.

[0323] 3) Wait until the cells adhere and grow to a density of 80%, then digest and passage.

[0324] 4) After each cell passage, culture again until reaching a density of 80%, and then perform the identification of the PD-L1 marker.

[0325] 5) Digest the adherent tumor cells with trypsin. After stopping with complete medium, centrifuge and discard the supernatant, then resuspend with PBS, centrifuge and wash once to remove the residual trypsin. Resuspend with the corresponding medium for tumor cells and count.

[0326] 6) Dilute the APC-anti-human CD274 (B7-H1, PD-L1) Antibody and the APC-Mouse IgG2bκ Isotype Ctrl Antibody 50-fold respectively with FACS buffer (1×PBS + 2% FBS) for later use.

[0327] 7) Parallel operation for the tumor cells to be tested: Take out 5×10 5 cells of each cell, resuspend with FACS buffer to 2×10 6 cells / mL. Take out 2 portions of 100 μL of cells. One portion is fully mixed with 100 μL of the 50-fold diluted APC-anti-human CD274 (B7-H1, PD-L1) Antibody as the PD-L1 positive group; one portion is fully mixed with 100 μL of the 50-fold diluted APC-Mouse IgG2b,κ Isotype Ctrl Antibody as the negative control, and incubate at 4°C for 60 min.

[0328] 8) Resuspend the cells with FACS buffer, centrifuge and discard the supernatant. After washing 3 times, resuspend and mix well with 200 μL of FACS buffer, and read the data with a flow cytometer. Adjust the positive rate of the negative control to 1% as the background to record the PD-L1 positive rate of the PD-L1 positive group.

[0329] The results are as Figure 12 shown. The PD-L1 antigen of EBC-1, NCI-H1975, HCC827, NCI-H1993, NCI-H596, A498, 786-O, Hs746T, HCT116, U-87MG cells has high expression, and the positive rate can reach over 95%.

[0330] Example 14. In vitro activity of KY-0118 combined with atezolizumab

[0331] The experimental steps are as follows:

[0332] Detection of the release amounts of IFN-γ, TNF-α and IL-10:

[0333] 1) Fresh hPBMCs were purchased from the manufacturer, centrifuged and resuspended, and then resuspended with the activation medium (X-Vivo15 medium containing anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody), counted, and adjusted to a density of 2×10 6 cells / mL, and activated in an environment of 37°C and 5% CO2 for 24 h.

[0334] 2) The hPBMCs after 24 h of activation were washed 4 times with 1×PBS, resuspended and counted with the co-incubation medium (1640 medium + 10% FBS + 1% P / S + 55 μM mercaptoethanol), and the cell density was adjusted to 5×10 5 cells / mL. 100 μL per well was added to a 96-well cell culture plate.

[0335] 3) Human renal cancer cells A498 in the logarithmic growth phase were digested, resuspended with the co-incubation medium and adjusted to a density of 1×10 6 cells / mL, and 50 μL per well was added to the 96-well cell culture plate containing hPBMCs.

[0336] 4) The test samples were prepared and 50 μL per well was added to the above 96-well cell culture plate, and cultured in an environment of 37°C and 5% CO2 for 72 h.

[0337] 5) After 72 h, the supernatant was obtained by centrifugation, appropriately diluted and then detected using the BD CBA detection kit.

[0338] 6) The beads in the kit were mixed evenly in equal volume to obtain mixed beads.

[0339] 7) 50 μL of the sample supernatant, mixed beads and PE detection antibody were mixed, and after mixing, they were protected from light at room temperature for 3 h.

[0340] 8) After washing 3 times with 1×Wash buffer, it was resuspended with 1×Wash buffer and read on the machine. Linear fitting was used to calculate the cytokine content in the supernatant.

[0341] The results are as Figure 13 shown. The IL-2 group was used as a positive control. Compared with Isotype and NC, KY-0118 alone could significantly enhance the release of IFN-γ in this experiment; however, atezolizumab alone could not (or weakly) promote the release of IFN-γ. The combination of atezolizumab + KY-0118 could significantly increase the release of IFN-γ compared with KY-0118 alone. At multiple dosing concentrations, it was observed that the increase in the release amount of IFN-γ caused by the combination therapy was higher than the sum of the two single-drug groups, indicating that the combination therapy of atezolizumab + KY-0118 had a synergistic effect. Within a certain range, as the concentration of atezolizumab increased, the release amount of IFN-γ also increased.

[0342] Compared with KY-0118 alone and atezolizumab alone, the combination of atezolizumab + KY-0118 stimulated a certain increase in the release of TNF-α and IL-10 from hPBMC, but the release amounts were both at relatively low levels.

[0343] Detection of IL-6 release amount:

[0344] 1) Fresh hPBMC were purchased from the manufacturer, centrifuged and resuspended, and then resuspended with activation medium (X-Vivo15 medium containing anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody), counted, and adjusted to a density of 2×10 6 cells / mL, and activated at 37°C in a 5% CO2 environment for 24 h.

[0345] 2) The hPBMC after 24 h of activation were washed 4 times with 1×PBS, resuspended and counted with co-incubation medium (X-Vivo15 medium + 1% P / S + 55 μM 2-mercaptoethanol), and the cell density was adjusted to 5×10 5 cells / mL. Added to a 96-well cell culture plate at 100 μL / well.

[0346] 3) The human renal cancer cell line A498 in the logarithmic growth phase was digested, resuspended with co-incubation medium and adjusted to a density of 1×10 6 cells / mL, and added to the 96-well cell culture plate containing hPBMC at 50 μL / well.

[0347] 4) Prepare the test samples and add 50 μL / well to the above 96-well cell culture plate, and culture at 37°C in a 5% CO2 environment for 72 h.

[0348] 5) After 72 h, centrifuge to obtain the supernatant, appropriately dilute it and then detect it using an ELISA detection kit.

[0349] 6) Mix 50 μL each of Biotin-Anti-IL-6 Antibody in the kit and the diluted sample supernatant (including the standard curve), and incubate at room temperature for 1 h. Wash 5 times with 1×Wash buffer.

[0350] 7) Add 50-fold diluted Streptavidin-HRP, 50 μL / well, and incubate at room temperature for 30 min. Wash 5 times with 1×Wash buffer

[0351] 8) Add 100 μL / well of the chromogenic solution and incubate in the dark at room temperature for 15 min.

[0352] 9) Add 50 μL / well of the stop solution to stop the color development, and measure the value of OD450 - OD630 with an enzyme-linked immunosorbent assay reader. After subtracting the value of the blank well, calculate the content of IL-6 in the supernatant by the method of four-parameter fitting.

[0353] Figure 14 The results showed that, compared with the KY-0118 monotherapy group, the atezolizumab + KY-0118 combination group did not show an increase in the release of IL-6. Compared with the NC group, there was a slight increase in the release of IL-6 in the atezolizumab + KY-0118 combination group.

[0354] In Example 15, in the MC38 tumor model of hPD-L1 mice, the efficacy of the combination of KY-0118 and atezolizumab was better than that of monotherapy.

[0355] The experimental procedures are as follows:

[0356] 1) Experimental mice: Female C57-hPD-1-hPD-L1 mice, 32 mice at the time of the experiment, 6 - 8 weeks old

[0357] 2) Experimental cells: Humanized PD-L1 mouse colon cancer MC38 cells (hPD-L1-MC38)

[0358] 3) Inoculation dose: 1×10 6 cells / mouse

[0359] 4) Inoculation site: Subcutaneous injection at the right anterior costal region of the mice

[0360] 5) Grouping and drug administration: When the tumors grew to about 80 - 120 mm, they were grouped, with 8 mice in each group, divided into 4 groups. The groupings and drug administration are shown in Table 6: 3 When the tumors grew to about 80 - 120 mm, they were grouped, with 8 mice in each group, divided into 4 groups. The groupings and drug administration are shown in Table 6:

[0361] Table 6 Information table of grouping and drug administration for mice

[0362]

[0363] 6) Intravenous injection of vehicle, KY-0118 in the monotherapy group, and KY-0118 in the combination group at a dose of 0.3 mg / kg once a week for 3 weeks; intraperitoneal injection of atezolizumab in the monotherapy group and atezolizumab in the combination group at a dose of 5 mg / kg twice a week for 3 weeks. The tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the tumor-bearing mice were recorded. The tumor volume and tumor growth inhibition rate were calculated. The tumor volume (V) was calculated as: (length × width 2 ) / 2, and the tumor growth inhibition rate = (1 - change in tumor volume in the drug treatment group / change in tumor volume in the control group) × 100%.

[0364] The experimental results showed ( Figure 15) In the hPD-L1-MC38 tumor cell C57-hPD-1-hPD-L1 mouse model, KY-0118 at a dose of 0.3 mg / kg and atezolizumab at a dose of 5 mg / kg can effectively inhibit the growth and proliferation of tumor cells hPD-L1-MC38 in mice. The tumor inhibition rates on Day 21 are 65.9% and 64.8% respectively. When KY-0118 at 0.3 mg / kg is combined with atezolizumab at 5 mg / kg, the tumor inhibition rate on Day 21 is 85.6%. The tumor inhibition effect of the combination group is significantly different from that of the two drugs used alone (p < 0.05).

[0365] Example 16. In the 786-O tumor model of huPBMC-NCG-dko mice, the combined efficacy of KY-0118 and atezolizumab is better than that of single drugs

[0366] The experimental steps are as follows:

[0367] 1) Experimental mice: Female huPBMC-NCG-dko mice, 32 in number, 6 - 8 weeks old at the time of experiment

[0368] 2) Experimental cells: Human renal carcinoma 786-O cells

[0369] 3) Inoculation dose: 1×10 7 cells / mouse

[0370] 4) Inoculation site: Subcutaneous in the right anterior costal region of the mouse

[0371] 5) Grouping and administration: When the tumor grows to about 100 - 150 mm 3 Group the mice, 8 in each group, divided into 4 groups. The groupings and administration are shown in the following table:

[0372] Table 7 Information table of mouse grouping and administration

[0373]

[0374]

[0375] 6) Administer the vehicle, single-drug group KY-0118 and combination group KY-0118 at 0.2 mg / kg by tail vein injection once a week for 3 weeks; administer the single-drug group atezolizumab and combination group atezolizumab at 3 mg / kg by intraperitoneal injection twice a week for 3 weeks. Measure the tumor volume and body weight twice a week, record the body weight and tumor volume of tumor-bearing mice, calculate the tumor volume and tumor growth inhibition rate. The tumor volume (V) is calculated as: (length × width 2 ) / 2, and the tumor growth inhibition rate = (1 - tumor volume change in the drug treatment group / tumor volume change in the control group) × 100%.

[0376] The experimental results show (Figure 16 ) In the 786 - O tumor cell huPBMC - NCG - dko mouse model, KY - 0118 at a dose of 0.2 mg / kg and atezolizumab at a dose of 3 mg / kg can effectively inhibit the growth and proliferation of tumor cells 786 - O in mice. The tumor inhibition rates on Day 18 were 69.2% and 65.7% respectively. When KY - 0118 at 0.2 mg / kg was combined with atezolizumab at 3 mg / kg, the tumor inhibition rate on Day 18 was 100%. There was a significant difference in the tumor inhibition effect between the combination group and the two drugs used alone (p < 0.05).

[0377] Table 8 Amino Acid Sequence List

[0378]

[0379]

[0380] All the documents mentioned in the present invention are cited in this application as references, as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A fusion protein, characterized in that, The fusion protein has the structure shown in Formula I from the N-terminus to the C-terminus: P-F-I(I) In the formula, each "-" is independently a linker peptide or a peptide bond; P is an anti-PD-1 antibody; F is an Fc fragment; I is an IL-2 mutant protein, and the amino acid sequence of the IL-2 mutant protein is as shown in SEQ ID NO: 6; The amino acid sequence of the fusion protein is as shown in SEQ ID NO:

1.

2. A polynucleotide, characterized in that, The polynucleotide encodes the fusion protein according to claim 1.

3. A carrier, characterized in that, The vector contains the polynucleotide according to claim 2.

4. An engineered host cell, characterized in that, The host cell contains the vector according to claim 3 or the polynucleotide according to claim 2 is integrated into the genome.

5. A method for preparing the fusion protein as claimed in claim 1, characterized in that, Comprising the steps of: (i) culturing the host cell according to claim 4 under suitable conditions to obtain a mixture containing the fusion protein according to claim 1; and (ii) purifying and / or separating the mixture obtained in step (i) to obtain the fusion protein according to claim 1.

6. An immunoconjugate, characterized in that, The immunoconjugate contains: (a) the fusion protein according to claim 1; and (b) a conjugate moiety selected from the group consisting of: a detectable label, or an enzyme capable of producing a detectable product.

7. A pharmaceutical composition, the pharmaceutical composition containing: (a) a first active ingredient, the active ingredient being selected from the group consisting of: the fusion protein according to claim 1, the host cell according to claim 4, the immunoconjugate according to claim 6, or a combination thereof; and (b) a pharmaceutically acceptable carrier.

8. Use of the fusion protein according to claim 1, the host cell according to claim 4, the immunoconjugate according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating solid tumors.

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