Bifunctional fusion protein comprising Anti-PD-1 antibody and il-2 mutant

Abstract

Provided is a bifunctional fusion protein comprising an anti-PD-1 antibody and an IL-2 mutant. The bifunctional fusion protein comprises the PD-1 antibody and the IL-2 mutant which are connected by a mutated FC. The bifunctional fusion protein has the effects of activating and amplifying PD-1 positive lymphocytes and regulating the activity of Treg cells, and can activate tumor immunity so as to treat cancers. Also provided is a use of the bifunctional fusion protein in the field of tumor treatment. Also provided is a combination therapy for treating tumors by combining the bifunctional fusion protein and atezolizumab.

Description

Bifunctional fusion protein of anti-pd-1 antibody and il-2 mutant TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine. Specifically, the present application relates to a bifunctional fusion protein of anti-PD-1 antibody and IL-2 mutant. BACKGROUND

[0002] Tumor immunotherapy has always been a hot spot 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. Tumor immunotherapy mainly includes cytokine therapy, immune checkpoint inhibitors and adoptive immune cell therapy, among which cytokine therapy is the earliest tumor immunotherapy and has great significance in tumor treatment.

[0003] The immune checkpoint inhibitor is represented by PD-1 (Programmed Death-1) inhibitor. PD-1 is an immune negative regulator, which, when combined with its ligand PD-L1 or PD-L2, down-regulates T cell activity, mediates immune tolerance, and mediates immune escape of cancer cells in the tumor microenvironment. The PD-1 inhibitor can block the binding of PD-1 and PD-L1, restore the recognition and killing of tumor cells by T cells. At present, the marketed PD-1 inhibitors include Nivolumab, Pembrolizumab, etc., which are used for first-line 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 stimulating effector T cells with cytokines.

[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, which binds to IL-2 receptor (IL-2R) to activate downstream JAK1-JAK3-STAT5, PI3K-mTOR1 and MAPK signaling pathways through CD122, CD132, and regulate immune response. When the immune environment is stable, CD4+ T cells secrete low concentration of IL-2. After immune activation, CD4+ and CD8+ T cells, NK cells, dendritic cells (DCs) and mast cells secrete IL-2 through autocrine and paracrine, and act on effector immune cells expressing IL-2 receptor (IL-2R+). IL-2 exerts immune regulation through the formation of a three / two-dimeric receptor IL-2α / β / γ. The three-dimeric IL-2R is composed of α chain (IL-2Rα; CD25), β chain (IL-2Rβ; CD122), and common chain (γc / CD132, shared with IL-4, etc.), has high affinity, and is mainly expressed on Treg cells, partially activated CD4+ and CD8+ T cells, and part of NK cells. The two-dimeric IL-2R composed of IL-2Rβ and γ chain has medium affinity, and is mainly expressed on CD8+ memory T cells and NK cells. IL-2 tends to bind to high-affinity receptors at low doses, and can be bound by medium-affinity receptors at high doses.

[0005] IL-2 combined with PD-1 inhibitor therapy is one of the new strategies for tumor immunotherapy. However, although IL-2 has good efficacy, it has a short half-life and needs to be administered at a high dose, which can easily lead to serious adverse reactions such as cytokine release syndrome and vascular leakage syndrome, limiting the application of IL-2 in clinical practice.

[0006] Therefore, there is a need in the art to develop an IL-2 / PD-1 inhibitor combined therapy drug with high efficacy and low side effects.

[0007] SUMMARY

[0008] The purpose of the present application is to provide an IL-2 / PD-1 inhibitor combined therapy drug with high efficacy and low side effects.

[0009] In a first aspect of the present application, an IL-2 mutant protein is provided, which has the following mutations relative to the wild-type IL-2 protein shown in SEQ ID NO: 7:

[0010] P at position 65 is mutated to K, R, H, D, E, N, Q, Y, W or F;

[0011] C at position 125 is mutated to S, A, G, T or V.

[0012] In another preferred embodiment, the IL-2 mutein has an activation ability on Treg cells of 20% (preferably 10%, more preferably 1%, more preferably 0.1%) or less than that of wild-type IL-2.

[0013] In another preferred embodiment, the IL-2 mutein has a binding ability on IL-2Rβγ of 80% (preferably 90%, more preferably 95%, more preferably 99%) or more than that of wild-type IL-2.

[0014] In another preferred embodiment, the IL-2 mutein has an activation ability on Treg cells of 20% (preferably 10%, more preferably 1%, more preferably 0.1%) or less than that of wild-type IL-2.

[0015] In another preferred embodiment, the IL-2 mutein has an activation ability on Treg cells of 20% (preferably 10%, more preferably 1%, more preferably 0.1%) or less than that of wild-type IL-2.

[0016] In a second aspect of the present application, there is provided a fusion protein comprising the IL-2 mutein as described in the first aspect of the present application.

[0017] In another preferred embodiment, the fusion protein comprises the following protein elements:

[0018] (i) an anti-PD-1 antibody;

[0019] (ii) an Fc fragment;

[0020] (iii) the IL-2 mutein as described in the first aspect of the present application.

[0021] In another preferred embodiment, the fusion protein has the following structure of Formula I from N-terminus to C-terminus:

[0022] P-F-I (I)

[0023] wherein each “-” is independently a connecting peptide or a peptide bond;

[0024] P is an anti-PD-1 antibody;

[0025] F is an Fc fragment;

[0026] I is the IL-2 mutein.

[0027] In another preferred embodiment, the PD-1 is human PD-1.

[0028] In another preferred embodiment, the anti-PD-1 antibody is selected from the group consisting of a Fab fragment, a single-chain antibody, a single-domain antibody, or a combination thereof.

[0029] In another preferred embodiment, the anti-PD-1 antibody has a light chain variable region and a heavy chain variable region.

[0030] In another preferred embodiment, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 9.

[0031] In another preferred embodiment, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 10.

[0032] In another preferred embodiment, the anti-PD-1 antibody has a structure from N-terminus to C-terminus as set forth in Formula IIa or IIb:

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

[0034] VL-L-VH (IIb);

[0035] wherein,

[0036] VL is a light chain variable region;

[0037] VH is a heavy chain variable region;

[0038] L is a connecting peptide or a peptide bond.

[0039] In another preferred embodiment, the L is a flexible linker or a rigid linker.

[0040] In another preferred embodiment, the flexible linker is a sequence of 1-6 (preferably, 3-5) consecutive G4S.

[0041] In another preferred embodiment, the anti-PD-1 antibody has an amino acid sequence as set forth in SEQ ID NO: 8, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0042] In another preferred embodiment, the Fc fragment is a wild-type IgG1 -derived Fc fragment, or a mutant Fc fragment.

[0043] In another preferred embodiment, the mutant Fc fragment has the following mutations relative to the wild-type IgG1 Fc fragment as set forth in SEQ ID NO: 12:

[0044] L at position 234 is mutated to A, L at position 235 is mutated to A, and P at position 329 is mutated to G.

[0045] In another preferred embodiment, the Fc fragment has an amino acid sequence as set forth in SEQ ID NO: 11.

[0046] In another preferred embodiment, the IL-2 mutein amino acid sequence is set forth in SEQ ID NO: 6.

[0047] In another preferred embodiment, the anti-PD-1 antibody and the Fc fragment are linked by a linker, preferably a flexible linker, more preferably a sequence set forth in GGS.

[0048] In another preferred embodiment, the Fc fragment and the IL-2 mutein are linked by a linker, preferably a flexible linker, more preferably a sequence set forth in GGGS (SEQ ID NO: 13).

[0049] In another preferred embodiment, the fusion protein has an amino acid sequence set forth in SEQ ID NO: 1, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0050] In a third aspect of the present application, a polynucleotide encoding a polypeptide selected from the group consisting of:

[0051] (1) the IL-2 mutein of the first aspect of the present application; or

[0052] (2) the fusion protein of the second aspect of the present application.

[0053] In a fourth aspect of the present application, a vector containing the polynucleotide of the third aspect of the present application is provided.

[0054] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentivirus vector, adenovirus vector, retrovirus vector, transposon, or a combination thereof.

[0055] In a fifth aspect of the present application, an engineered host cell containing the vector of the fourth aspect of the present application or the polynucleotide of the third aspect of the present application integrated into its genome is provided.

[0056] 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).

[0057] In another preferred embodiment, the host cell is a prokaryotic cell, such as E. coli.

[0058] In another preferred embodiment, the host cell is a 293 cell or a CHO cell.

[0059] In a sixth aspect of the present application, a method for preparing the IL-2 mutein according to the first aspect of the present application or the fusion protein according to the second aspect of the present application is provided, comprising the steps of:

[0060] (i) culturing the host cell according to the fifth aspect of the present application under suitable conditions, thereby obtaining a mixture containing the fusion protein according to the first aspect of the present application; and

[0061] (ii) purifying and / or isolating the mixture obtained in step (i), thereby obtaining the IL-2 mutein according to the first aspect of the present application or the fusion protein according to the second aspect of the present application.

[0062] In a seventh aspect of the present application, an immunoconjugate is provided, said immunoconjugate comprising:

[0063] (a) the fusion protein according to the second aspect of the present application; and

[0064] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0065] In another preferred embodiment, the conjugated moiety is selected from the group consisting of a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biological toxin, a cytokine, an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or benzyl-hydrolase-like protein (BPHL)), a chemotherapeutic agent (e.g., cisplatin), or any form of nanoparticle, etc.

[0066] In an eighth aspect of the present application, a pharmaceutical composition is provided, said pharmaceutical composition comprising:

[0067] (a) a first active ingredient selected from the group consisting of the IL-2 mutein according to the first aspect of the present application, the fusion protein according to the second aspect of the present application, the host cell according to the fifth aspect of the present application, the immunoconjugate according to the seventh aspect of the present application, or a combination thereof; and

[0068] (b) a pharmaceutically acceptable carrier.

[0069] In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

[0070] In another preferred embodiment, the pharmaceutical composition is an injection.

[0071] In another preferred embodiment, the pharmaceutical composition further comprises a second active ingredient, which is an anti-tumor drug.

[0072] In another preferred embodiment, the second active ingredient is selected from the group consisting of a chemotherapeutic drug, a targeted drug, an immune stimulator, an antibody conjugated drug, a polypeptide drug, and a nucleic acid drug.

[0073] In another preferred embodiment, the second active ingredient is a PD-1 antibody or a PD-L1 antibody.

[0074] In another preferred embodiment, the second active ingredient is atezolizumab.

[0075] In another preferred embodiment, the pharmaceutical composition is used for treating a disease.

[0076] In another preferred embodiment, the disease is a tumor with high expression of PD-L1.

[0077] In a ninth aspect of the present application, there is provided use of the IL-2 mutein according to the first aspect of the present application, the fusion protein according to the second aspect of the present application, the host cell according to the fifth aspect of the present application, the immunoconjugate according to the sixth aspect of the present application, or the pharmaceutical composition according to the eighth aspect of the present application, in the preparation of a medicament for treating a tumor.

[0078] In another preferred embodiment, the tumor is a tumor with high expression of PD-L1.

[0079] In another preferred embodiment, the tumor is selected from the group consisting of a hematological tumor, a solid tumor, or a combination thereof.

[0080] In another preferred embodiment, the hematological tumor is selected from the group consisting of acute myeloid leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma (MM), myelodysplastic syndrome, or a combination thereof.

[0081] 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's lymphoma, bladder cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, renal 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 carcinoma, thymus cancer, or a combination thereof.

[0082] In another preferred embodiment, the tumor is selected from the group consisting of colon cancer, renal cancer, or a combination thereof.

[0083] In another preferred embodiment, the medicament further comprises another anti-tumor agent.

[0084] In another preferred embodiment, the anti-tumor agent is selected from the group consisting of a chemotherapeutic agent, a targeted agent, an immune stimulator, an antibody conjugate, a polypeptide agent, a nucleic acid agent, or a combination thereof.

[0085] In another preferred embodiment, the anti-tumor agent is a PD-1 antibody or a PD-L1 antibody.

[0086] In another preferred embodiment, the anti-tumor agent is atezolizumab.

[0087] In a tenth aspect of the present application, 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 mutein of the first aspect of the present application, the fusion protein of the second aspect of the present application, the host cell of the fifth aspect of the present application, the immunoconjugate of the seventh aspect of the present application, or the pharmaceutical composition of the eighth aspect of the present application, or a combination thereof.

[0088] In another preferred embodiment, the tumor is a PD-L1 high tumor.

[0089] In another preferred embodiment, the method further comprises treating the subject with another disease treatment method.

[0090] In another preferred embodiment, the another 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 a combination thereof.

[0091] In another preferred embodiment, the another disease treatment method comprises administering to the subject in need thereof an effective amount of a PD-1 antibody or a PD-L1 antibody.

[0092] In another preferred embodiment, the anti-tumor agent is atezolizumab.

[0093] It should be understood that, within the scope of the present application, each of the technical features described above and in the following (e.g., in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0094] The following drawings are used to illustrate specific embodiments of the present application and are not used to limit the scope of the present application as defined by the claims.

[0095] Figure 1 shows a schematic diagram of a fusion protein structure.

[0096] Figure 2 shows the binding activity of the fusion protein to human PD-1 protein.

[0097] Figure 3 shows the binding difference of the fusion protein to human IL-2Rα (A), IL-2Rβγ (B), IL-2Rαβγ (C).

[0098] Figure 4 shows the functional study of KY-0118 in vitro activating immune cells.

[0099] Figure 5 shows the evaluation of KY-0118 on PBMC in vitro expansion activity.

[0100] Figure 6 shows the evaluation of ADCC effect of KY-0118.

[0101] Figure 7 shows KY-0118 inducing PBMC to release IFN-γ.

[0102] Figures 8A and 8B show KY-0118 inducing PBMC cell-tumor co-culture to release IFN-γ.

[0103] Figure 9 shows the body weight change and tumor volume change of tumor-bearing hPD-1 mice.

[0104] Figure 10 shows the 786-O transplanted tumor volume change in PBMC humanized mouse model.

[0105] Figure 11 shows the body weight change and tumor volume change of C57-hPD-1 mouse model.

[0106] Figure 12 shows the expression level of tumor cell PD-L1.

[0107] Figure 13 shows the detection results of IFN-γ, TNF-α and IL-10.

[0108] Figure 14 shows the detection results of IL-6.

[0109] Figure 15 shows the body weight change and tumor volume change of C57-hPD-1-hPD-L1 mouse model.

[0110] Figure 16 shows the 786-O transplanted tumor volume change in huPBMC-NCG-dko mouse model. DETAILED DESCRIPTION

[0111] The present inventors have first developed a bifunctional fusion protein of an anti-PD-1 antibody and an IL-2 mutant through extensive and intensive research. 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 an effect of activating and expanding PD-1 positive lymphocytes, modulating the activity of Treg cells, and can activate tumor immunity to treat cancer. On this basis, the present invention is completed.

[0112] Terms

[0113] For easier understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Before describing the present invention, it is to be understood that the present invention is not limited to the particular methodology and experimental conditions described, as such methodology and conditions can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, the scope of the present invention being limited only by the appended claims.

[0114] As used herein, the terms "comprise", "include", "contain" are used interchangeably and include not only the closed definition but also semi-closed and open definitions. In other words, the terms include "consist of", "consist essentially of".

[0115] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic response), i.e., a reasonable benefit / risk ratio.

[0116] As used herein, the term "therapeutically effective amount" refers to an amount that produces a functional or active effect on humans and / or animals and is acceptable to humans and / or animals. Those of ordinary skill in the art will appreciate that the "therapeutically effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and the use of other drugs in combination, etc.

[0117] Bifunctional fusion protein of the present invention

[0118] 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.

[0119] The fusion protein of the present invention comprises the following protein elements:

[0120] (i) an anti-human PD-1 antibody;

[0121] (ii) Fc fragment;

[0122] (iii) IL-2 mutein.

[0123] The anti-PD-1 antibody in the fusion protein of the present application can be any antibody or antigen-binding fragment thereof having PD-1 specific binding activity.

[0124] As used herein, "antigen-binding fragment" refers to a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a single Fv fragment having antigen binding activity. Fv antibodies contain the variable region of the heavy chain, the variable region of the light chain, but no constant region, and have the smallest antibody fragment with all antigen binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VHand VLdomains, and can form a structure required for antigen binding. In one embodiment, the anti-PD-1 antibody is a single chain antibody (scFv).

[0125] In the present application, the scFv of the present application also includes conservative variants thereof, which means that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar properties to form a polypeptide compared to the amino acid sequence of the scFv of the present application.

[0126] In a preferred embodiment, the amino acid sequence of the anti-PD-1 antibody is as shown in SEQ ID NO: 8.

[0127] As used herein, unless otherwise specified, Fc refers to the Fc fragment of a human immunoglobulin. The term "immunoglobulin Fc region" refers to a carboxy-terminal portion of an immunoglobulin chain constant region, particularly a heavy chain immunoglobulin constant region, or a portion thereof, for example, an immunoglobulin Fc region can include two or more domains of a heavy chain CH1, CH2, CH3 in combination with an immunoglobulin hinge region, in preferred examples, the Fc region of the immunoglobulin used includes at least one immunoglobulin hinge region, a CH2 domain and a CH3 domain, preferably lacking a CH1 domain.

[0128] In a preferred embodiment, the Fc fragment in the fusion protein of the present application is a mutant of the Fc fragment of human IgG1 immunoglobulin. In one embodiment, the Fc fragment of the present application has the following mutations relative to the wild-type IgG1 Fc fragment: L234A, L235A, P329G, but not limited to this. The Fc fragment of the present application can also include any other mutations, as long as the resulting fusion protein has reduced ADCC and CDC activity relative to the fusion protein having the wild-type Fc fragment.

[0129] In a preferred embodiment, the amino acid sequence of the Fc fragment is as shown in SEQ ID NO: 11.

[0130] As used herein, the terms "IL-2 mutant" and "IL-2 mutein" both refer to the IL-2 mutein of the first aspect of the present application. Conventional IL-2 drugs are terminated from pipeline due to excessive peripheral blood toxicity. The present application designs the mutation of IL-2 to make the mutated IL-2 preferentially bind to IL-2Rβγ receptor, thereby improving the safety of the drug.

[0131] In one embodiment, the IL-2 mutein of the present application has the following mutations relative to the wild-type IL-2 protein: P65K and C125S, but not limited thereto. The IL-2 mutein of the present application (or the fusion protein of the present application containing the same) can further include any other mutations, as long as the resulting mutant has reduced binding ability to IL-2Rα and activation ability to Treg cells, and has the same or substantially the same binding ability to IL-2Rβγ, relative to the wild-type IL-2 protein.

[0132] In a preferred embodiment, the amino acid sequence of the IL-2 mutein is shown as SEQ ID NO: 6.

[0133] In one embodiment, the structure of the fusion protein of the present application is shown as KY-0118 in FIG. 1, and the amino acid sequence is shown as SEQ ID NO: 1.

[0134] The fusion protein of the present application also includes variant forms of the above-mentioned 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 small 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, substitution with an amino acid having similar or similar performance usually does not change the function of the protein. For another example, addition or deletion of 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 multimeric forms of the polypeptide of the present application. The term also includes linear and nonlinear polypeptides (such as cyclic peptides).

[0135] The present application also includes active fragments, derivatives and analogs of the above-mentioned fusion protein. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially maintain the function or activity of the fusion protein of the present application.

[0136] The polypeptide fragments, derivatives, or analogs of the present application can be (i) a polypeptide having one or more conservative or non-conservative amino acid residue substitutions (preferably conservative amino acid residue substitutions), (ii) a polypeptide having a substituent group at one or more amino acid residues, (iii) a polypeptide fused to another compound (such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide having an additional amino acid sequence fused to the polypeptide sequence (a fusion protein fused to a leader sequence, a secretion sequence, or a 6His tag sequence). These fragments, derivatives, and analogs are within the scope of those skilled in the art in light of the teachings herein.

[0137] One preferred class of active derivatives are polypeptides having up to 5, preferably up to 3, and more preferably up to 1 amino acid replaced by a similar or conservative amino acid compared to the amino acid sequence of the present application. These conservative variant polypeptides are preferably generated by making amino acid substitutions in accordance with Table A.

[0138] Table A

[0139] The present application also provides analogs of the fusion proteins of the present application. These analogs can differ from the polypeptides of the present application in terms of the amino acid sequence, in terms of modifications that do not affect the sequence, or both. Analogues also include those having residues other than the naturally occurring L-amino acids (e.g., D-amino acids), as well as analogues having non-naturally occurring or synthetic amino acids (e.g., β, γ-amino acids). It is understood that the polypeptides of the present application are not limited to the representative polypeptides exemplified above.

[0140] In addition, the fusion proteins of the present application can be modified. Modifications (which generally do not alter the primary structure) include chemical derivatization of the polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides that are glycosylated during synthesis and processing or further processing steps. Such modifications can be accomplished by exposing the polypeptide to an enzyme that glycosylates (e.g., a mammalian glycosylating enzyme or deglycosylating enzyme). Modifications also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

[0141] The term "polynucleotide of the present application" can be a polynucleotide that encodes the fusion proteins of the present application, or a polynucleotide that further includes additional coding and / or non-coding sequences.

[0142] The present application also relates to variants of the above polynucleotides, which encode fragments, analogs and derivatives of the polypeptides or fusion proteins having the same amino acid sequence as the present application. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide which can be one or more nucleotides different from the corresponding sequence, but which do not change the functional properties of the fusion protein encoded by the polynucleotide.

[0143] The present application also relates to polynucleotides which hybridize to the above sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present application particularly relates to polynucleotides which hybridize to the polynucleotides of the present application under stringent conditions (or stringency conditions). In the present application, "stringent conditions" means: (1) hybridization and washing under low ionic strength and high temperature, such as 0.2 x SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization only when the identity between the two sequences is at least 90%, more preferably 95% or more.

[0144] The fusion proteins and polynucleotides of the present application are preferably provided in isolated form, more preferably, purified to homogeneity.

[0145] The full-length sequence of the polynucleotides of the present application can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed according to the nucleotide sequences disclosed herein, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art is used as a template for amplification. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

[0146] Once the relevant sequence is obtained, recombination can be used to obtain the relevant sequence in large quantities. This is usually done by cloning the sequence into a vector, which is then introduced into cells, and then the relevant sequence is isolated from the proliferated host cells by conventional methods.

[0147] In addition, artificial synthesis can also be used to synthesize the relevant sequence, especially when the length of the fragment is relatively short. Usually, a long fragment of the sequence can be obtained by first synthesizing a plurality of small fragments and then ligating them together.

[0148] At present, it is possible to obtain the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present application entirely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art.

[0149] The method for amplifying DNA / RNA using PCR technique is preferably used to obtain the polynucleotide of the present application. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE- rapid amplification of cDNA ends) can be preferably used, the primers for PCR can be appropriately selected based on the sequence information of the present application disclosed herein, and can be synthesized by a conventional method. The amplified DNA / RNA fragment can be separated and purified by a conventional method such as gel electrophoresis.

[0150] Expression vector

[0151] The present application also relates to a vector comprising the polynucleotide of the present application, and a host cell genetically engineered with the vector of the present application or the fusion protein coding sequence of the present application, and a method for producing the polypeptide of the present application by recombinant technology.

[0152] In the present application, the polynucleotide sequence encoding the fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vector well known in the art. Any plasmid and vector can be used as long as it can replicate and stabilize in the host. An important feature of the expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and a translation control element.

[0153] In the method for producing the fusion protein of the present application, any suitable vector can be used, which can be selected from one of pET, pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, and the fusion DNA sequence linked to the appropriate transcription and translation regulatory sequence is included in the expression vector.

[0154] Both eukaryotic and prokaryotic host cells can be used for the expression of the fusion protein of the present application, and the eukaryotic host cell is preferably a mammalian or insect host cell culture system, preferably COS, CHO, NS0, sf9, and sf21 cells; and the prokaryotic host cell is preferably one of DH5a, BL21(DE3), and TG1.

[0155] Methods well known to those skilled in the art can be used to construct expression vectors containing DNA sequences encoding the fusion proteins of the present application and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequences are operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of such promoters are the lac or trp promoter of E. coli, the PL promoter of bacteriophage lambda, 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 promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for initiation of translation and a transcription terminator.

[0156] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance for eukaryotic and prokaryotic cell culture, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E. coli.

[0157] Vectors containing the appropriate DNA sequences as described above, and appropriate promoters or control sequences, can be employed to transform appropriate host cells to enable them to express the proteins.

[0158] The host cells can be prokaryotic cells, such as bacterial cells, or lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as mammalian cells. Representative examples of useful host cells are prokaryotic cells, such as bacterial cells of E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast, plant cells (e.g., ginseng cells), and mammalian cells.

[0159] The polynucleotides of the present application, when expressed in higher eukaryotic cells, can be enhanced by the insertion of enhancer sequences into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, which act to increase the transcription of a gene. Examples of enhancers include the SV40 enhancer, which is in the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and enhancers associated with adenovirus.

[0160] The selection of appropriate vectors, promoters, enhancers, and host cells is well within the level of skill in the art.

[0161] Transformation of host cells with recombinant DNA can be performed using conventional techniques known to those of skill in the art. When the host is a prokaryote, such as E. coli, the transformation of the host cell can be effected by the use of techniques such as calcium chloride precipitation. If necessary, the transformation can be performed by electroporation. When the host is a eukaryote, the transformation can be effected by the use of techniques such as calcium phosphate precipitation, conventional mechanical procedures such as microinjection, electroporation, or the use of liposomes.

[0162] The resulting transformant can be cultured in conventional nutrient media to express the polypeptide encoded by the genes of the application. The culture conditions, such as temperature, pH and the like, can be selected by the use of known techniques. The resulting polypeptides can be recovered and purified using conventional procedures.

[0163] The recombinant polypeptides of the above methods can be expressed intracellularly, or intramembrane, or secreted from the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those of skill in the art. Examples of these methods include, but are not limited to, conventional renaturation procedures, treatment with protein precipitants (salting-out procedures), centrifugation, osmotic shock, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0164] The fusion proteins of the present application can be isolated and purified by affinity chromatography. Depending on the nature of the affinity column used, the fusion proteins bound to the affinity column can be eluted using conventional methods such as high salt buffer, change of pH, and the like.

[0165] Using the above methods, the fusion proteins can be purified to a substantially homogeneous material, for example, a single band on SDS-PAGE.

[0166] Pharmaceutical Compositions

[0167] In the present application, a pharmaceutical composition comprising the fusion protein of the present application or an immunoconjugate thereof is also provided.

[0168] The pharmaceutical composition of the present application contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the fusion protein (or conjugate thereof) of the present application and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition such as a needle, a solution is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present application can also be used with other therapeutic agents. The fusion protein or its immunoconjugate can be combined with a pharmaceutically acceptable adjuvant to form a pharmaceutical preparation to more stably exert the therapeutic effect, which can ensure the structural integrity of the amino acid core sequence of the fusion protein of the present application, and also protect the multifunctional groups of the protein from degradation (including but not limited to condensation, deamination, or oxidation). The preparation can be in various forms, and in general, for liquid preparations, it is generally stable at 2-8°C for at least one year, and for lyophilized preparations, it is stable at 30°C for at least six months. Here, the preparation can be a suspension, an aqueous needle, a lyophilized preparation commonly used in the pharmaceutical field, and preferably an aqueous needle or a lyophilized preparation.

[0169] For the pharmaceutical composition (e.g., aqueous needle or lyophilized preparation) of the present application, the pharmaceutically acceptable adjuvant includes one or a combination of a surfactant, a solution stabilizer, an isotonicity adjusting agent, and a buffer, wherein the surfactant includes a non-ionic surfactant such as polyoxyethylene sorbitan fatty acid ester (Tween 20 or 80); poloxamer (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; myristyl, linoleyl, or stearyl sarcosine; Pluronics; MONAQUAT™, etc., which is added in an amount to minimize the tendency of the protein to be granulated, the solution stabilizer can be a sugar including a reducing sugar and a non-reducing sugar, an amino acid including monosodium glutamate or histidine, an alcohol including a trihydric alcohol, a higher sugar alcohol, propylene glycol, polyethylene glycol, or a combination thereof, the solution stabilizer is added in an amount such that the finally formed preparation is considered by those skilled in the art to be stable for a stable period of time, the isotonicity adjusting agent can be one of sodium chloride, mannitol, and the buffer can be one of TRIS, histidine buffer, and phosphate buffer.

[0170] The pharmaceutical compositions are administered in a therapeutically effective amount, which is generally a safe and effective amount of the fusion protein of the present application or immunoconjugate thereof to be administered to a mammal, wherein the safe and effective amount is typically at least about 50 micrograms per kilogram of body weight, and in most cases not more than about 100 milligrams per kilogram of body weight, preferably the dosage is about 100 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight. Of course, the specific dose will also take into account a route of administration, the health condition of the patient, and the like, which are all within the skill of the art. Typically, the total amount administered will generally not exceed a certain range, for example, for intravenous injection, the dosage is 10 to 3000 mg / day / 50 kg, preferably 100 to 1000 mg / day / 50 kg.

[0171] The fusion protein of the present application and the pharmaceutical preparation containing the same can be used as an antitumor drug for tumor treatment. The antitumor drug as referred to in the present application means a drug having an inhibitory and / or therapeutic effect on a tumor, which can include a delay in the development of symptoms associated with tumor growth and / or a decrease in the severity of these symptoms, and further includes a reduction in symptoms associated with the growth of an already existing tumor and prevention of the occurrence of other symptoms, and also a reduction or prevention of metastasis.

[0172] The 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 combination administration include, but are not limited to: 1. Cytotoxic drugs (1) Drugs acting on the chemical structure of DNA: alkylating agents such as nitrogen mustards, nitrosoureas, methyl sulfonates; platinum compounds such as cisplatin, carboplatin and oxaliplatin, etc.; mytomycin (MMC); (2) Drugs affecting nucleic acid synthesis: dihydrofolate reductase inhibitors such as methotrexate (MTX) and Alimta, etc.; thymidine synthetase inhibitors such as fluorouracils (5FU, FT-207, capecitabine), etc.; purine nucleoside synthetase inhibitors such as 6-mercaptopurine (6-MP) and 6-TG, etc.; nucleotide reductase inhibitors such as hydroxyurea (HU), etc.; DNA polymerase inhibitors such as cytarabine (Ara-C) and gemcitabine (Gemz), etc.; (3) Drugs acting on nucleic acid transcription: drugs selectively acting on DNA templates, inhibiting DNA-dependent RNA polymerase, thereby inhibiting RNA synthesis such as: actinomycin D, daunorubicin, doxorubicin, epirubicin, aclacinomycin, and so on; (4) Drugs mainly acting on microtubulin synthesis: paclitaxel, taxotere, vinblastine, vinorelbine, podophyllotoxin, homoharringtonine; (5) Other cytotoxic drugs: asparaginase mainly inhibits protein synthesis; 2. Hormones Anti-estrogens: tamoxifen, droloxifene, exemestane, etc.; aromatase inhibitors: aminoglutethimide, lanthron, letrozole, and so on; anti-androgens: flutamide, RH-LH agonists / antagonists: leuprolide, and so on; 3. Biological response modifiers: interferons mainly inhibiting tumor through body immune function; other interleukins except IL-2; thymopeptides; 4. Monoclonal antibodies: rituximab (MabThera); Cetuximab (C225); Herceptin (Trastuzumab); Bevacizumab (Avastin); Yervoy (Ipilimumab); Pembrolizumab (Keytruda); Atezolizumab (Tecentriq); 5. Other drugs including some currently unknown mechanisms and to be further studied; cell differentiation inducers such as retinoids; apoptosis inducers.

[0173] The present application also includes a pharmaceutical composition and a treatment method for treating tumors by combining the fusion protein of the present application with Atezolizumab.

[0174] The main advantages of the present application include:

[0175] 1) In the bifunctional fusion protein of the present application, the IL-2 mutant is specially designed by modification (P65K and C125S mutation). Compared with wild-type IL-2, the bifunctional fusion protein of the present application essentially eliminates the affinity for human IL-2Rα and retains high IL-2Rβγ dimer binding activity. The bifunctional fusion protein of the present application preferentially activates lymphocytes expressing IL-2Rβγ, does not stimulate or stimulates at a low level Treg cells, and can significantly reduce systemic toxicity caused by IL-2 signaling pathway.

[0176] 2) The bifunctional fusion protein of the present application can induce higher concentration of IFN-γ release by PBMC. Compared with PD-1 / PD-L1 monoclonal antibody, the bifunctional fusion protein of the present application can promote PBMC to release a large amount of effector cytokines such as IFN-γ, while promoting high-efficiency expansion of T cells, and the bifunctional fusion protein of the present application has higher tumor cell killing ability.

[0177] 3) The bifunctional fusion protein of the present application has stronger tumor inhibition effect at animal experiment level, and the tumor inhibition effect is significantly better than Nivolumab and commercial IL-2 in various tumor models.

[0178] 4) The present application also provides a treatment method of the bifunctional fusion protein of the present application + atezolizumab combination, which has higher treatment effect and safety. Compared with the fusion protein single drug and atezolizumab single drug, the release amount of IFN-γ caused by the combination therapy is significantly increased, while the release amounts of IL-6, IL-10 and TNF-α are not significantly increased, and the safety is better.

[0179] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples are not specified, and the methods are usually carried out according to conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0180] Example 1. Structure design and expression preparation of fusion protein

[0181] The fusion protein structure involved in the present application is shown in Figure 1, the 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 structural form of KY-0118 is VL-VH-Fc-IL-2v, the amino acid sequence is shown as SEQ ID NO: 1, the amino acid sequence of aPD-1-Fc-IL-2 is shown as SEQ ID NO: 2, the amino acid sequence of aPD-1-Fc is shown as SEQ ID NO: 3, the amino acid sequence of Fc-IL-2v is shown as SEQ ID NO: 4, the amino acid sequence of Fc-IL-2 is shown as SEQ ID NO: 5, the amino acid sequence of IL-2v is shown as SEQ ID NO: 6, and the amino acid sequence of IL-2 is shown as SEQ ID NO: 7.

[0182] The plasmid of the above-mentioned fusion protein is transfected in 293 cells or CHO cells, the cells are lysed after culture, the fusion protein is purified, the eluate is collected and concentrated, and the purified fusion protein is expressed for subsequent experiments.

[0183] Table 1 Summary of basic information of fusion proteins

[0184] Example 2. The fusion protein can bind human PD-1 protein

[0185] The experimental steps are as follows:

[0186] 1) The target protein PD-1 (His tag) is diluted to 0.5 μg / mL with PBS, and 100 μl per well is added to the enzyme-labeled plate hole for overnight coating;

[0187] 2) Take out the enzyme-labeled plate, add 300 μL PBST per well to wash the plate once;

[0188] 3) Use PBS buffer to prepare 3% BSA blocking solution, add 300 μL blocking solution per well, cover the enzyme-labeled plate cover, and incubate at 37°C for 2 hours;

[0189] 4) Take out the enzyme-labeled plate, add 300 μL PBST per well to wash the plate twice;

[0190] 5) Dilute the detection samples (KY-0118, aPD-1-Fc-IL-2, Fc-IL-2v, Fc-IL-2, aPD-1-Fc), positive control (Pembrolizumab, Nivolumab) and negative control (Isotype) to 20nM start, dilute by 5 times ratio 8 gradient, add 100 μl per well, cover the enzyme-labeled plate cover, and incubate at 37°C for 1 hour;

[0191] 6) Take out the enzyme-labeled plate, add 300 μL PBST per well to wash the plate three times;

[0192] 7) Dilute the enzyme-labeled secondary antibody (Goat anti-human IgG Fc-HRP) by 1:10000, and add 100 μL to each well of the enzyme-labeled plate, cover the enzyme-labeled plate cover, and incubate at 37°C for 0.5 hours;

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

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

[0195] The experimental results are shown in FIG. 2, and KY-0118, aPD-1-Fc-IL-2, aPD-1-Fc and PD-1 (His tag) protein all have good binding activity.

[0196] Example 3. Binding differences of fusion proteins to human IL-2Rα, IL-2Rβγ, and 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, and add 100 μl to each well of the enzyme-labeled plate for overnight coating;

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

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

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

[0202] 5) The detection sample (KY-0118, aPD-1-Fc-IL-2, Fc-IL-2v, Fc-IL-2, aPD-1-Fc) and negative control (Isotype) were diluted to 100nM and 20nM respectively, and then diluted by 8 times with a 5 times ratio, wherein the sample with 20nM was added to the ELISA plate hole coated with IL-2Rα (His tag) and IL-2Rαβγ (His tag) at 100ul per hole, and the sample with 100nM was added to the ELISA plate hole coated with IL-2Rβγ (His tag) at 100ul per hole, and then covered with an ELISA plate cover and incubated at 37℃ for 1 hour;

[0203] 6) Take out the ELISA plate, and add 300ul PBST to each hole to wash the plate three times;

[0204] 7) After diluting the enzyme-labeled secondary antibody (Goat anti-human IgG Fc-HRP) by 1:10000, add 100ul to each hole of the ELISA plate, cover the ELISA plate cover, and incubate at 37℃ for 0.5 hours;

[0205] 8) Take out the ELISA plate, and add 300ul PBST to each hole to wash the plate four times;

[0206] 9) Add 100ul TMB color developing solution to each hole, color develop at room temperature for 5 minutes, and then add 50ul stop solution to stop and detect on the machine;

[0207] The experimental results are shown in Figure 3, and Figures 3A and 3B show that the aPD-1-Fc-IL-2 and Fc-IL-2 of the IL-2 segment without mutation have high binding activity to IL-2Rα and IL-2Rαβγ proteins; KY-0118 and Fc-IL-2v of the IL-2 segment after mutation do not show obvious binding activity to IL-2Rα and IL-2Rαβγ proteins; Figure 3C shows that the molecules before and after mutation of the IL-2 segment both maintain the same IL-2Rβγ binding activity.

[0208] IL-2 can efficiently activate immune cells and is the most potential target for tumor treatment, but it is also a target with high research risk. IL-2 can bind to IL-2Rα, IL-2Rβγ, and IL-2Rαβγ, and activate T cells, Treg cells, and NK cells without distinction, so the biggest research risk of IL-2 related drugs in clinical research is too much 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 to FcγRs, FcRn, and C1q proteins

[0210] The experimental procedure is as follows:

[0211] Affinity detection of KY-0118 to FcyRs:

[0212] 1) Chip preparation: Mouse anti-His antibody was diluted to 50 μg / mL with immobilization reagent (10 mM sodium acetate, pH 4.5). 950 μL of immobilization reagent was added to 50 μL of mouse anti-His antibody for immobilization of 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, 50 μg / mL of antibody was injected into the channels (channel 1-8, Fc 1,2) at a flow rate of 10 μL / min for about 420 s, and the immobilization amount was about 7000 to 14000 RU. After completing the immobilization, the chip was blocked with 1 M ethanolamine at 10 μL / min for 420 s.

[0213] 2) Protein reconstitution: The protein lyophilized powder was reconstituted according to the product COA, and was aliquoted at a specification of more than 10 μg per tube to avoid repeated freezing and thawing.

[0214] 3) Buffer exchange: The KY-0118 was subjected to buffer exchange using a desalting column and running reagent 1, and the sample after the exchange was subjected to concentration determination using a SPECTROstar Nano.

[0215] 4) Capture ligand: Three Fcy receptors were diluted to 0.25 μg / mL with running reagent 1, and were sequentially injected into the His capture chip experimental channel (Fc 2) at a flow rate of 10 μL / min for about 100 RU. The reference channel (Fc 1) did not require capture of the ligand.

[0216] 5) Analyte multi-cycle analysis: The KY-0118 was diluted with running reagent 1. The diluted KY-0118 was sequentially injected into the experimental channel and the reference channel at a flow rate of 30 μL / min, and the binding and dissociation times were correspondingly adjusted. The binding and dissociation steps were performed in running reagent 1. After each concentration analysis, the chip was 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 non-dissociated analyte. When the next concentration analysis was performed, the experimental channel required re-capture of the same amount of ligand.

[0217] Affinity detection of KY-0118 to FcyRs:

[0218] 1) Chip preparation: Dilute mouse anti-His antibody to 50 μg / mL with immobilization reagent (10 mM sodium acetate, pH 4.5), 950 μL immobilization reagent to 50 μL mouse anti-His antibody for immobilization of 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 50 μg / mL mouse anti-His antibody into channels (channel 1-8, Fc 1,2) 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 10 μL / min for 420 s.

[0219] 2) Protein reconstitution: Reconstitute the protein lyophilized powder according to the product COA, and sub-packaged at more than 10 μg per tube to avoid repeated freeze-thawing.

[0220] 3) Buffer exchange: Use desalting column and running reagent 2 to perform buffer exchange for KY-0118, and determine the concentration of the exchanged sample by SPECTROstarNano.

[0221] 4) Capture ligand: Dilute FcRn protein to 0.25 μg / mL with running reagent 2 and inject into the His capture chip experimental channel (Fc 2) at a flow rate of 10 μL / min for about 100 RU. The reference channel (Fc 1) does not need to be captured with ligand.

[0222] 5) Analyte multi-cycle analysis: Dilute KY-0118 with running reagent 2 by 2-fold dilution. Inject the diluted 3 antibodies into the experimental channel and the reference channel at a flow rate of 30 μL / min, and the corresponding binding and dissociation times. The binding and dissociation steps are performed 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 away the ligand and the non-dissociated analyte. When the next concentration analysis is performed, the experimental channel needs to be recaptured with the same amount of ligand.

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

[0224] 1) Protein reconstitution: Reconstitute the protein lyophilized powder according to the product COA, and sub-packaged at more than 10 μg per tube to avoid repeated freeze-thawing.

[0225] 2) Buffer exchange: Use desalting column and running reagent to perform buffer exchange for KY-0118 and C1q, and determine the concentration of the exchanged sample by SPECTROstarNano.

[0226] 3) Chip preparation: KY-0118 was diluted with immobilization reagent (10 mM sodium acetate, pH 4.5) to 10 μg / mL. The CM5 chip surface was activated with 400 mM EDC and 100 mM NHS for 420 s at a flow rate of 10 μL / min. Next, 10 μg / mL of KY-0118 was injected into the experimental channel (Fc2) for about 1000 RU at a flow rate of 10 μL / min, and finally, the chip was blocked with 1 M ethanolamine at 10 μL / min for 420 s. The reference channel (Fc1) was not injected with KY-0118, and the other operations were the same as the experimental channel (Fc2).

[0227] 4) Analyte multi-cycle analysis: C1q was diluted with running reagent 1 by 2-fold dilution. The diluted C1q was injected into the experimental channel and the reference channel in turn at a flow rate of 30 μL / min, and the corresponding binding and dissociation times were recorded. The binding and dissociation steps were performed in the running reagent. After each concentration analysis, the chip needed to be regenerated with the running reagent at a flow rate of 30 μL / min for 60 s to wash away the undissociated analyte.

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

[0229] Table 2 KY-0118 and human FcγRs, FcRn, C1q protein affinity test results

[0230] Example 5. Effect of KY-0118 on immune cell activation

[0231] The experimental steps are as follows:

[0232] 1) Dilute KY-0118 and wild-type IL-2 samples, 12 gradients, 0.00001-1000000 pM.

[0233] 2) PBMC cells were washed with PBS and resuspended; centrifuged at 400g for 8 min to remove the supernatant.

[0234] 3) PBMC cells were resuspended with 1640 basal medium, plated in a 96-well plate, 50 μL / well, and drug was added (50 μL drug + 50 μL PBMC), and incubated at 37°C for 15 min.

[0235] 4) Add BD Cytofix Buffer 1 mL, vortex well, fix at 37°C for 10 min, centrifuge at 2000 rpm for 5 min to remove the supernatant.

[0236] 5) Add 1 mL Stain buffer (FBS) per tube, resuspend and wash once, centrifuge at 2000 rpm for 5 min to remove supernatant.

[0237] 6) Add -20°C pre-cooled Perm Buffer III 0.5 mL per tube, vortex to mix, incubate at 4°C for 30 min for membrane breaking (or incubate on ice for 30 min).

[0238] 7) Centrifuge at 2000 rpm for 5 min to remove supernatant, add 1 mL Stain buffer (FBS), add Fc-blocker per 100 μL reaction system, incubate at room temperature in the dark for 10 min; after the reaction is completed, add 1 mL Stain buffer (FBS), wash the cells once, centrifuge at 2000 rpm for 5 min to remove supernatant.

[0239] 8) CD3 (FITC), CD8 (PerCP), CD56 (PE) and STAT5 (APC) are prepared into a Mix using Stain buffer (FBS), 100 μL / tube, incubate at room temperature in the dark for 40 min. Alexa 488 anti-human FOXP3 / CD25 PE / CD4 PerCP antibody mixture and STAT5 (APC) are prepared into a Mix. The staining method refers to the STAT5 antibody detection steps of BD, incubate at room temperature for 40 min.

[0240] 9) Add 1 mL Stain buffer (FBS), centrifuge at 2000 rpm for 5 min to remove supernatant; resuspend the cells with 200 μL volume of Stain buffer (FBS), and perform flow detection.

[0241] As shown in the results of Figure 4, the activation ability of KY-0118 on CD8+T cells and NK cells is similar to that of wild-type IL-2. The activation ability of KY-0118 on CD4+T cells is weaker than that of wild-type IL-2. The activation ability of KY-0118 on Treg cells is significantly weaker than that of wild-type IL-2, indicating that the mutation of the IL-2 site in KY-0118 effectively reduces the binding to IL-2Ra and weakens the activation on Treg cells. The EC50 value of KY-0118 on Treg cell activation is 190.6 pM, and the EC50 value of IL-2 on Treg cell activation is <0.0001 pM.

[0242] Example 6. Effect of KY-0118 on in vitro expansion activity of immune cells

[0243] The experimental steps are as follows:

[0244] 1) Centrifuge PBMC cells (4E6), wash once with PBS.

[0245] 2) Dilute 2mM CFSE (100ug dissolved in 90uL DMSO) stock with PBS 1:20, final concentration 0.1nM.

[0246] 3) Resuspend PBMC with pre-warmed PBS (10mL), add 10uL CFSE dilution, incubate at 37C for 15min.

[0247] 4) Stop reaction with 10mL pre-warmed complete media (1640 + 10% FBS + 1% tri-ant), centrifuge at 400g for 10min, remove supernatant, resuspend cells in 20mL fresh media, incubate at 37C for 30min.

[0248] 5) Centrifuge cells at 400g for 10min, remove supernatant, resuspend with 2.5mL fresh media. Cell density is about 1E6-1.5E6 / mL.

[0249] 6) Seed cells in 96-well plate at 1E5-1.5E5 / well in 100uL per well, add 100uL drug at corresponding concentration.

[0250] 7) Incubate culture for 5 days. Wash cells once with PBS containing FBS.

[0251] 8) Add flow antibody cocktail for staining, antibody cocktail: CD3 (PerCP), CD4 (APC), CD56 (PE),

[0252] 9) Incubate at room temperature for 40min in dark.

[0253] 10) Wash twice with PBS containing FBS, centrifuge at 400g for 5min, perform flow detection (population: CD8+ T cells defined as CD3+ CD4-, CD4+ T cells defined as CD3+ CD4+, NK cells defined as CD3- CD56+).

[0254] As shown in the experimental results of Figure 5, KY-0118 can promote the expansion of CD4+ T cells and CD8+ T cells. Based on the P65K and C125S mutations of IL-2V of KY-0118, the expansion activity of KY-0118 is weaker than that of IL-2, and the results are consistent with the design expectation.

[0255] Example 7. Evaluation of ADCC effect of KY-0118

[0256] The experimental procedure is as follows:

[0257] 1) Dilute sample protein in 10% FBS-containing medium to 200, 40, 10, 2.5, 0.625, 0.1562, 0.0390, and 0.0040 nM, transfer to cell plate, 50 μL per well.

[0258] 2) Transfer PD-1-CHO cells to centrifuge tube, centrifuge to remove supernatant, resuspend in 10% FBS-containing RPMI1640 medium, count, and adjust cell density to 4 x 10 5 cells / mL.

[0259] 3) Centrifuge CD16-NF-AT-jurkat cells to remove supernatant, resuspend in 10% FBS-containing RPMI1640 medium, count, and adjust cell density to 2 x 10 6 cells / mL.

[0260] 4) Mix PD-1-CHO cells and CD16-NF-AT-jurkat cells thoroughly, transfer to 96-well plate containing sample dilution, 50 μL per well, incubate in 37°C, 5% CO2 incubator for 6 hours.

[0261] 5) Take out 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 detection.

[0262] As shown in the experimental results of Figure 6, Anti-hPD-1-Ni-hIgG1 is an ADCC positive control antibody, and Anti-βGal-hIgG1 is an ADCC negative control antibody. Based on the LALA PG mutation of the Fc part in KY-0118, KY-0118 does not have ADCC effect, which is consistent with the weak binding phenomenon of CD32a and CD64 shown in Example 4.

[0263] Example 8. KY-0118 induces PBMC to release IFN-γ

[0264] The experimental procedure is as follows:

[0265] 1) Activation of hPBMC: Fresh hPBMC, after equalization, use centrifuge 400G, centrifuge for 5 min, pour out the supernatant, tilt the centrifuge tube for about 2 min, use a 200 μL pipette to suck out the liquid remaining in the tube opening. Add medium (X-Vivo 15 Medium + 10% inactivated FBS + 1% P / S) to resuspend, and adjust the cell density to 2 x 10 6 cells / mL. Transfer to T175 cell culture flask, add 10 nM Anti-CD3 mAb and 2 nM Anti-CD28 mAb, and incubate in a cell incubator for 24 h.

[0266] 2) After 24h, wash hPBMC three times with PBS solution. Resuspend with medium (RPMI 1640 + 10% Inactivated FBS + 1% P / S + 55 mM 2-Mercaptoethanol) for the last time, adjust cell density to 2 x 10 6 cells / mL. Seed in 96-well plate at 100 pL, 2 x 10 5 cells / well.

[0267] 3) Dissolve sample (KY-0118) in water for injection to 10 mg / mL. Dilute Isotype and KY-0118 samples using RPMI 1640 + 10% Inactivated FBS + 1% P / S + 55 mM 2-Mercaptoethanol. Isotype has two concentrations: 100 nM, 10 nM. KY-0118 has nine concentrations, starting concentration 2000 nM, dilute with 4-fold gradient. Set up negative control (Medium control). Add 100 pL samples and controls to wells, incubate in cell incubator for 72 h.

[0268] 4) After 72 h, centrifuge at 350 g for 5 min, collect 150 pL cell supernatant. Detect IFN-g content in cell supernatant using human IFN-g Elisa detection kit.

[0269] The experimental results are shown in Figure 7, KY-0118 can induce hPBMC cells to release IFN-g factor in a dose-dependent manner.

[0270] Example 9. KY-0118 can promote T cell-tumor co-culture to release IFN-g

[0271] The experimental steps are as follows:

[0272] 1) Activation of hPBMC: Fresh hPBMC, after balancing, centrifuge at 400 G for 5 min, pour out the supernatant, tilt the centrifuge tube for about 2 min, use a 200 pL pipette to suck out the liquid remaining in the tube opening. Resuspend with medium (X-Vivo 15 Medium + 10% Inactivated FBS + 1% P / S), adjust the cell density to 2 x 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, incubate in a cell incubator for 24 h.

[0273] 2) After 24h, wash the hPBMC three times with PBS solution. Resuspend with medium (RPMI 1640 + 10% inactivated FBS + 1% P / S + 55 μM 2-Mercaptoethanol) and adjust the density to 1 x 10 6 cells / mL, plant in 96-well plate at a density of 100 μL, 1 x 10 5 cells / well.

[0274] 3) Treat the tumor cells in logarithmic growth phase with trypsin at room temperature for 3-10 min to detach the adherent cells from the bottom of the culture bottle, add 4-6 times the volume of trypsin with 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). Plant the tumor cells in the hPBMC 96-well plate at a ratio of hPBMC: tumor cell = 2:1, 4:1, respectively, at 50 μL per well.

[0275] 4) Dissolve the sample (KY-0118) to 10 mg / mL with water for injection. Dilute the KY-0118 sample and Nivolumab using 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 control to the wells and incubate in the cell incubator for 72 h.

[0276] 5) After 72 h, centrifuge at 350 g for 5 min, and collect 150 μL of cell supernatant. Detect the IFN-γ content in the cell supernatant using a human IFN-γ Elisa detection kit.

[0277] Results: FIG. 8A is the effect of KY-0118 and Nivolumab on the release of IFN-γ under the co-incubation condition of PBMC with four tumor cells (H1993, A498, U-87MG, HCT116) at an effector target ratio of 2:1.

[0278] The results of FIG. 8A show that KY-0118 at 31.25 nM and 0.488 nM can significantly increase the release of IFN-γ in the co-incubation system of PBMC and tumor cells, and has a significant difference compared with the NC. Nivolumab weakly promotes the release of IFN-γ under this experimental system, and the release of IFN-γ in the experimental group of KY-0118 at 31.25 nM is obviously better than that in the experimental group of Nivolumab at 33 nM.

[0279] Figure 8B is the effect of KY-0118 and Nivolumab on the release of IFN-γ under the co-incubation condition of PBMC and four tumor cells (H1993, A498, U-87MG, HCT116) at an effector to target ratio of 4:1.

[0280] The results of Figure 8B show that KY-0118 at 31.25 nM and 0.488 nM can significantly increase the release of IFN-γ in the co-incubation system of PBMC and tumor cells, and has a significant difference compared with NC. Under this experimental system, Nivolumab has a weak promotion on the release of IFN-γ, and the release of IFN-γ in the experimental group of KY-0118 at 31.25 nM is obviously better than that in the experimental group of Nivolumab at 33 nM.

[0281] The results of Figure 8A and Figure 8B show that under the condition that other experimental conditions remain the same, the release of IFN-γ in the experimental group of effector to target ratio of 4:1 is lower than that in the experimental group of effector to target ratio of 2:1.

[0282] Example 10. KY-0118 can significantly inhibit the growth of MC38 transplanted tumor in hPD-1 mice at a low dose

[0283] 1) MC38 cells were inoculated subcutaneously on the right side of the female C57-hPD-1 mice (8-10 weeks old, 50 mice);

[0284] 2) When the tumor grew to 80-120 mm 3 left, the mice were divided into 7 groups, 6 mice in each group, and the group and administration are shown in Table 3:

[0285] Table 3 Information of administration of mice

[0286] 3) The solvent 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 0.29 mg / kg were given by tail vein injection, once a week, for 3 weeks; Nivolumab 3 mg / kg group was given by intraperitoneal injection, once a week, for 3 weeks; IL-2 group was given by subcutaneous injection, once a day, 5 times a week, for 3 weeks;

[0287] 4) The tumor volume and body weight were measured twice a week, and the relationship between the change of body weight and tumor volume of tumor-bearing mice and the administration time was recorded;

[0288] 5) The tumor volume and tumor growth inhibition rate were calculated:

[0289] The tumor volume (V) was calculated as: (length x width 2 ) / 2

[0290] Tumor growth inhibition rate (TGI%) was calculated using the following formula:

[0291] Tumor growth inhibition rate = (1 - tumor volume change of drug treatment group / tumor volume change of control group) x 100%

[0292] The experimental results are shown in Figure 9. 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 can effectively inhibit the growth and proliferation of tumor cells MC38 in mice, with tumor inhibition rates of 81.8%, 92.3%, and 99.2% on Day 20, respectively, in a dose-dependent manner. At a dose of 0.29 mg / kg of Fc-IL-2v, the tumor inhibition rate on Day 20 was -26.4%, showing no tumor inhibition effect. At a dose of 3 mg / kg of Nivolumab, the tumor inhibition rate on Day 20 was 95.2%, showing a significant tumor inhibition effect. After multiple doses of IL-2 (200,000 IU each, QD x 5 / week x 3), the tumor inhibition rate on Day 20 was 7.6%, showing almost no tumor inhibition effect. The above results show that KY-0118 at a dose of 0.05 mg / kg or above can effectively inhibit the growth and proliferation of mouse colon cancer MC38 cells in mice, and the effect is dose-dependent. The tumor inhibition effect of KY-0118 is better than that of Fc-IL-2v without targeting effect at an equimolar dose. The tumor inhibition effect of KY-0118 is better than that of high-dose IL-2. At a similar tumor inhibition effect, the dose of KY-0118 is much lower than that of Nivolumab.

[0293] Example 11. KY-0118 can effectively inhibit the growth of 786-O transplanted tumors in PBMC humanized mice

[0294] 1) 80 female PBMC humanized B2M mice were subcutaneously inoculated with human renal cancer 786-O cells on the right side of the front flank;

[0295] 2) When the tumor grew to 100-150 mm 3 left and right, the mice were randomly divided into 6 groups, 10 mice in each group, and the grouping information is shown in Table 4:

[0296] Table 4. Mouse grouping and drug administration information

[0297] 3) tail vein injection of solvent control group, KY-0118 0.08 mg / kg group, KY-0118 0.2 mg / kg group, KY-0118 0.5 mg / kg group, once a week, for 4 weeks; intraperitoneal injection of Nivolumab 3 mg / kg group, once a week, for 4 weeks; subcutaneous injection of IL-2 group, once a day, 5 times a week, for 4 weeks;

[0298] 4) tumor volume and body weight were measured twice a week, and the relationship between the changes of tumor volume and body weight of tumor-bearing mice and the administration time was recorded;

[0299] The experimental results show (Figure 10) that in the 786-O tumor cell PBMC humanized B2M mouse model, KY-0118 0.08 mg / kg, 0.2 mg / kg, and 0.5 mg / kg doses can effectively inhibit the growth and proliferation of tumor cells 786-O in mice, and the tumor inhibition rates on Day 28 are 45.75%, 62.93%, and 73.78%, respectively, showing a dose-dependent relationship; the tumor inhibition rate of Nivolumab 3 mg / kg dose on Day 28 is 6.41%, which does not show a tumor inhibition effect; the tumor inhibition rate of IL-2 after multiple administrations (200,000 IU / time, QD x 5 / week x 4) on Day 28 is 64.95%, which shows a tumor inhibition effect; the above results show that KY-0118 at a dose of 0.08 mg / kg and above can effectively inhibit the growth and proliferation of human renal cancer 786-O cells in mice, and shows a dose-dependent relationship; Nivolumab 3 mg / kg group with a higher dose of KY-0118 does not show a tumor inhibition effect; the tumor inhibition effect of KY-0118 is better than that of high-dose IL-2; under the approximate tumor inhibition effect (64.95% vs 62.93%), the usage amount of IL-2 (200,000 IU / time, QD x 5 / week x 4) is much higher than that of KY-0118 (0.2 mg / kg, QW x 4).

[0300] Example 12. KY-0118 can significantly inhibit the growth of Pan02-CDX transplanted tumors in hPD-1 mice

[0301] The experimental steps are as follows:

[0302] 1) Prepare female C57-hPD-1 mice (8-10 weeks old), 60; female C57 mice (8-10 weeks old), 20;

[0303] 2) Inoculate Panc02 cells subcutaneously on the right side of the mice;

[0304] 3) When the tumor grows to about 80-120 mm3, group the mice, 8 C57-hPD-1 mice per group, divided into 6 groups; 8 C57 mice per group, divided into 2 groups, group and administration are shown in Table 5:

[0305] Table 5 Information Table of Mouse Grouping and Administration

[0306] 4) C57-hPD-1 vehicle control group, C57-hPD-1 KY-0118 0.1 mg / kg group, C57-hPD-1 KY-0118 0.3 mg / kg group, C57-hPD-1 KY-0118 1 mg / kg group, and C57 vehicle control group and C57 KY-0118 1 mg / kg group were given by tail vein injection, once a week for 4 weeks; C57-hPD-1 Nivolumab 3 mg / kg group was given by intraperitoneal injection, once a week for 4 weeks; C57-hPD-1 IL-2 group was given by subcutaneous injection, once a day, 5 times a week for 4 weeks;

[0307] 5) Tumor volume and body weight were measured twice a week, and the relationship between tumor-bearing mouse body weight and tumor volume change and administration time was recorded;

[0308] 6) Calculate tumor volume and tumor growth inhibition rate:

[0309] Tumor volume (V) is calculated as: (length x width 2 ) / 2

[0310] Tumor growth inhibition rate (TGI%) is calculated using the following formula:

[0311] Tumor growth inhibition rate = (1 - tumor volume change of drug treatment group / tumor volume change of control group) x 100%

[0312] The experimental results show (Figure 11) that in the C57-hPD-1 mouse model of Panc02 tumor cells, KY-0118 0.1 mg / kg, 0.3 mg / kg, and 1 mg / kg doses can effectively inhibit the growth and proliferation of tumor cells Panc02 in mice, with tumor inhibition rates of 78.1%, 96.2%, and 100% on Day 27, respectively, in a dose-dependent manner, and KY-0118 1 mg / kg has a tumor inhibition rate of 100% on Day 17; Nivolumab 3 mg / kg has a tumor inhibition rate of 49.9% on Day 27, showing tumor inhibition effect; IL-2 has a tumor inhibition rate of 13.0% on Day 27 after multiple administration (200,000 IU / time, QD x 5 / week x 4), showing almost no tumor inhibition effect; In the C57 mouse model of Panc02 tumor cells, KY-0118 1 mg / kg has a tumor inhibition rate of 30.2% on Day 17.

[0313] The above results show that KY-0118 can effectively inhibit the growth and proliferation of mouse pancreatic cancer Panc02 cells in mice at a dose of 0.1 mg / kg and above, and in a dose-dependent manner; the tumor inhibition effect of 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 200,000 IU / time IL-2 (molar ratio of KY-0118 to IL-2 = 1:187); under the better tumor inhibition effect, the use dose of 0.1 mg / kg KY-0118 is lower than that of 3 mg / kg Nivolumab (molar ratio of KY-0118 to Nivolumab = 1:14). KY-0118 shows significant synergistic effect.

[0314] The results of Examples 10-12 show that KY-0118 has a stronger tumor inhibition effect than Nivolumab and commercial IL-2, and shows a dose-dependent trend.

[0315] Example 13. Screening of tumor cell lines with high expression of PD-L1 antigen

[0316] The experimental steps are as follows:

[0317] 1) Take the frozen tumor cells from liquid nitrogen, recover in a 37°C water bath, transfer to a centrifuge tube containing complete culture medium, centrifuge and discard the supernatant, resuspend with 1xPBS, centrifuge and wash once to remove DMSO in the frozen solution.

[0318] 2) Resuspend each tumor cell with the corresponding culture medium, transfer to a culture bottle and culture for 24 h before changing the medium.

[0319] 3) After the cells grow to 80% confluence, digest and passage.

[0320] 4) After each cell is passaged once, identify the PD-L1 marker again after the cells grow to 80% confluence.

[0321] 5) Digest the adherent tumor cells with trypsin, centrifuge and discard the supernatant after stopping with complete culture medium, resuspend with PBS and centrifuge to wash once to remove residual trypsin. Resuspend with the corresponding tumor cell culture medium and count.

[0322] 6) Dilute APC-anti-human CD274 (B7-H1, PD-L1) Antibody and APC-Mouse IgG2b kappa Isotype Ctrl Antibody 50 times with FACS buffer (1xPBS + 2% FBS) respectively and prepare for use.

[0323] 7) Parallel operation of the tumor cells to be tested: take 5x105 cells / mL, resuspended to 2 x 10 6 cells / mL, take out 2 x 100 μL of cells, mix one with 100 μL of 50-fold diluted APC-anti-human CD274 (B7-H1, PD-L1) Antibody as the PD-L1 positive group; mix the other with 100 μL of 50-fold diluted APC-Mouse IgG2b, κ Isotype Ctrl Antibody as the negative control, incubate at 4°C for 60 min.

[0324] 8) Resuspend the cells with FACS buffer, centrifuge and discard the supernatant, wash 3 times, resuspend with 200 μL of FACS buffer, mix well, and read the data with a flow cytometer, adjust the positive rate of the negative control to 1% as the background, and record the PD-L1 positive rate of the PD-L1 positive group.

[0325] The results are shown in Figure 12, which shows that 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 more than 95%.

[0326] Example 14. In vitro activity of KY-0118 in combination with atezolizumab

[0327] The experimental procedure is as follows:

[0328] Detection of IFN-γ, TNF-α and IL-10 release:

[0329] 1) Fresh hPBMC was purchased from the manufacturer, centrifuged and resuspended, resuspended with activation medium (X-Vivo 15 medium containing CD3 monoclonal antibody and CD28 monoclonal antibody), counted, and the density was adjusted to 2 x 10 6 cells / mL, activated at 37°C in a 5% CO2 environment for 24 h.

[0330] 2) The hPBMC activated for 24 h was washed 4 times with 1 x PBS, resuspended with co-incubation medium (1640 medium + 10% FBS + 1% P / S + 55 μM mercaptoethanol) and counted, and the cell density was adjusted to 5 x 10 5 cells / mL. Added to a 96-well cell culture plate at 100 μL / well.

[0331] 3) Digest the human renal cancer cells A498 in the logarithmic growth phase, resuspend with co-incubation medium and adjust the density to 1 x 10 6cells / mL, 50 μL / well to the 96-well cell culture plate containing hPBMC.

[0332] 4) Prepare the sample to be tested, 50 μL / well to the 96-well cell culture plate, incubate at 37°C, 5% CO2 for 72 h.

[0333] 5) After 72 h, centrifuge to get supernatant, dilute appropriately, and then use the BD CBA detection kit to detect.

[0334] 6) Mix the beads in the kit in equal volume to get mixed beads.

[0335] 7) Mix 50 μL of sample supernatant, mixed beads and PE detection antibody, mix well, and then incubate at room temperature for 3 h in the dark.

[0336] 8) Wash 3 times with 1x Wash buffer, resuspend with 1x Wash buffer, and then read on the machine. Use linear fitting to calculate the cytokine content in the supernatant.

[0337] The results are shown in FIG. 13, and IL-2 is the positive control. Compared with Isotype and NC, KY-0118 alone can significantly enhance the release of IFN-γ in this experiment; but atezolizumab alone cannot (or weakly) promote the release of IFN-γ. The combination of atezolizumab + KY-0118 can significantly improve the release of IFN-γ compared with KY-0118 alone. At multiple dosing concentrations, the level of increased release of IFN-γ caused by combination therapy is higher than the sum of the two single-drug groups, showing that the combination therapy of atezolizumab + KY-0118 has a synergistic effect. Within a certain range, the release of IFN-γ increases with the increase of the concentration of atezolizumab.

[0338] Compared with KY-0118 alone, atezolizumab alone, and the combination of atezolizumab + KY-0118, the release of TNF-α and IL-10 stimulated by the combination of atezolizumab + KY-0118 has a certain increase, but the release amount is at a low level.

[0339] IL-6 release amount detection:

[0340] 1) Fresh hPBMC was purchased from the manufacturer, centrifuged and resuspended, resuspended with activation medium (X-Vivo15 medium containing CD3 monoclonal antibody and CD28 monoclonal antibody), counted, and adjusted to a density of 2x10 6 cells / mL, and activated at 37°C, 5% CO2 for 24 h.

[0341] 2) Wash the activated hPBMC for 24h with 1xPBS for 4 times, resuspend with co-incubation medium (X-Vivo 15 medium + 1% P / S + 55μM mercaptoethanol) and count, adjust the cell density to 5x10 5 cells / mL. Add to 96-well cell culture plate with 100μL / well.

[0342] 3) Digest the human kidney cancer cell A498 in logarithmic growth phase, resuspend with co-incubation medium and adjust the density to 1x10 6 cells / mL, add to 96-well cell culture plate containing hPBMC with 50μL / well.

[0343] 4) Prepare the sample to be tested, add to the above 96-well cell culture plate with 50μL / well, incubate at 37℃ in 5% CO2 environment for 72h.

[0344] 5) After 72h, centrifuge to obtain supernatant, dilute appropriately and use ELISA kit for detection.

[0345] 6) Mix 50μL of Biotin-Anti-IL-6 Antibody in the kit and the diluted sample supernatant (including standard curve) at room temperature for 1h. Wash with 1xWash buffer for 5 times.

[0346] 7) Add 50-fold diluted Streptavidin-HRP with 50μL / well, incubate at room temperature for 30min. Wash with 1xWash buffer for 5 times

[0347] 8) Add 100μL / well of color developing solution, incubate at room temperature for 15min in the dark.

[0348] 9) Add 50μL / well of stop solution to stop color development, use microplate reader to obtain OD450-OD630 value. Subtract the value of blank well, calculate the content of IL-6 in supernatant by 4-parameter fitting method.

[0349] Figure 14 shows that compared with KY-0118 monotherapy group, the combination of atezolizumab + KY-0118 did not show an increase in IL-6 release. The combination of atezolizumab + KY-0118 showed a slight increase in IL-6 release compared with NC.

[0350] Example 15. The combination of KY-0118 and atezolizumab showed better efficacy than monotherapy in MC38 tumor model of hPD-L1 mice

[0351] The experimental procedure is as follows:

[0352] 1) Experimental mice: female C57-hPD-1-hPD-L1 mice, 32 mice at the time of experiment, 6-8 weeks old

[0353] 2) Experimental cells: humanized PD-L1 mouse colon cancer MC38 cells (hPD-L1-MC38)

[0354] 3) Inoculation amount: 1 x 10 6 cells / mouse

[0355] 4) Inoculation site: subcutaneous injection on the right side of the mouse

[0356] 5) Group administration: when the tumor grows to 80-120 mm 3 left and right, 8 mice in each group, divided into 4 groups, and the group and administration are shown in Table 6:

[0357] Table 6 Information of mouse grouping and administration

[0358] 6) Intravenous injection of solvent, single drug group KY-0118 and combination group KY-0118 0.3 mg / kg, once a week for 3 weeks; intraperitoneal injection of single drug group atezolizumab and combination group atezolizumab 5 mg / kg, twice a week for 3 weeks. Tumor volume and body weight were measured twice a week, tumor-bearing mouse body weight and tumor volume were recorded, tumor volume and tumor growth inhibition rate were calculated, tumor volume (V) was calculated as: (length x width 2 ) / 2, and tumor growth inhibition rate = (1-tumor volume change of drug treatment group / tumor volume change of control group) x 100%.

[0359] The experimental results (Figure 15) show that in the hPD-L1-MC38 tumor cell C57-hPD-1-hPD-L1 mouse model, KY-0118 0.3 mg / kg and atezolizumab 5 mg / kg can effectively inhibit the growth and proliferation of tumor cells hPD-L1-MC38 in mice, and the tumor inhibition rates on Day 21 are 65.9% and 64.8%, respectively; KY-0118 0.3 mg / kg and atezolizumab 5 mg / kg in combination, the tumor inhibition rate on Day 21 is 85.6%, and the tumor inhibition effect of the combination group is significantly different from that of the two drugs alone (p<0.05).

[0360] Example 16. In the 786-O tumor model of huPBMC-NCG-dko mice, the efficacy of KY-0118 combined with atezolizumab is better than that of single drug

[0361] The experimental procedure is as follows:

[0362] 1) Experimental mice: female huPBMC-NCG-dko mice, 32 mice at the age of 6-8 weeks

[0363] 2) Experimental cells: human renal cancer 786-O cells

[0364] 3) Inoculation amount: 1 x 10 7 cells / mouse

[0365] 4) Inoculation site: subcutaneous injection at the right flank of the mouse

[0366] 5) Group administration: when the tumor size reached 100-150 mm 3 left and right, the mice were divided into 8 groups, and the administration of each group is shown in the following table:

[0367] Table 7 Information of administration of mice in groups

[0368] 6) The solvent, KY-0118 in single-drug group and KY-0118 0.2 mg / kg in combination group were given by tail vein injection once a week for 3 weeks; atezolizumab in single-drug group and atezolizumab 3 mg / kg in combination group were given by intraperitoneal injection 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 tumor-bearing mice were recorded, and the tumor volume and tumor growth inhibition rate were calculated. The tumor volume (V) was calculated as: (length x width 2 ) / 2, and the tumor growth inhibition rate = (1-tumor volume change of drug treatment group / tumor volume change of control group) x 100%.

[0369] The experimental results (Figure 16) showed that in the huPBMC-NCG-dko mouse model of 786-O tumor cells, KY-0118 0.2 mg / kg and atezolizumab 3 mg / kg could effectively inhibit the growth and proliferation of 786-O tumor cells in mice, and the tumor inhibition rates on Day 18 were 69.2% and 65.7%, respectively; the combination of KY-0118 0.2 mg / kg and atezolizumab 3 mg / kg had a tumor inhibition rate of 100% on Day 18, and the tumor inhibition effect of the combination group was significantly different from that of the two drugs alone (p<0.05).

[0370] Table 8 Amino acid sequence table

[0371] All the documents mentioned in the present application are incorporated herein by reference as if each document were individually incorporated by reference. In addition, it should be understood that various changes and modifications can be made to the present application by those skilled in the art after reading the above description of the present application, and these equivalent forms also fall within the scope of the appended claims of the present application.

Claims

1. An IL-2 mutein, characterized in that, The mutant protein has the following mutations relative to the wild-type IL-2 protein set forth in SEQ ID NO: 7: P at position 65 is mutated to K, R, H, D, E, N, Q, Y, W, or F; C at position 125 is mutated to S, A, G, T, or V.

2. A fusion protein, characterized in that, The fusion protein comprises the IL-2 mutant protein of claim 1, the fusion protein has the following structure of Formula I from N-terminus to C-terminus: P-F-I (I) wherein each “-” is independently a linking peptide or a peptide bond; P is an anti-PD-1 antibody; F is an Fc fragment; I is the IL-2 mutant protein of claim 1.

3. The fusion protein of claim 2, wherein, The anti-PD-1 antibody has the amino acid sequence set forth in SEQ ID NO: 8, the Fc fragment has the amino acid sequence set forth in SEQ ID NO: 11, and the IL-2 mutant protein has the amino acid sequence set forth in SEQ ID NO:

6.

4. The fusion protein of claim 2 or 3, wherein, The IL-2 mutant protein has the amino acid sequence set forth in SEQ ID NO:

6.

5. The fusion protein according to any one of claims 2 to 4, wherein The Fc fragment has the amino acid sequence set forth in SEQ ID NO:

11.

6. The fusion protein of claim 2, wherein, The fusion protein has the amino acid sequence set forth in SEQ ID NO: 1, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

7. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-6. The polynucleotide encodes a polypeptide selected from the group consisting of: (1) the IL-2 mutant protein of claim 1; or (2) the fusion protein of any one of claims 2-6.

8. A vector, characterized in that, The vector contains the polynucleotide of claim 7.

9. An engineered host cell, characterized in that, The host cell contains the vector of claim 8 or the polynucleotide of claim 7 integrated in the genome.

10. A method of preparing the IL-2 mutein of claim 1 or the fusion protein of any one of claims 2-6, characterized in that, comprising the steps of: (i) culturing the host cell of claim 9 under suitable conditions, thereby obtaining a mixture containing the IL-2 mutant protein of claim 1 or the fusion protein of any one of claims 2-6; and (ii) purifying and / or isolating the mixture obtained in step (i), thereby obtaining the IL-2 mutant protein of claim 1 or the fusion protein of any one of claims 2-6.

11. An immunoconjugate, comprising, The immunoconjugate contains: (a) the fusion protein of any one of claims 2-6; and (b) a conjugating moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

12. A pharmaceutical composition containing: (a) a first active ingredient selected from the group consisting of the IL-2 mutant protein of claim 1, the fusion protein of any one of claims 2-6, the host cell of claim 9, the immunoconjugate of claim 11, or a combination thereof; and (b) a pharmaceutically acceptable carrier. ​ 13. The IL-2 mutein of claim 1, the fusion protein of any one of claims 2-6, the host cell of claim 9, the immunoconjugate of claim 11, or the pharmaceutical composition of claim 12, for use in a method of treating a disease, preferably the disease is a tumor that overexpresses PD-L1.

14. The IL-2 mutein, fusion protein, host cell, immunoconjugate, or pharmaceutical composition for use according to claim 13, wherein The method further comprises administering another anti-tumor agent, preferably a PD-1 antibody or a PD-L1 antibody, more preferably atezolizumab.

15. The IL-2 mutein, the fusion protein, the host cell, the immunoconjugate, or the pharmaceutical composition for use of claim 13 or 14, wherein the disease is selected from the group consisting of colon cancer, kidney cancer, or a combination thereof.

Images