IL2 variants and protein complexes containing them

Abstract

The present invention relates to an IL2 variant and a protein complex containing the same, as well as a method for producing and using the same. The protein complex can selectively increase the activity of effector T cells, thereby reducing side effects and maximizing anti-cancer activity, and can be used to prevent or treat various immune diseases related to cancer.

Description

[Technical Field]

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2022-0105643, filed on 23 August 2022, and Republic of Korea Patent Application No. 10-2023-0057361, filed on 2 May 2023, and the entire specification is a reference to this application.

[0002] This invention relates to IL2 variants and protein complexes containing them, as well as methods for producing the same and their applications. [Background technology]

[0003] Immune checkpoints are surface proteins of epidemic cells that cancer cells use to evade attack by the immune system. Cancer cells express PD-L1 on their cell surface and bind to PD-1, a type of immune checkpoint on T cells, thereby suppressing T cell activation and the ability of cancer cells to kill cancer cells. Immune checkpoint inhibitors activate the human immune system, causing epidemic cells to selectively attack cancer cells, thereby partially resolving the side effects and resistance problems of existing anticancer drugs that directly attack cancer cells. Furthermore, many immune checkpoint inhibitors have been approved for market after their efficacy has been confirmed in various types of cancer. However, their overall response rate (ORR) is around 30% on average, and there are still many problems that need to be overcome. To overcome these problems, interleukins are being considered important as a combination therapy with immune checkpoint inhibitors or as one of the dual-action agents to enhance their efficacy.

[0004] Among these, interleukin-2 (IL2) is a 15.5 kDa globular glycoprotein with a length of 133 amino acids that plays a central role in lymphocyte generation, survival, and homeostasis. IL2 is mainly biosynthesized by activated T cells, particularly CD4+ helper T cells, stimulating T cell proliferation and differentiation, and stimulating the generation of cytotoxic T lymphocytes (CTLs) and natural killer cells (NK cells), as well as their proliferation. Therefore, IL2 can increase the lymphocyte population in vivo and enhance the function of the aforementioned nematocysts. Currently, IL2-based therapy is approved and used in patients with metastatic renal cell carcinoma and malignant melanoma.

[0005] The lymphocyte activity of IL-2 is mediated by its binding to a combination of three distinct IL-2 receptors (IL2Rs): IL-2 receptor α (IL2Rα; CD25), IL-2 receptor β (IL2Rβ; CD122), and common cytokine receptor γ (IL2Rγ; CD132). The distribution of each subunit receptor differs from cell to cell, and the binding affinity to IL-2 also varies greatly from receptor to receptor. High-affinity IL2Rs consist of a trimer of three subunits (α, β, γ), while dimeric IL2 receptors consisting of β and γ subunits are called intermediate-affinity IL2Rs. Dimeric intermediate-affinity IL2Rs bind to IL-2 with an affinity approximately 100 times lower than that of trimmeric high-affinity receptors, but both dimeric and trimmeric IL2Rs can transmit signals upon IL-2 binding. Therefore, the α-subunit, CD25, is not essential for IL2 signaling. While the α-subunit provides high affinity binding to its receptor, β-subunit , and γ-subunit It is important for signal propagation. It is a trimer containing CD25. IL2RIt is expressed by regulatory T cells and endothelial cells. Furthermore, while the trimer IL2R is transiently induced on normally activated T cells, these T cells express only the dimer IL2R in a resting state (Nature Review Immunology. 2012 12:180-190).

[0006] Regulatory T cells are a group of T cells that regulate the immune system. They maintain tolerance to self-antigens, are involved in autoimmune diseases, and generally inhibit the activation and proliferation of effector T cells, or negatively regulate them, thereby impeding the efficacy of cancer treatment. Regulatory T cells continuously express the highest levels of CD25 and have a higher binding affinity for IL-2 than effector T cells, which raises concerns about their ability to inhibit the efficacy of cancer treatment using IL-2.

[0007] In connection with IL-2 immunotherapy, side effects generated by recombinant human IL-2 therapy have become a concern. Patients receiving high-dose IL-2 therapy frequently experience severe systemic side effects, including cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, cutaneous, and hematological disorders, which require intensive monitoring and hospitalization. The primary cause of these side effects can be explained by the development of vesicular leakage syndrome (VLS), which is a pathological increase in vascular permeability leading to extravascular fluid outflow (e.g., causing pulmonary and skin edema and hepatocyte damage) and intravascular fluid depletion (causing hypotension and compensatory heart rate increase) in multiple organs. There is no treatment for VLS other than discontinuing IL-2 administration. Low-dose IL-2 administration has been tested in patients to avoid VLS, but it has resulted in an inappropriate therapeutic effect of reduced cancer treatment efficiency. While VLS was previously thought to be triggered by the release of inflammatory cytokines, such as tumor necrosis factor (TNF)-α, from IL2-activated NK cells, it has recently been demonstrated that IL2-induced pulmonary edema is generated from the direct binding of IL2 to pulmonary endothelial cells (expressing low or intermediate levels of trimerized IL2R) (International Immunology, 2006 vol.18, no.10:1461-1471).

[0008] Therefore, despite the potential of IL2 as an immunosuppressant cancer treatment, in order to reduce toxicity and side effects and enhance therapeutic efficacy, IL2 variants that selectively activate effector T cells and their associated Fc (fragment crystallizable) regions are being considered. Immune checkpoints Furthermore, it is necessary to develop new treatment methods that can minimize the side effects of IL2-mediated cancer treatment and enhance its efficacy by utilizing antibodies against cancer cell-specific antigens. [Overview of the project] [Problems that the invention aims to solve]

[0009] One embodiment provides a protein containing an IL2 variant.

[0010] Other embodiments include an IL2 variant, a first polypeptide comprising a first CH3 antibody-invariant region, and a second polypeptide comprising a second CH3 antibody-invariant region; and Immunity checkpoint The objective is to provide an antibody or its antigen-binding fragment against a tumor, or a protein complex containing an antibody or its antigen-binding fragment against a tumor-associated antigen.

[0011] Another embodiment is to provide a method for producing the protein or the protein complex.

[0012] Another embodiment provides a pharmaceutical composition for the prevention or treatment of cancer, comprising the protein or the protein complex as an active ingredient.

[0013] Another embodiment provides a method for treating cancer, which includes the step of administering the protein or the protein complex.

[0014] Another aspect is to provide uses for the protein or protein complex for the manufacture of cancer therapeutic agents. [Means for solving the problem]

[0015] One embodiment provides a protein comprising an IL2 variant, wherein the IL2 variant contains one or more amino acids selected from the group consisting of glutamic acid (E), alanine (A), lysine (K), and serine (S) at one or more positions selected from the group consisting of 35, 38, 42, and 125.

[0016] The IL2 variant is also one in which one or more amino acids of wild-type IL2, including the amino acid of SEQ ID NO: 16, are substituted. The IL2 variant is also one in which one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids are substituted, as long as the activity of IL2 is maintained. For example, the IL2 variant is also one in which one or more positions selected from the group consisting of 18, 19, 35, 38, 42, 125, and 126 of wild-type IL2, including the amino acid of SEQ ID NO: 16, are substituted.

[0017] Specifically, the IL2 variant may contain one or more amino acids selected from the group consisting of methionine (M), arginine (R), alanine (A), leucine (L), serine (S), phenylalanine (F), valine (V), isoleucine (I), glutamine (Q), tryptophan (W), asparagine (N), threonine (T), glutamic acid (E), and lysine (K) at one or more positions selected from the group consisting of 18, 19, 35, 38, 42, 125, and 126.

[0018] The aforementioned IL2 variants also have amino acid substitutions that increase or decrease their binding affinity to the IL2 receptor. For example, they can decrease the binding affinity to IL2Rα and / or IL2Rβγ. Therefore, by reducing their binding affinity to IL2R, the IL2 variants can selectively activate effector T cells against regulatory T cells that inhibit IL2-mediated immune activity.

[0019] As a specific example, the IL2 variant may contain glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125.

[0020] In other specific examples, the IL2 variant may further contain one or more amino acids selected from the group consisting of leucine (L), methionine (M), arginine (R), alanine (A), serine (S), phenylalanine (F), valine (V), isoleucine (I), glutamine (Q), tryptophan (W), asparagine (N), and threonine (T) at one or more positions selected from the group consisting of 18 and 19.

[0021] In yet other specific examples, the IL2 variant may further contain one or more amino acids selected from the group consisting of threonine (T) and isoleucine (I) at position 126.

[0022] For example, the IL2 variant an amino acid selected from the group consisting of methionine (M), arginine (R), alanine (A), leucine (L), serine (S), phenylalanine (F), valine (V), isoleucine (I), and glutamine (Q) at position 18; an amino acid selected from the group consisting of serine (S), leucine (L), tryptophan (W), asparagine (N), isoleucine (I), threonine (T), alanine (A), methionine (M), and phenylalanine (F) at position 19; glutamic acid (E) at position 35; alanine (A) at position 38; lysine (K) at position 42; serine (S) at position 125; and an amino acid selected from the group consisting of threonine (T) and isoleucine (I) at position 126; and may contain an amino acid selected from the group consisting of the above.

[0023] Specifically, the IL2 variant glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125; methionine (M) at position 18, serine (S) at position 19, glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125; Alanine (A) is ranked 18th, serine (S) 19th, glutamic acid (E) 35th, alanine (A) 38th, lysine (K) 42nd, and serine (S) 125th; Arginine (R) is ranked 18th, serine (S) 19th, glutamic acid (E) 35th, alanine (A) 38th, lysine (K) 42nd, and serine (S) 125th; Methionine (M) is ranked 18th, leucine (L) 19th, glutamic acid (E) 35th, alanine (A) 38th, lysine (K) 42nd, and serine (S) 125th; Leucine (L) is ranked 18th, serine (S) 19th, glutamic acid (E) 35th, alanine (A) 38th, lysine (K) 42nd, and serine (S) 125th; Serine (S) is ranked 18th, tryptophan (W) 19th, glutamic acid (E) 35th, alanine (A) 38th, lysine (K) 42nd, and serine (S) 125th; Phenylalanine (F) is ranked 18th, asparagine (N) 19th, glutamic acid (E) 35th, alanine (A) 38th, lysine (K) 42nd, and serine (S) 125th; Valine (V) is ranked 18th, isoleucine (I) 19th, glutamic acid (E) 35th, alanine (A) 38th, lysine (K) 42nd, and serine (S) 125th; Isoleucine (I) is ranked 18th, threonine (T) 19th, glutamic acid (E) 35th, alanine (A) 38th, lysine (K) 42nd, and serine (S) 125th. Glutamine (Q) is ranked 18th, alanine (A) 19th, glutamic acid (E) 35th, alanine (A) 38th, lysine (K) 42nd, and serine (S) 125th; Methionine (M) is ranked 18th, methionine (M) 19th, glutamic acid (E) 35th, alanine (A) 38th, lysine (K) 42nd, and serine (S) 125th; Methionine (M) is ranked 18th, phenylalanine (F) 19th, glutamic acid (E) 35th, alanine (A) 38th, lysine (K) 42nd, and serine (S) 125th; Glutamic acid (E) is ranked 35th, alanine (A) 38th, lysine (K) 42nd, serine (S) 125th, and threonine (T) 126th; and This may include selection from the group consisting of glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, serine (S) at position 125, and isoleucine (I) at position 126.

[0024] In further specific examples, the IL2 variant may include an amino acid selected from the group consisting of SEQ ID NOs: 1 to 15. The IL2 variant may also include a polynucleotide coding the amino acid. Specifically, the polynucleotide may be selected from the group consisting of SEQ ID NOs: 56 to 70.

[0025] Furthermore, the protein may include an Fc region linked by a linker or carrier. For example, the linker may contain 1 to 50 amino acids, albumin or a fragment thereof, or a copolymer such as polyethylene glycol.

[0026] In one specific example, the linker may include the amino acid sequence of SEQ ID NO: 18.

[0027] In other specific examples, the Fc region may include one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 19 to 21; SEQ ID NOs: 22 to 24; SEQ ID NOs: 25 to 27; SEQ ID NOs: 28 to 30; SEQ ID NOs: 31 to 33; SEQ ID NOs: 34 to 36; SEQ ID NOs: 37 to 39; SEQ ID NOs: 40 to 42; SEQ ID NOs: 43 to 45; and SEQ ID NOs: 46 to 48.

[0028] Furthermore, the aforementioned protein is Immunity checkpoint The following may further include an antibody or antigen-binding fragment against the said. Immune checkpoints These include, for example, PD-L1, PD-1, LAG3, VISTA, BTLA, TIM3, TIGIT, and CTLA-4.

[0029] Furthermore, the protein may further contain an antibody or antigen-binding fragment against a tumor-associated antigen. Examples of tumor-associated antigens include PD-L1, EGFR, HER-2, B7H3, GPC3, CEA, TROP, and PSMA.

[0030] Therefore, other embodiments provide a protein complex comprising an IL2 variant and an Fc region.

[0031] Other embodiments include IL2 variants; and Immunity checkpoint The present invention provides a protein complex containing an antibody or an antigen-binding fragment against the antibody.

[0032] Another embodiment provides a protein complex comprising an IL2 variant and an antibody or antigen-binding fragment against a tumor-specific antigen.

[0033] In this specification, the term "protein complex" refers to a complex consisting of two or more related polypeptides, which is an artificial recombinant protein expressed after linking one or more genes of other proteins to one protein, and may be used interchangeably with "fusion protein." By linking two or more proteins, the protein complex can be expected to have a synergistic effect on its function. Therefore, the protein complex is a conjugate, protein complex, or fusion protein containing an Fc region, which can selectively increase the activity of regulatory T cells by replacing specific amino acids in IL2. Furthermore, the protein complex can effectively induce cancer cell death by specifically distributing itself in the tumor microenvironment, suppressing immune checkpoints, and activating nematocysts. Compared to conventional therapeutic agents, it can provide a therapeutic agent with fewer side effects and maximized anticancer activity.

[0034] Another embodiment comprises a first polypeptide containing a first CH3 antibody-invariant region and a second polypeptide containing a second CH3 antibody-invariant region, wherein the first polypeptide and the second polypeptide form a protein complex that forms a heterodimer, with one or more of the first or second polypeptides having a N-terminus. Immunity checkpoint The present invention provides a protein complex comprising an antibody or its antigen-binding fragment against, or an antibody or its antigen-binding fragment against a tumor-associated antigen, wherein one or more of the N-terminus or C-terminus of the first or second polypeptide contains an IL2 variant.

[0035] Figure 4 shows the structure of a protein complex containing an IL2 mutant, as a specific example.

[0036] Referring to Figure 4, the protein complex is located at the N-terminus of the first polypeptide containing the first CH3 antibody-invariant region. Immune checkpoints The product comprises an antibody against or an antigen-binding fragment thereof, or an antibody against a tumor-specific antigen or an antigen-binding fragment thereof, and may include an IL2 variant at the N-terminus of a second polypeptide containing a second CH3 antibody-invariant region.

[0037] Furthermore, the protein complex has a first polypeptide containing a first CH3 antibody-invariant region and a second polypeptide containing a second CH3 antibody-invariant region at its N-terminus. Immune checkpoints The product comprises an antibody against or an antigen-binding fragment thereof, or an antibody against a tumor-specific antigen or an antigen-binding fragment thereof, and may contain an IL2 variant at the C-terminus of the first or second polypeptide.

[0038] The aforementioned IL2 variant, Immune checkpoints The specific details regarding tumor-specific antigens are as described above. In the protein complex, Immune checkpoints The binding affinity of antibodies or antigen-binding fragments against cancer cells or T cells is superior to the binding affinity of T cells to IL2 receptors. Therefore, the above Immune checkpointsAntibodies or antigen-binding fragments against the tumor-specific antigen can reduce the side effects caused by IL2 receptor binding, which can induce systemic immune cell activation by first binding to cancer cells or T cells surrounding cancer cells. Furthermore, antibodies or antigen-binding fragments against the tumor-specific antigen not only specifically bind to cancer cells, but also exhibit superior binding affinity to cancer cells compared to the binding affinity between IL2 receptor-binding proteins. Therefore, antibodies or antigen-binding fragments against the tumor-specific antigen can reduce the side effects caused by IL2 receptor binding, which can induce systemic immune cell activation by first binding to cancer cells.

[0039] The aforementioned Immune checkpoints And / or antibodies against tumor-specific antigens or their antigen-binding fragments are, for example, antibodies, antigen-binding fragments (Fab), single-chain variable fragments (scFv), and nanobodies.

[0040] In this specification, the term "antibody" is used interchangeably with "immunoglobulin (Ig)". A complete antibody has a structure consisting of two full-length light chains and two full-length heavy chains, each light chain bound to a heavy chain by a disulfide bond (SS-bond). There are two types of light chains, λ and κ, and they consist of approximately 211 to 217 amino acids. Each human antibody similarly contains only one type of light chain. The light chain consists of a continuous invariant region and a variable region. There are five types of heavy chains (γ, δ, α, μ, ε), and the heavy chain determines the type of antibody. α and γ consist of 450 amino acids, while μ and ε consist of 550 amino acids. The heavy chain has two regions, namely a variable region and an invariant region. The variable region is the region on the antibody to which the antigen binds. The variable region may include a complementarity determining region (CDR) that confers binding specificity to the antigen.

[0041] The antibody may contain an antigen-binding fragment (Fab) region and a fragment crystallizable (Fc) region that binds to a cell surface receptor. When cleaved with papain, a complete antibody may be cleaved into two Fab regions and one Fc region. The Fab region is also a polypeptide consisting of a variable region (VH) domain and a heavy chain invariant region 1 (CH1) domain, linked by a disulfide bond to a polypeptide consisting of a variable region (VL) domain and a light chain invariant region (CL) domain. The Fc region is also a polypeptide consisting of two linked polypeptides, each containing a heavy chain invariant region 2 (CH2) domain and an invariant region 3 (CH3) domain. The Fc region may form a hinge region. The CH3 antibody invariant region refers to the heavy chain invariant region 3 domain of the antibody.

[0042] Generally, the effector function of an antibody involves the Fc region binding to its receptor, the Fcγ receptor, or to the complement component molecule C1q, thereby causing antibody-dependent cell cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Rabbit disease cells possess many Fcγ receptors, and since this can induce undesirable cell death, it is desirable to eliminate the effector function. Therefore, the CH3 antibody-invariant region has been modified to have high stability by reducing its binding affinity to the Fcγ receptor, eliminating or significantly reducing its effector function. Furthermore, the CH3 antibody-invariant region has also been modified to reduce antibody-dependent cell-mediated toxicity (ADCC).

[0043] In one specific example, the Fc region may contain tryptophan (W) at position 366 of the first CH3 antibody-invariant region, serine (S) at position 366 of the second CH3 antibody-invariant region, alanine (A) at position 368, and valine (V) at position 407, and may contain glycine (G) or phenylalanine (F) at position 351 of the second CH3 antibody-invariant region.

[0044] Furthermore, the Fc region may contain serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407 of the first CH3 antibody-invariant region, tryptophan (W) at position 366 of the second CH3 antibody-invariant region, and glycine (G) or phenylalanine (F) at position 351 of the first CH3 antibody-invariant region.

[0045] Furthermore, the Fc region contains tryptophan (W) at position 366 of the first CH3 antibody-invariant region, glycine (G) at position 351, serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407 of the second CH3 antibody-invariant region; and may contain phenylalanine (F) or tryptophan (W) at position 351 of the first CH3 antibody-invariant region.

[0046] Furthermore, the Fc region contains glycine (G) at position 351, serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407 of the first CH3 antibody-invariant region, and tryptophan (W) at position 366 of the second CH3 antibody-invariant region; and may contain phenylalanine (F) or tryptophan (W) at position 351 of the second CH3 antibody-invariant region.

[0047] In other specific examples, the first CH antibody-invariant region and the second CH antibody-invariant region may further contain one or more amino acids selected from the group consisting of alanine (A), glycine (G), glutamine (Q), phenylalanine (F), glutamic acid (E), and serine (S) at one or more positions selected from the group consisting of 234, 235, 329, 297, 331, and 265. Specifically, the CH antibody-invariant region is also the CH2 antibody-invariant region.

[0048] For example, the first CH2 antibody-invariant region and the second CH2 antibody-invariant region are also in which the leucine (L) at positions 234 and 235 are further replaced with alanine (A) (L234A / L235A).

[0049] Furthermore, the first CH2 antibody-invariant region and the second CH2 antibody-invariant region are also modified in which leucine (L) at positions 234 and 235 is replaced with alanine (A), and proline (P) at position 329 is further replaced with glycine (G) (L234A / L235A / P329G).

[0050] Furthermore, the first CH2 antibody-invariant region and the second CH2 antibody-invariant region are also obtained by further substituting the asparagine (N) at position 297 with alanine (A), glutamine (Q), or glycine (G) (N297A, N297Q, or N297G).

[0051] Furthermore, the first CH2 antibody-invariant region and the second CH2 antibody-invariant region are also obtained by substituting leucine (L) at positions 234 and 235 with phenylalanine (F) and glutamic acid (E), and further substituting proline (P) at position 331 with serine (S) (L234F / L235E / P331S).

[0052] Furthermore, the first CH2 antibody-invariant region and the second CH2 antibody-invariant region are also in which leucine (L) at positions 234 and 235 is replaced with phenylalanine (F) and glutamic acid (E), and aspartic acid (D) at position 265 is further replaced with alanine (A) (L234F / L235E / D265A).

[0053] In other specific examples, the Fc region may include one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 19 to 21; SEQ ID NOs: 22 to 24; SEQ ID NOs: 25 to 27; SEQ ID NOs: 28 to 30; SEQ ID NOs: 31 to 33; SEQ ID NOs: 34 to 36; SEQ ID NOs: 37 to 39; SEQ ID NOs: 40 to 42; SEQ ID NOs: 43 to 45; and SEQ ID NOs: 46 to 48.

[0054] In other specific examples, the first polypeptide or the second polypeptide; and the IL2 variant may be linked by a linker or carrier. The linker includes the amino acid sequence (GGGGS)n, where "n" is a natural number from 1 to 10. Specifically, the linker may include the amino acid sequence of SEQ ID NO: 18.

[0055] The first and second polypeptides can form the Fc region of the antibody.

[0056] The term "heterodimer" refers to a protein complex formed by the linking of two polypeptides that differ from each other in the order, number, or type of amino acid residues. The term "protein complex" also refers to a protein complex formed by the linking of two polypeptides that specifically bind to different targets.

[0057] The protein complex may also be an antibody or its antigen-binding fragment, a receptor-agonist conjugate, a receptor-antagonist conjugate, a receptor-ligand conjugate, or a ligand-decoy receptor conjugate. The protein complex may also include components selected from the group consisting of an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), an extracellular domain of a membrane receptor, an agonist, an antagonist, a ligand, a decoy receptor, a cytokine, a coagulation factor, and an affinity tag.

[0058] The antibody may be, for example, IgA, IgD, IgE, IgG, or IgM. The antibody may also be a monoclonal antibody or a polyclonal antibody. The antibody may also be an animal-derived antibody, a mouse-human chimeric antibody, a humanized antibody, or a human antibody.

[0059] In this specification, the term "antigen-binding fragment" means a fragment of the entire structure of rabbit globulin, which is a part of a polypeptide containing a portion to which an antigen can bind. For example, an antigen-binding fragment may also be scFv, (scFv)2, Fv, Fab, Fab', Fv F(ab')2, or a combination thereof.

[0060] In this specification, the term "receptor" means a substance that receives or transmits signals that can be transmitted to a biological system. The receptor is also a protein receptor. The receptor may bind to an agonist, antagonist, ligand, or cytokine. The agonist is a substance that binds to a receptor and activates it, thereby inducing a biological response. The antagonist is a substance that binds to a receptor and inhibits it, thereby suppressing a biological response. The ligand is a substance that binds to a receptor. The ligand may bind to a decoy receptor. A decoy receptor is a receptor that specifically binds to a ligand, thereby inhibiting signal transmission through the actual receptor. The cytokine is a small protein that acts on cellular signal transmission, regulation and maintenance of inflammatory processes.

[0061] The protein complex may also be deformed. For example, the protein complex may be deformed by conjugation or binding, glycosylation, tag attachment, or a combination thereof. The antibody may be conjugated with other drugs, such as anticancer agents. For example, the protein complex may also be conjugated with horseradish peroxidase (HRP), alkaline phosphatase, hapten, biotin, streptavidin, fluorescent substances, radioactive substances, quantum dots, polyethylene glycol (PEG), histidine tags, or a combination thereof. The fluorescent substances may also be Alexa Fluor532, Alexa Fluor546, Alexa Fluor568, Alexa Fluor680, Alexa Fluor750, Alexa Fluor790, or Alexa Fluor350 (all registered trademarks).

[0062] The amino acid positions in the Fc region and the CH2 and CH3 regions are determined according to the Kabat EU index (the EU index described in 'Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)'). The amino acid positions and corresponding amino acid types in the CH3 domain are based on human IgG1.

[0063] Other embodiments provide polynucleotides that code for proteins according to one embodiment or protein complexes according to other embodiments.

[0064] The specific contents of the aforementioned protein or protein complex are as described above.

[0065] In one specific example, the polynucleotide may include one or more polynucleotides selected from the group consisting of SEQ ID NOs. 56 to 70, and one or more polynucleotides selected from the group consisting of SEQ ID NOs. 74 to 76.

[0066] In other specific examples, the polynucleotide may include one or more polynucleotides selected from the group consisting of SEQ ID NOs. 56 to 70; a polynucleotide containing SEQ ID NOs. 73; and one or more polynucleotides selected from the group consisting of SEQ ID NOs. 74 to 76.

[0067] In further specific examples, the polynucleotide may include one or more polynucleotides selected from the group consisting of SEQ ID NOs. 56 to 70; a polynucleotide containing SEQ ID NOs. 73; one or more polynucleotides selected from the group consisting of SEQ ID NOs. 74 to 76; and one or more polynucleotides selected from the group consisting of SEQ ID NOs. 77 to 82.

[0068] Another embodiment provides a method for producing a protein or protein complex, comprising the step of transforming cells with an expression vector coding a protein according to one embodiment or a protein complex according to another embodiment to express the protein or protein complex. Another embodiment provides a protein or protein complex comprising an IL2 variant produced by the above method.

[0069] The specific contents of the aforementioned protein or protein complex are as described above.

[0070] An expression vector is an expression vector capable of expressing a target protein in a suitable host cell, and includes essential regulatory elements that are operably linked to express an inserted nucleic acid sequence. "Operaably linked" means that the nucleic acid expression regulatory sequence and the nucleic acid coding the target protein are functionally linked to perform a general function. The expression vector may also include polynucleotides coding the protein complex. The expression vector may include regulatory sites necessary for gene expression, such as enhancers, promoters, and poly(A) sequences.

[0071] The cells are also cancer cells. The cells are also in vitro cells. The cells are also bacteria, yeast, plant cells, or mammalian cells. The bacteria are also Intestinal bacteria. The mammalian cells mean cells derived from mice, rats, rabbits, dogs, cats, sheep, cattle, horses, monkeys, chimpanzees, or humans. The cells are also cell lines. The cells are selected from the group consisting of, for example, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, baby hamster kidney (BHK) cells, NS0 cells, PER.C6 cells, HeLa cells, MDCK (Madin-Darby Canine Kidney) cells, SP2 / 0 mouse myeloma cells, COS-7, and YB2 / 0 rat myeloma cells. The aforementioned CHO cells are also CHO DG44, CHO-K1, CHO-S, GS-CHO, or CHO DUKX(DXB11) cells. The aforementioned HEK cells are also HEK293 cells.

[0072] "Transformation" refers to a method of inserting a specific nucleic acid fragment into the genes of a cell and then expressing the inserted nucleic acid.

[0073] In one specific example, the method involves adding one or more N-terminuses of the first or second polypeptide. Immunity checkpoint The method may include the step of co-transfection cells with an expression vector encoding a first and / or second polypeptide containing an antibody or antigen-binding fragment against a tumor-associated antigen, or an antibody or antigen-binding fragment against a tumor-associated antigen, and an expression vector encoding a first or second polypeptide containing an IL2 variant at its N-terminus or C-terminus. Immunity checkpoint The process may include transforming two or more cells with an expression vector encoding a first polypeptide and / or a second polypeptide containing an antibody or antigen-binding fragment against a tumor-associated antigen, or an antibody or antigen-binding fragment against a tumor-associated antigen, and an expression vector encoding the first or second polypeptide containing IL2 at its N-terminus or C-terminus.

[0074] The cells may be cultured in a cell culture medium. A cell culture medium means a solution containing the nutrients necessary for culturing cells. The medium may include commercially available or manufactured media used for culturing cells. The cell culture medium may contain antibiotics. The cell culture medium may contain G418 (geneticin), puromycin, blasticidin, zeocin, or a combination thereof. The cell culture medium may include a chemically defined medium.

[0075] The cells may be cultured under conditions that allow for cell survival or proliferation. The conditions that allow for cell survival or proliferation may vary depending on the cell type. The cells may be cultured at approximately 25°C to 42°C, approximately 25°C to 40°C, approximately 30°C to 40°C, approximately 30°C to 37°C, or approximately 37°C. The cells may be cultured in the presence of air at approximately 1% CO2 to 10% CO2, or approximately 5% CO2 to 10% CO2. The cells may be cultured in a medium with a pH of approximately 6 to 8, approximately 6.2 to 7.8, approximately 6.4 to 7.6, approximately 6.6 to 7.4, or approximately 6.8 to 7.2. The cells may be cultured under conditions of approximately 10% to 80% dissolved oxygen, approximately 15% to 70%, or approximately 20% to 60% dissolved oxygen.

[0076] The culture method may vary depending on the cell type. Known methods can be used for the culture. The culture can be carried out using plates, flasks, etc. The culture can be carried out by attaching the cells to a substrate or by suspending them in a culture medium. The culture can be subculture, batch culture, fed-batch culture, perfusion culture, or a combination thereof. During the culture, the cell culture medium may be periodically replaced with fresh medium. The cells can be cultured for approximately 1 day or more, approximately 2 days or more, approximately 3 days or more, approximately 4 days or more, approximately 5 days or more, approximately 6 days or more, approximately 1 week or more, approximately 10 days or more, approximately 2 weeks or more, approximately 3 weeks or more, approximately 1 month or more, approximately 1 day to approximately 1 month, approximately 1 day to approximately 3 weeks, approximately 1 day to approximately 2 weeks, approximately 2 days to approximately 2 weeks, approximately 3 days to approximately 2 weeks, approximately 4 days to approximately 2 weeks, approximately 5 days to approximately 2 weeks, approximately 6 days to approximately 2 weeks, or approximately 1 week to approximately 2 weeks.

[0077] The process may include a step of obtaining a protein complex from the aforementioned cells or cell culture medium.

[0078] The aforementioned cell culture medium is also a culture medium without the aforementioned cells.

[0079] When the expression vector is co-transformed into cells, the N-terminus is transferred from the cells or cell culture medium. Immunity checkpoint A protein complex can be obtained comprising a first polypeptide containing an antibody or antigen-binding fragment against, or an antibody or antigen-binding fragment against a tumor-associated antigen, and a second polypeptide containing an IL2 variant at its N-terminus or C-terminus. Immunity checkpoint When transforming two or more cells with an expression vector encoding a first polypeptide containing an antibody or antigen-binding fragment against a tumor-associated antigen, or an antibody or antigen-binding fragment against a tumor-associated antigen, and an expression vector encoding a second polypeptide containing an IL2 variant at its N-terminus or C-terminus, the N-terminus is obtained from the cells or cell culture medium. Immunity checkpoint A first polypeptide containing an antibody or its antigen-binding fragment against a tumor-associated antigen, or an antibody or its antigen-binding fragment against a tumor-associated antigen; and a second polypeptide containing an IL2 variant at the N-terminus or C-terminus can be obtained.

[0080] The step of obtaining the protein complex involves the obtained N-terminus Immunity checkpoint The process may include a step of incubating a first polypeptide containing an antibody or antigen-binding fragment against a tumor-associated antigen, or an antibody or antigen-binding fragment against a tumor-associated antigen, with a second polypeptide containing an IL2 variant at its N-terminus or C-terminus to form a protein complex. The incubation may be carried out under reducing conditions. The reducing conditions may also be in the presence of 2-mercaptoethanol (2-ME), dithiothreitol (DTT), or a combination thereof.

[0081] The step of obtaining the protein complex may include a step of purifying the protein complex. The purification may be carried out by filtration, centrifugation, chromatography, dialysis, immunoprecipitation, or a combination thereof.

[0082] Other embodiments provide a pharmaceutical composition for the prevention or treatment of cancer comprising a protein according to one embodiment or a protein complex according to another embodiment. Yet another embodiment provides uses of a protein according to one embodiment or a protein complex according to another embodiment for the manufacture of a cancer preventive or therapeutic agent.

[0083] The specific details regarding the aforementioned protein or protein complex are as described above.

[0084] The aforementioned cancers may be solid or non-solid tumors. A solid tumor refers to a cancerous tumor that develops in an organ such as the liver, lungs, breast, or skin. A non-solid tumor is a cancer that develops in the blood and is also called a blood cancer. The aforementioned cancers may also be carcinomas, sarcomas, hematopoietic cell-derived carcinomas, germ cell tumors, or blastomas. The aforementioned cancers may be selected from the group consisting of breast cancer, skin cancer, head and neck cancer, pancreatic and intestinal cancer, lung cancer, intestinal cancer, colon cancer, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, pellucida sarcoma, melanoma, brain and spinal cord tumors, brain cancer, thymoma, mesothelioma, esophageal cancer, biliary tract cancer, testicular cancer, germ cell tumors, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, acute leukemia, late-onset leukemia, multiple myeloma, Hodgkin's disease, endocrine cancer, and sarcoma.

[0085] The term "prevention" refers to all actions that suppress or delay the onset of a disease by administering the pharmaceutical composition. The term "treatment" refers to all actions that improve or beneficially alter the symptoms of a disease by administering the pharmaceutical composition.

[0086] The pharmaceutical composition may include a pharmaceutically acceptable carrier. The carrier is used to mean an excipient, diluent, or auxiliary agent. The carrier may also be selected from the group consisting of, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, physiological saline, buffer such as PBS, methylhydroxybenzoic acid, propylhydroxybenzoic acid, talc, magnesium stearate, and mineral oil. The composition may include fillers, anti-flocculants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, or combinations thereof.

[0087] The aforementioned pharmaceutical composition can be prepared in any dosage form by conventional methods. The composition may be formulated, for example, as an oral dosage form (e.g., powder, tablet, capsule, syrup, pill, or granule) or as a parenteral dosage form (e.g., injection). The composition may also be manufactured as a systemic or topical dosage form.

[0088] The pharmaceutical composition may further comprise other anticancer agents. These anticancer agents may include cetuximab, panitumumab, erlotinib, gefitinib, trastuzumab, T-DM1, Perjeta, lapatinib, paclitaxel, taxol, tamoxifen, cisplatin, or combinations thereof. The pharmaceutical composition may also be a single composition or individual compositions. For example, the antibody or antigen-binding fragment composition may be a parenterally administered formulation, while the anticancer agent may be an orally administered formulation.

[0089] The pharmaceutical composition may contain the protein complex in an effective amount. The term "effective amount" means an amount sufficient to produce a preventive or therapeutic effect when administered to an individual in need of prevention or treatment of a disease. The effective amount can be appropriately selected by those skilled in the art depending on the selected cells or individuals. It may be determined by the severity of the disease, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration, the route of administration and elimination ratio, the duration of treatment, factors including drugs compounded or used concurrently with the composition used, and other factors well known in the medical field. The effective amount may also be about 0.5 μg to about 2 g, about 1 μg to about 1 g, about 10 μg to about 500 mg, about 100 μg to about 100 mg, or about 1 mg to about 50 mg per pharmaceutical composition.

[0090] The dosage of the pharmaceutical composition may be, for example, within the range of approximately 0.001 mg / kg to approximately 100 mg / kg, approximately 0.01 mg / kg to approximately 10 mg / kg, or approximately 0.1 mg / kg to approximately 1 mg / kg based on adult standards. The administration may be once daily, multiple times daily, or once a week, once every two weeks, once every three weeks, once every four weeks, or once a year.

[0091] Another embodiment provides a method for preventing or treating cancer, comprising the step of administering a protein according to one embodiment or a protein complex according to other embodiments to a cell or an organism.

[0092] The specific details regarding the aforementioned proteins, protein complexes, cells, cancer, prevention, or treatment are as described above.

[0093] The individual may also be a mammal, such as a human, cattle, horse, pig, dog, sheep, goat, or cat. The individual may also be suffering from cancer or be at high risk of developing cancer.

[0094] The method may further include the step of administering a second active ingredient to the individual. The second active ingredient is also an active ingredient for the prevention or treatment of cancer. The active ingredient may be administered simultaneously with, individually, or sequentially with the protein complex.

[0095] The protein or protein complex may be administered directly to an individual by any means, such as orally, intravenously, intramuscularly, transdermally, mucosally, intranasally, intratracheally, or subcutaneously. The protein complex may be administered systemically or topically, alone or in combination with other pharmaceutically active compounds.

[0096] The desired dose of the protein or protein complex may be appropriately selected by those skilled in the art, depending on the patient's condition and weight, the severity of the disease, the drug form, the route of administration, and the duration of administration. The dose may also be, for example, in the range of about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 1 mg / kg based on adult standards. The administration may be once daily, multiple times daily, or once a week, once every two weeks, once every three weeks, or once every four weeks or once a year. [Effects of the Invention]

[0097] One embodiment of the protein can selectively increase the activity of regulatory T cells by substituting a specific amino acid sequence in IL2. Furthermore, a pharmaceutical composition containing the protein has fewer side effects and maximizes anticancer activity compared to conventional therapeutic agents, and can therefore be used for the prevention or treatment of cancer. [Brief explanation of the drawing]

[0098] [Figure 1A] The results of purifying a PD-L1 antibody-IL2 mutant 2 protein complex using a specific protein-A affinity column purification method are shown below. [Figure 1B] The results of purifying a PD-L1 antibody-IL2 mutant 2 protein complex using a cation exchange resin column purification method are shown as a specific example. [Figure 1C] This paper presents the results of a purity analysis of a PD-L1 antibody-IL2 mutant 2 protein complex using SE-HPLC analysis as a specific example. [Figure 2A] This is a comparison of the anticancer effects of a PD-L1 antibody-IL2 mutant 2 protein complex and positive control groups (administered with Avelumab, and Avelumab and Aldesleukin) and negative control groups in mice transplanted with ulcer tumors, using one specific example. [Figure 2B] This is a specific example of the results confirming the individual-specific cancer cell growth inhibition and complete remission activity of the PD-L1 antibody-IL2 mutant 2 protein complex in mice transplanted with ulcer cancer. [Figure 2C] This report presents the results of confirming the individual-specific inhibition of cancer cell growth and complete remission effects in blepharocarcinoma-transplanted mice in negative and positive control groups (Avelumab and Avelumab combined with Aldesleukin). [Figure 3] This is a specific example of the results confirming the anticancer effect of the PD-L1 antibody-IL2 mutant 4 protein complex in mice transplanted with ulcer cancer. [Figure 4] The structure of a protein complex including an IL2 variant, as an example, is shown. [Figure 5A] This is the result of purifying the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c) using a specific example of a protein-A affinity column purification method. [Figure 5B] This is the result of purifying the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c) using a hydrophobic interaction column purification method as one specific example. [Figure 5C] The results of purity analysis of the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c) using one specific example are shown. [Figure 6A] This is a comparison of the anticancer effects of a mousePD-1 antibody-IL2 mutant 14 protein complex (a+c), negative control group, and positive control group in a mouse model of ulcer cancer using one specific example. [Figure 6B] This is a comparison of body weight changes in a blepharoplasty transplanted mouse model after administering a specific example of the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c), a negative control group, and a positive control group to the blepharoplasty transplanted mouse model. [Modes for carrying out the invention]

[0099] The following are preferred embodiments to aid in understanding the present invention. However, these embodiments are provided to further facilitate understanding of the present invention and do not limit the scope of the present invention. [Examples]

[0100] Example 1. Production and activity confirmation of IL2 mutant protein

[0101] 1-1. Cloning and culture of IL2 mutant proteins

[0102] An IL2 mutant was prepared by substituting some amino acids in the wild-type IL2 amino acid molecule (SEQ ID NO: 16). Specifically, a polynucleotide encoding the IL2 mutant tagged with 6XHis was synthesized using ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service. This polynucleotide was then inserted into the ApaII-NheI enzyme site of a pcDNA3.1 expression vector to produce an expression vector expressing the IL2 mutant. The amino acid substitution sites and the substituted amino acids in the IL2 mutant are shown in Table 1 below. Hereafter, ExpiFectamine TM The expression vector is converted to ExpiCHO-S using the CHO Transfection kit (ThermoFisher). TM Cell lines were transformed. The transformed cells were cultured at 32°C in a 5% CO2 incubator at 120 rpm for 12 days. After 12 days, the supernatant of the culture medium was separated and collected, and the IL2 mutant protein was obtained by filtering through a sterile filter.

[0103] [Table 1]

[0104] 1-2. Purification of IL2 mutant protein

[0105] After purification using His tagged to the IL-2 mutant protein obtained through the culture medium acquired in Example 1-1, a high-purity substance with a purity of 95% or higher was obtained.

[0106] First, the culture medium obtained in Example 1-1 was centrifuged to separate the cultured cells from the culture medium. Subsequently, the IL2 mutant protein in the separated medium was filtered through a 0.22 μm filter (Thermo Scientific) to remove any fine residues. The filtered medium was then purified using imidazole affinity chromatography (Ni-SepFast, Biotoolomics), followed by desalting and concentration to remove the imidazole buffer components present in the purified product. Subsequently, the content and purity of the final purified product were analyzed. Specifically, the filtered medium was placed on a Ni-SepFast column stabilized in Phosphate buffer saline (pH 7.4). Proteins that were not nonspecifically bound were washed away using the same buffer, and proteins that specifically bound to the Ni-SepFast column were eluted using imidazole buffer solutions (0M and 0.5M, pH 7.4) in an imidazole concentration increasing gradient manner. The secured IL2 mutant protein was stabilized by desalting in phosphate buffer saline (pH 7.4), and then the purity of the purified IL2 mutant protein was analyzed using size exclusion HPLC (TSK-3000SWxL, 7.8 mm × 30 cm, Tosoh Corporation). The molecular weight was also confirmed by SDS-PAGE analysis.

[0107] 1-3. Confirmation of activation of IL2 mutant protein in human immune cells

[0108] The activation of the IL2 mutant-protein complex purified in Examples 1-2 against human bunny plague cells was confirmed. Specifically, human peripheral blood mononuclear cells (Stemcell) and effector CD8+ T cells (CD3+, CD8+) and Treg cells (CD4+, CD25+) were reacted with antibodies that specifically fluorescently label these cells at 4°C for 30 minutes in the dark. Antibodies that did not adhere to the cells were then removed by centrifugation. Subsequently, the IL2 mutant 2, 4, and 14 proteins purified in Examples 1-2 were treated and reacted at 37°C for 20 minutes in the dark, and then fixed in 1 mL of Fixation buffer (BD,US) for 12 minutes. Aldesleukin (proleukin, Novartis, Switzerland) was used as a positive control group, and each drug was treated at concentrations ranging from 0.01 to 8,000 nM. Subsequently, 1.5 mL of Perm3 buffer (BD,US) was applied to the cytoplasm of fixed human peripheral blood mononuclear cells, and the cells were reacted at 4°C for 35 minutes to allow the fluorescent label to penetrate into the cytoplasm. Then, phosphorylated STAT-5 and Foxp3+ protein, a Treg cell marker, were fluorescently labeled and reacted for 30 minutes. Subsequently, the degree of STAT-5 phosphorylation in effector CD8+T and Treg cells treated with IL2 mutant protein was compared by fluid cell analysis.

[0109] [Table 2]

[0110] As a result, as shown in Table 2, the positive control group (Aldesleukin) showed CD8+ T cell / regulatory T cell EC 50 The ratio of values ​​to 16.3 confirmed that it induces Treg cell activity more strongly than CD8+ T cells.

[0111] On the other hand, IL2 variants 2, 4, and 14 proteins are found in CD8+ T cells / regulatory T cells. 50The ratios of these values, 0.6, 0.56, and 5.6 respectively, confirmed that the activity of CD8+ T cells was more selectively activated compared to the positive control group.

[0112] Example 2. Production and characterization of the IL2 mutant-Fc protein complex.

[0113] 2-1. Cloning and culture of IL2 mutant-Fc protein complex

[0114] Fc protein complexes containing IL2 mutants were prepared. Specifically, polynucleotides coding the Fc region were synthesized using ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service, and then inserted into the AvrII-BstZ17I enzyme site of the pCHO 1.0 expression vector to produce a first expression vector expressing the Fc region. Using the same service, polynucleotides coding the complex containing the linker and Fc region of IL2 mutants 1, 2, 4, and 14 prepared in Example 1 were synthesized, and then inserted into the AvrII-BstZ17I enzyme site of the pCHO 1.0 expression vector to produce a second expression vector expressing IL2 mutant-Fc. Subsequently, ExpiFectamine TM After mixing the first and second expression vectors in a 1:1 ratio using the CHO Transfection kit (ThermoFisher), ExpiCHO-S TM Cell lines were transformed. The transformed cells were cultured at 32°C in an 8% CO2 incubator at 125 rpm for 12 days. After 12 days, the culture supernatant was separated and collected, and filtered through a sterile filter to obtain protein complexes containing Fc and IL2 mutant-Fc.

[0115] 2-2. Purification of the IL2 mutant-Fc protein complex

[0116] The IL2 mutant-Fc protein complex obtained through the culture of the cell line produced in Example 2-1 was purified to obtain a high-purity substance.

[0117] First, the culture medium obtained in Example 2-1 was centrifuged to separate the cultured cells from the culture medium. Subsequently, the IL2 mutant-Fc protein complex in the separated medium was filtered using a 0.22 μm filter (Thermo Scientific) to remove any remaining residue. The filtered medium was then purified using Protein A affinity chromatography (MabSelect PrismA, Cytiva). Specifically, the filtered medium was placed on a Protein-A column stabilized with Phosphate buffer saline (pH 7.4), and proteins that were not nonspecifically bound were washed away using the same buffer. Proteins that specifically bound to the Protein-A column were then eluted using a pH gradient with two solutions containing 0.02 M Citric acid at pH 5.0 and pH 3.5, and the sample was neutralized at pH 7.2 using 1 M Tris. Subsequently, the secured IL2 mutant-Fc protein complex was stabilized with phosphate buffer saline (pH 7.4), and then the purity of the purified IL2 mutant-Fc protein complex was analyzed using size exclusion HPLC (TSK-3000SWxL, 7.8 mm × 30 cm, Tosoh Corporation).

[0118] 2-3. Confirmation of activation of the IL2 mutant-Fc protein complex in human immune cells.

[0119] The activation of the IL2 mutant-Fc protein complex purified in Example 2-2 against human bunny plague cells was confirmed. Specifically, human peripheral blood mononuclear cells (Stem Cell Inc.) and effector CD8+ T cells (CD3+, CD8+) and Treg cells (CD4+, CD25+) were reacted with antibodies that specifically fluorescently label these cells at 4°C for 30 minutes in the dark. Antibodies that did not adhere to the cells were then removed by centrifugation. Subsequently, the IL2 mutant 1-Fc protein complex, IL2 mutant 2-Fc protein complex, IL2 mutant 4-Fc protein complex, and IL2 mutant 14-Fc protein complex purified in Example 2-2 were treated and reacted at 37°C for 20 minutes in the dark, and then fixed in 1 mL of Fixation buffer (BD,US) for 12 minutes. Aldesleukin (proleukin, Novartis, Switzerland) was used as a positive control group, and each drug was treated at concentrations from 0.01 to 8,000 nM. Subsequently, 1.5 mL of Perm3 buffer (BD,US) was applied to the cytoplasm of fixed human peripheral blood mononuclear cells, and the cells were reacted at 4°C for 35 minutes to allow the fluorescent label to penetrate into the cytoplasm. Then, phosphorylated STAT-5 and Foxp3+ protein, a Treg cell marker, were fluorescently labeled and reacted for 30 minutes. Subsequently, the degree of STAT-5 phosphorylation in effector CD8+ T cells and Treg cells treated with the IL2 mutant-Fc protein complex was compared by fluid cell analysis.

[0120] [Table 3]

[0121] As a result, as shown in Table 3, the positive control group (Aldesleukin) showed CD8+ T cell / regulatory T cell EC 50 The ratio of values ​​to 33.3 confirmed that it induces Treg cell activity more strongly than CD8+ T cells.

[0122] On the other hand, the IL2 mutant 1-Fc protein complex, IL2 mutant 2-Fc protein complex, IL2 mutant 4-Fc protein complex, and IL2 mutant 14-Fc protein complex are found in CD8+ T cells / regulatory T cells. 50 The ratio of values ​​ranging from 0.79 to 3.2 confirmed that the activity of CD8+ T cells was more selectively activated compared to the positive control group.

[0123] In other words, it can be seen that one form of IL2 mutant retains the property of further selectively activating CD8+ cells than regulatory T cells, even in a structure with added Fc.

[0124] Example 3. Production and characterization of the PD-L1 antibody-IL2 mutant protein complex.

[0125] 3-1. Cloning and culture of PD-L1 antibody-IL2 mutant protein complexes

[0126] A protein complex containing a PD-L1 antibody and an IL2 variant was prepared. Specifically, using the same service as in Example 2-1, polynucleotides coding the PD-L1 antibody heavy chain variable region-heavy chain invariant region and polynucleotides coding the PD-L1 antibody light chain invariant region-light chain variable region were synthesized, respectively. Subsequently, the polynucleotides coding the PD-L1 antibody heavy chain variable region-heavy chain invariant region and the polynucleotides coding the PD-L1 antibody light chain invariant region-light chain variable region were inserted into the AvrII-BstZ17I enzyme site and EcoRV-PacI enzyme site of a pCHO1.0 expression vector, respectively, to produce a first expression vector expressing the PD-L1 antibody. Using the same service, a polynucleotide encoding a complex containing one of the IL2 mutants 1 to 15 produced in Example 1, along with a linker and an Fc region, was synthesized. This polynucleotide was then inserted into the AvrII-BstZ17I enzyme site of the pCHO 1.0 expression vector to produce a second expression vector expressing IL2 mutant-Fc. Subsequently, a PD-L1 antibody-IL2 mutant protein complex containing anti-PD-L1 Fab-Fc and IL2 mutant-Fc was obtained in the same manner as in Example 2-1, except that the first and second expression vectors were used.

[0127] 3-2. Purification of the PD-L1 antibody-IL2 mutant protein complex

[0128] The PD-L1 antibody-IL2 mutant 1 protein complex or the PD-L1 antibody-IL2 mutant 15 protein complex, obtained through the culture of the cell line produced in Example 3-1, was purified to obtain a high-purity substance.

[0129] First, the culture medium obtained in Example 3-1 was centrifuged to separate the cultured cells from the culture medium. Subsequently, the PD-L1 antibody-IL2 mutant protein complex in the separated medium was filtered using a 0.22 μm filter (Thermo Scientific) to remove any remaining residue. The filtered medium was then purified using Protein A affinity chromatography (MabSelect PrismA, Cytiva). Specifically, the filtered medium was placed on a Protein-A column stabilized with Phosphate buffer saline (pH 7.4), and proteins that were not nonspecifically bound were washed away using the same buffer. Proteins that specifically bound to the Protein-A column were then eluted using a buffer solution containing 0.05 M Citric acid (pH 3.9), and the sample was neutralized to pH 7.2 using 1 M Tris. Subsequently, secondary purification was performed using cation exchange chromatography (Source30S, Cytiva) to remove any remaining material-derived impurities in the sample separated through the affinity column. Specifically, the sample eluted and neutralized using a Protein-A column was titrated to pH 6.0 with 1 M Citric acid, then placed on a Source30S column stabilized with 20 mM sodium phosphate (pH 6.0) buffer. Nonspecifically bound proteins were washed away using the same buffer, and the PD-L1 antibody-IL2 mutant protein complex was secured by elution using an increasing gradient method with a buffer solution containing 0.3 M NaCl (pH 6.0). Subsequently, the secured PD-L1 antibody-IL2 mutant protein complex was stabilized with Phosphate buffer saline (pH 7.4), and the purity of the purified PD-L1 antibody-IL2 mutant protein complex was analyzed using size exclusion HPLC (TSK-3000SWxL, 7.8 mm × 30 cm, Tosoh).

[0130] Figure 1A shows the results of purifying a PD-L1 antibody-IL2 mutant 2 protein complex using a specific example of the Protein-A affinity column purification method.

[0131] Figure 1B shows the results of purifying a PD-L1 antibody-IL2 mutant 2 protein complex using a cation exchange resin column purification method as a specific example.

[0132] Figure 1C shows the results of a specific example of purity analysis of a PD-L1 antibody-IL2 mutant 2 protein complex via SE-HPLC analysis.

[0133] As a result, as shown in Figure 1A, we were able to identify proteins that specifically bind to the column using the elution buffer used in Protein-A affinity column purification. Specifically, we were able to identify PD-L1 antibody-IL2 mutant protein complexes that specifically bind to the column using 1500 to 1550 ml of eluate.

[0134] Furthermore, as shown in Figure 1B, we were able to confirm that proteins that nonspecifically bind to residual impurities in the eluate used with the Protein-A affinity column were removed.

[0135] Furthermore, as shown in Figure 1C, the PD-L1 antibody-IL2 variant 2 protein complex, obtained through Protein-A affinity column purification and cation exchange resin purification, was confirmed to have a purity of 99% with a retention time of 16.717 minutes.

[0136] In other words, it was found that the PD-L1 antibody-IL2 mutant protein produced by one embodiment could be purified to a high degree of purity.

[0137] 3-3. Confirmation of receptor binding affinity of PD-L1 antibody-IL2 mutant protein complex

[0138] The receptor binding affinity of a PD-L1 antibody-IL2 mutant protein complex according to one embodiment was confirmed through SPR (Surface Plasmon Resonance) analysis. Specifically, human PD-L1 and human IL2Rα, IL2Rβγ, and IL2Rαβγ were immobilized on a CM5 sensor chip via covalent bonds, respectively. Then, the PD-L1 antibody-IL2 mutant 2 protein complex and the PD-L1 antibody-IL2 mutant 4 protein complex prepared in Example 3-2 were deployed at various concentrations (serial dilutions of 2x in the concentration range of 0.391 to 400 nM), and their binding kinetics to human PD-L1 and IL2 receptor (IL2R) were confirmed. Subsequently, the binding affinity (KD) was calculated using the measured association constant (Ka) and dissociation constant (Kd) values. In this case, SPR sensogram analysis was performed using BIAlogue kinetics evaluation software. Avelumab and Aldesleukin were used as positive control groups, and the results are shown in Tables 4 and 5 below.

[0139] [Table 4]

[0140] [Table 5]

[0143] As a result, as shown in Figure 2A and Table 4, it was confirmed that the PD-L1 antibody-IL2 variant 2 protein complex and the positive control group produced in Example 3-2 bound to the human PD-L1 antigen. Specifically, the protein variant showed a binding affinity of 0.48 nM, while the positive control group showed a binding affinity of 0.12 nM, indicating a binding affinity approximately 4.1 times lower.

[0144] In other words, the positive control group has a bivalent anti-PD-L1 arm as a single antibody, while the PD-L1 antibody-IL2 mutant 2 protein complex has a monovalent anti-PD-L1 arm, which is thought to explain the difference in binding affinity.

[0145] Furthermore, as shown in Table 5, the PD-L1 antibody-IL2 mutant 2 protein complexes produced in Example 2 bound to IL2Rβγ and IL2Rαβγ with binding affinities of 37.9 nM and 17.3 nM, respectively, but did not react with IL2Rα. In addition, the PD-L1 antibody-IL2 mutant 2 protein complexes bound to IL2Rβγ and IL2Rαβγ with binding affinities of 23.8 nM and 30.6 nM, respectively, but did not react with IL2Rα.

[0146] In other words, it was confirmed that the PD-L1 antibody-IL2 mutant 2 protein complex and the PD-L1 antibody-IL2 mutant 4 protein complex according to one embodiment do not bind to the IL2 receptor α.

[0147] On the other hand, as shown in Table 5, the positive control group was confirmed to bind to IL2Rα, IL2Rβγ, and IL2Rαβγ with binding affinity gradations of 35.7 nM, 1.55 nM, and 0.10 nM, respectively.

[0148] On the other hand, it was confirmed that the binding affinity of the PD-L1 antibody-IL2 mutant 2 protein complex and the PD-L1 antibody-IL2 mutant 4 protein complex to IL2Rβγ decreased by approximately 24.5 times and 15.4 times, respectively, compared to the positive control group. Furthermore, when the βγ / αβγ ratio was calculated from the binding affinity results to IL2Rαβγ, the βγ / αβγ ratios of the PD-L1 antibody-IL2 mutant 2 protein complex and the PD-L1 antibody-IL2 mutant 4 protein complex were shown to be 2.19 and 0.78, respectively, while the positive control group showed a ratio of 15.5, confirming that the IL2Rβγ / αβγ ratio of the PD-L1 antibody-IL2 mutant protein complex was significantly lower compared to the positive control group. In other words, it can be seen that the PD-L1 antibody-IL2 mutant 2 protein complex and the PD-L1 antibody-IL2 mutant 4 protein complex not only failed to bind to IL2Rα, but also showed weakened binding to IL2Rβγ.

[0149] Therefore, one embodiment of the PD-L1 antibody-IL2 mutant protein complex is intended to induce higher activation in CD8+ T cells compared to regulatory T cells, and is characterized by the removal of the binding affinity to IL2Rα and the modification of the binding affinity to IL2Rβγ through the substitution of the specific amino acid, in order to selectively induce activation of CD8+ T cells expressing IL2Rβγ.

[0150] 3-4. Confirmation of the activity of the PD-L1 antibody-IL2 mutant protein complex on human immune cells.

[0151] The effects of the PD-L1 antibody-IL2 variant 1 protein complex or the PD-L1 antibody-IL2 variant 15 protein complex according to one aspect on the activation of human immune cells were confirmed. Specifically, human peripheral blood mononuclear cells (Stem cell) were reacted with specifically fluorescent-labeled effector CD8+ T cells (CD3+, CD8+) and regulatory T cells (CD4+, CD25+, FoxP3+) antibodies in the dark at 4°C for 30 minutes. Thereafter, the antibodies not attached to the cells were removed through centrifugation, and then the PD-L1 antibody-IL2 variant 1 protein complex or the PD-L1 antibody-IL2 variant 15 protein complex prepared in Example 3-2 was treated with human peripheral blood mononuclear cells at different concentrations (0.001 - 8000 nM) and reacted in the dark at 37°C for 20 minutes, and then fixed with 1 mL of Fixation buffer (BD, U.S.) for 12 minutes. In order to fluorescently label phosphorylated STAT-5 in the cytoplasm, 1.5 mL of Perm3 buffer (BD, U.S.) was treated and reacted at 4°C for 40 minutes to make the fluorescent label permeable. Thereafter, after fluorescently labeling the phosphorylated STAT-5 protein and reacting for 30 minutes, the ratio of cells in which STAT-5 was phosphorylated in effector CD8+ T and regulatory T cells was confirmed through FACS analysis. In the positive control group, Aldesleukin (Proleukin, Novartis, Switzerland) was used, and the results are shown in Table 6 below.

[0152]

Table 6

[0153] *N.S: Not Saturated at high concentration

[0154] As a result, as shown in Table 6, the EC of CD8+ T cells / regulatory T cells in the positive control group and the PD-L1 antibody-IL2 comparison group 50The ratios of the values ​​are shown as 62.9 and 22, respectively, and the EC of CD8+ T cells / regulatory T cells of the PD-L1 antibody-IL2 mutant 1 protein complex or the PD-L1 antibody-IL2 mutant 15 protein complex produced in Example 3-2 above. 50 The ratio of values ​​ranged from 0.16 to 2.88. In other words, in the case of the positive control group and the PD-L1 antibody-IL2 comparison group, regulatory T cells were strongly activated, while the PD-L1 antibody-IL2 mutant 1 protein complex or the PD-L1 antibody-IL2 mutant 15 protein complex produced in Example 3-2 further selectively activated CD8+ T cells, which are effector T cells, rather than regulatory T cells.

[0155] Therefore, one embodiment of the PD-L1 antibody-IL2 mutant protein complex can participate in the immune response involved in anticancer effects by more strongly inducing the activity of effector T cells than regulatory T cells.

[0156] 3-5. Confirmation of the anticancer activity of the PD-L1 antibody-IL2 mutant protein complex (1)

[0157] The anticancer activity of a PD-L1 antibody-IL2 mutant protein complex in one embodiment was confirmed. Specifically, in MC38 cells (1 × 10⁶ 6 A syngeneic mouse model of spleen tumor was prepared by subcutaneously administering 0.2 mL / mouse of PD-L1 antibody-IL2 mutant 2 protein complex prepared in Example 3-2 to the flank of C57BL / 6 mice (female, 6 weeks old, Jabio) via the flank. Five days after inoculation, the PD-L1 antibody-IL2 mutant 2 protein complex prepared in Example 3-2 was administered intraperitoneally at doses of 8 mg / kg and 16 mg / kg, each twice via Q2D. After administration of the complex, the tumor volume of the mice was measured (3 times / week) to compare anticancer activity. As a positive control group, Avelumab (Merck, Germany) was administered intraperitoneally at a dose of 10 mg / kg (twice via Q2D), and Avelumab was administered in combination with Aldesleukin (Aldesleukin 0.46 mg / kg, QD × 5 times, ip + Avelumab 10 mg / kg, Day 5, 7, ip).

[0158] Figure 2A shows the results of comparing the anticancer activity of a PD-L1 antibody-IL2 mutant protein complex and a positive control group using a specific example.

[0159] Figure 2B shows the results of confirming the individual-specific inhibitory effect on cancer cell growth and complete remission activity of the PD-L1 antibody-IL2 mutant 2 protein complex in mice transplanted with ulcer cancer, using one specific example.

[0160] Figure 2C shows the results of confirming the inhibitory effect on cancer cell growth and complete remission activity in blepharocarcinoma-transplanted mice in the negative control group and the positive control group (Avelumab, and Avelumab and Aldesleukin combination administration).

[0161] As a result, as shown in Figure 2A, the negative control group (vehicle) and the positive control group showed an increase in tumor volume over time after administration, while the PD-L1 antibody-IL2 mutant 2 protein complex produced in Example 3-2 demonstrated a potent inhibitory effect on cancer cell growth. Specifically, it was confirmed that the tumor almost completely disappeared at a dose of 16 mg / kg of the PD-L1 antibody-IL2 mutant 2 protein complex. Furthermore, unlike the PD-L1 antibody-IL2 control group protein complex, no weight loss or deaths were observed in individuals with the PD-L1 antibody-IL2 mutant 2 protein complex, regardless of the administered dose.

[0162] Furthermore, as shown in Figures 2B and 2C, in the negative and positive control groups, tumor volume increased over time after administration, and it was confirmed that the number of mice showing complete remission (CR) was relatively small, at 0 to 2 out of 10 experimental animals in the same administration group. On the other hand, in the case of the PD-L1 antibody-IL2 mutant 2 protein complex, the number of mice showing complete remission increased in a dose-dependent manner, at 6 to 9 out of 10 experimental animals in the same administration group, confirming that it exhibited significantly greater anticancer activity compared to the negative and positive control groups.

[0163] In other words, it can be seen that the PD-L1 antibody-IL2 mutant protein complex according to one embodiment exhibits superior anticancer activity compared to the combination of existing antibody therapeutic agents Avelumab and / or Avelumab and Aldesleukin.

[0164] 3-6. Confirmation of the anticancer activity of the PD-L1 antibody-IL2 mutant protein complex (2)

[0165] The anticancer activity of a PD-L1 antibody-IL2 mutant protein complex was confirmed in one embodiment. The anticancer activity was confirmed in the same manner as in Examples 3-5, except that a PD-L1 antibody-IL2 mutant 4 protein complex was used, and avelumab (Merck, Germany) was administered intraperitoneally at a dose of 10 mg / kg (Q2D x 2 times) as a positive control group.

[0166] Figure 3 shows the results of confirming the anticancer activity of a PD-L1 antibody-IL2 mutant 4 protein complex in one specific example.

[0167] As a result, as shown in Figure 3, the negative control group (vehicle) showed an increase in tumor volume over time after administration, while the PD-L1 antibody-IL2 mutant 4 protein complex produced in Example 3-2 showed a stronger inhibitory effect on cancer cell growth compared to the negative control group. Specifically, it was confirmed that the PD-L1 antibody-IL2 mutant 4 protein complex resulted in significantly less change in tumor volume at doses of 8 mg / kg and 16 mg / kg compared to the positive control group administered a dose of 10 mg / kg. Furthermore, in the positive control group, only one out of 10 experimental animals in the same administration group showed complete remission, while in the case of the PD-L1 antibody-IL2 mutant 4 protein complex, this increased to 4-5 out of 10 experimental animals in the same administration group, demonstrating significantly greater anticancer activity compared to the positive control group.

[0168] In other words, one embodiment of the PD-L1 antibody-IL2 mutant protein complex selectively increases the activity of regulatory T cells compared to existing immunosuppressive anti-cancer antibody therapies, and thus can be usefully used for the prevention or treatment of various immune diseases, including cancer.

[0169] Therefore, one embodiment of the PD-L1 antibody-IL2 mutant protein complex can provide a safer therapeutic agent compared to existing immunosuppressant cancer therapies by exhibiting excellent anticancer activity and reducing IL2-induced side effects.

[0170] Example 4. Production and activity confirmation of mousePD-1 antibody-IL2 mutant protein complex (a+b).

[0171] 4-1. Cloning and culture of mousePD-1 antibody-IL2 mutant protein complex (a+b)

[0172] A protein complex (a+b) containing anti-mousePD-1 Fab-Fc (hereinafter referred to as "a") and IL2 variant-Fc (hereinafter referred to as "b") was obtained in the same manner as in Example 3-1, except that a PD-1 antibody and IL2 variants 1 and 4 prepared in Example 1 were used (see Figure 4A).

[0173] 4-2. Purification of mousePD-1 antibody-IL2 mutant protein complex (a+b)

[0174] The mousePD-1 antibody-IL2 mutant protein complex (a+b) obtained through the culture of the cell line produced in Example 4-1 was purified using the same method as in Example 3-2 to obtain a high-purity substance.

[0175] 4-3. Confirmation of the activity of the mousePD-1 antibody-IL2 mutant protein complex (a+b) on human immune cells.

[0176] To confirm the effect of the mousePD-1 antibody-IL2 mutant protein complex (a+b) obtained in Example 4-2 on the activation of human rabbit disease cells, the experiment was carried out using the same method as in Example 3-4.

[0177] [Table 7]

[0178] As a result, as shown in Table 7, we were able to confirm that the CD8+ T cell / regulatory T cell ratios for the positive control group and the mousePD-1 antibody-IL2 mutant 4 protein complex (a+b) were 14.5 and 0.92, respectively. In other words, we were able to confirm that the positive control group strongly activated regulatory T cells, while the mousePD-1 antibody-IL2 mutant 4 protein complex (a+b) produced in Example 4-2 selectively activated CD8+ T cells, which are effector T cells, rather than regulatory T cells.

[0179] Therefore, it can be seen that the IL2 mutant protein, in one embodiment, retains its original properties well and can participate in the immune response by promoting the activity of effective T cells, even when it forms a complex not only with PD-L1 antibodies but also with other immune checkpoint inhibitors.

[0180] Example 5. Production and confirmation of activity of mousePD-1 antibody-IL2 mutant protein complex (a+c).

[0181] 5-1. Cloning and culture of mousePD-1 antibody-IL2 mutant protein complex (a+c)

[0182] Protein complexes containing mousePD-1 antibody and IL2 variants 1, 2, 3, 4, 7, 8, 14, and 15 were prepared. Specifically, polynucleotides coding the variable-invariant region of the mousePD-1 antibody heavy chain and polynucleotides coding the invariant-variable region of the mousePD-1 antibody light chain were synthesized using ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service. Subsequently, these polynucleotides were inserted into the AvrII-Bstz17I enzyme site and EcoRV-PacI enzyme site of a pCHO 1.0 expression vector, respectively, to produce a first expression vector expressing mousePD-1 antibody. Using the same service, a polynucleotide was synthesized that coded a complex containing one of the IL2 variants 1, 2, 3, 4, 7, 8, 14, and 15 from Example 1 and a linker at the C-terminus of the polynucleotide coding the variable-invariant region of the mousePD-1 antibody heavy chain. This complex was then inserted into the AvrII-BstZ17I enzyme site of the pCHO 1.0 expression vector, and the mousePD-1 antibody light chain invariant-variable region was inserted into the EcoRV-PacI enzyme site to produce a second expression vector. Subsequently, a protein complex (a+c) containing anti-mousePD-1 Fab-Fc(A) and anti-mousePD-1 Fab-Fc-C-terminal IL2 variant (hereinafter referred to as "c") was obtained using the same method as in Example 2-1 (see Figure 4b).

[0183] 5-2. Purification of mousePD-1 antibody-IL2 mutant protein complex (a+c)

[0184] The mousePD-1 antibody-IL2 mutant protein complex (a+c) obtained through the cell line culture produced in Example 5-1 was purified to obtain a high-purity substance (purity of 98% or higher).

[0185] First, the sample was purified by removing fine residues in the same manner as in Example 3-2, except that a mousePD-1 antibody-IL2 mutant protein complex (a+c) was used. Subsequently, the culture medium, which had undergone a filtration process, was placed on a Protein A column stabilized with Phosphate buffer saline (pH 7.4). Proteins that were not nonspecifically bound were washed away using the same buffer, and then washed again with a buffer solution containing 0.05 M Citric acid (pH 5.0). Thereafter, proteins that specifically bound to the Protein-A column were eluted using a pH-decreasing gradient method with 0.02 M Citric acid (pH 5.0 and pH 3.5), and the sample was neutralized to pH 7.2 using 1 M Tris.

[0186] Subsequently, secondary purification was performed using a hydrophobic interaction column (Phenyl HP, Cytiva) to remove any remaining substance-derived impurities in the sample separated via affinity column. Specifically, the sample, which had been eluted and neutralized with a Protein-A column, was added to a Phenyl HP column stabilized with 0.02 M sodium phosphate (pH 7.0) buffer and 0.8 M ammonium sulfate (pH 7.0). 1 M citric acid was added to the Phenyl HP column, and the mixture was titrated to pH 7.0. Proteins that were not nonspecifically bound were then washed out using the same buffer. Subsequently, the mousePD-1 antibody-IL2 mutant protein complex (a+c) was obtained by eluting using a gradually decreasing gradient method with a buffer solution containing 0.02 M sodium phosphate (pH 7.0). The secured mousePD-1 antibody-IL2 mutant protein complex (a+c) was stabilized in phosphate buffer saline (pH 7.4), and then purity analysis was performed using size exclusion HPLC (TSK-3000SWxL, 7.8 mm × 30 cm, Tosoh Corporation).

[0187] Figure 5A shows the results of purifying the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c) using a specific example of the Protein-A affinity column purification method.

[0188] Figure 5B shows the results of purifying the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c) using a hydrophobic interaction column purification method as one specific example.

[0189] Figure 5C shows the purity analysis results of a specific mousePD-1 antibody-IL2 mutant 14 protein complex (a+c).

[0190] As a result, as shown in Figure 5A, we were able to identify proteins that specifically bind to the column using the elution buffer used in Protein-A affinity column purification. Specifically, we were able to identify the mousePD-1 antibody-IL2 variant 14 protein complex (a+c) that specifically binds to the column using elution solutions of 2800 to 2900 ml.

[0191] Furthermore, as shown in Figure 5B, we were able to confirm that proteins that nonspecifically bind to residual impurities in the eluate used with the Protein A affinity column were removed.

[0192] Furthermore, as shown in Figure 5C, the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c) obtained through all the purification processes described above was confirmed to have a purity of 98.6% with a retention time of 15.639 minutes.

[0193] In other words, it can be seen that the mousePD-1 antibody-IL2 mutant protein complex (a+c) produced by one embodiment can be purified to a high degree of purity.

[0194] 5-3. Confirmation of receptor binding affinity of mousePD-1 antibody-IL2 mutant protein complex (a+c)

[0195] The receptor binding affinity of the mousePD-1 antibody-IL2 mutant protein complex (a+c) was confirmed in the same manner as in Example 3-3, except that the mousePD-1 antibody-IL2 mutant protein complex (a+c) and the mousePD-1 antibody-IL2 mutant protein complex (a+c) prepared in Example 5-2 were sequentially diluted 2-fold in a concentration range of 1.56 to 8000 nM and then developed.

[0196] [Table 8]

[0197] As a result, as shown in Table 8, the mousePD-1 antibody-IL2 mutant 1 protein complex (a+c) and the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c) showed binding affinity to IL2Rβ of 5,610 nM and 6,290 nM, respectively, confirming that they bound to IL2Rβ at similar levels. On the other hand, they showed binding affinity to IL2Rβγ of 4.51 nM and 35.3 nM, respectively, confirming that the binding affinity of the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c) was approximately 7.8 times lower compared to the mousePD-1 antibody-IL2 mutant 1 protein complex (a+c).

[0198] This means that while the binding affinity of the mousePD-1 antibody-IL2 mutant 1 protein complex (a+c) and the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c) to IL2Rβ was similar, the binding affinity of the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c) to IL2Rγ was approximately 7.8 times lower compared to the mousePD-1 antibody-IL2 mutant 1 protein complex (a+c).

[0199] 5-4. Confirmation of the activity of the mousePD-1 antibody-IL2 mutant protein complex (a+c) on human immune cells.

[0200] The effect of the mousePD-1 antibody-IL2 mutant protein complex (a+c) on the activation of human immune cells was confirmed in the same manner as in Example 3-4, except that the mousePD-1 antibody-IL2 mutant protein complexes (a+c),

[0201] [Table 9]

[0202] As a result, as shown in Table 9, in the positive control group (Aldesleukin), CD8+ T cells / regulatory T cells EC 50 While the ratio of values ​​was shown to be 21.4, it was confirmed that the mousePD-1 antibody-IL2 mutant protein complex (a+c) produced in Example 5-2 showed values ​​of 0.5 to 1.2. In other words, the positive control group activated regulatory T cells more strongly, while the mousePD-1 antibody-IL2 mutant protein complex (a+c) as a specific example selectively activated CD8+ T cells, which are effector T cells, rather than regulatory T cells. This indicates that the IL2 mutant retains its unique characteristic of selectively activating CD8+ T cells even in a structure where it is linked to the C-terminus of the FC region.

[0203] Furthermore, the activity of the mousePD-1 antibody-IL2 mutant 1 protein complex (a+c) towards Treg and CD8+ T cells was 11.91±8.02 nM and 9.61±1.44 nM, respectively, while the activity of the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c) towards Treg and CD8+ T cells was 320.7±279.1 nM and 161.1±70.3 nM, respectively. This confirmed that the activity of the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c) towards Treg and CD8+ T cells was decreased compared to the mousePD-1 antibody-IL2 mutant 1 protein complex (a+c). This can be attributed to a decrease in binding affinity to IL2Rγ (approximately 7.8 times), as confirmed in Example 5-3. In addition, the EC of effector T cells / regulatory T cells was also observed. 50 The ratio of values ​​is 0.5, indicating that it selectively activates effector T cells (CD8+ T cells) compared to regulatory T cells.

[0204] Therefore, one embodiment of the mousePD-1 antibody-IL2 mutant protein complex (a+c) can participate in the immune response involved in anticancer effects by selectively activating regulatory T cells and effector T cells.

[0205] 5-5. Confirmation of the anticancer activity of the mousePD-1 antibody-IL2 mutant protein complex (a+c)

[0206] The anticancer activity of a mousePD-1 antibody-IL2 mutant protein complex (a+c) in one embodiment was confirmed. Specifically, MC38 cells (1.0 × 10⁶) 6 A syngeneic mouse model of ulnar carcinoma was created by subcutaneously injecting (cell / mice) into the right flank of C57BL / 6 mice (female, 6 weeks old, Coretech Co., Ltd.) after a one-week acclimatization period. Subsequently, the tumor size in the mice was approximately 70-120 mm. 3At this stage, the mice were classified into groups of 3 to 6 according to the drug administration group, and then intraperitoneally administered 1, 3, 5, and 10 mg / kg of the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c) prepared in Example 5-2, once a week for a total of two doses. In this case, a vehicle was used as the negative control group, and the anti-PD1 antibody J43 (clone) 3 mg / kg and J43 3 mg / kg + IL2 comparison group-Fc complex 1.4 mg / kg were used as the positive control group. Subsequently, the tumor growth inhibitory effect of drug administration was observed from the first drug administration until 19 days later.

[0207] Figure 6A shows the results of comparing the anticancer activity of a mousePD-1 antibody-IL2 mutant 14 protein complex (a+c), negative control group, and positive control group in a blepharoplasty mouse model using one specific example.

[0208] Figure 6B shows the results of comparing body weight changes in a blepharoplasty transplant mouse model due to the anticancer activity of a mousePD-1 antibody-IL2 mutant 14 protein complex (a+c), negative control group, and positive control group, using one specific example.

[0209] As a result, as shown in Figures 6A and 6B, the negative and positive control groups showed an increase in tumor volume over time, while the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c) produced in Example 5-2 showed a dose-dependent inhibitory effect on cancer cell growth compared to immediately after administration. In particular, it was confirmed that the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c) showed excellent cancer cell growth effects when administered at 5 mg / kg and 10 mg / kg compared to the positive control group and the combined administration group. That is, it was confirmed that the mousePD-1 antibody-IL2 mutant 14 protein complex (a+c) exhibited significantly superior anticancer activity compared to the combined administration of PD-1 monoantibody and IL2 comparison group-Fc protein complex.

[0210] Therefore, one embodiment of the mousePD-1 antibody-IL2 mutant protein complex (a+c) specifically activates effective T cells compared to existing immunosuppressant cancer therapies, and thus can be usefully used for the prevention or treatment of various immune diseases related to cancer.

[0211] Example 6. Production of humanPD-1 antibody-IL-2 mutant protein complex (A+C)

[0212] 6-1. Cloning and culture of humanPD-1 antibody-IL2 mutant protein complex (A+C)

[0213] Protein complexes containing humanPD-1 antibody, IL2 comparison group, IL2 variant 1, and IL2 variant 14 were prepared, respectively. Specifically, protein complexes (A+C) containing anti-humanPD-1Fab-Fc (hereinafter referred to as "A") and anti-humanPD-1Fab-Fc-C-terminal IL2 variant (hereinafter referred to as "C") were obtained by the same method as in Example 5-1, except that polynucleotides coding the variable-invariant region and the variable-invariant region of the humanPD-1 antibody heavy chain were used.

[0214] 6-2. Purification of the human PD-1 antibody-IL2 mutant protein complex (A+C)

[0215] The humanPD-1 antibody-IL2 mutant protein complex (A+C) obtained through cell line culture prepared in Example 6-1 was purified using the same method as in Example 5-2, and then stabilized with Phosphate buffer saline (pH 7.4) to obtain high-purity humanPD-1 antibody-IL2 mutant protein complex (A+C). Purity analysis was then performed using the same method as in Example 5-2.

[0216] 6-3 . Confirmation of the binding affinity of the human PD-1 antibody-IL2 mutant protein complex (A+C).

[0217] The receptor binding affinity of the humanPD-1 antibody-IL2 mutant protein complex (A+C) was confirmed in the same manner as in Example 3-3, except that the humanPD-1 antibody-IL2 comparison protein complex (A+C), humanPD-1 antibody-IL2 mutant 1 protein complex (A+C), and humanPD-1 antibody-IL2 mutant 14 protein complex (A+C) prepared in Example 6-2 were sequentially diluted 2-fold in the concentration range of 1.56 to 8000 nM and then developed. In this case, Pembrolizumab was used as the positive control group for human PD-1, and humanPD-1 antibody-IL2 mutant 1 was used as the control group. The results are shown in Tables 10 and 11 below.

[0218] [Table 10]

[0219] [Table 11]

[0220] As a result, as shown in Table 10, the huPD-1 antibody-IL2 comparison protein complex (A+C), humanPD-1 antibody-IL2 variant 1 protein complex (A+C), and humanPD-1 antibody-IL2 variant 14 protein complex (A+C) produced in Example 6-2, as well as the positive control group, showed binding positivity of 2.25 to 2.95 nM, confirming that all of them exhibited similar levels of binding affinity to human PD-1.

[0221] On the other hand, as shown in Table 11, it was confirmed that the humanPD-1 antibody-IL2 mutant 1 protein complex (A+C) and the humanPD-1 antibody-IL2 mutant 14 protein complex (A+C) do not react with IL2Rα. Meanwhile, the humanPD-1 antibody-IL2 mutant 1 protein complex (A+C) and the humanPD-1 antibody-IL2 mutant 14 protein complex (A+C) showed similar levels of binding affinity to IL2Rβ, with binding affinity of 5.462 nM and 5.611 nM, respectively. It was also confirmed that the humanPD-1 antibody-IL2 mutant 1 protein complex (A+C) and the humanPD-1 antibody-IL2 mutant 14 protein complex (A+C) bind to IL2Rβγ with binding affinity of 2.92 nM and 68.3 nM, respectively. From the results described above, it can be seen that the humanPD-1 antibody-IL2 mutant 14 protein complex (A+C) had a similar binding affinity to IL2Rβ as the humanPD-1 antibody-IL2 mutant 1 protein complex (A+C), but its binding affinity to IL2Rγ was reduced. Specifically, it was confirmed that the binding affinity of the humanPD-1 antibody-IL2 mutant 14 protein complex (A+C) to IL2Rβγ and IL2Rαβγ was reduced by approximately 23.4 times and 32.6 times, respectively, compared to humanPD-1 antibody-IL2 mutant 1. In other words, it can be seen that the humanPD-1 antibody-IL2 mutant 1 protein complex (A+C) and the humanPD-1 antibody-IL2 mutant 14 protein complex (A+C) did not bind to ILRα and their binding to IL2Rγ was also weakened.

[0222] Therefore, one embodiment of the humanPD-1 antibody-IL2 mutant protein complex (A+C) is intended to induce higher activation in CD8+ T cells compared to regulatory T cells, and is characterized by removing the binding affinity to IL2Rα and regulating the binding affinity to IL2Rγ through the substitution of specific amino acids in order to induce selective activation of CD8+ T cells expressing IL2Rβγ.

[0223] The above-mentioned description of the present invention is illustrative, and a person with ordinary skill in the art to which the present invention pertains will understand that it can be easily modified into other specific forms without altering the technical idea or essential features of the present invention. Therefore, it should be understood that the above-mentioned embodiments are illustrative in all respects and not limiting.

Claims

1. A protein containing an IL2 variant, The IL2 variant, in wild-type IL2 containing the amino acid sequence of SEQ ID NO: 16, 1) Substitutes with glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125. 2) Substitutions in which glutamic acid (E) is substituted at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125, while one or more positions selected from the group consisting of positions 18 and 19 are substituted with one or more amino acids selected from the group consisting of methionine (M), arginine (R), alanine (A), leucine (L), serine (S), phenylalanine (F), valine (V), isoleucine (I), glutamine (Q), tryptophan (W), asparagine (N), and threonine (T), or 3) A molecule in which the 35th position is replaced by glutamic acid (E), the 38th position by alanine (A), the 42nd position by lysine (K), and the 125th position by serine (S), while the 126th position is replaced by one or more amino acids selected from the group consisting of threonine (T) and isoleucine (I). protein.

2. The IL2 variant, in the wild-type IL2, It is substituted with glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125, The 18th position is substituted with methionine (M), arginine (R), alanine (A), leucine (L), serine (S), phenylalanine (F), valine (V), isoleucine (I), or glutamine (Q). The 19th position is substituted with serine (S), leucine (L), tryptophan (W), asparagine (N), isoleucine (I), threonine (T), alanine (A), methionine (M), or phenylalanine (F). The protein according to claim 1.

3. The IL2 variant, in the wild-type IL2, It is substituted with glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125, The 18th position is replaced with methionine (M) or arginine (R), It is a compound that has been replaced with serine (S) at position 19. The protein according to claim 1.

4. The IL2 variant, in the wild-type IL2, Glutamic acid (E) is substituted at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125, It is a compound in which threonine (T) is substituted at position 126. The protein according to claim 1.

5. The IL2 mutant is, in the wild-type IL2, Glutamic acid (E) is substituted at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125, It is a compound in which isoleucine (I) is substituted at position 126. The protein according to claim 1.

6. The aforementioned IL2 variant is The protein according to claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 15.

7. The protein according to claim 1, wherein the protein comprises an Fc region linked by a linker or carrier.

8. A protein complex comprising a first polypeptide containing a first CH3 antibody-invariant region and a second polypeptide containing a second CH3 antibody-invariant region, wherein the first polypeptide and the second polypeptide form a heterodimer. The N-terminus of either the first polypeptide or the second polypeptide contains an antibody or antigen-binding fragment against an immune checkpoint, or an antibody or antigen-binding fragment against a tumor-associated antigen. The first polypeptide or the second polypeptide contains an IL2 variant at one or more of its N-terminus or C-terminus. The IL2 variant, in wild-type IL2 containing the amino acid sequence of SEQ ID NO: 16, 1) Substitutes with glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125. 2) Substitutions in which glutamic acid (E) is substituted at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125, while one or more positions selected from the group consisting of positions 18 and 19 are substituted with one or more amino acids selected from the group consisting of methionine (M), arginine (R), alanine (A), leucine (L), serine (S), phenylalanine (F), valine (V), isoleucine (I), glutamine (Q), tryptophan (W), asparagine (N), and threonine (T), or 3) A molecule in which the 35th position is replaced by glutamic acid (E), the 38th position by alanine (A), the 42nd position by lysine (K), and the 125th position by serine (S), while the 126th position is replaced by one or more amino acids selected from the group consisting of threonine (T) and isoleucine (I). Protein complex.

9. The first CH3 antibody-invariant region contains tryptophan (W) at position 366, and the second CH3 antibody-invariant region contains serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407. The protein complex according to claim 8, wherein the 351st position of the second CH3 antibody-invariant region contains glycine (G) or phenylalanine (F) (wherein the position of the amino acid is according to the Kabat EU index).

10. The first CH3 antibody-invariant region contains serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407, and the second CH3 antibody-invariant region contains tryptophan (W) at position 366. The protein complex according to claim 8, wherein the 351st position of the first CH3 antibody-invariant region contains glycine (G) or phenylalanine (F) (wherein the position of the amino acid is according to the Kabat EU index).

11. The first CH3 antibody-invariant region contains tryptophan (W) at position 366, and the second CH3 antibody-invariant region contains glycine (G) at position 351, serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407. The protein complex according to claim 8, wherein the 351st position of the first CH3 antibody-invariant region contains phenylalanine (F) or tryptophan (W) (wherein the position of the amino acid is according to the Kabat EU index).

12. The first CH3 antibody-invariant region contains glycine (G) at position 351, serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407, and the second CH3 antibody-invariant region contains tryptophan (W) at position 366. The protein complex according to claim 8, wherein the 351st position of the second CH3 antibody-invariant region contains phenylalanine (F) or tryptophan (W) (wherein the position of the amino acid is according to the Kabat EU index).

13. The protein complex according to claim 8, wherein the first CH antibody-invariant region and the second CH antibody-invariant region contain alanine (A) at positions 234 and 235.

14. The protein complex according to claim 8, wherein the immune checkpoint is one or more selected from the group consisting of PD-L1, PD-1, LAG-3, VISTA, BTLA, TIM-3, TIGIT, and CTLA-4.

15. The protein complex according to claim 8, wherein the tumor-specific antigen is one or more selected from the group consisting of PD-L1, EGFR, HER-2, B7H3, GPC3, CEA, TROP, and PSMA.

16. The protein complex according to claim 8, wherein the second polypeptide and the IL2 variant are linked by one or more linkers or carriers.

17. The protein complex according to claim 16, wherein the linker comprises the amino acid sequence of SEQ ID NO:

18.

18. A polynucleotide coding a protein or protein complex according to any one of claims 1 to 17.

19. A method for producing a protein or protein complex, comprising the step of transforming cells with an expression vector coding for a protein or protein complex according to any one of claims 1 to 17.

20. A pharmaceutical composition for the prevention or treatment of cancer, comprising a protein or protein complex as described in any one of claims 1 to 17 as an active ingredient.

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