IL2 variants and protein complexes comprising same

Abstract

The present disclosure relates to IL2 variants and protein complexes comprising the IL2 variants, methods of production and uses thereof. The protein complex can reduce side effects and improve anticancer activity as much as possible by selectively increasing the activity of effector T cells, and thus can be used for preventing or treating various immune diseases associated with cancer.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2022-0105643 filed on August 23, 2022 and Korean Patent Application No. 10-2023-0057361 filed on May 2, 2023, the specifications of which are incorporated herein by reference in their entirety.

[0002] The present application relates to IL2 variants and protein complexes comprising the IL2 variants, methods for producing the same, and uses thereof. Background Art

[0003] Immune checkpoints refer to surface proteins of immune cells used by cancer cells to avoid attacks by the immune system. Cancer cells inhibit T cell activation and cancer cell apoptosis by expressing programmed cell death-ligand 1 (PD-L1) on the cell surface and combining with programmed death receptor 1 (PD-1), a kind of immune checkpoint of T cells. Immune checkpoint inhibitors can partially solve the side effects and drug resistance problems of existing anticancer agents that directly attack cancer cells by activating the immune system in the body to make immune cells selectively attack cancer cells. In addition, by confirming the effectiveness of various cancers, multiple immune checkpoint inhibitors have been recognized for sales. However, its overall response rate (ORR) is an average of 30%, and many problems need to be overcome. In order to improve the effectiveness of immune checkpoint inhibitors to overcome the problems, interleukins are valued as a combination with immune checkpoint inhibitors and dual agonists.

[0004] Among them, interleukin-2 (IL2: interleukin-2) has a length of 133 amino acids and is a 15.5 kDa globular glycoprotein that plays a key role in the production of lymphocytes, survival, and homeostasis in the body. IL2 is biosynthesized by activated T cells, especially mainly CD4+ helper T cells, to stimulate the proliferation and differentiation of T cells, and stimulate the generation of cytotoxic T lymphocytes (CTL) and the generation and proliferation activity of natural killer cells (NK cells). Therefore, since IL2 can increase the lymphocyte population in the body and enhance the function of the immune cells, treatment with IL2 has been approved and used in patients with metastatic renal cell carcinoma and malignant melanoma.

[0005] The activity of IL2 on lymphocytes is mediated by binding to three different IL2 receptors (IL2R) combinations, namely IL2 receptor α (IL2 recepterα, IL2Rα; CD25), IL2 receptor β (IL2 receptorβ, IL2Rβ; CD122) and universal common cytokine receptor γ (IL2Rγ; CD132). The distribution of each subunit receptor varies depending on the cell, and the binding force with IL2 is also significantly different in each receptor. The high-affinity IL2R is composed of a trimer of three subunits (α, β, γ), and the dimeric IL2 receptor formed by the β and γ subunits is called the intermediate-affinity IL2R. The dimeric intermediate-affinity IL2R binds to IL2 with an affinity about 100 times lower than that of the trimeric high-affinity receptor, however, the dimeric IL2R and trimeric IL2R can transmit signals when IL2 binds. Therefore, the α-subunit, CD25 is not necessary for IL2 signaling. The α-subunit confers high affinity binding to its receptor, while the β-subunit and the γ-subunit are important for signal transduction. Trimeric IL2 including CD25 is expressed by regulatory T cells and endothelial cells. Moreover, the trimeric IL2R is transiently induced on conventional activated T cells, whereas the T cells only express dimeric IL2R in the 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 immune tolerance to self-antigens and participate in autoimmune diseases. They usually inhibit or downregulate the activation and proliferation of effector T cells to hinder the effectiveness of cancer treatment. Regulatory T cells continuously express the highest level of CD25 and show higher IL2 binding than effector T cells, so they become a problem that hinders the effectiveness of cancer treatment using IL2.

[0007] Regarding IL2 immunotherapy, the side effects of recombinant human IL2 treatment are causing problems. Patients receiving high-volume IL2 treatment usually experience severe cardiovascular, lung, kidney, liver, gastrointestinal, neurological, skin, blood diseases and other systemic negative effects, which require centralized supervision and patient hospitalization management. The main cause of this negative effect can be explained based on the occurrence of the so-called vascular (or capillary) leakage syndrome (vesicular leakage syndrom, VLS). The vascular leakage syndrome is a pathological increase in vascular permeability that reaches the extravascular outflow of body fluids in multiple organs (e.g., causing lung and skin edema and liver cell damage) and intravascular body fluid depletion (causing reduced blood pressure and compensatory heart rate increase). There is no other treatment for the VLS except for suspending IL2 administration. In order to avoid VLS, patients have been tested for low-volume administration of IL2, but the poor treatment cost of reduced cancer treatment efficiency has occurred instead. It was once believed that VLS was caused by the release of inflammatory cytokines, such as tumor necrosis factor (TNF)-α, from IL2-activated NK cells; however, it has recently been shown that IL2-induced pulmonary edema is generated by direct binding of IL2 to pulmonary endothelial cells, which express low or intermediate levels of trimeric IL2R (International Immunology, 2006 vol. 18, no. 10: 1461-1471).

[0008] Therefore, even if IL2 has the potential to be used as an immune anticancer therapeutic agent, in order to reduce toxicity and side effects and improve therapeutic efficacy, it is necessary to develop a new treatment method that can minimize the cancer treatment side effects caused by the IL2 and improve the efficacy by using IL2 variants that selectively activate effector T cells and their crystallizable fragment (Fc, fragment crystallizable) regions, immune checkpoint inhibitors, and antibodies to cancer cell-specific antigens. Summary of the invention

[0009] Technical issues

[0010] In one aspect, proteins are provided that include interleukin 2 (IL2) variants.

[0011] On the other hand, a protein complex is provided, comprising: an IL2 variant, a first polypeptide comprising a first CH3 antibody constant region, and a second polypeptide comprising a second CH3 antibody constant region; and an antibody or an antigen-binding fragment thereof to an immune checkpoint inhibitor or an antibody or an antigen-binding fragment thereof to a tumor associated antigen.

[0012] Another aspect provides a method for preparing the protein or the protein complex.

[0013] Another aspect provides a pharmaceutical composition for preventing or treating cancer, wherein the pharmaceutical composition comprises the protein or the protein complex as an effective ingredient.

[0014] Another aspect provides a method for treating cancer, which comprises the step of administering the protein or the protein complex.

[0015] Another aspect provides use of the protein or protein complex in preparing a cancer therapeutic agent.

[0016] Technical Solution

[0017] On the one hand, a protein comprising an interleukin (IL2) variant is provided, wherein the IL2 variant comprises 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 position 35, position 38, position 42, and position 125.

[0018] The IL2 variant may be one or more of the amino acids of the wild-type IL2 including the amino acids of SEQ ID NO: 16 substituted. As long as the activity of IL2 is maintained, the IL2 variant may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids substituted. For example, the IL2 variant may be one or more of the group consisting of the 18th, 19th, 35th, 38th, 42nd, 125th, and 126th positions of the wild-type IL2 amino acids selected from the amino acids of SEQ ID NO: 16 substituted.

[0019] Specifically, the IL2 variant may include 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 position 18, position 19, position 35, position 38, position 42, position 125, and position 126.

[0020] The binding force of the IL2 variant to the IL2 receptor can be increased or decreased by amino acid substitution. For example, the binding force to IL2Rα and / or IL2Rβγ can be reduced. Therefore, the IL2 reduces the binding force to IL2R, thereby selectively activating effector T cells compared to regulatory T cells that hinder the immune activity caused by IL2.

[0021] According to one embodiment, the IL2 variant may include glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125.

[0022] According to another embodiment, the IL2 variant may further include 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 position 18 and position 19.

[0023] According to another embodiment, the IL2 variant may further include one or more amino acids at position 126 selected from the group consisting of threonine (T) and isoleucine (I).

[0024] For example, the IL2 variant may include an amino acid selected from the group consisting of:

[0025] At position 18, 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);

[0026] at position 19, 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);

[0027] Glutamic acid (E) at position 35;

[0028] Alanine (A) at position 38;

[0029] Lysine (K) at position 42;

[0030] Serine (S) at position 125; and

[0031] The amino acid at position 126 is selected from the group consisting of threonine (T) and isoleucine (I).

[0032] Specifically, the IL2 variant may include amino acids selected from the group consisting of:

[0033] Glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125;

[0034] 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;

[0035] Alanine (A) 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;

[0036] Arginine (R) 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;

[0037] methionine (M) at position 18, leucine (L) 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;

[0038] Leucine (L) 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;

[0039] Serine (S) at position 18, tryptophan (W) 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; phenylalanine (F) at position 18, asparagine (N) 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;

[0040] Valine (V) at position 18, isoleucine (I) 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;

[0041] Isoleucine (I) at position 18, threonine (T) 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;

[0042] Glutamic acid (Q) at position 18, alanine (A) at position 19, glutamate (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125;

[0043] methionine (M) at position 18, methionine (M) 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;

[0044] methionine (M) at position 18, phenylalanine (F) 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;

[0045] Glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, serine (S) at position 125, and threonine (T) at position 126; and

[0046] 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.

[0047] According to another embodiment, the IL2 variant may include amino acids selected from the group consisting of SEQ ID NOs: 1 to 15. Furthermore, the IL2 variant may include a polynucleotide encoding the amino acids. Specifically, the polynucleotide may be selected from the group consisting of SEQ ID NOs: 56 to 70.

[0048] Furthermore, the protein may include an Fc region bound by a linker or a carrier. For example, the linker may include a copolymer of 1 to 50 amino acids, albumin or a fragment thereof, polyethylene glycol, etc.

[0049] According to one embodiment, the linker may include the amino acid sequence of SEQ ID NO:18.

[0050] According to another embodiment, 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.

[0051] Furthermore, the protein may further include an antibody or an antigen binding fragment thereof to an immune checkpoint inhibitor. The immune checkpoint inhibitor may be, for example, programmed cell death-ligand 1 (PD-L1), programmed death receptor 1 (PD-1), lymphocyte activation gene 3 (LAG-3), V domain Ig inhibitory factor (VISTA), B lymphocyte weakening factor (BTLA), T cell mina-globulin mucin 3 (TIM-3), T cell immunoglobulin and ITIM domain protein (TIGIT) and cytotoxic T lymphocyte associated protein 4 (CTLA-4), etc.

[0052] Furthermore, the protein may further include an antibody or an antigen-binding fragment thereof to a tumor associated antigen. The tumor associated antigen may be, for example, programmed cell death-ligand 1 (PD-L1), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor-2 (HER-2), B7H3, phosphatidylinositol proteoglycan 3 (GPC3), carcinoembryonic antigen (CEA), human trophoblast surface glycoprotein antigen (TROP), and prostate specific membrane antigen (PSMA).

[0053] Therefore, another aspect provides a protein complex comprising: an IL2 variant; and an Fc region.

[0054] In another aspect, a protein complex is provided, which includes: an IL2 variant; and an antibody or an antigen-binding fragment thereof against an immune checkpoint inhibitor.

[0055] In another aspect, a protein complex is provided, which includes: an IL2 variant; and an antibody or an antigen-binding fragment thereof against a tumor associated antigen.

[0056] In this specification, the term "protein complex" is a complex composed of two or more related polypeptides, refers to an artificial recombinant protein expressed after connecting one or more genes of other proteins to a protein gene, and can be used interchangeably with "fusion protein". Since the protein complex is formed by connecting two or more proteins, its functional increase effect can be expected. Therefore, as a conjugate, protein complex, or fusion protein including an Fc region, the protein complex can selectively increase the activity of effector T cells relative to regulatory T cells by replacing specific amino acids of IL2. In addition, the protein complex can be specifically distributed in the tumor microenvironment, inhibit immune checkpoints, and activate immune cells to effectively induce apoptosis of cancer cells. Therefore, a therapeutic agent can be provided, which has reduced side effects and maximized anti-cancer activity compared to existing therapeutic agents.

[0057] On the other hand, a protein complex is provided, which includes a first polypeptide and a second polypeptide, wherein the first polypeptide includes a first CH3 antibody constant region, the second polypeptide includes a second CH3 antibody constant region, and the first polypeptide and the second polypeptide form a heterodimer, wherein the N-terminus of any one or more of the first polypeptide or the second polypeptide includes an antibody or an antigen-binding fragment thereof to an immune checkpoint inhibitor, or an antibody or an antigen-binding fragment thereof to a tumor associated antigen, and any one or more of the N-terminus or C-terminus of the first polypeptide or the second polypeptide includes an IL2 variant.

[0058] Figure 4 Included is the structure of a protein complex including an IL2 variant according to one embodiment.

[0059] Reference Figure 4 The protein complex may include an antibody or an antigen-binding fragment thereof to an immune checkpoint inhibitor, or an antibody or an antigen-binding fragment thereof to a tumor-associated antigen, at the N-terminus of a first polypeptide including a first CH3 antibody constant region, and may include an IL2 variant at the N-terminus of a second polypeptide including a second CH3 antibody constant region.

[0060] Furthermore, the protein complex may include an antibody or an antigen-binding fragment thereof to an immune checkpoint inhibitor, or an antibody or an antigen-binding fragment thereof to a tumor-associated antigen, at the N-terminus of a first polypeptide including a first CH3 antibody constant region and a second polypeptide including a second CH3 antibody constant region, and may include an IL2 variant at the C-terminus of the first polypeptide or the second polypeptide.

[0061] The specific contents of the IL2 variant, immune checkpoint inhibitor, and tumor-associated antigen are as described above. In the protein complex, the binding force between the antibody or its antigen-binding fragment to the immune checkpoint inhibitor and the cancer cell or T cell is better than the binding force between the T cell and the IL2 receptor. Therefore, the antibody or its antigen-binding fragment to the immune checkpoint inhibitor mainly binds to the cancer cell or the T cell around the cancer cell, thereby reducing the side effects caused by the binding of the IL2 receptor that can induce immune cell activation throughout the body. In addition, the antibody or its antigen-binding fragment to the tumor-associated antigen not only specifically binds to the cancer cell, but also has a better binding force to the cancer cell than the binding force to the IL2 receptor binding protein. Therefore, the antibody or its antigen-binding fragment to the tumor-associated antigen can be combined with the cancer cell once to reduce the side effects caused by the IL2 receptor binding that is better than the IL2 receptor binding that may induce immune cell activity throughout the body.

[0062] The immune checkpoint inhibitor and / or the antibody or antigen-binding fragment thereof of the tumor-associated antigen can be, for example, an antibody, an antigen-binding fragment (Fab), a single-chain variable region fragment (scFv), or a nanobody.

[0063] In this specification, the term "antibody" and "immunoglobulin (Ig)" are used interchangeably. A complete antibody has a structure with two full-length light chains and two full-length heavy chains, and each light chain is bound to a heavy chain by a disulfide bond (SS-bond). The light chain has two types, λ and κ, and consists of approximately 211 to 217 amino acids. In each human antibody, there is only one light chain. The light chain consists of a continuous constant 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 are composed of 450 amino acids, and μ and ε are composed of 550 amino acids. The heavy chain has two regions, namely a variable region and a constant region. The variable region refers to the region in the antibody that binds to the antigen. The variable region may include a complementarity determining region (CDR), which confers binding specificity to the antigen.

[0064] The antibody may include an antigen-binding fragment (Fab) region and a crystallizable fragment (Fc) region, wherein the Fab region binds to an antigen and the Fc region binds to a cell surface receptor. When papain is used for cutting, a complete antibody may be cut into two Fab regions and one Fc region. The Fab region may be formed by connecting a polypeptide including a variable region (VH) domain of a heavy chain and a first constant region (CH1) domain of a heavy chain to a polypeptide including a variable region (VL) domain of a light chain and a constant region (CL) domain of a light chain through a disulfide bond. The Fc region may be formed by connecting two polypeptides including a second constant region (CH2) domain of a heavy chain and a third constant region (CH3) domain to each other. The Fc region may form a hinge region. The CH3 antibody constant region refers to the third heavy chain constant region domain in an antibody.

[0065] Generally, for the effector function of an antibody, the Fc region binds to the Fcγ receptor as its receptor or the C1q as a complement component molecule and causes antibody-dependent cell cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Immune cells may induce apoptosis of poor immune cells due to the presence of numerous Fcγ receptors, so it is preferred to remove the effector function. Therefore, the CH3 antibody constant region can be modified to reduce the binding force to the Fcγ receptor, have no effector function, or significantly reduce the function and have high stability. In addition, the CH3 antibody constant region can be modified to reduce antibody-dependent cellular cytotoxicity (ADCC).

[0066] According to one embodiment, the Fc region may include tryptophan (W) at position 366 of the first CH3 antibody constant region, serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407 of the second CH3 antibody constant region, and glycine (G) or phenylalanine (F) at position 351 of the second CH3 antibody constant region.

[0067] Furthermore, the Fc region may include serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407 of the first CH3 antibody constant region, tryptophan (W) at position 366 of the second CH3 antibody constant region; and glycine (G) or phenylalanine (F) at position 351 of the first CH3 antibody constant region.

[0068] Furthermore, the Fc region includes tryptophan (W) at position 366 of the first CH3 antibody constant region, glycine (G) at position 351 of the second CH3 antibody constant region, serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407; and includes phenylalanine (F) or tryptophan (W) at position 351 of the first CH3 antibody constant region.

[0069] Furthermore, the Fc region may include 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 constant region, tryptophan (W) at position 366 of the second CH3 antibody constant region; and phenylalanine (F) or tryptophan (W) at position 351 of the second CH3 antibody constant region.

[0070] According to another embodiment, the first CH antibody constant region and the second CH antibody constant region may further include 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 positions 234, 235, 329, 297, 331, and 265. Specifically, the CH antibody constant region may be a CH2 antibody constant region.

[0071] For example, in the first CH2 antibody constant region and the second CH2 antibody constant region, the leucine (L) at positions 234 and 235 may be further substituted with alanine (A) (L234A / L235A).

[0072] Furthermore, in the first CH2 antibody constant region and the second CH2 antibody constant region, leucine (L) at positions 234 and 235 may be substituted with alanine (A), and proline (P) at position 329 may be further substituted with glycine (G) (L234A / L235A / P329G).

[0073] Furthermore, in the first CH2 antibody constant region and the second CH2 antibody constant region, asparagine (N) at position 297 may be further substituted with alanine (A), glutamine (Q), or glycine (G) (N297A, N297Q, or N297G).

[0074] Furthermore, in the first CH2 antibody constant region and the second CH2 antibody constant region, leucine (L) at positions 234 and 235 are substituted by phenylalanine (F) and glutamic acid (E), and proline (P) at position 331 is further substituted by serine (S) (L234F / L235E / P331S).

[0075] Furthermore, in the first CH2 antibody constant region and the second CH2 antibody constant region, leucine (L) at positions 234 and 235 are substituted by phenylalanine (F) and glutamic acid (E), and aspartic acid (D) at position 265 is further substituted by alanine (A) (L234F / L235E / D265A).

[0076] And, according to another embodiment, 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.

[0077] And, according to another embodiment, the first polypeptide or the second polypeptide; and the IL2 variant may be combined through a linker or a carrier. The linker may include an amino acid sequence of (GGGGS)n, where "n" may be a natural number from 1 to 10. Specifically, the linker may include an amino acid sequence of SEQ ID NO: 18.

[0078] The first polypeptide and the second polypeptide may form an Fc region of an antibody.

[0079] The heterodimer refers to a combination of two polypeptides that differ in the order, number, or type of amino acid residues. The protein complex may be a protein complex formed by the combination of two polypeptides that specifically bind to different targets.

[0080] The protein complex can be an antibody or an antigen-binding fragment thereof, a conjugate of a receptor and an agonist, a conjugate of a receptor and an antagonist, a conjugate of a receptor and a ligand, or a conjugate of a ligand and a decoy receptor. Furthermore, the protein complex can include items in the group consisting of an antigen-binding fragment (Fab), a single-chain variable region 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.

[0081] The antibody may be, for example, IgA, IgD, IgE, IgG, or IgM. The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody may be an animal-derived antibody, a mouse-human chimeric antibody, a humanized antibody, or a human antibody.

[0082] In this specification, the term "antigen-binding fragment" is a fragment of the entire immunoglobulin structure, and refers to a portion of a polypeptide that includes a portion that can bind to an antigen. For example, the antigen-binding fragment can be scFv, (scFv)2, Fv, Fab, Fab', Fv F(ab)'2, or a combination thereof.

[0083] In this specification, the term "receptor" refers to a substance for receiving or transmitting a signal that can be transmitted through a biological system. The receptor may be a protein receptor. The receptor may be bound to an agonist, an antagonist, a ligand, or a cytokine. The agonist may be a substance that induces a biological response by binding to a receptor and activating the receptor. The antagonist may be a substance that inhibits a biological response by binding to a receptor and inhibiting the receptor. The ligand may be a substance that binds to a receptor. The ligand may bind to a decoy receptor. A decoy receptor refers to a receptor that hinders signal transmission through an actual receptor by specifically binding to a ligand. The cytokine refers to a small protein that acts on signal transmission, regulation and maintenance of inflammatory processes in cells.

[0084] The protein complex may be modified. For example, the protein complex may be modified by conjugation or binding, glycosylation, label attachment, or a combination thereof. The antibody may be conjugated with other drugs such as anticancer agents. For example, the protein complex may be conjugated with horseradish peroxidase (HRP), alkaline phosphate, hapten, biotin, streptavidin, fluorescent substances, radioactive substances, quantum dots, polyethylene glycol (PEG), histidine tags, or a combination thereof. The fluorescent substance may be Alexa Fluor. 532. Alexa 546. Alexa 568. Alexa 680、Alexa 750、Alexa 790 or Alexa 350.

[0085] The amino acid positions of the Fc region or CH2 and CH3 regions are based on the Kabat EU index (EU index recorded in 'Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)'). The amino acid positions of the CH3 domain and the amino acid types corresponding to the amino acid positions are based on human IgG1.

[0086] Another aspect provides a polynucleotide encoding a protein according to one aspect or a protein complex according to another aspect.

[0087] The details of the protein or protein complex are as described above.

[0088] According to one embodiment, 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.

[0089] According to another embodiment, the polynucleotide may include: one or more polynucleotides selected from the group consisting of SEQ ID NOs: 56 to 70; a polynucleotide including SEQ ID NO: 73; and one or more polynucleotides selected from the group consisting of SEQ ID NOs: 74 to 76.

[0090] According to another embodiment, the polynucleotide may include: one or more polynucleotides selected from the group consisting of SEQ ID NOs: 56 to 70; a polynucleotide including SEQ ID NO: 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.

[0091] Another aspect provides a method for preparing a protein or a protein complex, the method comprising the step of transfecting an expression vector encoding a protein according to one aspect or a protein complex according to another aspect into a cell to express the protein or the protein complex. And, another aspect provides a protein or a protein complex comprising an IL2 variant prepared by the method.

[0092] The details of the protein or protein complex are as described above.

[0093] An expression vector is an expression vector capable of expressing a target protein in an appropriate host cell, and refers to a vector that includes the necessary regulatory factors operably linked to express the inserted nucleic acid sequence. The "operably linked" means that the nucleic acid expression regulatory sequence is functionally linked to the nucleic acid encoding the target protein to perform a conventional function. The expression vector may include a polynucleotide encoding the protein complex. The expression vector may include regulatory sites required for expressing a gene, for example, an enhancer, a promoter, a poly (A) sequence, etc.

[0094] The cell may be a cancer cell. The cell may be an in vitro cell. The cell may be a bacterium, a yeast, a plant cell, or a mammalian cell. The bacterium may be Escherichia coli. The mammalian cell refers to a cell derived from a mouse, a rat, a rabbit, a dog, a cat, a sheep, a cow, a horse, a monkey, a chimpanzee, or a human. The cell may be a cell strain. The cell may be, for example, selected from the group consisting of Chinese hamster ovary cells (CHO), human embryonic kidney cells (HEK), baby hamster kidney (BHK), NSO cells, human embryonic retinal cells (PER.C6), human cervical cancer (HeLa), Madin-Darby dog ​​kidney epithelial cells (MDCK, Madin-Darby Canine Kidney), SP2 / 0 mouse myeloma cells, COS-7, and YB2 / 0 rat myeloma cells. The CHO cell may be a CHO DG44, CHO-K1, CHO-S, GS-CHO, or CHO DUKX (DXB11) cell. The HEK cell may be a HEK293 cell.

[0095] "Transfection" refers to a method of inserting a specific nucleic acid fragment into a cell gene to allow the inserted nucleic acid to be expressed.

[0096] According to one embodiment, the method may include the step of co-transfecting an expression vector encoding a first polypeptide and / or a second polypeptide including an antibody or an antigen-binding fragment thereof to an immune checkpoint inhibitor or an antibody or an antigen-binding fragment thereof to a tumor-associated antigen at the N-terminus of any one or more of the first polypeptide or the second polypeptide and an expression vector encoding a first polypeptide or a second polypeptide including an IL2 variant at the N-terminus or C-terminus to a cell. In addition, the method may include the step of transfecting an expression vector encoding a first polypeptide and / or a second polypeptide including an antibody or an antigen-binding fragment thereof to an immune checkpoint inhibitor or an antibody or an antigen-binding fragment thereof to a tumor-associated antigen at the N-terminus and an expression vector encoding a first polypeptide or a second polypeptide including IL2 at the N-terminus or C-terminus to two or more cells, respectively.

[0097] The cells may be cultured in a cell culture medium. A cell culture medium refers to a solution containing nutrients required for culturing cells. The culture medium includes commercial or manufactured culture mediums for culturing cells. The cell culture medium may include antibiotics. The cell culture medium may include G418 (geneticin), Puromycin, Blasticidin, Zeocin, or a combination thereof. The cell culture medium may include a chemically defined medium.

[0098] The cells are cultured under conditions that allow the survival or proliferation of the cells. The conditions that allow the survival or proliferation of the cells can be different according to the cell type. The cells can be cultured at about 25°C to about 42°C, about 25°C to about 40°C, about 30°C to about 40°C, about 30°C to about 37°C, or about 37°C. The cells can be cultured in the presence of air of about 1% CO2 to about 10% CO2, or about 5% CO2 to about 10% CO2. The cells can be cultured in a culture medium of about pH6 to about pH8, about pH6.2 to about pH7.8, about pH6.4 to about pH7.6, about pH6.6 to about pH7.4, or about pH6.8 to about pH7.2. The cells can be cultured under conditions of about 10% to about 80% dissolved oxygen, about 15% to about 70%, or about 20% to about 60%.

[0099] The culture can be different according to the cell type. The culture can be carried out using known methods. The culture can be carried out on a flat plate, a flask, etc. The culture can be carried out by attaching to a matrix or by suspending in a culture fluid. The culture can be subculture, batch culture, fed-batch culture, perfusion culture, or a combination thereof. During the culture, the cell culture medium can be regularly replaced with fresh culture medium. The cells can be cultured for about 1 day or longer, about 2 days or longer, about 3 days or longer, about 4 days or longer, about 5 days or longer, about 6 days or longer, about 1 week or longer, about 10 days or longer, about 2 weeks or longer, about 3 weeks or longer, about 1 month or longer, about 1 day to about 1 month, about 1 day to about 3 weeks, about 1 day to about 2 weeks, about 2 days to about 2 weeks, about 3 days to about 2 weeks, about 4 days to about 2 weeks, about 5 days to about 2 weeks, about 6 days to about 2 weeks, or about 1 week to about 2 weeks.

[0100] The step of obtaining the protein complex from the cells or cell culture fluid may be included.

[0101] The cell culture medium may be a culture medium that does not contain the cells.

[0102] In the case where the expression vector is co-transfected into a cell, a protein complex comprising a first polypeptide and a second polypeptide can be obtained from the cell or cell culture fluid, wherein the first polypeptide comprises an antibody or an antigen-binding fragment thereof to an immune checkpoint inhibitor, or an antibody or an antigen-binding fragment thereof to a tumor-associated antigen at the N-terminus, and the second polypeptide comprises an IL2 variant at the N-terminus or C-terminus. When an expression vector encoding a first polypeptide comprising an antibody or an antigen-binding fragment thereof to an immune checkpoint inhibitor, or an antibody or an antigen-binding fragment thereof to a tumor-associated antigen at the N-terminus and an expression vector encoding a second polypeptide comprising an IL2 variant at the N-terminus or C-terminus are transfected into two or more cells, respectively, the first polypeptide comprising an antibody or an antigen-binding fragment thereof to an immune checkpoint inhibitor, or an antibody or an antigen-binding fragment thereof to a tumor-associated antigen at the N-terminus; and a second polypeptide comprising an IL2 variant at the N-terminus or C-terminus can be obtained from the cell or cell culture fluid, respectively.

[0103] The step of obtaining the protein complex may include forming a protein complex by incubating a first polypeptide and a second polypeptide, wherein the first polypeptide includes an antibody or an antigen-binding fragment thereof to an immune checkpoint inhibitor or an antibody or an antigen-binding fragment thereof to a tumor associated antigen at the obtained N-terminus, and the second polypeptide includes an IL2 variant at the obtained N-terminus or C-terminus. The incubation may be performed under reducing conditions. The reducing conditions may be in the presence of 2-mercaptoethanol (2-ME), dithiothreitol (DTT), or a combination thereof.

[0104] The step of obtaining the protein complex may include the step of purifying the protein complex. The purification may be performed by filtration, centrifugation, chromatography, dialysis, immunoprecipitation, or a combination thereof.

[0105] Another aspect provides a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising the protein according to one aspect or the protein complex according to another aspect. Another aspect provides the use of the protein according to one aspect or the protein complex according to another aspect in the preparation of a medicament for preventing or treating cancer.

[0106] The details of the protein or the protein complex are as described above.

[0107] The cancer may be a solid tumor or a non-solid tumor. A solid tumor refers to a cancer that occurs in organs such as the liver, lungs, breasts, and skin. A non-solid tumor is a cancer that occurs in the blood, also known as a blood cancer. The cancer may be a carcinoma, a sarcoma, a cancer derived from blood cells, a germ cell tumor, or a blastoma. The cancer can be selected from the group consisting of breast cancer, skin cancer, head and neck cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, stomach cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, brain and spinal cord tumors, brain cancer, thymoma, mesothelioma, esophageal cancer, bile duct cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndrome (MDS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's Disease, neuroendocrine tumors and sarcoma.

[0108] The "prevention" refers to all actions to suppress a disease or delay the occurrence of the disease by administering the pharmaceutical composition. The "treatment" refers to all actions to improve or benefit the symptoms of a disease by administering the pharmaceutical composition.

[0109] The pharmaceutical composition may include a pharmaceutically acceptable carrier. The carrier is used to include an excipient, a diluent, or an adjuvant. The carrier may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, silicon phosphate, cellulose, methylcellulose, polyvinyl pyrrolidone, water, saline, a buffer solution such as PBS, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, or a combination thereof.

[0110] The pharmaceutical composition can be prepared into any dosage form according to conventional methods. The composition can be formulated into, for example, an oral dosage form (e.g., powder, tablet, capsule, syrup, pill, or granule) or a parenteral dosage form (e.g., injection). In addition, the composition can be formulated into a systemic dosage form or a local dosage form.

[0111] The pharmaceutical composition may further include other anticancer agents. The anticancer agent may be cetuximab, panitumumab, erlotinib, gefitinib, trastuzumab, T-DM1, Perjeta, lapatinib, paclitaxel, tamoxifen, cisplatin, or a combination thereof. The pharmaceutical composition may be a single composition or an individual composition. For example, the composition of the antibody or its antigen-binding fragment may be a composition of a parenteral administration dosage form, and the anticancer agent may be a composition of an oral administration dosage form.

[0112] The pharmaceutical composition may include an effective amount of the protein complex. The term "effective amount" refers to an amount sufficient to exert a preventive or therapeutic effect when applied to an individual who needs to prevent or treat a disease. The effective amount can be appropriately selected by a person of ordinary skill according to the selected cells or individuals. The effective amount can be determined based on factors including the severity of the disease, the patient's age, weight, health, sex, patient sensitivity to the drug, administration time, administration route, and excretion ratio, treatment period, and the drug used in conjunction with or simultaneously with the composition administered, as well as other well-known factors in the medical field. The effective amount can be about 0.5 μg to about 2g, about 1 μg to about 1g, about 10 μg to about 500mg, about 100 μg to about 100mg, or about 1mg to about 50mg in each pharmaceutical composition.

[0113] For example, the dosage of the pharmaceutical composition can be 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 an adult. The administration can be performed once a day, multiple times a day, once a week, once every two weeks, once every three weeks, or once every four weeks to once a year.

[0114] Another aspect provides a method for preventing or treating cancer, comprising the step of administering the protein according to one aspect or the protein complex according to another aspect to a cell or an individual.

[0115] The details regarding the protein, protein complex, cell, cancer, prevention, or treatment are as described above.

[0116] The individual may be a mammal, for example, a human, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat. The individual may be an individual who has cancer or has a high probability of developing cancer.

[0117] The method may further include administering a second active ingredient to the individual. The second active ingredient may be an active ingredient for preventing or treating cancer. The active ingredient may be administered simultaneously, separately, or sequentially with the protein complex.

[0118] The protein or protein complex can be directly administered to an individual, for example, by any means of oral, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration. The protein complex can be administered systemically or locally, and can be administered alone or together with other pharmaceutically active compounds.

[0119] The preferred amount of the protein or protein complex used varies depending on the patient's condition and body weight, disease severity, drug form, route of administration, and period, but can be appropriately selected by a person of ordinary skill. For example, the amount of administration can be 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 for an adult. The administration can be performed once a day, multiple times a day, or once a week, once every two weeks, once every three weeks, or once every four weeks to once a year.

[0120] Beneficial Effects

[0121] According to one aspect, the protein can selectively increase the activity of effector T cells relative to regulatory T cells by replacing a specific amino acid sequence of IL2. In addition, a pharmaceutical composition comprising the protein can reduce side effects and maximize anticancer activity compared to existing therapeutic agents, and thus can be used to prevent or treat cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1a The results of purifying the PD-L1 antibody-IL2 variant 2 protein complex according to one embodiment using a protein-A affinity column purification method are shown.

[0123] Figure 1b The results of purifying the PD-L1 antibody-IL2 variant 2 protein complex according to one embodiment using a cation exchange resin column purification method are shown.

[0124] Figure 1cThe results of analyzing the purity of the PD-L1 antibody-IL2 variant 2 protein complex according to one embodiment by size exclusion high performance liquid chromatography (SE-HPLC) analysis are shown.

[0125] Figure 2a The results of comparison of the anticancer effects of the PD-L1 antibody-IL2 variant 2 protein complex according to one embodiment, as well as a positive control group (Avelumab, and combined administration of Avelumab and Aldesleukin) and a negative control group in colorectal cancer-transplanted mice are shown.

[0126] Figure 2b The results of confirming the cancer cell growth inhibition and complete remission activity of the PD-L1 antibody-IL2 variant 2 protein complex according to one embodiment in each individual in mice transplanted with colorectal cancer are shown.

[0127] Figure 2c The negative control group and the positive control group (Avelumab and combined administration of Avelumab and Aldesleukin) show the cancer cell growth inhibition and complete remission effects of each individual in mice transplanted with colorectal cancer.

[0128] Figure 3 The results are shown for the anti-cancer effect of the PD-L1 antibody-IL2 variant 4 protein complex according to one embodiment in mice transplanted with colorectal cancer.

[0129] Figure 4 The structure of a protein complex including an IL2 variant according to one embodiment is shown.

[0130] Figure 5a The figure shows the results of purifying the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment using a protein-A affinity column purification method.

[0131] Figure 5b The figure shows the results of purifying the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment using a hydrophobic interaction column purification method.

[0132] Figure 5c The results of analyzing the purity of the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment are shown.

[0133] Figure 6a The figures show the results of comparison of the anticancer effects of the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment, a negative control group, and a positive control group in a colorectal cancer transplanted mouse model.

[0134] Figure 6b The results of comparing the changes in body weight of a colorectal cancer transplanted mouse model after administration of the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment, a negative control group, and a positive control group to the colorectal cancer transplanted mouse model are shown. DETAILED DESCRIPTION

[0135] Hereinafter, preferred embodiments are proposed to help understand the present invention. However, the following embodiments are only provided to make the present invention more understandable and clear, and the content of the present invention is not limited to the following embodiments.

[0136] [Example]

[0137] Example 1. Preparation and activity confirmation of IL2 variant protein

[0138] 1-1. Cloning and culture of IL2 variant proteins

[0139] The IL2 variant was prepared by replacing the amino acids at some positions in the wild-type IL2 amino acid (SEQ ID NO: 16). Specifically, the polynucleotide encoding the IL2 variant with 6X His tag was synthesized using the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific, and the synthesized polynucleotide was inserted into the APAINheI enzyme site of the pCDNA3.1 expression vector to prepare an expression vector expressing the IL2 variant. In the IL2 variant, each amino acid substitution site and the substituted amino acid are shown in Table 1 below. Then, the ExpiFectamine TM The expression vector was transfected into ExpiCHO-S TM Cell line. The transfected cells were cultured in an incubator at 32°C, 5% CO2, and 120 rpm for 12 days. After 12 days, the culture supernatant was separated and recovered, and filtered through a sterile filter to obtain the IL2 variant protein.

[0140] [Table 1]

[0141] 35 38 42 125 18 19 126 wt-IL2 K R F C L L Q IL2 control group E S IL2 variant 1 E A K S IL2 variant 2 E A K S M S IL2 variant 3 E A K S A S IL2 variant 4 E A K S R S IL2 variant 5 E A K S M L IL2 variant 6 E A K S L S IL2 variant 7 E A K S S W IL2 variant 8 E A K S F N IL2 variant 9 E A K S V I IL2 variant 10 E A K S I T IL2 variant 11 E A K S Q A IL2 variant 12 E A K S M M IL2 variant 13 E A K S M F IL2 variant 14 E A K S T IL2 variant 15 E A K S I

[0142] 1-2. Purification of IL2 variant protein

[0143] After purification using the His tag attached to the IL2 variant protein obtained in the culture solution obtained in Example 1-1, a highly pure substance with a purity of 95% or higher was obtained.

[0144] First, the culture fluid obtained in the embodiment 1-1 is centrifuged and separated into cultured cells and culture medium. Then, the IL2 protein in the separated culture medium is removed from fine residues by 0.22 μm filter (Thermo Scientific Society) filtration process. After the culture medium of the filtration process is purified once using imidazole affinity chromatography (Ni-SepFast, Biotoolomics Society), the imidazole buffer component present in the purified product is removed through desalting process and concentration process. Then, the content and purity of the final purified product are analyzed. Specifically, after the filtered culture medium is put into the Ni-SepFast column stabilized by phosphate buffer saline (phosphate buffer saline) (pH7.4), the non-specific protein without binding is washed with the same buffer solution, and the protein specifically bound to the Ni-SepFast column is dissolved by imidazole buffer solution (0M and 0.5M, pH7.4) by the imidazole concentration rising gradient mode. After the IL2 variant protein was stabilized by desalting with phosphate buffer (pH 7.4), the purity of the purified IL2 variant protein was analyzed by size exclusion high performance liquid chromatography (HPLC) (TSK-3000SWxL, 7.8 mm × 30 cm, Tosoh Corporation), and the molecular weight was confirmed by SDS-PAGE analysis.

[0145] 1-3. Confirmation of IL2 variant protein activation of human immune cells

[0146] The activation of human immune cells by the IL2 variant-protein complex purified in the embodiment 1-2 was confirmed. Specifically, human peripheral blood mononuclear cells (Stem cell company) and fluorescently labeled antibodies specifically attached to effector CD8+T cells (CD3+, CD8+) and Treg cells (CD4+, CD25+) were reacted at 4°C in the dark for 30 minutes, and then the antibodies not attached to the cells were removed by centrifugation. Then, the IL2 variants 2, 4, and 14 proteins purified in the embodiment 1-2 were treated to react at 37°C in the dark for 20 minutes, and fixed for 12 minutes using 1mL of the fixing buffer (BD, USA). At this time, aldesleukin (Aldesleukin) (Proleukin Note, Novartis, Switzerland) was used as a positive control group, and each drug was treated at a concentration of 0.01 to 8,000nM. Then, 1.5 mL of Perm3 buffer (BD, USA) was used to treat the cytoplasm of fixed human peripheral blood mononuclear cells and react at 4°C for 35 minutes to allow the fluorescent label to penetrate the cytoplasm, and the phosphorylated STAT-5 and Treg cell marker Foxp3+ protein were attached with fluorescent labels for 30 minutes. Then, the STAT-5 phosphorylation degree of the IL2 variant protein treatment group in effector CD8+T and Treg cells was compared by flow cytometry.

[0147] [Table 2]

[0148]

[0149] As shown in Figure 2, in the positive control group (aldesleukin), the EC of CD8+ T cells / regulatory T cells 50 The ratio of the values ​​was 16.3, which showed that the activity of inducing Treg cells was stronger than that of CD8+T cells.

[0150] In contrast, ECs of CD8+ T cells / regulatory T cells in IL2 variants 2, 4, and 14 proteins 50 The ratios of the values ​​were 0.6, 0.56, and 5.6, respectively, and it was confirmed that the activity of selectively activating CD8+ T cells was achieved compared with the positive control group.

[0151] Example 2. Preparation and characterization of IL2 variant-Fc protein complex

[0152] 2-1. Cloning and culture of IL2 variant-Fc protein complex

[0153] An Fc protein complex including IL2 variants was prepared. Specifically, the polynucleotide encoding the Fc region was synthesized using the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific, and the synthesized polynucleotide was then inserted into the AvrⅡ-BistZ17I enzyme site of the pCH01.0 expression vector to prepare a first expression vector expressing the Fc region. The same service was used to synthesize polynucleotides encoding the complex of IL2 variants 1, 2, 4, and 14 prepared in Example 1 and the linker and Fc region, and the synthesized polynucleotide was inserted into the AvrⅡ-BstZ17I enzyme site of the pCH01.0 expression vector to prepare a second expression vector expressing IL2 variant-Fc. Then, ExpiFectamine TM The first expression vector and the second expression vector were mixed at a ratio of 1:1 and transfected into ExpiCHO-S TM Cell line. The transfected cells were cultured in an incubator at 32°C, 8% CO2, and 125 rpm for 12 days. After 12 days, the culture supernatant was separated and recovered, and filtered through a sterile filter to obtain a protein complex including Fc and IL2 variant-Fc.

[0154] 2-2. Purification of IL2 variant-Fc protein complex

[0155] Purification The IL2 variant-Fc protein complex obtained by culturing the cell line prepared in Example 2-1 was purified to obtain a high-purity substance.

[0156] First, the culture solution obtained in the embodiment 2-1 was centrifuged and divided into culture cells and culture medium. Then, a 0.22 μm filter (Thermo Scientific) was used to remove fine residues from the IL2 variant-Fc protein complex in the separated culture medium. The culture medium after the filtration process was purified once using a protein A affinity column (MabSelect PrismA, Cytiva). Specifically, after the filtered culture medium was put into a protein-A column stabilized with a phosphate buffer solution (pH 7.4), the same buffer was used to wash non-specific proteins that were not bound, and the pH gradient formed by two solutions of pH 5.0 and pH 3.5 including 0.02M citric acid was used to dissolve the protein that specifically binds to the protein-A column, and the sample was neutralized to pH 7.2 using 1M Tris. The secured IL2 variant-Fc protein complex was then stabilized with phosphate buffer (pH 7.4), and the purity of the purified IL2 variant-Fc protein complex was analyzed using size exclusion high performance liquid chromatography (HPLC) (TSK-3000SWxL, 7.8 mm×30 cm, Tosoh).

[0157] 2-3. Confirmation of activation of human immune cells by IL2 variant-Fc protein complex

[0158] The activation of human immune cells by the IL2 variant-Fc protein complex purified in the embodiment 2-2 was confirmed. Specifically, human peripheral blood mononuclear cells (Stem cell company) and fluorescently labeled antibodies specifically attached to effector CD8+T cells (CD3+, CD8+) and Treg cells (CD4+, CD25+) were reacted at 4°C for 30 minutes in the dark, and then the antibodies not attached to the cells were removed by centrifugation. Then, the IL2 variant 1-Fc protein complex, IL2 variant 2-Fc protein complex, IL2 variant 4-Fc protein complex, and IL2 variant 14-Fc protein complex purified in the embodiment 2-2 were treated and reacted at 37°C in the dark for 20 minutes, and fixed for 12 minutes using 1mL of fixed buffer (BD, USA). At this time, aldesleukin (Aldesleukin) (Proleukin Note, Novartis, Switzerland) was used as a positive control group, and each drug was treated at a concentration of 0.01 to 8,000nM. Then, 1.5 mL of Perm3 buffer (BD, USA) was used to treat the cytoplasm of fixed human peripheral blood mononuclear cells and react at 4°C for 35 minutes to allow the fluorescent label to penetrate the cytoplasm, and the phosphorylated STAT-5 and Treg cell marker Foxp3+ protein were attached with fluorescent label for 30 minutes. Then, the STAT-5 phosphorylation degree of the IL2 variant-Fc protein complex treatment group in effector CD8+T cells and Treg cells was compared by flow cytometry.

[0159] [Table 3]

[0160]

[0161] Results, such as Figure 3 As shown, in the positive control group (aldesleukin), the EC of CD8+ T cells / regulatory T cells 50 The ratio of the values ​​was 33.3, from which it was confirmed that the activity of Treg cells was induced to be higher than that of CD8+T cells.

[0162] In contrast, ECs of CD8+ T cells / regulatory T cells in IL2 variant 1-Fc protein complex, IL2 variant 2-Fc protein complex, IL2 variant 4-Fc protein complex, and IL2 variant 14-Fc protein complex were significantly higher than those in IL2 variant 1-Fc protein complex. 50 The ratio of the values ​​was 0.79 to 3.2, and it was confirmed that the activity of CD8+T cells was more selectively activated than that of the positive control group.

[0163] That is, it was found that the IL2 variant according to one aspect selectively activates CD8+ cells more than regulatory T cells even in a structure to which Fc is attached.

[0164] Example 3. Preparation and characterization of PD-L1 antibody-IL2 variant protein complex

[0165] 3-1. Cloning and culture of PD-L1 antibody-IL2 variant protein complex

[0166] A protein complex including a PD-L1 antibody and an IL2 variant was prepared. Specifically, the same service as used in Example 2-1 was used to synthesize a polynucleotide encoding the heavy chain variable region-heavy chain constant region of the PD-L1 antibody and a polynucleotide encoding the light chain constant region-light chain variable region of the PD-L1 antibody, respectively. Then, the polynucleotide encoding the heavy chain variable region-heavy chain constant region of the PD-L1 antibody and the polynucleotide encoding the light chain constant region-light chain variable region of the PD-L1 antibody were inserted into the AvrⅡ-BstZ17I enzyme site and the EcoRV-PacI enzyme site of the pCH01.0 expression vector, respectively, to prepare a first expression vector expressing the PD-L1 antibody. The same service was used to synthesize a polynucleotide comprising a complex of any one of the IL2 variants 1 to 15 prepared in Example 1 and a linker and an Fc region, and then inserted into the AvrⅡ-BstZ17I enzyme site of the pCHO1.0 expression vector to prepare a second expression vector expressing IL2 variant-Fc. Then, a PD-L1 antibody-IL2 variant protein complex including anti-PD-L1 Fab-Fc and IL2 variant-Fc was obtained by the same method as that used in Example 2-1, except that the first expression vector and the second expression vector were used.

[0167] 3-2. Purification of PD-L1 Antibody-IL2 Variant Protein Complex

[0168] Purification The PD-L1 antibody-IL2 variant 1 protein complex to the PD-L1 antibody-IL2 variant 15 protein complex obtained by culturing the cell line prepared in Example 3-1 was used to obtain a highly pure substance.

[0169] First, the culture solution obtained in the embodiment 3-1 was centrifuged to separate the cultured cells and the culture medium. Then, a 0.22 μm filter (Thermo Scientific) was used to remove the fine residue from the PD-L1 antibody-IL2 variant protein complex in the separated culture medium. The culture medium after the filtration process was purified once using a protein A affinity column (MabSelect PrismA, Cytiva). Specifically, after the filtered culture medium was put into a protein-A column stabilized with a phosphate buffer (pH 7.4), the non-specific proteins that were not bound were washed with the same buffer, and the proteins that were specifically bound to the protein-A column were dissolved using a buffer solution including 0.05 M citric acid (pH 3.9), and the sample was neutralized to pH 7.2 using IM Tris. Then, in order to remove the residual material source impurities in the sample separated by the affinity column, a cation exchange chromatography (Source 30S, Cytiva) was used for secondary purification. Specifically, 1M citric acid was added to the sample eluted and neutralized in the protein-A column to titrate to pH 6.0, and the product was put into a Source30S column stabilized with a 20mM sodium phosphate (pH 6.0) buffer, and non-specific proteins that were not bound were washed with the same buffer, and eluted with a buffer solution including 0.3M sodium chloride (pH 6.0) in an ascending gradient manner to ensure multiple PD-L1 antibody-IL2 variant protein complexes. Then, the multiple PD-L1 antibody-IL2 variant protein complexes ensured were stabilized with a phosphate buffer (pH 7.4), and the purity of the purified PD-L1 antibody-IL2 variant protein complex was analyzed using size exclusion high performance liquid chromatography (HPLC) (TSK-3000SWxL, 7.8mm×30cm, Tosoh).

[0170] Figure 1a The results of purifying the PD-L1 antibody-IL2 variant 2 protein complex according to one embodiment using a protein-A affinity column purification method are shown.

[0171] Figure 1b The results of purifying the PD-L1 antibody-IL2 variant 2 protein complex according to one embodiment using a cation exchange resin column purification method are shown.

[0172] Figure 1c The results of analyzing the purity of the PD-L1 antibody-IL2 variant 2 protein complex according to one embodiment by SE-HPLC analysis are shown.

[0173] Results, such as Figure 1aAs shown, the protein that specifically binds to the column can be confirmed by the elution buffer used in the protein-A affinity column purification. Specifically, the PD-L1 antibody-IL2 variant protein complex that specifically binds to the column can be confirmed when the eluate is 1500 to 1550 ml.

[0174] And, if Figure 1b As shown, it was confirmed that impurities remaining in the eluate used in the protein-A affinity column and proteins non-specifically bound were removed.

[0175] And, if Figure 1c As shown, it was confirmed that the PD-L1 antibody-IL2 variant 2 protein complex purified by protein-A affinity column purification and cation exchange resin purification showed a purity of 99% at a retention time of 16.717 minutes.

[0176] That is, it was found that the PD-L1 antibody-IL2 variant protein according to one aspect was purified to a high purity.

[0177] 3-3. Confirmation of receptor binding ability of PD-L1 antibody-IL2 variant protein complex

[0178] The receptor binding force of the PD-L1 antibody-IL2 variant protein complex according to one aspect was confirmed by surface plasmon resonance (SPR) analysis. Specifically, human PD-L1 and human IL2Rα, IL2Rβγ, and IL2Rαβγ were covalently fixed to a CM5 sensor chip (sensor chip), and the PD-L1 antibody-IL2 variant 2 protein complex and PD-L1 antibody-IL2 variant 4 protein complex prepared in the embodiment 3-2 were expanded at different concentrations (2-fold serial dilution in the concentration range of 0.391 to 400nM) to confirm the binding kinetics of each of human PD-L1 and IL2 receptor (IL2receptor, IL2R). Afterwards, the binding affinity (binding affinity, KD) was calculated using the determined association constant (association constant, Ka) and dissociation constant (dissociation constant, Kd). At this point, 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.

[0179] [Table 4]

[0180]

[0181] [Table 5]

[0182]

[0183] Figure 2a The results of confirming the binding ability of the PD-L1 antibody-IL2 variant 2 protein complex according to one embodiment to the human PD-L1 antigen are shown.

[0184] Figure 2b The results of confirming the binding ability of Avelumab to the human PD-L1 antigen are shown.

[0185] Results, such as Figure 2a As shown in Table 4, it can be confirmed that the PD-L1 antibody-IL2 variant 2 protein complex prepared in Example 3-2 and the positive control group bind to the human PD-L1 antigen. Specifically, the protein variant exhibits a binding force of 0.48 nM, and the positive control group exhibits a binding force of 0.12 nM, which can be confirmed to exhibit a binding force that is about 4.1 times lower.

[0186] That is, it is considered that the positive control group exhibits a difference in binding ability because the monoclonal antibody has a bivalent anti-PD-L1 arm, while the PD-L1 antibody-IL2 variant 2 protein complex has a monovalent anti-PD-L1 arm.

[0187] Furthermore, as shown in Table 5, the PD-L1 antibody-IL2 variant 2 protein complex prepared in Example 2 binds to IL2Rβγ and IL2Rαβγ with binding affinities of 37.9 nM and 17.3 nM, respectively, but does not react with IL2Rα. Furthermore, the PD-L1 antibody-IL2 variant 2 protein complex binds to IL2Rβγ and IL2Rαβγ with binding affinities of 23.8 nM and 30.6 nM, respectively, but does not react with IL2Rα.

[0188] That is, it was confirmed that the PD-L1 antibody-IL2 variant 2 protein complex and the PD-L1 antibody-IL2 variant 4 protein complex according to one aspect do not bind to IL2 receptor α.

[0189] In contrast, as shown in Table 5, it was confirmed that the positive control group bound to IL2Rα, IL2Rβγ, and IL2Rαβγ with binding affinities of 35.7 nM, 1.55 nM, and 0.10 nM, respectively.

[0190] It can be confirmed that the binding force of the PD-L1 antibody-IL2 variant 2 protein complex and the PD-L1 antibody-IL2 variant 4 protein complex to IL2Rβγ is reduced by about 24.5 times and 15.4 times, respectively, compared with the positive control group. In addition, according to the results of calculating the βγ / αβγ ratio based on the binding force results for IL2Rαβγ, the βγ / αβγ ratios of the PD-L1 antibody-IL2 variant 2 protein complex and the PD-L1 antibody-IL2 variant 4 protein complex are 2.19 and 0.78, respectively, and 15.5 in the positive control group. It can be confirmed that the IL2Rβγ / αβγ ratio of the PD-L1 antibody-IL1 variant protein complex is significantly lower than that of the positive control group. That is, the PD-L1 antibody-IL2 variant 2 protein complex and the PD-L1 antibody-IL2 variant 4 protein complex not only do not bind to IL2Rα, but also weaken the binding to IL2Rβγ.

[0191] Therefore, according to one aspect, the PD-L1 antibody-IL2 variant protein complex is used to induce higher activation in CD8+T cells than regulatory T cells, characterized in that the binding ability to IL2Rα is removed and the binding ability to IL2Rβγ is adjusted by replacing the specific amino acids in order to selectively induce the activation of CD8+ cells expressing IL2Rβγ.

[0192] 3-4. Confirmation of the activity of PD-L1 antibody-IL2 variant protein complex on human immune cells

[0193] The effect of the PD-L1 antibody-IL2 variant 1 protein complex to the PD-L1 antibody-IL2 variant 15 protein complex on the activation of human immune cells according to one aspect was confirmed. Specifically, human peripheral blood mononuclear cells (Stem cell society) were reacted with effector CD8+T cells (CD3+, CD8+) and regulatory T cells (CD4+, CD25+, FoxP3+) antibodies that specifically attached fluorescent labels at 4°C for 30 minutes in a dark environment. Then, the antibodies that were not attached to the cells were removed by centrifugation, and human peripheral blood mononuclear cells were treated with the PD-L1 antibody-IL2 variant 1 protein complex to the PD-L1 antibody-IL2 variant 15 protein complex prepared in the embodiment 3-2 according to the concentration (0.001 to 8000nM), and the reaction was performed in a dark environment for 20 minutes at 37°C, and fixed for 12 minutes using 1mL of a fixing buffer (BD, USA). In order to attach the fluorescent marker to the phosphorylated STAT-5 in the cytoplasm, 1.5 mL of Perm buffer (BD, USA) was used for treatment and reacted at 4°C for 40 minutes to allow the fluorescent marker to penetrate. Then, the fluorescent marker was attached to the phosphorylated STAT-5 protein for reaction for 30 minutes, and the cell ratio of STAT-5 phosphorylated was confirmed in each of the effector CD8+T and regulatory T cells by FACS analysis. Aldesleukin (Proleukin, Norvatis, Switzerland) was used as a positive control group, and its results are shown in Table 6 below.

[0194] [Table 6]

[0195]

[0196]

[0197] *NS: Not Saturated at high concentration

[0198] 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 50 The ratios of the values ​​were 62.9 and 22, respectively, and the EC values ​​of CD8+ T cells / regulatory T cells of the PD-L1 antibody-IL2 variant 1 protein complex to the PD-L1 antibody-IL2 variant 15 protein complex prepared in Example 3-2 were 62.9 and 22, respectively. 50The ratio of the values ​​was 0.16 to 2.88. That is, it can be confirmed that regulatory T cells were highly activated in the positive control group and the PD-L1 antibody-IL2 comparison group, and the PD-L1 antibody-IL2 variant 1 protein complex to the PD-L1 antibody-IL2 variant 15 protein complex prepared in Example 3-2 more selectively activated CD8+T cells, i.e., effector T cells, compared with regulatory T cells.

[0199] Therefore, according to one aspect, the PD-L1 antibody-IL2 variant protein complex can participate in the immune response associated with the anti-cancer effect by inducing stronger activity of effector T cells than regulatory T cells.

[0200] 3-5. Confirmation of the anti-cancer activity of the PD-L1 antibody-IL2 variant protein complex (1)

[0201] The anticancer activity of the PD-L1 antibody-IL2 variant protein complex according to one aspect was confirmed. Specifically, MC38 cells (1×10 6 Cells) were subcutaneously administered to C57BL / 6 mice (female, 6 weeks old, Jabio) at a capacity of 0.2mL / mouse to prepare a colorectal cancer transplant mouse model (syngeneic mouse model). On the 5th day after inoculation, the PD-L1 antibody-IL2 variant 2 protein complex prepared in Example 3-2 was intraperitoneally administered at a capacity of 8mg / kg and 16mg / kg, respectively, twice Q2D×. After administration of the complex, the anticancer activity was compared by measuring the tumor volume of the mice (3 times / week). As a positive control group, Avelumab (Merck, Germany) was intraperitoneally administered at a dose of 10mg / kg (Q2D×twice), and Avelumab and Aldesleukin were co-administered (Aldesleukin 0.46 mg / kg, QD×5 times, intraperitoneal injection (ip) + Avelumab 10mg / kg / , day 5, day 7, intraperitoneal injection (ip)).

[0202] Figure 2a The results of comparing the anti-cancer activities of the PD-L1 antibody-IL2 variant 2 protein complex according to one embodiment and a positive control group are shown.

[0203] Figure 2b The results are shown for the cancer cell growth inhibitory effect and complete remission activity of the PD-L1 antibody-IL2 variant 2 protein complex according to one embodiment on colorectal cancer suppressor mice on an individual basis.

[0204] Figure 2cThe results of the cancer cell growth inhibitory effect and complete remission activity of the negative control group and the positive control group (Avelumab and combined administration of Avelumab and Aldesleukin) on colorectal cancer suppressor mice are shown.

[0205] As a result, it can be confirmed that Figure 2a As shown, the tumor volume in the negative control group (vehicle) and the positive control group increased over time after administration. On the contrary, the PD-L1 antibody-IL2 variant 2 protein complex prepared in Example 3-2 showed a strong cancer cell growth inhibitory effect. Specifically, it can be confirmed that most of the tumors disappeared when the PD-L1 antibody-IL2 variant 2 protein complex volume was 16 mg / kg. In addition, unlike the PD-L1 antibody-IL2 control group protein complex, for the PD-L1 antibody-IL2 variant 2 protein complex, there were no individuals with weight loss and death regardless of the administration volume.

[0206] And it can be confirmed that Figure 2b and Figure 2c As shown, the tumor volume in the negative control group and the positive control group increased over time after administration, and the number of mice showing complete remission (CR) was 0 to 2 out of 10 experimental animals in the same administration group, that is, less. On the contrary, for the PD-L1 antibody-IL2 variant 2 protein complex, it can be confirmed that the number of mice showing complete remission was 6 to 9 out of 10 experimental animals in the same administration group, that is, it increased in a volume-dependent manner, and showed significant anti-cancer activity compared with the negative control group and the positive control group.

[0207] That is, it was found that the combined administration of the PD-L1 antibody-IL2 variant protein complex according to one aspect and the existing antibody therapeutic agent Avelumab and / or Avelumab and Aldesleukin exhibited more excellent anti-cancer activity.

[0208] 3-6. Confirmation of the anticancer activity of the PD-L1 antibody-IL2 variant protein complex (2)

[0209] The anticancer activity of the PD-L1 antibody-IL2 variant protein complex according to one aspect was confirmed. The anticancer activity was confirmed by the same method as in Examples 3-5, except that the PD-L1 antibody-IL2 variant 4 protein complex was administered and Avelumab (Merck, Germany) was intraperitoneally administered at a volume of 10 mg / kg (Q2D×2 times) as a positive control group.

[0210] Figure 3 The results of confirming the anti-cancer activity of the PD-L1 antibody-IL2 variant 4 protein complex according to one embodiment are shown.

[0211] As a result, it can be confirmed that Figure 3 As shown, the tumor volume in the negative control group (vehicle) increased over time after administration. On the contrary, the PD-L1 antibody-IL2 variant 4 protein complex prepared in Example 3-2 showed a stronger cancer cell growth inhibitory effect than the negative control group. Specifically, it can be confirmed that the tumor volume change of the PD-L1 antibody-IL2 variant 4 protein complex was significantly less when the volume of 8 mg / kg and 16 mg / kg was administered than when the volume of 10 mg / kg was administered in the positive control group. In addition, the number of mice showing complete remission in the positive control was only 1 in 10 experimental animals in the same administration group. On the contrary, in the PD-L1 antibody-IL2 variant 4 protein complex, the number of mice showing complete remission increased to 4 to 5 in 10 experimental animals in the same administration group, thereby confirming that it showed significant anti-cancer activity compared with the positive control group.

[0212] That is, the PD-L1 antibody-IL2 variant protein complex according to one aspect selectively increases the activity of effector T cells relative to regulatory T cells compared to existing immune anti-cancer antibody therapeutics, and thus can be useful for preventing or treating various immune diseases including cancer.

[0213] Therefore, according to one aspect, the PD-L1 antibody-IL2 variant protein complex not only has excellent anti-cancer activity, but also reduces the side effects caused by IL2, thereby providing a safer therapeutic agent than existing immune anti-cancer therapeutic agents.

[0214] Example 4. Preparation and activity confirmation of mouse PD-1 antibody-IL2 variant protein complex (a+b)

[0215] 4-1. Cloning and culture of mouse PD-1 antibody-IL2 variant protein complex (a+b)

[0216] A protein complex (a+b) including anti-mouse PD-1 Fab-Fc (hereinafter referred to as "a") and IL2 variant-Fc (hereinafter referred to as "b") was obtained by the same method as in Example 3-1 except that the PD-1 antibody and the IL2 variants 1 and 4 prepared in Example 1 were used (refer to Figure 4 a).

[0217] 4-2. Purification of mouse PD-1 antibody-IL2 variant protein complex (a+b)

[0218] The mouse PD-1 antibody-IL2 variant protein complex (a+b) obtained by culturing the cell line prepared in Example 4-1 was purified by the same method as in Example 3-2 to obtain a high-purity substance.

[0219] 4-3. Confirmation of the activity of mouse PD-1 antibody-IL2 variant protein complex (a+b) on human immune cells

[0220] In order to confirm the effect of the mouse PD-1 antibody-IL2 variant protein complex (a+b) confirmed in Example 4-2 on the activation of human immune cells, an experiment was performed by the same method as in Example 3-4.

[0221] [Table 7]

[0222]

[0223] As a result, as shown in Table 7, it can be confirmed that the ratios of CD8+T cells / regulatory T cells in the positive control group and the mouse PD-1 antibody-IL2 variant 4 protein complex (a+b) were 14.5 and 0.92, respectively. That is, it can be confirmed that the regulatory T cells were highly activated in the positive control group, whereas the mouse PD-1 antibody-IL2 variant 4 protein complex (a+b) prepared in Example 4-2 selectively activated CD8+T cells, i.e., effector T cells, over regulatory T cells.

[0224] This shows that the IL2 variant according to one aspect can well maintain its original properties and promote the activity of effector T cells to participate in the immune response even when forming a complex with other immune checkpoint inhibitors other than PD-L1.

[0225] Example 5. Preparation and activity confirmation of mouse PD-1 antibody-IL2 variant protein complex (a+c)

[0226] 5-1. Cloning and culture of mouse PD-1 antibody-IL2 variant protein complex (a+c)

[0227] A protein complex including mouse PD-1 antibody and IL2 variants 1, 2, 3, 4, 7, 8, 14 and 15 was prepared. Specifically, the polynucleotide encoding the heavy chain variable region-constant region of the mouse PD-1 antibody and the polynucleotide encoding the light chain constant region-light chain variable region of the mouse PD-1 antibody were synthesized using the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific. Then, the polynucleotides were inserted into the AvrⅡ-Bstz17I enzyme site and the EcoRV-PacI enzyme site of the pCHO1.0 expression vector to prepare the first expression vector expressing the mouse PD-1 antibody. The same service was used to synthesize a polynucleotide comprising a complex of any one of the IL2 variants 1, 2, 3, 4, 7, 8, 14 and 15 of Example 1 and a linker at the C-terminus of the polynucleotide encoding the mouse PD-1 antibody heavy chain variable region-constant region, and then inserted into the AvrⅡ-BstZ17I enzyme site of the pCHO1.0 expression vector, and the mouse PD-1 antibody light chain constant region-light chain variable region was inserted into the EcoRV-PacI enzyme site to prepare a second expression vector. Then, a protein complex (a+c) comprising anti-mouse PD-1 Fab-Fc (a) and anti-mouse PD-1 Fab-Fc-C-terminal IL2 variant (hereinafter referred to as "c") was obtained by the same method as in Example 2-1 (refer to Figure 4 b).

[0228] 5-2. Purification of mouse PD-1 antibody-IL2 variant protein complex (a+c)

[0229] A high-purity substance (purity 98% or higher) was obtained by purifying the mouse PD-1 antibody-IL2 variant protein complex (a+c) obtained by culturing the cell line prepared in Example 5-1.

[0230] First, after removing fine residues in the same manner as in Example 3-2 except that the mouse PD-1 antibody-IL2 variant protein complex (a+c) was used, purification was performed once. Then, after the culture medium after the filtration process was put into a protein A column stabilized with a phosphate buffer (pH 7.4), the non-specific proteins that were not bound were washed with the same buffer, and washed again with a buffer solution including 0.05M citric acid (pH 5.0). Then, 0.02M citric acid (pH 5.0 and pH 3.5) was used to dissolve the protein that specifically bound to the protein-A column in a pH descending gradient, and the sample was neutralized to pH 7.2 using 1M Tris.

[0231] Then, in order to remove the material-derived impurities remaining in the sample separated by the affinity column, a hydrophobic interaction column (Phenyl HP, Cytiva) was used for secondary purification. Specifically, 1M citric acid was added to the sample dissolved and dissolved in the protein-A column and titrated to pH 7.0 to be put into the Phenyl HP column stabilized with 0.02M sodium phosphate (pH 7.0) buffer and 0.8M thiothreitol (pH 7.0), and the non-specific proteins that were not bound were washed with the same buffer. Then, a buffer solution including 0.02M sodium phosphate (pH 7.0) was used to dissolve in a salt concentration descending gradient to ensure the mouse PD-1 antibody-IL2 variant protein complex (a+c). The mouse PD-1 antibody-IL2 variant protein complex (a+c) was stabilized with phosphate buffer (pH 7.4), and the purity was analyzed using size exclusion HPLC (TSK-3000SWxL, 7.8 mm×30 cm, Tosoh Corporation).

[0232] Figure 5a The figure shows the results of purifying the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment using a protein-A affinity column purification method.

[0233] Figure 5b The figure shows the results of purifying the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment using a hydrophobic interaction column purification method.

[0234] Figure 5c The results of analyzing the purity of the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment are shown.

[0235] Results, such as Figure 5a As shown, the protein that specifically binds to the column by the elution buffer used in the protein-A affinity column purification can be confirmed. Specifically, the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) that specifically binds to the column can be confirmed at 2800 to 2900 ml of the elution solution.

[0236] And, if Figure 5b As shown, it was confirmed that impurities remaining in the eluate used in the protein-A affinity column and proteins non-specifically bound were removed.

[0237] And, if Figure 5c As shown, it was confirmed that the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) ensured by all the above purification processes showed a purity of 98.6% at the stop time of 15.639 minutes.

[0238] That is, it was found that the mouse PD-1 antibody-IL2 variant protein complex (a+c) according to one aspect was purified to a high purity.

[0239] 5-3. Confirmation of receptor binding ability of mouse PD-1 antibody-IL2 variant protein complex (a+c)

[0240] The receptor binding ability of the mouse PD-1 antibody-IL2 variant protein complex (a+c) was confirmed by the same method as in Example 3-3, except that the mouse PD-1 antibody-IL2 variant 1 protein complex (a+c) and the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) prepared in Example 5-2 were each serially diluted two-fold in the concentration range of 1.56 to 8000 nM.

[0241] [Table 8]

[0242]

[0243] As a result, as shown in Figure 8, the mouse PD-1 antibody-IL2 variant 1 protein complex (a + c) and the mouse PD-1 antibody-IL2 variant 14 protein complex (a + c) exhibited binding affinities of 5,610 nM and 6,290 nM for IL2Rβ, respectively, and thus it was confirmed that they bind at similar levels. On the contrary, the binding affinities for IL2Rβγ were 4.51 nM and 35.3 nM, respectively, and thus it was confirmed that the binding force in the mouse PD-1 antibody-IL2 variant 1 protein complex (a + c) was reduced by about 7.8 times compared to the mouse PD-1 antibody-IL2 variant 1 protein complex (a + c).

[0244] This indicates that, while the binding affinity of the mouse PD-1 antibody-IL2 variant 1 protein complex (a+c) and the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) to IL2Rβ is similar, the binding affinity of the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) to IL2Rγ is reduced by approximately 7.8 times compared with that of the mouse PD-1 antibody-IL2 variant 1 protein complex (a+c).

[0245] 5-4. Confirmation of the activity of mouse PD-1 antibody-IL2 variant protein complex (a+c) on human immune cells

[0246] In addition to using the mouse PD-1 antibody-IL2 variant 1 protein complex, mouse PD-1 antibody-IL2 variant 2 protein complex, mouse PD-1 antibody-IL2 variant 3 protein complex, mouse PD-1 antibody-IL2 variant 4 protein complex, mouse PD-1 antibody-IL2 variant 7 protein complex, mouse PD-1 antibody-IL2 variant 8 protein complex, mouse PD-1 antibody-IL2 variant 14 protein complex, mouse PD-1 antibody-IL2 variant 15 protein complex and mouse PD-1 antibody-IL2 variant comparison group prepared in Example 5-2, the effect of the mouse PD-1 antibody IL2 variant protein complex (a+c) on human immune cell activation was confirmed by the same method as in Example 3-4.

[0247] [Table 9]

[0248]

[0249] As a result, it was confirmed that the EC of CD8+ T cells / regulatory T cells in the positive control group (aldesleukin) was significantly higher than that in the positive control group (aldesleukin). 50 The ratio of the values ​​is 21.4, while it is 0.5 to 1.2 in the mouse PD-1 antibody-IL2 variant protein complex (a+c) prepared in Example 5-2. That is, it can be seen that, in contrast to the stronger activation of regulatory T cells in the positive control group, the mouse PD-1 antibody-IL2 variant protein complex (a+c) according to one embodiment selectively activates CD8+T cells of effector T cells compared with regulatory T cells. That is, it can be seen that the IL2 variant also maintains the inherent property of selectively activating CD8+T cells well in the structure connected to the C-terminus in the Fc region.

[0250] In addition, the activities of mouse PD-1 antibody-IL2 variant 1 protein complex (a+c) on T reg and CD8+T cells were 11.91±8.02nM and 9.61±1.44nM, respectively. In contrast, the activities of mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) on Treg and CD8+T cells were 320.7±279.1nM and 161.1±70.3nM, respectively, thus confirming that the activities of mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) on T reg and CD8+T cells were reduced compared with mouse PD-1 antibody-IL2 variant 1 protein complex (a+c). It can be seen that, as confirmed in Example 5-3, this reduction is caused by a reduction in binding force to IL2Rγ (about 7.8 times). Moreover, the EC of effector T cells / regulatory T cells 50 The ratio of the values ​​was 0.5, which showed that effector T cells (CD8+ T cells) were selectively activated compared with regulatory T cells.

[0251] Therefore, the mouse PD-1 antibody-IL2 variant protein complex (a+c) according to one aspect can participate in the immune response associated with the anti-cancer effect by selectively activating effector T cells compared to regulatory T cells.

[0252] 5-5. Confirmation of the anticancer activity of the mouse PD-1 antibody-IL2 variant protein complex (a+c)

[0253] The anticancer activity of the mouse PD-1 antibody-IL2 variant protein complex (a+c) according to one aspect was confirmed. Specifically, MC38 cells (1.0×10 6 The colorectal cancer transplant mouse model (syngeneic mouse model) was prepared by subcutaneously injecting 100 cells / mouse into the right flank of C57BL / 6 mice (female, 6 weeks old, strain Koatech) after a one-week acclimation period. Then, when the tumor size in the mouse reached about 70 to 120 mm 3 At this time, the mice were divided into 3 to 6 groups according to the drug administration group, and the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) prepared in Example 5-2 was intraperitoneally administered once a week at 1, 3, 5 and 10 mg / kg, respectively, twice in total. At this time, vehicle was used as a negative control group, and J43 (clone) 3 mg / kg and J43 3 mg / kg+IL2 comparison group-Fc complex 1.4 mg / kg as anti-PD1 antibodies were used as positive controls. Then, the tumor growth inhibitory effect caused by drug use was observed until the 19th day after the first drug use.

[0254] Figure 6a The figures show the results of comparison of the anticancer activity of the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment, a negative control group, and a positive control group in a colorectal cancer transplanted mouse model.

[0255] Figure 6b The results of comparing the changes in body weight of a colorectal cancer transplanted mouse model with the anti-cancer activity of the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment, a negative control group, and a positive control group are shown.

[0256] Results, such as Figure 6a and 6bAs shown, it can be confirmed that the tumor volume in the negative control group and the positive control group increases over time. On the contrary, the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) prepared in Example 5-2 shows a volume-dependent cancer cell growth inhibitory effect compared to after administration. In particular, compared with the positive control group and the combined administration group, when the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) is administered at 5 mg / kg and 10 mg / kg, it can be confirmed that an excellent cancer cell growth effect is exhibited. That is, it can be confirmed that the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) exhibits significantly excellent anti-cancer activity compared to the case of the combined administration of PD-1 monoclonal antibody and IL2 comparison group-Fc protein complex.

[0257] Therefore, the mouse PD-1 antibody-IL2 variant protein complex (a+c) according to one aspect specifically activates effector T cells compared to existing immune anti-cancer therapeutic agents, and thus can be used to prevent or treat various immune diseases associated with cancer.

[0258] Example 6. Preparation of human PD-1 antibody-IL2 variant protein complex (A+C)

[0259] 6-1. Cloning and culture of human PD-1 antibody-IL2 variant protein complex (A+C)

[0260] Protein complexes including human PD-1 antibody and IL2 comparison group, IL2 variant 1 and IL2 variant 14 were prepared respectively. Specifically, a protein complex (A+C) including anti-human PD-1 Fab-Fc (hereinafter referred to as "A") and anti-human PD-1 Fab-Fc-C-terminal IL2 variant (hereinafter referred to as "C") was obtained by the same method as Example 5-1, except that a polynucleotide encoding a human PD-1 antibody heavy chain variable region-constant region and a polynucleotide encoding a human PD-1 antibody light chain constant region-light chain variable region were used.

[0261] 6-2. Purification of human PD-1 antibody-IL2 variant protein complex (A+C)

[0262] After purifying the human PD-1 antibody-IL2 variant protein complex (A+C) obtained by culturing the cell line prepared in Example 6-1 by the same method as in Example 5-2, the highly pure human PD-1 antibody-IL2 variant protein complex (A+C) was stabilized using a phosphate buffer (pH 7.4). Thereafter, purity analysis was performed by the same method as in Example 5-2.

[0263] 6-3. Confirmation of the binding ability of the human PD-1 antibody-IL2 variant protein complex (A+C)

[0264] The human PD-1 antibody-IL2 comparative protein complex (A+C), human PD-1 antibody-IL2 variant 1 protein complex (A+C), and human PD-1 antibody-IL2 variant 14 protein complex (A+C) prepared in Example 6-2 were each serially diluted 2-fold in the concentration range of 1.56 to 8000 nM for development, and the receptor binding ability of the human PD-1 antibody-IL2 variant protein complex (A+C) was confirmed by the same method as in Example 3-3. At this time, Pembrolizumab was used as a positive control group for human PD-1, and human PD-1 antibody-IL2 variant 1 was used as a control group, and the results are shown in the following Tables 10 and 11.

[0265] [Table 10]

[0266]

[0267] [Table 11]

[0268]

[0269] As a result, as shown in Table 10, the huPD-1 antibody-IL2 comparison group protein complex (A+C), the human PD-1 antibody-IL2 variant 1 protein complex (A+C), the human PD-1 antibody-I2 variant 14 protein complex (A+C) and the positive control group prepared in Example 6-2 showed binding forces of 2.25 to 2.95 nM, which confirmed that they all showed similar levels of binding forces to human PD-1.

[0270] On the other hand, it can be confirmed that the human PD-1 antibody-IL2 variant 1 protein complex (A+C) and the human PD-1 antibody-IL2 variant 14 protein complex (A+C) do not react with IL2Rα as shown in Table 11. On the contrary, it can be confirmed that the human PD-1 antibody-IL2 variant 1 protein complex (A+C) and the human PD-1 antibody-IL2 variant 14 protein complex (A+C) exhibit binding affinities of 5.462nM and 5.611nM for IL2Rβ, respectively, that is, they exhibit similar levels of binding force, and the human PD-1 antibody-IL2 variant 1 protein complex (A+C) and the human PD-1 antibody-IL2 variant 14 protein complex (A+C) bind to IL2Rβγ with binding affinities of 2.92nM and 68.3nM, respectively. In view of the above results, it can be seen that the binding force of the human PD-1 antibody-IL2 variant 14 protein complex (A+C) to IL2Rβ is similar to that of the human PD-1 antibody-IL2 variant 1 protein complex (A+C), and on the contrary, the binding force to IL2Rγ is reduced. Specifically, it can be confirmed that the binding force of the human PD-1 antibody-IL2 variant 14 protein complex (A+C) to IL2Rβγ and IL2Rαβγ is reduced by about 23.4 times and 32.6 times compared with that of the human PD-1 antibody-IL2 variant 1, respectively. That is, it can be seen that the human PD-1 antibody-IL2 variant 1 protein complex (A+C) and the human PD-1 antibody-IL2 variant 14 protein complex (A+C) not only do not bind to ILRα, but also weaken the binding to IL2Rγ.

[0271] Therefore, the human PD-1 antibody-IL2 variant protein complex (A+C) according to the present invention is used to induce a higher degree of activation in CD8+T cells than regulatory T cells, and is characterized in that the binding ability to IL2Rα is removed to induce selective activation of CD8+T expressing IL2Rβγ and the binding ability to IL2Rγ is adjusted by replacing specific amino acids.

[0272] The above contents are intended to provide examples, and those skilled in the art to which the present invention belongs should be able to understand that the present invention can be easily changed into other specific forms without changing the technical ideas or essential features of the present invention. Therefore, the embodiments described above should be understood as exemplary from all angles, rather than restrictive.

Claims

1. A protein comprising an interleukin 2 (IL2) variant, wherein: The IL2 variant includes glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125.

2. The protein according to claim 1, wherein The IL2 variant further comprises 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) at one or more positions selected from the group consisting of position 18 and position 19.

3. The protein according to claim 2, wherein The IL2 variants include Methionine (M) or arginine (R) at position 18; and Serine (S) at position 19.

4. The protein according to claim 1, wherein The IL2 variant further comprises one or more amino acids at position 126 selected from the group consisting of threonine (T) and isoleucine (I).

5. The protein according to claim 1, wherein The IL2 variant comprises amino acids selected from the group consisting of SEQ ID NOs: 1-15. The protein of claim 1 , comprising an Fc region bound by a linker or a carrier.

7. A protein complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH3 antibody constant region, the second polypeptide comprises a second CH3 antibody constant region, the first polypeptide and the second polypeptide form a heterodimer, wherein: The N-terminus of any one or more of the first polypeptide or the second polypeptide comprises an antibody or an antigen-binding fragment thereof to an immune checkpoint inhibitor, or an antibody or an antigen-binding fragment thereof to a tumor-associated antigen, and, Either or both of the N-terminus or the C-terminus of the first polypeptide or the second polypeptide comprises an IL2 variant.

8. The protein complex according to claim 7, wherein The first CH3 antibody constant region includes a tryptophan (W) at position 366, the second CH3 antibody constant region includes a serine (S) at position 366, an alanine (A) at position 368, and a valine (V) at position 407; and The second CH3 antibody constant region includes glycine (G) or phenylalanine (F) at position 351 (wherein the amino acid position is according to the Kabat EU index).

9. The protein complex according to claim 7, wherein The first CH3 antibody constant region includes serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407, and the second CH3 antibody constant region includes tryptophan (W) at position 366; and Glycine (G) or phenylalanine (F) at position 351 in the first CH3 antibody constant region (wherein the amino acid position is based on the Kabat EU index).

10. The protein complex according to claim 7, wherein The first CH3 antibody constant region includes a tryptophan (W) at position 366, and the second CH3 antibody constant region includes a glycine (G) at position 351, a serine (S) at position 366, an alanine (A) at position 368, and a valine (V) at position 407; and Phenylalanine (F) or tryptophan (W) at position 351 in the first CH3 antibody constant region (wherein the amino acid position is based on the Kabat EU index).

11. The protein complex according to claim 7, wherein The first CH3 antibody constant region includes 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 constant region includes tryptophan (W) at position 366; and The second CH3 antibody constant region includes phenylalanine (F) or tryptophan (W) at position 351 (wherein the amino acid position is based on the Kabat EU index).

12. The protein complex according to claim 7, wherein The first CH3 antibody constant region includes 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 constant region includes tryptophan (W) at position 366; and The second CH3 antibody constant region includes phenylalanine (F) or tryptophan (W) at position 351 (wherein the amino acid position is based on the Kabat EU index).

13. The protein complex according to claim 7, wherein The immune checkpoint inhibitor is any one or more selected from the group consisting of programmed cell death-ligand 1 (PD-L1), programmed death receptor 1 (PD-1), lymphocyte activation gene 3 (LAG-3), V domain Ig inhibitory factor (VISTA), B lymphocyte weakening factor (BTLA), T cell mina-globulin mucin 3 (TIM-3), T cell immunoglobulin and ITIM domain protein (TIGIT) and cytotoxic T lymphocyte associated protein 4 (CTLA-4).

14. The protein complex according to claim 7, wherein The tumor-associated antigen is any one or more selected from the group consisting of programmed cell death-ligand 1 (PD-L1), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor-2 (HER-2), B7H3, phosphatidylinositol proteoglycan 3 (GPC3), carcinoembryonic antigen (CEA), human trophoblast surface glycoprotein antigen (TROP) and prostate-specific membrane antigen (PSMA).

15. The protein complex according to claim 7, wherein The second polypeptide; and the IL2 variant are bound by one or more linkers or carriers.

16. The protein complex according to claim 15, wherein The linker includes the amino acid sequence of SEQ ID NO:

18.

17. A polynucleotide encoding the protein or protein complex according to any one of claims 1 to 16.

18. A method for preparing a protein or a protein complex, comprising the step of transfecting an expression vector encoding the protein or protein complex according to any one of claims 1 to 17 into a cell.

19. A protein complex comprising an IL2 variant, wherein: The protein complex is prepared by the method according to claim 18.

20. A pharmaceutical composition for preventing or treating cancer, comprising the protein or protein complex according to any one of claims 1 to 17 as an effective ingredient.

21. A method for preventing or treating cancer, comprising the step of administering the protein or protein complex according to any one of claims 1 to 17 to an individual in need of the protein or protein complex.

22. Use of the protein or protein complex according to any one of claims 1 to 17 in the preparation of a cancer therapeutic agent.

Images