Cytokine fusion protein

Abstract

The present invention relates to a cytokine fusion protein comprising an immunocyte targeting domain and a cytokine action domain capable of specifically inducing the proliferation and / or activation of immunocytes. Specifically, the present invention relates to a fusion protein that specifically delivers a cytokine (e.g., IL-2 or IL-15) to exhausted immunocytes (e.g., exhausted T cells) in a tumor to induce and enhance the activation of immunocytes. The fusion protein of the present invention can selectively deliver a cytokine that promotes the proliferation or activation of immunocytes to effector T cells in the tumor microenvironment, etc., and can induce strong immune enhancement and an anti-cancer reaction in the tumor, and can significantly reduce the systemic toxicity or side effects caused by the cytokine through the unique structure of the cytokine action domain.

Description

Technical Field

[0001] The present invention relates to a cytokine fusion protein comprising an immunocyte targeting domain and a cytokine action domain capable of specifically inducing the proliferation and / or activation of immunocytes. Specifically, the present invention relates to a fusion protein that specifically delivers a cytokine (e.g., IL-2 or IL-15) to exhausted immunocytes (e.g., exhausted T cells) in a tumor to induce and enhance the activation of immunocytes. The present invention also relates to a polynucleotide encoding the fusion protein, a vector containing the same, and a host cell. The present invention also relates to a method for producing the fusion protein, and a pharmaceutical composition, method, and such uses for treating a disease, e.g., cancer, or enhancing an immune response in a mammal using the fusion protein.

Background Art

[0002] Cytokines are cell signaling molecules involved in the regulation of the immune system. For example, IL-2 is an essential cytokine involved in the proliferation and activation of T cells. IL-2 stimulates the proliferation and differentiation of T cells, induces the generation of cytotoxic T lymphocytes (CTLs), differentiates peripheral lymphocytes into cytotoxic cells and lymphokine-activated killer cells (LAKs), and stimulates the proliferation and activation of natural killer cells (NK cells).

[0003] Due to the actions of IL-2 as described above, research has been conducted with the expectation that IL-2 immunotherapy is effective in the treatment of metastatic cancer. As a result, recombinant human IL-2 (Proleukin) was approved by the US FDA. (R), Aldesleukin) was approved for the indications of metastatic renal cell carcinoma and metastatic melanoma. However, Proleukin causes serious side effects such as capillary leak syndrome (CLS), and the number of patients who can be administered and the amount that can be administered are limited. In addition, the half-life is short, less than 2 hours, and there is the inconvenience that a cycle of intravenous administration three times a day for 5 consecutive days must be repeated.

[0004] There are three types of IL-2 receptors: high-affinity, intermediate-affinity, and low-affinity receptors. The high-affinity receptor consists of three subunits: IL-2 receptor alpha (IL-2Rα; CD25), beta (IL-2Rβ; CD122), and gamma (IL-2Rγ; CD132). The intermediate-affinity receptor consists of IL-2Rβ and IL-2Rγ, and the low-affinity receptor consists of only IL-2Rα. The intermediate-affinity receptor consisting of the β and γ subunits has an affinity for IL-2 that is approximately 100 times lower than that of the high-affinity receptor consisting of the α, β, and γ subunits, but can transmit a signal when binding to IL-2. The α-subunit confers high-affinity binding ability to the receptor but is not essential for signal transmission.

[0005] Only the intermediate-affinity IL-2 receptor (IL-2Rβγ) is expressed in resting immune cells. When resting T cells are activated by an antigen, IL-2Rα is rapidly expressed, and if IL-2 binds to IL-2Rα, IL-2Rβ and IL-2Rγ will be involved. Binding of this IL-2Rαβγ complex to IL-2 causes signal transduction, promoting the growth of effector T cells (T eff ) including cytotoxic T cells that can kill virus-infected cells and tumor cells.

[0006] In addition, IL-2 mediates activation-induced cell death (AICD) of T cells. AICD is the process by which fully activated T cells experience programmed cell death, thereby inducing immune tolerance not only to normal self-antigens but also to antigens that persist, such as tumor antigens.

[0007] IL-2 is also involved in the maintenance of peripheral CD4 + CD25 + regulatory T cells (T reg regs). Regulatory T cells constitutively express IL-2Rα. Regulatory T cells suppress the function of cytotoxic T cells to attack self-antigens and tumor cells. Due to such diverse actions of IL-2, IL-2 is not suitable for showing an optimal tumor-suppressive effect.

[0008] Various attempts have been made to enhance the tumor-suppressive effect while reducing the toxicity of IL-2. As an example, a strategy to inhibit the binding of IL-2 to IL-2Rα has been attempted. This is because IL-2 acts on the intermediate-affinity IL-2Rβγ of T reg cells rather than on the high-affinity IL-2Rαβγ receptor of T eff cells. Examples thereof include combinations of anti-IL-2 monoclonal antibodies and IL-2 (Kamimura et al., J Immunol 177, 306-14 (2006); Boyman et al., Science 311, 1924-27 (2006)), IL-2 mutants with mutations introduced into the binding site to IL-2Rα (such as WO2012 / 107417), IL-2 with polyethylene glycol (PEG) groups introduced into the IL-2Rα binding site (WO2012 / 065086), and the like.

[0009] However, the IL-2 mutants developed to date still do not have a good therapeutic index as an anticancer agent, and even when administered at a high dose showing toxicity, the anticancer effect is not clear. The reason is considered to be that although the IL-2 mutants were made to act selectively on T reg regs rather than on T eff cells, they mainly show such an action peripherally, and their action in the tumor microenvironment (TME) is insufficient.

[0010] To overcome such drawbacks, research has been conducted to enable IL-2 variants to act in tumor tissues. A strategy has been attempted to bring IL-2 to tumor tissues by binding a tumor associated antigen (TAA)-specific antibody and IL-2. However, such a strategy can induce IL-2 to the tumor site, but it is difficult to effectively provide IL-2 to T eff cells. A method for selectively providing IL-2 to T cells among various tumor infiltrated lymphocytes (TIL) is needed. eff

[0011] Not only IL-2, but also other cytokines (e.g., IL-15) can play a role as important mediators in inducing immune activation or immunosuppression in the tumor microenvironment. Such cytokines are substances secreted by immune cells and mostly act locally on various targets to change the microenvironment. However, these cytokines also have the characteristics of acting locally and briefly and disappearing, making it difficult to develop them as therapeutic agents.

Summary of the Invention

Problems to be Solved by the Invention

[0012] The present inventors aim to develop an effective cytokine therapeutic agent that selectively activates exhausted immune cells in the tumor microenvironment, enhances anti-cancer activity, and reduces systemic side effects caused by exogenous cytokines.

Means for Solving the Problems

[0013] The present invention relates to a fusion protein comprising an immune cell targeting domain and a cytokine action domain capable of specifically inducing the proliferation and / or activation of immune cells. The present inventors have found that cytokines that promote the proliferation or activation of immune cells do not affect other immune cells or endothelial cells present in the periphery, but T in the TME environment eff ​To selectively deliver to cells, a cytokine - acting domain is linked to an antibody domain targeting immune cells, and a fusion protein that acts on T cells in the tumor in an "in - cis" manner is devised. eff A fusion protein that acts on T cells in the tumor in an "in - cis" manner is devised.

[0014] The present invention provides a fusion protein comprising one or more immune cell antigen - binding domains, an Fc domain consisting of two Fc chains, and a cytokine - acting domain. In one aspect of the present invention, the cytokine - acting domain is a cytokine that promotes the proliferation or activation of immune cells or a domain that binds thereto. The cytokine can be an exogenous cytokine injected from the outside or can utilize an endogenous cytokine present inside. Such cytokines can include IL - 2 polypeptide and IL - 15 polypeptide that can bind to the β - subunit of the IL - 2 receptor (IL - 2Rβ) and the common γ - chain receptor (also referred to as IL - 2Rγ) to induce signal transduction activation via IL - 2Rβγ.

[0015] In one aspect, the present invention provides a fusion protein comprising one or more immune cell antigen - binding domains, an Fc domain consisting of two Fc chains, and a cytokine - acting domain, wherein the cytokine - acting domain comprises an α - subunit (IL - 2Rα) polypeptide of the interleukin - 2 receptor and an IL - 2 polypeptide, and the IL - 2Rα polypeptide and the IL - 2 polypeptide are not covalently bonded to each other and are linked to different Fc chains of the Fc domain, respectively.

[0016] Since IL - 2 is pre - occupied by IL - 2Rα in the fusion protein of the present invention having such a structure, regulatory T cells (T reg) The binding affinity to Foxp3-negative CD4 T cells, some innate lymphoid cells, and other endothelial cells is weakened. On the other hand, cytotoxic CD8+ T cells, memory T cells, NK-T cells, etc. that express an intermediate-affinity IL-2 receptor (abbreviated as IL-2Rβγ) consisting of IL-2Rβ and IL-2Rγ without IL-2Rα can specifically bind, increasing the anti-cancer effect and significantly reducing systemic toxicity or side effects. eff Cells can specifically bind, increasing the anti-cancer effect and significantly reducing systemic toxicity or side effects.

[0017] In one aspect, the present invention provides a fusion protein comprising one or more immunocyte antigen-binding domains, an Fc domain consisting of two Fc chains, and a cytokine action domain, wherein the cytokine action domain is a complex in which an IL-2Rα polypeptide and an IL-2 polypeptide are covalently bonded to each other, and the complex is bound to an Fc chain on either side of the Fc domain via the IL-2Rα polypeptide or the IL-2 polypeptide.

[0018] In one aspect, the present invention provides a fusion protein comprising one or more immunocyte antigen-binding domains, an Fc domain consisting of two Fc chains, and a cytokine action domain, wherein the cytokine action domain comprises an α-subunit (IL-15Rα) polypeptide of the interleukin-15 receptor and an IL-15 polypeptide, and the IL-15Rα polypeptide and the IL-15 polypeptide are linked to different Fc chains of the Fc domain, respectively.

[0019] In one aspect, the present invention provides a fusion protein comprising one or more immunocyte antigen-binding domains, an Fc domain consisting of two Fc chains, and a cytokine action domain, wherein the cytokine action domain is a complex in which an IL-15Rα polypeptide and an IL-15 polypeptide are covalently bonded to each other, and the complex is bound to an Fc chain on either side of the Fc domain via the IL-15Rα polypeptide or the IL-15 polypeptide.

[0020] In one aspect, the present invention provides a fusion protein comprising one or more immunocyte antigen-binding domains, an Fc domain consisting of two Fc chains, and a cytokine-acting domain, wherein the cytokine-acting domain does not include IL-2 polypeptide and IL-15 polypeptide, and binds to endogenous IL-2 or IL-15 by including only IL-2Rα polypeptide or IL-15Rα polypeptide, and can induce these to T eff cells.

[0021] In one aspect of the present invention, the IL-2Rα polypeptide includes a wild-type mature IL-2Rα polypeptide having the amino acid sequence of SEQ ID NO: 3 in the sequence listing attached hereto, or an IL-2 binding fragment thereof, an extracellular domain or a Sushi domain, or a variant thereof.

[0022] In one aspect of the present invention, the IL-15Rα polypeptide includes a wild-type mature IL-15Rα polypeptide having the amino acid sequence of SEQ ID NO: 7 in the sequence listing attached hereto, or an IL-15 binding fragment thereof, an extracellular domain or a Sushi domain, or a variant thereof.

[0023] In one aspect of the present invention, the IL-2Rα and IL-15Rα polypeptides may include one or more amino acid modifications, desirably amino acid substitutions, that enhance the binding affinity for their respective binding ligands, IL-2 polypeptide and IL-15 polypeptide. In an exemplary aspect, the fusion protein of the present invention includes an IL-2Rα variant that includes one or more amino acid substitutions among L2D, L2E, L2Q, M25L, M25I, N27A, N27T, N27I, N27Y, S39K, L42F, L42I, L42A, L42V, L45R, N57E, I118R, H120A, H120D, H120K, H120Y, H120L, H120W, H120R, and K153G based on the amino acid sequence number of SEQ ID NO: 3. In a desirable aspect, the fusion protein of the present invention includes an IL-2Rα variant that includes an L42I amino acid substitution based on the amino acid sequence number of SEQ ID NO: 3.

[0024] In one aspect of the present invention, the IL-2 polypeptide includes a wild-type mature IL-2 polypeptide having the amino acid sequence of SEQ ID NO: 1 in the sequence listing attached hereto, or its C125S mutant (SEQ ID NO: 2), or a circular permutated mutant. In one aspect of the present invention, the IL-15 polypeptide includes a wild-type mature IL-15 polypeptide of SEQ ID NO: 6 in the sequence listing attached hereto, or a circular permutated mutant thereof. In one aspect of the present invention, the IL-2 polypeptide or IL-15 polypeptide may include one or more amino acid modifications that enhance the binding affinity for the α-subunit of their respective binding receptors. In a preferred aspect, the fusion protein of the present invention includes an IL-2 mutant containing an amino acid substitution of T37K based on the amino acid sequence of SEQ ID NO: 1.

[0025] In one aspect of the present invention, the immune cell antigen-binding domain includes a Fab molecule of an antibody that specifically binds to one or more T cell target antigens, or an antigen-binding fragment thereof. In one aspect of the present invention, the immune cell antigen-binding domain is a Fab molecule of an antibody that specifically binds to an immune checkpoint protein (e.g., PD-1 (programmed cell death-1) protein) expressed in T eff cells within the tumor microenvironment, or an antigen-binding fragment thereof. In one aspect of the present invention, the immune cell antigen-binding domain is a T effIt is also a component, antibody or antigen-binding fragment thereof that can bind to the surface marker of CD8+ effector T cells that confer binding specificity to cells. In a desirable aspect, the immune cell antigen-binding domain of the present invention is a Fab molecule of an anti-PD-1 antibody or an antigen-binding fragment thereof. In an exemplary aspect, the immune cell antigen-binding domain of the present invention is a Fab molecule of an anti-PD-1 antibody or an antigen-binding fragment thereof, or binds to an epitope identical to these antibodies or antigen-binding fragments, or is a Fab molecule of an antibody or an antigen-binding fragment thereof that competes with these antibodies or antigen-binding fragments. Desirably, the fusion protein of the present invention comprises complementarity-determining regions (CDRs) 1, 2, and 3 of the heavy-chain variable region containing the amino acid sequences of SEQ ID NOs: 38, 40, 42, or 44 in the sequence listing attached hereto, and CDRs 1, 2, and 3 of the light-chain variable region containing the amino acid sequences of SEQ ID NOs: 39, 41, 43, or 45, and an immune cell antigen-binding domain comprising a Fab molecule of an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0026] In one aspect of the present invention, the immune cell antigen-binding domain is also a multispecific binding domain comprising two or more different T cell target antigen-binding domains. In one aspect of the present invention, the fusion protein of the present invention may further comprise, as an additional antigen-binding domain, a Fab molecule of an antibody or an antigen-binding fragment thereof that specifically binds to one or more tumor cell target antigens.

[0027] In one aspect of the present invention, the Fc domain is a homodimer in which the first chain and the second chain are equal to each other or a heterodimer in which they are different from each other. In one aspect of the present invention, the Fc domain is a human IgG class Fc domain. In one aspect of the present invention, the Fc domain is an IgG1, IgG2, IgG3 or IgG4 subclass Fc domain. In one aspect of the present invention, when the Fc domain is an IgG4 Fc domain, it preferably contains an amino acid substitution of S228P (based on the EU numbering system of the Kabat literature). In one aspect of the present invention, the Fc domain may lack the CH2 domain and be composed of only the CH3 domain. Further, the Fc domain of the present invention may include one or more amino acid modifications that reduce the binding to Fc receptors or effector functions. In an exemplary aspect, such modifications of the Fc domain include amino acid modifications at one or more positions selected from E233, L234, L235, G236, G237, N297, L328, P329 and P331 (based on the EU numbering system of the Kabat literature), but are not limited thereto.

[0028] In one aspect of the present invention, the Fc domain may include a modification for promoting heterodimer formation between two Fc chains. In an exemplary aspect, such modifications of the Fc domain may include a knob-into-hole modification, a knob-into-hole disulfide modification, and the like. In a preferred aspect, the fusion protein of the present invention, together with the knob-into-hole modification or the knob-into-hole disulfide modification, further includes an amino acid substitution of K360E in one of the Fc chains and an amino acid substitution of Q347R in the other Fc chain, or one of the Fc chains further includes amino acid substitutions of K360E and Q347E, and the other Fc chain further includes an amino acid substitution of Q347R or amino acid substitutions of K360R and Q347R, and includes an Fc domain variant.

[0029] In one aspect of the present invention, the immune cell antigen-binding domain and the cytokine-acting domain can be linked directly or via a linker to the Fc domain. In one aspect of the present invention, the immune cell antigen-binding domain can be linked to the N-terminus of the Fc domain and the cytokine-acting domain can be linked to the C-terminus of the Fc domain, or vice versa. In one aspect of the present invention, the linker is preferably a peptide linker, but is not limited thereto.

[0030] Still other aspects of the present invention provide a polynucleotide encoding the fusion protein or each region thereof, a vector containing the polynucleotide, or a transformed cell into which the vector has been introduced. Still other aspects of the present invention provide a host cell containing the vector, and a method for culturing the host cell to produce the fusion protein of the present invention.

[0031] Still other aspects of the present invention provide a pharmaceutical composition for preventing, ameliorating or treating a tumor, cancer, metastatic tumor, metastatic cancer or infectious disease, which contains the fusion protein as an active ingredient. Still other aspects of the present invention provide a method for preventing, ameliorating or treating a tumor, cancer, metastatic tumor, metastatic cancer or infectious disease by administering the fusion protein to an individual in need of prevention, amelioration or treatment of a tumor, cancer, metastatic tumor, metastatic cancer or infectious disease.

Advantages of the Invention

[0032] According to the present invention, it is possible to provide an effective anticancer cytokine therapeutic agent that selectively activates cytotoxic effector T cells in the tumor microenvironment to enhance anticancer activity while reducing systemic side effects.

Brief Description of the Drawings

[0033]

Figure 1A

Figure 1B

Figure 1C

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 4

Figure 5

Figure 6A

Figure 6B

Figure 7A

Figure 7B

Figure 8A

Figure 8B

Figure 8C

Figure 8D

Figure 9

Figure 10A

Figure 10B

Figure 10C

Figure 11A

Figure 11B

Figure 11C

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16A

Figure 16B

Figure 17A

Figure 17B

Figure 18A

Figure 18B

Figure 18C

Figure 19A

Figure 19B

Figure 20

Embodiments for Carrying Out the Invention

[0034] In the present invention, "protein" or "polypeptide" is interpreted as a concept including at least two amino acids attached by covalent bonds and including oligopeptides. In the present invention, "protein A" or "polypeptide A" can be interpreted as a concept including all or part of the parent protein A or polypeptide A, their analogs and mutants. Also, "mutation", "modification" and "mutant" mean including substitution, insertion and / or deletion of amino acid residues, and preferably, the mutants of the present invention may include substitution of amino acid residues. Substitution of amino acid residues can be expressed in the order of amino acid residues, amino acid residue numbers, and substituted amino acid residues present in the parent wild-type protein.

[0035] In the present invention, "fusion protein" can be interpreted as a meaning including a form in which two or more proteins are bound to each other or a complex containing the same.

[0036] In the present invention, the terms "programmed cell death-1", "PD-1", and "PD-1 protein" are used interchangeably and include variants, isotypes, species homologs of human PD-1, and analogs having at least one common epitope with PD-1.

[0037] In the present invention, the term "antibody" includes whole antibodies and any "antigen-binding portion" or single chain thereof. "Antibody" refers to a protein comprising at least two heavy chains and two light chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region and a light chain constant region. The light chain constant region is composed of one domain, CL. The heavy chain variable region (VH) and the light chain variable region (VL) are further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which are arranged between regions called more conserved framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, and are arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen.

[0038] In the present invention, "antibody" includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. The term "antigen-binding portion" includes all antibody fragments that retain the antigen-binding function, i.e., the ability to specifically bind to an antigen, including any antibody fragment. Typically, such antigen-binding portions include antigen-binding fragments.

[0039] The term "antigen-binding domain" of the present invention generally refers to any domain capable of binding to a target antigen, including peptides, proteins, antibodies, aptamers, chemical residues, etc. When the fusion protein of the present invention contains an antibody as an antigen-binding domain, not only the whole antibody but also its antigen-binding portion, Fab molecule or antigen-binding fragment may be included as the antigen-binding domain. The term "Fab molecule" refers to a site having one constant region and one variable region each in a heavy chain and a light chain, which may partially contain a hinge region or may be partially deformed. The term "Fab molecule" used in the present invention is defined to include any possible antigen-binding site of an antibody, including Fab, Fab', F(ab')2, Fab'-SH, etc.

[0040] In the present invention, the "antigen-binding portion", "antigen recognition site", "antigen-binding domain", "antigen-binding fragment", and "binding fragment" of an antibody refer to a part of an antibody molecule containing amino acids that cause specific binding between the antibody and the antigen. For example, when the antigen is large, the antigen-binding fragment may bind only to a part of the antigen. The part that causes specific interaction between the antigen molecule and the antigen-binding fragment is referred to as an "epitope" or "antigenic determinant".Examples of "antigen-binding portion", "antigen recognition site", "antigen-binding domain", "antigen-binding fragment", and "binding fragment" that can be used in the present invention include Fab, Fab', F(ab')2, Fab'-SH, Fd, Fv, scFv, (scFv)2, scFv-Fc, (scFv)2-Fc, dsFv, (dsFv)2, dsFv-dsFv', VL, VH, diabody, ds-diabody, triabody, tetrabody, minibody ((scFV-CH3)2), nanobody, domain antibody, single domain antibody (sdAb), bivalent domain antibody, IgG delta CH2, Fynomer, FynomAbs (fynomers fused to antibodies), dual-affinity re-targeting (DART), AlbudAbs, BiTEs (bispecific T-cell engager), TandAbs (tandem diabodies), DAFs (dual acting Fab), two-in-one antibodies, SMIPs (small modular immunopharmaceuticals), anticalins, FN3 monobody, DARPins, Affibodies, Affilins, Affimers, Affitins, Alphabodies, Avimers, Im7, VLR, VNAR, Trimab, CrossMab, TRIDENT, binanobodies, di-sdFv, DVD-Igs (dual variable domain immunoglobulin), CovX-bodies (peptide modified antibodies), duo-body and triomAbs, etc., but are not limited thereto.

[0041] The antigen-binding fragment may include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), but does not necessarily have to include both. For example, the so-called Fd antibody fragment is composed of only the VH domain, but still retains some of the antigen-binding function of the complete antibody.

[0042] Fusion protein The fusion protein of the present invention includes one or more immune cell antigen-binding domains, an Fc domain consisting of two Fc chains, and a cytokine action domain. In the present invention, one or more immune cell antigen-binding domains are components of a fusion protein that bind to surface markers on desired target immune cells such as CD8+ effector T cells having an antitumor effect and transmit the cytokine region to the target cells. In the present invention, the immune cell antigen-binding domain is preferably an anti-PD-1 antibody or an antigen-binding fragment thereof. The fusion protein of the present invention can minimize the influence on peripheral blood NK cells and T cells by selectively transmitting cytokines to tumor infiltrated lymphocytes expressing the target due to the presence of the immune cell antigen-binding domain. Further, the immune cell antigen-binding domain is also a component, antibody or an antigen-binding fragment thereof that selectively binds to, for example, NKG2a, CD8a, FcRL6, CRTAM, LAG3, TIM3, CTLA4, TIGIT, etc.

[0043] The fusion protein of the present invention can have one or two Fab arms as shown in FIGS. 1A and 1B. Alternatively, as shown in FIG. 1C, the two Fab arms relate to different targets. For example, when the immune cell antigen-binding domain is one anti-PD-1 antibody or an antigen-binding fragment thereof, it may further include one or more Fab arms related to a target selected from the group consisting of NKG2a, CD8a, FcRL6, CRTAM, LAG3, TIM3, CTLA4, and TIGIT. Alternatively, the fusion protein of the present invention may further include a Fab arm targeting tumor cells as an antigen-binding domain.

[0044] In one embodiment of the present invention, the cytokine action domain is designed to preferentially activate cytotoxic T cells compared to T cells. In one embodiment of the present invention, the cytokine action domain is a fatigued T reg cells eff cells to preferentially activate cytotoxic T effIt includes a signal transduction activating moiety through IL-2Rβγ that is permanently expressed in cells, such as an IL-2 polypeptide or an IL-15 polypeptide. In one embodiment of the present invention, the cytokine action domain also includes IL-2Rα together with IL-2 in order to inhibit the binding of the high-affinity IL-2 receptor (IL-2Rαβγ) expressed in immune cells and endothelial cells of peripheral blood by IL-2. IL-2 in the form pre-occupied by IL-2Rα cannot bind to the high-affinity IL-2 receptor. In one embodiment of the present invention, the cytokine action domain includes an IL-2 protein and an IL-2Rα protein, and IL-2 and IL-2Rα are respectively linked to two Fc chains as shown in FIGS. 1A to 1C and are not covalently bonded to each other. IL-2 and IL-2Rα are linked to the Fc chain via a linker or directly. In one embodiment, the linker is also a peptide linker, for example, (GGGX)n, (XGGG)nXGG, (GGGGX)n, (GX)n (X = A or S, n = 1, 2, 3, or 4), SGGGSGGGSGGGSGG, GGGSGGGSGGGSGG, etc., but is not limited thereto. In one embodiment, the linker is also a peptide linker consisting of about 5 to 20 amino acids, but is not limited thereto, and any linker with any structure and length can be used as long as it preferably maintains the pre-occupied structure of the cytokine action domain and retains the function of transmitting the cytokine action domain to target cells. In one embodiment, the peptide linker can consist of one or more amino acids selected from the group consisting of G, S, A, P, E, T, D, and K.

[0045] In one embodiment of the present invention, the cytokine action domain may include IL-15 and IL-15Rα. FIG. 2B is an example when the cytokine action domain includes an IL-15 and IL-15Rα complex. In one embodiment of the present invention, furthermore, the cytokine action domain may include a covalently bonded IL-2 / IL-2Rα complex (FIG. 2A), or a covalently bonded IL-15 / IL-15Rα complex (FIG. 2B).

[0046] In one embodiment of the present invention, the cytokine action domain does not include IL-2 and IL-15 and may include only the IL-2Rα or IL-15Rα protein (FIGS. 3A to 3D). In this embodiment, the IL-2Rα protein binds to endogenous IL-2 and transports IL-2 to cells in which the target is expressed (e.g., PD-1-expressing T cells), but compared to T cells in which IL-2Rα is constitutively expressed, T cells expressing IL-2Rβ and γ are preferentially proliferated and activated. In this embodiment, IL-2Rα or IL-15Rα may be bound to both of the two Fc chains to form a homodimer or may be bound to only one Fc chain to form a heterodimer. reg Compared to T cells expressing IL-2Rβ and γ, T cells expressing IL-2Rβ and γ are preferentially proliferated and activated. eff In this embodiment, IL-2Rα or IL-15Rα may be bound to both of the two Fc chains to form a homodimer or may be bound to only one Fc chain to form a heterodimer.

[0047] In one embodiment of the present invention, the cytokine action domain is desirably not capable of sufficient cytokine signaling by itself, but in the form of a fusion protein bound to an immunocyte antigen-binding domain, it is selectively targeted to desired cells (cells expressing the target) to activate the cells. On the other hand, cells that express the cytokine receptor but do not express the target are not activated or have a reduced degree of activation. For example, in one embodiment of the present invention, the cytokine action domain containing IL-2 and IL-2Rα has a weaker binding affinity for the IL-2Rβγ receptor and substantially no binding affinity for the IL-2Rα receptor in T cells that do not express PD-1 compared to wild-type IL-2. However, in the form of a fusion protein bound to an anti-PD-1 antibody, after being targeted to cytotoxic CD8+ T cells in which PD-1 and the IL-2Rβγ receptor are simultaneously expressed, it stimulates cell proliferation and activation through sufficient signal transduction and exhibits excellent anti-cancer activity. However, for cells that do not express PD-1, the activity is weak or almost non-existent, and side effects due to cytokine activity in peripheral blood, such as capillary leak syndrome and systemic toxicity by over-activated NK cells, can be reduced.

[0048] IL-2 protein In the present invention, unless otherwise specified, the terms "interleukin-2", "IL-2", "IL-2 protein", or "IL-2 polypeptide" include IL-2 derived from vertebrates including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This includes any form of IL-2 that has been processed by cells as well as unprocessed IL-2. This term also includes naturally occurring variants or "IL-2 mutants" as defined below. Full-length human IL-2 means the mature, native-length human IL-2, which is a molecule having 133 amino acids of SEQ ID NO: 1. Unprocessed human IL-2 further includes 20 N-terminal amino acids not present in the mature IL-2 molecule.

[0049] The term "IL-2 mutant" includes mutant forms of various IL-2 molecules including full-length IL-2, truncated IL-2, or forms in which IL-2 is linked to other molecules. IL-2 mutants can include one or more amino acid modifications that affect the interaction with the IL-2Rα polypeptide or increase the binding to the IL-2Rα polypeptide. Such mutations can include substitution, deletion, truncation, or modification of wild-type amino acid residues. An example of an amino acid modification that increases the binding to the IL-2Rα polypeptide is the amino acid substitution of T37K based on the amino acid sequence of SEQ ID NO: 1.

[0050] In the present invention, the IL-2 protein is also full-length human IL-2 (SEQ ID NO: 1). It can also include, for example, a mutation in which the 125th cysteine of IL-2 of SEQ ID NO: 1 is substituted with serine (C125S, SEQ ID NO: 2) to prevent the formation of incorrect disulfide bonds. It is also in a circular permutated form in which the N-terminal and C-terminal positions of IL-2 are replaced to shorten the length of the linker.

[0051] In the present invention, the "IL-2 polypeptide" includes IL-2 proteins having an amino acid sequence with about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more sequence identity with the human IL-2 sequence of SEQ ID NO: 1.

[0052] IL-2Rα protein In the present invention, unless otherwise specified, the terms "IL-2Rα protein", "IL-2Rα polypeptide", "IL-2Rα", "CD25", or "interleukin-2 receptor α-subunit" mean the α-subunit of the IL-2 receptor derived from vertebrates including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term includes not only the unprocessed IL-2Rα but also any form of IL-2Rα processed intracellularly. This term also includes naturally occurring variants or "mutants of IL-2Rα" as defined below. The human IL-2Rα polypeptide used as a reference in the present invention has the amino acid sequence of positions 22 to 272 of UniProt P01589 consisting of 272 amino acids (SEQ ID NO: 3). In the present invention, the "IL-2Rα polypeptide" refers to an IL-2Rα polypeptide having the amino acid sequence of SEQ ID NO: 3 or its IL-2 binding fragment, the extracellular domain corresponding to positions 22 to 240 of UniProt P01589 (SEQ ID NO: 4) or a part thereof, or the Sushi domain. The term "IL-2 binding fragment of the IL-2Rα polypeptide" in the present invention means any length of fragment truncated from the extracellular domain of the IL-2Rα polypeptide in the C-terminal to N-terminal direction, and having at least 90% or more of the binding activity to IL-2, its binding ligand, compared to the original full binding activity.

[0053] In the present invention, the "IL-2Rα mutant" means an IL-2Rα protein that contains mutations at one or more positions that affect the interaction with IL-2 among various forms of IL-2Rα molecules, such as the extracellular domain of IL-2Rα (SEQ ID NO: 4), truncated IL-2Rα, or a form in which IL-2Rα is linked to other molecules. This mutation may include substitution, deletion, cleavage, or modification of wild-type amino acid residues.

[0054] In the present invention, the "IL-2Rα mutant" may contain mutations in the amino acid sequence of IL-Rα in order to enhance the affinity between IL-Rα and IL-2 and stabilize the complex. In the present invention, the positions of the mutations in IL-2Rα are based on the sequence of SEQ ID NO: 4. For example, the IL-2Rα mutant of the present invention may contain amino acid substitutions at one or more positions among L2, M25, N27, S39, L42, L45, N57, I118, H120, and K153 in the extracellular domain of IL-2Rα of SEQ ID NO: 4 or its truncated form. In the present invention, the IL-2Rα mutant may contain one or more amino acid substitutions among L2D, L2E, L2Q, M25L, M25I, N27A, N27T, N27I, N27Y, S39K, L42F, L42I, L42A, L42V, L45R, N57E, I118R, H120A, H120D, H120K, H120Y, H120L, H120W, H120R, and K153G. Further, the IL-2Rα mutant of the present invention may contain one or more mutations among F121A, V122A, and A114D.

[0055] An exemplary IL-2Rα mutant in the present invention is also the mutant of SEQ ID NO: 5 having an L42I amino acid substitution in the truncated form of the extracellular domain of IL-2Rα, but is not limited thereto.

[0056] IL-15 / IL-15Rα IL-15 is secreted by immune cells including monocytes after viral infection. IL-15 induces the proliferation of NK cells and other cells of the immune system and is involved in the death of virus-infected cells and cancer cells. IL-15 binds to the intermediate affinity IL-2Rβγ receptor, but binds to the IL-15Rα receptor with even higher affinity. IL-15Rα binds to IL-2βγ to form a functional high-affinity receptor αβγ.

[0057] In the present invention, "interleukin-15", "IL-15", "IL-15 protein", or "IL-15 polypeptide" includes IL-15 derived from vertebrates including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. In the present invention, the IL-15 polypeptide is also a human IL-15 polypeptide (SEQ ID NO: 6) having the amino acid sequence of positions 49 to 162 of Uniprot P40933 or a variant thereof that retains at least part of the activity of IL-15. Further, the IL-15 polypeptide includes one or more amino acid modifications that increase the binding to the IL-15Rα polypeptide or is circular permutated.

[0058] In the present invention, unless otherwise specified, the "IL-15Rα protein", "IL-15Rα polypeptide", "IL-15Rα", "CD215", or "interleukin-15 receptor α" means the α-subunit of the IL-15 receptor derived from vertebrates including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In the present invention, the IL-15Rα protein is also a cleaved form or mutant that retains at least part of the activity of human IL-15Rα or IL-15Rα. The reference human IL-15Rα polypeptide in the present invention has the amino acid sequence of positions 31 to 267 of UniProt Q13261 (SEQ ID NO: 7). In the present invention, the IL-15Rα polypeptide is also an IL-15Rα polypeptide having the amino acid sequence of SEQ ID NO: 7 or an IL-15 binding fragment thereof, an extracellular domain (SEQ ID NO: 8) or a Sushi domain, and may include one or more amino acid modifications that increase the binding to the binding ligand IL-15 polypeptide. The term "IL-15 binding fragment of the IL-15Rα polypeptide" in the present invention means any length of fragment truncated from the extracellular domain of the IL-15Rα polypeptide in the C-terminal to N-terminal direction, and the binding activity to its binding ligand IL-15 is at least 90% or more retained compared to the original complete binding activity.

[0059] An anti-PD-1 antibody or an antigen-binding fragment thereof In the present invention, the anti-PD-1 antibody or an antigen-binding fragment thereof means an antibody or an antigen-binding fragment thereof that can bind to PD-1, particularly the PD-1 polypeptide expressed on the cell surface. In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof binds to PD-1 with a KD value of 10 -7 M or less; in some embodiments, the KD value is 10 -8 , 10 -9 , 10 -10 or 10 -11 M or less. In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof has a KD value of 10 even in a low pH environment -9Less than M, desirably, KD value 10 -10 Less than M, more desirably, KD value 10 -11 Binds to PD-1 at less than M. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention binds to PD-1 with a KD of 9×10 -10 Less than M.

[0060] In the present invention, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region. Desirably, the heavy chain variable region comprises a heavy chain complementarity-determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 9, an HCDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 10-15, and an HCDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 16-31, or comprises an HCDR variant having conservative amino acid substitutions or 3 or fewer amino acid mutations as compared with these sequences; the light chain variable region comprises a light chain complementarity-determining region 1 (LCDR1) comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 32-35, an LCDR2 having the amino acid sequence of SEQ ID NO: 36, and an LCDR3 having the amino acid sequence of SEQ ID NO: 37, or comprises an LCDR variant having conservative amino acid substitutions or 3 or fewer amino acid mutations as compared with these sequences.

[0061] In the present invention, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region. Desirably, the heavy chain variable region comprises CDR1, CDR2 and CDR3 of a heavy chain variable region having the amino acid sequence of SEQ ID NO: 38, 40, 42 or 44, or the light chain variable region comprises CDR1, CDR2 and CDR3 of a light chain variable region having the amino acid sequence of SEQ ID NO: 39, 41, 43 or 45.

[0062] Methods for determining CDR sequences within a given VH or VL region are known to those skilled in the art and include the Kabat numbering system, the Chothia numbering system, the Martin numbering system, the Gelfand numbering system, and the IMGT numbering system, etc. (see Dondelinger et al., Front. Immunol. (2018) 9:2278, etc.). Antibodies or functional fragments thereof according to a preferred embodiment of the present invention may include, but are not limited to, CDR sequences according to the Kabat numbering system. Antibodies or functional fragments thereof containing CDR sequences that can be defined by known CDR determination methods for the variable region sequences of SEQ ID NOs: 38 to 45 are all included within the scope of the present invention.

[0063] In the present invention, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region. Desirably, the heavy chain variable region comprises a sequence as shown in SEQ ID NO: 38, 40, 42 or 44, or a mutant having 1 to 10 or fewer amino acid mutations compared to these sequences; the light chain variable region comprises a sequence as shown in SEQ ID NO: 39, 41, 43 or 45, or a mutant having 1 to 10 or fewer amino acid mutations compared to these sequences.

[0064] In the present invention, the heavy chain variable region of the anti-PD-1 antibody or antigen-binding fragment thereof may have an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 38, 40, 42 or 44.

[0065] In the present invention, the light chain variable region of the anti-PD-1 antibody or antigen-binding fragment thereof may have an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 39, 41, 43 or 45.

[0066] "Conservative amino acid substitution" refers to the substitution of an amino acid residue by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent identity or similarity can be adjusted upward so as to be corrected for the conservative nature of the substitution. Means for such adjustment are well known to those skilled in the art. See, for example, the literature incorporated herein by reference (see: Pearson (1994) Methods Mol. Biol. 24:307-331). Examples of amino acid groups having side chains with similar chemical properties are: 1) basic side chains: lysine, arginine, and histidine; 2) acidic side chains: aspartic acid and glutamic acid; 3) uncharged polar side chains: asparagine, glutamine, serine, threonine, tyrosine, and cysteine; 4) nonpolar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine; 5) sulfur-containing side chains: cysteine and methionine; 6) aliphatic side chains: glycine, alanine, valine, leucine, isoleucine, and proline; 7) aliphatic hydroxyl side chains: serine and threonine; 8) amide-containing side chains: asparagine and glutamine; and 9) aromatic side chains: phenylalanine, tyrosine, tryptophan, and histidine. Desirable conservative amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, conservative substitutions are any change having a positive value in the PAM250 log-likelihood matrix disclosed in the literature incorporated herein by reference (see: Gonnet et al. (1992) Science 256:1443-45). "Moderately conservative" substitutions are any change having a non-negative value in the PAM250 log-likelihood matrix. In the present invention, the scope of an antibody or an antigen-binding fragment thereof limited to a specific sequence is defined to further include sequences having conservative substitutions of the said sequence.

[0067] In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is also a recombinant antibody, preferably a murine antibody, a chimeric antibody, a humanized antibody or a human antibody.

[0068] In some embodiments, the heavy chain constant region of the chimeric or humanized anti-PD-1 antibody is derived from human IgG1, IgG2, IgG3 or IgG4 or mutant sequences thereof, and the light chain constant region can be derived from human κ chain, λ chain or mutant sequences thereof. For example, the heavy chain constant region is a human IgG4 heavy chain constant region (SEQ ID NO: 46) containing the S228P mutation and / or the light chain constant region is also a human immunoglobulin kappa constant region chain (SEQ ID NO: 47).

[0069] In some embodiments of the anti-PD-1 antibody or antigen-binding fragment thereof, the antibody is a chimeric antibody and the constant region of the antibody is derived from the constant region of a human antibody or a mutant thereof.

[0070] In some embodiments of the anti-PD-1 antibody or antigen-binding fragment thereof, the antibody is a humanized antibody, and the light chain framework region (FR) and heavy chain framework region of the antibody are each derived from a human germline light chain and heavy chain, or mutant sequences thereof.

[0071] In some embodiments of the anti-PD-1 antibody or antigen-binding fragment thereof, the antibody is a human antibody, and the entire sequence is derived from human germline light chain and heavy chain, or mutant sequences thereof.

[0072] In the present invention, the anti-PD-1 antibody or antigen-binding fragment thereof can be derived from a known anti-PD-1 antibody that binds to human PD-1 (SEQ ID NO: 48). For example, pembrolizumab (Keytruda (R) ), nivolumab (Opdivo (R)) tislelizumab (BeiGene and Novartis), cemiplimab (Libtayo (R) , Regeneron), dostarlimab (Jemperli (R) , GSK), BCD-100, camrelizumab, genolimzumab, MED10680 (AMP-514), sasanlimab (PF-06801591), sintilimab (IBI-308), spartalizumab (PDR-001), or STI-A1110, or an antibody or antigen-binding fragment thereof that comprises its heavy and light chains or complementarity-determining regions (CDRs), or an antibody or antigen-binding fragment thereof that binds to the same epitope as those or can compete with those.

[0073] Other antigen-binding domains Other immunocyte antigen-binding domains other than anti-PD-1 antibodies are also, for example, components, antibodies or antigen-binding fragments thereof that selectively bind to NKG2a, CD8a, FcRL6, CRTAM (CD355), LAG3, TIM3, CTLA4, TIGIT, etc.

[0074] Examples thereof include BMS-986315 (anti-NKG2a, WO2020 / 102501), monalizumab (anti-NKG2a); mAbs OKT8 or 51.1 (anti-CD8a antibodies, U.S. Patent No. 10,428,155, WO2020 / 060924), anti-CD8a antibodies disclosed in WO2019 / 023148 or U.S. Patent No. 10,072,080; anti-FcRL6 antibodies 1D8 or 7B7 disclosed in [Shreeder et al. (2010) J. Immunol. 185:23] and [Shreeder et al. (2008) Eur. J. Immunol. 38:3159] or WO2019 / 094743; anti-CRTAM antibody 5A11 disclosed in WO2019 / 086878 or WO2009 / 029883; relatlimab (BMS-986016, anti-LAG-3), anti-LAG-3 antibodies 25F7, 26H10, 25E3, 8B7, 11F2, or 17E5 disclosed in US2011 / 0150892 and WO2014 / 008218, or anti-LAG-3 antibody IMP731 disclosed in US2011 / 007023; ipilimumab or tremelimumab (anti-CTLA-4); TSR-022, MBG453, or LY3321367 (anti-TIM3); vibostolimab or tiragolumab (anti-TIGIT), etc., but are not limited thereto.

[0075] In addition to the immune cell antigen-binding domain, the fusion protein of the present invention can be composed of a bispecific effector protein further including an antigen-binding domain targeting tumor cells.

[0076] Fc region In the present invention, the term "Fc domain" or "Fc region" means the C-terminal region including the CH2 and CH3 domains (or the CH2, CH3, and CH4 domains) of the heavy-chain constant region of an antibody, and is used to mean including the wild-type Fc region and its variants. The boundary of the Fc region of the IgG heavy chain can be changed little by little. Generally, the human IgG heavy-chain Fc region can mean the region from Cys226 or Pro230 to the C-terminus of the heavy chain, or the region further including the hinge in the said region. The number of amino acid residues in the Fc region is based on the EU numbering system, also called the EU index, which defines the residue numbers in the human immunoglobulin heavy chain (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication, 1991).

[0077] In the present invention, the term "wild-type Fc region" includes an amino acid sequence that is identical to the amino acid sequence of the Fc region of a naturally-occurring immunoglobulin.

[0078] In the present invention, the term "variant of the Fc region" includes one or more amino acid residues that are different from the wild-type Fc region, and can also be abbreviated as "Fc variant".

[0079] In the present invention, the said Fc variant can have a homology of about 80% or more, desirably about 90% or more, with the wild-type Fc region sequence serving as the parent.

[0080] The fusion protein of the present invention includes the Fc region of an immunoglobulin consisting of a double strand of a first Fc chain and a second Fc chain. For example, the Fc domain of IgG is a dimer consisting of a double strand, and each chain includes the CH2 and CH3 IgG heavy-chain constant regions.

[0081] In the present invention, the Fc region is also a homodimer or a heterodimer. In a structure in which different protein molecules (e.g., IL-2 and IL-2Rα) are linked to the first and second chains of the Fc region, in order to obtain a desired fusion protein molecule in a high yield, no bond is formed between the Fc chains linked to the same protein molecule, and it is important to design such that a bond is formed between the Fc chains linked to different protein molecules. Thus, it is desirable to use an Fc heterodimer in which a modification is introduced into the Fc region so that the repulsive force between the same chains and the binding force between different chains can be improved.

[0082] In the present invention, the Fc heterodimer consists of a first Fc chain and a second Fc chain that are different from each other. The first Fc chain and the second Fc chain form a tertiary structure, and they can interact with each other by covalent bonds such as disulfide bonds and non-covalent interactions such as hydrogen bonds, ionic bonds, van der Waals bonds, and hydrophobic interactions.

[0083] In the Fc heterodimer of the present invention, in order to promote the binding between different double strands, a known technique of introducing mutations into the CH3 region can be used. For example, there is a technique using the knob-into-hole (KiH) method. In the KiH method, in the hydrophobic interaction region of the CH3 domain, on one strand, an amino acid with a large side chain is substituted with an amino acid with a small side chain, and on the other strand, a mutation of substituting a small amino acid with a large amino acid is utilized. For example, as described in WO96 / 27011 or the literature [Ridgway, J.B., et al., Protein Eng 9(1996)617-621;Merchant, A.M., et al., Nat Biotechnol 16(1998)677-681], a heteromeric polypeptide in which T366 (EU numbering standard, the same hereinafter) of one strand is substituted with a large residue such as Y or W, and Y407, T394, T366, etc. of the other strand are substituted with small residues such as T, A, S, etc. can be used. In one embodiment of the present invention, the CH3 domain of Fc can be further modified by introducing cysteine (C) at the corresponding position of each CH3 so that a disulfide bridge is formed between the two CH3 domains (KiHs-s).

[0084] For example, the CH3 domain of the knob chain may include a T366W mutation, and the CH3 domain of the hole chain may include T366S, L368A, and Y407V mutations (EU numbering standard). In addition, by introducing an E356C mutation or an S354C mutation into the CH3 domain of the knob chain and a Y349C mutation into the CH3 domain of the hole chain, a disulfide bridge can be further formed between the CH3 domains (Merchant, A.M., et al., Nature Biotech 16(1998)677-681). For example, the Fc heterodimer used in the present invention can use KiHs-s.

[0085] In the present invention, either one of the knob chain and the hole chain further includes an amino acid substitution of K360E, and the other chain further includes an amino acid substitution of Q347R, or either one of the chains further includes amino acid substitutions of K360E and Q347E, and the other chain may further include an amino acid substitution of Q347R or amino acid substitutions of K360R and Q347R.

[0086] A desirable Fc heterodimer used in the present invention includes a first Fc chain containing an amino acid substitution at T366 and a second Fc chain containing an amino acid substitution at one or more of T366, L368, and Y407. One of the first and second Fc chains further includes an amino acid substitution at K360, and the other chain further includes an amino acid substitution at Q347. Further, the chain containing the amino acid substitution at K360 among the first Fc chain and the second Fc chain may further include an amino acid substitution at Q347 (or includes it), and the other chain containing the amino acid substitution at Q347 may further include an amino acid substitution at K360.

[0087] Desirably, the Fc heterodimer in the present invention is (A1) the first Fc chain contains amino acid substitutions of T366W and K360E, and the second Fc chain contains amino acid substitutions of T366S, L368A, Y407V, and Q347R, or (A2) the first Fc chain contains amino acid substitutions of T366W and Q347R, and the second Fc chain contains amino acid substitutions of T366S, L368A, Y407V, and K360E, or (A3) the first Fc chain contains amino acid substitutions of T366W, K360R, and Q347R, and the second Fc chain contains amino acid substitutions of T366S, L368A, Y407V, K360E, and Q347E, or (A4) the first Fc chain contains amino acid substitutions of T366W, K360E, and Q347E, and the second Fc chain may contain amino acid substitutions of T366S, L368A, Y407V, K360R, and Q347R.

[0088] In the present invention, the Fc region may also be derived from human IgG1, IgG2, IgG3 or IgG4 or their mutant sequences, and may further contain other mutations for structural stabilization of the Fc region or for modifying the binding affinity for Fc receptors. For example, when the Fc region is derived from IgG4, it may further contain the S228P mutation. The S228P mutation reduces Fab arm exchange in which exchange occurs in the Fab region between two IgG4 antibodies. For example, when the Fc region is derived from IgG, it may contain an amino acid substitution at a position selected from E233, L234, L235, G236, G237, N297, L328, P329 and P331 (EU numbering standard) due to reduced effector function. For example, the Fc region may contain an amino acid substitution at a position selected from the group consisting of L234, L235 and P329. Alternatively, the Fc region may contain the amino acid substitutions of L234A and L235A, or may contain the amino acid substitutions of L234A, L235A and P329G. In the present invention, the Fc region may also lack the CH2 domain.

[0089] The amino acid sequences of the exemplary Fc chains used in the present invention are as shown in SEQ ID NOs: 49 and 50.

[0090] Method for producing a fusion protein The fusion protein of the present invention can be obtained by expressing an expression vector containing a polynucleotide sequence encoding the same in a host cell.

[0091] In the present invention, the term "polynucleotide" refers to an isolated nucleic acid molecule or structure composed of deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, such as mRNA, plasmid DNA, or virus-derived RNA. The polynucleotide may be constituted by or modified from the wild-type sequences of each domain forming the fusion protein by the fusion protein of the present invention, and may further contain elements (such as signal peptides) necessary for the expression of the fusion protein of the present invention. Also, a polynucleotide sequence encoding the entire amino acid sequence of the fusion protein of the present invention can be synthesized by PCR polymerization reaction and used.

[0092] In one embodiment of the present invention, when the fusion protein of the present invention adopts a heterodimeric structure, the expression vector may include a first expression vector containing a polynucleotide sequence encoding the amino acid sequence of the first monomer chain linked with IL-2 and a second expression vector containing a polynucleotide sequence encoding the amino acid sequence of the second monomer chain linked with IL-2Rα. In an exemplary embodiment of the present invention, the first expression vector may include a polynucleotide sequence encoding the amino acid sequences of SEQ ID NOs: 51 and 57, and the second expression vector may include a polynucleotide sequence encoding the amino acid sequences of SEQ ID NOs: 51 and 58.

[0093] In one embodiment of the present invention, the fusion protein of the present invention can be produced by mixing the proteins obtained after separately expressing the first and second expression vectors in host cells, or by co-expressing the first and second expression vectors in the same host cell to produce in a single cell. Desirably, the fusion protein of the present invention is produced in a single cell.

[0094] Therapeutic Administration and Dosage Forms In one aspect, the present invention provides a pharmaceutical composition comprising the fusion protein according to the present invention and a pharmaceutically acceptable excipient and carrier. The pharmaceutical composition can be used as an immunotherapeutic agent, for example, in the treatment of tumors, cancers, metastatic tumors or metastatic cancers. Also, the pharmaceutical composition can be used for the treatment of infectious diseases.

[0095] In one aspect, the present invention provides a pharmaceutical composition further comprising a second therapeutic agent in the pharmaceutical composition. In one embodiment, the second therapeutic agent is an immunomodulatory agent, a cell growth inhibitor, a cell adhesion inhibitor, a cytotoxic agent, a cell killing activator, or an agent that increases the sensitivity of cells to a cell killing inducer. In certain embodiments, the additional therapeutic agent is also an anti-cancer agent, such as a microtubule disrupting agent, an antimetabolite, a topoisomerase inhibitor, a DNA intercalating agent, an alkylating agent, hormone therapy, a kinase inhibitor, a receptor antagonist, a tumor cell killing activator, or an angiogenesis inhibitor.

[0096] In the present invention, a tumor, cancer, metastatic tumor or metastatic cancer is non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, kidney cancer, liver cancer, bone cancer, skin cancer, colon cancer, rectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, multiple myeloma, mycosis fungoides, Merkel cell carcinoma, and classical Hodgkin lymphoma (CHL), primary mediastinal large B cell lymphoma, T cell / histiocyte-rich large B cell lymphoma, Epstein-Barr virus (EBV)-positive and -negative post-transplant lymphoproliferative disorders (PTLD), or EBV-associated diffuse large B cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T cell lymphoma, nasopharyngeal carcinoma, or human herpesvirus 8 (HHV8)-associated primary effusion lymphoma, Hodgkin lymphoma, other hematological cancers, primary central nervous system (CNS) lymphoma, spinal tumors, central nervous system neoplasms including brainstem gliomas, and is not limited thereto.

[0097] In the present invention, the infectious disease is also selected from, but not limited to, late-onset viral infections including hepatitis B, hepatitis C virus infection, herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type 1, herpes simplex virus type 2, human papillomavirus, adenovirus, Kaposi's sarcoma associated with herpes virus infection, parvovirus (torque teno virus), JC virus, or BK virus infection.

[0098] The pharmaceutical composition according to the present invention can be used for the treatment of early or late stage cancer symptoms. In one aspect, the pharmaceutical composition according to the present invention can be used for the treatment of metastatic cancer. The pharmaceutical composition according to the present invention is useful for reducing, suppressing or shrinking tumor growth in both solid tumors and blood cancers. In certain aspects, treatment with the pharmaceutical composition according to the present invention results in a reduction of more than 50%, more than 60%, more than 70%, more than 80%, more than 90% of the tumor in the subject. In certain aspects, the pharmaceutical composition can be used to prevent tumor recurrence. In certain aspects, the pharmaceutical composition is useful for extending overall survival in subjects with cancer. In some aspects, the pharmaceutical composition is useful for reducing the toxicity of chemotherapy or radiotherapy while maintaining long-term survival in patients suffering from cancer.

[0099] In yet another aspect of the present invention, the pharmaceutical composition according to the present invention can be used as adjuvant therapy together with any other formulation or any other therapy known to those skilled in the art useful for the treatment of cancer.

[0100] The pharmaceutical composition according to the present invention can be administered together with a suitable carrier, excipient, and other formulations included within the dosage form to provide improved delivery, transmission, tolerance, etc. A number of suitable dosage forms can be identified in a formulary known to all pharmaceutical chemists (see: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA). These dosage forms are, for example, powders, pastes, ointments, jellies, waxes, oils, liquids, vesicles containing liquids (cationic or anionic) (e.g., LIPOFECTIN TM) DNA conjugates, anhydrous absorption pastes, water-in-oil and oil-in-water emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax (see Powell et al., “Compendium of excipients for parenteral formulations,” PDA (1998) J Pharm Sci Technol 52:238-311).

[0101] The dosage of the fusion protein is also variable depending on the age and weight of the subject to be administered, the target disease, the pathological condition, the route of administration, and the like. The fusion protein of the present invention is preferably administered to a patient once or over a series of treatments. The initial candidate dosage of the fusion protein can be about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) in either the case of one or more separate administrations or continuous infusion, depending on the type and severity of the disease. A typical daily dosage can also range from about 1 μg / kg to 100 mg / kg or more depending on the above factors. For administration repeated over several days, it can generally continue depending on the patient's condition until the desired degree of suppression of disease symptoms occurs. Exemplary dosages can also be from about 0.005 mg / kg to about 10 mg / kg. In still other non-limiting examples, the dosage can also include, per dosage, about 1 μg / kg, about 5 μg / kg, about 10 μg / kg, about 50 μg / kg, about 100 μg / kg, about 200 μg / kg, about 350 μg / kg, about 500 μg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 50 mg / kg, about 100 mg / kg, about 200 mg / kg, about 350 mg / kg, about 500 mg / kg to about 1000 mg / kg or more, and any range inferable therefrom above. Thus, one or more dosages of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to the patient. Such dosages can be administered intermittently, for example, weekly or every three weeks (e.g., such that the patient is administered the fusion protein about 2 to 20 times, or, for example, about 6 times the dosage). After an initial higher loading dose, one or more further lower doses can also be administered.

[0102] The pharmaceutical composition of the present invention can be administered via various delivery systems, such as encapsulation within liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). The introduction methods include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, transmucosal and oral routes. The composition can be administered by absorption through an epithelial or mucosal lining (e.g., oral mucosa, rectal and intestinal mucosa, etc.) via any conventional route, such as by infusion or bolus injection, and can be administered together with other biologically active agents. Administration can be systemic or local.

[0103] In addition, the pharmaceutical composition of the present invention can be delivered to vesicles, particularly liposomes (see, e.g., Langer (1990) Science 249:1527-1533). The use of nanoparticles for delivering the fusion protein of the present invention can also be considered. Antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and their manufacturing methods and uses are included by reference in the literature incorporated herein by reference (see: Arruebo, M., et al. 2009, “Antibody-conjugated nanoparticles for biomedical applications” in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi:10.1155 / 2009 / 439389). Nanoparticles can be developed and conjugated to antibodies contained in a pharmaceutical composition to target tumor cells or self-immune tissue cells or virus-infected cells. Nanoparticles for drug delivery are also described, for example, in US8257740 or US8246995, the full texts of which are each incorporated herein by reference.

[0104] In certain circumstances, the pharmaceutical composition of the present invention can be delivered to a controlled release system. In one aspect, a pump can be used. In still other aspects, polymeric substances can be used. In still other aspects, the controlled release system can be placed near the target of the composition, requiring only a portion of the systemic dose. Injectable formulations can include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal, and intramuscular injection, drip infusion, etc. These injectable formulations can be manufactured by officially known methods. Injectable formulations can be manufactured, for example, by dissolving, suspending, or emulsifying the antibody or its salt in a sterile aqueous medium or an oily medium commonly used for injection. As the aqueous injection medium, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, which can be used together with appropriate solubilizers such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 of hydrogenated castor oil (additive of polyoxyethylene (50 mol))], etc. As the oily medium, for example, sesame oil, soybean oil, etc. can be used, which can be used together with solubilizers such as benzyl benzoate, benzyl alcohol, etc. Thus, the manufactured injection solution is preferably filled into appropriate ampoules.

[0105] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously by a standard needle and syringe. Also, for subcutaneous delivery, a pen delivery device is easily used to deliver the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replacement cartridge containing the pharmaceutical composition. When all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replacement cartridge. Instead, the disposable pen delivery device is pre-charged with the pharmaceutical composition housed in a reservoir within the device. When the pharmaceutical composition in the reservoir is depleted, the entire device is discarded. A number of reusable pens and autoinjector delivery devices are used for subcutaneous delivery of the pharmaceutical composition of the present invention.

[0106] Advantageously, the pharmaceutical composition for oral or parenteral use is manufactured in a unit dosage form suitable for dosing the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injection solutions (ampoules), suppositories, and the like. The amount of the fusion protein contained is generally a unit dosage; particularly about 5 to about 500 mg per unit dosage form in the injectable form, and desirably about 5 to about 100 mg for antibodies and about 10 to about 250 mg for other dosage forms.

[0107] The present invention also provides a method for preventing, ameliorating, or treating a tumor, cancer, metastatic tumor, metastatic cancer, or infectious disease in an individual in need thereof, comprising administering the fusion protein of the present invention to the individual.

[0108] Examples Hereinafter, the present invention will be described in more detail through examples. These examples are merely illustrative for further specifying the present invention, and the scope of rights of the present invention is not limited by these examples. It is self-evident to those skilled in the art that various modifications to the examples are possible within the scope of rights of the present invention.

[0109] The molecular biology reagents used in this application were used according to the instructions of the manufacturer. Recombinant DNA technology was carried out using standard methods as described in the literature [Sambrook et al., Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1991]. The numbers of amino acid residues specified for immunoglobulin molecules or antibody molecules were indicated according to the EU index by [Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication, 1991] at the amino acid positions from the N-terminus.

[0110] Example 1. Production of cytokine fusion protein Example 1.1 Structural design of fusion protein For the specific activity increase of tumor-specific exhausted effector T cells (exhausted T eff ), it is important to selectively deliver cytokines that specifically induce the proliferation and / or activation of T eff cells to the T eff cells within the tumor. For this purpose, a cytokine that promotes the proliferation and / or activation of T eff cells and a T eff cell targeting domain were linked to each other, and the structure of the cytokine fusion protein was designed so as to act "in-cis" on the T eff cells. As cytokines that can induce the proliferation and / or activation of T eff cells, T effInterleukin-2 (hereinafter abbreviated as IL-2 or IL2) and interleukin-15 (hereinafter abbreviated as IL-15), which transmit signals via the βγ-subunit (IL-2Rβγ) of the interleukin-2 receptor expressed in cells, were considered, and in one embodiment, IL-2 was selected. This was configured as a bifunctional fusion protein by linking it to the Fc of an anti-PD-1 immune anti-cancer antibody as a cell targeting domain. eff It was composed of a bifunctional fusion protein by linking it to the Fc of an anti-PD-1 immune anti-cancer antibody as a cell targeting domain.

[0111] Cytokines such as IL-2, which are immune proteins present in the blood, are known to play an important role in intracellular signal transduction in various cells including immune cells. Therefore, a strategy is needed to increase selectivity so that it acts specifically only on T cells in the tumor while reducing the problems of toxicity or side effects caused by exogenous cytokines. eff A strategy is needed to increase selectivity so that it acts specifically only on T cells in the tumor while reducing the problems of toxicity or side effects caused by exogenous cytokines.

[0112] The inventor linked IL-2 to the C-terminus of the first Fc chain of the anti-PD-1 antibody, while linking the α-subunit of the interleukin-2 receptor (IL-2Rα or CD25) to the C-terminus of the second Fc chain, and induced an interaction between IL-2 and IL-2Rα linked to the ends of the first and second Fc chains of the anti-PD-1 antibody, respectively, thereby forming a form in which IL-2 is preoccupied by IL-2Rα. The IL-2 and IL-2Rα proteins are each linked only to the Fc terminus of the anti-PD-1 antibody, and IL-2 and IL-2Rα are not linked to each other by a covalent bond (see Figure 1).

[0113] Exemplary structures of the "Insis" fusion protein of the present invention are illustrated in FIGS. 1 to 3. As shown in FIG. 2, after covalently linking IL-2 and IL-2Rα via a linker to form a complex, a structure in which the cytokine complex is linked to the Fc of an anti-PD-1 antibody through either IL-2 or IL-2Rα is also possible. Further differently, a structure of a fusion protein in which only IL-2Rα or IL-15Rα is linked to an anti-PD-1 antibody with a cytokine action domain was also devised so as to utilize an endogenous cytokine in a solution for solving the problem of toxicity or side effects caused by an exogenous cytokine.

[0114] In the structure designed by the present inventors, since IL-2 is preoccupied by IL-2Rα, the α-subunit, β-subunit (IL-2Rβ or CD122), and γ-subunit (IL-2Rγ or CD132) of the IL-2 receptor are all expressed, and regulatory T cells (T reg ) that express a high-affinity IL-2 receptor (or abbreviated as IL-2Rαβγ), the binding affinity to Foxp3-negative CD4 T cells, some innate lymphoid cells, and other endothelial cells is weakened, while cytotoxic CD8+ T cells, memory T cells, NK-T cells, etc. that express an intermediate-affinity IL-2 receptor (or abbreviated as IL-2Rβγ) consisting of IL-2Rβ and IL-2Rγ without IL-2Rα eff Cells can bind more specifically.

[0115] Example 1.2 Preparation of the Sequence Composition of the Fusion Protein and the Expression Vector Anti-PD-1 Binding Domain As the anti-PD-1 binding domain, the Fab domain of the humanized anti-PD-1 antibody 1G1-h70 developed by the present inventors in previous research was used. The amino acid sequence of the Fab domain of anti-PD-1 1G1-h70 used in this study is as follows.

[0116] Light chain VL-CL (SEQ ID NO: 51) DIVMTQTPLSLSVTPGQPASISCRSSQNIVHSQGDTYLEWYLQKPGQSPQLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (The underlined part indicates the light chain variable region.) Heavy chain VH-CH1 (SEQ ID NO: 52) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGMIHPNSDTTTYNEKFKNRVTMTRDTSISTAYMELSRLRSDDTAVYYCAGTDQAAWFAFWGQGTTVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV (The underlined part indicates the heavy chain variable region.)

[0117] Linker design While maintaining the interaction between IL-2 / IL-2Rα in the form linked to the ends of each chain of the dimeric Fc domain of the anti-PD-1 immune anti-cancer antibody, and for the purpose of structural analysis for the binding of the said IL-2 to IL-2Rβγ expressed on the cell surface, a determination structure analysis was carried out to determine the appropriate lengths at the linking sites between the C-terminus of the Fc domain and the N-termini of IL-2 and IL-2Rα respectively.

[0118] Based on the overall determination structure of Keytruda, a known anti-PD-1 antibody, superimposed on the Fab domain determination structure of human PD-1 (hPD-1) / 1G1-h70, a strategy for the way the anti-PD-1 antibody binds to hPD-1 was planned. After positioning the C-terminus of the Fc domain of the anti-PD-1 antibody so that it does not collide with the IL-2 quaternary complex and is closest to the N-termini of IL-2 and IL-2Rα respectively, the distances from the C-terminus of the Fc domain to the N-termini of IL-2 and IL-2Rα were measured. As a result, the lengths of the linking sites between the C-terminus of the Fc domain and the N-termini of IL-2 and IL-2Rα were determined to be 59.2 Å and 53.6 Å respectively (Figure 4). Through such structural-based modeling, linkers of appropriate lengths for linking IL-2 and IL-2Rα to the Fc termini of the anti-PD-1 antibody were designed.

[0119] Improved heterodimeric Fc region In order to increase the production yield of the fusion protein of the present invention, the Fc region of the anti-PD-1 antibody was prepared with a heterodimeric Fc so that the heterodimeric binding between the Fc chain linked to IL-2 and the Fc chain linked to IL-2Rα is superior to the homodimeric binding between Fc chains having the same sequence. For this purpose, both a knob-into-hole (KiH) mutation and a charge / hydrophobic amino acid mutation were introduced into the Fc region.

[0120] The first Fc chain (SEQ ID NO: 49) included amino acid substitutions of T366W and Q347R, and the second Fc chain (SEQ ID NO: 50) included amino acid substitutions of T366S, L368A, Y407V, and K360E. These heterodimeric Fc mutants can significantly improve the production yield of the heterodimeric fusion protein to about 90% or more.

[0121] High-affinity mutant of IL-2Rα To enhance the binding force between IL-2 and IL-2Rα, a high-affinity IL-2Rα mutant was used. The IL-2Rα L42I mutant (SEQ ID NO: 5) in which the 42nd leucine amino acid of the IL-2Rα protein is substituted with isoleucine can bind to IL-2 with a further improved affinity (KD) value compared to wild-type IL-2Rα. Table 1 below shows the binding affinity and kinetics measured using SPR (surface plasmon resonance) Biacore 8K that observes the interaction between proteins in real time.

[0122]

Table 1

[0123] Preparation of expression vector The expression vector was prepared based on pTRIOZ_hIgG4(S228P) (InvivoGen) that expresses the human IgG4 constant region (S228P) (SEQ ID NOs: 46 and 47), such that the IL-2 C125S (SEQ ID NO: 2) or CD25 L42I (SEQ ID NO: 5) gene was inserted into the C-terminus of the Fc region. The C125S mutation of IL-2 was introduced to prevent cysteine mispairing in E. coli as in the FDA-approved IL-2 product Proleukin (Aldesleukin), and it does not affect biological activity (see Wang A, et al., Site-specific mutagenesis of the human interleukin-2 gene: structure-function analysis of the cysteine residues, Science, 224:1431-1433, 1984). In the present application, the term "wild-type IL-2" includes not only natural or recombinant human IL-2, but also IL-2 or Aldesleukin having the C125S mutation, or circular permutated form of IL-2 modified to have new N- and C-termini without deforming the binding properties of natural human IL-2. Non-limiting examples of circular permutated IL-2 include those in which the existing N-terminus (Ser4) and C-terminus (Thr133) are linked by a linker, cleaved to form a new N-terminus (Ser75) and C-terminus (Gln74), and the IL-2 helix is linked in the order of C-D-A-B.

[0124] The variable regions were prepared by including mouse immunoglobulin signal peptides in front of the heavy and light chain constant regions of the expression vector. The vector was basically prepared according to the instructions of the vector manufacturer. The signal peptide and the VH or VL sequence of the antibody were ligated by overlapping PCR and inserted into the pTRIOZ_hIgG4(S228P) vector. In the case of the VH sequence, the vector was cleaved using Mlu I / Nhe I restriction enzymes and then inserted. In the case of the VL sequence, the vector was cleaved using Asc I / BsiW I restriction enzymes and then inserted.

[0125] At this time, the first-chain (knob) expression vector was prepared by changing the nucleotide sequence so as to induce T366W and Q347R mutations in the Fc region, and the second-chain (hole) expression vector was prepared by using the PCR technique to obtain a nucleotide sequence that induces T366S, L368A, Y407V, and K360E mutations in the Fc region.

[0126] The first-chain (knob) expression vector was cleaved at the C-terminal portion using the Avr II restriction enzyme, and then amplified using the PCR technique so that the IL-2 C125S gene contained a 15-amino acid linker, and then prepared using the NEBuilder HiFi DNA Assembly Kit. The second-chain (hole) expression vector was cleaved at the C-terminal portion using the Avr II restriction enzyme, and then amplified using the PCR technique so that the CD25 L42I gene contained a 20-amino acid linker, and then prepared using the NEBuilder HiFi DNA Assembly Kit. At this time, the primers used were as shown in the following table.

[0127]

Table 2

[0128] The amino acid sequences encoded by each expression vector are as follows.

[0129] Light Chain: VL -CL (SEQ ID NO: 51) DIVMTQTPLSLSVTPGQPASISCRSSQNIVHSQGDTYLEWYLQKPGQSPQLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Table 3

[0130] Example 1.3 Expression and Purification of the Fusion Protein (Fusion Protein A) The first-chain (knob) expression vector (expression of the first heavy chain and light chain linked with IL2C125S) and the second-chain (hole) expression vector (expression of the second heavy chain and light chain linked with CD25 L42I) described in Example 1.2 were co-transfected into ExpiCHO TM cells to transiently express each protein.

[0131] The transfection was carried out under the conditions of an initial cell density of 6x10 6 cells / mL and an expression vector concentration of 0.8 μg DNA / mL. During this process, ExpiCHO TM Expression Medium, OptiPRO TM SFM and ExpiFectamine TM transfection kit (Gibco) was used according to the manufacturer's instructions.

[0132] The cells after the transfection process were cultured in a carbon dioxide incubator until the cell viability reached 75%, and then separated into the cell culture solution containing each protein and the cells through centrifugation.

[0133] To separate the target protein with the culture solution containing each recombinant protein, affinity chromatography was performed using a HiTrap MabselectXtra (Cytiva) column.

[0134] Specifically, it was purified using an AKTA avant25 FPLC. After equilibrating a HiTrap MabselectXtra column with PBS (3 mM Na2HPO4, 1.1 mM KH2PO4, 0.16 M NaCl, pH 7.2), the culture broth obtained through centrifugation was filtered through a 0.22 μm filter and loaded. After reacting with the culture broth, the column was sequentially washed with PBS (5 - 10 CV) and 50 mM NaOAc pH 5.2 (5 - 10 CV), and then the protein was eluted using an Elution buffer (0.1 M Glycine pH 3.0). After adding a 3M NaOAc pH 5.2 buffer to the eluted protein to a final concentration of 50 - 100 mM, centrifugation was performed to remove the co-extracted pigment. After pigment removal, it was concentrated using an Amicon tube (MWCO: 50K) and buffer-exchanged into ABS (0.1 M NaOAc, 0.15 M NaCl, pH 5.2) or PBS.

[0135] To confirm the production of the fusion protein, SDS-PAGE under non-reducing and reducing conditions was performed on 6% and 10% acrylamide-bisacrylamide (29:1) gels, respectively, and stained with Coomassie Blue (InstantBlue Coomassie Protein Stain (Abcam, ab119211)). Figure 5 shows the SDS-PAGE results confirming the purified fusion protein (fusion protein A). Fusion protein A has a heterodimeric structure in which IL-2 and IL-2Rα are respectively linked to the Fc region of the anti-PD-1 antibody and has two anti-PD-1 Fab arms (Figure 1A), and it was confirmed that it can be produced in a high yield.

[0136] Example 2. Various fusion protein designs and production Example 2.1 Production of a fusion protein having one anti-PD-1 Fab arm (fusion protein B, Figure 1B) The structural design and vector preparation are carried out in the same manner as in Example 1. However, the expression vector of Example 1 is used as the primary vector. At this time, the VH-CH1 part is removed using PCR with the first heavy chain (knob) vector and then used. The amino acid sequence encoded by the first heavy chain (knob) vector for the fusion protein B is as follows.

[0137]

Table 4

[0138] As the final expression vector, pG3.1, a self-prepared vector constructed to contain three MCSs (multiple cloning sites) from PcDNA3.1, was used. Each MCS has the same promoter (CMV), enhancer (CMV), and Poly A signal (BGH). The light chain, first heavy chain (knob), and second heavy chain (hole) genes cloned from the primary vector were inserted into the three MCSs respectively to prepare an expression vector. ExpiCHO TM The expression vector was transfected into cells to express each protein by the method as in Example 1, and the target protein was isolated and purified by the same method as in Example 1.

[0139] Example 2.2 Production of a heterodimeric fusion protein containing one IL-2Rα protein without IL-2 (fusion protein C, Figure 3C) The structural design, vector preparation, and protein expression and purification are carried out in the same manner as in Example 2.1, but it is used that the IL-2 C125S gene is not ligated to the C-terminus of the first heavy chain (knob) vector. The light chain and the second heavy chain (hole) are the same as in Example 1, and the amino acid sequence of the first heavy chain (knob) is as follows.

[0140]

Table 5

[0141] Example 2.3 Production of a homodimeric fusion protein containing two IL-2Rα proteins and not containing IL-2 (fusion protein D, Figure 3A) Structural design, preparation of an expression vector, and expression and purification of the protein were carried out in the same manner as in Example 1, but a homodimeric form of fusion protein D in which only the CD25 L42I (SEQ ID NO: 5) gene was linked to the C-terminus of the Fc region without mutation of the Fc region for improving the heterodimer production yield was prepared using the expression vector of Example 1. The light chain amino acid sequence of fusion protein D is the same as that in Example 1 (SEQ ID NO: 51), and the heavy chain amino acid sequence is as follows.

[0142] [Table 6]

[0143] To confirm the fusion proteins produced and purified in Examples 2.1 to 2.3, SDS-PAGE was performed using a 4 - 15% TGX Precast gel (Biorad, BR4561086), and the results are shown in Figure 6.

[0144] Example 3. Analysis of binding selectivity for IL-2 receptor proteins To confirm the binding selectivity of the IL-2 / IL-2Rα heterodimeric Fc region of the fusion protein of the present invention to the IL-2 receptor, binding ELISAs for IL-2Rα and IL-2Rβγ were performed. IL-2Rα-His (Acrobiosystems) and IL-2Rβγ-His (Acrobiosystems) were dispensed at 100 μl / well into a 96-well plate (Thermofisher scientific). After sealing, they were coated overnight at 4°C. After blocking and washing, the fusion protein A and the control group antibody (Fc-IL2; self-prepared) were diluted to concentrations of 0 to 1000 nM and dispensed at 100 μl / well, and incubated at room temperature for 2 hours. After washing, a secondary antibody (HRP-conjugated anti-His antibody) was added and incubated at room temperature for 30 minutes in the dark. After washing, a TMB substrate solution (Abcam) was added, and the color reaction was stopped with a STOP solution (Abcam). The absorbance at 450 nm was measured using a microplate reader (ThermoFisher).

[0145] The results are shown in FIGS. 7A and 7B, respectively. Fusion protein A did not show binding affinity for the IL-2Rα protein (FIG. 7A), while it showed excellent binding affinity for the IL-2Rβγ protein, although it was slightly weaker than wild-type IL-2 (FIG. 7B). This indicates that the IL-2 of the fusion protein of the present invention has excellent selectivity for IL-2Rβγ compared to IL-2Rα due to its unique structure in which IL-2 is preoccupied by IL-2Rα.

[0146] Example 4. Analysis of Binding Selectivity for IL-2 Receptor-Expressing Cells by PD-1 Expression Since the fusion protein of the present invention has an IL-2 / IL-2Rα complex structure fused with an anti-PD-1 binding domain, whether there is a change in IL-2Rβγ signal transduction in exhausted PD-1 + T eff cells present in the tumor microenvironment (TME) by the anti-PD-1 binding domain was analyzed. The analysis was performed using HEK-Blue TMPerformed using a reporter assay system (InvivoGen).

[0147] First, HEK-Blue TM CD122 / CD132 cells (InvivoGen) and HEK-Blue TM IL-2 cells (InvivoGen) were each transfected with the pCMV3-hPD1 (HG10377-CF) vector to generate PD-1-expressing cells. As a control group, the PCMV3-Empty vector (control group DNA) was transfected into the above two types of cells respectively for use. The transfected cells were dispensed into a 96-well plate at a concentration of 50,000 cells / well and cultured in a 37°C, 5% CO 2 chamber. Then, after removing the cell culture supernatant, fusion protein A at a concentration of 0 - 20 nM, recombinant human IL-2 (rIL2; Sigma), heterodimeric Fc-IL2 (Fc-IL2; self-prepared), and heterodimeric Fc-IL2 / IL2Rα (a structure in which IL-2 and IL-2Rα are respectively linked to heterodimeric Fc, Fc-IL2 / IL2Rα; self-prepared) were added to 100 μl of DMEM medium and dispensed to the cells, and cultured at 37°C for 8 hours. After adding 20 μl / well of the cell culture supernatant to a new 96-well plate, QUANTI-Blue TM solution (InvivoGen) was treated at 180 μl / well and incubated in a 37°C incubator for about 1 hour or more under light shielding. The activity of secreted alkaline phosphatase (SEAP) was measured at 620 - 655 nm using a Microplate reader. The results are shown in Figure 8.

[0148] The above procedure was repeated twice to obtain an average value. The EC 50 values of the fusion protein of the present invention according to the presence or absence of PD-1 expression and the type of IL-2R expression are shown in Table 2 below.

[0149]

Table 7

[0150] As shown in Table 2, the fusion protein of the present invention surprisingly exhibits an IL-2 responsiveness that is about 340-fold or more higher in IL-2Rβγ cells expressing PD-1 than in cells without PD-1 expression (see Table 2 and FIGS. 8A and 8B showing secondary results). On the other hand, the fusion protein of the present invention shows a weak IL-2 responsiveness to IL-2Rβγ cells without PD-1 expression (FIG. 8B), which is considered to be due to the fact that the fusion protein of the present invention has a no alpha but attenuated beta gamma affinity property, as also confirmed in the binding affinity test for the IL-2Rβγ protein in Example 3 (FIG. 7B). Without being bound by theory, such attenuated beta gamma affinity is thought to result in insufficient intracellular signaling in IL-2Rβγ-expressing cells without PD-1 expression. However, in cells co-expressing PD-1 (e.g., PD-1 in the TME + T eff cells), it is understood that the IL-2 of the fusion protein of the present invention can strongly bind to IL-2Rβγ while binding together in an "insis" structure with the help of the anti-PD-1 binding domain (the "switch-on" effect), and can cause intracellular signaling at a significant level.

[0151] The no alpha but attenuated beta gamma affinity property of the fusion protein of the present invention suggests that it does not bind well to peripheral blood NK cells that permanently express IL-2Rβγ but have a low PD-1 expression rate, which has a very advantageous advantage in terms of reducing systemic toxicity or side effects due to NK cell overactivity that conventional IL-2 proteins have.

[0152] In addition, for the fusion protein of the present invention, even when there is PD-1 expression in IL-2Rαβγ-expressing cells, the difference in reactivity to IL-2 was at a negligible level compared to IL-2Rβγ-expressing cells (340-fold vs 44-fold; see Table 2 and FIGS. 8C and 8D showing secondary results). In the case of cells engineered to express various receptor subunits such as IL-2Rαβγ, considering that the expression patterns of the subunits are diverse and that the expression of the IL-2Rβγ receptor can be partially accommodated to some extent, the actual difference in reactivity to IL-2 between IL-2Rαβγ-expressing cells and IL-2Rβγ-expressing cells would be even more pronounced. This means that the fusion protein of the present invention has a considerably low reactivity to IL-2 in T reg cells that permanently express IL-2Rαβγ, eosinophils that cause capillary leak syndrome, and other endothelial cells, thus solving the problem of systemic toxicity or serious side effects that may occur due to the appearance of exogenous IL-2 protein, while specifically binding to exhausted T eff cells present in the TME and being able to induce an excellent anti-cancer immune response.

[0153] Example 5. Evaluation of in vivo anti-cancer efficacy in a mouse tumor model Example 5.1 Test of anti-cancer efficacy against single administration of anti-PD-1 or Fc-IL2 / IL2Rα To evaluate the ability of the fusion protein of the present invention to selectively deliver and activate IL-2 to T eff cells expressing PD-1 in tumors and suppress tumor growth, an anti-cancer efficacy evaluation test of the fusion protein of the present invention was conducted using an MC38 colorectal cancer syngeneic mouse tumor model. The experimental procedure is summarized in FIG. 9.

[0154] MC38 cancer cells were cultured in DMEM (+10% FBS, 1% streptomycin - penicillin, 1% NEAA) medium at 37°C in a 5% CO2 chamber. MC38 cancer cells that reached Passage 9 were used for transplantation into mice. 1x10 6 MC38 cancer cells were injected subcutaneously using a 1 ml insulin syringe. At this time, the mice were kept anesthetized using isoflurane. The mice injected with MC38 cancer cells were observed for 7 days. All mice were purchased from Orient Bio and raised in a specific - pathogen - free condition animal facility (SPF facility).

[0155] After 7 days, the tumor size was measured, and mice that reached a size of 100 mm 3 (±30) were separately classified and used in the experiment. A total of 28 mice transplanted with MC38 cancer cells were secured and divided into 4 groups. Each group consisted of a fusion protein A treatment group of the present invention, an antibody treatment group having the same anti - PD - 1 binding domain as fusion protein A, a heterodimeric Fc - IL2 / IL2Rα treatment group, and a PBS treatment group as a control group. Each test substance was mixed with 100 μl of DPBS at the same dose (140 pmole) and injected into the mouse tail vein 2 times a week (BIW) for a total of 4 times starting from the 7th day (the PBS treatment group was injected with 100 μl of DPBS only intravenously). From the 7th day, the tumor size and body weight were measured and recorded 2 times a week. When the tumor size reached 2000 mm 3 euthanasia was carried out.

[0156] Figure 10 shows the diagrams measuring the change in tumor size over time (Figure 10A), mouse survival period (Figure 10B), and mouse body weight change (Figure 10C) for each group. As shown in Figures 10A and 10B, the fusion protein of the present invention exhibits significantly superior tumor growth inhibitory activity and extension of mouse survival period compared to anti-PD-1 binding antibody alone or Fc-IL-2 / IL-R2α alone. The fusion protein of the present invention achieved a high tumor growth inhibition rate (TGI) reaching 98%, and it was confirmed that 6 out of a total of 7 mice maintained a tumor-free state. On the other hand, no weight loss of mice was observed due to the administration of the fusion protein of the present invention (Figure 10C).

[0157] Example 5.2 Anti-cancer efficacy test for combined administration of anti-PD-1 and Fc-IL2 / IL2Rα Whether the fusion protein of the present invention exhibits improved anti-cancer efficacy due to its unique fusion structure was evaluated using the same mouse tumor model used in Example 5.1 above. The experiment was carried out according to the procedure described in Example 5.1, but a total of 21 mice transplanted with MC38 cancer cells were secured and divided into three groups: a group treated with the fusion protein A of the present invention, a combined treatment group administered with an antibody having the same anti-PD-1 binding domain as the fusion protein A and heterodimeric Fc-IL2 / IL2Rα in combination, and a PBS treatment group as a control group. The results are shown in Figure 11.

[0158] Surprisingly, the fusion protein of the present invention exerted much more excellent tumor growth inhibition and elimination effects (Figure 11A) compared to the group administered with an anti-PD-1 antibody containing the same anti-PD-1 binding domain in combination with Fc-IL2 / IL2Rα, and it was confirmed that the mouse survival period was significantly extended (Figure 11B). Such results indicate that the fusion protein of the present invention can effectively transmit IL-2 to T eff cells expressing PD-1 in tumors to induce the proliferation and activation of T eff cells, thereby exhibiting excellent anti-cancer efficacy.

[0159] Example 5.3 Long-term memory immune response test To confirm whether the fusion protein of the present invention can form long-term memory immunity and exhibit long-term effects even after the administration is interrupted, after the transplantation of MC38 cancer cells (primary transplantation), C57BL / 6 mice that became tumor-free by the administration of the fusion protein of the present invention were secured and a long-term memory immune response test was conducted. For this purpose, the procedure described in Example 5.1 was followed, but after administering the fusion protein A75 to 280 pmole once a week (QW) or twice a week (BIW) for a total of 1 to 4 times, a total of 26 mice that remained tumor-free until the 60th day after the primary transplantation (tumor-free mouse) were used in the experiment (75 pmole BIW 4 times: 6 mice, 140 pmole BIW 4 times: 6 mice, 150 pmole BIW 4 times: 5 mice, 280 pmole BIW 4 times: 4 mice, 150 pmole QW 1 time: 2 mice, 150 pmole QW 2 times: 3 mice).

[0160] On the 67th day after the MC38 primary transplantation, the 26 mice were divided into two groups and 1 x 10 6 individual MC38 cancer cells (14 mice) or B16F10 melanoma cells (12 mice) were transplanted subcutaneously into the right dorsal part (secondary transplantation, 2nd challenge), and tumor growth was observed. As a control group, 14 naive mice that had not undergone MC38 primary transplantation and fusion protein administration were divided into two groups, and MC38 or B16F10 cancer cells were transplanted in the same manner as above. The change in tumor size was observed until the 85th day after the primary transplantation. The experimental procedure is summarized in FIG. 12.

[0161] Fourteen mice that received a secondary transplantation of MC38 cancer cells after the first transplantation and administration of fusion protein A all remained tumor-free until the end of the observation period, while tumor growth was observed in the mice of the other groups (Figures 13 and 14). Among them, in the case of mice that received a secondary transplantation of B16F10 melanoma cells, it was observed that tumor growth was suppressed compared to naive mice even without additional administration of fusion protein A (Figure 13). Such results indicate that the fusion protein of the present invention induces a tumor-specific long-term immune response and retains the inhibitory effect (acquired immune response) against the same cancer for a long time even after discontinuation of dosing, and also partially retains the anti-cancer effect (innate immune response) against other cancers (tumor-nonspecific).

[0162] Example 5.4 Evaluation of Dose-Dependent Anti-Cancer Effect by Single Administration To confirm whether the anti-cancer effect of the fusion protein of the present invention changes dose-dependently, experiments were conducted using an MC38 colorectal cancer syngeneic mouse tumor model.

[0163] The experiment was carried out in the same manner as in Example 5.1. However, on the 7th day after transplantation, mice were divided into a total of 7 groups (5 or 6 mice per group) based on a tumor size of 100 mm 3 ±30, and each group was administered fusion protein A once at doses of 0 (PBS), 50, 150, 300, 600, 1200, and 3000 pmole. The tumor size and body weight were measured and recorded twice a week, and euthanasia was carried out when the tumor size reached 2000 mm 3 . The experimental procedure is summarized in Figure 15.

[0164] As shown in Fig. 16A, the fusion protein A of the present invention showed an increased tumor suppression efficacy in proportion to the dose in the range of 0 to 150 pmole, and at a dose of 300 pmole or more, the TGI was 99 to 100% at all doses. Therefore, it is judged that the dose at which saturation of the anti-cancer effect against MC38 occurs during single administration is 150 to 300 pmole. In terms of body weight change, after administration at 3000 pmole, a body weight loss phenomenon was shown on the 10th day, but it recovered again, and no body weight loss was observed at all other doses (Fig. 16B) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 in the drawing; multiple comparison using Tukey post hoc test compared to the PBS control group (comparison of each group), two-way ANOVA).

[0165] Example 5.5 Evaluation of anti-cancer effect in B16F10 melanoma syngeneic model The experiment was carried out in the same manner as in Example 5.1, but instead of MC38 cancer cells, the efficacy of the fusion protein was evaluated in 9-week-old C57BL / 6J mice transplanted with 7.5x10 5 B16F10 cells.

[0166] After transplantation, on the 7th day, when the tumor size reached an average of 96 mm 3 the mice were divided into three groups, and each group was administered PBS (10 mice), fusion protein A 300 pmole (5 mice), and fusion protein A 600 pmole (5 mice) once. The tumor size and body weight were measured and recorded twice a week.

[0167] Figure 17 shows the change in tumor size over time (Figure 17A) and the tumor growth inhibition rate (TGI%) (Figure 17B) for each group. As shown in Figures 17A and 17B, the fusion protein A treatment group showed a statistically significant tumor growth inhibitory effect compared to the PBS treatment control group (TGI of 70.2% (300 pmole) and 89.2% (600 pmole) relative to the PBS treatment group based on day 13 after dosing; in the figure, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA using Tukey's post hoc test).

[0168] Example 5.6 Evaluation of the anti-cancer effect in a Pan02 pancreatic cancer syngeneic model The experiment was carried out in the same manner as in Example 5.1, but instead of MC38 cancer cells, the efficacy of the fusion protein was evaluated in mice transplanted with 3x10 6 Pan02 cancer cells.

[0169] On the 7th day after transplantation, when the tumor size reached an average of 71 mm 3 the mice were divided into two groups, a fusion protein A treatment group (5 mice) and a PBS treatment group (5 mice), and the administration was continued. The fusion protein A was mixed in 100 μl of DPBS and injected into the mouse tail vein 4 times in total at a frequency of 75 pmole per mouse twice a week (BIW) (the PBS treatment group was injected with 100 μl of DPBS only intravenously). From the 7th day to the 40th day after transplantation, the tumor size and body weight were measured and recorded twice a week.

[0170] Figure 18 shows the measurement of the change in tumor size over time (Figure 18A), mouse survival period (Figure 18B), and mouse body weight change (Figure 18C) for each group. The fusion protein A showed excellent antitumor activity in which all 5 mice remained tumor-free from the third administration time point until the end of observation (Figure 18A; TGI 100%, ***p < 0.001 on the 17th day after dosing, ****p < 0.0001 on the 21st - 28th day after dosing; two-way ANOVA using Tukey's post hoc test compared to the PBS control group), and all mice survived until the end of observation (Figure 18B). In terms of body weight change, a temporary decrease in body weight was observed at the first administration of the fusion protein A, but then it recovered again and no further body weight decrease was observed (Figure 18C).

[0171] Example 5.7 Comparative Evaluation of the Anticancer Effect by the Number of Anti-PD-1 Fab Arms To confirm how the anticancer effect of the fusion protein of the present invention changes depending on the number of anti-PD-1 Fab arms, experiments were conducted using the MC38 colorectal cancer syngeneic mouse tumor model. The experiment was carried out in the same manner as in Example 5.1, but after transplanting MC38 cancer cells, on the 7th day, fusion protein A (2 PD-1 Fab arms) and fusion protein B (1 PD-1 Fab arm) were administered once at a dose of 450 pmole (5 mice per group), and PBS was administered to 7 mice as a control group.

[0172] Until the 17th day after dosing, the tumor size and body weight were measured and recorded twice a week. Figure 19 shows the change in tumor size over time (Figure 19A) and tumor growth inhibition rate (TGI%) (Figure 19B) for each group. As shown in Figure 19A and Figure 19B, the fusion protein A treatment group and the fusion protein B treatment group showed a statistically significant tumor growth inhibitory effect compared to the PBS treatment control group (TGI 100% (fusion protein A) and 76% (fusion protein B) based on the 17th day after dosing compared to the PBS treatment group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 in the figure, two-way ANOVA using Dunnett's post hoc test).

[0173] Example 6. Comparative Evaluation of Antigen Binding Affinity by pH Blood (approx. pH 7.4) and the tumor microenvironment (approx. pH 5.6 - 6.8) are known to have a pH difference. Therefore, the antibody binding affinity at the weakly acidic pH of the tumor microenvironment has an important impact on the therapeutic efficacy of immune anti-cancer antibodies. In the previous research of the present inventor, 1G1-h70 used as an anti-PD-1 binding domain in the fusion protein of the present invention showed much stronger binding affinity for human PD-1 compared to Keytruda and Opdivo, which are representative anti-PD-1 antibody drugs, at the low pH (6.0) of the tumor microenvironment (PCT / KR2022 / 001714). Therefore, in order to confirm whether the fusion protein A of the present invention retains excellent binding affinity within the tumor microenvironment, the binding kinetics to human PD-1 was measured through SPR (Biacore 8K, Cytiva) analysis. As a control group for comparison, anti-human PD-1 antibodies Keytruda (MSD) and Opdivo (BMS) were purchased from Shinwon Pharmaceutical as human pharmaceuticals and used.

[0174] Human PD-1 (Acrobiosystems, Cat#PD1-H5221) at a concentration of 1 μg / ml was immobilized on a CM5 chip (Cytiva) according to the manufacturer's instructions using an amine coupling kit (Cytiva). Control group antibodies (Keytruda, Opdivo), 1G1-h70, and fusion protein A at 7 concentrations (0 - 100 nM) were allowed to bind (association) to the CM5 chip at a flow rate of 30 μL / min for 180 seconds and dissociate (dissociation) for 1200 seconds and then flowed through the CM5 chip. After each experiment for each concentration cycle, the chip was regenerated with glycine at pH 1.5. At this time, the running buffer used was HBS-EP+ buffer (0.1 M HEPES, 1.5 M NaCl, 0.03 M EDTA and 0.5% v / v Surfactant P20) at pH 7.4 or pH 6.0, and the experimental proteins were diluted with the running buffer.

[0175] Analysis was performed using a 1:1 binding model with Biacore evaluation software version 3.0, and the resulting kinetic constants (ka and kd) and affinity (KD) values are shown in Table 3 below.

[0176]

Table 8

[0177] As shown in Table 3, the fusion protein A and 1G1-h70 antibody of the present invention exhibit a binding affinity (KD) level similar to that of Keytruda and Opdivo at pH 7.4 (blood), but at pH 6.0 (tumor microenvironment), they show a binding affinity approximately 15 - 16 times stronger than that of Keytruda and Opdivo. Therefore, it was confirmed that the fusion protein of the present invention retains the excellent binding affinity at the low pH of the tumor microenvironment possessed by the anti-PD-1 antibody 1G1-h70.

[0178] Example 7. Confirmation analysis of PD-1-mediated IL-2 signal transduction To verify whether the fusion protein of the present invention exhibits PD-1-mediated IL-2 signal transduction, the IL-2 signal transduction activity with or without pretreatment with an anti-PD-1 antibody was analyzed using the HEK-Blue TM reporter assay system (InvivoGen).

[0179] HEK-Blue TM CD122 / CD132 cells (InvivoGen) expressing IL-2Rβγ were transfected with the pCMV3-hPD1 vector or the PCMV3-Empty vector (control group). The transfected cells were dispensed into a 96-well plate at a concentration of approximately 50,000 cells / well and incubated at 37°C, 5% CO 2They were cultured in a chamber. Then, after removing the cell culture supernatant, 20 μL of a 1 μM anti-PD-1 antibody (the 1G1-h70 antibody used in Example 6 above) or a control group isotype (human IgG4) antibody was added per well and incubated for 40 minutes. Thereafter, they were treated with 1 nM concentration of recombinant human IL-2 (rhIL-2) or fusion protein A and cultured at 37 °C for 8 hours. After adding 20 μl / well of the cell culture supernatant to a new 96-well plate, QUANTI-Blue TM solution (InvivoGen) was treated at 180 μl / well and incubated in a 37 °C incubator for about 1 hour or more under light shielding. After the reaction started, the OD was measured at 620 nm using a Microplate reader every 30 minutes. The results are shown in Figure 20.

[0180] As shown in Figure 20, the fusion protein of the present invention shows a weaker signal transduction reactivity than IL-2 in cells that do not express PD-1 (PD-1-CD122+CD132+ cells) (attenuated IL-2Rβγ binding characteristics), while in cells that express PD-1 (PD-1+CD122+CD132+ cells), it shows much better reactivity than IL-2. Thus, it can be seen that the fusion protein of the present invention acts specifically and selectively on PD-1-expressing cells. Also, when the cells were pretreated with an anti-PD-1 antibody to pre-block the PD-1 of the cells before treating with the fusion protein of the present invention, it was confirmed that the IL-2 reactivity of the fusion protein of the present invention was completely neutralized. This suggests that the IL-2 signal transduction shown by the fusion protein of the present invention is mediated by PD-1.

[0181] As described above, exemplary specific embodiments have been described in detail for a clear understanding of the present invention. However, the above description and examples should not be construed as limiting the scope of the present invention. The scope of the rights of the present invention is defined by the claims and their equivalents. The disclosures of all patents and scientific documents cited in this application are hereby expressly incorporated by reference in their entirety.

[0182] Sequence Listing

Table 9

Table 10

Table 11

Table 12

Table 13

Table 14

Table 15

Table 16

Claims

1. It comprises one or more immune cell antigen-binding domains, an Fc domain consisting of two Fc chains, and a cytokine-acting domain. The immune cell antigen-binding domain is ligated to the N-terminus of the Fc domain and the cytokine-acting domain is ligated to the C-terminus of the Fc domain, or the immune cell antigen-binding domain is ligated to the C-terminus of the Fc domain and the cytokine-acting domain is ligated to the N-terminus of the Fc domain, The aforementioned immune cell antigen-binding domain is the Fab molecule of an antibody that binds to the PD-1 (Programmed cell death-1) protein or its antigen-binding fragment. The cytokine action domain comprises (i) an interleukin-2 receptor α-subunit (IL-2Rα) polypeptide and (ii) an IL-2 polypeptide, wherein the IL-2Rα polypeptide and the IL-2 polypeptide are each individually linked to different Fc chains of the Fc domain, resulting in a fusion protein.

2. The fusion protein according to claim 1, wherein the IL-2Rα polypeptide and the IL-2 polypeptide are not covalently bonded to each other.

3. The fusion protein according to claim 1, wherein the IL-2Rα polypeptide is an IL-2Rα polypeptide having the amino acid sequence of SEQ ID NO: 3, an IL-2 binding fragment thereof, an extracellular domain, or a sushi domain, and can include one or more amino acid modifications that increase binding to the binding ligand IL-2 polypeptide.

4. The fusion protein according to claim 3, wherein the IL-2Rα polypeptide contains the L42I amino acid substitution based on the amino acid sequence of SEQ ID NO: 3, or contains the amino acid sequence of SEQ ID NO:

5.

5. The fusion protein according to claim 1, wherein the IL-2 polypeptide contains or is circulatingly permuted a C125S amino acid substitution based on the amino acid sequence number of SEQ ID NO:

1.

6. The aforementioned immune cell antigen-binding domain, Heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 9, HCDR2 containing any one amino acid sequence selected from the group consisting of SEQ ID NOs: 10-15, HCDR3 containing any one amino acid sequence selected from the group consisting of SEQ ID NOs: 16-31, Light chain complementarity determination region 1 (LCDR1) containing any one amino acid sequence selected from the group consisting of SEQ ID NOs. 32-35, LCDR2 containing the amino acid sequence of SEQ ID NO: 36, and The fusion protein according to claim 1, comprising a Fab molecule of an anti-PD-1 antibody containing the amino acid sequence of SEQ ID NO: 37, or an antigen-binding fragment thereof.

7. The fusion protein according to claim 1, wherein the immune cell antigen-binding domain comprises a Fab molecule of an anti-PD-1 antibody or an antigen-binding fragment thereof, comprising complementarity-determining regions CDR1, CDR2, and CDR3 of a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 38, 40, 42, or 44, and CDR1, CDR2, and CDR3 of a light chain variable region containing the amino acid sequence of SEQ ID NO: 39, 41, 43, or 45.

8. The fusion protein according to claim 6, wherein the anti-PD-1 antibody comprises a heavy chain variable region having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 38, 40, 42, or 44, and a light chain variable region having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 39, 41, 43, or 45.

9. The fusion protein according to claim 7, wherein the anti-PD-1 antibody comprises a heavy chain variable region having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 38, 40, 42, or 44, and a light chain variable region having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 39, 41, 43, or 45.

10. The fusion protein according to claim 1, wherein the Fc domain is an IgG subcategory Fc domain or an IgG1, IgG2, IgG3, or IgG4 subcategory Fc domain, and in the case of an IgG4Fc domain, it may include an amino acid substitution of S228P (according to the EU numbering system standards of Kabat literature).

11. The fusion protein according to claim 1, wherein the Fc domain includes a modification that promotes heterodimer formation between two Fc chains.

12. The fusion protein according to claim 11, wherein the deformation is a knob-into-hole deformation, comprising a knob deformation in one of the Fc chains of the Fc domain and a hole deformation in the other Fc chain, wherein the knob deformation comprises the amino acid substitution T366W based on Kabat's EU numbering system, and the hole deformation comprises the amino acid substitutions T366S, L368A, and Y407V.

13. The fusion protein according to claim 12, wherein the knob deformation further comprises the amino acid substitution of S354C, and the hole deformation further comprises the amino acid substitution of Y349C.

14. The fusion protein according to claim 1, wherein the cytokine action domain is bound to the Fc domain via a linker.

15. The fusion protein according to claim 14, wherein the linker is a peptide of the general formula (GX)n, (GGGX)n, (XGGG)nXGG, or (GGGGX)n, where X is A or S and n is a natural number from 1 to 4.

16. The fusion protein according to claim 1, wherein the antigen-binding fragment is scFv, (scFv)2, Bis-scFv, dsFv, (dsFv)2, Fv, dsFv-dsFv', diabody, ds-diabody, triabody, tetrabody, nanobody, domain antibody, single-domain antibody (sdAb), or bivalent domain antibody.

17. A polynucleotide for encrypting a fusion protein according to any one of claims 1 to 16.

18. An expression vector comprising the polynucleotide described in claim 17.

19. A host cell comprising the polynucleotide described in claim 17 or an expression vector containing the same.

20. A method for producing a fusion protein according to any one of claims 1 to 16, comprising the step of culturing a host cell containing an expression vector comprising a polynucleotide that encodes the fusion protein according to any one of claims 1 to 16.

21. The expression vector, A first expression vector comprising a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 51 and a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 57, and A method for producing a fusion protein according to claim 20, comprising a second expression vector containing a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 51 and a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 58.

Images