Cytohormone fusion proteins

Abstract

The present invention relates to a cytohormone fusion protein comprising a cytohormone domain that can specifically induce an immune cell targeting domain and proliferation and / or activation of immune cells. Specifically, the present invention relates to a fusion protein that specifically delivers a cytohormone (e.g., IL-2 or IL-15) to a depleted immune cell (e.g., a depleted T cell) within a tumor, thereby inducing and enhancing the activation of the immune cell. The fusion protein according to the present invention can selectively deliver a cytohormone that promotes the proliferation or activation of immune cells to effector T cells or the like in a tumor microenvironment, thereby inducing a strong immune enhancement and anti-cancer response in a tumor. Moreover, through the unique structure of the cytohormone action domain, systemic toxicity and even side effects caused by the cytohormone can be obviously reduced.

Description

Technical Field

[0001] The present invention relates to a cytokine fusion protein comprising an immune cell targeting domain and a cytokine action domain that can specifically induce the proliferation and / or activation of immune cells. Specifically, the present invention relates to a fusion protein that specifically delivers cytokines (e.g., IL-2 or IL-15) to exhausted immune cells (e.g., exhausted T cells) in a tumor, thereby inducing and enhancing the activation of immune cells. In addition, the present invention relates to a polynucleotide encoding the fusion protein, a vector comprising the polynucleotide, and a host cell. In addition, the present invention relates to a method for producing the fusion protein, and a pharmaceutical composition, method, and use thereof for using the fusion protein to treat a disease (e.g., cancer) or enhance an immune response in a mammal. 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 (CTL), and differentiates peripheral lymphocytes into cytotoxic cells and lymphokine-activated killer cells (LAK), stimulating the proliferation and activation of natural killer cells (NK cells).

[0003] Due to the effects of IL-2 as described above, IL-2 immunotherapy is expected to be effective in treating metastatic cancer. After research, the U.S. Food and Drug Administration (FDA) approved human recombinant IL-2 (Proleukin Aldesleukin is used for the indications of metastatic renal cell carcinoma and metastatic melanoma. However, proleukin causes serious side effects such as capillary leak syndrome (CLS), which limits the patients to whom it can be administered and the amount of drug that can be administered. In addition, the half-life is shorter than 2 hours, so there is the inconvenience of repeating the cycle of IV administration three times a day for 5 consecutive days.

[0004] There are three types of IL-2 receptors: high affinity, medium affinity, and low affinity. The high affinity receptor is composed of three subunits: IL-2 receptor alpha (IL-2Rα, CD25), beta (IL-2Rβ, CD122), and gamma (IL-2Rγ, CD123). The medium affinity receptor is composed of IL-2Rβ and IL-2Rγ, and the low affinity receptor is composed of IL-2Rα only. Although the medium affinity receptor composed of β and γ subunits has an affinity for IL-2 that is about 100 times lower than the high affinity receptor composed of α, β, and γ subunits, it can transmit signals when bound to IL-2. Although the α-subunit gives the receptor high affinity binding ability, it is not necessary for transmitting signals.

[0005] Only the medium-affinity IL-2 receptor (IL-2Rβγ) is expressed in resting immune cells. When resting T cells are activated by antigens, IL-2Rα is rapidly expressed, and when IL-2 binds to IL-2Rα, IL-2Rβ and IL-2Rγ are involved. Signaling occurs through the binding of the IL-2Rαβγ complex to IL-2, thereby promoting the activation of effector T cells (T cells), including cytotoxic T cells that can kill virus-infected cells or tumor cells. eff )’s growth.

[0006] In addition, IL-2 mediates activation-induced cell death (AICD) of T cells. AICD is a process in which fully activated T cells undergo programmed cell death, thereby becoming immune tolerant not only to normal self-antigens but also to persistent antigens such as tumor antigens.

[0007] IL-2 is also involved in maintaining peripheral CD4 + CD25 + Regulatory T cells (T reg ). IL-2Rα is persistently expressed in regulatory T cells. Regulatory T cells inhibit the function of cytotoxic T cells attacking self-antigens or tumor cells. Due to this multifaceted role of IL-2, IL-2 is not suitable for showing the best tumor suppression effect.

[0008] Various attempts have been made to reduce the toxicity of IL-2 and enhance its tumor suppressor effect. For example, strategies to inhibit the binding of IL-2 to IL-2Rα have been tried to allow IL-2 to act on T cells. eff The medium affinity of cells for IL-2Rβγ but not T regHigh affinity IL-2Rβγ receptors for cells. Examples thereof include a combination 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 variants with mutations introduced into the binding site with IL-2Rα (WO2012 / 107417, etc.), and IL-2 with a polyethylene glycol (PEG) group introduced into the IL-2Rα binding site (WO2012 / 065086).

[0009] However, the IL-2 variants developed so far still have a poor therapeutic index as anticancer agents, and even when administered at high doses that show toxicity, the anticancer effect is not obvious. This is believed to be because IL-2 variants act selectively on T cells. eff Cells rather than T reg However, this effect is mainly manifested peripherally and is not sufficient in the tumor microenvironment (TME).

[0010] In order to overcome this shortcoming, research has been conducted to make IL-2 variants work in tumor tissues, and a strategy has been tried to make tumor associated antigen (TAA)-specific antibodies bind to IL-2 to bring IL-2 into tumor tissues. However, although this strategy can induce IL-2 to the tumor site, it is difficult to induce IL-2 in the T cells within the tumor. eff It is necessary to selectively provide IL-2 to various tumor infiltrating lymphocytes (TILs). eff Methods for providing IL-2 to cells.

[0011] Not only IL-2, but other cytokines (e.g., IL-15) can also play an important role as mediators in inducing immune activity or immunosuppression in the tumor microenvironment. Such cytokines are substances secreted by immune cells and generally act locally on a variety of targets, thereby changing the microenvironment. However, these cytokines also have the characteristics of short local effects and disappearance, making them difficult to develop into therapeutic agents. Summary of the invention

[0012] Problems to be solved by the invention

[0013] The present inventors intend to develop an effective cytokine therapeutic agent that selectively activates exhausted immune cells in the tumor microenvironment, thereby enhancing anti-cancer activity while reducing systemic side effects caused by exogenous cytokines.

[0014] Means of solving problems

[0015] The present invention relates to a fusion protein comprising an immune cell targeting domain and a cytokine action domain that can specifically induce the proliferation and / or activation of immune cells. The present inventors aim to prevent cytokines that promote the proliferation or activation of immune cells from affecting other immune cells or endothelial cells present in the periphery and selectively deliver them to T cells in the TME environment. eff The two domains act in an “in-cis” manner on T cells in tumors. eff Cellular fusion proteins.

[0016] The present invention provides a fusion protein comprising one or more immune cell antigen binding domains, an Fc domain formed by two Fc chains, and a cytokine domain. In one aspect of the present invention, the cytokine domain is a cytokine that promotes the proliferation or activation of immune cells or a domain that binds to the cytokine. The cytokine can use an exogenous cytokine injected from the outside or an endogenous cytokine present inside. As such cytokines, IL-2 polypeptides and IL-15 polypeptides that can bind to the β-subunit (IL-2Rβ) and common γ chain receptor (also referred to as IL-2Rγ) of the IL-2 receptor and induce activation of signaling through IL-2Rβγ can be used.

[0017] In one aspect, the present invention provides a fusion protein, which comprises one or more immune cell antigen binding domains, an Fc domain formed by two Fc chains, and a cytokine domain, wherein the cytokine domain comprises an α-subunit of interleukin-2 receptor (IL-2Rα) polypeptide and an IL-2 polypeptide, wherein the IL-2Rα polypeptide and the IL-2 polypeptide are not covalently bonded to each other and are respectively connected to different Fc chains of the Fc domain.

[0018] Since IL-2 is occupied by IL-2Rα, the fusion protein of the present invention with such a structure binds to regulatory T cells (T cells) expressing high affinity IL-2 receptor (or IL-2Rαβγ for short). reg), Foxp3-negative CD4 T cells, some innate lymphocytes or other endothelial cells. In contrast, in the absence of IL-2Rα, it can bind to cytotoxic CD8+ T cells, memory T cells, NK-T cells, etc., which express the medium-affinity IL-2 receptor composed of IL-2Rβ and IL-2Rγ (or IL-2Rβγ for short). eff Cell-specific binding can enhance the anti-cancer effect and significantly reduce systemic toxicity or side effects.

[0019] In one aspect, the present invention provides a fusion protein comprising one or more immune cell antigen binding domains, an Fc domain formed by two Fc chains, and a cytokine domain, wherein the cytokine 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 the Fc chain on either side of the Fc domain via the IL-2Rα polypeptide or the IL-2 polypeptide.

[0020] In one aspect, the present invention provides a fusion protein, which comprises one or more immune cell antigen binding domains, an Fc domain formed by two Fc chains, and a cytokine domain, wherein the cytokine domain comprises an α-subunit of interleukin-15 receptor (IL-15Rα) polypeptide and an IL-15 polypeptide, wherein the IL-15Rα polypeptide and the IL-15 polypeptide are respectively connected to different Fc chains of the Fc domain.

[0021] In one aspect, the present invention provides a fusion protein comprising one or more immune cell antigen binding domains, an Fc domain formed by two Fc chains, and a cytokine domain, wherein the cytokine 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 the Fc chains on either side of the Fc domain via the IL-15Rα polypeptide or the IL-15 polypeptide.

[0022] In one aspect, the present invention provides a fusion protein, the fusion protein comprising one or more immune cell antigen binding domains, an Fc domain formed by two Fc chains, and a cytokine domain, wherein the cytokine domain does not include an IL-2 polypeptide and an IL-15 polypeptide but only includes an IL-2Rα polypeptide or an IL-15Rα polypeptide, thereby being able to bind to endogenous IL-2 or IL-15 and induce it to T cells. eff cell.

[0023] In one aspect of the present invention, the IL-2Rα polypeptide comprises a wild-type mature IL-2Rα polypeptide having the amino acid sequence of SEQ ID NO: 3 in the sequence list attached to this specification, or an IL-2 binding fragment, an extracellular domain or a Sushi domain of an IL-2Rα polypeptide, or a variant thereof.

[0024] In one aspect of the present invention, the IL-15Rα polypeptide comprises a wild-type mature IL-15Rα polypeptide having the amino acid sequence of SEQ ID NO: 7 in the sequence list attached to this specification, or an IL-15 binding fragment, extracellular domain or Sushi domain of an IL-15Rα polypeptide, or a variant thereof.

[0025] In one aspect of the invention, the IL-2Rα polypeptide and IL-15Rα polypeptide may include one or more amino acid modifications, preferably amino acid substitutions, that improve the binding affinity with the respective binding ligands, i.e., IL-2 polypeptide and IL-15 polypeptide. In an exemplary aspect, the fusion protein of the invention includes an IL-2Rα variant, wherein the IL-2Rα variant includes one or more of the following amino acid substitutions based on the amino acid sequence number of SEQ ID NO: 3: 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. In a preferred aspect, the fusion protein of the present invention includes an IL-2Rα variant, wherein the IL-2Rα variant includes an L42I amino acid substitution based on the amino acid sequence number of SEQ ID NO: 3.

[0026] In one aspect of the present invention, the IL-2 polypeptide comprises a wild-type mature IL-2 polypeptide having an amino acid sequence of SEQ ID NO: 1 in the sequence list attached to this specification, or a C125S variant thereof (SEQ ID NO: 2) or a circularly permuted (circularly permutated) variant thereof. In one aspect of the present invention, the IL-15 polypeptide comprises a wild-type mature IL-15 polypeptide having SEQ ID NO: 6 in the sequence list attached to this specification, or a circularly permuted variant 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 increase the binding affinity to the α-subunit of the respective binding receptor. In a preferred aspect, the fusion protein of the present invention comprises an IL-2 variant, and the IL-2 variant comprises a T37K amino acid substitution based on the amino acid sequence number of SEQ ID NO: 1.

[0027] In one aspect of the invention, the immune cell antigen binding domain comprises a Fab molecule or an antigen binding fragment thereof of an antibody that specifically binds to one or more T cell target antigens. In one aspect of the invention, the immune cell antigen binding domain comprises a Fab molecule or an antigen binding fragment thereof that specifically binds to T cell target antigens in the tumor microenvironment. eff Immunocheck points expressed in cells ( immunecheckpoint ) protein (e.g., programmed cell death-1 (PD-1) protein). In one aspect of the present invention, the immune cell antigen binding domain may be a Fab molecule or an antigen-binding fragment of an antibody that specifically binds to a T cell in the tumor microenvironment. effCells confer binding specificity to CD8+ effector T cells surface marker binding components, antibodies or their antigen-binding fragments. In a preferred 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 of an antibody. In exemplary aspects, the immune cell antigen binding domain of the present invention is a Fab molecule or an antigen-binding fragment of a pembrolizumab or nivolumab antibody, or a Fab molecule or an antigen-binding fragment of an antibody that binds to the same antigenic determinant region (epitope) as these antibodies or antigen-binding fragments or competes with these antibodies or antigen-binding fragments. Preferably, the fusion protein of the present invention includes an immune cell antigen binding domain, which includes a Fab molecule of an anti-PD-1 antibody or an antigen-binding fragment thereof comprising: complementarity determining region (CDR) 1, CDR2 and CDR3 of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38, 40, 42 or 44 in the sequence list attached to the specification; and CDR1, CDR2 and CDR3 of a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39, 41, 43 or 45.

[0028] In one aspect of the invention, the immune cell antigen binding domain may be a multispecific binding domain comprising two or more different T cell target antigen binding domains. In one aspect of the invention, the fusion protein of the invention may include a Fab molecule or an antigen binding fragment thereof of an antibody that specifically binds to more than one tumor cell target antigen as an additional antigen binding domain.

[0029] In one aspect of the present invention, the Fc domains may be homodimers identical to each other in the first chain and the second chain or heterodimers different from each other. In one aspect of the present invention, the Fc domains are human IgG class Fc domains. In one aspect of the present invention, the Fc domains are IgG1, IgG2, IgG3 or IgG4 subclass Fc domains. In one aspect of the present invention, in the case where the Fc domains are IgG4 Fc domains, it is preferably an amino acid substitution including S228P (based on the EU numbering system of Kabat literature). In one aspect of the present invention, the Fc domains lack CH2 domains and may be composed only of CH3 domains. In addition, the Fc domains of the present invention may include one or more amino acid modifications that reduce the binding or effector function of Fc receptors. In exemplary aspects, 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.

[0030] In one aspect of the invention, the Fc domain may include a modification for promoting heterodimer formation between two Fc chains. In exemplary aspects, 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 invention includes the knob-into-hole modification or the knob-into-hole disulfide modification, and an Fc domain variant in which either Fc chain further includes an amino acid substitution of K360E and the other Fc chain further includes an amino acid substitution of Q347R, or either Fc chain further includes an amino acid substitution of K360E and Q347E and the other Fc chain further includes an amino acid substitution of Q347R or an amino acid substitution of K360R and Q347R.

[0031] In one aspect of the invention, the immune cell antigen binding domain and the cytokine domain can be directly connected to the Fc domain or connected to the Fc domain through a linker. In one aspect of the invention, the immune cell antigen binding domain is connected to the N-terminus of the Fc domain, and the cytokine domain is connected to the C-terminus of the Fc domain, or a structure opposite thereto can be adopted. In one aspect of the invention, the linker is preferably a peptide linker, but is not limited thereto.

[0032] Another aspect of the present invention provides a polynucleotide encoding the fusion protein or its regions, a vector comprising the polynucleotide, or a transformed cell introduced with the vector. Another aspect of the present invention provides a host cell comprising the vector, and provides a method for culturing the host cell to produce the fusion protein of the present invention.

[0033] Another aspect of the present invention provides a pharmaceutical composition for preventing, improving or treating tumors, cancers, metastatic tumors, metastatic cancers or infectious diseases, comprising the fusion protein as an effective ingredient. Another aspect of the present invention provides a method for preventing, improving or treating tumors, cancers, metastatic tumors, metastatic cancers or infectious diseases by administering the fusion protein to an individual who needs to prevent, improve or treat tumors, cancers, metastatic tumors, metastatic cancers or infectious diseases.

[0034] Effects of the Invention

[0035] According to the present invention, an effective anticancer cell hormone therapeutic agent can be provided, which selectively activates cytotoxic effector T cells in the tumor microenvironment, thereby enhancing anticancer activity while reducing systemic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figures 1a to 1c The structure of a fusion protein including IL-2 and IL-2Rα according to one embodiment of the present invention is schematically shown.

[0037] Figure 2a and 2b The structure of a fusion protein according to one embodiment of the present invention, which includes a complex of IL-2 and IL-2Rα or a complex of IL-15 and IL-15Rα, is schematically shown.

[0038] Figures 3a to 3d The structure of a fusion protein according to one embodiment of the present invention that does not include IL-2 and IL-15 but includes IL-2Rα or IL-15Rα is schematically shown.

[0039] Figure 4 The crystal structure analysis results show how the fusion protein according to one embodiment of the present invention binds to hPD-1 and IL-2Rβγ based on the structure-designed linker.

[0040] Figure 5 The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to confirm the fusion protein A according to one embodiment of the present invention.

[0041] Figure 6a and 6b The figure is the SDS-PAGE result confirming the fusion proteins B, C and D according to one embodiment of the present invention.

[0042] FIG. 7 is an enzyme-linked immunosorbent assay (ELISA) to determine whether the IL-2Rα protein ( Figure 7a ) and IL-2Rβγ protein ( Figure 7b ) were tested for selective binding.

[0043] FIG. 8 is a series of HEK-Blue test result curves showing whether or not the PD-1 fusion protein according to one embodiment of the present invention is expressed and the difference in IL-2 signaling responsiveness according to the type of IL-2 receptor expression.

[0044] Fig. 9 The following is a schedule of animal experiments for confirming the in vivo anticancer effect of the fusion protein according to one embodiment of the present invention in the MC38 mouse tumor model.

[0045] FIG. 10 is a graph showing changes in tumor size over time when a fusion protein according to an embodiment of the present invention is administered in a MC38 mouse tumor model ( Fig.10a ), survival rate( Fig.10b ) and weight changes ( Fig.10c ) is a graph comparing the results of administering anti-PD-1 antibody alone and administering heterodimeric Fc-IL2 / IL2Rα alone.

[0046] FIG. 11 is a graph showing changes in tumor size over time when a fusion protein according to an embodiment of the present invention is administered in a MC38 mouse tumor model ( Fig.11a ), survival rate( Fig.11b ) and weight changes ( Fig.11c ) is a graph comparing the results based on the combined administration of anti-PD-1 antibody and heterodimer Fc-IL2 / IL2Rα.

[0047] Fig.12 The following is a schedule of animal experiments for confirming the long-term memory immune response of the fusion protein according to one embodiment of the present invention.

[0048] Fig.13 is a graph showing changes in tumor size over time in a long-term memory immune response experiment in which a fusion protein according to an embodiment of the present invention is administered.

[0049] Fig.14 The figure is a photograph of an animal model showing the change in tumor size after administration of the fusion protein according to one embodiment of the present invention in a long-term memory immune response experiment.

[0050] Fig.15 The animal experiment schedule for evaluating the volume-dependent anticancer effect of a single administration of a fusion protein according to one embodiment of the present invention in the MC38 mouse tumor model is shown.

[0051] FIG. 16 is a graph showing changes in tumor size over time after a single administration of a fusion protein according to an embodiment of the present invention in a MC38 mouse tumor model ( Fig.16a ) and weight changes ( Fig.16b ) curve graph.

[0052] FIG. 17 is a graph showing the change in tumor size over time after administration of a fusion protein according to one embodiment of the present invention in a B16F10 mouse melanoma model ( Fig.17a ) and tumor growth inhibition rate (TGI%) ( Fig.17b ) curve graph.

[0053] FIG. 18 is a graph showing changes in tumor size over time in a Pan02 mouse pancreatic cancer model in which a fusion protein according to an embodiment of the present invention is administered ( Fig.18a ), survival rate( Fig.18b ) and weight changes ( Fig.18c ) curve graph.

[0054] FIG. 19 is a graph showing the change in tumor size over time after a single administration of fusion proteins A and B according to one embodiment of the present invention in a MC38 mouse tumor model ( Fig.19a ) and tumor growth inhibition rate (TGI%) ( Fig.19b ) are compared and shown in the graph.

[0055] Fig. 20 FIG. 4 is a graph of HEK-Blue test results, showing the difference in IL-2 signaling responsiveness depending on whether or not the fusion protein according to one embodiment of the present invention is pre-treated with an anti-PD-1 antibody. DETAILED DESCRIPTION

[0056] In the present invention, "protein" or "polypeptide" is interpreted as a concept including oligopeptides as amino acids attached by at least two covalent bonds. In the present invention, "A protein" or "A polypeptide" can be interpreted as a concept including all or part of the A protein or A polypeptide as a parent, its analogs and mutants. In addition, "mutation", "modification" and "variant" are the meanings including replacement, insertion and / or deletion of amino acid residues, and preferably the mutant of the present invention may include replacement of amino acid residues. The replacement of amino acid residues can be expressed according to the amino acid residues present in the wild-type protein as a parent, the numbering of the amino acid residues and the order of the replaced amino acid residues.

[0057] In the present invention, the term "fusion protein" may be interpreted as meaning a form in which two or more proteins are bound to each other or a complex including such a form.

[0058] 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 antigenic determining region with PD-1.

[0059] In the present invention, the term "antibody" includes the whole antibody and any "antigen binding portion" or a single chain thereof. "Antibody" refers to a protein or an antigen binding portion thereof comprising at least two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain is formed by a heavy chain variable region and a heavy chain constant region. The heavy chain constant region is formed by three domains, namely CH1, CH2 and CH3. Each light chain is formed by a light chain variable region and a light chain constant region. The light chain constant region is formed by one domain, namely CL. The heavy chain variable region (VH) and the light chain variable region (VL) can be additionally subdivided into hypervariability regions called complementarity determining regions (CDRs), which are arranged between more conservative regions called framework regions (FRs). Each VH and VL is formed by three CDRs and four FRs, and is arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0060] In the present invention, "antibody" includes polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies, but is not limited thereto. The term "antigen-binding portion" includes any antibody fragment and includes all antibody fragments that retain antigen-binding function, i.e., the ability to specifically bind to an antigen. Typically, such an antigen-binding portion includes an antigen-binding fragment.

[0061] The term "antigen binding domain" of the present invention is a general term for any domain that can bind to a target antigen, including peptides, proteins, antibodies, aptamers, chemical residues, etc. In the case where the fusion protein of the present invention includes an antibody as an antigen binding domain, it may include not only the entire antibody, but also its antigen binding portion, Fab molecule or antigen binding fragment as an antigen binding domain. The term "Fab molecule" refers to a site in the heavy chain and light chain that has a constant region and a variable region, respectively, and may partially include a hinge region or partially deformed. The term "Fab molecule" used in the present invention includes Fab, Fab', F(ab')2, Fab'-SH, etc. and is defined as including the meaning of all possible antigen binding sites of antibodies.

[0062] 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 portion of an antibody molecule that includes amino acids that are responsible for the specific binding between the antibody and the antigen. For example, in the case of a large antigen, an antigen binding fragment may only bind to a portion of the antigen. The portion that is responsible for the specific interaction of the antigen binding fragment with the antigen molecule is referred to as an "antigenic determinant" 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, single chain variable fragment (scFv), (scFv)2, scFv-Fc, (scFv)2-Fc, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, dsFv-dsFv′, VL, VH, diabody, double strand (ds) diabody, triabody, tetrabody, minibody (scFV-CH3)2), nanobody, domain antibody, single domain antibody (sdAb), bivalent domain antibody, IgG delta CH2, fynomer, and fynomers fused to antibodies. antibodies, FynomAbs), dual-affinity re-targeting (DART), albumin-binding domain antibodies (AlbudAbs), bispecific T-cell engager (BiTE), tandem diabodies (TandAbs), dual acting Fab (DAF), two-in-one antibodies, small modular immunopharmaceuticals (SMIP), anticalins, FN3 monomers (monobody), designed ankyrin repeat proteins (designed ankyrin repeat proteins,DARPin), Affibodies, Affilins, Affimers, Affitins, Alphabodies, Avimers, Im7, variable lymphocyte receptor (VLR), variable new antigen receptor (VNAR), Trimab, Cross monoclonal antibody (Cross monoclonal antibody, CrossMab), Trident (TRIDENT), binanobodies, Bi-sdFv, dual variable domain immunoglobulin (dvd-Ig), CovX-bodies (peptide modified antibodies), Duobody and triomAbs, etc., but not limited thereto. ,

[0063] Although the antigen-binding fragment may include the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH), it does not necessarily include both. For example, although the so-called Fd antibody fragment is formed only by the VH domain, it still retains the antigen-binding function of the complete antibody to some extent.

[0064] Fusion Protein

[0065] The fusion protein of the present invention includes one or more immune cell antigen binding domains, an Fc domain formed by two Fc chains, and a cytokine domain. In the present invention, one or more immune cell antigen binding domains are components of a fusion protein that binds to surface markers on desired target immune cells such as CD8+ effector T cells with anti-tumor effects and delivers 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 of an anti-PD-1 antibody. The fusion protein of the present invention can selectively deliver cytokines to tumor infiltrated lymphocytes expressing the corresponding targets due to the presence of immune cell antigen binding domains, thereby minimizing the effects on NK cells or T cells in peripheral blood. In addition, the immune cell antigen binding domain can be a component, antibody, or antigen binding fragment thereof that selectively binds to, for example, NKG2a, CD8a, FcRL6, CRTAM, LAG3, TIM3, CTLA4, TIGIT, etc.

[0066] The fusion protein of the present invention is Figure 1a and Figure 1bAs shown, there may be one or two Fab arms. Figure 1c As shown, the two Fab arms may target different targets. For example, in the case where the immune cell antigen binding domain is an anti-PD-1 antibody or an antigen binding fragment of an anti-PD-1 antibody, it may further include a Fab arm targeting one or more targets 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.

[0067] In one embodiment of the present invention, the cytokine domain is designed to bind to T reg Cells can preferentially activate cytotoxic T cells eff In one embodiment of the present invention, the cytokine domain includes a cytokine domain that activates T cells by depleting the T cells. eff The signaling activation part of IL-2Rβγ persistently expressed in the cell, such as IL-2 polypeptide or IL-15 polypeptide. In one embodiment of the present invention, the cytokine domain also includes IL-2Rα and IL-2 to hinder the binding of IL-2 to the high-affinity IL-2 receptor (IL-2Rαβγ) expressed in the immune cells or endothelial cells in the peripheral blood. IL-2 in a form pre-occupied by IL-2Rα cannot bind to the high-affinity IL-2 receptor. In one embodiment of the present invention, the cytokine domain includes IL-2 protein and IL-2Rα protein, and IL-2 and IL-2Rα such as Figure 1a to Figure 1c As shown, they are connected to two Fc chains respectively and are not covalently bonded to each other. IL-2 and IL-2Rα are connected to the Fc chain or directly to the Fc chain through a linker. In one example, the linker may be a peptide linker, such as (GGGX)n, (XGGG)nXGG, (GGGGX)n, (GX)n (X=A or S, n=1,2,3 or 4), SGGGSGGGSGGGSGG, GGGSGGGSGGGSGG, etc., but are not limited to those. In one example, the linker may be a peptide linker formed by about 5 to 20 amino acids, but is not limited thereto, and a linker of any structure and length that appropriately maintains the preemptive structure of the cytokine domain and maintains the function of delivering the cytokine domain to the target cell can be used. In one example, the peptide linker may be composed of one or more amino acids selected from the group consisting of G, S, A, P, E, T, D and K.

[0068] In one embodiment of the present invention, the cytokine domain may include IL-15 and IL-15Rα. Figure 2bThis is an example of a case where the cytokine domain includes a complex of IL-15 and IL-15Rα. In one embodiment of the present invention, the cytokine domain may also include a covalently bonded IL-2 / IL-2α complex ( Figure 2a ) or covalently bound IL-15 / IL-15α complex ( Figure 2b ).

[0069] In one embodiment of the present invention, the cytokine domain may not include IL-2 and IL-15 but only include IL-2Rα protein or IL-15Rα protein ( Figure 3a to Figure 3d In this example, the IL-2Rα protein binds to endogenous IL-2 and delivers IL-2 to cells expressing the target (e.g., T cells expressing PD-1), but does not bind to T cells that always express IL-2Rα. reg Compared with cells expressing IL-2Rβ and γ, T cells eff Cell proliferation and activation. In this example, IL-2Rα or IL-15Rα may bind to both Fc chains to form a homodimer, or may bind to only one Fc chain to form a heterodimer.

[0070] In one example of the present invention, the cytokine domain is preferably incapable of sufficient cytokine signaling by itself, but selectively targets the desired cells (cells expressing the target) in the form of a fusion protein bound to an immune cell antigen binding domain, thereby activating the cells. In contrast, cells expressing cytokine receptors but not expressing the target are not activated or their activation is weakened. For example, in one example of the present invention, the cytokine domain comprising IL-2 and IL-2Rα has weaker binding to the IL-2Rβγ receptor than wild-type IL-2 in T cells that do not express PD-1, and substantially has no binding to the IL-2Rα receptor. However, in the form of a fusion protein bound to an anti-PD-1 antibody, after targeting cytotoxic CD8+T cells that simultaneously express PD-1 and IL-2Rβγ receptors, the proliferation and activation of cells are stimulated by sufficient signaling, thereby exhibiting excellent anti-cancer activity. However, cells that do not express PD-1 have weak or almost no activity, which can reduce side effects caused by cytokine activity in peripheral blood (such as capillary leak syndrome) and systemic toxicity caused by overactivated NK cells.

[0071] IL-2 protein

[0072] In the present invention, unless otherwise specified, "interleukin-2", "IL-2", "IL-2 protein", or "IL-2 polypeptide" includes IL-2 from vertebrates including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats). This includes not only unprocessed IL-2, but also any form of IL-2 processed in cells. This term also includes naturally occurring variants or "IL-2 mutants" defined below. Full-length human IL-2 refers to mature, natural-length human IL-2, which is a molecule with 133 amino acids of SEQ ID NO: 1. Unprocessed human IL-2 further includes 20 N-terminal amino acids that are not present in mature IL-2 molecules.

[0073] The term "IL-2 mutant" includes mutant forms of IL-2 molecules in various forms, including full-length IL-2, truncated forms of IL-2, or forms of IL-2 connected to other molecules. IL-2 mutants may include one or more amino acid deformations that affect the interaction with the IL-2Rα polypeptide or increase the binding to the IL-2Rα polypeptide. The mutation may include substitution, deletion, truncation or modification of wild-type amino acid residues. The amino acid substitution of T37K based on the amino acid sequence number of SEQ ID NO: 1 can be cited as an example of an amino acid modification that increases binding to the IL-2Rα polypeptide.

[0074] In the present invention, the IL-2 protein may be full-length human IL-2 (SEQ ID NO: 1). In addition, for example, in order to prevent the formation of an inaccurate disulfide bond, a mutation may be included in which the cysteine ​​at position 125 of the IL-2 of SEQ ID NO: 1 is replaced with serine (C125S, SEQ ID NO: 2). In addition, in order to shorten the length of the linker, a circular permutated form may be used in which the N-terminus and the C-terminus of IL-2 are replaced.

[0075] In the present invention, "IL-2 polypeptide" includes IL-2 proteins having an amino acid sequence that is 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, or about 99% or more identical to the human IL-2 sequence of SEQ ID NO:1.

[0076] IL-2Rα protein

[0077] In the present invention, unless otherwise specified, "IL-2Rα protein", "IL-2Rα polypeptide", "IL-2Rα", "CD25" or "interleukin-2 receptor α-subunit" means the α-subunit of the IL-2 receptor from vertebrates including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes not only unprocessed IL-2Rα but also any form of IL-2Rα processed in cells. The term also includes naturally occurring variants or "mutants of IL-2Rα" defined below. In the present invention, the human IL-2Rα polypeptide used as a benchmark has an amino acid sequence from 22 to 272 of the protein database (Universal Protein, UniProt) P01589 consisting of 272 amino acids (SEQ ID NO: 3). In the present invention, the "IL-2Rα polypeptide" may be an IL-2Rα polypeptide having the amino acid sequence of SEQ ID NO: 3 or an IL-2 binding fragment of an IL-2Rα polypeptide, an extracellular domain corresponding to positions 22 to 240 of UniProt P01589 (SEQ ID NO: 4) or a portion thereof, or a Sushi domain. The term "IL-2 binding fragment of an IL-2Rα polypeptide" as used herein means a fragment of any length truncated from the C-terminus to the N-terminus in the extracellular domain of an IL-2Rα polypeptide, which retains at least 90% of the binding activity to the binding ligand of the IL-2Rα polypeptide, i.e., IL-2, compared to the original complete binding activity.

[0078] In the present invention, "IL-2Rα mutant" means an IL-2Rα protein including a mutation at one or more positions that affect the interaction with IL-2 in various forms of IL-2Rα molecules including the extracellular domain of IL-2Rα (SEQ ID NO: 4), truncated forms of IL-2Rα, or forms in which IL-2Rα is linked to other molecules. The mutation may include substitution, deletion, truncation or modification of wild-type amino acid residues.

[0079] In the present invention, the "IL-2Rα mutant" may include a mutation in the amino acid sequence of IL-Rα to stabilize the complex by increasing the affinity of IL-Rα to IL-2. In the present invention, the position of the mutation of IL-2Rα is based on the sequence of SEQ ID NO: 4. For example, the IL-2Rα mutant of the present invention may include an amino acid substitution at one or more positions of L2, M25, N27, S39, L42, L45, N57, I118, H120 and K153 in the extracellular domain of IL-2Rα of SEQ ID NO: 4 or a truncated form thereof. In the present invention, the IL-2Rα mutant may include 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. In addition, the IL-2Rα mutant of the present invention may include one or more mutations among F121A, V122A and A114D.

[0080] In the present invention, an exemplary IL-2Rα mutant may be a mutant of SEQ ID NO: 5 having an L42I amino acid substitution in a truncated form of the extracellular domain of IL-2Rα, but is not limited thereto.

[0081] IL-15 / IL-15Rα

[0082] IL-15 is secreted by immune cells including mononuclear phagocytes after viral infection. IL-15 induces the proliferation of NK cells and other cells of the immune system and participates in killing virus-infected cells and cancer cells. IL-15 also binds to the medium affinity IL-2Rβγ receptor, but binds to the IL-15Rα receptor with much higher affinity. IL-15Rα binds to IL-2βγ and forms a functional high affinity receptor αβγ.

[0083] In the present invention, unless otherwise specified, "interleukin-15", "IL-15", "IL-15 protein", or "IL-15 polypeptide" includes IL-15 from vertebrates including mammals (e.g., primates (e.g., humans)) and rodents (e.g., mice and rats). In the present invention, the IL-15 polypeptide may be a human IL-15 polypeptide having an amino acid sequence from 49 to 162 of Uniprot P40933 (SEQ ID NO: 6) or a variant of the IL-15 polypeptide that at least partially retains the activity of IL-15. In addition, the IL-15 polypeptide may include one or more amino acid modifications that increase the binding to the IL-15Rα polypeptide or be circularly permutated.

[0084] In the present invention, unless otherwise specified, "IL-15Rα protein", "IL-15Rα polypeptide", "IL-15Rα", "CD215" or "interleukin-15 receptor α" means the α-subunit of the IL-15 receptor 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 may be a truncated form or mutant that retains at least a portion of the activity of human IL-15Rα or IL-15Rα. In the present invention, the human IL-15Rα polypeptide used as a reference has the amino acid sequence from 31 to 267 of UniProt Q13261 (SEQ ID NO: 7). In the present invention, the IL-15Rα polypeptide may be an IL-15Rα polypeptide having an amino acid sequence of SEQ ID NO: 7 or an IL-15 binding fragment of an IL-15Rα polypeptide, 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 an IL-15Rα polypeptide" as used herein means a fragment of any length truncated from the C-terminus to the N-terminus in the extracellular domain of an IL-15Rα polypeptide, which retains at least 90% of the binding activity to the binding ligand of the IL-15Rα polypeptide, i.e., IL-15, compared to the original complete binding activity.

[0085] Anti-PD-1 antibody or antigen-binding fragment thereof

[0086] In the present invention, anti-PD-1 antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof that can bind to PD-1, especially PD-1 polypeptide expressed on the cell surface. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof has a KD value of 10 -7M or less binds to PD-1; in some examples, the KD value is 10 -8 M, 10 -9 M, 10 -10 M or 10 -11 In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment binds to PD-1 with a KD value of 10 even in a low pH environment. - 9 M or less, preferably KD value 10 -10 M or less, preferably KD value 10 -11 In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention binds to PD-1 at 9×10 -10 Binds to PD-1 with a KD below M.

[0087] 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, preferably the heavy chain variable region comprises: a heavy chain complementarity-determining region 1 (HCDR1) comprising an amino acid sequence of SEQ ID NO: 9; a HCDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 15; and a HCDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 16 to 31, or comprising a HCDR variant having a conservative amino acid substitution or three or less amino acid mutations compared to 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 to 35; a LCDR2 comprising an amino acid sequence of SEQ ID NO: 36; and a LCDR3 comprising an amino acid sequence of SEQ ID NO: 37. The amino acid sequence of NO:37, or a LCDR variant having a conservative amino acid substitution or three or less amino acid mutations compared to these sequences.

[0088] In the present invention, the anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain variable region and / or a light chain variable region, preferably, the heavy chain variable region comprises CDR1, CDR2 and CDR3 of the heavy chain variable region comprising 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 the light chain variable region comprising the amino acid sequence of SEQ ID NO: 39, 41, 43 or 45.

[0089] Methods for determining CDR sequences in a specific VH region or VL region are known in the relevant art, including the Kabat numbering system, the Chothia numbering system, the Martin numbering system, the Gelfand numbering system, and the international immunogenetics information system (IMGT) numbering system (see Dondelinger et al., Front. Immunol. (2018) 9: 2278, etc.). Antibodies or functional fragments thereof according to preferred embodiments of the present invention may include CDR sequences according to the Kabat numbering system, but are not limited thereto. Antibodies or functional fragments thereof that include CDR sequences that can be defined according to known CDR determination methods in the variable region sequences of SEQ ID NO: 38 to 45 are all included in the scope of the present invention.

[0090] In the present invention, the anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain variable region and / or a light chain variable region, preferably, the heavy chain variable region comprises a sequence as represented by SEQ ID NO: 38, 40, 42 or 44, or comprises a variant having 1 to 10 amino acid mutations compared with these sequences; the light chain variable region comprises a sequence as represented by SEQ ID NO: 39, 41, 43 or 45, or comprises a variant having 1 to 10 amino acid mutations compared with these sequences.

[0091] In the present invention, the heavy chain variable region of the anti-PD-1 antibody or its antigen-binding fragment may have an amino acid sequence that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity to the amino acid sequence of SEQ ID NO: 38, 40, 42 or 44.

[0092] In the present invention, the light chain variable region of the anti-PD-1 antibody or its antigen-binding fragment may have an amino acid sequence that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity to the amino acid sequence of SEQ ID NO: 39, 41, 43 or 45.

[0093] The so-called "conservative amino acid substitution" is an amino acid residue replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. In the case where two or more amino acid sequences differ from each other due to conservative substitutions, the similarity or similarity can be adjusted upward to correct the conservative nature of the substitution. The means for making such adjustments are well known to those skilled in the relevant art. For example, refer to the literature included in this case as a reference (reference: Pearson (1994) Methods Mol.Biol. 24: 307-331). Examples of amino acid groups having side chains with similar chemical properties include: 1) basic side chains: lysine, arginine, and histidine; 2) acidic side chains: aspartic acid and glutamic acid; 3) uncharged polar side chains: asparagine, glutamic acid, serine, threonine, tyrosine, and cysteine; 4) non-polar 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-hydroxy side chains: serine and threonine; 8) amide-containing side chains: asparagine and glutamic acid; and 9) aromatic side chains: phenylalanine, tyrosine, tryptophan, and histidine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamate. Alternatively, a conservative substitution is any change with a positive value in the PAM250 log-likelihood matrix of acceptable point mutations disclosed in the literature included herein by reference (reference: Gonnet et al. (1992) Science 256: 1443-45). A "moderately conservative" substitution is any change with a non-negative value in the PAM250 log-likelihood matrix. In the present invention, the scope of antibodies or antigen-binding fragments thereof limited to a specific sequence is defined as also including sequences having conservative substitutions of the corresponding sequence.

[0094] In some examples, the anti-PD-1 antibody or antigen-binding fragment thereof may be a recombinant antibody, preferably a murine antibody, a chimeric antibody, a humanized antibody or a human antibody.

[0095] In some examples, 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 may be derived from human kappa, lambda chain or mutant sequences thereof. For example, the heavy chain constant region is a human IgG4 heavy chain constant region including an S228P mutation (SEQ ID NO: 46) and / or the light chain constant region may be a human immunoglobulin kappa constant region chain (SEQ ID NO: 47).

[0096] 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.

[0097] In some examples of anti-PD-1 antibodies or antigen-binding fragments thereof, the antibodies are humanized antibodies, and the light chain framework region (FR) and heavy chain framework region of the antibodies are derived from human germline light chain and heavy chain or mutant sequences thereof, respectively.

[0098] In an example of a portion of an anti-PD-1 antibody or an antigen-binding fragment thereof, the antibody is a human antibody, and the entire sequence is derived from human gonadal light and heavy chains or mutant sequences thereof.

[0099] In the present invention, the anti-PD-1 antibody or antigen-binding fragment thereof may also be derived from a known anti-PD-1 antibody that binds to human PD-1 (SEQ ID NO: 48). For example, it may be pembrolizumab (Keytruda ), nivolumab (Opdivo ), tislelizumab (BeiGene and Novartis), cemiplimab (Ribitac Regeneron), dostarlimab (Jemperli Glaxo Smith Kline (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 comprising a heavy chain and a light chain thereof or comprising a complementarity determining region (CDR), or an antibody or antigen-binding fragment thereof that can bind to the same antigenic determining region as these antibodies or compete with these antibodies.

[0100] Other antigen binding domains

[0101] As other immune cell antigen binding domains other than anti-PD-1 antibodies, it can be components, antibodies or antigen-binding fragments thereof that selectively bind to, for example, NKG2a, CD8a, FcRL6, CRTAM (CD355), LAG3, TIM3, CTLA4, TIGIT, etc.

[0102] As examples of the other immune cell antigen binding domains, there are BMS-986315 (anti-NKG2a, WO 2020 / 102501), monalizumab (anti-NKG2a); mAbs OKT8 or 51.1 (anti-CD8a antibody, U.S. Patent No. 10,428,155, WO 2020 / 060924), WO 2019 / 023148 ​​or U.S. Patent No. 10,072,080; the anti-FcRL6 antibody 1D8 or 7B7 disclosed in the literature [Shreeder et al., (2010) Journal of Immunology (J.Immunol.) 185:23] and [Shreeder et al., (2008) European Journal of Immunology (Eur.J.Immunol.) 38:3159] or WO2019 / 094743; the anti-CRTAM antibody 5A11 disclosed in WO 2019 / 086878 or WO 2009 / 029883; relatlimab (BMS-986016, anti-LAG-3), US 2011 / 0150892 and WO The anti-LAG-3 antibodies 25F7, 26H10, 25E3, 8B7, 11F2 or 17E5 disclosed in 2014 / 008218, or the 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.

[0103] The fusion protein of the present invention includes, in addition to the immune cell antigen binding domain, an antigen binding domain that targets tumor cells and can be formed by a bispecific protein.

[0104] Fc region

[0105] In the present invention, the term "Fc domain" or "Fc region" means the C-terminal region including the CH2 domain and the CH3 domain (or the CH2 domain, the CH3 domain and the CH4 domain) in the heavy chain constant region of the antibody, and is used to cover the meaning of the wild-type Fc region and its variants. Although the boundaries of the Fc region of the IgG heavy chain may vary slightly, the human IgG heavy chain Fc region may generally refer to the region from Cys226 or Pro230 to the C-terminal region of the heavy chain, or the region further including the hub in the region. The numbering of amino acid residues in the Fc region is based on the EU numbering system also known as the EU index according to the Kaba document (Kaba et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes of Health (NIH) Press, 1991) that defines the residue numbering in the human immunoglobulin heavy chain.

[0106] In the present invention, the term "wild-type Fc region" includes an amino acid sequence identical to the amino acid sequence of the Fc region of an immunoglobulin found in nature.

[0107] In the present invention, the term "Fc region variant" includes one or more amino acid residues that are different from the wild-type Fc region, and may be simply referred to as "Fc variant".

[0108] In the present invention, the Fc variant may have about 80% or more, preferably about 90% or more identity with the wild-type Fc region sequence as a parent.

[0109] The fusion protein of the present invention includes an immunoglobulin Fc region formed by two chains, a first Fc chain and a second Fc chain. For example, the Fc domain of IgG is a dimer formed by two chains, and each chain includes CH2 and CH3 IgG heavy chain constant regions.

[0110] In the present invention, the Fc region can be a homodimer or a heterodimer. In a structure in which the first chain and the second chain of the Fc region are connected to different protein molecules (e.g., IL-2 and IL-2Rα), in order to obtain the desired fusion protein molecule with a high yield, it is important to design the Fc chains connected to the same protein molecule so that no bond is formed between them, but bonds are formed between the Fc chains connected to different protein molecules. Therefore, it is preferred to use an Fc heterodimer in which modifications are introduced into the Fc region so as to increase the repulsive force between the same chains and the binding force between different chains.

[0111] In the present invention, the Fc heterodimer is formed by a first Fc chain and a second Fc chain that are different from each other. The first Fc chain and the second Fc chain may form a tertiary structure and may interact with each other through covalent bonds such as disulfide bonds, or hydrogen bonds, ionic bonds, Van der Waals bonds, non-covalent interactions such as hydrophobic interactions, etc.

[0112] The Fc heterodimer in the present invention can also use known techniques for introducing mutations into the CH3 region in order to promote the binding between the two different chains. 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, an amino acid with a large branch size is replaced with an amino acid with a small branch size at one chain, and a small amino acid is replaced with a large amino acid at the other chain. For example, as described in WO96 / 27011 or in the literature [Ridgway, JB et al., Protein Eng 9 (1996) 617-621; Merchant, AM et al., Nat Biotechnol 16 (1998) 677-681], a heteromeric polypeptide in which T366 (based on EU numbering, the same below) of one chain is replaced with a large residue such as Y or W, and Y407, T394, T366, etc. of the opposite chain are replaced 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 introduce cysteine ​​(C) at the corresponding position of each CH3 and further deform it so that a disulfide crosslink (KiHs-s) can be formed between the two CH3 domains.

[0113] 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 (based on EU numbering). In addition, by introducing an E356C mutation or an S354C mutation into the CH3 domain of the knob chain and introducing a Y349C mutation into the CH3 domain of the hole chain, additional disulfide crosslinks may be formed between the CH3 domains (Merchant, AM et al., Nature Biotech 16 (1998) 677-681). For example, the Fc heterodimer used in the present invention may use KiHs-s.

[0114] In addition, in the present invention, either 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 chain further includes an amino acid substitution of K360E and Q347E, and the other chain may further include an amino acid substitution of Q347R or an amino acid substitution of K360R and Q347R.

[0115] The preferred Fc heterodimer used in the present invention comprises a first Fc chain and a second Fc chain, wherein the first Fc chain comprises an amino acid substitution at T366, the second Fc chain comprises an amino acid substitution at one or more of T366, L368 and Y407, one of the first Fc chain and the second Fc chain further comprises an amino acid substitution at K360, and the other chain further comprises an amino acid substitution at Q347. In addition, the chain comprising the amino acid substitution at K360 of the first Fc chain and the second Fc chain further comprises an amino acid substitution at Q347 and (or) the other chain comprising the amino acid substitution at Q347 may further comprise an amino acid substitution at K360.

[0116] In the present invention, preferably in the Fc heterodimer,

[0117] (A1) the first Fc chain comprises amino acid substitutions of T366W and K360E, and the second Fc chain comprises amino acid substitutions of T366S, L368A, Y407V and Q347R, or

[0118] (A2) the first Fc chain comprises amino acid substitutions of T366W and Q347R, and the second Fc chain comprises amino acid substitutions of T366S, L368A, Y407V and K360E, or

[0119] (A3) the first Fc chain comprises amino acid substitutions of T366W, K360R and Q347R, and the second Fc chain comprises amino acid substitutions of T366S, L368A, Y407V, K360E and Q347E, or

[0120] (A4) The first Fc chain may include amino acid substitutions of T366W, K360E and Q347E, and the second Fc chain may include amino acid substitutions of T366S, L368A, Y407V, K360R and Q347R.

[0121] In addition, in the present invention, the Fc domain may be derived from human IgG1, IgG2, IgG3 or IgG4 or a mutant sequence thereof, and may further include other mutations for changing the structural stabilization of the Fc domain or the binding affinity to the Fc receptor. For example, in the case where the Fc region is derived from IgG4, the S228P mutation may be further included. The S228P mutation reduces the exchange of Fab arms that occurs in the Fab region between two IgG4 antibodies. For example, in the case where the Fc region is derived from IgG, in order to reduce the effector function (based on EU numbering), an amino acid substitution may be included at a position selected from E233, L234, L235, G236, G237, N297, L328, P329 and P331. For example, the Fc region may include an amino acid substitution at a position selected from the group consisting of L234, L235 and P329. Alternatively, the Fc region may include the amino acid substitutions of L234A and L235A, or include the amino acid substitutions of L234A, L235A and P329G. In addition, in the present invention, the Fc region may lack a CH2 domain.

[0122] The amino acid sequences of exemplary Fc chains used in the present invention are shown in SEQ ID NO:49 and SEQ ID NO:50.

[0123] Method for producing fusion protein

[0124] The fusion protein of the present invention can be obtained by expressing an expression vector including a polynucleotide sequence encoding the fusion protein in a host cell.

[0125] In the present invention, the term "polynucleotide" means an isolated nucleic acid molecule or structure composed of deoxyribonucleotides or ribonucleotides in a single-stranded or double-stranded form, such as messenger ribonucleic acid (mRNA), plasmid deoxyribonucleic acid (DNA), and virus-derived RNA. The polynucleotide can be generated by constituting the fusion protein of the present invention or by changing the wild-type sequence of each domain that forms the fusion protein, and can further include elements required for the expression of the fusion protein of the present invention (e.g., a signal peptide). In addition, a polynucleotide sequence encoding the entire amino acid sequence of the fusion protein of the present invention can be synthesized by polymerase chain reaction (PCR) polymerization reaction for use.

[0126] In one example of the present invention, when the fusion protein of the present invention adopts a heterodimer structure, the expression vector may include a first expression vector and a second expression vector, wherein the first expression vector includes a polynucleotide sequence encoding an amino acid sequence of a first monomer chain connected to IL-2, and the second expression vector includes a polynucleotide sequence encoding an amino acid sequence of a second monomer chain connected to IL-2Rα. In an exemplary embodiment of the present invention, the first expression vector includes a polynucleotide sequence encoding an amino acid sequence of SEQ ID NO: 51 and SEQ ID NO: 57, and the second expression vector includes a polynucleotide sequence encoding an amino acid sequence of SEQ ID NO: 51 and SEQ ID NO: 58.

[0127] In one embodiment of the present invention, the fusion protein of the present invention is produced by expressing the first expression vector and the second expression vector in a host cell separately and then mixing the obtained proteins, or by expressing the first expression vector and the second expression vector simultaneously in the same host cell and producing them in a single cell. Preferably, the fusion protein of the present invention is produced in a single cell.

[0128] Therapeutic administration and dosage forms

[0129] In one aspect, the present invention provides a fusion protein according to the present invention and a pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier. The pharmaceutical composition can be used as an immunotherapeutic agent in the treatment of, for example, a tumor, a cancer, a metastatic tumor, or a metastatic cancer. The pharmaceutical composition can also be used to treat infectious diseases.

[0130] 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 agent that enhances the sensitivity of cells to immunomodulators, cell growth inhibitors, cell attachment inhibitors, cytotoxic agents, cell death agents, or cell death inducing agents. In a specific embodiment, the additional therapeutic agent may be an anticancer agent, such as a microtubule disintegrator, a metabolic antagonist, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormone therapy, a kinase inhibitor, a receptor antagonist, a tumor cell cell death agent, or an angiogenesis inhibitor.

[0131] In the present invention, the tumor, cancer, metastatic tumor or metastatic cancer can be 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, stomach cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma and classic Hodgkin's lymphoma (classic Hodgkin's lymphoma, CHL), primary mediastinal large B cell lymphoma, T cell / histiocyte-rich B cell lymphoma, human herpes virus (Epstein-Barr virus, EBV)-positive and -negative post transplant lymphoproliferative disorder (PTLD), or EBV-associated diffuse large B cell lymphoma (diffuse large B-cell lymphoma). The invention relates to non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer,

[0132] In the present invention, the infectious disease may be a disease selected from the group consisting of chronic viral infections including hepatitis B, hepatitis C, herpes virus, Epstein-Barr virus, human immunodeficiency virus (HIV), cytomegalovirus, herpes simplex virus type 1, herpes simplex virus type 2, human papillomavirus, adenovirus, Kaposi's sarcoma associated with herpes virus infection, Torque teno virus, JC virus or BK virus infection, and is not limited thereto.

[0133] The pharmaceutical composition according to the present invention can be used to treat early stage or late stage symptoms of cancer. In one aspect, the pharmaceutical composition according to the present invention can be used to treat metastatic cancer. The pharmaceutical composition according to the present invention is useful in reducing or inhibiting or shrinking tumor growth of both solid tumors and blood cancers. In a specific aspect, the treatment of the pharmaceutical composition according to the present invention causes a reduction of more than 50% of the tumor in the subject, a reduction of more than 60%, a reduction of more than 70%, a reduction of more than 80%, and a reduction of more than 90%. In a specific aspect, the pharmaceutical composition can be used to prevent the recurrence of tumors. In a specific aspect, the pharmaceutical composition is useful in prolonging overall survival in subjects with cancer. In a part of the aspect, the pharmaceutical composition is useful in maintaining long-term survival and reducing the toxicity brought by chemotherapy or radiotherapy in patients with cancer.

[0134] In another aspect of the present invention, the pharmaceutical composition according to the present invention can be used as an adjuvant therapy together with any other agent or any other therapy known to those skilled in the relevant art that is useful in treating cancer.

[0135] The pharmaceutical composition according to the present invention can be administered with other agents contained in the dosage form to provide suitable carriers, excipients, and improved handling, delivery, tolerance, etc. A plurality of suitable dosage forms can be identified in the formulary known to all pharmaceutical chemists (reference: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania (PA)). Such dosage forms include, for example, powders, pastes, ointments, gels, waxes, oils, liquids, liquids (cationic or anionic) containing vesicles (e.g., transfection reagents (LIPOFECTIN TM )), DNA conjugates, anhydrous absorption slurries, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax (reference: Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol (1998) 52:238-311).

[0136] The dosage of the fusion protein can be variable according to the age and weight of the subject to be administered, the target disease, the condition, the route of administration, etc. The fusion protein of the present invention is administered to the patient in a suitable manner after one or a series of treatments. The initial preparatory 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) according to the type and severity of the disease, whether it is administered separately or continuously injected more than once. The typical daily dosage can be about 1 μg / kg to 100 mg / kg or more than 100 mg / kg according to the factors mentioned. After repeated administration for more than a few days, according to the patient's state, it can usually be sustained until the desired degree of disease symptom suppression occurs. An exemplary dosage can be about 0.005 mg / kg to about 10 mg / kg. In another non-limiting example, the dosage may also include 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 and any range derivable therein. Thus, the patient may be administered 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). The dosages described above may be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives about 2 to about 20, or for example about 6, doses of the fusion protein). Following an initial higher loading dose, more than one lower dose may also be administered.

[0137] The pharmaceutical compositions of the present invention can be administered using a variety of delivery systems such as liposome encapsulation, microparticles, microcapsules, recombinant cells expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al. (1987) J. Biol. Chem. 262: 4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any common route, such as by injection or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0138] The pharmaceutical compositions of the present invention may also be delivered via vesicles, particularly liposomes (see, e.g., Langer (1990) Science 249: 1527-1533). It is also contemplated that nanoparticles may be used to deliver the fusion proteins of the present invention. Antibody-conjugated nanoparticles may be used for both therapeutic and diagnostic purposes. Antibody-conjugated nanoparticles and methods of preparation and use are described in detail in the literature included herein by reference (see, Arruebo, M. et al. 2009, "Antibody-conjugated nanoparticles for biomedical applications" in J. Nanomat Volume 2009, Article ID 439389, p. 24, doi: 10.1155 / 2009 / 439389). Nanoparticles are developed and coupled to antibodies contained in pharmaceutical compositions, so that tumor cells or autoimmune tissue cells or virus-infected cells can be targeted. Nanoparticles for drug delivery are also described in, for example, US 8257740 or US 8246995, each of which is incorporated herein in its entirety.

[0139] Under certain conditions, the pharmaceutical composition of the present invention can be delivered using a controlled release system. In one aspect, a pump can be used. In another aspect, a polymeric substance can be used. In another aspect, a controlled release system can be configured near the target of the composition and only a portion of the systemic dose is required. Injectable preparations may include administration types for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared using formally known methods. Injectable preparations can be prepared, for example, by dissolving or suspending or emulsifying the described antibody or its salt in a sterile water-soluble medium or an oily medium commonly used for injection. As water-soluble medium for injection, there are physiological saline, isotonic solution containing glucose and other auxiliary agents that can be used together with appropriate solubilizing agents, such as alcohol (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) additive) of hydrogenated castor oil]. As oily medium, sesame oil, soybean oil, etc. that can be used together with solubilizing agents such as benzyl benzoate and benzyl alcohol can be used. Therefore, the prepared injection is preferably loaded into an appropriate ampoule.

[0140] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously via a standard needle and syringe. In addition, 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. Reusable pen delivery devices generally use a replaceable cartridge containing the pharmaceutical composition. If all the pharmaceutical composition in the cartridge is applied and the cartridge becomes empty, the empty cartridge is easily discarded and can be 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 replaceable cartridge. In contrast, a disposable pen delivery device is pre-filled with a pharmaceutical composition contained in a storage place in the device. If the pharmaceutical composition in the storage place is exhausted, the entire device is discarded. Multiple reusable pen and autoinjector delivery devices are used for subcutaneous delivery of the pharmaceutical composition of the present invention.

[0141] Advantageously, the pharmaceutical composition for oral or non-oral use described above is prepared into a unit dosage form suitable for matching the dosage of the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of fusion protein contained is generally about 5 mg to about 500 mg per unit dosage, especially the injection form of the administration form, and preferably contains about 5 mg to about 100 mg of the antibody and about 10 mg to about 250 mg of the antibody relative to other administration forms.

[0142] The present invention also provides a method for preventing, improving or treating a tumor, cancer, metastatic tumor, metastatic cancer or infectious disease in an individual, the method comprising administering the fusion protein of the present invention to the individual in need of preventing, improving or treating a tumor, cancer, metastatic tumor, metastatic cancer or infectious disease.

[0143] Example

[0144] Hereinafter, the present invention will be described in more detail by way of examples. These examples are provided only for the purpose of describing the present invention in more detail as examples, and the scope of the present invention is not limited to these examples. It is self-evident to those skilled in the art that various modifications may be made to the examples within the scope of the present invention.

[0145] The molecular biology reagents used in this case are used according to the instructions of the manufacturer. Recombinant DNA technology is described in the literature [Sambrook et al., Molecular Cloning: Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1991], using standard methods to implement. The numbering of the amino acid residues specific to immunoglobulin molecules or antibody molecules is represented by the amino acid position starting from the N-terminus based on the EU index obtained by the literature [Kaba et al., Immunology Protein Sequences (Sequences of Proteins of Immunological Interest), the fifth edition, NIH Press, 1991].

[0146] Example 1. Production of cytokine fusion protein

[0147] Example 1.1 Structural design of fusion protein

[0148] In order to increase tumor-specific exhausted effector T cells (exhausted T eff ) specific activity, and importantly, the specific induction of T eff Cytokines that promote cell proliferation and / or activation are selectively delivered to T cells within the tumor. eff To this end, the structure of the cytokine fusion protein is designed to promote T eff Cytokines and T cell proliferation and / or activation eff The cell targeting domains are linked to each other and are expressed in cis in the T eff As an inducible T eff Cell proliferation and / or activation of cytokines, considered through the T eff The βγ-subunit of the interleukin-2 receptor (IL-2Rβγ) expressed in the cells transmits signals to interleukin-2 (hereinafter referred to as IL-2 or IL2) and interleukin-15 (hereinafter referred to as IL-15 or IL15), and IL-2 is selected as an embodiment. IL-2 is used as T eff The cell targeting domain is connected to the Fc of the anti-PD-1 immune anticancer antibody to form a bifunctional fusion protein.

[0149] It is known that immune proteins, such as IL-2, present in the blood play an important role in intracellular signaling in various cells including immune cells. Therefore, it is necessary to increase the selectivity of strategies to reduce the toxicity or side effects caused by exogenous cytokines and to target only the T cells in the tumor. eff Cells play a specific role.

[0150] The present inventors linked IL-2 to the C-terminus of the first Fc chain of the anti-PD-1 antibody, and linked the α-subunit of the IL-2 receptor (IL-2Rα or CD25) to the C-terminus of the second Fc chain, thereby inducing an interaction between IL-2 and IL-2Rα, which are respectively linked to the first Fc chain and the second Fc chain of the anti-PD-1 antibody, thereby forming a form in which IL-2 is seized by IL-2Rα. The IL-2 protein and the IL-2Rα protein are respectively linked only to the Fc terminal of the anti-PD-1 antibody, and IL-2 and IL-2Rα are not covalently linked to each other (see FIG. 1 ).

[0151] Figures 1 to 3 show exemplary structures of the "cis" fusion protein of the present invention. As shown in Figure 2, after IL-2 and IL-2Rα are covalently bonded to form a complex through a linker, a structure in which the cytokine complex is connected to the Fc of an anti-PD-1 antibody through either side of IL-2 and IL-2Rα can also be achieved. In addition, as a solution to the problem of toxicity or side effects caused by exogenous cytokines, the structure of a fusion protein in which only IL-2Rα or IL-15Rα is connected as a cytokine domain to an anti-PD-1 antibody so that endogenous cytokines can be used has also been studied.

[0152] In the structure designed by the inventors, since IL-2 is occupied by IL-2Rα, the regulatory T cells (T cells) expressing the high affinity IL-2 receptor (or IL-2Rαβγ for short) expressed by the α-subunit, β-subunit (IL-2Rβ or CD122) and γ-subunit (IL-2Rγ or CD132) of the IL-2 receptor are activated. reg ), Foxp3-negative CD4 T cells, some innate lymphocytes or other endothelial cells, but can bind to cytotoxic CD8+ T cells, memory T cells, NK-T cells, etc. that express the medium-affinity IL-2 receptor composed of IL-2Rβ and IL-2Rγ without IL-2Rα (or IL-2Rβγ for short). eff The cells further bind specifically.

[0153] Example 1.2 Sequence structure of fusion protein and preparation of expression vector

[0154] Anti-PD-1 binding domain

[0155] The present inventors used the Fab domain of the humanized anti-PD-1 antibody 1G1-h70 developed in previous studies as the anti-PD-1 binding domain. The amino acid sequence of the Fab domain of anti-PD-1 1G1-h70 used in this study is shown below.

[0156] Light chain VL-CL (SEQ ID NO: 51)

[0157] DIVMTQTPLSLSVTPGQPASISCRSSQNIVHSQGDTYLEWYLQKPGQSPQLLIYKVSKRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (The underlined part indicates the light chain variable region.)

[0158] Heavy chain VH-CH1 (SEQ ID NO: 52)

[0159] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGMIHPNSDTTTYNEKFKNRV TMTRDTSISTAYMELSRLRSDDTAVYYCAGTDQAAWFAFWGQGTTVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV (The underlined part indicates the heavy chain variable region.)

[0160] Linker design

[0161] The interaction between IL-2 / IL-2Rα is maintained in the form of each chain end connected to the dimeric Fc domain of the anti-PD-1 immune anticancer antibody. At the same time, in order to analyze the structure of the IL-2 used to bind to IL-2Rβγ expressed on the cell surface, a crystal structure analysis was performed to determine the appropriate length of the connection site between the C-terminus of the Fc domain and the N-terminus of each IL-2 and IL-2Rα.

[0162] The overall crystal structure of the known anti-PD-1 antibody, Keytruda, was superimposed on the crystal structure of the Fab domain of human PD-1 (hPD-1) / 1G1-h70 to simulate the way in which the anti-PD-1 antibody binds to hPD-1. The C-terminus of the Fc domain of the anti-PD-1 antibody was positioned closest to the N-terminus of each of IL-2 and IL-2Rα without colliding with the IL-2 quaternary complex, and then the distance from the C-terminus of the Fc domain to the N-terminus of each of IL-2 and IL-2Rα was measured. Therefore, the length of the connection site between the C-terminus of the Fc domain and the N-terminus of each of IL-2 and IL-2Rα was determined to be 59.2A and 53.6A, respectively. Figure 4 ). By modeling based on this structure, linkers of appropriate lengths were designed to connect IL-2 and IL-2Rα to the Fc terminus of anti-PD-1 antibodies, respectively.

[0163] Improved heterodimeric Fc region

[0164] In order to improve the manufacturing yield of the fusion protein of the present invention, the Fc region of the anti-PD-1 antibody is prepared using heterodimeric Fc so that the heterodimeric binding between the Fc chain linked to IL-2 and the Fc chain linked to IL-2Rα is dominant over the homodimeric binding between Fc chains with the same sequence. To this end, knob-into-hole (KiH) mutations are introduced into the Fc region together with charge / hydrophobic amino acid mutations.

[0165] The amino acid substitutions T366W and Q347R are included in the first Fc chain (SEQ ID NO: 49), and the amino acid substitutions T366S, L368A, Y407V and K360E are included in the second Fc chain (SEQ ID NO: 50). These heterodimeric Fc variants significantly increase the production yield of heterodimeric fusion proteins to more than about 90%.

[0166] High affinity variant of IL-2Rα

[0167] In order to enhance the binding force between IL-2 and IL-2Rα, a high affinity IL-2Rα variant is used. The IL-2RαL42I variant (SEQ ID NO: 5) in which the leucine amino acid at position 42 of the IL-2Rα protein is replaced 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 Biacore 8K, a surface plasmon resonance (SPR) for real-time observation of protein-protein interactions.

[0168] [Table 1]

[0169]

[0170] Production of expression vector

[0171] An expression vector was prepared based on pTRIOZ_hIgG4(S228P) (InvivoGen) expressing the human IgG4 constant region (S228P) (SEQ ID NO: 46 and SEQ ID NO: 47) so that the IL-2C125S (SEQ ID NO: 2) or CD25 L42I (SEQ ID NO: 5) gene was placed at the C-terminus of the Fc region. The C125S mutation of IL-2 is introduced to prevent cysteine ​​mispairing in Escherichia coli (E. coli), as shown in the FDA-approved IL-2 product, i.e., Proleukin (aldesleukin), and does not affect biological activity (see Wang A et al., Site-specific mutagenesis of the human interleukin-2 gene: structure-function analysis of the cysteineresidues, Science, 224: 1431-1433, 1984). In the present case, the term "wild-type IL-2" includes not only natural or recombinant human IL-2, but also all IL-2 in a cyclic substitution form with new N-terminal and C-terminal modifications while not modifying the binding properties of IL-2 or aldesleukin with the C125S mutation or natural human IL-2. As a non-limiting example of cyclic replacement of IL-2, there is a method in which the existing N-terminal part (Ser4) and the C-terminal part (Thr133) are connected by a linker, and truncated to form a new N-terminal part (Ser75) and C-terminal part (Gln74), and the IL-2 helix is ​​connected in the order of CDAB, etc.

[0172] The variable regions of each were prepared by including a mouse immunoglobulin signal peptide in front of the heavy chain constant region and the light chain constant region of the expression vector. The vector was prepared basically according to the instructions of the vector manufacturer. The signal peptide was connected to the VH or VL sequence of the antibody using overlapping PCR and placed in the pTRIOZ_hIgG4(S228P) vector. In the case of the VH sequence, the vector was cut with Mlu I / Nhe I restriction enzymes and then inserted, and in the case of the VL sequence, the vector was cut with Asc I / BsiW I restriction enzymes and then inserted.

[0173] At this time, the first chain (knob) expression vector was prepared by changing the nucleotide sequence in a manner that induces T366W and Q347R mutations in the Fc region, and the second chain (hole) expression vector was prepared by using PCR technology to induce T366S, L368A, Y407V and K360E mutations in the Fc region.

[0174] The first chain (knob) expression vector was prepared by amplifying the IL-2C125S gene by PCR after cutting the C-terminal part with the Avr II restriction enzyme to include a 15-amino acid linker, and then using the NEBuilder HiFi DNA Assembly Kit. The second chain (hole) expression vector was prepared by amplifying the CD25 L42I gene by PCR after cutting the C-terminal part with the Avr II restriction enzyme to include a 20-amino acid linker, and then using the NEBuilder HiFi DNA Assembly Kit. The primers used at this time are shown in the following table.

[0175]

[0176] The amino acid sequences encoded by each expression vector are shown below.

[0177] Light chain: VL-CL (SEQ ID NO: 51)

[0178] DIVMTQTPLSLSVTPGQPASISCRSSQNIVHSQGDTYLEWYLQKPGQSPQLLIYKVSKRFSGVPDRFSG SGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0179] Heavy chain 1_knob_with_IL2C125S: VH-CH1-Hinge-CH2-CH3-Linker-IL2C125S (SEQ ID NO: 57)

[0180]

[0181] Heavy chain 2_hole_with_CD25L42I: VH-CH1-hinge-CH2-CH3-linker-CD25L42I (SEQ ID NO: 58)

[0182]

[0183]

[0184] Example 1.3 Expression and purification of fusion protein (fusion protein A)

[0185] The first chain (knob) expression vector (linking the first heavy chain and light chain expression of IL2C125S) and the second chain (hole) expression vector (linking the second heavy chain and light chain expression of CD25L42I) described in Example 1.2 were co-expressed in ExpiCHO TM ) cells were transfected to temporarily express each protein.

[0186] The initial cell density of the transfection was 6×10 6 The cells / ml and expression vector concentration were 0.8 μg DNA / ml. ExpiCHO was used according to the manufacturer's instructions. TM )Expression Medium, OptiPRO TM SFM and ExpiFectamine TM ) transfection kit (Gibco).

[0187] After the transfected cells were cultured in a carbon dioxide incubator until the cell viability reached 75%, they were separated by centrifugation into cell culture fluid containing each protein and cells.

[0188] In order to separate the target protein from the culture solution containing each recombinant protein, affinity chromatography using HiTrap MabselectXtra (Cytiva) column was performed.

[0189] Specifically, after purification by AKTA pioneer (avant) 25 fast protein liquid chromatography (FPLC), phosphate buffered saline (PBS) (3 mmol / L Na2HPO4, 1.1 mmol / L KH2PO4, 0.16 mol / L NaCl, pH 7.2) was used to balance the Helepu MabselectXtra column, and the culture solution obtained by centrifugation was filtered and added using a 0.22 micron filter. After the reaction with the culture solution was completed, the column was cleaned in sequence using PBS (5CV~10CV), 50 mmol / L NaOAc pH 5.2 (5CV~10CV), and the protein was eluted using elution buffer (0.1 mol / L glycine pH 3.0). After adding 3 mol / L NaOAc pH 5.2 buffer to the eluted protein to make the final concentration reach 50 mmol / L~100 mmol / L, the extracted pigment was removed together by centrifugation. After pigment removal, the cells were concentrated using an Amicon tube (molecular weight cut off (MWCO): 50K) and buffer exchanged using acrylonitrile butadiene styrene (ABS) (0.1 mol / L NaOAc, 0.15 mol / L NaCl, pH 5.2) or PBS.

[0190] To confirm the generation of 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 This is the SDS-PAGE result of confirming the purified fusion protein (fusion protein A). It is confirmed that fusion protein A is a form in which IL-2 and IL-2Rα are respectively connected to the Fc region of the anti-PD-1 antibody and has two anti-PD-1 Fab arms ( Figure 1a ) and can be produced in high yield.

[0191] Example 2. Design and production of various fusion proteins

[0192] Example 2.1 Production of a fusion protein with an anti-PD-1 Fab arm (fusion protein B, Figure 1b )

[0193] The structure design and vector preparation were performed in the same manner as in Example 1. The expression vector of Example 1 was used as the primary vector. In this case, the VH-CH1 portion was removed from the heavy chain first chain (knob) vector using PCR. The amino acid sequence encoded by the heavy chain first chain (knob) vector in fusion protein B is shown below.

[0194] Fc_knob_with_IL2C125S: hub-CH2-CH3-linker-IL2C125S (SEQ ID NO: 59)

[0195]

[0196] As the final expression vector, a self-made vector, i.e., pG3.1, was used, which was constructed in a manner that included three multiple cloning sites (MCS) from pcDNA3.1. Each MCS has the same promoter (cytomegalovirus (CMV)), enhancer (CMV) and polyadenylation signal (Poly Asignal) (bovine growth hormone (BGH)). The light chain, heavy chain first chain (knob) and heavy chain second chain (hole) genes cloned in the primary vector were placed in the three MCSs to prepare the expression vector. In ExpiCHO TM ) cells, the expression vector is transfected, each protein is expressed using the same method as in Example 1, and the target protein is separated and purified using the same method as in Example 1.

[0197] Example 2.2 Production of a heterodimeric fusion protein that does not include IL-2 but includes an IL-2Rα protein (fusion protein C, Figure 3c )

[0198] The same method as in Example 2.1 was used for structure design, vector preparation, protein expression and purification, except that the IL-2C125S gene was not linked to the C-terminus of the heavy chain first chain (knob) vector. The light chain and heavy chain second chain (hole) were the same as in Example 1, and the amino acid sequence of the heavy chain first chain (knob) is shown below.

[0199] Heavy chain 1_knob_without_IL2C125S: VH-CH1-hub-CH2-CH3 (SEQ ID NO: 60)

[0200]

[0201] Example 2.3 Production of a homodimeric fusion protein that does not include IL-2 but includes two IL-2Rα proteins (fusion protein D, Figure 3a )

[0202] The same method as in Example 1 was used for structural design, preparation of expression vector, expression and purification of protein, but the expression vector of Example 1 was used to prepare a homodimer 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 a variant Fc region for improving the yield of heterodimer production. The light chain amino acid sequence of fusion protein D was the same as in Example 1 (SEQ ID NO: 51), and the heavy chain amino acid sequence was as follows.

[0203] Heavy chain_with_CD25L42I:VH-CH1-hub-CH2-CH3-linker-CD25L42I (SEQ ID NO:61)

[0204]

[0205] In order to confirm the fusion proteins produced and purified in Examples 2.1 to 2.3, SDS-PAGE was performed on 4% to 15% TGX precast gel (Biorad, BR4561086). The results are shown in FIG6 .

[0206] Example 3. Analysis of Binding Selectivity to IL-2 Receptor Protein

[0207] In order to confirm the binding selectivity of the IL-2 / IL-2Rα heterodimer Fc region of the fusion protein of the present invention to the IL-2 receptor, binding ELISA for IL-2Rα and IL-2Rβγ was performed. 100 μl / well of IL-2Rα-His (Acrobiosystems) and IL-2Rβγ-His (Acrobiosystems) were injected into a 96-well plate (ThermoFisher scientific), sealed and coated overnight at 4°C. After blocking and washing, fusion protein A and control antibody (Fc-IL2, self-made) were diluted to a concentration of 0 to 1000 nanomoles / liter and injected at 100 μl / well, respectively, and incubated at room temperature for two hours. After washing, a secondary antibody (Horse Radish Peroxidase (HRP)-conjugated anti-His antibody) was added and incubated at room temperature for 30 minutes in the dark. After washing, tetramethylbenzidine (TMB) substrate solution (Abcam) was added, and the color reaction was terminated with stop solution (Abcam). The absorbance at 450 nm was measured using a microplate reader (ThermoFisher).

[0208] The results are shown in Figure 7a and Figure 7b Fusion protein A showed no binding affinity to IL-2Rα protein ( Figure 7a ), in contrast, for IL-2Rβγ protein, it exhibited slightly weaker but superior binding to wild-type IL-2 ( Figure 7b ). This situation shows that the fusion protein of the present invention exhibits excellent selectivity for IL-2Rβγ compared with IL-2Rα due to the unique structure in which IL-2 is occupied by IL-2Rα.

[0209] Example 4. Analysis of Binding Selectivity to IL-2 Receptor-Expressing Cells Expressing PD-1

[0210] Since the fusion protein of the present invention has an IL-2 / IL-2Rα complex structure fused with an anti-PD-1 binding domain, the anti-PD-1 binding domain can deplete PD-1 in the tumor microenvironment (TME). + T eff HEK-Blue TM ) reporter gene assay system (Invitrogen) to perform the analysis.

[0211] First, HEK-Blue expressing IL-2Rβγ was used to TM )CD122 / CD132 cells (Invitrogen) and HEK-Blue expressing IL-2Rαβγ TM )IL-2 cells (Invitrogen) were transfected with pCMV3-hPD1 (HG10377-CF) vector to produce PD-1 expressing cells. As a control group, the two cells were transfected with pCMV3-Empty vector (control group DNA) respectively. The transfected cells were injected into 96-well plates at a concentration of 50,000 cells / well and cultured in a 37°C, 5% CO2 chamber. Next, after removing the cell culture supernatant, 100 μl of Dulbecco's Modified Eagle Medium (DMEM) medium was added with 0 to 20 nanomoles / liter of fusion protein A, recombinant human IL-2 (rIL2, Sigma), heterodimer Fc-IL2 (Fc-IL2, self-made), and heterodimer Fc-IL2 / IL2Rα (a structure in which IL-2 and IL-2Rα are linked to heterodimer Fc, Fc-IL2 / IL2Rα; self-made), respectively, and injected into 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, 180 μl / well of a colorimetric assay reagent, i.e., QUANTI-Blue, was used to analyze the cells. TM ) solution (Invitrogen) and incubated in a 37°C incubator in the dark for more than 1 hour. The optical density (OD) of the secreted alkaline phosphatase (secreted embryonic kaline phosphatase, SEAP) activity was measured at 620 nm to 655 nm using a microplate reader. The results are shown in FIG8 .

[0212] The above procedure was repeated twice and the average value was calculated. 50 The values ​​are reported in Table 2 below.

[0213] [Table 2]

[0214]

[0215] As shown in Table 2, surprisingly, the fusion protein of the present invention showed about 340 times higher IL-2 reactivity in IL-2Rβγ cells expressing PD-1 than in cells not expressing PD-1 (see Table 2 and the secondary results shown in Figure 8a and Figure 8b On the other hand, the fusion protein of the present invention exhibits weak IL-2 reactivity to IL-2Rβγ cells that do not express PD-1 ( Figure 8b ), which is believed to be due to the fact that the fusion protein of the present invention has no α but has a weakened βγ affinity, as shown in the binding test of IL-2Rβγ protein in Example 3 ( Figure 7b ). While not being bound by theory, it appears that such reduced βγ affinity does not allow for adequate intracellular signaling in IL-2Rβγ expressing cells that do not express PD-1. However, it should be understood that in cells that also express PD-1 (e.g., PD-1 in the TME), + T eff In cells), the IL-2 of the fusion protein of the present invention adheres together in a "cis" structure with the help of the anti-PD-1 binding domain and can strongly bind to IL-2Rβγ ("switch-on" effect), thereby causing intracellular signal transduction at a significant level.

[0216] The property of the fusion protein of the present invention that it has no α but has a weakened βγ affinity suggests that although IL-2Rβγ is permanently expressed, it will not bind well to NK cells in peripheral blood with a low PD-1 expression rate, and therefore has a very advantageous advantage in reducing systemic toxicity and even side effects caused by excessive NK cell activity that the previous IL-2 protein has.

[0217] In addition, the difference in IL-2 reactivity of the fusion protein of the present invention to IL-2Rαβγ expressing cells was very small compared with IL-2Rβγ expressing cells (340 times vs 44 times; see Table 2 and Table 3 showing secondary results). Figure 8c and Figure 8d ). In the case of cells produced by expressing various receptor subunits such as IL-2Rαβγ, the actual difference in responsiveness to IL-2 between IL-2Rαβγ expressing cells and IL-2Rβγ expressing cells is more significant, considering that the expression forms of the subunits can be diverse and can also partially accept the expression of IL-2Rβγ receptors to a certain extent. This situation means that the fusion protein of the present invention has a significant effect on T cells that permanently express IL-2Rαβγ. reg The IL-2 reactivity of eosinophils and other endothelial cells, which cause capillary leak syndrome, is quite low, thus solving the problem of systemic toxicity and even serious side effects that may occur due to the presence of exogenous IL-2 protein, and showing the depleted T cells in the TME.eff Cell-specific binding to induce a prominent immune anti-cancer response.

[0218] Example 5. In vivo anticancer efficacy evaluation in mouse tumor models

[0219] Example 5.1 Anti-cancer efficacy test comparing anti-PD-1 or Fc-IL2 / IL2Rα alone

[0220] In order to evaluate the ability of the fusion protein of the present invention to selectively deliver IL-2 to T cells expressing PD-1 in tumors, eff The anticancer efficacy of the fusion protein of the present invention was evaluated by using the MC38 colorectal cancer syngeneic mouse tumor model. Fig. 9 The experimental procedures are briefly summarized in .

[0221] MC38 cancer cells were cultured in a 37°C, 5% CO2 chamber using DMEM (+10% fetal bovine serum (FBS), 1% streptomycin-penicillin (streptomycin-penicillin), 1% non-essential amino acid (NEAA)) medium. MC38 cancer cells reaching passage 9 were transplanted into mice. 1×10 cells were injected subcutaneously between the mid-back and right ribs (right back) of 8-week-old C57BL / 6 female mice using a 1 ml insulin syringe. 6 MC38 cancer cells. At this time, the mice were anesthetized with isoflurane. The mice injected subcutaneously with MC38 cancer cells were observed for 7 days. All mice were purchased from Orient Bio and raised in a specific-pathogen-free (SPF) facility.

[0222] After 7 days, the tumor size was determined and mice reaching a size of 100 cubic millimeters (±30) were classified separately for the experiment. A total of 28 mice transplanted with MC38 cancer cells were obtained and divided into four groups. Each group included 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 heterodimer Fc-IL2 / IL2Rα treatment group, and a PBS treatment group as a control group. Each test substance was mixed in the same amount (140 pmoles) in 100 microliters of Dulbecco's Phosphate-Buffered Saline (DPBS), and injected into the tail vein of the mouse four times twice a week (BIW) from the 7th day (PBS treatment group only intravenously injected 100 microliters of DPBS). Tumor size and body weight were measured and recorded twice a week from the 7th day. Euthanasia was performed when the tumor size reached 2000 cubic millimeters.

[0223] FIG10 measures and shows the changes in tumor size over time in each group ( Fig.10a ), mouse survival period ( Fig.10b ) and changes in mouse body weight ( Fig.10c ).like Fig.10a and Fig.10b As shown, the fusion protein of the present invention exhibits significantly superior tumor growth inhibition activity and prolonged mouse survival compared to the use of anti-PD-1 binding antibody alone or Fc-IL-2 / IL-R2α alone. It was confirmed that the fusion protein of the present invention achieved a tumor growth inhibition rate (TGI) of up to 98%, and 6 of a total of 7 mice remained tumor-free. On the other hand, no weight loss of mice due to the administration of the fusion protein of the present invention was observed ( Fig.10c ).

[0224] Example 5.2 Comparative anticancer efficacy test of combined administration of anti-PD-1 and Fc-IL2 / IL2Rα

[0225] The same mouse tumor model used in Example 5.1 was used to evaluate whether the fusion protein of the present invention exhibits improved anticancer efficacy due to its unique fusion structure. The experiment was performed according to the procedure described in Example 5.1, and a total of 21 mice transplanted with MC38 cancer cells were obtained, which were divided into three groups: a fusion protein A treatment group of the present invention, a combined treatment group of an antibody having the same anti-PD-1 binding domain as fusion protein A and heterodimer Fc-IL2 / IL2Rα, and a PBS treatment group as a control group. The results are shown in Figure 11.

[0226] Surprisingly, it was confirmed that the fusion protein of the present invention exerted significantly superior tumor growth inhibition and removal effects compared to the group in which the anti-PD-1 antibody including the same anti-PD-1 binding domain and Fc-IL2 / IL2Rα were co-administered ( Fig.11a ) and significantly prolonged the survival of mice ( Fig.11b ). This result shows that the fusion protein of the present invention can effectively deliver IL-2 to T cells expressing PD-1 in tumors. eff cells, thereby inducing T eff It promotes cell proliferation and activation, thus exerting excellent anti-cancer effects.

[0227] Example 5.3 Long-term memory immune response test

[0228] In order to confirm whether the fusion protein of the present invention forms long-term memory immunity and thus shows long-term effects even after administration is interrupted, a long-term memory immune response test was performed on C57BL / 6 mice that were tumor-free by administering the fusion protein of the present invention after MC38 cancer cell transplantation (first transplantation). For this purpose, according to the procedure described in Example 5.1, 75 to 280 pmol of fusion protein A was administered once to four times at a frequency of once a week (QW) or twice a week (BIW), and a total of 26 mice that remained tumor-free until the 60th day after the first transplantation (tumor-free mouse) were used for the experiment (75 pmol BIW four times: 6 mice, 140 pmol BIW four times: 6 mice, 150 pmol BIW four times: 5 mice, 280 pmol BIW four times: 4 mice, 150 pmol QW once: 2 mice, 150 pmol QW twice: 3 mice).

[0229] On day 67 after the first MC38 transplantation, the 26 mice were divided into two groups and 1×10 6 MC38 cancer cells (14 mice) or B16F10 melanoma cells (12 mice) were transplanted into the right back (second transplantation, second challenge), and tumor growth was observed. As a control group, 14 mice that did not undergo the first transplantation of MC38 and administration of fusion protein (naive state) were ) were divided into two groups and transplanted with MC38 or B16F10 cancer cells using the same method as above. The changes in tumor size were observed until day 85 after the first transplantation. Fig.12 Brief summary.

[0230] After the first transplantation of MC38 and administration of fusion protein A, all 14 mice transplanted with MC38 cancer cells for the second time remained tumor-free until the end of observation. On the contrary, tumor growth was observed in mice of other groups ( Fig.13 and Fig.14 In the case of mice transplanted with B16F10 melanoma cells for the second time, tumor growth was inhibited compared with the original mice, even though no fusion protein A was additionally administered ( Fig.13 ). Such results mean that the fusion protein of the present invention induces a tumor-specific long-term immune response, and maintains the inhibitory effect on the same cancer type for a long time even after administration is discontinued (acquired immune response), and also partially maintains the (tumor-nonspecific) anti-cancer effect (innate immune response) on other cancer types.

[0231] Example 5.4 Evaluation of the dose-dependent anticancer effect brought about by a single administration

[0232] In order to confirm whether the anticancer effect of the fusion protein of the present invention changes in a dose-dependent manner, an experiment was conducted using the MC38 colorectal cancer syngeneic mouse tumor model.

[0233] The experiment was conducted in the same manner as in Example 5.1. The mice were divided into 7 groups (5 or 6 mice in each group) based on the tumor size of 100 mm3±30 on the 7th day after transplantation. Fusion protein A was administered once to each group at a dosage of 0 (PBS), 50 pmol, 150 pmol, 300 pmol, 600 pmol, 1200 pmol, and 3000 pmol. The tumor size and body weight were measured and recorded twice a week. When the tumor size reached 2000 mm3, the mice were euthanized. The experimental procedure was as follows. Fig.15 Brief summary in.

[0234] like Fig.16a As shown, the fusion protein A of the present invention increases the tumor inhibition effect in proportion to the dosage in the range of 0 to 150 pmoles, and at a dosage of 300 pmoles or more, the TGI is 99% to 100% in all dosages. Therefore, in a single administration, the dosage that produces a saturated anticancer effect on MC38 is judged to be 150 pmoles to 300 pmoles. In terms of body weight change, weight loss occurred on the 10th day after administration at 3000 pmoles but recovered, and no weight loss was observed at all other dosages ( Fig.16b ) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 in the figure; multiple comparisons (group comparisons) compared with the PBS control group were performed using Tukey's post hoc test, two-way analysis of variance (two-way ANOVA)).

[0235] Example 5.5 Evaluation of anticancer effects in a syngeneic model of B16F10 melanoma

[0236] The experiment was carried out in the same manner as in Example 5.1. 5 The efficacy of the fusion protein was evaluated in 9-week-old C57BL / 6J mice expressing B16F10 cells but not MC38 cancer cells.

[0237] On the 7th day after transplantation, when the tumor size reached an average of 96 cubic millimeters, the mice were divided into three groups, and each group was given a single administration of PBS (10 mice), 300 pmol of fusion protein A (5 mice), and 600 pmol of fusion protein A (5 mice). The tumor size and body weight were measured and recorded twice a week.

[0238] Figure 17 shows the changes in tumor size over time in each group ( Fig.17a ) and tumor growth inhibition rate (TGI%) ( Fig.17b ).like Fig.17a and Fig.17b As shown, the fusion protein A-treated group showed a statistically significant tumor growth inhibition effect compared with the PBS-treated control group (based on the 13th day after administration, the TGI was 70.2% (300 pmol) and 89.2% (600 pmol) compared with the PBS-treated group; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 in the figure, two-way ANOVA using Tukey's post hoc test).

[0239] Example 5.6 Evaluation of anticancer effects in the Pan02 pancreatic cancer syngeneic model

[0240] The experiment was carried out in the same manner as in Example 5.1. 6 The efficacy of the fusion protein was evaluated in mice bearing Pan02 cancer cells but not MC38 cancer cells.

[0241] On the 7th day after transplantation, when the tumor size reached an average of 71 cubic millimeters, the mice were divided into two groups: a fusion protein A treatment group (5 mice) and a PBS treatment group (5 mice) for administration. Fusion protein A was mixed in 100 μl DPBS and injected into the tail vein of the mice four times at a frequency of 75 pmol per mouse twice a week (BIW) (the PBS treatment group was only intravenously injected with 100 μl DPBS). The tumor size and body weight were measured twice a week from the 7th day after transplantation to the 40th day and recorded.

[0242] Figure 18 shows the changes in tumor size over time in each group ( Fig.18a ), mouse survival period ( Fig.18b ) and changes in mouse body weight ( Fig.18c) was measured and shown. Fusion protein A showed excellent antitumor activity from the third administration to the end of observation, with all five mice remaining tumor-free ( Fig.18a ; TGI 100%, ***p<0.001 on the 17th day after administration, ****p<0.0001 on the 21st to 28th day after administration; two-way ANOVA with Tukey's post hoc test compared with the PBS control group), all mice survived until the end of observation ( Fig.18b In terms of body weight changes, a temporary decrease in body weight was observed after a single administration of fusion protein A, but this was subsequently restored and no further weight loss was observed ( Fig.18c ).

[0243] Example 5.7 Comparative evaluation of anticancer effects according to the number of anti-PD-1 Fab arms

[0244] In order to confirm how the fusion protein of the present invention changes the anticancer effect according to the number of anti-PD-1Fab arms, an experiment was conducted using the MC38 colorectal cancer syngeneic mouse tumor model. The experiment was conducted using the same method as in Example 5.1. On the 7th day after transplantation of MC38 cancer cells, fusion protein A (2 PD-1Fab arms) and fusion protein B (1 PD-1Fab arm) were administered once at a dose of 450 pmol (5 mice per group), and PBS was administered to 7 mice as a control group.

[0245] Until the 17th day after administration, tumor size and body weight were measured and recorded twice a week. Figure 19 shows the changes in tumor size over time in each group ( Fig.19a ) and tumor growth inhibition rate (TGI%) ( Fig.19b ).like Fig.19a and Fig.19b As shown, the fusion protein A-treated group and the fusion protein B-treated group showed statistically significant tumor growth inhibition effects compared with the PBS-treated control group (based on the 17th day after administration, TGI was 100% (fusion protein A) and 76% (fusion protein B) compared with the PBS-treated group; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 in the figure, two-way ANOVA with Dunnett's post hoc test).

[0246] Example 6 Comparative Evaluation of Antigen Binding Ability Based on pH

[0247] It is known that there is a pH difference between blood (about pH 7.4) and tumor microenvironment (tumor microenvironment) (about pH 5.6-6.8). Therefore, the antibody binding force at the weakly acidic pH of the tumor microenvironment has an important influence on the therapeutic efficacy of immune anticancer antibodies. In previous studies by the inventors, it was confirmed that 1G1-h70 used as an anti-PD-1 binding domain in the fusion protein of the present invention exhibits a stronger binding affinity to human PD-1 at a low pH value (6.0) of the tumor microenvironment compared with representative anti-PD-1 antibody drugs, iReida and Opdivo (PCT / KR2022 / 001714). Therefore, in order to confirm whether the fusion protein A of the present invention maintains excellent binding affinity in the tumor microenvironment, the binding kinetics (Kinetics) with human PD-1 are measured by SPR (Biaco 8K, Si Tuo Fan) analysis. As a control group for comparison, anti-human PD-1 antibodies Keytruda (MSD) and Opdivo (BMS) were purchased from SG Pharmaceuticals and used as human medicines.

[0248] According to the manufacturer's instructions, 1 μg / ml concentration of human PD-1 (Bipsys, cat # PD1-H5221) was immobilized in a CM5 chip (Situofan) using an amine coupling kit (Situofan). 7 concentrations (0-100 nmol / L) of control antibodies (Keyrida, Opdivo) were combined with 1G1-h70 and fusion protein A at a flow rate of 30 μl / min for 180 seconds and dissociated for 1200 seconds and flowed to the CM5 chip. In each concentration cycle, after each experiment, the chip was regenerated using glycine at pH 1.5. At this time, the running buffer uses HBS-EP+ buffer (0.1 mol / L hydroxyethylpiperazineethanesulfonic acid (HEPES), 1.5 mol / L NaCl, 0.03 mol / L ethylenediaminetetraacetic acid (EDTA) and 0.5% v / v surfactant (Surfactant) P20) at pH 7.4 or pH 6.0, and the experimental protein is diluted in the running buffer.

[0249] The analysis was performed using a 1:1 binding model in Biacore Evaluation Software Version 3.0, and the resulting kinetic constants (ka and kd) and affinity (KD) values ​​are reported in Table 3 below.

[0250] [Table 3]

[0251]

[0252] As shown in Table 3, the fusion protein A and 1G1-h70 antibody of the present invention exhibited a binding affinity (KD) similar to that of Keytruda and Opdivo at pH 7.4 (blood), but exhibited a binding affinity about 15 to 16 times stronger than that of Keytruda and Opdivo at pH 6.0 (tumor microenvironment). Therefore, it was confirmed that the fusion protein of the present invention maintains excellent binding affinity at the low pH of the tumor microenvironment of the anti-PD-1 antibody 1G1-h70.

[0253] Example 7. Confirmation analysis of PD-1-mediated IL-2 signaling

[0254] In order to verify whether the fusion protein of the present invention exhibits PD-1-mediated IL-2 signaling, HEK-Blue TM ) reporter gene assay system (Invivogen) was used to analyze IL-2 signaling activity according to the presence or absence of pre-treatment with anti-PD-1 antibody.

[0255] In HEK-Blue expressing IL-2Rβγ TM )CD122 / CD132 cells (Invivogen) were transfected with pCMV3-hPD1 vector or pCMV3-Empty vector (control group). The transfected cells were injected into 96-well plates at a concentration of approximately 50,000 cells / well and cultured in a 37°C, 5% CO2 chamber. Then, after removing the cell culture supernatant, 1 μmol / L of anti-PD-1 antibody (1G1-h70 antibody used in Example 6) or control group isotype (human IgG4) antibody was added per 20 μL per well and incubated for 40 minutes. Thereafter, recombinant human IL-2 (rhIL-2) or fusion protein A was treated at a concentration of 1 nanomolar / L and cultured at 37°C for 8 hours. After adding 20 μL / well of the cell culture supernatant to a new 96-well plate, 180 μL / well of the colorimetric assay reagent, QUANTI-Blue TM ) solution (Invivogen) and incubated in a 37°C incubator in the dark for more than 1 hour. After the reaction started, the OD was measured at 620 nm using a microplate reader at 30-minute intervals. The results are shown in Fig. 20 .

[0256] like Fig. 20 As shown, the fusion protein of the present invention is expressed in cells that do not express PD-1 (PD-1 - CD122 + CD132 +cells) showed weaker signaling responsiveness compared with IL-2 (reduced IL-2Rβγ binding properties), while in contrast, in cells expressing PD-1 (PD-1 + CD122 + CD132 + The fusion protein of the present invention showed a significantly more prominent reactivity than IL-2 in PD-1 expressing cells, which shows that the fusion protein of the present invention is specific and selectively acts on PD-1 expressing cells. In addition, when the fusion protein of the present invention is pre-treated with an anti-PD-1 antibody to pre-block the PD-1 of the cells before the treatment, it was confirmed that the IL-2 reactivity of the fusion protein of the present invention was completely neutralized. This situation suggests that the IL-2 signaling expressed by the fusion protein of the present invention is mediated by PD-1.

[0257] In order to clearly understand the present invention, the above exemplary specific implementations are described in detail, but the description and embodiments should not be interpreted as limiting the scope of the present invention. The scope of the present invention is defined by the attached application scope and its equivalents. The disclosures of all patents and scientific literature cited in this case are explicitly cited as a reference in their entirety.

[0258] Sequence Listing

[0259] [Table 4]

[0260]

[0261]

[0262]

Claims

1. A fusion protein comprising one or more immune cell antigen binding domains, an Fc domain formed by two Fc chains, and a cytokine domain, wherein the cytokine domain comprises an α-subunit polypeptide of an interleukin-2 receptor or an α-subunit polypeptide of an interleukin-15 receptor.

2. The fusion protein according to claim 1, wherein the cytokine domain comprises an IL-2Rα polypeptide and an IL-2 polypeptide, wherein the IL-2Rα polypeptide and the IL-2 polypeptide are not covalently bonded to each other and are respectively connected to different Fc chains of the Fc domain.

3. The fusion protein according to claim 1, wherein the cytokine domain is a complex comprising an IL-2Rα polypeptide and an IL-2 polypeptide, the IL-2Rα polypeptide and the IL-2 polypeptide in the complex are covalently bonded to each other, and the complex is connected to one of the Fc chains of the Fc domain via the IL-2Rα polypeptide or the IL-2 polypeptide.

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

5. The fusion protein of claim 4, wherein the IL-2Rα polypeptide comprises one or more amino acid substitutions selected from the group consisting of 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. 6 . The fusion protein according to claim 4 , wherein the IL-2Rα polypeptide comprises an amino acid substitution of L42I based on the amino acid sequence number of SEQ ID NO: 3 or comprises an amino acid sequence of SEQ ID NO:

5.

7. The fusion protein according to claim 2 or 3, wherein the IL-2 polypeptide comprises an amino acid substitution of C125S based on the amino acid sequence number of SEQ ID NO: 1 or one or more amino acid modifications that increase binding to the IL-2Rα polypeptide, or the IL-2 polypeptide is cyclically substituted.

8. The fusion protein according to claim 7, wherein the IL-2 polypeptide comprises an amino acid substitution of T37K based on the amino acid sequence number of SEQ ID NO:

1.

9. The fusion protein according to claim 1, wherein the cytokine domain comprises an IL-15Rα polypeptide and an IL-15 polypeptide, and the IL-15Rα polypeptide and the IL-15 polypeptide are respectively linked to different Fc chains of the Fc domain.

10. The fusion protein according to claim 1, wherein the cytokine domain is a complex comprising an IL-15Rα polypeptide and an IL-15 polypeptide, the IL-15Rα polypeptide and the IL-15 polypeptide in the complex are covalently bonded to each other, and the complex is connected to one of the Fc chains of the Fc domain via the IL-15Rα polypeptide or the IL-15 polypeptide.

11. The fusion protein according to any one of claims 1, 9 and 10, wherein the IL-15Rα polypeptide is an IL-15Rα polypeptide having an amino acid sequence of SEQ ID NO: 7 or an IL-15 binding fragment, an extracellular domain or a ScL domain of an IL-15Rα polypeptide, and the IL-15Rα polypeptide may include one or more amino acid modifications that increase binding to a ligand IL-15 polypeptide.

12. The fusion protein according to claim 9 or 10, wherein the IL-15 polypeptide comprises one or more amino acid modifications that increase binding to the IL-15Rα polypeptide or the IL-15 polypeptide is cyclically substituted.

13. The fusion protein according to claim 1, wherein the immune cell antigen binding domain comprises a Fab molecule of an antibody that specifically binds to more than one T cell target antigen or an antigen binding fragment of the antibody. The fusion protein according to claim 13 , further comprising a Fab molecule of an antibody or an antigen-binding fragment of the antibody that specifically binds to more than one tumor cell target antigen. 15 . The fusion protein according to claim 13 , wherein the immune cell antigen-binding domain comprises a Fab molecule of an antibody that specifically binds to programmed cell death-1 protein or an antigen-binding fragment of the antibody.

16. The fusion protein according to claim 15, wherein the immune cell antigen binding domain is a Fab molecule of pembrolizumab or nivolumab antibody or an antigen binding fragment thereof, or a Fab molecule of an antibody that binds to the same antigenic determining region as pembrolizumab or nivolumab antibody or an antigen binding fragment thereof or competes with pembrolizumab or nivolumab antibody or an antigen binding fragment thereof.

17. The fusion protein according to claim 15, wherein the immune cell antigen binding domain is a Fab molecule or an antigen binding fragment thereof of an anti-PD-1 antibody comprising: A heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain complementarity determining region 2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 15, a heavy chain complementarity determining region 3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 to 31, A light chain complementarity determining region 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 to 35, a light chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 36, and The light chain complementarity determining region 3 comprises the amino acid sequence of SEQ ID NO:

37.

18. The fusion protein according to claim 15, 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 region 1, complementarity determining region 2 and complementarity determining region 3 of a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 38, 40, 42 or 44; and complementarity determining region 1, complementarity determining region 2 and complementarity determining region 3 of a light chain variable region comprising an amino acid sequence of SEQ ID NO: 39, 41, 43 or 45.

19. The fusion protein of claim 17 or 18, wherein the immune cell antigen binding domain is a Fab molecule of an anti-PD-1 antibody or an antigen binding fragment thereof comprising: a heavy chain variable region comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 38, 40, 42 or 44; and a light chain variable region comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 39, 41, 43 or 45.

20. The fusion protein according to claim 1, wherein the Fc domain is an IgG class Fc domain or an IgG1, IgG2, IgG3 or IgG4 subclass Fc domain, and in the case of an IgG4 Fc domain, can include an amino acid substitution of S228P (based on the EU numbering system of Kabat literature).

21. The fusion protein according to claim 1, wherein the Fc domain comprises one or more amino acid modifications that reduce the binding or functional activity to an Fc receptor, or the Fc domain lacks a CH2 domain.

22. The fusion protein of claim 21, wherein the Fc domain comprises an amino acid modification 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).

23. The fusion protein of claim 1, wherein the Fc domain comprises modifications that promote the formation of heterodimers between the two Fc chains.

24. The fusion protein of claim 23, wherein the modification is a knob-into-hole modification comprising a knob modification in either Fc chain of the Fc domain and a hole modification in the other Fc chain.

25. The fusion protein of claim 24, wherein the knob modification comprises an amino acid substitution of T366W based on the EU numbering system of carbachol, and the hole modification comprises an amino acid substitution of T366S, L368A and Y407V.

26. The fusion protein of claim 25, wherein the knob chain of the Fc domain further comprises an amino acid substitution of S354C, and the hole chain of the Fc domain further comprises an amino acid substitution of Y349C.

27. The fusion protein according to any one of claims 24 to 26, wherein Either Fc chain of the Fc domain further comprises an amino acid substitution of K360E, and the other Fc chain further comprises an amino acid substitution of Q347R, or Either Fc chain of the Fc domain further comprises amino acid substitutions of K360E and Q347E, and the other Fc chain further comprises amino acid substitution of Q347R or amino acid substitutions of K360R and Q347R. The fusion protein according to claim 1 , wherein the cytokine action domain is bound to the Fc domain via a linker. The fusion protein of claim 28 , wherein the linker is a peptide linker.

30. The fusion protein according to claim 29, 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.

31. The fusion protein of any one of claims 13 to 19, wherein the antigen binding fragment is a single-chain variable region fragment, (scFV)2, a dual single-chain variable region fragment, a disulfide-stabilized Fv fragment, (dsFv)2, Fv, dsFv-dsFv', a diabody, a two-chain diabody, a triabody, a tetrabody, a nanobody, a domain antibody, a single domain antibody, or a bivalent domain antibody.

32. A polynucleotide encoding the fusion protein of any one of claims 1 to 3, 9 and 10.

33. An expression vector comprising the polynucleotide of claim 32.

34. A host cell comprising the polynucleotide of claim 32 or an expression vector comprising the polynucleotide.

35. A method for producing a fusion protein according to any one of claims 1 to 3, 9 and 10, comprising the following steps: A host cell comprising an expression vector comprising a polynucleotide encoding the fusion protein of any one of claims 1 to 3, 9 and 10 is cultured.

36. The method for producing a fusion protein according to claim 35, wherein the expression vector comprises: A first expression vector comprising a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 51 and a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 57, and The second expression vector includes a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:51 and a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:

58.

37. The method for producing a fusion protein according to claim 36, wherein the first expression vector and the second expression vector are simultaneously expressed in a host cell to obtain the fusion protein.

38. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 3, 9 and 10, for preventing, improving or treating tumors, cancers, metastatic tumors, metastatic cancers or infectious diseases.

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