Heterodimeric Fc fusion proteins and related compositions, uses and methods

Abstract

Disclosed are heterodimeric Fc fusion proteins that are not only quick, simple, and efficient to purify, but also exhibit properties that are desirable as therapeutic agents, such as increased FcRn binding ability and increased FcγR binding ability, as well as compositions, uses, and methods related thereto.

Description

[Technical field]

[0001] The present invention relates to Fc fusion technology, and more particularly to heterodimeric Fc fusion proteins, and compositions, uses and methods related thereto. [Background technology]

[0002] Immunoglobulin Fc fusion technology Fc fusion technology, in which a protein of interest is linked to the Fc residue of human immunoglobulin (typically IgG), is a useful tool for improving the therapeutic properties of biologically active proteins. The fusion protein in which the Fc region is linked to the protein of interest binds to neonatal Fc receptor (FcRn) and reduces endosomal degradation, thereby extending the half-life in blood. The Fc region can also mediate the reaction of Fcγ receptors on immune cells, thereby enhancing the immune effector function of the fusion protein.

[0003] The naturally occurring Fc region of IgG tends to form homodimers, so the easiest way to develop therapeutic proteins based on Fc fusion technology is to conjugate biologically active target proteins (i.e., ligands, extracellular domain receptors, cytokines) to the Fc region and express them in suitable host cells to produce homodimeric Fc fusion proteins, which can be easily isolated using affinity reagents specific for IgG Fc.

[0004] In some situations, heterodimeric Fc fusions are desired. Many proteins, including cytokines and their receptors, naturally exist and function as heterodimers, and some proteins exhibit enhanced pharmacological properties when produced as heterodimers with a partner protein of choice for therapeutic purposes.

[0005] The knob-into-hole mutation technique for constructing an asymmetric bispecific antibody of human IgG1 type is accompanied by the generation of many by-products such as knob-knob and hole-hole homodimers, as well as monomers. The purification to remove such by-products and secure the heterodimer requires complicated steps. In particular, when the physicochemical properties of the heterodimer are similar to those of the homodimer and monomer, it is very difficult to separate and purify the heterodimer to a level suitable for use as a human therapeutic agent. As a strategy to overcome this, in the case of antibodies, antibody variable region engineering that makes the physicochemical properties of the heterodimer different from those of the homodimer and monomer, and a method of purely separating the heterodimer by using specific resins for the κ light chain and the λ light chain by constructing the light chain portion of the heterodimer with κ light chain and λ light chain, respectively (e.g., International Publication WO2016-146594) are known. However, unlike antibodies, in the case of heterodimeric Fc fusion proteins in which two different proteins existing in natural phase are asymmetrically bound to Fc, there are many constraints on protein sequence engineering for separating the heterodimer.

[0006] Interleukin-1 (IL-1) Interleukin-1 (IL-1) is a potent pro-inflammatory cytokine produced by a variety of cell types, including mononuclear phagocytes, in response to infection and inflammation. The IL-1 family consists of seven agonists, including IL-1α and IL-1β, and three naturally occurring receptor antagonists, including the IL-1 receptor antagonist (IL-1Ra). Two IL-1 receptors (IL-1R type I and IL-1R type II) have been identified. These two receptors can interact with all three forms of IL-1 family molecules. IL-1RI mediates IL-1-induced cellular activities, but the IL-1 / IL-1RI complex cannot signal by itself and is dependent on the participation of a second receptor chain, the IL-1R accessory protein (IL1RAcP). Besides IL1-signaling, IL1RAcP plays a critical role in mediating the effects of IL33 via the ST2 / IL1RAP complex and IL36 via the IL1Rrp2 / IL1RAcP complex. IL-1 is known to be involved in a variety of diseases such as joint, bone and muscle diseases such as rheumatoid arthritis and osteoarthritis; inherited systemic autoinflammatory diseases such as familial Mediterranean fever; systemic juvenile idiopathic arthritis and adult-onset Still's disease; general inflammatory diseases such as gout and type 2 diabetes; acute onset ischemic diseases such as myocardial infarction; and cancer. Numerous therapies to block IL-1 activity are approved and under development. IL-1 targeting began in 1993 with the introduction of anakinra (Kineret, Amgen). Anakinra is a recombinant form of the natural IL-1 receptor antagonist (IL-1Ra) that blocks the activity of both IL-1α and IL-1β. Neutralization of IL-1 with antibodies or soluble receptors has also proven effective, leading to the approval of the soluble receptor rilonacept (Arcalyst (Regeneron)) and the anti-IL-1β neutralizing monoclonal antibody canakinumab (Ilaris (Novartis)).Furthermore, International Publication WO2014 / 126582 discloses a heterodimeric protein that contains the extracellular domains of human IL-1R1 and human IL-1RAcP, in which the IL-1R1 domain and the IL-1RAcP domain are fused to other mutants of the Fc portion of human IgG1, respectively.

[0007] Inflammasome Inflammasomes are intracellular protein complexes that promote the secretion of IL-1β and IL-18 in response to pathogens or cell damage, and induce inflammatory cell death. The secretion of IL-1β and IL-18 by inflammasomes occurs mainly from myeloid cells, and when inflammatory cell death is induced, the secretion of alarmin proteins IL-1α and HMGB1 via gasdermin pores is also promoted. Normal inflammasomes play an important role in innate immunity, but abnormal activity of inflammasomes acts as a cause of various diseases such as inflammatory diseases, degenerative neurological diseases, and cancer. Therapeutic strategies for diseases associated with inflammasome activity include 1) inhibition of inflammasome sensor proteins such as Nlrp3, 2) inhibition of caspases, which are enzymes that mediate gastrointestinal pore formation and cytokine secretion, and 3) inhibition of effector proteins. In particular, inhibition of effector proteins is expected to be effective in a variety of related diseases because it can control the inflammatory response caused by inflammasomes independently of upper signaling pathways.

[0008] Transforming growth factor beta (TGF-β) In human tumors, not only cancer cells but also immune cells such as cytotoxic T cells and natural killer cells that suppress tumor growth are observed, as well as regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC), which are immune suppressor cells that promote tumor growth. MDSCs are generated by abnormal differentiation of myeloid cells due to chronic inflammation, and pro-inflammatory cytokines such as IL-1 and IL-6, which induce chronic inflammation, and chemokines play a key role in the generation and infiltration of MDSCs into tumors. The distribution and activity of immune cells and immune suppressor cells in tumors is called the tumor immune microenvironment (TIME), and is known to greatly contribute to the response of immune anticancer drugs such as PD-1 antibodies. Intratumoral tumor immunosuppressive cells MDSC and Treg are known to suppress the functions of cytotoxic T cells and natural killer cells through various mechanisms. In particular, it has been revealed that these immunosuppressive cells and TGF-β (transforming growth factor β), a cytokine secreted by cancer cells, play a key role in immunosuppression. Recently, it has been reported that TGF-β inhibition significantly improves the immune response to anticancer drugs in mice and humans. However, TGF-β is an anti-inflammatory cytokine, and it has been reported that deficiency of TGF-β1 induces severe inflammatory disease in mice, and inhibition of the TGF-β signaling pathway in cancer cells increases intratumoral infiltration of MDSC. The transforming growth factor beta (TGF-β) receptor family is composed of TGF-βRI, TGF-βRII, and TGF-βRIII. In particular, TGF-βRII is known to bind with high affinity to TGF-β1 and TGF-β3, and with relatively low binding affinity to TGF-β2. Summary of the Invention [Problem to be solved by the invention]

[0009] It is an object of the present disclosure to provide heterodimeric Fc fusion proteins that are not only fast, simple and efficient to purify, but also exhibit desirable therapeutic properties such as increased FcRn binding and increased FcγR binding. Another object of the present disclosure is to provide compositions relating to said heterodimeric Fc-fusion proteins. Yet another object of the present disclosure is to provide uses related to said heterodimeric Fc fusion protein. It is yet another object of the present disclosure to provide methods relating to said heterodimeric Fc-fusion proteins. [Means for solving the problem]

[0010] A first aspect of the present disclosure is i) a first polypeptide comprising, in N-terminal to C-terminal direction, a first protein of interest, a first linker, and a first Fc portion of an immunoglobulin; ii) a second polypeptide comprising, in N-terminal to C-terminal direction, a second protein of interest, a second linker, and a second Fc portion of an immunoglobulin; the first target protein and the second target protein are different proteins; The first linker and the second linker each include a κ-type light chain constant region (C Lκ ), or a mutant thereof, and a lambda light chain constant region (C Lλ ), or a variant thereof.

[0011] A second aspect of the present disclosure provides a pharmaceutical composition comprising, as an active ingredient, said heterodimeric Fc fusion protein and a pharma- ceutically acceptable carrier.

[0012] A third aspect of the present disclosure is a method for the preparation of a κ-type light chain constant site (C Lκ ), or a mutant thereof, and a lambda light chain constant region (CLλ The present invention provides a composition for use as a linker between a protein of interest and an Fc portion in a heterodimeric Fc fusion protein comprising the protein of interest and an immunoglobulin Fc portion, the composition comprising:

[0013] A fourth aspect of the present disclosure provides a heterodimeric Fc fusion protein comprising a protein of interest and an immunoglobulin Fc region, comprising a kappa light chain constant region (KLC) for use as a linker between the protein of interest and the Fc region. Lκ ), or a mutant thereof, and a lambda light chain constant region (C Lλ ), or a variant thereof.

[0014] A fifth aspect of the present disclosure relates to a κ-type light chain constant site (C Lκ ), or a mutant thereof, and a lambda light chain constant region (C Lλ ), or a variant thereof, as a linker between a protein of interest and an Fc portion in a heterodimeric Fc fusion protein comprising the protein of interest and an immunoglobulin Fc portion.

[0015] In a sixth aspect of the present disclosure, a κ-type light chain constant site (K ... Lκ ), or a mutant thereof, and a lambda light chain constant region (C Lλ ), or a mutant thereof, is introduced into the heterodimeric Fc fusion protein.

[0016] A seventh aspect of the present disclosure provides a heterodimeric Fc fusion protein comprising a kappa light chain constant site (KLC) and a .alpha.-amino acid sequence similar to that of the .alpha.-amino acid sequence ... Lκ ) and the lambda light chain constant region (C Lλ The present invention also provides a method for purifying the heterodimeric Fc fusion protein, comprising purifying the heterodimeric Fc fusion protein based on its affinity to the Fc fusion protein. In an eighth aspect of the present disclosure, a κ-type light chain constant site (KCL) is used as a linker between a target protein and an Fc site. Lκ ), or a mutant thereof, and a lambda light chain constant region (C Lλ The present invention provides a method for increasing the FcRn-binding ability of a heterodimeric Fc fusion protein, comprising the step of introducing an FcRn-binding domain of a heterodimeric Fc fusion protein into a heterodimeric Fc fusion protein.

[0017] In a ninth aspect of the present disclosure, a κ-type light chain constant site (K ... Lκ ), or a mutant thereof, and a lambda light chain constant region (C Lλ ), or a variant thereof, respectively.

[0018] In a tenth aspect of the present disclosure, a kappa light chain constant site (KLC) is used as a linker between a target protein and an Fc site. Lκ ), or a mutant thereof, and a lambda light chain constant region (C Lλ The present invention provides a method for increasing the FcγR binding ability of a heterodimeric Fc fusion protein, comprising the step of introducing an FcγR-binding domain into a heterodimeric Fc fusion protein, or a mutant thereof, into the heterodimeric Fc fusion protein.

[0019] In an eleventh aspect of the present disclosure, a κ-type light chain constant site (K ... Lκ ), or a mutant thereof, and a lambda light chain constant region (C Lλ The present invention provides a method for increasing antibody-dependent cell-mediated cytotoxicity (ADCC) of a heterodimeric Fc-fusion protein, comprising introducing into the heterodimeric Fc-fusion protein a polypeptide having a nucleotide sequence similar to that of the Fc-fusion protein of the present invention, or a variant thereof, respectively. Effect of the Invention

[0020] According to the present disclosure, there are provided heterodimeric Fc fusion proteins, and compositions, uses and methods relating thereto, which are not only rapidly, easily and efficiently purified but also exhibit properties that are desirable as therapeutic agents, such as increased FcRn binding ability and / or increased FcγR binding ability. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 is a schematic diagram showing a method for producing an Fc fusion protein in which two different proteins are linked together. [Diagram 2] FIG. 1 is a schematic diagram of a method for producing an IL-1 receptor Fc fusion protein. [Figure 3A]1 shows the results of SDS-PAGE analysis of IL1T, a heterodimeric IL-1 trap. [Figure 3B] Size exclusion chromatography analysis of IL1T, a heterodimeric IL-1 trap. [Figure 4A] FIG. 1 is a schematic diagram of major and by-products at each purification step of PET101, a κλ heterodimeric IL-1 trap. [Figure 4B] 1 shows the results of SDS-PAGE analysis of the eluates from each purification step of PET101. [Figure 4C] FIG. 4C(A) shows the results of size exclusion chromatography analysis of the eluate from the first purification step of PET101, and FIG. 4C(B) shows the results of size exclusion chromatography analysis of the eluate from the third purification step of PET101. [Diagram 5] 1 shows the results of hydrophobic interaction chromatography (HIC) analysis of PET101. [Figure 6] These are the results of an analysis of the binding ability of IL1T, PET101, canakinumab, and rilonacept to human IL-1β using ELISA. [Figure 7A] This shows the results of an analysis of the binding ability of PET101 to human IL-1α and human IL-1β using surface plasmon resonance (SPR). [Figure 7B] This shows the results of an analysis of the binding ability of PET101 to monkey IL-1β using surface plasmon resonance (SPR). [Figure 7C] This is the result of an analysis of the binding ability of PET101 to mouse IL-1α and mouse IL-1β using surface plasmon resonance (SPR). [Figure 7D] This shows the results of an analysis of the binding ability of rilonacept to human IL-1α and human IL-1β using surface plasmon resonance (SPR). [Figure 7E] This shows the results of an analysis of the binding ability of rilonacept to monkey IL-1β using surface plasmon resonance (SPR). [Figure 7F] These are the results of an analysis of the binding ability of rilonacept to mouse IL-1α and mouse IL-1β using surface plasmon resonance (SPR). [Figure 7G] These are the results of an analysis of the binding ability of canakinumab to human IL-1α and human IL-1β using surface plasmon resonance (SPR). [Figure 7H] This is the result of an analysis of the binding ability of canakinumab to monkey IL-1β using surface plasmon resonance (SPR). [Figure 7I] This is the result of an analysis of the binding ability of canakinumab to mouse IL-1α and mouse IL-1β using surface plasmon resonance (SPR). [Figure 8] (A) and (B) show the results of an analysis of the ability of PET101, rilonacept, and canakinumab to inhibit the IL-1α signal pathway in an IL-1 reporter cell line. [Figure 9] This is the result of analyzing the ability of PET101, rilonacept, and canakinumab to inhibit IL-1β-mediated IL-6 secretion in the A549 non-small cell lung cancer cell line. [Figure 10] This shows the results of a protein thermal stability analysis of rilonacept, IL1T, and PET101. [Figure 11A] The stability of PET101 in PBS at 37°C for 4 weeks was analyzed using SDS-PAGE. [Figure 11B] The stability of PET101 in PBS at 37° C. for 4 weeks was analyzed using size exclusion chromatography (B). [Figure 12A] This is a BLI sensogram result of IL1T and PET101 against human FcRn. [Figure 12B] These are the BLI sensogram results of rilonacept and canakinumab against human FcRn. [Figure 13A] This shows the BLI sensogram results of PET101 and rilonacept against human FcγRI. [Figure 13B] This shows the BLI sensogram results of PET101 and rilonacept against human FcγRIIIA. [Figure 14] These are the results of an analysis of the ability of PET101 and rilonacept to inhibit human IL-1β-mediated mouse IL-6 secretion depending on the administered concentration. [Figure 15] This shows the results of an analysis of the ability of PET101 and rilonacept to inhibit human IL-1β-mediated mouse IL-6 secretion depending on the time after administration. [Figure 16] FIG. 16 shows the results of plasma concentration analysis of the test substances over time when PET101 and rilonacept were administered subcutaneously at 3 mg / kg in cynomolgus monkeys. [Figure 17A] Schematic diagram of IL-1 / IL-18 bispecific heterodimer trap 118T3 (A) and SDS-PAGE results of 118T3 purified products under reducing and non-reducing conditions (B). [Figure 17B] Schematic diagram of IL-1 / IL-18 bispecific κλ heterodimer trap 118T3v01 (A) and SDS-PAGE results of stepwise purification products of 118T3v01 under reducing and non-reducing conditions (B). [Figure 18] Binding analysis of 118T3 and 118T3v01 to human IL-1β (A) and human IL-18 (B) using ELISA. [Figure 19] This shows the results of a simultaneous binding analysis of 118T3v01 to human IL-1β and human IL-18 using surface plasmon resonance (SPR). [Figure 20] (A) is a schematic diagram of 118T3B obtained by removing one IL18BP from 118T3v01, and (B) shows the results of SDS-PAGE of the 118T3B purification products in stages under reducing and non-reducing conditions. [Figure 21A] These are BLI sensogram results of 118T3B for human IL-1α, human IL-1β, monkey IL-1β, and mouse IL-1β. [Figure 21B] This shows the BLI sensogram results of 118T3B against monkey IL-1β and mouse IL-1β. [Figure 21C] BLI sensogram results of 118T3B against human IL-18, monkey IL-18, and mouse IL-18. [Figure 22] This shows the results of an analysis of the ability of 118T3v01 and 118T3B to inhibit the signal transduction pathways of human IL-1β (A) and human IL-18 (B) in an IL-1 reporter cell line. [Diagram 23] Schematic diagram of the IL-1 / TGF-β bispecific heterodimer PET301 (A) and SDS-PAGE results of the products of each purification step of PET301 under reducing and non-reducing conditions (B). [Figure 24A] Binding analysis of PET301 to human IL-1β (A) and human TGF-β1 (B) using ELISA. [Figure 24B] Binding analysis of PET301 to human TGF-β3 (A) and human TGF-β2 (B) using ELISA. [Figure 25A] (A) shows the results of an analysis of the ability of PET301 to suppress human IL-1β using an IL-1 reporter cell line, and (B) shows the results of an analysis of the ability of PET301 to suppress human TGF-β1 using a TGF-β reporter cell line. [Figure 25B] 1 shows the results of analyzing the inhibitory ability of human TGF-β3 (A) and the inhibitory ability of human TGF-β2 (B) using a TGF-β reporter cell line. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly used in the art to which this disclosure belongs. For the purposes of understanding this disclosure, the following definitions apply, and terms used in the singular include the plural and vice versa.

[0023] As used herein, the terms "polypeptide" and "protein" may be used interchangeably to refer to a longer peptide having the amino acid sequence of a native protein or an amino acid sequence having one or more mutations, such as deletion, substitution, addition and / or insertion of one or more amino acid residues.

[0024] The term "fusion protein" refers to a protein having two or more moieties covalently linked together, where each moiety is derived from a different protein.

[0025] The terms "Fc", "Fc site" and "Fc region" refer to the hinge region or a part thereof, the CH2 region and the CH3 region in an antibody molecule. The Fc region of the IgG class refers to, for example, from cysteine ​​at position 226 to the C-terminus, or from proline at position 230 to the C-terminus, according to the EU numbering (also called EU INDEX), but is not limited thereto. The Fc site can be appropriately obtained by partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc. with a protease such as pepsin or papain, and then re-eluting the fraction adsorbed on a protein A column or a protein G column.

[0026] The term "heterodimer" refers to a polypeptide that is composed of two polypeptides that differ in their amino acid sequences.

[0027] The term "linker" refers to one or more molecules, e.g., nucleic acid, amino acid, or non-peptide residues, that can be inserted between one or more component regions. For example, the linker is used to provide a desired site of interest between the components to facilitate manipulation. The linker is also provided to enhance expression of the fusion protein from the host cell, reduce steric hindrance so that the component can assume its optimal tertiary structure and / or properly interact with the target molecule. The linker sequence may comprise one or more amino acids naturally linked to the receptor component, or it may be an added sequence used to provide a desired site of interest to enhance expression of the fusion protein, to allow the component region to form an optimal tertiary structure, and / or to enhance the interaction of the component with its target molecule. Desirably, the linker does not interfere with the structure of each functional component in the fusion protein, and increases the flexibility of the fusion protein components.

[0028] Heterodimeric Fc fusion proteins One aspect of the present disclosure is i) a first polypeptide comprising, in N-terminal to C-terminal direction, a first protein of interest, a first linker, and a first Fc portion of an immunoglobulin; ii) a second polypeptide comprising, in N-terminal to C-terminal direction, a second protein of interest, a second linker, and a second Fc portion of an immunoglobulin; the first target protein and the second target protein are different proteins; The first linker and the second linker each include a κ-type light chain constant region (C Lκ ), or a mutant thereof, and a lambda light chain constant region (C Lλ ) or variant heterodimeric Fc fusion proteins are provided.

[0029] In order to overcome the problem of purifying a heterodimeric Fc fusion protein, the present inventors have inserted an antibody κ light chain constant site (C ) between the C-terminus of two different target proteins and the N-terminus of the hinge region of the Fc region. Lκ ) and antibody lambda light chain constant region (C Lλ The κλFc fusion protein technology of the present disclosure is a commercially produced C Lκ Specific resin and C Lλ Utilizing a specific resin, it is possible to rapidly and simply isolate heterodimeric Fc fusion proteins in high purity.

[0030] C Lκ Area and C Lλ Each of the regions has a molecular weight of about 12.5 kDa, and the increase in molecular weight of the Fc fusion protein due to their introduction is expected to be at a level of about 25 kDa. The κλFc fusion protein technology according to the present disclosure can improve the convenience and efficiency of separation and purification of the heterodimer without causing a significant change in the molecular weight, stability, and safety of the fusion protein.

[0031] In one embodiment, C LλThe mutant also has a deletion of the C-terminal serine (Ser), specifically, the C-terminal serine (Ser) at position 215 according to the Kabat numbering system.

[0032] In one embodiment, C Lκ Mutants and / or C Lλ The mutant also has a cysteine ​​(Cys) that forms a disulfide bond with the heavy chain constant region CH1 substituted with serine (Ser), alanine (Ala) or valine (Val). Specifically, the cysteine ​​(Cys) that forms a disulfide bond with the heavy chain constant region CH1 is at position 214 according to the Kabat numbering system.

[0033] In FIG. 1, (A) shows an in-line homodimeric Fc fusion protein in which a polypeptide in which two different proteins (proteins 1 and 2) are linked in a single chain is bound to the N-terminus of the hinge region of the Fc region of human immunoglobulin G (IgG (immunoglobulin G)), (B) shows a heterodimeric Fc fusion protein in which two different proteins are linked to the N-terminus of the hinge region of the IgG Fc region, and (C) shows the C of IgG Fc. H3 (D) shows a knob-into-hole heterodimeric Fc fusion protein (KiH) in which a mutation was introduced into the region that promotes heterodimer formation, and (E) shows two different proteins fused to the human immunoglobulin kappa light chain constant region (C Lκ ) and the human immunoglobulin lambda light chain constant region (C Lλ (E) shows the κλ heterodimeric Fc fusion protein in which the κλ heterodimer is linked to the κλ-Fc fusion protein in a single chain and then attached to the N-terminus of the hinge region of the Fc region. H31 shows a knob-into-hole κλ heterodimer Fc fusion protein (KiH κλ-Fc fusion protein) in which a mutation that promotes heterodimer formation has been introduced into the region. In Fig. 1, (A), (B), and (C) correspond to the prior art, and (D) and (E), particularly (E), belong to the scope of the present disclosure.

[0034] In one embodiment, the heterodimeric Fc-fusion protein additionally comprises a third protein of interest at one or more of the C-terminus of the first polypeptide or the C-terminus of the second polypeptide.

[0035] In one embodiment, the heterodimeric Fc-fusion protein may have mono-specificity or multi-specificity. The term "mono-specificity" means that it is capable of specifically binding to one target, and "multi-specificity" means that it is capable of specifically binding to two or more distinct targets. For example, the fusion protein may be bi-specific, comprising a region capable of specifically binding to a first target and a region capable of specifically binding to a second target.

[0036] In one embodiment, the target protein is a ligand, a receptor, a cytokine, an enzyme, or a peptide. In a specific example, the receptor is a cytokine receptor. In a specific example, the cytokine and / or the receptor naturally exists and functions in a heterodimer, such as interleukin-1 (IL1), interleukin-2 (IL2), interleukin-3 (IL3), interleukin-4 (IL4), interleukin-5 (IL5), interleukin-6 (IL6), interleukin-11 (IL11), interleukin-13 (IL13), interleukin-15 (IL15), interleukin-18 (IL18), interleukin-23 (IL23), interleukin-31 (IL31), interleukin-42 (IL42), interleukin-51 (IL42), interleukin-52 (IL42), interleukin-63 (IL42), interleukin-64 (IL42), interleukin-65 (IL42), interleukin-66 (IL42), interleukin-67 (IL42), interleukin-68 (IL42), interleukin-69 (IL42), interleukin-70 (IL42), interleukin-71 (IL42), interleukin-72 (IL42), interleukin-73 (IL42), interleukin-74 (IL42), interleukin-75 (IL42), interleukin-76 (IL42), interleukin-77 (IL42), interleukin-78 (IL42), interleukin-79 ... The antibody may be one or more selected from the group consisting of, but not limited to, IL-31, interleukin-33 (IL33), interleukin-35 (IL35), interleukin-36 (IL36), leukemia inhibitory factor (LIF), oncostatin M (OSM), granulocyte macrophage colony stimulating factor (GM-CSF), gamma-interferon (IFN-γ), thymic stromal lymphopoietin (TSLP), transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF), and tumor necrosis factor-alpha (TNF-α).

[0037] As a specific example, the present inventors constructed an Fc fusion protein that binds the extracellular region of the IL-1 receptor (IL-1R1, IL-1RAcP), which is naturally a heterodimer, using three types of fusion proteins that are expected to have a high formation rate and are relatively easy to purify, namely, an in-line homodimer (Figure 1A), a KiH heterodimer (Figure 1C), and a KiHκλ heterodimer (Figure 1E)). In FIG. 2, (A) is an IL-1-specific in-line homodimeric Fc fusion protein in which the extracellular region of the IL-1 receptor is bound to Fc by the structure A in FIG. 1. As an example of an IL-1-specific in-line homodimeric Fc fusion protein, a fusion protein having the same sequence as rilonacept (prior art) was constructed. (B) is an IL-1-specific heterodimeric Fc fusion protein (IL1T) (prior art) in which the extracellular region of the IL-1 receptor is bound to Fc by the structure (C) in FIG. 1. (C) is an example of an IL-1-specific heterodimeric Fc fusion protein in which the extracellular region of the IL-1 receptor is bound to Fc by the structure E in FIG. 1. Lκ and C Lλ and then bound to Fc to form an IL-1 specific κλ heterodimeric Fc fusion protein (PET101) (one specific example of the present disclosure).

[0038] As in the above specific example, one of the first and second target proteins is interleukin-1 receptor type 1 (IL1R1), and the other is interleukin-1 receptor accessory protein (IL1RAcP). For example, the first target protein is interleukin-1 receptor type 1 (IL1R1), and the first linker is a kappa light chain constant region (KLC). Lκ ), or a variant thereof, the second protein of interest is interleukin-1 receptor accessory protein (IL1RAcP), and the second linker is a lambda light chain constant region (C Lλ), or variants thereof. For example, the interleukin-1 receptor type 1 (IL1R1) and the interleukin-1 receptor accessory protein (IL1RAcP) are also their extracellular domains (ectodomains), respectively.

[0039] As another specific example, the present inventors constructed a dual inhibitor in the form of IL18BP (interleukin-18-binding protein), a natural human IL-18 inhibitor, fused to the C-terminal portion of an IL-1 receptor Fc fusion protein in order to develop a simultaneous inhibitor of the inflammasome effector cytokines IL-1α, IL-1β, and IL-18. Among IL-1 inhibitors, the in-line homodimer rilonacept has a molecular weight of about 250 kDa, and when IL18BP is fused, a problem of reduced tissue permeability due to a further increase in molecular weight may occur. Therefore, the present inventors prepared an IL-1 / IL-18 dual inhibitor in the form of IL18BP fused to the heterodimeric IL-1 inhibitors IL1T and PET101, which have relatively small molecular weights.

[0040] As yet another specific example, the present inventors focused on the possibility that inhibition of IL-1 may reduce inflammation and MDSC formation caused by TGF-β inhibition and increase anti-cancer efficacy, and produced an IL-1 / TGF-β dual inhibitor based on PET101. Specifically, in order to produce an Fc fusion protein with dual specificity for human IL-1 and TGF-β, the PET301 structure was designed in a form in which the extracellular domain of TGF-βRII was bound to the C-terminus of the Fc region of PET101 (Figure 23(A)).

[0041] Thus, according to one embodiment, the heterodimeric Fc fusion protein also comprises interleukin-18 binding protein (IL18BP) or type II transforming growth factor-beta receptor (TGFβRII) as a third protein of interest.

[0042] Specifically, it may contain interleukin-18 binding protein (IL18BP) at either the C-terminus of the first polypeptide or the C-terminus of the second polypeptide, or more specifically, it may contain interleukin-18 binding protein (IL18BP) at the C-terminus of the second polypeptide.

[0043] In another example, the heterodimeric Fc fusion protein also contains a type II transforming growth factor-beta receptor (TGFβRII) at both the C-terminus of the first polypeptide and the C-terminus of the second polypeptide.

[0044] In one embodiment, the heterodimeric Fc fusion protein also has a CH3 region mutation such that the first Fc moiety and the second Fc moiety are capable of promoting heterodimer formation. Mutations for promoting heterodimer formation are known in the art and include, without limitation, such mutations. For example, the CH3 regions of the first Fc moiety and the second Fc moiety also include knob-into-hole mutations.

[0045] In one embodiment, the heterodimeric Fc fusion protein is one in which the first linker, the second linker, or both are attached to the N-terminus of the Fc region hinge. "Hinge" refers to the portion of the antibody heavy chain polypeptide that connects the CH1 domain and the CH2 domain in a wild-type antibody heavy chain (e.g., from about position 216 to about position 320, or from about position 226 to about position 230 according to the Kabat EU numbering system), and is generally flexible enough to allow the associated binding sites to move independently, with 25 or fewer amino acid residues. For example, the hinge may have, but is not limited to, the amino acid sequence DKTHTCPXCP, HTCPXCP, or CPXCP (wherein X is S or P).

[0046] In one embodiment, the heterodimeric Fc fusion protein also further comprises a third linker between the C-terminus of the first and / or second polypeptide and the third protein of interest. In a specific embodiment, the linker residue is a peptide linker having a length of 2 to 100 amino acids. Exemplary linkers include linear peptides having at least two amino acid residues, such as Gly-Gly, Gly-Ala-Gly, Gly-Pro-Ala, Gly(G)n and Gly-Ser (GS) linkers. A GS linker is a (GS) n , (GSGSG) n , (G2S) n , G2S2G, (G2SG) n , (G3S) n , (G4S) n , (GGSGG) n G n and GSG4SG4SG, where n is greater than or equal to 1. In certain embodiments, the linker is also (G4S)2.

[0047] In one embodiment, the immunoglobulin Fc region is an Fc region derived from IgG, IgA, IgD, IgE, IgM, a combination thereof, or a hybrid thereof, specifically, an IgG Fc region, in particular, an IgG1 Fc region, but is not limited thereto.

[0048] Compositions, Uses and Methods The above description of the heterodimeric Fc fusion protein equally applies to the compositions, uses, methods, etc. described below.

[0049] In another aspect of the present disclosure, there is provided a pharmaceutical composition comprising, as an active ingredient, the heterodimeric Fc-fusion protein and a pharma- ceutically acceptable carrier.

[0050] Pharmaceutically acceptable carriers enhance or stabilize the composition or facilitate its manufacture. Pharmaceutically acceptable carriers include physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

[0051] The pharmaceutical composition of the present disclosure may be administered by a variety of methods known in the relevant art. The route and / or mode of administration may vary depending on the desired result. Administration may be intravenous, intramuscular, intraperitoneal, or subcutaneous, or may be administered adjacent to the target site. The pharma- ceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). The composition should be sterile and fluid. The composition may also be in lyophilized form. Isotonicity agents, such as sugars, polyalcohols, such as mannitol or sorbitol, and sodium chloride, may be included in the composition. The pharmaceutical composition of the present invention may also be manufactured by methods widely known and commonly practiced in the relevant art.

[0052] Dosage levels of the active ingredient in the pharmaceutical composition may vary to obtain an amount of the active ingredient that is not toxic to the patient and is effective for a particular patient, composition, and administration method to achieve the desired therapeutic response. The selected dosage level will depend on a variety of pharmacokinetic factors, such as the particular composition of the present disclosure used, or the route of administration, the time of administration, the rate of excretion, the duration of treatment, other drugs, compounds, and / or substances used in combination, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and other factors. As a non-limiting example, the dosage ranges from about 0.0001 to 100 mg / kg body weight, more commonly 0.1 to 20 mg / kg body weight. Exemplary treatment regimens involve administration once per week, or once every two weeks, or once per month, or once every 3 to 6 months.

[0053] In one embodiment, the composition is for preventing or treating an immune-related disease or disorder, hi a specific embodiment, the immune-related disease or disorder is cancer, an autoimmune disease or an inflammatory disease.

[0054] In one embodiment, the composition further comprises one or more therapeutic agents, hi particular embodiments, the therapeutic agent is an immune checkpoint inhibitor, such as one or more of a PD1 inhibitor, a PD-L1 inhibitor, and a CTLA4 inhibitor.

[0055] Yet another aspect of the present disclosure is a method for the preparation of a κ-type light chain constant site (C Lκ ), or a mutant thereof, and a lambda light chain constant region (C Lλ The present invention provides a composition for use as a linker between a protein of interest and an Fc portion in a heterodimeric Fc fusion protein comprising the protein of interest and an immunoglobulin Fc portion, the composition comprising:

[0056] Yet another aspect of the present disclosure provides a heterodimeric Fc fusion protein comprising a protein of interest and an immunoglobulin Fc portion, comprising a kappa light chain constant region (KLC) for use as a linker between the protein of interest and the Fc portion. Lκ ), or a mutant thereof, and a lambda light chain constant region (C Lλ ), or a variant thereof.

[0057] Yet another aspect of the present disclosure is a method for the preparation of a κ-type light chain constant site (C Lκ ), or a mutant thereof, and a lambda light chain constant region (C Lλ ), or a variant thereof, as a linker between a protein of interest and an Fc portion in a heterodimeric Fc fusion protein comprising the protein of interest and an immunoglobulin Fc portion.

[0058] Yet another embodiment of the present disclosure is a method for the preparation of a κ-type light chain constant site (KCL) as a linker between a protein of interest and an Fc site. Lκ ), or a mutant thereof, and a lambda light chain constant region (C Lλ), or a mutant thereof, is introduced into the heterodimeric Fc fusion protein.

[0059] Yet another aspect of the present disclosure is a heterodimeric Fc fusion protein comprising a kappa light chain constant site (KLC) and a .alpha.-amino acid sequence similar to that of the .alpha.-amino acid sequence ... Lκ ) and the lambda light chain constant region (C Lλ In a specific embodiment, the purification method comprises purifying the heterodimeric Fc fusion protein based on affinity for the Fc fusion protein. 1) performing immunoglobulin affinity chromatography; 2) κ light chain invariant region (C Lκ ) performing affinity chromatography; 3) λ-type light chain invariant region (C Lλ ) performing affinity chromatography.

[0060] Various Fc variants are also used to increase the binding to neonatal Fc receptor (FcRn) and increase serum half-life. The binding of the Fc region to FcRn prevents endosome degradation upon endocytosis of antibodies or Fc fusion proteins, and induces their release back into the blood via pH-dependent dissociation, thereby mediating the pharmacokinetic properties of antibodies and Fc fusion proteins, such as long drug half-life and high blood exposure. However, many Fc fusion proteins are characterized by low FcRn binding compared to antibodies due to interference between the Fc region and binding proteins, and low FcRn binding strength correlates with a short half-life of the Fc fusion protein.

[0061] The present inventors have demonstrated that the κλFc fusion protein technology has the following advantages in addition to the advantages of purification: Lκ Area and C LλThe inventors noticed that by positioning the region at the N-terminus of the hinge region, the distance between the Fc region and the binding protein can be increased, thereby reducing interference between them and providing the advantage of FcRn binding, and confirmed this through experiments.

[0062] In accordance with yet another aspect of the present disclosure, a linker between a protein of interest and an Fc site is a kappa light chain constant site (KLC). Lκ ), or a mutant thereof, and a lambda light chain constant region (C Lλ The present invention provides a method for increasing the FcRn-binding ability of a heterodimeric Fc fusion protein, comprising the step of introducing an FcRn-binding domain of a heterodimeric Fc fusion protein into a heterodimeric Fc fusion protein.

[0063] In addition, the κ light chain constant region (C Lκ ), or its mutant m, and a λ-type light chain constant region (C Lλ ), or a variant thereof, respectively.

[0064] The Fc region of an antibody mediates the immune effector function of the antibody by binding to Fc gamma receptors (Fcγ receptors) on the surface of immune cells in addition to FcRn. The present inventors tested the possibility that the κλFc fusion protein technology has the advantage of Fc gamma receptor binding in addition to FcRn, and confirmed that it has increased binding ability to FcγRI and FcγRIIIa.

[0065] Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibodies on target cells and subsequently cause lysis of the target cells. The ADCC correlates with binding to FcγRIIIa, and increased binding to FcγRIIIa induces increased ADCC activity.

[0066] Thus, yet another aspect of the present disclosure provides a method for the preparation of a κ-type light chain constant site (KCL) as a linker between a protein of interest and an Fc site. Lκ), or a mutant thereof, and a lambda light chain constant region (C Lλ ), or a mutant thereof, respectively, are provided.

[0067] In addition, the κ light chain constant region (C Lκ ), or a mutant thereof, and a lambda light chain constant region (C Lλ The present invention provides a method for increasing antibody-dependent cell-mediated cytotoxicity (ADCC) of a heterodimeric Fc-fusion protein, comprising introducing into the heterodimeric Fc-fusion protein a polypeptide having a nucleotide sequence similar to that of the Fc-fusion protein of the present invention, or a variant thereof, respectively.

[0068] Hereinafter, the present disclosure will be described in more detail with reference to specific examples. However, these examples are intended to aid in understanding the present disclosure and are not intended to limit the scope of the present disclosure in any way.

[0069] Comparative Example 1: Preparation of heterodimeric Fc fusion protein with IL-1 specificity (IL1T) To realize the IL-1 specific heterodimeric Fc fusion protein having the structure shown in FIG. 2(B), knob-into-hole mutation technology was applied to the Fc region (SEQ ID NO: 1) of human immunoglobulin G1 (IgG1) to obtain gene sequences encoding the polypeptides of knob mutation-containing Fc (S354C, T366W (SEQ ID NO: 2)) and hole mutation-containing Fc (Y349C, T366S, L368A, Y407V (SEQ ID NO: 3)). Gene sequences encoding amino acid residues 1 to 333 constituting the signal sequence and extracellular portion of human IL-1R1 (GenBank: AAM88423.1) or amino acid residues 1 to 359 constituting the signal sequence and extracellular portion of human IL-1RAcP (GenBank: BAA25421.1) were chemically synthesized (Integrated DNA Technologies, IA, USA) and cloned into the respective vectors. The vectors capable of expressing polypeptides corresponding to IL-1R-hinge region-CH2-CH3 (S354C, T366W) (sequence number 4) and IL-1RAcP-hinge region-CH2-CH3 (Y349C, T366S, L368A, Y407V) (sequence number 5) were designated P001 and P002, respectively.

[0070] Co-transfection of P001 and P002 vectors was performed in the Chinese hamster ovary-derived cell line ExpiCHO-S. TM The transient expression was carried out in a 5'-cell culture medium (A29127 (Gibco)) and the heterodimeric IL-1 trap expressed through this transient expression was named IL1T. All reagents used in the transient expression process were prepared according to the manufacturer's (Gibco) manual and carried out according to the maximum titer production protocol in the manual. Sartoclear Dynamics 登録商標The 12-day culture medium was filtered using a LabV (SDLV-1000-40C-E (Sartorius)) filter system, and then purified using a HiTrap MabSelect SuRe (11003493 (GE Healthcare)) purification column, followed by sample concentration using an Amicon Ultra-15 Centrifugal Filter Unit (Merck Millipore). The concentration of the final purified product was quantified based on the extinction coefficient and molecular weight specific to the sample. The purified product was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) (Figures 3A and 3B). SDS-PAGE analysis was performed under non-reducing and reducing conditions, and bands of each size were confirmed by Coomassie Brilliant Blue staining (Figure 3A). Under reducing conditions, a mixture of IL-1R-hinge region-CH2-CH3 (S354C, T366W) and IL-1RAcP-hinge region-CH2-CH3 (Y349C, T366S, L368A, Y407V) monomers was observed at approximately 70 kDa. Under non-reducing conditions, a product estimated to be a mixture of heterodimers and homodimers was observed at approximately 150 kDa, and unassembled monomers were observed at approximately 70 kDa (Figure 3A). Size exclusion chromatography analysis was performed using an Alliance HPLC system equipped with an Agilent Bio SEC-3 HPLC column (5190-2511 (Agilent)). 登録商標 An HPLC-e 2695 Separations Module (Waters, 2695) was used. The analysis confirmed the presence of 82.88% of a mixture of heterodimer (IL1T) and homodimer at a retention time of 7.142 min, aggregates were detected at retention times of 5.470 min and 6.344 min, and a monomer mixture was detected at a retention time of 7.893 min (Figure 3B).

[0071] Example 1: Production of IL-1 specificity-bearing κλ heterodimeric Fc fusion protein (PET101) To produce a heterodimeric Fc fusion protein with IL-1 specificity using the κλFc fusion protein technology, an antibody light chain κ constant site was introduced between the IL-1R extracellular site and the hinge region (SEQ ID NO: 6), and an antibody light chain λ constant site was introduced between the IL-1RAcP extracellular site and the hinge region (SEQ ID NO: 7). In order to prevent unnecessary assembly during this process, the cysteine ​​present at the C-terminus of the natural κ and λ constant sites was replaced with serine. The IL-1R-C produced in this manner was Lκ -hinge region-CH2-CH3 (S354C, T366W) and IL-1RAcP-C Lλ The expression vectors encoding the amino acid sequences (sequence numbers 8 and 9) of the -hinge region-CH2-CH3 (Y349C, T366S, L368A, Y407V) were designated P003 and P004, respectively, and the heterodimer produced by co-transfection in the ExpiCHO-S cell line was designated PET101.

[0072] The expected main products and by-products for each purification process and purification step of PET101 are shown in Figure 4A. More specifically, affinity chromatography-based purification was performed in the same manner as in Comparative Example 1. Affinity chromatography was performed on the primary purified product using KappaSelect resin (17545801 (GE Healthcare)) followed by LambdaFabSelect resin (17548201 (GE Healthcare)). Dialysis, concentration, quantification, SDS-PAGE analysis, and size exclusion chromatography analysis of the primary (MabSelect), secondary (KappaSelect), and tertiary (LambdaFabSelect) purified products were performed in the same manner as in Comparative Example 1. In the non-reducing SDS-PAGE analysis of the MabSelect primary purification eluate, an unassembled monomer was confirmed at about 70 kDa. When the tertiary purification was completed, the monomer was removed and a protein expected to be PET101 at about 180 kDa was confirmed (Figure 4B "1"). In the purity analysis using size exclusion chromatography, the primary purification product was found to contain heterodimers, homodimers, monomers, etc., and the dimer peak was confirmed at a retention time of 6.836 seconds and the area of ​​the mixture was confirmed to be 87.57% (Figure 4B "2"). In the primary purification product, secondary (KappaSelect) and tertiary (LambdaFabSelect) affinity chromatography consecutive purification was performed to separate the heterodimer. In the SDS-PAGE result of the final purification product, the LambdaFabSelect tertiary purification product, no monomer was confirmed, and a peak of the predicted heterodimer product was confirmed (Figure 4B "3"). In size exclusion chromatography analysis, the putative heterodimer product peak eluted with a retention time of 6.838 min, and the purity of the final purified product was confirmed to be 99.31% ("3" in Figure 4B).

[0073] Test Example 1: PET101 Heterodimer Content Analysis To confirm the content of heterodimer in the final purified product of PET101 obtained in Example 1, hydrophobic interaction chromatography analysis capable of separating heterodimers and homodimers was performed. The chromatography column used was a MAbPac HIC-10 column (4.6 x 250 mm, 5 μm (Thermo Fisher Scientific)). For homodimer analysis, IL-1R-C Lκ -hinge region-CH2-CH3 homodimer (PET101 knob κκ) and IL-1RAcP-C Lλ The -hinge region-CH2-CH3 homodimer (PET101 hole λλ) was expressed and purified in the manner described in Example 1 and compared with the final purified product of PET101. In hydrophobic interaction chromatography, as shown in FIG. 5, it was confirmed that the PET101 knob ββ homodimer was eluted first, followed by the final purified product of PET101 and the PET101 hole ββ homodimer in that order. In the final purified product of PET101, the ratio of the heterodimer (PET101 κλ) that is not a homodimer was measured to be 98.02%, proving that the purification process described in Example 1 can be used to separate a high-purity heterodimer.

[0074] Test Example 2: Analysis of IL-1β binding ability using enzyme-linked immunosorbent assay (ELISA) Binding assay of three IL-1 specific Fc fusion proteins (rilonacept, IL1T, and PET101) to human IL-1β was performed using ELISA. For comparison of binding ability, human IL-1β antibody canakinumab was used as a control. 96 half-well immunosorbent assay plates (3690, Corning) were treated with human IL-1β (10139-HNAE, Sino Biological) at a concentration of 30 ng / well and allowed to coat overnight at 4°C. The next day, plates were washed with Phosphate Buffered Saline with Tween-20 (TEKNOVA, P1192, PBST). All washing steps in this assay were performed using an AquaMax 2000 plate washer (0310-5363, Molecular Devices). IL1T, PET101, rilonacept, and canakinumab were diluted to the appropriate concentration in PBST containing 1% (w / v) skim milk and applied to each well. Binding was induced at room temperature for 1 hour and the plate was washed three times. Peroxidase-conjugated anti-human IgGFc chlorine antibody (31413 (Invitrogen)) was then diluted (1:10,000) in PBST containing 1% (w / v) skim milk to induce binding. After washing three times with PBST, the color reaction was induced in a dark room by treatment with TMB substrate solution (34028 (Thermo Fisher Scientific)) at room temperature. The reaction was then terminated by treatment with 1N sulfuric acid (255 (DUKSAN)), and the absorbance was measured at 450 nm using a SpctraMax i3 multimode plate reader (10192-220 (Molecular Devices)). PET101 showed similar EC values ​​for IL1T, rilonacept, and human IL-1β. 50 When treated with the same mole concentration, canakinumab showed a higher OD value (Figure 6). 450 The results of this study show that the C-terminal end of the C-terminal region of the Fc hinge region, which is introduced between the human IL-1 receptor and the N-terminus of the Fc hinge region, is effective in suppressing the binding of IL-1 to the human IL-1 receptor. Lκ Area and C LλThis suggests that the region does not affect the binding of the human IL-1 receptor to human IL-1β.

[0075] Test Example 3: Measurement of binding constant via surface plasmon resonance (SPR) To quantitatively analyze the binding strength of PET101 to IL-1α and IL-1β, surface plasmon resonance analysis was performed using Biacore T200 (GE Healthcare). For binding constant measurement, a Series S CM5 sensor chip (BR100530 (GE Healthcare)) was used, and the anti-human Fc antibody included in the Human Antibody Capture Kit (BR100839 (GE Healthcare)) was immobilized on the sensor chip using an amine coupling kit (BR100050 (GE Healthcare)) according to the manufacturer's manual. Flow cell 1 of the CM5 sensor with immobilized anti-human Fc antibody was used as a blank cell, and flow cells 2, 3, and 4 were loaded with 100 nM PET101 (18 μg / ml), 100 nM rilonacept (25 μg / ml), and 100 nM canakinumab (15 μg / ml), respectively, at a flow rate of 10 μl / min for 1 minute. After all flow cells were prepared, 0.25, 0.5, 1, 2, 4, and 8 nM of human IL-1α (200-LA (R&D Systems)), human IL-1β (201-LB (R&D Systems)), mouse IL-1α (400-ML (R&D Systems)), mouse IL-1β (401-ML (R&D Systems)), and monkey (Rhesus macaque) IL-1β (1318-RL (R&D Systems)) antigens were added at a flow rate of 30 μl / min, and binding reactions were carried out for 4 minutes. Subsequently, 1X HBS-EP+ buffer (BR100669 (GE Healthcare)) was added at a flow rate of 30 μl / min, and dissociation reactions were carried out for 10 minutes. Each acquired sensorgram was compared to a blank cell, normalization and subtraction were performed, and affinity was calculated. The affinity was calculated using BIAevaluation software (GE Healthcare) using a 1:1 Langmuir model analysis to calculate the binding rate constant and dissociation rate constant. D ) is the dissociation rate constant (K d ) to the binding rate constant (Ka ) and PET101 has high levels of K of 6.97 pM, 3.46 pM, and 1.43 pM for human IL-1α, human IL-1β, and monkey IL-1β, respectively. D The K values ​​for mouse IL-1α and mouse IL-1β were 19.42 pM and 304.54 pM, respectively. D It was confirmed that the binding constants of rilonacept were 68.39 pM for human IL-1β (Figures 7A, 7B, and 7C, Table 1). Rilonacept showed a similar binding pattern to PET101, and the binding constants were as shown in Table 1 (Figures 7D, 7E, and 7F). The human IL-1β-specific antibody canakinumab had a K of 68.39 pM for human IL-1β. D The K values ​​of canakinumab measured against monkey IL-1β were 0.01, 0.01, 0.02, 0.01, 0.02, 0.01, 0.02, and 0.03, respectively, and confirmed that it did not bind to human IL-1α and had no interspecies cross-binding ability against mouse antigens (Figures 7G, ​​7H, and 7I, Table 1). D The value was 2.847 μM, which showed a rapid dissociation pattern and was analyzed to have a very low binding affinity (FIGS. 7G, 7H, and 7I, Table 1). [Table 1]

[0076] Test Example 4: Evaluation of IL-1 signal transduction pathway suppression ability using reporter cell lines The IL-1β reporter HEK293 cell line (hkb-il1b (Invivogen)) secretes the secreted reporter protein secreted embryonic alkaline phosphatase (SEAP) in a concentration-dependent manner in response to human IL-1α and IL-1β (https: / / www.invivogen.com / hek-blue-il1b). The cell line was simultaneously treated with IL-1 and an IL-1 inhibitor, and the SEAP in the culture medium was quantified to measure the inhibitory ability of the signal transduction pathway. The IL-1β reporter HEK293 cell line was cultured in DMEM (11965-092 (Gibco)) + 10% FBS (16140-071 (Gibco)). The cells were cultured in a 96-well plate (167008 (Thermo Fisher Scientific)) at 1 × 10 cells / well in complete culture medium. 4Cells / well were plated and incubated overnight in a humidified incubator at 37°C with 5% (v / v) CO2. Human IL-1α (10128-HNCH, Sino Biological) or human IL-1β (10139-HNAE, Sino Biological) at 20ng / ml in serum-free DMEM medium was premixed with IL-1 inhibitors (PET101, rilonacept or canakinumab) at 0.0001024nM, 0.000512nM, 0.00256nM, 0.0128nM, 0.064nM, 0.3nM2, 1.6nM, 8nM, 40nM, 200nM in serum-free DMEM medium at a 1:1 ratio for 30 minutes. In this case, human IL-1α or human IL-1β at a final concentration of 10 ng / ml was mixed with 0.0000512 nM, 0.000256 nM, 0.00128 nM, 0.0064 nM, 0.032 nM, 0.16 nM, 0.8 nM, 4 nM, 20 nM, and 100 nM of IL-1 inhibitor (PET101, rilonacept, or canakinumab), and the culture medium of the cell culture plate prepared through overnight culture was aspirated, and the premixed medium was treated to the cells so that 100 μl per well was cultured for 5 hours at 37°C in a 5% (v / v) CO2 humidified incubator. The supernatant was analyzed, and the inhibitory ability of the IL-1 inhibitor on the IL-1β signal transduction pathway was determined using a SEAP reporter gene analysis kit (T1017 (Invitrogen)). However, the average luminescence value measured after treatment with 10 ng / ml human IL-1α or human IL-1β alone was converted to 100% SEAP secretion, and the average luminescence value measured after treatment with the same volume of PBS (10010 (Gibco)) as the treated substance was converted to 0% SEAP secretion, and all values ​​were calculated based on this. Analysis of IL-1α suppression ability in IL-1β reporter HEK293 cell line showed that PET101 had an EC 50 Rilonacept had an EC value of 0.308 nM. 50 In this case, canakinumab was unable to induce effective inhibition (FIG. 8(A)). Analysis of human IL-1β inhibition in the same cells confirmed that PET101 had an EC value of 0.275 nM. 50Rilonacept had an EC value of 0.243 nM. 50 It was confirmed that the SEAP secretion was suppressed by about 99.5% in the reporter cell line when 4 nM PET101 or rilonacept was treated, but only about 30% of the SEAP secretion was suppressed in the reporter cell line when 4 nM canakinumab was treated (FIG. 8(B)).

[0077] Test Example 5: Evaluation of IL-1 signal transduction pathway suppression ability using A549 non-small cell lung cancer cell line The A549 non-small cell lung cancer cell line is responsive to IL-1β and induces the secretion of interleukin-6 (IL-6) through activation of phosphoinositide 3-kinase (PI3K) and interleukin-1 receptor-associated kinase 4 (IRAK4) (Hiroyuki Eda et al. Cell Biol Int. 2011). The efficacy of PET101 was evaluated in a human lung cancer cell line that was artificially created using this mechanism and was not a reporter cell line. The A549 non-small cell lung cancer cell line (CCL-185 (ATCC)) was cultured in RPMI-1640 (LM001-03 (Welgene)) + 10% FBS (16140-071 (Gibco)) medium. Cells were plated in 96-well plates at 2 × 10 5After plating the cells / well, the wells were incubated at 37°C in a 5% (v / v) CO2 humidified incubator for 7 hours to induce cell attachment. The medium was then removed from the wells, and 100 μl of serum-free RPMI-1640 medium (LM001-03 (Welgene)) was added, followed by fasting for 18 hours in a humidified incubator at 37°C in a 5% (v / v) CO2 humidified incubator. Each well was treated with 100 μl of premixed IL-1β (10139-HNAE (Sino Biological)) at a final concentration of 1 ng / ml and appropriate concentrations of IL-1 inhibitors (PET101, rilonacept or canakinumab) in serum-free RPMI-1640 medium at room temperature for 30 minutes. After 3 hours of treatment, the amount of IL-6 in the supernatant was quantified using a mouse IL-6 DuoSet ELISA kit (DY406 (R&D Systems)) according to the manufacturer's manual. PET101 showed an EC value of 44 pM to inhibit IL-1β in A549 cell lines. 50 value, and rilonacept had a similar EC value of 37 pM. 50 Canakinumab had an EC value of 2678 pM under the same conditions. 50 It was confirmed that it had value (Figure 9).

[0078] Test Example 6: Thermal stability analysis of PET101 The thermal stability of PET101 was analyzed by Protein Thermal Shift TM The assay was performed using a dye kit (4461146 (Applied Biosystems)) according to the manufacturer's instructions. The results showed that rilonacept, IL1T, and PET101 had similar T m It was confirmed that the values ​​were significant (Figure 10).

[0079] Test Example 7: Stability analysis of PET101 at 37℃ Stability analysis of PET101 dissolved in PBS (pH 7.4) (10010 (Gibco)) was performed at 37°C. 1mg / ml PET101 was incubated at 37°C, and on days 0, 1, 2, 3, 4, 7, 10, 14, 21, and 28, samples were transferred to -80°C and SDS-PAGE analysis was performed as described in Example 1 (Figure 11A). No fragmentation or aggregation was observed in the analysis results under reducing and non-reducing conditions. Analysis of the presence or absence of aggregates using size exclusion chromatography revealed an aggregate peak at a retention time of about 6.2, and a very low peak was detected up to the 7th day sample at 37°C (Figure 11B), and no aggregates were detected in the area analysis using Empower software (Waters) (Table 2). In the sample at 37°C on the 10th day, 99.12% PET101 was present, with 0.88% aggregates (Table 2). In the sample at 37°C on the 28th day, 98.85% PET101 was present, with 1.15% aggregates (Table 2). [Table 2]

[0080] Test Example 8: Evaluation of FcRn binding ability of PET101 The binding ability of PET101 and rilonacept to FcRn was evaluated using a bio-layer interferometry (BLI) analyzer, Octet Red96e (ForteBio), at 30°C. A HIS1K (anti-penta-HIS) biosensor (18-5120 (ForteBio)) was used, and the analysis buffer was prepared by mixing 10x Kinetics Buffer (18-1042 (ForteBio)) and PBS pH 7.4 (10010 (Gibco)) at a ratio of 1:9 to a final pH of 6.0, and used as the running buffer. The rotation speed of the analysis plate was 1,000 rpm under all conditions. Human FcRn containing a His tag (FCM-H82W4 (AcroBiosystems)) was diluted in the running buffer to a concentration of 2 μg / ml and loaded onto the biosensor. 31.25-1000 nM IL1T, PET101, rilonacept or canakinumab have binding rate constants (K a To measure the dissociation rate constant (K), the association time was run for 120 seconds, followed by a dissociation time of 120 seconds in the running buffer. d ) was determined. a (0 seconds to 120 seconds) value and K d (120 s to 125 s) values ​​were calculated using a 1:1 binding model in the Octet analysis software (ForteBio), and the equilibrium dissociation constant (K D ) values ​​were determined (Figures 12A and 12B).

[0081] Test Example 9: Evaluation of the binding ability of PET101 to Fc gamma receptors The binding ability of PET101 to Fc gamma receptors was evaluated using a bio-layer interferometry (BLI) analysis device, Octet Red96e (ForteBio), at 30°C. A HIS1K (anti-penta-HIS) biosensor (18-5120 (ForteBio)) was used, and the analysis buffer was prepared by diluting 10x Kinetics Buffer (18-1042 (ForteBio)) with PBS pH 7.4 (10010 (Gibco)) to prepare a running buffer, and the analysis plate was rotated at 1,000 rpm. Human FcγRI (1257-FC (R&D Systems)) or human FcγRIIIA (4325-FC (R&D Systems)) containing a His tag was diluted in the running buffer to a concentration of 2 μg / ml and loaded onto the biosensor. The binding rate constant (K a To measure the dissociation rate constant (K), binding was performed for 120 seconds for 80 to 1,280 nM PET101 or rilonacept, followed by dissociation for 120 seconds in the running buffer. d ) was determined. D was calculated using the 1:1 binding model in the Octet analysis software (ForteBio), and the interval in which the curve fitting was performed was K a (0 seconds to 120 seconds) and K d (120 seconds to 240 seconds). In this case, the K D The K values ​​for FcγRIIIA were 19.82 nM and 45.79 nM, respectively (Figure 13A). D was calculated using a 1:1 binding model in the Octet analysis software (ForteBio), and the interval in which curve fitting was performed was K a (0 seconds to 120 seconds) and K d (120 to 125 seconds). In this case, the K binding between PET101 and rilonacept related to FcγRIIIA was D The values ​​were found to be 661 nM and 913 nM, respectively (Figure 13B).

[0082] Test Example 10: Evaluation of in vivo neutralizing activity according to the administration concentration of PET101 For the in vivo neutralizing ability evaluation experiment (n=5 / group) by concentration of PET101, 6-week-old C57BL / 6 female mice weighing approximately 20g per individual were used. All experimental procedures related to the progress of animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Medytox before initiation. The in vivo neutralizing ability evaluation of PET101 was carried out using a method established in previous literature, utilizing the phenomenon that IL-6 in mouse blood increases when human or monkey IL-1α or IL-1β is administered to C57BL / 6 female mice (Economides et al., Nat Med. 2003 & Lacy et al. mAbs. 2015). Specifically, the extent to which PET101 can suppress the increase in IL-6 after artificially administering exogenous human IL-1β to mice was compared with rilonacept. First, mice were subcutaneously (sc) administered 0.001 (0.3x), 0.003 (1x), 0.01 (3x), 0.03 (10x), 0.1 (30x), 0.3 (100x), or 1 (300x) mg / kg of PET101 or 0.0014 (0.3x), 0.0047 (1x), 0.14 (3x), 0.465 (10x), 0.140 (30x), 0.465 (100x), or 1.40 (300x) mg / kg of rilonacept (number in brackets = moles of anti-IL-1 trap / moles of human IL-1β corresponding to 300 ng / kg administered later). 24 hours after subcutaneous administration, human IL-1β (10139-HNAE (Sino Biological)) was administered intraperitoneally (ip) at 300 ng / kg (5 ml / kg). Two hours after administration, serum was collected from each mouse. IL-6 quantification in mouse serum was performed using a Mouse IL-6 DuoSet ELISA Kit (DY406 (R&D Systems)).Without administering anti-IL-1 trap, the same amount of PBS was administered subcutaneously, and after 24 hours, the same amount of PBS was administered intraperitoneally, and the quantified IL-6 value of the serum was calculated as 0%. Without administering anti-IL-1 trap, the same amount of PBS was administered subcutaneously, and after 24 hours, 300ng / kg (5ml / kg) IL-1β was administered intraperitoneally, and the quantified IL-6 value of the serum was calculated as 100%. Based on this, the quantitative value of each group was converted into inhibitory ability (%). Analysis results: EC of PET101. 50 The EC value for rilonacept was 3.32 μg / kg. 50 The value was 10.52μg / kg, confirming that PET101 had approximately 3.17 times greater activity than rilonacept (Figure 14). When PET101 or rilonacept was administered in a 3-fold (molar) overdose compared to the administered IL-1β, PET101 showed approximately 80% inhibitory activity, while rilonacept showed approximately 50% inhibitory activity (Figure 14).

[0083] Test Example 11: Evaluation of in vivo neutralizing activity according to administration time of PET101 For the in vivo neutralization ability evaluation experiment of PET101 over time (n=5 / group (total 16 groups)), 6-week-old C57BL / 6 females weighing approximately 20g per individual were used. All experimental procedures related to the progress of animal experiments were approved by the Medytox Animal Experiment Ethics Committee (IACUC) before initiation. The experiment was carried out by quantifying the amount of IL-6 generated by artificially injecting human IL-1β at various times after administration of PET101 to mice, specifically, the extent to which PET101 remaining on the first, second, third, and sixth days after administration can suppress IL-6 was compared with rilonacept. First, PBS, 10mg / kg PET101, or 10mg / kg rilonacept was administered subcutaneously (sc) to mice. After subcutaneous administration, 1, 2, 3, and 6 days later, PBS or human IL-1β (10139-HNAE (Sino Biological)) was administered intraperitoneally (ip) at 300 ng / kg (5 ml / kg). Two hours after administration, serum from each mouse was collected, and IL-6 in the mouse serum was quantified using a mouse IL-6 DuoSet ELISA kit (DY406 (R&D Systems)) as described in Test Example 5. As a result of quantitative analysis, mouse IL-6 was quantified at 0 to 6 pg / ml in the group administered PBS (sc) + PBS (ip) regardless of the time (Figure 15). This means that IL-6 is not present or is present in trace amounts in the blood of normal C57BL / 6 female mice (Figure 15). In the case of PBS (sc) + hIL-1β (ip) administration, the administered human IL-1β induced the expression of mouse IL-6, and 3,352 to 3,769 pg / ml of IL-6 was quantified. In the case of PET101 administration, the induction of IL-6 was completely suppressed until the third day, and 6 days after PET101 administration, when human IL-1β was treated, about 2,005 pg / ml of IL-6 was quantified (Figure 15). In the case of the rilonacept administration group, the induction of IL-6 was completely suppressed only on the first day, and 8 pg / ml, 31 pg / ml, and 2,348 pg / ml of IL-6 were measured in the blood on the second, third, and sixth days, respectively (Figure 15).Through this analysis, it was confirmed that PET101 was superior to rilonacept in terms of the duration of efficacy in suppressing human IL-1β in mice (FIG. 15).

[0084] Study Example 12: Primate PK analysis of PET101 using cynomolgus monkeys PK analysis of PET101 was performed using cynomolgus monkeys. Rilonacept was used as a control substance. PET101 and rilonacept were administered subcutaneously (sc) at 3 mg / kg (n=3 / group). Blood samples were obtained at 0 min, 30 min, 1 h, 2 h, 4 h, 8 h, 1 day, 2 days, 3 days, 6 days, 10 days, 14 days, 21 days, and 28 days, and plasma was separated. The concentrations of PET101 and rilonacept in plasma were measured by ELISA. Briefly, a 96-well ELISA plate (3590 (Corning)) was coated with 1 μg / ml high affinity chimeric anti-human IL-1R mouse antibody by storing it overnight at 4°C, and the plate was washed with PBST the next day and blocked with 3% BSA solution (Sigma) at room temperature for 1 h, followed by washing with PBST. The plasma collected at each time point was appropriately diluted and subjected to binding reaction with coated anti-human IL-1R rabbit antibody. After washing with PBST, the plate was treated with high affinity chimeric anti-human IL-1RAcP rabbit antibody and binding reaction was induced at room temperature for 1 hour. After washing with PBST, the plate was treated with peroxidase-conjugated anti-rabbit IgG antibody (7074S (Cell Signaling Technology)) and binding reaction was induced at room temperature for 1 hour. The plate was washed three times with PBST, treated with TMB substrate solution (34028 (Thermo Fisher Scientific)) and reaction was induced for 20 minutes in a dark room at room temperature. The reaction was terminated by treatment with 1N sulfuric acid solution (255 (DUKSAN)), and absorbance at 450 nm was measured using a SpectraMax i3x multimode plate reader (i3x (Molecular Devices)). Standard assay samples for PET101 and rilonacept were prepared and used for quantification, and the concentrations of the analytes were quantified at each time point via standard curves generated from naive cynomolgus serum containing each concentration. At 3 mg / kg subcutaneous administration, PET101 and rilonacept had similar half-lives (T 1 / 2) (Table 3). However, the area under the blood concentration-time curve (AUCinf (area under the curve from time 0 to infinity)) of PET101 was measured to be 2.17 times higher than that of rilonacept, confirming that PET101 has superiority in terms of drug exposure in the monkey body (Figure 16, Table 3). [Table 3]

[0085] Comparative Example 2: Preparation of a heterodimeric polypeptide (118T3) with dual specificity for IL-1 and IL-18 First, a heterodimer was prepared by fusing human IL18BP to the C-terminal region of the Fc region of IL1T (Figure 17A (A)). The gene sequence of amino acid residues 31 to 194 constituting human IL18BP (GenBank: BAA76374.1) was chemically synthesized (Integrated DNA Technologies, IA, USA) and cloned into each expression vector. Specifically, a heterodimer expressed by co-transfection of vectors capable of expressing a polypeptide corresponding to IL1R-hinge region-CH2-CH3(S354C, T366W, K447del)-(G4S)2-IL18BP (SEQ ID NO: 10) and IL1RAcP-hinge region-CH2-CH3(Y349C, T366S, L368A, Y407V, K447del)-IL18BP (SEQ ID NO: 11) is called 118T3 (FIG. 17A (A)). The primary expression of 118T3 was carried out in the same manner as in Comparative Example 1, and the culture medium on the 12th day was cultured in Sartoclear Dynamics. 登録商標The product was filtered using a LabV (SDLV-1000-40C-E (Sartorius)) filter system, and then purified using a HiTrap MabSelect SuRe (11003493 (GE Healthcare)) purification column. The final purified product was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the same manner as in Comparative Example 1 (FIG. 17A (B)). As a result, a monomer mixture was confirmed at about 110 kDa under reducing conditions, and a product estimated to be a mixture of heterodimers and homodimers at about 220 kDa under non-reducing conditions was confirmed as well as unassembled monomers at about 110 kDa (FIG. 17B (B)).

[0086] Example 2: Production of IL-1 and IL-18 bispecific κλ heterodimeric polypeptide (118T3v01) A heterodimer was prepared by fusing human IL18BP to the C-terminal region of the Fc region of PET101 (FIG. 17B(A)). Specifically, the heterodimer expressed by co-transfection of vectors capable of expressing a polypeptide corresponding to IL1R-κ constant region-hinge region-CH2-CH3(S354C, T366W, K447del)-(G4S)2-IL18BP (SEQ ID NO: 12) and IL1RAcP-λ constant region-hinge region-CH2-CH3(Y349C, T366S, L368A, Y407V, K447del)-IL18BP (SEQ ID NO: 13) was named 118T3v01 (FIG. 17B(A)). Specifically, the nutrient solution was collected on the 12th day after expression was performed in the manner described in Example 1, and the nutrient solution was cultured in a Sartoclear Dynamics CT scanner. 登録商標After filtration using a LabV (SDLV-1000-40C-E (Sartorius)) filter system, affinity chromatography-based purification was performed using a HiTrap MabSelect SuRe (11003493 (GE Healthcare)) purification column. The primary purification product was subjected to affinity chromatography using KappaSelect resin (17545801 (GE Healthcare)) and then LambdaFabSelect resin (17548201 (GE Healthcare)). Dialysis, concentration, quantification, and SDS-PAGE analysis of the primary (MabSelect) purification product, secondary (KappaSelect) purification product, and tertiary (LambdaFabSelect) purification product were performed according to the method described in Example 1. In non-reducing SDS-PAGE analysis of the MabSelect primary purification eluate, an unassembled monomer was confirmed at approximately 130 kDa. When the tertiary purification was completed, no monomer was observed, but a dimeric product estimated to be 118T3v01 was confirmed at approximately 250 kDa (Figure 17B (B)).

[0087] Test Example 13: Analysis of the binding ability of IL-1 / IL-18 bispecific heterodimer to human IL-1β and human IL-18 using enzyme-linked immunosorbent assay (ELISA) Binding analysis of IL-1 / IL-18 bispecific heterodimers 118T3 and 118T3v01 to human IL-1β and human IL-18 was carried out using ELISA in the same manner as in Test Example 2. The analysis results confirmed that 118T3 and 118T3v01 have similar human IL-1β binding abilities (FIG. 18(A)), and that their human IL-18 binding abilities are also similar (FIG. 18(B)).

[0088] Test Example 14: Analysis of binding properties of 118T3v01 via surface plasmon resonance (SPR) To confirm whether 118T3v01 binds to human IL-1β and human IL-18 simultaneously, surface plasmon resonance analysis was performed using Biacore T200 (GE Healthcare). For all samples used in the following analysis, 1x HBS-EP+ buffer (BR100669 (GE Healthcare)) was used for dilution and as a mobile phase. In detail, the anti-human Fc antibody contained in the Human Antibody Capture Kit (BR100839 (GE Healthcare)) was immobilized on a Series S CM5 sensor chip (BR100530 (GE Healthcare)) using an amine coupling kit (BR100050 (GE Healthcare)) according to the manufacturer's manual. 100 nM 118T3v01 (25 μg / ml) was loaded onto the sensor with anti-human Fc immobilized at a flow rate of 10 μl / min for 1 minute, and equilibration was maintained for 2 minutes, confirming that 1766.1 RU of 118T3v01 was loaded onto the sensor surface (FIG. 19). Then, the binding reaction of 200 nM human IL-18 (10119-HNCE (Sino Biological)) was induced for 3 minutes, and the dissociation reaction was induced for 5 minutes. At equilibrium, it was confirmed that 134.5 RU of human IL-18 was bound (FIG. 19). Then, the binding reaction and dissociation reaction of 200 nM human IL-1β (10139-HNAE (Sino Biological)) were allowed to proceed for 3 minutes and 5 minutes, respectively, confirming that 137.5 RU of human IL-1β was bound. The RU value in surface plasmon resonance analysis is proportional to the mass of the substance loaded on the sensor surface, and 1 RU is 1 pg / mm 2This means that the substance is fixed to the sensor or bound by interaction. The molecular weight of 118T3v01 is about 250 kDa, and the molecular weights of human IL-1β and human IL-18 are about 18.4 kDa (Figure 19). Based on this, it was confirmed that one 118T3v01 protein binds to 1.03 human IL-18 and simultaneously binds to 1.06 human IL-1β (Figure 19). 118T3v01 has two human IL18BP proteins that can bind to human IL-18 bound to the C-terminus of the Fc region, but it was confirmed that only one of them binds to IL-18 (Figure 19). This suggests that steric hindrance occurs between the two bound IL18BP proteins.

[0089] Example 3: Optimization of IL-1 and IL-18 bispecific κλ heterodimers (118T3B) In Test Example 14, it was confirmed that one of the two human IL18BP proteins present in 118T3v01 was not involved in target binding. In order to remove one unnecessary bound IL18BP and to reduce the molecular weight, 118T3B was prepared by removing the IL18BP in the knob portion (FIG. 20(A)). To express 118T3B, expression was performed using an expression vector encoding IL1R-κ constant region-hinge region-CH2-CH3 (S354C, T366W) (SEQ ID NO: 8) and IL1RAcP-λ constant region-hinge region-CH2-CH3 (Y349C, T366S, L368A, Y407V, K447del)-IL18BP (SEQ ID NO: 13) in the manner described in Comparative Example 1, and purification was performed in the manner described in Example 1 (FIG. 20(B)). As a result of SDS-PAGE analysis, monomers of approximately 90 kDa and approximately 130 kDa were confirmed under reducing conditions, and it was confirmed that after the tertiary purification under non-reducing conditions, the monomers were removed and a highly pure substance estimated to be the 118T3B heterodimer was obtained at approximately 220 kDa (Figure 20(B)).

[0090] Test Example 15: Evaluation of the binding ability of 118T3B to IL-1 and IL-18 The binding ability of 118T3B to human IL-1α (200-LA (R&D Systems)), human IL-1β (201-LB (R&D Systems)), monkey IL-1β (1318-RL (R&D Systems)), mouse IL-1β (401-ML (R&D Systems)), human IL-18 (10139-HNAE (Sino Biological)), monkey IL-18 (2548-RM (R&D Systems)), and mouse IL-18 (50073-MNCE (Sino Biological)) was evaluated at 30°C using a bio-layer interferometry (BLI) analyzer, Octet Red96e (ForteBio). An anti-human IgG Fc Capture (AHC) biosensor (ForteBio, 18-5063) was used, and the analysis buffer was 10x Kinetics Buffer (18-1042 (ForteBio)) diluted with PBS pH 7.4 (10010 (Gibco)) to prepare the running buffer, and the analysis plate was rotated at 1,000 rpm. 118T3B was diluted in the running buffer at a concentration of 20 μg / ml and loaded onto the biosensor for 5 minutes. 0.25 to 8 nM human IL-1α, human IL-1β, monkey IL-1β, and mouse IL-1β showed binding rate constants (K a To measure the dissociation rate constant (K), the association time was run for 300 seconds, followed by a dissociation time of 1,200 seconds with running buffer. d ) was determined (Figures 21A and 21B). The binding rate constants (K a To measure the dissociation rate constant (K), the association time was advanced to 300 s, followed by a dissociation time of 600 s in running buffer. d ) was determined (Figure 21C). a Value and K d The equilibrium dissociation constant (K D ) values ​​were determined (Figures 21A, 21B, and 21C).

[0091] Test Example 16: Evaluation of the ability of IL-1 / IL-18 bispecific heterodimers to inhibit IL-1 and IL-18 signal transduction pathways using reporter cell lines To evaluate the ability of the IL-1 / IL-18 bispecific heterodimers 118T3v01 and 118T3B to inhibit the human IL-1β signaling pathway, the human IL-1β reporter cell line (hkb-il1b (Invivogen)) and the experimental method described in Test Example 4 were used. As a result, 118T3v01 and 118T3B had similar EC values ​​of 123 pM and 135 pM, respectively. 50 It was confirmed that the inhibitory activity of 118T3v01 and 118T3B against human IL-18 was similar to that of Test Example 4, using an IL-18 reporter HEK293 cell line (hkb-hmil18 (Invivogen)) and 1 ng / ml human IL-18 (10139-HNAE (Sino Biological)). As a result, 118T3v01 and 118T3B had similar EC values ​​of 392 pM and 305 pM, respectively. 50 It was confirmed that the value was (Figure 22(B)).

[0092] Example 4: Production of IL-1 and TGF-β bispecific κλ heterodimeric polypeptide (PET301) A gene sequence encoding amino acid residues 24 to 184 constituting the extracellular region of human TGFβRII (GenBank: ACZ58377.1) was chemically synthesized (Integrated DNA Technologies, IA, USA). The synthesized gene was cloned into each expression vector via the XhoI and XbaI restriction sites, and the obtained positive clones were verified by DNA sequencing. Specifically, the heterodimer expressed in a production cell line by co-transfection of vectors expressing polypeptides corresponding to IL1R-κ constant region-hinge region-CH2-CH3(S354C, T366W, K447A)-(G4S)4G-TGFβRII (SEQ ID NO: 14) and IL1RAcP-λ constant region-hinge region-CH2-CH3(Y349C, T366S, L368A, Y407V, K447A)-(G4S)4G-TGFβRII (SEQ ID NO: 15) is called PET301 (FIG. 23(A)). The IL-1 / TGF-β bispecific heterodimer was expressed primarily according to the method described in Comparative Example 1 and purified according to the method described in Example 1. The products at each purification step were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (FIG. 23(B)). The analysis results showed that under reducing conditions, a monomer was confirmed at approximately 110 kDa, whereas under non-reducing conditions, after the tertiary purification, the monomer was removed and a product presumed to be a PET301 heterodimer was confirmed at approximately 220 kDa (Figure 23(B)).

[0093] Test Example 17: Analysis of the binding ability of PET301 to human IL-1β and human TGF-β using enzyme-linked immunosorbent assay (ELISA) Binding analysis of PET301 to human IL-1β and human TGF-β was performed using ELISA. Human IL-1β (10139-HNAE (Sino Biological)), human TGF-β1 (R&D Systems, 7754-BH) or human TGF-β3 (8420-B3 (R&D Systems)) was coated on a 96-well immunosorbent assay plate (3590 (Corning)) at a concentration of 60 ng / well. The next day, the plate was washed with Phosphate Buffered Saline with Tween-20 (TEKNOVA, P1192 (PBST)). Blocking was performed with PBST supplemented with 1% (w / v) bovine serum albumin (BSA), and the plate was incubated at room temperature for 1 hour, followed by washing three times. PET301 was diluted to an appropriate concentration in 1% (w / v) BSA-containing PBST and treated in each well. Binding was induced at room temperature for 1 hour, and the plate was washed three times. Peroxidase-conjugated anti-human IgGFc chlorine antibody (31413 (Invitrogen)) was diluted (1:10,000) in 1% (w / v) BSA-containing PBST to induce binding, washed three times with PBST, reacted with TMB substrate solution (34028 (Thermo Fisher Scientific)), and then the absorbance at 450 nm was measured. Based on the measured absorbance, EC 50 Values ​​were calculated using Prism7 software (GraphPad Software) (Figure 24A(A), Figure 24A(B), Figure 24B(A), Table 4).

[0094] The binding ability of PET301 to human TGF-β2 was analyzed by coating PET301 at a concentration of 60 ng / well on a 96-well immunosorbent assay plate (3590 (Corning)). Blocking was performed at room temperature for 1 hour in PBST containing 1% (w / v) BSA. Human TGF-β2 (100-35B (Peprotech)) of an appropriate concentration was diluted in PBST containing 1% (w / v) BSA and the binding reaction was induced at room temperature for 1 hour. The plate was then treated with anti-TGF-β2 biotinylated antibody (BAF302 (R&D Systems)) and the binding reaction was induced at room temperature for 1 hour. The plate was then treated with streptavidin-HRP (R&D Systems) and the binding reaction was induced at room temperature for 30 minutes. The subsequent process was carried out in the same manner as described above (Figure 24B (B), Table 4).

[0095] The analysis results showed that PET301 had EC values ​​of 0.226 nM, 0.799 nM, and 0.357 nM for human IL-1β, human TGF-β1, and human TGF-β3, respectively. 50 PET301 had an EC value of 3.081 nM for human TGF-β2, confirming its high affinity for these antigens (Table 4). 50 It was confirmed that the mAbs had relatively low affinity (Table 4). [Table 4]

[0096] Test Example 18: Evaluation of the ability of PET301 to inhibit the signal transduction pathway between IL-1 and TGF-β using a reporter cell line The ability of PET301 to inhibit the human IL-1β signal transduction pathway was evaluated using the IL-1β reporter HEK293 cell line (hkb-il1b (Invivogen)) in the same manner as described in Test Example 4. The analysis results showed that PET301 had an EC 50It was confirmed that the TGF-β inhibitory activity of PET301 was high (Figure 25A (A)). To analyze the inhibitory activity of PET301 against human TGF-β1, human TGF-β2, and human TGF-β3, a TGF-β reporter HEK293 cell line (hkb-tgfb (Invivogen)) was used, and 1 ng / ml human TGF-β1 (7754-BH (R&D Systems)), 10 ng / ml human TGF-β2 (100-35B (Peprotech)), or 1 ng / ml human TGF-β3 (8420-B3 (R&D Systems)) was used to activate the cell line. To analyze the TGF-β inhibitory activity of PET301, Quanti-Blue TM The expression of SEAP was quantified using a solution (rep-qbs (Invivogen)). The analysis results showed that PET301 had EC values ​​of 30 pM and 89 pM for human TGF-β1 and human TGF-β3, respectively. 50 It was confirmed that human TGF-β2 could not be efficiently suppressed (FIG. 25A (B), FIG. 25B (A) and (B)).

Claims

1. i) a first polypeptide comprising, in N-terminal to C-terminal direction, a first protein of interest, a first linker, and a first Fc portion of an immunoglobulin; ii) a second polypeptide comprising, in N-terminal to C-terminal direction, a second protein of interest, a second linker, and a second Fc portion of an immunoglobulin; the first target protein and the second target protein are different proteins; The first linker and the second linker each include a κ-type light chain constant region (C Ｌκ ), or a mutant of the κ light chain constant site and a λ light chain constant site (C Ｌλ ), or a mutant of the lambda light chain constant site, The mutant kappa light chain constant site comprises a kappa light chain constant site in which the cysteine ​​that forms a disulfide bond with CH1 of the heavy chain constant site is mutated to prevent unwanted assembly; The mutant lambda light chain constant site is a lambda light chain constant site in which a cysteine ​​that forms a disulfide bond with CH1 of the heavy chain constant site is mutated to prevent unnecessary assembly, or a C-terminal serine is deleted, and the cysteine ​​that forms a disulfide bond with CH1 of the heavy chain constant site is at position 214 according to Kabat numbering. Heterodimeric Fc fusion proteins.

2. λ light chain constant region (C Ｌλ 2. The heterodimeric Fc fusion protein of claim 1, wherein the mutant of is a C-terminal serine deleted mutant.

3. κ light chain constant region (C Ｌκ ), a mutant of the λ-type light chain constant region (C Ｌλ 2. The heterodimeric Fc fusion protein of claim 1, wherein the cysteine ​​that forms a disulfide bond with the heavy chain constant region CH1 of either of the heavy chains is substituted with serine, alanine or valine.

4. The heterodimeric Fc fusion protein of claim 1, further comprising a third target protein at one or more of the C-terminus of the first polypeptide or the C-terminus of the second polypeptide.

5. The heterodimeric Fc-fusion protein of claim 1 having a monospecific or multispecific structure.

6. The heterodimeric Fc-fusion protein of claim 1, wherein the target protein is a ligand, a receptor, a cytokine, an enzyme or a peptide.

7. The heterodimeric Fc fusion protein of claim 6 , wherein the receptor is a cytokine receptor.

8. The cytokines include interleukin-1 (IL1), interleukin-2 (IL2), interleukin-3 (IL3), interleukin-4 (IL4), interleukin-5 (IL5), interleukin-6 (IL6), interleukin-11 (IL11), interleukin-13 (IL13), interleukin-15 (IL15), interleukin-18 (IL18), interleukin-23 (IL23), interleukin-31 (IL31), interleukin-33 (IL33), interleukin-41 (IL41), interleukin-42 (IL42), interleukin-43 (IL43), interleukin-44 (IL44), interleukin-45 (IL45), interleukin-46 (IL46), interleukin-47 (IL47), interleukin-48 (IL48), interleukin-49 (IL49), interleukin-50 (IL49), interleukin-51 (IL49), interleukin-52 (IL49), interleukin-53 (IL49), interleukin-54 (IL49), interleukin-55 (IL49), interleukin-56 (IL49), interleukin-57 (IL49), interleukin-58 (IL49), interleukin-59 (IL49), interleukin-60 (IL49), interleukin-61 (IL49), interleukin-62 (IL49), interleukin-63 (IL49), interleukin-64 (IL49), interleukin-65 (IL49), interleukin-66 (IL49), interleukin-67 (IL49), interleukin-68 (IL49), interleukin-69 (IL49), interleukin-70 (IL49), interleuk 8. The heterodimeric Fc fusion protein of claim 7, which is one or more selected from the group consisting of interleukin-35 (IL35), interleukin-36 (IL36), leukemia inhibitory factor (LIF), oncostatin M (OSM), granulocyte macrophage colony stimulating factor (GM-CSF), gamma interferon (IFN-γ), thymic stromal lymphopoietin (TSLP), transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF), and tumor necrosis factor-alpha (TNF-α).

9. The heterodimeric Fc fusion protein of claim 1, wherein one of the first and second target proteins is interleukin-1 receptor type 1 (IL1R1) and the other is interleukin-1 receptor accessory protein (IL1RAcP).

10. The first target protein is interleukin-1 receptor type 1 (IL1R1), and the first linker is a kappa light chain constant region (K Ｌκ ), or a mutant of a mutant of a κ-type light chain constant region, the second target protein is interleukin-1 receptor accessory protein (IL1RAcP), and the second linker is a λ-type light chain constant region (C Ｌλ ), or a mutant of the lambda light chain constant site, The mutant κ light chain constant site is a κ light chain constant site in which the cysteine ​​that forms a disulfide bond with CH1 of the heavy chain constant site is replaced with serine, alanine, or valine; The mutant λ light chain constant site is a λ light chain constant site in which the cysteine ​​that forms a disulfide bond with CH1 of the heavy chain constant site is replaced with serine, alanine, or valine, or the C-terminal serine is deleted; The cysteine ​​that forms a disulfide bond with CH1 of the heavy chain constant site is at position 214 according to the Kabat numbering system. The heterodimeric Fc fusion protein of claim 1.

11. 10. The heterodimeric Fc fusion protein of claim 9, wherein the interleukin-1 receptor type 1 (IL1R1) and the interleukin-1 receptor accessory protein (IL1RAcP) are their extracellular domains, respectively.

12. The heterodimeric Fc fusion protein of claim 1, comprising interleukin-18 binding protein (IL18BP) or type II transforming growth factor-beta receptor (TGFβRII) as a third target protein.

13. The heterodimeric Fc fusion protein according to claim 12, comprising interleukin-18 binding protein (IL18BP) at either the C-terminus of the first polypeptide or the C-terminus of the second polypeptide.

14. The heterodimeric Fc fusion protein of claim 13, which comprises interleukin-18 binding protein (IL18BP) at the C-terminus of the second polypeptide.

15. The heterodimeric Fc fusion protein of claim 12, which contains a type II transforming growth factor-beta receptor (TGFβRII) at both the C-terminus of the first polypeptide and the C-terminus of the second polypeptide.

16. 2. The heterodimeric Fc fusion protein of claim 1, wherein the first Fc moiety and the second Fc moiety have mutated CH3 regions to promote heterodimer formation, and the CH3 regions of the first Fc moiety and the second Fc moiety comprise knob-into-hole mutations.

17. The heterodimeric Fc fusion protein of claim 1, wherein the linker is attached to the N-terminus of the hinge within the Fc site.

18. The heterodimeric Fc fusion protein of claim 1, further comprising a third linker between the C-terminus of the polypeptide and the target protein.

19. The heterodimeric Fc fusion protein of claim 18, wherein the third linker is a Gly-Ser (GS) linker.

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