A fusion protein preparation comprising an IL-2 protein and a CD80 protein

Abstract

The present invention relates to a pharmaceutical formulation with improved stability of a fusion protein dimer comprising a modified IL-2 protein and a CD80 protein. The fusion protein dimer comprising IL-2 protein and a CD80 protein not only activates immune cells through IL-2 but also efficiently regulates Treg cells through CD80. When the formulation according to the present invention is applied to a fusion protein dimer comprising IL-2 protein and a CD80 protein, the stability of the fusion protein dimer is significantly increased, allowing it to be used as a liquid formulation. This increases the commercial availability of the fusion protein dimer.

Description

Technical Field

[0001] The present invention relates to a liquid preparation with improved stability of a fusion protein containing an IL-2 protein and a CD80 protein.

Background Art

[0002] IL-2, also called T cell growth factor, is a globular glycoprotein that plays a central role in lymphocyte production, survival, and homeostasis. IL-2 has a protein size of about 15.5 kDa to about 16 kDa and consists of 133 amino acids. IL-2 mediates various immune effects by binding to an IL-2 receptor composed of three different subunits. Furthermore, IL-2 is mainly synthesized by activated T cells, particularly CD4+ helper T cells. IL-2 stimulates the proliferation and differentiation of T cells and induces the production of cytotoxic T lymphocytes and the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer cells.

[0003] Furthermore, IL-2 is involved in the proliferation and differentiation of B cells and promotes immunoglobulin synthesis by B cells. Furthermore, IL-2 promotes the production, proliferation, and activation of natural killer cells. Therefore, since IL-2 can increase the lymphocyte population and enhance the function of immune cells in vivo, it is used as an anticancer agent. Currently, treatment with IL-2 is approved for patients with metastatic renal cell carcinoma or malignant melanoma.

[0004] However, IL-2 has a dual function of not only mediating an increase in the number and activity of immune cells but also being important for maintaining immune tolerance. It has also been reported that IL-2 may not be optimal for inhibiting tumor growth. The reason is that in the presence of IL-2, activation-induced cell death (AICD) may occur in the resulting cytotoxic T lymphocytes, and the immune response may be inhibited by IL-2-dependent regulatory T cells (Treg cells) (Imai et al., Cancer Sci 98, 416-423, 2007).

[0005] In addition, in patients administered with IL-2, severe cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, dermatological, hematological, and systemic side effects occur. Therefore, various IL-2 variants have been studied in order to improve the therapeutic effect of IL-2 and minimize its side effects (U.S. Patent No. 5,229,109). However, many problems still remain to be solved for the pharmacological use of IL-2.

[0006] On the other hand, CD80, also known as B7-1, is a member of the membrane-bound protein B7 family involved in immune regulation by delivering co-stimulatory and co-inhibitory responses and binding to its ligands. CD80 is a transmembrane protein expressed on the surfaces of T cells, B cells, dendritic cells, and monocytes. CD80 is known to bind to CD28, CTLA4 (CD152), and PD-L1. CD80, CD86, CTLA4, and CD28 are involved in the co-stimulation-co-inhibition system. For example, CD80 is known to control the activity of T cells and be involved in their proliferation, differentiation, and survival.

[0007] For example, when CD80 and CD86 interact with CD28, a co-stimulatory signal is generated, activating T cells. Ultimately, CD80 binds to CTLA4 expressed on the surface of activated T cells, stimulating the upregulation of CTLA4. As a result, CD80 inhibits the T cell response prior to the manipulation of the immune response caused by the CD80 / CD28 interaction. This feedback loop enables fine regulation of the immune response.

[0008] Furthermore, CD80 is known to bind to PD-L1 with an affinity similar to that of the binding of CD28, another B7 family member, to PD-L1. PD-L1 is known as one of the two ligands of the programmed cell death-1 (PD-1) protein, and PD-L1 is known to be involved in the regulation of T cells. The binding of CD80 to PD-L1 is another mechanism that can block the PD-1 / PD-L1 interaction, which can prevent the inhibition of T cell responses in tumors. However, an increase in CD80 levels causes the binding of CD80 to CD28, thereby inducing a T cell response. At the same time, CD80 can inhibit T cell responses by binding to CTLA4.

[0009] A fusion protein containing a CD80 fragment, an immunoglobulin Fc, and an IL-2 variant was confirmed to be able to activate immune cells and at the same time control the immunomodulatory activity of regulatory T cells, and thus can efficiently treat cancer and infectious diseases (Korean Patent No. 10-2201086). In order to efficiently apply such a protein to the treatment of cancer diseases and infectious diseases, it is necessary to develop a stable high-concentration protein formulation that provides dosage and dosing advantages. Detailed Description of the Invention

[0010] [Technical Problem] The inventors of the present invention developed a formulation that enhances the stability of a novel fusion protein dimer containing an IL-2 protein and a CD80 protein in one molecule, thereby completing the present invention. [Means for Solving the Problems]

[0011] In order to achieve the above object, in one aspect of the present invention, a pharmaceutical preparation containing a fusion protein dimer containing an IL-2 protein and a CD80 protein is provided. [Effects of the Invention]

[0012] The fusion protein dimer containing the IL-2 protein and the CD80 protein can not only activate immune cells by IL-2, but also efficiently control Treg cells by CD80. In order to clinically use such a fusion protein dimer, it is necessary to ensure the stability of the protein preparation. When the pharmaceutical preparation according to the present invention is applied to the fusion protein dimer containing the IL-2 protein and the CD80 protein, the stability of the fusion protein dimer is significantly improved and it can be used as a liquid preparation. Therefore, the commercial availability of the fusion protein dimer can be enhanced.

Brief Description of Drawings

[0013]

Figure 1

Figure 2

Figure 3

Best Mode for Carrying Out the Invention

[0014] A pharmaceutical preparation containing a fusion protein containing the IL-2 protein and the CD80 protein In one aspect of the present invention, there is provided a pharmaceutical preparation comprising: (i) a fusion protein dimer containing the IL-2 protein and the CD80 protein at a concentration of 3.0 mg / mL to 5.0 mg / mL; (ii) a buffer at a concentration of 10 mM to 30 mM; and (iii) a surfactant at a concentration of 0.155 w / w% to 0.185 w / w%, wherein the pH of the preparation is 6.5 to 7.5.

[0015] Here, the pharmaceutical preparation may be a liquid preparation.

[0016] A fusion protein containing the IL-2 protein and the CD80 protein As used herein, the term "IL-2" or "interleukin-2" refers to any wild-type IL-2 obtained from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. IL-2 may be obtained from animal cells and also includes that obtained from recombinant cells capable of producing IL-2. Furthermore, IL-2 may be wild-type IL-2 or a variant thereof.

[0017] In this specification, IL-2 or a variant thereof may be collectively referred to by the term "IL-2 protein" or "IL-2 polypeptide". IL-2, IL-2 protein, IL-2 polypeptide, and IL-2 variant specifically bind to, for example, the IL-2 receptor. This specific binding can be identified by methods known to those skilled in the art.

[0018] One embodiment of IL-2 may have the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. Here, IL-2 may be in a mature form. Specifically, mature IL-2 may not contain a signal sequence and may have the amino acid sequence of SEQ ID NO: 10. Here, IL-2 may be used under the concept of encompassing a fragment of wild-type IL-2 in which a part of the N-terminus or C-terminus of wild-type IL-2 is cleaved.

[0019] Furthermore, a fragment of IL-2 can be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive amino acids are cleaved from the N-terminus of a protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. Furthermore, a fragment of IL-2 can be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive amino acids are cleaved from the C-terminus of a protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

[0020] As used herein, the term "IL-2 variant" refers to a form in which some of the amino acids in the full-length IL-2 or the above-mentioned fragment of IL-2 are substituted. That is, the IL-2 variant may have an amino acid sequence different from that of wild-type IL-2 or its variant. However, the IL-2 variant may have an activity equivalent to or similar to that of wild-type IL-2. Here, "IL-2 activity" may refer to, for example, specific binding to the IL-2 receptor, and this specific binding can be measured by methods known to those skilled in the art.

[0021] Specifically, the IL-2 variant can be obtained by substituting some of the amino acids in wild-type IL-2. One embodiment of the IL-2 variant obtained by amino acid substitution can be obtained by substituting at least one of the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10.

[0022] Specifically, the IL-2 variant can be obtained by substituting at least one of the 38th, 42nd, 45th, 61st, or 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 with another amino acid. Also, when IL-2 is in a form in which a part of the N-terminus in the amino acid sequence of SEQ ID NO: 35 is cleaved, the amino acids at positions corresponding complementarily to those in the amino acid sequence of SEQ ID NO: 10 may be substituted with other amino acids. For example, when IL-2 has the amino acid sequence of SEQ ID NO: 35, the IL-2 variant can be obtained by substituting at least one of the 58th, 62nd, 65th, 81st, or 92nd amino acids in the amino acid sequence of SEQ ID NO: 35 with another amino acid. These amino acid residues correspond to the 38th, 42nd, 45th, 61st, and 72nd amino acid residues in the amino acid sequence of SEQ ID NO: 10, respectively. According to one embodiment, as long as such an IL-2 variant maintains IL-2 activity, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be substituted. According to other embodiments, 1 to 5 amino acids may be substituted.

[0023] In one embodiment, the IL-2 variant may be in a form in which two amino acids are substituted. Specifically, the IL-2 variant can be obtained by substituting the 38th and 42nd amino acids in the amino acid sequence of SEQ ID NO: 10. Further, in one embodiment, the IL-2 variant can be obtained by substituting the 38th and 45th amino acids in the amino acid sequence of SEQ ID NO: 10. Further, in one embodiment, the IL-2 variant can be obtained by substituting the 38th and 61st amino acids in the amino acid sequence of SEQ ID NO: 10. Further, in one embodiment, the IL-2 variant can be obtained by substituting the 38th and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10. Further, in one embodiment, the IL-2 variant can be obtained by substituting the 42nd and 45th amino acids in the amino acid sequence of SEQ ID NO: 10. Further, in one embodiment, the IL-2 variant can be obtained by substituting the 42nd and 61st amino acids in the amino acid sequence of SEQ ID NO: 10. Further, in one embodiment, the IL-2 variant can be obtained by substituting the 42nd and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10. Further, in one embodiment, the IL-2 variant can be obtained by substituting the 45th and 61st amino acids in the amino acid sequence of SEQ ID NO: 10. Further, in one embodiment, the IL-2 variant can be obtained by substituting the 45th and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10. Further, in one embodiment, the IL-2 variant can be obtained by substituting the 61st and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10.

[0024] Furthermore, the IL-2 variant may be in a form in which three amino acids are substituted. Specifically, the IL-2 variant can be obtained by substituting the 38th, 42nd, and 45th amino acids in the amino acid sequence of SEQ ID NO: 10. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting the 38th, 42nd, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting the 38th, 42nd, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting the 38th, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting the 38th, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting the 38th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting the 42nd, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting the 42nd, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting the 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10.

[0025] Furthermore, the IL-2 variant may be in a form in which four amino acids are substituted. Specifically, the IL-2 variant can be obtained by substituting the 38th, 42nd, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting the 38th, 42nd, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting the 38th, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting the 38th, 42nd, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting the 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10.

[0026] Furthermore, the IL-2 variant may be in a form in which five amino acids are substituted. Specifically, the IL-2 variant can be obtained by substituting each of the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 with another amino acid.

[0027] Here, the "other amino acid" introduced by the substitution may be any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. However, with respect to the amino acid substitution of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 38th amino acid cannot be substituted with arginine, the 42nd amino acid cannot be substituted with phenylalanine, the 45th amino acid cannot be substituted with tyrosine, the 61st amino acid cannot be substituted with glutamic acid, and the 72nd amino acid cannot be substituted with leucine.

[0028] Regarding the amino acid substitution of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 38th amino acid, arginine, may be substituted with an amino acid other than arginine. Preferably, regarding the amino acid substitution of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 38th amino acid, arginine, may be substituted with alanine (R38A).

[0029] Regarding the amino acid substitution of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 42nd amino acid, phenylalanine, may be substituted with an amino acid other than phenylalanine. Preferably, regarding the amino acid substitution of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 42nd amino acid, phenylalanine, may be substituted with alanine (F42A).

[0030] Regarding the amino acid substitution of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 45th amino acid, tyrosine, may be substituted with an amino acid other than tyrosine. Preferably, regarding the amino acid substitution of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 45th amino acid, tyrosine, may be substituted with alanine (Y45A).

[0031] Regarding the amino acid substitution of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 61st amino acid, glutamic acid, may be substituted with an amino acid other than glutamic acid. Preferably, regarding the amino acid substitution of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 61st amino acid, glutamic acid, may be substituted with arginine (E61R).

[0032] Regarding the amino acid substitution of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, leucine, which is the 72nd amino acid, may be substituted with an amino acid other than leucine. Preferably, regarding the amino acid substitution of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, leucine, which is the 72nd amino acid, may be substituted with glycine (L72G).

[0033] Specifically, the IL-2 variant can be obtained by at least one substitution selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO: 10.

[0034] Specifically, the IL-2 variant can be obtained by amino acid substitutions at 2, 3, 4, or 5 positions selected from the positions consisting of R38A, F42A, Y45A, E61R, and L72G.

[0035] Furthermore, the IL-2 variant may be in a form where two amino acids are substituted. Specifically, the IL-2 variant can be obtained by the substitutions of R38A and F42A. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of R38A and Y45A. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of R38A and E61R. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of R38A and L72G. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of F42A and Y45A. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of F42A and E61R. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of F42A and L72G. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of E61R and L72G.

[0036] Furthermore, the IL-2 variant may be in a form in which three amino acids are substituted. Specifically, the IL-2 variant can be obtained by the substitutions of R38A, F42A, and Y45A. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of R38A, F42A, and E61R. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of R38A, F42A, and L72G. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of R38A, Y45A, and E61R. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of R38A, Y45A, and L72G. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of F42A, Y45A, and E61R. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of F42A, Y45A, and L72G. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of F42A, E61R, and L72G. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of Y45A, E61R, and L72G.

[0037] Furthermore, the IL-2 variant may be in a form in which four amino acids are substituted. Specifically, the IL-2 variant can be obtained by the substitutions of R38A, F42A, Y45A, and E61R. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of R38A, F42A, Y45A, and L72G. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of R38A, F42A, E61R, and L72G. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of R38A, Y45A, E61R, and L72G. Furthermore, in one embodiment, the IL-2 variant can be obtained by the substitutions of F42A, Y45A, E61R, and L72G.

[0038] Furthermore, the IL-2 variant can be obtained by the substitutions of R38A, F42A, Y45A, E61R, and L72G.

[0039] Preferably, embodiments of the IL-2 variant can include any one selected from the following combinations (a) to (d) of substitutions in the amino acid sequence of SEQ ID NO: 10: (a) R38A / F42A (b) R38A / F42A / Y45A (c) R38A / F42A / E61R (d) R38A / F42A / L72G.

[0040] Here, when IL-2 has the amino acid sequence of SEQ ID NO: 35, there may be amino acid substitutions at positions that complementarily correspond to the amino acid sequence of SEQ ID NO: 10. Furthermore, even when IL-2 is a fragment of the amino acid sequence of SEQ ID NO: 35, there may be amino acid substitutions at positions that complementarily correspond to the amino acid sequence of SEQ ID NO: 10.

[0041] Specifically, the IL-2 variant may have the amino acid sequence of SEQ ID NO: 6, 22, 23, or 24.

[0042] Furthermore, the IL-2 variant may be characterized by having low in vivo toxicity. Here, the low in vivo toxicity may be a side effect caused by the binding of IL-2 to the interleukin-2 receptor alpha chain (IL-2Rα). To improve the side effects caused by the binding of IL-2 to IL-2Rα, various IL-2 variants have been developed, and such IL-2 variants may be those disclosed in US Patent No. 5,229,109 and Korean Patent No. 1667096. In particular, the IL-2 variant described in the present application has a lower binding ability to the interleukin-2 receptor alpha chain (IL-2Rα), and thus has lower in vivo toxicity than wild-type IL-2.

[0043] As used herein, the term "CD80" is also referred to as "B7-1" and is a membrane protein present in dendritic cells, activated B cells, and monocytes. CD80 provides a co-stimulatory signal essential for the activation and survival of T cells. CD80 is known as a ligand for two different proteins, CD28 and CTLA-4, present on the surface of T cells. CD80 is composed of 288 amino acids and, in particular, can have the amino acid sequence of SEQ ID NO: 11. Further, as used herein, the term "CD80 protein" refers to full-length CD80 or a CD80 fragment.

[0044] As used herein, the term "CD80 fragment" refers to a cleaved form of CD80. Further, the CD80 fragment can be the extracellular domain of CD80. One embodiment of the CD80 fragment can be obtained by removing amino acids 1 to 34 from the N-terminus, which is the signal sequence of CD80. In particular, one embodiment of the CD80 fragment may be a protein composed of amino acids 35 to 288 in SEQ ID NO: 11. Further, one embodiment of the CD80 fragment may be a protein composed of amino acids 35 to 242 in SEQ ID NO: 11. Further, one embodiment of the CD80 fragment may be a protein composed of amino acids 35 to 232 in SEQ ID NO: 11. Further, one embodiment of the CD80 fragment may be a protein composed of amino acids 35 to 139 in SEQ ID NO: 11. Further, one embodiment of the CD80 fragment may be a protein composed of amino acids 142 to 242 in SEQ ID NO: 11. In one embodiment, the CD80 fragment may have the amino acid sequence of SEQ ID NO: 2.

[0045] Further, the IL-2 protein and the CD80 protein may be bound to each other via a linker or a carrier. In particular, IL-2 or a variant thereof, and CD80 (B7-1) or a fragment thereof, may be bound to each other via a linker or a carrier. As used herein, linkers and carriers can be used interchangeably.

[0046] The linker connects two proteins. One embodiment of the linker may include 1 to 50 amino acids, albumin or a fragment thereof, the Fc domain of an immunoglobulin, etc. Here, the Fc domain of an immunoglobulin refers to a protein that includes the constant region 2 (CH2) and constant region 3 (CH3) of the heavy chain of the immunoglobulin and does not include the variable regions of the heavy and light chains of the immunoglobulin and the constant region 1 (CH1) of the light chain. The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM, and preferably may be IgG4. Here, the Fc domain of wild-type immunoglobulin G4 may have the amino acid sequence of SEQ ID NO: 4.

[0047] Furthermore, the Fc domain of an immunoglobulin may be an Fc domain variant and a wild-type Fc domain. Furthermore, as used herein, the term "Fc domain variant" may refer to a form that is different from the wild-type Fc domain in terms of glycosylation pattern, has higher glycosylation compared to the wild-type Fc domain, or has lower glycosylation compared to the wild-type Fc domain, or a deglycosylated form. Furthermore, an aglycosylated Fc domain is included therein. The Fc domain or its variant can be adapted so that the number of sialic acid, fucosylation, or glycosylation is adjusted by the culture conditions of the host or genetic manipulation.

[0048] Furthermore, the glycosylation of the Fc domain of an immunoglobulin can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Furthermore, the Fc domain variant may be a mixed form of the Fc regions of immunoglobulins, IgG, IgA, IgE, IgD, and IgM, respectively. Furthermore, the Fc domain variant may be a form in which some amino acids of the Fc domain are substituted with other amino acids. One embodiment of the Fc domain variant may have the amino acid sequence of SEQ ID NO: 12.

[0049] The fusion protein may have a structure in which the Fc domain is used as a linker (or carrier), and the CD80 protein and the IL-2 protein, or the IL-2 protein and the CD80 protein are respectively linked to the N-terminus and C-terminus of the linker or carrier. The linkage between the N-terminus or C-terminus of the Fc domain and CD-80 or IL-2 may optionally be achieved by a linker peptide.

[0050] Specifically, the fusion protein may have the following structural formula (I) or (II): N’-X-[Linker(1)] n -Fc domain-[Linker(2)] m -Y-C’(I) N’-Y-[Linker(1)] n -Fc domain-[Linker(2)] m -X-C’(II), and may consist of Here, in structural formulas (I) and (II), N’ is the N-terminus of the fusion protein, C’ is the C-terminus of the fusion protein, X is the CD80 protein, Y is the IL-2 protein, Linkers (1) and (2) are peptide linkers, and n and m are each independently 0 or 1.

[0051] Preferably, the fusion protein may consist of structural formula (I). The IL-2 protein is as described above. Furthermore, the CD80 protein is as described above. According to one embodiment, the IL-2 protein may be an IL-2 variant having 1 to 5 amino acid substitutions compared to wild-type IL-2. The CD80 protein may be a fragment obtained by cleaving up to about 34 consecutive amino acid residues from the N-terminus or C-terminus of wild-type CD80. Alternatively, the CD protein may be an extracellular immunoglobulin-like domain having the activity of binding to the T cell surface receptors CTLA-4 and CD28.

[0052] Specifically, the fusion protein may have the amino acid sequence of SEQ ID NO: 9, 26, 28, or 30. According to other embodiments, the fusion protein includes a polypeptide having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9, 26, 28, or 30. Here, the identity is, for example, the percentage of homology, which can be determined by homology comparison software such as the Blast N software of the National Center for Biotechnology Information (NCBI) in the United States.

[0053] Peptide linker (1) may be included between the CD80 protein and the Fc domain. Peptide linker (1) may consist of 5 to 80 consecutive amino acids, 20 to 60 consecutive amino acids, 25 to 50 consecutive amino acids, or 30 to 40 consecutive amino acids. In one embodiment, peptide linker (1) may consist of 30 amino acids. Furthermore, peptide linker (1) may contain at least one cysteine. Specifically, peptide linker (1) may contain 1, 2, or 3 cysteines. Furthermore, peptide linker (1) may be derived from the hinge of an immunoglobulin. In one embodiment, peptide linker (1) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 3.

[0054] Peptide linker (2) may consist of 1 to 50 consecutive amino acids, 3 to 30 consecutive amino acids, or 5 to 15 consecutive amino acids. In one embodiment, peptide linker (2) is (G4S) n (wherein n is an integer from 1 to 10). Here, in (G4S) n , n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, peptide linker (2) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 5.

[0055] In another aspect of the present invention, a dimer obtained by the binding of two fusion proteins, each containing an IL-2 protein and a CD80 protein, is provided. The fusion proteins containing IL-2 or a variant thereof and CD80 or a fragment thereof are as described above.

[0056] Here, the binding between the fusion proteins constituting the dimer can be achieved by a disulfide bond formed by a cysteine present in the linker, but is not limited thereto. The fusion proteins constituting the dimer may be the same or different from each other. Preferably, the dimer may be a homodimer. One embodiment of the fusion protein constituting the dimer may be a protein having the amino acid sequence of SEQ ID NO: 9.

[0057] Pharmaceutical preparation In one aspect of the present invention, a pharmaceutical preparation containing a fusion protein dimer containing an IL-2 protein and a CD80 protein is provided.

[0058] As used herein, the term "pharmaceutical preparation" refers to a preparation in which the biological activity of the active ingredient is clearly effective and which does not contain components that cause side effects in the subject to which the preparation is administered.

[0059] The term "subject" may be a mammal such as a human, dog, cow, horse, pig, sheep, goat, cat, mouse, rabbit, rat, etc., and preferably may be a human, dog, or cat.

[0060] As used herein, the term "pharmaceutical preparation" refers to a pharmaceutical preparation using a suitable aqueous solvent such as water, or an aqueous / oily mixture (for example, a water-alcohol mixture). The preparation can maintain stability such as chemical or physical stability and biological activity.

[0061] The term "stability" refers to the property of maintaining a certain state and is generally related to minimizing the degradation, denaturation, aggregation, or unfolding of biologically active substances such as proteins, peptides, or biologically active polymers.

[0062] On the other hand, the formulation may be a liquid formulation. The liquid formulation is an aqueous solution or suspension and can be stably maintained at room temperature, refrigeration (e.g., 2°C - 8°C), or freezing (e.g., -20°C or -70°C) during storage.

[0063] The pharmaceutical formulation of the present invention can be administered parenterally. Here, parenteral administration can be carried out by methods such as subcutaneous administration, intravenous administration, mucosal administration, and intramuscular administration. In one embodiment of the present invention, the formulation can preferably be administered by intravenous injection.

[0064] The fusion protein dimer in the pharmaceutical formulation can have a concentration of 3.0 mg / mL to 5.0 mg / mL. Furthermore, the fusion protein dimer can have a concentration of 3.0 mg / mL to 4.8 mg / mL, 3.0 mg / mL to 4.6 mg / mL, 3.0 mg / mL to 4.4 mg / mL, 3.0 mg / mL to 4.2 mg / mL, 3.2 mg / mL to 4.8 mg / mL, 3.2 mg / mL to 4.6 mg / mL, 3.2 mg / mL to 4.4 mg / mL, 3.2 mg / mL to 4.2 mg / mL, 3.4 mg / mL to 4.8 mg / mL, 3.4 mg / mL to 4.6 mg / mL, 3.4 mg / mL to 4.4 mg / mL, 3.4 mg / mL to 4.2 mg / mL, 3.6 mg / mL to 4.8 mg / mL, 3.6 mg / mL to 4.6 mg / mL, 3.6 mg / mL to 4.4 mg / mL, 3.6 mg / mL to 4.2 mg / mL, 3.8 mg / mL to 4.8 mg / mL, 3.8 mg / mL to 4.6 mg / mL, 3.8 mg / mL to 4.4 mg / mL, 3.8 mg / mL to 4.2 mg / mL, or 3.9 mg / mL to 4.1 mg / mL. Specifically, the fusion protein dimer may have a concentration of 4.0 mg / mL.

[0065] Furthermore, the buffer may be a histidine buffer. Here, histidine may have a concentration of 10 mM to 30 mM. Furthermore, histidine may have a concentration of 10 mM to 28 mM, 10 mM to 26 mM, 10 mM to 24 mM, 10 mM to 22 mM, 10 mM to 21 mM, 12 mM to 28 mM, 12 mM to 26 mM, 12 mM to 24 mM, 12 mM to 22 mM, 12 mM to 21 mM, 14 mM to 28 mM, 14 mM to 26 mM, 14 mM to 24 mM, 14 mM to 22 mM, 14 mM to 21 mM, 16 mM to 28 mM, 16 mM to 26 mM, 16 mM to 24 mM, 16 mM to 22 mM, 16 mM to 21 mM, 18 mM to 28 mM, 18 mM to 26 mM, 18 mM to 24 mM, 18 mM to 22 mM, 18 mM to 21 mM, 19 mM to 28 mM, 19 mM to 26 mM, 19 mM to 24 mM, 19 mM to 22 mM, or 19 mM to 21 mM. Specifically, histidine may have a concentration of 20 mM.

[0066] Furthermore, the pH of the pharmaceutical preparation may be 6.5 to 7.5. Furthermore, the pH of the pharmaceutical preparation may be 6.5 to 7.3, 6.5 to 7.2, 6.5 to 7.1, 6.7 to 7.3, 6.7 to 7.2, 6.7 to 7.1, 6.8 to 7.3, 6.8 to 7.2, 6.8 to 7.1, 6.9 to 7.3, 6.9 to 7.2, or 6.9 to 7.1. Preferably, the pH of the pharmaceutical preparation may be 7.0.

[0067] Furthermore, the surfactant of the pharmaceutical preparation may contain any one selected from the group consisting of polysorbates (such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85); poloxamers (such as poloxamer 181, poloxamer 188, and poloxamer 407); polyethylene glycol (PEG); and combinations thereof. Preferably, the pharmaceutical preparation may contain two of the surfactants.

[0068] Furthermore, the surfactant may be contained in the formulation at a concentration of 0.065 w / w% to 0.2 w / w%. Furthermore, the surfactant may be contained in the formulation at a concentration of 0.155 w / w% to 0.185 w / w%. In one embodiment, the surfactant may be poloxamer 188. Here, the surfactant may be contained in the formulation at a concentration of 0.065 w / w% to 0.075 w / w%. Furthermore, in one embodiment, the surfactant may be polysorbate 80. Here, the surfactant may be contained in the formulation at a concentration of about 0.09 w / w% to about 0.11 w / w%. Preferably, poloxamer 188 and polysorbate 80 may be contained in the formulation, and they may be contained at concentrations of 0.065 w / w% to 0.075 w / w% and 0.09 w / w% to about 0.11 w / w% respectively. Specifically, poloxamer 188 and polysorbate 80 may be contained in the formulation at concentrations of 0.07 w / w% and 0.1 w / w% respectively.

[0069] Furthermore, the pharmaceutical formulation may further contain an amino acid. The amino acid may be any one selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan.

[0070] Here, the amino acid may have a concentration of 10 mg / mL to 30 mg / mL. Further, the amino acid may have a concentration of 10 mg / mL to 25 mg / mL, 10 mg / mL to 20 mg / mL, 10 mg / mL to 18 mg / mL, 10 mg / mL to 16 mg / mL, 12 mg / mL to 25 mg / mL, 12 mg / mL to 20 mg / mL, 12 mg / mL to 18 mg / mL, 12 mg / mL to 16 mg / mL, 14 mg / mL to 25 mg / mL, 14 mg / mL to 20 mg / mL, 14 mg / mL to 18 mg / mL, or 14 mg / mL to 16 mg / mL. Specifically, the amino acid may have a concentration of 15 mg / mL.

[0071] In one embodiment, the amino acid may be arginine, preferably arginine-HCl. Here, arginine may be contained at a concentration of 14 mg / mL to 16 mg / mL, preferably at a concentration of 15 mg / mL.

[0072] Furthermore, the pharmaceutical preparation may further contain sugar. The sugar may be any one selected from the group consisting of sucrose, sorbitol, glycerol, trehalose, and mannitol. Here, the sugar may be contained at a concentration of 120 mg / mL to 180 mg / mL. Further, the sugar may have a concentration of 120 mg / mL to 170 mg / mL, 120 mg / mL to 160 mg / mL, 120 mg / mL to 155 mg / mL, 130 mg / mL to 170 mg / mL, 130 mg / mL to 160 mg / mL, 130 mg / mL to 155 mg / mL, 135 mg / mL to 170 mg / mL, 135 mg / mL to 160 mg / mL, 135 mg / mL to 155 mg / mL, 140 mg / mL to 170 mg / mL, 140 mg / mL to 160 mg / mL, 140 mg / mL to 155 mg / mL, 145 mg / mL to 170 mg / mL, 145 mg / mL to 160 mg / mL, or 145 mg / mL to 155 mg / mL.

[0073] In one embodiment, the sugar may be sucrose, and the sucrose may have a concentration of 150 mg / mL.

[0074] In one embodiment of the present invention, the pharmaceutical preparation may contain: (i) a fusion protein dimer containing IL-2 protein and CD80 protein at a concentration of 3.0 mg / mL to 5.0 mg / mL; (ii) histidine at a concentration of 10 mM to 30 mM; (iii) poloxamer 188 at a concentration of 0.065 w / w% to 0.075 w / w%; (iv) polysorbate 80 at a concentration of 0.09 w / w% to 0.11 w / w%; (v) arginine at a concentration of 10 mg / mL to 30 mg / mL; and (vi) sucrose at a concentration of 120 mg / mL to 180 mg / mL, where the pH of the pharmaceutical preparation may be 6.5 to 7.5.

[0075] The pharmaceutical preparation may be stored in a container selected from the group consisting of vials, cartridges, syringes, and autoinjectors.

[0076] Furthermore, the container in which the preparation is stored can be stored at room temperature, 2°C to 8°C, or 25°C to 40°C until it is administered to a subject in need of treatment.

[0077] The subject may be a mammal such as a human, dog, cow, horse, pig, sheep, goat, cat, mouse, rabbit, and rat, and preferably may be a human.

[0078] The preparation can be administered by parenteral administration such as subcutaneous administration, intravenous administration, mucosal administration, intramuscular administration, or intraperitoneal administration, but is not limited thereto. Preferably, it may be administered intravenously.

Mode for Carrying Out the Invention

[0079] Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for explaining the present invention, and the scope of the present invention is not limited thereto.

[0080] Preparation Example 1. Preparation of hCD80-Fc-IL-2 variant (2M): GI-101 To produce a fusion protein dimer containing a human CD80 fragment, an Fc domain, and an IL-2 variant, a polynucleotide was synthesized through the Invitrogen GeneArt gene synthesis service of ThermoFisher Scientific. Specifically, the polynucleotide contains a nucleotide sequence (SEQ ID NO: 8) encoding a fusion protein that includes, in this order from the N-terminus, a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3) to which a linker is attached, an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) having two amino acid substitutions. The polynucleotide was inserted into the pcDNA3_4 vector. Further, this vector was introduced into CHO cells (Expi-CHO (trademark)), and the fusion protein of SEQ ID NO: 9 was expressed. After introducing the vector, culturing was carried out for 7 days under the conditions of 37 °C, 125 RPM, and 8% CO2. Then, the culture was collected, and the fusion protein was purified from it. The purified fusion protein was named "GI-101".

[0081] Purification was performed using chromatography with MabSelect SuRe protein A resin. The fusion protein was bound to this under the conditions of 25 mM Tris, 25 mM NaCl, and pH 7.4. Then, elution was carried out with 100 mM NaCl and 100 mM acetic acid (pH 3). 20% of 1 M Tris-HCl was placed in the collection tube at pH 9, and then the fusion protein dimer was collected. For the collected fusion protein dimer, the buffer was exchanged by dialysis with PBS buffer for 16 hours.

[0082] Subsequently, size exclusion chromatography using a TSKgel G3000SWXL column (Tosoh Bioscience) was performed to measure the absorbance at a wavelength of 280 nm over time, and a highly concentrated fusion protein dimer was obtained. Here, the isolated and purified fusion protein dimer was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions and stained with Coomassie Brilliant Blue to confirm the purity (Figure 1). When detected with a NanoDrop, it was confirmed that the fusion protein dimer was contained at a concentration of 2.78 mg / ml (Figure 2). The results obtained by analysis using size exclusion chromatography are shown in Figure 3.

[0083] Example 1. Evaluation of Optimal Buffer / pH Conditions To determine the optimal buffer / pH for a liquid formulation containing a fusion protein dimer (GI-101) of CD80 protein and IL-2 protein, a total of eight buffer / pH screenings were performed. A stability test (40 °C, 2 weeks) was carried out to select the optimal buffer / pH.

[0084] After storing the buffer / pH screening samples at 40 °C for 2 weeks, the samples were analyzed using size exclusion chromatography (SEC). To identify the size exclusion profile of the GI-101 protein, SEC was performed using HPLC (Waters, e2695 and Thermo scientific, Ultimate 3000). The % area of the monomer was calculated using the chromatogram at 214 nm.

[0085] During formulation development, the SEC profile was used with the aim of maximizing the % monomer. The stability test results of the buffer / pH screening are summarized in Table 1. Sample #6 containing a histidine buffer, pH 7.0, showed fewer changes than the other samples.

[0086]

Table 1

[0087] Based on the stability test data, histidine buffer at pH 7.0 was determined as the buffer for GI-101.

[0088] [Table 2]

[0089] Example 2. Excipient Screening Test Example 2.1 Excipient Screening To determine the excipients for the liquid formulation containing GI-101, an excipient screening test was conducted under the buffer / pH (histidine buffer, pH 7.0) conditions selected in Example 1.

[0090] For eight different excipients [polysorbate 80, poloxamer 188, arginine-HCl (L-arginine monohydrochloride), L-methionine, D-mannitol, sorbitol, sucrose, and D-(+)-trehalose dihydrate], the Tm and Tagg, SEC, and the results of visual particle tests obtained under five different test conditions were statistically analyzed, and poloxamer 188, arginine-HCl, and sucrose were selected as excipients.

[0091] [Table 3]

[0092] Example 2.2. Screening of Optimal Excipient Concentrations According to Example 2.1, poloxamer 188, arginine-HCl, and sucrose were selected as excipients for the liquid formulation containing GI-101.

[0093] To determine the optimal concentrations of the combination of the three excipients, the concentrations of each excipient were varied, and screening tests were conducted under 16 conditions.

[0094] Example 2.2.1. Thermal Stability Test (40°C, 2 weeks) After storing 16 excipient screening test samples at 40°C for 2 weeks, the samples were analyzed by protein concentration (A280) test and SEC test. The results are shown in Table 4.

[0095] [Table 4]

[0096] Example 2.2.2. Results of RSM and Simulation Using RSM (Response Surface Model), the optimal concentration of each excipient was determined. By adjusting the concentration of each excipient in the prediction profiler, the optimal response could be found. In the results predicted by the prediction profiler, the concentration of sucrose was set to the optimal value of 150 mg / mL, which is the maximum value of the experiment. Therefore, only the concentrations of arginine-HCl and poloxamer 188 were set by simulation. The predicted optimal concentration ranges of each excipient are shown in Table 5.

[0097] [Table 5]

[0098] Example 2.3 Final Stability Test (4 weeks) Based on the optimal concentration ranges of each excipient in Table 5, the final formulation candidates in Table 6 were obtained. For the formulation candidates in Table 6, the final 4-week stability test was carried out.

[0099] [Table 6]

[0100] The stability of the formulation candidates was tested under a total of five different conditions. An initial release test (t = 0) was conducted immediately after sample preparation, and the candidate samples were stored at 5°C (long-term condition), -70°C (second long-term condition), 25°C (accelerated condition), and 40°C (severe condition) for 4 weeks, respectively. In particular, for the 40°C stability test (severe condition), additional samples were taken at the 2-week time point to identify the trend of change. After storage for 4 weeks under each condition, the samples were analyzed by SEC, protein concentration (A280), and pH test. The results of the final stability test for the GI-101 formulation candidate are shown in Table 7.

[0101]

Table 7

[0102] As a result of the stability test, the formulation candidates showed a stable state under the conditions of -70°C, 5°C, and 25°C. From these results, the final formulation for GI-101 was determined to be GI-101 8 mg / mL, histidine buffer (pH 7.0) 20 mM, poloxamer 188 0.07 w / w%, arginine-HCl 15 mg / mL, and sucrose 150 mg / mL.

[0103]

Table 8

[0104] Example 3. Polysorbate 80 Addition Test In the process of manufacturing the GI-101 active pharmaceutical ingredient using the formulation candidates determined in Example 2.3, visible particles were found, and the formulation was further developed. It was confirmed that visible particles appear when the silicone present in the active pharmaceutical ingredient binds to proteins due to physical stress and forms a silicone-protein complex. Since there are limitations in removing silicone or alleviating physical stress during the manufacturing process of the active pharmaceutical ingredient, a new formulation that inhibits the formation of the silicone-protein complex was developed to solve the problem. Poloxamer 188 has been reported to be inefficient as a surfactant for inhibiting the formation of the silicone-protein complex. Based on this, a test was conducted to add polysorbate 80 (PS80) as a surfactant.

[0105] Specifically, the test was carried out by adding polysorbate 80 at multiple concentrations (0 w / w%, 0.02 w / w%, 0.04 w / w%, 0.06 w / w%, 0.08 w / w%, and 0.1 w / w%) to GI-101 DS (GI-101 4 mg / mL, 20 mM histidine buffer (pH 7.0), 0.07 w / w% poloxamer 188, 15 mg / mL arginine-HCl, and 150 mg / mL sucrose). To enhance long-term stability, the concentration of GI-101 was adjusted to 4 mg / mL.

[0106] Example 3.1 Visual Particle Observation Each of the three tubes was tested under each condition, and even if particles were observed in only one of the three tubes, it was marked as O (i.e., "particle observed") (Table 9).

[0107]

Table 9

[0108] In the samples with 0.08 w / w% or more of PS80 added, no particles were observed up to 4 weeks. In the case of samples where particles were observed at the 3rd week, the test at the 4th week was not conducted.

[0109] Example 3.2 Evaluation of Quality Effects To investigate the quality effect by the addition of polysorbate 80 (PS80), the protein concentration, charge fluctuation, and purity of the samples with 0.1 w / w% of PS80 added were measured. As a result of observing the samples with 0.1 w / w% of PS80 added for up to 4 weeks, it was confirmed that the quality was maintained. The measurement results under each condition are shown in Table 10.

[0110]

Table 10

[0111] Considering that adding PS80 at a high concentration is effective in reducing visible particles, the concentration of PS80 was determined to be 0.1 w / w%. Combining these results, the composition of the final formulation of GI-101 was determined as shown in Table 11 below.

[0112]

Table 11

Claims

1. (i)A fusion protein dimer containing IL-2 protein and CD80 protein at a concentration of 3.0 mg / mL to 5.0 mg / mL; (ii)Histidine at a concentration of 10 mM to 30 mM; (iii)Poloxamer 188 at a concentration of 0.065 w / w% to 0.075 w / w%; (iv)Polysorbate 80 at a concentration of 0.09 w / w% to 0.11 w / w%; (v)Arginine at a concentration of 10 mg / mL to 30 mg / mL; and (vi)Sucrose at a concentration of 120 mg / mL to 180 mg / mL A pharmaceutical preparation comprising: The pharmaceutical preparation, wherein the pH of the preparation is 6.5 to 7.

5.

2. The pharmaceutical preparation according to Claim 1, wherein the concentration of the fusion protein dimer is 3.6 mg / mL to 4.4 mg / mL.

3. The pharmaceutical preparation according to Claim 1, wherein the concentration of the histidine is 20 mM.

4. The pharmaceutical preparation according to Claim 1, wherein the pH of the preparation is 6.8 to 7.

2.

5. The pharmaceutical preparation according to Claim 4, wherein the pH of the preparation is 7.

0.

6. The pharmaceutical preparation according to Claim 1, wherein the concentration of the arginine is 14 mg / mL to 16 mg / mL.

7. The pharmaceutical preparation according to Claim 1, wherein the concentration of the arginine is 15 mg / mL.

8. The pharmaceutical preparation according to Claim 1, wherein the concentration of the sucrose is 140 mg / mL to 160 mg / mL.

9. The pharmaceutical preparation according to Claim 1, wherein the concentration of the sucrose is 150 mg / mL.

10. The pharmaceutical preparation according to Claim 1, which is for intravenous administration.

11. The pharmaceutical preparation according to Claim 1, which is a preparation for the prevention or treatment of cancer or infectious diseases.

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