Fusion protein containing anti-LAG-3 antibody and IL-2 and uses thereof

Abstract

The present invention relates to a fusion protein comprising an anti-LAG-3 antibody and IL-2 or a variant thereof. In the fusion protein of the present invention, the anti-LAG-3 antibody moiety can bind to LAG-3, thereby regulating the binding of LAG-3 to MHCII. Furthermore, the IL-2 protein or a variant thereof can regulate T cell activity. Therefore, a pharmaceutical composition comprising the fusion protein can enhance immune activity in vivo and be effectively used as an anticancer agent, and thus has high industrial applicability.

Description

[Technical Field]

[0001] The present invention relates to a fusion protein comprising an anti-LAG-3 antibody and IL-2, and uses thereof. In particular, the present invention relates to a novel fusion protein that is effective in treating or preventing cancer. [Background technology]

[0002] Cancer immunotherapy is a method of treating cancer using the body's immune response. Cancer immunotherapy can induce the immune system to attack cancer cells by targeting antigens, such as surface proteins, on cancer cells. In particular, it has been reported that anti-cancer immunity can be activated by blocking immune checkpoint pathways. Immune checkpoints are one of the main mechanisms by which tumor cells evade the immune system. Therefore, inhibiting or blocking immune checkpoints can enhance T cell activation, thereby enhancing anti-tumor immunity.

[0003] On the other hand, IL-2 is synthesized mainly by activated T cells, especially CD4+ helper T cells. IL-2 stimulates the proliferation and differentiation of T cells, induces the production of cytotoxic T cells (CTLs), and induces the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer cells (LAK cells).

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

[0005] On the other hand, LAG-3 is known to have a mechanism similar to that of PD-1. LAG-3 is an immune checkpoint inhibitor expressed in T cells and NK cells and has a structure similar to that of CD4. However, LAG-3 has an additional 30 amino acids in the D1 domain, and is therefore known to have high affinity for MHC class II (MHCII). Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, the present inventors conducted research to develop a fusion protein with a novel combination that enhances the activity of immune cells. As a result, the present inventors confirmed that a fusion protein comprising an anti-LAG-3 antibody and an IL-2 variant effectively controls immune cells. Based on these results, the present inventors confirmed that the fusion protein is effective as an anti-cancer agent, thereby completing the present invention. [Means for solving the problem]

[0007] To achieve the above object, one aspect of the present invention provides a fusion protein comprising an antibody that specifically binds to LAG-3 and an IL-2 protein or a variant thereof.

[0008] In another aspect, the present invention provides a polynucleotide encoding the fusion protein, an expression vector containing the polynucleotide, and a transformed cell into which the expression vector has been introduced.

[0009] In yet another aspect of the invention, there is provided a method for preparing a fusion protein, comprising the steps of culturing the transformed cell and collecting the fusion protein.

[0010] In yet another aspect of the present invention, there is provided the fusion protein and its medical uses.

[0011] In yet another aspect of the present invention, there is provided a use of the fusion protein for treating or preventing cancer.

[0012] In yet another aspect of the present invention, there is provided a method for treating or preventing cancer, comprising the step of administering the fusion protein to a subject.

[0013] In yet another aspect of the present invention, there is provided a use of the fusion protein for the manufacture of a medicament for the treatment or prevention of cancer. [Effects of the Invention]

[0014] The fusion protein comprising the anti-LAG-3 antibody and an IL-2 variant according to the present invention not only regulates LAG-3-related mechanisms but also has the same or similar functions as IL-2. That is, the fusion protein not only regulates the binding of LAG-3 to MHCII but also activates immune cells. Therefore, the fusion protein can be used as an anticancer agent. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows the structure of an anti-hLAG-3 antibody-hIgG4 Fc-hIL-2v2 fusion protein (GI-104E1) according to one embodiment. [Figure 2] FIG. 1 shows the structure of an anti-hLAG-3 antibody-hIgG4 Fc-hIL-2v3 fusion protein (GI-104E2) according to one embodiment. [Figure 3] This is a diagram showing the production of GI-104E1 followed by confirmation by SDS-PAGE. [Figure 4] This is a diagram showing the production of GI-104E2 followed by confirmation by SDS-PAGE. [Figure 5] GI-104E1 was produced and then confirmed by Western blot. [Figure 6] GI-104E2 was produced and then confirmed by Western blot. [Figure 7] FIG. 1 shows the mechanism of action of the fusion protein and experimental methods for GI-104E1 or GI-104E2. [Figure 8] 1 is a graph verifying whether GI-104E1 binds to LAG-3 and inhibits LAG-3-MHCII-mediated signaling by a LAG-3 blocking assay. [Figure 9] 1 is a graph examining whether GI-104E2 binds to LAG-3 and inhibits LAG-3-MHCII-mediated signaling by a LAG-3 blocking assay. [Figure 10] 1 is a graph showing the tumor volume observed in mice subcutaneously implanted with CT26 cancer cells, which were administered vehicle (PBS), anti-LAG-3 antibody, Fc-IL-2v2, a combination of anti-LAG-3 antibody and Fc-IL-2v2, and a fusion protein (GI-104E1) containing anti-LAG-3 antibody and IL-2v2 once a week for three weeks. [Figure 11] 1 is a graph showing tumor volume in mice subcutaneously implanted with CT26 cancer cells after administration of vehicle (PBS), anti-LAG-3 antibody, Fc-IL-2v2, a combination of anti-LAG-3 antibody and Fc-IL-2v2, and a fusion protein (GI-104E1) comprising anti-LAG-3 and IL-2v2. [Figure 12] 1 is a graph showing the tumor volume of a group of mice subcutaneously implanted with CT26 cancer cells and administered with vehicle (PBS). [Figure 13] 10 is a graph showing the tumor volume of a group of mice subcutaneously implanted with CT26 cancer cells and administered with an anti-LAG-3 antibody. [Figure 14] 10 is a graph showing the tumor volume of subjects in a group of mice subcutaneously implanted with CT26 cancer cells and administered Fc-IL-2v2. [Figure 15] 10 is a graph showing the tumor volume of subjects in a group of mice subcutaneously implanted with CT26 cancer cells and administered anti-LAG-3 antibody and Fc-IL-2v2 simultaneously. [Figure 16]10 is a graph showing the tumor volume of subjects in a group of mice subcutaneously implanted with CT26 cancer cells and administered a fusion protein (GI-104E1) comprising anti-LAG-3 and IL-2v2. [Figure 17] 1 is a graph showing the number of subjects in each group that experienced 30% or more, 50% or more, and 80% or more tumor growth inhibition based on the mean tumor volume growth in the vehicle (PBS)-treated group after vehicle (PBS), anti-LAG-3 antibody, Fc-IL-2v2, a combination of anti-LAG-3 antibody and Fc-IL-2v2, and a fusion protein (GI-104E1) containing anti-LAG-3 and IL-2v2 were administered to mice subcutaneously implanted with CT26 cancer cells once a week for 3 weeks and 32 days after the first administration. [Figure 18] 1 is a graph showing the survival rate observed in mice subcutaneously implanted with CT26 cancer cells, which were administered vehicle (PBS), anti-LAG-3 antibody, Fc-IL-2v2, a combination of anti-LAG-3 antibody and Fc-IL-2v2, and a fusion protein (GI-104E1) containing anti-LAG-3 and IL-2v2 once a week for three weeks. [Figure 19] 1 is a graph showing the tumor volume observed in mice subcutaneously implanted with CT26 cancer cells, which were administered vehicle (PBS), anti-LAG-3 antibody, Fc-IL-2v3, a combination of anti-LAG-3 antibody and Fc-IL-2v3, and a fusion protein (GI-104E2) containing anti-LAG-3 and IL-2v3 once a week for three weeks. [Figure 20] 1 is a graph showing the tumor volume of subjects in each treatment group after administration of vehicle (PBS), anti-LAG-3 antibody, Fc-IL-2v3, a combination of anti-LAG-3 antibody and Fc-IL-2v3, and a fusion protein (GI-104E2) containing anti-LAG-3 and IL-2v3 to mice subcutaneously implanted with CT26 cancer cells. [Figure 21] 1 is a graph showing the tumor volume of a group of mice subcutaneously implanted with CT26 cancer cells and administered with vehicle (PBS). [Figure 22] 10 is a graph showing the tumor volume of a group of mice subcutaneously implanted with CT26 cancer cells and administered with an anti-LAG-3 antibody. [Figure 23] 10 is a graph showing the tumor volume of subjects in a group of mice subcutaneously implanted with CT26 cancer cells and administered Fc-IL-2v3. [Figure 24] 10 is a graph showing the tumor volume of subjects in a group of mice subcutaneously implanted with CT26 cancer cells and administered anti-LAG-3 antibody and Fc-IL-2v3 simultaneously. [Figure 25] 10 is a graph showing the tumor volume of subjects in a group of mice subcutaneously implanted with CT26 cancer cells and administered a fusion protein (GI-104E2) comprising anti-LAG-3 and IL-2v3. [Figure 26] 1 is a graph showing the number of subjects in each group that experienced 30% or more, 50% or more, and 80% or more tumor growth inhibition based on the mean tumor volume growth in the vehicle (PBS)-treated group after vehicle (PBS), anti-LAG-3 antibody, Fc-IL-2v3, a combination of anti-LAG-3 antibody and Fc-IL-2v3, and a fusion protein (GI-104E2) containing anti-LAG-3 and IL-2v3 were administered to mice subcutaneously implanted with CT26 cancer cells once a week for 3 weeks and 32 days after the first administration. [Figure 27] 1 is a graph showing the survival rate observed in mice subcutaneously implanted with CT26 cancer cells, which were administered once a week for three weeks with vehicle (PBS), anti-LAG-3 antibody, Fc-IL-2v3, a combination of anti-LAG-3 antibody and Fc-IL-2v3, and a fusion protein (GI-104E2) comprising anti-LAG-3 and IL-2v3. DETAILED DESCRIPTION OF THE INVENTION

[0016] BEST MODE FOR CARRYING OUT THE INVENTION Fusion protein containing anti-LAG-3 antibody and IL-2 In one aspect of the present invention, a fusion protein is provided comprising an antibody that specifically binds to LAG-3 and an IL-2 protein.

[0017] In this case, the fusion protein may be in a form in which at least one IL-2 protein is linked to an anti-LAG-3 antibody. In one embodiment, the fusion protein may contain one or two IL-2 proteins or variants thereof. In this case, the anti-LAG-3 antibody and IL-2 may be linked to each other via a linker.

[0018] As used herein, the term "LAG-3" refers to CD223 or lymphocyte activation gene 3. This protein is encoded by the LAG-3 gene. LAG-3 is known to have a mechanism similar to that of PD-1. Furthermore, LAG-3 is an immune checkpoint inhibitor expressed in T cells and NK cells, and has a structure similar to that of CD4. However, LAG-3 has an additional 30 amino acids in the D1 domain, thus giving it high affinity for MHC class II. Due to such structural features, LAG-3 is also known to inhibit T cell activation.

[0019] The anti-LAG-3 antibody of the present invention may be an antibody that specifically binds to LAG-3. Furthermore, any form of antibody fragment may be used as long as it contains an antigen-binding domain capable of specifically binding to LAG-3. In this case, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the anti-LAG-3 antibody may contain the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. Furthermore, the LCDR1, LCDR2, and LCDR3 of the light chain variable region of the anti-LAG-3 antibody may contain the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.

[0020] In one embodiment, an antibody that specifically binds to LAG-3 may comprise a heavy chain variable region containing the amino acid sequence of SEQ ID NO:4 and a light chain variable region containing the amino acid sequence of SEQ ID NO:8.

[0021] As used herein, the term "IL-2" or "interleukin-2," unless otherwise specified, refers to any wild-type IL-2 obtained from any vertebrate source, including mammals such as primates (such as humans) and rodents (such as mice and rats). IL-2 may be obtained from animal cells, and also includes IL-2 obtained from recombinant cells capable of producing IL-2. Furthermore, IL-2 may be wild-type IL-2 or a variant thereof.

[0022] As used herein, IL-2 or variants thereof may be collectively referred to as "IL-2 protein" or "IL-2 polypeptide." IL-2, IL-2 protein, IL-2 polypeptide, and IL-2 variants specifically bind, for example, to the IL-2 receptor. This specific binding can be determined by methods known to those skilled in the art.

[0023] IL-2 may be in a mature form. Specifically, mature IL-2 may not contain a signal sequence and may contain a fragment of wild-type IL-2 in which a portion of the N-terminus or C-terminus of wild-type IL-2 is truncated. In this case, IL-2 may have the amino acid sequence of SEQ ID NO: 22.

[0024] As used herein, the term "IL-2 variant" refers to a form of full-length IL-2 or the above-mentioned fragments of IL-2 in which some amino acids have been substituted. That is, an IL-2 variant may have an amino acid sequence different from that of wild-type IL-2 or a fragment thereof. However, an IL-2 variant may have activity equivalent to or similar to that of wild-type IL-2. In this case, "IL-2 activity" may refer, for example, to specific binding to the IL-2 receptor, which can be measured by methods known to those skilled in the art.

[0025] Specifically, the IL-2 variant may be obtained by substituting some of the amino acids of wild-type IL-2. An embodiment of the IL-2 variant obtained by amino acid substitution may be obtained by substituting at least one of the amino acids at positions 38, 42, 45, 61, and 72 of the amino acid sequence of SEQ ID NO: 22.

[0026] Specifically, an IL-2 variant may be obtained by substitution of at least one of the amino acids at positions 38, 42, 45, 61, or 72 of the amino acid sequence of SEQ ID NO: 22 with another amino acid. According to one embodiment, one, two, or three amino acids may be substituted, as long as such an IL-2 variant retains IL-2 activity.

[0027] In one embodiment, the IL-2 variant may be in the form of a substitution of two amino acids. Specifically, the IL-2 variant may be obtained by substitution of the amino acids at positions 38 and 42 of the amino acid sequence of SEQ ID NO: 22. Furthermore, in one embodiment, the IL-2 variant may be obtained by substitution of the amino acids at positions 38 and 45 of the amino acid sequence of SEQ ID NO: 22. Furthermore, in one embodiment, the IL-2 variant may be obtained by substitution of the amino acids at positions 38 and 61 of the amino acid sequence of SEQ ID NO: 22. Furthermore, in one embodiment, the IL-2 variant may be obtained by substitution of the amino acids at positions 38 and 72 of the amino acid sequence of SEQ ID NO: 22. Furthermore, in one embodiment, the IL-2 variant may be obtained by substitution of the amino acids at positions 42 and 45 of the amino acid sequence of SEQ ID NO: 22. Furthermore, in one embodiment, the IL-2 variant may be obtained by substitution of the amino acids at positions 42 and 61 of the amino acid sequence of SEQ ID NO: 22. Furthermore, in one embodiment, an IL-2 variant may be obtained by substitution of the amino acids at positions 42 and 72 in the amino acid sequence of SEQ ID NO: 22. Furthermore, in one embodiment, an IL-2 variant may be obtained by substitution of the amino acids at positions 45 and 61 in the amino acid sequence of SEQ ID NO: 22. Furthermore, in one embodiment, an IL-2 variant may be obtained by substitution of the amino acids at positions 45 and 72 in the amino acid sequence of SEQ ID NO: 22. Furthermore, in one embodiment, an IL-2 variant may be obtained by substitution of the amino acids at positions 61 and 72 in the amino acid sequence of SEQ ID NO: 22.

[0028] Furthermore, the IL-2 variant may be in a form in which three amino acids are substituted. Specifically, the IL-2 variant may be obtained by substitution of the amino acids at positions 38, 42, and 45 of the amino acid sequence of SEQ ID NO: 22. Furthermore, in one embodiment, the IL-2 variant may be obtained by substitution of the amino acids at positions 38, 42, and 61 of the amino acid sequence of SEQ ID NO: 22. Furthermore, in one embodiment, the IL-2 variant may be obtained by substitution of the amino acids at positions 38, 42, and 72 of the amino acid sequence of SEQ ID NO: 22. Furthermore, in one embodiment, the IL-2 variant may be obtained by substitution of the amino acids at positions 38, 45, and 61 of the amino acid sequence of SEQ ID NO: 22. Furthermore, in one embodiment, the IL-2 variant may be obtained by substitution of the amino acids at positions 38, 45, and 72 of the amino acid sequence of SEQ ID NO: 22. In one embodiment, the IL-2 variant may be obtained by substitution of the amino acids at positions 38, 61, and 72 of the amino acid sequence of SEQ ID NO: 22. Furthermore, in one embodiment, an IL-2 variant may be obtained by substitution of the amino acids at positions 42, 45, and 61 of the amino acid sequence of SEQ ID NO: 22. Furthermore, in one embodiment, an IL-2 variant may be obtained by substitution of the amino acids at positions 42, 45, and 72 of the amino acid sequence of SEQ ID NO: 22. Furthermore, in one embodiment, an IL-2 variant may be obtained by substitution of the amino acids at positions 45, 61, and 72 of the amino acid sequence of SEQ ID NO: 22.

[0029] In this case, the "another amino acid" introduced by substitution may be any amino acid 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, with the proviso that, with regard to the amino acid substitutions of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 22, the amino acid at position 38 cannot be substituted with arginine, the amino acid at position 42 cannot be substituted with phenylalanine, the amino acid at position 45 cannot be substituted with tyrosine, the amino acid at position 61 cannot be substituted with glutamic acid, and the amino acid at position 72 cannot be substituted with leucine.

[0030] Regarding the amino acid substitution of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 22, the amino acid arginine at position 38 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: 22, the amino acid arginine at position 38 may be substituted with alanine (R38A).

[0031] Regarding the amino acid substitution of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 22, the amino acid phenylalanine at position 42 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: 22, the amino acid phenylalanine at position 42 may be substituted with alanine (F42A).

[0032] Regarding the amino acid substitution of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 22, 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: 22, the 45th amino acid tyrosine may be substituted with alanine (Y45A).

[0033] Regarding the amino acid substitution of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 22, the amino acid glutamic acid at position 61 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: 22, the amino acid glutamic acid at position 61 may be substituted with arginine (E61R).

[0034] Regarding the amino acid substitution of the IL-2 variant, in the amino acid sequence of SEQ ID NO: 22, the amino acid leucine at position 72 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: 22, the amino acid leucine at position 72 may be substituted with glycine (L72G).

[0035] Specifically, the IL-2 variant may 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: 22. Preferably, the IL-2 variant may have amino acid substitutions at two or three positions selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G.

[0036] The IL-2 variant may be in the form of two amino acid substitutions. Specifically, the IL-2 variant may be obtained by the substitutions R38A and F42A. Furthermore, in one embodiment, the IL-2 variant may be obtained by the substitutions R38A and Y45A. Furthermore, in one embodiment, the IL-2 variant may be obtained by the substitutions R38A and E61R. Furthermore, in one embodiment, the IL-2 variant may be obtained by the substitutions R38A and L72G. Furthermore, in one embodiment, the IL-2 variant may be obtained by the substitutions F42A and Y45A. Furthermore, in one embodiment, the IL-2 variant may be obtained by the substitutions F42A and E61R. Furthermore, in one embodiment, the IL-2 variant may be obtained by the substitutions F42A and L72G. Furthermore, in one embodiment, the IL-2 variant may be obtained by the substitutions E61R and L72G.

[0037] Furthermore, the IL-2 variant may be in the form of three amino acid substitutions. Specifically, the IL-2 variant may be obtained by the substitutions R38A, F42A, and Y45A. Furthermore, in one embodiment, the IL-2 variant may be obtained by the substitutions R38A, F42A, and E61R. Furthermore, in one embodiment, the IL-2 variant may be obtained by the substitutions R38A, F42A, and L72G. Furthermore, in one embodiment, the IL-2 variant may be obtained by the substitutions R38A, Y45A, and E61R. Furthermore, in one embodiment, the IL-2 variant may be obtained by the substitutions R38A, Y45A, and L72G. Furthermore, in one embodiment, the IL-2 variant may be obtained by the substitutions F42A, Y45A, and E61R. Furthermore, in one embodiment, the IL-2 variant may be obtained by the substitutions F42A, Y45A, and L72G. Furthermore, in one embodiment, the IL-2 variant may be obtained by the substitutions F42A, E61R, and L72G. Furthermore, in one embodiment, the IL-2 variant may be obtained by the substitutions Y45A, E61R, and L72G.

[0038] In one embodiment, the IL-2 variant may have the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:21.

[0039] Furthermore, the IL-2 variant may be characterized by low in vivo toxicity. In this case, low in vivo toxicity may refer to side effects caused by IL-2 binding to the IL-2 receptor alpha chain (IL-2Rα). Various IL-2 variants have been developed to improve side effects caused by IL-2 binding to IL-2Rα, and such IL-2 variants may be those disclosed in U.S. Pat. No. 5,229,109 and Korean Patent No. 10-1667096. In particular, the IL-2 variants described in the present application have a reduced ability to bind to the IL-2 receptor alpha chain (IL-2Rα) and therefore have lower in vivo toxicity than wild-type IL-2.

[0040] The fusion protein may comprise an immunoglobulin Fc region, where the immunoglobulin Fc domain comprises immunoglobulin heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3). The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM, preferably IgG4.

[0041] Furthermore, the Fc domain of an immunoglobulin may be not only a wild-type Fc domain but also an Fc domain variant. Furthermore, as used herein, the term "Fc domain variant" may refer to a form that differs from the wild-type Fc domain in terms of glycosylation pattern, having higher glycosylation than the wild-type Fc domain or lower glycosylation than the wild-type Fc domain, or a deglycosylated form. Furthermore, an unglycosylated Fc domain is also encompassed in Fc domain variants. The Fc domain or its variant may be configured to have the number of sialic acids, fucosylation, or glycosylation adjusted by host culture conditions or genetic engineering.

[0042] Furthermore, glycosylation of the Fc domain of an immunoglobulin may be modified by standard methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Furthermore, the Fc domain variant may be a hybrid form of the Fc region of each of immunoglobulins, IgG, IgA, IgE, IgD, or IgM. Furthermore, the Fc domain variant may be a form in which some amino acids in the Fc domain are substituted with other amino acids.

[0043] In one embodiment, the Fc domain may have the amino acid sequence of SEQ ID NO:14.

[0044] The fusion protein according to the present invention may contain a fusion protein including the light chain variable region and light chain constant region (CL1) of an anti-LAG-3 antibody, the heavy chain variable region and heavy chain constant region (CH1) of an anti-LAG-3 antibody, an Fc domain, and an IL-2 protein. In this case, at least one IL-2 protein may be contained in the anti-LAG-3 antibody. In one embodiment, the fusion protein contains two IL-2 proteins. Furthermore, in one embodiment, the IL-2 protein may be linked to the C-terminus of the anti-LAG-3 antibody.

[0045] Specifically, the fusion protein containing an Fc domain and an IL-2 protein may be a dimer in which two fusion proteins represented by structural formula (1) are linked together. N'-X-[linker (1)]o-Fc region fragment or variant thereof-[linker (2)]pY-C'(1) In this case, in structural formula (1), N' is the N-terminus of the fusion protein, C' is the C-terminus of the fusion protein; X is an anti-LAG-3 antibody or fragment thereof; Y is the IL-2 protein, Linker (1) and linker (2) are peptide linkers, o and p each independently represent 0 or 1;

[0046] In this case, the anti-LAG-3 antibody and IL-2 protein are as described above. Furthermore, in the fusion protein, IL-2 or a variant thereof may be linked to an Fc region linked to the C-terminus of the anti-LAG-3 antibody. In this case, the IL-2 or a variant thereof and the Fc region may be linked via a linker.

[0047] The peptide linker (1) may consist of 1 to 50 consecutive amino acids, 3 to 30 consecutive amino acids, or 5 to 15 amino acids. In one embodiment, the peptide linker (1) may consist of 12 amino acids. Furthermore, the peptide linker (1) may contain at least one cysteine. Specifically, the peptide linker (1) may contain one, two, or three cysteines. Furthermore, the peptide linker (1) may be derived from an immunoglobulin hinge. In one embodiment, the peptide linker (1) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 13.

[0048] The peptide linker (2) may consist of 1 to 30 consecutive amino acids, 2 to 20 consecutive amino acids, or 2 to 10 amino acids. In one embodiment, the peptide linker (2) may be (G4S)n (wherein n is an integer from 1 to 10). In this case, in (G4S), n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the peptide linker may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 19.

[0049] Furthermore, the fusion protein represented by structural formula (1) may include proteins represented by structural formula (1-1) and structural formula (1-2). N'-X'-[linker (1)]o-Fc region fragment or variant thereof-[linker (2)]pY-C'(1-1) N'-X''-C'(1-2) In this case, in the structural formulas (1-1) and (1-2), N' is the N-terminus of the fusion protein, C' is the C-terminus of the fusion protein; X' is a heavy chain region of an anti-LAG-3 antibody, including a heavy chain variable region and CH1; X'' is the light chain region of an anti-LAG-3 antibody, including the light chain variable region and CL; Y is the IL-2 protein, Linker (1) and linker (2) are peptide linkers, o and p each independently represent 0 or 1;

[0050] Furthermore, the heavy chain variable region and the light chain variable region are as described above.

[0051] The amino acid sequences of each of the regions constituting the fusion protein are as shown in Tables 1 and 2 below. Specifically, Table 1 shows the amino acid sequence of anti-hLAG-3(1E09)VL-kappa+CL. Table 2 shows the amino acid sequences of anti-hLAG-3(1E09)VH+CH1, hIgG4Fc, and hIL-2v2 / hIL-2v3.

[0052] [Table 1]

[0053] [Table 2] TIFF0007814329000003.tif148149

[0054] Polynucleotide encoding the fusion protein In another aspect of the invention, a polynucleotide encoding the fusion protein is provided.

[0055] In this case, the polynucleotide may contain a nucleotide sequence encoding the fusion protein represented by structural formula (1-1) and structural formula (1-2).

[0056] Specifically, the polynucleotide encoding the heavy chain region may contain the nucleotide sequence of SEQ ID NO: 17 or SEQ ID NO: 23. Furthermore, the polynucleotide encoding the light chain region may contain SEQ ID NO: 18.

[0057] As long as the polynucleotides encode the same polypeptide, one or more nucleotides may be changed by substitution, deletion, insertion, or a combination thereof. When preparing a nucleotide sequence by chemical synthesis, synthesis methods known in the art, such as the synthesis method described by Engels and Uhlmann (Angew Chem Int Ed Engl., 37:73-127, 1988), may be used. Such methods include the triester method, the phosphite method, the phosphoramidite method, and the H-phosphonate method, PCR and other autoprimer methods, and oligonucleotide synthesis on a solid support.

[0058] According to one embodiment, the polynucleotide may contain a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to SEQ ID NO: 17, 18, or 23.

[0059] The polynucleotide may further contain a nucleic acid encoding a signal sequence or leader sequence. As used herein, the term "signal sequence" refers to a signal peptide that induces secretion of a protein of interest. The signal peptide is translated in the host cell and then cleaved. Specifically, a signal sequence is an amino acid sequence that initiates translocation of a protein across the endoplasmic reticulum (ER) membrane.

[0060] Characteristics of signal sequences are known in the art. Such signal sequences typically contain 16 to 30 amino acid residues, but may contain more or fewer amino acid residues. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal sequence during translocation of the immature polypeptide across the membrane lipid bilayer.

[0061] After initiation, the signal sequence is cleaved in the lumen of the ER by a cellular enzyme known as a signal peptidase. In this case, the signal sequence may be a secretory signal sequence of tPa (tissue plasminogen activator), HSV gD (herpes simplex virus glycoprotein D), IgG signal sequence, or growth hormone. Preferably, a secretory signal sequence used in higher eukaryotic cells, including mammals, may be used. Furthermore, a signal sequence contained in wild-type IL-2 may be used, or a signal sequence substituted with a codon frequently expressed in the host cell may be used. In one embodiment, the signal sequence includes the light chain signal sequence of the 14.18 antibody (Gillies et al., J. Immunol. Meth. 1989. 125:191-202), the heavy chain signal sequence of the MOPC141 antibody (Sakano et al., Nature. 1980. 286:676-683), and other signal sequences known in the art (see, for example, Watson et al., Nucleic Acid Research. 1984. 12:5145-5164).

[0062] A vector carrying a polynucleotide encoding a fusion protein In yet another aspect of the invention, there is provided a vector comprising the polynucleotide.

[0063] In this case, the polynucleotide encoding the heavy chain region and the polynucleotide encoding the light chain region may be contained in different vectors, or alternatively, the polynucleotide encoding the heavy chain region and the polynucleotide encoding the light chain region may be contained in a single vector.

[0064] The polynucleotides are as described above. In this case, the polynucleotide encoding the heavy chain region may contain the nucleotide sequence of SEQ ID NO: 17 or 23, and the polynucleotide encoding the light chain region may contain the nucleotide sequence of SEQ ID NO: 18. In one embodiment, the polynucleotide encoding the heavy chain region and the polynucleotide encoding the light chain region may contain the nucleotide sequences of SEQ ID NO: 17 and SEQ ID NO: 18, respectively. In one embodiment, the polynucleotide encoding the heavy chain region and the polynucleotide encoding the light chain region may contain the nucleotide sequences of SEQ ID NO: 23 and SEQ ID NO: 18, respectively. In this case, the vector may be two vectors containing each polynucleotide of the heavy chain and light chain polynucleotide combination, or may be a bicistronic vector containing both polynucleotides of the heavy chain and light chain polynucleotide combination.

[0065] A vector may be introduced into a host cell, where it may recombine with the genome of the host cell and be inserted into the genome of the host cell. Alternatively, a vector may be understood as a nucleic acid means containing a polynucleotide sequence that replicates autonomously as an episome. In this case, the vector may be operably linked to an appropriate promoter so that the polynucleotide can be expressed in the host cell. Vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, minichromosomes, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0066] Specifically, vectors include plasmid DNA, phage DNA, and commercially developed plasmids (e.g., pUC18, pBAD, pIDTSAMRT-AMP), Escherichia coli-derived plasmids (e.g., pYG601BR322, pBR325, pUC118, pUC119), Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5), yeast-derived plasmids (e.g., YEp13, YEp24, YCp50), phage DNA (e.g., Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP), animal virus vectors (e.g., retrovirus, adenovirus, vaccinia virus), and insect virus vectors (e.g., baculovirus). Because vectors exhibit different protein expression levels and modifications depending on the host cell, it is preferable to select and use the host cell most suitable for the purpose.

[0067] As used herein, the term "gene expression" or "expression" of a protein of interest is understood to mean the transcription of a DNA sequence, the translation of an mRNA transcript, and the secretion of a fusion protein product or a fragment thereof. One useful expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. The expression vector may further contain a human cytomegalovirus (CMV) promoter to promote continuous transcription of the target gene in mammalian cells and a bovine growth hormone polyadenylation signal sequence to increase the steady-state level of RNA after transcription.

[0068] Transformed cells expressing the fusion protein In another aspect of the present invention, a transformed cell into which the vector is introduced is provided.

[0069] Host cells for transformation include, but are not limited to, prokaryotic cells, eukaryotic cells, and cells of mammalian, plant, insect, fungal, or bacterial origin. An example of a prokaryotic cell is Escherichia coli. An example of a eukaryotic cell is yeast. Mammalian cells include CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, Hela cells, 911 cells, AT1080 cells, A549 cells, SP2 / 0 cells, human lymphoblastoid cells, NSO cells, HT-1080 cells, PERC.6 cells, HEK293 cells, and HEK293T cells. However, mammalian cells are not limited to these, and any cells known to those skilled in the art to be usable as mammalian host cells may be used.

[0070] Furthermore, expression vectors can be introduced into host cells using CaCl precipitation, the Hanahan method with increased efficiency achieved by using a reducing agent such as dimethyl sulfoxide (DMSO) in CaCl precipitation, electroporation, calcium phosphate precipitation, protoplast fusion, stirring using silicon carbide fibers, Agrobacterium transformation, transformation using PEG, dextran sulfate, or lipofectamine, and dry / inhibition-mediated transformation. Furthermore, infection can be used as a means to deliver the target substance into cells using viral particles. Furthermore, vectors can be introduced into host cells using gene bombardment, etc.

[0071] As noted above, to optimize the properties of the fusion protein as a therapeutic agent or for other purposes, the glycosylation pattern (e.g., sialic acid, fucosylation, and glycosylation) of the fusion protein may be adjusted by manipulating glycosylation-related genes harbored by the host cell by methods known to those skilled in the art. Methods for preparing fusion proteins In yet another aspect of the present invention, there is provided a method for preparing a fusion protein comprising an anti-LAG-3 antibody and IL-2 or a variant thereof, the method comprising culturing a transformed cell and collecting the fusion protein from the culture medium.

[0072] As used herein, the term "culturing" refers to the process of artificially growing microorganisms under appropriately controlled environmental conditions.

[0073] The transformed cells may be cultured using a method known in the art. Specifically, the culture is not particularly limited as long as the fusion protein of the present invention can be produced by expression. Specifically, the culture may be performed in a batch process or continuously in a fed-batch or repeated fed-batch process.

[0074] Furthermore, collection of the fusion protein from the culture medium may be carried out by a method known in the art. Specifically, the collection method is not particularly limited as long as it allows collection of the fusion protein produced by the present invention. Preferably, the collection method may be centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), or chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion). Use of fusion proteins In yet another aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing cancer, comprising the fusion protein.

[0075] As used herein, the term "cancer" is classified as a disease in which normal tissue cells continue to proliferate and develop indefinitely for some reason, regardless of the life events of the organism or the condition of the surrounding tissues. The cancer in the present invention may be any one selected from the group consisting of various cancers of the human body, such as stomach cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma, but is not limited to the above types. Furthermore, the cancer in the present invention may be, but is not limited to, a radiation-resistant cancer.

[0076] As used herein, the term "prevention" refers to any action that inhibits the development of or delays the onset of cancer by administering a pharmaceutical composition. As used herein, the term "treatment" refers to any action that improves or beneficially alters the symptoms of cancer by administering a pharmaceutical composition.

[0077] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any carrier as long as it is a non-toxic substance suitable for delivery to a patient. Distilled water, alcohol, fats, waxes, and inert solids may be included as carriers. A pharmaceutically acceptable adjuvant (buffer or dispersant) may also be included in the pharmaceutical composition.

[0078] Specifically, by including a pharmaceutically acceptable carrier, the pharmaceutical composition can be prepared as a parenteral formulation depending on the administration route using a standard method known in the art. In this case, the term "pharmaceutically acceptable" means that the toxicity of the carrier is not more than the level that can be tolerated by the subject to be applied (prescribed) without inhibiting the activity of the active ingredient.

[0079] When the pharmaceutical composition is prepared as a parenteral preparation, it may be formulated into an injection, transdermal patch, nasal inhalant, or suppository formulation with a suitable carrier according to a method known in the art. When prepared as an injection, sterile water, ethanol, a polyol such as glycerol or propylene glycol, or a mixture thereof, can be used as a suitable carrier, and preferably, an isotonic solution such as Ringer's solution, or phosphate-buffered saline (PBS) containing triethanolamine or sterile water for injection and 5% dextrose can be used. The formulation of pharmaceutical compositions is known in the art, and for details, refer to Remington's Pharmaceutical Sciences (19th Edition, 1995), etc. This document is deemed to be a part of this specification.

[0080] Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "administration" refers to introducing a predetermined substance into a subject by an appropriate method, and the administration route of the pharmaceutical composition may be any common route as long as the pharmaceutical composition can reach the target tissue. Administration may be, but is not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, or rectal administration.

[0081] The term "subject" refers to all animals, including humans, rats, mice, livestock, etc. Preferably, the subject may be a mammal, including a human.

[0082] The term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit-risk ratio applicable to any medical treatment and that does not cause adverse side effects. The level of a pharmaceutically effective amount can be readily determined by one skilled in the art, depending on factors including the patient's sex, age, weight, and health condition, the type and severity of the disease, the activity of the drug, sensitivity to the drug, the method and time of administration, the route of administration, the rate of excretion, the duration of treatment, other drugs used in combination or simultaneously, and other factors known in the medical field. The daily dose may range from 0.01 μg / kg to 10 g / kg, or from 0.01 mg / kg to 1 g / kg. Administration may be once a day or several times a day. Such doses should not be construed as limiting the scope of the present invention in any manner.

[0083] In yet another aspect of the present invention, there is provided a use of the fusion protein for treating or preventing cancer, wherein the fusion protein, cancer, treatment, and prevention are as defined above.

[0084] In yet another aspect of the present invention, there is provided a method for treating or preventing cancer, comprising administering a fusion protein to a subject, wherein the fusion protein, administration, cancer, treatment, and prevention are as described above. The subject may be a mammal, preferably a human. Furthermore, the subject may be a patient suffering from cancer or a subject susceptible to cancer.

[0085] The administration route, dose, and administration frequency of the fusion protein or fusion protein dimer may vary depending on the patient's condition and the presence or absence of side effects, and therefore the fusion protein or fusion protein dimer may be administered to a subject in various ways and amounts. The optimal administration method, dose, and administration frequency can be selected within an appropriate range by those skilled in the art. Furthermore, the fusion protein or fusion protein dimer may be administered in combination with other drugs or physiologically active substances known to have a therapeutic effect on the disease to be treated, or may be formulated in the form of a mixed preparation with other drugs. [Example]

[0086] [Mode for carrying out the present invention] The present invention will now be described in more detail by the following examples, which are intended to illustrate the present invention only, and it will be apparent to those skilled in the art that the scope of the present invention should not be construed as being limited by these examples.

[0087] I. Preparation of Fusion Proteins Preparation Example 1. Preparation of anti-hLAG-3 antibody-hIgG4 Fc-hIL-2v2 fusion protein: GI-104E1 To prepare a fusion protein containing anti-hLAG-3 antibody and hIL-2v2, polynucleotides were inserted into the pcDNA3_4 vector (Invitrogen) using GenScript's Expression and Optimization service. Specifically, the polynucleotide (SEQ ID NO: 18) contained the nucleotide sequence (SEQ ID NO: 16) encoding a fusion protein containing, from the N-terminus, anti-hLAG-3 VL (anti-hLAG-3 (1E09) VL-kappa + CL), and the polynucleotide (SEQ ID NO: 17) contained the nucleotide sequence (SEQ ID NO: 27) encoding a fusion protein containing, from the N-terminus, anti-hLAG-3 VH (anti-hLAG-3 (1E09) VH + CH1), an Fc domain (F02 (hIgG4Fc)), a linker (G4S linker), and human IL-2v2, in this order. This vector was introduced into CHO cells (HD CHO-S). After vector introduction, the cells were cultured in serum-free HD CHO-S™ expression medium at 37°C and 8% CO for 14 days. The culture medium was then harvested and the fusion protein was purified using affinity chromatography (affinity purification column) with MabSelect SuRe™ LX.

[0088] The isolated and purified fusion protein was subjected to SDS-PAGE and Western blotting under reducing (R) or non-reducing (NR) conditions, and its molecular weight and purity were confirmed by HPLC analysis (Figures 3 and 5). The concentration was measured by Bradford assay, and it was confirmed that the fusion protein was contained at a concentration of 0.69 mg / ml.

[0089] Preparation Example 2. Preparation of control antibodies (anti-hLAG-3 antibody and α-LAG-3) To generate a control anti-hLAG-3 antibody, polynucleotides were inserted into the pcDNA3_4 vector (Invitrogen) using GenScript's Expression and Optimization service. Specifically, the polynucleotide (SEQ ID NO: 18) contained the nucleotide sequence (SEQ ID NO: 16) encoding a fusion protein containing, from the N-terminus, anti-hLAG-3 VL (anti-hLAG-3 (1E09) VL-kappa + CL), and the polynucleotide (SEQ ID NO: 29) contained the nucleotide sequence (SEQ ID NO: 15) encoding a fusion protein containing, from the N-terminus, anti-hLAG-3 VH (anti-hLAG-3 (1E09) VH + CH1) and an Fc domain (F02 (hIgG4 Fc)). These vectors were then introduced into CHO cells (HD CHO-S). After vector introduction, the cells were cultured in serum-free HD CHO-S™ expression medium at 37°C and 8% CO for 14 days. The culture medium was then harvested and the fusion protein was purified using affinity chromatography (affinity purification column) with MabSelectSure™ LX.

[0090] Preparation Example 3. Preparation of hIgG4 Fc-hIL-2v2:Fc-IL-2v2 To prepare a fusion protein dimer containing an Fc domain and an IL-2 variant, a polynucleotide was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific. Specifically, the polynucleotide (SEQ ID NO: 25) contained a nucleotide sequence (SEQ ID NO: 11) encoding a fusion protein containing, from the N-terminus, an Fc domain (SEQ ID NO: 14), a linker (SEQ ID NO: 19), and an IL-2 variant (2M) with two amino acid substitutions (R38A and F42A) (SEQ ID NO: 20), in this order. This polynucleotide was inserted into the pcDNA3_4 vector (Invitrogen). Furthermore, the vector was transfected into CHO cells (EXPI-CHO™) to express the fusion protein of SEQ ID NO: 11. After vector transfection, the cells were cultured at 37°C, 125 RPM, and 8% CO2 for 7 days. The culture medium was then harvested, and the fusion protein was purified from the culture medium.

[0091] Preparation Example 4. Preparation of hIgG4 Fc-hIL-2v3:Fc-IL-2v3 To generate a fusion protein containing an Fc domain and an IL-2 variant, a polynucleotide was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific. Specifically, the polynucleotide (SEQ ID NO: 26) contained a nucleotide sequence (SEQ ID NO: 24) encoding a fusion protein containing, from the N-terminus, an Fc domain (SEQ ID NO: 14), a linker (SEQ ID NO: 19), and an IL-2 variant (3M) with three amino acid substitutions (R38A, F42A, and E61R) (SEQ ID NO: 21), in this order. This polynucleotide was inserted into the pcDNA3_4 vector (Invitrogen). Furthermore, the vector was transfected into CHO cells (Expi-CHO™) to express the fusion protein of SEQ ID NO: 24. After vector transfection, the cells were cultured at 37°C, 125 RPM, and 8% CO2 for 7 days. The culture medium was then harvested, and the fusion protein was purified from the culture medium.

[0092] Preparation Example 5. Preparation of anti-hLAG-3 antibody-hIgG4 Fc-hIL-2v3 fusion protein: GI-104E2 To generate a fusion protein containing an anti-LAG-3 antibody and hIL-2v3, polynucleotides were inserted into the pcDNA3_4 vector (Invitrogen) using GenScript's Expression and Optimization service. Specifically, the polynucleotide (SEQ ID NO: 18) contained the nucleotide sequence (SEQ ID NO: 16) encoding a fusion protein containing, from the N-terminus, anti-hLAG-3 VL (anti-hLAG-3 (1E09) VL-kappa + CL), and the polynucleotide (SEQ ID NO: 23) contained the nucleotide sequence (SEQ ID NO: 28) encoding a fusion protein containing, from the N-terminus, anti-hLAG-3 VH (anti-hLAG-3 (1E09) VH + CH1), an Fc domain (F02 (hIgG4Fc)), a linker (G4S linker), and human IL-2v3, in this order. This vector was introduced into CHO cells (HD CHO-S). After vector introduction, the cells were cultured in serum-free HD CHO-S™ expression medium at 37°C and 8% CO for 14 days. The culture medium was then collected and the fusion protein was purified using affinity chromatography (affinity purification column) with MabSelectSure™ LX.

[0093] The isolated and purified fusion protein was subjected to SDS-PAGE and Western blotting under reducing (R) or non-reducing (NR) conditions, and its molecular weight and purity were confirmed by HPLC analysis (Figures 4 and 6). The concentration was measured by Bradford assay, and it was confirmed that the fusion protein was contained at a concentration of 0.51 mg / ml.

[0094] II. Identification of the immunoreactivity of the fusion protein Experimental Example 1. Identification of the T cell activation function of fusion proteins using LAG-3 blocking assay Experimental Example 1.1. Identifying the effect of GI-104E1 on T cell function LAG-3 is an immune checkpoint inhibitor expressed in T cells and NK cells and has a structure similar to that of CD4. However, LAG-3 has an additional 30 amino acids in the D1 domain, which gives it high affinity to MHC class II. Due to this structural feature, LAG-3 is known to inhibit T cell activation.

[0095] In this experiment, we used the LAG-3 blocking bioassay system (Promega, JA1115) to evaluate the T cell activation function of the fusion protein. Specifically, one vial of MHCII APC cells stored in liquid nitrogen was thawed in a 37°C incubator for 2 minutes and then added to 14.5 ml of cell recovery medium (90% DMEM + 10% FBS) containing TCR activation antigen and mixed thoroughly. 100 μl of the MHCII APC suspension was added to each well of a 96-well white cell culture plate (Corning catalog number 3917) and cultured for 24 hours in a 37°C, 5% CO2 incubator.

[0096] The 96-well white cell culture plate containing the 24-hour-cultured MHCII APC cells was removed and the culture medium was removed. Then, 40 μl of GI-104E1 and 1E09 (a surrogate for Relatlimab) as a positive control were added at various concentrations per well, and 40 μl of assay buffer was added as a negative control. The white cell culture plate was then covered and placed at room temperature until the LAG-3 effector cells were ready.

[0097] One vial of LAG-3 effector cells stored in liquid nitrogen was thawed in a 37°C incubator for 2 minutes, then added to 7 ml of assay buffer (90% RPMI + 10% FBS) and mixed thoroughly. 40 μl of this mixed suspension was added to each well of a 96-well white cell culture plate stored at room temperature and reacted for 5 hours in an incubator at 37°C and 5% CO2. After the reaction was completed, the reaction mixture was left at room temperature for 10 minutes, and then 80 μl of Bio-Glo Reagent was added to each well, taking care to avoid air bubbles. Bio-Glo Reagent was also added to the two outermost wells, and these wells were used as blanks to correct for background signals. The reaction was allowed to proceed at room temperature for 10 minutes, and then luminescence was measured using a GloMax® Discover System (Promega, USA).

[0098] As a result, it was confirmed that GI-104E1 was able to activate T cell function by binding to LAG-3 expressed in effector T cells and inhibiting the function of LAG-3 (Fig. 8). Experimental Example 1.2. Identifying the effect of GI-104E2 on T cell function An LAG-3 blocking assay for GI-104E2 was performed using the same method as in Experimental Example 1.1, and the results confirmed that GI-104E2 was able to activate T cell function by binding to LAG-3 expressed in effector T cells and inhibiting the function of LAG-3 (Figure 9).

[0099] Experimental Example 2. Identification of the anti-cancer effect of the fusion protein Experimental Example 2.1. Identification of the anti-cancer effect of GI-104E1 in mice implanted with mouse-derived colorectal cancer cells BALB / c mice (female, 7 weeks old) obtained from Orient Bio Inc. were given a 7-day acclimation period. Then, 5 × 10 6Allografting was performed by diluting individual CT26 cancer cell lines (ATCC, USA) with 1 ml of PBS and subcutaneously administering 100 μl of this dilution to the right dorsal flank of each mouse. After cancer cell transplantation, tumor volume was measured over a certain period of time, and tumors were approximately 50 mm 3 ~120mm 3 Subjects who reached the target size were selected, and the selected mice were then equally divided into groups containing 8 mice each based on tumor size and body weight. Test groups were then composed as shown in Table 3, and the test substance was administered intraperitoneally. After the first administration, the administration was performed once a week for a total of three times. Regarding the size of the transplanted tumor, the tumor volume of all subjects was measured twice a week for four weeks.

[0100] [Table 3]

[0101] The measurement results were shown in graphs with the mean and standard deviation (SD) values ​​for each group. Furthermore, the statistical significance of tumor volume reduction compared to the vehicle (#p≦0.05, ####p≦0.0001) and the statistical significance of tumor volume differences compared to the GI-104E1-treated group (*p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001) were analyzed using two-way ANOVA and Dunnett's T3 test.

[0102] As a result, in mice implanted with CT26 cancer cell line, the group administered GI-104E1 at a dose of 3 mg / kg showed significant inhibition of tumor growth at the midpoint and end of the observation period compared to the negative control (p≦0.05, p≦0.0001, two-way analysis of variance (ANOVA)). Furthermore, GI-104E1 administration significantly inhibited tumor growth compared to administration of anti-LAG-3 antibody or Fc-IL-2v2 at the same molar concentration as GI-104E1. In particular, GI-104E1 showed significant tumor inhibitory effects at the midpoint and end of the observation period, even when compared with the experimental group in which anti-LAG-3 antibody and Fc-IL-2v2 were co-administered (*p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001, two-way analysis of variance (ANOVA)) (Figures 10-16).

[0103] When changes in tumor growth rate were observed at the end of the study, the number of subjects showing a 50% or greater reduction in tumor growth rate was 1 in the negative control group, 1 in the anti-LAG-3 antibody-administered group, 0 in the Fc-IL-2v2-administered group, and 0 in the anti-LAG-3 antibody and Fc-IL-2v2 co-administered group. On the other hand, in the experimental group administered GI-104E1, a 50% or greater reduction in tumor growth rate was observed in 7 subjects, and 5 subjects showed an 80% or greater reduction in tumor growth rate (Figure 17).

[0104] Furthermore, the survival rates of the experimental group administered GI-104E1 and the other experimental control and negative control groups were evaluated at the time of death of the experimental subjects or the time of 2,000 mm 3 The survival rates of the experimental groups were analyzed based on the incidence of tumor volume, and the statistical significance of the difference in survival rate between the experimental groups was analyzed using the Mantel-Cox test. As a result, the experimental group administered with GI-104E1 showed a higher survival rate than the other experimental control groups and the negative control group, and it was confirmed that the difference in survival rate between the experimental groups was statistically significant (****p≦0.0001) (Figure 18).

[0105] Experimental Example 2.2. Identification of the anti-cancer effect of GI-104E2 in mice implanted with mouse-derived colorectal cancer cells BALB / c mice (female, 7 weeks old) obtained from Orient Bio Inc. were given a 7-day acclimation period. 6 Allografting was performed by diluting individual CT26 cancer cell lines (ATCC, USA) with 1 ml of PBS and subcutaneously administering 100 μl of this dilution to the right dorsal flank of each mouse. After cancer cell transplantation, tumor volume was measured over a certain period of time, and tumors were approximately 50 mm 3 ~120mm 3 Subjects who reached the target size were selected, and the selected mice were then equally divided into groups containing three mice each based on tumor size and body weight. Test groups were then constructed as shown in Table 4, and the test substance was administered intraperitoneally. After the first administration, administration was performed once a week for a total of three times. Regarding the size of the transplanted tumor, the tumor volume of all subjects was measured twice a week for four weeks.

[0106] [Table 4]

[0107] As a result, in mice implanted with CT26 cancer cell line, the group administered GI-104E2 at a dose of 6 mg / kg showed tumor growth inhibition at the midpoint and end of the experiment compared to the negative control. Furthermore, the GI-104E2-administered group showed superior tumor growth inhibition compared to the administration of anti-LAG-3 antibody or Fc-IL-2v3 at the same molar concentration as 6 mg / kg of GI-104E2. In particular, GI-104E2 showed superior tumor growth inhibition at the midpoint and end of the experiment compared to the experimental group co-administered with anti-LAG-3 antibody and Fc-IL-2v3 (Figures 19-25).

[0108] Furthermore, when changes in tumor growth rate were observed at the end of the study, the number of subjects showing a 50% or greater reduction in tumor growth rate was 1 in the negative control group, 0 in the anti-LAG-3 antibody-administered group, 1 in the Fc-IL-2v3-administered group, and 1 in the anti-LAG-3 antibody and Fc-IL-2v3 co-administered group. On the other hand, in the experimental group administered GI-104E2, a 50% or greater reduction in tumor growth rate was observed in all subjects, and only 1 subject showed an 80% or greater reduction in tumor growth rate (Figure 26).

[0109] The survival rates of the experimental group administered GI-104E2 and the other experimental control and negative control groups were determined based on the time of death of the experimental subjects or the time of 2,000 mm 3 The results were analyzed based on the incidence of tumor volume. The results confirmed that the experimental group administered with GI-104E2 showed a higher survival rate than the other experimental control groups and the negative control group (Figure 27).

Claims

1. an antibody or antibody fragment thereof that specifically binds to LAG-3; IL-2 variants and A fusion protein comprising: The fusion protein is a dimer formed by linking two fusion proteins represented by the following structural formula (1): N'-X-[linker (1)]o-Fc region fragment or variant thereof-[linker (2)]p-Y-C' (1) [In structural formula (1), N' is the N-terminus of the fusion protein; C' is the C-terminus of the fusion protein; X is the antibody or the antibody fragment, Y is the IL-2 variant; the linker (1) and the linker (2) are peptide linkers, o and p are each independently 0 or 1. The antibody or the antibody fragment a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 1, an HCDR2 of SEQ ID NO: 2, and an HCDR3 of SEQ ID NO: 3; a light chain variable region comprising an LCDR1 of SEQ ID NO: 5, an LCDR2 of SEQ ID NO: 6, and an LCDR3 of SEQ ID NO: 7; Including, A fusion protein, wherein the IL-2 variant has an amino acid sequence of SEQ ID NO: 22 with substitutions at positions selected from the group consisting of R38A, F42A, Y45A, E61R, L72G, and combinations thereof.

2. The antibody that specifically binds to LAG-3 is a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; The fusion protein of claim 1 , comprising:

3. The fusion protein of claim 1, wherein the IL-2 variant has the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:

21.

4. A polynucleotide encoding the fusion protein according to any one of claims 1 to 3.

5. An expression vector comprising the polynucleotide of claim 4.

6. A transformed cell into which the expression vector according to claim 5 has been introduced.

7. Culturing the transformed cell according to claim 6; collecting the fusion protein from the culture medium; A method for preparing a fusion protein, comprising:

8. A pharmaceutical composition for treating or preventing cancer, comprising the fusion protein according to any one of claims 1 to 3.

9. 9. The pharmaceutical composition of claim 8, wherein the cancer is selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

10. Use of the fusion protein according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment or prevention of cancer.

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