Modified MFG-e8 protein

Abstract

The present disclosure relates to a composition and a method for treating cancer, a composition and a method for activating phagocytic cells associated therewith, a composition and a method for activating immune responses, and a modified MFG-E8 protein or nucleic acid encoding the same, which are used in said compositions and methods.

Description

Modified MFG-E8 protein

[0001] The present disclosure relates to compositions and methods for treating cancer, compositions and methods for activating phagocytes related thereto, compositions and methods for activating immune responses, and modified MFG-E8 proteins or nucleic acids encoding the same that are used in these compositions and methods.

[0002] Cancer remains the leading cause of death, and new treatments are constantly being sought. Cancer treatments include surgery, radiation therapy, chemotherapy (anticancer drugs), and immunotherapy, but anticancer drug treatment is also used after surgery. Anticancer drugs used include alkylating agents, antimetabolites, alkaloid anticancer drugs, antibiotic anticancer drugs, and platinum preparations, but their therapeutic effects are still insufficient and they also have the problem of a high incidence of side effects. From this perspective, there is a need to develop pharmaceuticals for better cancer treatment.

[0003] On the other hand, MFG-E8 (milk fat globule-EGF factor 8) has been identified as a factor secreted from the mammary gland that promotes mammary gland differentiation and lactation stimulation (Non-Patent Document 1: Stubbs T et al. 1990. Proc. Natl. Acad. Sci. USA 87:8417-8421). MFG-E8 has been shown to have not only the above-mentioned effects but also various other functions. One of these important functions is to recognize phosphatidylserine (PtdSer) on the membrane of apoptotic cells as an opsonin, promoting phagocytosis by macrophages and dendritic cells, thereby maintaining subsequent immune tolerance (Non-Patent Document 2: Hanayama R. et al. 2002. Nature 417:182-187; Non-Patent Document 3: Hanayama R., et al. 2004. Science 304:1147-1150). Furthermore, MFG-E8 has been shown to induce immune tolerance by promoting the proliferation of Foxp3-positive regulatory T cells, thereby negatively modulating the anti-tumor immunity of tumor vaccines (Non-Patent Document 4: Jinushi M., et al. 2007. J Clin Invest 117:1902-1913). Based on this finding, attempts have been made to develop therapeutic methods using MFG-E8 decoy genes or inhibitory antibodies, with the assumption that they will be used in combination with tumor antigens such as cancer vaccines (Patent Document 1: International Publication No. 2008 / 043018). Furthermore, it has been shown that MFG-E8 is not only expressed in antigen-presenting cells such as dendritic cells, but also in a wide range of tumor cells, including those in breast cancer, colon cancer, and melanoma (Non-Patent Document 5: Carmon L., et al. 2002. J Clin Invest 110:453-462), that it has tumor-promoting effects by promoting angiogenesis and tumor metastasis, and that it is positively correlated with the clinical progression of melanoma (Non-Patent Document 6: Neutzner M., et al. 2007. Cancer Res 67:6777-6785).

[0004] The inventors of the present disclosure previously developed a method for enhancing the therapeutic effects of other cancer therapies using an anti-MFG-E8 antibody (Patent Document 2: International Publication No. 2009 / 147781; Non-Patent Document 7: Jinushi and Tahara et al., JEM, 2009). This invention utilizes an antibody against the integrin-binding motif RGD to inhibit the phagocytosis of apoptotic cells, which is initiated by MFG-E8 cross-linking PtdSer with integrins, and instead induces phagocytosis via the Fc portion of the antibody, thereby activating the subsequent immune response. This invention provides a novel method for cancer treatment, providing significant technical benefits. However, this method has the following drawbacks: 1) MFG-E8 is not the only molecule that binds to PtdSer to induce immune tolerance; and 2) the immunostimulatory effect is dependent on Fcγ receptors on phagocytes, and some Fcγ receptors not only activate but also suppress immune responses. Therefore, the development of new pharmaceuticals for cancer treatment has been desired.

[0005] International Publication No. WO 2008 / 043018 International Publication No. WO 2009 / 147781

[0006] Stubbs T et al. 1990. Proc. Natl. Acad. Sci. USA 87:8417-8421 Hanayama R. et al. 2002. Nature 417:182-187 Hanayama R. , et al. 2004. Science 304:1147-1150 Jinushi M. , et al. 2007. J Clin. Invest. 117:1902-1913) Carmon L. , et al. 2002. J Clin. Invest. 110:453-462 Neutzner M. , et al. 2007. Cancer Res. 67:6777-6785 Jinushi and Tahara et al. , JEM, 2009.206:1317-1326

[0007] There is still a need to develop new pharmaceuticals for cancer treatment. The present disclosure provides a novel composition and method for cancer treatment, a composition and method for activating phagocytes, a composition and method for stimulating immune responses, and a modified MFG-E8 protein or a nucleic acid encoding the same, for use in these compositions and methods.

[0008] While examining the results obtained through intensive research into MFG-E8, the inventors of the present disclosure came up with the idea of ​​utilizing the PtdSer-binding ability of MFG-E8 in a cancer treatment method, unlike conventional methods in which MFG-E8 itself was inhibited with an antibody. After much trial and error, the inventors of the present disclosure discovered that, whereas MFG-E8 antibodies are limited to inhibiting the binding of MFG-E8 to integrins on phagocytes, by utilizing the PtdSer-binding ability of MFG-E8, it is possible to inhibit the binding of PtdSer to PtdSer-binding molecules other than MFG-E8, thereby suppressing immune tolerance induction. For example, by inhibiting the binding of PtdSer to Gas6 / protein S-TAM receptor, C1q-C1q receptor, β2-GPI, and TIM-4, which are known as PtdSer-binding molecules, immune tolerance induction can be inhibited for all of these. Furthermore, we found that fusing the PtdSer-binding portion of MFG-E8 with RAP (receptor-associated protein), a binding protein for CD91 / LRP1, can control the phagocytic pathway and reliably activate the subsequent immune response. Furthermore, we found that using this fusion form with RAP can eliminate the instability of the immune response caused by the presence of a wide variety of Fcγ receptors, which was observed when using the MFG-E8 antibody.

[0009] In one aspect, the present disclosure provides a protein having the PtdSer binding site of MFG-E8, such as integrin α ｖ β ３／５ , α ８ β １ The binding site, i.e., MFG-E8 binds to integrin α ｖ β ３／５ or integrin α ８ β１ The present invention provides a protein that does not have a site that binds to

[0010] In one aspect, the present disclosure provides a protein having the PtdSer binding site of MFG-E8, such as integrin α ｖ β ３／５ , α ８ β １ A nucleic acid encoding a protein without a binding site is provided.

[0011] In one aspect, the present disclosure provides a protein having the PtdSer binding site of MFG-E8, such as integrin α ｖ β ３／５ , α ８ β １ Compositions are provided that include proteins that do not have binding sites or nucleic acids that encode the same.

[0012] In one aspect, the present disclosure provides a protein having the PtdSer binding site of MFG-E8, such as integrin α ｖ β ３／５ , α ８ β １ Methods are provided that use compositions that include proteins that do not have a binding site or nucleic acids that encode the same.

[0013] More specifically, the present disclosure provides the following: [Item 1] A protein having a phosphatidylserine (PtdSer) binding site of milk fat globule membrane glycoprotein (milk fat globule-EGF factor 8: MFG-E8), which binds to integrin α ｖ β ３／５ , α ８ β １ [Item 2] The protein according to Item 1, wherein the protein has the C1C2 domain of MFG-E8 or a part thereof as a PtdSer-binding site. [Item 3] The protein according to Item 1, wherein the protein has the C1C2 domain of MFG-E8 or a part thereof as a PtdSer-binding site. ｖ β ３／５ , α ８ β １The protein according to item 1, which does not have the RGD motif (Arg-Gly-Asp integrin binding motif) of MFG-E8 or a part thereof as a binding site. [Item 4] The protein is an integrin α ｖ β ３／５ , α ８ β １ The protein according to item 1, which does not have the EGF-like domain (epidermal growth factor-like domain) of MFG-E8 or a part thereof as a binding site. [Item 5] The protein does not bind to phagocyte integrin α ｖ β ３／５ or integrin α ８ β １ The protein according to item 1, further having a site that binds to a receptor different from that of the phagocyte receptor. [Item 6] The protein according to item 5, wherein the phagocyte receptor is a receptor that stimulates the phagocytosis of phagocytes. [Item 7] The protein according to item 6, wherein the receptor that stimulates the phagocytosis of phagocytes is CD91 / LRP1. [Item 8] The protein according to item 5, wherein the site that binds to the phagocyte receptor is receptor-associated protein (RAP) or a part thereof. [Item 9] A nucleic acid encoding the protein according to any one of items 1 to 8. [Item 10] A composition for stimulating the phagocytosis of phagocytes, comprising the protein according to any one of items 1 to 8 or a nucleic acid encoding the same. [Item 11] A composition for stimulating an immune response, comprising the protein according to any one of items 1 to 8 or a nucleic acid encoding the same. [Item 12] A composition for treating cancer, comprising the protein according to any one of items 1 to 8 or a nucleic acid encoding the same. [Item 13] The composition for treating cancer according to Item 12, to be used in combination with an anti-cancer therapy that exhibits a cytocidal effect. [Item 14] The composition for treating cancer according to Item 12, to be used in combination with an anti-cancer therapy that uses an immune checkpoint inhibitor. [Item 15] A protein having the PtdSer binding site of MFG-E8, which binds to integrin α ｖ β ３／５ , α ８ β１ [Item 16] A method for activating the phagocytic activity of phagocytes, comprising the step of contacting a protein having no binding site or a nucleic acid encoding the same with PtdSer of apoptotic cells. [Item 17] A method for activating the phagocytic activity of phagocytes, comprising the step of administering to a subject having a disease, symptom, or condition requiring activation of the phagocytic activity of phagocytes a protein having the PtdSer binding site of MFG-E8, which is integrin α ｖ β ３／５ , α ８ β １ [Item 17] A method for stimulating the phagocytic activity of phagocytes in a subject, comprising the step of administering a composition containing a protein having no binding site or a nucleic acid encoding the same. [Item 18] The method according to items 15 or 16, wherein the protein has the C1C2 domain of MFG-E8 or a part thereof as a PtdSer-binding site. [Item 19] The method according to item 16, wherein the protein has the C1C2 domain of MFG-E8 or a part thereof as a PtdSer-binding site. ｖ β ３／５ , α ８ β １ The method according to item 15 or 16, wherein the protein does not have the RGD motif (Arg-Gly-Asp integrin binding motif) of MFG-E8 or a part thereof as a binding site. ｖ β ３／５ , α ８ β １ The method according to item 15 or 16, wherein the binding site does not include the epidermal growth factor-like domain of MFG-E8 or a part thereof. ｖ β ３／５ or integrin α ８ β １The method of item 15 or 16, wherein the phagocyte receptor further has a site that binds to a receptor different from that of the phagocyte receptor. [Item 21] The method of item 15 or 16, wherein the phagocyte receptor is a receptor that activates the phagocytosis of phagocytes. [Item 22] The method of item 15 or 16, wherein the receptor that activates the phagocytosis of phagocytes is CD91 / LRP1. [Item 23] The method of item 15 or 16, wherein the site that binds to the phagocyte receptor is receptor-associated protein (RAP) or a portion thereof. [Item 23] The method of item 16, wherein the disease, symptom, or condition requiring activation of the phagocyte activity of phagocytes is cancer. [Item 28] The method of item 23, wherein the method is used in combination with an anti-cancer therapy that exhibits a cytocidal effect. [Item 29] The method of item 23, wherein the method is used in combination with an anti-cancer therapy that uses an immune checkpoint inhibitor.

[0014] The present disclosure has the effect of providing a new composition and method for cancer treatment, a composition and method for activating phagocytes related thereto, a composition and method for activating immune responses, and a modified MFG-E8 protein or a nucleic acid encoding the same for use in these compositions and methods.

[0015] Figure 1 is a schematic diagram showing the mechanism of action of the present disclosure. (A) In normal cells, MFG-E8 recognizes PtdSer on the membrane of apoptotic cells and binds to integrin α of phagocytes. ｖ β ３／５ , α ８ β １ (B) A protein having a PtdSer binding site according to the present disclosure, which binds to integrin α, thereby promoting phagocytosis by phagocytes and suppressing the subsequent immune response. ｖ β ３／５ , α ８ β １Proteins lacking a binding site inhibit the binding of MFG-E8 and other PtdSer-binding phagocytosis-promoting factors to PtdSer, thereby restricting the immunosuppressive phagocytic pathway of phagocytes. This allows phagocytes activated by a PtdSer-independent pathway to subsequently activate immune responses. (C) By further fusing RAP, which binds to the CD91 / LRP1 receptor of phagocytes, to a protein of the present disclosure, the phagocytes are regulated and induced to a phagocytic pathway favorable for immune response induction, resulting in the subsequent strong activation of immune responses. Figure 2 is a schematic diagram showing an example of the structure of the modified human MFG-E8 protein or modified mouse MFG-E8 protein of the present disclosure. Wild-type MFG-E8 (A) shows the structure of wild-type mouse MFG-E8 protein. C1C2 (B) shows the structure of a modified mouse MFG-E8 protein lacking the EGF-like domain. PStRAP(C) shows the structure of a modified MFG-E8 protein in which a portion of RAP (Y38-L360) is fused to the C-terminus of a modified mouse MFG-E8 protein lacking the EGF-like domain. FIG. 3 shows experimental results confirming the anti-cancer effects of C1C2 and PStRAP in the modified MFG-E8 protein of the present disclosure. FIG. 4 shows experimental results confirming the properties of LNP. FIG. 5 shows the results of an evaluation of liver dysfunction caused by LNP administration. FIG. 6 shows the results of an evaluation of the binding specificity of PStRAP to cell membrane lipids. FIG. 7 shows the results of measuring the amount of PStRAP in serum after administration of LNP-PStRAP. FIG. 8 shows an outline of an experimental method for evaluating liver dysfunction caused by LNP administration. FIG. 9 shows the results of an experimental evaluation of liver dysfunction caused by LNP administration.

[0016] Typically, removal of apoptotic cells suppresses the immune response to self cells without inflammation, and they are quickly digested by phagocytes (avoiding autoimmune diseases). However, conversely, it also suppresses the immune response to cancer cells, where the boundary between self and non-self is unclear. The inhibitory mechanism associated with this apoptotic cell removal begins with the binding of factors such as MFG-E8, Gas6, β2-GPI, and C1q to PtdSer expressed on the apoptotic cell membrane. This disclosure focuses on the use of the C1C2 domain, the PtdSer-binding domain of MFG-E8, and its basic operating principle is to mask PtdSer with a molecule lacking the integrin-binding domain from the C1C2 domain, thereby inhibiting recognition of PtdSer-binding molecules (Figures 1, 2A, and 2B). Furthermore, we used a fusion protein (PStRAP; ​​PtdSer-targeted RAP) in which the C1C2 domain was linked to the CD91 / LRP1-binding protein, RAP, to control the phagocytic process of phagocytes (Figures 1 and 2C). CD91 / LRP1 is a common receptor for many heat shock proteins, including RAP, and CD91 / LRP1-mediated phagocytosis is known to induce strong immune responses.

[0017] The concept of "in situ vaccination" is a method for activating immune responses using dead cells in vivo, and research is currently underway worldwide. However, most of these studies have focused on how to kill cancer cells, which has been systematized as immunogenic cell death. Meanwhile, there has been little research focused on how to process (phagocytose) cancer cells, regardless of the method of killing. Related applications by the present inventors (Japanese Patent Application No. 2010-515738, International Publication No. 2009 / 147781) represent pioneering research into this strategy. This disclosure was completed with a focus on the phagocytosis of cancer cells. Compared to the above-mentioned related applications by the inventors of this disclosure, this method has a broader range of applicable cases and can be used in combination with almost all standard anti-cancer treatments, excluding surgical treatment, and is expected to enhance therapeutic efficacy.

[0018] Furthermore, immune checkpoint inhibitors, which have been recognized as a new standard of care, share the same strategy of "activating anti-cancer immune responses," but conventional immune checkpoint inhibitors have a therapeutic strategy of releasing the suppression of immune cells after activation, and therefore have a different mode of action from the present disclosure, which activates the induction of immune responses. Therefore, the cancer treatment composition and cancer treatment method of the present disclosure are also considered to be effective as combination drugs with immune checkpoint inhibitors.

[0019] DEFINITIONS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0020] In this specification, when multiple ranges of numerical values ​​are shown, the same applies to ranges formed by combining any lower limit value and upper limit value of those multiple ranges.

[0021] As used herein, the term "substantially" has the same meaning as commonly understood by a person skilled in the art to which the present disclosure pertains, but is intended to encompass a desired state and a state that is unavoidably not achieved due to biological or chemical properties, taking into consideration, for example, that a biological or chemical phenomenon may not completely achieve a desired state.

[0022] As used herein, the term "about" in connection with a numerical value means that the value can vary within, for example, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01%.

[0023] As used herein, the terms "comprise" and "contain" have the same meaning as commonly understood by a person skilled in the art to which the present disclosure belongs, and include, for example, "comprises" and "consists of." Specifically, a composition that "comprises" or "contains" A may contain another component, B, in addition to only A.

[0024] As used herein, the term "having" has the same meaning as commonly understood by a person skilled in the art to which the present disclosure pertains, for example, it has the same meaning as "comprises" or "contains" above, and a protein that "has" A may have another component, B, in addition to having only A.

[0025] As used herein, the terms "consisting of" or "composed of" when referring to a composition have the same meaning as commonly understood by a person skilled in the art to which the present disclosure pertains, but are used to indicate components that exclusively constitute the composition. For example, a composition "consisting of" A contains exclusively A. However, in one embodiment, a composition "consisting of" A encompasses an embodiment in which a contaminant other than A is contained that is unavoidable in production due to biological and chemical properties.

[0026] As used herein, "identity" refers to the degree to which two or more comparable amino acid sequences or nucleotide sequences are identical to each other. Thus, the higher the identity between two amino acid sequences or nucleotide sequences, the higher the identity or similarity between those sequences. The level of identity between amino acid sequences or nucleotide sequences is usually determined using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (e.g., Karlin S, Altschul S F. Proc. Natl Acad Sci USA. 87:2264-2268 (1990), Karlin S, Altschul S F. Natl Acad Sci USA. 90:5873-7 (1993), etc.). Programs called BLASTN and BLASTX based on such BLAST algorithms have been developed (e.g., Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. J Mol Biol. 215:403-10 (1990)). Specific techniques for these analysis methods are known, and can be found on the NCBI website. For example, when a certain amino acid sequence A is identical to another amino acid sequence B by a certain percentage, this means that amino acid sequence A and amino acid sequence B have the specified percentage of identity.

[0027] As used herein, the term "conservative substitution" means that the amino acid residue is replaced with an amino acid residue having a similar side chain.

[0028] For example, conservative substitutions include substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine. Conservative substitutions also include substitutions between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0029] As used herein, "treatment" or "therapy" of a disease, symptom, or condition has the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains, and includes any action beneficial to a subject with the disease, symptom, or condition. In one aspect, "treatment" or "therapy" of a disease, symptom, or condition includes suppression, inhibition of progression, delay of progression, amelioration, or prevention of the disease, symptom, or condition.

[0030] As used herein, "activation" of physiological activity, such as the phagocytic action of phagocytes or immune response, has the same meaning as commonly understood by those skilled in the art to which the present disclosure pertains, and encompasses all manner of inducing a state in which the physiological activity is exerted more effectively, including inducing the activity from a state in which the physiological activity is absent and enhancing an already existing physiological activity.

[0031] (A protein having the PtdSer binding site of MFG-E8, and integrin α ｖ β ３／５ , α ８ β １ Proteins with no binding site

[0032] In one aspect, the present disclosure provides a protein having the PtdSer binding site of MFG-E8, such as integrin α ｖ β ３／５ , α ８ β １The present disclosure provides a protein that does not have a binding site. PtdSer displayed on the surface of apoptotic cells is usually recognized by factors such as MFG-E8, Gas6, β2-GPI, C1q, and TIM-4, which triggers the phagocytosis of phagocytes, promoting the rapid digestion of apoptotic cells and suppressing subsequent immune responses. However, the protein of the present disclosure binds to PtdSer displayed on the surface of apoptotic cells via the PtdSer-binding site of MFG-E8, thereby inhibiting the binding of PtdSer to factors such as MFG-E8, Gas6, β2-GPI, C1q, and TIM-4, thereby regulating the phagocytic pathway of phagocytes and activating subsequent immune responses.

[0033] In one embodiment, the PtdSer-binding site of MFG-E8 in the protein of the present disclosure is an amino acid sequence contained in MFG-E8, and has a sequence region necessary and sufficient for binding to PtdSer. The sequence region can be identified by those skilled in the art based on literature or by conducting routine experiments.

[0034] In one embodiment, the PtdSer-binding site of MFG-E8 in the protein of the present disclosure has the full length or a part of the C1C2 domain, which is the C domain of coagulation factors VIII and V. The C1C2 domain of MFG-E8 is widely known to those skilled in the art, and for example, when MFG-E8 is human MFG-E8 (NCBI Gene ID: 4240), the C1C2 domain is identified as the region of amino acid sequence 70 to 387.

[0035] In one embodiment, the PtdSer-binding site of MFG-E8 in the protein of the present disclosure may have a mutation relative to the amino acid sequence of wild-type MFG-E8, as long as the ability to bind to PtdSer is maintained. For example, the PtdSer-binding site of MFG-E8 in the protein of the present disclosure may have a conservative substitution relative to the amino acid sequence of wild-type MFG-E8. In one embodiment, the PtdSer-binding site of MFG-E8 in the protein of the present disclosure has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of wild-type MFG-E8 at said site. In one embodiment, the protein of the present disclosure comprises a portion of MFG-E8. In one embodiment, the protein of the present disclosure consists essentially of a portion of MFG-E8.

[0036] The protein of the present disclosure is integrin α ｖ β ３／５ , α ８ β １ The site is an amino acid sequence contained in MFG-E8, which does not have a binding site for integrin α ｖ β ３／５ , α ８ β １ The sequence region is a sequence region necessary for binding to the target protein. Such a sequence region can be identified by a person skilled in the art based on literature or by performing routine experiments.

[0037] In one aspect, the protein of the present disclosure is integrin α ｖ β ３／５ , α ８ β １It does not have the full length or a part of the RGD motif (Arg-Gly-Asp integrin motif) as a binding site. The RGD motif of MFG-E8 is widely known to those skilled in the art. For example, when MFG-E8 is human MFG-E8 (NCBI Gene ID: 4240), the RGD motif is specified as the amino acid sequence arginine-glycine-aspartic acid contained in the region of amino acid sequence 46-48. > Amino acid sequence of human MFG-E8 (NCBI Gene ID: 4240) (SEQ ID NO: 3)

[0038] In one aspect, the protein of the present disclosure is integrin α ｖ β ３／５ , α ８ β １ It does not have the full length or a part of an EGF-like domain (epidermal growth factor-like domain) as a binding site. The EGF-like domain of MFG-E8 is widely known to those skilled in the art. For example, when MFG-E8 is human MFG-E8 (NCBI Gene ID: 4240), the EGF-like domain is identified as the region of amino acid sequence 24 to 67.

[0039] In one embodiment, the protein of the present disclosure further comprises a site that binds to a receptor on a phagocyte. In one embodiment, the receptor on a phagocyte is integrin α ｖ β ３／５ or α ８ β １ In one embodiment, the phagocyte receptor is not integrin α ｖ β ３／５ , α ８ β １ Or it is not a TAM receptor (Tyro3, ​​Axl, Mer). In one embodiment, the protein of the present disclosure is provided as a fusion protein of a portion of MFG-E8 and a site that binds to a receptor on a phagocyte. In one embodiment, the protein of the present disclosure may include the signal peptide of MFG-E8.

[0040] In one embodiment, the receptor of the phagocyte is a receptor that activates the phagocytic action of the phagocyte. The receptor that activates the phagocytic action of the phagocyte includes a receptor that has the effect of inducing phagocytosis in phagocytes that do not exhibit phagocytosis, and a receptor that enhances phagocytosis in phagocytes that exhibit phagocytosis.

[0041] The receptor that activates the phagocytic activity of phagocytes is not particularly limited, and can be appropriately selected from receptors known to those skilled in the art as receptors that activate the phagocytic activity of phagocytes, such as, but not limited to, CD91 / LRP, Fc receptors, and complement receptors (CR1, CR3, and CR4).

[0042] The site that the protein of the present disclosure additionally has that binds to a receptor on a phagocyte is not particularly limited, and any compound known to those skilled in the art that binds to a receptor that activates the phagocytic activity of a phagocyte can be appropriately selected. The structure of the compound is not particularly limited, and compounds such as proteins, peptides, and nucleic acids can be used. For example, receptor-associated protein (RAP), heat shock proteins (Hsp70, Hsp90, gp96, calreticulin), the Fc region of an antibody, C3b, iC3b, or parts thereof can be used.

[0043] When RAP is used in the present disclosure, the full length of RAP may be used, or a portion thereof that binds to a receptor in phagocytes may be used. In one embodiment, the region of RAP used in the present disclosure is not particularly limited as long as it maintains the ability to stimulate phagocytosis in phagocytes. In one embodiment, the RAP used in the present disclosure preferably comprises the D1 to D3 domains. For example, when human RAP (NCBI Gene ID: 4043) is used, it is preferable to use a portion comprising the region Y35 to L357. When mouse RAP (NCBI Gene ID: 16976) is used, it is preferable to use a portion comprising the region Y38 to L360. In one embodiment, the RAP used in the present disclosure comprises the D3 domain (R237 to R353 in human RAP, R240 to R356 in mouse RAP) but does not comprise D1 or D2. In one embodiment, the RAP used in the present disclosure substantially comprises only the portion necessary to maintain the ability to stimulate phagocytosis in phagocytes. For example, RAP used in the present disclosure does not contain the endoplasmic reticulum retention sequence consisting of the C-terminal four amino acids (HNEL) (Bu G. et al., EMBO J. 1995; 14(10):2269-80).

[0044] In one embodiment, the RAP contained in the protein of the present disclosure may have a mutation in the amino acid sequence of wild-type RAP, as long as the mutation maintains the ability to stimulate phagocytosis in phagocytes. For example, the RAP contained in the protein of the present disclosure may have a conservative substitution in the amino acid sequence of wild-type RAP. In one embodiment, the RAP contained in the protein of the present disclosure has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of wild-type RAP.

[0045] The additional phagocyte receptor-binding domain of the protein of the present disclosure can be linked to the PtdSer-binding domain directly or via a linker. In one embodiment, the protein having the additional phagocyte receptor-binding domain is provided as a fusion protein.

[0046] The phagocytes are not particularly limited, and any cells known to those skilled in the art as cells exhibiting phagocytosis can be appropriately selected. In one embodiment, the phagocytes are cells that activate immune responses through phagocytosis. For example, monocytes, macrophages, and dendritic cells can be selected as the phagocytes, but are not limited to these.

[0047] A protein having the PtdSer binding site of MFG-E8 of the present disclosure, ｖ β ３／５ , α ８ β １ The MFG-E8-derived portion of a protein that does not have a binding site, or the additional site that binds to a phagocyte receptor, may be derived from proteins of various organisms, such as humans, primates such as monkeys, and mammals such as dogs, cats, and mice.

[0048] A protein having the PtdSer binding site of MFG-E8 of the present disclosure, ｖ β ３／５ , α ８ β １ Proteins without binding sites can have additional modifications. For example, they may be modified with various compounds to improve storage stability, control blood retention time, etc. Examples of such compounds include, but are not limited to, PEG (polyethylene glycol). Proteins of the present disclosure may include sites with other functions in addition to the PtdSer binding site and the site that binds to a receptor on phagocytes.

[0049] (A protein having the PtdSer binding site of MFG-E8, and integrin α ｖ β ３／５ , α ８ β １ a nucleic acid encoding a protein without a binding site

[0050] In one aspect, the present disclosure provides a protein having the PtdSer binding site of MFG-E8, comprising integrin α ｖ β ３／５, α ８ β １ The present invention provides a nucleic acid encoding a protein that does not have a binding site. Typically, the protein is a protein that has the PtdSer binding site of MFG-E8 (as described above), and is a protein that binds to integrin α ｖ β ３／５ , α ８ β １ In one embodiment, the nucleic acid of the present disclosure encodes a fusion protein consisting essentially of a portion of MFG-E8 or a portion of MFG-E8 and a portion that binds to a receptor on a phagocyte.

[0051] The nucleic acid of the present disclosure may be a ribonucleotide or a deoxynucleotide. Furthermore, the form of the nucleic acid is not particularly limited, and may be a single-stranded or double-stranded form. The codons used in the nucleic acid sequence are not particularly limited, and various codons can be appropriately selected and used depending on the purpose. For example, the codons can be appropriately selected in consideration of codon frequency and the like depending on the type of host cell, expression system, etc. used in producing the protein.

[0052] The nucleic acid of the present disclosure is a protein having the PtdSer binding site of MFG-E8, and is a protein that binds to integrin α ｖ β ３／５ , α ８ β １ In addition to the portion encoding a protein without a binding site, any additional portion for controlling the transcription, replication, etc. of the nucleic acid may be included. In one embodiment, the nucleic acid of the present disclosure is provided as a vector.

[0053] In one embodiment, the nucleic acid of the present disclosure is used to express and produce a protein of the present disclosure. In one embodiment, the nucleic acid of the present disclosure is introduced in the form of DNA into a host cell such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or human embryonic kidney (HEK293) cells, and used to produce a protein of the present disclosure from the recombinant host cell.

[0054] In one aspect, the nucleic acid of the present disclosure is used to express the protein of the present disclosure in a living body and induce a physiological effect in the living body. In one aspect, the nucleic acid of the present disclosure is delivered to the living body in the form of DNA or mRNA using various drug delivery systems and used to express the protein of the present disclosure at a desired location in the living body at a desired time.

[0055] (A protein having the PtdSer binding site of MFG-E8, and integrin α ｖ β ３／５ , α ８ β １ a composition comprising a protein having no binding site or a nucleic acid encoding the same)

[0056] In one aspect, the present disclosure provides a protein having the PtdSer binding site of MFG-E8, such as integrin α ｖ β ３／５ , α ８ β １ The present disclosure provides a composition comprising a protein having no binding site or a nucleic acid encoding the same. Typically, the composition of the present disclosure is a protein having the PtdSer binding site of MFG-E8, which is a protein having integrin α ｖ β ３／５ , α ８ β １ a protein described in (1) above that does not have a PtdSer binding site for MFG-E8, or a protein that has the PtdSer binding site for integrin α ｖ β ３／５ , α ８ β １ Nucleic acids encoding proteins without binding sites are included.

[0057] In one embodiment, the composition of the present disclosure is used to stimulate phagocytosis of phagocytes. Thus, in one embodiment, the present disclosure provides a protein having the PtdSer binding site of MFG-E8, which binds to integrin α ｖ β ３／５ , α ８ β １A composition for stimulating the phagocytic activity of phagocytes is provided, which comprises a protein having no binding site or a nucleic acid encoding the same.

[0058] In one aspect, the composition for stimulating the phagocytic activity of phagocytes of the present disclosure can be used to stimulate the phagocytic activity of phagocytes in vivo or ex vivo, hi one aspect, the composition for stimulating the phagocytic activity of phagocytes of the present disclosure can be used as a reagent for analyzing the function of phagocytes in vivo or ex vivo.

[0059] In one aspect, the composition for stimulating the phagocytic activity of phagocytes disclosed herein is administered to a subject having a disease, symptom, or condition that requires the stimulation of the phagocytic activity of phagocytes to stimulate the phagocytic activity of phagocytes.

[0060] In one aspect, the composition for activating the phagocytic activity of phagocytes of the present disclosure is used to treat a disease, symptom, or condition in a subject having the disease, symptom, or condition that requires activation of the phagocytic activity of phagocytes.

[0061] In one aspect, the composition for stimulating the phagocytic activity of phagocytes of the present disclosure is used as a composition for stimulating an immune response or a composition for treating cancer, as described below.

[0062] In one aspect, the compositions of the present disclosure are used to stimulate an immune response. Thus, in one aspect, the present disclosure provides a protein having the PtdSer binding site of MFG-E8, which binds to integrin α ｖ β ３／５ , α ８ β １ A composition for stimulating an immune response is provided, which comprises a protein having no binding site or a nucleic acid encoding the same.

[0063] In one aspect, the composition for stimulating an immune response of the present disclosure is used to stimulate an immune response in a subject having a disease, symptom, or condition that requires stimulation of the immune response.

[0064] In one aspect, the composition for stimulating an immune response of the present disclosure is used to treat a disease, symptom, or condition in a subject having said disease, symptom, or condition that requires stimulation of an immune response.

[0065] In one aspect, the composition for stimulating an immune response of the present disclosure is used as a composition for treating cancer, as described below.

[0066] In one embodiment, the compositions of the present disclosure are used to treat cancer. Accordingly, in one embodiment, the present disclosure provides a protein having the PtdSer binding site of MFG-E8, which is integrin α ｖ β ３／５ , α ８ β １ A composition for treating cancer is provided, which comprises a protein having no binding site or a nucleic acid encoding the same.

[0067] The cancer treatment composition of the present disclosure can be used in combination with other cancer treatment methods, as long as its effectiveness is not lost. The other cancer treatment methods are not particularly limited, and any known cancer treatment methods known to those skilled in the art can be used. Examples of such treatment methods include, but are not limited to, surgery, radiation therapy (including gamma knife therapy, cyber knife therapy, boron neutron capture therapy, proton therapy, and heavy ion therapy), MR-guided focused ultrasound surgery, cryotherapy, radiofrequency coagulation therapy, ethanol injection therapy, and arterial embolization therapy.

[0068] The cancer therapeutic agent used in the cancer treatment method in combination with the cancer treatment composition of the present disclosure is not particularly limited. For example, non-limiting examples of the cancer therapeutic agent include alkylating agents, antimetabolites, microtubule inhibitors, antibiotic anticancer agents, topoisomerase inhibitors, platinum compounds, molecular targeted drugs, hormone agents, and biological agents. Examples of alkylating agents include cyclophosphamide, ifosfamide, nitrosoureas, dacarbazine, temozolomide, nimustine, busulfan, melphalan, procarbazine, and ranimustine. Examples of antimetabolites include enocitabine, carmofur, capecitabine, tegafur, tegafur-uracil, tegafur-gimeracil-oteracil potassium, gemcitabine, cytarabine, cytarabine ocfosfate, nelarabine, fluorouracil, fludarabine, pemetrexed, pentostatin, methotrexate, cladribine, doxifluridine, hydroxycarbamide, mercaptopurine, etc. Examples of microtubule inhibitors include alkaloid anticancer drugs such as vincristine, and taxane anticancer drugs such as docetaxel and paclitaxel. Examples of antibiotic anticancer agents include mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, actinomycin D, aclarubicin, idarubicin, pirarubicin, peplomycin, mitoxantrone, amrubicin, zinostatin stimalamer, etc. Examples of topoisomerase inhibitors include CPT-11, irinotecan, and nogitecan, which have topoisomerase I inhibitory activity, and etoposide and sobuzoxane, which have topoisomerase II inhibitory activity. Examples of platinum agents include cisplatin, nedaplatin, oxaliplatin, and carboplatin. Examples of hormone agents include dexamethasone, finasteride, tamoxifen, astrozole, exemestane, ethinylestradiol, chlormadinone, goserelin, bicalutamide, flutamide, prednisolone, leuprorelin, letrozole, estramustine, toremifene, fosfestrol, mitotane, methyltestosterone, medroxyprogesterone, and mepitiostane.Examples of biological preparations include interferon α, β, and γ, interleukin 2, ubenimex, dried BCG, etc. Examples of molecular targeted drugs include rituximab, alemtuzumab, trastuzumab, cetuximab, panitumumab, imatinib, dasatinib, nilotinib, gefitinib, erlotinib, temsirolimus, bevacizumab, VEGF trap, sunitinib, sorafenib, tosituzumab, bortezomib, gemtuzumab ozogamicin, ibritumomab ozogamicin, ibritumomab tiuxetan, tamibarotene, tretinoin, etc. Other examples include inhibitors that target angiogenesis, such as human epidermal growth factor receptor 2 inhibitors, epidermal growth factor receptor inhibitors, Bcr-Abl tyrosine kinase inhibitors, epidermal growth factor tyrosine kinase inhibitors, mTOR inhibitors, and vascular endothelial growth factor receptor 2 inhibitors (α-VEGFR-2 antibodies), various tyrosine kinase inhibitors such as MAP kinase inhibitors, inhibitors that target cytokines, proteasome inhibitors, molecular targeted drugs such as antibody-anticancer drug combinations, complement targeted drugs, etc. These inhibitors also include antibodies.It may also be used in combination with the following drugs: thalidomide, everolimus, Elplat, ABI-007, ixabepilone, miriplatin, lapatinib, pemetrexed, cladribine, liposomal doxorubicin, Z-100, hycamtin, vandedanib, ZD4054, anastrozole, GSK1572932A, pazopanib, denosumab, S-1, mogamulizumab, Tesanib, trastuzumab, Enzastaurin, Immucyst, NIK-333, axitinib, bosutinib, E7080, sobridotin, degarelix, fulvestrant, Zoladex, cediranib, eribulin, TSU-68, TAC-101, TAS-108, NK911, NK105, elotinib, LBH589, MK-0457, tamibarotene, Nalidomide, BNP1350, AZD0530, AZD1152, AZD2281, AZD4877, ABT-869, ONO-4538, OTS102, KW-0761, ARQ197, ofatumumab, AMG655, TAK-700, TAK-683, TAK-448, CBP501, TAK-285, TAK-593, MLN8054, MLN4924, pe Rtuzumab, R1507, NK012, BIBF1120, BIBW2992, Patupilone, MK-2461, CP751,871, PF-00299804, satraplatin, CMC-544, YM155, GPI21016, YHO-13351, mogamulizumab, secukinumab, eculizumab, ravulizumab, avacopan, pegcetacoplan.

[0069] In one aspect, the composition for treating cancer of the present disclosure is preferably used in combination with other cancer therapies that exhibit cytocidal effects. By using the composition for treating cancer of the present disclosure in combination with other cancer therapies that exhibit cytocidal effects, the composition for treating cancer of the present disclosure can induce an adaptive immune response associated with cancer cell killing, thereby enhancing the cancer therapeutic effect.

[0070] The other cancer therapies that exhibit the cytocidal effect are not particularly limited, and any known cancer therapies known to those skilled in the art as cancer therapies that exhibit the cytocidal effect can be used. For example, chemotherapy, radiation therapy, CAR-T therapy, oncolytic virus therapy, etc. can be used as cancer therapies that exhibit the cytocidal effect, but are not limited to these. In one aspect, alkylating agents characterized by cytotoxic activity, antimetabolites, microtubule inhibitors, antibiotic anticancer agents, topoisomerase inhibitors, platinum preparations, molecularly targeted drugs, etc. can be particularly preferred, but are not limited to these. Specific examples that can be used include, but are not limited to, gemcitabine, 5-FU, CPT-11, etoposide, cisplatin, oxaliplatin, paclitaxel, docetaxel, dacarbazine, doxorubicin, bevacizumab, cetuximab, anti-vascular endothelial growth factor receptor 2 inhibitory antibodies, epidermal growth factor tyrosine kinase inhibitors, etc.

[0071] When the composition for treating cancer of the present disclosure is used in combination with other cancer therapies that exhibit cytocidal effects, the timing of administration of the composition for treating cancer of the present disclosure and the timing of use of the other cancer therapies that exhibit cytocidal effects can be appropriately combined. In one embodiment, the composition for treating cancer of the present disclosure is used simultaneously with other cancer therapies that exhibit cytocidal effects. In one embodiment, the composition for treating cancer of the present disclosure is used before other cancer therapies that exhibit cytocidal effects. In one embodiment, the composition for treating cancer of the present disclosure is used after other cancer therapies that exhibit cytocidal effects.

[0072] In one embodiment, the composition for treating cancer of the present disclosure is preferably used in combination with a cancer treatment method using an immune checkpoint inhibitor. The composition for treating cancer of the present disclosure enhances the induction of adaptive immune responses, thereby enhancing the effect of immune checkpoint inhibitors, which deregulate immune responses after they have been induced and activated. The immune checkpoint inhibitor is not particularly limited, and any known immune checkpoint inhibitor known to those skilled in the art can be used. Examples of immune checkpoint inhibitors that can be used include, but are not limited to, anti-CTLA-4 antibodies, PD1 blockers, PDL1 blockers, LAG-3 inhibitors, B7-H3 inhibitors, B7-H4 inhibitors, and TIM3 inhibitors. In one embodiment, the anti-CTLA-4 antibody can be, but is not limited to, ipilimumab or tremelimumab. In one embodiment, the PD1 blocker can be, but is not limited to, nivolumab, lambrolizumab, cemiplimab, CT-011, or AMP-224. In one embodiment, PDL1 blockers may include, but are not limited to, durvalumab, avelumab, atezolizumab, BMS-936559, and FAZ053. In one embodiment, LAG-3 inhibitors may include, but are not limited to, IMP321. In one embodiment, B7-H3 inhibitors may include, but are not limited to, MGA271.

[0073] When the composition for treating cancer of the present disclosure is used in combination with a cancer treatment method using an immune checkpoint inhibitor, the timing of administration of the composition for treating cancer of the present disclosure and the timing of use of the cancer treatment method using an immune checkpoint inhibitor can be appropriately combined. In one embodiment, the composition for treating cancer of the present disclosure is used simultaneously with the cancer treatment method using an immune checkpoint inhibitor. In one embodiment, the composition for treating cancer of the present disclosure is used before the cancer treatment method using an immune checkpoint inhibitor. In one embodiment, the composition for treating cancer of the present disclosure is used after the cancer treatment method using an immune checkpoint inhibitor.

[0074] The cancer treatment composition of the present disclosure is effective against a wide variety of cancers, including, but not limited to, epithelial cancers such as pharyngeal cancer, laryngeal cancer, tongue cancer, lung cancer, breast cancer, esophageal cancer, stomach cancer, colon cancer, uterine cancer, ovarian cancer, liver cancer, pancreatic cancer, gallbladder cancer, kidney cancer, prostate cancer, malignant melanoma, and thyroid cancer; and non-epithelial cancers such as osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, fibrosarcoma, leukemia, malignant lymphoma, and myeloma.

[0075] The compositions for activating the phagocytic activity of phagocytes and the compositions for treating cancer disclosed herein can be formulated by mixing, dissolving, granulating, tableting, emulsifying, encapsulating, lyophilizing, etc. with pharmaceutically acceptable carriers well known in the art.

[0076] For oral administration, the protein or nucleic acid of the present disclosure can be formulated together with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc. into dosage forms such as tablets, pills, sugar-coated tablets, soft capsules, hard capsules, solutions, suspensions, emulsions, gels, syrups, slurries, etc.

[0077] For parenteral administration, the protein or nucleic acid of the present disclosure can be formulated into dosage forms such as injectable solutions, suspensions, emulsions, creams, ointments, inhalants, suppositories, etc. together with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc. For injection formulations, the protein or nucleic acid of the present disclosure can be dissolved in an aqueous solution, preferably a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiological saline buffer.

[0078] The compositions of the present disclosure can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle. Alternatively, the protein or nucleic acid of the present disclosure can be prepared in powder form together with a carrier, etc., and an aqueous solution or suspension can be prepared using sterile water, etc., before use. For administration by inhalation, the protein or nucleic acid of the present disclosure can be powdered together with a carrier, etc., and a powder mixture can be prepared with a suitable base, such as lactose or starch. Suppository formulations can be prepared by mixing the protein or nucleic acid of the present disclosure with a carrier, etc., and a conventional suppository base, such as cocoa butter. Furthermore, the compositions for treating cancer of the present disclosure can be encapsulated in a polymer matrix, etc., and formulated as sustained-release preparations.

[0079] When the composition of the present disclosure contains a nucleic acid encoding a protein, in one embodiment, the nucleic acid is administered to the body in the form of mRNA, which is expressed as a protein in the body, and can be expressed in the circulating blood or near a tumor. The dosage form used for administration in the form of mRNA is not particularly limited, and dosage forms known to those skilled in the art can be used. For example, mRNA used as a nucleic acid of the present disclosure can be encapsulated in liposomes. The method for forming liposomes is not particularly limited, and any method known to those skilled in the art can be used. Liposomes are unilamellar or multilamellar vesicles with a membrane formed of a lipophilic material and an internal aqueous compartment. The aqueous compartment is used to contain the polynucleotide material to be delivered to the target site. In one embodiment, the liposomes can contain one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids. In one aspect, the one or more cationic lipids can be selected from the group consisting of C12-200, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE (imidazole-based), HGT5000, HGT5001, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA, and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and combinations thereof.

[0080] The concentration of the composition of the present disclosure may be adjusted appropriately. For example, when a protein is used, the concentration may be 1 μg / mL to 50 mg / mL, 10 μg / mL to 50 mg / mL, 20 μg / mL to 50 mg / mL, 30 μg / mL to 50 mg / mL, 40 μg / mL to 50 mg / mL, 50 μg / mL to 50 mg / mL, 60 μg / mL to 50 mg / mL, 70 μg / mL to 50 mg / mL, 80 μg / mL to 50 mg / mL, 90 μg / mL to 50 mg / mL, 0.1 mg / mL to 50 mg / mL, 0.2 mg / mL to 50 mg / mL, 0.3 mg / mL to 50 mg / mL, 0.4 mg / mL to 50 mg / mL, 0.5 mg / mL to 50 mg / mL, 0.6 mg / mL to 50 mg / mL, or 100 mg / mL to 50 mg / mL. 0mg / mL, 0.7mg / mL~50mg / mL, 0.8mg / mL~50mg / mL, 0.9mg / mL~50mg / mL, 1mg / mL~50 mg / mL, 2 mg / mL to 50 mg / mL, 3 mg / mL to 50 mg / mL, 4 mg / mL to 50 mg / mL, 5 mg / mL to 50 mg / mL, 6m g / mL ~ 50 mg / mL, 7 mg / mL ~ 50 mg / mL, 8 mg / mL ~ 50 mg / mL, 9 mg / mL ~ 50 mg / mL, 10 mg / mL ~ 50 It can be used at concentrations of mg / mL, 20 mg / mL to 50 mg / mL, 30 mg / mL to 50 mg / mL, 40 mg / mL to 50 mg / mL.For example, when a nucleic acid is used, the concentration may be 20 μg / mL to 30 mg / mL, 30 μg / mL to 30 mg / mL, 40 μg / mL to 30 mg / mL, 50 μg / mL to 30 mg / mL, 60 μg / mL to 30 mg / mL, 70 μg / mL to 30 mg / mL, 80 μg / mL to 30 mg / mL, 90 μg / mL to 30 mg / mL, 100 μg / mL to 30 mg / mL, 200 μg / mL to 30 mg / mL, 300 μg / mL to 30 mg / mL, 400 μg / mL ~ 30 mg / mL, 500 μg / mL ~ 30 mg / mL, 600 μg / mL ~ 30 mg / mL, 700 μg / mL ~ 30 mg / mL, 80 0 μg / mL to 30 mg / mL, 900 μg / mL to 30 mg / mL, 1 mg / mL to 30 mg / mL, 2 mg / mL to 30 mg / mL, 3 mg / mL to 30 mg / mL, 4 mg / mL to 30 mg / mL, 5 mg / mL to 30 mg / mL, 6 mg / mL to 30 mg / mL, 7 mg / mL to 30 mg / mL, 8 mg / m It can be used at concentrations of L to 30 mg / mL, 9 mg / mL to 30 mg / mL, 10 mg / mL to 30 mg / mL, 20 mg / mL to 30 mg / mL.

[0081] When the composition of the present disclosure is used as a reagent, the amount of protein or nucleic acid used can be adjusted appropriately depending on the purpose. For example, when a protein is used, the amount is preferably 1 μg to 500 mg, and when a nucleic acid is used, the amount is preferably 20 μg to 300 μg.

[0082] When administering a composition of the present disclosure to a living body, the dosage of the protein or nucleic acid varies depending on the patient's symptoms, administration route, body weight, age, etc., but is preferably, for example, 1 μg to 500 mg of protein and 20 μg to 300 μg of nucleic acid per day for an adult. Furthermore, when used in combination with other cancer treatment methods, the dosage of the composition of the present disclosure can be adjusted appropriately depending on the effectiveness of the other treatment, the patient's condition, etc. In one aspect, when administering a composition of the present disclosure in combination with other cancer treatment drugs, the dosage of each drug is preferably an effective amount, or 0.01 to 1 times the effective amount. Furthermore, when used in combination with a composition of the present disclosure, the radiation dose in other cancer treatment methods, such as radiation therapy, can be reduced to 0.1 to 0.8 times.

[0083] The administration route of the composition of the present disclosure is not particularly limited. For example, the composition of the present disclosure can be administered parenterally, for example, by injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.), or via transdermal, transmucosal, nasal, pulmonary, or oral routes, but is not limited thereto.

[0084] In one embodiment, the target organism to which the composition of the present disclosure is administered is not particularly limited, and examples of the target organism include, but are not limited to, humans, primates such as monkeys, and mammals such as dogs and cats.

[0085] (A protein having the PtdSer binding site of MFG-E8, and integrin α ｖ β ３／５ , α ８ β １ Methods using compositions containing proteins with no binding site or nucleic acids encoding the same)

[0086] In one aspect, the present disclosure provides a protein having the PtdSer binding site of MFG-E8, such as integrin α ｖ β ３／５ , α ８ β １ The present disclosure provides a method for using a composition comprising a protein having no binding site or a nucleic acid encoding the same. Typically, the method of the present disclosure provides a method for using a protein having the PtdSer binding site of MFG-E8, such as integrin α ｖ β ３／５ , α ８ β １ The composition described in "Composition containing a protein having no binding site or a nucleic acid encoding the same" is used, and the protein is a protein having the PtdSer binding site of MFG-E8, and ｖ β ３／５ , α ８ β １ The protein is described in (protein with no binding site).

[0087] In one aspect, the method of the present disclosure is a method for activating the phagocytic activity of phagocytes in vivo or in vitro. Accordingly, in one aspect, the present disclosure provides a protein having the PtdSer binding site of MFG-E8, which is integrin α ｖ β ３／５ , α ８ β １ In one aspect, the present disclosure provides a method for stimulating the phagocytosis of phagocytes, comprising contacting a protein having no binding site with PtdSer of an apoptotic cell. ｖ β ３／５ , α ８ β １ The present invention provides a method for activating the phagocytic activity of phagocytes, which comprises contacting a protein that does not have a binding site with PtdSer of an apoptotic cell, and which uses a composition containing the protein or a nucleic acid encoding the protein.

[0088] In one aspect, the method of the present disclosure comprises administering to a subject having a disease, symptom, or condition requiring activation of phagocytosis of phagocytes a protein having the PtdSer binding site of MFG-E8, which is integrin α ｖ β ３／５ , α ８ β １ The present invention provides a method for activating the phagocytic activity of phagocytes in a subject, the method comprising administering a composition comprising a protein having no binding site or a nucleic acid encoding the protein.

[0089] In one aspect, the method of the present disclosure comprises administering to a subject having a disease, symptom, or condition requiring activation of phagocytosis of phagocytes a protein having the PtdSer binding site of MFG-E8, which is integrin α ｖ β ３／５ , α ８ β １ A method of treating said disease, symptom or condition in said subject is provided, comprising administering a composition comprising a protein having no binding site or a nucleic acid encoding the same.

[0090] In one aspect, the method of the present disclosure is the use of the composition of the present disclosure in the treatment of a disease, symptom, or condition requiring activation of the phagocytic activity of phagocytes. Thus, in one aspect, the present disclosure provides a method for activating the phagocytic activity of phagocytes in a subject having a disease, symptom, or condition requiring activation of the phagocytic activity of phagocytes, comprising administering to the subject a protein having the PtdSer binding site of MFG-E8, wherein the protein is integrin α ｖ β ３／５ , α ８ β １ The use of a composition comprising a protein that does not have a binding site or a nucleic acid encoding the same is provided.

[0091] In one aspect, the present disclosure provides a method for treating a disease, symptom, or condition requiring activation of phagocytosis in a subject having the disease, symptom, or condition, the method comprising: ｖ β ３／５ , α ８ β １ The use of a composition comprising a protein that does not have a binding site or a nucleic acid encoding the same is provided.

[0092] In one aspect, the method of the present disclosure is a method for stimulating an immune response. Thus, in one aspect, the method of the present disclosure comprises administering to a subject having a disease, symptom, or condition requiring stimulation of an immune response, a protein having the PtdSer binding site of MFG-E8, which is integrin α ｖ β ３／５ , α ８ β １ A method of stimulating an immune response in a subject is provided, comprising administering a composition comprising a protein that does not have a binding site or a nucleic acid encoding the same.

[0093] In one aspect, the method of the present disclosure is the use of the composition of the present disclosure in stimulating an immune response. Thus, in one aspect, the present disclosure provides a protein having the PtdSer binding site of MFG-E8, which binds to integrin α, in a method of stimulating an immune response in a subject having a disease, symptom, or condition requiring such stimulation. ｖ β ３／５ , α８ β １ The use of a composition comprising a protein that does not have a binding site or a nucleic acid encoding the same is provided.

[0094] In one embodiment, the method of the present disclosure is a method of treating cancer. Accordingly, in one embodiment, the method of the present disclosure comprises administering to a subject in need of cancer treatment a protein having the PtdSer binding site of MFG-E8, such as integrin α ｖ β ３／５ , α ８ β １ A method of treating cancer in a subject is provided, comprising administering a composition comprising a protein that does not have a binding site or a nucleic acid encoding the same.

[0095] In one aspect, the method of the present disclosure is the use of a composition of the present disclosure in a method of treating cancer. Thus, in one aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a protein having the PtdSer binding site of MFG-E8, the protein comprising integrin α ｖ β ３／５ , α ８ β １ The use of a composition comprising a protein that does not have a binding site or a nucleic acid encoding the same is provided.

[0096] The composition used in the method of the present disclosure is a protein having the PtdSer binding site of MFG-E8, which is integrin α ｖ β ３／５ , α ８ β １ The composition, dosage, administration method, administration route, and administration target are described in the section "Composition containing a protein having no binding site or a nucleic acid encoding the same." In addition, other cancer therapeutic agents can be used in combination as described above.

[0097] Hereinafter, the present disclosure will be described in more detail with reference to examples, but these are merely illustrative and do not limit the present disclosure.

[0098] Example 1: Confirmation of the efficacy of cancer therapy in a mouse model Materials and Methods Experimental Method: Cell lines were generated by overexpressing a modified mouse MFG-E8 protein C1C2 (Figure 2B) lacking the EGF-like domain, or a modified MFG-E8 protein PStRAP (Figure 2C) in which a portion of RAP (Y38-L360) was fused to the C-terminus of the modified mouse MFG-E8 protein lacking the EGF-like domain, using the mouse fibrosarcoma cell line MCA205 (provided by Dr. Steven A. Rosenberg of the NIH), which constitutively expresses wild-type MFG-E8. The amino acid sequences of the Y38-L360 portions of mouse RAP protein (mRAP) and the corresponding Y35-L357 portions of human RAP protein (hRAP) are shown below.

[0099] An overview is shown on the left side of Figure 3. The MCA205 parent strain or mutant overexpressing strains were treated with mitomycin C to stop growth, and 5 × 10 dying cells were injected into the left ventral region of wild-type C57BL / 6 mice. ５ Seven days after administration, 5 × 10 ５ Each mouse was subcutaneously implanted into the right flank, and tumor growth was measured over time. The tumor growth curve shows the mean value, and the error bars show the standard error (SD). Statistical analysis was performed using a 2-way ANOVA followed by a Turkey's test as a post-hoc test, with p<0.05 considered significant.

[0100] Results: The results are shown in the graph on the right side of Figure 3. The complete rejection rates of transplanted tumor cells in the groups administered with the parental cell line, the C1C2-expressing cell line, and the PStRAP-expressing cell line as dead cells were 1 / 8, 2 / 8, and 3 / 8, respectively. The group administered with the PStRAP-expressing cell line showed a clearer delay in tumor growth than the groups administered with the parental cell line or the C1C2-expressing cell line. Although no statistically significant difference was detected in the tumor growth curves of the C1C2-expressing cell line compared to the parental cell line, the number of mice that showed complete rejection was higher.

[0101] Discussion: The results of this experiment show that the protein having the PtdSer binding site of MFG-E8 of the present disclosure binds to integrin α ｖ β ３／５ , α ８ β １These results indicate that proteins without binding sites enhance the immune response to cancer cells by controlling the phagocytic pathway of phagocytes, and have excellent cancer therapeutic effects. In particular, the modified MFG-E8 protein, which is fused with a portion of RAP (Y38-L360), showed extremely excellent cancer therapeutic effects.

[0102] Example 2: Characteristics of LNP Materials and Methods Commercially available ALC-0315 was used as LNP (lipid nanoparticles). mRNA expressing firefly luciferase (Fluc) was encapsulated in LNP and administered intravenously to wild-type mice. As a negative control group, HEPES, used as a solvent, was administered intravenously. Six and 24 hours after administration, luciferin was administered and the mice were euthanized, and each organ was collected. Luminescence from the luciferase protein was detected using an IVIS Imaging System. Three mice were used in each group.

[0103] Results: The results of detecting luminescence from the luciferase protein are shown in Figure 4. It was found that LNP was delivered primarily to the liver 6 hours after administration, and mRNA was translated and expressed as protein. 24 hours after administration, the amount of protein decreased. In the graph on the right side of Figure 4, the error bars indicate standard deviation (SD).

[0104] Example 3: Evaluation of liver dysfunction caused by LNP administration Materials and Methods LNP-mRNA prepared in the same manner as in Example 2 was intravenously administered to wild-type mice. As a negative control group, HEPES used as a solvent was intravenously administered. Blood samples were taken serially 6, 24, and 48 hours after administration, and blood ALT and AST concentrations were measured. Four mice were used in each group.

[0105] Results: The administered LNP accumulated in the liver 6 hours after administration, but no liver dysfunction was observed (Figure 5, error bars represent SD).

[0106] Example 4: Evaluation of the binding specificity of PStRAP to cell membrane lipids Materials and Methods: Hepatocytes were isolated from wild-type mice and transfected in vitro with PStRAP mRNA. PStRAP secreted into the supernatant after 6 hours of culture was used. The binding activity of PStRAP was evaluated using membrane lipid strips (P-6002, ECHELON BIOSCIENCES), in which 15 types of lipids were spotted on a hydrophobic membrane.

[0107] Results: The results are shown in Figure 6. PStRAP was found to bind specifically to phosphatidylserine (Ptd-serine).

[0108] Example 5: Measurement of serum PStRAP levels after LNP-PStRAP administration Materials and Methods mRNA expressing PStRAP (Flag-tagged at the C-terminus) was encapsulated in LNP (ALC-0315) and intravenously administered to wild-type mice. HEPES, used as a solvent, was administered intravenously to a negative control group. Blood samples were collected serially 6, 24, 72, and 120 hours after administration to obtain serum. The amount of PStRAP in the serum was semiquantified using an ELISA method targeting the Flag-tag. Four mice were used in each group. A significance test was performed on the semiquantitative results using the Student's t-test (*: p<0.05, ***: p<0.0001).

[0109] Results: The results are shown in Figure 7 (error bars indicate SD). It was found that serum PStRAP was detected at the highest level 6 hours after administration and decreased over time.

[0110] Example 6: Confirmation of the antitumor effect of combined use of LNP-PStRAP and anticancer drugs. Materials and Methods. An outline is shown in Figure 8. Wild-type mice were subcutaneously implanted with the mouse fibrosarcoma cell line MCA205 to create tumor-bearing mouse models. Seven days after tumor implantation, mRNA expressing PStRAP was encapsulated in LNP (ALC-0315 as described above) and administered intravenously. As a negative control, empty LNP (Empty) containing no mRNA was administered intravenously. Six hours after administration, the anticancer drug oxaliplatin (L-OHP) or PBS as a negative control was administered intraperitoneally (Figure 8). Subsequently, tumor diameter was measured, tumor volume was calculated, and tumor growth rate was measured. Six mice were used in each group. The measurement results were subjected to a 2-way ANOVA followed by a Tukey's test to determine significance.

[0111] Results: The results are shown in Figure 9 (error bars represent SD). A significant antitumor effect was observed in the oxaliplatin and LNP-PStRAP combination group. These results indicate that the modified MFG-E8 protein of the present disclosure or a nucleic acid encoding the same can be used in combination with other anticancer therapies to achieve particularly excellent anticancer effects.

[0112] The present disclosure has extremely high industrial value by providing new cancer therapeutic agents and cancer treatment methods, as well as related agents and methods for activating phagocytes, and agents and methods for activating immune responses.

Claims

1. A protein having a phosphatidylserine (PtdSer) binding site of milk fat globule-EGF factor 8 (MFG-E8), which binds to integrin α ｖ β ３／５ , α ８ β １ A protein with no binding site.

2. The protein according to claim 1, wherein the protein has the C1C2 domain of MFG-E8 or a part thereof as a PtdSer binding site.

3. The protein is integrin α ｖ β ３／５ , α ８ β １ The protein according to claim 1, which does not have the RGD motif (Arg-Gly-Asp integrin binding motif) of MFG-E8 or a part thereof as a binding site.

4. The protein is integrin α ｖ β ３／５ , α ８ β １ The protein according to claim 1, which does not have the epidermal growth factor like domain of MFG-E8 or a part thereof as a binding site.

5. The protein binds to phagocyte integrin α ｖ β ３／５ or integrin α ８ β １ The protein of claim 1, further comprising a site that binds to a receptor different from that of the 6. The protein according to claim 5, wherein the receptor of a phagocyte is a receptor that activates the phagocytosis of a phagocyte.

7. The protein according to claim 6, wherein the receptor that activates the phagocytosis of the phagocyte is CD91 / LRP1.

8. The protein according to claim 5, wherein the site that binds to a receptor on a phagocyte is a receptor-associated protein (RAP) or a part thereof.

9. A nucleic acid encoding a protein according to any one of claims 1 to 8.

10. A composition for activating the phagocytic activity of phagocytes, comprising the protein according to any one of claims 1 to 8 or a nucleic acid encoding the same.

11. A composition for stimulating an immune response, comprising a protein according to any one of claims 1 to 8 or a nucleic acid encoding the protein.

12. A composition for treating cancer, comprising the protein according to any one of claims 1 to 8 or a nucleic acid encoding the protein.

13. The composition for treating cancer according to claim 12, for use in combination with an anti-cancer therapy exhibiting a cytocidal effect.

14. The composition for treating cancer described in claim 12, for use in combination with an anti-cancer therapy using an immune checkpoint inhibitor.

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