JP-7856314-B2 - Macrophage activators

Inventors

• 乾 利夫

Assignees

• 再生ファーマ株式会社

Dates

Publication Date

20260511

Application Date

20211208

Priority Date

20201208

Claims (5)

1. Contains Gc protein without N-acetylgalactosamine added. The Gc protein is a macrophage activator having more than 90% sequence identity with the amino acid sequence shown in Sequence ID No. 1 .
2. The macrophage activator according to claim 1, wherein the Gc protein is a purified product from human, bovine, goat, or mouse serum or milk.
3. The macrophage activator according to claim 1 or 2, wherein the amino acid not to which N-acetylgalactosamine is attached is the threonine at position 418 or 420 in SEQ ID NO: 1.
4. A pharmaceutical composition comprising a macrophage activator according to any one of claims 1 to 3, for use in anticancer, anti-infective, anti-autoimmune disease, anti-autism, anti-inflammatory disease, anti-brain/neurodegenerative disease, skin improvement, or treatment of heart disease.
5. A step of contacting Gc protein with N-acetylgalactosaminidase, A method for producing a macrophage activator, comprising the step of contacting a Gc protein with neuraminidase before contacting it with N-acetylgalactosaminidase , The method for producing a macrophage activator, wherein the Gc protein has 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1.

Description

This invention relates to macrophage activators. Macrophages are a type of white blood cell found in all tissues of the body, including the skin, lungs, intestines, and brain. Macrophages are known to play an important role in both innate and adaptive immunity. In innate immunity, they engulf foreign substances that invade the body along with neutrophils and digest them using proteolytic enzymes and lipases stored within the macrophage. In adaptive immunity, they present molecules derived from engulfed foreign substances as antigens on the cell surface, activating helper T cells. Furthermore, macrophages are known to be involved in the formation of various pathological conditions such as inflammatory diseases, arteriosclerosis, obesity, and cancer. Gc protein is a protein present in plasma and is also called vitamin D-binding protein because it binds to vitamin D. Gc protein has the ability to activate macrophages, but conventionally, the glycan structure added to Gc protein after translation was considered important for macrophage activation. Gc protein is expressed with an O-linked glycan consisting of a trisaccharide of GalNAc (N-acetylgalactosamine) bound to sialic acid and galactose, attached to the threonine (Thr) at position 418 or 420. It was believed that macrophages could not be activated when sialic acid or galactose was bound to GalNAc, or when GalNAc was not bound to the threonine at position 418 or 420. Therefore, Patent Documents 1 and 2 disclose a method for removing sialic acid and galactose by acting on Gc protein with β-galactosidase or sialidase. Recombinant expression of Gc protein in specific cells can produce Gc protein in which only GalNAc is bound to the threonine (Thr) at position 418 or 420 (Patent Document 3). However, the types of cells that could be used were limited. Furthermore, the relationship between the glycosylation structure of Gc protein and its macrophage activation ability had not been sufficiently clarified. Special Publication No. 6-510908Special Publication No. 11-511962International Publication WO2019/117295 The results of the macrophage phagocytic activity test are shown.The results of the NO production test are shown.The results of the NO suppression test are shown.The results of the TNF-α suppression test are shown.The results of the M2 differentiation ability evaluation test are shown. <<Macrophage activator>> Gc protein is a protein found in mammalian body fluids such as plasma and milk, and is also called vitamin D-binding protein because it binds to vitamin D. In body fluids such as plasma and milk, Gc protein exists in a form in which an O-linked sugar chain consisting of GalNAc (N-acetylgalactosamine) bonded to threonine (Thr) at position 418 or 420 is attached. This form is not considered to have the ability to activate macrophages. On the other hand, a form in which only GalNAc is attached to threonine at position 418 or 420 is known to have the ability to activate macrophages and is called GcMAF (Gc protein-derived macrophage activating factor). In contrast, the macrophage activator of the present invention is characterized by containing Gc protein without the addition of N-acetylgalactosamine. <GC Protein> Gc protein is not particularly limited as long as it is derived from mammals; for example, it can be derived from humans, cattle, goats, or mice. However, human-derived Gc protein is preferred because it does not cause an immune response when administered to humans. Specific Gc proteins include polypeptides that exhibit 85% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1 of the sequence listing, or polypeptides consisting of amino acid sequences in which one or more amino acids are deleted, inserted, substituted, and/or added to the amino acid sequence shown in Sequence ID No. 1 of the sequence listing. The sequence identity with the amino acid sequence shown in Sequence ID No. 1 is preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more. In the amino acid sequence shown in Sequence ID No. 1, the number of deleted, inserted, substituted and/or added amino acids is preferably 68 or less, more preferably 45 or less, even more preferably 22 or less, even more preferably 9 or less, and particularly preferably 4, 3, or 2 or less. The position of the amino acid to which N-acetylgalactosamine is not attached is not particularly limited, but it is preferably a threonine in the amino acid sequence of the Gc protein, and preferably the threonine at position 418 or 420 in SEQ ID NO: 1. <Serum, milk> Gc protein can be obtained from bodily fluids such as the milk or serum of the aforementioned mammal. The milk or serum containing Gc protein can be used as is, or the Gc protein can be purified from the milk or serum before use. The serum is not particularly limited as long as it is prepared from blood collected from a mammal. The serum can be prepared by conv