Anti-n-glycolylneuraminic acid monoclonal antibody or antigen-binding fragment thereof

Abstract

The present invention provides an anti-N-glycolylneuraminic acid monoclonal antibody or an antigen-binding fragment thereof having: a heavy chain variable region that includes HCDR1, HCDR2, and HCDR3; and a light chain variable region that includes LCDR1, LCDR2, and LCDR3. The amino acid sequences constituting the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from sets 1-15 of specific CDR sequences, or are selected, in the sets 1-15 of the specific CDR sequences, from sets of amino acid sequences in which one or two amino acids are substituted, deleted, and / or added in at least one amino acid sequence, and the amino acid sequences bind to N-glycolylneuraminic acid.

Description

Anti-N-glycolylneuraminic acid monoclonal antibody or antigen-binding fragment thereof

[0001] The present invention relates to an anti-N-glycolylneuraminic acid monoclonal antibody or an antigen-binding fragment thereof.

[0002] Sialic acids are generally located at the terminal (non-reducing end) of the sugar chains that cover the cell surface, and in mammals, two molecular species exist: N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Neu5Gc is biosynthesized by CMP-Neu5Ac hydroxylase (CMah) from Neu5Ac as a precursor at the glycosyl donor stage (Fig. 1).

[0003] However, humans lack Cmah, which is responsible for the biosynthesis of Neu5Gc, and there is no alternative biosynthetic pathway for Neu5Gc. Therefore, humans cannot biosynthesize Neu5Gc. On the other hand, sialic acid transporters present in the cell membrane and Golgi apparatus membrane, and sialyltransferases responsible for adding sialic acid to the terminals of glycans in the Golgi apparatus, do not distinguish between Neu5Ac and Neu5Gc, transporting both in the same way and adding them to glycans. Therefore, humans add Neu5Gc ingested from their diet over many years to their own glycan terminals and express it in tissues. For example, Neu5Gc has been detected in various human cancer tissues (Non-Patent Document 1), and its presence has also been reported in normal tissues, albeit in smaller amounts than in cancer tissues (Non-Patent Document 2).

[0004] Neu5Gc expressed in human tissues is a "xenoantigen" that combines the properties of a "xenoantigen" that cannot be biosynthesized by the body itself and an "autoantigen" that is expressed in self-tissues. Therefore, the human immune system can produce antibodies against Neu5Gc upon exposure to self-tissue expression of Neu5Gc or bacteria that express Neu5Gc. In relation to the production of such antibodies, it has been proposed that the immune response between Neu5Gc expressed in self-tissues and anti-Neu5Gc antibodies can cause systemic chronic inflammation, which may contribute to the exacerbation of various diseases (Non-Patent Documents 2, 3, etc.).

[0005] Malykh YN, Schauer R, Shaw L. N-Glycolylneuraminic acid in human tumours. Biochimie. 2001;83(7):623- 34.Tangvoranuntakul P, Gagneux P, Diaz S, Bardor M, Varki N, Varki A, Muchmore E. Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid. Proc Natl Acad Sci US A. 2003;100(21):12045- 50.Taylor RE, Gregg CJ, Padler-Karavani V, et al. Novel mechanism for the generation of human xeno-autoantibodies against the nonhuman sialic acid N-glycolylneuraminic acid. J Exp Med. 2010;207(8):1637-46.

[0006] Anti-Neu5Gc monoclonal antibodies can be used in the detection of Neu5Gc in cells or tissues, or in research into the immune response between Neu5Gc and anti-Neu5Gc antibodies and diseases that may result from them, etc. However, at the time of filing this application, there are no widely available anti-Neu5Gc monoclonal antibodies.

[0007] A primary object of the present invention is to provide a novel anti-Neu5Gc monoclonal antibody.

[0008] [1] One aspect of the present invention provides an anti-N-glycolylneuraminic acid monoclonal antibody or an antigen-binding fragment thereof, which binds to N-glycolylneuraminic acid and has a heavy chain variable region including heavy chain complementarity-determining region 1 (HCDR1), heavy chain complementarity-determining region 2 (HCDR2), and heavy chain complementarity-determining region 3 (HCDR3), and a light chain variable region including light chain complementarity-determining region 1 (LCDR1), light chain complementarity-determining region 2 (LCDR2), and light chain complementarity-determining region 3 (LCDR3), wherein the amino acid sequences constituting the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from Sets 1 to 15 of CDR sequences listed in Table 1 below, or from a set of amino acid sequences in which at least one amino acid sequence in Sets 1 to 15 of CDR sequences has one or two amino acid substitutions, deletions, and / or additions. [2] In the monoclonal antibody or antigen-binding fragment thereof according to [1] above, the heavy chain variable region and the light chain variable region comprise: a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 36; a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 37; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 38; and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 39; a heavy chain variable region comprising an amino acid sequence having 100% to 100% identity to the amino acid sequence of SEQ ID NO: 41 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 42 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 43; a heavy chain variable region comprising an amino acid sequence having 100% to 100% identity to the amino acid sequence of SEQ ID NO: 44 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 45;a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 47; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 48 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 49; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 86 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 87; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 88 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 89; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 90, and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:91; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:94 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:95; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:96 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:97 a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:98 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:99; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:100 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:101;Alternatively, the present invention may comprise a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 102 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 93. [3] The monoclonal antibody or antigen-binding fragment thereof according to [1] or [2] above may be linked to N-glycolylneuraminic acid located at the non-reducing terminal of a sugar chain. [4] In the monoclonal antibody or antigen-binding fragment thereof according to [3] above, the sugar chain may comprise two Neu5Gc residues, α2,3-linked and α2,8-linked, located in this order from opposite sides of the non-reducing terminal. [5] According to another aspect of the present invention, there is provided a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [4] above. [6] According to another aspect of the present invention, there is provided a vector or plasmid comprising the nucleic acid molecule according to [5] above. [7] According to another aspect of the present invention, there is provided a host cell transformed with the vector or plasmid according to [6] above. [8] According to another aspect of the present invention, there is provided a hybridoma producing the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [4] above. [9] According to another aspect of the present invention, there is provided a composition for immunohistochemical staining comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [4] above.

[10] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [4] above.

[11] According to another aspect of the present invention, there is provided a method for producing a chronic inflammation model mouse using N-glycolylneuraminic acid as an antigen, the method comprising administering peracetylated N-glycolylmannosamine to a Cmah-knockout mouse to express a sugar chain containing N-glycolylneuraminic acid, and administering the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [4] above to the mouse to induce an immune response with the sugar chain.

[0009] According to the present invention, a novel anti-Neu5Gc monoclonal antibody is provided.

[0010] 1 is a schematic diagram illustrating the biosynthesis of Neu5Gc from Neu5Ac. Specifically, CMP-Neu5Ac is hydroxylated by Cmah to produce CMP-Neu5Gc. During this reaction, R in the diagram corresponds to CMP. This is a schematic diagram illustrating the structure of a sugar chain containing Neu5Gc. This is a schematic diagram illustrating the structure of the gene sequence ST8Sia6-GFP, in which the GFP gene is linked to the rear of the ST8Sia6 gene via an IRES sequence. This is a schematic diagram illustrating the detection of an anti-Neu5Gc antibody using cells. The anti-Neu5Gc antibody does not bind to cells into which the Cmah gene has not been introduced (left), but does bind to cells into which the Cmah gene has been introduced (right). This is a schematic diagram illustrating the detection of an anti-Neu5Gc antibody using flow cytometry. This is a schematic diagram illustrating a bypass pathway for Neu5Gc biosynthesis by administration of PA-ManNGc. 1 is a diagram showing the reaction results between an anti-Neu5Gc antibody in the culture supernatant of hybridoma cells established using flow cytometry and cells expressing Neu5Gc. 2 is a diagram showing the reaction results between an anti-Neu5Gc antibody in the culture supernatant of hybridoma cells established using flow cytometry and cells not expressing Neu5Gc. 3 is a diagram showing the results of cell staining with an anti-Neu5Gc monoclonal antibody. 4 is an observation image showing the results of mouse tissue staining with an anti-Neu5Gc monoclonal antibody.

[0011] Preferred embodiments of the present invention will be described below, but the present invention is not limited to these embodiments. Furthermore, unless inappropriate in the context, the embodiments can be combined as appropriate. Furthermore, in this specification, the term "to" indicating a range of values ​​includes the upper and lower limits.

[0012] A. Antibodies Antibodies are proteins that recognize and specifically bind to specific substances (antigens), and are also called immunoglobulins (Ig). Antibodies typically have a basic Y-shaped four-chain structure consisting of two heavy chains and two light chains linked by disulfide bonds. There are five types of heavy chains: γ chain, μ chain, α chain, δ chain, and ε chain. There are two types of light chains: λ chain and κ chain. Antibodies are classified into five isotypes, IgG, IgM, IgA, IgD, and IgE, depending on the type of heavy chain.

[0013] Each of the two heavy chains contains a heavy chain constant (CH) region and a heavy chain variable (VH) region. Each of the two light chains contains a light chain constant (CL) region and a light chain variable (VL) region. The heavy and light chain variable regions form an antigen-binding site.

[0014] The heavy chain variable (VH) region contains three hypervariable regions (HCDR1, HCDR2, and HCDR3), also called complementarity-determining regions (CDRs), that determine antigen specificity, and four highly conserved regions (HFR1, HFR2, HFR3, and HFR4), also called framework regions (FRs). These regions are arranged from the amino terminus to the carboxy terminus in the following order: HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, and HFR4.

[0015] The light chain variable (VL) region contains three complementarity determining regions (LCDR1, LCDR2, and LCDR3) and four framework regions (LFR1, LFR2, LFR3, and LFR4), arranged from the amino terminus to the carboxy terminus in the following order: LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, and LFR4.

[0016] The anti-Neu5Gc monoclonal antibodies according to embodiments of the present invention bind to Neu5Gc, preferably Neu5Gc contained in a glycan (in other words, a glycan containing Neu5Gc), for example, Neu5Gc located at the terminal (non-reducing end) of a glycan (in other words, a glycan containing Neu5Gc at its terminal). In one embodiment, the epitope of the anti-Neu5Gc monoclonal antibody according to embodiments of the present invention includes Neu5Gc contained in a glycan, for example, one or more Neu5Gc located at the terminal of a glycan. The anti-Neu5Gc monoclonal antibodies according to embodiments of the present invention specifically bind to Neu5Gc and typically do not bind to Neu5Ac (e.g., Neu5Ac located at the terminal of a glycan). For example, when an anti-Neu5Gc monoclonal antibody according to an embodiment of the present invention is subjected to immunohistochemical staining of cells expressing sugar chains that contain other sialic acids, such as Neu5Ac, but do not contain Neu5Gc, no significant staining occurs.

[0017] In the sugar chain to which the anti-Neu5Gc monoclonal antibody binds, one or more Neu5Gc units are preferably attached to the non-reducing end of a sugar such as galactose (Gal). The bond type of Neu5Gc typically includes an α2,3 bond, an α2,6 bond, an α2,8 bond, etc. The glycan to which the anti-Neu5Gc monoclonal antibody binds is preferably at least one selected from the group consisting of a glycan containing one α2,3-linked Neu5Gc at the non-reducing end (hereinafter sometimes referred to as an "α2,3 monosialo glycan"), a glycan containing one α2,6-linked Neu5Gc at the non-reducing end (hereinafter sometimes referred to as an "α2,6 monosialo glycan"), and a glycan containing two sialic acids, α2,3-linked and α2,8-linked, located in this order from the opposite side of the non-reducing end, at least one of which is Neu5Gc (hereinafter sometimes referred to as an "α2,8 disialo glycan"), more preferably an α2,3 monosialo glycan and / or an α2,8 disialo glycan (see Figure 2; however, the sugar to which the sialic acid is bound is not limited to Gal).

[0018] An anti-Neu5Gc monoclonal antibody according to one embodiment of the present invention has a heavy chain variable region including HCDR1, HCDR2, and HCDR3, and a light chain variable region including LCDR1, LCDR2, and LCDR3, and the amino acid sequences constituting the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from CDR sequence sets 1 to 15 shown in Table 1.

[0019]

[0020] An anti-Neu5Gc monoclonal antibody according to one embodiment of the present invention has a heavy chain variable region including HCDR1, HCDR2, and HCDR3, and a light chain variable region including LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from a set of amino acid sequences in which one or two amino acids are substituted, deleted, and / or added to at least one amino acid sequence in CDR sequence sets 1 to 15 shown in Table 1. Specifically, the set of CDR sequences in the anti-Neu5Gc monoclonal antibody is a set of CDR sequences in which one or more, preferably one to four, more preferably one to three, for example one or two amino acid substitutions, deletions, and / or additions are selected from the six amino acid sequences (CDR sequences) in each of CDR sequence sets 1 to 15 shown in Table 1.

[0021] In this specification, amino acid substitutions are preferably conservative substitutions. Conservative substitution means replacing a certain amino acid with another amino acid having similar chemical properties and / or structure. Examples of substitutions between amino acids having similar chemical properties include substitutions between amino acids having similar polarity (hydrophobic or hydrophilic) and / or charge (basic, acidic, neutral). Examples of substitutions between amino acids having similar structures include substitutions between aromatic amino acids or between aliphatic amino acids. Those skilled in the art can appropriately select what type of substitution constitutes a conservative substitution depending on the amino acid to be substituted.

[0022] Specific examples of conservative substitutions include substitution of Ala with Ser or Thr, substitution of Arg with Gln, His, or Lys, substitution of Asn with Glu, Gln, Lys, His, or Asp, substitution of Asp with Asn, Glu, or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitution of Glu with Asn, Gln, Lys, or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg, or Tyr, substitution of Ile with Leu, M substitution of Lys with Asn, Glu, Gln, His or Arg; substitution of Met with Ile, Leu, Val or Phe; substitution of Phe with Trp, Tyr, Met, Ile or Leu; substitution of Ser with Thr or Ala; substitution of Thr with Ser or Ala; substitution of Trp with Phe or Tyr; substitution of Tyr with His, Phe or Trp; and substitution of Val with Met, Ile or Leu.

[0023] Anti-Neu5Gc monoclonal antibodies according to preferred embodiments of the present invention include the following antibodies: Antibody 1 having a heavy chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 36, and a light chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 37; Antibody 2 having a heavy chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 38, and a light chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 39; Antibody 3 having a heavy chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 40, and a light chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 41; Antibody 4 having a heavy chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 42, and a light chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 43;Antibody 5 having a heavy chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 44, and a light chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 45; Antibody 6 having a heavy chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 46, and a light chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 47; Antibody 7 having a heavy chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 48, and a light chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 49; Antibody 8 having a heavy chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 86, and a light chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 87;Antibody 9 having a heavy chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 88, and a light chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 89; Antibody 10 having a heavy chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 90, and a light chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 91; Antibody 11 having a heavy chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 94, and a light chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 95; Antibody 12 having a heavy chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 96, and a light chain variable region comprising an amino acid sequence that is 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identical to the amino acid sequence of SEQ ID NO: 97;antibody 13 having a heavy chain variable region comprising an amino acid sequence having 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identity to the amino acid sequence of SEQ ID NO: 98, and a light chain variable region comprising an amino acid sequence having 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identity to the amino acid sequence of SEQ ID NO: 99; antibody 14 having a heavy chain variable region comprising an amino acid sequence having 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identity to the amino acid sequence of SEQ ID NO: 100, and a light chain variable region comprising an amino acid sequence having 70% or more, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identity to the amino acid sequence of SEQ ID NO: 101; Antibody 15 having a heavy chain variable region comprising an amino acid sequence having 70% or more, for example 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identity to the amino acid sequence of SEQ ID NO: 102, and a light chain variable region comprising an amino acid sequence having 70% or more, for example 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identity to the amino acid sequence of SEQ ID NO: 93; and Antibody 16 having a heavy chain variable region comprising an amino acid sequence having 70% or more, for example 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identity to the amino acid sequence of SEQ ID NO: 103, and a light chain variable region comprising an amino acid sequence having 70% or more, for example 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, or 98% or more identity to the amino acid sequence of SEQ ID NO: 37.

[0024] Preferably, in each of the above antibodies 1 to 16, the amino acid sequences constituting HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the above sets of CDR sequences 1 to 15, or are selected from a set of amino acid sequences in which at least one amino acid sequence in the above sets of CDR sequences 1 to 15 has one or two amino acid substitutions, deletions, and / or additions.

[0025] The identity of the amino acid sequences is the ratio of identical amino acids appearing at corresponding positions when two amino acid sequences are aligned, with gaps introduced as necessary. The "identity" of two amino acid sequences can be determined, for example, using the BLAST (Basic Local Alignment Search Tool) program (Altschul et al., J. Mol. Biol., (1990), 215(3):403-10).

[0026] Specific examples of anti-Neu5Gc monoclonal antibodies according to preferred embodiments of the present invention include the following antibodies: an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 103 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 37; an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39; an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 40 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 41; an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 43; an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 44 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 45; an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 47; an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 48 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 49; an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 86 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 87; an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 88 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 89; an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 90 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 91; an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 95; an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 96 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 97; an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 98 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 99; an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 100 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 101; and an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 102 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 93.

[0027] The class of the anti-Neu5Gc monoclonal antibody is not limited. Specifically, the class of the anti-Neu5Gc monoclonal antibody may be IgG, IgM, IgA, IgD, or IgE, and is preferably IgG or IgM.

[0028] In one embodiment, the anti-Neu5Gc monoclonal antibody is a murine antibody.

[0029] The anti-Neu5Gc monoclonal antibody can be produced by any suitable method. An example of a method for producing an anti-Neu5Gc monoclonal antibody will be described below.

[0030] [Immunization] Monoclonal antibodies can generally be obtained by a method including immunizing a non-human mammal with an immunogen to obtain antibody-producing cells, fusing the antibody-producing cells with myeloma cells to obtain hybridoma cells, and screening for hybridoma cells that produce the desired antibody.

[0031] However, as mentioned above, non-human mammals express Cmah and are capable of biosynthesizing Neu5Gc, and therefore do not produce anti-Neu5Gc antibodies. Therefore, immunization must be performed on non-human mammals in which Cmah has been knocked out (KO), preferably Cmah-KO mice. Cmah-KO non-human mammals can be created, for example, using genome editing techniques.

[0032] Furthermore, because glycans are widely present in the body, their immunogenicity is generally low, making it extremely difficult to generate antibodies against glycans as antigens. Humans can produce anti-Neu5Gc antibodies when exposed to bacteria expressing glycans containing Neu5Gc. Therefore, it is conceivable to use such bacteria as immunogens in Cmah-KO non-human mammals. However, when such bacterial cells are used as immunogens, there is a possibility that unintended immune responses other than those against the Neu5Gc antigen may occur.

[0033] Therefore, in the method for producing an anti-Neu5Gc monoclonal antibody, it is preferable to immunize Cmah-KO mice using CHO cells that overexpress Neu5Gc as an immunogen. CHO cells are derived from Chinese hamsters, which are genetically similar to mice but belong to the same order Rodentia, and therefore can minimize undesired immune responses. Furthermore, genetic modification is easy, and they grow well and are easy to culture. Subclone strains such as CHO-K1 cells and CHO-S cells may also be used as CHO cells.

[0034] CHO cells that overexpress Neu5Gc can be obtained by introducing an expression vector incorporating the Cmah gene into CHO cells. CMP-Neu5Gc is produced in large quantities by the Cmah expressed by the introduction of the Cmah gene, and as a result of addition to sugar chains by sialyltransferase, sialo-glycans containing Neu5Gc at the sugar chain terminus can be overexpressed.

[0035] In one embodiment, the CHO cells are transfected with the ST8Sia6 gene in addition to the Cmah gene. ST8Sia6 is an α2,8-sialyltransferase that adds sialic acid (e.g., Neu5Gc) via an α2,8 linkage to glycans containing α2,3-linked sialic acid at the non-reducing end (specifically, having a Siaα2-3Gal structure) in glycoproteins, glycolipids, and the like. While CHO cells naturally express α2,3 monosialoglycans abundantly, CHO cells transfected with the Cmah gene and the ST8Sia6 gene are able to express α2,8 disialoglycans abundantly. Therefore, by using CHO cells transfected with the Cmah gene and the ST8Sia6 gene as an immunogen, antibodies against α2,8 disialoglycans can be suitably induced, which may be useful for generating a chronic inflammation mouse model, as described below. In another embodiment, the CHO cells are transfected with the ST6Gal1 gene in addition to the Cmah gene. ST6Gal1 is an α2,6-sialyltransferase that adds sialic acid to the galactose terminus via an α2,6 linkage in glycoproteins, glycolipids, etc. CHO cells transfected with the Cmah gene and the ST6Gal1 gene can abundantly express α2,6 monosialo-glycans, and therefore can be used as an immunogen to effectively induce antibodies against α2,6 monosialo-glycans.

[0036] Cmah-KO mice can be immunized by administering the above-mentioned CHO cells overexpressing Neu5Gc together with an adjuvant, carrier, diluent, etc., as necessary. The dose of the immunogen is, for example, 1.0 × 10 ４ cells / mouse ~1.0 x 10 ７ The immunogen is administered intravenously, subcutaneously, or intraperitoneally. The interval and number of immunizations are not particularly limited and can be determined depending on factors such as the antibody titer in the serum. The interval between immunizations is, for example, several days to several weeks, preferably 1 to 2 weeks. The number of immunizations is, for example, 2 to 10 times, preferably 3 to 8 times.

[0037] The antibody titer in serum can be measured by an enzyme immunoassay (EIA) such as an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), etc. In measuring the antibody titer, cells expressing a sugar chain containing Neu5Gc, for example, human-derived cells into which the Cmah gene has been introduced, can be used as an antigen to capture the antibody.

[0038] In one embodiment, the anti-Neu5Gc antibody titer can be measured using a human-derived cell line (e.g., U937 cells) and a human-derived cell line (e.g., U937-Cmah-ST8Sia6 cells) into which the gene sequence ST8Sia6-GFP (see Figure 3 ), in which the GFP gene is linked to the Cmah gene and the ST8Sia6 gene via an IRES sequence (Internal Ribosome Entry Sites), has been introduced. The genes before and after the IRES sequence are transcribed as a single mRNA and then translated individually. Therefore, since the expression level of the ST8Sia6 gene can be correlated with the expression level of the GFP gene, the fluorescence intensity of GFP can be regarded as the expression level of ST8Sia6 (and, consequently, the expression level of α2,8 disialoglycans). Furthermore, human-derived cells are Neu5Gc negative and do not bind to anti-Neu5Gc antibodies. On the other hand, human cells transfected with the Cmah gene are Neu5Gc-positive and can be bound by anti-Neu5Gc antibodies (see Figure 4). These characteristics facilitate the detection of anti-Neu5Gc antibodies that bind to sialoglycans of various binding modes by flow cytometry. For example, in a plot where the horizontal axis represents the fluorescence intensity of GFP (which may correspond to the expression level of ST8Sia6) and the vertical axis represents the fluorescence intensity of a labeled anti-immunoglobulin antibody (PE-labeled in the illustrated example, which may correspond to the expression level of anti-Neu5Gc antibody), a shift of the plot to the upper right indicates the production of a large number of antibodies that bind to α2,8-disialoglycans (see Figure 5). A shift of the plot to the lower right indicates the production of a large number of antibodies that bind to sialoglycans whose expression decreases with increased expression of α2,8-disialoglycans, such as α2,3-monosialoglycans. In addition, because the human-derived cells are derived from a different animal species than the CHO cells used as the immunogen, cross-reactions with anti-CHO cell antibodies are suppressed. Furthermore, by using whole cells, it is possible to detect antibodies that recognize a wide variety of Neu5Gc-containing epitopes expressed on the cell surface. Therefore, this method is considered suitable for antibody screening in addition to serum antibody titers.In another embodiment, the anti-Neu5Gc antibody titer can be measured using a human-derived cell line (e.g., U937 cells) or a human-derived cell line (e.g., U937-Cmah-ST6Gal1 cells) into which a gene sequence ST6Gal1-GFP, in which the GFP gene is linked to the rear of the Cmah gene and the ST6Gal1 gene via an IRES sequence, has been introduced. In cells into which the above genes have been introduced, the expression levels of ST6Gal1 and GFP can be correlated, and therefore the fluorescence intensity of GFP can be regarded as the expression level of ST6Gal1 (and consequently, the expression level of α2,6-monosialoglycans). Therefore, for example, by analyzing the expression levels of anti-Neu5Gc antibodies and ST6Gal1 using flow cytometry in the same manner as described above, it may be possible to easily detect anti-Neu5Gc antibodies that bind to Neu5Gc of each binding type (e.g., anti-Neu5Gc antibodies that bind to α2,3-linked or α2,6-linked Neu5Gc).

[0039] [Preparation of Hybridoma Cells] Antibody-producing cells are collected from Cmah-KO mice whose serum has been confirmed to have a sufficient anti-Neu5Gc antibody titer, and then fused with myeloma cells. Examples of antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells. Of these, spleen cells and lymph node cells are preferred.

[0040] As myeloma cells to be fused with antibody-producing cells, myeloma cells in which Cmah has been knocked out are typically used. Commonly available myeloma cells derived from non-human mammals such as mice can synthesize Neu5Gc. Therefore, hybridoma cells (especially hybridoma cells producing cytotoxic anti-Neu5Gc antibodies) produced using such myeloma cells may be attacked by the anti-Neu5Gc antibodies they produce. In contrast, hybridoma cells produced using myeloma cells in which Cmah has been knocked out have the advantage of not experiencing the problem of self-attack.

[0041] The myeloma cells used preferably have drug selectivity, for example, the property of being unable to survive in HAT selection medium containing hypoxanthine, aminopterin, and thymidine when unfused with antibody-producing cells, but being able to survive when fused with antibody-producing cells. Specific examples of preferred myeloma cells include mouse myeloma cell lines in which Cmah has been knocked out, such as P3-x63-Ag8U.1, SP2 / O-Ag14, PAI, P3U1, NSI / 1-Ag4-1, and NSO / 1.

[0042] Cell fusion between antibody-producing cells and myeloma cells can be carried out, for example, by mixing the antibody-producing cells and myeloma cells in a cell number ratio of approximately 1:1 to 10:1 in a serum-free medium and reacting them in the presence of a fusion promoter. Polyethylene glycol with an average molecular weight of 1,000 Da to 4,000 Da is preferably used as the fusion promoter. Alternatively, antibody-producing cells and myeloma cells can be fused by, for example, electroporation.

[0043] After the cell fusion treatment, the cells are cultured in a selective medium such as HAT medium, and the grown cells can be obtained as hybridoma cells.

[0044] [Screening of Hybridoma Cells] Screening of hybridoma cells producing a target antibody can be performed, for example, by collecting a portion of the culture supernatant of the hybridoma cells and evaluating the specificity, affinity, etc. using enzyme immunoassay, radioimmunoassay, etc. For example, similar to the measurement of the anti-Neu5Gc antibody titer in serum, hybridoma cells that produce a monoclonal antibody with high affinity for Neu5Gc and have stable cell growth can be screened by flow cytometry using a human-derived cell line (e.g., U937 cells) and a human-derived cell line into which the Cmah gene has been introduced (e.g., a human-derived cell line into which the Cmah gene and the ST8Sia6-GFP gene sequence have been introduced (e.g., U937-Cmah-ST8Sia6 cells), a human-derived cell line into which the Cmah gene and the ST6Gal1-GFP gene sequence have been introduced (e.g., U937-Cmah-ST6Gal1 cells), etc.), and then isolated by limiting dilution, etc.

[0045] [Obtaining Monoclonal Antibodies] Anti-Neu5Gc monoclonal antibodies can be obtained from isolated hybridoma cells using conventional cell culture methods, ascites formation methods, etc. In the cell culture method, the cells are cultured under conventional culture conditions (e.g., 37°C, 5% CO) using an animal cell culture medium. ２ Hybridoma cells are cultured at a concentration of 1000 mM (1000 mM NaCl) and antibodies are isolated from the culture supernatant. In the ascites formation method, hybridoma cells are administered intraperitoneally to an animal of the same species (e.g., a mouse) as the mammal from which the myeloma cells were derived, and after mass proliferation of the hybridoma cells, ascites is collected and antibodies are isolated from the ascites. If antibody purification is required, it can be performed by appropriately selecting or combining known methods such as ammonium sulfate precipitation, ion exchange chromatography, gel filtration chromatography, and affinity chromatography.

[0046] [Cloning of Monoclonal Antibodies] Cloning of monoclonal antibodies can be performed according to standard methods. For example, cloning of monoclonal antibodies can be performed by culturing isolated hybridoma cells to extract mRNA, synthesizing cDNA encoding the light chain variable region and heavy chain variable region from the mRNA by reverse transcription, and amplifying the cDNA by 5' RACE-PCR and incorporating it into a cloning vector or plasmid. By analyzing the nucleotide sequence of the cDNA, the nucleotide sequence and amino acid sequence encoding the light chain variable region and heavy chain variable region of the anti-Neu5Gc monoclonal antibody can be identified.

[0047] The antibody can also be produced by genetic engineering using a nucleotide sequence or amino acid sequence encoding the light chain variable region and / or heavy chain variable region of the anti-Neu5Gc monoclonal antibody (e.g., using a nucleic acid molecule encoding them). Alternatively, a vector, plasmid, or the like for expressing each component of a desired antibody can be produced from genetic information based on the nucleotide sequence or amino acid sequence, and then introduced into host cells (mammalian cells, insect cells, plant cells, yeast cells, microbial cells, etc.) to produce the antibody.

[0048] [Modified Antibody] The anti-Neu5Gc monoclonal antibody may be modified as needed. Examples of the modified anti-Neu5Gc monoclonal antibody include chimeric antibodies, humanized antibodies, and functionally modified antibodies.

[0049] A chimeric antibody can be an antibody in which the light chain, the heavy chain, or both are composed of a variable region derived from a non-human mammal and a constant region derived from a human. Examples of chimeric antibodies include chimeric antibodies in which the variable region of a mouse antibody is joined to the constant region of a human antibody. For example, an expression vector for a chimeric antibody can be prepared by excising the variable region from the gene for a mouse anti-Neu5Gc monoclonal antibody and excising and ligating the constant region from the gene for a human antibody. By transforming host cells with such an expression vector, transformed cells that produce chimeric antibodies can be obtained, and the desired chimeric antibody can be obtained from the culture supernatant by culturing the transformed cells.

[0050] A humanized antibody can be an antibody consisting of a variable region composed of the complementarity-determining regions of a non-human mammal antibody and a framework region derived from a human antibody, and a constant region derived from a human antibody. Humanized antibodies include antibodies consisting of a variable region composed of the complementarity-determining regions of a mouse antibody and a framework region derived from a human antibody, and a constant region derived from a human antibody. Humanized antibodies can be obtained by using so-called CDR grafting technology to graft the complementarity-determining regions of a non-human mammal antibody onto a human antibody so that they are linked between the framework regions of the human antibody.

[0051] A functionally modified antibody refers to an antibody in which a function other than the antigen-binding function has been modified, such as a cell-killing function, a complement-activating function, a blood retention function, etc. Functionally modified antibodies can be produced, for example, by modifying the amino acid sequence, sugar chain, etc. of the constant region.

[0052] B. Antigen-binding Fragments The antigen-binding fragments according to embodiments of the present invention are fragments of the anti-Neu5Gc monoclonal antibodies described in Section A, and typically have the same antigen-binding ability as the monoclonal antibodies. Specifically, the antigen-binding fragments bind to Neu5Gc, preferably Neu5Gc contained in glycans (in other words, glycans containing Neu5Gc), for example, Neu5Gc located at the terminal (non-reducing end) of a glycan (in other words, glycans containing Neu5Gc at the terminal). The antigen-binding fragments typically do not bind to Neu5Ac (more specifically, Neu5Ac located at the terminal of a glycan).

[0053] Antigen-binding fragments include, for example, Fab, Fab', and F(ab'). ２ , double-chain fragments such as diabodies (dsFv) and scFv, sc(Fv) ２ and the like.

[0054] The antigen-binding fragment can be obtained by cleaving the anti-Neu5Gc monoclonal antibody with a protease appropriate for the purpose. Specifically, Fab can be obtained by treating the antibody with papain. F(ab') ２ can be obtained by treating an antibody with pepsin. Fab' is F(ab') ２ scFv, sc(Fv) ２ and dsFv can be obtained by obtaining cDNAs encoding the heavy chain variable region and light chain variable region of an antibody, respectively, and expressing them in host cells by genetic engineering techniques.

[0055] C. Use of the antibody or antigen-binding fragment [Immunohistochemical staining composition] The anti-Neu5Gc monoclonal antibody and its antigen-binding fragment described in Section A bind to Neu5Gc, preferably Neu5Gc contained in glycans, and can therefore be used for the detection by immunohistochemical staining of cells or tissues expressing glycans containing Neu5Gc. Therefore, immunohistochemical staining compositions containing the above-mentioned anti-Neu5Gc monoclonal antibody and its antigen-binding fragment are also included in the present invention.

[0056] The immunohistochemical staining may be a direct method using a labeled anti-Neu5Gc monoclonal antibody or its antigen-binding fragment as the primary antibody, or an indirect method (indirect staining) using an unlabeled anti-Neu5Gc monoclonal antibody or its antigen-binding fragment as the primary antibody and a labeled antibody that specifically binds to the primary antibody as the secondary antibody.

[0057] As described above, humans cannot biosynthesize Neu5Gc, but express glycans containing Neu5Gc taken in from the diet on their cell surfaces. Human cells or tissues (e.g., cancer cells or tissues, vascular endothelial cells, etc.) that actively take up substances via the salvage pathway express higher amounts of glycans containing Neu5Gc than normal tissues. Therefore, the immunohistochemical staining composition can be useful for immunohistochemical staining of human cancer cells or tissues, vascular endothelial cells, etc.

[0058] [Pharmaceutical Composition] The anti-Neu5Gc monoclonal antibody or its antigen-binding fragment can be used for the treatment, prevention, research, etc. of diseases associated with human cells or human tissues that express Neu5Gc-containing sugar chains in greater amounts than normal cells or normal tissues. Therefore, pharmaceutical compositions containing the anti-Neu5Gc monoclonal antibody and its antigen-binding fragment are also included in the present invention.

[0059] In the pharmaceutical composition, the anti-Neu5Gc monoclonal antibody and its antigen-binding fragment may be in any suitable form, such as the form of the anti-Neu5Gc monoclonal antibody or its antigen-binding fragment itself, the form of an antibody-drug conjugate (ADC) conjugated with a drug, the form bound as a target binding site to a drug delivery carrier such as a polymeric micelle, lipid nanoparticle, or liposome, or the form expressed as a part of a CAR in chimeric antigen receptor T cells (CAR-T cells).

[0060] Specific examples of the above diseases include cancer (e.g., brain tumor, cervical cancer, esophageal cancer, tongue cancer, lung cancer, breast cancer, pancreatic cancer, stomach cancer, small intestine cancer, duodenal cancer, colon cancer, bladder cancer, kidney cancer, liver cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, thyroid cancer, gallbladder cancer, pharyngeal cancer, sarcoma, melanoma, leukemia, lymphoma, multiple myeloma, etc.) and cardiovascular diseases (e.g., vascular inflammation, arteriosclerosis, etc.).

[0061] [Creation of chronic inflammation model mice] As described above, in humans, the immune response between Neu5Gc expressed in autologous tissues and anti-Neu5Gc antibodies can cause systemic chronic inflammation, which can be associated with various diseases. For example, people with a high intake of red meat have a higher risk of cardiovascular disease, Neu5Gc expression is observed in vascular endothelial cells of humans who have long-term dietary intake of Neu5Gc, and macrophage accumulation is observed around them, and further, chimpanzees with Cmah rarely develop arteriosclerosis. Therefore, the immune response between Neu5Gc expressed in human autologous tissues and anti-Neu5Gc antibodies can be involved in vascular inflammation, arteriosclerosis, etc.

[0062] Regarding the above-mentioned cardiovascular diseases observed in humans, the immune response to the foreign autoantigen Neu5Gc is an event that occurs specifically in humans, so it is not easy to perform detailed experimental analysis of the inflammatory state in humans.

[0063] On the other hand, although mouse models have been powerful and widely used tools in the study of human pathologies, there are currently no mouse models available for analyzing the above-mentioned inflammatory conditions (immune responses).

[0064] When constructing the above-mentioned mouse model using Cmah-KO mice, shortening the time required for heterologous autoantigen formation is an issue. This is because, due to the short lifespan of mice and the efficiency of experiments, feeding them a diet containing Neu5Gc and waiting for Neu5Gc to accumulate takes too long, making the mouse model insufficient for practical use.

[0065] It has been reported that the above-mentioned problem can be solved by bypassing the Neu5Gc biosynthetic pathway using peracetylated N-glycolylmannosamine (Per-acetyl ManNGc (hereinafter referred to as PA-ManNGc)), which is obtained by peracetylating ManNGc to enhance its cell membrane permeability, as shown in Figure 6 (Kayser et al., J Biol Chem. 1992;267(24):16934-8., Gagiannis et al., Biochim Biophys Acta. 2007;1770(2):297-306., Wratil et al., Angew Chem Int Ed Engl. 2016;55(33):9482-512.). Furthermore, it has been revealed that Neu5Gc, which is expressed as a heterologous autoantigen in vascular endothelial cells of Cmah-KO mice following administration of PA-ManNGc, mainly has an α2,8 disialoglycan structure.

[0066] Furthermore, even if the above-mentioned bypass pathway can shorten the time required for the formation of heterologous autoantigens, there are problems in that when mice are immunized with a Neu5Gc-containing antigen, immune responses to antigens other than the Neu5Gc antigen may also occur, and the produced antibodies may not necessarily cause inflammation, resulting in individual differences in immune responses (which may make it difficult to analyze the inflammatory state).

[0067] To solve the above problems, direct administration of an anti-Neu5Gc monoclonal antibody to Cmah-KO mice is effective. This is because it is possible to induce a reproducible inflammatory state against the heterologous autoantigen Neu5Gc, and to stably reproduce a human-specific heterologous autoantigen immune response state in mice. In this case, the anti-Neu5Gc monoclonal antibody is preferably an antibody capable of inducing inflammation and / or an antibody that binds to α2,8 disialoglycans.

[0068] Therefore, the present invention also provides a method for producing a chronic inflammation model mouse that uses Neu5Gc as an antigen, which comprises administering PA-ManNGc to a Cmah-knockout mouse to express a glycan containing Neu5Gc (e.g., an α2,8 disialo glycan), and administering the anti-Neu5Gc monoclonal antibody or its antigen-binding fragment to the mouse to induce an immune reaction with the glycan.

[0069] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0070] The materials and methods used in the experiments are as follows. (Mice) C57 / BL6J Cmah-KO mice established as previously reported (Naito et al., Germinal center marker GL7 probes activation-dependent repression of N-glycolylneuraminic acid, a sialic acid species involved in the negative modulation of B-cell activation. Mol Cell Biol. 2007;27(8):3008-22) were used in the experiments. Cmah-KO mice were raised in a specific pathogen-free (SPF) environment.

[0071] (Cells and Culture Method) Human monocyte-derived cell line U937 cells, U937-Cmah cells (U937 cells stably expressing Cmah), U937-Cmah-ST8Sia6 cells (U937 cells stably expressing Cmah and ST8Sia6), U937-Cmah-ST6Gal1 cells (U937 cells stably expressing Cmah and ST6Gal1), Sp2 / O Cmah-KO cells (a mouse myeloma cell line in which Cmah has been knocked out), and anti-Neu5Gc antibody-producing hybridoma cells were cultured in PRMI-1640 medium (FUJIFILM Wako) supplemented with 10% FBS, pyruvate, non-essential amino acids, and 2-mercaptoethanol. CHO-Cmah-ST8Sia6 cells (CHO cells stably expressing Cmah and ST8Sia6) were cultured in α-MEM (FUJIFILM Wako) supplemented with 10% FBS at 37°C and 5% CO. ２ The cells are commercially available or can be prepared according to the methods described herein and / or known methods using commercially available cells and materials.

[0072] (Antibodies and Lectins) For flow cytometry, PE-labeled anti-mouse IgG (H&L) antibody (Jackson Immuno Research), PE-labeled anti-mouse IgM antibody (Southern Biotech), FITC-labeled anti-mouse IgG1 antibody (BD Bioscience), FITC-labeled anti-mouse IgG2 antibody (BD Bioscience), FITC-labeled anti-mouse IgG3 antibody (PharMingen), FITC-labeled anti-mouse IgM antibody (PharMingen), and FITC-labeled LCA (Seikagaku Corporation) were used. For immunohistochemistry, anti-mouse CD31 antibody (MEC13.3) (BD Bioscience), Alexa488-labeled anti-mouse IgG1 antibody (Invitrogen), and Alexa568-labeled anti-rat IgG antibody (Invitrogen) were used.

[0073] (Flow cytometry) Cell staining and measurement were performed using FACS buffer (1% BSA / 0.01% NaN3 / PBS). Cells were collected, washed with PBS, suspended in FACS buffer, and blocked at room temperature for 30 minutes. After reacting with mouse serum or hybridoma cell culture supernatant at room temperature for 60 minutes, the cells were washed with PBS and reacted with PE-labeled anti-mouse IgM antibody or PE-labeled anti-mouse IgG antibody at room temperature for 30 minutes. After washing with PBS, the cells were resuspended in FACS buffer and measured using a FACSCaliber (BD Biosciences). Data were analyzed using FlowJo (BD Biosciences).

[0074] (Preparation and purification of monoclonal antibody) Hybridoma cells producing anti-Neu5Gc monoclonal antibody were cultured in Hybridoma-SFM medium (GIBCO) for 3-4 days, and the culture supernatant was collected and dialyzed against Tris-buffered saline (TBS) buffer (pH 7.6) using Spectra / Pore Biotech CE dialysis tubing MWCO 50,000 (Funakoshi). Na was added to a final concentration of 0.4-0.5M. ２ SO ４ The antibody-captured thiophilic resin was packed into a column and 50 mM NaCl was added to the column. ２ HPO ４ / NaH ２ P.O. ４ The antibody was eluted with .

[0075] Experimental Example 1 Preparation of Immunogens CHO-K1 cells were transformed with an expression vector incorporating a gene encoding Cmah (cDNA sequence of mouse-derived Cmah: NCBI Gene ID 12763) and a gene encoding ST8Sia6 (cDNA sequence of mouse-derived ST8Sia6: NCBI Gene ID 338596) to overexpress Neu5Gc and ST8Sia6 (CHO-Cmah-ST8Sia6 cells), or CHO-K1 cells were transformed with an expression vector incorporating a gene encoding Cmah (cDNA sequence of mouse-derived Cmah: NCBI Gene ID 12763) and a gene encoding ST6Gal1 (cDNA sequence of rat-derived ST6Gal1: NCBI Gene ID 338596) to overexpress Neu5Gc and ST8Sia6 (CHO-Cmah-ST8Sia6 cells). CHO-K1 cells (CHO-Cmah-ST6Gal1 cells) overexpressing Neu5Gc and ST6Gal1 by transformation with an expression vector incorporating Neu5Gc (SEQ ID NO: 25197) were used as immunogens. CHO-Cmah-ST8Sia6 cells or CHO-Cmah-ST6Gal1 cells that had been cultured for 2-3 days after subculture and reached 100% confluence were harvested with a scraper and washed several times with PBS. The harvested cells were stored frozen in a pelleted state at -30°C until use.

[0076] [Immunization and Blood Collection] Eight-week-old Cmah-KO mice were immunized after collecting pre-immune serum. Blood collection and immunization were performed six times at one-week intervals. All immunizations were performed using approximately 1.0 × 10 6 The immunogen sample containing 100 μL of immunogen cells was administered subcutaneously to the base of the tail. For the first immunization, the immunogen cells were suspended in complete Freud's adjuvant (CFA) and PBS, mixed at a 1:1 (v / v) ratio, and emulsified through a needle for 30 minutes or more. For the second and subsequent booster immunizations, immunogen cells suspended in PBS were used. The final immunization was performed in the same manner as the first immunization to enlarge the lymph nodes. Blood was collected from the tail vein, and serum was prepared. The serum was stored frozen at -30°C.

[0077] [Detection of anti-Neu5Gc antibodies in mouse serum] Anti-Neu5Gc antibodies in mouse serum were detected by flow cytometry using human monocyte-derived cell line U937 cells, U937-Cmah-ST8Sia6 cells, or U937-Cmah-ST6Gal1 cells. Specifically, serum was collected 7 days after immunization, and immunohistochemical staining was performed on U937 cells, U937-Cmah-ST8Sia6 cells, or U937-Cmah-ST6Gal1 cells using Cmah-KO mouse serum (1:50) as the primary antibody, and PE-labeled anti-mouse IgM antibody (1:200) and PE-labeled anti-mouse IgG antibody (1:1000) as the secondary antibodies. GFP fluorescence intensity and PE fluorescence intensity were measured using a flow cytometer.

[0078] Compared with pre-immune serum from Cmah-KO mice, the PE fluorescence intensity in post-immune serum was increased. This indicates that anti-Neu5Gc IgM antibodies were induced by immunization with CHO-Cmah-ST8Sia6 cells or CHO-Cmah-ST6Gal1 cells. Furthermore, class switching from IgM to IgG antibodies occurred with increasing immunization frequency.

[0079] In addition, U937-Cmah-ST8Sia6 cells, mouse-derived Cmah cDNA sequence and mouse-derived ST8Sia6 gene after IRES sequence (Internal Ribosome Entry Sites) linked to the GFP gene via a base sequence (ST8Sia6-GFP) and have been produced by introducing an MSCV vector into U937 cells, GFP fluorescence intensity and ST8Sia6 expression intensity correlate. Similarly, U937-Cmah-ST6Gal1 cells, mouse-derived Cmah cDNA sequence and rat-derived ST6Gal1 gene after IRES sequence linked to the GFP gene via a MSCV vector (ST6Gal1-GFP) and have been produced by introducing an MSCV vector into U937 cells, GFP fluorescence intensity and ST6Gal1 expression intensity correlate.

[0080] [Preparation of Hybridoma Cells] Four days after the final immunization, Cmah-KO mice were euthanized, and lymphocytes were prepared from the iliac lymph nodes. The obtained lymphocytes were washed three times with serum-free medium and then mixed with Sp2 / O Cmah-KO cells at a 1:1 ratio. Cell fusion was performed using the HVJ Envelope Cell Fusion Kit GenomeONE. ＴＭ After cell fusion, the cells were cultured overnight in RPMI-1640 medium.

[0081] [Screening of Hybridoma Cells] Hybridoma selection and cloning were performed using a semi-solid medium containing HAT (hypoxanthine, aminopterin, thymidine). Cells cultured overnight were collected and resuspended in 5 mL of RPMI-1640 medium, and then cultured in the semi-solid medium ClonaCell. ＴＭ The cells were gently mixed with Neu5Gc-HY medium D (STEMCELL) at a 1:9 (v / v) ratio. Ten mL of this cell mixture was placed in 10-cm plates and cultured for 7-10 days. Formed single colonies were randomly picked and transferred to 96-well plates containing 200 μL of RPMI-1640 medium supplemented with 50x HAT Medium Supplement Hybri-Max (Sigma) and 50x Hybridoma Fusion and Cloning Supplement (Santa Cruz Biotechnology), and cultured for 4-7 days. Hybridomas producing antibodies with binding to Neu5Gc were then screened by flow cytometry using the culture supernatant.

[0082] When the immunogen was CHO-Cmah-ST8Sia6 cells, 40 clones (approximately 4.6%) out of a total of 864 clones picked were hybridoma cells producing anti-Neu5Gc antibodies. Among them, nine clones (Z43-1C4, Z43-1H9, Z43-2B10, Z43-2D9, Z39-3A2, Z43-4E10, Z43-6C1, Z43-7G4, Z43-8C1) were established that produced antibodies with particularly high affinity to Neu5Gc and had stable cell growth. Of these, six clones (Z43-1C4, Z43-1H9, Z43-4E10, Z43-6C1, Z43-7G4, Z43-8C1) produced anti-α2,8-type Neu5Gc antibodies. Of the remaining three clones, two clones (Z43-2D9, Z39-3A2) produced anti-a2,3 type Neu5Gc antibodies, and one clone (Z43-2B10) produced an anti-Neu5Gc antibody of unknown binding mode. Figure 7A shows the results of flow cytometry analysis of seven of the clones used for cloning the monoclonal antibodies described below. Furthermore, as shown in Figure 7B, none of these clones reacted with U937 cells (i.e., cells lacking the Cmah gene and not expressing Neu5Gc). When the immunogen was CHO-Cmah-ST6Gal1 cells, 102 of the 2,304 clones picked (approximately 4.4%) were hybridoma cells producing anti-Neu5Gc antibodies. Among them, six clones (CZ48-1H2, CZ48-5A10, CZ51-2D3, CZ60-6F2, CZ60-7C9, CZ60-7H1) were established that produced antibodies with high affinity, particularly for Neu5Gc, and showed stable cell growth. Of these, one clone (CZ51-2D3) produced anti-α2,6 type Neu5Gc antibodies. The remaining five clones (CZ48-1H2, CZ48-5A10, CZ60-6F2, CZ60-7C9, CZ60-7H1) produced anti-α2,3 type Neu5Gc antibodies. None of these clones reacted with U937 cells (i.e., cells lacking the Cmah gene and not expressing Neu5Gc).

[0083] From the above results, it is believed that the CHO-Cmah-ST8Sia6 cells used as immunogens were cells that overexpressed α2,8 disialoglycans by introducing glycosyltransferase genes, but not all of the expressed Neu5Gc constituted α2,8 disialoglycans. B cells producing antibodies against Neu5Gc contained in glycans with linkages other than α2,8 as epitopes fused with myeloma cells to form hybridomas. Similarly, CHO-Cmah-ST6Gal1 cells express both α2,3 monosialoglycans and α2,6 monosialoglycans, and it is believed that antibodies were produced against each of the Neu5Gc contained in each sialoglycan as epitopes.

[0084] [Cloning of Monoclonal Antibodies] The anti-Neu5Gc antibodies produced by the 15 established clones were cloned according to standard methods. Briefly, each clone was cultured to extract mRNA, cDNA was synthesized by reverse transcription, and DNA encoding the light chain variable region and heavy chain variable region was amplified from the cDNA by 5' RACE, and then ligated into a cloning vector (pCRBlunt-RV vector (Invitrogen)) for cloning. The gene sequences and amino acid sequences encoding the light chain variable region and heavy chain variable region of the cloned anti-Neu5Gc monoclonal antibody were analyzed.

[0085] The amino acid sequences constituting the light chain variable region and heavy chain variable region of each of the cloned anti-Neu5Gc monoclonal antibodies are shown in Tables 2A and 2B. Furthermore, the CDR sequences of the amino acid sequences constituting each light chain variable region and each heavy chain variable region were identified by analysis using the IgBLAST database. The identified CDR sequences are shown in Table 3.

[0086]

[0087]

[0088] Furthermore, isotype analysis revealed that all of the above monoclonal antibodies were composed of a κ light chain and a γ1 heavy chain (clones Z43-1H9, Z43-2B10, Z43-4E10, Z43-7G4, CZ48-5A10, CZ51-2D3, CZ60-6F2), a μ heavy chain (Z43-2D9, Z39-3A2, Z43-8C1, CZ48-1H2, CZ60-7C9, CZ60-7H1, Z43-6C1), or a γ2b heavy chain (clone Z43-1C4).

[0089] [Evaluation of the specificity of anti-Neu5Gc monoclonal antibodies for Neu5Gc] As shown in Figure 7A, the results of flow cytometry using U937-Cmah-ST8Sia6 cells confirmed that the monoclonal antibodies produced by clones Z43-1H9 and Z43-7G4 (hereinafter referred to as "mab_No. Z43-1H9" and "mab_No. Z43-7G4," respectively) recognize a 2,8-linked disialoglycan structure as an epitope. Therefore, we examined whether these antibodies exhibit binding affinity to the heterologous autoantigen Neu5Gc expressed by administration of PA-ManNGc.

[0090] First, evaluation was performed using a system using cultured cells. Specifically, the procedure is as follows. 80% confluent U937 cells were diluted 1:10 and cultured for 2 days in 0.5 mM PA-ManNGc-supplemented medium to express Neu5Gc as a heterologous autoantigen. Additionally, as a negative control, U937 cells were similarly cultured in a medium without PA-ManNGc. Next, the binding of the two anti-Neu5Gc monoclonal antibodies (MAB No. Z43-1H9 and MAB No. Z43-7G4) was confirmed by flow cytometry.

[0091] As a result, U937 cells cultured in a medium without PA-ManNGc (left) did not bind to the anti-Neu5Gc monoclonal antibody, whereas U937 cells cultured in a medium with PA-ManNGc (center) showed similar binding to U937-Cmah cells expressing Neu5Gc by Cmah gene introduction (right, positive control, cultured in a medium without PA-ManNGc) (Fig. 8).

[0092] Next, evaluation was performed using a mouse system. Specifically, the procedure is as follows. PA-ManNGc was dissolved in PBS and administered intravenously to 8-10 week-old Cmah-KO mice via the orbital venous plexus at a dose of 200 mg / kg weight once daily for a total of two days. This resulted in the expression of Neu5Gc as a heterologous autoantigen in the vascular endothelium. Heart tissue sections were prepared from these mice and subjected to immunohistochemical staining using the two anti-Neu5Gc monoclonal antibodies described above. Specifically, the mice were anesthetized under abdominal and thoracotomy, an incision was made in the right atrial appendage of the heart, and PBS was perfused through the left ventricle to remove blood. After that, 10% neutral buffered formalin (FUJIFILM Wako) was perfused for at least 10 minutes for formalin fixation. The heart, kidney, abdominal aorta, and inferior vena cava were excised, post-fixed overnight in 10% neutral buffered formalin, and then replaced with 30% sucrose. They were then embedded in OCT compound (SAKURA Fintechical), and 40 μm-thick frozen cardiac sections were prepared using a freezing microtome (Leica). For immunohistochemistry, 5% BSA / 0.05% Tween-20 / TBS ​​was used as the blocking buffer. After blocking for 30 minutes at room temperature, the sections were incubated with purified anti-Neu5Gc monoclonal antibodies (mab No. Z43-1H9, mab No. Z43-7G4) and anti-CD31 antibodies for 2 hours at room temperature. After washing with TBS-T (0.05% Tween-20 / TBS), the sections were reacted with Alexa488-labeled anti-mouse IgG1 antibody and Alexa568-labeled anti-rat IgG antibody at room temperature for 30 minutes. After washing with TBS-T, the nuclei were stained with Hoechst 33342, washed with distilled water, air-dried, and mounted using an anti-fading mounting medium. Observations were performed using an all-in-one microscope imaging system BZ-9000 (Keyence). The observation results are shown in Figure 9.

[0093] As a result, the anti-Neu5Gc monoclonal antibody did not react with sections from control Cmah-KO mice, but did react with sections from Cmah-KO mice in which Neu5Gc was expressed in the vascular endothelium due to administration of PA-ManNGc (scale bar in the figure is 200 μm).

[0094] These results demonstrate that the two anti-Neu5Gc monoclonal antibodies are specific to the heterologous autoantigen Neu5Gc formed by administration of PA-ManNGc. These anti-Neu5Gc monoclonal antibodies are considered to be useful for constructing mouse models.

[0095] The anti-Neu5Gc monoclonal antibody according to the embodiment of the present invention can be suitably used in research or treatment of immune reactions to Neu5Gc as an antigen or inflammatory reactions caused by the immune response, detection of cells or tissues expressing Neu5Gc, etc. Furthermore, the anti-Neu5Gc monoclonal antibody or antigen-binding fragment thereof can also be useful for targeting human cancer cells.

Claims

1. An anti-N-glycolylneuraminic acid monoclonal antibody or antigen-binding fragment thereof, which binds to N-glycolylneuraminic acid, comprising: a heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3); and a light chain variable region comprising light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), and light chain complementarity determining region 3 (LCDR3), wherein the amino acid sequences comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the following sets of CDR sequences 1 to 15, or from the set of amino acid sequences in which at least one amino acid sequence in the following sets of CDR sequences has one or two amino acid substitutions, deletions, and / or additions.

2. The heavy chain variable region and the light chain variable region comprise: a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 36 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 37; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 38 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 39; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 40 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 41; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 42 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 43; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 44 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 45; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 47; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 48 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 49; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 86 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 87; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 88 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 89;a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:90 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:91; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:94 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:95; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:96 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:97; a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:98 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:99; The monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising: a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 100 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 101; or a heavy chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO: 102 and a light chain variable region comprising an amino acid sequence having 70% to 100% identity to the amino acid sequence of SEQ ID NO:

93.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, which binds to N-glycolylneuraminic acid located at the non-reducing end of a sugar chain.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 3, wherein the sugar chain contains two Neu5Gc of the α2,3-linked and α2,8-linked types located in this order from opposite sides at the non-reducing end.

5. A nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof of claim 1.

6. A vector or plasmid comprising the nucleic acid molecule of claim 5.

7. A host cell transformed with the vector or plasmid of claim 6.

8. A hybridoma producing the monoclonal antibody or antigen-binding fragment thereof according to claim 1.

9. A composition for immunohistochemical staining comprising the monoclonal antibody or antigen-binding fragment thereof according to claim 1.

10. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of claim 1.

11. A method for producing a chronic inflammation model mouse using N-glycolylneuraminic acid as an antigen, comprising: administering peracetylated N-glycolylmannosamine to a Cmah knockout mouse to express a sugar chain containing N-glycolylneuraminic acid; and administering the monoclonal antibody or its antigen-binding fragment according to claim 1 to the mouse to induce an immune reaction with the sugar chain.

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