RU2789757C2 - Antibody specifically binding to icam-1 and its use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: antibody to ICAM-1 or its antigen-binding fragment are proposed. Nucleic acid molecules encoding the specified antibody, a recombinant expression vector, and a recombinant cell for the production of an antibody according to the invention are also proposed. In addition, a pharmaceutical composition for the prevention or treatment of a disease mediated with immune cells, as well as a composition for detection of ICAM-1 are provided. Compositions contain an antibody to ICAM-1 or its antigen-binding fragment as an active ingredient.

EFFECT: invention provides regulation of differentiation and function of dendritic cells for more efficient control of an immune response.

14 cl, 39 dwg, 11 tbl, 6 ex

Description

The field of technology to which the present invention relates

The present disclosure relates to the specific binding of an anti-ICAM-1 antibody to ICAM-1, or an antigen-binding fragment thereof, and their use, more specifically, to an anti-ICAM-1 antibody, or antigen-binding fragment thereof, and a pharmaceutical composition for the prevention and/or treatment of a disease mediated by immune cells, and a pharmaceutical composition for regulating the function and/or differentiation of dendritic cells, containing an antibody or antigen-binding fragment.

Background of the Invention

Dendritic cells (DCs) are well-characterized antigen-presenting cells that integrate a variety of immune responses and comprise a heterogeneous family of antigen-presenting cells that are involved in immune initiation and immune tolerance. It is now known that immature dendritic cells induce the transition of anergic T cells, and dendritic cells transformed into mature dendritic cells by active stimulants such as lipopolysaccharide (LPS) induce primary T cell responses. In addition, semi-mature dendritic cells with unique cytokine production profiles can perform immunotolerant functions.

ICAM-1 (Intercellular adhesion molecule 1) denotes domain 1 - domain 5, as well as a 90 kDa type I cell surface glycoprotein consisting of five extracellular domains of the immunoglobulin superfamily, numbered from N-terminus to C-terminus, transmembrane region and intracellular region .

ICAM-1 mediates leukocyte-leukocyte interactions such as interactions between T cells and antigen presenting cells. It also mediates the outflow of leukocytes into tissues during the inflammatory process. According to an in vitro study, it has been suggested that antibodies that interfere with the interaction of ICAM-1 and leukocyte function-associated antigen-1 (LFA-1) may interfere with T cell adhesion to endothelial cells, and it has been suggested that that T cell activation by these antibodies can also be significantly reduced in mixed lymphocytes (Proc Natl Acad Sci USA. 1988, 85:3095-3099). In a monkey study using R6-5-D6 (enlimomab), a mouse monoclonal antibody that binds to the human ICAM-1 domain, renal allograft survival was improved and T-cell penetration into the graft was reduced compared to controls (J Immunol. 1990, 144:4604-4612). In addition, enlimomab has been shown to have an inhibitory effect on disease activity in patients with rheumatoid arthritis (Arthritis Rheum. 1994, 37:992-999; J Rheumatol. 1996, 23:1338-1344). However, treatment with enlimomab after kidney transplantation is not very effective in reducing the incidence of acute rejection or the risk of delayed graft function. In addition, enrimomab blocks the adhesion of neutrophils as well as T cells to vascular endothelial cells, and thus disruption of neutrophil migration has been reported to potentially increase susceptibility to infection (J. Immunol. 1999, 162:2352-2357). There is a need to develop a substance that specifically binds to ICAM-1 to regulate the differentiation and function of dendritic cells, thereby more effectively controlling the immune response.

Detailed Description of the Invention

Technical problem

In one example, an anti-ICAM-1 antibody, or antigen-binding fragment thereof, is provided that specifically recognizes ICAM-1.

An anti-ICAM-1 antibody or antigen-binding fragment thereof may comprise heavy chain complementarity determining regions (CDRs) containing a polypeptide (CDR-H1) containing the amino acid sequence of SEQ ID NO: 1 (GYTFTDYA), a polypeptide (CDR-H2), containing the amino acid sequence under SEQ ID NO: 2 (ISTYSGNT), and a polypeptide (CDR-H3) containing the amino acid sequence under SEQ ID NO: 3 (ARSLYFGSSGFDY), or a heavy chain variable region containing the complementarity determining region of the heavy chain described higher; and light chain complementarity determining regions containing a polypeptide (CDR-L1) containing the amino acid sequence under SEQ ID NO: 4 (QTLVYRNGNTY), a polypeptide (CDR-L2) containing the amino acid sequence under SEQ ID NO: 5 (KVS), and a polypeptide (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 6 (SQNTHFPYT), or a light chain variable region comprising a light chain complementarity determining region as described above.

According to one embodiment, the heavy chain variable region may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 11-34, and the light chain variable region may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs. : 8 and 35-38.

In another example, a nucleic acid molecule encoding a complementarity determining region of a heavy chain, a heavy chain variable region, or an anti-ICAM-1 antibody heavy chain is provided.

In another example, a nucleic acid molecule is provided that encodes a light chain complementarity determining region, a light chain variable region, or an anti-ICAM-1 antibody light chain.

In another example, a recombinant vector is provided comprising a nucleic acid molecule encoding a heavy chain complementarity determining region, a heavy chain variable region, or an anti-ICAM-1 antibody heavy chain, and a nucleic acid molecule encoding a light chain complementarity determining region, variable region light chain or light chain of an anti-ICAM-1 antibody, in one vector or each in separate vectors.

In another example, a recombinant cell containing a recombinant vector may be provided.

In another example, a pharmaceutical composition for the prevention and/or treatment of a disease may be provided, comprising an anti-ICAM-1 antibody or antigen-binding fragment thereof as an active ingredient. The disease may be a disease mediated by immune cells. In another example, a method is provided for preventing and/or treating an immune cell mediated disease, the method comprising the step of administering a pharmaceutically effective amount of an anti-ICAM-1 antibody and/or antigen binding fragment to a subject in need of preventing and/or treating an immune cell mediated disease. The method may further include the step of identifying a subject in need of prevention and/or treatment of an immune cell-mediated disease prior to the step of administering. In another example, the use of an anti-ICAM-1 antibody or antigen-binding fragment thereof for the prevention and/or treatment of a disease mediated by immune cells, or the use of an antibody to ICAM-1 or an antigen-negative fragment thereof in the manufacture of a medicament for the prevention or treatment of a disease mediated by immune cells . In the pharmaceutical composition, method and use, the disease mediated by immune cells can be selected from transplant rejection, graft versus host disease, asthma, autoimmune disease, and the like.

In another example, a pharmaceutical composition is provided for regulating dendritic cell function and/or differentiation, comprising an anti-ICAM-1 antibody or antigen-binding fragment thereof as an active ingredient. In another example, a method for regulating dendritic cell function and/or differentiation is provided, comprising the step of administering a pharmaceutically effective amount of an anti-ICAM-1 antibody or antigen-binding fragment thereof to a subject in need of regulation of dendritic cell function and/or differentiation. In another example, the use of an anti-ICAM-1 antibody or antigen-binding fragment thereof in the manufacture of a medicament for the regulation of function and/or differentiation of an immune cell-mediated disease is contemplated.

Technical solution

Provided herein is an anti-ICAM-1 antibody, or antigen-binding fragment thereof, that specifically recognizes ICAM-1, and uses thereof.

An anti-ICAM-1 antibody or antigen-binding fragment thereof is characterized in that it specifically recognizes and/or binds to domain 2 among ICAM-1 domains. An anti-ICAM-1 antibody, or antigen-binding fragment thereof, may be an antagonist having inhibitory activity against ICAM-1. However, the antibody does not inhibit ICAM-1 and LFA-1 binding itself by binding to ICAM-1 domain 2 rather than ICAM-1 domain 1, which is the binding site for LFA-1, an ICAM-1 ligand that is an antigen. This can be confirmed by the fact that the antibody does not inhibit T cell transmigration. The antibody has the potential to influence the activation of the spatial function of immune synapses for transmission of T cell activity and binding and rearrangement between related proteins, since it suppresses the immune activity of T cells when co-cultured with dendritic cells and T cells and functionally induces immune suppression by inducing differentiation into tolerogenic dendritic cells. In this regard, an antibody may be an antagonist that functionally inhibits ICAM-1 activity rather than physically inhibiting receptor and ligand binding. In addition, the antibody can induce the result of suppression of the immune activity of T cells containing the ligand, as a result of the above activity. In other words, an anti-ICAM-1 antibody or antigen-binding fragment provided herein can inhibit dendritic cell function and/or differentiation and can have a prophylactic and/or therapeutic effect on an immune cell-mediated disease. by binding to domain 2 of ICAM-1 (eg, human ICAM-1) and inducing antigen-specific T-cell tolerance.

ICAM-1 (intercellular adhesion molecule 1), also referred to as CD54 (cluster of differentiation 54), is a cell surface glycoprotein expressed on an endothelial cell or a cell of the immune system.

ICAM-1 serving as an antigen of an antibody provided herein may be mammalian, for example, human ICAM-1 (for example, accession numbers in NCBI NP_000192.2, etc.), simian ICAM-1 ( for example, access numbers in NCBI NP_001266532, etc.). In one example, an antibody may be cross-reactive with human ICAM-1 and simian ICAM-1.

In the present description, the term "antibody" refers to a substance produced by stimulation of an antigen in the immune system, or a protein that specifically binds to a particular antigen, and is used as a concept to include a protein derived recombinantly or synthetically based on the substance or protein. The antibody may not be produced naturally, for example, recombinantly or synthetically. The antibody can be an animal antibody (eg, a mouse antibody, etc.), a chimeric antibody (eg, a mouse-human chimeric antibody), a humanized antibody, or a human antibody. The antibody may be a monoclonal antibody or a polyclonal antibody.

In addition, in the present description, you can understand that the antibody includes all fragments of antibodies with antigennegative ability, unless otherwise indicated. As used herein, the term "complementarity determining regions (CDRs)" refers to a region that confers antigen-binding specificity among the variable regions of an antibody. An antigen-binding fragment of an antibody described above may be an antibody fragment containing one or more complementarity-determining regions.

In one example, an anti-ICAM-1 antibody, or antigen-binding fragment thereof, is provided that specifically recognizes ICAM-1 or specifically binds to ICAM-1.

An anti-ICAM-1 antibody or antigen-binding fragment thereof may comprise heavy chain complementarity determining regions (CDRs) containing a polypeptide (CDR-H1) containing the amino acid sequence of SEQ ID NO: 1 (GYTFTDYA), a polypeptide (CDR-H2), containing the amino acid sequence under SEQ ID NO: 2 (ISTYSGNT), and a polypeptide (CDR-H3) containing the amino acid sequence under SEQ ID NO: 3 (ARSLYFGSSGFDY), or a heavy chain variable region containing the complementarity determining region of the heavy chain described higher; and light chain complementarity determining regions containing a polypeptide (CDR-L1) containing the amino acid sequence under SEQ ID NO: 4 (QTLVYRNGNTY), a polypeptide (CDR-L2) containing the amino acid sequence under SEQ ID NO: 5 (KVS), and a polypeptide (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 6 (SQNTHFPYT), or a light chain variable region comprising a light chain complementarity determining region as described above.

In the heavy chain variable region, heavy chain framework 1 (VH-FR1; N-terminal contiguous region CDRH1) may comprise the amino acid sequence of SEQ ID NO: 97 (QVQLX1QSGAEX2X3X4PGX5SVKX6SCKX7S; X1 is Q or V, X2 is L or V, X3 is is V or K, X4 is R or K, X5 is V or A, X6 is I or V, X7 is G or A) or under SEQ ID NO: 98 (QVQLX8QSGAEVX9KPGASVKX10SCKX11S; X8 is V or Q, X9 is K or V, X10 is I or V, X11 is G or A) e.g. amino acid sequence selected from SEQ ID NOs: 39, 40, 41, 42, 43, 44, 45, 46, 47 and 48, heavy chain framework 2 (VH-FR2; the region between the C-terminus of CDRH1 and the N-terminus of CDRH2) may contain the amino acid sequence under SEQ ID NO: 99 (LHWVX12QX13X14X15X16X17LEWX18GV; X12 is K or R, X13 is S or A , X14 is H or P, X15 is pr is A or G, X16 is K or Q, X17 is S or R, X18 is I or M) or under SEQ ID NO: 100 (LHWVX19QAPGQX20LEWX21GV; X19 is R or K, X20 is R or S, X21 is I or M) e.g. amino acid sequence selected from SEQ ID NOs: 49, 50, 51, 52, 53, 54 and 55, frame 3 heavy chain (VH-FR3; the region between the C-terminus of CDRH2 and the N-terminus of CDRH3) may contain the amino acid sequence under SEQ ID NO: 101 (X22YX23QKFX24GX25X26TX27TX28DX29SX30X31TAYX32ELX33X34LX35SEDX36AX37X38YC; X22 is D or K, X23 is N or S, X23 is N or S, Q, X25 is K or R, X26 is A or V, X27 is M or I, X28 is V or R, X29 is K or T, X30 is S or A, X31 is T or S , X32 is L or M, X33 is A or S, X34 is R or S, X35 is T or R, X36 is S or T, X37 is I or V, X38 is H or Y) or under SEQ ID NO: 102 (X39YX40QKFX41GX 42x43TX4445RX46SAX47TAYX48LSSLRSEDTAX49X50YC; X39 is D or K X40 is N or S X41 is R or Q X42 is R or K X43 is A or V X44 is I or M X45 is R or V X46 is T or K, X47 is S or T, X48 is M or L, X49 is V or I, X50 is Y or H), e.g., an amino acid sequence selected from SEQ ID NOs: 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 and 80, and/or frame 4 heavy chain (VH-FR4; region adjacent to the C-terminus of CDRH3) may contain the amino acid sequence of SEQ ID NO: 81.

In the light chain variable region, light chain framework 1 (VL-FR1; N-terminal contiguous region of CDRL1) may comprise the amino acid sequence of SEQ ID NO: 103 (DVVLTQX51PLSX52PVX53LGX54X55ASISCRSS; X51 is T or S, X52 is L or S, X53 is is N or T, X54 is D or Q, X55 is Q or P) or under SEQ ID NO: 104 (DVVLTQX56PLSX57PVTLGQPASISCRSS; X56 is S or T, X57 is L or S), for example, the amino acid sequence selected of SEQ ID NOs: 82, 83 and 84, light chain framework 2 (VL-FR2; the region between the C-terminus of CDRL1 and the N-terminus of CDRL2) may contain the amino acid sequence under SEQ ID NO: 105 (LHWYX58QX59X60GQX61PX62LLIX63; X58 is L or Q, X59 is K or R, X60 is A or P, X61 is S or P, X62 is K or R, X63 is Y or absent), e.g., amino acid sequence, selected from SEQ ID NOs: 85, 86, 87, 88 and 89 light chain framework 3 (VL-FR3; the region between the C-terminus of CDRL2 and the N-terminus of CDRL3) may contain the amino acid sequence under SEQ ID NO: 106 (NRFSGVPDRFSGSGX64GTDFTLKISRVEAEDX65GVYFC; X64 is S or A, X65 is L or V), for example, an amino acid sequence selected from SEQ ID NO : 90, 91 and 93, and/or light chain framework 4 (VL-FR4; region adjacent to the C-terminus of CDRL3) may contain the amino acid sequence of SEQ ID NO: 107 (FGGGTKX66X67X68X69X70; X66 is I or L, X67 is is K or E, X68 is R or I, X69 is Q or K, X70 is R or absent), e.g. the amino acid sequence of SEQ ID NO: 93 or 94.

In an anti-ICAM-1 antibody or antigen-binding fragment thereof, the heavy chain variable region may comprise a VH-FR1 containing the amino acid sequence of SEQ ID NO: 97 or 98 (e.g., SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48), CDRH1 containing the amino acid sequence under SEQ ID NO: 1, VH-FR2 containing the amino acid sequence under SEQ ID NO: 99 or 100 (for example, SEQ ID NO: 49, 50, 51, 52 , 53, 54 or 55), CDRH2 containing the amino acid sequence under SEQ ID NO: 2, VH-FR3 containing the amino acid sequence under SEQ ID NO: 101 or 102 (for example, SEQ ID NO: 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80), a CDRH3 containing the amino acid sequence under SEQ ID NO: 3, and VH-FR4 containing the amino acid sequence under SEQ ID NO: 81; and the light chain variable region may comprise VL-FR1 containing the amino acid sequence of SEQ ID NO: 103 or 104 (e.g. SEQ ID NO: 82, 83 or 84), CDRL1 containing the amino acid sequence of SEQ ID NO: 4, VL- FR2 containing the amino acid sequence under SEQ ID NO: 105 (for example, SEQ ID NO: 85, 86, 87, 88 or 89), CDRL2 containing the amino acid sequence under SEQ ID NO: 5, VL-FR3 containing the amino acid sequence under SEQ ID NO: 106 (for example, SEQ ID NO: 90, 91 or 93), CDRL3 containing the amino acid sequence under SEQ ID NO: 6, and VL-FR4 containing the amino acid sequence under SEQ ID NO: 107 (for example, SEQ ID NO : 93 or 94).

According to one embodiment, the heavy chain variable region may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 11-34, and the light chain variable region may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs. : 8 and 35-38.

Animal-derived antibodies obtained by immunizing an immunized animal with the desired antigen can generally elicit an immune rejection response when administered therapeutically to humans, and a chimeric antibody has been developed to suppress this immune rejection response. Chimeric antibodies are antibodies obtained by replacing the constant region of an animal-derived antibody that elicits an antiisotypic response with a constant region of a human antibody using genetic engineering techniques. Chimeric antibodies significantly improved anti-isotypic response compared to animal-derived antibodies, but animal-derived amino acids are still present in the variable region, which has side effects on a potential anti-idiotypic response. To reduce these side effects, a humanized antibody has been developed. It is obtained by grafting a CDR region (complementarity determining regions), which plays an important role in antigen binding among the variable regions of a chimeric antibody, into a human antibody framework.

The most important thing in CDR grafting technology to obtain a humanized antibody is the selection of an optimized human antibody that can best accept the CDR regions of an animal-derived antibody, for this purpose, the use of antibody databases, crystal structure analysis, molecular modeling technology, and the like can be used.

According to one embodiment, the antibody can be an animal antibody (eg, a mouse antibody), a chimeric antibody (eg, a mouse-human chimeric antibody), or a humanized antibody.

In an antibody or antigen-binding fragment, a heavy chain framework and/or a heavy chain constant region, except for the heavy chain CDRs of SEQ ID NO: 1-3, may be a heavy chain framework and/or a heavy chain constant region derived from an immunoglobulin (IgG (IgG1 , IgG2, IgG3, IgG4, etc.), IgM, IgA, IgD, or IgE), such as human immunoglobulin (IgG (IgG1, IgG2, IgG3, IgG4, etc.), IgM, IgA, IgD, or IgE ) and the light chain framework and/or light chain constant region, except for the light chain CDRs of SEQ ID NO: 4-6, may be a light chain framework and/or kappa (κ) or lambda (λ) type light chain constant region.

A complete antibody has two full length light chains and two full length heavy chains, in which each light chain is linked to the heavy chain via a disulfide bond. The antibody constant region is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region is of gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) type, and has gamma subclasses. 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The light chain constant region is of kappa (κ) and lambda (λ) type.

The term "heavy chain" is intended to cover full-length heavy chains and fragments thereof, wherein the full-length heavy chain contains the V _H variable region domain, including amino acid sequences sufficient to provide antigen specificity, three constant regions, C _H1 , C _H2 and C _H3 , and hinge. In addition, the term "light chain" is intended to cover full length light chains and fragments thereof, full length light chains containing a V _L variable region domain, including amino acid sequences sufficient to provide antigen specificity, and a C _L constant region.

The term "complementarity determining region (CDR)" refers to an amino acid sequence occurring in the hypervariable region of an immunoglobulin heavy chain or light chain. The heavy and light chains may respectively contain three CDRs (CDRH1, CDRH2 and CDRH3; and CDRL1, CDRL2 and CDRL3). The CDR may provide a contact residue that plays an important role in the binding of antibodies to antigens or epitopes. On the other hand, in the present description, the terms "specific binding" and "specific recognition" have the same general meaning known to the person skilled in the art, and indicate that the antibody and antigen specifically interact with each other, causing an immunological reaction.

The term "antigen-binding fragment" refers to fragments of an intact immunoglobulin containing a portion of a polypeptide constituting an antigen-binding site, for example, a portion of an antibody containing a CDR. For example, the antigen binding fragment may be scFv, (scFv) ₂ , scFvFc, Fab, Fab' or F(ab') ₂ .

The Fab is a structure having a light chain and heavy chain variable region, a light chain constant region and a heavy chain first constant region (CH1), and has one antigen binding site. Fab' differs from Fab in that Fab' contains a hinge region with at least one cysteine residue at the C-terminus C _H1 . The F(ab')2 antibody is formed by disulfide binding of cysteine residues in the Fab' hinge region. Fv is the minimum antibody fragment consisting of only the heavy chain variable region and the light chain variable region. Recombination techniques for generating an Fv fragment are widely known in the art. The double chain Fv contains a heavy chain variable region and a light chain region that are linked to each other via a non-covalent bond. A single chain Fv typically contains a heavy chain variable region and a light chain variable region that are linked to each other by a covalent bond via a peptide linker or linked at the C-terminus to form a dimeric structure similar to a double chain Fv.

Antigen-binding fragments can be generated by a protease (for example, a Fab can be generated by restrictive digestion of the whole antibody with papain and an F(ab')2 fragment can be generated by digestion with pepsin) or can be generated by using a genetic recombination technique.

The term "hinge region" as used herein refers to the region between the CH1 and CH2 domains in an antibody heavy chain that provides flexibility to the antigen-binding site.

The anti-ICAM-1 antibody may be a monoclonal antibody. A monoclonal antibody can be obtained using a method widely known in the art, for example, using the phage display method. Alternatively, an anti-ICAM-1 antibody can be constructed in the form of an animal (eg, mouse) monoclonal antibody using a conventional method.

Meanwhile, individual monoclonal antibodies can be screened based on the ability to bind to ICAM-1 using a typical ELISA (enzyme-linked immunosorbent assay) format. Inhibitory activity can be determined using functional assays, such as cell assays, or functional assays, such as competitive ELISA (competitive ELISA) to analyze molecular interactions in the case of conjugates. The corresponding affinity (Kd values) for ICAM-1 can then be analyzed for selected representatives of monoclonal antibodies based on strong inhibitory activity.

In another example, a pharmaceutical composition for the prevention and/or treatment of a disease is provided, comprising an anti-ICAM-1 antibody or antigen-binding fragment thereof as an active ingredient. The disease may be a disease mediated by immune cells. In another example, a method is provided for preventing and/or treating an immune cell mediated disease, the method comprising the step of administering a pharmaceutically effective amount of an anti-ICAM-1 antibody or antigen-binding fragment thereof to a subject in need of preventing and/or treating an immune cell mediated disease. The method may further include the step of identifying a patient in need of prevention and/or treatment of an immune cell-mediated disease prior to the step of administration. In another example, the use of an anti-ICAM-1 antibody or antigen-binding fragment thereof for the prevention and/or treatment of a disease mediated by immune cells, or the use of an antibody to ICAM-1 or an antigen-negative fragment thereof in the manufacture of a medicament for the prevention or treatment of a disease mediated by immune cells .

In the case of a pharmaceutical composition, method and use, the disease mediated by immune cells may be selected from all diseases associated with immune cells. The immune cell may be at least one selected from the group consisting of a T cell, a B cell, a dendritic cell, a macrophage, a monocyte, and the like. In one example, an immune cell-mediated disease can be selected from transplant rejection (e.g., rejection that occurs during allogeneic cell transplantation, allogeneic organ transplantation, xenogeneic cell transplantation, xenogeneic organ transplantation), graft-versus-host disease (e.g., graft-versus-host disease that occurs during allogeneic bone marrow transplantation), asthma, obesity, type 2 diabetes, autoimmune disease (eg, encephalomyelitis (eg, allergic encephalomyelitis), rheumatoid arthritis, systemic lupus erythematosus (lupus), atopic dermatitis, multiple sclerosis, type 1 diabetes mellitus, chronic inflammatory disease (eg, inflammatory bowel disease (eg, Crohn's disease, ulcerative colitis)), Behçet's disease, Sjögren's syndrome, myasthenia gravis, scleroderma, polyarteritis nodosa, Kikuchi disease, collagenosis, thyroid ita Hashimoto, psoriasis, vitiligo, hyperthyroidism, fibromyalgia, alopecia areata, allergies, etc.), inflammation mediated by immune cells (eg, T cells, B cells, dendritic cells, macrophages, monocytes, etc.) (eg, macrophage-mediated inflammation, etc.), inflammatory disease caused by immune cell-mediated inflammation, and the like.

In another example, a pharmaceutical composition is provided for regulating differentiation and/or function of dendritic cells, wherein the composition contains an anti-ICAM-1 antibody or antigen-binding fragment thereof as an active ingredient. In another example, a method is provided for regulating dendritic cell differentiation and/or function, the method comprising the step of administering a pharmaceutically effective amount of an anti-ICAM-1 antibody or antigen-binding fragment thereof to a subject in need of regulation of dendritic cell differentiation and function. In another example, the use of an anti-ICAM-1 antibody or antigen-binding fragment thereof in regulating dendritic cell differentiation and/or function, or the use of an anti-ICAM-1 antibody or antigen-binding fragment thereof in the manufacture of a medicament for regulating differentiation and/or function of dendritic cells.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may generally be used in medicaments and one or more of them is selected from the group consisting of lactose, dextrose, sucrose, trehalose, arginine, histidine, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin , calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc., but not limited to. The pharmaceutical composition may additionally contain one or more components selected from the group consisting of a diluent, excipients, lubricant, wetting agent, sweetener, flavoring agent, emulsifier, suspending agent, preservative, and the like, which are usually used in the manufacture of a pharmaceutical composition.

The pharmaceutical composition or an effective amount of the antibody or antigen-binding fragment thereof can be administered orally or parenterally. In the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, intranasal administration, intrapulmonary administration, intrarectal administration, or topical administration to the affected area can be administered. Because oral administration results in digestion of proteins or peptides, the active ingredient in oral compositions may be coated or formulated to prevent stomach digestion. In addition, the composition can be administered using an optional device that allows the active ingredient to be delivered to the target cell.

As described herein, a "pharmaceutically effective amount" may refer to the amount or dosage of the active ingredient (i.e., an anti-ICAM-1 antibody or antigen-binding fragment thereof) and may be determined in various ways by factors such as formulation, route of administration, age of the patient. , weight, sex, disease state, nutrition, time of administration, interval of administration, route of administration, elimination rate, and response sensitivity.

The content of the anti-ICAM-1 antibody or antigen-binding fragment thereof, or the dosage of the anti-ICAM-1 antibody or antigen-binding fragment thereof in a pharmaceutical composition may be administered in various ways, depending on factors such as formulation method, route of administration, patient age, weight, sex, pathological condition, nutrition, time of administration, interval of administration, route of administration, rate of elimination, and response sensitivity. For example, a daily dose of an anti-ICAM-1 antibody or antigen-binding fragment thereof may be in the range of 0.001 to 1000 mg/kg, especially 0.01 to 100 mg/kg, more specifically 0.1 to 50 mg/kg and even more. specifically from 0.1 to 20 mg/kg, but not limited to this. The daily dose may be formulated in a single formulation in unit dosage form, or formulated in suitably divided dosage forms, or it may be formulated to be contained in a multi-dose container.

The pharmaceutical composition may be administered in combination with other drugs, such as other anti-cancer agents, and appropriate prescriptions may be given in terms of dose, route of administration, and types of other drugs, depending on the condition of the patient.

The pharmaceutical composition may be formulated as a solution in an oil or an aqueous medium, suspension, syrup, emulsion, extract, powder, granule, tablet or capsule, and may additionally contain a dispersing or stabilizing agent for formulation.

The patient to whom the pharmaceutical composition is administered may be a mammal, including a primate, including a human and a monkey.

Meanwhile, since an anti-ICAM-1 antibody or an antigen-binding fragment thereof specifically binds to ICAM-1, ICAM-1 can be detected or confirmed by them. Accordingly, in another example of the present invention, a composition for detecting ICAM-1 is provided, wherein the composition comprises an anti-ICAM-1 antibody or an antigen-binding fragment thereof. In another example, a method for detecting ICAM-1 is provided, the method comprising the step of treating a biological sample with an anti-ICAM-1 antibody or antigen-binding fragment thereof; and a step of identifying the presence of an antigen and antibody reaction. If an antigen-antibody reaction is detected, it can be determined (concluded) that ICAM-1 is present in the biological sample. Therefore, the detection method may further include the step of determining that ICAM-1 is present in the biological sample when the antigen-antibody reaction is detected after the step of determining the presence of the antigen-antibody reaction. The biological sample may be selected from the group consisting of cells, tissues, and biological fluids derived (isolated) from mammals such as humans (e.g., transplant patients or those who have received a transplant, patients with autoimmune diseases, etc.), and their cultures.

The step of identifying the presence of an antigen and antibody reaction can be carried out using various methods known in the art. For example, it can be determined by conventional enzymatic reaction, fluorescence, luminescence and/or radiation detection, and in particular can be measured by a method selected from the group consisting of immunochromatography, immunohistochemistry, enzyme immunoassay (ELISA), radioimmunoassay. (RIA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA), luminescent immunoassay (LIA), Western blotting, microarray analysis, etc., but without limitation.

In another example, a polypeptide molecule is provided, wherein the polypeptide molecule comprises a heavy chain complementarity determining region, a light chain complementarity determining region, or a combination thereof of an anti-ICAM-1 antibody described above; or a heavy chain variable region, a light chain variable region, or a combination thereof. The polypeptide molecule can be used to make antibodies as an antibody precursor and can be part of a protein backbone (eg, a peptibody), a bispecific antibody, or a multispecific antibody having a structure similar to that of an antibody.

In another example, a nucleic acid molecule encoding a complementarity determining region of a heavy chain, a heavy chain variable region, or an anti-ICAM-1 antibody heavy chain is provided.

In another example, a nucleic acid molecule is provided that encodes a light chain complementarity determining region, a light chain variable region, or an anti-ICAM-1 antibody light chain.

In another example, a recombinant vector is provided comprising a nucleic acid molecule encoding a heavy chain complementarity determining region, a heavy chain variable region, or an anti-ICAM-1 antibody heavy chain, and a nucleic acid molecule encoding a light chain complementarity determining region, variable region light chain or light chain of an anti-ICAM-1 antibody, in a single vector or in separate vectors carrying each of the polynucleotides.

The term "vector" refers to a method of expressing a target gene in a host cell, for example, a plasmid vector, a cosmid vector, and a viral vector such as a bacteriophage vector, an adenoviral vector, a retroviral vector, and an adeno-associated viral vector. The recombinant vector can be constructed from plasmids commonly used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1 pEX, pHV14, pET series and pUC19), phages (eg, λgt4λB, λ-Charon, λδz1, and M13), or by virus manipulation (eg, SV40, etc.).

In a recombinant vector, a polynucleotide may be operably linked to a promoter. The term "operably linked" is intended to mean a functional link between a nucleotide sequence of interest and an expression control sequence (eg, a promoter sequence). By being "operably linked", a regulatory element can control transcription and/or translation of other nucleotide sequences.

The recombinant vector may be constructed, usually as a cloning vector or an expression vector. In the case of recombinant expression vectors, a vector commonly available in the art can be used to express the foreign protein in plant, animal or microbial cells. Various methods well known in the art can be used to construct recombinant vectors.

For use in hosts, such as prokaryotic or eukaryotic cells, the recombinant vector can be designed accordingly. For example, when a vector is constructed as an expression vector for use in a prokaryotic host, the vector typically contains a strong promoter for transcription (e.g., pLκλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a binding site ribosomes for translation initiation and transcription/translation termination sequences. On the other hand, an expression vector for use in a eukaryotic host contains an origin of replication functional in a eukaryotic cell, such as an f1 origin, an SV40 origin, a pMB1 origin, an adenovirus origin, an AAV origin, and a BBV replication, but not limited to. In addition, the expression vector typically contains a promoter derived from mammalian cell genomes (eg, metallothionein promoter) or from mammalian viruses (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and HSV tk promoter), and the sequence polyadenylation as a transcription termination sequence.

In another example, a recombinant cell containing a recombinant vector is provided.

A recombinant cell can be obtained by introducing a recombinant vector into a suitable host cell. As long as it provides stable sequential cloning and expression of the recombinant vector, any host cell known in the art can be used in the present disclosure. Examples of a prokaryotic host cell available to the present disclosure include E. coli , E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus spp. such as Bacillus subtilis and Bacillus thuringiensis , and enterobacteria strains such as Salmonella typhimurium, Serratia marcescens and various Pseudomonas species. Eukaryotic host cells that may be used for transformation may include, but are not limited to, Saccharomyces cerevisiae , insect cells, and animal cells such as the Sp2/0 cell line, CHO (Chinese Hamster Ovary) K1, CHO DG44, CHO-S, PER.C6 , W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, MDCK, HEK293, etc., but not limited to.

The nucleic acid molecule, or a recombinant vector carrying it, can be introduced (transfected) into a host cell using a method well known in the art. This transfection can be performed using CaCl ₂ or the electroporation method if the host cell is prokaryotic. In the case of eukaryotic host cells, genetic introduction can be achieved by, but not limited to, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection and particle bombardment, and the like.

The method for selecting a transformed host cell can be easily carried out according to a method well known in the art using the phenotype expressed by the selectable marker. For example, when the selectable marker is a gene conferring resistance to a particular antibiotic, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

In another example, a method is provided for producing an anti-ICAM-1 antibody or antigen-binding fragment thereof, the method comprising the step of expressing a polynucleotide or recombinant vector containing it in a host cell. The production method may include the step of culturing the recombinant cell containing the recombinant vector, and optionally may further include the step of isolating and/or purifying the antibody from the culture medium.

Positive effects

An anti-ICAM-1 antibody or antigen-binding fragment provided herein binds to domain 2 of human ICAM-1 and has the property of inducing antigen-specific resistance in T cells, so that it can be effectively used to regulate differentiation and/or function of dendritic cells and/or for the prevention and/or treatment of a disease mediated by immune cells.

A brief description of the drawings

On FIG. 1 shows the result of confirmation of binding of the SI9 chimeric antibody prepared in one example to Du145 cells, a cell line expressing ICAM-1, by flow cytometry.

On FIG. 2a-2d are graphs showing the results of quantification of purified antibodies by measuring OD (optical density) at 280 nm in one example.

On FIG. 3a-3d are graphs showing the results of peak symmetry factor measurement by performing size exclusion HPLC (hereinafter SE-HPLC) analysis on an anti-ICAM-1 antibody prepared according to one example.

On FIG. 4a-4d are graphs showing the results of measuring the change in fluorescence with the ANS reagent when an anti-ICAM-1 antibody prepared according to one example was held at 61°C for 1 hour.

On FIG. 5 is a graph showing the results of ANS reactivity measurement after exposure of an anti-ICAM-1 antibody prepared according to one example at 67° C. for 1 hour.

On FIG. 6 is a graph comparing the binding capacity of an anti-ICAM-1 antibody to an ICAM-1 antigen prepared according to one example using an ELISA test (y-axis: OD value of 450 nm; x-axis: antibody concentration (ng/mL)).

On FIG. 7a-7c are graphs showing the melting point of an anti-ICAM-1 antibody prepared according to one example (7a: chimeric antibody, 7b: H17L1, 7c: H17L4).

On FIG. 8 is a graph showing the affinity (KD) as measured by binding constant (Kon) and dissociation constant (Koff) of an anti-ICAM-1 antibody made according to one example.

On FIG. 9 is a heat map plot showing changes in gene expression profiles upon administration of a humanized antibody prepared according to one example under various peripheral blood conditions.

On FIG. 10 is a graph showing changes in the gene expression profile obtained by administration of the humanized DNP007 antibody, prepared according to one example, under various peripheral blood conditions by KEGG pathway analysis (-10log (adjusted p-value)).

On FIG. 11 is a graph showing changes in the gene expression profile obtained by administering the humanized DNP007 antibody, prepared according to one example, under various peripheral blood conditions by GO biological process analysis (-10log (adjusted p-value)).

On FIG. 12a and 12b are graphs showing changes in the gene expression profile obtained by administering a humanized DNP007 antibody, prepared according to one example, in various peripheral blood conditions by analyzing the increase and decrease in cytokine gene expression.

On FIG. 13 is a graph showing changes in cytokine levels obtained by administering the humanized antibody DNP007, prepared according to one example, under various peripheral blood conditions by analyzing the change in increase and decrease in protein level.

On FIG. 14 is a graph showing the changes in cytokine levels obtained when a humanized DNP007 antibody, prepared according to one example, was administered under co-culture conditions with dendritic cells and their own T cells by analyzing the change in increase and decrease in protein levels.

On FIG. 15 is a graph showing the level of expression of maturation cofactors in dendritic cells treated with the humanized DNP007 antibody prepared according to one example.

On FIG. 16 is a graph showing the extent of secretion of inflammatory cytokines by dendritic cells treated with the humanized antibody DNP007 prepared according to one example.

On FIG. 17 is a graph showing the degree of apoptosis of dendritic cells treated with the humanized antibody DNP007 prepared according to one example.

On FIG. 18a and 18b are graphs showing expression levels of dendritic cell surface factors treated with the humanized DNP007 antibody prepared according to one example and showing whether tolerogenic dendritic cells are induced.

On FIG. 19a and 19b are graphs showing the level of expression of IDO (indoleamine-2,3-dioxygenase) in dendritic cells treated with a humanized DNP007 antibody prepared according to one example, FIG. 19a shows the result of the canonical path, FIG. 19b shows the result of the non-canonical path. Showing every figure.

On FIG. 20 is a graph showing the level of production of interferon gamma (IFN-r), an inflammatory cytokine, and the degree of proliferation of T cells cultured with dendritic cells treated with a humanized DNP007 antibody prepared according to one example.

On FIG. 21 shows the degree of T cell proliferation according to different dendritic cell maturation pathways (LPS stimulation, TNF-a stimulation, and CD40L stimulation) and the degree of production of interferon gamma (IFN-r), an inflammatory cytokine, by T cells cultured with dendritic cells, treated with a humanized DNP007 antibody prepared according to one example.

On FIG. 22a-22c show the results of the confirmation of the therapeutic effect on rheumatoid arthritis of the humanized DNP007 antibody produced according to one example in an animal model of rheumatoid arthritis. On FIG. 22a and 22b show arthritis severity scores before and after antibody administration, and FIG. 22c shows levels of anti-collagen antibodies, respectively.

On FIG. 23a and 23b show the results of confirmation of the inhibitory effect of the humanized DNP007 antibody against graft versus host disease, obtained according to one example in an animal model of graft versus host disease. On FIG. 22a shows survival and FIG. 22b shows the degree of recovery of T cells, respectively.

Ways of carrying out the present invention

Hereinafter, the present invention will be described in more detail by means of examples, however, they are only illustrative and are not intended to limit the scope of the present invention.

Example 1 Obtaining a mouse monoclonal antibody to ICAM-1

To develop an antibody specific for ICAM-1, the following experiment was performed.

1-1: mouse immunity

Recombinant human ICAM-1 protein (0.5 mg/mL; NCBI accession number NP_000192.2) was mixed with an equal volume of adjuvant (Invivogen, # vac-adx-10) to form an immune substance. In the intraperitoneal cavity (IP) of a 6-week-old female Balb/c mouse, 200 μl of the prepared immune substance was injected three times with an interval of 3 weeks.

1-2: Obtaining a mouse monoclonal antibody to ICAM-1

The spleen of the immunized mouse described above was excised to obtain a single cell suspension. The resulting cells were washed twice with RPMI (GIBCO #21875034) and then mixed with 0.4% (w/v) trypan blue (Sigma) at a ratio of 1:1 (v/v) and then cells were counted. . trypan blue staining (Sigma-Aldrich, no. T8154). The mouse myeloma cell line X63 (ATCC CRL-1580) was washed, counted and used as a cell fusion partner.

Myeloma cells and splenocytes were mixed in a ratio of 1:5, and the supernatant was removed after centrifugation. 1 ml of 50% (w/v) PEG (polyethylene glycol) 1500 preheated to 37°C was added slowly over 1 minute. After holding for approximately 1 minute, RPMI medium was slowly added and gradually diluted. After centrifugation, it was suspended in RPMI (20% FBS, hypoxanthine-aminopterin-thymidine; Gibco) containing 1x HAT (hypoxanthine, aminopterin, thymidine), dispensed into a 96-well plate at 150 µl/well, and then cultured in an incubator at 37 °C and 5% CO ₂ . After confluence, HAT feeding was performed for a certain period of time, and when a well-formed colony was observed, 150 μl of HT medium (hypoxanthine, thymidine) was added and cultured in an incubator at 37 °C and 5% CO ₂ for 48 hours.

100 µl of culture medium was removed from the 96-well hybridoma culture plate to confirm whether ICAM-1 was reactive. ICAM-1 (R&D system, no. ADP4-050) was diluted in PBS at a concentration of 1.0 µg (microgram)/ml, then dispensed into a Nunc immunoplate (Thermo, no. 439454) at 100 µl/well, stored in an incubator at 37° C and 5% CO ₂ for 1 hour and covered. After complete removal of the coating solution, 1× casein blocking solution (Sigma-Aldrich, no. B6429) was dispensed at 200 µl/well and stored in an incubator at 37°C for 1 hour to complete the blocking. After complete removal of the blocking solution, the hybridoma culture solution was dispensed at 100 µl/well and stored in an incubator at 37°C for 1 hour to induce an antigen-antibody reaction. After the culture solution was completely removed, it was washed three times with a wash solution (0.02% Tween 20 in PBS). Secondary HRP-labeled goat anti-mouse IgG (Jackson) was diluted 1:10,000 (v/v) in blocking solution, dispensed at 100 µl/well, and stored in an incubator at 37°C for 30 minutes to induce secondary reactions. antibodies. After washing three times, TMB (Thermo) was dispensed at 80 µl/well to induce color development, and then 1.0 N was added. sulfuric acid (H ₂ SO ₄ ) to complete the reaction. Additionally, flow cytometry was performed to confirm whether the resulting anti-ICAM-1 antibody recognizes ICAM-1 expressed on the cell surface. To the prostate cancer cell line Du145 (ATCC # HTB-81), 1 x 10 ⁶ cells expressing ICAM-1, 100 µl of culture solution was added and allowed to stand at room temperature for 15 minutes to induce antibody binding. After washing with PBS, 100 µl of secondary FITC-labeled goat anti-mouse IgG (Jackson) diluted 1:100 (v/v) in PBS was added and the reaction was carried out at room temperature for 10 minutes. After washing to remove unreacted secondary antibody, reactivity was confirmed by flow cytometry. As in the test method described above, a positive antibody that binds to the ICAM-1 antigen was first selected, and a monoclonal antibody capable of fluorescent staining in the Du145 cell line was additionally selected. The selected monoclonal antibody (denoted SI9) showed high reactivity to the ICAM-1 protein antigen and the ability to efficiently bind to the surface of the Du145 cell line.

Example 2. Obtaining a chimeric monoclonal antibody to ICAM-1

2-1. Cloning of the gene sequence of an antibody to ICAM-1

The mouse monoclonal antibody SI9 gene selected in Example 1 was cloned using the Mouse Ig-Primer Set (Millipore, Cat # 69831). The hybridoma producing the SI9 monoclonal antibody developed in Example 1 was cultured, and total RNA was extracted from the fused cell line. PCR was performed using RNA extracted with the Mouse Ig-Primer Set as a template, and after inserting it into the pGem-T vector (Promega, Cat # A3600), the DNA sequence was confirmed by sequencing and the mouse antibody gene was identified through the site IMGT (www.imgt.org). The amino acid sequences of the variable regions of the heavy and light chains and the nucleic acid coding sequences of the analyzed SI9 antibody are presented in Table. 1:

Table 1

Name Subsequence SEQID NO Chimeric V _H -CDR1 SI9 GYTFTDYA 1 Chimeric V _H -CDR2 SI9 ISTYSGNT 2 Chimeric V _H -CDR3 SI9 ARSLYFGSSGFDY 3 Chimeric V _L -CDR1 SI9 QTLVYRNGNTY 4 Chimeric V _L -CDR2 SI9 KVS 5 Chimeric V _L -CDR3 SI9 SQNTHFPYT 6 Chimeric V _H SI9 (amino acid sequence) QVQLQQSGAELVRPGVSVKISCKGS GYTFTDYA LHWVKQSHAKSLEWIGV ISTYSGNT DYNQKFRGKATMTVDKSSTTAYLELARLTSEDSAIHYC ARSLYFGSSGFDY WGQGTALTVSS 7 Chimeric V _L SI9 (amino acid sequence) DVVLTQTPLSLPVNLGDQASISCRSS QTLVYRNGNTY LHWYLQKAGQSPKLLIY KVS NRFSGVPDRFSGSGSGTDFTLKISRVAEDLGVYFC SQNTHFPYT FGGGTKIKR 8 Chimeric V _H SI9 (nucleic acid coding sequence) caggtgcagctgcagcagagcggcgcggaactggtgcgcccgggcgtgagcgtgaaaattagctgcaaaggcagcggctatacctttaccgattatgcgctgcattgggtgaaacagagccatgcgaaaagcctggaatggattggcgtgattagcacctatagcggcaacaccgattataaccagaaatttcgcggcaaagcgaccatgaccgtggataaaagcagcaccaccgcgtatctggaactggcgcgcctgaccagcgaagatagcgcgattcattattgcgcgcgcagcctgtattttggcagcagcggctttgattattggggccagggcaccgcgctgaccgtgagcagctaa 9 Chimeric V _L SI9 (nucleic acid coding sequence) gatgtggtgctgacccagaccccgctgagcctgccggtgaacctgggcgatcaggcgagcattagctgccgcagcagccagaccctggtgtatcgcaacggcaacacctatctgcattggtatctgcagaaagcgggccagagcccgaaactgctgatttataaagtgagcaaccgctttagcggcgtgccggatcgctttagcggcagcggcagcggcaccgattttaccctgaaaattagccgcgtggaagcggaagatctgggcgtgtatttttgcagccagaacacccattttccgtatacctttggcggcggcaccaaaattaaacgc 10

2-2. Obtaining a chimeric antibody

Based on the amino acid sequence of the obtained mouse anti-ICAM-1 antibody SI9 above, a chimeric anti-ICAM-1 antibody was obtained.

2-2-1. Plasmid design

To express the chimeric anti-ICAM-1 antibody, a heavy chain expression plasmid and a light chain expression plasmid, respectively, were prepared. The light chain expression plasmid was generated using the pOptiVEC vector (Invitrogen) and the heavy chain expression plasmid was generated using the pcDNA3.3 vector (Invitrogen).

To express each cDNA encoding the variable region and cDNA encoding the constant region of an antibody as a contiguous amino acid sequence without additional amino acid insertion, each of the genes obtained by linking the cloned nucleic acid sequence encoding the variable region (see Table 1) was synthesized with a known nucleic acid sequence encoding a human IgG4 constant region (heavy chain; GenBank_AIC59040.1) or a nucleic acid sequence encoding a kappa constant region (light chain; GenBank_ AAA58989.1) (Bioneer). The "CPSCP" sequence of the middle hinge region of the light chain constant region was further modified to "CPPCP", similar to the IgG1 isotype, to prevent antibody shuffling. The heavy and light chain expression genes synthesized as described above were cut with restriction enzymes Xho I and Sal I, and then the light chain expression gene was ligated into the pOptiVec vector, and the heavy chain expression gene was ligated into the pcDNA3.3 vector, respectively, and a plasmid was constructed for full antibody expression (pcDNA3.3 expression plasmid-anti-ICAM-1 antibody heavy chain and pOptiVEC expression plasmid-anti-ICAM-1 antibody light chain).

2-2-2. Transformation

The resulting pcDNA3.3-anti-ICAM-1 antibody heavy chain expression plasmid and pOptiVEC-anti-ICAM-1 antibody light chain expression plasmid were transfected into CHO-derived DG44 cells (Invitrogen) to carry out the transformation process.

First, 3 days before transfection, suspended DG44 cells were cultured in MEMa medium containing 5% FBS to induce adherent cells. Transformation was performed in a 6-well plate using the ViaFect transfection regent (Promega, cat no.: E4981). The day before transformation, DG44 cells adapted to the adhesive state were obtained by subculturing at a concentration of 1 x 10 ⁵ cells/well. The amount of DNA used for transformation was used in a 1:2 combination of pcDNA3.3-anti-ICAM-1 antibody heavy chain expression plasmids and pOptiVEC-anti-ICAM-1 antibody light chain expression plasmids, respectively, 1.0 μg and 2.0 μg . The transformation was carried out within 48 hours. The transformed cell population was analyzed using a flow cytometer and the results are shown in FIG. 1. As shown in FIG. 1, it is possible to confirm the phenomenon of binding of a chimeric antibody obtained by inserting a gene encoding the variable region of a mouse antibody SI9 into a gene encoding a constant region of a human antibody with a cell line expressing ICAM-1.

Example 3 Preparation of Humanized Anti-ICAM-1 Antibody

3.1. Sequence selection of recombinant antibodies using in silico humanization

A humanized antibody sequence obtained by recombining a germline framework region encoding a human antibody gene while maintaining the amino acid sequence of each of the heavy and light chain CDRs (CDRH1: GYTFTDYA (SEQ ID NO: 1), CDRH2: ISTYSGNT (SEQ ID NO: 2) , CDRH3: ARSLYFGSSGFDY (SEQ ID NO: 3), CDRL1: QTLVYRNGNTY (SEQ ID NO: 4), CDRL2: KVS (SEQ ID NO: 5), CDRL3: SQNTHFPYT (SEQ ID NO: 6) chimeric anti-ICAM-1 antibody (SI9) were selected using the in silico method.

As a result, 24 kinds of heavy chain variable regions and 4 kinds of light chain variable regions, respectively, were selected as sequences for the humanized DNP007 antibody. The amino acid sequences of the heavy chain variable region, light chain variable region, CDR, and framework of the selected humanized antibody are shown in Table 1. 2, 3, 4 and 5 below. In Table. 2 and 3 below, the bold underlined regions are the amino acid sequences of the CDRs (CDR1, CDR2, and CDR3 in order).

table 2

Sequence of the heavy chain variable region of the humanized antibody DNP007

Classification Name Amino acid sequence SEQID NO vh VH1 QVQLVQSGAEVKKPGASVKISCKGS GYTFTDYA LHWVRQAPGQRLEWIGV ISTYSGNT DYNQKFRGRATIRDTSASTAYMELSSLRSEDTAVYYC ARSLYFGSSGFDY WGQGTALTVSS eleven VH2 QVQLVQSGAEVKKPGASVKVSCKGS GYTFTDYA LHWVRQAPGQRLEWMGV ISTYSGNT DYNQKFRGRVTMTVDTSASTAYMELSSLRSEDTAVHYC ARSLYFGSSGFDY WGQGTALTVSS 12 VH3 QVQLVQSGAEVKKPGASVKVSCKAS GYTFTDYA LHWVRQAPGQRLEWMGV ISTYSGNT DYNQKFRGRATIRDKSASTAYLELSSLRSEDTAVHYC ARSLYFGSSGFDY WGQGTALTVSS 13 VH4 QVQLVQSGAEVKKPGASVKVSCKAS GYTFTDYA LHWVRQAPGQRLEWIGV ISTYSGNT DYNQKFRGRVTMTRDTSATTAYLELSSLRSEDTAVYYC ARSLYFGSSGFDY WGQGTALTVSS 14 VH5 QVQLVQSGAEVKKPGASVKISCKAS GYTFTDYA LHWVRQAPGQRLEWMGV ISTYSGNT DYNQKFRGRVTITVDKSATTAYMELSSLRSEDTAVYYC ARSLYFGSSGFDY WGQGTALTVSS 15 VH6 QVQLVQSGAEVVKPGASVKVSCKGS GYTFTDYA LHWVRQAPGQRLEWIGV ISTYSGNT KYSQKFQGKATITRDKSASTAYLELSSLRSEDTAVYYC ARSLYFGSSGFDY WGQGTALTVSS 16 VH7 QVQLVQSGAEVKKPGASVKVSCKGS GYTFTDYA LHWVKQAPGQRLEWMGV ISTYSGNT KYSQKFQGRATMTRDTSATTAYMELSSLRSEDTAIHYC ARSLYFGSSGFDY WGQGTALTVSS 17 VH8 QVQLQQSGAEVKKPGASVKISCKGS GYTFTDYA LHWVRQAPGQSLEWMGV ISTYSGNT KYSQKFQGRATMTRDKSASTAYMELSSLRSEDTAVYYC ARSLYFGSSGFDY WGQGTALTVSS 18 VH9 QVQLQQSGAEVVKPGASVKVSCKAS GYTFTDYA LHWVRQAPGQSLEWMGV ISTYSGNT KYSQKFQGKVTITVDTSATTAYMELSSLRSEDTAVHYC ARSLYFGSSGFDY WGQGTALTVSS 19 VH10 QVQLVQSGAEVVKPGASVKISCKAS GYTFTDYA LHWVKQAPGQSLEWMGV ISTYSGNT KYSQKFQGRVTITVDTSASTAYLELSSLRSEDTAIYYC ARSLYFGSSGFDY WGQGTALTVSS 20 VH11 QVQLQQSGAEVKKPGASVKISCKAS GYTFTDYA LHWVRQAPGQRLEWIGV ISTYSGNT KYSQKFQGKVTITRDTSASTAYLELSSLRSEDTAIHYC ARSLYFGSSGFDY WGQGTALTVSS 21 VH12 QVQLQQSGAEVKKPGASVKVSCKAS GYTFTDYA LHWVKQAPGQRLEWIGV ISTYSGNT KYSQKFQGRVTMTVDKSATTAYMELSSLRSEDTAVYYC ARSLYFGSSGFDY WGQGTALTVSS 22 VH13 QVQLVQSGAEVKKPGASVKISCKGS GYTFTDYA LHWVRQAPGQRLEWMGV ISTYSGNT DYNQKFRGRVTITRDKSASTAYLELSSLRSEDTAVYYC ARSLYFGSSGFDY WGQGTALTVSS 23 VH14 QVQLVQSGAEVKKPGASVKVSCKGS GYTFTDYA LHWVRQAPGQRLEWMGV ISTYSGNT DYNQKFRGRVTITVDTSATTAYMELSSLRSEDTAVHYC ARSLYFGSSGFDY WGQGTALTVSS 24 VH15 QVQLVQSGAEVKKPGASVKVSCKGS GYTFTDYA LHWVRQAPGQRLEWIGV ISTYSGNT DYNQKFRGRVTMTRDTSASTAYMELSSLRSEDTAVHYC ARSLYFGSSGFDY WGQGTALTVSS 25 VH16 QVQLVQSGAEVKKPGASVKVSCKGS GYTFTDYA LHWVRQAPGQRLEWMGV ISTYSGNT DYNQKFRGRATMTRDTSASTAYLELSSLRSEDTAVYYC ARSLYFGSSGFDY WGQGTALTVSS 26 VH17 QVQLVQSGAEVKKPGASVKISCKGS GYTFTDYA LHWVRQAPGQRLEWMGV ISTYSGNT DYNQKFRGRATITVDTSASTAYMELSSLRSEDTAVYYC ARSLYFGSSGFDY WGQGTALTVSS 27 VH18 QVQLVQSGAEVKKPGASVKVSCKGS GYTFTDYA LHWVRQAPGQRLEWIGV ISTYSGNT DYNQKFRGRVTITRDKSATTAYMELSSLRSEDTAVYYC ARSLYFGSSGFDY WGQGTALTVSS 28 VH19 QVQLVQSGAEVKKPGASVKISCKGS GYTFTDYA LHWVRQAPGQRLEWIGV ISTYSGNT KYSQKFQGRATITVDTSATTAYLELSSLRSEDTAVYYC ARSLYFGSSGFDY WGQGTALTVSS 29 VH20 QVQLVQSGAEVKKPGASVKISCKGS GYTFTDYA LHWVRQAPGQRLEWIGV ISTYSGNT KYSQKFQGRATMTRDTSASTAYLELSSLRSEDTAVHYC ARSLYFGSSGFDY WGQGTALTVSS thirty VH21 QVQLVQSGAEVKKPGASVKVSCKGS GYTFTDYA LHWVRQAPGQRLEWIGV ISTYSGNT KYSQKFQGRVTMTVDKSATTAYLELSSLRSEDTAVYYC ARSLYFGSSGFDY WGQGTALTVSS 31 VH22 QVQLVQSGAEVKKPGASVKISCKGS GYTFTDYA LHWVRQAPGQRLEWMGV ISTYSGNT KYSQKFQGRATMTRDKSATTAYMELSSLRSEDTAVHYC ARSLYFGSSGFDY WGQGTALTVSS 32 VH23 QVQLVQSGAEVKKPGASVKVSCKGS GYTFTDYA LHWVRQAPGQRLEWMGV ISTYSGNT KYSQKFQGRATMTVDKSASTAYLELSSLRSEDTAVHYC ARSLYFGSSGFDY WGQGTALTVSS 33 VH24 QVQLVQSGAEVKKPGASVKISCKGS GYTFTDYA LHWVRQAPGQRLEWIGV ISTYSGNT KYSQKFQGRVTITVDKSATTAYMELSSLRSEDTAVHYC ARSLYFGSSGFDY WGQGTALTVSS 34

Table 3

Sequence of the light chain variable region of the humanized antibody DNP007

Classification Name Amino acid sequence SEQID NO VL VL1 DVVLTQSPLSLPVTLGQPASISCRSS QTLVYRNGNTY LHWYQQRPGQSPRLLIY KVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFC SQNTHFPYT FGGGTKLEIK 35 VL2 DVVLTQSPLSLPVTLGQPASISCRSS QTLVYRNGNTY LHWYQQRAGQSPRLLIY KVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFC SQNTHFPYT FGGGTKLEIK 36 VL3 DVVLTQTPLSSPVTLGQPASISCRSS QTLVYRNGNTY LHWYLQRAGQPPRLLIY KVS NRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYFC SQNTHFPYT FGGGTKLEIK 37 VL4 DVVLTQTPLSSPVTLGQPASISCRSS QTLVYRNGNTY LHWYQQRPGQPPRLLIY KVS NRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYFC SQNTHFPYT FGGGTKLEIK 38

Table 4

Heavy chain variable region framework sequence of the humanized antibody DNP007

Classification Subsequence SEQID NO Applicable VH number VH-FR1 QVQLQQSGAELVRPGVSVKISCKGS 39 Chimera QVQLVQSGAEVKKPGASVKISCKGS 40 VH1, VH13, VH17, VH19, VH20, VH22, VH24 QVQLVQSGAEVKKPGASVKVSCKAS 41 VH3, VH4 QVQLVQSGAEVKKPGASVKISCKAS 42 VH5 QVQLVQSGAEVVKPGASVKVSCKGS 43 VH6 QVQLQQSGAEVKKPGASVKISCKGS 44 VH8 QVQLQQSGAEVVKPGASVKVSCKAS 45 VH9 QVQLVQSGAEVVKPGASVKISCKAS 46 VH10 QVQLQQSGAEVKKPGASVKISCKAS 47 VH11 QVQLQQSGAEVKKPGASVKVSCKAS 48 VH12 VH-FR2 LHWVKQSHAKSLEWIGV 49 Chimera LHWVRQAPGQRLEWIGV 50 VH1, VH4, VH6, VH11, VH15, VH18, VH19, VH20, VH21, VH24 LHWVRQAPGQRLEWMGV 51 VH2, VH3, VH5, VH13, VH14, VH16, VH17, VH22, VH23 LHWVKQAPGQRLEWMGV 52 VH7 LHWVRQAPGQSLEWMGV 53 VH8, VH9 LHWVKQAPGQSLEWMGV 54 VH10 LHWVKQAPGQRLEWIGV 55 VH12 VH-FR3 DYNQKFRGKATMTVDKSSTTAYLELARLTSEDSAIHYC 56 Chimera DYNQKFRGRATIRDTSASTAYMELSSLRSEDTAVYYC 57 VH1 DYNQKFRGRVTMTVDTSASTAYMELSSLRSEDTAVHYC 58 VH2 DYNQKFRGRATIRDKSASTAYLELSSLRSEDTAVHYC 59 VH3 DYNQKFRGRVTMTRDTSATTAYLELSSLRSEDTAVYYC 60 VH4 DYNQKFRGRVTITVDKSATTAYMELSSLRSEDTAVYYC 61 VH5 KYSQKFQGKATITRDKSASTAYLELSSLRSEDTAVYYC 62 VH6 KYSQKFQGRATMTRDTSATTAYMELSSLRSEDTAIHYC 63 VH7 KYSQKFQGRATMTRDKSASTAYMELSSLRSEDTAVYYC 64 VH8 KYSQKFQGKVTITVDTSATTAYMELSSLRSEDTAVHYC 65 VH9 KYSQKFQGRVTITVDTSASTAYLELSSLRSEDTAIYYC 66 VH10 KYSQKFQGKVTITRDTSASTAYLELSSLRSEDTAIHYC 67 VH11 KYSQKFQGKVTITRDTSASTAYLELSSLRSEDTAIHYC 68 VH12 DYNQKFRGRVTITRDKSASTAYLELSSLRSEDTAVYYC 69 VH13 DYNQKFRGRVTITVDTSATTAYMELSSLRSEDTAVHYC 70 VH14 DYNQKFRGRVTMTRDTSASTAYMELSSLRSEDTAVHYC 71 VH15 DYNQKFRGRATMTRDTSASTAYLELSSLRSEDTAVYYC 72 VH16 DYNQKFRGRATITVDTSASTAYMELSSLRSEDTAVYYC 73 VH17 DYNQKFRGRVTITRDKSATTAYMELSSLRSEDTAVYYC 74 VH18 KYSQKFQGRATITVDTSATTAYLELSSLRSEDTAVYYC 75 VH19 KYSQKFQGRATMTRDTSASTAYLELSSLRSEDTAVHYC 76 VH20 KYSQKFQGRVTMTVDKSATTAYLELSSLRSEDTAVYYC 77 VH21 KYSQKFQGRATMTRDKSATTAYMELSSLRSEDTAVHYC 78 VH22 KYSQKFQGRATMTVDKSASTAYLELSSLRSEDTAVHYC 79 VH23 KYSQKFQGRVTITVDKSATTAYMELSSLRSEDTAVHYC 80 VH24 VH-FR4 WGQGTALTVSS 81 Chimeric, VH1, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH11, VH12, VH13, VH14, VH15, VH16, VH17, VH18, VH19, VH20, VH21, VH22, VH23, VH24

Table 5

Framework sequence of the light chain variable region of the humanized antibody DNP007

Classification Subsequence SEQID NO Applicable VL number VL-FR1 DVVLTQTPLSLPVNLGDQASISCRSS 82 Chimera DVVLTQSPLSLPVTLGQPASISCRSS 83 VL1, VL2 DVVLTQTPLSSPVTLGQPASISCRSS 84 VL3, VL4 VL-FR2 LHWYLQKAGQSPKLLIY 85 Chimera LHWYLQKAGQSPKLLI 86 VL1 LHWYQQRAGQSPRLLIY 87 VL2 LHWYLQRAGQPPRLLIY 88 VL3 LHWYQQRPGQPPRLLIY 89 VL4 VL-FR3 NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC 90 Chimera, VL1 NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFC 91 VL2 NRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYFC 92 VL3, VL4 VL-FR4 FGGGTKIKRQ 93 Chimera FGGGTKLEIKR 94 VL1, VL2, VL3, VL4

3.2. Expression and analysis of humanized recombinant antibodies

Selected antibody sequences were ligated to the human IgG4 heavy chain constant region and kappa light chain constant region, respectively, and expressed in HEK293 cells (ATCC CRL-1573) as human IgG4.

Table 6

The sequence of the constant area of the humanized antibodies DNP007

Classification Amino acid sequence SEQID NO CH ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 95 CL Tvaapsvfifpsdeqsgtasvclnnfypreakvqwkwkvdnsgnsqsvteqdstyskdstysstlskvycevthqglsspvtksfnrges 96

7 days after transfection, the humanized recombinant antibody was purified from the culture solution using KanCap A resin (Kaneca).

The purified antibody was quantified by measuring OD (optical density) at 280 nm and the results are shown in FIG. 2a-2d. As shown in FIG. 2a-2d, most of the antibodies produced regardless of the type of combined heavy and light chains showed relatively high productivity.

To analyze the purity of the purified antibody, a size exclusion HPLC (hereinafter SE-HPLC) analysis was performed using a Sepax Zenix-C SEC-300 size exclusion column (Sepax technologies) to measure the peak symmetry factor. The results obtained are shown in FIG. 3a-3d. When highly purified antibody was analyzed by SE-HPLC, one symmetrical peak was observed, but when heterogeneous substances such as a macromolecule/small molecule were included, two or more peaks were observed or a low peak symmetry factor was observed. As shown in FIG. 3a-3d, in most cases, the humanized anti-ICAM-1 antibody showed a relatively high peak symmetry factor.

3-3. Selection of humanized antibodies according to physicochemical/biological properties

3-3-1. Screening of highly stable humanized antibodies

3-3-1-1. Primary screening of highly stable humanized antibodies

In order to preferentially select antibodies with high stability, humanized antibodies were selected showing resistance to heat denaturation by leaving the antibodies under severe high temperature conditions and observing changes in their physical properties.

The measurement of heat denaturation was confirmed by a binding experiment using 8-anilino-1-naphthalenesulfonic acid (hereinafter ANS; Sigma). ANS is a compound that can confirm the presence or absence of protein denaturation because it binds to a hydrophobic site exposed during protein denaturation and emits light at a wavelength of 470 nm.

The 96 kinds of humanized recombinant antibodies obtained in Example 2-1 were constantly diluted to a concentration of 0.2 mg/ml using PBS (phosphate buffered saline) and left at a high temperature (61 °C) for a suitable time (1 hour ). 20 μl of 0.2 mg/ml ANS solution was added to 200 μl of antibody sample, mixed, reacted for 15 minutes and analyzed under excitation at 360 nm and emission at 460 nm. The wavelength of 460 nm emitted from the ANS reaction was measured with a fluorometer (BioTek, SynergyHT), and the relative score based on the H1L1 antibody (100%) was shown in FIG. 4a-4d. In the case of VH7 and VH22 heavy chains, regardless of the light chain to be combined, the fluorescence emission at 470 nm ANS seemed to increase rapidly and was confirmed to be slightly denatured by heating, however, other antibodies showed relative resistance to heating.

3-3-1-2. Secondary screening of highly stable humanized antibodies

A total of 47 humanized antibodies were initially selected showing relatively high antibody productivity, relatively high peak symmetry factor, and/or relatively high thermal stability under high temperature conditions (61°C) in FIG. 2a-2d, 3a-3d and 4a-4d (see Fig. 5).

For a more rigorous stability assessment, 47 humanized antibodies were constantly diluted to a concentration of 0.2 mg/mL, kept at 67°C for 1 hour, ANS reactivity was measured, and the relative reactivity was shown based on the H1L1 antibody in FIG. 5. Compared to ANS reactivity at 61°C (4a-4d), ANS reactivity at 67°C tended to be slightly higher, however, the 6 types of humanized combination antibodies H17L1, H4L3, H5L3, H11L3, H17L3, and H17L4 (see Table 7) showed relatively low ANS reactivity and were judged to be highly resistant to heat denaturation.

Table 7

Name of the antibody Combination of HC and LC H17L1 VH17+VL1 H4L3 VH4+VL3 H5L3 VH5+VL3 H11L3 VH11+VL3 H17L3 VH17+VL3 H17L4 VH17+VL4

3-3-2. Analysis of antigen binding

As an assessment of the stability test, as shown in table. 7, humanized antibodies of the 6 lead antibodies were selected, and the binding capacity of the 5 antibodies, except for H11L3, was compared with the parent antibody (chimeric antibody; see Example 1.3).

ICAM-1 antigen (ICAM-1; R&D System) was applied to a 96-well plate at 100 ng/well followed by blocking. The primary antibody was serially diluted twice from 80 ng/mL and bound for 1 hour at 37°C, and the secondary antibody diluted 1:10,000 with HRP-labelled goat anti-human Ig conjugate (Jackson ImmunoResearch) was pooled at 37°C in within 30 minutes. Each step was washed three times and reacted with TMB (3,3',5,5'-tetramethylbenzidine). After stopping the reaction by treatment with 1 N. H ₂ SO ₄ solution in the same amount (100 μl) as the TMB solution, the OD value was measured at 450 nm.

The binding capacity of the parent antibody and the five humanized recombinant antibodies prepared as described above to the ICAM-1 antigen is shown in FIG. 6 and in Tab. 8.

Table 8

Antibody concentration
(ng/ml) chimeric H17L1 H4L3 H5L3 H17L3 H17L4 80 1.138 1.108 1.229 1.246 1.197 1.238 40 0.724 0.802 0.832 0.862 0.848 0.983 20 0.473 0.506 0.520 0.592 0.567 0.637 10 0.298 0.297 0.305 0.35 0.344 0.349 5 0.182 0.186 0.189 0.208 0.201 0.236 2.5 0.118 0.119 0.115 0.128 0.135 0.136

All 5 types of humanized antibodies evaluated generally showed higher reactivity than the chimeric parent antibody, and among them, the antibody from the H17L4 combination showed the highest reactivity.

3-3-3. Analysis of melting temperature

In particular, the melting points of the humanized H17L1 and H17L4 antibodies exhibiting stable physical properties were compared with the parent antibody (chimeric antibody; see Example 1.3) to assess stability.

Protein Thermal Shift Dye (Lifetechnologies, #4466038) was added to the antibody sample to prepare the reaction sample, and the temperature was continuously raised from 25°C to 95°C to induce denaturation of the antibody sample. The denaturation of the antibodies was confirmed by irradiating the reaction solution with a wavelength of 580 nm and measuring the wavelength of 623 nm emitted by the dye to determine the thermal shift of the protein, and the melting point was analyzed using the ViiATM 7 software. The results are shown in Table. 9 below and in FIG. 7a (chimeric parental antibody), 7b (H17L1) and 7C (H17L4):

Table 9

Melting point 1 Melting point 2 chimeric 67.26°C 71.47°C H17L1 67.14°C 82.33°C H17L4 66.91°C 82.57°C

As shown in Table. 9 and in FIG. 7A-7C, in each sample antibodies identified two melting temperatures. Melting point 1 was similarly measured at approximately 67°C for all three antibodies, including the chimeric parent antibody, however, melting point 2 was 10°C or more in the two humanized antibodies compared to the chimeric parent antibody. This means that the 2 types of humanized antibodies have significant resistance to heat denaturation compared to the chimeric parental antibodies.

3-3-4. Affinity measurement

The Octet (Fortebio) system was used to compare the affinity of humanized antibodies H17L1 and H17L4 in relation to antigen with parental antibodies. Each of the three antibodies was attached to the AR2G (Fortebio) reactionary to the amine of the amino and 5 different concentrations of the ICAM-1 antigen solution were added to indict the antigen and antibodies reaction. Using the results of the reaction of antigen and antibodies, they measured the binding constant (Kon) and the dissociation constant (KOFF) and calculated affinity (KD), the results are shown in table. 10 and in FIG. 8:

Table 10

Kon(1/Ms) Koff(1/s) Rmax(nm) KD(M) Full X ² Full R ² chimeric 1.65× ¹⁰⁵ 1.69 × 10 ^-3 0.3511 1.03 × 10 ^-8 0.0635 0.9869 H17L1 2.49× ¹⁰⁵ 8.53 × 10 ^-4 0.4283 3.43 × 10 ^-9 0.0487 0.9927 H17L4 2.56× ¹⁰⁵ 9.29 × 10 ^-4 0.4282 3.62 × 10 ^-9 0.1555 0.9702

As shown in Table. 10 and in FIG. 8, the affinity (KD) of the chimeric parent antibody was 1.03 x 10 ^-8 M and that of the humanized antibodies H17L1 and H17L4 were 3.43 x 10 ^-9 and 3.62 x 10 ^-9 M, respectively. It was possible to confirm the increased affinity of the humanized antibody.

Example 4 Peripheral Blood Immunomodulatory Efficacy Test ( in vitro )

4-1. Isolation of peripheral blood mononuclear cells and measurement of changes in IFNg

To confirm the immunomodulatory efficacy of the antibodies provided herein, peripheral blood mononuclear cells were treated with the DNP007 antibody (H17L4 antibody) obtained in Example 3, and the level of secretion of IFN-gamma, a typical pro-inflammatory cytokine, was measured. Blood was collected from healthy volunteers and peripheral blood mononuclear cells were isolated using Ficoll-Paque Plus (GE healthcare, no. 17144002). Isolated peripheral blood mononuclear cells were suspended in RPMI medium supplemented with 10% FBS, and GM-CSF (Creagene) and IL-4 (Creagene) were added at a concentration of 100 ng/ml, respectively, so that dendritic cells could differentiate from monocytes. The DNP007 antibody was added to peripheral blood mononuclear cells at a concentration of 10.0 μg/ml.

To confirm the effectiveness of the DNP007 antibody against monocyte progenitor dendritic cells, the isolated peripheral blood mononuclear cells were cultured in a culture dish for 2 hours to induce adhesion to the culture dish, and then the adherent cells and suspended cells were separated respectively, GM-CSF and IL-4 at a concentration of 100 ng/ml, respectively, and along with this was added the antibody DNP007 (10.0 μg/ml).

On culture day 6, peripheral blood mononuclear cells, adherent cells, and suspension cells were washed, respectively, and lipopolysaccharides (LPS, Sigma-Aldrich, No. L2630) were added at a concentration of 5.0 μg/ml, and cultured. The next day, the culture solution was taken and the concentration of secreted IFN-gamma was measured using the Human IFN gamma Uncoated ELISA kit (Invitrogen, no. 88-7316-77).

In the group of samples treated with DNP007 antibody, under all conditions of peripheral blood mononuclear cells, adherent cells and suspended cells, the level of INF-gamma secretion was markedly reduced (Table 11). These results were perceived as a phenomenon that occurs when the DNP007 antibody suppresses the immune cell activation mechanism by LPS.

Table 11

Treatment IFN-gamma (pg/ml) Relative change (%) All PBMCs (-) 559.7 100% DNP007 (10.0 µg/ml) 166.9 29.8% Adhesive PBMCs (-) 612.7 100% DNP007 (10.0 µg/ml) 425.9 69.5% Suspended PBMCs (-) 513.9 100% DNP007 (10.0 µg/ml) 130.0 25.3%

4-2. Analysis of the effect of the DNP007 antibody on peripheral blood mononuclear cells - analysis of the gene expression profile (transcriptome analysis by RNA sequencing)

To determine whether the DNP007 immune response is regulated by dendritic cells alone or by contact with immune cells, including dendritic cells and T cells, human peripheral blood mononuclear cells were isolated and separated into 5 types of peripheral blood groups and 100 ng/ml GM was added. -CSF and 100 ng/ml IL-4. Cells were obtained by culturing for 6 days under conditions with or without the addition of 10 μg/ml of DNP007 antibody. To induce transition to mature dendritic cells, cells were washed on day 6 of culture and treated with 5 μg/ml LPS (Sigma-Aldrich) for one day to obtain cells for each group, total RNA was isolated, and expression profiles of analyzed genes were determined.

(1) All PBMCs: human peripheral blood mononuclear cells were isolated from the blood of healthy volunteers by concentration gradient centrifugation with Ficoll-Paque (GE Healthcare).

(2) Dendritic cells derived from CD14+ monocytes: CD14+ monocytes were isolated from human peripheral blood mononuclear cells separated by concentration gradient centrifugation of blood from healthy volunteers with Ficoll-Paque (GE Healthcare), by magnetic separation (using magnetic beads) .

(3) CD14-depleted PBMCs: CD14+ monocytes from (2) were separated by magnetic separation (using magnetic beads) and the remaining peripheral blood population was used.

(4) Attached PBMC: Human peripheral blood mononuclear cells isolated by concentration gradient centrifugation with Ficoll-Paque (GE Healthcare) from healthy volunteers were attached to the cells in an incubator at 37°C, 5% CO ₂ for 2 hours in medium RPMI supplemented with 10% FBS and then the supernatant (including suspension cells) were removed and only attached cells were separated.

(5) Used the top layer of suspension cells removed from the experiment in (4).

The results are shown in FIG. 9. As shown in FIG. 9, dendritic cells differentiated only from CD14+ monocytes (A) and peripheral blood populations without CD14+ monocytes (B) showed a gene expression profile similar to that of the control group not treated with DNP007. On the other hand, in the state (C, D, E) in which several other immune cells are present together, rather than in the state in which CD14+ derived dendritic cells exist alone, when DNP007 was added, the gene expression profile was significantly different from the gene expression profile of the control group that did not receive DNP007.

In addition, as a result of the analysis of the KEGG pathway and the biological process of GO, by dividing the upstream and downstream genes twice or more compared to the control group not treated with DNP007, into groups of genes with similar functions in these general gene expression profiles, gene expression profiles altered DNP007 mainly included genes involved in the regulation of immune responses, including inflammatory responses (FIGS. 10 and 11). A schematic diagram of a portion of cytokines among genes having a clear pattern of change is shown in FIG. 12a and 12b. As a result of analysis of upper layer gene expression of suspension cells and whole peripheral blood mononuclear cell (whole PBMC) cell samples, excluding attached cells in peripheral blood, as shown in FIG. 12a and 12b, it was found that the expression of genes for cytokines that are targets of typical autoimmune diseases such as IL6, IL17, IL23 and IL36 was suppressed by treatment with the DNP007 antibody.

Due to these results, the anti-ICAM-1 antibody DNP007 provided herein affects not only dendritic cells, but acts on the contact process or cross-reaction between dendritic cells and other immune cells, whereby it can be observed that the expression of various immune response genes regulated, resulting in an immunosuppressive response.

4-3. Analysis of the effect of the DNP007 antibody on peripheral blood mononuclear cells - cytokine analysis

In the preparation of Experiment 4-2 above, in order to confirm whether the change in gene expression showed a correlation with the actual change in protein expression, in Experiment 4-2, the supernatant was separated and the actual cytokines were quantified.

IFN-gamma, IL1-beta, IL-6, IL-10, IL-12(p70), IL-17, IL-27 were analyzed using the Human Luminex Screening assay (LXSAH, R&D) and TGF-beta was analyzed with using a TGF-beta premix assay kit (TGTBMAG-64K-03; Merck Millipore).

All cytokines analyzed as shown in FIG. 13 demonstrated a 40 to 80% reduction in the actual level of protein expression upon administration of the DNP007 antibody. This result is consistent with the result of the gene expression profile in Example 4-2, and it can be noted that the immunosuppressive response of the DNP007 antibody was reconfirmed.

In addition, even when immune system activation was induced by LPS treatment under co-culture conditions with T cells from the same individual after separation into normal human peripheral blood and differentiation into dendritic cells, the sample injected with the DNP007 antibody showed marked inhibition of immune cytokine expression at the protein level. , as shown in FIG. 14.

Example 5 Antibody Effect Test on Dendritic Cells

5-1. Assay for the induction of activity of semi-mature dendritic cells in peripheral blood mononuclear cells

In this example, the effect of the antibody provided herein on the differentiation and maturation of dendritic cells was confirmed. More specifically, after attaching CD14 microbeads (Miltenyi Biotec, 130-050-201) to peripheral blood obtained by blood collection from healthy volunteers, human peripheral blood mononuclear cells were separated using a cell harvester (Miltenyi Biotec., cat. no. 000403).

To these was added an RPMI culture solution containing 10% FBS treated with GM-CSF (Creagene) and IL-4 (Creagene) at a concentration of 100 ng/ml, respectively, to induce differentiation into immature dendritic cells (immature DC) for 5 days. On day 5 of culture, 5 μg/ml LPS (lipopolysaccharides, Sigma-Aldrich, no. L2630) was added and dendritic cells were stimulated for 24 hours to differentiate into mature dendritic cells (mature DC). DNP007 antibody (anti-H17L4 antibody) and control antibody (hIgG) provided herein were added at a concentration of 5.0 μg/ml on days 0 and 3 of the dendritic cell differentiation induction process. At differentiation day 6, expression levels of CD80 (Invitrogen, cat. no. 11-0809-42), CD86 (BD, cat. no. 555657), CD40 (BD, cat. no. 555588), CD54 (BD, cat. no. 347977) and HLA-DR (BioLegend, catalog # 307616) (antibodies to factor above in parentheses), surface dendritic cell maturation factor, were verified and compared using a flow cytometer.

The resulting expression level (mean fluorescence intensity, MFI) of each factor was converted to a relative value for the LPS-only group and is shown in FIG. 15.

As shown in FIG. 15, in the case of control antibody (hIgG) treated dendritic cells, the expression of all cofactors such as CD80, CD54, CD40 and HLA-DR was increased by LPS stimulation, it was confirmed that the expression of these cofactors was significantly reduced in the test antibody treated group DNP007 and LPS. These results show that the above antibodies limit the maturation of dendritic cells as a result of antigen stimulation and induce an immunosuppressive environment.

5-2. Pro-inflammatory cytokine secretion test in dendritic cells

To confirm whether the antibody provided herein affects the stage of differentiation and maturation of dendritic cells, human peripheral blood mononuclear cells isolated from peripheral blood were cultured with GM-CSF and IL-4 for 5 days to differentiate into dendritic cells and then stimulating factors such as LPS were added and cultured for 24 hours to induce maturation, the amount of pro-inflammatory cytokines in the culture solution was analyzed to measure the maturity of dendritic cells.

More specifically, after attaching CD14 microbeads (Miltenyi Biotec, 130-050-201) to human peripheral blood, human peripheral blood mononuclear cells were isolated using a cell harvester (Miltenyi Biotec., cat. no. 000403). To these was added an RPMI culture solution containing 10% FBS treated with GM-CSF and IL-4 at 100 ng/ml to induce differentiation into immature DCs for 5 days. On day 5 of culture, 5 μg/ml LPS was added and dendritic cells were stimulated for 24 hours to induce differentiation into mature dendritic cells (mature DC). DNP007 antibody (anti-H17L4 antibody) and control antibody (hIgG) provided herein were added at concentrations of 0.1 to 10.0 μg/ml, respectively, on days 0 and 3 of the dendritic cell differentiation induction process. On day 6 of differentiation, the amount of pro-inflammatory cytokines (IL-6 and TNF-a) in the culture solution was analyzed by ELISA technique.

On FIG. 16 shows the results obtained above. As shown in FIG. 16, in mature dendritic cells treated with control antibody, the secretion of IL-6 and TNF-α, which are typical pro-inflammatory cytokines, was significantly increased as a result of LPS stimulation, while in dendritic cells treated with test antibody DNP007, the ability to secrete pro-inflammatory cytokines was significantly reduced. Dendritic cell maturation refers to the process by which dendritic cells acquire the ability to act as antigen-presenting cells and mature dendritic cell functions induce antigen-specific T cell activity. As a result of treatment with the antibodies provided herein, the secretion of the pro-inflammatory cytokines IL-6 and TNF-α, which are an important indicator of the maturity of dendritic cells, was significantly inhibited, and as a result, it was confirmed that the antibodies provided herein have the function inhibition of maturation of dendritic cells.

5-3. Dendritic cell apoptosis test

To determine whether the antibody provided herein affects apoptosis at the stage of dendritic cell differentiation, the degree of death of dendritic cells differentiated and matured within 6 days according to antibody treatment was measured. The extent of apoptosis was confirmed by flow cytometry after staining with 7AAD.

More specifically, after attaching CD14 microbeads (Miltenyi Biotec, 130-050-201) to human peripheral blood, human peripheral blood mononuclear cells were isolated using a cell harvester (Miltenyi Biotec., cat. no. 000403). RPMI culture solution containing 10% FBS treated with GM-CSF and IL-4 at 100 ng/ml respectively was added to induce differentiation into immature DCs for 5 days. On day 5 of culture, 5 μg/ml LPS was added and dendritic cells were stimulated for 24 hours to differentiate mature dendritic cells. The DNP007 antibody (anti-H17L4 antibody) provided herein and the control antibody (hIgG) were added twice at a concentration of 5.0 μg/ml on days 0 and 3 of the dendritic cell differentiation induction process. On day 6 after induction of differentiation, mature dendritic cells (mature DC) were stained with 7AAD (7-amino-actinomycin D, BD, 51-68981E 5 μl/test) and analyzed with a flow cytometer.

The results obtained above are shown in FIG. 17. As shown in FIG. 17, as a result of confirming the degree of apoptosis of dendritic cells that had undergone differentiation and maturation, the control group treated with antibodies showed an apoptosis of 2.33%, while the apoptosis rate of dendritic cells treated with test antibody DNP007 was 36.3%, which indicated a higher effect of apoptosis than in the control group.

The cytotoxic effect of the test antibody on dendritic cells can be assessed as apoptosis.

5-4. Immune tolerogenic dendritic cell (tolerogenic DC) induction test

To confirm whether an antibody provided herein induces tolerogenic dendritic cells, expression of tolerogenic dendritic cell markers was confirmed by flow cytometry in semi-mature dendritic cells that had undergone differentiation and maturation for 6 days.

More specifically, the DNP007 antibody (anti-H17L4 antibody) provided herein and the control antibody (hIgG) were added to semi-mature dendritic cells that had undergone differentiation and maturation from human peripheral blood mononuclear cells for 6 days in the same manner as in Examples. 5-2 or 5-3 twice at a concentration of 5.0 μg/ml on days 0 and 3 of the process of induction of dendritic cell differentiation, and the expression of tolerogenic dendritic cell markers was confirmed by flow cytometry on day 6 after induction of differentiation.

Cell surface expression factors PDL1 (Invitrogen, cat. no. 17-5983-42), PDL2 (Miltenyi biotec, cat. no. 130-098-528), and adenosine receptor A2b (Novus, cat. no. NBP2-41312PE) (above in brackets indicated antibodies to the factor) were stained by treatment with the antibody at the appropriate concentration and reacted for 20 minutes in the refrigerator. 2 ml flow cytometry buffer (0.5% BSA, 0.01% NaN ₃ in 1x PBS) was added, centrifuged at 2200 rpm. within 3 minutes, the supernatant was removed, washed and analyzed.

Cell fluid substances, IDO (indoleamine 2,3-dioxygenase; Invitrogen, cat. no. 12-9477-42), TGF-β (R&D systems, cat. no. IC240P), IL-10 (Miltenyi biotec, cat. no. 130 -112-729) (antibodies to the factor above in brackets) were stained according to the manufacturer's instructions for the intracellular antigen staining method kit. 100 μl of IC Fixation Buffer (Invitrogen, Cat # 00-8222-49) was added to the cell pellet, the light was blocked at room temperature, and the reaction was carried out for 30 minutes to fix the cells. 2 ml of 1× permeabilization buffer (Invitrogen, Cat # 00-8333-56) was added, centrifuged at 600 g for 5 minutes and the supernatant was discarded. The appropriate concentration of each antibody was added to 100 μl of 1x permeabilization buffer, treated with the remaining cell pellet, and reacted for 30 minutes after light blocking at room temperature. 2 ml of 1× permeabilization buffer was added, centrifuged at 600 g for 5 minutes, washed, and 2 ml of flow cytometry buffer was added, followed by centrifugation to perform an additional wash. Cells were fixed by adding 1% paraformaldehyde and expression levels of each antigen were analyzed using a flow cytometer.

The results obtained are shown in Fig. 18a and 18b. As shown in FIG. 18a and 18b, dendritic cells treated with the test antibody DNP007 showed a significant increase in the expression of IDO, a typical immune tolerance factor, compared to dendritic cells treated with control antibody (control), as well as an increase in the expression levels of PDL1, PDL2 and adenosine R A2b, known as other immunosuppressive factors, compared with the control group.

Thus, it was confirmed that the expression of some factors associated with IDO and immune tolerance was increased based on the analysis of dendritic cells treated with the antibodies provided herein. In particular, IDO, expressed on dendritic cells, is known to inhibit T cell proliferation and play an important role in immune tolerance. Thus, increasing IDO expression with the antibodies provided herein suggests a high likelihood of inducing immune tolerance.

5-5. IDO expression test in dendritic cells

To determine which of the two known maturation pathways inhibited dendritic cell maturation by the antibodies provided herein, LPS and CD40L were used to induce dendritic cell maturation. Expression of IDO, a marker of tolerogenic dendritic cells, was confirmed by flow cytometry in semi-mature dendritic cells that had undergone differentiation and maturation for 6 days.

More specifically, LPS stimulation, known as the canonical pathway, was examined under the same conditions as in Examples 5-2 or 5-3 by obtaining semi-mature dendritic cells that underwent differentiation and maturation for 6 days.

In the CD40L stimulation test, known as the non-canonical pathway, immature dendritic cells were differentiated for 5 days by adding RPMI culture solution containing 10% FBS treated with GM-CSF and IL-4 at 100 ng/mL, respectively. On culture day 5, 1 μg/ml CD40L (Enzo, ALX-522-110) was added to RPMI containing 10% FBS treated with GM-CSF and 100 ng/ml IL-4 and stimulated for 48 hours to induce mature dendritic cells (mature DC). The DNP007 antibody (anti-H17L4 antibody) provided herein and the control antibody (hIgG) were treated twice at a concentration of 5.0 μg/ml on days 0 and 3 during the process of induction of dendritic cell differentiation, and CD40L was treated at 5.0 mcg/ml per day 5.

On day 7 of differentiation induction, mature dendritic cells (mature DCs) were stained with the aforementioned staining intracellular antigens and analyzed using a flow cytometer.

The results obtained are shown in Fig. 19a (canonical path) and 19b (non-canonical path). As shown in FIG. 19a and 19b, test antibody DNP007 caused a decrease in IDO expression in cells during the maturation process of dendritic cells with CD40L, while dendritic cell maturation with LPS showed a significant difference in IDO expression between individuals. As a pathway for dendritic cell maturation, there are canonical pathways induced by LPS and TNF-α stimulation and non-canonical pathways induced by CD40L. The above results show that the antibodies provided herein affect both pathways of dendritic cell maturation, specifically limiting maturation by CD40L and inducing differentiation into tolerogenic dendritic cells.

5-6. T cell immunosuppression test with water soluble substances

In order to confirm whether a factor influencing T cells of dendritic cells whose maturation is inhibited by the treatment of the antibodies provided herein, intercellular conjugation, or mediators secreted by the cells is caused, dendritic cells and T cells were cultured under different conditions, and the ability to to inhibition of T-cell activity.

More specifically, human T cells isolated using a cell harvester after attaching CD3 microbeads (Miltenyi, 130-050-201) to human peripheral blood, and mature dendritic cells (mature DC) obtained under the same conditions as in Examples 5-1, for 5-5, were cultured under separate conditions to confirm the ability to inhibit the activity of T cells. The bottom chamber of the plate, designed to pass through the culture medium, was coated with 1 μg/ml of anti-CD3 antibody (BD, 557052) and 1 μg/ml of anti-CD28 antibody (BD, 555725), and placed autologous T cells isolated from peripheral blood. At this time, autologous T cells were labeled with CFSE as a condition for confirmation of proliferation. Dendritic cells differentiated by treatment with the DNP007 antibody (anti-H17L4 antibody) provided herein were placed in the upper chamber. The two cell types were cultured for 4 days, making a common culture medium in each chamber. The number of dendritic cells and autologous T cells were treated in a ratio of 1:10.

The results obtained are shown in Fig. 20. As shown in FIG. 20, in the absence of direct contact between the two cell types, T cell proliferation was inhibited by dendritic cells treated with test antibody, and the production of interferon gamma (IFN-r), a pro-inflammatory cytokine, was reduced. The above results show that the antibody-treated dendritic cells provided herein can inhibit T cell activity and proliferation by exchanging material with T cells, and indicate that a substance secreted by dendritic cells acts as a mediator for the inhibition T cell activity, and induces immune tolerance.

5-7. T cell immunosuppression test with antibody-treated dendritic cells

To evaluate the ability to inhibit the T cell activity of dendritic cells whose maturation was inhibited by treatment with the antibodies provided herein, the activity and proliferation of T cells were confirmed under conditions in which dendritic cells and T cells were cultured together. First, dendritic cell maturation was induced using LPS, TNF-a, and CD40L, which are stimulators of two known maturation pathways, and T cell proliferation and intracellular pro-inflammatory cytokine expression were measured. More specifically, dendritic cell maturation was induced using two known maturation pathways (canonical pathway: LPS, and TNF-alpha/non-canonical pathway: CD40L). Stimulation with LPS and CD40L was performed as in Example 5-5. TNF-alpha stimulation was performed by treatment with RPMI containing 10% FBS treated with GM-CSF and IL-4 at 100 ng/ml, respectively, 13 ng/ml TNF-a, 10 ng/ml IL-1β, and 350 ng/ml PGE2 on day 5 in a state of inducing immature dendritic cells and stimulating for 48 hours differentiation into mature DCs. The DNP007 antibody (anti-H17L4 antibody) provided herein and the control antibody (hIgG) were treated twice at a concentration of 5.0 μg/ml on days 0 and 3 of the dendritic cell differentiation induction process.

To induce T cell activity, autologous T cells and mature dendritic cells differentiated in the same manner as above were added to a plate coated with 1 μg/ml anti-CD3 antibody in a 1:10 ratio. After 3 days, the expression level of intracellular inflammatory cytokines and T cell proliferation were measured. At this time, CFSE labeling was performed in a similar manner to confirm T cell proliferation.

The results obtained are shown in Fig. 21. As shown in FIG. 21, T cells cultured with control antibody-treated dendritic cells showed active cell proliferation and high expression of IFN-γ and IL-10, which are typical of pro-inflammatory cytokines. In contrast, T cells treated with maturation-limited dendritic cells by treatment with test antibody DNP007 inhibited proliferation despite being stimulated by anti-CD3 antibody and markedly reduced the secretion of pro-inflammatory cytokines.

These results indicate that the antibodies provided herein affect both dendritic cell maturation pathways (canonical pathway induced by LPS and TNF-α stimulation and non-canonical pathway induced by CD40L) and dendritic cell-sensitized T cells. , whose maturation is inhibited by limiting the proliferation and expression of pro-inflammatory cytokines. In other words, semi-mature dendritic cells induced by the antibodies provided herein inhibit the activity and proliferation of T cells.

Example 6 Effect of Antibodies on the Treatment of Immune Related Diseases ( in vivo )

6-1. Rheumatoid Arthritis Treatment Effectiveness Test

In order to confirm whether the antibody provided herein is effective in rheumatoid arthritis, a mild to moderate rheumatoid arthritis model was prepared as follows: on the first day, 4 mg of bovine type II collagen (Chodrex inc, cat no. 20021) was prepared using CFA (Sigma, Cat No. F5881) in an amount of 0.5 ml + 0.5 ml in a 1:1 volume ratio, divided into 10 parts of 0.1 ml each, and injected into the epidermis of primate animals (Javanese macaques ( Macaca Fascicularis), Paul: female, age: 2.5-5 years, body weight: 2-6 kg, source: Primgen). On days 21 and 45, the injection was carried out in the same manner, except that the second and third injections were replaced with IFA (Sigma, Cat # F5506) instead of CFA. The control group was introduced in the same way. The RA score, which measures the onset of arthritis, was assessed semi-quantitatively by the degree of swelling or red color change in each joint of the animal (FIG. 22a). The DNP007 antibody (anti-H17L4 antibody) provided herein was administered at a dose of 8 mg/kg twice a week starting from the 70th day after the first collagen treatment, and arthritis of 64 joints was evaluated according to the evaluation criteria. In addition, bovine collagen-specific antibodies were tested on the basis of the blood plasma of the administered animals. The change in bovine collagen-specific antibody was measured using the Chondrex ELISA Kit (Cat. No. 2052T).

The results obtained are shown in Fig. 22b and 22c. As a result, the arthritis score decreased in the group (n=3) treated with test DNP007 antibody compared to the non-treated group (n=2) (Fig. 22b), and the anti-collagen antibody, a direct immunological factor in the disease, was also significantly decreased (Fig. 22c). These results indicate that the antibodies provided herein are effective in the treatment of rheumatoid arthritis.

6-2. Test for the effectiveness of the treatment of graft-versus-host disease

Graft-versus-host disease (GVHD) is one of the side effects in transplant patients (recipients) of allogeneic organ (e.g. bone marrow) transplantation and a phenomenon that occurs when NK cells or T cells from a donor attack the recipient's organs. To confirm whether the antibody provided herein is effective in inhibiting graft-versus-host disease during allogeneic bone marrow transplantation, peripheral blood mononuclear cells (PBMC) were added to NOD-SCID mice, and T cell recovery and survival Compared with such a control group.

More specifically, a graft-versus-host (GVHD) model was generated by irradiating NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) female mice with 1.6 Gy followed by human PBMC. Human PBMCs were obtained by centrifugation in a Leuosisp™ tube (Greiner bio-one, 227290) using Ficoll (GE Healthcare, 17544202) and separation of the middle buffy layer. Isolated human PBMCs were injected intraperitoneally at 1 x 10 ⁷ cells/200 μl per mouse.

A test group was prepared by administering the DNP007 antibody (Anti-H17L4 antibody) provided herein at a dose of 10 mg/kg twice a week for a total of 12 times for 6 weeks, and a positive control was prepared by administering CTLA-4-Ig (Bio X cell, BE0099) at 10 mg/kg twice a week for a total of 12 times for 6 weeks. G1 (n=3) is the negative control group without PBMC administration, and G2 (n=7) is the negative control group in which GVHD is induced and PBS is administered as vehicle. In addition, G3 (n=7) is the positive control group injected with CTLA-4-Ig, and G4 (n=7) is the test group injected with test antibody DNP007. Measurement of GVHD induction and treatment effect was recorded by evaluating five clinical criteria (weight loss, posture, activity, coat, skin).

The results obtained are shown in FIG. 23a and 23b. As a result, it was confirmed that the test group (G4) administered with the antibody provided herein had a significantly improved survival rate than the negative control group G2 (Fig. 23a), and it was confirmed that the recovery of human T cells was usually carried out effectively (Fig. 23b). These results show that the antibody provided herein has the effect of preventing graft-versus-host disease that occurs during allogeneic bone marrow transplantation and allows recovery of normal donor T cells.

--->

SEQUENCE LIST

<110> DINONA

<120> ANTIBODY SPECIFIC BINDING TO ICAM-1 AND ITS USE

<130>OPP20195065KR

<150> KR 10-2018-0173725

<151> December 31, 2018

<160> 107

<170> KopatentIn 3.0

<210> 1

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-CDR1)

<400> 1

Gly Tyr Thr Phe Thr Asp Tyr Ala

15

<210> 2

<211> 8

<212> PROTEIN

<123> Artificial sequence

<220>

<223> Synthetic (VH-CDR2)

<400> 2

Ile Ser Thr Tyr Ser Gly Asn Thr

15

<210> 3

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-CDR3)

<400> 3

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr

1 5 10

<210> 4

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-CDR1)

<400> 4

Gln Thr Leu Val Tyr Arg Asn Gly Asn Thr Tyr

1 5 10

<210> 5

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-CDR2)

<400> 5

Lys Val Ser

1

<210> 6

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-CDR3)

<400> 6

Ser Gln Asn Thr His Phe Pro Tyr Thr

15

<210> 7

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (chimeric VH)

<400> 7

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Val

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile His Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 8

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (chimeric VL)

<400> 8

Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Asn Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Leu Val Tyr Arg

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Ala Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Asn

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Ile Lys Arg

100 105 110

<210> 9

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic (nucleic acid sequence,

encoding chimeric VH)

<400> 9

caggtgcagc tgcagcagag cggcgcggaa ctggtgcgcc cgggcgtgag cgtgaaaatt 60

agctgcaaag gcagcggcta tacctttacc gattatgcgc tgcattgggt gaaacagagc 120

catgcgaaaa gcctggaatg gattggcgtg attagcacct atagcggcaa caccgattat 180

aaccagaaat ttcgcggcaa agcgaccatg accgtggata aaagcagcac caccgcgtat 240

ctggaactgg cgcgcctgac cagcgaagat agcgcgattc attattgcgc gcgcagcctg 300

tattttggca gcagcggctt tgattattgg ggccagggca ccgcgctgac cgtgagcagc 360

taa 363

<210> 10

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic (nucleic acid sequence encoding chimeric VL)

<400> 10

gatgtggtgc tgacccagac cccgctgagc ctgccggtga acctgggcga tcaggcgagc 60

attagctgcc gcagcagcca gaccctggtg tatcgcaacg gcaacaccta tctgcattgg 120

tatctgcaga aagcggggcca gagcccgaaa ctgctgattt

agcggcgtgc cggatcgctt tagcggcagc ggcagcggca ccgattttac cctgaaaatt 240

agccgcgtgg aagcggaaga tctgggcgtg tatttttgca gccagaacac ccattttccg 300

tatacctttg gcggcggcac caaaattaaa cgc 333

<210> 11

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH1)

<400> 11

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Arg Ala Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 12

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH2)

<400> 12

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Arg Val Thr Met Thr Val Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val His Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 13

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH3)

<400> 13

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Arg Ala Thr Ile Thr Arg Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val His Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 14

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH4)

<400> 14

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ala Thr Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 15

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH5)

<400> 15

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Arg Val Thr Ile Thr Val Asp Lys Ser Ala Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 16

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH6)

<400> 16

Gln Val Gln Leu Val Gln Ser Gly Glu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Sers Lys Gly Ser Gly Thr Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Arg Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 17

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH7)

<400> 17

Gln Val Gln Leu Val Gln Ser Gly Glu Val Lys les Pro Gly Ala

1 5 10 15

Ser Val Lys Val Sers Lys Gly Ser Gly Thr Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Lys Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Met Thr Arg Asp Thr Ser Ala Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Ile His Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 18

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH8)

<400> 18

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Met

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Met Thr Arg Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 19

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH9)

<400> 19

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Met

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Lys Val Thr Ile Thr Val Asp Thr Ser Ala Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val His Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 20

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH10)

<400> 20

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Lys Gln Ala Pro Gly Gln Ser Leu Glu Trp Met

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Val Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 21

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH11)

<400> 21

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Lys Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Ile His Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 22

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH12)

<400> 22

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Lys Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Ala Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 23

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH13)

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Arg Val Thr Ile Thr Arg Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 24

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH14)

<400> 24

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Arg Val Thr Ile Thr Val Asp Thr Ser Ala Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val His Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 25

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH15)

<400> 25

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val His Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 26

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH16)

<400> 26

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Arg Ala Thr Met Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 27

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH17)

<400> 27

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Arg Ala Thr Ile Thr Val Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 28

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH18)

<400> 28

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Arg Val Thr Ile Thr Arg Asp Lys Ser Ala Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 29

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH19)

<400> 29

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Ile Thr Val Asp Thr Ser Ala Thr Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 30

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH20)

<400> 30

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Met Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val His Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 31

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH21)

<400> 31

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Ala Thr Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 32

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH22)

<400> 32

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Met Thr Arg Asp Lys Ser Ala Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val His Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 33

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH23)

<400> 33

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Met Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val His Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 34

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH24)

<400> 34

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Val Asp Lys Ser Ala Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val His Tyr Cys

85 90 95

Ala Arg Ser Leu Tyr Phe Gly Ser Ser Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Leu Thr Val Ser Ser

115 120

<210> 35

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL1)

<400> 35

Asp Val Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Leu Val Tyr Arg

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Asn

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 36

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL2)

<400> 36

Asp Val Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Leu Val Tyr Arg

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Ala Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Asn

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 37

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL3)

<400> 37

Asp Val Val Leu Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Leu Val Tyr Arg

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Arg Ala Gly Gln Pro

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Asn

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 38

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL4)

<400> 38

Asp Val Val Leu Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Leu Val Tyr Arg

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Pro

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser asn Arg Phe Ser Gly Val Pro

50 55 60

ASP Arg Phe Ser Gly Ser Gly Ala Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Asn

85 90 95

Thr HIS PHE PRE THR PHE GLY GLY THR LYS LEU GLU ILE LYS

100 105 110

<210> 39

<211> 25

<212> PROTEIN

<123> Artificial sequence

<220>

<223> Synthetic (VH-FR1)

<400> 39

GLN VAL GLN LEU GLN GLN SER GLU GLU LEU VAL Arg Pro Gly Val

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser

20 25

<210> 40

<211> 25

<212> PROTEIN

<123> Artificial sequence

<220>

<223> Synthetic (VH-FR1)

<400> 40

Gln Val Gln Leu Val Gln Ser Gly Glu Val Lys les Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser

20 25

<210> 41

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR1)

<400> 41

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

<210> 42

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR1)

<400> 42

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser

20 25

<210> 43

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR1)

<400> 43

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Gly Ser

20 25

<210> 44

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR1)

<400> 44

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser

20 25

<210> 45

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR1)

<400> 45

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

<210> 46

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR1)

<400> 46

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser

20 25

<210> 47

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR1)

<400> 47

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser

20 25

<210> 48

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR1)

<400> 48

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

<210> 49

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR2)

<400> 49

Leu His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile Gly

1 5 10 15

Val

<210> 50

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR2)

<400> 50

Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile Gly

1 5 10 15

Val

<210> 51

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR2)

<400> 51

Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met Gly

1 5 10 15

Val

<210> 52

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR2)

<400> 52

Leu His Trp Val Lys Gln Ala Pro Gly Gln Arg Leu Glu Trp Met Gly

1 5 10 15

Val

<210> 53

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR2)

<400> 53

Leu His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Met Gly

1 5 10 15

Val

<210> 54

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR2)

<400> 54

Leu His Trp Val Lys Gln Ala Pro Gly Gln Ser Leu Glu Trp Met Gly

1 5 10 15

Val

<210> 55

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR2)

<400> 55

Leu His Trp Val Lys Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile Gly

1 5 10 15

Val

<210> 56

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 56

Asp Tyr Asn Gln Lys Phe Arg Gly Lys Ala Thr Met Thr Val Asp Lys

1 5 10 15

Ser Ser Thr Thr Ala Tyr Leu Glu Leu Ala Arg Leu Thr Ser Glu Asp

20 25 30

Ser Ala Ile His Tyr Cys

35

<210> 57

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 57

Asp Tyr Asn Gln Lys Phe Arg Gly Arg Ala Thr Ile Thr Arg Asp Thr

1 5 10 15

Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 58

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 58

Asp Tyr Asn Gln Lys Phe Arg Gly Arg Val Thr Met Thr Val Asp Thr

1 5 10 15

Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val His Tyr Cys

35

<210> 59

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 59

Asp Tyr Asn Gln Lys Phe Arg Gly Arg Ala Thr Ile Thr Arg Asp Lys

1 5 10 15

Ser Ala Ser Thr Ala Tyr Leu Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val His Tyr Cys

35

<210> 60

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 60

Asp Tyr Asn Gln Lys Phe Arg Gly Arg Val Thr Met Thr Arg Asp Thr

1 5 10 15

Ser Ala Thr Thr Ala Tyr Leu Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 61

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 61

Asp Tyr Asn Gln Lys Phe Arg Gly Arg Val Thr Ile Thr Val Asp Lys

1 5 10 15

Ser Ala Thr Thr Ala Tyr Met Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 62

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 62

Lys Tyr Ser Gln Lys Phe Gln Gly Lys Ala Thr Ile Thr Arg Asp Lys

1 5 10 15

Ser Ala Ser Thr Ala Tyr Leu Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 63

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 63

Lys Tyr Ser Gln Lys Phe Gln Gly Arg Ala Thr Met Thr Arg Asp Thr

1 5 10 15

Ser Ala Thr Thr Ala Tyr Met Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Ile His Tyr Cys

35

<210> 64

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 64

Lys Tyr Ser Gln Lys Phe Gln Gly Arg Ala Thr Met Thr Arg Asp Lys

1 5 10 15

Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 65

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 65

Lys Tyr Ser Gln Lys Phe Gln Gly Lys Val Thr Ile Thr Val Asp Thr

1 5 10 15

Ser Ala Thr Thr Ala Tyr Met Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val His Tyr Cys

35

<210> 66

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 66

Lys Tyr Ser Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Val Asp Thr

1 5 10 15

Ser Ala Ser Thr Ala Tyr Leu Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Ile Tyr Tyr Cys

35

<210> 67

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 67

Lys Tyr Ser Gln Lys Phe Gln Gly Lys Val Thr Ile Thr Arg Asp Thr

1 5 10 15

Ser Ala Ser Thr Ala Tyr Leu Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Ile His Tyr Cys

35

<210> 68

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 68

Lys Tyr Ser Gln Lys Phe Gln Gly Lys Val Thr Ile Thr Arg Asp Thr

1 5 10 15

Ser Ala Ser Thr Ala Tyr Leu Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Ile His Tyr Cys

35

<210> 69

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 69

Asp Tyr Asn Gln Lys Phe Arg Gly Arg Val Thr Ile Thr Arg Asp Lys

1 5 10 15

Ser Ala Ser Thr Ala Tyr Leu Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 70

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 70

Asp Tyr Asn Gln Lys Phe Arg Gly Arg Val Thr Ile Thr Val Asp Thr

1 5 10 15

Ser Ala Thr Thr Ala Tyr Met Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val His Tyr Cys

35

<210> 71

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 71

Asp Tyr Asn Gln Lys Phe Arg Gly Arg Val Thr Met Thr Arg Asp Thr

1 5 10 15

Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val His Tyr Cys

35

<210> 72

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 72

Asp Tyr Asn Gln Lys Phe Arg Gly Arg Ala Thr Met Thr Arg Asp Thr

1 5 10 15

Ser Ala Ser Thr Ala Tyr Leu Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 73

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 73

Asp Tyr Asn Gln Lys Phe Arg Gly Arg Ala Thr Ile Thr Val Asp Thr

1 5 10 15

Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 74

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 74

Asp Tyr Asn Gln Lys Phe Arg Gly Arg Val Thr Ile Thr Arg Asp Lys

1 5 10 15

Ser Ala Thr Thr Ala Tyr Met Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 75

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 75

Lys Tyr Ser Gln Lys Phe Gln Gly Arg Ala Thr Ile Thr Val Asp Thr

1 5 10 15

Ser Ala Thr Thr Ala Tyr Leu Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 76

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 76

Lys Tyr Ser Gln Lys Phe Gln Gly Arg Ala Thr Met Thr Arg Asp Thr

1 5 10 15

Ser Ala Ser Thr Ala Tyr Leu Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val His Tyr Cys

35

<210> 77

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 77

Lys Tyr Ser Gln Lys Phe Gln Gly Arg Val Thr Met Thr Val Asp Lys

1 5 10 15

Ser Ala Thr Thr Ala Tyr Leu Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 78

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 78

Lys Tyr Ser Gln Lys Phe Gln Gly Arg Ala Thr Met Thr Arg Asp Lys

1 5 10 15

Ser Ala Thr Thr Ala Tyr Met Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val His Tyr Cys

35

<210> 79

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 79

Lys Tyr Ser Gln Lys Phe Gln Gly Arg Ala Thr Met Thr Val Asp Lys

1 5 10 15

Ser Ala Ser Thr Ala Tyr Leu Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val His Tyr Cys

35

<210> 80

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<400> 80

Lys Tyr Ser Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Val Asp Lys

1 5 10 15

Ser Ala Thr Thr Ala Tyr Met Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val His Tyr Cys

35

<210> 81

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR4)

<400> 81

Trp Gly Gln Gly Thr Ala Leu Thr Val Ser Ser

1 5 10

<210> 82

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR1)

<400> 82

Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Asn Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser

20 25

<210> 83

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR1)

<400> 83

Asp Val Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser

20 25

<210> 84

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR1)

<400> 84

Asp Val Val Leu Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser

20 25

<210> 85

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR2)

<400> 85

Leu His Trp Tyr Leu Gln Lys Ala Gly Gln Ser Pro Lys Leu Leu Ile

1 5 10 15

Tyr

<210> 86

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR2)

<400> 86

Leu His Trp Tyr Leu Gln Lys Ala Gly Gln Ser Pro Lys Leu Leu Ile

1 5 10 15

<210> 87

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR2)

<400> 87

Leu His Trp Tyr Gln Gln Arg Ala Gly Gln Ser Pro Arg Leu Leu Ile

1 5 10 15

Tyr

<210> 88

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR2)

<400> 88

Leu His Trp Tyr Leu Gln Arg Ala Gly Gln Pro Pro Arg Leu Leu Ile

1 5 10 15

Tyr

<210> 89

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR2)

<400> 89

Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

1 5 10 15

Tyr

<210> 90

<211> 36

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR3)

<400> 90

Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly

20 25 30

Val Tyr Phe Cys

35

<210> 91

<211> 36

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR3)

<400> 91

Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly

20 25 30

Val Tyr Phe Cys

35

<210> 92

<211> 36

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR3)

<400> 92

Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly

1 5 10 15

Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly

20 25 30

Val Tyr Phe Cys

35

<210> 93

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR4)

<400> 93

Phe Gly Gly Gly Thr Lys Ile Lys Arg Gln

1 5 10

<210> 94

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR4)

<400> 94

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

1 5 10

<210> 95

<211> 327

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (heavy chain)

<400> 95

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 96

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (light chain)

<400> 96

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

1 5 10 15

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

20 25 30

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

35 40 45

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

65 70 75 80

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

85 90 95

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 97

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR1)

<220>

<221> MOD_RES

<222> (5)

<223> Xaa is Gln or Val

<220>

<221> MOD_RES

<222> (11)

<223> Xaa represents Leu or Val

<220>

<221> MOD_RES

<222> (12)

<223> Xaa is Val or Lys

<220>

<221> MOD_RES

<222> (13)

<223> Xaa is Arg or Lys

<220>

<221> MOD_RES

<222> (16)

<223> Xaa represents Val or Ala

<220>

<221> MOD_RES

<222> (20)

<223> Xaa represents Ile or Val

<220>

<221> MOD_RES

<222> (24)

<223> Xaa represents Gly or Ala

<400> 97

Gln Val Gln Leu Xaa Gln Ser Gly Ala Glu Xaa Xaa Xaa Pro Gly Xaa

1 5 10 15

Ser Val Lys Xaa Ser Cys Lys Xaa Ser

20 25

<210> 98

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR1)

<220>

<221> MOD_RES

<222> (5)

<223> Xaa is Val or Gln

<220>

<221> MOD_RES

<222> (12)

<223> Xaa is Lys or Val

<220>

<221> MOD_RES

<222> (20)

<223> Xaa represents Ile or Val

<220>

<221> MOD_RES

<222> (24)

<223> Xaa represents Gly or Ala

<400> 98

Gln Val Gln Leu Xaa Gln Ser Gly Ala Glu Val Xaa Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Xaa Ser Cys Lys Xaa Ser

20 25

<210> 99

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR2)

<220>

<221> MOD_RES

<222> (5)

<223> Xaa is Lys or Arg

<220>

<221> MOD_RES

<222> (7)

<223> Xaa represents Ser or Ala

<220>

<221> MOD_RES

<222> (8)

<223> Xaa is His or Pro

<220>

<221> MOD_RES

<222> (9)

<223> Xaa represents Ala or Gly

<220>

<221> MOD_RES

<222> (10)

<223> Xaa is Lys or Gln

<220>

<221> MOD_RES

<222> (11)

<223> Xaa is Ser or Arg

<220>

<221> MOD_RES

<222> (15)

<223> Xaa represents Ile or Met

<400> 99

Leu His Trp Val Xaa Gln Xaa Xaa Xaa Xaa Xaa Leu Glu Trp Xaa Gly

1 5 10 15

Val

<210> 100

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR2)

<220>

<221> MOD_RES

<222> (5)

<223> Xaa is Arg or Lys

<220>

<221> MOD_RES

<222> (11)

<223> Xaa is Arg or Ser

<220>

<221> MOD_RES

<222> (15)

<223> Xaa represents Ile or Met

<400> 100

Leu His Trp Val Xaa Gln Ala Pro Gly Gln Xaa Leu Glu Trp Xaa Gly

1 5 10 15

Val

<210> 101

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<220>

<221> MOD_RES

<222> (1)

<223> Xaa is Asp or Lys

<220>

<221> MOD_RES

<222> (3)

<223> Xaa represents Asn or Ser

<220>

<221> MOD_RES

<222> (7)

<223> Xaa is Arg or Gln

<220>

<221> MOD_RES

<222> (9)

<223> Xaa is Lys or Arg

<220>

<221> MOD_RES

<222> (10)

<223> Xaa represents Ala or Val

<220>

<221> MOD_RES

<222> (12)

<223> Xaa represents Met or Ile

<220>

<221> MOD_RES

<222> (14)

<223> XAA is Val or Arg

<220>

<221> MOD_RES

<222> (16)

<223> XAA is Lys or Thr

<220>

<221> MOD_RES

<222> (18)

<223> XAA is Ser or Ala

<220>

<221> MOD_RES

<222> (19)

<223> XAA is Thr or Ser

<220>

<221> MOD_RES

<222> (23)

<223> XAA is Leu or Met

<220>

<221> MOD_RES

<222> (26)

<223> XAA is ALA or Ser

<220>

<221> MOD_RES

<222> (27)

<223> XAA is an arg or ser

<220>

<221> MOD_RES

<222> (29)

<223> XAA is Thr or Arg

<220>

<221> MOD_RES

<222> (33)

<223> XAA is a ser or thr

<220>

<221> MOD_RES

<222> (35)

<223> XAA is Ile or Val

<220>

<221> MOD_RES

<222> (36)

<223> Xaa is His or Tyr

<400> 101

Xaa Tyr Xaa Gln Lys Phe Xaa Gly Xaa Xaa Thr Xaa Thr Xaa Asp Xaa

1 5 10 15

Ser Xaa Xaa Thr Ala Tyr Xaa Glu Leu Xaa Xaa Leu Xaa Ser Glu Asp

20 25 30

Xaa Ala Xaa Xaa Tyr Cys

35

<210> 102

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VH-FR3)

<220>

<221> MOD_RES

<222> (1)

<223> Xaa is Asp or Lys

<220>

<221> MOD_RES

<222> (3)

<223> Xaa represents Asn or Ser

<220>

<221> MOD_RES

<222> (7)

<223> Xaa is Arg or Gln

<220>

<221> MOD_RES

<222> (9)

<223> Xaa is Arg or Lys

<220>

<221> MOD_RES

<222> (10)

<223> Xaa represents Ala or Val

<220>

<221> MOD_RES

<222> (12)

<223> Xaa represents Ile or Met

<220>

<221> MOD_RES

<222> (14)

<223> Xaa is Arg or Val

<220>

<221> MOD_RES

<222> (16)

<223> Xaa is Thr or Lys

<220>

<221> MOD_RES

<222> (19)

<223> Xaa is Ser or Thr

<220>

<221> MOD_RES

<222> (23)

<223> Xaa represents Met or Leu

<220>

<221> MOD_RES

<222> (35)

<223> Xaa represents Val or Ile

<220>

<221> MOD_RES

<222> (36)

<223> Xaa is Tyr or His

<400> 102

Xaa Tyr Xaa Gln Lys Phe Xaa Gly Xaa Xaa Thr Xaa Thr Xaa Arg Xaa

1 5 10 15

Ser Ala Xaa Thr Ala Tyr Xaa Glu Leu Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Xaa Xaa Tyr Cys

35

<210> 103

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR1)

<220>

<221> MOD_RES

<222> (7)

<223> Xaa is Thr or Ser

<220>

<221> MOD_RES

<222> (11)

<223> Xaa represents Leu or Ser

<220>

<221> MOD_RES

<222> (14)

<223> Xaa is Asn or Thr

<220>

<221> MOD_RES

<222> (17)

<223> Xaa is Asp or Gln

<220>

<221> MOD_RES

<222> (18)

<223> Xaa is Gln or Pro

<400> 103

Asp Val Val Leu Thr Gln Xaa Pro Leu Ser Xaa Pro Val Xaa Leu Gly

1 5 10 15

Xaa Xaa Ala Ser Ile Ser Cys Arg Ser Ser

20 25

<210> 104

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR1)

<220>

<221> MOD_RES

<222> (7)

<223> Xaa is Ser or Thr

<220>

<221> MOD_RES

<222> (11)

<223> Xaa represents Leu or Ser

<400> 104

Asp Val Val Leu Thr Gln Xaa Pro Leu Ser Xaa Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser

20 25

<210> 105

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR2)

<220>

<221> MOD_RES

<222> (5)

<223> Xaa represents Leu or Gln

<220>

<221> MOD_RES

<222> (7)

<223> Xaa is Lys or Arg

<220>

<221> MOD_RES

<222> (8)

<223> Xaa is Ala or Pro

<220>

<221> MOD_RES

<222> (11)

<223> Xaa is Ser or Pro

<220>

<221> MOD_RES

<222> (13)

<223> Xaa is Lys or Arg

<220>

<221> MOD_RES

<222> (17)

<223> Xaa is Tyr or absent

<400> 105

Leu His Trp Tyr Xaa Gln Xaa Xaa Gly Gln Xaa Pro Xaa Leu Leu Ile

1 5 10 15

xaa

<210> 106

<211> 36

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR3)

<220>

<221> MOD_RES

<222> (15)

<223> Xaa represents Ser or Ala

<220>

<221> MOD_RES

<222> (31)

<223> Xaa represents Leu or Val

<400> 106

Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Xaa Gly

1 5 10 15

Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Xaa Gly

20 25 30

Val Tyr Phe Cys

35

<210> 107

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic (VL-FR4)

<220>

<221> MOD_RES

<222> (7)

<223> Xaa represents Ile or Leu

<220>

<221> MOD_RES

<222> (8)

<223> Xaa is Lys or Glu

<220>

<221> MOD_RES

<222> (9)

<223> Xaa represents Arg or Ile

<220>

<221> MOD_RES

<222> (10)

<223> Xaa is Gln or Lys

<220>

<221> MOD_RES

<222> (11)

<223> Xaa is Arg or absent

<400> 107

Phe Gly Gly Gly Thr Lys Xaa Xaa Xaa Xaa Xaa

1 5 10

<---

Claims (44)

1. Anti-ICAM-1 antibody or antigen-binding fragment containing the following complementarity determining regions (CDRs): CDR-H1 containing the amino acid sequence under SEQ ID NO: 1, CDR-H2 containing the amino acid sequence under SEQ ID NO: 2, CDR-H3 containing the amino acid sequence under SEQ ID NO: 3, CDR-L1 containing the amino acid sequence under SEQ ID NO: 4, CDR-L2 containing the amino acid sequence under SEQ ID NO: 5 and CDR-L3 containing the amino acid sequence under SEQ ID NO: 6. 2. An antibody to ICAM-1 or its antigen-binding fragment according to claim 1, containing as a scaffold: heavy chain framework 1 comprising the amino acid sequence of SEQ ID NO: 97 or SEQ ID NO: 98; heavy chain framework 2 comprising the amino acid sequence of SEQ ID NO: 99 or SEQ ID NO: 100; frame 3 heavy chain containing the amino acid sequence under SEQ ID NO: 101 or SEQ ID NO: 102; heavy chain framework 4 comprising the amino acid sequence of SEQ ID NO: 81; frame 1 light chain containing the amino acid sequence under SEQ ID NO: 103 or SEQ ID NO: 104; frame 2 light chain containing the amino acid sequence under SEQ ID NO: 105; frame 3 light chain containing the amino acid sequence under SEQ ID NO: 106; And light chain framework 4 containing the amino acid sequence of SEQ ID NO: 107. 3. An antibody to ICAM-1 or its antigen-binding fragment according to claim 1, containing as a scaffold: heavy chain framework 1 comprising an amino acid sequence selected from SEQ ID NOs: 39, 40, 41, 42, 43, 44, 45, 46, 47 and 48; heavy chain framework 2 comprising an amino acid sequence selected from SEQ ID NOs: 49, 50, 51, 52, 53, 54 and 55; heavy chain framework 3 comprising an amino acid sequence selected from SEQ ID NOs: 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 , 74, 75, 76, 77, 78, 79 and 80; heavy chain framework 4 comprising the amino acid sequence of SEQID NO: 81; frame 1 light chain containing the amino acid sequence selected from SEQ ID NO: 82, 83 and 84; frame 2 light chain containing the amino acid sequence selected from SEQ ID NO: 85, 86, 87, 88 and 89; frame 3 light chain containing the amino acid sequence selected from SEQ ID NO: 90, 91 and 92; And light chain framework 4 comprising the amino acid sequence of SEQ ID NO: 93 or SEQ ID NO: 94. 4. An antibody to ICAM-1 or its antigen-binding fragment according to claim 1, containing a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 11-34; And a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 35-38. 5. Antibody to ICAM-1 or its antigennegative fragment according to any one of paragraphs. 1-4, wherein the anti-ICAM-1 antibody is an animal antibody, a chimeric antibody, or a humanized antibody. 6. Antibody to ICAM-1 or antigennegative fragment according to any one of paragraphs. 1-4, wherein the fragment is an anti-ICAM-1 antibody scFv, (scFv)2, Fab, Fab' or F(ab')2. 7. Pharmaceutical composition for the prevention or treatment of a disease mediated by immune cells, containing an antibody to ICAM-1 or antigennegative fragment according to any one of paragraphs. 1-4, wherein the immune cell-mediated disease is transplant rejection, graft-versus-host disease, asthma, obesity, type 2 diabetes mellitus, immune cell-mediated inflammation, or an autoimmune disease. 8. The pharmaceutical composition according to claim 7, where the autoimmune disease is encephalomyelitis, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, multiple sclerosis, type 1 diabetes mellitus, Crohn's disease, ulcerative colitis, Behcet's disease, Sjögren's syndrome, myasthenia gravis, scleroderma , polyarteritis nodosa, Kikuchi disease, collagenosis, Hashimoto's thyroiditis, psoriasis, vitiligo, hyperthyroidism, fibromyalgia, alopecia areata or allergies. 9. A nucleic acid molecule encoding a heavy chain variable region containing a CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, a CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 containing the amino acid sequence of SEQ ID NO: 2 SEQ ID NO: 3; light chain variable region containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6; or both of them. 10. Nucleic acid molecule encoding amino acid sequence selected from SEQ ID NOs: 7 and 11-34; amino acid sequence selected from SEQ ID NOs: 8 and 35-38; or both of them. 11. A recombinant vector containing a nucleic acid molecule according to paragraph 9 or 10 for the expression of an antibody to ICAM-1 or its antigen-binding fragment according to paragraph 1. 12. A recombinant cell containing a recombinant vector according to paragraph 11 for obtaining an antibody to ICAM-1 or its antigen-binding fragment according to paragraph 1. 13. A method for producing an antibody to ICAM-1 or its antigen-binding fragment, comprising the step of cultivating a recombinant cell according to claim 12, containing a recombinant vector containing a nucleic acid molecule encoding the amino acid sequences under SEQ ID NO: 1-6; and the step of isolating and purifying said antibody from the culture medium containing said recombinant cell. 14. Composition for the detection of ICAM-1, containing an antibody to ICAM-1 or antigennegative fragment according to any one of paragraphs. 1-4.

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