Anti-CD40L / anti-CD28 bispecific antibody and its applications

Abstract

The anti-CD40L / anti-CD28 bispecific antibody of this application contains anti-CD40L scFv and anti-CD28 scFv, does not exhibit thromboembolic side effects, prevents T cell activation due to CD28 dimerization, and can demonstrate excellent therapeutic effects for autoimmune diseases and / or graft-versus-host diseases, thus exhibiting the excellent physical properties of a bispecific antibody.

Description

[Technical Field] 【0001】 Cross-reference of related applications This application claims priority under Korean Patent Application No. 10-2023-0064576 dated 18 May 2023, and all contents disclosed in the said Korean Patent Application are incorporated herein by reference. This application relates to anti-CD40L antibodies, anti-CD28 antibodies, anti-CD40L / anti-CD28 bispecific antibodies, and their uses. [Background technology] 【0002】 There are approximately 80 types of autoimmune diseases worldwide, and it is estimated that more than 300 million people suffer from these diseases globally. Traditionally, treatment for autoimmune diseases has involved the use of immunosuppressants or anti-inflammatory drugs that suppress immune cells in general. However, these are symptomatic treatments that only suppress the final tissue damage and do not eliminate the cause of the disease, making it difficult to halt its progression. Furthermore, the use of general immunosuppressants can lead to infections as a side effect by reducing immunity to external pathogens, making their sustained use difficult. Therefore, instead of general immunosuppression, there is active development of treatments (immune tolerance inducers) that selectively suppress and eliminate the immune response to specific autoantigens, thereby suppressing autoimmune responses while maintaining the immune response to external pathogens. 【0003】 Therefore, the development of antibody therapies targeting CD40L and CD28, which are known as representative T lymphocyte costimulatory molecules, is underway. However, conventional anti-CD40L antibodies have been reported to have side effects such as the Fc region of the antibody cross-binding with FcγRIIa located on platelets, promoting platelet activation and causing thromboembolic symptoms. Furthermore, conventional anti-CD28 antibodies have the problem of activating T cells through dimerization alone. Therefore, the inventors of this application propose novel anti-CD40L antibodies, anti-CD28 antibodies, bispecific antibodies containing the aforementioned antibodies, and new applications for these as therapeutic agents for autoimmune diseases and / or graft-versus-host diseases, which solve the aforementioned problems. [Overview of the project] [Problems that the invention aims to solve] 【0004】 One embodiment provides an anti-CD40L / anti-CD28 bispecific antibody comprising the following: (1) an anti-CD40L antibody or its antigen-binding fragment as a CD40L-targeting moiety capable of specifically recognizing and / or binding to the CD40L protein, and (2) An anti-CD28 antibody or its antigen-binding fragment, which can specifically recognize and / or bind to the CD28 protein as a CD28-targeted moiety. Another embodiment provides an anti-CD40L antibody or an antigen-binding fragment thereof. Another embodiment provides an anti-CD28 antibody or an antigen-binding fragment thereof. Another embodiment provides one or more pharmaceutical compositions for prevention, improvement, or treatment selected from the group consisting of autoimmune diseases and graft-versus-host diseases, comprising the anti-CD40L / anti-CD28 bispecific antibody, the anti-CD40L antibody or its antigen-binding fragment, and / or the anti-CD28 antibody or its antigen-binding fragment. 【0005】 Other embodiments provide uses for the anti-CD40L / anti-CD28 bispecific antibody, the anti-CD40L antibody or its antigen-binding fragment, and / or the anti-CD28 antibody or its antigen-binding fragment for the prevention, improvement, or treatment of one or more conditions selected from the group consisting of autoimmune diseases and graft-versus-host diseases. Other embodiments provide uses for the anti-CD40L / anti-CD28 bispecific antibody, the anti-CD40L antibody or its antigen-binding fragment, and / or the anti-CD28 antibody or its antigen-binding fragment for the manufacture of one or more pharmaceutical compositions for prevention, improvement, or treatment selected from the group consisting of autoimmune diseases and graft-versus-host diseases. Another embodiment provides a method for preventing, improving, or treating one or more autoimmune diseases and graft-versus-host diseases, comprising the step of administering the anti-CD40L / anti-CD28 bispecific antibody, the anti-CD40L antibody or its antigen-binding fragment, and / or the anti-CD28 antibody or its antigen-binding fragment to a subject who requires prevention, improvement, or treatment of one or more autoimmune diseases and graft-versus-host diseases, selected from the group consisting of autoimmune diseases and graft-versus-host diseases. The method may further include a step of identifying a subject who requires prevention, improvement, or treatment of one or more autoimmune diseases and graft-versus-host diseases, selected from the group consisting of autoimmune diseases and graft-versus-host diseases, prior to the administration step. 【0006】 Another embodiment provides the anti-CD40L / anti-CD28 bispecific antibody, the anti-CD40L antibody or its antigen-binding fragment, and / or a polynucleotide that encodes the anti-CD28 antibody or its antigen-binding fragment. Another embodiment provides a recombinant vector comprising the polynucleotide. The recombinant vector can be used as an expression vector for the anti-CD40L / anti-CD28 bispecific antibody, the anti-CD40L antibody or its antigen-binding fragment, and / or the anti-CD28 antibody or its antigen-binding fragment, comprising a polynucleotide. 【0007】 Another embodiment provides cells comprising the anti-CD40L / anti-CD28 bispecific antibody, the anti-CD40L antibody or its antigen-binding fragment, and / or polynucleotides encoding the anti-CD28 antibody or its antigen-binding fragment. The cells may be recombinant cells transformed with a recombinant vector comprising the polynucleotide. Another embodiment provides a method for producing the anti-CD40L / anti-CD28 bispecific antibody, the anti-CD40L antibody or its antigen-binding fragment, and / or the anti-CD28 antibody or its antigen-binding fragment, comprising the step of expressing the polynucleotide in cells. The step of expressing the polynucleotide can be carried out by culturing cells containing the polynucleotide in an environment in which the polynucleotide can be expressed (for example, in a recombinant vector). [Means for solving the problem] 【0008】 Definition of Terms In the present specification, "consisting of an array", "essentially consisting of an array", or "including an array" can mean all cases including the said array, but does not intend to exclude cases including other arrays than the said array. In the present specification, the terms "protein or polypeptide comprising or consisting of an amino acid sequence recognized as SEQ ID NO." and "gene or polynucleotide comprising or consisting of a nucleic acid sequence recognized as SEQ ID NO." can refer to a protein (or polypeptide) or a gene (or polynucleotide) that consists essentially of the said amino acid sequence or nucleic acid sequence, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or more, at least 95% or more, at least 96% or more, at least 97% or more, at least 98% or more, or at least 99% or more sequence identity with the said amino acid sequence or nucleic acid sequence while maintaining its inherent activity and / or function. 【0009】 The term "antibody" as used in the present application can include a wide variety of biochemically distinguishable classes of polypeptides. Those skilled in the art will understand that the heavy chains are classified into gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), some of which have subclasses (e.g., γ1-γ4), and the light chains are classified into kappa or lambda (κ, λ). Determining the "class" of an antibody as IgG, IgM, IgA or IgE respectively is a characteristic of this chain. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, etc., are well characterized and are known to confer functional specialization. A complete antibody contains two full-length light chains and two full-length heavy chains, where each light chain can be linked to the heavy chain by a disulfide bond. Antibodies have heavy chain constant regions and light chain constant regions. The heavy chain constant region is of the gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, and these can be further typed as gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), or alpha 2 (α2). The light chain constant region may be of the kappa (κ) or lambda (λ) type. 【0010】 The term "heavy chain" refers to a full-length heavy chain or a fragment thereof that includes a variable region V that contains an amino acid sequence sufficient to provide specificity for an antigen H , and three constant regions C H1 , C H2 and C H3 , and a hinge region. The term "light chain" can refer to a full-length light chain or a fragment thereof that includes a variable region V that contains an amino acid sequence sufficient to provide specificity for an antigen L- , and a constant region C L . The term "complementary determining region (CDR)" refers to the amino acid sequences found in the hypervariable regions of the heavy or light chains of immunoglobulins. The heavy and light chains can each contain three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). CDRs can provide residues that play an important role in the binding of an antibody to an antigen or epitope. The terms "specifically binds" or "specifically recognized" are well known to those skilled in the art and indicate that an antibody and an antigen interact specifically to induce immunological activity. 【0011】 The antibody may be an animal-derived antibody (e.g., a mouse-derived antibody or a chicken-derived antibody), a chimeric antibody (e.g., a mouse-human chimeric antibody or a chicken-human chimeric antibody), or a humanized antibody. The antibody or antigen-binding fragment may be isolated from a living organism or produced non-naturally. The antibody or antigen-binding fragment may be produced recombinantly or synthetically (not spontaneously). In other examples, the antibody may be derived from (isolated from) any animal, including mammals such as humans, birds, etc. For example, the antibody may be from a human, mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken. Here, the human antibody is an antibody having the amino acid sequence of a human immunoglobulin, and includes antibodies isolated from a human immunoglobulin library, or antibodies isolated from an animal that has been transplanted to one or more human immunoglobulins and does not express endogenous immunoglobulins. The antibody may be a monoclonal antibody or a polyclonal antibody, for example, a monoclonal antibody. Monoclonal antibodies can be manufactured by methods widely known in the art. For example, they can be manufactured using the phage display technique. Animal-derived antibodies, produced by immunizing animals with a desired antigen, can generally induce immune rejection when administered to humans for therapeutic purposes. Chimeric antibodies were developed to suppress such immune rejection. Chimeric antibodies are formed by using genetic engineering techniques to replace the invariant region of an animal-derived antibody, which is responsible for the anti-isotype reaction, with the invariant region of a human antibody. While chimeric antibodies show significantly improved anti-isotype reactions compared to animal-derived antibodies, potential side effects due to anti-idiotype reactions still exist because animal-derived amino acids remain in the variable region. Humanized antibodies were developed to mitigate these side effects. These are manufactured by transplanting the CDR (complementarity-determining region), which plays a crucial role in antigen binding within the variable region of a chimeric antibody, into a human antibody framework. 【0012】 In this specification, the term “antigen-binding fragment” refers to a fragment derived from a complete immunoglobulin structure that includes a portion capable of binding to an antigen, such as a CDR. For example, an antigen-binding fragment may be, but is not limited to, scFv, (scFv)2, Fab, Fab', or F(ab')2. In the present invention, the antigen-binding fragment may be an antibody-derived fragment that includes, for example, at least one complementarity-determining region selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2. 【0013】 In one example, the antigen-binding fragment is an antigen-binding fragment without an Fc region. 、 In one example, the antibody may be in the form of a single-domain antibody lacking scFv, Fv, (scFv)2, Fab, Fab', F(ab')2, or Fc region, but is not limited to these. In one example, the antigen-binding fragment may be in the form of a monovalent antibody, or, in one example, a single-domain antibody in the form of scFv, Fv, Fab, Fab', or a monovalent antibody, but is not limited to these. 【0014】 Among the antigen-binding fragments, Fab has a structure that includes variable regions of the light chain and heavy chain, an invariant region of the light chain, and the first invariant region (CH1) of the heavy chain, and possesses one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is formed through disulfide bonds of cysteine ​​residues in the hinge region of Fab'. Fv is the smallest antibody fragment possessing only a heavy chain variable region and a light chain variable region, and recombinant techniques for producing Fv fragments are well known in the industry. In two-chain Fv, the heavy chain variable region and the light chain variable region are linked by non-covalent bonds, while in single-chain Fv, the heavy chain variable region and the single chain variable region are generally linked by covalent bonds via a peptide linker, or directly linked at the C-terminus, and thus, like two-chain Fv, they can form dimer-like structures. 【0015】 The antigen-binding fragment can be obtained using a proteolytic enzyme (for example, Fab can be obtained by restricting the entire antibody with papain, and the F(ab')2 fragment can be obtained by cleaving it with pepsin), or it can be produced by genetic engineering. The term "hinge region" refers to a region within the heavy chain of an antibody that is located between the CH1 and CH2 regions and has the function of providing flexibility to the antigen-binding site within the antibody. 【0016】 The immunoglobulins (e.g., human immunoglobulins) or antibody molecules described herein may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, IgY, etc.), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc.), or subclass of immunoglobulin molecules. In an antibody or antibody fragment, the portion excluding the CDR or variable region (e.g., the invariant region) may be derived from a human antibody, and in particular, these may be derived from, for example, IgG, IgA, IgD, IgE, IgM, or IgY, such as IgG1, IgG2, IgG3, or IgG4. 【0017】 As used herein, "peptide linker" can mean an oligopeptide containing 1 to 100 amino acids, particularly 2 to 50 amino acids, each of which can be any type of amino acid without limitation. Any conventional peptide linker can be used with appropriate modifications or without modifications to meet specific purposes. In certain embodiments, the peptide linker can contain, for example, Gly, Asn and / or Ser residues, and / or can contain neutral amino acids such as Thr and / or Ala. Amino acid sequences suitable for peptide linkers may be known. In the related art, the length of the peptide linker can be appropriately determined within the limit that the functions of the antibody and / or scFv are not affected. For example, the peptide linker can be formed of 1 to 100 amino acids, 2 to about 50 amino acids, or 5 to 25 amino acids, each of which may be selected from the group consisting of Gly, Asn, Ser, Thr and Ala. In one embodiment, the peptide linker can be represented by (G m S l ) n (where m, l and n are the numbers of "G", "S" and "(G m S l )" respectively, and each can be independently selected from integers of about 1 to about 10, particularly 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). In one example, the peptide linker can contain the amino acid sequence of GQSSRSSGGGGSSGGGGS (SEQ ID NO: 93) or GQSSRSSSGGGSSGGGGS (SEQ ID NO: 118). 【0018】 CD40L targeting moiety CD40L is a member of the TNF family of molecules primarily expressed on activated T cells (including Th0, Th1, and Th2 subtypes), and is known to form homotrimers similar to other members of this family. CD40L has also been found to be expressed on mast cells, as well as on activated basophils and eosinophils. CD40L binds to the CD40 receptor (CD40R) on antigen-presenting cells (APCs), inducing diverse effects depending on the type of target cell. Generally, CD40L acts as a co-stimulator, inducing APC activation in conjunction with T cell receptor stimulation by MHC molecules on APCs. The term "CD40L" can refer to both full-length CD40L and soluble fragments of CD40L generated from protein lysis, such as the extracellular domain form. Amino acid sequences for both the membrane-bound and soluble forms of human CD40L (e.g., Swissprot:P29965) can be obtained from known databases. 【0019】 CD40L (also known as CD154, CD40 ligand, gp39, or TBAM) may be a type II membrane glycoprotein (Swiss-ProtAcc-No P29965) of approximately 33 kDa. Furthermore, a shorter 18 kDa soluble form of CD40L exists (also known as sCD40L or soluble CD40L). These soluble forms of CD40L are generated by the proteolytic processing of membrane-bound proteins, but the cellular activity of the soluble species is known to be weak in the absence of higher oligomerization (e.g., trimerization). CD40L (Cluster of Differentiation 40 ligand) is a type II transmembrane protein with a variable molecular weight ranging from 32 to 39 kDa due to post-translational modification. CD40L belongs to the tumor necrosis factor superfamily and exists as a CD40L trimer in a sandwich extracellular structure. CD40L is mainly expressed in activated T cells, but is also known to be expressed in activated B cells and platelets. The aforementioned CD40L may be, but is not limited to, human CD40L, mouse CD40L, monkey CD40L, donkey CD40L, sheep CD40L, rabbit CD40L, goat CD40L, guinea pig CD40L, camel CD40L, horse CD40L, or chicken CD40L. 【0020】 In one example, the anti-CD40L antibody or its antigen-binding fragment can exhibit a beneficial effect of preventing thrombotic side effects by not containing the Fc region. In one example, the antigen-binding fragment of the anti-CD40L antibody may not contain an Fc region and / or may be in the form of a monovalent antibody. In one example, the antigen-binding fragment of the anti-CD40L antibody may be an antigen-binding fragment without an Fc region, and in one example, it may be in the form of scFv, Fv, (scFv)2, Fab, Fab', F(ab')2, or a single-domain antibody without an Fc region, but is not limited to these. In one example, the anti-CD40L antibody or its antigen-binding fragment may be anti-CD40L scFv. 【0021】 In one embodiment, an anti-CD40L antibody or its antigen-binding fragment may include: L-CDR1 containing the amino acid sequence of SEQ ID NO: 1, 2, or 3, L-CDR2 containing the amino acid sequence of SEQ ID NO: 4, 5, or 6, L-CDR3 containing the amino acid sequence of SEQ ID NO: 7, 8, or 9, H-CDR1 containing the amino acid sequence of SEQ ID NO: 10, 11, or 12, H-CDR2 containing the amino acid sequence of SEQ ID NO: 13, 14, or 15, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 16, 17, or 18. The amino acid sequences of the complementarity-determining regions (CDRs) of the anti-CD40L antibody or its antigen-binding fragment are exemplified in Table 1 below. 【0022】 [Table 1] 【0023】 For example, the anti-CD40L antibody or its antigen-binding fragment may include: (1) L-CDR1 containing the amino acid sequence of SEQ ID NO: 1, L-CDR2 containing the amino acid sequence of SEQ ID NO: 4, L-CDR3 containing the amino acid sequence of SEQ ID NO: 7, H-CDR1 containing the amino acid sequence of SEQ ID NO: 10, H-CDR2 containing the amino acid sequence of SEQ ID NO: 13, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 16. (2) L-CDR1 containing the amino acid sequence of SEQ ID NO: 2, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, L-CDR3 containing the amino acid sequence of SEQ ID NO: 8, H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 14, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 17, or (3) L-CDR1 containing the amino acid sequence of SEQ ID NO: 3, L-CDR2 containing the amino acid sequence of SEQ ID NO: 6, L-CDR3 containing the amino acid sequence of SEQ ID NO: 9, H-CDR1 containing the amino acid sequence of SEQ ID NO: 12, H-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 18. 【0024】 In one embodiment, an anti-CD40L antibody or its antigen-binding fragment may include: L-CDR1 containing the amino acid sequence of SEQ ID NO: 1 or 2, L-CDR2 containing the amino acid sequence of SEQ ID NO: 4 or 5, L-CDR3 containing the amino acid sequence of SEQ ID NO: 7 or 8, H-CDR1 containing the amino acid sequence of SEQ ID NO: 10 or 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 13 or 14, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 16 or 17. For example, the anti-CD40L antibody or its antigen-binding fragment may include the following: (1) L-CDR1 containing the amino acid sequence of SEQ ID NO: 1, L-CDR2 containing the amino acid sequence of SEQ ID NO: 4, L-CDR3 containing the amino acid sequence of SEQ ID NO: 7, H-CDR1 containing the amino acid sequence of SEQ ID NO: 10, H-CDR2 containing the amino acid sequence of SEQ ID NO: 13, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 16, or (2) L-CDR1 containing the amino acid sequence of SEQ ID NO: 2, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, L-CDR3 containing the amino acid sequence of SEQ ID NO: 8, H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 14, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 17. 【0025】 The anti-CD40L antibody or its antigen-binding fragment comprises a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 1, 2, or 3, L-CDR2 containing the amino acid sequence of SEQ ID NO: 4, 5, or 6, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 7, 8, or 9, and The heavy chain variable region may include H-CDR1 containing the amino acid sequence of SEQ ID NO: 10, 11, or 12, H-CDR2 containing the amino acid sequence of SEQ ID NO: 13, 14, or 15, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 16, 17, or 18. In one example, the amino acid sequence of the variable region framework of the anti-CD40L antibody or its antigen-binding fragment is illustrated in Table 2 below. 【0026】 [Table 2] 【0027】 [Table 3] 【0028】 For example, the anti-CD40L antibody or its antigen-binding fragment may include the following: Light chain variable region containing the amino acid sequence of SEQ ID NOs. 47, 48, 49, 50, or 128, and A heavy chain variable region containing the amino acid sequence of SEQ ID NOs. 51, 52, 53, 54, 55, 56, or 57. 【0029】 For example, the anti-CD40L antibody or its antigen-binding fragment may include the following: Light chain variable region containing the amino acid sequence of SEQ ID NOs. 47, 48, 49, or 128, and A heavy chain variable region containing the amino acid sequence of SEQ ID NOs. 51, 52, 53, 54, 55, or 56. 【0030】 The amino acid sequences of the variable region of the anti-CD40L antibody or its antigen-binding fragment are exemplified in Table 3 below. 【0031】 [Table 4] 【0032】 [Table 5] 【0033】 [Table 6] 【0034】 In the light chain variable region and heavy chain variable region of Table 3, each complementarity determination region is indicated by an underline. 【0035】 In one example, the anti-CD40L antibody or its antigen-binding fragment may include the following: (1) A light chain variable region containing the amino acid sequence of SEQ ID NO: 47, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 51, (2) A light chain variable region containing the amino acid sequence of SEQ ID NO: 48, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 52, 53, 54, or 55, (3) A light chain variable region containing the amino acid sequence of SEQ ID NO: 49, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 56, or (4) A light chain variable region containing the amino acid sequence of SEQ ID NO: 50, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 57. In one example, the anti-CD40L antibody or its antigen-binding fragment may include the following: (1) A light chain variable region containing the amino acid sequence of SEQ ID NO: 47, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 51, (2) Light chain variable region containing the amino acid sequence of SEQ ID NO: 48, and heavy chain variable region containing the amino acid sequence of SEQ ID NO: 52, 53, 54, or 55, (3) A light chain variable region containing the amino acid sequence of SEQ ID NO: 49, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 56. 【0036】 In one example, the anti-CD40L antibody or its antigen-binding fragment may be an scFv (single-chain variable region fragment) comprising the following: A light chain variable region comprising L-CDR1 containing the amino acid sequence of SEQ ID NO: 1, 2, or 3, L-CDR2 containing the amino acid sequence of SEQ ID NO: 4, 5, or 6, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 7, 8, or 9, and A heavy chain variable region comprising H-CDR1 containing the amino acid sequence of SEQ ID NO: 10, 11, or 12, H-CDR2 containing the amino acid sequence of SEQ ID NO: 13, 14, or 15, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 16, 17, or 18. 【0037】 In one example, the anti-CD40L scFv may include the following: A light chain variable region comprising L-CDR1 containing the amino acid sequence of SEQ ID NO: 1 or 2, L-CDR2 containing the amino acid sequence of SEQ ID NO: 4 or 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 7 or 8, and A heavy chain variable region comprising H-CDR1 containing the amino acid sequence of SEQ ID NO: 10 or 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 13 or 14, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 16 or 17. 【0038】 In one example, the anti-CD40L scFv may include the following: Light chain variable region containing the amino acid sequence of SEQ ID NOs. 47, 48, 49, 50, or 128, and A heavy chain variable region containing the amino acid sequence of SEQ ID NOs. 51, 52, 53, 54, 55, 56, or 57. In one example, the anti-CD40L scFv may include the following: Light chain variable region containing the amino acid sequence of SEQ ID NOs. 47, 48, 49, or 128, and A heavy chain variable region containing the amino acid sequence of SEQ ID NOs. 51, 52, 53, 54, 55, or 56. 【0039】 In one example, the anti-CD40L scFv may include the following: (1) A light chain variable region containing the amino acid sequence of SEQ ID NO: 47, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 51, (2) A light chain variable region containing the amino acid sequence of SEQ ID NO: 48 or 128, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 52, 53, 54 or 55, (3) A light chain variable region containing the amino acid sequence of SEQ ID NO: 49, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 56, or (4) A light chain variable region containing the amino acid sequence of SEQ ID NO: 50, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 57. 【0040】 In one example, the anti-CD40L scFv may include the following: (1) A light chain variable region containing the amino acid sequence of SEQ ID NO: 47, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 51, (2) Light chain variable region containing the amino acid sequence of SEQ ID NO: 48 or 128, and heavy chain variable region containing the amino acid sequence of SEQ ID NO: 52, 53, 54 or 55, (3) A light chain variable region containing the amino acid sequence of SEQ ID NO: 49, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 56. 【0041】 At this time, the light chain variable region and the heavy chain variable region can be linked to each other directly (for example, without a linker) or via a peptide linker in any order. The peptide linker is as described above. For example, the anti-CD40L scFv may include a light chain variable region and a heavy chain variable region from the N'-terminus to the C'-terminus. Alternatively, the anti-CD40L scFv may include a heavy chain variable region and a light chain variable region from the N'-terminus to the C'-terminus. In one example, the anti-CD40L scFv may contain the amino acid sequence of SEQ ID NOs. 58, 59, 60, 61, 62, 63, or 64. In one example, the anti-CD40L scFv may contain the amino acid sequence of SEQ ID NOs. 58, 59, 60, 61, 62, or 63. 【0042】 CD28 Targeted Moiety The CD28 (Cluster of Differentiation 28) molecule is a 44kDa glycoprotein specifically expressed on the surface of T cells and is known to be an essential factor for T cell activity and survival. CD28 costimulation-mediated cell signaling is one of the important factors that regulate T cell activity and is essential for T cell responses to antigens and T cell-mediated B cell responses. The aforementioned CD28 may be, but is not limited to, human CD28, mouse CD28, monkey CD28, donkey CD28, sheep CD28, rabbit CD28, goat CD28, guinea pig CD28, camel CD28, horse CD28, or chicken CD28. In one example, the antigen-binding fragment of the anti-CD28 antibody has the form of a monovalent antibody, thereby preventing CD28 dimerization and preventing T cell activation. In one example, the antigen-binding fragment of the anti-CD28 antibody may not contain an Fc region and / or may be in the form of a monovalent antibody. In one example, the antigen-binding fragment of the anti-CD28 antibody may be in the form of a monovalent antibody, or, in one example, a single-domain antibody in the form of scFv, Fv, Fab, Fab', or a monovalent antibody, but is not limited to these. In one example, the antigen-binding fragment of the anti-CD28 antibody may be anti-CD28 scFv. In other embodiments, an anti-CD28 antibody or its antigen-binding fragment may include: L-CDR1 containing the amino acid sequence of SEQ ID NO: 65, L-CDR2 containing the amino acid sequence of SEQ ID NO: 66, L-CDR3 containing the amino acid sequence of SEQ ID NO: 67, H-CDR1 containing the amino acid sequence of SEQ ID NO: 68 or 69, H-CDR2 containing the amino acid sequence of SEQ ID NO: 70, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 71. 【0043】 The amino acid sequences of the complementarity-determining regions (CDRs) of the anti-CD28 antibody or its antigen-binding fragment are exemplified in Table 4 below. 【0044】 [Table 7] 【0045】 For example, the anti-CD28 antibody or its antigen-binding fragment may include the following: (1) L-CDR1 containing the amino acid sequence of SEQ ID NO: 65, L-CDR2 containing the amino acid sequence of SEQ ID NO: 66, L-CDR3 containing the amino acid sequence of SEQ ID NO: 67, H-CDR1 containing the amino acid sequence of SEQ ID NO: 68, H-CDR2 containing the amino acid sequence of SEQ ID NO: 70, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 71, or (2) L-CDR1 containing the amino acid sequence of SEQ ID NO: 65, L-CDR2 containing the amino acid sequence of SEQ ID NO: 66, L-CDR3 containing the amino acid sequence of SEQ ID NO: 67, H-CDR1 containing the amino acid sequence of SEQ ID NO: 69, H-CDR2 containing the amino acid sequence of SEQ ID NO: 70, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 71. 【0046】 The anti-CD28 antibody or its antigen-binding fragment comprises a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 65, L-CDR2 containing the amino acid sequence of SEQ ID NO: 66, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 67, and The heavy chain variable region may include H-CDR1 containing the amino acid sequence of SEQ ID NO: 68 or 69, H-CDR2 containing the amino acid sequence of SEQ ID NO: 70, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 71. In one example, the amino acid sequence of the variable region framework of the anti-CD28 antibody or its antigen-binding fragment is illustrated in Table 5 below. 【0047】 [Table 8] 【0048】 For example, the anti-CD28 antibody or its antigen-binding fragment may include the following: Light chain variable region containing the amino acid sequence of SEQ ID NOs. 87, 88, 98, or 100, and A heavy chain variable region containing the amino acid sequence of SEQ ID NOs. 89, 90, 99, or 101. The amino acid sequences of the variable region of the anti-CD28 antibody or its antigen-binding fragment are exemplified in Table 6 below. 【0049】 [Table 9] 【0050】 [Table 10] 【0051】 In the light chain variable region and heavy chain variable region of Table 6, each complementarity determination region is indicated by an underline. 【0052】 In one example, the anti-CD28 antibody or its antigen-binding fragment may include the following: (1) A light chain variable region containing the amino acid sequence of SEQ ID NO: 87, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89, (2) A light chain variable region containing the amino acid sequence of SEQ ID NO: 88, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 90, (3) Light chain variable region containing the amino acid sequence of SEQ ID NO: 98, and heavy chain variable region containing the amino acid sequence of SEQ ID NO: 99, (4) A light chain variable region containing the amino acid sequence of SEQ ID NO: 100, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 99, or (5) A light chain variable region containing the amino acid sequence of SEQ ID NO: 98, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 101. 【0053】 In one example, the anti-CD28 antibody or its antigen-binding fragment may be an scFv comprising the following: Light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 65, L-CDR2 containing the amino acid sequence of SEQ ID NO: 66, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 67, and A heavy chain variable region comprising H-CDR1 containing the amino acid sequence of SEQ ID NO: 68 or 69, H-CDR2 containing the amino acid sequence of SEQ ID NO: 70, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 71. In one example, the anti-CD28 scFv may include the following: Light chain variable region containing the amino acid sequence of SEQ ID NO: 87 or 88, and A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89 or 90. In one example, the anti-CD28 scFv may include the following: (1) A light chain variable region containing the amino acid sequence of SEQ ID NO: 87, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89, or (2) A light chain variable region containing the amino acid sequence of SEQ ID NO: 88, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 90. 【0054】 At this time, the light chain variable region and the heavy chain variable region can be linked to each other directly (for example, without a linker) or via a peptide linker in any order. The peptide linker is as described above. For example, the anti-CD28 scFv may include a light chain variable region and a heavy chain variable region extending from the N'-terminus to the C'-terminus. Alternatively, the anti-CD28 scFv may include a heavy chain variable region and a light chain variable region extending from the N'-terminus to the C'-terminus. In one example, the anti-CD28 scFv may contain the amino acid sequence of SEQ ID NO: 91 or 92. 【0055】 Anti-CD40L / anti-CD28 bispecific antibody Another embodiment provides an anti-CD40L / anti-CD28 bispecific antibody comprising the following: (1) an anti-CD40L antibody or its antigen-binding fragment as a CD40L-targeted moiety capable of specifically recognizing and / or binding to CD40L, and (2) An anti-CD28 antibody or its antigen-binding fragment as a CD28-targeted moiety capable of specifically recognizing and / or binding to CD28. The anti-CD40L / anti-CD28 bispecific antibody may include the following: (1) CD40L-targeting moieties that can specifically recognize and / or bind to CD40L include antigen-binding fragments of anti-CD40L antibodies that do not contain an Fc site (or Fc region), and (2) An antigen-binding fragment of an anti-CD28 antibody in the form of a monovalent antibody, as a CD28-targeted moisture that can specifically recognize and / or bind to CD28. In one example, the antigen-binding fragment of the anti-CD40L antibody is 、 Anti-CD40L may also be in the form of a single-domain antibody lacking the scFv, Fv, (scFv)2, Fab, Fab', F(ab')2, or Fc region. In one example, the antigen-binding fragment of the anti-CD28 antibody may be in the form of a single-domain antibody such as anti-CD28 scFv, Fv, Fab, Fab', or a monovalent antibody. 【0056】 In one example, the anti-CD40L / anti-CD28 bispecific antibody may include the following: (1) Antigen-binding fragment of an anti-CD40L antibody that does not contain the Fc region, and (2) Antigen-binding fragment of an anti-CD28 antibody in the form of a monovalent antibody. The antigen-binding fragment of the anti-CD40L antibody that does not contain the Fc region may be in the form of anti-CD40L scFv, Fv, (scFv)2, Fab, Fab', F(ab')2, or a single-domain antibody that lacks the Fc region. The antigen-binding fragment of the anti-CD28 antibody in the form of a monovalent antibody may also be in the form of an anti-CD28 scFv, Fv, Fab, Fab', or a single-domain antibody in the form of a monovalent antibody. 【0057】 The antigen-binding fragment of the anti-CD40L antibody contained in the aforementioned bispecific antibody does not contain an Fc region. As a result, it is possible to achieve advantageous effects such as exhibiting equivalent or superior binding affinity to the CD40L antigen, suppression of co-stimulatory molecules and cytokine secretion compared to conventional anti-CD40L antibodies containing an Fc region, while without showing thromboembolic side effects. Furthermore, because the antigen-binding fragment of the anti-CD28 antibody in the form of a monovalent antibody is an antigen-binding fragment in the form of a monovalent antibody, it can achieve the advantageous effect of preventing T cell activation due to CD28 dimerization. 【0058】 In one example, the anti-CD40L / anti-CD28 bispecific antibody may include the following: (1) Anti-CD40L antibody or its antigen-binding fragment, and (2) L-CDR1 containing the amino acid sequence of SEQ ID NO: 65, L-CDR2 containing the amino acid sequence of SEQ ID NO: 66, L-CDR3 containing the amino acid sequence of SEQ ID NO: 67, H-CDR1 containing the amino acid sequence of SEQ ID NO: 68 or 69, H-CDR2 containing the amino acid sequence of SEQ ID NO: 70, and An anti-CD28 antibody or its antigen-binding fragment containing H-CDR3, which includes the amino acid sequence of SEQ ID NO: 71. The anti-CD40L antibody or its antigen-binding fragment, and the anti-CD28 antibody or its antigen-binding fragment are as described above. 【0059】 In one example, the anti-CD40L / anti-CD28 bispecific antibody may include the following: (1) Antigen-binding fragment of an anti-CD40L antibody that does not contain the Fc region, and (2) Antigen-binding fragment of an anti-CD28 antibody in the form of a monovalent antibody. 【0060】 The antigen-binding fragment of the anti-CD40L antibody is L-CDR1 containing the amino acid sequence of SEQ ID NO: 1, 2, or 3, L-CDR2 containing the amino acid sequence of SEQ ID NO: 4, 5, or 6, L-CDR3 containing the amino acid sequence of SEQ ID NO: 7, 8, or 9, H-CDR1 containing the amino acid sequence of SEQ ID NO: 10, 11, or 12, H-CDR2 containing the amino acid sequence of SEQ ID NO: 13, 14, or 15, and It may contain H-CDR3 containing the amino acid sequence of SEQ ID NO: 16, 17, or 18. 【0061】 The antigen-binding fragment of the anti-CD28 antibody is L-CDR1 containing the amino acid sequence of SEQ ID NO: 65, L-CDR2 containing the amino acid sequence of SEQ ID NO: 66, L-CDR3 containing the amino acid sequence of SEQ ID NO: 67, H-CDR1 containing the amino acid sequence of SEQ ID NO: 68 or 69, H-CDR2 containing the amino acid sequence of SEQ ID NO: 70, and It may contain H-CDR3 containing the amino acid sequence of SEQ ID NO: 71. The antigen-binding fragments of the anti-CD40L antibody and the anti-CD28 antibody are as described above. 【0062】 In one example, the anti-CD40L / anti-CD28 bispecific antibody is Is the anti-CD40L antibody or its antigen-binding fragment anti-CD40L scFv? The anti-CD28 antibody or its antigen-binding fragment is anti-CD28 scFv, or The anti-CD40L antibody or its antigen-binding fragment may be anti-CD40L scFv, and the anti-CD28 antibody or its antigen-binding fragment may be anti-CD28 scFv. 【0063】 In one example, the anti-CD40L / anti-CD28 bispecific antibody may include an anti-CD40L antibody in scFv form and an anti-CD28 antibody in scFv form. The anti-CD40L scFv contained in the bispecific antibody does not contain an Fc region, and therefore, compared to conventional anti-CD40L antibodies containing an Fc region, it can achieve the advantageous effect of exhibiting equivalent or superior binding affinity to the CD40L antigen, suppression of costimulatory molecules, and suppression of cytokine secretion, while not showing thromboembolic side effects. Furthermore, since the anti-CD28 scFv contained in the bispecific antibody is a monovalent antibody, it can achieve the advantageous effect of preventing T cell activation due to CD28 dimerization. 【0064】 The bispecific antibody may include anti-CD40L scFv and anti-CD28 scFv, wherein the anti-CD40L scFv may be linked directly (e.g., without a peptide linker) or via a peptide linker to the N'-terminus, C'-terminus, or both of the anti-CD28 scFv. The anti-CD28 scFv may be linked directly (e.g., without a peptide linker) or via a peptide linker to the N'-terminus, C'-terminus, or both of the anti-CD40L scFv. In one example, the anti-CD40L scFv or anti-CD28 scFv contained in the bispecific antibody may include heavy chain variable regions and light chain variable regions in any order. For example, the scFv contained in the bispecific antibody may include light chain variable regions and heavy chain variable regions in the direction from the N' terminus to the C' terminus, and may selectively include a peptide linker between them. Alternatively, the scFv contained in the bispecific antibody may include light chain variable regions and heavy chain variable regions in the direction from the C' terminus to the N' terminus, and may selectively include a peptide linker between them. 【0065】 The anti-CD40L scFv and anti-CD28 scFv of the aforementioned bispecific antibody may be linked through the antibody's invariant region. The invariant region of the antibody may refer to the invariant region of the heavy chain (γ, δ, α, μ, or ε) or light chain (λ or κ) of the antibody (e.g., IgG, IgM, IgA, or IgE). In one example, the invariant region of the antibody may refer to the invariant region (C kappa) of the light chain of an IgG antibody. In one example, the invariant region of the antibody may include the amino acid sequence of SEQ ID NO: 95. The invariant region of the antibody can be linked to anti-CD40L scFv directly (e.g., without a peptide linker) or via a peptide linker. The invariant region of the antibody can be linked to anti-CD28 scFv directly (e.g., without a peptide linker) or via a peptide linker. 【0066】 In one example, the bispecific antibody may contain the following from the N' terminus to the C' terminus: anti-CD40L scFv, Selectively, the first peptide linker, Invariant region of antibody, Selectively, the second peptide linker, and Anti-CD28 scFv. 【0067】 In one example, the bispecific antibody may contain the following from the N' terminus to the C' terminus: anti-CD28 scFv, Selectively, the first peptide linker, Invariant region of antibody, Selectively, the second peptide linker, and Anti-CD40L scFv. The first peptide linker and the second peptide linker may be present independently or absent in the bispecific antibody, and may be identical or different from each other. 【0068】 In one example, the bispecific antibody may contain the amino acid sequence of SEQ ID NO: 96 or 97. The aforementioned bispecific antibody can achieve significantly superior physical properties (e.g., thermal stability) compared to conventional bispecific antibodies. For example, the bispecific antibody can maintain excellent binding affinity to antigens even when exposed to high temperatures for extended periods, and can exhibit excellent suppression of co-stimulatory molecules and cytokine secretion. 【0069】 Production of polynucleotides, recombinant vectors, and antibodies Other embodiments provide polynucleotides encoding the anti-CD40L / anti-CD28 bispecific antibody, the anti-CD40L antibody or its antigen-binding fragment, and / or the anti-CD28 antibody or its antigen-binding fragment. Another embodiment provides a recombinant vector containing polynucleotides. For example, the recombinant cells may be cells that have been phenotype-infected with the recombinant vector. 【0070】 Another embodiment provides a method for producing the anti-CD40L / anti-CD28 bispecific antibody, the anti-CD40L antibody or its antigen-binding fragment, and / or the anti-CD28 antibody or its antigen-binding fragment, comprising the step of expressing a polynucleotide in cells. The step of expressing a polynucleotide can be carried out by culturing cells containing the polynucleotide under conditions that allow for the expression of the polynucleotide (e.g., in a recombinant vector). The method may further include the step of separating and / or purifying the antibody or its antigen-binding fragment from the cell culture after the expression or culturing step. 【0071】 The term "vector" can refer to a means of expressing a target gene in a host cell, as exemplified by plasmid vectors, cosmid vectors, and viral vectors such as bacteriophage vectors, adenovirus vectors, and retrovirus vectors. Adenovirus vectors and recombinant vectors can consist of plasmids commonly used in the industry (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pIJ61, pLAFR1, pET series, and pUC19), phages (e.g., λgt4λB, λ-Charon, λΔz1, and M13), or manipulated viruses (e.g., SV40, etc.). 【0072】 In recombinant vectors, polynucleotides can be operably ligated to a promoter. The term “operably ligated” is intended to refer to a functional linkage between a nucleotide sequence of interest and a regulatory element (e.g., a promoter sequence). When “operably ligated,” the regulatory element can control the transcription and / or translation of the nucleotide of interest. Recombinant vectors can typically be constructed as cloning vectors or expression vectors. In the case of recombinant expression vectors, commonly available vectors in the industry can be used to express foreign proteins in plant, animal, or microbial cells. Various methods known in the industry can be used to prepare recombinant vectors. 【0073】 Recombinant vectors can be prepared accordingly for use in hosts such as prokaryotic or eukaryotic cells. For example, when a vector is constructed as an expression vector for use in a prokaryotic host, the vector may typically include a strong promoter for transcription (e.g., pLκλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for translation initiation, and a transcription / translation termination sequence. On the other hand, an expression vector for use in a eukaryotic host may include, but is not limited to, origins of replication that are operable in eukaryotic cells, such as the f1 origin of replication, SV40 origin of replication, pMB1 origin of replication, adeno origin of replication, AAV origin of replication, and BBV origin of replication. Furthermore, the expression vector may also contain a promoter typically derived from the genome of a mammalian cell (e.g., the metallothionein promoter), or a promoter derived from a mammalian virus (e.g., the late adenovirus promoter, the vaccinia virus 7.5K promoter, the SV40 promoter, the cytomegalovirus promoter, and the HSV tk promoter), and a polyadenylated sequence as a transcription termination sequence. Recombinant cells can be produced by introducing a recombinant vector into a suitable host cell. Any host cell known to the art can be used in this invention, as long as the recombinant vector can be sequentially cloned and stably expressed. Examples of prokaryotic host cells usable in this invention can be selected from Bacillus spp. such as E. coli, Bacillus subtilis and Bacillus thuringiensis, and Enterobacteriaceae strains such as Salmonella typhimurium, Serratia marcescens and various Pseudomonas genera. Eukaryotic host cells that can be used for transformation can be selected from, but are not limited to, Saccharomyce cerevisiae, insect cells and animal cells such as Sp2 / 0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN and MDCK. Polynucleotides or recombinant vectors containing them can be introduced (transfected) into host cells using methods known in the art. For example, if the host cell is a prokaryote, this transfection can be carried out using CaCl2 or electroporation. In the case of eukaryotic host cells, gene transfer can be achieved using, but is not limited to, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, or particle impaction. To select transformed host cells, a phenotype associated with a selection marker can be used, following methods known in the art. For example, if the selection marker is a gene that confers resistance to a specific antibiotic, host cells can be grown in culture medium in the presence of the antibiotic to select transformants of interest. Another embodiment provides a method for producing bispecific antibodies, comprising the step of expressing polynucleotides or recombinant vectors in host cells. In one embodiment, the production method may include culturing recombinant cells having polynucleotides or recombinant vectors therein, and optionally isolating and / or purifying antibodies from the culture medium. 【0074】 Pharmaceutical use Other embodiments provide pharmaceutical uses for the prevention, improvement, or treatment of one or more autoimmune diseases and graft-versus-host diseases selected from the group consisting of the anti-CD40L antibody, the anti-CD28 antibody, or the anti-CD40L / anti-CD28 bispecific antibody. More specifically, this application provides one or more pharmaceutical compositions for prevention, improvement, or treatment selected from the group consisting of autoimmune diseases and graft-versus-host diseases, comprising the anti-CD40L antibody, the anti-CD28 antibody, or the anti-CD40L / anti-CD28 bispecific antibody as an active ingredient. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. 【0075】 Other embodiments provide uses for the anti-CD40L / anti-CD28 bispecific antibody, the anti-CD40L antibody or its antigen-binding fragment, and / or the anti-CD28 antibody or its antigen-binding fragment for the prevention, improvement, or treatment of one or more conditions selected from the group consisting of autoimmune diseases and graft-versus-host diseases. Other embodiments provide uses for the anti-CD40L / anti-CD28 bispecific antibody, the anti-CD40L antibody or its antigen-binding fragment, and / or the anti-CD28 antibody or its antigen-binding fragment for the manufacture of one or more pharmaceutical compositions for prevention, improvement, or treatment selected from the group consisting of autoimmune diseases and graft-versus-host diseases. 【0076】 Another embodiment provides a method for preventing, improving, or treating one or more autoimmune diseases and graft-versus-host diseases, comprising the step of administering the anti-CD40L / anti-CD28 bispecific antibody, the anti-CD40L antibody or its antigen-binding fragment, and / or the anti-CD28 antibody or its antigen-binding fragment to a subject who requires prevention, improvement, or treatment of one or more autoimmune diseases and graft-versus-host diseases, selected from the group consisting of autoimmune diseases and graft-versus-host diseases. The method may further include a step of identifying a subject who requires prevention, improvement, or treatment of one or more autoimmune diseases and graft-versus-host diseases, selected from the group consisting of autoimmune diseases and graft-versus-host diseases, prior to the administration step. 【0077】 In this application, “prevention” means all actions that suppress or delay the onset of a disease by administration of the composition according to one example; “treatment” means all actions that improve or beneficially alter the symptoms of an individual suspected of having a disease or who has developed a disease by administration of the composition according to one example; and “improvement” can mean all actions that at least reduce the severity of symptoms, for example, parameters related to the state in which the disease is treated by administration of the composition according to one example. The disease may mean autoimmune diseases and / or graft-versus-host diseases. In one example, the autoimmune disease and / or graft-versus-host disease may be caused by the overexpression and / or overactivation of T cells. 【0078】 The aforementioned autoimmune diseases include rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, type 1 diabetes, Crohn's disease, scleroderma, Sjögren's syndrome, psoriasis, inflammatory bowel disease, ulcerative colitis, ankylosing spondylitis, interstitial lung disease, uveitis, optic neuritis, peripheral neuropathies, sarcoidosis, antiphospholipid syndrome, and inflammatory myopathy. One or more conditions may be selected from the group consisting of myopathies, Behcet's disease, alopecia totalis / universalis, pemphigus vulgaris, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, Guillain-Barré syndrome, celiac disease, and pernicious anemia, but is not limited to these. The aforementioned graft-versus-host disease (GVHD) may be one or more selected from the group consisting of acute graft-versus-host disease and chronic graft-versus-host disease, but is not limited to these. 【0079】 The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, and / or excipient. The pharmaceutically acceptable carrier, diluent, and / or excipient may be any selected from those commonly used for antibody formulation. For example, pharmaceutically acceptable carriers include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoic acid, propylhydroxybenzoic acid, talc, magnesium stearate, and mineral oil. 【0080】 The aforementioned pharmaceutical composition may further contain one or more substances selected from the group consisting of lubricants, wetting agents, sweeteners, flavor enhancers, emulsifiers, suspending agents, preservatives, and the like. The pharmaceutical composition may be administered to a subject orally or parenterally. Parenteral administration may be by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial injection, local injection, intranasal injection, intrapulmonary injection, or rectal injection. Since oral administration leads to the digestion of proteins and peptides, the active ingredient of the oral administration composition may be coated or formulated to prevent digestion in the stomach. The pharmaceutical composition may also be administered using any device that allows the active ingredient to be delivered to target cells. 【0081】 In this specification, the term "pharmaceutical effective dose" may mean the amount of the active ingredient (the anti-CD40L / anti-CD28 bispecific antibody, anti-CD40L antibody, and / or anti-CD28 antibody) that exerts a pharmaceutically significant effect in the prevention, improvement, or treatment of autoimmune diseases and / or graft-versus-host diseases. The pharmaceutical effective dose of an antibody, or the appropriate dosage of a pharmaceutical composition expressed in terms of the amount of antibody, can be formulated in a variety of ways depending on various factors such as age, weight, sex, pathological condition, diet, excretion rate, patient response sensitivity, dosage form type, administration time, route of administration, and method of administration. For example, the pharmaceutical effective dose of an antibody or the appropriate dosage of a pharmaceutical composition for an adult may range from about 0.001 to about 1000 mg (amount of antibody) / kg (body weight) per day, from about 0.01 to about 100 mg, or from 0.1 to 50 mg / kg. 【0082】 As used herein, the pharmaceutically effective amount means the amount of active ingredient present in a quantity or dose that produces the desired effect. The active ingredient may be the anti-CD40L / anti-CD28 bispecific antibody, the anti-CD40L antibody, and / or the anti-CD28 antibody. The amount of active ingredient present in the pharmaceutical composition can be formulated in various ways depending on factors such as the formulation method, administration method, the patient's age, weight, sex, medical condition, diet, administration time, administration interval, route of administration, excretion rate, and response sensitivity. For example, if the active ingredient is an antibody, the single dose may be in the range of 0.001-1000 mg / kg, 0.01-100 mg / kg, 0.01-50 mg / kg, 0.01-20 mg / kg, 0.01-10 mg / kg, 0.01-5 mg / kg, 0.1-100 mg / kg, 0.1-50 mg / kg, 0.1-20 mg / kg, 0.1-10 mg / kg, 0.1-5 mg / kg, 1-100 mg / kg, 1-50 mg / kg, 1-20 mg / kg, 1-10 mg / kg, or 1-5 mg / kg, but is not limited to these ranges. 【0083】 In one example, the pharmaceutical composition may be administered to the subject once a day or divided into two or more doses per day, and in one example, it may be administered at intervals of 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 1 week, 2 weeks, 1 month and / or 3 months. The aforementioned dosage and / or administration cycle shall not limit the scope of this application in any way. 【0084】 In other examples, the content of the active ingredient in a pharmaceutical composition is, on a weight basis of the entire pharmaceutical composition, 0.01% to 99.9% by weight, 0.01% to 90% by weight, 0.01% to 80% by weight, 0.01% to 70% by weight, 0.01% to 60% by weight, 0.01% to 50% by weight, 0.01% to 40% by weight, 0.01% to 30% by weight, 1% to 99.9% by weight, 1% to 90% by weight, 1% to 80% by weight, 1% to 70% by weight, 1% to 60% by weight, 1% to 50% by weight, and 1% to 80% by weight. It may be, but is not limited to, 40% by weight, 1% to 30% by weight, 5% to 99.9% by weight, 5% to 90% by weight, 5% to 80% by weight, 5% to 70% by weight, 5% to 60% by weight, 5% to 50% by weight, 5% to 40% by weight, 5% to 30% by weight, 10% to 99.9% by weight, 10% to 90% by weight, 10% to 80% by weight, 10% to 70% by weight, 10% to 60% by weight, 10% to 50% by weight, 10% to 40% by weight, or 10% to 30% by weight. 【0085】 Furthermore, the pharmaceutical composition may further contain, in addition to the active ingredient, a pharmaceutically acceptable carrier and / or adjuvant. The pharmaceutically acceptable carrier may mean a carrier that is commonly used in the formulation of drugs containing proteins, nucleic acids, or cells, and that does not irritate living organisms and does not inhibit the biological activity and / or properties of the active ingredient. For example, the carrier may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoic acid, propylhydroxybenzoic acid, talc, magnesium stearate, mineral oil, etc. The pharmaceutical composition may also further contain one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., which are commonly used in the manufacture of pharmaceutical compositions. 【0086】 In one example, the pharmaceutically acceptable carrier is one that is commonly used in formulation and includes, but is not limited to, physiological saline, sterile water, Ringer's solution, buffered physiological saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc., and may further include other common additives such as antioxidants and buffers as needed. Diluents, dispersants, surfactants, binders, lubricants, etc. may be added in addition, and the formulation may be prepared as an injectable dosage form such as aqueous solution, suspension, emulsion, pills, capsules, granules, or tablets. Regarding appropriate pharmaceutically acceptable carriers and formulations, formulations can be preferably prepared according to each component using the methods disclosed in Remington's Pharmaceutical Sciences (19th edition, 1995). In one embodiment, the pharmaceutical composition may include a pharmaceutically acceptable carrier, such as a binder like lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin; an excipient like dicalcium phosphate; a disintegrant like corn starch or sweet potato starch; a lubricant like magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax; a sweetener; a fragrance; a syrup; a liquid carrier like fatty oil; a sterile aqueous solution; an injectable ester like propylene glycol; polyethylene glycol; ethyl oleate; a suspension; an emulsion; a lyophilized preparation; a topical preparation; a stabilizer; a buffer; animal oils; vegetable oils; waxes; paraffin; starch; tracant; cellulose derivatives; polyethylene glycol; silicone; bentonite; silica; talc; zinc oxide; or a suitable combination thereof. 【0087】 The subjects of the pharmaceutical compositions provided herein may be mammals including humans, dogs, cats, horses, cattle, pigs, goats, rabbits, mice, rats, etc., or cells, tissues, or cultures thereof isolated from these. For example, the subjects may be individuals (mammals such as humans) that require the prevention, improvement, and / or treatment of autoimmune diseases and / or graft-versus-host diseases as described above, or that have autoimmune diseases and / or graft-versus-host diseases, or cells, tissues, or cultures thereof isolated from these. 【0088】 The pharmaceutical composition may be administered orally or parenterally, or by contact with cells, tissues, or body fluids. Specifically, in the case of parenteral administration, it can be administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, endothelial injection, local injection, intranasal injection, intrapulmonary injection, and rectal injection. When administered orally, the oral composition needs to be coated with the active agent or formulated in a way that protects it from digestion in the stomach, as the protein or peptide will be digested. When administered intranasally, the pharmaceutical composition can be diluted and administered by nasal spray so that it is absorbed into the nasal cavity through a sprayer or spray system. Examples of nasal sprays or respiratory formulations for nasal spraying include aerosols. Furthermore, the pharmaceutical composition may be formulated in the form of a solution in an oil or aqueous medium, an injection, a suspension, a syrup, an emulsion, a topical application, a patch, an extract, a powder, a granule, a tablet, a capsule, or an aerosol, and may further contain a dispersant or stabilizer for formulation. 【0089】 In one embodiment, the pharmaceutical composition may be formulated as troches, lozenges, tablets, water-soluble suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs. In another embodiment, the pharmaceutical composition may be formulated as an injection solution, suppositories, respiratory inhalation powders, spray aerosols, ointments, topical powders, oils, or creams. In yet another embodiment, the pharmaceutical composition can be formulated as an injection solution. Specifically, a therapeutically effective amount of antiviral peptide can be mixed with a stabilizer or buffer in water to produce a solution or suspension, which can then be formulated as a unit dose in ampoules or vials. In yet another embodiment, the pharmaceutical composition can be formulated as an aerosol after producing a water-dispersed concentrate or wet powder by combining it with propellants or other additives. In another embodiment, when the pharmaceutical composition is formulated for transdermal use, an effective amount of antiviral peptide can be combined with animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc., as a carrier to produce ointments, creams, topical powders, oils, skin preparations, etc. [Effects of the Invention] 【0090】 The anti-CD40L / anti-CD28 bispecific antibody of this application contains anti-CD40L scFv and anti-CD28 scFv, does not exhibit thromboembolic side effects, prevents T cell activation due to CD28 dimerization, and can demonstrate excellent therapeutic effects for autoimmune diseases and / or graft-versus-host diseases, thus exhibiting the excellent physical properties of a bispecific antibody. [Brief explanation of the drawing] 【0091】 [Figure 1] This shows the genetic information of 49 clones that bind to human CD40L in enzyme immunoassay. [Figure 2a]This figure shows the results of flow cytometry, confirming that the 15 expressed and purified anti-CD40L antibodies mentioned above bind to the Jurkat D1.1 (hCD40L+) cell line expressing human CD40L. [Figure 2b] This figure shows the results of flow cytometry, confirming that the 15 expressed and purified anti-CD40L antibodies mentioned above bind to the Jurkat D1.1 (hCD40L+) cell line expressing human CD40L. [Figure 3] This figure shows the results of flow cytometry, confirming that the aforementioned anti-CD40L antibodies (1G-2, 1F-7, 3B-7) bind to the human CD40L recombinant protein and inhibit its binding to the Ragi(CD40L+) cell line. [Figure 4a] This figure shows the results of comparing the ability of 17 types of anti-CD40L scFv antibodies to suppress B cell costimulatory molecule expression with that of a positive control group (hu5C8 clone). The results confirm that clones 1G-2 and 3F-9 showed superior suppressive activity compared to the positive control group. [Figure 4b] This figure shows the results of comparing the ability of 17 types of anti-CD40L scFv antibodies to suppress B cell costimulatory molecule expression with that of a positive control group (hu5C8 clone). The results confirm that clones 1G-2 and 3F-9 showed superior suppressive activity compared to the positive control group. [Figure 4c] This figure shows the results of comparing the ability of 17 types of anti-CD40L scFv antibodies to suppress B cell costimulatory molecule expression with that of a positive control group (hu5C8 clone). The results confirm that clones 1G-2 and 3F-9 showed superior suppressive activity compared to the positive control group. [Figure 5] This figure shows that when the dose-response (dose-response, 5 or 10 μg / ml) of the anti-CD40L antibody (1G-2) was examined among 17 clones, it showed similar inhibitory activity to the positive control group. [Figure 6] This figure confirms that high-concentration (5 μg / ml) treatment with anti-CD40L antibody (1G-2) exhibits more than 50% inhibitory activity on B cell division. [Figure 7]This figure confirms that the aforementioned anti-CD40L antibody (1G-2) significantly reduces antibody production in a concentration-dependent manner. [Figure 8] This figure shows that the expression of costimulatory molecules decreases as the concentration of anti-CD40L antibody (1G-2) increases. [Figure 9] This figure confirms that IL-12 production in dendritic cells is effectively suppressed by treatment with anti-CD40L antibody (1G-2). [Figure 10] This figure shows the antigen-binding affinity of 1G-2 clones. [Figure 11] This figure compares the ability of 15 types of anti-CD40L scFv antibodies to suppress costimulatory molecules in Daudi cells with that of a positive control group (hu5C8). The results confirm that the 1H-8 and 3E-3 clones exhibited similar suppressive activity to the positive control group. [Figure 12] This figure shows the T cell epitope content of selected chicken-derived anti-CD40L antibodies (1G-2, 1H-8, 3E-3) evaluated using immunoinformatics tools via in silico antibody sequence analysis by Abzena, a UK company, for immunogenicity analysis. [Figure 13a] This figure shows the results of selecting 41, 40, and 38 clones, respectively, that exhibited higher affinity than conventional wild-type antibody clones during the deimmunization process of antibodies (1G-2, 1H-8, 3E-3), and then obtaining their genetic information through nucleotide sequence analysis. [Figure 13b] This figure shows the results of selecting 41, 40, and 38 clones, respectively, that exhibited higher affinity than conventional wild-type antibody clones during the deimmunization process of antibodies (1G-2, 1H-8, 3E-3), and then obtaining their genetic information through nucleotide sequence analysis. [Figure 13c] This figure shows the results of selecting 41, 40, and 38 clones, respectively, that exhibited higher affinity than conventional wild-type antibody clones during the deimmunization process of antibodies (1G-2, 1H-8, 3E-3), and then obtaining their genetic information through nucleotide sequence analysis. [Figure 14]This figure shows the process of purifying recombinant antibodies from cell culture medium using affinity chromatography after overexpression of antibodies by transforming human embryonic kidney (Human embryonic kidney 293F, HEK293F) cells. [Figure 15] This figure shows the results of flow cytometry confirming that the three anti-CD40L antibodies (3E-3 clone, immunized 2E9F clone, and 2E9F VH G49S clone) bind to the human CD40L-expressing Jurkat D1.1 (hCD40L+) cell line. [Figure 16] This figure shows the results of enzyme immunoassay confirming that the three aforementioned anti-CD40L antibodies bind to human CD40L (recombinant protein in the form of hCD40L-Isoleucine zipper-Hemagglutinin). [Figure 17a] This figure confirms that both the 2E9F clone and the 2E9F VH G49S clone suppress the CD40 / CD40L interaction. [Figure 17b] This figure confirms that both the 2E9F clone and the 2E9F VH G49S clone suppress the CD40 / CD40L interaction. [Figure 18] This figure shows the selection of 79 clones that exhibited higher affinity compared to conventional wild-type antibody clones during the humanization process of 3E-3 clones, and their genetic information obtained through nucleotide sequence analysis. [Figure 19] This figure shows the alignment results of the humanized sequence of the framework region and the secured clone sequence 91. [Figure 20] The figure shows the result of creating a combination of eight clones containing human and chicken-derived residues and cloning them into a phagemid vector. [Figure 21] This figure shows that eight combinations of clones demonstrated binding ability to human CD40L recombinant protein in phage enzyme immunoassay. [Figure 22]This figure shows the results of flow cytometry, confirming that eight expressed and purified antibody clones bind to a human CD40L-overexpressing L cell line. [Figure 23] This figure confirms that the binding inhibitory ability of the TSS, TSG, TIS, and TIG clones was superior among the eight clones manufactured (LSS, LSG, LIS, LIG, TSS, TSG, TIS, and TIG clones). [Figure 24] The figure shows the results of testing the ability of the TSS, TSG, TIS, and TIG clones to suppress the expression of B cell costimulatory molecules (CD80, CD86) at different concentrations (2, 4, 6, 8, 10 μg / ml), confirming that the TSG clone had the highest ability to suppress B cell costimulatory molecule expression. [Figure 25] A schematic diagram of bond strength measurement using Biacore is shown. [Figure 26a] The results of measuring CD40L bond strength using the multi-kinetics method and the single-kinetics method are shown. [Figure 26b] The results of measuring CD40L bond strength using the multi-kinetics method and the single-kinetics method are shown. [Figure 27] This diagram shows the results of selecting 49 clones that bind to human CD28 and analyzing their genetic information. [Figure 28] This figure confirms that 12 expressed and purified anti-CD28 antibodies bind to the Jurkat E6.1 (Human CD28+) cell line. [Figure 29] This figure shows the results of testing the IL-2 secretion inhibitory activity of 12 types of anti-CD28 antibodies in the Jurkat E6.1 cell line, confirming that the J6-2 and J6-3 clones showed similar inhibitory activity to the positive control group. [Figure 30] This figure confirms that among 12 types of anti-CD28 antibodies, the J6-3 clone showed higher IL-2 inhibitory activity than the positive control group (FR104) and exhibited similar IFN gamma secretion inhibitory activity to the positive control group. [Figure 31]This figure shows that cells treated with the J6-3 clone exhibit a significantly reduced binding affinity for CD80 / CD86 proteins compared to cells not treated with the J6-3 clone. [Figure 32] This figure confirms that clones 4-25, 6-15, and 6-23 bind similarly to the positive control group (FR104). [Figure 33] This figure shows the results of an immunogenicity analysis conducted via in silico antibody analysis by Abzena, a UK company, to confirm the immunogenicity of the J6-3 clone. [Figure 34] This is a schematic diagram for comparing the human germline sequence with the highest homology to the J6-3 clone for both deimmunization and humanization purposes. [Figure 35] This figure shows the results of sequence analysis of various genes that bind to human CD28 in enzyme immunoassay. [Figure 36] This figure shows the final acquisition of clone 95 (G12 clone) through sequencing analysis after enzyme immunoassay. [Figure 37] This figure shows the results of the analysis of the G12 clone, confirming the presence of immunogenicity at a total of two locations. [Figure 38] This figure shows the immunogenicity of the conventional J6-3 clone and the final clone after humanization has been established. [Figure 39] This figure shows the results of flow cytometry confirming that expressed and purified anti-CD28 antibodies (J6-3 clone and humanized 2-G12 clone) bind to the Jurkat E6.1 (hCD28+) cell line. [Figure 40] This figure shows that the J6-3 clone and the humanized 2-G12 clone were confirmed to bind to human CD28 recombinant protein (hCD28) by enzyme immunoassay. [Figure 41]The results of examining the cytokine secretion inhibitory ability of the humanized 2-G12 clone on CD4 T cells showed that the 2-G12 clone exhibited superior IL-2 (interleukin-2) secretion inhibitory ability compared to the positive control group (FR104), and confirmed that it showed similar IFN gamma inhibitory ability to the positive control group (FR104). [Figure 42] The diagram shows a schematic representation of the TSG clone, a humanized anti-CD40L scFv antibody produced in Examples 1-4, and the 2-G12 antibody, a humanized anti-CD28 scFv antibody produced in Examples 2-3, after being cloned in the form of bispecific antibodies containing a Ck-linking domain and then expressed in a mammalian expression system. [Figure 43] This figure shows the successful purification of recombinant antibodies from cell culture medium using affinity chromatography after overexpression of antibodies by transformation of human embryonic kidney (Human embryonic kidney 293F, HEK293F) cells. [Figure 44] This figure shows the successfully purified bispecific antibody (TSGxHu2G12 clone) produced in Example 3.2.1. [Figure 45] This graph shows that the bispecific antibody (TSGxHu2G12 clone) exhibited CD28 binding affinity comparable to that of TSGx2-G12 and CD28. [Figure 46] This graph shows that the bispecific antibody (TSGxHu2G12 clone) exhibited binding affinity to cell surface CD28 to a similar degree as TSGx2-G12 and CD28. [Figure 47] A schematic diagram of the experiment to confirm the CD28-CD80 interaction inhibitory ability of Example 3.2.4 is shown. [Figure 48] This graph shows that a bispecific antibody containing the Hu2G12 clone (TSGxHu2G12) suppresses the binding of recombinant CD80 protein at a concentration of 1000 nM, to a similar degree to that of a bispecific antibody containing the 2-G12 clone (TSGx2-G12). [Figure 49]In Example 3-3, among the six anti-CD40L / anti-CD28 bispecific antibodies produced by an additional humanization process, the clone with the S42Q mutation introduced into the light chain (VL) (TSGxHu2G12-S42Q) and the clone with the S76N mutation introduced into the heavy chain (VH) (TSGxHu2G12-S76N) showed a binding affinity to CD28 that was comparable to that of the anti-CD40L / anti-CD28 bispecific antibody (TSGx2-G12, Table 16) produced in Example 3-1. [Figure 50] This graph shows that the TSGxHu2G12-S42Q and TSGxHu2G12-S76N clones have similar binding affinity to cell surface CD28 compared to the TSGx2-G12 clone. [Figure 51] A schematic diagram of the experiment to confirm the CD28-CD80 interaction inhibitory ability of Example 3.3.4 is shown. [Figure 52] The graph shows that the TSGxHu2G12-S42Q and TSGxHu2G12-S76N clones exhibited a similar level of recombinant CD80 protein binding inhibitory ability as the TSGx2-G12 clone. [Figure 53] This schematic diagram illustrates a positive feedback mechanism in which activated T cells activate APCs, and activated APCs then increase the activity of T cells again. [Figure 54] A schematic diagram of the experimental process in Example 3-4 is shown. [Figure 55] This graph shows that both the biantibody (TSGx2-G12) and the CD28 mono-inhibitory antibody (aCot-Ck-aCD28; aCot-Ck-2-G12) exhibit IFNr inhibitory effects. [Figure 56] This schematic diagram illustrates the phenomenon where, when self-antigen-specific T cells recognize a self-antigen presented by an antigen-presenting cell and receive a TCR signal (signal 1), they will not be activated even if they are subsequently stimulated again with the self-antigen, unless they also receive a co-stimulation (signal 2) from the antigen-presenting cell. [Figure 57] A schematic diagram of the experimental process in Example 3-5 is shown. [Figure 58]In the group treated with the anti-CD40L / anti-CD28 bispecific antibody, the amount of IFNr was significantly reduced compared to the negative control group. This experimental result confirmed that CD4 T cells treated with the bispecific antibody did not respond to antigen restimulation. [Figure 59] These experimental results demonstrate that the anti-CD40L / anti-CD28 bispecific antibody of this application possesses excellent T-cell suppressive ability regardless of the order in which the anti-CD40L antibody and the anti-CD28 antibody are administered. [Figure 60] Schematic diagrams of the simultaneous suppression method and the individual suppression method are shown. [Figure 61] The experimental results confirm that CD40L and CD28 are co-localized at the same position only in the group treated with the anti-CD40L-Ck-CD28 bispecific antibody, suggesting that the anti-CD40L / anti-CD28 bispecific antibody of this application acts in a simultaneous inhibitory manner. [Figure 62] The experimental results confirm that CD40L and CD28 are co-localized at the same position only in the group treated with the anti-CD40L-Ck-CD28 bispecific antibody, suggesting that the anti-CD40L / anti-CD28 bispecific antibody of this application acts in a simultaneous inhibitory manner. [Figure 63] In the group treated with the anti-CD40L-Ck-CD28 bispecific antibody, the CD28-mNeonGreen fluorescence in the cell membrane region was significantly altered, and the FRET efficacy value was statistically significant compared to the other groups. These experimental results suggest that the anti-CD40L / anti-CD28 bispecific antibody of this application acts in a simultaneous inhibitory manner. [Figure 64] In the group treated with the anti-CD40L-Ck-CD28 bispecific antibody, the CD28-mNeonGreen fluorescence in the cell membrane region was significantly altered, and the FRET efficacy value was statistically significant compared to the other groups. These experimental results suggest that the anti-CD40L / anti-CD28 bispecific antibody of this application acts in a simultaneous inhibitory manner. [Figure 65]The group treated with the anti-CD40L / anti-CD28 bispecific antibody showed no weight loss during the experimental period, and disease severity was not assessed, confirming that the disease did not develop. Furthermore, since all mice survived the experimental period, this figure confirms that disease progression was completely suppressed only in the group treated with the bispecific antibody. [Figure 66] The group treated with the anti-CD40L / anti-CD28 bispecific antibody showed no weight loss during the experimental period, and disease severity was not assessed, confirming that the disease did not develop. Furthermore, since all mice survived the experimental period, this figure confirms that disease progression was completely suppressed only in the group treated with the bispecific antibody. [Figure 67] This shows one exemplary form of the anti-CD40L / anti-CD28 bispecific antibody of this application (anti-CD40L scFv - Ck - anti-CD28 scFv). [Figure 68] This graph shows that among mouse anti-CD40L antibodies that bind to mouse CD40L, clones 1-1-H, 1-12C, 1-12H, 2-12C, and 3-11-C effectively bind to mouse CD40L expressed on the cell surface in a concentration-dependent manner. [Figure 69] This graph shows that the PV1x2-12-C antibody binds to activated mouse CD4 T cells, after which antibody binding was measured using flow cytometry with a fluorescently labeled anti-Ck antibody. [Figure 70] In Example 3, similar to the experimental results for the humanized anti-CD40L / anti-CD28 bispecific antibody, the mouse anti-CD40L / anti-CD28 bispecific antibody suppressed T cell activity by blocking the CD40L and CD28 signaling pathways of CD4 T cells. [Figure 71] This graph shows that the form of bispecific antibody provided in this application effectively suppresses autoimmune demyelinating diseases, as confirmed by the fact that the clinical severity of the disease was significantly lower in the group treated with the mouse anti-CD40L / anti-CD28 bispecific antibody (PV1x2-12-C). [Modes for carrying out the invention] 【0092】 The present invention will be described more specifically below with reference to the following examples. However, these are merely illustrative examples of the present invention, and the scope of the present invention is not limited by these examples. [Examples] 【0093】 Example 1. Anti-CD40L antibody Example 1-1. Selection of chicken-derived anti-CD40L antibodies (Lead antibody) Three chickens (biofore, White leghorn chicken) were immunized with human CD40L recombinant protein (R&D Systems, Minneapolis, MN, USA), and an antibody library in scFv form was constructed using a conventionally reported method. The complexity of the three chicken antibody libraries produced was 2.4 × 10⁶. 9 , 2.4×10 9 , 2.7×10 9 Bio-panning against human CD40L was performed using a chicken antibody library, and 49 clones that bound to human CD40L were selected by enzyme immunoassay. Their genetic information was then obtained by nucleotide sequence analysis (Figure 1). 【0094】 Fifteen antibody clones that bind to human CD40L recombinant protein were cloned into an animal cell expression vector (pCEP4 vector, Invitrogen, Carlsbad, CA, USA) to produce the scFv-Ck-scFv-HIS-HA fusion protein [anti-CD40L scFv antibody (N'-VL-linker (SEQ ID NO: 93)-VH-C')-Ck (SEQ ID NO: 95)-anti-cotinin scFv antibody (N'-VL-linker (SEQ ID NO: 93)-VH-C')-HIS-HA]. After transforming human embryonic kidney (human embryonic kidney 293F, HEK293F) cells (Invitrogen, Carlsbad, CA, USA), the antibodies were purified from the cell culture medium by affinity chromatography. In order to confirm the effect of the anti-CD40L antibody in the form of a bispecific antibody in which two monovalent antibodies are linked, the anti-CD40L scFv antibody was linked to an anti-cotinine scFv antibody that binds to cotinine, a substance not present in the body, and the following experiment was conducted. The anti-cotinine antibody was obtained from the Korean Patent Publication (KR 10-2019-0023084 A), and the CDR and variable region sequence of the anti-cotinine antibody are as shown in SEQ ID NOs. 120-126 and SEQ ID NOs. 127-128. 【0095】 [Table 11] 【0096】 1.1.1. Confirmation of binding to human CD40L The 15 anti-CD40L antibodies expressed and purified as described above were confirmed to bind to human CD40L-expressing Jurkat D1.1 (hCD40L+) cell lines (ATCC, Manassas, VA, USA) by flow cytometry (Figures 2a and 2b). Furthermore, flow cytometry confirmed that the aforementioned anti-CD40L antibodies (1G-2, 1F-7, 3B-7) bind to human CD40L recombinant protein and inhibit its binding to Raji(CD40L+) cell lines (ATCC, Manassas, VA, USA) (Figure 3). 【0097】 1.1.2. Suppression of B cell and dendritic cell activity CD40L expressed on the surface of T cells is known to bind to CD40 on the surface of antigen-presenting cells (APCs), such as B cells and dendritic cells, thereby activating these APCs. Therefore, we evaluated the ability of anti-CD40L antibodies to inhibit CD40L / CD40 interaction and suppress the activity of B cells and dendritic cells. To obtain human B cells and mononuclear cells, leukocyte apheresis was performed on healthy adults, and B cells and mononuclear cells were separated from the resulting leukocytes using the MACS cell separation method (Miltenyi Biotec, 130-050-201). The mononuclear cells were differentiated into dendritic cells using GM-CSF (Granulocyte-macrophage colony-stimulating factor) and IL-4 (interleukin 4). 【0098】 Mouse-derived fibroblast cell lines expressing human CD40L (CD154L cells) (Sim et al. Arthritis Research & Therapy (2015) 17:190 DOI 10.1186 / s13075-015-0687-1) were co-cultured with B cells or dendritic cells to construct a system in which CD40 stimulation was transmitted between B cells and dendritic cells. The manufactured anti-CD40L antibody was then used to confirm whether it suppressed the activity of B cells or dendritic cells. In this study, CD154L cells were treated with 60 Gy of radiation to suppress cell division. As a positive control group, a conventionally known anti-CD40L antibody (hu5C8 clone) (Karpusas et al. Structure of CD40 Ligand in Complex with the Fab Fragment of a Neutralizing Humanized Antibody Structure. 2001 Apr 4;9(4):321-9.) was expressed and purified in scFv form. 【0099】 After culturing B cells, CD154L cells, and anti-CD40L antibody (5 μg / ml) together, the extent to which the increase in expression of costimulatory molecules (CD80, CD86) induced by B cell activation was suppressed by the anti-CD40L antibody was confirmed by flow cytometry, and the results are shown in Figures 4a to 4c (Figures 4b and 4c show the results of Figure 4a quantitatively). As can be seen in Figures 4a, 4b, and 4c, when comparing the ability of 17 anti-CD40L scFv antibodies to suppress B cell costimulatory molecule expression with that of a positive control group (hu5C8 clone), it was confirmed that clones 1G-2 and 3F-9 showed superior suppressive ability compared to the positive control group (hu5C8) (Figure 4c). Furthermore, when the dose-response (5 or 10 μg / ml) of the anti-CD40L antibody (1G-2) was examined among the 17 clones, it showed similar inhibitory activity to the positive control group (hu5C8) (Figure 5). 【0100】 1.1.3. Suppression of B cell division When B cells and CD154L cells are co-cultured, the CD40 of the B cells is stimulated, causing the B cells to divide. At this time, the degree of cell division can be confirmed by treating the dividing cells with Edu and detecting it with an anti-Edu antibody. To confirm the B cell division inhibitory ability of the anti-CD40L antibody (1G-2), Edu (Thermo Fisher Scientific, C10637) was added to the co-culture system (culture medium of B cells, CD154L cells, and anti-CD40L antibody), and the amount of Edu inserted into the DNA of dividing B cells was analyzed using an anti-Edu antibody (Thermo Fisher Scientific, C10637) with a flow cytometer to measure the degree of B cell division. As a result, we confirmed that high-concentration (5 μg / ml) treatment with anti-CD40L antibody (1G-2) suppressed B cell division by more than 50% (Figure 6). 【0101】 1.1.4. Suppression of antibody production by B cells When B cells and CD154 L cells are co-cultured, B cells secrete IgM in response to CD40 stimulation, and the degree of B cell activation can be measured by measuring the amount of secreted IgM. Therefore, we used IgM ELISA to confirm whether antibody production by B cells was suppressed during treatment with the anti-CD40L antibody (1G-2), and the results are shown in Figure 7. As can be seen in Figure 7, the anti-CD40L antibody (1G-2) was confirmed to significantly reduce antibody production in a concentration-dependent manner (Figure 7). 【0102】 1.1.5. Suppression of co-stimulatory molecule expression in dendritic cells In addition to inhibiting B cell activity, anti-CD40L antibodies also inhibited dendritic cell (DC) activity. To verify this, dendritic cells, CD154L cells, and anti-CD40L antibodies were cultured together. The expression levels of dendritic cell costimulatory molecules (CD80, CD86) were then measured by flow cytometry, and the results are shown in Figure 8. As can be seen in Figure 8, we confirmed that the expression of costimulatory molecules (CD80, CD86) decreased as the concentration of anti-CD40L antibody (1G-2) increased (Figure 8). 【0103】 1.1.6. Suppression of cytokine secretion by dendritic cells When dendritic cells and CD154 L cells are co-cultured, the dendritic cells are activated by CD40 stimulation, leading to increased cytokine production. Therefore, the degree of dendritic cell activation can be measured by measuring the amount of secreted cytokines. To confirm whether cytokine production in dendritic cells is suppressed during anti-CD40L antibody treatment, the amount of IL-12 (interleukin-12) secreted into the supernatant of the co-culture system (culture medium of dendritic cells, CD154L cells, and anti-CD40L antibody) was measured by IL-12 ELISA, and the results are shown in Figure 9. As can be seen in Figure 9, we confirmed that IL-12 production is effectively suppressed by treatment with anti-CD40L antibody (1G-2) (Figure 9). 【0104】 1.1.7. Confirmation of binding force to CD40L Through Examples 1.1.1 to 1.1.6 described above, it was confirmed that the 1G-2 clone of the anti-CD40L antibody can effectively suppress the function of CD40L. 【0105】 To measure the binding affinity between the 1G-2 clone and the CD40L protein, surface plasmon resonance (SPR) was measured at 25°C using a Biacore T200 (GE Healthcare). Binding affinity measurement using the Biacore T200 was performed by injecting an analyte onto a link-immobilized chip surface, followed by association, dissociation, and regeneration. Affinity values ​​were calculated using the 1:1 binding model of BIAevaluation software version 1.0, with the antibody immobilized on the CM5 chip surface. 2B1-Ck-1G2 and 1G2-Ck-COT 300RU were immobilized on the CM5 chip surface at pH 4.5 and pH 5.0, respectively. Similar to the anti-cotinin antibody, 2B1 was used as a partner antibody to confirm the effect of the 1G2 clone in the form of a bispecific antibody. CD40L protein was diluted by half starting from 5 nM, and the binding strength was measured with an association / dissociation time of 3 minutes each at a flow rate of 30 μl / min. After each cycle, 10 mM Glycine pH 2.0 containing 500 mM NaCl was regenerated at a flow rate of 30 μl / min for 30 seconds. Figure 10 shows the results of measuring the binding affinity between the 1G-2 clone and the CD40L protein. The binding affinity measurement showed a KD value of 0.1-0.2 nM, indicating a high on-rate (Figure 10). 【0106】 Examples 1-2. Selection of anti-CD40L antibodies (backup antibodies) To further select anti-CD40L antibodies with superior B-cell activity inhibitory capabilities, we used a peripheral blood B-lymphoma cell line expressing human CD40 (Daudi cell) (KCLB, Seoul, Republic of Korea) and a mouse fibroblast cell line expressing human CD40L (CD154L cell). We evaluated the B-cell activity inhibitory capabilities of 15 antibodies that bind to the human CD40L recombinant protein and have different complementarity determining region (CDR) sequences. After culturing the aforementioned Daudi cells, CD154L cells, and anti-CD40L antibody (2 μg / ml) together, the expression levels of the costimulatory molecules (CD80, CD86) in the Daudi cells were confirmed by flow cytometry, and the results are shown in Figure 11. The ability of 15 types of anti-CD40L scFv antibodies to suppress costimulatory molecules in Daudi cells was compared with that of a positive control group (hu5C8). The results showed that 1H-8 and 3E-3 clones exhibited similar suppressive activity to the positive control group (Figure 11). 【0107】 Based on a combination of Examples 1-1 and 1-2, clones 1G-2, 1H-8, and 3E-3 were selected as anti-CD40L antibodies, and the CDR and variable region sequences of the antibodies are summarized in the table below. 【0108】 [Table 12] 【0109】 [Table 13] 【0110】 [Table 14] 【0111】 In the table above, the CDR sequences (L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2, H-CDR3) included in the light chain variable region and heavy chain variable region are underlined. 【0112】 Examples 1-3. Antibody deimmunization Antibodies selected from the animal immunity library may exhibit immunogenicity when administered to humans; therefore, the antibodies were optimized to have low immunogenicity and high affinity for the target antigen. To analyze the immunogenicity of the selected chicken-derived anti-CD40L antibodies (1G-2, 1H-8, 3E-3), T cell epitope content was evaluated using immunoinformatics tools via in silico antibody sequence analysis by Abzena, UK (Figure 12). To select antibodies that have low immunogenicity to humans while having high affinity for CD40L, a mutant non-immunogenic antibody library was created in which highly immunogenic regions in the antibody sequence were replaced with human germline amino acids. 【0113】 The complexity of the three non-immunogenic antibody libraries (1G-2, 1H-8, 3E-3) that were prepared was 5.4 × 10⁶. 8 , 4.3×10 9 , 2.6×10 8 Bio-panning against human CD40L recombinant protein was performed on the prepared non-immunogenic antibody library, and 52, 50, and 54 clones that bound to human CD40L recombinant protein were secured by enzyme immunoassay. Based on the conventional wild-type antibody clones, 41, 40, and 38 clones showing higher affinity were selected, and their genetic information was secured by nucleotide sequence analysis (Figures 13a-13c). In the candidate antibody, 2E9F derived from the 3E-3 clone has 15 of its 16 immunogenic residues replaced with residues identical to human germline amino acids, while the remaining V H By introducing serine, the same amino acid as human germline, at amino acid residue G49, non-immunogenic antibody genes were synthesized. Selected non-immunogenic antibodies were 2E9F and 2E9F V. HTo express G49S in the scFv-Ck-scFv morphology, the mammalian cell expression vector was cloned. After overexpression of the antibody by transforming human embryonic kidney (Human embryonic kidney 293F, HEK293F) cells, the recombinant antibody was purified from the cell culture medium using affinity chromatography (Figure 14). 【0114】 1.3.1. Confirmation of binding to human CD40L The three anti-CD40L antibodies (3E-3 clone, immunized 2E9F clone, and 2E9F VH G49S clone) were confirmed to bind to human CD40L-expressing Jurkat D1.1 (hCD40L+) cell lines by flow cytometry (Figure 15). Furthermore, it was confirmed by enzyme immunoassay that the three anti-CD40L antibodies mentioned above bind to human CD40L (recombinant protein in the form of hCD40L-Isoleucine zipper-Hemagglutinin) (R&D Systems, Minneapolis, MN, USA) (Figure 16). 【0115】 1.3.2. Ability to inhibit CD40 / CD40L binding After treating Jurkat D1.1 (hCD40L+) cell lines or CD154L (hCD40L+) cell lines with the aforementioned immunized 2E9F clones and 2E9F VH G49S clones, and then treating them with CD40 antigen, analysis by flow cytometry confirmed that both the 2E9F clones and 2E9F VH G49S clones suppressed the CD40 / CD40L interaction (Figures 17a and 17b). The anti-CD40L antibodies (1G-2, 1H-8) selected in Examples 1-2 were also deimmunized using the method described above. 【0116】 Examples 1-4. Humanization of antibodies To improve the safety of the antibody, humanization was performed by replacing the framework region (excluding the complementarity-determining region (CDR)) of the chicken-derived anti-CD40L antibody in a manner similar to that of the human germline sequence. Simultaneously with advancing antibody humanization, a trimer-controlled library (GENEWIZ, NJ, USA) was used to create a mutant antibody library containing both human germline amino acids and the amino acid sequences of conventional antibodies in order to select antibodies with high affinity for CD40L. 【0117】 To further humanize the 10 chicken-derived residues remaining in the 3E-3 clone, a secondary humanization library was created using the trimer-controlled library provided by GENEWIZ. The resulting humanization library has a complexity of 5.0 × 10⁶. 8 The total number of amino acid residues to be substituted was 31. Bio-panning against human CD40L recombinant protein was performed on the humanized antibody library, and a total of 87 clones that bound to human CD40L recombinant protein were secured by enzyme immunoassay. Based on the conventional wild-type antibody clones, 79 clones showing higher affinity were selected, and their genetic information was obtained by nucleotide sequence analysis (Figure 18). As a result, we secured clone 91 in which 28 of the 31 residues were substituted in a manner similar to the human germline amino acid sequence. 【0118】 Figure 19 shows the alignment results of the humanized framework region sequence and the secured clone 91 sequence. Three unhumanized chicken-derived residues (highlighted in red in Figure 19) were generated in combinations of eight clones, each containing both human and chicken-derived residues, via polymerase chain reaction using the secured clones, and then cloned into the phagemid vector (Figure 20). 【0119】 The three non-humanized chicken-derived residues correspond to the 12th residue from the N' end of the L-FR2 region (humanized sequence: L, sequence of clone 91: T), the 21st residue from the N' end of the H-FR1 region (humanized sequence: S, sequence of clone 91: I), and the 14th residue from the N' end of the H-FR2 region (humanized sequence: S, sequence of clone 91: G). By combining the number of possible combinations, eight clones were named LSS, LSG, LIS, LIG, TSS, TSG, TIS, and TIG. For example, the "LSS" clone is the one in which the 12th residue from the N' end of the L-FR2 region is "L", the 21st residue from the N' end of the H-FR1 region is "S", and the 14th residue from the N' end of the H-FR2 region is "S". 【0120】 1.4.1. Confirmation of binding to human CD40L Phage enzyme immunoassay was performed on the eight clones (LSS, LSG, LIS, LIG, TSS, TSG, TIS, and TIG clones) that had been cloned. The results confirmed that all combinations of clones showed binding ability to human CD40L recombinant protein in phage enzyme immunoassay (Figure 21). 【0121】 1.4.2. Confirmation of binding to L cell lines In phage enzyme immunoassay, eight humanized antibody clones exhibiting binding ability to human CD40L recombinant protein were cloned using a mammalian cell expression vector to express them in the scFv-Ck-scFv morphology. After overexpression of the antibodies by transforming human embryonic kidney (Human embryonic kidney 293F, HEK293F) cells, the recombinant antibodies were purified from the cell culture medium using affinity chromatography. The binding of the eight expressed and purified antibody clones to an L cell line overexpressing human CD40L was confirmed by flow cytometry (Figure 22). 【0122】 1.4.3. Confirmation of inhibition of human CD40 and L cell line binding. Flow cytometry was performed to confirm whether the aforementioned anti-CD40L antibody inhibits the binding of human CD40 protein and hCD40L to L cell lines. Of the eight clones manufactured (LSS, LSG, LIS, LIG, TSS, TSG, TIS, and TIG clones), it was confirmed that the TSS, TSG, TIS, and TIG clones exhibited superior binding inhibition ability (Figure 23). 【0123】 1.4.4. Confirmation of suppression of B cell costimulatory molecule expression The ability of the TSS, TSG, TIS, and TIG clones to suppress the expression of B cell costimulatory molecules (CD80, CD86) was confirmed at different concentrations (2, 4, 6, 8, and 10 μg / ml). The TSG clone was found to have the highest ability to suppress B cell costimulatory molecule expression (Figure 24). As a result, the TSG clone was selected as the lead antibody (hu3E-3) for the humanized antibody, and the TSS, TIS, and TIG clones were secured as backup antibodies. The humanized TSG clone contained only two chicken-derived residues in its framework region, and the remaining region was entirely substituted in the same manner as the human germline sequence. Of the eight clones mentioned above, the CDR and variable region sequences of the TSG clones are summarized in the table below as an example. 【0124】 [Table 15] 【0125】 In the table above, the CDR sequences (L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2, H-CDR3) included in the light chain variable region and heavy chain variable region are underlined. In addition, the 12th residue from the N' end of the L-FR2 region, the 21st residue from the N' end of the H-FR1 region, and the 14th residue from the N' end of the H-FR2 region are shown in square brackets and bold underline. 【0126】 1.4.5. Measurement of the binding affinity of anti-CD40L antibody The binding affinity of three anti-CD40L antibodies (1H-8 clone, TSG clone, and 3E-3 clone) to CD40L was measured. Antigen-antibody binding affinity was measured using a Biacore T200 (GE Healthcare) at 25°C by surface plasmon resonance (SPR). Biacore-based binding affinity measurement involves injecting an analyte into a chip surface immobilized with a ligand, followed by association, dissociation, and regeneration (see schematic diagram in Figure 25). Affinity values ​​were calculated using the 1:1 binding model of BIAevaluation software version 1.0. Binding strength was measured by immobilizing the CD40L (rhCD40 Ligand, R&D system, 6420-CL / CF) protein on the CM5 chip surface. The rhCD40 Ligand was immobilized on the CM5 chip surface at a level of 100 RU under pH 5.5 conditions. HBS-EP (GE Healthcare, BR-10-0669) was used as the sample and system buffer. Three types of bivalent antibodies in the form of scFv-Ck-scFv [structure of anti-CD40L scFv antibody (N'-VL-VH-C')-Ck-anticotinine antibody (N'-VL-VH-C'), see Example 1-1] were measured at a flow rate of 30 μl / min with association / dissociation times of 4 minutes and 3 minutes, respectively. Regeneration with 10 mM Glycine-HCl (pH 2.0) at a flow rate of 30 μl / min for 30 seconds was performed after each cycle. Table 12 below shows the conditions for measuring the binding affinity of the anti-CD40L antibody. 【0127】 [Table 16] 【0128】 As a result of measuring the CD40L binding force by the Multi-kinetics method, the regeneration of the antibody was not stably performed. Therefore, additional experiments were conducted using the single kinetics method without the regeneration step, and the results are shown in FIGS. 26a and 26b, respectively. It is known that the K value does not change between the Sigle kinetics and multi kinetics methods. In this example, it was also confirmed that there is no significant difference in the K value of the 1H8 clone, TSG clone, and 3E-3 clone between the two methods. D D 【0129】 Example 2. Anti-CD28 antibody Example 2-1. Selection of anti-CD28 antibody (lead antibody) Three White leghorn chickens (Biofore) were immunized with human CD28 recombinant protein (R&D Systems, Minneapolis, MN, USA) to prepare an antibody library in scFv form. The complexities of the three prepared chicken antibody libraries were 1.5×10 9 , 1.3×10 9 , and 9.4×10 8 . Bio-panning against human CD28 was performed using the chicken antibody library. Subsequently, 49 clones that bind to human CD28 were selected by enzyme immunoassay, and their gene information was secured by nucleotide sequence analysis (FIG. 27). 【0130】 ​​To produce 12 antibody clones that bind to human CD28 protein in the form of scFv-Ck-scFv-HIS-HA fusion protein [anti-CD28 scFv antibody (N'-VL-linker-VH-C')-Ck (SEQ ID NO: 95)-anti-cotinine scFv antibody (N'-VL-linker-VH-C')-HIS-HA, see Example 1-1], cloning was performed into an animal cell expression vector. After transforming human embryonic kidney 293F (HEK293F) cells, the antibody was purified from the cell culture supernatant by affinity chromatography. 【0131】 【Table 17】 【0132】 2.1.1. Confirmation of binding to human CD28 It was confirmed by flow cytometry that the expressed and purified anti-CD28 antibody binds to the Jurkat E6.1 (Human CD28+) cell line (ATCC, Manassas, VA, USA). As a positive control group, an antibody that specifically binds to CD28 (FR104 clinical candidate substance from OSE Immunotherpies, J Immunol. 2016. 197(12):4593-4602) was cloned and purified in the same form for comparison. As a result, it was confirmed that the 12 expressed and purified anti-CD28 antibodies bind to the Jurkat E6.1 (Human CD28+) cell line (Figure 28). 【0133】 2.1.2. Ability to inhibit cytokine secretion of Jurkat E6.1 cell line (in vitro) For T cells to be activated, the T cell receptor (TCR) on the surface of the T cell must recognize the antigen presented on the surface of an antigen-presenting cell (APC). It is known that T cells will not be activated unless they receive a signal transmitted via the TCR (signal 1) as well as a costimulatory signal (signal 2) transmitted when B7 molecules (B7.1 [CD80], B7.2 [CD86]) on the surface of the APC bind to CD28 on the surface of the T cell (Cancer Res. 2001 61(5): 1976-82). Therefore, we evaluated whether the anti-CD28 antibody could block CD28-B7 binding and suppress the activation of human T cells. When Jurkat E6.1 cells, a CD28-positive T cell line, were cultured together with OKT3-Ck-HA antibody (US 5885573 A), a TCR-stimulating antibody, and the Raji cell line, a CD80 / CD86-positive B cell line, the T cells were activated and secreted IL-2 (interleukin-2). We then treated the co-culture system with the anti-CD28 antibody to confirm whether the IL-2 secretion ability of T cells was inhibited. Specifically, Jurkat E6.1 cell line, Raji cell line, OKT3 (10 nM), and anti-CD28 antibody (1 μM) were cultured together, and after 48 hours, the supernatant of the culture medium was collected and IL-2 ELISA was performed. As the positive control group, the FR104 antibody expressed in the form of FR104 scFv-Ck-Cot scFv [structure of FR104 scFv antibody (N'-VL-VH-C')-Ck (SEQ ID NO: 95)-anti-cotinin antibody (N'-VL-VH-C'), see Example 1-1] was used. The ability of 12 anti-CD28 antibodies to suppress IL-2 secretion in the Jurkat E6.1 cell line was examined, and it was confirmed that the J6-2 and J6-3 clones showed similar suppressive activity to the positive control group (Figure 29). 【0134】 To confirm whether the aforementioned experimental results could be reproduced in human primary T cells, additional experiments were conducted. CD4-positive T cells and mononuclear cells were isolated from healthy adult leukocytes obtained by leukocyte apheresis using the MACS cell separation method. The isolated mononuclear cells were treated with GM-CSF (Granulocyte-macrophage colony-stimulating factor) and IL-4 (interleukin-4) to differentiate them into immature dendritic cells, and then treated with LPS (lipopolysaccharide, 1 μg / ml) for 24 hours to mature them, and used as antigen-presenting cells for T cell stimulation. When CD4 T cells were cultured together with the aforementioned dendritic cells and an anti-CD3 antibody for TCR stimulation, T cell activation was induced. At this time, the anti-CD28 antibody was added, and its effect of suppressing T cell activation was verified. Specifically, CD4 T cells, dendritic cells, anti-CD3 antibody (2 μg / ml), and purified anti-CD28 antibody (10 μg / ml) were cultured together. After 72 hours, the supernatant was collected and IL-2 and IFN gamma (interferon gamma) ELISA was performed. As a result, among the 12 anti-CD28 antibodies, the J6-3 clone showed higher IL-2 inhibitory activity than the positive control group (FR104) and exhibited similar IFN gamma secretion inhibitory activity to the positive control group (Figure 30). 【0135】 2.1.3. Confirmation of competitive binding with CD80 / CD86 proteins To reconfirm the binding specificity of the J6-3 clone to T cell surface CD28, competitive binding affinity was investigated using recombinant CD80 and CD86 proteins. Jurkat E6.1 cells were treated with the J6-3 clone for 1 hour (1500 nM) to completely mask CD28 expressed on the surface of Jurkat E6.1 cells. After washing the Jurkat E6.1 cells, they were treated with recombinant CD80 and CD86 proteins (500 nM) to confirm whether the J6-3 clone inhibited the binding of these recombinant proteins. The CD80 / CD86 proteins were in the form of Fc-fusion proteins, and their binding was analyzed using a flow cytometer with a fluorescently labeled anti-Fc secondary antibody. 【0136】 As a result, we confirmed that the binding affinity of CD80 / CD86 proteins was significantly reduced in cells treated with the J6-3 clone compared to cells not treated with the J6-3 clone. On the other hand, this reduction in CD80 / CD86 protein binding affinity was not observed in cells treated with the negative control antibody (J6-85 clone), thus demonstrating its specificity (Figure 31). Therefore, we confirmed that the J6-3 clone competitively binds to cell surface CD28 with CD80 / CD86 proteins. The CDR and variable region sequences of the aforementioned J6-3 clone are summarized in the table below. 【0137】 [Table 18] 【0138】 In the table above, the CDR sequences (L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2, H-CDR3) included in the light chain variable region and heavy chain variable region are underlined. 【0139】 Example 2-2. Selection of anti-CD28 antibodies (backup antibodies) To secure additional backup antibodies other than the J6-3 clone from the anti-CD28 antibody library, clones that bind to CD28 were selected through screening. Numerous additional antibody clones with binding affinity to CD28 were selected, gene sequencing analysis was completed, and recombinant proteins were purified in the same morphology as the J6-3 clone. Anti-CD28 antibodies (1000 nM) in the scFv-Ck-scFv morphology [anti-CD28 scFv antibody (N'-VH-VL-C')-Ck-anticotinine antibody (N'-VH-VL-C', see Example 1-1] were reacted with CD28-positive Jurkat E6.1 cell lines, and their binding affinity to CD28 was confirmed by flow cytometry. As a result, we confirmed that clones 4-25, 6-15, and 6-23 bound in the same way as the positive control group (FR104) (Figure 32). Therefore, three backup antibody clones (clones 4-25, 6-15, and 6-23) that bind to the Jurkat E6.1 cell line were secured, similar to the positive control group. 【0140】 Examples 2-3. Non-immunization of antibodies and production of humanized antibodies (1) To confirm the immunogenicity of the J6-3 clones selected through flow cytometry and interleukin-2 inhibitory activity experiments, immunogenicity analysis was performed using in silico antibody analysis from Abzena, UK (Figure 33). After confirming immunogenicity, the human germline sequence with the highest homology to the J6-3 clone selected in Example 2-1 was compared for deimmunization and humanization simultaneously (see schematic diagram in Figure 34). A library was prepared using degenerate codons to replace the immunogenic portion of the complementary-determining region (CDR) with a human germline sequence. 【0141】 Using the prepared random library containing human germlines, bio-panning against human CD28 was performed, and in enzyme immunoassay, various gene information binding to human CD28 was secured by nucleotide sequence analysis (Figure 35). As a result of nucleotide sequence analysis, clones that completely matched the results of conventional immunogenicity analysis could not be selected, but it was confirmed that many complementarity-determining regions were changed to human germlines compared to the conventional 6-3 clone. 【0142】 The clone with the most positions changed compared to the J6-3 clone was selected, and a library was prepared considering only the unchanged parts by the same method performed above. After enzyme immunoassay of the newly prepared library by bio-panning again, the 95th clone (G12 clone) was finally secured through nucleotide sequence analysis (Figure 36). The presence or absence of immunogenicity removal of the selected clone G12 was confirmed again, and it was confirmed that there were a total of two immunogenicities in the analysis results of the G12 clone (Figure 37). A library for changing this to human germlines was prepared, and through bio-panning, enzyme immunoassay, and nucleotide sequence analysis, a clone with completed immunogenicity and humanization of the conventional J6-3 clone was established (Figure 38). The clone that underwent the non-immunization and humanization processes was named the 2-G12 clone. The CDR and variable region sequences of the 2-G12 clone are summarized in the following table. 【0143】 【Table 19】 【0144】 In the above table, the CDR sequences (L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2, H-CDR3) included in the light chain variable region and heavy chain variable region are underlined. 【0145】 2.3.1. Binding ability to human CD28 In phage enzyme immunoassay, a humanized antibody clone (2-G12) that binds to human CD28 recombinant protein was cloned into a mammalian cell expression vector to express it in the scFv-Ck-scFv morphology [anti-CD28 scFv antibody (N'-VH-VL-C')-Ck-anti-cotinine antibody (N'-VH-VL-C')]. After overexpression of the antibody by transforming human embryonic kidney (Human embryonic kidney 293F, HEK293F) cells, the recombinant antibody was purified from the cell culture medium using affinity chromatography. The expressed and purified anti-CD28 antibodies (J6-3 clone and humanized 2-G12 clone) were found to be Jurkat E6.1 (hCD28). + Binding to the cell line was confirmed by flow cytometry (Figure 39). Furthermore, the J6-3 clone and the humanized 2-G12 clone were confirmed to bind to human CD28 recombinant protein (hCD28) (R&D Systems, Minneapolis, MN, USA) by enzyme immunoassay (Figure 40). 【0146】 2.3.2. CD4 T cell cytokine secretion inhibitory ability As a result of examining the cytokine secretion inhibitory ability of CD4 T cells from the humanized 2-G12 clone, it was confirmed that the 2-G12 clone had superior IL-2 (interleukin-2) secretion inhibitory ability compared to the positive control group (FR104) and exhibited similar IFN gamma inhibitory ability to the positive control group (FR104) (Figure 41). 【0147】 Examples 2-4. Production of humanized antibodies (2) To improve antibody stability, additional humanization was performed by replacing the chicken-derived amino acid sequence remaining in the framework region of the 2-G12 clone with a sequence identical to that of the human germline. As a result, a Hu2G12 clone was obtained in which 13 of the 23 chicken-derived residues in the conventional 2-G12 framework region were replaced with human germline sequences. The substituted residues were light chain (V L ) A13V, N14S, E17Q, V19A, K20R, S60D, T76S, R79Q and heavy chain (V H These are P1E, K3Q, T13Q, H105Q, and Q108E. The CDR and variable region sequences of the aforementioned Hu2G12 clone are summarized in Table 16 below. 【0148】 [Table 20] 【0149】 In the table above, the CDR sequences (L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2, H-CDR3) included in the light chain variable region and heavy chain variable region are underlined, and the 10 non-humanized residues are shown in bold. 【0150】 Examples 2-5. Production of humanized antibodies (3) In the Hu2G12 clone produced in Example 2-4, in order to further humanize the 10 unhumanized residues, the light chain (V) was selected from the remaining 10 chicken residues. L ) S42Q and T46L, and heavy chain (V H Single mutations to human germline sequences were introduced into the G49S, S76N, and K94R residues of ) respectively, resulting in a total of 5 clones and heavy chains (V H Fully humanized V (Vitamin V) with all three residues replaced. H They created clones. The aforementioned clones were named Hu2G12-S42Q (VL with S42Q mutation), Hu2G12-T46L (VL with T46L mutation), Hu2G12-G49S (VH with G49S mutation), Hu2G12-S76N (VH with S76N mutation), Hu2G12-K94R (VH with K94R mutation), and Hu2G12-G49S / S76N / K94R (VH with G49S, S76N, and K94R mutations). In one example, the CDR and variable region sequences of the following Hu2G12-S42Q and Hu2G12-S76N clones are summarized in Tables 17 and 18 below. 【0151】 [Table 21] 【0152】 [Table 22] 【0153】 Example 3. Anti-CD40L / anti-CD28 bispecific antibody Example 3-1. Anti-CD40L / anti-CD28 bispecific antibody (1) Anti-CD40L / anti-CD28 bispecific antibodies were prepared by conjugating each anti-CD40L antibody and anti-CD28 antibody in the form of a monovalent antibody. To produce exemplary anti-CD40L / anti-CD28 bispecific antibodies in the form described above, the TSG clone, which is a humanized anti-CD40L scFv antibody produced in Examples 1-4, and the 2-G12 antibody, which is a humanized anti-CD28 scFv antibody produced in Examples 2-3, were cloned in the form of bispecific antibodies containing a Ck linkage domain, and then expressed in a mammalian expression system (see schematic diagrams in Figures 42 and 67). Specifically, the antibodies were overexpressed by transformation of human embryonic kidney (Human embryonic kidney 293F, HEK293F) cells, and then recombinant antibodies were successfully purified from the cell culture medium using affinity chromatography (Figure 43). Figure 43 shows the SDS-PAGE results for the anti-CD40L / anti-CD28 bispecific antibody. 【0154】 Tables 19 and 20 below show the complete sequences of the anti-CD40L / anti-CD28 bispecific antibodies. The bispecific antibody in Table 19 contains, from the N' terminus, the anti-CD40L antibody (TSG clone), the Ck region (SEQ ID NO: 95), and the anti-CD28 antibody (2-G12 clone). The bispecific antibody in Table 20 contains, from the N' terminus, the anti-CD28 antibody (2-G12 clone), the Ck region (SEQ ID NO: 95), and the anti-CD40L antibody (TSG clone). The bispecific antibody in Table 19 is named TSGx2-G12, and the bispecific antibody in Table 20 is named 2-G12xTSG. 【0155】 [Table 23] 【0156】 [Table 24] 【0157】 [Table 25] 【0158】 [Table 26] 【0159】 Example 3-2. Anti-CD40L / anti-CD28 bispecific antibody (2) 3.2.1. Production of anti-CD40L / anti-CD28 bispecific antibodies Using the same method as in Example 3-1, the TSG clone, a humanized anti-CD40L scFv antibody produced in Example 1-4, and the Hu2G12 clone, an additionally humanized anti-CD28 scFv antibody produced in Example 2-4, were cloned into a mammalian cell-expressing pCEP4 vector (Invitrogen, USA) in the form of a bispecific antibody containing a Ck linking domain ([N'-(TSG scFv) - linker-Ck-linker - (Hu2G12 scFv)-C']; TSGxHu2G12), and transformed into Expi293F (Gibco, USA) cells. Subsequently, recombinant antibodies were purified from the culture medium using affinity chromatography, and the purified recombinant antibodies were confirmed by SDS-PAGE (Figure 44). The bispecific antibody produced above was named TSGxHu2G12. 【0160】 3.2.2. Confirmation of binding force to CD28 We confirmed whether the humanized anti-CD28 antibody site, obtained by adding the bispecific antibody produced in Example 3-2, retained its binding affinity to CD28 and its ability to inhibit CD28-CD80 (CD28 ligand) interaction. Therefore, the anti-CD40L / anti-CD28 bispecific antibody (TSGx2-G12, Table 19) produced in Example 3-1 and the anti-CD40L / anti-CD28 bispecific antibody (TSGxHu2G12) produced in Example 3-2 were used to treat recombinant human CD28 protein (R&D Systems, USA), and the binding affinity was confirmed by enzyme immunosorbent assay (ELISA). Specifically, recombinant CD28 protein (in a form bound to the human Fc region) was coated onto a 96-well plate (Corning, USA), and then blocked with 3% BSA / PBS (w / v). After that, TSGx2-G12 and TSGxHu2G12 were reacted for 2 hours, washed three times with 0.05% PBST (v / v), and treated with anti-hCk-HRP (Millipore, USA) for 1 hour. After the reaction was complete, three additional washes were performed, color development was carried out using ABTS (Thermo Scientific, USA), and absorbance was measured at 405 nm using a microplate reader (Thermo Scientific, USA). The results are shown in Figure 45. As can be seen from Figure 45, TSGxHu2G12 exhibited CD28 binding strength comparable to that of TSGx2-G12 and CD28. 【0161】 3.2.3. Confirmation of binding affinity to cell surface CD28 The TSGxHu2G12 clone was reacted with CD28-positive Jurkat E6.1 cell line, and its binding ability to CD28 expressed on the cell surface was confirmed by flow cytometry. Specifically, the 2G-12 and Hu2G12 clones were reacted with Jurkat E6.1 cell line for 1 hour, followed by three washes with 1% BSA / PBS (w / v) with 0.02% NaN3 (v / v), and then treatment with anti-hCk-APC (AbCam, UK) for 1 hour. After the reaction was complete, three more washes were performed, and fluorescence was detected using a flow cytometer (Becton Dickinson, USA). The results are shown in Figure 46. As shown in Figure 46, we confirmed that the bispecific antibody containing the Hu2G12 clone (TSGxHu2G12) has a binding affinity similar to that of the bispecific antibody containing the 2-G12 clone (TSGx2-G12) (Figure 46). 【0162】 3.2.4. Confirmation of CD28-CD80 interaction inhibitory ability To evaluate the CD28-CD80 interaction inhibitory ability of the TSGxHu2G12 clone, the 2G-12 and Hu2G12 clones were bound (100 nM or 1000 nM) for 1 hour to CD28-positive Jurkat E6.1 cell lines. The cells were then washed three times with 1% BSA / PBS (w / v) with 0.02% NaN3 (v / v), and 1 mg of recombinant CD80 protein (Sino Biological, China) was reacted for 1 hour. After the reaction, three more washes were performed, followed by reaction with anti-hFc-FITC (Invitrogen, USA), which can detect recombinant CD80 protein, and then three more washes. Fluorescence was then measured using a flow cytometer (Beckton Dickinson, USA) to confirm whether the TSGxHu2G12 clone could inhibit CD28 binding of recombinant CD80 protein. The results are shown in Figure 48. Figure 47 shows a schematic diagram of the experiment. As a result, we confirmed that a bispecific antibody containing the Hu2G12 clone (TSGxHu2G12) suppressed the binding of recombinant CD80 protein to a similar extent as a bispecific antibody containing the 2-G12 clone (TSGx2-G12) at an antibody concentration of 1000 nM (Figure 48). 【0163】 Example 3-3. Anti-CD40L / anti-CD28 bispecific antibody (3) 3.3.1. Production of anti-CD40L / anti-CD28 bispecific antibodies Using the same method as in Example 3-1, the TSG clone, a humanized anti-CD40L scFv antibody produced in Example 1-4, and the Hu2G12-S42Q, Hu2G12-T46L, Hu2G12-G49S, Hu2G12-S76N, Hu2G12-K94R, or Hu2G12-G49S / S76N / K94R clones, additionally humanized anti-CD28 scFv antibodies produced in Example 2-5, were cloned into a mammalian cell-expressing pCEP4 vector (Invitrogen, USA) in the form of bispecific antibodies containing a Ck-linking domain, and transformed into Expi293F (Gibco, USA) cells. Subsequently, recombinant antibodies were purified from the culture medium using affinity chromatography, and the purified recombinant antibodies were confirmed by SDS-PAGE. The bispecific antibodies produced were named TSGxHu2G12-S42Q, TSGxHu2G12-T46L, TSGxHu2G12-G49S, TSGxHu2G12-S76N, TSGxHu2G12-K94R, and TSGxHu2G12-G49S / S76N / K94R, respectively. 【0164】 3.3.2. Confirmation of binding force to CD28 We confirmed whether the humanized anti-CD28 antibody site, obtained by adding the bispecific antibody produced in Example 3-3, retained its binding affinity to CD28 and its ability to inhibit CD28-CD80 (CD28 ligand) interaction. Therefore, the anti-CD40L / anti-CD28 bispecific antibody (TSGx2-G12, Table 16) produced in Example 3-1 and the anti-CD40L / anti-CD28 bispecific antibodies (TSGxHu2G12-S42Q, TSGxHu2G12-T46L, TSGxHu2G12-G49S, TSGxHu2G12-S76N, TSGxHu2G12-K94R, TSGxHu2G12-G49S / S76N / K94R) produced in Example 3-3 were used to treat recombinant human CD28 protein (R&D systems, USA), and the binding affinity was confirmed by enzyme immunosorbent assay (ELISA) using the same method as in Example 3.2.2. The results are shown in Figure 49. 【0165】 As can be seen from Figure 49, in Example 3-3, an additional humanization process was carried out, and of the six anti-CD40L / anti-CD28 bispecific antibodies produced, the light chain (V L A clone (TSGxHu2G12-S42Q) into which the S42Q mutation was introduced, and the heavy chain (V H A clone (TSGxHu2G12-S76N) into which the S76N mutation was introduced showed a binding affinity to CD28 comparable to that of the anti-CD40L / anti-CD28 bispecific antibody (TSGx2-G12, Table 16) produced in Example 3-1. 【0166】 3.3.3. Confirmation of binding affinity to cell surface CD28 In Example 3.3.2, TSGxHu2G12-S42Q and TSGxHu2G12-S76N clones, which showed CD28 binding affinity comparable to that of the TSGx2-G12 clone, were reacted with the CD28-positive Jurkat E6.1 cell line, and their binding ability to CD28 expressed on the cell surface was confirmed by flow cytometry. Specifically, the Jurkat E6.1 cell line was reacted with the TSGx2-G12, TSGxHu2G12-S42Q, and TSGxHu2G12-S76N clones for 1 hour, then washed three times with 1% BSA / PBS (w / v) with 0.02% NaN3 (v / v), treated with anti-hCk-APC (AbCam, UK), and reacted for 1 hour. After the reaction was complete, three further washes were performed, and fluorescence was detected using a flow cytometer (Becton Dickinson, USA). The results are shown in Figure 50. As can be seen in Figure 50, the TSGxHu2G12-S42Q and TSGxHu2G12-S76N clones showed a slightly reduced binding affinity to cell surface CD28 compared to the TSGx2-G12 clone, but still exhibited a comparable binding affinity. 【0167】 3.3.4. Confirmation of CD28-CD80 interaction inhibitory ability To evaluate the CD28-CD80 interaction inhibitory ability of the aforementioned anti-CD40L / anti-CD28 bispecific antibody, CD28-positive Jurkat E6.1 cell lines were conjugated with TSGx2-G12, TSGxHu2G12-S42Q, and TSGxHu2G12-S76N clones (100nM, 250nM, or 1000nM) for 1 hour. The cells were then washed three times with 1% BSA / PBS (w / v) with 0.02% NaN3 (v / v), and 1 mg of recombinant CD80 protein (Sino Biological, China) was reacted for 1 hour. After the reaction, three washes were performed, followed by reaction with anti-hFc-Alexa Flour 594 (Jackson Laboratory, USA), which can detect recombinant CD80 protein, and then three washes. Fluorescence was then measured using a flow cytometer (Beckton Dickinson, USA), and the results are shown in Figure 52. Figure 51 shows a schematic diagram of the experiment described above. As can be seen from Figure 52, the TSGxHu2G12-S42Q and TSGxHu2G12-S76N clones exhibited recombinant CD80 protein binding inhibitory ability to a degree comparable to that of the TSGx2-G12 clone. 【0168】 Example 3-4. In vitro activity of anti-CD40L / anti-CD28 bispecific antibody (T cell suppressive ability) For T cells to be activated, T cell receptors (TCRs) on the surface of T cells must recognize antigens presented on the surface of antigen-presenting cells (APCs). At this time, T cells are known to be activated not only by signals transmitted via the TCR (Signal 1), but also by costimulatory signals (signal 2) transmitted when B7 molecules (B7.1[CD80], B7.2[CD86]) on the surface of APCs bind to CD28 on the surface of T cells. Activated T cells induce CD40L expression, and when CD40L from CD4 T cells binds to CD40 on the surface of APCs, CD40 transmits an activation signal to antigen-presenting cells. As a result, the expression of MHC molecules and costimulatory molecules (CD80, CD86, 41BBL, etc.) on the surface of APCs increases, as does the expression of T cell-stimulating cytokines such as IL-12. This activated APC is known to have a positive feedback mechanism that further enhances T cell activity (schematic diagram in Figure 53). Therefore, we evaluated whether the anti-CD40L / anti-CD28 bispecific antibody could simultaneously bind to CD40L and CD28 on CD4 T cells, block CD40L-CD40 and CD28-B7 binding, and consequently suppress CD4 T cell activation. 【0169】 CD4-positive T cells and mononuclear cells were isolated from healthy adult leukocytes obtained by leukocyte apheresis using the MACS cell separation method (CD14 microbead, 130-050-201, Miltenyi Biotech). Mononuclear cells were differentiated into dendritic cells by treatment with GM-CSF (20 ng / ml, JW Creagene, rhGM-CSF) and IL-4 (20 ng / ml, JW Creagene, rhIL-4) for 5 days. CD4-positive T cells were isolated from the leukocyte fraction of the same donor using the MACS cell separation method (CD4 microbead, 130-097-048, Miltenyi Biotech), added to differentiated dendritic cells, and then co-cultured with an anti-CD3 antibody (clone OKT3, 1 μg / ml, 50-561-956, Bio X cell) to stimulate the TCR. In the culture space, to maintain sufficient distance between T cells, B cells isolated from the leukocyte fraction of the same donor using the MACS cell separation method (CD19 microbead, 130-050-301, Miltenyi Biotech) were irradiated to prevent division and added as bystander cells for culture. The T cell suppressive efficacy of the bispecific antibody was evaluated by adding it to the above co-culture conditions. A schematic diagram of the experimental process described above is shown in Figure 54. 【0170】 The experimental results are shown in Figure 55. At this time, the CD28 mono-inhibitory antibody (aCot-Ck-aCD28; aCot-Ck-2-G12), which was also tested, showed an IFNr inhibitory effect to a similar degree as the bi-antibody (TSGx2-G12), but no IFNr inhibitory effect was observed with the CD40L mono-inhibitory antibody (aCD40L-Ck-aCot; TSG-Ck-aCot). Therefore, the T cell activation inhibitory effect of the bispecific antibody under these experimental conditions is considered to be mainly due to its inhibitory effect on CD28. This is interpreted as the fact that CD40L is expressed only in activated T cells, and therefore, no CD40L inhibitory effect was observed under the 48-hour culture conditions used to observe the initial activation of T cells. 【0171】 Examples 3-5. In vitro activity of anti-CD40L / anti-CD28 bispecific antibodies (induction of T cell immune tolerance) Immune tolerance is a mechanism by which the human immune system prevents autoimmune diseases. It refers to the phenomenon where, when self-antigen-specific T cells recognize self-antigens presented by antigen-presenting cells and receive a TCR signal (signal 1), they will not be activated even if they are subsequently stimulated again by self-antigens, unless they receive a co-stimulation (signal 2) from the antigen-presenting cells (see schematic diagram in Figure 56). In this example, it was confirmed that the anti-CD40L antibody (TSG clone), anti-CD28 antibody (2-G12 clone), and anti-CD40L / anti-CD28 bispecific antibody (TSGx2-G12 clone) simultaneously block CD40L and CD28 co-stimulation during the T cell activation step, and then promote immune tolerance in which these T cells are not activated again even when stimulated for activation. 【0172】 Similar to the initial T cell activation step experiment described above, human CD4 T cells, dendritic cells, and irradiated B cells were co-cultured with anti-CD3 antibody to induce CD4 T cell activity. Bispecific antibodies were then added to block co-stimulation (induction of immune tolerance). After 7 days, these CD4 T cells were stimulated again with new dendritic cells and anti-CD3 antibody, and their unresponsiveness was evaluated. T cell unresponsiveness was assessed by measuring IFNr secretion in the culture supernatant 48 hours after antigen restimulation using ELISA. A schematic diagram of the experimental process described above is shown in Figure 57. 【0173】 The experimental results are shown in Figure 58. In the case of CD40L monoantibodies or CD28 monoantibodies, a partial decrease in IFNr secretion compared to the negative control group antibody confirmed that monoantibodies also have an immune tolerance-inducing effect. In the group treated with the anti-CD40L / anti-CD28 bispecific antibody, the amount of IFNr decreased significantly compared to the negative control group, confirming that CD4 T cells treated with the bispecific antibody did not react to antigen restimulation (Figure 58). Therefore, it was confirmed that the bispecific antibody has a significant immune tolerance-inducing effect. 【0174】 Furthermore, the efficacy of various forms of anti-CD40L / anti-CD28 bispecific antibodies (anti-CD40L-Ck-CD28 bispecific antibody and anti-CD28-Ck-CD40L bispecific antibody) was compared. Two forms of anti-CD40L / anti-CD28 bispecific antibodies (anti-CD40L antibody-anti-CD28 antibody and anti-CD28 antibody-anti-CD40L antibody) were prepared. Then, the same treatment as described above was performed using these bispecific antibodies. After 48 hours, the IFNr production inhibitory ability was measured by ELISA, and it was confirmed that both antibodies effectively suppressed T cell activation (Figure 59), thus confirming that they have excellent T cell suppression ability regardless of the order in which the anti-CD40L antibody and anti-CD28 antibody are applied. 【0175】 Examples 3-6. Confirmation of cis interaction between anti-CD40L / anti-CD28 bispecific antibodies. The anti-CD40L / anti-CD28 bispecific antibody provided in this application can suppress the function of both CD40L and CD28 expressed on T cells independently by binding to them separately. In addition, it can simultaneously suppress both CD40L and CD28 expressed on a single T cell by simultaneously binding to them (cis-interaction). This linked inhibition method shows superior efficacy compared to separate inhibition. Schematic diagrams of the linked inhibition method and the separate inhibition method are shown in Figure 60. In this example, to confirm that the anti-CD40L / anti-CD28 bispecific antibody acts on T cells via cis interactions in a linked inhibition manner, a fluorescence resonance energy transfer (FRET) experiment was performed. 【0176】 The fluorescent protein-conjugated CD28 recombinant receptor [N'-Extracellular domain-Transmembrane domain-Cytoplasmic domain-linker-mNeongreen-C' morphology] (Addgene, [#113400] pEF6a-CD28-PafA) and CD40L recombinant receptor [N'-mScarlet-linker-Cytoplasmic domain-Transmembrane domain-Extracellular domain-C'] (Addgene, [#52337] CD154-His-bio) were cloned into pRK5 expression vectors. Human embryonic kidney (Human embryonic kidney 293A, HEK293A) cells cultured with 0.5 mg and 1 mg of the respective DNA for 24 hours were then simultaneously transformed using Lipofectamine 2000 (Invitrogen, USA). After 24 hours, the cells were treated with PMA (Phorbol myristate acetate) and cultured for a further 12 hours to induce cell activation. The activated HEK293A cells were then treated with 75 nM of anti-CD40L-Ck-CD28 bispecific antibody in scFv-Ck-scFv form, anti-CD40L (anti-CD40L scFv-Ck-cotinine scFv) or anti-CD28 (anti-cotinine scFv-Ck-CD28 scFv) antibody, or a mixture thereof (anti-CD40L antibody + anti-CD28 antibody), and two types of control group antibodies in the same form, and observed for 10 minutes. 【0177】 Subsequently, using an mCherry filter (Excitation 562 / 40, dichroic mirror 593, Emission 641 / 75), the light intensity was adjusted using a neutral density (ND) filter, and the cells were irradiated with an ND4 filter for 1 minute to photobleach the CD40L-mScarlet fluorescence. The mCherry fluorescence change after photobleaching was approximately -80% on average. The degree of fluorescence change of mNeonGreen in the cell membrane region (FITC intensity) was measured, and the degree of Fφrster resonance energy transfer (FRET) [(FITC intensity after photobleaching - FITC intensity before photobleaching) / FITC intensity after photobleaching] was calculated. The experimental results are shown in Figures 61 and 62. The experimental results confirmed that co-localization of CD40L and CD28 at the same position occurred only in the group treated with the anti-CD40L-Ck-CD28 bispecific antibody. 【0178】 Furthermore, after photobleaching, the degree of fluorescence change of CD28-mNeonGreen in the cell membrane region was measured, and the FRET efficacy was calculated. The results are shown in Figures 63 and 64. Only in the group treated with the anti-CD40L-Ck-CD28 bispecific antibody did the CD28-mNeonGreen fluorescence in the cell membrane region change significantly, and the FRET efficacy value was statistically significant compared to the other groups. This means that the anti-CD40L-Ck-CD28 bispecific antibody expressed on the cell surface binds to CD40L and CD28 expressed on the same cell surface through cis interaction. Therefore, this suggests that the anti-CD40L-Ck-CD28 bispecific antibody may act not only through individual inhibition of CD40L and CD28 via cis interactions, but also through a simultaneous inhibition mechanism. 【0179】 Examples 3-7. In vivo activity of anti-CD40L / anti-CD28 bispecific antibodies. While it is difficult to create in vivo autoimmune disease models using human T cells in experimental animals such as mice, a mouse model similar to autoimmune diseases exists: the xenogeneic GVHD (graft-versus-host disease) mouse model, in which inflammatory immune disease is induced by human T cells. In this mouse model, when human T cells are injected into immunodeficient mice, the human T cells recognize and attack the mouse's xeno-antigens, thereby inducing T cell-mediated inflammatory disease. This model shares a similar mechanism with autoimmune diseases in that it induces inflammation through excessive activation of T cells. Therefore, by evaluating the degree of inflammatory disease induction in mice, the degree of excessive activation of human T cells can be evaluated, and by observing the suppression of human T cell activity by T cell inhibitors and the resulting alleviation of inflammatory disease, it is possible to mimic the inhibitory effect on autoimmune diseases. Consequently, it can be used as an appropriate model for evaluating the in vivo functions of human anti-CD40L antibodies and human CD28 antibodies. 【0180】 Furthermore, this model can also be used to mimic allogeneic GVHD that can occur in hematological malignancy patients after hematopoietic stem cell transplantation due to transplanted donor T cells. Therefore, it can be used not only for autoimmune diseases but also as a model to evaluate the efficacy of antibodies as therapeutic agents for GVHD caused by T cell hyperactivation. Therefore, in this example, we aimed to establish a xenogeneic GVHD mouse model using human T cells and to verify the inflammatory disease suppressive effects of anti-CD40L antibodies and anti-CD28 antibodies. NSG mice (immunodeficient mice) (NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ), Jackson Laboratory, #005557) were subjected to 1 × 10⁻¹⁶ mononuclear cells (PBMCs, 1 × 10⁻¹⁶) isolated from the peripheral blood of healthy adults. 7 After intravenous injection of the drug, disease severity was scored based on weight loss and clinical symptoms in the mice, and disease progression was monitored. 【0181】 The experimental groups were divided into four groups: an untreated group (PBMC), a group treated with anti-CD40L antibody (TSG clone) alone (PBMC+aCD40L), a group treated with anti-CD28 antibody (2-G12 clone) alone (PBMC+aCD28), a group treated with a combination of anti-CD40L and anti-CD28 antibodies (PBMC+aCD40L+aCD28), and a group treated with anti-CD40L / anti-CD28 bispecific antibodies (PBMC+aCD40L-aCD28, PBMC+aCD28-aCD40L). Antibodies were administered intraperitoneally to mice three times a week, with 400 μg of antibody injected each time, for a total of nine injections. The weight results were based on the weight of the mice until their death. The experimental results are shown in Figures 65 and 66. In the group that did not receive antibody treatment (negative control group, PMBC), we confirmed that the disease developed due to weight loss after 30 days. 【0182】 The groups treated with either anti-CD40L antibody or anti-CD28 antibody alone showed similar levels of weight loss as the negative control group, similar disease progression in disease severity assessment, and all mice died within a similar timeframe as the negative control group. This indicates that, under these GVHD model conditions, suppression of a single co-stimulatory molecule alone is insufficient to inhibit disease progression. However, considering the in vitro inhibitory effects of the two individual antibodies on T cells and antigen-presenting cells, this does not mean that they have no efficacy in vivo. The group treated with both anti-CD40L and anti-CD28 antibodies showed slower weight loss compared to the control group, and slower disease progression in disease severity assessment. Furthermore, increased survival time was observed in mice treated with each antibody alone, indicating that combined therapy with the two antibodies can more effectively suppress disease progression. This result validates the efficacy of some individual antibodies and demonstrates that combined therapy with these two antibodies can produce a synergistic effect. However, the current antibody administration regimen could not completely suppress disease progression, resulting in weight loss and eventual death of the mice. 【0183】 On the other hand, the group treated with the anti-CD40L / anti-CD28 bispecific antibody showed no weight loss during the experimental period, and disease severity assessment confirmed that the disease did not develop. Furthermore, since all mice survived the experimental period, it was confirmed that disease progression was completely suppressed only in the group treated with the bispecific antibody. This result explains the superiority of the bispecific antibody morphology, in which two antibody sites exist simultaneously within a single molecule, compared to combined treatment with two single antibodies, and demonstrates the conceptual originality of the linked inhibition effect against CD40L and CD28. However, this result does not negate the partial efficacy of each single antibody or the individual efficacy of each single antibody when produced in a form other than scFv. 【0184】 Example 4. Anti-CD40L / anti-CD28 bispecific antibody for mice. Example 4-1. Production of anti-CD40L / anti-CD28 bispecific antibody for mice As mentioned above, animal models of autoimmune diseases using hyperactivation of human T cells are theoretically difficult to realize in experimental animals. In this example, in order to further support the anti-CD40L / anti-CD28 bispecific antibody provided in this application and its therapeutic effect on autoimmune diseases, we attempted to confirm the therapeutic effect on autoimmune diseases by treating an autoimmune disease mouse model with an anti-CD40L / anti-CD28 bispecific antibody (surrogate antibody) against mouse CD28 and mouse CD40L. This application proposes a novel anti-CD40L / anti-CD28 bispecific antibody in which each anti-CD40L antibody and anti-CD28 antibody are linked in the form of a monovalent antibody, and its use in the treatment of autoimmune diseases. Accordingly, the following example of the production of a mouse anti-CD40L / anti-CD28 bispecific antibody in which each antibody is linked in the form of a monovalent antibody, and its excellent therapeutic effect on autoimmune diseases, should be interpreted as supporting the anti-CD40L / anti-CD28 bispecific antibody and its therapeutic effect on autoimmune diseases provided by this application. 【0185】 4.1.1. Anti-mouse CD28 antibody The anti-mouse CD28 antibody used the scFv sequence of the PV-1 clone (Plos One 2017; 12(3):e0171822, Novel CD28 antagonist mPEG PV1-Fab' mitigates experimental autoimmune uveitis by suppressing CD4+ T lymphocyte activation and IFN-γ production). The CDR and variable region sequences of the aforementioned PV-1 clone (scFv) are shown in Table 21 below. 【0186】 [Table 27] 【0187】 4.1.2. Anti-mouse CD40L antibody The anti-mouse CD40L antibody was newly manufactured and used from chickens immunized with the mouse CD40L protein. Chickens were immunized four times with mouse CD40L recombinant protein (Sino Biological, China), and an antibody library in scFv form was constructed using a conventionally reported method. The complexity of the three chicken antibody libraries produced was 5.0 × 10⁶. 9 , 5.8×10 9 , 6.3×10 9 Bio-panning was performed against mouse CD40L (R&D systems, USA) using a chicken antibody library, and 16 clones that bound to mouse CD40L were selected using phage enzyme immunoassay. Their genetic information was then obtained by nucleotide sequence analysis. Of the 16 antibody clones that bind to the mouse CD40L recombinant protein, eight clones with different HCDR3 groups were cloned into a pCEP4 (Invitrogen, USA) vector for animal cell expression to produce scFv-hFc fusion proteins [anti-mouse CD40L scFv(N'-VL-linker-VH-C')-human Fc]. After transforming these clones into Expi293F (Gibco, USA) cells, the antibodies were purified from the cell culture medium by affinity chromatography, and the purified recombinant antibodies were confirmed by SDS-PAGE. 【0188】 The eight anti-mouse CD40L antibodies expressed above were confirmed to bind to L(mCD154+) cell lines expressing mouse CD40L (L4.5 cell line, DSMZ, Cat No. ACC 772) by flow cytometry. Specifically, L(mCD154+) cell lines were conjugated with anti-mouse CD40L antibodies at concentrations of 10, 100, and 1000 nM. After washing three times with 1% BSA / PBS (w / v) with 0.02% NaN3 (v / v), anti-hFc-FITC (Invitrogen, USA) was diluted 1:100 and conjugated, followed by three more washes. Fluorescence was detected using a flow cytometer (BD bioscience, USA). The results are shown by comparing mean fluorescence intensity (MFI) values, confirming that the 1-1-H, 1-12C, 1-12H, 2-12C, and 3-11-C clones effectively bind to mouse CD40L expressing the cell surface in a concentration-dependent manner (Figure 68). Based on the results described above, the 2-12-C clone with the highest binding affinity to mouse CD40L was selected. The CDR and variable region sequences of the 2-12-C clone are shown in Table 22 below. 【0189】 [Table 28] 【0190】 4.1.3. Anti-CD40L / anti-CD28 bispecific antibody for mouse The PV1 antibody, an anti-mouse CD28 scFv antibody, and the 2-12-C clone, an anti-mouse CD40L scFv antibody developed as described above, were cloned in the form of a bispecific antibody containing a human Ck linkage domain [anti-mouse CD28 scFv antibody (N'-VL-VH-C')-Ck-anti-mouse CD40L scFv antibody (N'-VL-VH-C')]. These were then cloned into a mammalian cell-expressing pCEP4 (Invitrogen, USA) vector, transformed into Expi293F (Gibco, USA) cells, and recombinant antibodies were purified from the culture medium using affinity chromatography. The purified recombinant antibodies were then confirmed by SDS-PAGE. The mouse anti-CD40L / anti-CD28 bispecific antibody produced as described above was named PV1x2-12-C clone, and its sequence is shown in Table 23 below. 【0191】 [Table 29] 【0192】 [Table 30] 【0193】 Example 4-2. Confirmation of mouse CD4 T cell binding ability (in vitro) Experiments were conducted to evaluate the T cell binding ability of the mouse anti-CD40L / anti-CD28 bispecific antibody (PV1x2-12-C) prepared in Example 4-1. CD4 T cells were isolated from mouse spleens and lymph nodes by MACS (mouse CD4 microbead, 130-117-043 Miltenyi Biotech), and then stimulated in vitro with plate-bound aCD3 (clone 145-2C11, BE0001-1, Bio X Cell, 10 μg / ml) and aCD28 (clone 37.51, 553294 BD bioscience, 2 μg / ml) for 48 hours to induce CD40L expression in the CD4 T cells. After binding the PV1x2-12-C antibody to activated CD4 T cells, antibody binding was measured by flow cytometry using a fluorescently labeled anti-Ck antibody. The results confirmed that the PV1x2-12-C antibody binds to activated mouse CD4 T cells (Figure 69). 【0194】 Example 4-3. Confirmation of T cell suppression ability (in vitro) We evaluated whether the aforementioned mouse anti-CD40L / anti-CD28 bispecific antibody (PV1x2-12-C) could simultaneously bind to CD40L and CD28 on CD4 T cells, block CD40L-CD40 and CD28-B7 binding, and thereby suppress CD4 T cell activation. Mouse bone marrow cells were isolated and cultured with GMCSF (20 ng / ml, JW Creagene, rmGM-CSF) and IL-4 (20 ng / ml, JW Creagene, rmIL-4) for 5 days to differentiate into dendritic cells. CD4 T cells were isolated from mouse spleens and lymph nodes by MACS, and then mouse CD4 T cells and dendritic cells were cultured together. Anti-mouse CD3 antibody (1 μg / ml) was then added to stimulate the TCR. Under the above co-culture conditions, the T cell suppressive efficacy of the bispecific antibody (PV1x2-12-C), an anti-CD40L / anti-CD28 bispecific antibody for mouse use, was evaluated by adding the bispecific antibody. 【0195】 After 48 hours, the supernatant was separated, and T cell-activating cytokines IFNr (IFN-r ELISA set, BDB555138, BD bioscience) and IL2 (IL-2 ELISA set, BDB555148, BD bioscience) were measured using the ELISA method. The results confirmed that IFNr secretion and IL2 secretion were significantly reduced in the group treated with PV1x2-12-C compared to the negative control group. This suggests that, similar to the experimental results of the anti-CD40L / anti-CD28 bispecific antibody that underwent the humanization process in Example 3, the mouse anti-CD40L / anti-CD28 bispecific antibody suppressed T cell activity by blocking the CD40L and CD28 signaling pathways of CD4 T cells (Figure 70). 【0196】 Example 4-4. Evaluation of the ability to suppress autoimmune diseases. In Example 4-3, it was confirmed that the mouse anti-CD40L / anti-CD28 bispecific antibody (PV1x2-12-C) suppressed T cells in the same way as the humanized anti-CD40L / anti-CD28 bispecific antibody. Therefore, experiments were conducted to confirm whether the mouse anti-CD40L / anti-CD28 bispecific antibody could suppress in vivo autoimmune T cells and thus suppress autoimmune diseases. 【0197】 We established an experimental autoimmune encephalomyelitis (EAE) mouse model, an animal model of multiple sclerosis, a central nervous system autoimmune disease, and verified the efficacy of an anti-CD40LxCD28 antibody. We conducted an experiment to induce autoimmune demyelinating disease in WT C57Bl / 6 mice (C57BL / 6, Orient Bio) by subcutaneously injecting a large amount of neuromyelin autoantigen peptide (MOG peptide, Peptron, 200 μg / mouse) along with Complete Freuds Adjuvant (Incomplete Freuds Adjuvant (IFA) (F5506, Sigma-Aldrich) + Killed mycobacterium tuberculosis (231141, BD Biosciences), 1000 μg / mouse) and pertussis toxin (P7208, Sigma-Aldrich). We then confirmed that disease progression could be suppressed by intraperitoneal injection of a mouse anti-CD40L / anti-CD28 bispecific antibody (PV1x2-12-C). As a control group for single antibodies, bispecific antibodies were used, which were obtained by linking each single antibody (PV1 and 2-12-C clone, scFv) with an anti-cotinine antibody scFv (Korean Patent Publication KR 10-2019-0023084 A) (Cotx2-12-C; PV1xCot, see Example 1-1). Figure 71 shows the results of monitoring the progression of the disease over time after administering 200 μg of antibody to each mouse via intraperitoneal injection a total of six times at two-day intervals. 【0198】 As shown in Figure 71, in the control group (not treated with antibodies), disease progression was confirmed by an increase in mean clinical score over time. When treated with a single antibody control group (Cotx2-12-C; PV1xCot), disease progression was partially suppressed compared to the untreated group, confirming the partial efficacy of the single antibody. In the group treated with the bispecific antibody (PV1x2-12-C), the clinical severity of the disease was significantly lower, confirming that the bispecific antibody effectively suppresses autoimmune demyelinating disease. These experimental results should be interpreted as supporting the excellent effect of the anti-CD40L / anti-CD28 bispecific antibody, newly proposed in this application, which is linked in the form of an antigen-binding fragment of an anti-CD40L antibody without an Fc region and an antigen-binding fragment of a monovalent anti-CD28 antibody. This antibody does not exhibit side effects such as thrombosis, and simultaneously suppresses both types of costimulatory molecules, effectively suppressing autoimmune diseases. 【0199】 From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. In this regard, the embodiments described above should be understood to be illustrative and not limiting in all respects. The scope of the present invention should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

Claims

[Claim 1] It is an anti-CD40L / anti-CD28 bispecific antibody, (1) Antigen-binding fragment of an anti-CD40L antibody that does not contain the Fc region, and (2) comprising an antigen-binding fragment of an anti-CD28 antibody in the form of a monovalent antibody, The aforementioned anti-CD40L / anti-CD28 bispecific antibody. [Claim 2] (1) The antigen-binding fragment of the anti-CD40L antibody is anti-CD40L scFv, Fv, (scFv) 2 , Fab, Fab', F(ab') 2 Alternatively, it may be in the form of a single-domain antibody lacking an Fc region. (2) The antigen-binding fragment of the anti-CD28 antibody is in the form of a single-domain antibody, such as anti-CD28 scFv, Fv, Fab, Fab', or a monovalent antibody. The anti-CD40L / anti-CD28 bispecific antibody according to claim 1. [Claim 3] (1) The antigen-binding fragment of the anti-CD40L antibody is anti-CD40L scFv, (2) The antigen-binding fragment of the anti-CD28 antibody is anti-CD28 scFv. The anti-CD40L / anti-CD28 bispecific antibody according to claim 1. [Claim 4] The anti-CD40L / anti-CD28 bispecific antibody according to claim 1, wherein the anti-CD40L / anti-CD28 bispecific antibody is in a form in which anti-CD40L scFv and anti-CD28 scFv are linked by the invariant region (C kappa) of the light chain of an IgG antibody. [Claim 5] 1) The antigen-binding fragment of the anti-CD40L antibody is L-CDR1 containing the amino acid sequence of SEQ ID NO: 1 or 2, L-CDR2 containing the amino acid sequence of SEQ ID NO: 4 or 5, L-CDR3 containing the amino acid sequence of SEQ ID NO: 7 or 8, H-CDR1 containing the amino acid sequence of SEQ ID NO: 10 or 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 13 or 14, and Contains H-CDR3 containing the amino acid sequence of SEQ ID NO: 16 or 17, 2) The antigen-binding fragment of the anti-CD28 antibody is L-CDR1 containing the amino acid sequence of SEQ ID NO: 65, L-CDR2 containing the amino acid sequence of SEQ ID NO: 66, L-CDR3 containing the amino acid sequence of SEQ ID NO: 67, H-CDR1 containing the amino acid sequence of SEQ ID NO: 68 or 69, H-CDR2 containing the amino acid sequence of SEQ ID NO: 70, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 71 The anti-CD40L / anti-CD28 bispecific antibody according to claim 1. [Claim 6] 1) The antigen-binding fragment of the anti-CD40L antibody is (1) L-CDR1 containing the amino acid sequence of SEQ ID NO: 1, L-CDR2 containing the amino acid sequence of SEQ ID NO: 4, L-CDR3 containing the amino acid sequence of SEQ ID NO: 7, H-CDR1 containing the amino acid sequence of SEQ ID NO: 10, H-CDR2 containing the amino acid sequence of SEQ ID NO: 13, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 16, or (2) comprising L-CDR1 containing the amino acid sequence of SEQ ID NO: 2, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, L-CDR3 containing the amino acid sequence of SEQ ID NO: 8, H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 14, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 17, 2) The antigen-binding fragment of the anti-CD28 antibody is L-CDR1 containing the amino acid sequence of SEQ ID NO: 65, L-CDR2 containing the amino acid sequence of SEQ ID NO: 66, L-CDR3 containing the amino acid sequence of SEQ ID NO: 67, H-CDR1 containing the amino acid sequence of SEQ ID NO: 68 or 69, H-CDR2 containing the amino acid sequence of SEQ ID NO: 70, and H-CDR3 containing the amino acid sequence of SEQ ID NO:

71. The anti-CD40L / anti-CD28 bispecific antibody according to claim 5. [Claim 7] 1) The antigen-binding fragment of the anti-CD40L antibody is (1) A light chain variable region containing the amino acid sequence of SEQ ID NOs. 47, 48, 49, or 128, and (2) Includes a heavy chain variable region containing the amino acid sequence of SEQ ID NOs. 51, 52, 53, 54, 55, or 56, 2) The antigen-binding fragment of the anti-CD28 antibody is (1) A light chain variable region containing the amino acid sequence of SEQ ID NOs. 87, 88, 98, or 100, and (2) A heavy chain variable region including the amino acid sequence of SEQ ID NOs. 89, 90, 99, or 101, The anti-CD40L / anti-CD28 bispecific antibody according to claim 5. [Claim 8] 1) The antigen-binding fragment of the anti-CD40L antibody is (1) A light chain variable region containing the amino acid sequence of SEQ ID NO: 47, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 51, (2) Light chain variable region containing the amino acid sequence of SEQ ID NO: 48 or 128, and heavy chain variable region containing the amino acid sequence of SEQ ID NO: 52, 53, 54 or 55, (3) A light chain variable region containing the amino acid sequence of SEQ ID NO: 49 and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 56, 2) The antigen-binding fragment of the anti-CD28 antibody is (1) A light chain variable region containing the amino acid sequence of SEQ ID NO: 87, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89, (2) A light chain variable region containing the amino acid sequence of SEQ ID NO: 88, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 90, (3) Light chain variable region containing the amino acid sequence of SEQ ID NO: 98, and heavy chain variable region containing the amino acid sequence of SEQ ID NO: 99, (4) A light chain variable region containing the amino acid sequence of SEQ ID NO: 100, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 99, or (5) A light chain variable region containing the amino acid sequence of SEQ ID NO: 98, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 101, The anti-CD40L / anti-CD28 bispecific antibody according to claim 5. [Claim 9] The anti-CD40L / anti-CD28 bispecific antibody according to claim 5, wherein the anti-CD40L / anti-CD28 bispecific antibody is in a form in which anti-CD40L scFv and anti-CD28 scFv are linked by the invariant region (C kappa) of the light chain of an IgG antibody. [Claim 10] The anti-CD40L / anti-CD28 bispecific antibody according to claim 9, wherein the invariant region of the light chain of the IgG antibody includes the amino acid sequence of SEQ ID NO:

95. [Claim 11] A pharmaceutical composition for prevention or treatment of one or more diseases selected from the group consisting of autoimmune diseases and graft-versus-host diseases, comprising an anti-CD40L / anti-CD28 bispecific antibody as described in any one of claims 1 to 10. [Claim 12] The aforementioned autoimmune disease is caused by T cell overexpression, as described in claim 11. [Claim 13] The aforementioned autoimmune diseases include rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, type 1 diabetes, Crohn's disease, scleroderma, Sjögren's syndrome, psoriasis, inflammatory bowel disease, ulcerative colitis, ankylosing spondylitis, interstitial lung disease, uveitis, optic neuritis, peripheral neuropathies, sarcoidosis, antiphospholipid syndrome, and inflammatory myopathy. The pharmaceutical composition according to claim 11, which is one or more selected from the group consisting of myopathies, Behcet's disease, alopecia totalis / universalis, pemphigus vulgaris, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, Guillain-Barré syndrome, celiac disease, and pernicious anemia. [Claim 14] The pharmaceutical composition according to claim 11, wherein the graft-versus-host disease is one or more selected from the group consisting of acute graft-versus-host disease and chronic graft-versus-host disease. [Claim 15] An anti-CD40L antibody or its antigen-binding fragment, L-CDR1 containing the amino acid sequence of SEQ ID NO: 1 or 2, L-CDR2 containing the amino acid sequence of SEQ ID NO: 4 or 5, L-CDR3 containing the amino acid sequence of SEQ ID NO: 7 or 8, H-CDR1 containing the amino acid sequence of SEQ ID NO: 10 or 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 13 or 14, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 16 or 17, The aforementioned anti-CD40L antibody or its antigen-binding fragment. [Claim 16] (1) L-CDR1 containing the amino acid sequence of SEQ ID NO: 1, L-CDR2 containing the amino acid sequence of SEQ ID NO: 4, L-CDR3 containing the amino acid sequence of SEQ ID NO: 7, H-CDR1 containing the amino acid sequence of SEQ ID NO: 10, H-CDR2 containing the amino acid sequence of SEQ ID NO: 13, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 16, or (2) L-CDR1 containing the amino acid sequence of SEQ ID NO: 2, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, L-CDR3 containing the amino acid sequence of SEQ ID NO: 8, H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 14, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 17 The anti-CD40L antibody or antigen-binding fragment thereof according to claim 15. [Claim 17] Light chain variable region containing the amino acid sequence of SEQ ID NOs. 47, 48, 49, or 128, and The anti-CD40L antibody or antigen-binding fragment thereof according to claim 15, comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 51, 52, 53, 54, 55, or 56. [Claim 18] (1) A light chain variable region containing the amino acid sequence of SEQ ID NO: 47, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 51, (2) Light chain variable region containing the amino acid sequence of SEQ ID NO: 48 or 128, and heavy chain variable region containing the amino acid sequence of SEQ ID NO: 52, 53, 54 or 55, (3) The anti-CD40L antibody or antigen-binding fragment thereof according to claim 15, comprising a light chain variable region containing the amino acid sequence of SEQ ID NO: 49 and a heavy chain variable region containing the amino acid sequence of SEQ ID NO:

56. [Claim 19] The anti-CD40L antibody or antigen-binding fragment according to claim 15, characterized in that the anti-CD40L antibody or antigen-binding fragment thereof does not contain an Fc region. [Claim 20] The anti-CD40L antibody or its antigen-binding fragment is anti-CD40L scFv, Fv, (scFv) 2 , Fab, Fab', F(ab') 2 Alternatively, the anti-CD40L antibody or antigen-binding fragment thereof according to claim 15, which is in the form of a single-domain antibody lacking an Fc region. [Claim 21] The anti-CD40L antibody or antigen-binding fragment thereof according to claim 15, wherein the anti-CD40L antibody or antigen-binding fragment thereof is anti-CD40L scFv. [Claim 22] A pharmaceutical composition for the prevention or treatment of one or more diseases selected from the group consisting of autoimmune diseases and graft-versus-host diseases, comprising an anti-CD40L antibody or an antigen-binding fragment thereof as described in any one of claims 15 to 21. [Claim 23] The aforementioned autoimmune disease is caused by excessive activation of T cells, as described in claim 22. [Claim 24] An anti-CD28 antibody or its antigen-binding fragment, L-CDR1 containing the amino acid sequence of SEQ ID NO: 65, L-CDR2 containing the amino acid sequence of SEQ ID NO: 66, L-CDR3 containing the amino acid sequence of SEQ ID NO: 67, H-CDR1 containing the amino acid sequence of SEQ ID NO: 68 or 69, H-CDR2 containing the amino acid sequence of SEQ ID NO: 70, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 71 The aforementioned anti-CD28 antibody or its antigen-binding fragment. [Claim 25] (1) A light chain variable region containing the amino acid sequence of SEQ ID NOs. 87, 88, 98, or 100, and (2) A heavy chain variable region including the amino acid sequence of SEQ ID NOs. 89, 90, 99, or 101, The anti-CD28 antibody or antigen-binding fragment thereof according to claim 24. [Claim 26] (1) A light chain variable region containing the amino acid sequence of SEQ ID NO: 87, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89, (2) A light chain variable region containing the amino acid sequence of SEQ ID NO: 88, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 90, (3) Light chain variable region containing the amino acid sequence of SEQ ID NO: 98, and heavy chain variable region containing the amino acid sequence of SEQ ID NO: 99, (4) A light chain variable region containing the amino acid sequence of SEQ ID NO: 100, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 99, or (5) A light chain variable region containing the amino acid sequence of SEQ ID NO: 98, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 101, The anti-CD28 antibody or antigen-binding fragment thereof according to claim 24. [Claim 27] The anti-CD28 antibody or its antigen-binding fragment according to claim 24, wherein the antigen-binding fragment of the anti-CD28 antibody is in the form of a monovalent antibody. [Claim 28] The anti-CD28 antibody or its antigen-binding fragment according to claim 24, wherein the antigen-binding fragment of the anti-CD28 antibody is in the form of a single-domain antibody in the form of an anti-CD28 scFv, Fv, Fab, Fab', or monovalent antibody. [Claim 29] The anti-CD28 antibody or antigen-binding fragment thereof according to claim 24, wherein the anti-CD28 antibody or antigen-binding fragment thereof is anti-CD28 scFv. [Claim 30] A pharmaceutical composition for the prevention or treatment of one or more diseases selected from the group consisting of autoimmune diseases and graft-versus-host diseases, comprising an anti-CD40L antibody or an antigen-binding fragment thereof as described in any one of claims 24 to 29. [Claim 31] The aforementioned autoimmune disease is caused by excessive activation of T cells, as described in claim 30.

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