WO2022169269A1 - Anti-ctla-4 antibody and use thereof - Google Patents

Abstract

The present invention relates to an anti-CTLA-4 antibody and, more specifically, to an anti-CTLA-4 antibody and a multi-specific antibody designed on the basis of the anti-CTLA-4 antibody. It has been identified that an anti-CTLA-4 antibody according to the present invention exhibits excellent binding to CTLA-4 and remarkably reduces tumor size in animal models. In addition, the multi-specific antibody designed on the basis of the anti-CTLA-4 antibody also exhibits excellent binding to antigen 4-1BB, and thus can more effectively inhibit tumors. Therefore, the anti-CTLA-4 antibody and the multi-specific antibody designed on the basis of the anti-CTLA-4 antibody, of the present invention, can be effectively used in the prevention or treatment of tumors or cancer and inflammatory diseases of interest.

Description

Anti-CTLA-4 antibodies and uses thereof

The present invention relates to an anti-CTLA-4 antibody, and more particularly, to the anti-CTLA-4 antibody and a multispecific antibody designed based thereon.

CTLA-4 (cytotoxic T lymphocyte-associated protein 4) is expressed in activated T cells and transmits an inhibitory signal to T cells. CTLA-4 is a co-inhibitory molecule and its counterpart to co-stimulatory B7-CD28. When the surface expression of CTLA-4 is up-regulated and binds to B7 and higher avidity, the co-stimulatory signal of CD28 is blocked, so that the negative signal by CTLA-4 reduces T cell proliferation and reduces IL-2 production. Therefore, induction of a co-stimulatory signal by the interaction of B7 and CD28 by blocking CTLA-4 can be expected to have anticancer efficacy by tumor rejection.

Accordingly, the present inventors have completed the present invention by developing an anti-CTLA-4 antibody and a dual-specific antibody designed based thereon.

Accordingly, an object of the present invention is to provide an antibody or antigen-binding fragment thereof that specifically binds to CTLA-4 (cytotoxic T lymphocyte-associated protein 4).

Another object of the present invention is to provide a multi-specific antibody comprising a heavy chain variable region, an IgG4 isotype, and an scFv of an anti 4-1BB antibody.

Another object of the present invention, the anti-CTLA-4 antibody, a binding fragment thereof; or the multispecific antibody; to provide a nucleic acid encoding the nucleic acid, a recombinant expression vector containing the nucleic acid, and a host cell transfected with the expression vector.

Another object of the present invention is to generate an antibody by culturing the host cell; And to provide a method for producing an antibody that specifically binds to CTLA-4, an antigen-binding fragment thereof, or a multispecific antibody comprising the step of isolating and purifying the generated antibody.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising an antibody that specifically binds to CTLA-4, an antigen-binding fragment thereof, or a multispecific antibody.

Another object of the present invention is to provide a method for preventing or treating cancer comprising administering to an individual in need thereof an antibody, antigen-binding fragment thereof, or multispecific antibody that specifically binds to CTLA-4. .

In order to achieve the above object, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CTLA-4 (cytotoxic T lymphocyte-associated protein 4), comprising:

a heavy chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 9, 15, 23, 31, 39, 46, 54, 62, 68, 75, 88, 100 and 115;

SEQ ID NO: 2. heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of 10, 16, 24, 32, 40, 47, 55, 63, 69, 76, 81, 89, 95, 101, 108 and 116;

a heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11, 17, 25, 33, 41, 48, 56, 64, 70, 77, 82, 90, 96, 102, 109 and 117; and

a light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 18, 26, 34, 42, 49, 57, 71, 83, 103 and 110;

a light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 19. 27, 35, 50, 58, 84, 104 and 111; and

A light chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 20, 28, 36, 43, 51, 59, 65, 72, 78, 85, 92, 97, 105, 112 and 118.

The present invention also provides (a) heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9 or 148, heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 149, 152 and 155, and amino acids of SEQ ID NO: 11 a heavy chain variable region comprising a heavy chain CDR3 comprising a sequence; IgG4 isotype represented by the amino acid sequence of SEQ ID NO: 139; and a scFv of an anti 4-1BB antibody comprising the amino acid sequence shown in SEQ ID NOs: 141 to 145; a heavy chain variable region of a multispecific antibody comprising; (b) a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or 158, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; Multispecific antibodies are provided.

The present invention also provides an antibody that specifically binds to CTLA-4, an antigen-binding fragment thereof or a nucleic acid encoding a multispecific antibody, a recombinant expression vector comprising the nucleic acid, and a host cell transfected with the expression vector.

The present invention also comprises the steps of culturing the host cell to generate an antibody; And it provides a method for producing an antibody that specifically binds to CTLA-4, an antigen-binding fragment thereof, or a multispecific antibody comprising the step of isolating and purifying the generated antibody.

The present invention also provides a pharmaceutical composition for preventing or treating cancer comprising an antibody that specifically binds to CTLA-4, an antigen-binding fragment thereof, or a multispecific antibody.

The present invention also provides a method for preventing or treating cancer, comprising administering an antibody, antigen-binding fragment thereof, or multispecific antibody that specifically binds to CTLA-4 to an individual in need thereof.

It was confirmed that the anti-CTLA-4 antibody according to the present invention exhibits excellent binding ability to CTLA-4 and significantly reduces tumor size in animal models. In addition, the multispecific antibody designed based on the anti-CTLA-4 antibody exhibits excellent binding ability to the 4-1BB antigen, and thus can more effectively inhibit tumors. Therefore, the anti-CTLA-4 antibody of the present invention and a multispecific antibody designed based thereon can be usefully used for the prevention or treatment of a desired tumor, cancer, or infectious disease.

1 is a diagram showing the results of primary selection of candidate clones through ELISA screening.

2 is a diagram showing the sequencing results of the primary selected candidate clones.

3 is a diagram showing the results of analyzing the CLTA-4 antigen binding force of the clones primarily selected through flow cytometry.

4 is a diagram showing the in vitro functional assay results of clones (secondary selection) having binding affinity to CTLA-4.

5A is a diagram showing the results of SDS-PAGE analysis of anti-CTLA-4-8 antibodies finally selected through in vitro functional assay.

5B is a diagram showing the results of analyzing the purity of the anti-CTLA-4-8 antibody through SEC-HPLC.

5C is a diagram showing the results of confirming the antigen-binding ability of the anti-CTLA-4-8 antibody through SPR analysis.

Figure 5D is a diagram showing the result of confirming the antigen-binding ability of the anti-CTLA-4-8 antibody through flow cytometry.

6 is a diagram showing the sequence and purification results of the dual specific antibody against CTLA-4 and 4-1BB.

7A is a diagram showing the results of SDS-PAGE analysis of the produced bispecific antibody.

7B is a diagram showing the results of confirming the antigen-binding ability of the bispecific antibody quantified through Q-ELISA.

8 is a diagram showing the results of confirming the antigen binding force of the dual specific antibody through flow cytometry.

9 is a diagram showing the results of confirming the antigen binding force of the dual specific antibody in a clinical sample through flow cytometry.

10 is a diagram showing the results of confirming the antigen binding force of the dual specific antibody through SPR analysis.

11 is a diagram showing the results of analysis of tumor size according to treatment with a dual specific antibody in an animal model.

12A and 12B are diagrams showing the sequence of a dual specific antibody with improved antigen-binding ability.

13 is a diagram showing the results of mutation analysis and antigen-binding ability of a dual-specific antibody with improved antigen-binding ability.

Hereinafter, the present invention will be described in detail.

According to an aspect of the present invention, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CTLA-4 (cytotoxic T lymphocyte-associated protein 4), comprising:

a heavy chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 9, 15, 23, 31, 39, 46, 54, 62, 68, 75, 88, 100 and 115;

SEQ ID NO: 2. heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of 10, 16, 24, 32, 40, 47, 55, 63, 69, 76, 81, 89, 95, 101, 108 and 116;

a heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11, 17, 25, 33, 41, 48, 56, 64, 70, 77, 82, 90, 96, 102, 109 and 117; and

a light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 18, 26, 34, 42, 49, 57, 71, 83, 103 and 110;

a light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 19. 27, 35, 50, 58, 84, 104 and 111; and

A light chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 20, 28, 36, 43, 51, 59, 65, 72, 78, 85, 92, 97, 105, 112 and 118.

As used herein, the term “antibody” refers to an anti-CTLA-4 antibody that specifically binds to CTLA-4. The scope of the present invention includes not only complete antibody forms that specifically bind to CTLA-4, but also antigen-binding fragments of the antibody molecule.

A complete antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain linked to the heavy chain by a disulfide bond.

As used herein, the term "heavy chain" refers to a full-length heavy chain comprising a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and three constant region domains CH1, CH2 and CH3. and fragments thereof. In addition, as used herein, the term "light chain" refers to a full-length light chain comprising a variable region domain VL and a constant region CL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and fragments thereof. all means

The whole antibody includes subtypes of IgA, IgD, IgE, IgM and IgG, in particular, IgG includes IgG1, IgG2, IgG3 and IgG4. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types and subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3). ), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has a kappa (κ) and a lambda (λ) type.

As used herein, the term "variable region" of an antibody refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of complementarity determining regions (CDRs; ie, CDR1, CDR2, and CDR3) and framework regions (FR). VH refers to the variable domain of a heavy chain. VL refers to the variable domain of the light chain.

"Complementarity determining region (CDR)" refers to amino acid residues of an antibody variable domain, which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.

In an embodiment of the present invention, the antibody or antigen-binding fragment thereof

(1) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 2 and the heavy chain CDR3 of SEQ ID NO: 3, and the light chain CDR1 of SEQ ID NO: 4, the light chain CDR2 of SEQ ID NO: 5 and the light chain of SEQ ID NO: 6 a light chain variable region comprising CDR3;

(2) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 9, the heavy chain CDR2 of SEQ ID NO: 10 and the heavy chain CDR3 of SEQ ID NO: 11, and the light chain CDR1 of SEQ ID NO: 12, the light chain CDR2 of SEQ ID NO: 5 and the light chain of SEQ ID NO: 6 a light chain variable region comprising CDR3;

(3) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 15, the heavy chain CDR2 of SEQ ID NO: 16 and the heavy chain CDR3 of SEQ ID NO: 17, and the light chain CDR1 of SEQ ID NO: 18, the light chain CDR2 of SEQ ID NO: 19 and the light chain of SEQ ID NO: 20 a light chain variable region comprising CDR3;

(4) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 23, the heavy chain CDR2 of SEQ ID NO: 24 and the heavy chain CDR3 of SEQ ID NO: 25, and the light chain CDR1 of SEQ ID NO: 26, the light chain CDR2 of SEQ ID NO: 27 and the light chain of SEQ ID NO: 28 a light chain variable region comprising CDR3;

(5) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 31, the heavy chain CDR2 of SEQ ID NO: 32 and the heavy chain CDR3 of SEQ ID NO: 33, and the light chain CDR1 of SEQ ID NO: 34, the light chain CDR2 of SEQ ID NO: 35 and the light chain of SEQ ID NO: 36 a light chain variable region comprising CDR3;

(6) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 39, the heavy chain CDR2 of SEQ ID NO: 40 and the heavy chain CDR3 of SEQ ID NO: 41, and the light chain CDR1 of SEQ ID NO: 42, the light chain CDR2 of SEQ ID NO: 5 and the light chain of SEQ ID NO: 43 a light chain variable region comprising CDR3;

(7) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 46, the heavy chain CDR2 of SEQ ID NO: 47 and the heavy chain CDR3 of SEQ ID NO: 48, and the light chain CDR1 of SEQ ID NO: 49, the light chain CDR2 of SEQ ID NO: 50 and the light chain of SEQ ID NO: 51 a light chain variable region comprising CDR3;

(8) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 54, the heavy chain CDR2 of SEQ ID NO: 55 and the heavy chain CDR3 of SEQ ID NO: 56, and the light chain CDR1 of SEQ ID NO: 57, the light chain CDR2 of SEQ ID NO: 58 and the light chain of SEQ ID NO: 59 a light chain variable region comprising CDR3;

(9) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 62, the heavy chain CDR2 of SEQ ID NO: 63 and the heavy chain CDR3 of SEQ ID NO: 64, and the light chain CDR1 of SEQ ID NO: 57, the light chain CDR2 of SEQ ID NO: 58 and the light chain of SEQ ID NO: 65 a light chain variable region comprising CDR3;

(10) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 68, the heavy chain CDR2 of SEQ ID NO: 69 and the heavy chain CDR3 of SEQ ID NO: 70, and the light chain CDR1 of SEQ ID NO: 71, the light chain CDR2 of SEQ ID NO: 58 and the light chain of SEQ ID NO: 72 a light chain variable region comprising CDR3;

(11) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 75, the heavy chain CDR2 of SEQ ID NO: 76 and the heavy chain CDR3 of SEQ ID NO: 77, and the light chain CDR1 of SEQ ID NO: 57, the light chain CDR2 of SEQ ID NO: 58 and the light chain of SEQ ID NO: 78 a light chain variable region comprising CDR3;

(12) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 81 and the heavy chain CDR3 of SEQ ID NO: 82, and the light chain CDR1 of SEQ ID NO: 83, the light chain CDR2 of SEQ ID NO: 84 and the light chain of SEQ ID NO: 85 a light chain variable region comprising CDR3;

(13) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 88, the heavy chain CDR2 of SEQ ID NO: 89 and the heavy chain CDR3 of SEQ ID NO: 90, and the light chain CDR1 of SEQ ID NO: 57, the light chain CDR2 of SEQ ID NO: 58 and the light chain of SEQ ID NO: 92 a light chain variable region comprising CDR3;

(14) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 9, the heavy chain CDR2 of SEQ ID NO: 95 and the heavy chain CDR3 of SEQ ID NO: 96, and the light chain CDR1 of SEQ ID NO: 57, the light chain CDR2 of SEQ ID NO: 58 and the light chain of SEQ ID NO: 97 a light chain variable region comprising CDR3;

(15) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 100, the heavy chain CDR2 of SEQ ID NO: 101 and the heavy chain CDR3 of SEQ ID NO: 102, and the light chain CDR1 of SEQ ID NO: 103, the light chain CDR2 of SEQ ID NO: 104 and the light chain of SEQ ID NO: 105 a light chain variable region comprising CDR3;

(16) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 46, the heavy chain CDR2 of SEQ ID NO: 108 and the heavy chain CDR3 of SEQ ID NO: 109, and the light chain CDR1 of SEQ ID NO: 110, the light chain CDR2 of SEQ ID NO: 111 and the light chain of SEQ ID NO: 112 a light chain variable region comprising CDR3; or

(17) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 115, the heavy chain CDR2 of SEQ ID NO: 116 and the heavy chain CDR3 of SEQ ID NO: 117, and the light chain CDR1 of SEQ ID NO: 57, the light chain CDR2 of SEQ ID NO: 58 and the light chain of SEQ ID NO: 118 It is preferable to include; a light chain variable region comprising CDR3.

In the present invention, a "framework region (FR)" is a variable domain residue other than a CDR residue. Each variable domain typically has four FRs: FR1, FR2, FR3 and FR4.

In an embodiment of the present invention, the primary antibody or antigen-binding fragment thereof is SEQ ID NO: 7, 13, 21, 29, 37, 44, 52, 60, 66, 73, 79, 86, 93, 98, 106, 113 and 119 It may include a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of.

In an embodiment of the present invention, the antibody or antigen-binding fragment thereof is SEQ ID NO: 8, 14, 22, 30, 38, 45, 53, 61, 67, 74, 80, 87, 94, 99, 107, 114 and 120 It may include a light chain variable region comprising one or more amino acid sequences selected from the group consisting of.

In an embodiment of the present invention, the antigen or antigen-binding fragment thereof,

a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 8;

a heavy chain variable region of SEQ ID NO: 13 and a light chain variable region of SEQ ID NO: 14;

a heavy chain variable region of SEQ ID NO: 21 and a light chain variable region of SEQ ID NO: 22;

a heavy chain variable region of SEQ ID NO: 29 and a light chain variable region of SEQ ID NO: 30;

a heavy chain variable region of SEQ ID NO: 37 and a light chain variable region of SEQ ID NO: 38;

a heavy chain variable region of SEQ ID NO: 44 and a light chain variable region of SEQ ID NO: 45;

a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 53;

a heavy chain variable region of SEQ ID NO: 60 and a light chain variable region of SEQ ID NO: 61;

a heavy chain variable region of SEQ ID NO: 66 and a light chain variable region of SEQ ID NO: 67;

a heavy chain variable region of SEQ ID NO: 73 and a light chain variable region of SEQ ID NO: 74;

a heavy chain variable region of SEQ ID NO: 79 and a light chain variable region of SEQ ID NO: 80;

a heavy chain variable region of SEQ ID NO: 86 and a light chain variable region of SEQ ID NO: 87;

a heavy chain variable region of SEQ ID NO: 93 and a light chain variable region of SEQ ID NO: 94;

a heavy chain variable region of SEQ ID NO: 98 and a light chain variable region of SEQ ID NO: 99;

a heavy chain variable region of SEQ ID NO: 106 and a light chain variable region of SEQ ID NO: 107;

a heavy chain variable region of SEQ ID NO: 113 and a light chain variable region of SEQ ID NO: 114; or

It may include; a heavy chain variable region of SEQ ID NO: 119 and a light chain variable region of SEQ ID NO: 120.

In an embodiment of the present invention, the antibody or antigen-binding fragment thereof is preferably represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 121 to 132, 134 to 138.

In an embodiment of the present invention, the antigen-binding fragment is preferably single-chain Fvs (scFv), single-chain antibody, Fab, F(ab'), or disulfide-bound Fvs (sdFv).

In the present invention, the antigen-binding fragment refers to a fragment having an antigen-binding function.

Among the antibody fragments, Fab has a structure having variable regions of light and heavy chains, a constant region of a light chain and a first constant region (CH1) of a heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge-region comprising one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 is formed when a cysteine residue in the hinge region of Fab' forms a disulfide bond.

Fv corresponds to the smallest antibody fragment having only a heavy chain variable region and a light chain variable region. In a double-chain Fv (two-chain Fv), the heavy chain variable region and the light chain variable region are connected by a non-covalent bond, and single-chain Fv (scFv) is generally a heavy chain variable region and light chain variable region through a peptide linker. Since the regions are covalently linked or linked directly at the C-terminus, they can form a dimer-like structure like a double-stranded Fv. These antibody fragments can be prepared using proteolytic enzymes (for example, by restriction digestion of the intact antibody with papain to obtain Fab, and by digestion with pepsin to obtain F(ab')2), or by genetic recombination technology. can be produced using

An “Fv” fragment is an antibody fragment that contains a complete antibody recognition and binding site. This region is a dimer in which one heavy chain variable domain and one light chain variable domain are bound.

A "Fab" fragment comprises the variable and constant domains of a light chain and the variable and first constant domains (CH1) of a heavy chain. F(ab')2 antibody fragments generally comprise a pair of Fab' fragments covalently linked by cysteines in the hinge region present at the C-terminus of the Fab' fragment.

A “single chain Fv (scFv)” antibody fragment is a construct consisting of a single polypeptide chain comprising the VH and VL domains of an antibody. It may further comprise a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the desired structure for antigen binding.

In a preferred embodiment of the present invention, the scFv may be one in which the heavy chain variable region and the light chain variable region are linked through a linker, preferably, the VH domain and the VL domain are linked by a polypeptide linker.

The linker may be a peptide linker and may have a length of about 10-25 aa. For example, hydrophilic amino acids such as glycine and/or serine may be included, but are not limited thereto.

Specifically, the linker may include, for example, (GS) _n , (GGS) _n , (GSGGS) _n or (G _n S) _m (n,m is 1 to 10, respectively), but the linker is For example, it may be (G _n S) _m (n and m are each 1 to 10). Specifically, the linker may include GGGGS. Preferably, the linker may be one represented by SEQ ID NO: 91, 133, 140 or 160.

Antibodies of the invention include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, scFvs, Fab fragments, F(ab′)2 fragments, disulfide-linked Fvs (sdFv) and anti-idiotypes. (anti-Id) antibodies or epitope-binding fragments of such antibodies, and the like.

Said monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to conventional (polyclonal) antibodies, which typically include different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

"Epitope" refers to a protein determinant to which an antibody can specifically bind. Epitopes are usually composed of a group of chemically active surface molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics as well as specific charge properties. Conformational and non-steric epitopes are distinguished in that binding to the former is lost but not to the latter in the presence of a denaturing solvent.

Such "humanized" forms of non-human (eg, mouse) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are of a non-human species (donor antibody) that retain the desired specificity, affinity and ability for residues from the hypervariable region of the recipient, eg, mouse, rat, rabbit or non-human primate. Human immunoglobulin (recipient antibody) replaced with residues from the hypervariable region of

The "human antibody" is a molecule derived from human immunoglobulin, and means that the entire amino acid sequence constituting the antibody, including the complementarity determining region and structural region, is composed of human immunoglobulin.

A portion of the heavy and/or light chain is identical to or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remaining chain(s) are derived from another species or are of another antibody class or subclass. Included are "chimeric" antibodies (immunoglobulins) that are identical to or homologous to the corresponding sequence in antibodies belonging to the subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

The antibody or antibody fragment of the present invention may include not only the sequence of the anti-CTLA-4 antibody of the present invention described herein, but also a biological equivalent thereof to the extent that it can specifically recognize CTLA-4. For example, additional changes may be made to the amino acid sequence of an antibody to further improve the binding affinity and/or other biological properties of the antibody. Such modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody. Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. According to the analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine can be said to be biologically functional equivalents.

In consideration of the above-described mutations having biological equivalent activity, it is construed that the antibody or nucleic acid molecule encoding the same of the present invention includes a sequence exhibiting substantial identity to the sequence set forth in SEQ ID NO:. The substantial identity is at least 90% when the above-described sequence of the present invention and any other sequences are aligned as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. refers to a sequence exhibiting homology, most preferably at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, at least 99% homology. Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) can be accessed from NBCI, etc. BLAST can be accessed at www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

Based on this, the antibody or antigen-binding fragment thereof of the present invention is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% compared to the specified sequence or all of the sequences described in the specification. , 99% or more homology. Such homology can be determined by sequence comparison and/or alignment by methods known in the art. For example, a sequence comparison algorithm (ie, BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine the percent sequence homology of a nucleic acid or protein of the invention.

According to another aspect of the present invention, (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9 or 148, a heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 10, 149, 152 and 155, and the sequence a heavy chain variable region comprising a heavy chain CDR3 comprising the amino acid sequence of No. 11; IgG4 isotype represented by the amino acid sequence of SEQ ID NO: 139; and a scFv of an anti 4-1BB antibody comprising the amino acid sequence shown in SEQ ID NOs: 141 to 145; a heavy chain variable region of a multispecific antibody comprising; (b) a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or 158, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; Multi-specific antibodies are provided.

In the present invention, the multi-specific antibody refers to an antibody having binding ability or antagonistic ability to two or more targets, and is a form in which antibodies having binding ability or antagonistic ability to two different targets are bound or binding ability to one target. It refers to an antibody to which an antibody and a substance having antagonistic ability to another target are bound.

The multispecific antibody of the present invention may be a bi-specific antibody, a tri-specific antibody, a tetra-specific antibody, or an antibody targeting more than one target, preferably Preferably, it may be a bispecific antibody.

Antibodies belonging to the multispecific antibody may be classified into scFv-based antibodies, Fab-based antibodies, and IgG-based antibodies. In the case of multispecific antibodies, since two or more signals can be simultaneously inhibited or amplified, it can be more effective than the case of inhibiting/amplifying one signal. , low-dose dosing is possible, and suppress/amplify two or more signals in the same time and space.

Methods for making multispecific antibodies are well known. Traditionally, recombinant production of multispecific antibodies is based on the co-expression of two or more immunoglobulin heavy/light chain pairs under conditions in which the two or more heavy chains have different specificities.

In the case of multispecific antibodies based on scFvs, a diabody can be made by preparing a hybrid scFv in a heterodimeric form by combining the VL and VH of different scFvs with each other, and linking different scFvs with each other to form a tendem ScFv. A heterodimeric miniantibody can be prepared by expressing CH1 and CL of the Fab at the ends of each scFv, and a 'knob into hole' form by substituting some amino acids in the CH3 domain, which is the homodimeric domain of Fc By changing the heterodimeric structure of , these altered CH3 domains can be expressed at different ends of each scFv, thereby preparing a heterodimeric scFv-type minibody.

In the case of a Fab-based multispecific antibody, individual Fab's directed against a specific antigen can be combined with each other using a disulfide bond or a mediator to form a heterodimeric Fab, and different antigens are located at the ends of the heavy or light chains of the specific Fab. It can be prepared to have two antigen valencies by expressing the scFv for , or to have four antigen valencies in homodimeric form by providing a hinge region between the Fab and scFv. In addition, by fusing scFvs for different antigens to the light and heavy chain ends of the Fab, a dual-target bibody with three antigen binding values, and different scFvs to the light and heavy chain ends of the Fab. It can be obtained by chemically conjugating three different Fabs, a triple-targeted bibody having three valencies for the two.

In the case of multispecific antibodies based on IgG, hybrid hybridomas, also known as quadromas, were prepared by re-crossing mouse and rat hybridomas by Trion Pharma. Methods for producing antibodies are known. In addition, while sharing the light chain portion, a heterodimeric form by modifying some amino acids of the CH3 homodimeric domain of Fc with respect to different heavy chains can produce a bispecific antibody in the so-called 'Holes and Knob' form. In addition to the heterodimeric form of the bispecific antibody, two different scFvs can be fused to the constant domains instead of the variable domains of the light and heavy chains of IgG to produce homodimeric (scFv)4-IgG. In addition, ImClone, based on IMC-1C11, a chimeric monoclonal antibody against human VEGFR-2, has a mouse platelet-derived growth factor receptor-α at the amino terminus of the light chain of this antibody. ), a bispecific antibody was produced and reported by fusion of only a single variable domain. In addition, through the so-called 'dock and lock (DNL)' method using the dimerization and docking domain (DDD) of the protein kinase A (PKA) R subunit and the anchoring domain of PKA, a large number of antigen binding agents for CD20 It can be prepared with an antibody having

In an embodiment of the present invention, the heavy chain variable region may be represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 13, 150, 153 and 156.

In an embodiment of the present invention, the scFv of the anti 4-1BB antibody may be one represented by the amino acid sequence of SEQ ID NO: 146.

In an embodiment of the present invention, the heavy chain variable region of the multispecific antibody is preferably operably linked as shown in Structural Formula 1 below.

[Structural Formula 1]

Heavy chain variable region - IgG4 isotype - scFv of anti 4-1BB antibody

In a preferred embodiment of the present invention, the scFv of the IgG4 isotype and the anti 4-1BB antibody may be linked by a linker.

In a more preferred embodiment of the present invention, the heavy chain variable region of the multispecific antibody may be one in which the scFv of the IgG4 isotype and the anti 4-1BB antibody are linked by a linker, which is an amino acid of SEQ ID NO: 147, 151, 154 or 157 It is preferably represented by a sequence.

In an example of the present invention, a linker represented by the amino acid sequence of SEQ ID NO: 140 was used when preparing a multispecific antibody.

In an embodiment of the present invention, the heavy chain variable region of the multispecific antibody may be one represented by the amino acid sequence of SEQ ID NO: 147, 151, 154 or 157.

In an embodiment of the present invention, the light chain variable region may be represented by the amino acid sequence of SEQ ID NO: 14 or 159.

In an embodiment of the present invention, it is preferred that the multispecific antibody specifically binds to CTLA-4 and 4-1BB.

Antibodies that specifically bind to CTLA-4 and 4-1BB of the present invention are herein defined as hCTLA-4-#8/HC-hIgG4 x EU101-scFv, B01.01-hCTLA-4-8/HC-hIgG4 x EU101-scFv, B01.02-hCTLA-4-8/HC-hIgG4 x EU101-scFv, B01.03-hCTLA-4-8/HC-hIgG4 x EU101-scFv or B01.09-hCTLA-4-8/ HC-hIgG4 x EU101-scFv may be named and used.

According to another aspect of the present invention, an antibody that specifically binds to CTLA-4, an antigen-binding fragment thereof, or a nucleic acid encoding a multispecific antibody, a recombinant expression vector comprising the nucleic acid, and a host cell transfected with the expression vector provides

In the present invention, nucleic acid has a meaning comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acids, are natural nucleotides as well as analogues in which sugar or base regions are modified. also includes The sequences of the nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions or non-conservative substitutions or conservative substitutions of nucleotides.

In the present invention, a vector is a means for expressing a target gene in a host cell, and includes a plasmid vector, a cosmid vector, a bacteriophage vector, an adenoviral vector, a retroviral vector, and a viral vector such as an adeno-associated viral vector. . Components of a vector generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more antibiotic resistance marker genes, an enhancer element, a promoter, a transcription termination sequence. Nucleic acids encoding antibodies are operatively linked, such as promoters and transcription termination sequences.

DNA encoding the antibody is easily isolated or synthesized using conventional molecular biological techniques (eg, by using an oligonucleotide probe capable of specifically binding to the DNA encoding the antibody and the heavy and light chains) Alternatively, the nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or further expression.

"Operably linked" refers to a functional association between a nucleic acid expression control sequence (eg, an array of promoter, signal sequence or transcriptional regulator binding sites) and another nucleic acid sequence, such that the control sequence is the other nucleic acid sequence. to regulate transcription and/or translation of

When using a prokaryotic cell as a host, a strong promoter capable of propagating transcription (eg, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. Further, for example, when a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (eg, a metallotionine promoter, a β-actin promoter, a human heglobin promoter, and a human muscle creatine promoter) or a mammalian cell. Promoters derived from animal viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV , the promoter of Moloney virus, the promoter of Epstein Barr virus (EBV) and the promoter of Loose Sacoma virus (RSV)), and generally has a polyadenylation sequence as a transcription termination sequence.

Optionally, the vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).

The vector contains an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. There is a resistance gene.

The host cell used to produce the antibody of the present invention may be, but is not limited to, a prokaryotic, yeast or higher eukaryotic cell.

The host cells include Escherichia coli , Bacillus subtilus and Bacillus thuringiensis , such as Bacillus genus strains, Streptomyces , Pseudomonas (eg Pseudomonas ) For example, using prokaryotic host cells such as Pseudomonas putida ), Proteus mirabilis and Staphylococcus (eg Staphylocus carnosus ) can

In an embodiment of the present invention, the host cell is an animal cell, for example, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK , TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS- 0, U20S or HT1080 is preferred.

According to another aspect of the present invention, the present invention comprises the steps of culturing the above-described host cell to generate an antibody; And it provides a method for producing an antibody that specifically binds to CTLA-4, an antigen-binding fragment thereof, or a multispecific antibody comprising the step of isolating and purifying the generated antibody.

The host cells may be cultured in various media. Among commercially available media, it can be used as a culture medium without limitation. All other essential supplements known to those skilled in the art may be included in appropriate concentrations. Culture conditions, such as temperature, pH, etc., are already in use with the host cells selected for expression, as will be apparent to those skilled in the art.

For the recovery of the antibody or antigen-binding fragment thereof, impurities may be removed by, for example, centrifugation or ultrafiltration, and the resultant product may be purified using, for example, affinity chromatography. Additional other purification techniques may be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, and the like.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an antibody that specifically binds to CTLA-4, an antigen-binding fragment thereof, or a multispecific antibody.

In addition, the present invention provides a method for preventing or treating cancer, comprising administering to an individual in need thereof an antibody, antigen-binding fragment thereof, or multispecific antibody that specifically binds to CTLA-4.

In the present invention, prevention means any action that suppresses or delays the progression of clinical symptoms of a disease by administering the composition according to the present invention, and treatment is inhibition of the development of clinical symptoms for a disease, alleviation of clinical symptoms for a disease or removal.

The cancer is, for example, Hodgkin's lymphoma, non-Hodgkin's lymphoma (eg, B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, marginal area B-cell lymphoma, Burkitt's lymphoma) , lymphoid cell lymphoma, hairy cell leukemia), acute myeloid leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, multiple myeloma or acute lymphocytic leukemia.

Said cancer is, for example, ovarian cancer, rectal cancer, stomach cancer, testicular cancer, anal region cancer, uterine cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, esophageal cancer, melanoma, Kaposi's sarcoma, Endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, brain stem glioma, pituitary adenocarcinoma, epidermal cancer, carcinoma of the squamous cell carcinoma of the cervix, fallopian tubes Carcinoma, endometrial carcinoma, vaginal carcinoma, soft tissue sarcoma, urethral cancer, vulvar carcinoma, penile cancer, bladder cancer, kidney or ureter cancer, renal pelvic carcinoma, spinal tumor, neoplasm of central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis neonatal, metastatic lesions of the above cancers, or combinations of the above cancers.

The cancer may be, for example, glioblastoma, lung cancer, bladder cancer, oral cancer, head and neck squamous cell cancer, gallbladder cancer or cervical cancer. In particular, in the case of glioblastoma, the antibody or antigen-binding fragment thereof is a barrier formed by a tight junction in the capillary endothelial cell membrane of the brain that exists between the brain and spine and the surrounding circulatory system, the blood-brain barrier (blood-brain barrier). barrier) needs to be passed. It can be used in conjunction with a transporter to cross the BBB. In order to deliver a drug through the BBB, for example, there is a method of disrupting the osmotic pressure of the BBB using a method such as bradkinin or HIFU (Hign density fucues ultrasound). It may also include the use of delivery systems such as receptor-mediated transcytosis of glucose and amino acid transporters, insulin or transferrin, or blocking the active efflux transporter of glycoproteins in cells.

In an embodiment of the present invention, the subject is a subject expected to develop cancer; affected individuals; Or it may be an individual who has been determined to be cured, but is not limited thereto.

Pharmaceutically acceptable carriers included in the composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate. , microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration etc. can be administered.

When administered orally, the protein or peptide is digestible and therefore oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the pharmaceutical composition may be administered by any device capable of transporting the active agent to a target cell.

A suitable dosage of the composition according to the present invention varies depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient, usually Thus, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. For example, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg. As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to prevent or treat cancer or infectious diseases.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or may be prepared by inclusion in a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally include a dispersant or stabilizer.

Duplicate content is omitted in consideration of the complexity of the present specification, and terms not defined otherwise in the present specification have the meanings commonly used in the technical field to which the present invention pertains.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Development of anti-CTLA-4 antibody

The human anti-CTLA-4 antibody was developed using the phage display human antibody library (DPALS, a contract for material transfer and technology transfer from Ewha Womans University Industry-University Foundation on December 1, 2017).

The selection process for a phage antibody that binds to recombinant human CTLA-4 antigen (sino, Cat No. 11159-H08H) is as follows.

Recombinant human CTLA-4 antigen at a concentration of 5ug/ml was added to an immunotube (Nunc, Maxisorp) and coated at 4°C overnight. The next day, the coating solution is removed, and after blocking with 3% non-fat skim milk at room temperature for 1 hour, the Phage antibody library is treated, and the coated recombinant human CTLA-4 antigen is reacted with the Phage antibody library for at least 2 hours at room temperature. Immunotube was washed once with 0.5% PBS-Tween and washed 4 times with 0.1% PBS-Tween. Elution buffer (AmicoGEN, ProA Elution buffer) was added and reacted for 10 minutes to separate phage antibody bound to recombinant human CTLA-4 protein, and the isolated phage antibody was neutralized with 1M Tris-HCl, pH9.0 solution. Neutralized phage was infected with 10ml TG1 host cells in mid-log phase for 1 hour, 100ul was titrated by serial dilution, and the rest were harvested and cultured O/N on 15cm LB agar/Amp./2% glucose plate. The next day, the culture medium was added to the plate, the bacteria were scraped with a spreader, and 50% sterile glycerol was added for storage (primary panning) or amplification & phage rescue for secondary panning.

1st panning stock 200ml 2YT/Amp. It was inoculated into the culture medium and cultured until OD600=0.7~08. VCSM13 helper phage was added and cultured at 37°C for 1 hour, treated with kanamycin (final concentration, 70ug/ml), and then incubated O/N at 30°C. The next day, the culture solution was collected by centrifugation at 5000 g for 20 minutes and filtered through a 0.22um filter. 1/5 volume of PEG8000 (Sigma, manufactured by 25 mM) and 1/10 volume of NaCl (5M) were added to the culture medium and mixed, then precipitated at 0° C. and ice for 2 hours or more, and centrifuged at 12,000 rpm for 1 hour. . The possible culture medium was completely removed and the precipitated phage pellet was suspended in PBS/1% BSA. For phage tittering, 500ul of mid-log phase TG1 host cells were infected for 30 minutes, followed by serial dilution to LB agar/Amp. After culturing O/N at 37°C, the colonies were counted and titrated by multiplying the dilution value. This process was repeated at least 3 times (3 round panning) to select a phage antibody that binds to the recombinant human CTLA-4 antigen.

Example 2. Selection of candidate clones through ELISA screening (primary selection)

As a result of the third panning using the phage antibody library against human recombinant CTLA-4 antigen, each single colony was inoculated into each well of a 96-well plate, incubated at 37°C for about 3 hours, treated with 0.5mM IPTG, and heated at 30°C. The phage antibody was induced by O/N. An ELISA plate (Nunc) was coated with 2ug/ml of human recombinant CTLA-4-His(sino) antigen. The next day, after centrifugation, cold 1X TES buffer (20% w/v sucrose, 50 mM Tris, 1 mM EDTA, pH 8.0) was added to the cell pellet, mixed, and treated on ice for 30 minutes. Thereafter, 0.2X TES buffer was added and mixed, placed on ice for 30 minutes, and centrifuged to extract the periplasmic fraction. The coated ELISA plate was blocked with 3% non-fat skim milk, and then the extracted periplasmic fraction was treated to bind the coated antigen, and the result of color development was confirmed by treatment with anti-HA-HRP (sigmaaldrich). The ELISA results are shown in FIG. 1 .

As shown in FIG. 1 , 45 candidate clones with high color development were selected.

Example 3. Sequencing of the first selected clones

By proliferating the bacteria of the clone selected in Example 2, DNA was extracted and the gene sequence was analyzed. The analysis results are shown in FIG. 2 .

As shown in Figure 2, including two clones having the same sequence as clone #4, 17 clones having the same sequence as clone #8, clone #13, clone #29, clone #38, clone #41, Gene sequences corresponding to 6 types of kappa clones and antibody variable regions were identified. In addition, 4 clones having the same sequence as Clone # 1, 2 clones having the same sequence as clone #51, clone #55, clone #61, clone # 63, 2 clones having the same sequence as clone #66, # Genes corresponding to 11 types of lambda clone antibody variable region, including 3 clones having the same sequence as 80 clone, #86 clone, 2 clones having the same sequence as clone #87, #94 clone, and #64 clone sequence was obtained. Clones with the same gene sequence were found most frequently in clone #8.

Based on the sequencing results, the sequences of CDRs, heavy chains and light chains of 6 types of kappa clones and 11 types of lambda clones are shown in Table 1. In the sequences in Table 1, CDRs are indicated in bold.

Clone	division	order	SEQ ID NO:	order	SEQ ID NO:
hCTLA-4-kappa
4	VH	DYAMA	One	EVQLLESGGGLVQPGGSLRLSCAASGFTFS DYAMA WVRQAPGKGLEWVS SIHYSGTT YYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RDRIYPAFDY WGQGTLVTVSS	7
		SIHYSGTT	2
		RDRIYPAFDY	3
	VL		RASQSIGNYLA	4	EIVLTQSPGTLSLSPGKRATLSC RASQSIGNYLA WYQQKPGQAPRLLIY GAST RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQSYSTPIT FGQGTKVEIKG	8
		GAST	5
		QQSYSTPIT	6
8	VH		SYAM	9	EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVS SLYYSGYT YYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RSAPGWGFDY WGQGTLVTVSS	13
		SLYYSGYT	10
		RSAPGWGFDY	11
	VL		QASRDITNYLN	12	EIVLTQSPGTLSLSPGERATLSC QASRDITNYLN WYQQKPGQAPRLLIY GAST RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQSYSTPIT FGQGTKVEIKG	14
		GAST	5
		QQSYSTPIT	6
13	VH		SYAIS	15	EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAIS WVRQAPGKGLEWVS TISGSGGQ TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RGGIGFDI WGQGTLVTVSS	21
		TISGSGGQ	16
		RGGGIGFDI	17
	VL		RAASSISSYLN	18	EIVLTQSPGTLSLSPGERATLSC RAASSISSYLN WYQQKPGQAPRLLIY KASN LATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYNWPPIT FGQGTKVEIKG	22
		KASN	19
		QQYNWPPIT	20
29	VH	SYYWS	23	EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYYWS WVRQAPGKGLEWVS AISGSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RGYLGFDS WGQGTLVTVSS	29
		AISGSGGS	24
		RGYLGFDS	25
	VL	RASQSIRTYLN	26	EIVLTQSPGTLSLSPGERATLSC RASQSIRTYLN WYQQKPGQAPRLLIY SASS RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYHC QQYYGTPLT FGQGTKVEIKG	30
		SASS	27
		QQYYGTPLT	28
38	VH		SNYMS	31	EVQLLESGGGLVQPGGSLRLSCAASGFTFS SNYMS WVRQAPGKGLEWVS SIHNSGKT YYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TRGVLGFDS WGQGTLVTVSS	37
		SIHNSGKT	32
		TRGVLGFDS	33
	VL	RTSQSISSYLN	34	EIVLTQSPGTLSLSPGERATLSC RTSQSISSYLN WYQQKPGQAPRLLIY GSSS RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYYSFPIT FGQGTKVEIKG	38
		GSSS	35
		QQYYSFPIT	36
41	VH	SYWIG	39	EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYWIG WVRQAPGKGLEWVS VISHSGNN KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARLEEGKSFDI RGQGTLVTVSS	44
		VISHSGNN	40
		ARLEEGKSFDI	41
	VL	RASQDISNYLA	42	EIVLTQGPGTLSLSPGERATLSC RASQDISNYLA WYQQRPGQAPRLLIY GAST RATGIPDRFSGSGSGTDFTLTISRLEPEDFGVYYC QQSYSTPPST FGQGTKVEIKG	45
		GAST	5
		QQSYSTPPST	43
hCTLA-4-lamda
One	VH	DYAMS	46	EVQLLESGGGLVQPGGSLRLSCAASGFTFS DYAMS WVRQAPGKGLEWVS GISYSGGS TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RTKNRFDY WGQGTLVTVSS	52
		GISYSGGS	47
		RTKNRFDY	48
	VL		TGSSSNIGSNYVS	49	QSVLTQPPSASGTPGQRVTISC TGSSSNIGSNYVS WYRQLPGTAPKLLIY SDSN RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC ATWDASLSAYV FGGGTKLTVLG	53
		SDSN	50
		ATWDASLSAYV	51
51	VH	TYGMH	54	EVQLLESGGGLVQPGGSLRLSCAASGFTFS TYGMH WVRQAPGKGLEWVS SISGSGAY TYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RGQNRYGFPNFDY WGQGTLVTVSS	60
		SISGSGAY	55
		RGQNRYGFPNFDY	56
	VL	SGSSSNIGSNYVY	57	QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVY WYQQLPGTAPKLLIY RNNQ RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC ASWSDSLSGYV FGGGTKLTVLG	61
		RNNQ	58
		ASWSDSSLSGYV	59
55	VH	LHAMH	62	EVQLLESGGGLVQPGGSLRLSCAASGFTFS LHAMH WVRQAPGKGLEWVS SISSGSSYI YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RGGSQGFDY WGQGTLVTVSS	66
		SISSGSSYI	63
		RGGSQGFDY	64
	VL	SGSSSNIGSNYVY	57	QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVY WYQQLPGTAPKLLIY RNNQ RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC AAWDDSLSGWV FGGGTKLTVLG	67
		RNNQ	58
		AAWDDSLSGWV	65
61	VH	SYPMT	68	EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYPMT WVRQAPGKGLEWVS VISYSGGLT YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RGFGRFDY WGQGTLVTVSS	73
		VISYSGGLT	69
		RGFGRFDY	70
	VL	SGSSSNIGNNAVH	71	QSVLTQPPSASGTPGQRVTISC SGSSSNIGNNAVH WYQQLPGTAPKLLIY RNNQ RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC AAWDDSLSGVV FGGGTKLTVLG	74
		RNNQ	58
		AAWDDSLSGVV	72
63	VH	TYAM	75	EVQLLESGGGLVQPGGSLRLSCAASGFTFS TYAMH WVRQAPGKGLEWVS SISSSSNYI YYADSVKGRFTISRDNSKNTLYLQMDSLRAEDTAVYYCA RGYATGLDY WGQGTLVTVSS	79
		SISSSSNYI	76
		RGYATGLDY	77
	VL	SGSSSNIGSNYVY	57	QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVY WYQQLPGTAPKLLIY RNNQ RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC EAWDDSLSGYV FGGGTKLTVLG	80
		RNNQ	58
		EAWDDSLSGYV	78
66	VH	DYAMA	One	EVQLLESGGGLVQPGGSLRLSCAASGFTFS DYAMA WVRQAPGKGLEWVS SIYYGGST YYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RDRTFGYFDY WGQGTLVTVSS	86
		SIYYGGST	81
		RDRTFGYFDY	82
	VL	SGSSSNIGNDYVS	83	QSVLTQPPSASGTPGQRVTISC SGSSSNIGNDYVS WYQQLPGTAPKLLIY QDSK RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC QSYDSSVPV FGGGTKLTVLG	87
		QDSK	84
		QSYDSSVPV	85
80	VH	HYAM	88	EVQLLESGGGLVQPGGSLRLSCAASGFTFS HYAMT WVRQAPGKGLEWVS SISSSSSET YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TRSLPRHFDI WGQGTLVTVSS	93
		SISSSSSET	89
		TRSLPRHFDI	90
	VL	SGSSSNIGSNYVY	57	QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVY WYQQLPGTAPKLLIY RNNQ RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC ATWDDSLSGYV FGGGTKLTVLG	94
		RNNQ	58
		ATWDDSSLSGYV	92
86	VH		SYAM	9	EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVS AISYSGNNK YYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RGLQGFDY WGQGTLVTVSS	98
		AISYSGNNK	95
		RGLQGFDY	96
	VL	SGSSSNIGSNYVY	57	QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVY WYQQLPGTAPKLLIY RNNQ RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC SAWDDSLSGYV FGGGTKLTVLG	99
		RNNQ	58
		SAWDDSSLSGYV	97
87	VH		SYAMN	100	EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMN WVRQAPGKGLEWVS SISGRGGTT YYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RGPWGYFDH WGQGTLVTVSS	106
		SISGRGGTT	101
		RGPWGYFDH	102
	VL		TRSSGSTASNYVQ	103	QSVLTQPPSASGTPGQRVTISC TRSSGSTASNYVQ WYQQLPGTAPKLLIY DNSK RPSGVPYRFSGSKSGTSASLAISGLRSEDEADYYC AT C DDSLSGYV FGGGTKLTVLG	107
		DNSK	104
AT C DDSLSGYV	105
94	VH	DYAMS	46	EVQLLESGGGLVQPGGSLRLSCAASGFTFS DYAMS WVRQAPGKGLEWVS SISSSSSY IHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RFNVRTRYQSYAFDY WGQGTLVTVSS	113
		SISSSSSY	108
		RFNVRTRYQSYAFDY	109
	VL		TGSSGSIANNDVQ	110	QSVLTQPPSASGTPGQRVTISC TGSSGSIANNDVQ WYQQLPGTAPKLLIY KNNK RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC AAWDDSLNNPV FGGGTKLTVLG	114
		KNNK	111
		AAWDDSLNNPV	112
64	VH	NYGM	115	EVQLLESGGGLVQPGGSLRLSCAASGFTFS NYGMH WVRQAPGKGLEWVS AISGSGRA TYYADNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TRTSGYYSVPHYTFDS WGQGTLVTVSS	119
		AISGSGRA	116
		TRTSGYYSVPHYTFDS	117
	VL	SGSSSNIGSNYVY	57	QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVY WYQQLPGTAPKLLIY RNNQ RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC AAWDDSLSGYV FGGGTKLTVLG	120
		RNNQ	58
		AAWDDSLSGYV	118

In addition, the entire sequence of the antibody described in Table 1 is shown in Table 2. The following sequence includes the linker sequence as shown in FIG. 2 . The linker may be one represented by the amino acid sequence of SEQ ID NO: 91, 133, 140 or 160.

Clone	order	SEQ ID NO:
hCTLA-4-kappa
4	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMAWVRQAPGKGLEWVSSIHYSGTTYYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRIYPAFDYWGQGTLVTVSSSGGGSGGGGSGGGGSEIVLTQSPGTLSAPLSPGKRATLSGTDFGIPDRLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRIYPAFDYWGQGTLVTVSSSGGGSGGGGSGGGGSEIVLTQSPGTLSQGSRPGKRATLSGTDFGIPDRLELAWYQTLQG	121
8	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSSLYYSGYTYYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAPGWGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCQASRDITITNYLNWYGTDFQQSRKLEPGQAPRLVYGTDFQQSRKPGQAPRLNWYDFQK	122
13	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAISWVRQAPGKGLEWVSTISGSGGQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGIGFDIWGQGTLVTVSSSGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRAASSTIYLNWYDFGTGSNQQSRQAPLSPGERATLSCRAASSTIYLNWYDFGSNQQYLNWYDFGQN	123
29	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYYWSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYLGFDSWGQGTLVTVSSGGGSGGSGGGGSGGGGSEIVLTQSPGTLSQGSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYLGFDSWGQGTLVTVSSGGGSGGSGGGGSGGGGSEIVLTQSPGTLSQYLSPGERATLSCRASQSIRTYPGLENWYGTSPGGSQGGSQAPGERATLSCRASQSIRTYLNWYGTQG	124
38	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSNYMSWVRQAPGKGLEWVSSIHNSGKTYYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGVLGFDSWGQGTLVTVSSSGGGSGGGGSGGGGSEIVLTQSPGTLSQLSPGERATLSCRTSQTSQSISSYLNWYDFQQKSRPGIGVEPIGVEQVE	125
41	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWIGWVRQAPGKGLEWVSVISHSGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLEEGKSFDIRGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQGPGTLSLSPGERATLSCRASQTINYLAWVSVISHSGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLEEGKSFDIRGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQGPGTLSLSPGERATLSCRASQTINYLAWYQQRPGQKGTPKGTPKGTPKG	126
hCTLA-4-lamda
One	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVSGISYSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTKNRFDYWGQGTLVTVSSGGGGSGGSGSGGGGSQSVLTQPGSDYYQSGTPGQRVTISCTGSSSLPSGLRSGCATGTSWKLSWDLGDRSGCATS	127
51	EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVSSISGSGAYTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGQNRYGFPNFDYWGQGTLVTVSSSGGGSGGGGSGGGSGQGSQLSVLTQPPSASGGTTPGQRVTISCSKSSRPGGSLGFSLGYRNYGSGTSGGTSGLSGLSED	128
55	EVQLLESGGGLVQPGGSLRLSCAASGFTFSLHAMHWVRQAPGKGLEWVSSISSGSSYIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSQGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSGSSSNIGSNYGTISGTAVYYCARGGSQGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASSGTPGQRVTISCSGSGSSSNIGSNYGTISGTAVYWDKLWDASKLAAGTIS	129
61	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMTWVRQAPGKGLEWVSVISYSGGLTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFGRFDYWGQGTLVTVSSSGGGSGGGGSGGGGSQSVLTSGVGGGTRNQKSLGPGQRVTISCSGSGSSSNIVPGNNAVHAPSEGTAVYYCARGFGRFDYWGQGTLVTVSSSGGGSGGGGSGGGGSQSVLTQGGGTRNQRPGQRVTISCSGSGSSSNIVPGNNAVHAPSE	130
63	EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMHWVRQAPGKGKGLEWVSSISSSSNYIYYADSVKGRFTISRDNSKNTLYLQMDSLRAEDTAVYYCARGYATGLDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSGSSSNIGSNYGTAPSEGGTTQLQLQSLYRVTISCSGSSSNIGSNYGTAPYW	131
66	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMAWVRQAPGKGLEWVSSIYYGGSTYYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRTFGYFDYWGQGTLVTVSSGPVGGGSGGGGSGSGGGGSQSVLTQPPGSKLSQRPSGQRVTISCSKGSSSNIGNDYVGTSGTSWKLSGQLPSGQRVTISCSKGSSSNIGNDYVGTSAPDY	132
80	EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYAMTWVRQAPGKGLEWVSSISSSSSETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRSLPRHFDIWGQGTLVTVSSSGGGSGGGGSGGGGSQSVLTQPPSASGQLPSGQRVTISCSGSSSSAVPSGLRSGDRGSGTGGTGQLPSGQRVTISCSGSSSSAVPSGNYVYSEWGTSG	134
86	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSAISYSGNNKYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLQGFDYWGQGTLVTVSSSGGGSGGGGPGGGGSQSVLTKLQPPYYYSASGTPGQRVTISCSGSGSSSNIGSNYGTAPGSLRGGSGDDDDD	135
87	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSSISGRGGTTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGPWGYFDHWGQGTLVTVSSGGGGSGGGGSGSGGGGSQSVLTQPPSGQDNWVRQRPGKGLEWVSSISGRGGTTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGPWGYFDHWGQGTLVTVSSGGGGSGGGGSGSGGGGSQSVLTQPPSGQDNWVRQRVTISCTRSSGVPYLPYGTAPGGGDLSGQDNWVSTGSLPYGTAPG	136
94	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVSSISSSSSYIHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARFNVRTRYQSYAFDYWGQGTLVTVSSSGGGSGGYGGSGGGGSQSGQSVKLLTQPPSASGTPGQRVTISCTGSSGRPSGQSLVDRKSLRGSNKSENKSENKSENKDE	137
64	EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSAISGSGRATYYADNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRTSGYYSVPHYTFDSWGQGTLVTVSSGGGGSGGSGSGGGSGGSQSVKLLTQPPSASGTPGQRVTISCSGSSSRPGTSLSGDLVLGYKSGSGSRPGTSLGSLVLGVYKSGSSSAWQSLATYYADNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY	138

Example 4. CLTA-4 antigen binding analysis of the first selected clones by FACS

The gene sequence of 17 clones selected by phage antibody library screening was synthesized and cloned into an antibody containing the heavy chain of the hIgG1 isotype and the constant region of the kappa- or lambda-light chain. For antibody production, the plasmid of each cloned antibody was expressed using the Expi293F transfection system (Gibco, ThermoFisher), and the antibody was purified using protein A resin (AmicoGEN). The concentration was quantified.

For each purified candidate antibody, 293 cells expressing human CTLA-4 were prepared, and binding ability to CTLA-4 antigen was analyzed. After transfection of human CTLA-4 full-length cDNA (sino) containing Mammalian CMV promoter into 293 cells, primary selection was performed with Hygromycin antibiotic, and 293 cells expressing strongly CTLA-4 were separated with a secondary FACS sorter to obtain cell lines. produced. 293 cells expressing CTLA-4 were treated with each purified candidate antibody and then treated with anti-hIgG-PE as a secondary antibody to confirm the binding effect of the anti-CTLA-4 antibody by flow cytometry. The flow cytometry results are shown in FIG. 3 .

As shown in FIG. 3 , it was confirmed that the antigen-binding ability of clones 4, 8, 13, 61, 63, 64 and 94 was excellent. The clones 4, 8, 13, 61, 63, 64 and 94 were secondarily selected and used in the experiments described below.

Example 5. In vitro functional assay

CTLA-4 Blockade Bioassay designed to block the interaction between CTLA-4 and its ligands CD80 and CD86 for an in vitro functional assay of 7 clones having binding to CTLA-4 selected in Example 4 (Promega) was used. Effector cells expressing CTLA-4/CD28 were laid on a 96-well plate, and the candidate human CTLA-4 antibody was treated with a 1/2 serial dilution from a concentration of 30 ug/ml, followed by aAPC/Raji cells expressing CD80/86 and 37 Co-incubation at ℃ for 6 hours. The analysis principle is that the binding of CTLA-4 to CD80/86 interferes with the activity of CD28, so that luminescence does not develop, and the human anti-CTLA-4 antibody activates CD28 by interfering with the binding of CTLA-4 and CD80/86. luminescence is developed. The in vitro functional assay results are shown in FIG. 4 .

As shown in FIG. 4 , as a result of treatment with the selected candidate antibody, the anti-CTLA-4-8 antibody (clone #8, CTLA4-8) was compared with the functional grade positive control anti-CTLA-4 antibody (Biolegend). ) was confirmed to have similar efficacy. Accordingly, the anti-CTLA-4-8 antibody was selected as a clone leader antibody.

Example 6. Characterization of anti-CTLA-4-8 antibody

The properties of the anti-CTLA-4-8 antibody selected in Example 5 were analyzed.

First, SDS-PAGE analysis of the produced anti-CTLA-4-8 antibody was performed. Specifically, 5ug of anti-CTLA-4-8 antibody was mixed with sample buffer (Invitrogen, Cat#B0007), heated to prepare a denaturated sample, and SDS-PAGE gel (Mini PROTEIN TGX Stain Free Gel, Bio-rad). After running, the gel was stained. The SDS-PAGE results are shown in FIG. 5A.

As shown in FIG. 5A , a band of anti-CTLA-4-8 antibody was identified in the stained gel.

SEC-HPLC for the analysis of the purity of the antibody can determine the oligomerization, aggregation, conjugation and conformation of the produced antibody. The IgG antibody was analyzed by HPLC (Agilent Technologies, 1260 infinity II LC system) using a size exclusion column (Tosoh, TSKgel G3000 SWXL, 7.8×300 mm, Part No. 0008541, Column No. 023D08819D). For mobile phase, 10X Phosphate Buffered Saline buffer (wellgene, Cat#LB204 02) was diluted with 1X PBS in tertiary distilled water, and Corning 1000 mL Vacuum Filter/Storage Bottle System, 0.22μm Pore 54.5cm² PES Membrane, Sterile, 12/Case was used for filtration. As for the analysis method, flow rate 1ml/min, 280nm wavelength band was analyzed for 20min, and 20ul of the sample was injected. As a standard sample, a gel filtration standard (BIO RAD, Cat. #151-1901) was used. The SEC-HPLC results are shown in FIG. 5B.

As shown in Figure 5B, the purity, structure and morphology of the anti-CTLA-4-8 antibody were confirmed.

SPR analysis of anti-CTLA-4-8 antibody was performed. Specifically, a CM5 chip (GE Healthcare, Cat#BR-1005-30) immobilized with an anti-human Fc antibody was prepared using a human capture kit (GE healthcare, Cat#BR-1008-39). The produced antibody was prepared by diluting it to 1ug/mL. The diluted antibody was fixed by flowing it through the CM5 chip at a flow rate of 10 uL/min for 60 seconds, and the CTLA-4-His (Sino) antigen was 100, 50, 25, 12.5, 6.25, 3.125 nM for an association time of 150 seconds, The dissociation time was injected for 240 seconds. The binding force was analyzed by fitting the sensorgram measured through the Biacore T200 (GE healthcare) equipment to a 1:1 binding model. The SPR analysis result is shown in FIG. 5C.

As shown in FIG. 5C , the binding affinity of the anti-CTLA-4-8 antibody with the recombinant human CTLA-4-His antigen was analyzed as kD=7.053E ^-10 .

293 cells expressing CTLA-4 were treated with 5 ug of anti-CTLA-4-8 antibody, and treated with anti-hlgG-PE as a secondary antibody. Thereafter, the binding effect of the anti-CTLA-4 antibody was confirmed once more by flow cytometry. The flow cytometry results are shown in Figure 5D.

As shown in FIG. 5D , the anti-CTLA-4-8 antibody showed binding affinity similar to that of the positive control antibody (Biolegend).

Example 7. Bispecific antibody production and purification

In order to increase the efficacy of CD8 T cells, an increase in anticancer efficacy can be expected by blocking CTLA-4, an inhibitory molecule, with an anti-CTLA-4 antibody and activating the 4-1BB signal with an EU101 antibody.

For the production of the anti-CTLA-4-8 antibody and the dual specific antibody of EU101, the hIgG4 isotype is linked to the heavy chain of the whole form of the anti-CTLA-4-8 antibody, and (GGGGS) ₃ linker of the hIgG4 constant region A heavy chain was prepared by connecting EU101-scFv (Eutilex's self-developed antibody, SEQ ID NO: 146) to the C-terminus. The heavy chain and the light chain of the CTLA-4-8 antibody were used together to produce a bispecific antibody using the Expi293F transfection system (Gibco, ThermoFisher). The produced bispecific antibody was purified using Protein A resin (AmicoGen). The sequence and purification results of the produced bispecific antibody are shown in FIG. 6 .

As shown in FIG. 6 , the CDR of the anti-CTLA-4-8 antibody is underlined, the hIgG1 isotype in the sequence of the produced bispecific antibody is indicated in light color, and the linker is indicated in light color and underlined. . In addition, as a result of purification, it was confirmed that the production of the produced bispecific antibody was about 5.3 mg in 180 ml of the culture medium.

The configuration and specific sequence of the bispecific antibody heavy chain are shown in Table 3.

division	order	SEQ ID NO:	order	SEQ ID NO:
Anti-CTLA-4-8 VH		SYAM	9	EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVS SLYYSGYT YYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RSAPGWGFDY WGQGTLVTVSS	13
	SLYYSGYT	10
	RSAPGWGFDY	11
hIgG4 isotype	ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK			139
(GGGGS) 3 linker	GGGGSGGGGSGGGGS			140
EU101-scFv	SYWMH	141	QVQLVQSGAEVKKPGASVKLSCKASGYTFS SYWMH WVRQAPGQGLEWIG EINPGNGHT NYNEKFKSRVTMTRDTSTSTAYMELSSLRSEDTAVYYCA RSFKTARAFAY WGQGTLVTVSSGGGGSGSGASQGSGGGGSDIVMTKLSPAFYQAPGQGLEWIG EINPGNGHT NYNEKFKSRVTMTRDTSTSTAYMELSSLRSEDTAVYYCA RSFKTARAFAY WGQGTLVTVSSGGGGSGSGASQGSGGGGSDIVMTKLSPAFLSVTPGEKVTITC RGPHSKFTAG	146
	EINPGNGHT	142
	RSFKTARAFAY	143
	RASQTISDYLH	144
	QDGHSWPPT	145
Full sequence of bispecific antibody heavy chain	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSSLYYSGYTYYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAPGWGFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK GGGGSGGGGSGGGGS QVQLVQSGAEVKKPGASVKLSCKASGYTFSSYWMHWVRQAPGQGLEWIGEINPGNGHTNYNEKFKSRVTMTRDTSTSTAYMELSSLRSEDTAVYYCARSFKTARAFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPAFLSVTPGEKVTITCRASQTISDYLHWYQQKPDQAPKLLIKYASQSISGIPSRFSGSGSGTDFTFTISSLEAEDAATYYCQDGHSWPPTFGQGTKLEIK			147

Example 8. Characterization of bispecific antibodies

For SDS-PAGE analysis of the bispecific antibody produced in Example 7, 5ug was mixed with each reducing sample buffer and non-reducing sample buffer (Invitrogen, Cat#B0007) and then heated to prepare a denaturated sample and SDS-PAGE After running on gel (Mini PROTEIN TGX Stain Free Gel, Bio-rad), it was stained. The SDS-PAGE results are shown in FIG. 7A.

As shown in FIG. 7A , it was confirmed that the size of the heavy chain of the bispecific antibody was increased in the bispecific antibody as compared to the single antibody.

The antigen-binding ability of the quantified bispecific antibody was confirmed by the Q-ELISA method. Human recombinant 4-1BB-His antigen (sino) and human recombinant CTLA-4-His antigen (sino) were coated on an ELISA plate at a concentration of 1ug/ml, turned on at 4°C, and 3% non-fat skim milk at room temperature. After blocking for 1.5 hours, the antibody (single antibody EU101, anti-CTLA-4-8 antibody, or dual specific antibody) was serially diluted from 500 ng to 1/2 to 4-1BB antigen and CTLA-4 antigen, respectively, at room temperature. After treatment for 2 hours and secondary antibody hIgG-HPR at room temperature for 1 hour, color was developed with ABTs substrate (sigma). The Q-ELISA results are shown in Figure 7B.

As shown in FIG. 7B , it was confirmed that the antigen binding capacity of the dual specific antibody was somewhat reduced than that of the single antibody.

Example 9. Confirmation of antigen binding affinity of dual specific antibodies by flow cytometry

Jurkat 8-1 cells expressing h4-1BB and CTLA-4-293 cells expressing hCTLA-4 were treated with a dual-specific antibody and treated with a secondary hIgG-PE antibody for flow cytometry. The flow cytometry results are shown in FIG. 8 .

As shown in Figure 8, the binding affinity of the bispecific antibody in 293 cells expressing CTLA-4 was slightly lower than that of the positive control anti-CTLA-4 antibody (Biolegend), and in Jurkat 8-1 expressing h4-1BB. The binding affinity of the bispecific antibody was slightly higher than that of the positive control anti-4-1BB antibody (BD) (histogram analysis, arrow).

Example 10. Identification of antigen binding ability of bispecific antibodies in clinical samples

hPBMCs were isolated from healthy donor blood by density purchase (Ficoll paque), and each T cell was MACS-separated using hCD8 and hCD4 Mocrobeads (Miltenybiotec). The isolated hCD8 and hCD4 cells were treated with hCD3/28 T cell activator Beads (Thermo Fisher) and activated for 48 hours. The flow cytometry results are shown in FIG. 9 .

As shown in Figure 9, activated hCD8 cells, 　 and hCD4 cells; and hCD4 Treg cells (CD4+Foxp3+ cells); the binding capacity of the bispecific antibody was higher than that of the single antibody CTLA-4 or 4-1bb antibody. Also, in CD4 cells and Treg cells, the binding affinity of the bispecific antibody was higher than that of the CTLA-4 antibody (histogram analysis, arrow).

Activated CD8 T cells showed high avidity in the order of anti-CTLA-4 < anti-4-1BB < bispecific antibody, and in CD4 and CD4Treg cells, the avidity of the bispecific antibody was higher than that of anti-CTLA-4.

Example 11. Confirmation of antigen binding ability of dual specific antibodies through SPR analysis

Binding affinity to rhCTLA-4 and antigen of the control (BsAb. Yervoy) and the bispecific antibody (CTLA-4-#8x EU101 BsAb) was analyzed through SPR analysis. The Yervoy (Ipilimumab, BMS) antibody used as a control is a drug prescribed by BMS for adult melanoma patients over 12 years of age, and was used as a control antibody for anti-CTLA-4-8, and its sequence is patent appl. Cited from US20150283234. In addition, EU101-scFv was connected to the N-terminus of Yervoy's heavy chain to be used as a control of the same structure as the dual specific antibody of CTLA-4-#8x EU101. The analysis results are shown in FIG. 10 .

As shown in FIG. 10 , the binding capacity of the bispecific antibody (CTLA-4-#8 x EU101 BsAb) to rhCTLA-4 was higher than that of the Yervoy x EU101 BsA antibody (control), whereas the bispecific antibody to rh4-1BB was bispecific. It was confirmed that the antibody (CTLA-4-#8 x EU101 BsAb) was low.

Example 12. Tumor size analysis according to bispecific antibody treatment

The in vivo anticancer efficacy of single and dual specific antibodies was analyzed using C57BL/6 double Knock-In mice into which h4-1BB and hCTLA-4 genes were inserted. Specifically, after colon cancer was generated by injecting mouse MC38 cells, as a negative control group, hIgG serum administration group, each EU101 and CTLA-4-8 single antibody administration group, each CTLA-4-8 x EU101 and Yervoy x EU101 dual specificity The antibody at a concentration of 5 mg/kg was administered to the antibody administration group 5 times at 3-day intervals. The in vivo anticancer efficacy of each antibody was analyzed for each individual's cancer size by group, and the results are shown in FIG. 11 .

As shown in FIG. 11 , the anti-CTLA-4-8 single antibody showed the strongest anticancer efficacy, and the CTLA-4-8x EU101 bispecific antibody had better anticancer efficacy than the Yervoy x EU101 bispecific antibody. Anti-CTLA-4 antibody showed a relatively increased ratio of mCD8 and mCD4 of immune cells (TIL, tumor infiltrated lymphocytes) infiltrating the tumor, and showed a similar tendency in PBMC.

Example 13. Preparation of dual-specific antibodies with improved antigen-binding affinity

A CDR yeast library was prepared using the entire sequence of the heavy chain of the dual specific antibody prepared in Example 7 as a template, and clones with improved binding ability were screened. As a result, B01.01 (T05.01), B01.02 (T05.02), B01.03 (T05.04) and B01.09 (T05L06) were selected. The amino acid sequences of the four clones are shown in FIG. 12 .

As shown in FIG. 12 , the CDR region of each antibody is underlined, the IgG4 region is indicated in light color, and the (GGGGS) ₃ linker region is indicated in light color and underlined. In addition, the amino acid sequence changed to improve binding strength is indicated in light color and upper line.

Based on the sequencing results of FIG. 12 , the regions where the amino acid sequence was changed in the bispecific antibody with improved binding are shown in Table 4. More specifically, the anti-CTLA-4-8 antibody of (i) B01.01 (T05.01), B01.02 (T05.02) and B01.03 (T05.04) with amino acids changed to improve binding strength heavy chain region; and (ii) and light chain regions of anti-CTLA-4-8 antibodies of B01.09 (T05L06) are summarized in Table 4. In the sequence of Table 4, CDRs are shown in bold, and amino acids changed to improve binding are underlined.

Clone	division	order	SEQ ID NO:	order	SEQ ID NO:
B01.01 (T05.01)	Anti-CTLA-4-8 antibody VH region	RYAM	148	EVQLLESGGGLVQPGGSLRLSCAASGFTFS R YAMH WVRQAPGKGLEWVS A LYYSGYT YYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RSAPGWGFDY WGQGTLVTVSS	150
		ALYYSGYT	149
		RSAPGWGFDY	11
	bispecific antibody heavy chain	EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYAMHWVRQAPGKGLEWVSALYYSGYTYYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAPGWGFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK GGGGSGGGGSGGGGS QVQLVQSGAEVKKPGASVKLSCKASGYTFSSYWMHWVRQAPGQGLEWIGEINPGNGHTNYNEKFKSRVTMTRDTSTSTAYMELSSLRSEDTAVYYCARSFKTARAFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPAFLSVTPGEKVTITCRASQTISDYLHWYQQKPDQAPKLLIKYASQSISGIPSRFSGSGSGTDFTFTISSLEAEDAATYYCQDGHSWPPTFGQGTKLEIK			151
B01.02 (T05.02)	Anti-CTLA-4-8 antibody VH region		SYAM	9	EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVS SLYYS RW T YYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RSAPGWGFDY WGQGTLVTVSS	153
		SLYYSRWT	152
		RSAPGWGFDY	11
	bispecific antibody heavy chain	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSSLYYSRWTYYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAPGWGFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK GGGGSGGGGSGGGGS QVQLVQSGAEVKKPGASVKLSCKASGYTFSSYWMHWVRQAPGQGLEWIGEINPGNGHTNYNEKFKSRVTMTRDTSTSTAYMELSSLRSEDTAVYYCARSFKTARAFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPAFLSVTPGEKVTITCRASQTISDYLHWYQQKPDQAPKLLIKYASQSISGIPSRFSGSGSGTDFTFTISSLEAEDAATYYCQDGHSWPPTFGQGTKLEIK			154
B01.03 (T05.04)	Anti-CTLA-4-8 antibody VH region		SYAM	9	EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVSS LYYS RWM YYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RSAPGWGFDY WGQGTLVTVSS	156
		SLYYSRWM	155
		RSAPGWGFDY	11
	bispecific antibody heavy chain	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSSLYYSGYTYYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAPGWGFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK GGGGSGGGGSGGGGS QVQLVQSGAEVKKPGASVKLSCKASGYTFSSYWMHWVRQAPGQGLEWIGEINPGNGHTNYNEKFKSRVTMTRDTSTSTAYMELSSLRSEDTAVYYCARSFKTARAFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPAFLSVTPGEKVTITCRASQTISDYLHWYQQKPDQAPKLLIKYASQSISGIPSRFSGSGSGTDFTFTISSLEAEDAATYYCQDGHSWPPTFGQGTKLEIK			157
B01.09 (T05L06)	Anti-CTLA-4-8 antibody VL region		QASRDITNYLN	12	EIVLTQSPGTLSLSPGERATLSC QASRDITNYLN WYQQKPGQAPRLLIY RG ST RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQSYSTPIT FGQGTKVEIK	159
		RGST	158
		QQSYSTPIT	6

Example 14. Mutation site analysis and antigen binding strength confirmation of dual specific antibody with improved binding

The mutation site of the clone with improved binding strength was summarized, and the improved binding affinity analysis value (KD) was compared with the KD value of the anti-CTLA-4-8 antibody. The results of analyzing the mutation site and confirming the antigen binding ability are shown in FIG. 13 .

As shown in FIG. 13 , the bispecific antibody with improved binding affinity is about 1.15 times B01.01, about 2.43 times B01.02, about 2.8 times B01.03, and about 1.2 times B01.09 compared to the bispecific antibody as a template. It was confirmed that there was a double increase.

Above, specific parts of the present invention have been described in detail, for those of ordinary skill in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

Claims (23)

1. An antibody or antigen-binding fragment thereof that specifically binds to CTLA-4 (cytotoxic T lymphocyte-associated protein 4), comprising:

   a heavy chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 9, 15, 23, 31, 39, 46, 54, 62, 68, 75, 88, 100 and 115;

   SEQ ID NO: 2. heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of 10, 16, 24, 32, 40, 47, 55, 63, 69, 76, 81, 89, 95, 101, 108 and 116;

   a heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11, 17, 25, 33, 41, 48, 56, 64, 70, 77, 82, 90, 96, 102, 109 and 117; and

   a light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 18, 26, 34, 42, 49, 57, 71, 83, 103 and 110;

   a light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 19. 27, 35, 50, 58, 84, 104 and 111; and

   A light chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 20, 28, 36, 43, 51, 59, 65, 72, 78, 85, 92, 97, 105, 112 and 118.
2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof

   (1) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 2 and the heavy chain CDR3 of SEQ ID NO: 3, and the light chain CDR1 of SEQ ID NO: 4, the light chain CDR2 of SEQ ID NO: 5 and the light chain of SEQ ID NO: 6 a light chain variable region comprising CDR3;

   (2) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 9, the heavy chain CDR2 of SEQ ID NO: 10 and the heavy chain CDR3 of SEQ ID NO: 11, and the light chain CDR1 of SEQ ID NO: 12, the light chain CDR2 of SEQ ID NO: 5 and the light chain of SEQ ID NO: 6 a light chain variable region comprising CDR3;

   (3) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 15, the heavy chain CDR2 of SEQ ID NO: 16 and the heavy chain CDR3 of SEQ ID NO: 17, and the light chain CDR1 of SEQ ID NO: 18, the light chain CDR2 of SEQ ID NO: 19 and the light chain of SEQ ID NO: 20 a light chain variable region comprising CDR3;

   (4) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 23, the heavy chain CDR2 of SEQ ID NO: 24 and the heavy chain CDR3 of SEQ ID NO: 25, and the light chain CDR1 of SEQ ID NO: 26, the light chain CDR2 of SEQ ID NO: 27 and the light chain of SEQ ID NO: 28 a light chain variable region comprising CDR3;

   (5) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 31, the heavy chain CDR2 of SEQ ID NO: 32 and the heavy chain CDR3 of SEQ ID NO: 33, and the light chain CDR1 of SEQ ID NO: 34, the light chain CDR2 of SEQ ID NO: 35 and the light chain of SEQ ID NO: 36 a light chain variable region comprising CDR3;

   (6) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 39, the heavy chain CDR2 of SEQ ID NO: 40 and the heavy chain CDR3 of SEQ ID NO: 41, and the light chain CDR1 of SEQ ID NO: 42, the light chain CDR2 of SEQ ID NO: 5 and the light chain of SEQ ID NO: 43 a light chain variable region comprising CDR3;

   (7) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 46, the heavy chain CDR2 of SEQ ID NO: 47 and the heavy chain CDR3 of SEQ ID NO: 48, and the light chain CDR1 of SEQ ID NO: 49, the light chain CDR2 of SEQ ID NO: 50 and the light chain of SEQ ID NO: 51 a light chain variable region comprising CDR3;

   (8) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 54, the heavy chain CDR2 of SEQ ID NO: 55 and the heavy chain CDR3 of SEQ ID NO: 56, and the light chain CDR1 of SEQ ID NO: 57, the light chain CDR2 of SEQ ID NO: 58 and the light chain of SEQ ID NO: 59 a light chain variable region comprising CDR3;

   (9) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 62, the heavy chain CDR2 of SEQ ID NO: 63 and the heavy chain CDR3 of SEQ ID NO: 64, and the light chain CDR1 of SEQ ID NO: 57, the light chain CDR2 of SEQ ID NO: 58 and the light chain of SEQ ID NO: 65 a light chain variable region comprising CDR3;

   (10) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 68, the heavy chain CDR2 of SEQ ID NO: 69 and the heavy chain CDR3 of SEQ ID NO: 70, and the light chain CDR1 of SEQ ID NO: 71, the light chain CDR2 of SEQ ID NO: 58 and the light chain of SEQ ID NO: 72 a light chain variable region comprising CDR3;

   (11) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 75, the heavy chain CDR2 of SEQ ID NO: 76 and the heavy chain CDR3 of SEQ ID NO: 77, and the light chain CDR1 of SEQ ID NO: 57, the light chain CDR2 of SEQ ID NO: 58 and the light chain of SEQ ID NO: 78 a light chain variable region comprising CDR3;

   (12) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 81 and the heavy chain CDR3 of SEQ ID NO: 82, and the light chain CDR1 of SEQ ID NO: 83, the light chain CDR2 of SEQ ID NO: 84 and the light chain of SEQ ID NO: 85 a light chain variable region comprising CDR3;

   (13) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 88, the heavy chain CDR2 of SEQ ID NO: 89 and the heavy chain CDR3 of SEQ ID NO: 90, and the light chain CDR1 of SEQ ID NO: 57, the light chain CDR2 of SEQ ID NO: 58 and the light chain of SEQ ID NO: 92 a light chain variable region comprising CDR3;

   (14) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 9, the heavy chain CDR2 of SEQ ID NO: 95 and the heavy chain CDR3 of SEQ ID NO: 96, and the light chain CDR1 of SEQ ID NO: 57, the light chain CDR2 of SEQ ID NO: 58 and the light chain of SEQ ID NO: 97 a light chain variable region comprising CDR3;

   (15) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 100, the heavy chain CDR2 of SEQ ID NO: 101 and the heavy chain CDR3 of SEQ ID NO: 102, and the light chain CDR1 of SEQ ID NO: 103, the light chain CDR2 of SEQ ID NO: 104 and the light chain of SEQ ID NO: 105 a light chain variable region comprising CDR3;

   (16) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 46, the heavy chain CDR2 of SEQ ID NO: 108 and the heavy chain CDR3 of SEQ ID NO: 109, and the light chain CDR1 of SEQ ID NO: 110, the light chain CDR2 of SEQ ID NO: 111 and the light chain of SEQ ID NO: 112 a light chain variable region comprising CDR3; or

   (17) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 115, the heavy chain CDR2 of SEQ ID NO: 116 and the heavy chain CDR3 of SEQ ID NO: 117, and the light chain CDR1 of SEQ ID NO: 57, the light chain CDR2 of SEQ ID NO: 58 and the light chain of SEQ ID NO: 118 A light chain variable region comprising CDR3; comprising, an antibody or antigen-binding fragment thereof.
3. According to claim 1, wherein the antibody or antigen-binding fragment thereof is SEQ ID NO: 7, 13, 21, 29, 37, 44, 52, 60, 66, 73, 79, 86, 93, 98, 106, 113 and 119 An antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of.
4. According to claim 1, wherein the antibody or antigen-binding fragment thereof is SEQ ID NO: 8, 14, 22, 30, 38, 45, 53, 61, 67, 74, 80, 87, 94, 99, 107, 114 and 120 An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising one or more amino acid sequences selected from the group consisting of.
5. According to claim 1, wherein the antigen or antigen-binding fragment thereof,

   a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 8;

   a heavy chain variable region of SEQ ID NO: 13 and a light chain variable region of SEQ ID NO: 14;

   a heavy chain variable region of SEQ ID NO: 21 and a light chain variable region of SEQ ID NO: 22;

   a heavy chain variable region of SEQ ID NO: 29 and a light chain variable region of SEQ ID NO: 30;

   a heavy chain variable region of SEQ ID NO: 37 and a light chain variable region of SEQ ID NO: 38;

   a heavy chain variable region of SEQ ID NO: 44 and a light chain variable region of SEQ ID NO: 45;

   a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 53;

   a heavy chain variable region of SEQ ID NO: 60 and a light chain variable region of SEQ ID NO: 61;

   a heavy chain variable region of SEQ ID NO: 66 and a light chain variable region of SEQ ID NO: 67;

   a heavy chain variable region of SEQ ID NO: 73 and a light chain variable region of SEQ ID NO: 74;

   a heavy chain variable region of SEQ ID NO: 79 and a light chain variable region of SEQ ID NO: 80;

   a heavy chain variable region of SEQ ID NO: 86 and a light chain variable region of SEQ ID NO: 87;

   a heavy chain variable region of SEQ ID NO: 93 and a light chain variable region of SEQ ID NO: 94;

   a heavy chain variable region of SEQ ID NO: 98 and a light chain variable region of SEQ ID NO: 99;

   a heavy chain variable region of SEQ ID NO: 106 and a light chain variable region of SEQ ID NO: 107;

   a heavy chain variable region of SEQ ID NO: 113 and a light chain variable region of SEQ ID NO: 114; or

   A heavy chain variable region of SEQ ID NO: 119 and a light chain variable region of SEQ ID NO: 120; comprising, an antibody or antigen-binding fragment thereof.
6. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 121 to 132, 134 to 138.
7. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is a single-chain Fvs (scFv), a single-chain antibody, Fab, F(ab'), or a disulfide-bonded Fvs (sdFv).
8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the scFv has a heavy chain variable region and a light chain variable region connected through a linker.
9. (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9 or 148, a heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 149, 152 and 155 and an amino acid sequence of SEQ ID NO: 11 a heavy chain variable region comprising a heavy chain CDR3;

   IgG4 isotype represented by the amino acid sequence of SEQ ID NO: 139; and

   a heavy chain variable region of a multispecific antibody comprising;

   (b) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12;

   a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or 158;

   A multi-specific antibody comprising a; a light chain variable region comprising a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.
10. The antibody of claim 9, wherein the heavy chain variable region is represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 13, 150, 153 and 156.
11. The antibody of claim 9, wherein the scFv of the anti 4-1BB antibody is represented by the amino acid sequence of SEQ ID NO: 146.
12. The antibody of claim 9, wherein the heavy chain variable region of the multispecific antibody is operably linked as shown in Structural Formula 1 below.

    [Structural Formula 1]

    Heavy chain variable region - IgG4 isotype - scFv of anti 4-1BB antibody
13. The antibody of claim 12, wherein the scFv of the IgG4 isotype and the anti 4-1BB antibody are linked by a linker.
14. The antibody of claim 13, wherein the heavy chain variable region of the multispecific antibody is represented by the amino acid sequence of SEQ ID NO: 147, 151, 154 or 157.
15. The antibody of claim 9, wherein the light chain variable region is represented by the amino acid sequence of SEQ ID NO: 14 or 159.
16. 10. The antibody of claim 9, wherein the multispecific antibody specifically binds to CTLA-4 and 4-1BB.
17. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8; or

    16. A nucleic acid encoding a multispecific antibody according to any one of claims 9 to 15.
18. A recombinant expression vector comprising the nucleic acid of claim 17 .
19. A host cell transfected with the recombinant expression vector of claim 18 .
20. 20. The method of claim 19, wherein the host cell is COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK , BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS-0, U20S or HT1080 host cells .
21. 20. A method comprising: culturing the host cell of claim 19 to generate an antibody; And a method for producing an antibody that specifically binds to CTLA-4, an antigen-binding fragment thereof, or a multispecific antibody comprising the step of isolating and purifying the generated antibody.
22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8; or

    A pharmaceutical composition for preventing or treating cancer, comprising a multi-specific antibody according to any one of claims 9 to 15.
23. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8; Or the multispecific antibody according to any one of claims 9 to 15; comprising administering to an individual in need thereof, a method for preventing or treating cancer.

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