KR20210154940A - Conformational epitope of ceacam1 and anti-ceacam1 antibody or fragment thereof specifically binding thereto - Google Patents

Abstract

The present invention relates to conformational epitope of CEACAM1 and an anti-CEACAM1 antibody or a fragment thereof specifically binding to the same. The conformational epitope of CEACAM1 of the present invention includes all amino acids at positions which are important for specific binding with an anti-CEACAM1 antibody and maintains an adequate three dimensional structure, thereby exhibiting high affinity with the anti-CEACAM1 antibody. Also, an antibody or a fragment thereof specifically binding to the conformational epitope according to the present invention can effectively inhibit CEACAM1-CEACAM1 interaction and CEACAM1-CEACAM6 interaction.

Description

Stereoscopic epitope of CEACAM1 and anti-CEACAM1 antibody or fragment thereof specifically binding thereto

The present invention relates to a stereoscopic epitope of CEACAM1 and an anti-CEACAM1 antibody or fragment thereof that specifically binds thereto.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is one of the transmembrane glycoproteins belonging to the carcinoembryonic antigen group. CEACAM1 is mainly expressed in activated T cells and natural killer cells, and shows high expression in cancer cells as well.

CEACAM1 plays a role in regulating innate and adaptive immune responses. In this regard, in the case of CEACAM1-overexpressing cancer cells, CEACAM1-CEACAM1 interaction occurs with CEACAM1-expressing T cells. When the CEACAM1-CEACAM1 interaction occurs, in the ITIM (immunoreceptor tyrosine-based inhibition motif) portion of CEACAM1 phosphorylated by LCK (lymphocyte-specific protein tyrosine kinase) bound to the CD4 terminus of the T cell receptor (TCR) of the T cell. Src homology region 2 domain-containing phosphatase-1 (SHP1) binds. Furthermore, it has been reported that the CD3ζ end is dephosphorylated by the SHP1 protein and the RAS-MAPK signaling mechanism is not activated and thus T cells are not activated, thereby evading the immune response in cancer cells (Scott D. Gray). -Owen & Richard S. Blumberg, Nature Reviews Immunologyvolume 6 , pages433-446, 2006).

In addition, CEACAM1 has not only a homophilic interaction, but also a heterophilic interaction with CEACAM5 or CEACAM6. Among them, CEACAM6 is expressed in various carcinomas such as breast tumors, pancreatic tumors, ovarian adenocarcinomas, and lung adenocarcinoma (Blumenthal et al . BMC Cancer ., 2007 Jan 3;7: 2). CEACAM1 expressed in activated T cells inhibits TCR signal through binding to CEACAM6, which prevents T cells from being activated by the same mechanism as CEACAM1-CEACAM1 interaction, thereby evading the immune response of CEACAM6 expressing cancer cells. Results have been reported (Witzens-Harig et al ., Blood 2013 May 30:121(22):4493-503).

Accordingly, inhibiting the CEACAM1-CEACAM1 interaction in cancer cells has emerged as a promising anticancer therapy, and in order to produce and verify an antibody against CEACAM1 that can effectively inhibit the CEACAM1-CEACAM1 interaction, an accurate stereoscopic epitope of CEACAM1 has to be developed. The need for clarification is emerging.

Scott D. Gray-Owen & Richard S. Blumberg, Nature Reviews Immunologyvolume 6, pages433-446, 2006 Blumenthal et al. BMC Cancer., 2007 Jan 3;7:2 Wiitzens-Harig et al., Blood 2013 May 30:121(22):4493-503

Accordingly, in order to identify the stereoscopic epitope of CEACAM1, the present inventors crystallized the structure of a complex in which an anti-CEACAM1 antibody and CEACAM1 were bound through XRD (X-ray diffraction), and the antibody specifically binding to the stereoscopic epitope was CEACAM1 The present invention was completed by confirming that -CEACAM1 interaction and CEACAM1-CEACAM6 interaction were inhibited.

In order to achieve the above object, one aspect of the present invention consists of 4 to 69 amino acids in the amino acid sequence of positions 35 to 141 of CEACAM1, 63, 64, 66, 68, 75, 76, 78, 83, It provides a stereoscopic epitope comprising any one amino acid selected from the group consisting of amino acids at positions 86, 90, 123, 125, 129, and 131 and combinations thereof.

In another aspect of the present invention, as consisting of 4 to 69 amino acids in the amino acid sequence of positions 35 to 141 of CEACAM1, 63, 64, 66, 68, 75, 76, 78, 83, 86, 90, 123, 125 , provides an anti-CEACAM1 antibody or fragment thereof that specifically binds to a stereoscopic epitope comprising any one amino acid selected from the group consisting of amino acids at positions 129 and 131 and combinations thereof.

The stereoscopic epitope of CEACAM1 of the present invention shows high affinity with the anti-CEACAM1 antibody by maintaining an appropriate three-dimensional structure while including all amino acids at positions important for specific binding to the anti-CEACAM1 antibody. In addition, the antibody or fragment thereof that specifically binds to the stereoscopic epitope according to the present invention can effectively inhibit the CEACAM1-CEACAM1 interaction and the CEACAM1-CEACAM6 interaction.

1 is a diagram showing the crystal structure of the Fab and CEACAM1 N-domain complex of the anti-CEACAM1 antibody of an embodiment by XRD (X-ray diffraction) analysis.
2 is a diagram showing the structure of the N-domain complex of CEACAM1 and the variable region of the anti-CEACAM1 antibody of an embodiment (left) and the dimer binding structure between the N-domain of CEACAM1 (right).
3 is a diagram showing the amino acid of the portion where the Fab and CEACAM1 N-domain of the anti-CEACAM1 antibody of one embodiment bind.
4 is a diagram showing amino acids constituting a dimer bond between CEACAM1 N-domains.
5A to 5D are diagrams showing amino acids having an intermolecular distance between the Fab of the anti-CEACAM1 antibody and the CEACAM1 N-domain within 4.5 Å.
6 is a diagram showing the amino acid of the hCEACAM1 extracellular domain and sequence information of the DNA encoding the same.
7 is a diagram showing amino acids of the hIgG4 Fc region and sequence information of DNA encoding the same.
8 is a view confirming the homophilic interaction between CEACAM1-Fc and CEACAM1-Fc according to the concentration of CEACAM1-Fc.
9 is a view confirming the inhibitory effect of the homophilic interaction between CEACAM1-Fc and CEACAM1-Fc according to the concentration of the anti-CEACAM1 antibody of one embodiment.
10 is a diagram showing sequence information of DNA encoding hCEACAM6(ECD)-Fc.
11 is a view confirming the heterophilic interaction between CEACAM1-Fc and CEACAM6-Fc according to the concentration of CEACAM1-Fc.
12 is a view confirming the inhibitory effect of the heterophilic interaction between CEACAM1-Fc and CEACAM6-Fc according to the concentration of the anti-CEACAM1 antibody of one embodiment.
13 is a view confirming the effect of increasing ZAP70 phosphorylation of the anti-CEACAM1 antibody of an embodiment in Jurkat cells overexpressing CEACAM1.
14 is a view confirming the NFAT expression increase effect of the anti-CEACAM1 antibody of an embodiment in Jurkat cells overexpressing NFAT and CEACAM1.
15 is a view confirming the effect of increasing IL-2 expression of the anti-CEACAM1 antibody of an embodiment in Jurkat cells overexpressing CEACAM1.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention, as consisting of 4 to 69 amino acids in the amino acid sequence of positions 35 to 141 of CEACAM1, 63, 64, 66, 68, 75, 76, 78, 83, 86, 90, 123, 125 , provides a stereoscopic epitope comprising any one amino acid selected from the group consisting of amino acids at positions 129 and 131 and combinations thereof.

The Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is one of the transmembrane glycoproteins belonging to the carcinoembryonic antigen group. The CEACAM1 is mainly expressed in activated T cells and natural killer cells, and shows high expression in cancer cells as well. The amino acid sequence at positions 35 to 141 of CEACAM1 corresponds to the N-domain of CEACAM1, and is known to be involved in CEACAM1-CEACAM1 interaction or CEACAM1-CEACAM6 interaction. The CEACAM1 may be human CEACAM1, and the amino acid sequence of the human CEACAM1 may be the amino acid sequence of SEQ ID NO: 1.

The 63rd amino acid is phenylalanine (Phe), the 64th amino acid is glycine (Gly), the 66th amino acid is serine (Ser), the 68th amino acid is tyrosine (tyrosine, Tyr), 75 The th amino acid is glycine, the 76th amino acid is asparagine (Asn), the 78th amino acid is glutamine (Gln), the 83rd amino acid is alanine (Ala), and the 86th amino acid is threonine (threonine, Thr), the 90th amino acid is threonine, the 123th amino acid is glutamine, the 125th amino acid is isoleucine (Ile), the 129th amino acid is leucine (Leu), and the 131th amino acid is asparagine. to be.

The stereoscopic epitope may include adjacent amino acids or amino acids that are not adjacent due to three-dimensional folding of a protein. Specifically, a stereoscopic epitope may include at least 4, 5, 9, or 10 amino acids in a three-dimensional structure of an independent space.

Specifically, the stereoscopic epitope may include amino acids at positions 63, 64, 66 and 68 of the amino acid sequence at positions 35 to 141 of CEACAM1. In addition, the stereoscopic epitope may include amino acids at positions 75, 76, 78, 83, 86, and 90 of the amino acid sequence at positions 35 to 141 of CEACAM1. In addition, the stereoscopic epitope may include amino acids at positions 123, 125, 129, and 131 in the amino acid sequence at positions 35 to 141 of CEACAM1.

Furthermore, the stereoscopic epitope may include amino acids at positions 63, 66, 68, 78, 123, 125, 129 and 131 of the amino acid sequence at positions 35 to 141 of CEACAM1. In addition, the stereoscopic epitope may include amino acids at positions 64, 75, 76, 83, 86, and 90 in the amino acid sequence at positions 35 to 141 of CEACAM1. Preferably, the stereoscopic epitope consists of 4 to 69 amino acids in the amino acid sequence of positions 35 to 141 of CEACAM1, 63, 64, 66, 68, 75, 76, 78, 83, 86, 90, 123, It may include amino acids at positions 125, 129 and 131 .

In the present invention, in order to identify a novel stereoscopic epitope of CEACAM1, the structure of a complex in which CEACAM1 and an anti-CEACAM1 antibody of an embodiment are bound was crystallized at a resolution of 1.8 Å ( FIGS. 1 to 5d ). As a result, the novel epitope of CEACAM1 and the paratope of anti-CEACAM1 of one example were identified, and are shown in Table 1 below.

CEACAM1 Anti-CEACAM1 antibody Type of interaction VH VL Phe63 Thr101 Van der Waals Phe63 Tyr104 Hydrophobic Phe63 Ala105 Van der Waals Phe63 Tyr33 Van der Waals Phe63 Tyr50 Hydrophobic Phe63 Ala51 Van der Waals Gly64 Tyr104 Van der Waals Ser66 Tyr104 H-bonding Tyr68 Asn31 H-bonding Gly75 Asn31 Hydrophobic Asn76 Asn74 Ionic interaction Gln78 Lys102 Ionic interaction Gln78 Tyr104 Ionic interaction Ala83 Tyr104 Hydrophobic Thr86 Leu94 Hydrophobic Thr90 Gly54 Hydrophobic Thr90 Gly56 Hydrophobic Gln123 Asn31 Ionic interaction Ile125 Lys102 Van der Waals Ile125 Tyr104 Hydrophobic Leu129 Tyr50 Van der Waals Leu129 Pro100 Van der Waals Leu129 Thr101 Van der Waals Asn131 Tyr32 Ionic interaction

As shown in Table 1 above, the anti-CEACAM1 antibody contains 63, 64, 66, 68, 75, 76, 78, 83, 86, 90, 123, 125, 129 and It was confirmed that it binds to the amino acid at position 131.

In another aspect of the present invention, as consisting of 4 to 69 amino acids in the amino acid sequence of positions 35 to 141 of CEACAM1, 63, 64, 66, 68, 75, 76, 78, 83, 86, 90, 123, 125 , provides an anti-CEACAM1 antibody or fragment thereof that specifically binds to a stereoscopic epitope comprising any one amino acid selected from the group consisting of amino acids at positions 129 and 131 and combinations thereof.

The stereoscopic epitope is the same as described above.

Specifically, the antibody or fragment thereof may specifically bind to a stereoscopic epitope including amino acids at positions 63, 64, 66 and 68 of the amino acid sequence at positions 35 to 141 of CEACAM1. In addition, the antibody or fragment thereof may specifically bind to a stereoscopic epitope including amino acids at positions 75, 76, 78, 83, 86, and 90 in the amino acid sequence at positions 35 to 141 of CEACAM1. Furthermore, the antibody or fragment thereof may specifically bind to a stereoscopic epitope including amino acids at positions 123, 125, 129, and 131 in the amino acid sequence at positions 35 to 141 of CEACAM1.

Furthermore, the antibody or fragment thereof specifically binds to a stereoscopic epitope comprising amino acids at positions 63, 66, 68, 78, 123, 125, 129 and 131 of the amino acid sequence at positions 35 to 141 of CEACAM1. can In addition, the antibody or fragment thereof may specifically bind to a stereoscopic epitope including amino acids at positions 64, 75, 76, 83, 86 and 90 in the amino acid sequence at positions 35 to 141 of CEACAM1. Preferably, the antibody or fragment thereof consists of 4 to 69 amino acids in the amino acid sequence of positions 35 to 141 of CEACAM1, 63, 64, 66, 68, 75, 76, 78, 83, 86, 90, It may specifically bind to a stereoscopic epitope including amino acids at positions 123, 125, 129 and 131.

The antibody or fragment thereof may bind to CEACAM1 within an intermolecular distance of 4.5 Å. The antibody or fragment thereof may have a van der Waals bond, a hydrophobic bond, or an electrostatic bond with CEACAM1.

The antibody or fragment thereof may have a binding affinity ^{with CEACAM1 of less than 1×10 −8} K _{D (M).} Specifically, the antibody or fragment thereof is CEACAM1 and 9×10 ^-9 , 8×10 ^-9 , 7×10 ^-9 , 6×10 ^-9 , 5×10 ^-9 or 4×10 ^-9 K _D (M ) or less than the binding affinity. In one embodiment of the present invention, the binding affinity of the antibody of Example 1 to CEACAM1 was measured to be ^{3.36×10 −9} K _{D (M).}

The antibody or fragment thereof is a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 2, heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 3, heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: It may include a light chain CDR1 comprising the amino acid sequence shown in 5, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 6, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 7.

The antibody or fragment thereof may include a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9.

The antibody fragment may be any one selected from the group consisting of Fab, scFv, F(ab') _{2 and Fv.} Antibody fragments refer to antigen-binding domains excluding the determinable region (Fc region) that has an effector function to transmit antigen-binding stimulation to cells or complement, single domain antibody or small antibody ( minibody) and the like).

Another aspect of the present invention provides an anticancer agent comprising the antibody or fragment thereof as an active ingredient.

An anticancer agent comprising the antibody or fragment thereof as an active ingredient may be used to treat CEACAM1 overexpressed cancer or tumor. Specifically, when the T cell receptor (TCR) of cytotoxic T cells, which plays a role in eliminating cancer cells, recognizes the antigenic determinant of cancer or tumor cells, it is located at the end of CD4 (cluster of differentiation 4), one of the components of TCR. The bound LCK (lymphocyte-specific protein tyrosine kinase) protein phosphorylates another component of TCR, cluster of differentiation 3ζ (CD3ζ). When the ZAP70 (Zeta-chain-associated protein kinase 70) protein is bound to the phosphorylated CD3ζ portion, the end of the ZAP70 protein is phosphorylated again by the LCK protein, and the RAS-MAPK signaling mechanism (Ras-MAP kinase signal transduction) is activated and T cells are activated

However, in the case of CEACAM1-overexpressing cancer cells or tumor cells, the immunoreceptor tyrosine-based inhibition motif (ITIM) portion of CEACAM1 phosphorylated by the LCK protein bound to the CD4 terminus of the TCR due to the CEACAM1-CEACAM1 interaction occurs in the Src homology (SHP1) portion. region 2 domain-containing phosphatase-1) protein is bound. In addition, the CD3ζ terminus is dephosphorylated by the SHP1 protein, and the RAS-MAPK signaling mechanism is not activated, so that the T cells are not activated.

Therefore, the antibody or fragment thereof can be used as an anticancer agent by binding to CEACAM1 expressed in cytotoxic T cells, natural killer cells and cancer cells and blocking the CEACAM1-CEACAM1 interaction in advance.

In addition, the term "anticancer" as used in the present invention includes 'prevention' and 'treatment', where 'prevention' refers to any action that inhibits the growth of cancer or delays the progression of cancer by administering the anticancer agent. means, and 'treatment' refers to any action in which the symptoms of cancer are improved or beneficially changed by administration of the antibody of the present invention.

In addition, the term "cancer" used in the present invention may be characterized as selected from the group consisting of pancreatic cancer, melanoma, lung cancer and myeloma, but is not limited thereto, and has CEACAM1 as a receptor and immune checkpoint pathway abnormally The cancer or tumor to be operated is not particularly limited, and may include solid cancer and hematological cancer.

Another aspect of the present invention provides a polynucleotide encoding the antibody or fragment thereof. Specifically, the polynucleotide may include the nucleotide sequence represented by SEQ ID NOs: 10 and/or 11.

The polynucleotide may be mutated by substitution of one or more bases, deletion, insertion, or a combination thereof. When the nucleotide sequence is prepared by chemical synthesis, a synthesis method well known in the art, for example, the method described in the literature (Engels and Uhlmann, Angew Chem Int Ed Engl., 37:73-127, 1988) can be used. , triester, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis methods on a solid support.

In addition, another aspect of the present invention provides an expression vector comprising the polynucleotide. The expression vector may be plasmid DNA, phage DNA, etc., commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), Escherichia coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis-derived Plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal viral vectors (retrovirus), adenovirus, vaccinia virus, etc.), insect virus vectors (baculovirus, etc.). Since the expression vector shows different protein expression levels and modifications depending on the host cell, it is preferable to select and use the most suitable host cell for the purpose.

Another aspect of the present invention provides a transformed cell into which the expression vector is introduced. The host cell of the transformed cell may include, but is not limited to, cells of mammalian, plant, insect, fungal or cellular origin. As the mammalian cells, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells or HEK293T cells, etc. may be used. It is not limited, and any cell that can be used as a mammalian host cell known to those skilled in the art can be used.

In addition, when introducing an expression vector into a host cell, the CaCl ₂ precipitation method, the CaCl ₂ precipitation method using a reducing material such as DMSO (dimethyl sulfoxide), the Hanahan method, electroporation method, calcium phosphate precipitation method , protoplast fusion method, agitation method using silicon carbide fiber, agrobacterium mediated transformation method, transformation method using PEG, dextran sulfate, lipofectamine and drying/inhibition mediated transformation method can be used.

Another aspect of the present invention provides a method for producing an antibody or fragment thereof comprising culturing the transformed cell. Specifically, the production method includes the steps of: i) culturing the transformed cells to obtain a culture; and ii) recovering the antibody or fragment thereof from the culture.

The method of culturing the transformed cells can be performed using a method well known in the art. Specifically, the culture may be continuously cultured in a batch process or in a fed batch or repeated fed batch process.

The step of recovering the antibody or fragment thereof from the culture may be performed by a method known in the art. Specifically, the recovery method includes centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (eg, ammonium sulfate precipitation), chromatography (eg, ion exchange, affinity, hydrophobicity and size exclusion) can be used.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

I. Confirmation of binding structure of CEACAM1 and anti-CEACAM1 antibody

Example 1. CEACAM1 N-domain production

In order to bind and express the amino acid sequence at positions 35 to 141 corresponding to the CEACAM1 N-domain with a maltose-binding protein (MBP) tag linked to 10 histidine, pET28-MBP-TEV vector (addgene, Plasmid #69929) derived 10His-MBP-TEV-X-pJA vector was cloned. E. coli BL21(DE3)RIPL strain was transformed using the cloned vector, and the protein was expressed by treatment with IPTG (isopropyl β-D-1-thiogalactopyranoside) at a concentration of 200 μM at 18°C. Protein-expressed E. coli was sonicated in 100 mM NaCl and 20 mM Tris-HCl (pH 7.5) buffer, and only the lysate was separated through centrifugation. After that, affinity chromatography for the 10His tag was performed using cobalt resin. The resulting eluate was treated with TEV protease to cut the 10His-MBP tag. The 10His-MBP tag was isolated and removed using a HitrapQ anion exchange column and a Histrap affinity chromatography column, and high-purity CEACAM1 N-domain was purified.

Example 2. Fab construction of anti-CEACAM1 antibody

Example 2.1. Preparation of anti-CEACAM1 antibody in which the heavy chain is substituted with IgG1 type

To construct a Fab of the anti-CEACAM1 antibody, the IgG4 heavy chain of the anti-CEACAM1 antibody (SEQ ID NOs: 12 and 13) was converted into human IgG1 (SEQ ID NOs: 14 and 15). In order to convert to human IgG1 type, a gene encoding a CH1-hinge-CH2-CH3 fragment of human IgG1 type was obtained through PCR. The primers used at this time are shown in Table 2 below.

primer Sequence information (5'-> 3') SEQ ID NO: VH-NotI-L_F primer GCG GCC GCC ATG TAC TTG GGA CTG AAC TAT GTA TTC ATA GTT TTT CTC TTA AAT GGT GTC CAG AGT 16 VH-ApaI-CH1_R primer ATG GGC CCT TGG TGG AGG CTG AGG AGA CGG TGA C 17 CH1-Fc_ApaI_F primer GCC TCC ACC AAG GGC CCA 18 IgG1_Fc_HindIII_R primer NNN NGG ATC CAA GCT TAC TAT TTA CCC GGA 19

The gene encoding the CH1-hinge-CH2-CH3 fragment of the IgG1 type obtained through the PCR and the gene encoding the heavy chain variable region of the anti-CEACAM1 antibody were not I (NEB, CAT. NO. R0189L) and Hind III ( NEB, CAT. NO. R0104T) A gene encoding a heavy chain of anti-CEACAM1 antibody-IgG1 type was obtained through overlap PCR using a primer having a cut portion. In this case, the primers used are shown in Table 3 below.

primer Sequence information (5'-> 3') SEQ ID NO: IgG1_HC_NotI_F primer GCG GCC GCC ATG TAC TTG G 20 IgG1_HC_HindIII_R primer NNN NGG ATC CAA GCT TAC TAT TTA CCC GGA 19

The gene (DNA) obtained by the PCR was loaded on a 1% agarose gel and separated using a DNA Gel extraction kit (QIAGEN, CAT. NO. 28706).

Not I (NEB, CAT. NO. R0189L) and HindIII (NEB, CAT. NO. R0104T) restriction enzymes were added to the isolated DNA and reacted at 37°C for 4 hours, followed by QIAquick PCR Purification Kit (QIAGEN, CAT. No. 28106) was used to obtain DNA.

In addition, the obtained DNA and T4 DNA ligase (ligase, NEB, CAT. NO. M0203S) were added to a linearized pCIW vector treated with NotI and HindIII restriction enzymes, and reacted at 16° C. for 4 hours. After 4 hours, 1 μl of the ligation mixture was taken, put into 100 μl of XL1-Blue Electroporation-Competent Cells, mixed, and then transformed using an electroporation system.

A single colony from the transformed plate was inoculated into SB/car medium and cultured overnight. DNA was obtained from the transformed cells using the QIAprep Spin Miniprep Kit (QIAGEN, CAT. NO. 127106), and sequencing was requested from an external company, Cosmogenetech. As a result, a gene encoding a heavy chain of the anti-CEACAM1 antibody-IgG1 type represented by the nucleotide sequence of SEQ ID NO: 33 was identified. After culturing the identified transformed cells, a large amount of DNA was obtained using the QIAGEN Plasmid Plus Midi Kit (QIAGEN, CAT. NO. 12945).

Example 2.2. Production of anti-CEACAM1 antibody in which the heavy chain is substituted with IgG1 type

Expi293F ^TM Cells (Gibco, CAT. NO. A14527) were seeded one day before transfection at a concentration of ^{2.0×10 6 cells/ml in Expi293 TM} Expression Medium (Gibco, CAT. NO. A1435101). After culturing at 37° C. temperature, 8% CO ₂ , and 125 rpm conditions for ^{24 hours, 25.5 ml was prepared at a concentration of 2.5×10 6} cells/ml (viability = 95%).

30 μg of the obtained CEACAM1 antibody-IgG1-type heavy chain DNA (pCIW_MG1124HC_IgG1: 15 μg, pCIW_MG1124LC: 15 μg) was diluted in 1.5 ml of OptiPro™ SEM media (Gibco, CAT. NO. 12309019) and reacted at room temperature for 5 minutes. . Then, 80 μl of ExpiFectamine ^TM 293 reagent (Gibco, CAT. NO. A14524) was also added to 1.5 ml of OptiPro TM SEM media (Gibco, CAT. NO. 12309019) and reacted at room temperature for 5 minutes. Then, each diluted DNA and ExpiFectamine ^TM 293 reagent were mixed well, and 3 ml of the mixture was reacted at room temperature for 30 minutes.

The 3 ml of the mixture was put into 30 ml ^{of Expi293F TM Cells having a concentration of 2.5 × 10 6} cells/ml (viability = 95%). After incubation for 16 to 18 ^{hours, ExpiFectamine TM 293 enhancer 1 (Gibco} , CAT. NO. A14524) 150 ㎕ and ^{ExpiFectamine TM 293 enhancer 2 (Gibco,} CAT. NO. A14524) 6 ilgan CO ₂ was shaken with a 1.5 ㎖ added It was cultured in an incubator (Shaking Incubator, MB-206CXXL).

After the incubation was completed, the cell pellet was removed by centrifugation at 4,000 rpm for 20 minutes, and the supernatant was filtered through a 0.45 μm filter. 100 μl of CaptivA Protein A Resin (REPLIGEN, CA-PRI-0100), a Protein A resin, is prepared per 30 ml of each culture medium, and centrifuged at 1000 rpm for 2 minutes to remove the storage buffer, and Protein A Binding buffer (Pierce, CAT. NO. 21007) was washed 3 times with 1 ml each.

Put Protein A resin in the prepared culture solution, rotate incubation for 2 hours at room temperature, put the mixture in Pierce Spin column snap-cap (Thermo, CAT. NO. 69725), and QIAvac 24 plus (QIAGEN, CAT. NO) 19413) The column was filled with resin using a vacuum manifold. Put 5 ml of Protein A binding buffer (Pierce, CAT. NO. 21007) into the resin and wash, add 200 μl of Protein A elution buffer (Pierce, CAT. NO. 21009), incubate at room temperature for 2 minutes, and then 1,000 rpm It was eluted by centrifugation for 1 minute under the conditions.

Example 2.3. Fab isolation and purification of anti-CEACAM1 antibody

In order to purify only Fab from the anti-CEACAM1 antibody-IgG1 type prepared in Example 2.2, papain protease was treated at a ratio of 1:100. Papain protease was used to separate the Fab and Fc by cutting the sequence between the Fab and Fc of the IgG1 heavy chain. Papain protease treatment was reacted in PBS buffer, and then the buffer conditions were changed by dialysis with NaCl at 0 mM concentration and Tris-HCl at 20 mM concentration (pH 7.5). Since the Fab portion of the anti-CEACAM1 antibody had a theoretical pI value of 8.8, it was separated and purified using a HitrapSP cation exchange column. Anti-CEACAM1 Fc came out without being attached to the column, and anti-CEACAM1 Fab was separated and purified with high purity under conditions of about 50 mM to 70 mM NaCl. After the purified anti-CEACAM1 Fab and the CEACAM1 N-domain isolated in Example 1 were mixed at a ratio of 1:2, final purification was performed using a HiLoad 26/60 Superdex 75 gel-filtration column.

Example 3. Crystallization and structural analysis of the Fab complex of CEACAM1 N-domain and anti-CEACAM1 antibody

Screening was performed under about 800 conditions to optimize the crystallization conditions.

Finally, the Fab of the anti-CEACAM1 antibody and the CEACAM1 N-domain complex (36.6 mg/ml) were prepared using 0.1 M lithium sulfate monohydrate and 0.1 M ADA (N-(2-acetamido) iminodiacetic acid). acid, pH 6.5), 14% (w/v) polyethylene glycol 4000 (polyethylene glycol 4000), and 2% (v/v) isopropanol were crystallized under conditions containing. The resulting crystals were treated under conditions in which 17.5% ethylene glycol was additionally added to prevent freezing.

XRD (X-ray diffraction) data were collected at the 5C beamline of the Pohang Accelerator Laboratory and processed with the HKL2000 suit(1). The structure of the complex was determined using the Molecular replacement (2) method of the Phenix program, and the model used for this was a structure having high sequence homology with an anti-CEACAM antibody (PDB entry: 4EVN) and a structure corresponding to the CEACAM1 N-domain (PDB entry: 4WHD) was used. Refinement of the structure used Phenix's Refinement function (3), CNS program (4), and COOT program (5). Crystallographic data statistics are shown in Table 4 below.

Data Collection space group P212121 Unit cell dimensions a, b, c (Å) 70.985, 140.722, 206.120 α, β, γ (°) 90, 90, 90 Wavelength (Å) 1.0000 Resolution (Å) 50.0-1.79 R _sym 9.9 (27.0) I /( I ) 14.6 (1.9) Completeness (%) 73.8 (23.8) Redundancy 5.6 (1.3) Refinement Resolution (Å) 50.0-1.8 No. of reflections 142860 R _work / R _free (%) 22.83 / 25.76 R.m.s deviations bond lengths (Å) / angles (º) 0.008 / 0.979 Average B-values (Å ² ) 29.6 Ramachandran plot (%) Favored / Additional allowed 89.8 / 10.0 Generously allowed 0.0 ^a The numbers in parentheses are the statistics from the highest resolution shell.

After the crystallization process, XRD data were acquired to finally identify the complex structure of the Fab of the anti-CEACAM1 antibody with a resolution of 1.8 Å and the CEACAM1 N-domain. Based on the crystal structure, the Fab paratope and CEACAM1 N-domain epitope of the anti-CEACAM1 antibody that bind within an intermolecular distance of 4.5 Å were confirmed.

As a result, the heavy chain variable region of the anti-CEACAM1 antibody showed more binding to CEACAM1 than the light chain variable region. In the case of the heavy chain variable region, Asn31, Tyr32, Gly54, Gly56, Ser57, Asn74, Pro100, Thr101, Lys102, Tyr104, and Ala105 amino acids were hydrophobically or electrostatically bound to the CEACAM1 N-domain. In the case of the light chain variable region, amino acids Tyr33, Tyr50, Ala51, and Leu94 were hydrophobic or electrostatically bound to the CEACAM1 N-domain.

Based on the CEACAM1 N-domain, amino acids Phe63, Gly64, Ser66, Tyr68, Gly75, Asn76, Gln78, Ala83, Thr86, Thr90, Gln123, Ile125, Leu129, Asn131 amino acids were bound to the anti-CEACAM1 antibody. The total surface area of antigen-antibody binding was 791.98 Å ² ( FIGS. 1 and 2 ). Amino acids that play an important role in dimer formation of the previously known CEACAM1 N-domain are Phe63, Ser66, Tyr68, Gln78, Gln123, Ile125, Leu129 and Asn131, as shown in FIGS. 3 and 4 . This confirms that it can also be bound by the Fab of the anti-CEACAM1 antibody ( FIGS. 5A to 5D ). Through this, the epitope of the CEACAM1 N-domain to which the anti-CEACAM1 antibody binds overlaps the dimer interface of CEACAM1, confirming that anti-CEACAM1 can effectively inhibit the activity of CEACAM1.

Experimental Example 4. Measurement of binding affinity between anti-CEACAM1 antibody and CEACAM1

Quantitative binding ability of the anti-CEACAM1 antibody to CEACAM1 was measured using Octet QKe (Pall ForteBio). Anti-CEACAM1 antibody at 400 nM concentration was diluted 6 times to 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM and 6.25 nM antibody concentrations in Greiner 96-well-plates (Greiner). , CAT. NO. 655209) in a row, and put into the last well at a concentration of 0 nM. In another column, hCEACAM1 (R&D Systems, CAT. NO. 2244-CM) was diluted to a concentration of 6.25 μg/μl, and 200 μl of each was added in a row.

Washing buffer, neutralization buffer and baseline buffer were diluted 10-fold with Reagent/Kinetics buffer (10X) (Fortebio, CAT. NO. 18-1092), and 200 μl of each was put in a row in a row, and regeneration buffer ( regeneration buffer) was added in a row by 200 μl. Prepare a separate Greiner 96-well-plate, put 200 μl of each well in the same number of wells as the number of biosensors to be used with Reagent/Kinetics buffer (1X) in a row, and Biosensors/Anti-His(His1K)(Fortebio, CAT. NO. 18 -5120) cassette was installed. Association and dissociation times were set to 300 seconds and 600 seconds, respectively, and KD values were measured.

As a result, as shown in Table 5 below, it was confirmed to have an affinity (KD) value of 3.90 nM for CEACAM1.

K _D (M) K _on (1/Ms) K _off (1/s) 3.39 ×10 ^-9 1.63 × 10 ⁵ 5.67 ×10 ^-4

II. Confirmation of activity of anti-CEACAM1 antibody

Example 5.1. hCEACAM 1-Fc production

PCR was performed for the extracellular domain (Gln35-Gly428) of CEACAM1 from human CEACAM1 cDNA (R&D systems, RDC0951) using NotI-SS-CEACAM1 forward primer and CEACAM1-Hinge reverse primer. In this case, the primers used are shown in Table 6 below.

primer Sequence information (5'-> 3') SEQ ID NO: NotI-SS-CEACAM1 F primer GCG GCC GCC ATG TAC TTG GGA CTG AAC TAT GTA TTC ATA GTT TTT CTC TTA AAT GGT GTC CAG AGT CAG CTC ACT ACT GAA TCC ATG 21 CEACAM1-Hinge R primer GCA AGG AGG GCC GTA CTT AGA CTC CCC AGG TGA GAG GCC ATT TTC 22

The DNA obtained through the PCR was loaded on a 1% agarose gel, and DNA encoding the hCEACAM1 extracellular domain (FIG. 6 and SEQ ID NO: 23) was obtained using a DNA Gel extraction kit.

In addition, in order to obtain a gene of the human IgG4 Fc region, PCR was performed using the heavy chain DNA of the anti-CEACAM1 antibody of SEQ ID NO: 12 as a template, the forward primer hIgG4-Hinge and the reverse primer hIgG4-CH3-stop-HindIII did In this case, the primers used are shown in Table 7 below.

primer Sequence information (5'-> 3') SEQ ID NO: hIgG4-Hinge F primer GAG TCT AAG TAC GGC CCT CC T TGC 24 hIgG4-CH3-stop HindIII R primer GGA TCC AAG CTT ACT ACT TTC CCA GTG ACA GTG A 25

DNA obtained through PCR was loaded on a 1% agarose gel, and DNA encoding the hIgG4 Fc region (FIG. 7 and SEQ ID NO: 26) was obtained using a DNA Gel extraction kit. The DNA encoding the hCEACAM1 extracellular domain and the DNA encoding the hIgG4 Fc region were subjected to PCR using NotI-SS-CEACAM1 forward primer and hIgG4-CH3-stop HindIII reverse primer having a restriction enzyme cleavage site.

DNA obtained through PCR was loaded on a 1% agarose gel, and hCEACAM1-Fc DNA was obtained using a DNA Gel extraction kit. The obtained hCEACAM1-Fc DNA was treated with NotI and HindIII restriction enzymes and reacted at 37°C for 4 hours, then hCEACAM1-Fc insert DNA fragments were separated using QIAquick PCR Purification Kit. After that, In order to clone the prepared hCEACAM1-Fc insert DNA fragments into the pcIW vector, the pcIW vector was also treated with NotI and HindIII restriction enzymes to separate it into a linear pCIW vector. The hCEACAM1-Fc insert DNA fragments cut with NotI and HindIII restriction enzymes and T4 DNA ligase were added to the linear pcIW vector and reacted at 16°C for 4 hours. After 4 hours, 1 μl of the ligation mixture was taken, put into 100 μl of XL1-Blue Electroporation-Competent Cells, mixed, and then transformed using electroporation.

A single colony from the transformed plate was inoculated into SB/car medium and cultured overnight. DNA was obtained from the transformed cells using the QIAprep Spin Miniprep Kit (QIAGEN, CAT. NO. 127106), and sequencing was requested from an external company, Cosmogenetech. As a result, a gene encoding hCEACAM1-Fc represented by the nucleotide sequence of SEQ ID NO: 34 was identified. After culturing the identified transformed cells, a large amount of DNA was obtained using the QIAGEN Plasmid Plus Midi Kit (QIAGEN, CAT. NO. 12945).

Thereafter, hCEACAM1-Fc protein was produced and isolated using the transient overexpression system using Expi293F animal cells in the same manner as in Example 2.2.

Example 5.2. Confirmation of hCEACAM1-Fc and hCEACAM1-Fc interaction

The hCEACAM1-Fc prepared in Example 5.1 was diluted with PBS to 2.5 μg/ml, and then put into a NUNC immuno module (NUNC, 468667) plate by 100 μl and coated at 4° C. overnight. The next day, after washing with PBS containing 0.05% Tween20, 300 μl of PBS containing 1% BSA was added to each well and reacted at room temperature for 2 hours to block.

In addition, after washing 3 times with PBS containing 0.05% Tween20, biotin-labeled hCEACAM1-Fc was diluted 3 times from a concentration of 150 μg/ml on the hCEACAM1-Fc-coated plate, and put into 11 wells, and finally The wells were reacted at 37° C. for 1 hour by putting only PBS with 1% BSA added thereto. After 1 hour, 300 μl of PBS with 0.05% Tween20 was added to each well and washed 3 times. Then, a streptavidin-peroxidase polymer (Sigma, S2438-250UG) was added to PBS with 1% BSA. : Diluted at a ratio of 5000, put in 100 μl each, and reacted at 37° C. for 40 minutes. After the reaction was completed, 300 μl of PBS with 0.05% Tween20 was added to each well, washed 3 times, and 100 μl of TMB microwell peroxidase substrate (KPL, 50-76-03) was added and reacted for 5 minutes. To terminate the reaction, an equal amount of sulfuric acid (Sigma-aldrich, 339741) was added and the O.D value was measured at a wavelength of 450 nm using an ELISA reader.

As a result, it was confirmed that the binding depends on the hCEACAM1-Fc protein concentration. At this time, the EC50 value was 0.286 μg/ml ( FIG. 8 ).

Example 5.3. Confirmation of the inhibitory effect of the anti-CEACAM1 antibody on the hCEACAM1-hCEACAM1 interaction

The hCEACAM1-Fc prepared in Example 5.1 was diluted with PBS to 2.5 μg/ml, and then put into a NUNC immuno module (NUNC, 468667) plate by 100 μl and coated at 4° C. overnight. The next day, 300 μl of PBS with 1% BSA added to the coated plate was put into each well and reacted at room temperature for 2 hours to block (blocking).

In another 96-well-plate, dilute the anti-CEACAM1 antibody two-fold to a concentration of 60 μg/ml and put it into 11 wells. In the last well, only PBS with 1% BSA was added, and then biotin-labeled hCEACAM1-Fc was put in the same amount with the anti-CEACAM1 antibody diluted to 0.5 μg/ml, and reacted at room temperature for 1 hour. After 1 hour, 100 μl of a mixture of anti-CEACAM1 antibody and biotin-labeled hCEACAM1-Fc was added to the blocked plate, followed by reaction at room temperature for 1 hour.

After 1 hour, 300 μl of PBS with 0.05% Tween20 was added to each well and washed 3 times. Then, a streptavidin-peroxidase polymer (Sigma, S2438-250UG) was added to PBS with 1% BSA. : Diluted at a ratio of 5000, put in 100 μl each, and reacted at 37° C. for 40 minutes. After the reaction was completed, 300 μl of PBS with 0.05% Tween20 was added to each well, washed 3 times, and 100 μl of TMB microwell peroxidase substrate (KPL, 50-76-03) was added and reacted for 5 minutes. To terminate the reaction, the same amount of sulfuric acid (Sigma-aldrich, 339741) was added and the OD value was measured at a wavelength of 450 nm using an ELISA reader.

As a result, as the concentration of the anti-CEACAM1 antibody increased, it was found that the homophilic interaction of hCEACAM1-Fc was inhibited. At this time, the IC50 value was 1.055 μg/ml (FIG. 9).

Example 6.1. hCEACAM 6-Fc production

PCR was performed for the extracellular domain (Lys35-Gly320) of CEACAM6 from human CEACAM6 cDNA (R&D systems, RDC0955) using SS-hCEACAM6 forward primer and CEACAM6-Hinge reverse primer. In this case, the primers used are shown in Table 8 below.

primer Sequence information (5'-> 3') SEQ ID NO: SS_hCEACAM6_F AAT GGT GTC CAG AGT AAG CTC ACT ATT GAA TCC 27 hCEACAM6_R_hinge (HIgG1) GTC ACA AGA TTT GGG CTC TCC AGA GAC TGT GAT CAT 28

The DNA obtained through the PCR was loaded on a 1% agarose gel, and DNA encoding the hCEACAM6 extracellular domain was obtained using a DNA Gel extraction kit.

In addition, in order to obtain the human IgG1 Fc region gene, PCR was performed using a hinge (HIgG1) forward primer and a CH3 (HIgG1)_HindIII reverse primer as a template using an anti-CEACAM1 antibody-IgG1 type heavy chain vector as a template. In this case, the primers used are shown in Table 9 below.

primer Sequence information (5'-> 3') SEQ ID NO: hinge(HIgG1)_F primer GAG CCC AAA TCT TGT GAC 29 CH3(HIgG1)_HindIII_R primer TTG GAT CCA AGC TTA CTA TTT ACC CGG AGA CAG GGA 30

The DNA obtained through the PCR was loaded on a 1% agarose gel, and DNA encoding the hIgG1 Fc region was obtained using a DNA Gel extraction kit. The DNA encoding the hCEACAM6 extracellular domain and the DNA encoding the hIgG1 Fc region were subjected to PCR using a NotI-SS forward primer having a restriction enzyme cleavage site and a CH3(HIgG1)_HindIII reverse primer. In this case, the primers used are shown in Table 10 below.

primer Sequence information (5'-> 3') SEQ ID NO: NotI-SS_F primer NNN NNN GCG GCC GCC ATG TAC TTG GGA CTG AAC TAT GTA TTC ATA GTT TTT CTC TTA AAT GGT GTC CAG AGT 31 CH3(HIgG1)_HindIII_R primer TTG GAT CCA AGC TTA CTA TTT ACC CGG AGA CAG GGA 30

The DNA obtained through the PCR was loaded on a 1% agarose gel, and a DNA encoding hCEACAM6(ECD)-Fc was obtained using a DNA Gel extraction kit (FIG. 10 and SEQ ID NO: 32). The obtained hCEACAM6-Fc DNA was treated with NotI and HindIII restriction enzymes and reacted at 37° C. for 4 hours, then hCEACAM6-Fc insert DNA fragments were separated using the QIAquick PCR Purification Kit. Then, in order to clone the prepared hCEACAM6-Fc insert DNA fragments into the pcIW vector, the pcIW vector was also treated with NotI and HindIII restriction enzymes to separate it into a linear pCIW vector. The hCEACAM6-Fc insert DNA fragments cut with NotI and HindIII restriction enzymes and T4 DNA ligase were added to the linear pcIW vector and reacted at 16°C for 4 hours. After 4 hours, 1 μl of the ligation mixture was taken, put into 100 μl of XL1-Blue Electroporation-Competent Cells, mixed, and then transformed using electroporation.

A single colony from the transformed plate was inoculated into SB/car medium and cultured overnight. DNA was obtained from the transformed cells using the QIAprep Spin Miniprep Kit (QIAGEN, CAT. NO. 127106), and sequencing was requested from an external company, Cosmogenetech. As a result, a gene encoding hCEACAM6-Fc was identified. After culturing the identified transformed cells, a large amount of DNA was obtained using the QIAGEN Plasmid Plus Midi Kit (QIAGEN, CAT. NO. 12945).

Thereafter, hCEACAM6-Fc protein was produced and isolated using the transient overexpression system using Expi293F animal cells in the same manner as in Example 2.2.

Example 6.2. Confirmation of interaction between hCEACAM1-Fc and hCEACAM6-Fc

The hCEACAM6-Fc prepared in Example 6.1 was diluted with PBS to 2.5 μg/ml, and then put into a NUNC immuno module (NUNC, 468667) plate by 100 μl and coated at 4° C. overnight. The next day, 300 μl of PBS with 1% BSA added to the coated plate was put into each well and reacted at room temperature for 2 hours to block (blocking).

In addition, biotin-labeled hCEACAM1-Fc was diluted 3-fold from a concentration of 150 μg/ml on the hCEACAM6-Fc-coated plate and put into 11 wells, and in the last well, only PBS with 1% BSA was added for 1 hour. The reaction was carried out at a temperature of 37°C. After 1 hour, 300 μl of PBS with 0.05% Tween20 was added to each well and washed 3 times. Then, a streptavidin-peroxidase polymer (Sigma, S2438-250UG) was added to PBS with 1% BSA. : Diluted at a ratio of 5000, put in 100 μl each, and reacted at 37° C. for 40 minutes. After the reaction was completed, 300 μl of PBS with 0.05% Tween20 was added to each well, washed 3 times, and 100 μl of TMB microwell peroxidase substrate (KPL, 50-76-03) was added and reacted for 5 minutes. To terminate the reaction, an equal amount of sulfuric acid (Sigma-aldrich, 339741) was added and the O.D value was measured at a wavelength of 450 nm using an ELISA reader.

As a result, it was confirmed that as the concentration of hCEACAM1-Fc protein increased, it was associated with hCEACAM6-Fc. At this time, the EC50 value was 0.305 μg/ml ( FIG. 11 ).

Example 6.3. Confirmation of the inhibitory effect of the anti-CEACAM1 antibody on the hCEACAM1-hCEACAM1 interaction

The hCEACAM6-Fc prepared in Example 6.1 was diluted with PBS to 2.5 μg/ml, and then put into a NUNC immuno module (NUNC, 468667) plate by 100 μl and coated at 4° C. overnight. The next day, 300 μl of PBS with 1% BSA added to the coated plate was put into each well and reacted at room temperature for 2 hours to block (blocking).

In another 96-well-plate, dilute the anti-CEACAM1 antibody two-fold to a concentration of 60 μg/ml and put it into 11 wells. In the last well, only PBS with 1% BSA was added, and then biotin-labeled hCEACAM1-Fc was put in the same amount with the anti-CEACAM1 antibody diluted to 0.5 μg/ml, and reacted at room temperature for 1 hour. After 1 hour, 100 μl of a mixture of anti-CEACAM1 antibody and biotin-labeled hCEACAM1-Fc was added to the blocked plate, followed by reaction at room temperature for 1 hour.

After 1 hour, 300 μl of PBS with 0.05% Tween20 was added to each well and washed 3 times. Then, a streptavidin-peroxidase polymer (Sigma, S2438-250UG) was added to PBS with 1% BSA. : Diluted at a ratio of 5000, put in 100 μl each, and reacted at 37° C. for 40 minutes. After the reaction was completed, 300 μl of PBS with 0.05% Tween20 was added to each well, washed 3 times, and 100 μl of TMB microwell peroxidase substrate (KPL, 50-76-03) was added and reacted for 5 minutes. To terminate the reaction, an equal amount of sulfuric acid (Sigma-aldrich, 339741) was added and the O.D value was measured at a wavelength of 450 nm using an ELISA reader.

As a result, it was shown that the binding between hCEACAM1-Fc and hCEACAM6-Fc was inhibited as the concentration of the anti-CEACAM1 antibody increased. At this time, the IC50 value was 0.795 μg/ml (FIG. 12).

III. Confirmation of TCR signaling activation effect of anti-CEACAM1 antibody

To check whether the anti-CEACAM1 antibody activates TCR signaling by blocking the inhibition of T cell activation due to the CEACAM1-CAECAM1 interaction, phosphorylation of ZAP70, one of the TCR signaling pathways, and NFAT (nuclear factor) of activated T-cells), it was confirmed whether the expression levels of transcription factors and IL-2 were increased.

Example 7.1. Confirmation of ZAP70 phosphorylation effect

First, in order to construct Jurkat cells overexpressing CEACAM1, a CEACAM1 coding nucleotide sequence was inserted into pEF1α-AcGFP-N1 plasmid (Clontech, Cat. No. 631973) using restriction enzymes to insert pEF1α-AcGFP-N1-CCM1. A plasmid was constructed.

Specifically, the pEF1α-AcGFP-N1-CCM1 plasmid was PCR from human CEACAM1 cDNA (R&D, CAT. NO. RDC0951) using a CEACAM1 forward primer with a HindIII restriction enzyme cleavage site and a reverse primer with a SalI restriction enzyme cleavage site. was performed. In this case, the primers used are shown in Table 11 below.

primer Sequence information (5'-> 3') SEQ ID NO: CEACAM1_HindIII_F primer GAC AAG CTT ATG GGG CAC CTC TCA GCC C 35 CEACAM1_SalI_R primer GAC GTC GAC GTC TGC TTT TTT ACT TCT GAA TAA 36

The DNA obtained through the PCR was loaded on a 0.8% agarose gel, and HindIII/SalI hCEACAM1 DNA was obtained using a DNA gel extraction kit (Promega, CAT. NO. A9282). The obtained hCEACAM1 DNA was treated with HindIII and SalI restriction enzymes and reacted at 37°C for 2 hours, then hCEACAM1 insert DNA fragments were isolated using Gel and PCR clean up system (Promega, CAT. NO. A9282). Then, to clone the prepared hCEACAM1 insert DNA fragments into the pEF1α-AcGFP-N1 vector (Clontech, Cat No. 631973), the pEF1α-AcGFP-N1 vector was also treated with HindIII and SalI restriction enzymes to obtain a linear pEF1α-AcGFP-N1 vector. separated into The hCEACAM1 insert DNA fragments cut with HindIII and SalI restriction enzymes and T4 DNA ligase were added to the linear pEF1α-AcGFP-N1 vector, and reacted at 16°C for 4 hours. After 4 hours, 1 μl of the ligation mixture was taken, and 100 μl of the DH-5α E. coli strain was transformed into competent cells. A single colony was inoculated into the medium from the transformed plate and cultured overnight. DNA was obtained from the transformed cells using DNA plasmid SV (Geneall, CAT. NO. 101-102), and sequencing was requested from an external company, Cosmogenetech.

As a result, a gene encoding hCEACAM1 represented by the nucleotide sequence of SEQ ID NO: 37 was identified. After culturing the identified transformed cells, a large amount of DNA was obtained using the QIAGEN Plasmid Plus Midi Kit (QIAGEN, CAT. NO. 12945).

Thereafter, 10 μg of pEF1α-AcGFP-N1-CCM1 plasmid was transfected into 3×10 ⁶ Jurkat E6.1 cells (ATCC) using the Neon transfection system (1400 voltage / 20 ms / 2 pulses). . After 72 hours, the transfected Jurkat cells were harvested and the GFP-expressing Jurkat cells were sorted using flow cytometry (FACSAria). Sorted Jurkat cells were cultured in a culture medium containing 1 mg/ml of G418 (Sigma, Cat. No. G8168). At this time, as the culture medium, cIMDM (complete IMDM) medium containing Pen/Strep (1x), NEAA (1x), sodium pyruvate (1x) and 10% FBS was used.

Jurkat cells and Jurkat cells overexpressing CEACAM1 were ^{aliquoted as much as 3×10 6} cells per well, treated with 10 μg/ml hIgG4 (Sigma, Cat. No. I4639) or anti-CEACAM1 antibody, and then for 5 hours It was cultured at a temperature of 37° C. and 5% CO _{2 conditions.} Thereafter, some of the cultured Jurkat cells and Jurkat cells overexpressing CEACAM1 were mixed with an anti-CD3 antibody (eBioscience, Cat. No. 16-0037-85) and an anti-CD28 antibody (eBioscience, Cat. No. 16-0289-85) and stimulated for 1 min.

Each Jurkat cell and the Jurkat cell overexpressing CEACAM1 were lysed using RIPA buffer (ice-cold lysis buffer). The cell lysate was then centrifuged, mixed with 6X Laemmli buffer, and the mixture was loaded onto a Novex 4-12% Bis-Tris gradient gel using MES running buffer to transfer the proteins in the mixture onto a nitrocellulose membrane. . Thereafter, non-specific reactions were blocked by treatment with 5% bovine serum albumin (BSA), and western blotting was performed using a primary antibody and a secondary antibody conjugated with HRP. At this time, the primary antibody used was anti-phosphorylation-ZAP70 (Y319, Cell signaling, Cat. No. 2717S), anti-ZAP70 antibody (Cell signaling, Cat. No. 2705S), anti-CEACAM1 antibody (ORIGENE, Cat. No. TA350817), an anti-Actin antibody (Cell signaling, Cat. No. 49671) was used.

At this time, Jurkat cells that did not express CEACAM1 that were not stimulated with anti-CD3 antibody and anti-CD28 antibody were set as a negative control, stimulated with anti-CD3 antibody and anti-CD28 antibody, and treated with hIgG4 did not express CEACAM1. Jurkat cells, which were not present, were set as positive controls. In addition, Jurkat cells overexpressing CEACAM1 stimulated with anti-CD3 antibody and anti-CD28 antibody and treated with hIgG4 or anti-CEACAM1 antibody were set as the experimental group.

As a result, as shown in FIG. 13 , in the case of the negative control group, it was confirmed that ZAP70 was not phosphorylated, and in the case of the positive control group, it was confirmed that ZAP70 was phosphorylated (p-ZAP70). In addition, in the experimental group treated with hIgG4, the amount of phosphorylated ZAP70 was smaller than that of the positive control group. On the other hand, in the case of the experimental group treated with the anti-CEACAM1 antibody, a degree of phosphorylated ZAP70 similar to that of the positive control was measured.

Example 7.2. Confirmation of increased expression of NFAT transcription factor

To construct Jurkat cells overexpressing NFAT and CEACAM1 ^{, 10 μg of the CEACAM1 overexpressing Jurkat cells prepared in Example 7.1 with 3×10 6} cell numbers were pGL4.30 [luc2p / NFAT-RE / Hygro] and 1 ㎍ of pTurbo RFP-C plasmid was transfected using the Neon transfection system (1400 voltage / 20 ms / 2 pulses). After 72 hours, the transfected Jurkat cells were harvested and RFP-expressing Jurkat cells were sorted using flow cytometry (FACSAria). Sorted Jurkat cells were cultured in culture medium containing 1 mg/ml of G418 (Sigma, Cat. No. G8168) and 0.5 mg/ml of Hygromycin B (Invitrogen, Cat No. 10687010). At this time, the same culture medium as used in Example 7.1 was used.

Jurkat cells overexpressing NFAT and CEACAM1 were seeded in each well by 3×10 ⁶ cells, treated with hIgG4 (Sigma, Cat. No. I4639) or anti-CEACAM1 antibody, and then 0.05 μg/ml of anti-CD3 antibody (eBioscience, Cat. No.16-0037-85). At this time, the hIgG4 (Sigma, Cat. No. I4639) or anti-CEACAM1 antibody was diluted three-fold starting with a concentration of 30 μg/ml and used. After 24 hours, 100 μl of the supernatant was transferred to a 96-well-plate, and 100 μl of the Bright-Glo luciferase assay system (Promega, Cat. No. E2610) was treated. Thereafter, fluorescence was measured using a GloMax Discover GloMax Discover multimode microplate reader.

At this time, NFAT and CEACAM1 overexpressing Jurkat cells stimulated with anti-CD3 antibody and treated with hIgG4 were set as a control group, and NFAT and CEACAM1 overexpressing Jurkat cells treated with anti-CEACAM1 antibody were set as an experimental group.

As a result, as shown in FIG. 14 , the fluorescence intensity increased in a concentration-dependent manner in the experimental group, and through this, it was confirmed that the expression level of NFAT increased.

Example 7.3. Confirmation of increase in IL-2 expression level

First, 96-well-plates were coated with 1 μg/ml of anti-CD3 antibody (OKT-3) at 4° C. overnight. Each well was washed twice with cold DPBS before adding cells. ^{Thereafter, Jurkat cells overexpressing CEACAM1 in the number of 1×10 5} cells prepared in Example 7.1 were dispensed into each well, and 200 μl of an anti-CEACAM1 antibody at a concentration of 10 μg/ml was added thereto for 3 days. cultured. Then, the supernatant was collected, and the expression level of IL-2 was measured using an IL-2 ELISA kit (BD Bioscience, Cat#: 550611).

At this time, Jurkat cells overexpressing CEACAM1 stimulated with anti-CD3 antibody and treated with hIgG4 were set as a control group, and Jurkat cells overexpressing CEACAM1 treated with anti-CEACAM1 antibody were set as an experimental group.

As a result, as shown in FIG. 15 , it was confirmed that the IL-2 expression level of the experimental group was significantly increased compared to the control group.

<110> Green Cross Corporation Mogam Institute for Biomedical Research <120> CONFORMATIONAL EPITOPE OF CEACAM1 AND ANTI-CEACAM1 ANTIBODY OR FRAGMENT THEREOF SPECIFICALLY BINDING THERETO <130> FPD/202111-0089 <160> 37 <170> KoPatentIn 3.0 <210> 1 <211> 526 <212> PRT <213> Homo sapiens <400> 1 Met Gly His Leu Ser Ala Pro Leu His Arg Val Arg Val Pro Trp Gln 1 5 10 15 Gly Leu Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr 20 25 30 Thr Ala Gln Leu Thr Thr Glu Ser Met Pro Phe Asn Val Ala Glu Gly 35 40 45 Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln Gln Leu Phe Gly 50 55 60 Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Val 65 70 75 80 Gly Tyr Ala Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Asn Ser 85 90 95 Gly Arg Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val 100 105 110 Thr Gln Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp 115 120 125 Leu Val Asn Glu Glu Ala Thr Gly Gin Phe His Val Tyr Pro Glu Leu 130 135 140 Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Lys 145 150 155 160 Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Thr Thr Tyr 165 170 175 Leu Trp Trp Ile Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln 180 185 190 Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Leu Ser Val Thr Arg Asn 195 200 205 Asp Thr Gly Pro Tyr Glu Cys Glu Ile Gln Asn Pro Val Ser Ala Asn 210 215 220 Arg Ser Asp Pro Val Thr Leu Asn Val Thr Tyr Gly Pro Asp Thr Pro 225 230 235 240 Thr Ile Ser Pro Ser Asp Thr Tyr Tyr Arg Pro Gly Ala Asn Leu Ser 245 250 255 Leu Ser Cys Tyr Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Leu 260 265 270 Ile Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn 275 280 285 Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys His Ala Asn Asn Ser 290 295 300 Val Thr Gly Cys Asn Arg Thr Thr Val Lys Thr Ile Ile Val Thr Glu 305 310 315 320 Leu Ser Pro Val Val Ala Lys Pro Gln Ile Lys Ala Ser Lys Thr Thr 325 330 335 Val Thr Gly Asp Lys Asp Ser Val Asn Leu Thr Cys Ser Thr Asn Asp 340 345 350 Thr Gly Ile Ser Ile Arg Trp Phe Phe Lys Asn Gln Ser Leu Pro Ser 355 360 365 Ser Glu Arg Met Lys Leu Ser Gln Gly Asn Thr Thr Leu Ser Ile Asn 370 375 380 Pro Val Lys Arg Glu Asp Ala Gly Thr Tyr Trp Cys Glu Val Phe Asn 385 390 395 400 Pro Ile Ser Lys Asn Gln Ser Asp Pro Ile Met Leu Asn Val Asn Tyr 405 410 415 Asn Ala Leu Pro Gln Glu Asn Gly Leu Ser Pro Gly Ala Ile Ala Gly 420 425 430 Ile Val Ile Gly Val Val Ala Leu Val Ala Leu Ile Ala Val Ala Leu 435 440 445 Ala Cys Phe Leu His Phe Gly Lys Thr Gly Arg Ala Ser Asp Gln Arg 450 455 460 Asp Leu Thr Glu His Lys Pro Ser Val Ser Asn His Thr Gln Asp His 465 470 475 480 Ser Asn Asp Pro Pro Asn Lys Met Asn Glu Val Thr Tyr Ser Thr Leu 485 490 495 Asn Phe Glu Ala Gln Gln Pro Thr Gln Pro Thr Ser Ala Ser Pro Ser 500 505 510 Leu Thr Ala Thr Glu Ile Ile Tyr Ser Glu Val Lys Lys Gln 515 520 525 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence for anti-CEACAM1 antibody of CDR1 (VH) <400> 2 Gly Phe Thr Phe Ser Asn Tyr Ala 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence for anti-CEACAM1 antibody of CDR2 (VH) <400> 3 Ile Ser His Gly Gly Gly Ser Ile 1 5 <210> 4 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence for anti-CEACAM1 antibody of CDR3 (VH) <400> 4 Ala Arg Asp Pro Thr Lys Gly Tyr Ala Pro Thr Phe Asp Tyr 1 5 10 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence for anti-CEACAM1 antibody of CDR1 (VL) <400> 5 Ser Ser Asn Ile Gly Asn Asn Tyr 1 5 <210> 6 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence for anti-CEACAM1 antibody of CDR2 (VL) <400> 6 Ala Asp Ser Arg Arg Pro 1 5 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence for anti-CEACAM1 antibody of CDR3 (VL) <400> 7 Gly Ala Trp Asp Leu Ser Leu Asn Gly Tyr Val 1 5 10 <210> 8 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence for anti-CEACAM1 antibody of variable heavy region <400> 8 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser His Gly Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Thr Lys Gly Tyr Ala Pro Thr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 9 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence for anti-CEACAM1 antibody of variable light region <400> 9 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asp Ser Arg Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Leu Ser Leu 85 90 95 Asn Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 10 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence for anti-CEACAM1 antibody of variable heavy region <400> 10 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc aattatgcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagtg atctctcatg gtggtggtag tatatattac 180 gctgattctg taaaaggtcg gctcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagatcct 300 actaaggggt atgctcctac tttcgactac tggggccagg gtacactggt caccgtgagc 360 tca 363 <210> 11 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence for anti-CEACAM1 antibody of variable light region <400> 11 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtagtg gctcttcatc caatattggc aataattatg tctcctggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat gctgatagta ggcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtggt gcttgggatc tgagcctgaa tggttatgtc 300 ttcggcggag gcaccaagct gaccgtccta ggt 333 <210> 12 <211> 1401 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence for anti-CEACAM1 antibody of heavy chain(IgG4) <400> 12 atgtacttgg gactgaacta tgtattcata gtttttctct taaatggtgt ccagagtgag 60 gtgcagctgt tggagtctgg gggaggcttg gtacagcctg gggggtccct gagactctcc 120 tgtgcagcct ctggattcac ctttagcaat tatgctatga gctgggtccg ccaggctcca 180 gggaaggggc tggagtgggt ctcagtgatc tctcatggtg gtggtagtat atattacgct 240 gattctgtaa aaggtcggct caccatctcc agagacaatt ccaagaacac gctgtatctg 300 caaatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgag agatcctact 360 aaggggtatg ctcctacttt cgactactgg ggccagggta cactggtcac cgtgagctca 420 gccagcacca agggaccctc cgtgttccca ctggcaccct gctcccggag caccagcgag 480 agcaccgccg ccctgggatg tctggtgaaa gactacttcc ctgaaccagt caccgtgtcc 540 tggaactctg gcgcactgac ttccggagtc cacacctttc ccgccgtgct ccagagcagc 600 ggactgtact ctctgtcaag cgtggtcaca gtcccctcct ctagtctggg aacaaagact 660 tatacctgca acgtggatca taaaccttcc aatactaagg tggacaaaag agtggagtct 720 aagtacggcc ctccttgccc accatgtcca gcacctgaat ttctgggcgg accctccgtg 780 ttcctgtttc ctccaaagcc taaagataca ctgatgatct cccgcacccc tgaggtcaca 840 tgcgtggtcg tggacgtgag ccaggaggac ccagaagtcc agttcaactg gtatgtggac 900 ggcgtcgaag tgcacaatgc taagaccaaa cctagggagg aacagtttaa ctctacttac 960 agagtcgtga gtgtcctgac cgtgctgcat caggattggc tgaacggcaa ggagtataag 1020 tgcaaagtga gcaataaggg actgccatca agcatcgaga aaaccattag caaggcaaaa 1080 ggccagcctc gcgaaccaca ggtgtacaca ctgcccccta gtcaggagga aatgaccaag 1140 aaccaggtca gcctgacatg tctggtgaaa gggttctatc catcagatat tgctgtggag 1200 tgggaaagca atggtcagcc cgagaacaat tacaagacta ccccacccgt gctggacagt 1260 gatgggtcat tctttctgta ttctcgactg accgtggaca aaagtcggtg gcaggagggt 1320 aatgtctttt catgcagcgt gatgcacgag gcactgcaca accattacac tcagaagtca 1380 ctgtcactgt cactgggaaa g 1401 <210> 13 <211> 467 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence for anti-CEACAM1 antibody of heavy chain(IgG4) <400> 13 Met Tyr Leu Gly Leu Asn Tyr Val Phe Ile Val Phe Leu Leu Asn Gly 1 5 10 15 Val Gln Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Asn Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Val Ile Ser His Gly Gly Gly Ser Ile Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Pro Thr Lys Gly Tyr Ala Pro Thr Phe Asp 115 120 125 Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu 145 150 155 160 Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn 210 215 220 Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser 225 230 235 240 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 275 280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Leu Gly Lys 465 <210> 14 <211> 1059 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence for CH1-hinge-CH2-CH3 (hIgG1) <400> 14 gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgaaccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact tccttggggg 360 gaccgtcaat tcttcctctt ccccccaaaa cccaaggaac cccctcttga tgaactcctt 420 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 480 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 540 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 600 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 660 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 720 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 780 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 840 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 900 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 960 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1020 tacaccgcaga agagcctctc cctgtctccg ggtaaatag 1059 <210> 15 <211> 352 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence for CH1-hinge-CH2-CH3 (hIgG1) <400> 15 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Pro Trp Gly Asp Arg Gln Phe Phe Leu Phe Pro 115 120 125 Pro Lys Pro Lys Glu Pro Pro Leu Asp Glu Leu Leu Gly Gly Pro Ser 130 135 140 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 145 150 155 160 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 165 170 175 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 180 185 190 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 195 200 205 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 210 215 220 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 225 230 235 240 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 245 250 255 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 260 265 270 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 275 280 285 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 290 295 300 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 305 310 315 320 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 325 330 335 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 <210> 16 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for VH-NotI-L <400> 16 gcggccgcca tgtacttggg actgaactat gtattcatag tttttctctt aaatggtgtc 60 cagagt 66 <210> 17 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for VH-ApaI-CH1 <400> 17 atgggccctt ggtggaggct gaggagacgg tgac 34 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for CH1-Fc_ApaI <400> 18 gcctccacca agggccca 18 <210> 19 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for IgG1_Fc_HindIII <400> 19 nnnnggatcc aagcttacta tttacccgga 30 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for IgG1_HC_NotI <400> 20 gcggccgcca tgtacttgg 19 <210> 21 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for NotI-SS-CEACAM1 <400> 21 gcggccgcca tgtacttggg actgaactat gtattcatag tttttctctt aaatggtgtc 60 cagagtcagc tcactactga atccatg 87 <210> 22 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for CEACAM1-Hinge <400> 22 gcaaggaggg ccgtacttag actccccagg tgagaggcca ttttc 45 <210> 23 <211> 1182 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence for human CEACAM1 extracellular domain <400> 23 cagctcacta ctgaatccat gccattcaat gttgcagagg ggaaggaggt tcttctcctt 60 gtccacaatc tgccccagca actttttggc tacagctggt acaaagggga aagagtggat 120 ggcaaccgtc aaattgtagg atatgcaata ggaactcaac aagctacccc agggcccgca 180 aacagcggtc gagagacaat ataccccaat gcatccctgc tgatccagaa cgtcacccag 240 aatgacacag gattctacac cctacaagtc ataaagtcag atcttgtgaa tgaagaagca 300 actggacagt tccatgtata cccggagctg cccaagccct ccatctccag caacaactcc 360 aaccctgtgg aggacaagga tgctgtggcc ttcacctgtg aacctgagac tcaggacaca 420 acctacctgt ggtggataaa caatcagagc ctcccggtca gtcccaggct gcagctgtcc 480 aatggcaaca ggaccctcac tctactcagt gtcacaagga atgacacagg accctatgag 540 tgtgaaatac agaacccagt gagtgcgaac cgcagtgacc cagtcacctt gaatgtcacc 600 tatggcccgg acacccccac catttcccct tcagacacct attaccgtcc aggggcaaac 660 ctcagcctct cctgctatgc agcctctaac ccacctgcac agtactcctg gcttatcaat 720 ggaacattcc agcaaagcac acaagagctc tttatcccta acatcactgt gaataatagt 780 ggatcctata cctgccacgc caataactca gtcactggct gcaacaggac cacagtcaag 840 acgatcatag tcactgagct aagtccagta gtagcaaagc cccaaatcaa agccagcaag 900 accacagtca caggagataa ggactctgtg aacctgacct gctccacaaa tgacactgga 960 atctccatcc gttggttctt caaaaaccag agtctcccgt cctcggagag gatgaagctg 1020 tcccagggca acaccaccct cagcataaac cctgtcaaga gggaggatgc tgggacgtat 1080 tggtgtgagg tcttcaaccc aatcagtaag aaccaaagcg accccatcat gctgaacgta 1140 aactataatg ctctaccaca agaaaatggc ctctcacctg gg 1182 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for hIgG4-Hinge <400> 24 gagtctaagt acggccctcc ttgc 24 <210> 25 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for hIgG4-CH3-stop HindIII <400> 25 ggatccaagc ttactacttt cccagtgaca gtga 34 <210> 26 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence for Human Ig gamma-4 chain C region (codon optimization in Ab team) <400> 26 gagtctaagt acggccctcc ttgcccacca tgtccagcac ctgaatttct gggcggaccc 60 tccgtgttcc tgtttcctcc aaagcctaaa gatacactga tgatctcccg cacccctgag 120 gtcacatgcg tggtcgtgga cgtgagccag gaggacccag aagtccagtt caactggtat 180 gtggacggcg tcgaagtgca caatgctaag accaaaccta gggaggaaca gtttaactct 240 acttacagag tcgtgagtgt cctgaccgtg ctgcatcagg attggctgaa cggcaaggag 300 tataagtgca aagtgagcaa taagggactg ccatcaagca tcgagaaaac cattagcaag 360 gcaaaaggcc agcctcgcga accacaggtg tacacactgc cccctagtca ggaggaaatg 420 accaagaacc aggtcagcct gacatgtctg gtgaaagggt tctatccatc agatattgct 480 gtggagtggg aaagcaatgg tcagcccgag aacaattaca agactacccc acccgtgctg 540 gacagtgatg ggtcattctt tctgtattct cgactgaccg tggacaaaag tcggtggcag 600 gagggtaatg tcttttcatg cagcgtgatg cacgaggcac tgcacaacca ttacactcag 660 aagtcactgt cactgtcact gggaaagtag taaaagctt 699 <210> 27 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for SS_hCEACAM6 <400> 27 aatggtgtcc agagtaagct cactattgaa tcc 33 <210> 28 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for hCEACAM6_R_hinge (HIgG1) <400> 28 gtcacaagat ttgggctctc cagagactgt gatcat 36 <210> 29 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for hinge (HIgG1) <400> 29 gagcccaaat cttgtgac 18 <210> 30 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for CH3(HIgG1)_HindIII <400> 30 ttggatccaa gcttactatt tacccggaga caggga 36 <210> 31 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for NotI-SS <400> 31 nnnnnngcgg ccgccatgta cttgggactg aactatgtat tcatagtttt tctcttaaat 60 ggtgtccaga gt 72 <210> 32 <211> 1556 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence for hCEACAM6(ECD)-Fc <400> 32 aagctcacta ttgaatccac gccattcaat gtcgcagagg ggaaggaggt tcttctactc 60 gcccacaacc tgccccagaa tcgtattggt tacagctggt acaaaggcga aagagtggat 120 ggcaacagtc taattgtagg atatgtaata ggaactcaac aagctacccc agggcccgca 180 tacagtggtc gagagacaat ataccccaat gcatccctgc tgatccagaa cgtcacccag 240 aatgacacag gattctatac cctacaagtc ataaagtcag atcttgtgaa tgaagaagca 300 accggacagt tccatgtata cccggagctg cccaagccct ccatctccag caacaactcc 360 accccgtgga ggacaaggat gctgtggcct tcacctgtga acctgaggtt cagaacacaa 420 cctacctgtg gtgggtaaat ggtcagagcc tcccggtcag tcccaggctg cagctgtcca 480 atggcaacat gaccctcact ctactcagcg tcaaaaggaa cgatgcagga tcctatgaat 540 gtgaaataca gaacccagcg agtgccaacc gcagtgaccc agtcaccctg aatgtcctct 600 atggcccaga tgtccccacc atttccccct caaaggccaa ttaccgtcca ggggaaaatc 660 tgaacctctc ctgccacgca gcctctaacc cacctgcaca gtactcttgg tttatcaatg 720 ggacgttcca gcaatccaca caagagctct ttatccccaa catcactgtg aataatagcg 780 gatcctatat gtgccaagcc cataactcag ccactggcct caataggacc acagtcacga 840 tgatcacagt ctctggagag cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc 900 cagcacctga actccttggg ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca 960 ccctcatgat ctccccggacc cctgaggtca catgcgtggt ggtggacgtg agccacgaag 1020 accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat gccaagacaa 1080 agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc accgtcctgc 1140 accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa gccctcccag 1200 cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca caggtgtaca 1260 ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc tgcctggtca 1320 aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag ccggagaaca 1380 actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc tacagcaagc 1440 tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg 1500 aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt aaatag 1556 <210> 33 <211> 1419 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence for anti-CEACAM1 antibody HC_IgG1 type <400> 33 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc aattatgcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagtg atctctcatg gtggtggtag tatatattac 180 gctgattctg taaaaggtcg gctcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagatcct 300 actaaggggt atgctcctac tttcgactac tggggccagg gtacactggt caccgtgagc 360 tcagcctcca ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct 420 gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480 tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 540 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 600 acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgaa 660 cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga acttccttgg 720 ggggaccgtc aattcttcct cttcccccca aaacccaagg aaccccctct tgatgaactc 780 cttgggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 840 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 900 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 960 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 1020 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1080 accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1140 cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1200 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1260 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1320 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1380 cactacacgc agaagagcct ctccctgtct ccgggtaaa 1419 <210> 34 <211> 1929 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence for pCIW_HC_CCM1-FcIgG4 <400> 34 atgtacttgg gactgaacta tgtattcata gtttttctct taaatggtgt ccagagtcag 60 ctcactactg aatccatgcc attcaatgtt gcagagggga aggaggttct tctccttgtc 120 cacaatctgc cccagcaact ttttggctac agctggtaca aaggggaaag agtggatggc 180 aaccgtcaaa ttgtaggata tgcaatagga actcaacaag ctaccccagg gcccgcaaac 240 agcggtcgag agacaatata ccccaatgca tccctgctga tccagaacgt cacccagaat 300 gacacaggat tctacaccct acaagtcata aagtcagatc ttgtgaatga agaagcaact 360 ggacagttcc atgtataccc ggagctgccc aagccctcca tctccagcaa caactccaac 420 cctgtggagg acaaggatgc tgtggccttc acctgtgaac ctgagactca ggacacaacc 480 tacctgtggt ggataaacaa tcagagcctc ccggtcagtc ccaggctgca gctgtccaat 540 ggcaacagga ccctcactct actcagtgtc acaaggaatg acacaggacc ctatgagtgt 600 gaaatacaga acccagtgag tgcgaaccgc agtgacccag tcaccttgaa tgtcacctat 660 ggcccggaca cccccaccat ttccccttca gacacctatt accgtccagg ggcaaacctc 720 agcctctcct gctatgcagc ctctaaccca cctgcacagt actcctggct tatcaatgga 780 acattccagc aaagcacaca agagctcttt atccctaaca tcactgtgaa taatagtgga 840 tcctatacct gccacgccaa taactcagtc actggctgca acaggaccac agtcaagacg 900 atcatagtca ctgagctaag tccagtagta gcaaagcccc aaatcaaagc cagcaagacc 960 acagtcacag gagataagga ctctgtgaac ctgacctgct ccacaaatga cactggaatc 1020 tccatccgtt ggttcttcaa aaaccagagt ctcccgtcct cggagaggat gaagctgtcc 1080 cagggcaaca ccaccctcag cataaaccct gtcaagaggg aggatgctgg gacgtattgg 1140 tgtgaggtct tcaacccaat cagtaagaac caaagcgacc ccatcatgct gaacgtaaac 1200 tataatgctc taccacaaga aaatggcctc tcacctgggg agtctaagta cggccctcct 1260 tgcccaccat gtccagcacc tgaatttctg ggcggaccct ccgtgttcct gtttcctcca 1320 aagcctaaag atacactgat gatctcccgc acccctgagg tcacatgcgt ggtcgtggac 1380 gtgagccagg aggacccaga agtccagttc aactggtatg tggacggcgt cgaagtgcac 1440 aatgctaaga ccaaacctag ggaggaacag tttaactcta cttacagagt cgtgagtgtc 1500 ctgaccgtgc tgcatcagga ttggctgaac ggcaaggagt ataagtgcaa agtgagcaat 1560 aagggactgc catcaagcat cgagaaaacc attagcaagg caaaaggcca gcctcgcgaa 1620 ccacaggtgt acacactgcc ccctagtcag gaggaaatga ccaagaacca ggtcagcctg 1680 acatgtctgg tgaaagggtt ctatccatca gatattgctg tggagtggga aagcaatggt 1740 cagcccgaga acaattacaa gactacccca cccgtgctgg acagtgatgg gtcattcttt 1800 ctgtattctc gactgaccgt ggacaaaagt cggtggcagg agggtaatgt cttttcatgc 1860 agcgtgatgc acgaggcact gcacaaccat tacactcaga agtcactgtc actgtcactg 1920 ggaaagtag 1929 <210> 35 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for CEACAM1_HindIII <400> 35 gacaagctta tggggcacct ctcagccc 28 <210> 36 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for CEACAM1_SalI <400> 36 gacgtcgacg tctgcttttt tacttctgaa taa 33 <210> 37 <211> 1562 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence for human CEACAM1 <400> 37 atggggcacc tctcagcccc acttcacaga gtgcgtgtac cctggcaggg gcttctgctc 60 acagcctcac ttctaacctt ctggaacccg cccaccactg cccagctcac tactgaatcc 120 atgccattca atgttgcaga ggggaaggag gttcttctcc ttgtccacaa tctgccccag 180 caactttttg gctacagctg gtacaaaggg gaaagagtgg atggcaaccg tcaaattgta 240 ggatatgcaa taggaactca acaagctacc ccagggcccg caaacagcgg tcgagagaca 300 atatacccca atgcatccct gctgatccag aacgtcaccc agaatgacac aggattctac 360 accctacaag tcataaagtc agatcttgtg aatgaagaag caactggaca gttccatgta 420 tacccggagc tgcccaagcc ctccatctcc agcaacaact ccaaccctgt ggaggacaag 480 gatgctgtgg ccttcacctg tgaacctgag actcaggaca caacctacct gtggtggata 540 aacaatcaga gcctcccggt cagtcccagg ctgcagctgt ccaatggcaa caggaccctc 600 actctactca gtgtcacaag gaatgacaca ggaccctatg agtgtgaaat acagaaccca 660 gtgagtgcga accgcagtga cccagtcacc ttgaatgtca cctatggccc ggacaccccc 720 accatttccc cttcagacac ctattaccgt ccaggggcaa acctcagcct ctcctgctat 780 gcagcctcta acccacctgc acagtactcc tggcttatca atggaacatt ccagcaaagc 840 acacaagagc tctttatccc taacatcact gtgaataata gtggatccta tacctgccac 900 gccaataact cagtcactgg ctgcaacagg accacagtca agacgatcat agtcactgag 960 ctaagtccag tagtagcaaa gccccaaatc aaagccagca agaccacagt cacaggagat 1020 aaggactctg tgaacctgac ctgctccaca aatgacactg gaatctccat ccgttggttc 1080 ttcaaaaacc agagtctccc gtcctcggag aggatgaagc tgtcccaggg caacaccacc 1140 ctcagcataa accctgtcaa gagggaggat gctgggacgt attggtgtga ggtcttcaac 1200 ccaatcagta agaaccaaag cgaccccatc atgctgaacg taaactataa tgctctacca 1260 caagaaaatg gcctctcacc tggggccatt gctggcattg tgattggagt agtggccctg 1320 gttgctctga tagcagtagc cctggcatgt tttctgcatt tcgggaagac cggcagggca 1380 agcgaccagc gtgatctcac agagcacaaa ccctcagtct ccaaccacac tcaggaccac 1440 tccaatgacc cacctaacaa gatgaatgaa gttacttatt ctaccctgaa ctttgaagcc 1500 cagcaaccca cacaaccaac ttcagcctcc ccatccctaa cagccacaga aataatttat 1560 aa 1562

Claims (14)

It consists of 4 to 69 amino acids in the amino acid sequence corresponding to positions 35 to 141 of CEACAM1, and is composed of amino acids 63, 64, 66, 68, 75, 76, 78, 83, 86, 90, 123, 125, 129, 131 th A stereoscopic epitope comprising any one amino acid selected from the group consisting of amino acids at positions and combinations thereof. The method of claim 1,
The CEACAM1 is a stereoscopic epitope consisting of the amino acid sequence of SEQ ID NO: 1. The method of claim 1,
The 63rd amino acid is phenylalanine (Phe), the 64th amino acid is glycine (Gly), the 66th amino acid is serine (Ser), the 68th amino acid is tyrosine (tyrosine, Tyr), 75 The th amino acid is glycine, the 76th amino acid is asparagine (Asn), the 78th amino acid is glutamine (Gln), the 83th amino acid is alanine (Ala), and the 86th amino acid is threonine (threonine, Thr), the 90th amino acid is threonine, the 123th amino acid is glutamine, the 125th amino acid is isoleucine (Ile), the 129th amino acid is leucine (Leu), and the 131th amino acid is asparagine which is a stereoscopic epitope. The method of claim 1,
wherein the stereogenic epitope comprises amino acids at amino acid positions 63, 64, 66, 68, 75, 76, 78, 83, 86, 90, 123, 125, 129 and 131 of CEACAM1. It consists of 4 to 69 amino acids in the amino acid sequence at positions 35 to 141 of CEACAM1, and is composed of amino acids at positions 63, 64, 66, 68, 75, 76, 78, 83, 86, 90, 123, 125, 129, and 131 An anti-CEACAM1 antibody or fragment thereof that specifically binds to a stereoscopic epitope comprising any one amino acid selected from the group consisting of amino acids and combinations thereof. 6. The method of claim 5,
The antibody or fragment thereof is composed of 4 to 69 amino acids in the amino acid sequence of positions 35 to 141 of CEACAM1, 63, 64, 66, 68, 75, 76, 78, 83, 86, 90, 123, 125, An antibody or fragment thereof that specifically binds to a stereoscopic epitope comprising amino acids at positions 129 and 131. 6. The method of claim 5,
The antibody or fragment thereof, wherein the antibody or fragment thereof binds to CEACAM1 within a 4.5 Å intermolecular distance. 6. The method of claim 5,
The antibody or fragment thereof is a van der Waals bond, hydrophobic bond or electrostatic bond with CEACAM1, the antibody or fragment thereof. 6. The method of claim 5,
An antibody or fragment thereof, characterized in that the antibody or fragment thereof has a binding affinity ^{for CEACAM1 of less than 1×10 −8} K _{D (M).} A polynucleotide encoding the antibody of claim 5 or a fragment thereof. An expression vector comprising the polynucleotide of claim 10. An isolated transformed cell into which the expression vector of claim 11 was introduced. A method for producing an antibody or fragment thereof comprising culturing the transformed cell of claim 12 . An anticancer agent comprising the antibody or fragment thereof according to any one of claims 5 to 9 as an active ingredient.

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