KR20180108183A - Anti-ceacam1 antibody and use thereof - Google Patents

Abstract

The present invention relates to an anti-CEACAM1 antibody having improved specific binding to CEACAM1 and to uses thereof, wherein the anti-CEACAM1 antibody according to the present invention is excellent in the specific binding ability to CEACAM1, and activates an anticancer immune function of cytotoxic T cells and natural killer cells, thereby being useful as an anticancer agent and a composition for treating cancer.

Description

ANTI-CEACAM1 ANTIBODY AND USE THEREOF < RTI ID = 0.0 >

The present invention relates to anti-CEACAM1 antibodies that specifically bind to CEACAM1 and uses thereof.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) belongs to the carcinoembryonic antigen group and is one of membrane-bound glycoproteins in living body. In humans, CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7 and CEACAM8 are expressed. At this time, CEACAM1 is mainly expressed in activated T cells and natural killer cells and shows high expression in cancer cells. In addition, low expression is seen in epithelial cells, endothelial cells, and hematopoietic stem cells. In addition, CEACAM1 also plays a role in the regulation of innate and adaptive immune responses. Specifically, CEACAM1 has been shown to be an inhibitory receptor for activated T cells contained within the human intestinal epithelium (Morales et al ., J. Immunol , 1999, 163, 1363-1370).

CEACAM1 also activates the immune checkpoint pathway as an immune checkpoint agent such as PD-1 and CTLA-4. The immune checkpoint pathway consists of a variety of co-stimulatory and inhibitory molecules that cooperate to protect tissue from damage by the immune system under physiological conditions and maintain self-tolerance. When CEACAM1 is activated, homologous affinity interaction of T cells and natural killer cells can induce inhibition of lymphocyte cytotoxic effect, thereby avoiding immunity. Therefore, suppression of the immune checkpoint pathway in cancer cells has emerged as a promising anticancer strategy.

In addition, studies on several human tumor types have shown that tumors can avoid immunity by using the CEACAM1 signaling pathway, and monoclonal antibodies (mAbs) are used in preclinical animal models of tumors to induce CEACAM1 interaction , It was shown that they could enhance the immune response to tumors.

On the other hand, existing anticancer drugs have a much higher side effect than anticancer effects. On the other hand, therapeutic agents that block immunity checkpoints, which have recently begun to suffer from the spotlight, activate cancer cells by activating inactivated immune cells instead of directly killing cancer cells. In the case of Merck's Keytruda, the side effects are minimal compared to conventional anticancer drugs and the anticancer effect is excellent. Specifically, the kit luda therapeutic agent uses Pembrolizumab, which is a PD-1 inhibiting antibody. As a result of chemotherapy, the chemotherapy response rate was 4%, but the therapeutic dose of kitrude was 25% (2 ㎎ / ㎏) to 87% (10 ㎎ / ㎏) Respectively.

Meanwhile, in the case of CEACAM1 inhibiting antibody acting on CEACAM1 expressed on the surface of cytotoxic T cells and natural killer cells and cancer cells, which have a main role of anticancer immune function, in addition to CEACAM1, CEACAM3 and CEACAM5 . This is due to the property of binding to the N domain, which is highly homologous between CEACAM1, CEACAM3 and CEACAM5.

 Morales et al., J. Immunol, 1999, 163, 1363-1370

Thus, to develop an anti-CEACAM1 antibody that specifically binds to CEACAM1, the present inventors confirmed that the anti-CEACAM1 antibody binds to the N and B domains of CEACAM1 and is not cross-reactive with CEACAM3, CEACAM5, CEACAM6 and CEACAM8 Thus completing the present invention.

The present invention relates to a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, A light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 5, a light chain CDR2 including the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 including the amino acid sequence shown in SEQ ID NO: 6, or an antibody fragment thereof .

Furthermore, the present invention provides an anticancer agent comprising the anti-CEACAM1 antibody of the present invention or an antibody fragment thereof as an active ingredient.

In addition, the present invention provides an anti-cancer adjuvant comprising the anti-CEACAM1 antibody of the present invention or an antibody fragment thereof as an active ingredient.

Further, the present invention provides a composition for treating cancer, which comprises a chemotherapeutic adjuvant and a cell therapeutic agent according to the present invention.

The present invention also relates to a method for the treatment of cancer, comprising the steps of: i) contacting a lymphocyte isolated from an individual suffering from cancer with the anti-CEACAM1 antibody or antibody fragment of the present invention; ii) administering to the individual a lymphocyte contacted with the anti- CEACAM1 antibody or antibody fragment thereof The method comprising the steps of:

Furthermore, the present invention provides a method of inhibiting the proliferation of CEACAMl expressing tumor cells, comprising contacting the tumor cells expressing CEACAM1 with an anti-CEACAM1 antibody or antibody fragment of the invention.

The anti-CEACAM1 antibody according to the present invention has excellent specific binding ability to CEACAM1 and can be usefully used as an anticancer agent and cancer therapeutic composition by activating the anti-cancer immunity function of cytotoxic T cells and natural killer cells.

Figure 1 A is a schematic representation of the structure of a recombinant CEACAMl prepared according to one example.
1B is a graph showing the binding strength of the anti-CEACAM1 antibody (hereinafter referred to as C25) according to the constitutive domain of the recombinant CEACAM1.
Figure 2 is a comparison of the binding strength of recombinant CEACAM1 and C25 according to the concentration of C25.
FIG. 3 is a graph showing the binding strength between CEACAM1 and C25 expressed on the surface of Jurkat E6.1 T cell line according to the concentration of C25. FIG.
FIG. 4 shows the affinity between C25 and CEACAM1. FIG. The affinity was obtained with kinetic constant K _on and K _off and equilibrium dissociation constant K _D.
5 is a photograph showing the isoelectric point of C25. The first lane is IEF-Markers 3-10, the second lane is huIgG4, and the third lane is C25.
Figure 6a shows the activation of Jurkat E6.1 T cell line by C25 through CD69, CD25 and Ki67 marker expression. The concentration of C25 and huIgG4 is 10 [mu] g / ml.
Figure 6b shows the number of activated Jurkat E6.1 T cells and secretion of IL-2 by C25. The concentration of C25 and huIgG4 is 10 [mu] g / ml.
Figure 6c shows activation of Jurkat E6.1 T cell line by C25 through CD69, CD25 and Ki67 marker expression. The concentration of C25 and huIgG4 is 25 [mu] g / ml.
Figure 6d shows the number of activated Jurkat E6.1 T cells and secretion of IL-2 by C25. The concentration of C25 and huIgG4 is 25 [mu] g / ml.
Figure 7a shows activation of Jurkat E6.1 T cell line by C25 through CD69, CD25 and Ki67 marker expression. The concentration of? CD3 is 0.1 占 퐂 / ml.
Fig. 7B shows the number of activated Jurkat E6.1 T cells by C25 and secretion amount of IL-2. Fig. The concentration of? CD3 is 0.1 占 퐂 / ml.
Figure 7c shows activation of Jurkat E6.1 T cell line by C25 through CD69, CD25 and Ki67 marker expression. The concentration of? CD3 is 1 占 퐂 / ml.
Figure 7d shows the number of activated Jurkat E6.1 T cells and secretion of IL-2 by C25. The concentration of? CD3 is 1 占 퐂 / ml.
FIG. 8A is a photograph of C25 staining to confirm the expression level of CEACAM1 in normal tissues of humans and monkeys.
8B is a photograph of staining of human and monkey tissues using huIgG4
Figure 9 shows that C25 is not cross-reactive with the CEACAM-3, 5, 6 and 8 receptors.
FIG. 10 shows that C25 binds not only to human but also to CEACAM1 of a monkey.
FIG. 11A is a graph showing the enhancement of anti-cancer effect of TALL-104 T cells by C25. FIG.
FIG. 11B is a graph showing the enhancement of the anti-cancer effect of NK92MI cells by C25. FIG.

The present invention relates to a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, A light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or an antibody fragment thereof do.

The term "antibody" used in the present invention means an immune protein that binds to an antigen and interferes with the action of the antigen or eliminates the antigen. There are five types of antibodies, IgM, IgD, IgG, IgA and IgE, and they each contain a heavy chain made up of heavy chain constant region genes μ, δ, γ, α, and ε. In antibody technology, IgG is mainly used. There are four kinds of isotypes of IgG1, IgG2, IgG3 and IgG4, and their structural and functional characteristics may be different.

The IgG also has a Y-shaped, highly stable structure (molecular weight, about 150 kDa) made up of two heavy chain (about 50 kDa) proteins and two light chains (about 25 kDa) The light and heavy chains of the antibody are divided into constant regions having the same amino acid sequence as the variable region in which the amino acid sequences of the antibodies differ from each other. The heavy chain constant region includes CH1, H (hinge), CH2, CH3 domains Lt; / RTI > Each domain consists of two β-sheets and is linked to a disulfide bond in the molecule. Two variable regions of the heavy chain and the light chain are combined to form an antigen binding site. Each of these regions is present in two Y-shaped arms. An antibody binding site (Fab fragment), and the part that can not bind antigen is called Fc (crystalizable fragment). The Fab and Fc are connected by a flexible hinge region.

The term "CDR" used in the present invention means a hypervariable region that has a different amino acid sequence for each antibody in the heavy chain and light chain variable regions of the antibody, and means a site binding to an antigen. As for the stereostructure of the antibody, the CDR has a loop shape on the surface of the antibody, and a framework region (FR) exists under the loop for structurally supporting the CDR. There are three ring structures in each of the heavy chain and the light chain, and these six ring structures combine with each other to directly contact the antigen.

In addition, the anti-CEACAM1 antibody or antibody fragment thereof comprises a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 including an amino acid sequence represented by SEQ ID NO: 2, and an amino acid sequence represented by SEQ ID NO: 3 Lt; RTI ID = 0.0 > CDR3. ≪ / RTI > Preferably, the antibody or antibody fragment thereof may comprise a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 7. However, if the CDR1, CDR2, and CDR3 of the heavy chain variable domain are the same, the framework portion can be modified. In particular, the amino acid sequences of some of the backbone moieties can be modified to produce humanized antibodies.

The anti-CEACAM1 antibody or antibody fragment thereof may further comprise a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 including an amino acid sequence represented by SEQ ID NO: 5, an amino acid sequence represented by SEQ ID NO: Lt; RTI ID = 0.0 > CDR3. ≪ / RTI > Preferably, the antibody or antibody fragment thereof may comprise a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8. However, if the CDR1, CDR2, and CDR3 of the light chain variable domain are the same, the skeletal part can be modified. In particular, the amino acid sequences of some of the backbone moieties can be modified to produce humanized antibodies.

In addition, the anti-CEACAM1 antibody or antibody fragment thereof of the present invention may comprise a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: However, if the CDR1, CDR2, and CDR3 of the heavy and light chain variable domains are the same, the skeletal part can be modified. In particular, the amino acid sequences of some of the backbone moieties can be modified to produce humanized antibodies.

In addition, the antibody fragment may be any one selected from the group consisting of Fab, scFv and Fv. Antibody fragments are antigen binding domains except for the Fc region, which is an effector function for transferring the binding stimulus to an antigen to a cell or a complement, and a single domain antibody or a minibody ), ≪ / RTI > and the like.

In addition, since the antibody fragment has a small size as compared with a full-length IgG, it has good permeability to tissues and tumors, can be produced in bacteria, has an advantage of low production cost, and has no Fc, This function is used when you do not want the function of transferring to the complement or the complement. Antibody fragments are suitable for biopsy because of their short half-life in the human body. However, when replacing some basic, acidic or neutral amino acids among the amino acids constituting the antibody, the unique isoelectric point (pI) have. The change in isoelectric point of the antibody may induce changes such as reducing the toxic side effects of the antibody in vivo or increasing the water solubility. Therefore, in the case of the therapeutic antibody, a full structure IgG can be used considering the affinity and the structural form.

In addition, the anti-CEACAM1 antibody or antibody fragment of the present invention may have a heavy chain variable domain sequence comprising the amino acid sequence shown in SEQ ID NO: 7, or a peptide having 97%, 98% or 99% homology with the heavy chain variable domain sequence .

In addition, the anti-CEACAM1 antibody or antibody fragment thereof of the present invention comprises a light chain variable domain sequence comprising the amino acid sequence shown in SEQ ID NO: 8, or a light chain variable domain sequence having at least 97%, 98% or 99% . ≪ / RTI >

In addition, the anti-CEACAM1 antibody or antibody fragment thereof of the present invention has a heavy chain variable domain sequence comprising the amino acid sequence represented by SEQ ID NO: 7 and a light chain variable domain sequence comprising the amino acid sequence represented by SEQ ID NO: 98% or 99%.

In addition, the anti-CEACAM1 antibody refers to an antibody that binds to CEACAM1. As used herein, the term "C25" is an embodiment of the anti-CEACAM1 antibody.

In addition, the anti-CEACAM1 antibody or antibody fragment thereof recognizes the N domain and the B domain of CEACAM1 as an epitope. The antibody is also not cross-reactive with CEACAM3, CEACAM5, CEACAM6 and CAECAM8.

In addition, the anti-CEACAM1 antibody of the present invention can be easily produced by a known monoclonal antibody production technique. Methods for producing monoclonal antibodies can be performed by preparing hybridomas using B lymphocytes from immunized animals, or by using phage display techniques, but are not limited thereto.

The present invention also provides an anticancer agent comprising an anti-CEACAM1 antibody or an antibody fragment thereof as an active ingredient.

An anticancer agent comprising the anti-CEACAM1 antibody of the present invention or an antibody fragment thereof as an active ingredient can be used for treating cancer or tumor overexpressing CEACAM1. Specifically, when recognizing an antigenic determinant of a cancer or tumor cell in a TCR (T cell receptor) of a cytotoxic T cell that serves to remove cancer cells, the antigen is bound to the CD4 (cluster of differentiation 4) The lymphocyte-specific protein tyrosine kinase (LCK) protein phosphorylates CD3ζ (cluster of differentiation 3ζ), another component of the TCR. When the ZAP70 (Zeta-chain-associated protein kinase 70) protein binds to the phosphorylated CD3ζ portion, the terminal of the ZAP70 protein is phosphorylated by the LCK protein and the RAS-MAP kinase signal transduction is activated. Cells are activated.

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

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

As used herein, the term "anti-cancer" includes prevention and treatment. Herein, " prevention " means prevention of cancer proliferation by administration of the anticancer agent, , And " treatment " means all the actions of improving or improving the symptoms of cancer by the administration of the antibody of the present invention.

The term "cancer" used in the present invention is characterized by being selected from the group consisting of pancreatic cancer, melanoma, lung cancer and myeloma. However, the present invention is not limited thereto. The cancer or tumor to be operated is not particularly limited, and may include solid cancer and blood cancer.

In addition, the present invention provides an anti-cancer adjuvant comprising an anti-CEACAM1 antibody or an antibody fragment thereof as an active ingredient.

The present invention also provides a composition for treating cancer comprising the above-mentioned anticancer adjuvant and a cell therapeutic agent. The cell treatment agent may include cytotoxic T cells or natural killer cells.

The term "cell therapeutic agent" used in the present invention refers to an agent capable of proliferating and selecting living autologous, allogenic, and xenogenic cells in vitro to restore the functions of cells and tissues, Which is used for preventive or therapeutic purposes through a series of actions that change the behavior of the patient. Specifically, it may be a cytotoxic T cell, a natural killer cell.

Also provided is a method of anti-cancer immunotherapy using the anti-CEACAM1 antibody or antibody fragment thereof of the present invention. Specifically, the method comprises: i) contacting an anti-CEACAM1 antibody or antibody fragment thereof with a lymphocyte isolated from an individual suffering from cancer; ii) administering to the subject a lymphocyte contacted with an anti-CEACAM1 antibody or antibody fragment thereof.

In addition, the lymphocyte is a kind of leukocyte, which accounts for about 25% of all white blood cells, and may be NK cells (natural killer cells), T cells and B cells. And preferably at least one of cytotoxic T cells or natural killer cells.

The term "individual" used in the present invention means a person who is in a condition capable of alleviating, inhibiting or treating a disease by administering the anticancer adjuvant of the present invention or having a disease.

The term "contacting" as used herein also means mixing an anti-CEACAM1 antibody or antibody fragment thereof with cells expressing CEACAM1.

The term "administering " as used herein refers to the introduction of an effective amount of a substance into an individual by an appropriate method, and the administration route of the composition comprising the anti-CEACAM1 antibody or antibody fragment thereof of the present invention, Can be administered via a common route that can be reached. Specifically, parenteral administration (for example, intravenous, subcutaneous, intraperitoneal or local administration) may be used depending on the intended use, and intravenous administration may preferably be employed. In some cases, administration to solid tumors may be preferable to local administration in order to facilitate the access of the antibody. The dosage varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. The single dose may be administered on a daily or weekly basis in an amount of about 0.001 to 10 mg / kg. The effective amount may be adjusted according to the discretion of the physician treating the patient.

In addition, the composition for cancer treatment according to the present invention can be administered in a pharmaceutically effective amount to treat cancer cells or their metastasis, or to inhibit cancer growth. The type of cancer, the age and weight of the patient, the nature and severity of symptoms, the type of current treatment, the number of treatments, the route and route of administration, and can be readily determined by those skilled in the art. The composition of the present invention may be administered together with the above-described pharmacological or physiological components or may be administered sequentially, or may be administered in combination with additional conventional therapeutic agents, and administered sequentially or concurrently with conventional therapeutic agents. Such administration may be single or multiple administrations. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The present invention also provides a method of inhibiting the proliferation of CEACAMl expressing tumor cells using the anti-CEACAM1 antibody or antibody fragment thereof. Specifically, it may comprise contacting tumor cells expressing CEACAM1 with an anti-CEACAM1 antibody or an antibody fragment thereof.

 Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

Example  1. The anti- CEACAM1  The antibody (C25)

Example  Section 1.1 - CEACAM1  Preparation of antibodies

The C25 antibody fragment inserted in scFv form in the Pcomb3x vector (Addgene; Cat. # 63891) was amplified by PCR to obtain light and heavy chain genes containing sequences recognized by each restriction enzyme. The heavy chain gene of C25 was treated with kpnI and apaI restriction enzyme, and the light chain gene of C25 was treated with kpnI and BsiWI restriction enzyme.

Then, the heavy and light chain genes were inserted into the pcIW vector (Promega; Cat. # E1731) digested with the same restriction enzymes. Then, using a restriction enzyme, a vector containing the heavy chain transcription unit and the light chain transcription unit at the same time was selected. The selected vector was extracted with Endofree plasmid maxi kit (QIAGEN; Cat. # 12362), and the base sequence of the antibody was finally confirmed by sequencing using some of the extracted DNA.

The extracted 30 항 antibody DNA was transfected into 30 ml of Expi293F cell (ThermoFisher scientific; Cat. # A14527) at a concentration of 2.5 × 10 ⁶ / ml. Day after transfection, enhancer; by culturing for 7 days in (ThermoFisher. Cat # A14524) the transfection and added to the infected cells at 37 ℃ stirred incubator, CO ₂ 8%, 125 rpm conditions to produce C25 antibody.

After culturing, the supernatant separated by centrifugation in the culture was reacted with 100 μl of protein A beads (Repligen; Cat. # CA-PRI-0100) for 2 hours. The beads were then washed with 10 ml of binding buffer (ThermoFisher scientific; Cat. # 21019) and 200 μl of elution buffer (ThermoFisher scientific; Cat. # 21004) was added to the beads to separate the C25 antibody conjugated to the beads. The separated antibody was neutralized by the addition of 10 [mu] l of 1.5 M Tris-HCl pH 8.8 (Bio-rad; Cat. # 210005897).

Experimental Example  1. The anti- CEACAM1  Confirmation of antibody binding capacity

Experimental Example  1.1. Of CEACAM1  Domain-specific anti- CEACAM1  Confirmation of antibody binding capacity

DNAs of CEACAM1 mutant proteins conjugated with the human immunoglobulin Ckappa domain were transfected in 30 ml of ExpiHEK293F (ThermoFisher scientific; Cat. # A14527) at a concentration of 2.5 x 10 ⁶ / ml in 30 μg. enhancer (ThermoFisher; Cat. # A14524) was added to the transfected cells and cultured at 37 ° C, 8% CO ₂ , 125 rpm for 7 days in a stirred culture. The supernatant of the culture was then separated and reacted with a kappa selection bead (GE Healthcare; Cat. # 17-5458-01) for 2 hours. Thereafter, the beads were washed with 10 ml of a binding buffer (ThermoFisher scientific; Cat. # 21019) and 200 μl of elution buffer (ThermoFisher scientific; Cat. # 21004) was added to the beads to separate and purify the CEACAM1 mutant protein from the beads 1a).

2.5 μg of each purified CEACAM1 mutant protein was diluted in 1000 μl PBS, and 20 μl of each of the purified CEACAM1 mutant proteins was dispensed into each well, followed by reaction at 4 ° C for 16 hours. 1 < / RTI > After dilution in 1000 μl of PBS, 25 μl of each was dispensed into each well and allowed to react at 37 ° C for 1 hour. Then, 10 쨉 l of Tween 20 (Sigma-aldrich; Cat. # P9146) was washed three times with wash buffer diluted in 990 쨉 l PBS, and then washed with human radish peroxidase (HRP) One μl of IgG (HRP-conjugated anti-human IgG: Sigma; Cat. # A0170) was diluted in 5000 μl of PBS, and 25 μl of each was added to each well and reacted at 37 ° C for 1 hour. Then, each well was washed three times with the washing buffer, and 25 μl of TMB solution (KPL; Cat. # 52-00-03) was added to each well to induce color development. Then, 2M concentration of H ₂ SO ₄ 25 μl was added to each well to stop the reaction and the absorbance at 450 nm was measured.

As a result, N domain and B domain were found to be essential sites for complete affinity binding. A1 and A2 did not appear to be required for direct coupling. Therefore, C25 is mainly bound to the N domain of CEACAM1, and the B domain is additionally required to bind with complete affinity (FIG. 1B).

Experimental Example  1.2. CEACAM1  Protein and anti- CEACAM1  Confirmation of antibody binding capacity

2 μg of the recombinant CEACAM1 protein was diluted in 1000 μl of PBS, and 50 μl of each was added to a 96-well plate (Nunc; Cat. # 467679) and reacted at 4 ° C for 16 hours. Subsequently, 300 μl of 3% (v / v) bovine serum albumin (Bovine Serum Albumin) was added to each well, followed by reaction at 37 ° C for 1 hour. 0.75 [mu] g of C25 was diluted in 1000 [mu] l PBS. The diluted C25 solution was again subjected to sequential dilution 14 times in PBS and 1: 1 volume ratio. Fifteen of the diluted C25 were dispensed into each well and reacted at 37 캜 for 1 hour. Then, 10 μl of Tween 20 (Sigma-aldrich; Cat. # P9146) was washed 3 times with washing buffer diluted in 990 μl PBS, 1 μl of human IgG conjugated with HRP was diluted in 5000 μl PBS, well, and reacted for 1 hour. Thereafter, each well was washed 3 times with the washing buffer, and 50 mu l of TMB solution After adding 50 μl of 2M H ₂ SO ₄ to each well, the reaction was stopped and absorbance was measured at 450 nm.

As a result, the EC ₅₀ (Half maximal effective concentration) value was measured to be 0.35 nM (FIG. 2).

Experimental Example  1.3. Expressed on the cell surface CEACAM1 and  Confirmation of bond strength of C25

CEACAM1 cDNA was transfected into Jurkat cell (Jurkat E6.1 (ATCC; TIB-152TM) stably expressing CEACAM1 and treated with 700 μg / ml of antibiotic G418. The selected cell line was treated with 2% (v / v ) Was resuspended in FBS-supplemented DPBS (hereinafter referred to as FACS buffer), centrifuged at 1,500 rpm, resuspended in FACS buffer such that the number of cells was 3 × 10 ⁶ / (BD Pharmingen; Cat. # 564220) solution was added to 0.5 [mu] l of 0.5 [mu] L of each well. Cells and culture medium of each well were recovered and centrifuged at 1,500 rpm and the supernatant was discarded. Ml of FACS buffer and then incubated at 4 DEG C for 15 minutes.

5 μg / ml, 2.5 μg / ml, 1.25 μg / ml, 0.625 μg / ml, and 0.3125 μg / ml of human IgG4 (Sigma; Cat. # I4639) in 50 μl of FACS buffer / Ml and 0.15625 占 퐂 / ml, respectively. In the presence of the Fc blocker, 50 [mu] l of diluted C25 or human IgG4 was added to the cells in the presence of 20 [mu] g / ml, 10 g / ml, 5 g / ml, 2.5 g / ml, 1.25 g / ml, 0.625 g / 0.3125 ㎍ / ㎖ and 0.15625 ㎍ / ㎖, respectively, and reacted at 4 캜 for 1 hour and 30 minutes.

Cells reacted with C25 were resuspended in FACS buffer and centrifuged at 1,500 rpm for washing. Phycoerithrin-conjugated goat anti-human Fab'2 (Sigma; Cat. # P8047) labeled with phycoerythrin (hereinafter referred to as PE) was diluted in FACS buffer at a volume ratio of 1: 200 100 μl of each well was added to each well and reacted in the dark for 30 minutes at 4 ° C.

The cells reacted with PE were collected, resuspended in FACS buffer, centrifuged at 1,500 rpm, and the supernatant was discarded and washed twice. The cells were then resuspended in 100 占 퐇 of immobilized buffer (BD Cytofix 占 Cat. # 554655) and incubated in the dark for 30 minutes at 4 占 폚.

Cells reacted with the immobilized buffer were collected, resuspended in FACS buffer, centrifuged at 1,500 rpm and washed twice to discard the supernatant. The washed cells were resuspended in 200 [mu] l of FACS buffer and compared to the mean fluorescence intensity (MFI) of the labeled PE in cells in a FACS LSR-Fortessa kit. All FACS analyzes were performed using FlowJo software.

As a result, (MFI 1800 or higher) at the highest level of binding force up to 5 μg / ml, and decreased rapidly from the lower concentration (FIG. 3).

Experimental Example  1.4. term- CEACAM1  Quantitative binding assay of antibodies

Quantitative binding of C25 to CEACAM1 was measured using a BIAcore T-100 (GE Healthcare, USA) biosensor. Purified CEACAM1-Fc from HEK293 (ThermoFisher scientific; Cat. # A14527) cells was diluted to a concentration of 10 μg / ml in acetate buffer (10 mM, pH 4.0) Healthcare, US) at 200 Rmax.

C25 was serially diluted 4 times in 1: 1 volume ratio in HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% surfactant P20) , and dissociation was performed at a flow rate of 30 μl / min for 1,200 seconds. The dissociation of the antibody bound to CEACAM1-Fc was induced by flowing 10 mM Glycine-HCl pH 1.5 at a flow rate of 30 μl / min for 180 seconds. Affinity was calculated using the BIAcore T-100 evaluation software using the kinetic constant (K _on And K _off ) and the equilibrium dissociation constant (K _D ).

As a result, an affinity (K _D ) value of 11.4 nM was obtained at a concentration range of 12.5 nM to 200 nM (Fig. 4).

Experimental Example  2. The anti- CEACAM1  Identification of antibody properties

Experimental Example  2.1. term- CEACAM1  Antibody Isoelectric point  Confirm

IEF anode buffer (20x) was mixed with 980 ml of deionized water (DW) to make a 1xIEF anode buffer, and 20 ml of IEF anode buffer pH 3-10 (10x) was mixed with 180 ml of DW to make a 1xIEF negative electrode buffer. 1xIEF anode buffer and 1xIEF cathode buffer were used after cooling to 4 ° C.

Using an IEF gel (Invitrogen / pH 3-10 IEF gel, 1.0 mmx10 well / EC6655BOX), the upper chamber was filled with 200 ml 1xIEF cathode buffer and the lower chamber was filled with 600 ml 1xIEF anode buffer. 15 μl of 20 μl of the mixture prepared by mixing 10 μl of C25 at a concentration of 1.12 mg / ml and 10 μl of an IEF sample buffer pH 3-10 (2 ×) was loaded and IEF Markers 3-10 (SERVA / 10 mg / mL / SERVA Liquid Mix, 39212.01).

The electrophoresis was carried out by varying the voltage in three steps of 100V: 1 hour, 200V: 1 hour and 500V: 30 minutes, and fixed with 12% TCA solution for 30 minutes. After fixing, the isoelectric point was confirmed using Coomassie Blue R-250 Intron Biotechnology (IBS-BC006).

As a result, the actual isoelectric point value measured to optimize the buffer pH of the antibody was 9.8, which was significantly higher than the theoretical value of 7.76 (FIG. 5).

Experimental Example  3. The anti- CEACAM1  The antibody T cell  Measuring the Effect on Activation

Experimental Example  3.1. term- CEACAM1  Following antibody treatment T cell  Activation measurement

Jurkat E6.1 (ATCC; TIB-152TM) cells were resuspended in 200 μl of FACS buffer to a cell number of 1 × 10 ⁵ , and then plated in plate-coated anti-CD3 (0.1 μg / ml; eBioscience; 16-0037). C25 (1 mg / ml) was treated with 2 占 퐇 or 5 占 퐇 to a final concentration of 10 占 퐂 / ml or 25 占 퐂 / ml, followed by stimulation for 96 hours. HuIgG4 was used as a control.

After 96 hours, the cells and culture medium were recovered and centrifuged at 1,500 rpm. The supernatant was stored at -80 ° C for IL-2 secretion, and the remaining cells were collected, replaced with FACS buffer, Blocking Fc receptors for a few minutes.

CD25-PE-Cy7 antibody or anti-CD69-APC antibody (eBioscience; anti-CD25-PE-Cy7: Cat. # 25-0259; anti-CD69-APC: Cat. # 06-0699) And reacted at 4 캜 for 15 minutes.

Cells and culture medium were collected and filled up to 200 μl with FACS buffer, and centrifuged at 1,200 rpm for 5 minutes to remove supernatant. After resuspending the cells in a fresh FACS buffer, this procedure was repeated three times to completely remove unbound antibody. The cells were resuspended in DPBS supplemented with 1% (v / v) paraformaldehyde and fixed at 4 ° C for 30 minutes.

Cells were resuspended in lxFoxP3 staining buffer (eBioscience; Cat. # 00-5523-00) and centrifuged. After repeating this procedure twice, anti-Ki67 antibody (eBioscience; Cat. # 350520) labeled with different dyes was diluted 1: 100 volume ratio in 1xFoxP3 staining buffer and treated with 50 μl total volume and incubated at 4 ° C And reacted for 30 minutes. Cells and culture were harvested, filled with FoxP3 staining buffer and centrifuged. After repeating this procedure three times, the FACS buffer was finally replaced with FACS buffer, and CD25 + CD69 + activated cells and Ki67 + proliferating cells were identified in FACS LSR-Fortessa equipment and analyzed using FlowJo software.

As a result, when Jurkat E6.1 was treated with 10 μg / ml of antibody and 25 μg / ml of C25 in CCM1 induction expression condition (α-CD3 0.1 μg / ml, culture for 4 days) Was more than twice as high as that of CD25 + CD69 + Jurkat T cells expressing CD25 and CD69 that expressed activated cells compared with the control group, huIgG4, and the ratio of Ki67 + Jurkat T cells expressing Ki67 And the cell number increased more than twice (Figs. 6A to 6D).

CEACAM1 + Jurkat cells (EF1 promoter-CEACAM1-GFP vector), which was constructed to overexpress CEACAM1, were transfected with Jurkat E6.1 and treated with a selectable marker G418 disulfate salt solution (Sigma; G8168) at a concentration of 700 μg / GFP + CEACAM1 + cells were isolated using Fluorescence-Activated Cell Sorter.

Jurkat E6.1 (ATCC; TIB-152TM) cells were resuspended in 200 μl of FACS buffer to a cell density of 1 × 10 ⁵ , and then plate-coated anti-CD3 (0.1 μg / ml; eBioscience; 16-0037) anti-CD3 (1 [mu] g / ml). C25 (1 mg / ml) was treated with 2 占 퐇 or 5 占 퐇 to a final concentration of 10 占 퐂 / ml or 25 占 퐂 / ml and stimulated for 48 hours. HuIgG4 was used as a control.

After 48 hours, the cells and culture medium were recovered and centrifuged at 1,500 rpm. The supernatant was stored at -80 ° C for IL-2 ELISA, and the remaining cells were collected, replaced with FACS buffer, Fc receptors were blocked. CD25-PE-Cy7 antibody or anti-CD69-APC antibody was treated on the plate and reacted at 4 ° C for 15 minutes.

Cells and culture medium were collected, filled up to 200 μl with FACS buffer, and centrifuged at 1,200 rpm for 5 minutes to remove supernatant. After resuspending the cells in a fresh FACS buffer, this procedure was repeated three times to completely remove unbound antibody. The cells were resuspended in DPBS supplemented with 1% (v / v) paraformaldehyde and fixed at 4 ° C for 30 minutes.

Cells were filled up to 200 μl in 1x FoxP3 staining buffer and centrifuged at 1,200 rpm for 5 minutes to remove supernatant. This procedure was repeated twice, followed by treatment with 50 μl of a different dye-labeled anti-Ki67 antibody at a volume ratio of 1: 100, followed by reaction at 4 ° C for 30 minutes.

Cells were filled up to 200 μl in 1x FoxP3 staining buffer and centrifuged at 1,200 rpm for 5 minutes to remove supernatant. After repeating this procedure three times, the FACS buffer was finally replaced with FACS buffer, and CD25 + CD69 + activated cells and Ki67 + proliferating cells were identified in FACS LSR-Fortessa equipment and analyzed using FlowJo software.

As a result, the degree of activation of CEACAM1-Jurkat T cell was 1.5 times (C25 25 μg / ml) or more than four times (C25 10 μg / ml) in the CD25 + CD69 + CEACAM1-Jurkat T cell compared to the huIgG4- The ratio and number of Jurkat T cells were both increased more than twice (C25 25 占 퐂 / ml) and up to 10 times (C25 10 占 퐂 / ml) (Figs. 7a to 7d).

In addition, anti-IL-2 antibody (capture Ab: eBioscience; Cat. # 14-7029-85) was diluted to 1 μg / ml in coating buffer (50 mM carbonate / bicarbonate buffer, pH 9.6). Then, 200 μl of anti-IL-2 antibody solution diluted in 96-well microplate was added to each well and reacted at 4 ° C for 16 hours to 18 hours. Then, 96-well microplates were washed with DPBS and 200 μl of blocking buffer (SuperBlock ™ Blocking Buffer: ThermoFisher Scientific; Cat. # 37515) was added to each well and reacted at room temperature for 30 minutes.

IL-2 recombinant protein (R & D Systems; Cat. # P60568) was diluted in blocking buffer to a concentration of 20 [mu] g / ml to obtain a standard curve. The diluted IL-2 recombinant protein solution was further diluted 11 times in a 1: 1 volume ratio. Twelve IL-2 recombinant protein samples, supernatants from Jurkat E6.1 cells stored at -80 ° C or supernatants from CEACAM1 + Jurkat E6.1 cells were seeded in 96 wells coated with anti-IL-2 antibody 100 μl of each well was added to each well of the microplate and reacted at room temperature for 2 hours.

 10 μl of Tween 20 (Sigma-aldrich; Cat. # P9146) was washed 4 times with 96-well microplates in washing buffer diluted in 990 μl PBS, and then incubated with biotin-conjugated anti-IL-2 antibody (Detection antibody: eBioscience; Cat . # 13-7028) was diluted in blocking buffer at a ratio of 1: 1,000 by volume, and each well was reacted at room temperature for 2 hours.

After 96-well microplates were washed four times with washing buffer, 100 μl of peroxidase-labeled streptavidin (Sigma; Cat. # S5512) diluted 1: 1,000 in blocking buffer was added to each well And allowed to react at room temperature for 2 hours.

100 μl of TMB peroxidase substrate solution (KPL; Cat. # 52-00-02) was added to each well and incubated for 10 minutes. TMB stop solution (KPL; Cat. # 50-85-05) was added to each well to stop the reaction. The O.D values were measured at a wavelength of 450 nm using a molecular dynamics reader. Measurements were analyzed using SoftMax Pro 5.4.1 (Figures 6b, 6d, 7b and 7d).

As a result, when the antibody concentration was 10 μg / ml and 25 μg / ml of C25 was treated in the CEACAM1 induction expression condition (a-CD3 0.1 μg / ml, 4 days culture) of Jurkat E6.1, One IL-2 secretion level was three times higher than that of the control group (Fig. 6b, 6d).

In addition, Jurkat cells stably expressing CEACAM1 were incubated with 10 μg / ml antibody and 25 μg / ml anti-CD3 (0.1 μg / ml) or high concentrations of anti-CD3 / Ml of C25 was treated for 48 hours, the level of IL-2 secretion was higher than that of the control (FIGS. 7b and 7d), and the effect on C25 secretion of IL-2 specifically for low concentration anti-CD3 stimulation It was found to be very large and increased from ten times to twenty times that of the control (Fig. 7B).

Experimental Example  4. In humans and monkeys by organization CEACAM1  Confirmation of expression

A 10 mM solution of EZ-Link ™ Sulo-NHS-LC-LC-Biotin (ThermoFisher Scientific; Cat. # 21338) was prepared and 13.3 μl per 1 mg of C25 was added and biotinylated at 4 ° C for 2 hours. Human and monkey TMA slides were deparaffinized in an oven at < RTI ID = 0.0 > 60 C < / RTI > Hydration was gradually carried out in the order of xylene (100%), alcohol (90%), alcohol (80%) and alcohol-DW.

For antigen retrieval, TMA slides were treated in a constant temperature water bath containing 1.5 L Tris-EDTA pH 9.0 buffer, and reacted at 100 for 20 minutes. To prevent intracellular peroxidase activity, 100 μl of 3% hydrogen peroxide was added and reacted for 6 minutes. 100 μl of 5% (v / v) normal horse serum was treated and reacted for 30 minutes .

The primary antibody (biotin-conjugated hIgG4 or C25; 1 mg / ml) was diluted 1: 1 or 1: 5 by volume and treated with 100 μl of the solution. 100 ㎕ of ABC reagent was treated and reacted for 30 minutes. DAB substrate kit (VECTOR LABORATORIES; Cat # SK-4100) was treated, developed for 2 minutes, and stained with hematoxylin for comparison. Since CEACAM1 expression in normal cells is very low, the antibody was used at a relatively high concentration.

The degree of staining for normal tissues of Cynomolgus monkeys of C25 was similar to that of human normal tissues, and the level of CEACAM1 expression in normal tissues was very low, indicating that they were stained when treated at high concentrations (FIG. 8A) . Human IgG4 was used at the same concentration as a control for C25 (Fig. 8B).

Experimental Example  5. The anti- Of CEACAM1  Cross-reaction confirmation

In the HEK293 cells, 10 μg of pEF1? -AcGFP-N1 vector (Clontech, Cat No. 631973), CEACAM1, CEACAM3, CEACAM5, CEACAM6 and CEACAM8 plasmid vectors were used as control vectors, respectively. Using a Neon transfection system, width 20 ms and pulse number 2. After 48 h of transfection, GFP expression was confirmed by fluorescence microscopy and the cells were treated with 1 ml of TrypLE Express Solution (ThermoFisher Scientific; Cat. # 12605010), and the cells were washed with DMEM supplemented with 10% (v / v) FBS (Gibco, Cat No. 11995-065) was diluted to 9 mL and centrifuged at 1,200 rpm for 5 minutes to remove supernatant.

After washing once with PBS, the cells were resuspended in FACS buffer to a concentration of ⁵ x 10 cells / 100 μl. Human Fc Block solution was added at 1 μl per sample and reacted at 4 ° C for 10 minutes. huIgG4 or C25 were treated at a concentration of 1 / / 5x10 ⁵ cells and reacted at 4 째 C for 1 hour. After washing with FACS buffer, PE-labeled goat-anti-huIgG4 antibody was diluted 1: 200 in FACS buffer, and 100 μl of each sample was added and reacted at 4 ° C for 30 minutes. FACS buffer was filled up to 200 μl, and the supernatant was removed by centrifugation at 1,200 rpm for 5 minutes. 300 μl of the fixed buffer was added and the cells were resuspended. Cells expressing GFP were selected from FACS LSR-Fortessa kit and their expression was confirmed by specific CEACAM members according to CEACAM members (Table 1). The binding of C25 was also confirmed by PE fluorescence channel and analyzed using FlowJo software.

Kinds shape matter  persons get  wife CEACAM1 cDNA clone CEACAM1 R & D Systems, RDC0951 Isotype Mouse IgG1 Isotype Control PE R & D Systems, IC002P Anti-CEACAM1 antibody Human CEACAM1 / CD66a PEconjugated Antibody R & D Systems, FAB2244P CEACAM3 cDNA clone CEACAM3-GFP Origene, RG217469 Isotype Sheep IgG R & D systems, 5-001-A Anti-CEACAM3 antibody HUMAN CEACAM-3 / CD66d Antibody R & D systems, AF4166P Secondary antibody Donkey Anti-Sheep IgG (H + L) Phycoerythrin R & D Systems, F0126 CEACAM5 cDNA clone CEACAM5-GFP Origene, RG206434 Isotype Mouse IgG2a Isotype Control PE R & D Systems, IC003P Anti-CEACAM5 antibody HUMAN CEACAM-5 / CD66e PE-conjugated Antibody R & D systems, FAB41281P CEACAM6 cDNA clone CEACAM6-GFP Origene, RG202454 Isotype Mouse IgG1 K Isotype Control PE eBioscience, 12-4714-82 Anti-CEACAM6 antibody Human CD66c-PE eBioscience, 12-0667-42 CEACAM8 cDNA clone CEACAM8 Origene / RG204740 Isotype Mouse IgG1 Isotype Control PE R & D Systems, IC002P Anti-CEACAM8 antibody Human CEACAM8 PE-conjugated Antibody R & D systems / FAB4246P

As a result, C25 showed the characteristic that CEACAM1 binds only to CEACAM1 in the CEACAM member (FIG. 9).

In addition, 10 μg of each control vector, Cynomolgus CEACAM1 plasmid vector and human CEACAM1 plasmid vector was added to HEK293 cells, and transfection was carried out using a Neon transfection system under the conditions of pulse voltage 1100 V, pulse width 20 ms and pulse number 2. After 48 h of transfection, GFP expression was confirmed by fluorescence microscopy. The cells were treated with TrypLE Express solution, and then transferred to FACS buffer to stop the reaction.

After washing once with PBS, the cells were resuspended in FACS buffer to a concentration of ⁵ x 10 cells / 100 μl. Human Fc Block solution was added at a rate of 1 μl per sample and reacted at 4 ° C for 10 minutes. Human IgG4 or C25 was treated at 1 / / 5x10 ⁵ cell concentration and reacted at 4 째 C for 1 hour. After washing with FACS buffer, the PE-labeled goat-anti-huIgG4 antibody was diluted 1: 200 in FACS buffer, and 100 μl of each was added at 4 ° C for 30 minutes. FACS buffer was filled up to 200 μl, and the supernatant was removed by centrifugation at 1,200 rpm for 5 minutes. Cells were resuspended in 300 [mu] l of fixed buffer. Cells expressing GFP were selected from the FACS LSR-Fortessa kit and their expression was confirmed by specific CEACAM members according to CEACAM members. The binding of C25 was also confirmed by PE fluorescence channel and analyzed using FlowJo software.

As a result, it was confirmed that C25 recognizes not only human CEACAM1 but also monkey (Cynomolgus) CEACAM1 (FIG. 10).

Experimental Example  6. The anti- CEACAM1  Antibody Anticancer effect  Confirm

Cancer cells (MKN45:. JCRB Cell Bank; Cat # JCRB0254 ;, MKN1:. JCRB Cell Bank; Cat # JCRB0252) to RPMI 1640 medium (. ThermoFisher Scientific; Cat # 11875093 ) to 1x10 ⁴ / 200㎕ concentration and resuspended in 96 200 μl of each well was added to each well and incubated overnight at 37 ° C in 5% CO ₂ . Untouched cells were removed with the medium and TALL-104 (ATCC; Cat. # CRL-11386TM) or NK92MI (ATCC; Cat. # CRL-2408TM) cells were seeded at various ratios (0: 1, 0.1: 1: 1, 10: 1). At this time, C25 was added to each well at a concentration of 10 μg / ml. HuIgG4 was treated in the same manner as a control group.

The cells were co-cultured for 6 hours, and the non-conjugated lysis cells were removed with the medium, and the MTS assay reagent Cell Titer 96Aqueous One Solution (Promega; Cat. # G3582) was diluted 1: 4 with RPMI 1640 medium. 200 μl of the diluted solution was added to each well and further incubated in the dark for 3 hours. After that, O.D values of each sample were measured with a spectrophotometer at a wavelength of 490 nm, and the measured values were analyzed using SoftMax Pro 5.4.1 program.

As a result, when the anti-cancer effect of CTL (FIG. 11A: TALL-104) or NK (FIG. 11B: NK-92MI) cells was confirmed by confirming the in vitro efficacy of C25, CEACAM1 was expressed at a high level In the case of the MKN45 gastric cell line, C25 increased the anti-cancer immunity of CTL and NK cells, whereas the gastric cell line in which CEACAM1 was not expressed at all (MKN1) showed no anti-cancer effect (FIGS. 11A and 11B) .

<110> Mogam Institute for Biomedical Research <120> ANTI-CEACAM1 ANTIBODY AND USE THEREOF <130> FPD / 201701-0007 <160> 22 <170> KoPatentin 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequnce for anti-CEACAM1 antibody of CDR1 (VH) <400> 1 Asn Tyr Ala Met Ser   1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequnce for anti-CEACAM1 antibody of CDR2 (VH) <400> 2 Val Ile Ser His Gly Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequnce for anti-CEACAM1 antibody of CDR3 (VH) <400> 3 Asp Pro Thr Lys Gly Tyr Ala Pro Thr Phe Asp Tyr   1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequnce for anti-CEACAM1 antibody of CDR1 (VL) <400> 4 Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser   1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequnce for anti-CEACAM1 antibody of CDR2 (VL) <400> 5 Ala Asp Ser Lys Arg Pro Ser   1 5 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequnce for anti-CEACAM1 antibody of CDR3 (VL) <400> 6 Gly Ala Trp Asp Ala Ser Leu Asn Gly Tyr Val   1 5 10 <210> 7 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> amino acid seqeunce for anti-CEACAM1 antibody of variable region          (VH) <400> 7 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 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg      50 55 60 Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met  65 70 75 80 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp                  85 90 95 Pro Thr Lys Gly Tyr Ala Pro Thr Phe Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 8 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> amino acid seqeunce for anti-CEACAM1 antibody of variable region          (VL) <400> 8 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 Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Ala Asp Ser Lys 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 Ala Ser Leu                  85 90 95 Asn Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly             100 105 110 <210> 9 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> amino acid seqeunce for anti-CEACAM1 antibody of heavy chain <400> 9 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 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 Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly     210 215 220 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg                 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro             260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala         275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val     290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr                 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu             340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys         355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser     370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser                 405 410 415 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala             420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys         435 440 445 <210> 10 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> amino acid seqeunce for anti-CEACAM1 antibody of light chain <400> 10 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 Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Ala Asp Ser Lys 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 Ala Ser Leu                  85 90 95 Asn Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln             100 105 110 Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu         115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr     130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr                 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His             180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys         195 200 205 Thr Val Ala Pro Thr Glu Cys     210 215 <210> 11 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> amino acid seqeunce for anti-CEACAM1 antibody of signal seqeunce          (heavy chain) <400> 11 Met Tyr Leu Gly Leu Asn Tyr Val Phe Ile Val Phe Leu Leu Asn Gly   1 5 10 15 Val Gln Ser             <210> 12 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> amino acid seqeunce for anti-CEACAM1 antibody of signal seqeunce          (light chain) <400> 12 Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp Val Ser   1 5 10 15 Gly Thr Cys Gly              20 <210> 13 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequnce for anti-CEACAM1 antibody of CDR1 (VH) <400> 13 aattatgcta tgagc 15 <210> 14 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequnce for anti-CEACAM1 antibody of CDR2 (VH) <400> 14 gtgatctctc atggtggtgg tagtatatat tacgctgatt ctgtaaaagg t 51 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequnce for anti-CEACAM1 antibody of CDR3 (VH) <400> 15 gatcctacta aggggtatgc tcctactttc gactac 36 <210> 16 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequnce for anti-CEACAM1 antibody of CDR1 (VL) <400> 16 agtggctctt catccaatat tggcaataat tatgtctcc 39 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequnce for anti-CEACAM1 antibody of CDR2 (VL) <400> 17 gctgatagta agcggccaag c 21 <210> 18 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequnce for anti-CEACAM1 antibody of CDR3 (VL) <400> 18 ggtgcttggg atgctagcct gaatggttat gtc 33 <210> 19 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> nucleotide seqeunce for anti-CEACAM1 antibody of variable region          (VH) <400> 19 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> 20 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> nucleotide seqeunce for anti-CEACAM1 antibody of variable region          (VL) <400> 20 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtagtg gctcttcatc caatattggc aataattatg tctcctggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat gctgatagta agcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtggt gcttgggatg ctagcctgaa tggttatgtc 300 ttcggcggag gcaccaagct gaccgtccta ggt 333 <210> 21 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> nucleotide seqeunce for anti-CEACAM1 antibody of signal sequence          (heavy chain) <400> 21 atgtacttgg gactgaacta tgtattcata gtttttctct taaatggtgt ccagagt 57 <210> 22 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> nucleotide seqeunce for anti-CEACAM1 antibody of signal sequence          (light chain) <400> 22 atggatagcc aggctcaggt gctgatgctg ctgctgctgt gggtgtcagg gacttgcggg 60                                                                           60

Claims (16)

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR3 including the amino acid sequence represented by SEQ ID NO: 3, an amino acid sequence represented by SEQ ID NO: 4 A light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6, or an antibody fragment thereof. The method according to claim 1,
A heavy chain variable domain comprising an amino acid sequence represented by SEQ ID NO: 7; or an anti-CEACAM1 antibody or antibody fragment thereof. The method according to claim 1,
Wherein the light chain variable domain comprises an amino acid sequence represented by SEQ ID NO: 8. 10. An anti-CEACAM1 antibody or antibody fragment thereof, which is a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: The method according to claim 1,
A heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 8. 2. The anti-CEACAM1 antibody or antibody fragment thereof according to claim 1, The method according to claim 1,
Wherein the antibody fragment is any one selected from the group consisting of Fab, scFv and Fv. The method according to claim 1,
A heavy chain variable domain sequence comprising the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 97% homology with the heavy chain variable domain sequence. The method according to claim 1,
A light chain variable domain sequence comprising the amino acid sequence of SEQ ID NO: 8, or a human CEACAM1-recognizing monoclonal antibody or antibody fragment thereof, which is at least 97% homologous to said light chain variable domain sequence. The method according to claim 1,
A heavy chain variable domain sequence comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain sequence comprising the amino acid sequence of SEQ ID NO: 8. Antibody fragment. An anticancer agent comprising the anti-CEACAM1 antibody or an antibody fragment thereof according to any one of claims 1 to 8 as an active ingredient. 10. The method of claim 9,
Wherein said cancer is selected from the group consisting of pancreatic cancer, melanoma, lung cancer and myeloma. 10. An anti-cancer adjuvant comprising the anti-CEACAM1 antibody or an antibody fragment thereof according to any one of claims 1 to 8 as an active ingredient. A composition for treating cancer, comprising the anti-cancer adjuvant and the cell therapeutic agent of claim 11. 13. The method of claim 12,
Wherein the cell therapeutic agent is a cytotoxic T cell or a natural killer cell. i) contacting an anti-CEACAM1 antibody or antibody fragment thereof of claim 1 with a lymphocyte isolated from an individual suffering from cancer;
ii) administering to the subject a lymphocyte contacted with an anti-CEACAM1 antibody or antibody fragment thereof. 15. The method of claim 14,
Wherein the lymphocyte comprises at least one of a cytotoxic T cell and a natural killer cell. A method for inhibiting proliferation of CEACAMl expressing tumor cells, comprising contacting a tumor cell expressing CEACAM1 with the anti-CEACAM1 antibody or antibody fragment of claim 1.

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