KR102019913B1 - Anti-ceacam1 antibody and use thereof - Google Patents

Abstract

The present invention relates to an anti-CEACAM1 antibody having improved specific binding ability to CEACAM1 and its use. The anti-CEACAM1 antibody according to the present invention has excellent specific binding ability to CEACAM1, cytotoxic T cells and natural killer cells. It can be usefully used as an anticancer agent and a composition for treating cancer by activating its anticancer immune function.

Description

Anti-CEACAM1 Antibody and Uses thereof {ANTI-CEACAM1 ANTIBODY AND USE THEREOF}

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

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) belongs to a group of carcinoembryonic antigens and is one of the transmembrane glycoproteins in vivo. 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, epithelial cells, endothelial cells, and hematopoietic stem cells show low expression. In addition, CEACAM1 also plays a role in the regulation of the innate and adaptive immune responses. Specifically, CEACAM1 has been shown to be an inhibitory receptor for activated T cells contained in human intestinal epithelium (Morales et al ., J. Immunol , 1999, 163, 1363-1370).

CEACAM1 also activates the immune checkpoint pathway as immune checkpoint agents such as PD-1 and CTLA-4. The immune checkpoint pathway consists of various co-stimulatory and inhibitory molecules that work in concert to protect tissues from damage by the immune system under physiological conditions and to maintain self-tolerance. When CEACAMl is activated, homologous affinity interactions of T cells and natural killer cells can induce inhibition of lymphocyte cytotoxic effects, thereby avoiding immunity. Thus, inhibiting the immune checkpoint pathway in cancer cells has emerged as a promising anticancer treatment strategy.

In addition, studies on several human tumor types have reported that tumors can evade immunity by using the CEACAM1 signaling pathway, and CEACAM1 interactions with monoclonal antibodies (mAb) in preclinical animal models of tumors. Has been shown to enhance the immune response to tumors.

On the other hand, the existing anti-cancer drugs, the side effects are very large compared to the anti-cancer effect. On the other hand, therapies that block immune checkpoints that have recently come into the spotlight attack cancer cells by activating inactivated immune cells instead of killing cancer cells directly. In the case of Merck's Keytruda treatment, the side effects are minimal compared to existing anticancer drugs, and the anticancer effect is excellent. Specifically, the kitruda therapeutic agent uses pembrolizumab, a PD-1 inhibitor. As a result of conducting Phase 2 clinical trial in the melanoma patient group, the anticancer response rate was 4% in the case of chemotherapy, but the Kitruda treatment was 25% (2 mg / kg) to 87% (10 mg / kg) anticancer The reaction rate is shown.

On the other hand, CEACAM1 inhibitory antibodies act on the cytotoxic T cells that play a major role in anti-cancer immune function, and on CEACAM1 expressed on the surface of natural killer cells and cancer cells, and in addition to CEACAM1, CEACAM3 and CEACAM5 are also present. Recognize. This is due to the characteristic of binding to the N domain with high homology between CEACAMl, CEACAM3 and CEACAM5.

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

In order to develop an anti-CEACAM1 antibody that specifically binds to CEACAM1, the present inventors have confirmed that the anti-CEACAM1 antibody binds to the N and B domains of CEACAM1 and has no cross-reactivity with CEACAM3, CEACAM5, CEACAM6 and CEACAM8. The present invention has been completed.

The present invention is represented by heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4 It provides an anti-CEACAM1 antibody or antibody fragment comprising a light chain CDR1 comprising an amino acid sequence, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6 .

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.

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

Furthermore, the present invention provides a composition for treating cancer, comprising an anticancer adjuvant and a cell therapy agent according to the present invention.

In addition, the present invention comprises the steps of: i) contacting a lymphocyte isolated from an individual with cancer with an anti-CEACAM1 antibody or antibody fragment thereof, ii) administering a lymphocyte in contact with the anti-CEACAM1 antibody or antibody fragment thereof. It provides an anticancer immunity treatment method comprising the step of.

Furthermore, the present invention provides a method of inhibiting proliferation of CEACAMl expressing tumor cells, comprising contacting CEACAMl expressing tumor cells with an anti-CEACAMl antibody or antibody fragment of the present 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 a cancer treatment composition by activating anticancer immune function of cytotoxic T cells and natural killer cells.

1A illustrates the structure of recombinant CEACAM1 prepared according to one embodiment.
Figure 1b is a diagram showing the binding force of the anti-CEACAM1 antibody (hereinafter, C25) according to the constituent domain of recombinant CEACAM1.
2 is a view comparing the binding force of recombinant CEACAM1 and C25 according to the concentration of C25.
3 is a diagram showing the binding force of CEACAM1 and C25 expressed on the surface of the Jurkat E6.1 T cell line according to the concentration of C25.
4 shows the affinity of C25 and CEACAM1. Affinity was obtained by the kinematic constants K _on and K _off and the equilibrium dissociation constant K _D.
5 is a photograph showing an isoelectric point of C25. Lane 1 is IEF-Markers 3-10, lane 2 is huIgG4, lane 3 is C25.
Figure 6a confirms the activation of Jurkat E6.1 T cell line by C25 through the expression of CD69, CD25 and Ki67 markers. The concentrations of C25 and huIgG4 are 10 μg / ml.
Figure 6b is a view showing the number of activated Jurkat E6.1 T cells and the amount of IL-2 secreted by C25. The concentrations of C25 and huIgG4 are 10 μg / ml.
Figure 6c confirms the activation of the Jurkat E6.1 T cell line by C25 through the CD69, CD25 and Ki67 marker expression. The concentrations of C25 and huIgG4 are 25 μg / ml.
Figure 6d is a diagram showing the number of activated Jurkat E6.1 T cells and the amount of IL-2 secreted by C25. The concentrations of C25 and huIgG4 are 25 μg / ml.
Figure 7a confirms the activation of the Jurkat E6.1 T cell line by C25 through the expression of CD69, CD25 and Ki67 markers. The concentration of αCD3 is 0.1 μg / ml.
Figure 7b is a diagram showing the number of activated Jurkat E6.1 T cells and the amount of IL-2 secreted by C25. The concentration of αCD3 is 0.1 μg / ml.
Figure 7c confirms the activation of the Jurkat E6.1 T cell line by C25 through CD69, CD25 and Ki67 marker expression. The concentration of αCD3 is 1 μg / ml.
Figure 7d is a diagram showing the number of activated Jurkat E6.1 T cells and the amount of IL-2 secreted by C25. The concentration of αCD3 is 1 μg / ml.
Figure 8a is a picture stained using C25 to confirm the degree of CEACAM1 expression in normal tissues of humans and monkeys.
8b is a photograph stained with huIgG4 in normal tissues of humans and monkeys
9 shows that C25 has no cross-reactivity with the CEACAM-3, 5, 6 and 8 receptors.
10 shows that C25 binds to CEACAM1 of monkeys as well as humans.
Figure 11a is a diagram showing the antitumor effect of TALL-104 T cells by C25.
Figure 11b is a diagram showing the antitumor effect of NK92MI cells by C25.

The present invention is represented by heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4 Providing an anti-CEACAM1 antibody or antibody fragment thereof comprising a light chain CDR1 comprising an amino acid sequence of interest, a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 6 do.

In addition, the term "antibody" as used in the present invention means an immunoprotein that binds to the antigen and interferes with the action of the antigen or removes the antigen. There are five kinds of antibodies, IgM, IgD, IgG, IgA, and IgE, and include heavy chains made from the heavy chain constant region genes μ, δ, γ, α, and ε, respectively. In antibody technology, IgG is mainly used, and there are four kinds of isotypes, IgG1, IgG2, IgG3, and IgG4, and their structural and functional characteristics may be different.

In addition, the IgG forms a Y-shaped highly stable structure (molecular weight, about 150 kDa) consisting of two heavy chain (about 50 kDa) proteins and two light chain (about 25 kDa) proteins. The light and heavy chains of an antibody are divided into variable regions with different amino acid sequences and constant regions with the same amino acid sequence, and the CH1, H (hinge), CH2, and CH3 domains in the heavy chain constant region. This exists. Each domain consists of two β-sheets and is connected by intramolecular disulfide bonds. The two variable regions of the heavy and light chains are combined to form an antigen binding site, which is present in each of the two Y-shaped arms. A fragment that is not able to bind an antigen is called a Fc (crystalizable fragment). Fab and Fc are connected by a flexible hinge region.

In addition, the term "CDR" used in the present invention is a hypervariable region, which is a site having an amino acid sequence for each antibody in the heavy and light chain variable regions of the antibody, and means a site that binds to the antigen. Looking at the conformation of the antibody, the CDRs have a loop shape on the surface of the antibody, and under the ring, there is a framework region (FR) that structurally supports it. There are three ring structures in the heavy and light chains, respectively, and these six ring region structures merge with each other to make direct contact with the antigen.

In addition, the anti-CEACAM1 antibody or antibody fragment thereof includes a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence represented by SEQ ID NO: 3 To have a heavy chain variable domain comprising a heavy chain CDR3. Preferably the antibody or antibody fragment thereof may comprise a heavy chain variable domain comprising the amino acid sequence represented by SEQ ID NO: 7. However, if the CDR1, CDR2, CDR3 of the heavy chain variable domain is the same, the framework (framework) portion can be modified. In particular, the amino acid sequences of some framework regions can be modified to produce humanized antibodies.

In addition, the anti-CEACAM1 antibody or antibody fragment thereof includes a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and an amino acid sequence represented by SEQ ID NO: 6 May have a light chain variable domain comprising a light chain CDR3. Preferably the antibody or antibody fragment thereof may comprise a light chain variable domain comprising the amino acid sequence represented by SEQ ID NO: 8. However, if the CDR1, CDR2, CDR3 of the light chain variable domain is the same, the backbone may be modified. In particular, the amino acid sequences of some framework regions can be modified to produce humanized antibodies.

In addition, the anti-CEACAM1 antibody or antibody fragment thereof of the present invention may include a heavy chain variable domain comprising an amino acid sequence represented by SEQ ID NO: 7 and a light chain variable domain comprising an amino acid sequence represented by SEQ ID NO: 8. However, if the CDR1, CDR2, CDR3 of the heavy and light chain variable domains are the same, the backbone may be modified. In particular, the amino acid sequences of some framework regions 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 refer to antigen-binding domains, except for the Fc region, which functions as an effector function to transfer antigen-stimulating binding to cells, complement, etc., and is a single domain antibody or a minibody. And third generation antibody fragments).

In addition, since the antibody fragment is smaller in size compared to the IgG of the complete structure, the penetration rate into tissues or tumors is good, and since the antibody fragments can be produced by bacteria, the production cost is low. Use it when you do not want the function to transfer the complement. Antibody fragments are suitable for diagnostic purposes because of their short half-life in the human body, but the replacement of some basic, acidic or neutral amino acids among the amino acids that make up an antibody can alter its unique isoelectric point (pI). have. Since the isoelectric point change of the antibody can induce changes such as reducing the toxic side effects of the antibody in vivo or increasing the water solubility, the therapeutic antibody can be used with a fully structured IgG in consideration of affinity or structural form.

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, or 97%, 98% or 99% homology with the heavy chain variable domain sequence It can be characterized.

In addition, an anti-CEACAM1 antibody or antibody fragment thereof of the present invention has a light chain variable domain sequence comprising the amino acid sequence represented by SEQ ID NO: 8, or at least 97%, 98% or 99% homology with the light chain variable domain sequence It may be characterized by.

In addition, the anti-CEACAM1 antibody or antibody fragment thereof of the present invention is at least 97% of the heavy chain variable domain sequence comprising the amino acid sequence represented by SEQ ID NO: 7 and the light chain variable domain sequence comprising the amino acid sequence represented by SEQ ID NO: 8, It may be characterized by having 98% or 99% homology.

In addition, the anti-CEACAM1 antibody means an antibody that binds to CEACAM1. The term "C25" as used in the present invention is one embodiment of the anti-CEACAM1 antibody.

In addition, the anti-CEACAM1 antibody or antibody fragment thereof recognizes the N and B domains of CEACAM1 as antigenic epitopes. 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 prepared by known monoclonal antibody production techniques. The method for preparing monoclonal antibodies can be performed by preparing hybridomas using B lymphocytes obtained from immunized animals, or can be performed by using phage display technology, but is 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 or antibody fragment thereof of the present invention as an active ingredient can be used to treat cancer or tumors overexpressing CEACAM1. Specifically, when the antigenic determinant of cancer or tumor cells is recognized by the TCR (T cell receptor) of cytotoxic T cells, which serve to remove cancer cells, they bind to one of the components of the TCR (cluster of differentiation 4). The lymphocyte-specific protein tyrosine kinase (LCK) protein phosphorylates the cluster of differentiation 3ζ (CD3ζ), another component of TCR. When ZAP70 (Zeta70-associated protein kinase 70) protein is bound to the phosphorylated CD3ζ, the end of ZAP70 protein is phosphorylated by LCK protein to activate RAS-MAPK signal transduction. The cell is activated.

However, in the case of cancer cells or tumor cells in which CEACAM1 is overexpressed, SHP1 (Src homology region) in the immunoreceptor tyrosine-based inhibition motif (ITIM) portion of CEACAM1 phosphorylated by LCK protein bound to the CD4 end of TCR due to CEACAM1-CEACAM1 interaction. 2 domain-containing phosphatase-1) proteins bind, and CD3ζ ends up dephosphorylation by SHP1 protein, which prevents RAS-MAPK signaling from activating T cells.

Thus, 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.

In addition, the term "anticancer" used in the present invention includes 'prevention' and 'treatment', where 'prevention' refers to any action that inhibits the proliferation of cancer or delays the progression of cancer by administration of the anticancer agent. As used herein, the term “treatment” refers to any behavior in which cancer symptoms 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 in that it is selected from the group consisting of pancreatic cancer, melanoma, lung cancer and myeloma, but is not limited thereto, and has an immune checkpoint pathway abnormally with CEACAM1 as a receptor. The cancer or tumor to be operated is not particularly limited and may include solid and hematological cancers.

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

The present invention also provides a cancer treatment composition comprising the anticancer adjuvant and a cell therapy agent. The cell therapy agent may include cytotoxic T cells or natural killer cells.

In addition, the term "cell therapeutic agent" used in the present invention refers to the biological characteristics of cells by proliferating and selecting in vitro viable autologous, allogenic, and xenogenic cells to restore the function of cells and tissues. By means of a series of acts that change the meaning of medicines used for prevention and treatment purposes. Specifically, it may be cytotoxic T cells, natural killer cells.

The present invention also provides an anti-cancer immunotherapy method using the anti-CEACAM1 antibody or antibody fragment thereof. Specifically, the method comprises the steps of: i) contacting lymphocytes isolated from an individual suffering from cancer with an anti-CEACAM1 antibody or antibody fragment thereof; ii) administering to the subject lymphocytes in contact with an anti-CEACAMl antibody or antibody fragment thereof.

In addition, the lymphocytes are a type of white blood cells, which are about 25% of all white blood cells, and may be NK cells, T cells, and B cells. Preferably it may be characterized by comprising at least one of cytotoxic T cells or natural killer cells.

In addition, the term "individual" used in the present invention refers to a person suffering from or in a condition in which the disease can be alleviated, suppressed or treated by administering the anticancer adjuvant of the present invention.

In addition, the term "contact" as used in the present invention means mixing an anti-CEACAM1 antibody or antibody fragment thereof with a cell expressing CEACAM1.

In addition, the term "administration" as used herein means introducing an effective amount of a substance into a subject in an appropriate manner, and the route of administration of the composition comprising the anti-CEACAM1 antibody or antibody fragment thereof of the invention is directed to the target tissue. Administration can be by any general route that can be reached. Specifically, it may be parenteral administration (eg, intravenous, subcutaneous, intraperitoneal, or topical administration) depending on the purpose of use, and preferably intravenous administration. In some cases, for administration to solid cancers, local administration may be desirable to facilitate and quick access of the antibody. Dosage varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the patient. Single doses may be administered in daily or weekly amounts in an amount from about 0.001 to 10 mg / kg. The effective amount can be adjusted at the discretion of the physician treating the patient.

In addition, the composition for treating cancer according to the present invention may be administered in a pharmaceutically effective amount to treat cancer cells or their metastases, or to inhibit the growth of cancer. It may vary depending on various factors such as the type of cancer, the age, weight of the patient, the nature and extent of symptoms, the type of current treatment, the number of treatments, the dosage form and the route, and can be easily determined by those skilled in the art. The compositions of the present invention may be administered together or sequentially with the pharmacological or physiological components described above, and may also be administered in combination with additional conventional therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. Such administration may be single or multiple administration. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

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

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

Example  1. CEACAM1  Antibody (C25)

Example  Paragraph 1.1 CEACAM1  Preparation of Antibodies

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

Thereafter, the heavy and light chain genes were inserted into the pcIW vector (Promega; Cat. # E1731) digested with the same restriction enzyme. Then, a restriction enzyme was used to select vectors containing both heavy and light chain transcription units. The selected vector was extracted using Endofree plasmid maxi kit (QIAGEN; Cat. # 12362), and finally, the nucleotide sequence of the antibody was confirmed by sequencing using some of the extracted DNA.

The extracted 30 μg antibody DNA was transfected into 30 mL of 2.5 × 10 ⁶ / mL Expi293F cell (ThermoFisher scientific; Cat. # A14527). The day after transfection, an enhancer (ThermoFisher; Cat. # A14524) was added to the transfected cells to produce C25 antibodies by incubating for 7 days at 37 ° C., 8% CO ₂ , and 125 rpm in a stirred incubator.

After incubation, 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. Thereafter, the beads were washed with 10 ml of binding buffer (ThermoFisher scientific; Cat. # 21019) and 200 μl of the eluting buffer (ThermoFisher scientific; Cat. # 21004) was added to the beads to separate C25 antibodies conjugated to the beads. The isolated antibody was neutralized by adding 10 μl of 1.5 M Tris-HCl pH 8.8 (Bio-rad; Cat. # 210005897).

Experimental Example  1. CEACAM1  Confirmation of antibody binding

Experimental Example  1.1. CEACAM1  Anti- by domain CEACAM1  Confirmation of antibody binding

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

2.5 μg of each purified CEACAM1 mutant protein was diluted in 1000 μl PBS, and then 20 μl of each CEACAM1 mutant protein was reacted at 4 ° C. for 16 hours. 1 μl of C25 After diluting in 1000 μl PBS, 25 μl was dispensed into each well and reacted at 37 ° C. for 1 hour. Thereafter, each well was washed three times with a washing buffer diluted with 10 µl of Tween20 (Sigma-aldrich; Cat. # P9146) in 990 µl PBS, and then human-conjugated with horse radish peroxidase (HRP). 1 μl of IgG (HRP-conjugated anti-human IgG: Sigma; Cat. # A0170) was diluted in 5000 μl PBS and 25 μl of each well was reacted at 37 ° C. for 1 hour. Then, to the wash buffer and washing each well 3 times, TMB solution (KPL;. Cat # 52-00-03) to 25 and then put ㎕ by inducing color development in each well, the concentration of 2M H ₂ SO ₄ 25 μl was added to each well to stop the reaction and the absorbance was measured at 450 nm.

As a result, N and B domains have been shown to be essential sites for binding with full affinity. A1 and A2 did not appear to be required for direct binding. Therefore, C25 mainly binds to the N domain of CEACAM1, and it was shown that an additional B domain is required to bind with complete affinity (FIG. 1B).

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

After diluting 2 μg of recombinant CEACAM1 protein in 1000 μl of PBS, 50 μl of 96-well-plate (Nunc; Cat. # 467679) was dispensed and reacted at 4 ° C. for 16 hours. Thereafter, 300 μl of 3% (v / v) bovine serum albumin was added to each well, followed by reaction at 37 ° C. for 1 hour. 0.75 μg of C25 was diluted in 1000 μl PBS. The diluted C25 solution was again diluted 14 times with PBS in a 1: 1 volume ratio. 50 μl of diluted C25 was dispensed into each well and reacted at 37 ° C. for 1 hour. Thereafter, each well was washed three times with a washing buffer diluted with 10 µl of Tween20 (Sigma-aldrich; Cat. # P9146) in 990 µl PBS, and 1 µl of HRP conjugated human IgG was diluted in 5000 µl PBS. 50 μl each well was added and allowed to react for 1 hour. Then, wash each well three times with the wash buffer, and 50 μl of TMB solution. After inducing color development in each well, 50 μl of ₂ M H ₂ SO 에 was added to each well to stop the reaction, and the absorbance was measured at 450 nm.

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

Experimental Example  1.3. Expressed on the cell surface With CEACAM1  Checking the binding force of C25

CEACAM1 cDNA was transfected into Jurkat cells (Jurkat E6.1 (ATCC; TIB-152TM) stably expressing CEACAM1 and selected by treatment with the antibiotic G418 700 μg / ml. ) Resuspend in FBS-added DPBS (hereinafter referred to as FACS buffer), centrifuge at 1,500 rpm, resuspend in FACS buffer so that the number of cells is 3x10 ⁶ / ml, and then each well of U-bottom 96-well plat 100 µl of each well was collected, and the cells and culture medium of each well were collected, centrifuged at 1,500 rpm, and the supernatant was discarded.The recovered cells were washed with a solution of human Fc block (BD Pharmingen; Cat. # 564220) in 0.5 ml. Resuspended in 50 μl FACS buffer, and incubated at 4 ° C. for 15 minutes.

C25 or human IgG4 (Sigma; Cat. # I4639) was added to 50 μl FACS buffer at 20 μg / ml, 10 μg / ml, 5 μg / ml, 2.5 μg / ml, 1.25 μg / ml, 0.625 μg / ml, 0.3125 μg / Ml, diluted twice to a concentration of 0.15625 μg / ml. 50 μl of diluted C25 or human IgG4 was added to the cells with Fc blocker, and 20 μg / ml, 10 μg / ml, 5 μg / ml, 2.5 μg / ml, 1.25 μg / ml, 0.625 μg / ml, 0.3125 [mu] g / ml and 0.15625 [mu] g / ml were adjusted to concentration, and the reaction was carried out at 4 DEG C for 1 hour and 30 minutes.

The cells reacted with C25 were resuspended in FACS buffer, and then washed twice by centrifugation at 1,500 rpm. Phycoerithrin-conjugated goat anti-human Fab'2 (Sigma; Cat. # P8047) labeled with phycoerythrin (hereinafter referred to as PE) was diluted 1: 200 in a FACS buffer. After 100 μl into each well, the reaction was performed in a dark place at 4 ° C. for 30 minutes.

The cells reacted with PE were recovered, resuspended in FACS buffer, and washed twice to centrifuge at 1,500 rpm to discard the supernatant. Cells were then resuspended in 100 μl fixed buffer (BD Cytofix ™; Cat. # 554655) and incubated in the dark at 4 ° C. for 30 minutes.

The cells reacted with the fixed buffer were recovered, resuspended in FACS buffer, and the washing was repeated twice to centrifuge at 1,500 rpm to discard the supernatant. The washed cells were resuspended in 200 [mu] l FACS buffer, and then compared to mean fluorescence intensity (MFI) of PE labeled cells on FACS LSR-Fortessa instrument. All FACS analysis used FlowJo software.

As a result, Up to 5 μg / ml showed the highest level of binding force (MFI 1800 and above), and then decreased sharply from the concentration below (FIG. 3).

Experimental Example  1.4. term- CEACAM1  Quantitative binding of antibodies

Quantitative binding of C25 to CEACAM1 was measured using a BIAcore T-100 (GE Healthcare, USA) biosensor. CEACAM1-Fc purified from HEK293 (ThermoFisher scientific; Cat. # A14527) cells was diluted to 10 μg / ml in acetate buffer (10 mM, pH 4.0), and then CM5 chip (GE) was prepared using an amine-carboxyl reaction. Healthcare, US) to 200 Rmax.

C25 was serially diluted four times in a 1: 1 volume ratio in HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20), followed by association for 120 seconds with CM5 chip. The flow rate was 30 μl / min for 1,200 seconds dissociation. 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. Using the BIAcore T-100 evaluation software, the affinity was calculated using the kinematic 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. Anti- CEACAM1  Identification of Antibody Properties

Experimental Example  2.1. term- CEACAM1  Antibody Isoelectric point  Confirm

20 ml of IEF anode buffer (50x) was mixed with 980 ml of deionized water (DW) to make 1xIEF anode buffer, and 20 ml of IEF cathode buffer pH3-10 (10x) was mixed with 180 ml of DW to make 1xIEF cathode buffer. 1xIEF anode buffer and 1xIEF cathode buffer were used after cooling to 4 ° C.

An IEF gel (Invitrogen / pH 3-10 IEF gel, 1.0 mmx10 well / EC6655BOX) was used to fill a 200 ml 1xIEF cathode buffer in the upper chamber and a 600 ml 1xIEF anode buffer in the lower chamber. 15 μl of a mixture of 20 μl of a mixture of 10 μl of 1.25 mg / ml C25 and 10 μl of IEF sample buffer pH 3-10 (2 ×) was loaded, and IEF Markers 3-10 (SERVA / 10mg) was used as a marker. / mL / SERVA Liquid Mix, 39212.01) 5 μl was used.

The electrophoresis was performed in three stages at 100V: 1hr, 200V: 1hr and 500V: 30min, and fixed for 30 minutes using 12% TCA solution. After fixation, the isoelectric point was confirmed after staining 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 is very high unlike the theoretical value of 7.76 (FIG. 5).

Experimental Example  3. Anti- CEACAM1  Antibodies T cell  Measure impact on activation

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

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

After 96 hours, cells and cultures were recovered and centrifuged at 1,500 rpm, and the supernatants were stored at -80 ° C for IL-2 secretion measurement, and the remaining cells were recovered, replaced with FACS buffer, and then at 15 ° C. The Fc receptor was blocked for a minute.

After treatment with a CD25-PE-Cy7 antibody or an anti-CD69-APC antibody (eBioscience; anti-CD25-PE-Cy7: Cat. # 25-0259; anti-CD69-APC: Cat. # 17-0699) The reaction was carried out at 4 ° C. for 15 minutes.

Cells and cultures were collected, filled with 200 μl of FACS buffer, and centrifuged at 1,200 rpm for 5 minutes to remove supernatant. After resuspending the cells in fresh FACS buffer, this procedure was repeated three times to completely remove the antibodies that did not adhere to the cells. The cells were resuspended in DPBS to which 1% (v / v) paraformaldehyde was added and then fixed at 4 ° C. for 30 minutes.

Cells were resuspended in 1 × FoxP3 staining buffer (eBioscience; Cat. # 00-5523-00) and centrifuged. After repeating this process twice, another dye-labeled anti-Ki67 antibody (eBioscience; Cat. # 350520) was diluted in a 1 × FoxP3 staining buffer at a ratio of 1: 100 by volume to 50 μl of the total volume and at 4 ° C. The reaction was carried out for 30 minutes. Cells and cultures were recovered, filled with FoxP3 staining buffer, and centrifuged. After repeating this process three times, the CD25 + CD69 + activated cells and Ki67 + proliferative cells were identified on the FACS LSR-Fortessa equipment and finally replaced with FACS buffer, and analyzed using FlowJo software.

As a result, the degree of activation of Jurkat T cells when CCM1 induced expression of Jurkat E6.1 (α-CD3 0.1 μg / ml, 4 days culture) was treated with 10 μg / ml and 25 μg / ml of C25. The ratio of CD25 + CD69 + Jurkat T cells expressing CD25 and CD69 expressing activated cells and the number of cells increased more than double in the control group huIgG4, and the ratio of Ki67 + Jurkat T cells expressing Ki67 expressing proliferating cells. The number of cells increased more than two times (Figs. 6A to 6D).

In addition, after transfecting CEACAM1 + Jurkat cells (EF1 promoter-CEACAM1-GFP vector prepared to CEACAM1 over Jurkat E6.1), treatment with the selection marker G418 disulfate salt solution (Sigma; G8168) at a concentration of 700 μg / ml. GFP + CEACAM1 + cells were isolated using a Fluorescence-Activated Cell Sorter.

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

After 48 hours, cells and cultures were recovered and centrifuged at 1,500 rpm, and the supernatants were stored at -80 ° C for IL-2 ELISA, and the remaining cells were recovered and replaced with FACS buffer for 15 minutes at 4 ° C. The Fc receptor was 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 cultures were collected, filled with FACS buffer to 200 μl, and centrifuged at 1,200 rpm for 5 minutes to remove supernatant. After resuspending the cells in fresh FACS buffer, this procedure was repeated three times to completely remove the antibodies that did not adhere to the cells. Cells were resuspended in DPBS to which 1% (v / v) formaldehyde (paraformaldehyde) was added and fixed at 4 ° C. for 30 minutes.

Cells were charged to 200 μl in a 1 × FoxP3 staining buffer and centrifuged at 1,200 rpm for 5 minutes to remove supernatant. After repeating this process twice, 50 μl of the other dye-labeled anti-Ki67 antibody was diluted at a volume ratio of 1: 100, and reacted at 4 ° C. for 30 minutes.

Cells were charged to 200 μl in a 1 × FoxP3 staining buffer and centrifuged at 1,200 rpm for 5 minutes to remove supernatant. After repeating this process three times, the CD25 + CD69 + activated cells and Ki67 + proliferating cells were identified on FACS LSR-Fortessa equipment after the last replacement with FACS buffer, and analyzed using FlowJo software.

As a result, the degree of activation of CEACAM1-Jurkat T cells increased by 1.5 times (C25 25 ㎍ / ml) or more than four times (C25 10 ㎍ / ml) in CD25 + CD69 + CEACAM1-Jurkat T cells compared to huIgG4 treatment group, Ki67 CEACAM1- Both ratios and numbers of Jurkat T cells were found to be more than doubled (C25 25 μg / ml) and up to 10 times (C25 10 μg / ml) (FIG. 7A to FIG. 7D).

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

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

 After washing 96 well microplates four times with a wash buffer diluted with 10 μl of Tween20 (Sigma-aldrich; Cat. # P9146) in 990 μl PBS, a biotinylated anti-IL-2 antibody (Detection antibody: eBioscience; Cat # 13-7028) in a blocking buffer diluted 1: 1,000 in a volume ratio of 100 ㎕ each well in each well was allowed to react for 2 hours at room temperature.

After washing the 96 well microplate four times with a washing buffer, 100 μl of a solution obtained by diluting a peroxidase-labeled streptavidin (Sigma; Cat. # S5512) in a blocking buffer at a ratio of 1: 1,000 by volume was added to each well. Put and reacted at room temperature for 2 hours.

After washing the 96 well microplate four times with a washing buffer, 100 μl of TMB peroxidase substrate solution (KPL; Cat. # 52-00-02) was added to each well and reacted for 10 minutes. (KPL; Cat. # 50-85-05) was added to 100 μl to stop the reaction. O.D values were measured at 450 nm using a molecular dynamics reader. The measurements were analyzed via SoftMax Pro 5.4.1 (Figures 6b, 6d, 7b and 7d).

As a result, it was measured in the culture supernatant when C25 at the antibody concentration of 10 μg / ml and 25 μg / ml was treated under CEACAM1 induced expression conditions (a-CD3 0.1 μg / ml, 4 days culture) of Jurkat E6.1. One IL-2 secretion was three times higher than the control (Fig. 6b, 6d).

In addition, antibody concentrations of 10 μg / ml and 25 μg of Jurkat cells stably expressing CEACAM1 were stimulated at low concentrations of anti-CD3 (0.1 μg / ml) or high concentrations of anti-CD3 (1 μg / ml). When treated with / ml of C25 for 48 hours, IL-2 secretion was higher than that of the control group (FIG. 7B, 7D), and especially for low concentrations of anti-CD3 stimulation, the effect of C25 on IL-2 secretion was Very large, it was confirmed that the increase from 10 times to 20 times than the control (Fig. 7b).

Experimental Example  4. Organizational in Humans and Monkeys CEACAM1  Expression confirmation

A 10 mM solution of EZ-Link ™ Sulo-NHS-LC-LC-LC-Biotin (ThermoFisher Scientific; Cat. # 21338) was made and biotinylated at 4 ° C. for 13.3 μl per 1 mg of C25. Human and monkey TMA slides were deparaffinized for 1 hour in a 60 ° C. oven. Xylene (100% Xylene), alcohol (alcohol 90%), alcohol (alcohol-80%), alcohol (alcohol-DW) in order to gradually hydration (hydration).

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

Primary antibodies (Primary antibody, biotin-conjugated hIgG4 or C25; 1 mg / ㎖) was diluted in a 1: 1 or 1: 5 volume ratio of 100 μl and then reacted at room temperature for 2 hours. After 100 µl of the ABC reagent was reacted for 30 minutes. After treatment with DAB substrate kit (VECTOR LABORATORIES; Cat # SK-4100), color development was performed for 2 minutes, followed by control staining with hematoxylin. Since CEACAM1 expression in normal cells is very low, antibodies were used at relatively high concentrations.

The degree of staining of normal tissues of Cynomolgus monkeys of C25 was similar to that of human normal tissues, and CEACAM1 expression levels in the normal tissues were very low, so it was confirmed that staining was carried out at high concentration (FIG. 8A). . Human IgG4 was used at the same concentration as a control for C25 (FIG. 8B).

Experimental Example  5. Anti- CEACAM1  Cross Reaction Check

10 μg of the pEF1? -AcGFP-N1 vector (Clontech, Cat No. 631973), CEACAM1, CEACAM3, CEACAM5, CEACAM6 and CEACAM8 plasmid vectors were added to HEK293 cells as a control vector, and pulse voltage 1100V, pulse was performed using a Neon transfection system. Transfection was performed under the conditions of width 20 ms and pulse number 2. After 48 hours of transfection, GFP expression was confirmed by fluorescence microscopy, and then cells were detached by 1 ml of TrypLE Express Solution (ThermoFisher Scientific; Cat. # 12605010) and added DMEM (Gibco, 10% (v / v) FBS). 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 × 10 ⁵ cells / 100 μl. 1 μl of human Fc Block solution was added to each sample and reacted at 4 ° C. for 10 minutes. huIgG4 or C25 was treated at a concentration of 1 μg / ⁵ × 10 ⁵ cells and reacted at 4 ° 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. The supernatant was removed by filling the FACS buffer to 200 μl and centrifuging for 5 minutes at 1,200 rpm. 300 μl of fixed buffer was added and the cells were resuspended. Cells expressing GFP were selected from FACS LSR-Fortessa equipment, and the expression was confirmed with specific antibodies for each CEACAM member 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 MouseIgG1 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 MouseIgG2a 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 MouseIgG1 Isotype Control PE R & D Systems, IC002P Anti-CEACAM8 antibody Human CEACAM8 PE-conjugated Antibody R & D systems / FAB4246P

As a result, C25 was characterized by binding only to CEACAM1 in the CEACAM member to which CEACAM1 belongs (Fig. 9).

In addition, 10 μg of each control vector, Cynomolgus CEACAM1 plasmid vector, and human CEACAM1 plasmid vector were added to HEK293 cells, and transfected under the conditions of pulse voltage 1100V, pulse width 20ms and pulse number 2 using a Neon transfection system. After 48 hours of transfection, GFP expression was confirmed by fluorescence microscopy, and then cells were removed by treatment with TrypLE Express solution and 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 × 10 ⁵ cells / 100 μl. 1 μl of human Fc Block solution was added per sample and reacted at 4 ° C. for 10 minutes. Human IgG4 or C25 was treated at a concentration of 1 μg / ⁵ × 10 ⁵ cells and reacted at 4 ° 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 each was reacted at 4 ° C. for 30 minutes. The supernatant was removed by filling the FACS buffer to 200 μl and centrifuging for 5 minutes at 1,200 rpm. The cells were resuspended in 300 μl fixed buffer. Cells expressing GFP were selected by FACS LSR-Fortessa equipment, and the expression was confirmed by specific antibodies for each CEACAM member according to CEACAM members. The binding of C25 was also confirmed by PE fluorescence channel and analyzed using FlowJo software.

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

Experimental Example  6. Anti- CEACAM1  Antibody Anticancer effect  Confirm

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

After incubation for 6 hours, the non-conjugated lysed 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 ㎕ of the diluted solution was added to each well and incubated for 3 hours in the dark. Thereafter, the O.D values of each sample were measured at a wavelength of 490 nm on a spectrophotometer, and the measured values were analyzed using the SoftMax Pro 5.4.1 program.

As a result, when the antitumor effect by CTL (FIG. 11A: TALL-104) or NK (FIG. 11B: NK-92MI) cells was confirmed as a method of 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 anticancer immune response of CTL and NK cells, whereas in the case of the (MKN1) gastric cell line in which CEACAM1 was not expressed at all, it showed no anticancer 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 seqeunce 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 seqeunce 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 seqeunce 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 seqeunce 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 seqeunce 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 seqeunce 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 Tyr 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 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 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 Tyr 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 seqeunce for anti-CEACAM1 antibody of CDR1 (VH) <400> 13 aattatgcta tgagc 15 <210> 14 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> nucleotide seqeunce 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 seqeunce 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 seqeunce 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 seqeunce 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 seqeunce 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)

Heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, and amino acid sequence represented by SEQ ID NO: 4 An anti-CEACAM1 antibody or antibody fragment thereof comprising a light chain CDR1 comprising a, a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 5, a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 6. The method of claim 1,
An anti-CEACAM1 antibody or antibody fragment thereof, characterized in that it is a heavy chain variable domain comprising the amino acid sequence represented by SEQ ID NO: 7. The method of claim 1,
An anti-CEACAM1 antibody or antibody fragment thereof, characterized in that it is a light chain variable domain comprising the amino acid sequence represented by SEQ ID NO: 8. The method of claim 1,
An anti-CEACAM1 antibody or antibody fragment thereof, comprising a heavy chain variable domain comprising an amino acid sequence represented by SEQ ID NO: 7 and a light chain variable domain comprising an amino acid sequence represented by SEQ ID NO: 8. The method of claim 1,
The antibody fragment is characterized in that any one selected from the group consisting of Fab, scFv and Fv, anti-CEACAM1 antibody or antibody fragment thereof. delete delete delete An anticancer agent comprising the anti-CEACAM1 antibody of claim 1 or an antibody fragment thereof as an active ingredient. The method of claim 9,
The cancer is characterized in that selected from the group consisting of pancreatic cancer, melanoma, lung cancer and myeloma, anticancer agent. An anticancer adjuvant comprising the anti-CEACAM1 antibody or antibody fragment thereof according to any one of claims 1 to 5 as an active ingredient. Claim 11 anticancer adjuvant and cell therapy comprising a composition for cancer treatment. The method of claim 12,
The cell therapy agent is characterized in that the cytotoxic T cells or natural killer cells, cancer treatment composition. An anti-cancer immunotherapy composition comprising lymphocytes isolated from an individual suffering from cancer and the anti-CEACAM1 antibody of claim 1 or an antibody fragment thereof. The method of claim 14,
The lymphocyte is characterized in that it comprises at least one of cytotoxic T cells and natural killer cells, anticancer immunotherapy composition. A composition for inhibiting proliferation of CEACAMl expressing tumor cells, comprising the anti-CEACAMl antibody or antibody fragment of claim 1.

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