KR20230061710A - Recombinant protein which recognizes CADM1 and the composition comprising the same for treating cancer - Google Patents
Recombinant protein which recognizes CADM1 and the composition comprising the same for treating cancer Download PDFInfo
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- KR20230061710A KR20230061710A KR1020210146231A KR20210146231A KR20230061710A KR 20230061710 A KR20230061710 A KR 20230061710A KR 1020210146231 A KR1020210146231 A KR 1020210146231A KR 20210146231 A KR20210146231 A KR 20210146231A KR 20230061710 A KR20230061710 A KR 20230061710A
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- cadm1
- recombinant protein
- cancer
- cell
- crtam
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Abstract
본 발명은 CADM1을 발현하는 세포에 독성을 갖는 재조합 단백질에 관한 것으로, 구체적으로 CADM1에 높은 친화력을 가진 CRTAM 단백질을 포함하는 재조합 단백질 및 이를 이용하여 CADM1을 발현하는 암에 대한 예방 또는 치료용 약학 조성물에 관한 것이다.
본 발명의 재조합 단백질 중 CRTAM의 세포외 도메인은 CADM1을 인지하여 CADM1이 발현되는 특정 암세포와 결합하고, 재조합 단백질의 인간 이뮤노글로불린 G1 중쇄 불변 영역부위(human IgG1 heavy chain constant region)는 자연살생세포의 IgG 수용체(FcγRIIIa, CD16a)와 결합하여 자연살생세포 내부로 활성화 신호를 전달해 줌으로써 CADM1이 발현되는 세포에 대한 세포독성을 가질 수 있게 할 뿐만 아니라 상기 재조합 단백질에 약물 접합체를 결합시켜 이용할 수도 있다. 따라서, 본 발명에 의한 재조합 단백질은 CADM1이 발현되는 세포에 특이적으로 세포독성을 갖는 치료제로서 유용하게 사용될 수 있다. The present invention relates to a recombinant protein having toxicity to cells expressing CADM1, and specifically, a recombinant protein containing a CRTAM protein having high affinity for CADM1, and a pharmaceutical composition for preventing or treating cancer expressing CADM1 using the same. It is about.
Among the recombinant proteins of the present invention, the extracellular domain of CRTAM recognizes CADM1 and binds to specific cancer cells expressing CADM1, and the human immunoglobulin G 1 heavy chain constant region of the recombinant protein is natural. It binds to the IgG receptors (FcγRIIIa, CD16a) of killer cells and transmits an activation signal to the inside of natural killer cells, enabling CADM1-expressing cells to have cytotoxicity, as well as combining drug conjugates to the recombinant protein for use. there is. Therefore, the recombinant protein according to the present invention can be usefully used as a therapeutic agent having cytotoxicity specifically to cells expressing CADM1.
Description
본 발명은 CRTAM 세포외 도메인을 포함하는 재조합 단백질에 관한 것으로, 구체적으로 CADM1에 높은 친화력을 가진 CRTAM 단백질을 포함하는 재조합 단백질 및 이를 이용하여 CADM1을 발현하는 암에 대한 치료용 약학 조성물에 관한 것이다.The present invention relates to a recombinant protein containing a CRTAM extracellular domain, and more particularly, to a recombinant protein containing a CRTAM protein having high affinity for CADM1 and a pharmaceutical composition for treating cancer expressing CADM1 using the same.
Cell adhesion molecule 1 (CADM1)은 Necl2, TSLC1, IGSF4, RA175, 또는 SynCam이라고도 불리우며, 면역 글로불린(Ig) 수퍼 패밀리(immunoglobulin superfamily)에 속하는 내재성 막 단백질(integral membrane protein)이(Kuramochi et al., 2001; Murakami, 2005; Takai et al., 2008). CADM1은 다양한 세포에서 관찰되며, 세포와 세포 간의 부착에 관여하여 여러가지 생명 현상을 조절할 수 있다고 알려져 있다(Murakami, 2005). CADM1은 비소세포 폐암(non-small lung cancer cell, NSCLC)에서 처음으로 발견되었고, 다양한 상피조직 유래 암종에서 발현되며, 몇몇 암종에서는 발현이 증가되어 암종의 분자 표지 또는 치료용 타겟으로 사용된다(Kuramochi et al., 2001; Murakami, 2005; Takai et al., 2008). 지금까지 보고된 CADM1을 발현하는 암종은 비소세포 폐암(non-small lung cancer cell, NSCLC)(Kuramochi et al., 2001), 폐 선암종(lung adenocarcinoma)(Murakami, 2005), 사람T세포림프친화바이러스 1형에 감염된 T 세포 백혈병 (HTLV-1-infected T cell leukemia)(Nakahata et al., 2012), 성인T세포백혈병(adult T cell leukemia)(Nakahata et al., 2021), 난소암(ovarian cancer)(Si et al., 2020), 유방암(breast cancer)(Takahashi et al., 2012), 자궁경부암(cervical cancer)(Steenbergen et al., 2004), 전립선암(prostate cancer)(Fukuhara et al., 2002), 상인두암(nasopharyngeal carcinoma)(Hui et al., 2003), 위암(stomach cancer)(Honda et al., 2002), 췌장암(pancreatic cancer)(Jansen et al., 2002) 으로 알려져 있다.Cell adhesion molecule 1 (CADM1), also called Necl2, TSLC1, IGSF4, RA175, or SynCam, is an integral membrane protein belonging to the immunoglobulin (Ig) superfamily (Kuramochi et al., 2001; Murakami, 2005; Takai et al., 2008). CADM1 is observed in various cells and is known to be involved in cell-to-cell adhesion to regulate various life phenomena (Murakami, 2005). CADM1 was first discovered in non-small lung cancer cell (NSCLC) and is expressed in various epithelial-derived carcinomas. et al., 2001; Murakami, 2005; Takai et al., 2008). Carcinomas expressing CADM1 reported so far are non-small lung cancer cell (NSCLC) (Kuramochi et al., 2001), lung adenocarcinoma (Murakami, 2005), human T-cell lymphotropic virus Type 1 infected T cell leukemia (HTLV-1-infected T cell leukemia) (Nakahata et al., 2012), adult T cell leukemia (Nakahata et al., 2021), ovarian cancer ) (Si et al., 2020), breast cancer (Takahashi et al., 2012), cervical cancer (Steenbergen et al., 2004), prostate cancer (Fukuhara et al. , 2002), nasopharyngeal carcinoma (Hui et al., 2003), stomach cancer (Honda et al., 2002), and pancreatic cancer (Jansen et al., 2002).
효과적인 암 치료를 위해서 직접적으로 암 세포를 표적하는 자연살생세포(natural killer cell, NK cell)가 중요하다는 사실이 보고되어왔고, 주요 기전으로 항체 의존성 세포 독성(antibody dependent cellular cytotoxicity, ADCC)을 유발하고자 하였다(Lu et al., 2020). 또한, 항체에 직접 독소 역할을 하는 화합물을 결합시켜, 항체가 인식하는 암을 직접 죽이는 항체-약물 접합체(antibody-drug conjugate)에 의한 세포독성을 유도하는 시도가 연구되고 있다(Alley et al., 2010). It has been reported that natural killer cells (NK cells) that directly target cancer cells are important for effective cancer treatment, and to induce antibody dependent cellular cytotoxicity (ADCC) as a major mechanism. (Lu et al., 2020). In addition, an attempt to induce cytotoxicity by an antibody-drug conjugate that directly kills cancer recognized by the antibody by binding a compound that acts as a toxin to the antibody is being studied (Alley et al., 2010).
한편, class-I MHC-restricted T cell associated molecule (CRTAM)은 내재성 막 단백질(integral membrane protein)로, 활성화된 림프구에서 발현되며(Kennedy et al., 2000), 세포독성 T 림프구(cytotoxic T lymphocyte)나 자연살생세포(natural killer cell, NK cell)가 표적세포의 세포자살을 유도할 때, 표적세포에 좀 더 잘 부착되게 하는 역할을 한다(Boles et al., 2005; Takeuchi et al., 2009). 또한 보조 T 림프구(helper T cell)의 IFN-γ와 IL-22 분비량을 촉진시킨다(Yeh et al., 2008). On the other hand, class-I MHC-restricted T cell associated molecule (CRTAM) is an integral membrane protein, expressed in activated lymphocytes (Kennedy et al., 2000), and cytotoxic T lymphocytes. ) or natural killer cells (NK cells), when they induce apoptosis of target cells, they play a role in better adhesion to target cells (Boles et al., 2005; Takeuchi et al., 2009). ). It also promotes the secretion of IFN-γ and IL-22 by helper T cells (Yeh et al., 2008).
현재, 항-CADM1 항체를 이용한 항체-약물 접합체(antibody drug conjugate, ADC)(한국공개특허 제10-2011-0126739호)나 CADM1의 특정 부분인 v9을 인식하는 항-CADM1 항체(미국공개특허 제2021-0079092호)는 공개되어 있으나, CRTAM을 포함하는 재조합 단백질을 이용하여 CADM1을 발현하는 단백질에 세포독성을 보임으로써 면역 항암 치료제로서 이용하는 발명에 대해서는 기재된 바 없다. Currently, an antibody-drug conjugate (ADC) using an anti-CADM1 antibody (Korean Patent Publication No. 10-2011-0126739) or an anti-CADM1 antibody recognizing v9, a specific part of CADM1 (US Patent Publication No. 2021-0079092) has been published, but there is no description about the invention of using a recombinant protein containing CRTAM as an immunocancer therapeutic agent by showing cytotoxicity to a protein expressing CADM1.
이러한 배경하에, 본 발명자들은 다양한 암세포에서의 CADM1 발현이 확인 또는 유도된다는 점과, 상기 CADM1과 CRTAM이 높은 친화도로 결합 가능하다는 점을 이용하여 CRTAM의 세포외 도메인(extracelular domain)을 사용한 CADM1을 발현하는 암 세포에 특이적으로 세포독성을 가지는 재조합 단백질을 제작함으로써 본 발명을 완성하였다.Under this background, the present inventors expressed CADM1 using the extracellular domain of CRTAM, taking advantage of the fact that CADM1 expression is confirmed or induced in various cancer cells and that the CADM1 and CRTAM can bind with high affinity. The present invention was completed by preparing a recombinant protein having cytotoxicity specifically to cancer cells.
따라서 본 발명의 목적은 CADM1에 특이적으로 결합하는 재조합 단백질을 제공하는 것이다.Accordingly, an object of the present invention is to provide a recombinant protein that specifically binds to CADM1.
본 발명의 다른 목적은 상기 재조합 단백질을 코딩하는 폴리 뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 벡터 및 상기 벡터로부터 형질전환된 세포를 제공하는 것이다.Another object of the present invention is to provide a polynucleotide encoding the recombinant protein, a vector containing the polynucleotide, and a cell transformed from the vector.
본 발명의 또 다른 목적은 상기 재조합 단백질을 유효성분으로 포함하여 CADM1을 발현하는 암의 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for treating cancer expressing CADM1, including the recombinant protein as an active ingredient.
상기와 같은 본 발명의 목적을 달성하기 위해서, 본 발명은 항원 결합 부위인 CRTAM(class-I MHC-restricted T cell associated molecule)의 세포외 도메인; Igκ(Ig kappa) 리더 펩타이드(reader peptide); 및 인간 이뮤노글로불린 G1 중쇄 불변 영역부위(human IgG1 heavy chain constant region)를 포함하는 재조합 단백질을 제공한다. In order to achieve the object of the present invention as described above, the present invention is an antigen binding site, the extracellular domain of CRTAM (class-I MHC-restricted T cell associated molecule); Ig kappa (Ig kappa) leader peptide (reader peptide); and a human immunoglobulin G1 heavy chain constant region.
또한, 본 발명은 상기 재조합 단백질을 코딩하는 폴리 뉴클레오티드, 상시 폴리 뉴클레오티드를 포함하는 벡터 및 상기 벡터로 형질전환된 세포를 제공한다.In addition, the present invention provides a polynucleotide encoding the recombinant protein, a vector always containing the polynucleotide, and a cell transformed with the vector.
또한, 본 발명은 상기 재조합 단백질이 CADM1을 발현하는 암세포의 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for treating cancer cells in which the recombinant protein expresses CADM1.
본 발명의 재조합 단백질 중 CRTAM의 세포외 도메인은 CADM1을 인지하여 CADM1이 발현되는 특정 세포와 결합하고, 재조합 단백질의 인간 이뮤노글로불린 G1 중쇄 불변 영역부위(human IgG1 heavy chain constant region)는 자연살생세포의 IgG 수용체(FcγRIIIa, CD16a)와 결합하여 자연살생세포 내부로 활성화 신호를 전달해 줌으로써 CADM1을 발현하는 암종에 특이적으로 세포독성을 갖게 되므로 암 치료를 위한 면역세포 치료제로서 유용하게 이용될 수 있다.Among the recombinant proteins of the present invention, the extracellular domain of CRTAM recognizes CADM1 and binds to specific cells expressing CADM1, and the human IgG1 heavy chain constant region of the recombinant protein is a natural killer cell. It binds to the IgG receptor (FcγRIIIa, CD16a) of natural killer cells and transmits an activation signal to the inside of natural killer cells, resulting in specific cytotoxicity to carcinoma expressing CADM1, so it can be usefully used as an immune cell therapeutic agent for cancer treatment.
도 1은 본 발명의 일 실시예에 따른 재조합 CRTAM 융합 단백질을 발현시키는 각 도메인의 cDNA 구역을 나타낸 모식도이다.
도 2는 본 발명의 일 실시예에 따른 발현 벡터(레트로바이럴 벡터)의 모식도이다.
도 3은 본 발명의 일 실시예에 따른 재조합 CRTAM 융합 단백질의 모식도이다.
도 4는 본 발명에서 제공하는 재조합 단백질의 타겟인 CADM1을 발현하는 암세포에서 CADM1의 발현량을 유세포 분석을 이용해 측정한 결과를 나타낸 도이다.
도 5a는 본 발명에서 제공하는 재조합 단백질을 Chinese hamster ovary (CHO) 세포에서 발현시킨 후, protein A column을 이용한 친화크로마토그래피(affinity chromatography)로 정제한 결과를 나타낸 도이다.
도 5b는 본 발명에서 제공하는 재조합 단백질을 인간 IgG 중쇄의 불변 영역을 인식하는 항체(항 인간 IgG 중쇄 불변영역 항체, anti-human IgG Fc region antibody)를 이용한 웨스턴 블로팅으로 확인한 결과를 나타낸 도이다.
도 5c는 본 발명에서 제공하는 재조합 단백질이 타겟인 CADM1을 발현하는 암세포를 인식하는지 여부를 유세포 분석을 이용해 나타낸 도이다.
도 6은 본 발명의 일 실시예에 따른 CD16a 발현 벡터(레트로바이럴 벡터)의 모식도이다.
도 7은 본 발명의 일 실시예에 따른 CD16a 발현 벡터를 레트로바이러스 시스템을 이용하여 형질도입시킨 자연살생세포(CD16a.NK92.MI)에서의 CD16a의 발현 비율을 공벡터(empty vector)를 형질도입시킨 자연살생세포(Mock.NK92.MI)와 비교한 결과를 나타낸 도이다.
도 8은 본 발명의 일 실시예에 따른 재조합단백질이 CADM1을 발현하는 암세포에 항체 의존성 세포 독성(antibody dependent cellular cytotoxicity, ADCC)을 유발하는지 여부를 비방사성 세포독성 분석법(non-radioactive cytotoxicity assay)으로 측정한 결과를 나타낸 도이다.1 is a schematic diagram showing cDNA regions of each domain expressing a recombinant CRTAM fusion protein according to an embodiment of the present invention.
2 is a schematic diagram of an expression vector (retroviral vector) according to an embodiment of the present invention.
3 is a schematic diagram of a recombinant CRTAM fusion protein according to an embodiment of the present invention.
4 is a diagram showing the results of measuring the expression level of CADM1 in cancer cells expressing CADM1, which is the target of the recombinant protein provided in the present invention, using flow cytometry.
Figure 5a is a diagram showing the results of purification by affinity chromatography using a protein A column after expressing the recombinant protein provided in the present invention in Chinese hamster ovary (CHO) cells.
Figure 5b is a view showing the results of Western blotting using an antibody (anti-human IgG heavy chain constant region antibody, anti-human IgG Fc region antibody) recognizing the human IgG heavy chain constant region of the recombinant protein provided in the present invention. .
5c is a diagram showing whether the recombinant protein provided in the present invention recognizes cancer cells expressing CADM1 as a target, using flow cytometry.
6 is a schematic diagram of a CD16a expression vector (retroviral vector) according to an embodiment of the present invention.
7 shows the expression ratio of CD16a in natural killer cells (CD16a.NK92.MI) transduced with a CD16a expression vector using a retroviral system according to an embodiment of the present invention. It is a diagram showing the results compared with the natural killer cells (Mock.NK92.MI).
8 is a non-radioactive cytotoxicity assay to determine whether the recombinant protein according to an embodiment of the present invention induces antibody dependent cellular cytotoxicity (ADCC) in cancer cells expressing CADM1. It is a diagram showing the measured results.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
본 발명은 하나의 양태로서, CRTAM의 세포외 도메인; Igκ(Ig kappa) 리더 펩타이드(reader peptide); 및 인간 이뮤노글로불린 G1 중쇄 불변 영역부위(human IgG1 heavy chain constant region)를 포함하는 재조합 단백질을 제공한다.The present invention, as one aspect, the extracellular domain of CRTAM; Ig kappa (Ig kappa) leader peptide (reader peptide); and a human immunoglobulin G 1 heavy chain constant region.
본 발명에서 제공하는 "재조합 단백질" 중 CRTAM의 세포외 도메인은 CADM1이 발현되는 특정 세포와 결합하고, 재조합 단백질의 인간 이뮤노글로불린 G1 중쇄 불변 영역부위(human IgG1 heavy chain constant region)는 자연살생세포의 IgG 수용체(FcγRIIIa, CD16a)와 결합하여 자연살생세포 내부로 활성화 신호를 전달해 준다. 결국, 이러한 신호는 자연살생세포를 활성화시켜 CADM1이 발현되는 세포에 대한 세포독성을 가질 수 있게 한다. 또한, 상기 재조합 단백질에 약물 접합체를 결합시켜, 상기 재조합 단백질이 CADM1이 발현되는 특정 세포와 결합하고, 상기 재조합 단백질에 결합된 약물 접합체에 의해 CADM1이 발현되는 세포에 대한 세포독성을 가질 수 있게 한다. Among the "recombinant proteins" provided by the present invention, the extracellular domain of CRTAM binds to specific cells in which CADM1 is expressed, and the human immunoglobulin G 1 heavy chain constant region of the recombinant protein is natural. It binds to the IgG receptor (FcγRIIIa, CD16a) of the killer cell and transmits the activation signal to the inside of the natural killer cell. Ultimately, these signals activate natural killer cells, allowing them to have cytotoxicity against cells expressing CADM1. In addition, by binding a drug conjugate to the recombinant protein, the recombinant protein binds to a specific cell in which CADM1 is expressed, and has cytotoxicity to cells in which CADM1 is expressed by the drug conjugate bound to the recombinant protein. .
본 발명에서 "CADM1"은 다양한 암종(malignant cells)에서 관찰된다. 지금까지 "CADM1"이 발현되는 암종은 비소세포 폐암(non-small lung cancer cell, NSCLC), 폐 선암종(lung adenocarcinoma), 사람T세포림프친화바이러스 1형에 감염된 T 세포 백혈병(HTLV-1-infected T cell leukemia), 성인T세포백혈병(adult T cell leukemia), 난소암(ovarian cancer), 유방암(breast cancer), 자궁경부암(cervical cancer), 전립선암(prostate cancer), 상인두암(nasopharyngeal carcinoma), 위암(stomach cancer) 및 췌장암(pancreatic cancer)으로 이루어지는 군으로부터 선택될 수 있으나, 이에 제한되지 않는다.In the present invention, "CADM1" is observed in various malignant cells. So far, carcinomas in which "CADM1" is expressed are non-small lung cancer cell (NSCLC), lung adenocarcinoma, and human T-cell lymphotropic virus type 1-infected T-cell leukemia (HTLV-1-infected). T cell leukemia), adult T cell leukemia, ovarian cancer, breast cancer, cervical cancer, prostate cancer, nasopharyngeal carcinoma, It may be selected from the group consisting of stomach cancer and pancreatic cancer, but is not limited thereto.
본 발명의 재조합 단백질은 CADM1에 특이적으로 결합하는 것을 특징으로 하며, 상기 "CRTAM"은 서열번호 1 또는 이와 95% 이상의 상동성을 나타내는 아미노산 서열로 이루어진 것일 수 있으나, 이에 제한되는 것은 아니다. 또한, 본 발명에 따른 재조합 단백질의 범위는 서열번호 1로 표시되는 아미노산 서열을 갖는 단백질 및 상기 단백질의 기능적 동등물을 포함한다. '기능적 동등물’이란 아미노산의 부가, 치환, 또는 결실의 결과, 상기 서열번호 1로 표시된 아미노산 서열과 적어도 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 서열번호 1로 표시되는 단백질과 실질적으로 동질의 생리활성을 나타내는 단백질을 말한다. '실질적으로 동질의 생리활성'이란 CADM1에 특이적으로 결합할 수 있는 활성을 가진 것을 의미한다.The recombinant protein of the present invention is characterized in that it specifically binds to CADM1, and the "CRTAM" may consist of SEQ ID NO: 1 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto. In addition, the scope of the recombinant protein according to the present invention includes a protein having the amino acid sequence represented by SEQ ID NO: 1 and functional equivalents of the protein. 'Functional equivalent' means at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably It refers to a protein that has 95% or more sequence homology and exhibits substantially the same physiological activity as the protein represented by SEQ ID NO: 1. 'Substantially homogeneous physiological activity' means having an activity capable of specifically binding to CADM1.
상기 CRTAM의 세포외 도메인에 위치한 CADM1 결합부위의 N'말단에 Igκ(Ig kappa) 리더 펩타이드(reader peptide)가 연결되며, 상기 CADM1 결합부위의 C'말단에 인간 이뮤노글로불린 G1 중쇄 불변 영역부위(human IgG1 heavy chain constant region)가 연결될 수 있으나, 이에 한정되는 것은 아니다. 상기 Igκ(Ig kappa) 리더 펩타이드(reader peptide)는 서열번호 2로 표시되는 아미노산 서열을 포함하며, 인간 이뮤노글로불린 G1 중쇄 불변 영역부위(human IgG1 heavy chain constant region)는 서열번호 3으로 표시되는 아미노산 서열을 포함할 수 있으며, 서열번호 2 또는 서열번호 3으로 표시된 아미노산 서열과 적어도 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 서열번호 2 또는 서열번호 3으로 표시되는 단백질과 실질적으로 동질의 생리활성을 나타내는 단백질을 말한다.An Igκ (Ig kappa) reader peptide is linked to the N' end of the CADM1 binding site located in the extracellular domain of the CRTAM, and a human immunoglobulin G 1 heavy chain constant region at the C' end of the CADM1 binding site. (human IgG 1 heavy chain constant region) may be linked, but is not limited thereto. The Ig kappa (Ig kappa) leader peptide includes the amino acid sequence represented by SEQ ID NO: 2, and the human immunoglobulin G 1 heavy chain constant region is represented by SEQ ID NO: 3 It may include an amino acid sequence that is, at least 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 It refers to a protein having a homology and exhibiting substantially the same physiological activity as the protein represented by SEQ ID NO: 2 or SEQ ID NO: 3.
또한, 본 발명의 다른 하나의 양태는 상기 기술한 ‘CADM1'에 특이적으로 결합하는 재조합 단백질’을 코딩 (암호화)할 수 있는 폴리 뉴클레오티드이다. 본 발명의 재조합 단백질을 암호화하는 폴리 뉴클레오티드는 코돈의 축퇴성 (degeneracy)으로 인하여 또는 상기 항원 수용체를 발현시키고자 하는 생물에서 선호되는 코돈을 고려하여, 코딩 영역으로부터 발현되는 항원 수용체의 아미노산 서열을 변화시키지 않는 범위 내에서 코딩영역에 다양한 변형이 이루어질 수 있고, 코딩영역을 제외한 부분에서도 유전자의 발현에 영향을 미치지 않는 범위 내에서 다양한 변형 또는 수식이 이루어질 수 있으며, 그러한 변형 유전자 역시 본 발명의 범위에 포함됨을 당업자는 잘 이해할 수 있을 것이다. 즉, 본 발명의 폴리 뉴클레오티드는 이와 동등한 활성을 갖는 단백질을 코딩하는 한, 하나 이상의 핵산 염기가 치환, 결실, 삽입 또는 이들의 조합에 의해 변이될 수 있으며, 이들 또한 본 발명의 범위에 포함된다. In addition, another aspect of the present invention is a polynucleotide capable of encoding (encoding) the above-described 'recombinant protein that specifically binds to CADM1'. The polynucleotide encoding the recombinant protein of the present invention changes the amino acid sequence of the antigen receptor expressed from the coding region due to codon degeneracy or in consideration of codons preferred in organisms intended to express the antigen receptor. Various modifications may be made to the coding region within a range not specified, and various modifications or modifications may be made to a part other than the coding region within a range that does not affect gene expression, and such modified genes are also within the scope of the present invention. It will be well understood by those skilled in the art. That is, as long as the polynucleotide of the present invention encodes a protein having an activity equivalent thereto, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention.
또한, 본 발명의 다른 하나의 양태는 상기 폴리 뉴클레오티드를 포함하는 벡터, 상기 벡터로 형질전환된 세포이다.Another aspect of the present invention is a vector containing the polynucleotide and a cell transformed with the vector.
본 발명에서 사용되는 벡터는 당 분야에 공지된 벡터를 다양하게 사용할 수 있고, 상기 재조합 단백질을 생산하고자 하는 숙주세포의 종류에 따라 프로모터 (promoter), 종결자 (terminator), 인핸서 (enhancer) 등과 같은 발현조절 서열, 막 표적화 또는 분비를 위한 서열 등을 적절히 선택하고 목적에 따라 다양하게 조합할 수 있다. 본 발명의 벡터는 플라스미드 벡터, 코즈미드 벡터, 박테리오 파아지 벡터 및 바이러스 벡터 등을 포함하나 이에 제한되지 않는다. 적합한 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 같은 발현 조절 엘리먼트 외에도 막 표적화 또는 분비를 위한 시그널 서열 또는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있다.Vectors used in the present invention may use various vectors known in the art, and depending on the type of host cell to produce the recombinant protein, such as a promoter, terminator, enhancer, etc. Expression control sequences, sequences for membrane targeting or secretion, etc. may be appropriately selected and combined in various ways depending on the purpose. Vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors and viral vectors. Suitable vectors include expression control elements such as promoters, operators, initiation codons, stop codons, polyadenylation signals and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and may be prepared in various ways depending on the purpose.
본 발명의 용어, "치료"는 상기 조성물의 투여에 의해 CADM1을 발현하는 암에 의한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term "treatment" refers to all activities that improve or beneficially change symptoms caused by cancer expressing CADM1 by administration of the composition.
상기 조성물은 약학적으로 허용 가능한 담체를 포함할 수 있다.The composition may include a pharmaceutically acceptable carrier.
상기 "약학적으로 허용 가능한 담체"란 생물체를 자극하지 않으면서, 주입되는 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 의미할 수 있다. 본 발명에 사용 가능한 상기 담체의 종류는 특별히 제한되지 아니하며 당해 기술 분야에서 통상적으로 사용되고 약학적으로 허용되는 담체라면 어느 것이든 사용할 수 있다. 상기 담체의 비제한적인 예로는, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사 용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 등을 들 수 있다. 이들은 단독으로 사용되거나 2종 이상을 혼합하여 사용될 수 있다.The "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not inhibit the biological activity and properties of the compound to be injected without irritating living organisms. The type of the carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more.
약학적으로 허용 가능한 담체를 포함하는 상기 조성물은 경구 또는 비경구의 여러 가지 제형일 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다.The composition containing a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
상세하게는, 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 화합물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로오스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 흔히 사용되는 단순 희석제인 물, 액체 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 오일, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.Specifically, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose, and lactose in the compound. , can be prepared by mixing gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions for internal use, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.
상기 조성물은 약학적으로 유효한 양으로 투여할 수 있다.The composition can be administered in a pharmaceutically effective amount.
상기 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 감염된 바이러스 종류, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.The "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is dependent on the type and severity of the subject, age, sex, infected virus type, and drug activity, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitantly used drugs and other factors well known in the medical arts.
상기 투여는 어떠한 적절한 방법으로 환자에게 본 발명의 조성물을 도입하는 것을 의미하며, 상기 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 복강 내 투여, 정맥내 투여, 근육 내 투여, 피하 투여, 피내 투여, 경구 투여, 국소 투여, 비 내 투여될 수 있으나, 이에 제한되지는 않는다.The administration means introducing the composition of the present invention to a patient by any suitable method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration may be administered, but is not limited thereto.
본 발명의 조성물을 매일 투여 또는 간헐적으로 투여해도 좋고, 1일당 투여 횟수는 1회 또는 2~3회로 나누어 투여하는 것이 가능하다. 두 유효성분이 각각 단제인 경우의 투여횟수는 같은 횟수여도 좋고, 다른 횟수로 해도 된다. 또한, 본 발명의 조성물은 혈액암의 예방 또는 치료를 위하여 단독으로, 또는 다른 약물 치료와 병용하여 사용할 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.The composition of the present invention may be administered daily or intermittently, and the number of administrations per day may be administered once or divided into 2 to 3 times. The number of administrations when the two active ingredients are each a single agent may be the same or may be different. In addition, the composition of the present invention can be used alone or in combination with other drug treatments for the prevention or treatment of blood cancer. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, and can be easily determined by those skilled in the art.
상기 개체란, CADM1을 발현하는 암이 발병하였거나 발병할 수 있는 인간과, 원숭이, 소, 말, 양, 돼지, 닭, 칠면조, 메추라기, 고양이, 개, 마우스, 쥐, 토끼 또는 기니아 피그를 포함한 모든 동물을 의미한다. 본 발명의 약학적 조성물을 개체에게 투여함으로써 상기 질환을 효과적으로 예방 또는 치료할 수 있다면 개체의 종류는 제한 없이 포함된다.The subject refers to all humans, including humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs that have or may develop cancer expressing CADM1. means animals. The type of subject is included without limitation as long as the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject.
본 발명에 있어 치료 대상이 되는 질환은 종양이 있다. 상기 종양의 종류로는 지금까지 "CADM1"이 발현되는 암종은 비소세포 폐암(non-small lung cancer cell, NSCLC), 폐 선암종(lung adenocarcinoma), 사람T세포림프친화바이러스 1형에 감염된 T 세포 백혈병(HTLV-1-infected T cell leukemia), 성인T세포백혈병(adult T cell leukemia), 난소암(ovarian cancer), 유방암(breast cancer), 자궁경부암(cervical cancer), 전립선암(prostate cancer), 상인두암(nasopharyngeal carcinoma), 위암(stomach cancer) 및 췌장암(pancreatic cancer)으로 이루어지는 군으로부터 선택될 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the disease to be treated is a tumor. As for the types of tumors, carcinomas in which "CADM1" is expressed so far include non-small lung cancer cell (NSCLC), lung adenocarcinoma, and T-cell leukemia infected with human T-cell lymphotropic virus type 1. (HTLV-1-infected T cell leukemia), adult T cell leukemia, ovarian cancer, breast cancer, cervical cancer, prostate cancer, merchant It may be selected from the group consisting of nasopharyngeal carcinoma, stomach cancer and pancreatic cancer, but is not limited thereto.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited to these examples.
실시예Example 1. 유전자 합성 방법에 의한 수용성 재조합 1. Soluble recombination by gene synthesis method CRTAMCRTAM 융합 단백질 유전자의 of the fusion protein gene 클로닝cloning
본 발명의 수용성 재조합 CRTAM 융합 단백질을 제조하기 위해서 CRTAM의 세포외 도메인(extracellular domain) 각각을 인간 이뮤노글로불린 G1 중쇄 불변 영역(IgG1 heavy chain constant region)에 연결시킨 단백질 암호화 서열을 유전자 데이터베이스(database)에서 확인하였다. 그리고 상기 도메인들을 이루는 염기서열의 종결 코돈 부분을 제외한 나머지 서열들을 하나로 이어 유전자 합성을 진행하였다. 유전자 합성의 정확도는 단백질 발현 플라스미드를 제작한 후 시퀀싱을 통하여 확인하였다. 상기 재조합 단백질을 발현시키는 각 도메인의 cDNA 구역을 나타낸 모식도를 도 1에 나타내었다.In order to prepare the water-soluble recombinant CRTAM fusion protein of the present invention, the protein coding sequence in which each of the extracellular domains of CRTAM is linked to the human immunoglobulin G1 heavy chain constant region (IgG1 heavy chain constant region) is genetic database confirmed in In addition, gene synthesis was performed by connecting the remaining sequences except for the stop codon portion of the base sequence constituting the domains into one. The accuracy of gene synthesis was confirmed through sequencing after constructing a protein expression plasmid. A schematic diagram showing the cDNA region of each domain expressing the recombinant protein is shown in FIG. 1 .
실시예 2. 수용성 재조합 CRTAM 융합 단백질 발현 플라스미드 제조Example 2. Preparation of water soluble recombinant CRTAM fusion protein expression plasmid
인간 CADM1을 인식하는 CRTAM의 세포외 도메인과 인간 이뮤노글로불린 G1 중쇄 불변 영역을 융합시킨 재조합 단백질을 Chinese hamster ovary (CHO) 세포에 발현시키기 위해, 해당 유전자를 레트로바이러스 유래 발현벡터인 pLNCX2(Addgene)에 클로닝하였다. To express in Chinese hamster ovary (CHO) cells a recombinant protein in which the extracellular domain of CRTAM recognizing human CADM1 is fused with the human immunoglobulin G 1 heavy chain constant region, the corresponding gene was used as a retrovirus-derived expression vector, pLNCX2 (Addgene ) was cloned into.
먼저, pLNCX2 벡터에 클로닝하기 위해서 5′말단에 제한효소 XhoI의 절단 부위 서열을 만드는 프라이머를 합성하여 중합효소연쇄반응(polymerase chain reaction, PCR)으로 제한효소 절단 부위를 만들고, 3′말단에도 제한효소 NotI의 절단 부위 서열을 만드는 프라이머를 합성하여 중합효소연쇄반응으로 제한효소 절단 부위를 만들었다. 그 후 중합효소연쇄반응으로 인해 제한효소 절단 부위를 가진 유전자를 5′말단은 XhoI을 처리하였으며, 3′말단은 NotI을 처리하였다. 그리고 발현 벡터의 다중클로닝 자리를 xhoI과 NotI을 처리하여 유전자가 삽입될 수 있게 만들었다. 제한효소가 처리된 유전자와 발현 벡터를 섞어 준 다음, 결합 효소(ligase)를 처리하여 연결하였다. First, in order to clone into the pLNCX2 vector, a primer that creates a restriction enzyme Xho I cleavage site sequence is synthesized at the 5' end, a restriction enzyme cleavage site is created by polymerase chain reaction (PCR), and restriction is also made at the 3' end. A restriction enzyme cleavage site was prepared by synthesizing a primer that creates the cleavage site sequence of the enzyme Not I and polymerase chain reaction. After that, the 5' end of the gene having a restriction enzyme cleavage site due to polymerase chain reaction was treated with Xho I, and the 3' end was treated with Not I. In addition, the multicloning site of the expression vector was treated with xho I and Not I so that the gene could be inserted. After mixing the restriction enzyme-treated gene and the expression vector, they were ligated by treatment with a ligase.
그 결과 인간 CADM1을 인식하는 CRTAM의 세포외 도메인과 인간 이뮤노글로불린 G1 중쇄 불변 영역을 융합시킨 단백질(도 3 참조)을 발현하는 재조합 벡터 CRTAM-IgG.pLNCX2를 완성하였다(도 2 참조). As a result, a recombinant vector CRTAM-IgG.pLNCX2 expressing a protein obtained by fusing the extracellular domain of CRTAM recognizing human CADM1 with the human immunoglobulin G 1 heavy chain constant region (see FIG. 3) was completed (see FIG. 2).
실시예Example 3. 3. CADM1을CADM1 발현하는 사람 세포주 screening screening human cell lines that express
CADM1을 세포 표면에 발현하는 사람 세포주를 screening하기 위해서, 폐 선암종(lung adenocarcinoma) 유래 세포주인 A549 (ATCC)에서 CADM1의 발현을 확인하였다. 발현 확인을 위해 형광단백질이 부착된 CADM1과 결합이 가능한 항체(Biobyt)를 상기 세포주들에 처리한 후, 유세포 분석(fluorescence-activated cell sorting)을 이용하였다. 분석결과, A549 세포주에서 CADM1을 발현하는 것을 확인하였다(도 4 참조). To screen human cell lines expressing CADM1 on the cell surface, expression of CADM1 was confirmed in A549 (ATCC), a cell line derived from lung adenocarcinoma. To confirm expression, the cell lines were treated with an antibody (Biobyt) capable of binding to CADM1 to which a fluorescent protein was attached, and then fluorescence-activated cell sorting was used. As a result of the analysis, it was confirmed that CADM1 was expressed in the A549 cell line (see FIG. 4).
상기 결과를 근거로 CADM1을 발현하는 A549 세포주를 타겟 세포로 이용하여, 제작한 수용성 재조합 CRTAM 융합 단백질(CRTAM-Ig)에 대한 친화도를 측정하였다. Based on the above results, the affinity for the prepared water-soluble recombinant CRTAM fusion protein (CRTAM-Ig) was measured using the A549 cell line expressing CADM1 as a target cell.
실시예Example 4. 수용성 재조합 4. Water Soluble Recombination CRTAMCRTAM 융합 단백질과 fusion protein and CADM1을CADM1 발현하는 사람 세포주의 친화도 측정 Affinity measurement of expressing human cell lines
제작한 수용성 재조합 CRTAM 융합 단백질이 CADM1을 발현하는 사람 세포주와 친화도가 있는지 확인하기 위하여, 수용성 재조합 CRTAM 융합 단백질을 CHO 세포에서 발현시킨 후, protein A column을 이용하여 친화크로마토그래피(affinity chromatography) (GE healthcare)로 정제하였다(도 5a 참조).In order to confirm the affinity of the prepared soluble recombinant CRTAM fusion protein with a human cell line expressing CADM1, after expressing the soluble recombinant CRTAM fusion protein in CHO cells, affinity chromatography was performed using a protein A column ( GE healthcare) was purified (see Fig. 5a).
또한, 정제한 수용성 키메릭 항원 수용체를 인간 IgG 중쇄의 불변 영역을 인식하는 항체(항 인간 IgG 중쇄 불변영역 항체, anti-human IgG Fc region antibody, abcam)를 이용하여 웨스턴 블로팅으로 확인하였다(도 5b 참조).In addition, the purified water-soluble chimeric antigen receptor was confirmed by Western blotting using an antibody (anti-human IgG heavy chain constant region antibody, anti-human IgG Fc region antibody, abcam) recognizing the human IgG heavy chain constant region (Fig. see 5b).
그 후, 상기 정제한 수용성 재조합 CRTAM 융합 단백질을 CADM1을 발현하는 세포주(A549 세포주)에 처리하여 수용성 재조합 CRTAM 융합 단백질이 해당 세포와 결합이 가능한지 유세포 분석을 통하여 확인한 결과를 도 5c에 나타내었다.Thereafter, the purified water-soluble recombinant CRTAM fusion protein was treated with CADM1-expressing cell line (A549 cell line), and it was confirmed through flow cytometry whether the water-soluble recombinant CRTAM fusion protein could bind to the cells. The result is shown in FIG. 5C.
도 5c에 나타난 바와 같이, CADM1을 발현하는 A549 세포는 control Ig (isotype control)과 결합하지 않았으나, 수용성 CRTAM 융합 단백질(CRTAM-Ig)과는 결합하는 것을 확인하였다.As shown in FIG. 5c , it was confirmed that A549 cells expressing CADM1 did not bind to control Ig (isotype control), but bound to soluble CRTAM fusion protein (CRTAM-Ig).
상기 결과에서 확인된 바와 같이, 제작한 수용성 CRTAM 융합 단백질이 CADM1을 발현하는 사람 세포주와 친화도가 높음을 확인하였고, 이는 CADM1 발현 세포주인 A549 세포를, 제작한 수용성 CRTAM 융합 단백질(CRTAM-Ig)의 CADM1 단백질 특이적 세포독성 검증을 위해 사용할 수 있다는 것을 의미한다. 따라서, A549 세포를 표적세포로 하여 CRTAM-Ig를 이용한 항체 의존성 세포 독성(antibody dependent cell cytotoxicity, ADCC)을 측정하였다.As confirmed from the above results, it was confirmed that the prepared water-soluble CRTAM fusion protein had high affinity with the human cell line expressing CADM1, which was confirmed by using A549 cells, a CADM1-expressing cell line, with the produced water-soluble CRTAM fusion protein (CRTAM-Ig). This means that it can be used for the verification of CADM1 protein-specific cytotoxicity. Therefore, antibody dependent cell cytotoxicity (ADCC) was measured using CRTAM-Ig using A549 cells as target cells.
실시예Example 5. 자연살생세포에 5. To natural killer cells IgGIgG 수용체( receptor ( FcγRIIIaFcγRIIIa , , CD16aCD16a ) 발현) expression
상기 제작한 수용성 재조합 CRTAM 융합 단백질이 CADM1을 발현하는 세포에 항체 의존성 세포 독성(antibody dependent cellular cytotoxicity, ADCC)을 유발하는지를 시험하기 위해, 자연살생세포(Natural killer cell, NK cell)인 NK92.MI 세포(ATCC)에 IgG 수용체(FcγRIIIa, CD16a)를 발현시켰다. In order to test whether the prepared water-soluble recombinant CRTAM fusion protein induces antibody dependent cellular cytotoxicity (ADCC) in cells expressing CADM1, natural killer cell (NK cell) NK92.MI cells (ATCC) expressed IgG receptors (FcγRIIIa, CD16a).
우선, 인간 CD16a의 유전자 합성을 진행하였고, 유전자 합성의 정확도는 단백질 발현 플라스미드를 제작한 후 시퀀싱을 통하여 확인하였다(GenBank bumber: NM_000569.7). 그 후, 인간 CD16a를 NK92.MI 세포에 발현시키기 위해, 해당 유전자를 레트로바이러스 유래 발현벡터인 pLNCX2(Addgene)에 제한효소 HindIII와 NotI의 절단 부위를 이용하여 인간 CD16a.pLNCX2를 클로닝하였다(도 6참조). 그 후, 인간 CD16a를 NK92.MI 세포에 발현시킨 후, 항체와 유세포분석기를 이용하여, NK92.MI 세포에서 인간 CD16a의 발현을 항 인간 CD16a 항체(anti-human CD16a antibody, BD biosciences)와 유세포분석기(flow cytometry)로 확인하였다. 이때 음성 대조군으로는 NK92.MI 세포에 공벡터(empty pLNCX2)만을 발현시킨 세포(Mock.NK92.MI)를 사용하였다(도 7 참조). First, gene synthesis of human CD16a was performed, and the accuracy of gene synthesis was confirmed by sequencing after constructing a protein expression plasmid (GenBank bumber: NM_000569.7). Then, in order to express human CD16a in NK92.MI cells, human CD16a.pLNCX2 was cloned into pLNCX2 (Addgene), a retrovirus-derived expression vector, using restriction enzymes Hind III and Not I cleavage sites ( see Figure 6). Then, after expressing human CD16a in NK92.MI cells, using an antibody and flow cytometry, the expression of human CD16a in NK92.MI cells was measured by anti-human CD16a antibody (BD biosciences) and flow cytometry. It was confirmed by flow cytometry. At this time, as a negative control, cells (Mock.NK92.MI) in which only the empty vector (empty pLNCX2) was expressed in NK92.MI cells were used (see FIG. 7).
상기 결과에서 확인된 바와 같이, 인간 CD16a이 세포 표면에 발현하는 NK92.MI 세포(CD16a.NK92.MI)를 제작한 수용성 융합 단백질의 CADM1 단백질 특이적 항체 의존성 세포독성(ADCC, antibody dependent cellular cytotoxicity, ADCC) 검증을 위해 사용할 수 있다는 것을 확인하였다.As confirmed from the above results, the CADM1 protein-specific antibody dependent cellular cytotoxicity (ADCC) of the soluble fusion protein prepared in NK92.MI cells (CD16a.NK92.MI) expressing human CD16a on the cell surface ADCC) confirmed that it can be used for verification.
실시예Example 6. 수용성 재조합 6. Soluble recombination CRTAMCRTAM 융합 단백질의 of fusion proteins CADM1CADM1 발현 세포 특이적 항체 의존성 세포 독성 검증 Verification of cell-specific antibody-dependent cytotoxicity
상기 제작한 수용성 재조합 CRTAM 융합 단백질이 CADM1을 발현하는 세포에 항체 의존성 세포 독성(antibody dependent cellular cytotoxicity, ADCC)을 유발하는지를 시험하기 위해, 상기 실시예 3과 4에서 선정한 표적 세포(target cell)인 CADM1을 발현하는 A549 세포를 상기 실시예 5에서 제작된 인간 CD16a를 발현하는 NK92.MI 세포(CD16a.NK cell)를 작동 세포(effector)로 하여 제작한 수용성 재조합 CRTAM 융합 단백질(1 μg/ml)을 배양액에 넣고 각각 6시간 동안 공배양 하였다. 이때, 융합 단백질의 음성 대조군으로 control Ig (human IgG, 1 μg/ml)를 사용하였다. 공배양 후 상층액에 존재하는 젖산 탈수소 효소(lactate dehydrogenase)의 양으로 각각의 자연살생세포(CD16a.NK cell 또는 mock.NK cell)의 표적 세포에 대한 세포독성 정도를 측정하는 비방사성 세포독성 분석법(non-radioactive cytotoxicity assay)을 이용하였다. In order to test whether the prepared water-soluble recombinant CRTAM fusion protein induces antibody dependent cellular cytotoxicity (ADCC) in cells expressing CADM1, the target cell selected in Examples 3 and 4, CADM1 The water-soluble recombinant CRTAM fusion protein (1 μg/ml) prepared by using A549 cells expressing human CD16a-expressing NK92.MI cells (CD16a.NK cells) prepared in Example 5 as an effector was It was put into the culture medium and co-cultured for 6 hours each. At this time, control Ig (human IgG, 1 μg/ml) was used as a negative control for the fusion protein. A non-radioactive cytotoxicity assay that measures the degree of cytotoxicity of each natural killer cell (CD16a.NK cell or mock.NK cell) on target cells by the amount of lactate dehydrogenase present in the supernatant after co-culture. (non-radioactive cytotoxicity assay) was used.
먼저, 96 well 세포 배양 접시의 well에 작동 세포(1x105 cell)와 표적 세포(1x104 cell)를 각각 넣어 effector: target ratio를 10:1로 맞추고, well당 부피는 100μl가 되도록 접종하였다. 그 후 제작한 수용성 재조합 CRTAM 융합 단백질(1 μg/ml) 또는 control Ig (human IgG, 1 μg/ml)를 넣어주고, 원심분리기를 이용해 250g에서 4분 동안 원심분리하여 세포 간 간격을 가깝게 만든다. 6시간 배양 후 각 well의 상층액을 50μl씩 걷어내어 흡광도 측정용 투명 96 well 접시에 옮긴 후 분석용액 및 1M 염산용액을 처리하여 효소 반응을 진행 및 정지시킨다. 효소 반응을 정지시키고 난 후에는 형광/발광/흡광 측정기(multi-detection plate reader)를 이용하여 490nm 파장대의 흡광도를 측정 및 수치 변환하여 각 well의 작동 세포에 의한 표적 세포의 세포독성 정도를 정량화하였다.First, effector cells (1x10 5 cell) and target cells (1x10 4 cell) were put into wells of a 96-well cell culture dish, respectively, and the effector: target ratio was adjusted to 10:1, and the volume per well was inoculated to be 100 μl. Thereafter, the prepared water-soluble recombinant CRTAM fusion protein (1 μg/ml) or control Ig (human IgG, 1 μg/ml) was added and centrifuged at 250 g for 4 minutes using a centrifuge to close the cell spacing. After 6 hours of incubation, 50 μl of the supernatant from each well is removed and transferred to a transparent 96-well dish for measuring absorbance, and then the enzyme reaction is progressed and stopped by treatment with analysis solution and 1M hydrochloric acid solution. After stopping the enzyme reaction, the absorbance in the 490 nm wavelength band was measured and converted into numbers using a fluorescence/luminescence/absorption meter (multi-detection plate reader) to quantify the degree of cytotoxicity of target cells by effector cells in each well. .
그 결과 도 8에서 나타낸 바와 같이, 제작한 재조합 수용성 CRTAM 단백질을 넣어주고, 표적 세포(target cell)로 CADM1을 발현하는 A549 세포를 공배양 했을 때는 32%의 매우 높은 독성을 보인 반면, control Ig (human IgG)를 넣어주고, 표적 세포(target cell)로 A549 세포를 공배양 했을 때는 7.6%의 독성을 보였다. As a result, as shown in FIG. 8, when the recombinant soluble CRTAM protein was added and co-cultured with A549 cells expressing CADM1 as target cells, a very high toxicity of 32% was shown, whereas control Ig ( human IgG) was added, and when A549 cells were co-cultured as target cells, toxicity was 7.6%.
상기 결과를 통해 제작한 수용성 재조합 CRTAM 융합 단백질이 CADM1 단백질을 발현하는 세포에 특이적인 항체 의존성 세포 독성(antibody dependent cellular cytotoxicity, ADCC)을 유발한다는 것을 확인함으로써, 상기 재조합 단백질을 이용하여 효과적으로 관련된 암 질환 치료가 가능함을 확인할 수 있었다.Through the above results, it was confirmed that the prepared water-soluble recombinant CRTAM fusion protein induces antibody dependent cellular cytotoxicity (ADCC) specific to cells expressing the CADM1 protein, thereby effectively related cancer diseases using the recombinant protein It was confirmed that treatment was possible.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.
<110> IMMUNOLOGICAL DESIGNINGLAB CO., LTD <120> Recombinant protein which recognizes CADM1 and the composition comprising the same for treating cancer <130> KP21-0047-ILDL <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 270 <212> PRT <213> Artificial Sequence <220> <223> CRTAM extracellular domain <400> 1 Ser Leu Thr Asn His Thr Glu Thr Ile Thr Val Glu Glu Gly Gln Thr 1 5 10 15 Leu Thr Leu Lys Cys Val Thr Ser Leu Arg Lys Asn Ser Ser Leu Gln 20 25 30 Trp Leu Thr Pro Ser Gly Phe Thr Ile Phe Leu Asn Glu Tyr Pro Ala 35 40 45 Leu Lys Asn Ser Lys Tyr Gln Leu Leu His His Ser Ala Asn Gln Leu 50 55 60 Ser Ile Thr Val Pro Asn Val Thr Leu Gln Asp Glu Gly Val Tyr Lys 65 70 75 80 Cys Leu His Tyr Ser Asp Ser Val Ser Thr Lys Glu Val Lys Val Ile 85 90 95 Val Leu Ala Thr Pro Phe Lys Pro Ile Leu Glu Ala Ser Val Ile Arg 100 105 110 Lys Gln Asn Gly Glu Glu His Val Val Leu Met Cys Ser Thr Met Arg 115 120 125 Ser Lys Pro Pro Pro Gln Ile Thr Trp Leu Leu Gly Asn Ser Met Glu 130 135 140 Val Ser Gly Gly Thr Leu His Glu Phe Glu Thr Asp Gly Lys Lys Cys 145 150 155 160 Asn Thr Thr Ser Thr Leu Ile Ile His Thr Tyr Gly Lys Asn Ser Thr 165 170 175 Val Asp Cys Ile Ile Arg His Arg Gly Leu Gln Gly Arg Lys Leu Val 180 185 190 Ala Pro Phe Arg Phe Glu Asp Leu Val Thr Asp Glu Glu Thr Ala Ser 195 200 205 Asp Ala Leu Glu Arg Asn Ser Leu Ser Ser Gln Asp Pro Gln Gln Pro 210 215 220 Thr Ser Thr Val Ser Val Thr Glu Asp Ser Ser Thr Ser Glu Ile Asp 225 230 235 240 Lys Glu Glu Lys Glu Gln Thr Thr Gln Asp Pro Asp Leu Thr Thr Glu 245 250 255 Ala Asn Pro Gln Tyr Leu Gly Leu Ala Arg Lys Lys Ser Gly 260 265 270 <210> 2 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Human Ig kappa leader peptide <400> 2 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp 20 <210> 3 <211> 234 <212> PRT <213> Artificial Sequence <220> <223> IgG1 heavy chain constant region <400> 3 Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 1 5 10 15 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 20 25 30 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 35 40 45 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 50 55 60 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 65 70 75 80 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 85 90 95 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 100 105 110 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 115 120 125 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 130 135 140 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 145 150 155 160 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 165 170 175 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 180 185 190 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 210 215 220 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 4 <211> 810 <212> DNA <213> Artificial Sequence <220> <223> CRTAM extracellular domain <400> 4 tctctgacta accacacaga aaccatcacc gtggaggaag gccagacgct cactctaaag 60 tgtgtcactt ctctgaggaa gaactcctcc ctccagtggc tgaccccctc agggttcacc 120 atttttttaa atgagtatcc tgctttaaaa aattccaaat accagcttct tcatcactcg 180 gccaatcagc tctccatcac tgtgcctaac gtaaccctgc aagatgaagg cgtgtacaag 240 tgcttacatt acagcgactc tgtaagcaca aaggaagtga aagtgattgt gctggcaact 300 cctttcaagc caatcctgga agcttcagtt atcagaaagc aaaatggaga agaacatgtt 360 gtactcatgt gctccaccat gagaagcaag ccccctccgc agataacctg gctacttggg 420 aatagcatgg aagtgtccgg tggaacgctc catgaatttg aaactgatgg gaagaaatgt 480 aatactacca gcactctcat aatccacact tatggcaaaa attcaacggt ggactgcatt 540 atccgacaca gaggcctgca agggagaaaa ctagtagcac ccttccggtt tgaagatttg 600 gttactgatg aagagacagc ttcagatgct ctggagagaa actctctatc ctctcaagac 660 ccacagcagc ccaccagtac tgtctcagta acggaagatt ctagtacatc ggagattgac 720 aaggaagaga aagaacaaac cactcaagat cctgacttga ccaccgaagc aaatcctcag 780 tatttaggac tggcaagaaa gaaaagtggc 810 <210> 5 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Human Ig kappa leader peptide <400> 5 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 gac 63 <210> 6 <211> 705 <212> DNA <213> Artificial Sequence <220> <223> IgG1 heavy chain constant region <400> 6 ggatccgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 60 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 120 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 180 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 240 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 300 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 360 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 420 tcacgagatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 480 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 540 acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 600 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 660 aaccactaca cgcagaagag cctctccctg tctccgggta aatga 705 <210> 7 <211> 765 <212> DNA <213> Artificial Sequence <220> <223> Human CD16a <400> 7 atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact 60 gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagggt gctcgagaag 120 gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg 180 tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca 240 gtcgacgaca gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg 300 cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360 gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca 420 tatttacaga atggcaaagg caggaagtat tttcatcata attctgactt ctacattcca 480 aaagccacac tcaaagacag cggctcctac ttctgcaggg ggcttgttgg gagtaaaaat 540 gtgtcttcag agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca 600 tcattctttc cacctgggta ccaagtctct ttctgcttgg tgatggtact cctttttgca 660 gtggacacag gactatattt ctctgtgaag acaaacattc gaagctcaac aagagactgg 720 aaggaccata aatttaaatg gagaaaggac cctcaagaca aatga 765 <110> IMMUNOLOGICAL DESIGNINGLAB CO., LTD <120> Recombinant protein which recognizes CADM1 and the composition including the same for treating cancer <130> KP21-0047-ILDL <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 270 <212> PRT <213> artificial sequence <220> <223> CRTAM extracellular domain <400> 1 Ser Leu Thr Asn His Thr Glu Thr Ile Thr Val Glu Glu Gly Gln Thr 1 5 10 15 Leu Thr Leu Lys Cys Val Thr Ser Leu Arg Lys Asn Ser Ser Leu Gln 20 25 30 Trp Leu Thr Pro Ser Gly Phe Thr Ile Phe Leu Asn Glu Tyr Pro Ala 35 40 45 Leu Lys Asn Ser Lys Tyr Gln Leu Leu His His Ser Ala Asn Gln Leu 50 55 60 Ser Ile Thr Val Pro Asn Val Thr Leu Gln Asp Glu Gly Val Tyr Lys 65 70 75 80 Cys Leu His Tyr Ser Asp Ser Val Ser Thr Lys Glu Val Lys Val Ile 85 90 95 Val Leu Ala Thr Pro Phe Lys Pro Ile Leu Glu Ala Ser Val Ile Arg 100 105 110 Lys Gln Asn Gly Glu Glu His Val Val Leu Met Cys Ser Thr Met Arg 115 120 125 Ser Lys Pro Pro Pro Gln Ile Thr Trp Leu Leu Gly Asn Ser Met Glu 130 135 140 Val Ser Gly Gly Thr Leu His Glu Phe Glu Thr Asp Gly Lys Lys Cys 145 150 155 160 Asn Thr Thr Ser Thr Leu Ile Ile His Thr Tyr Gly Lys Asn Ser Thr 165 170 175 Val Asp Cys Ile Ile Arg His Arg Gly Leu Gln Gly Arg Lys Leu Val 180 185 190 Ala Pro Phe Arg Phe Glu Asp Leu Val Thr Asp Glu Glu Thr Ala Ser 195 200 205 Asp Ala Leu Glu Arg Asn Ser Leu Ser Ser Gln Asp Pro Gln Gln Pro 210 215 220 Thr Ser Thr Val Ser Val Thr Glu Asp Ser Ser Thr Ser Glu Ile Asp 225 230 235 240 Lys Glu Glu Lys Glu Gln Thr Thr Gln Asp Pro Asp Leu Thr Thr Glu 245 250 255 Ala Asn Pro Gln Tyr Leu Gly Leu Ala Arg Lys Lys Ser Gly 260 265 270 <210> 2 <211> 21 <212> PRT <213> artificial sequence <220> <223> Human Ig kappa leader peptide <400> 2 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp 20 <210> 3 <211> 234 <212> PRT <213> artificial sequence <220> <223> IgG1 heavy chain constant region <400> 3 Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 1 5 10 15 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 20 25 30 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 35 40 45 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 50 55 60 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 65 70 75 80 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 85 90 95 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 100 105 110 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 115 120 125 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 130 135 140 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 145 150 155 160 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 165 170 175 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 180 185 190 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 210 215 220 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 4 <211> 810 <212> DNA <213> artificial sequence <220> <223> CRTAM extracellular domain <400> 4 tctctgacta accacacaga aaccatcacc gtggaggaag gccagacgct cactctaaag 60 tgtgtcactt ctctgaggaa gaactcctcc ctccagtggc tgaccccctc agggttcacc 120 atttttttaa atgagtatcc tgctttaaaa aattccaaat accagcttct tcatcactcg 180 gccaatcagc tctccatcac tgtgcctaac gtaaccctgc aagatgaagg cgtgtacaag 240 tgcttacatt acagcgactc tgtaagcaca aaggaagtga aagtgattgt gctggcaact 300 cctttcaagc caatcctgga agcttcagtt atcagaaagc aaaatggaga agaacatgtt 360 gtactcatgt gctccaccat gagaagcaag ccccctccgc agataacctg gctacttggg 420 aatagcatgg aagtgtccgg tggaacgctc catgaatttg aaactgatgg gaagaaatgt 480 aatactacca gcactctcat aatccacact tatggcaaaa attcaacggt ggactgcatt 540 atccgacaca gaggcctgca agggagaaaa ctagtagcac ccttccggtt tgaagatttg 600 gttactgatg aagagacagc ttcagatgct ctggagagaa actctctatc ctctcaagac 660 ccacagcagc ccaccagtac tgtctcagta acggaagatt ctagtacatc ggagattgac 720 aaggaagaga aagaacaaac cactcaagat cctgacttga ccaccgaagc aaatcctcag 780 tatttaggac tggcaagaaa gaaaagtggc 810 <210> 5 <211> 63 <212> DNA <213> artificial sequence <220> <223> Human Ig kappa leader peptide <400> 5 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 gac 63 <210> 6 <211> 705 <212> DNA <213> artificial sequence <220> <223> IgG1 heavy chain constant region <400> 6 ggatccgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 60 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 120 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 180 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcggggag 240 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 300 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 360 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 420 tcacgagatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 480 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 540 acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 600 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 660 aaccactaca cgcagaagag cctctccctg tctccgggta aatga 705 <210> 7 <211> 765 <212> DNA <213> artificial sequence <220> <223> Human CD16a <400> 7 atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact 60 gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagggt gctcgagaag 120 gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg 180 tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca 240 gtcgacgaca gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg 300 cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360 gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca 420 tatttacaga atggcaaagg caggaagtat tttcatcata attctgactt ctacattcca 480 aaagccacac tcaaagacag cggctcctac ttctgcaggg ggcttgttgg gagtaaaaat 540 gtgtcttcag agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca 600 tcattctttc cacctgggta ccaagtctct ttctgcttgg tgatggtact cctttttgca 660 gtggacacag gactatattt ctctgtgaag acaaacattc gaagctcaac aagagactgg 720 aaggaccata aatttaaatg gagaaaggac cctcaagaca aatga 765
Claims (9)
상기 항원 결합 도메인은 CRTAM의 세포외 도메인 한 쌍이 이합체(dimer)의 형태로 각각 인간 이뮤노글로블린 G1 중쇄 불변 영역(IgG1 heavy chain constant region)에 연결된 것을 특징으로 하는 재조합 단백질.According to claim 1,
The antigen-binding domain is a recombinant protein, characterized in that a pair of extracellular domains of CRTAM are linked to a human immunoglobulin G 1 heavy chain constant region, respectively, in the form of a dimer.
상기 CRTAM의 세포외 도메인은 서열번호 1로 표시되는 아미노산 서열을 포함하는 것인, 재조합 단백질.According to claim 2,
The extracellular domain of the CRTAM is a recombinant protein comprising the amino acid sequence represented by SEQ ID NO: 1.
Igκ(Ig kappa) 리더 펩타이드(reader peptide)는 서열번호 2로 표시되는 아미노산 서열을 포함하며, 인간 이뮤노글로불린 G1 중쇄 불변 영역부위(human IgG1 heavy chain constant region)는 서열번호 3으로 표시되는 아미노산 서열을 포함하는 것인, 재조합 단백질.According to claim 1,
The Ig kappa (Ig kappa) leader peptide includes the amino acid sequence represented by SEQ ID NO: 2, and the human immunoglobulin G 1 heavy chain constant region is represented by SEQ ID NO: 3 A recombinant protein comprising an amino acid sequence.
상기 CADM1을 발현하는 암은 비소세포 폐암(non-small lung cancer cell, NSCLC), 폐 선암종(lung adenocarcinoma), 사람T세포림프친화바이러스 1형에 감염된 T 세포 백혈병(HTLV-1-infected T cell leukemia), 성인T세포백혈병(adult T cell leukemia), 난소암(ovarian cancer), 유방암(breast cancer), 자궁경부암(cervical cancer), 전립선암(prostate cancer), 상인두암(nasopharyngeal carcinoma), 위암(stomach cancer) 및 췌장암(pancreatic cancer)으로 이루어지는 군으로부터 선택되는 것을 특징으로 하는 조성물.According to claim 8,
Cancers expressing the CADM1 include non-small lung cancer cell (NSCLC), lung adenocarcinoma, and human T-cell lymphotropic virus type 1-infected T-cell leukemia (HTLV-1-infected T cell leukemia). ), adult T cell leukemia, ovarian cancer, breast cancer, cervical cancer, prostate cancer, nasopharyngeal carcinoma, stomach cancer cancer) and pancreatic cancer (pancreatic cancer) characterized in that the composition is selected from the group consisting of.
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KR20110126739A (en) | 2009-03-05 | 2011-11-23 | 옥스포드 바이오테라퓨틱스 리미티드 | Fully human antibodies specific to cadm1 |
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