KR102115236B1 - Chimera antigen receptors for treating pancreatic cancer or biliary tract cancer - Google Patents

Chimera antigen receptors for treating pancreatic cancer or biliary tract cancer Download PDF

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KR102115236B1
KR102115236B1 KR1020190009135A KR20190009135A KR102115236B1 KR 102115236 B1 KR102115236 B1 KR 102115236B1 KR 1020190009135 A KR1020190009135 A KR 1020190009135A KR 20190009135 A KR20190009135 A KR 20190009135A KR 102115236 B1 KR102115236 B1 KR 102115236B1
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유다경
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Abstract

본 발명은 신규한 키메라 항원 수용체와, 이의 용도로 췌장암 또는 담관계암 치료에 관한 것으로, 보다 상세하게는 항원 결합 도메인; 힌지 영역; 및 신호전달 도메인을 포함하며, 상기 항원 결합 도메인은 카보하이드레이트 항원 19-9(carbohydrate antigen 19-9, CA 19-9)에 결합하는 것인, 키메라 항원 수용체에 관한 것이다.
본 발명에서 제공하는 키메라 항원 수용체를 발현할 수 있도록 형질 전환된 T 세포 또는 NK세포는 췌장암 또는 담관계암 세포를 특이적으로 인식하고, 그 사멸을 유도하여 최종적으로는 췌장암 또는 담관계암을 효과적으로 예방 또는 치료할 수 있다. The present invention relates to a novel chimeric antigen receptor and its use for the treatment of pancreatic cancer or cholangiocarcinoma, more specifically an antigen binding domain; Hinge area; And a signaling domain, wherein the antigen-binding domain binds to a carbohydrate antigen 19-9, CA 19-9, and is related to a chimeric antigen receptor.
T cells or NK cells transformed to express the chimeric antigen receptor provided by the present invention specifically recognize pancreatic cancer or bile duct cancer cells, and induce their death, thereby effectively reducing pancreatic cancer or bile duct cancer. It can be prevented or treated.

Figure 112019008649483-pat00009
Figure 112019008649483-pat00009

Description

췌장 또는 담관계암 치료를 위한 키메라 항원 수용체{Chimera antigen receptors for treating pancreatic cancer or biliary tract cancer}Chimeric antigen receptors for treating pancreatic cancer or biliary tract cancer}

본 발명은 신규한 키메라 항원 수용체와, 이의 용도로 췌장암 또는 담관계암의 치료에 관한 것이다. The present invention relates to novel chimeric antigen receptors and treatment of pancreatic cancer or cholangiocarcinoma for their use.

췌장암은 췌장에 발생하는 암을 의미한다. 담관계암은 간에서 생성된 담즙이 배출되는 통로인 담관계에 발생하는 암을 총칭하는 것으로 간내담관암 (Intrahepatic cholangiocarcinoma), 간외담관암 (extrahepatic bile duct cancer), 담낭암(gallbladder cancer)과 바터팽대부암 (ampulla of Vater cancer)을 포함한다. 이들 암종은 기전상 암 줄기세포에서 발생하는 것으로 생각된다 (Sell and Dunsford Am J. Pathol. 134:1347-1363, 1989). 세계적으로 췌장담관계암의 발생은 국가와 지역에 따른 차이를 보인다. 췌장암의 경우에는 미주, 유럽 등 서구와 일본에서 보다 빈발하나 담관계암은 서구보다 국내에서 보다 호발한다. 췌장담관계암은 진행된 상태에서 발견되는 경우가 많아 외과적 절제율이 매우 낮고 절제 후에도 높은 재발율을 보이기 때문에 난치성 암종에 포함된다. 더구나, 항암화학요법이나 방사선요법에 대한 반응율이 낮은 특징을 가지며 (Pederson et al Cancer Res. 4325-4332, 1997), 이들 암종의 진단이 쉽지 않아 조기에 발견되는 비율이 낮아 예후가 극히 불량하다 Pancreatic cancer refers to cancer that occurs in the pancreas. Bile duct cancer refers to cancer that occurs in the bile ducts, which is a channel through which bile produced in the liver is discharged, and intrahepatic cholangiocarcinoma, extrahepatic bile duct cancer, gallbladder cancer and barter swelling cancer ( ampulla of Vater cancer). These carcinomas are thought to originate in cancer stem cells in a mechanism (Sell and Dunsford Am J. Pathol. 134: 1347-1363, 1989). The incidence of pancreatic bile duct cancer worldwide varies by country and region. Pancreatic cancer is more frequent in the West, including the Americas and Europe, and in Japan, but bile duct cancer is more prevalent in Korea than in the West. Pancreatic cholangiocarcinoma is often included in advanced conditions and is included in refractory carcinoma due to its very low surgical resection rate and high recurrence rate after resection. Moreover, it has characteristics of low response rate to chemotherapy or radiotherapy (Pederson et al Cancer Res. 4325-4332, 1997), and it is difficult to diagnose these carcinomas.

췌장담관계암이 발생하는 원인에는 유전적 요인과 후천적 요인이 있다. 유전적 요인은 여러 암발생에 관련된 유전자 변이에 의해 발생위험이 증가되는 경우가 해당하며, 후천적인 요인은 식습관, 만성염증, 발암물질에 대한 노출 등이 관여한다. 췌장담관계암의 발생에 관여하는 세포생물학 및 분자유전학적 기전이 많이 밝혀졌음에도 불구하고 암세포특이적 치료효과를 보일 수 있는 타겟분자는 별로 알려져 있지 않다. The causes of pancreatic bile duct cancer are genetic and acquired. Genetic factors include cases where the risk of occurrence is increased by genetic mutations related to various cancer occurrences, and acquired factors include eating habits, chronic inflammation, and exposure to carcinogens. Although many cell biological and molecular genetic mechanisms involved in the development of pancreatic bile duct cancer have been revealed, few target molecules are known to have cancer cell specific therapeutic effects.

한편, 키메라 항원 수용체(이하 본 명세서에서는 이를 「CAR」라 약칭하는 경우가 있다)를 발현하는 T 세포(이하 본 명세서에서는 이를 「CAR-T 세포」라 약칭하는 경우가 있다)는, 암 세포의 표면에 특이적으로 발현되는 암 세포 표면 항원을 인식하는 수용체를 코딩하는 유전자를 T 세포에 도입하여 암 세포를 사멸시킬 수 있도록 유전자가 재조합된 T 세포를 의미한다. 이스라엘의 Weizmann Institute of Science의 화학자이면서 면역학자인 Dr. Zelig Eshhar 등이 암세포에서 특이적으로 발현하는 항원과 결합하는 수용체를 갖는 T세포를 인위적으로 만들면 암세포만 표적하여 면역반응을 일으켜 암세포를 죽일 수 있다는 지견을 도출하여, 키메라 항원 수용체를 장착한 T 세포를 만드는데 성공하였고 1989년 PNAS에 발표한 바 있다. On the other hand, T cells expressing a chimeric antigen receptor (hereinafter sometimes referred to as "CAR" in this specification) are referred to as "CAR-T cells" in the present specification. Refers to a T cell whose gene has been recombined to kill cancer cells by introducing a gene encoding a receptor that recognizes a cancer cell surface antigen specifically expressed on the surface into a T cell. Dr. chemist and immunologist at the Weizmann Institute of Science in Israel When Zelig Eshhar et al. Artificially make T cells with receptors that bind antigens specifically expressed in cancer cells, they derive the knowledge that they can target cancer cells and cause an immune response to kill cancer cells. T cells equipped with chimeric antigen receptors And succeeded in making it to PNAS in 1989.

그러나, 초창기에 제조된 CAR-T 세포, 즉 1세대 CAR-T 세포는 신호 전달 도메인으로서 CD3ζ만을 이용하였는데, 그 치료 효과가 미미했고, 또한, 지속시간도 짧다는 단점이 있었다. 이에, CAR-T 세포의 반응성을 향상시키기 위한 노력이 행해졌으며, 보조 자극 도메인(CD28 또는 CD137/4-1BB)과 CD3ζ를 결합한 2세대 CAR-T 세포가 제조되었는데 1세대 CAR-T 세포와 비교하여 체내에 잔존하는 CAR-T 세포의 수가 현저히 증가하였다. 한편, 2세대 CAR-T 세포는 한 가지의 보조 자극 도메인을 이용하였는데, 두 가지의 보조 자극 도메인을 이용하는 CAR-T 세포를 3세대 CAR-T라 지칭하며, 현재 연구는 2세대 및 3세대 CAR-T 세포에 집중되어 있다. However, CAR-T cells produced in the early stages, that is, first-generation CAR-T cells, used only CD3ζ as a signal transduction domain. However, the therapeutic effect was insignificant and the duration was short. Thus, efforts have been made to improve the reactivity of CAR-T cells, and second-generation CAR-T cells that combine an auxiliary stimulation domain (CD28 or CD137 / 4-1BB) and CD3ζ were prepared, compared to the first-generation CAR-T cells. Thus, the number of CAR-T cells remaining in the body was significantly increased. On the other hand, the second generation CAR-T cells used one auxiliary stimulation domain, and CAR-T cells using two auxiliary stimulation domains are referred to as the third generation CAR-T, and the current study is the second generation and third generation CAR. -T cells are concentrated.

CAR-T 세포를 이용해서 암을 치료하는 방법과 관련하여, 3명의 말기 만성림프성백혈병(CCL, chronic lymphoid leukemia) 환자에게 CD19를 인지할 수 있도록 변형된 세포독성 T세포(Cytotoxic T cell)을 주사했더니, 그 중 2명에서 백혈병이 완전히 치료되었고, 그 상태가 10개월 정도 지속되었다는 보고가 있고, (N. Engl J Med 2011; 365:725-733 August 25, 2011, Sic. Transl. Med 2011 Aug 10;3(95):95ra73) 여기에서 사용된 CAR-T 세포는 2세대에 해당되는 것으로서 보조 자극 도메인으로서 4-1BB를, 신호 전달 도메인으로서 CD3ζ를 이용한 것이다. 상기 CAR-T 세포의 항원 결합 도메인은 백혈병 암세포의 표면에서 발견되는 CD19를 항원으로 인식한다. Regarding the method of treating cancer using CAR-T cells, cytotoxic T cells (Cytotoxic T cells) that have been modified to recognize CD19 in three patients with chronic lymphoid leukemia (CCL) are used. After injection, 2 of them had completely cured leukemia, and there were reports that the condition lasted for about 10 months, (N. Engl J Med 2011; 365: 725-733 August 25, 2011, Sic. Transl. Med 2011 Aug 10; 3 (95): 95ra73) CAR-T cells used herein correspond to the second generation and use 4-1BB as an auxiliary stimulation domain and CD3ζ as a signal transduction domain. The antigen-binding domain of the CAR-T cell recognizes CD19 found on the surface of leukemia cancer cells as an antigen.

또한, 급성 백혈병 환자에게 CTL019를 투여하여 치료하였더니, 환자 30명중 27명이 완전 관해를 경험하였고, 전체 환자의 67%가 2년동안 완전 관해, 78%가 2년간 생존하였다는 보고가 있으며, 대상 환자가 재발성 혹은 불응성 환자였음을 고려한다면, 이는 매우 놀라운 것이다.(N Engl J Med 2014; 371:1507-1517, October 16, 2014)In addition, when CTL019 was administered to acute leukemia patients, 27 out of 30 patients experienced complete remission, and 67% of all patients reported complete remission for 2 years and 78% survived for 2 years. This is quite surprising, considering that the patient was a recurrent or refractory patient (N Engl J Med 2014; 371: 1507-1517, October 16, 2014).

현재, 다양한 CAR-T 세포를 이용한 치료법에 대하여 lymphoma, myeloma 등 다양한 암을 대상으로 임상시험이 진행중에 있고, 사용가능한 의약품으로서의 CAR-T 세포가 시장에 등장할 것으로 예상된다. CAR-T 세포를 이용한 암치료는 자가 유래 방식이어서 대량생산되는 제품은 아니지만, 환자 맞춤형으로서 그 치료효과가 기존의 항암제와 비교할 수 없을 정도로 높다.Currently, clinical trials are underway for various cancers such as lymphoma and myeloma for treatments using various CAR-T cells, and CAR-T cells as usable pharmaceutical products are expected to appear on the market. Cancer treatment using CAR-T cells is a self-derived method, but is not a mass-produced product, but as the patient is customized, its therapeutic effect is so high that it cannot be compared with existing anticancer drugs.

본 발명의 일 목적은 췌장암 또는 담관계암의 예방 또는 치료에 사용할 수 있는 키메라 항원 수용체를 제공하고자 한다. One object of the present invention is to provide a chimeric antigen receptor that can be used for the prevention or treatment of pancreatic cancer or bile duct cancer.

본 발명의 다른 목적은 췌장암 또는 담관계의 예방 또는 치료에 사용할 수 있는 것으로 본 발명에 따른 키메라 항원 수용체를 발현하는 T 세포 또는 NK 세포를 제공하고자 한다. Another object of the present invention is to provide a T cell or NK cell expressing the chimeric antigen receptor according to the present invention, which can be used for the prevention or treatment of pancreatic cancer or bile ducts.

본 발명의 또 다른 목적은 본 발명에 따른 T 세포 또는 NK 세포를 이용하여 췌장암 또는 담관계암 예방 또는 치료용 약학 조성물에 관한 것이다. Another object of the present invention relates to a pharmaceutical composition for preventing or treating pancreatic cancer or bile duct cancer using the T cells or NK cells according to the present invention.

본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

본 발명의 일 구현 예에 따르면, 항원 결합 도메인; 힌지 영역; 및 신호전달 도메인을 포함하며, 상기 항원 결합 도메인은 카보하이드레이트 항원 19-9(carbohydrate antigen 19-9, CA 19-9)에 결합하는 것인, 키메라 항원 수용체에 관한 것이다. According to one embodiment of the invention, the antigen binding domain; Hinge area; And a signaling domain, wherein the antigen-binding domain binds to a carbohydrate antigen 19-9, CA 19-9, and a chimeric antigen receptor.

본 발명에서 상기 항원 결합 도메인은 서열번호 2로 표시되는 VH 도메인 및 서열번호 3으로 표시되는 VL 도메인을 포함할 수 있다. In the present invention, the antigen binding domain may include a VH domain represented by SEQ ID NO: 2 and a VL domain represented by SEQ ID NO: 3.

본 발명에서 상기 VH 도메인과 VL 도메인은 가요성 링커를 통해 연결될 수 있다.In the present invention, the VH domain and the VL domain may be linked through a flexible linker.

본 발명에서 상기 가요성 링커는 서열번호 4로 표시될 수 있다.In the present invention, the flexible linker may be represented by SEQ ID NO: 4.

본 발명에서 상기 키메라 항원 수용체는 신호 펩타이드(signal peptide)를 더 포함할 수 있다.In the present invention, the chimeric antigen receptor may further include a signal peptide.

본 발명에서 상기 신호 펩타이드는 서열번호 1로 표시되는 n-터미널 CD8α 신호 펩타이드(n-terminal CD8α signal peptide)일 수 있다.In the present invention, the signal peptide may be an n-terminal CD8α signal peptide represented by SEQ ID NO: 1.

본 발명에서 상기 힌지 영역은 CD-8 알파, CD28, 4-1BB, OX40, CD3 제타(*?*) 사슬의 전부 또는 일부, T 세포 수용체 α 또는

Figure 112019008649483-pat00001
사슬, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, 이들의 기능적 유도체 또는 이들의 조합을 포함한 인간 단백질의 힌지 영역일 수 있다.In the present invention, the hinge region is CD-8 alpha, CD28, 4-1BB, OX40, CD3 zeta (*? *) All or part of the chain, T cell receptor α or
Figure 112019008649483-pat00001
Human proteins, including chains, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof or combinations thereof It may be a hinge region.

본 발명에서 상기 힌지 영역은 서열번호 5로 표시될 수 있다.In the present invention, the hinge region may be represented by SEQ ID NO: 5.

본 발명에서 상기 신호전달 도메인은 막통과 도메인을 더 포함할 수 있다.In the present invention, the signaling domain may further include a transmembrane domain.

본 발명에서 상기 막통과 도메인은 T 세포 수용체 α 또는 β 사슬, CD3 제타(

Figure 112019008649483-pat00002
) 사슬의 전부 또는 일부, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, 이들의 기능적 유도체 또는 이들의 조합을 포함할 수 있다.In the present invention, the transmembrane domain is a T cell receptor α or β chain, CD3 zeta (
Figure 112019008649483-pat00002
) All or part of the chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, their functional derivatives or their Combinations.

본 발명에서 상기 막통과 도메인은 서열번호 6으로 표시될 수 있다.In the present invention, the transmembrane domain may be represented by SEQ ID NO: 6.

본 발명에서 상기 신호전달 도메인은 보조자극 도메인을 더 포함할 수 있다.In the present invention, the signal transduction domain may further include an auxiliary stimulation domain.

본 발명에서 상기 보조자극 도메인은 4-1BB (CD137), OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, 자연살해 그룹 2 멤버 C (NKG2C), 자연살해 그룹 2 멤버 (NKG2D), B7-H3, CD83, ICAM-1, LFA-1 (CD11a/CD18) 또는 ICOS에 결합하는 리간드, 이들의 활성 단편, 이들의 기능 유도체, 또는 이들의 조합을 포함할 수 있다.In the present invention, the co-stimulatory domain is 4-1BB (CD137), OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, natural killing group 2 member C (NKG2C), natural killing group 2 member (NKG2D), B7-H3, CD83, ICAM-1, LFA-1 (CD11a / CD18) or ligands that bind to ICOS, active fragments thereof, functional derivatives thereof, or combinations thereof.

본 발명에서 상기 보조자극 도메인은 서열번호 7로 표시될 수 있다.In the present invention, the co-stimulatory domain may be represented by SEQ ID NO: 7.

본 발명에서 상기 신호전달 도메인은 CD3 제타(ζ)의 전부 또는 일부, 공통 FcR 감마 (FcER1G), Fc감마RIIIa, FcR베타 (Fc 엡실론립), CD3감마, CD3델타, CD3 엡실론, CD79a, CD79b, DNAX- 활성화 단백질 10 (DAP10), DNAX- 활성화 단백질 12 (DAP12), 이의 활성 단편, 이의 기능적 유도체 또는 이들의 조합을 포함할 수 있다. In the present invention, the signaling domain is all or part of CD3 zeta (ζ), common FcR gamma (FcER1G), Fc gamma RIIIa, FcR beta (Fc epsilonlip), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DNAX-activated protein 10 (DAP10), DNAX-activated protein 12 (DAP12), active fragments thereof, functional derivatives thereof, or combinations thereof.

본 발명에서 상기 신호전달 도메인은 서열번호 8로 표시되는 폴리펩타이드를 포함할 수 있다.In the present invention, the signal transduction domain may include a polypeptide represented by SEQ ID NO: 8.

본 발명에서 상기 키메라 항원 수용체는 서열번호 9로 표시될 수 있다.In the present invention, the chimeric antigen receptor may be represented by SEQ ID NO: 9.

본 발명의 다른 구현 예에 따르면, 본 발명에서 제공하는 키메라 항원 수용체를 암호화하는 폴리뉴클레오타이드에 관한 것이다. According to another embodiment of the present invention, it relates to a polynucleotide encoding a chimeric antigen receptor provided by the present invention.

본 발명에서 상기 폴리뉴클레오타이드는 서열번호 10으로 표시될 수 있다. In the present invention, the polynucleotide may be represented by SEQ ID NO: 10.

본 발명의 또 다른 구현 예에 따르면, 본 발명에서 제공하는 폴리뉴클레오타이드를 포함하는 발현 카세트에 관한 것이다. According to another embodiment of the present invention, it relates to an expression cassette comprising a polynucleotide provided in the present invention.

본 발명에서 상기 발현 카세트는 프로모터를 더 포함할 수 있다.In the present invention, the expression cassette may further include a promoter.

본 발명에서 상기 프로모터는 SFFV 프로모터, 인간연장 인자 1α (EF) 프로모터 또는 CAG 프로모터일 수 있다.In the present invention, the promoter may be an SFFV promoter, a human extension factor 1α (EF) promoter, or a CAG promoter.

본 발명에서 상기 발현 카세트는 절단된 EGFR 유전자(EGFRt)를 부가 유전자로 더 포함할 수 있다.In the present invention, the expression cassette may further include a truncated EGFR gene (EGFRt) as an additional gene.

본 발명에서 상기 폴리뉴클레오타이드와 상기 절단된 EGFR 유전자는 절단 가능한 링커로 연결될 수 있다.In the present invention, the polynucleotide and the truncated EGFR gene may be linked by a cleavable linker.

본 발명에서 상기 절단 가능한 링커는 T2A 리보솜 스킵 요소일 수 있다.In the present invention, the cleavable linker may be a T2A ribosome skip element.

본 발명의 또 다른 구현 예에 따르면, 본 발명에서 제공하는 발현 카세트를 포함하는, 벡터에 관한 것이다.According to another embodiment of the present invention, it relates to a vector comprising the expression cassette provided by the present invention.

본 발명에서 상기 벡터는 플라스미드, 트랜스포손, 코스미드 또는 바이러스 벡터일 수 있다.In the present invention, the vector may be a plasmid, transposon, cosmid, or viral vector.

본 발명에서 상기 벡터는 레트로바이러스 벡터, 아데노바이러스 벡터, 아데노-관련 바이러스(AAC) 벡터, 렌티바이러스 벡터, 폭스바이러스 벡터, 시미안 바이러스 벡터, 백시니아 바이러스 벡터 또는 센다이 바이러스 벡터, 엡스타인-바르(Epstein-Barr) 바이러스(EBV) 벡터 또는 HSV 벡터일 수 있다.In the present invention, the vector is a retrovirus vector, adenovirus vector, adeno-associated virus (AAC) vector, lentivirus vector, poxvirus vector, simian virus vector, vaccinia virus vector or Sendai virus vector, Epstein-Bar (Epstein) -Barr) virus (EBV) vector or HSV vector.

본 발명의 또 다른 구현 예에 따르면, 본 발명에서 제공하는 벡터가 형질 전환된 숙주 세포에 관한 것이다. According to another embodiment of the present invention, it relates to a host cell transformed with the vector provided by the present invention.

본 발명에서 상기 숙주 세포는 T 세포 또는 NK 세포일 수 있다. In the present invention, the host cell may be a T cell or NK cell.

본 발명의 또 다른 구현 예에 따르면, 본 발명에서 제공하는 숙주 세포를 유효 성분으로 포함하는, 암의 예방 또는 치료용 약학 조성물에 관한 것이다. According to another embodiment of the present invention, the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising the host cell provided as an active ingredient.

본 발명에서 상기 암은 췌장암 또는 담관계암일 수 있다.In the present invention, the cancer may be pancreatic cancer or bile duct cancer.

본 발명에서 제공하는 키메라 항원 수용체를 발현할 수 있도록 형질 전환된 T 세포 또는 NK 세포는 췌장암 또는 담관계암 세포를 특이적으로 인식하고, 그 사멸을 유도하여 최종적으로는 췌장암 또는 담관계암을 효과적으로 예방 또는 치료할 수 있다. T cells or NK cells transformed to express the chimeric antigen receptor provided by the present invention specifically recognize pancreatic cancer or bile duct cancer cells, and induce their death to finally effectively treat pancreatic cancer or bile duct cancer. It can be prevented or treated.

도 1은 본 발명의 일 실시예에 따른 키메라 항원 수용체의 구조를 개략적으로 나타낸 것이다.
도 2는 본 발명의 일 실시예에 따른 키메라 항원 수용체 카세트의 구조를 나타낸 것으로 개략적 구조와 췌장담도암 세포막 특이 항원인 CA19-9을 타겟하기 위하여 제작된 CAR 콘스트럭트를 나타낸 것이다.
도 3은 본 발명의 실시예 1에서 벡터에 제한 효소 처리 후 전기 영동하여 제작 삽입된 키메라 항원 수용체 카세트를 확인한 결과를 나타낸 것이다.
도 4는 본 발명의 실시예 2에서 본 발명의 키메라 항원 수용체가 타겟하고자 하는 CA 19-9 세포막 항원이 췌장 담도암 조직 세포주에서 발현되는 지 여부를 면역형광염색을 이용하여 분석한 것으로서, 여러 췌장 담도암 조직 세포주에서 제작된 키메라 항원 수용체 카세트가 타겟하는 CA 19-9 항원에서의 발현을 나타내는 결과이다.
도 5는 본 발명의 실시예 3에서 유세포 분석기를 이용하여 말초혈액에서 분리한 T 세포의 순도를 확인한 결과를 나타낸 것이다. T 세포의 순도는 97~98% 로 확인되었다.
도 6은 본 발명의 실시예 3에서 유세포 분석기를 이용하여 말초혈액에서 분리한 활성화된 T 세포의 현미경 사진을 나타낸 것이다.
도 7은 본 발명의 실시예 3에서 렌티바이러스(lentivirus)를 이용하여 제작된 키메라 항원 수용체 카세트가 T 세포에 안정적으로 형질 전환(transfection) 되었음을 나타낸 결과이다.1 schematically shows the structure of a chimeric antigen receptor according to an embodiment of the present invention.
FIG. 2 shows the structure of a chimeric antigen receptor cassette according to an embodiment of the present invention, and shows a schematic structure and a CAR construct produced to target CA19-9, a pancreatic cholecyst cancer cell membrane specific antigen.
Figure 3 shows the results of confirming the inserted chimeric antigen receptor cassette produced by electrophoresis after restriction enzyme treatment in the vector in Example 1 of the present invention.
Figure 4 is an example 2 of the present invention, the chimeric antigen receptor of the present invention, the target CA 19-9 cell membrane antigen is analyzed in the pancreatic biliary cancer tissue cell line using immunofluorescence staining, and analyzed by various pancreas It is a result showing the expression in the target CA 19-9 antigen of the chimeric antigen receptor cassette produced in the biliary cancer tissue cell line.
Figure 5 shows the results of confirming the purity of T cells isolated from peripheral blood using a flow cytometer in Example 3 of the present invention. The purity of T cells was found to be 97-98%.
6 is a micrograph of activated T cells isolated from peripheral blood using a flow cytometer in Example 3 of the present invention.
7 is a result showing that the chimeric antigen receptor cassette prepared using lentivirus in Example 3 of the present invention was stably transfected into T cells.

본 발명에서 달리 정의되지 않은 한, 본 명세서에서 사용된 모든 기술 및 과학 용어는 본 발명이 속하는 분야의 당업자에 의해 흔히 이해되는 것과 동일한 의미를 가진다. 본 명세서에 기재된 것과 유사하거나 동등한 임의의 방법 및 재료가 본 발명의 시험을 위해 실행 시 사용될 수 있지만, 바람직한 재료 및 방법이 본 명세서에 기재된다. 본 발명의 기재 및 청구 시, 하기 정의가 이용될 것이다. 본 명세서에서 사용된 전문용어는 제한인 것으로 의도되지 않고, 오히려 특정한 실시형태를 기술할 목적을 위해 본 명세서에서 사용되는 것으로 또한 이해될 것이다. 관사 "하나" 및 "일"은 관사의 문법상 목적어의 하나 또는 하나 초과(즉, 적어도 하나 또는 하나 이상)를 의미하도록 본 명세서에서 사용된다.Unless defined otherwise in the present invention, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in practice for the testing of the present invention, preferred materials and methods are described herein. In the description and claims of the present invention, the following definitions will be used. It is to be understood that the terminology used herein is not intended to be limiting, but rather is used herein for the purpose of describing particular embodiments. The articles “one” and “one” are used herein to mean one or more (ie, at least one or more than one) of an object's grammatical object.

본 발명의 일 구현 예에 따르면, 항원 결합 도메인; 힌지 영역 (또는 스페이서); 및 신호전달 도메인을 포함하는, 키메라 항원 수용체에 관한 것이다.According to one embodiment of the invention, the antigen binding domain; Hinge region (or spacer); And a signaling domain.

본 발명에서 용어 "키메라 항원 수용체" 또는 "CAR"이란 세포외 항원 결합 도메인 및 세포내 신호전달 도메인을 포함하는 조작된 수용체를 의미한다. CAR의 가장 흔한 유형이 CD3ζ 사슬과 같은 T 세포 동시수용체의 막통과 및 세포내 도메인에 융합된 단일 클론 항체로부터 유래된 단쇄 가변 단편(scFv)을 포함하지만, 본 명세서에 기재된 본 발명은 이들 도메인으로 제한되지 않는다. 오히려, 본 명세서에 사용된 바와 같은 "키메라 항원 수용체" 또는 "CAR"은 임의의 세포 내 신호전달 분자를 발현하도록 조작된 임의의 수용체 및 이들에 융합되거나 연결된 세포외 항원 결합 도메인을 의미한다.In the present invention, the term "chimeric antigen receptor" or "CAR" means an engineered receptor comprising an extracellular antigen binding domain and an intracellular signaling domain. The most common types of CARs include transmembrane of T cell co-receptors such as the CD3ζ chain and single chain variable fragments (scFv) derived from monoclonal antibodies fused to intracellular domains, but the invention described herein includes these domains. It is not limited. Rather, “chimeric antigen receptor” or “CAR” as used herein refers to any receptor engineered to express any intracellular signaling molecule and an extracellular antigen binding domain fused or linked to them.

본 발명의 키메라 항원 수용체에서 상기 항원 결합 도메인의 다른 일 말단에 신호 펩타이드(signal peptide)를 더 포함할 수 있다. In the chimeric antigen receptor of the present invention, a signal peptide may be further included at the other end of the antigen binding domain.

본 발명에서 상기 "신호 펩타이드"는 펩타이드 서열을 포함하는 데, 이는 임의의 분비된 또는 막통과 단백질의 일종 펩타이드로, 본 발명의 키메라 항원 수용체를 세포막 및 세포 표면으로의 수송을 지시하고, 본 발명의 키메라 항원 수용체의 정확한 위치 선정을 제공할 수 있다. 특히, 본 발명에서 상기 신호 펩타이드는 본 발명의 키메라 항원 수용체를 세포막에 지지시키고, 그 점에서 상기 키메라 항원 수용체의 세포 외 부분이 세포 표면 상에 표시되며, 막통과 부분이 원형질 막에 걸쳐 있고, 활성 도메인은 세포질 부분, 또는 세포의 내부에 있게 된다.In the present invention, the "signal peptide" includes a peptide sequence, which is a peptide of any secreted or transmembrane protein, directs the transport of the chimeric antigen receptor of the present invention to the cell membrane and cell surface, and the present invention Can provide accurate positioning of chimeric antigen receptors. In particular, in the present invention, the signal peptide supports the chimeric antigen receptor of the present invention on the cell membrane, at which point the extracellular portion of the chimeric antigen receptor is displayed on the cell surface, the transmembrane portion spans the plasma membrane, The active domain will be in the cytoplasmic part, or inside the cell.

본 발명에서 상기 신호 펩타이드로는 상기의 기능을 하는 펩타이드라면 제한없이 사용될 수 있지만, 예를 들면, 서열번호 1로 표시되는 n-터미널 CD8α 신호 펩타이드(n-terminal CD8α signal peptide)일 수 있다. In the present invention, the signal peptide may be used without limitation as long as it is a peptide that functions as described above, but may be, for example, an n-terminal CD8α signal peptide represented by SEQ ID NO: 1.

본 발명에서 상기 항원 결합 도메인은 카보하이드레이트 항원 19-9(carbohydrate antigen 19-9)을 특이적으로 인식할 수 있는 단쇄 가변 단편(scFv)를 포함할 수 있다. In the present invention, the antigen binding domain may include a single chain variable fragment (scFv) capable of specifically recognizing carbohydrate antigen 19-9.

본 발명에서 상기 "단쇄 가변 단편" 또는 "scFv"란, VL과 VH 사이의 펩타이드 링커에 의한, 항체의 가변 중쇄(VH) 및 가변 경쇄(VL)의 융합 단백질을 의미한다. In the present invention, the "single chain variable fragment" or "scFv" means a fusion protein of the variable heavy chain (VH) and variable light chain (VL) of an antibody, by a peptide linker between VL and VH.

본 발명에서 상기 항원 결합 도메인은 서열번호 2로 표시되는 VH 도메인 및 서열번호 3으로 표시되는 VL 도메인을 포함할 수 있다. In the present invention, the antigen binding domain may include a VH domain represented by SEQ ID NO: 2 and a VL domain represented by SEQ ID NO: 3.

또한, 본 발명에서 상기 VH 도메인과 VL 도메인은 가요성 링커를 통해 연결될 수 있다. 본 발명에서 상기 가요성 링커는 약 10개 내지 30개의 아미노산(예를 들어, 30개, 25개, 20개, 19개, 18개, 17개, 16개, 15개, 14개, 13개, 12개, 11개, 10개, 9개, 8개, 7개, 6개 또는 5개의 아미노산)의 글라이신/세린 링커일 수 있고, 바람직하게는 15개의 아미노산 길이일 수 있다. 본 발명에서 상기 링커 길이는 키메라 항원 수용체의 중요한 결정부위로 작용할 수 있어, 상기 범위보다 더 짧은 링커는 친화도를 증대시킬 수 있지만, 또한 세포내 다합체 형성을 발생시켜 CAR의 발현을 손상시킬 수 있는 반면, 상기 범위보다 더 긴 링커는 VL 및 VH CDR을 공간상 더 멀리 이동시킴으로써 항원 친화도를 감소시킬 수 있다. In addition, in the present invention, the VH domain and the VL domain may be linked through a flexible linker. In the present invention, the flexible linker is about 10 to 30 amino acids (for example, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids) glycine / serine linker, preferably 15 amino acids in length. In the present invention, the linker length can act as an important determinant of the chimeric antigen receptor, so that a linker shorter than the above range can increase affinity, but can also cause intracellular multimer formation to impair CAR expression. Whereas, linkers longer than the above range can reduce antigen affinity by moving the VL and VH CDRs further away in space.

본 발명에서 상기 가요성 링커는 서열번호 4로 표시될 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the flexible linker may be represented by SEQ ID NO: 4, but is not limited thereto.

본 발명에서 상기 힌지 영역은 항원 결합 도메인과 막통과 도메인을 연결하는 부분으로 '스페이서'라고도 불리우는데, T 세포막 또는 NK 세포막으로부터 항원 결합 도메인을 확장하기 위한 목적을 갖는다. In the present invention, the hinge region is a part that connects the antigen-binding domain and the transmembrane domain and is also referred to as a 'spacer', and has the purpose of expanding the antigen-binding domain from the T cell membrane or NK cell membrane.

본 발명에서 상기 힌지 영역은 예를 들어 인간 또는 그의 일부를 포함하는 임의의 속으로부터의 임의의 적합한 서열로부터 얻어질 수 있으며, 혹은 본 기술분야에서 통상적으로 사용하는 CD-8 알파, CD28, 4-1BB, OX40, CD3 제타(ζ) 사슬의 전부 또는 일부, T 세포 수용체 α 또는 β 사슬, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, 이들의 기능적 유도체 또는 이들의 조합을 포함한 인간 단백질의 힌지 영역을 포함할 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the hinge region can be obtained from any suitable sequence from any genera including, for example, a human or a part thereof, or CD-8 alpha, CD28, 4- conventionally used in the art. 1BB, OX40, all or part of the CD3 zeta chain, T cell receptor α or β chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86 , CD134, CD137, ICOS, CD154, functional derivatives thereof, or combinations thereof, but may include hinge regions of human proteins.

또한, 본 발명에서 상기 힌지 영역은 면역글로불린에 제한하지 않으면서 면역글로불린 (예: IgG1, IgG2, IgG3, IgG4 및 IgD)으로부터 선택되는 하나를 포함할 수 있으나, 이에 제한되는 것은 아니다. In addition, in the present invention, the hinge region may include one selected from immunoglobulins (eg, IgG1, IgG2, IgG3, IgG4 and IgD) without being limited to immunoglobulins, but is not limited thereto.

본 발명에서 상기 힌지 영역은 서열번호 5로 표시될 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the hinge region may be represented by SEQ ID NO: 5, but is not limited thereto.

본 발명에서 상기 신호전달 도메인은 T 세포 내부에서 발견되거나 발견되도록 조작된 키메라 항원 수용체의 부분을 의미한다. 본 발명에서 상기 신호전달 도메인은 T 세포의 혈장 막에서 키메라 항원 수용체를 고정하는 역할을 하는 막통과 도메인을 포함하거나 포함하지 않을 수 있다. In the present invention, the signaling domain refers to a portion of a chimeric antigen receptor that has been found or engineered to be found inside T cells. In the present invention, the signaling domain may or may not include a transmembrane domain that serves to fix the chimeric antigen receptor in the plasma membrane of T cells.

본 발명에서 상기 막통과 도메인과 상기 신호전달 도메인은 동일한 단백질(예를 들어, CD3ζ)로부터 유래할 수 있고, 혹은 상기 막통과 도메인과 상기 신호전달 도메인은 상이한 단백질(예를 들어, CD28의 막관통 도메인 및 CD3ζ 분자의 세포내 신호전달 도메인, 또는 그 반대)로부터 유래할 수 있다.In the present invention, the transmembrane domain and the signaling domain may be derived from the same protein (eg, CD3ζ), or the transmembrane domain and the signaling domain may be different proteins (eg, transmembrane of CD28) Domain and the intracellular signaling domain of the CD3ζ molecule, or vice versa.

본 발명에서 상기 막통과 도메인은 세포막에 걸친 소수성 폴리펩타이드를 포함한다. 특히, 막통과 도메인은 세포막의 한면 (세포 외)에서 세포막의 다른면 (세포 내 또는 세포질)을 통하여 걸쳐 있을 수 있다.In the present invention, the transmembrane domain comprises a hydrophobic polypeptide across the cell membrane. In particular, the transmembrane domain can span from one side of the cell membrane (extracellular) to the other side of the cell membrane (intracellular or cytoplasmic).

본 발명에서 상기 막통과 도메인은 알파 나선 또는 베타 배럴, 또는 이들의 조합의 형태일 수 있다. 또한, 본 발명에서 상기 막통과 도메인은 다수의 막통과 조각, 각 알파-나선형, 베타 시트, 또는 이들의 조합을 갖는 다원 단백질을 포함할 수 있다.In the present invention, the transmembrane domain may be in the form of an alpha helix or beta barrel, or a combination thereof. In addition, in the present invention, the transmembrane domain may include a plural transmembrane protein having multiple transmembrane fragments, each alpha-helix, beta sheet, or a combination thereof.

본 발명에서 상기 막통과 도메인으로는, 예를 들면 T 세포 수용체 α 또는 β 사슬, CD3 제타(ζ) 사슬의 전부 또는 일부, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, 이들의 기능적 유도체 및 이들의 조합을 포함할 수 있으나, 이에 제한되는 것은 아니다. In the present invention, as the transmembrane domain, for example, all or part of the T cell receptor α or β chain, CD3 zeta chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, and combinations thereof, but are not limited thereto.

본 발명에서 상기 막통과 도메인으로 예를 들어 인공적으로 설계된 것은 주로 류신 및 발린과 같은 소수성 잔기를 포함하는 폴리펩타이드일 수 있다. 본 발명의 일 실시양태에서, 페닐알라닌, 트립토판 및 발린의 삼중체는 합성 막 변이 도메인의 각 말단에서 발견될 수 있다.In the present invention, artificially designed, for example, as the transmembrane domain may be a polypeptide mainly containing hydrophobic residues such as leucine and valine. In one embodiment of the invention, triplets of phenylalanine, tryptophan and valine can be found at each end of the synthetic membrane mutation domain.

본 발명에서 상기 막통과 도메인은 서열번호 6으로 표시될 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the transmembrane domain may be represented by SEQ ID NO: 6, but is not limited thereto.

본 발명에서 상기 막통과 도메인과 상기 신호전달 도메인 사이에 적어도 하나의 보조자극 도메인을 더 포함할 수 있다. 본 발명에서 상기 보조자극 도메인은 보조자극 신호가 전달되는 부위로서 항원 결합 도메인과 결합한 특정 항원을 인식한 CAR-T 세포 또는 CAR-NK 세포가 면역반응을 일으키며 자가 증식을 돕고, 체내에 잔존하는 시간을 늘리도록 신호를 전달하는 역할을 할 수 있다. In the present invention, at least one auxiliary stimulation domain may be further included between the transmembrane domain and the signal transduction domain. In the present invention, the co-stimulatory domain is a site where a co-stimulatory signal is transmitted, and CAR-T cells or CAR-NK cells that recognize a specific antigen bound to the antigen-binding domain cause an immune response, help auto-proliferation, and remain in the body. It can serve to convey a signal to increase.

본 발명의 일 실시양태에서, 상기 보조자극 도메인은 4-1BB (CD137), OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, 자연살해 그룹 2 멤버 C (NKG2C), 자연살해 그룹 2 멤버 (NKG2D), B7-H3, CD83, ICAM-1, LFA-1 (CD11a/CD18) 또는 ICOS에 결합하는 리간드, 이들의 활성 단편, 이들의 기능 유도체, 및 이들의 조합을 포함하는 폴리펩타이드로부터 유래한 기능 신호전달 도메인을 포함할 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the invention, the co-stimulatory domain is 4-1BB (CD137), OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, natural killing group 2 member C (NKG2C), Natural Killing Group 2 members (NKG2D), including B7-H3, CD83, ICAM-1, LFA-1 (CD11a / CD18) or ligands binding to ICOS, active fragments thereof, functional derivatives thereof, and combinations thereof It may include a functional signaling domain derived from a polypeptide, but is not limited thereto.

본 발명에서 상기 보조자극 도메인은 서열번호 7로 표시될 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the auxiliary stimulation domain may be represented by SEQ ID NO: 7, but is not limited thereto.

본 발명에서 상기 신호전달 도메인은 면역세포 신호전달 경로의 적어도 일부 면을 자극 또는 활성화시키기 위해 면역세포의 활성화를 제공하는 폴리펩타이드를 포함할 수 있다.In the present invention, the signaling domain may include a polypeptide that provides activation of immune cells to stimulate or activate at least a portion of the immune cell signaling pathway.

본 발명의 일 실시양태에서, 상기 신호전달 도메인은 CD3 제타(ζ)의 전부 또는 일부, 공통 FcR 감마 (FcER1G), Fc감마RIIIa, FcR베타 (Fc 엡실론 립), CD3감마, CD3델타, CD3 엡실론, CD79a, CD79b, DNAX- 활성화 단백질 10 (DAP10), DNAX- 활성화 단백질 12 (DAP12), 이의 활성 단편, 이의 기능적 유도체 및 이들의 조합을 포함하는 폴리펩타이드로부터 유래한 기능 신호전달 도메인을 포함할 수 있으나, 이에 제한되는 것은 아니며, 이러한 신호전달 도메인은 당업계에 공지되어 있다. In one embodiment of the invention, the signaling domain is all or part of CD3 zeta (ζ), common FcR gamma (FcER1G), Fc gamma RIIIa, FcR beta (Fc epsilon lip), CD3 gamma, CD3 delta, CD3 epsilon , CD79a, CD79b, DNAX-activating protein 10 (DAP10), DNAX-activating protein 12 (DAP12), active fragments thereof, functional derivatives thereof and functional combinations derived from polypeptides comprising combinations thereof. However, it is not limited thereto, and such a signaling domain is known in the art.

본 발명에서 상기 신호전달 도메인은 서열번호 8로 표시되는 폴리펩타이드를 포함할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the signaling domain may include the polypeptide represented by SEQ ID NO: 8, but is not limited thereto.

도 1은 본 발명의 일 실시예에 따른 키메라 항원 수용체의 구조를 개략적으로 나타낸 것으로, 상기 키메라 항원 수용체는 서열번호 9로 표시되는 폴리펩티드로, n-터미널 CD8α 신호 펩타이드(signal peptide)-CA 19-9 특이적 단쇄 가변 단편(scFv)-CD8 힌지-CD28 막통과 도메인-4-1BB-CD3ζ로 구성될 수 있다. Figure 1 schematically shows the structure of a chimeric antigen receptor according to an embodiment of the present invention, the chimeric antigen receptor is a polypeptide represented by SEQ ID NO: 9, n-terminal CD8α signal peptide (signal peptide) -CA 19- 9 specific single-chain variable fragment (scFv) -CD8 hinge-CD28 transmembrane domain-4-1BB-CD3ζ.

본 발명의 다른 구현 예에 따르면, 본 발명에 따른 키메라 항원 수용체를 암호화하는 폴리뉴클레오타이드에 관한 것이다. 본 발명에서 상기 키메라 항원 수용체를 암호화하는 폴리 뉴클레오타이드는 임의의 재래적인 방법에 의해 특정된 키메라 항원 수용체의 아미노산 서열로부터 용이하게 제조된다. 아미노산 서열을 부호화하는 염기 서열은 앞서 말한 NCBI RefSeq ID 또는 GenBenk의 가입 번호로부터 각 도메인의 아미노산 서열에 대해 얻을 수 있으며, 본 공개의 핵산은 표준 분자 생물학적 및/또는 화학적 절차를 사용하여 제작될 수 있다. 예를 들어, 염기 서열에 기초한 폴리 뉴클레오타이드는 합성될 수 있고, 중합효소연쇄반응 (PCR)을 이용하여 cDNA 라이브러리로부터 얻은 DNA 단편을 조합하는 것을 통해 본 공개의 폴리뉴클레오타이드를 제조할 수 있다.According to another embodiment of the present invention, it relates to a polynucleotide encoding a chimeric antigen receptor according to the present invention. In the present invention, the polynucleotide encoding the chimeric antigen receptor is easily prepared from the amino acid sequence of the chimeric antigen receptor specified by any conventional method. The nucleotide sequence encoding the amino acid sequence can be obtained for the amino acid sequence of each domain from the aforementioned NCBI RefSeq ID or GenBenk accession number, and the nucleic acids of the present disclosure can be prepared using standard molecular biological and / or chemical procedures. . For example, polynucleotides based on base sequences can be synthesized and polynucleotides of the present disclosure can be prepared by combining DNA fragments obtained from cDNA libraries using polymerase chain reaction (PCR).

본 발명의 폴리뉴클레오타이드는 유전자 또는 발현 또는 클로닝 카세트의 일부일 수 있으나, 이에 제한되는 것은 아니다. The polynucleotide of the present invention may be a gene or part of an expression or cloning cassette, but is not limited thereto.

본 발명에서 용어 "폴리뉴클레오타이드"는 뉴클레오티드의 사슬로서 정의된다. 폴리뉴클레오타이드는 DNA 및 RNA를 포함한다. 더욱이, 핵산은 뉴클레오타이드의 중합체이다. 따라서, 여기서 사용된 핵산 및 폴리 누클레오티드는 상호 교환 가능하다. 당업계의 기술자는 핵산이 폴리뉴클레오타이드이고 이것이 단량체 "뉴클레오타이드"로 가수 분해될 수 있다는 일반적인 지식을 갖는다. 단량체 뉴클레오타이드는 뉴클레오사이드로 가수 분해 될 수 있다. 여기서 사용된 폴리뉴클레오타이드는 제한없는 재조합 수단, 즉 통상적인 클로닝 기술 및 중합 효소 연쇄 반응 (PCR) 등을 사용한 재조합 라이브러리 또는 세포유전체로부터의 핵산 서열 클로닝을 포함하는 당업계에서 이용 가능한 임의의 수단 그리고 합성 수단에 의해 획득된 모든 핵산 서열을 포함 하나 그것에 국한되는 것이 아니다.In the present invention, the term "polynucleotide" is defined as a chain of nucleotides. Polynucleotides include DNA and RNA. Moreover, nucleic acids are polymers of nucleotides. Thus, the nucleic acids and polynucleotides used herein are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide and it can be hydrolyzed to a monomer “nucleotide”. Monomeric nucleotides can be hydrolyzed to nucleosides. The polynucleotides used herein are any means available in the art and synthetic, including, but not limited to, recombinant means using conventional cloning techniques and polymerase chain reaction (PCR), etc., or cloning nucleic acid sequences from cytogenes. Includes, but is not limited to, all nucleic acid sequences obtained by means.

본 발명에서 상기 폴리뉴클레오타이드는 서열번호 10으로 표시될 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the polynucleotide may be represented by SEQ ID NO: 10, but is not limited thereto.

본 발명의 또 다른 구현 예에 따르면, 본 발명에서 제공하는 폴리뉴클레오타이드를 포함하는 발현 카세트(cassette)에 관한 것이다. According to another embodiment of the present invention, it relates to an expression cassette (cassette) comprising a polynucleotide provided in the present invention.

본 발명의 발현 카세트는 본 발명의 폴리뉴클레오타이드의 발현을 조절할 수 있는 프로모터, 인핸서, 폴리아데닐화 신호, 전사 종결요소 또는 내부 리보솜 진입 부위(IRES) 등과 같은 발현 제어 서열을 포함할 수 있다. The expression cassette of the present invention may include an expression control sequence such as a promoter, an enhancer, a polyadenylation signal, a transcription termination element, or an internal ribosome entry site (IRES), which can regulate the expression of the polynucleotide of the present invention.

본 발명에서 상기 프로모터로는 예를 들어, SFFV 프로모터, 인간연장 인자 1α (EF) 프로모터 또는 CAG (CMV 증강인자를 갖는 닭 베타-액틴 프로모터) 프로모터를 포함할 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the promoter may include, for example, SFFV promoter, human extension factor 1α (EF) promoter or CAG (chicken beta-actin promoter having CMV enhancer) promoter, but is not limited thereto.

본 발명의 발현 카세트는 그 발현을 모니터링하는 데 유용한 추가 서열로, 절단된 EGFR 유전자(EGFRt)를 부가 유전자로 더 포함할 수 있다. 본 발명에서 상기 EGFRt는 비-면역성 선별 툴(tool)로, 예를 들면, 비오티닐화된 세툭시맙(biotinylated cetuximab)과 항-비오틴 마이크로비드를 이용한 면역자기 선별(immunomagnetic selection) 시 EGFRt-를 포함하는 카세트가 형질 전환된 T 세포 또는 NK 세포를 선별하는 데에 활용할 수 있을 뿐만 아니라, T 세포 또는 NK 세포 추적을 위한 유세포 분석 시 추적 마커로 사용할 수 있으며, 세툭시맙/얼비툭스® 중재 항체 의존적 세포 상해 활성(antibody dependent cellular cytotoxicity, ADCC) 경로를 통한 자살 유전자로 활용될 수 있다. The expression cassette of the present invention is an additional sequence useful for monitoring its expression, and may further include a truncated EGFR gene (EGFRt) as an additional gene. In the present invention, the EGFRt is a non-immune selection tool, for example, EGFRt- during immunomagnetic selection using biotinylated cetuximab and anti-biotin microbeads. Not only can the cassette containing it be used to select transformed T cells or NK cells, but also can be used as a tracking marker in flow cytometry for tracking T cells or NK cells, and dependent on cetuximab / Erbitux® mediated antibody It can be utilized as a suicide gene through the path of cytotoxicity (antibody dependent cellular cytotoxicity, ADCC).

본 발명에서 상기 절단된 EGFRt는 서열번호 11로 표시될 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the truncated EGFRt may be represented by SEQ ID NO: 11, but is not limited thereto.

또한, 본 발명에서 상기 절단된 EGFRt과 본 발명의 폴리뉴클레오타이드의 신호전달 도메인의 일 말단과 절단 가능한 링커인 리보솜 스킵 요소에 의해 연결될 수 있다. In addition, in the present invention, the cleaved EGFRt may be linked to one end of the signaling domain of the polynucleotide of the present invention by a ribosomal skip element that is a cleavable linker.

본 발명에서 상기 "리보솜 스킵"이란 특이적 펩타이드가 새로운 삽입된 아미노산을 공유로 연결하는 것으로부터 세포의 리보솜을 막고, 대신에 이것이 계속해서 번역되는 것을 허용하여서 폴리단백질의 동시번역 절단을 생성시키는, 번역의 대안적인 기전을 의미한다. 이 과정은 "2A 리보솜 스킵" 요소 또는 시스 작용 하이드롤라제 요소(예를 들어, CHYSEL 서열)에 의해 유도된다. 몇몇 실시형태에서, 이 서열은 강한 알파 나선 경향을 가지는 비보존적 아미노산 서열, 이어서 공통 서열 -D(V/I)ExNPG P(여기서, x는 임의의 아미노산임)를 포함한다. G와 P 사이에 명확한 절단이 발생한다. 본 발명에서 상기 리보솜 스킵 요소는 2A 리보솜 스킵 요소일 수 있고, 바람직하게는 5' T2A 리보솜 스킵 요소일 수 있으나 이에 제한되는 것은 아니다.In the present invention, the "ribosomal skip" prevents the ribosome of the cell from the specific peptide covalently linking the new inserted amino acid, and instead allows it to be continuously translated, resulting in simultaneous translational cleavage of the polyprotein, It means an alternative mechanism of translation. This process is driven by a “2A ribosomal skip” element or a cis acting hydrolase element (eg CHYSEL sequence). In some embodiments, this sequence comprises a non-conservative amino acid sequence with a strong alpha helix tendency, followed by the consensus sequence -D (V / I) ExNPG P, where x is any amino acid. Clear cuts occur between G and P. In the present invention, the ribosome skip element may be a 2A ribosome skip element, preferably a 5 'T2A ribosome skip element, but is not limited thereto.

본 발명에서 상기 절단 가능한 링커는 서열번호 12로 표시될 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the cleavable linker may be represented by SEQ ID NO: 12, but is not limited thereto.

도 2는 본 발명의 일 실시예에 따른 키메라 항원 수용체 카세트의 구조를 개략적으로 나타낸 것으로, 상기 키메라 항원 수용체 카세트는 서열번호 13으로 표시되는 폴리뉴클레오타이드로, EF-1α 프로모터-n-터미널 CD8α 신호 펩타이드(signal peptide)-CA 19-9 특이적 단쇄 가변 단편(scFv)-CD8 힌지-CD28 막통과 도메인-4-1BB-CD3ζ-T2A를 암호화하는 폴리뉴클레오타이드로 구성될 수 있고, 혹은 서열번호 14로 표시되는 폴리뉴클레오타이드로, EF-1α 프로모터-n-터미널 CD8α 신호 펩타이드(signal peptide)-CA 19-9 특이적 단쇄 가변 단편(scFv)-CD8 힌지-CD28 막통과 도메인-4-1BB-CD3ζ-T2A-EGFRt를 암호화하는 폴리뉴클레오타이드로 구성될 수 있으나, 이에 제한되는 것은 아니다. Figure 2 schematically shows the structure of a chimeric antigen receptor cassette according to an embodiment of the present invention, wherein the chimeric antigen receptor cassette is a polynucleotide represented by SEQ ID NO: 13, EF-1α promoter-n-terminal CD8α signal peptide (signal peptide) -CA 19-9 specific short chain variable fragment (scFv) -CD8 hinge-CD28 can be composed of polynucleotides encoding transmembrane domain-4-1BB-CD3ζ-T2A, or represented by SEQ ID NO: 14 As a polynucleotide, EF-1α promoter-n-terminal CD8α signal peptide-CA 19-9 specific single chain variable fragment (scFv) -CD8 hinge-CD28 transmembrane domain-4-1BB-CD3ζ-T2A- It may be composed of a polynucleotide encoding EGFRt, but is not limited thereto.

본 발명의 또 다른 구현 예에 따르면, 본 발명에서 제공하는 발현 카세트를 포함하는 벡터에 관한 것이다. According to another embodiment of the present invention, it relates to a vector comprising an expression cassette provided by the present invention.

본 발명에서 상기 벡터로는 예를 들어, 플라스미드, 트랜스포손, 코스미드 또는 바이러스 벡터(예를 들면, 파아지, 레트로바이러스, 렌티바이러스 또는 아데노바이러스)를 사용할 수 있다In the present invention, as the vector, for example, a plasmid, transposon, cosmid, or viral vector (eg, phage, retrovirus, lentivirus, or adenovirus) may be used.

본 발명의 일 실시양태에서, 상기 벡터는 레트로바이러스 벡터(온코레트로바이러스(oncoretrovirus) 벡터, 렌티바이러스 벡터, 및 슈도 타입 벡터 포함), 아데노바이러스 벡터, 아데노-관련 바이러스(AAC) 벡터, 렌티바이러스 벡터, 폭스바이러스 벡터, 시미안 바이러스 벡터, 백시니아 바이러스 벡터 또는 센다이 바이러스 벡터, 엡스타인-바르(Epstein-Barr) 바이러스(EBV) 벡터 또는 HSV 벡터를 사용할 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the invention, the vector is a retroviral vector (including oncoretrovirus vector, lentiviral vector, and pseudo type vector), adenovirus vector, adeno-associated virus (AAC) vector, lentiviral vector , Poxvirus vector, simian virus vector, vaccinia virus vector or sendai virus vector, Epstein-Barr virus (EBV) vector or HSV vector, but is not limited thereto.

본 발명의 또 다른 구현 예에 따르면, 본 발명에서 제공하는 벡터가 형질 전환된 숙주 세포에 관한 것이다. According to another embodiment of the present invention, it relates to a host cell transformed with the vector provided by the present invention.

본 발명에서 용어 "숙주 세포"란 발현 벡터를 함유할 수 있는 임의의 유형의 세포를 말한다. 숙주 세포는 진핵생물 세포(예를 들면, 식물, 동물, 진균 또는 조류(algae)), 원핵생물 세포(예를 들면, 세균 또는 원생동물) 또는 바이러스 또는 레트로바이러스 벡터일 수 있다. 숙주 세포는 배양된 또는 "기성 제품(off-the-shelf)" 세포 또는 1차 세포(즉, 대상체로부터 직접 단리됨)일 수 있다. 숙주 세포는 부착성 세포 또는 현탁된 세포, 즉, 현탁액 중에서 성장하는 세포일 수 있다. 적합한 숙주 세포는 당업계에 공지되어 있고, 예를 들면, DH5α 이. 콜라이(E. coli) 세포, 차이니즈 햄스터 난소 세포, 원숭이 VERO 세포, COS 세포, 및 HEK293 세포 등이 포함된다. 재조합 발현 벡터를 증폭시키거나 복제할 목적을 위해, 숙주 세포는 원핵 생물 세포, 예를 들면, DH5α 세포일 수 있다. 또한, 본 발명에서는 재조합체 CAR을 생산할 목적을 위해, 상기 숙주 세포는 포유동물 세포, 바람직하게는 인간 세포일 수 있다. 본 발명에서 상기 숙주 세포는 임의의 세포 유형일 수 있고, 임의의 유형의 조직으로부터 유래될 수 있고, 임의의 발단 단계의 것으로, 예를 들면, 체액, 조직 또는 기관, 예를 들면, 혈액(말초혈, 제대혈 등) 또는 골수로부터 수집되거나, 단리되거나, 정제되거나, 또는 유도된 세포를 사용할 수 있다. The term "host cell" in the present invention refers to any type of cell that may contain an expression vector. The host cell can be a eukaryotic cell (eg, a plant, animal, fungus or algae), a prokaryotic cell (eg, a bacterial or protozoan) or a viral or retroviral vector. The host cell can be cultured or “off-the-shelf” cells or primary cells (ie, isolated directly from the subject). The host cell may be adherent cells or suspended cells, ie cells that grow in suspension. Suitable host cells are known in the art, for example, DH5α E. E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, and HEK293 cells. For purposes of amplifying or replicating a recombinant expression vector, the host cell can be a prokaryotic cell, such as a DH5α cell. In addition, in the present invention, for the purpose of producing a recombinant CAR, the host cell may be a mammalian cell, preferably a human cell. In the present invention, the host cell can be of any cell type, can be derived from any type of tissue, and is of any stage of development, eg, body fluid, tissue or organ, such as blood (peripheral blood , Umbilical cord blood, etc.) or cells collected, isolated, purified, or derived from bone marrow.

본 발명에서 상기 숙주 세포로는 면역 세포[T 세포, 수지상 세포, B 세포, 조혈 줄기세포, 대식세포, 단핵구, NK 세포 또는 조혈 세포(호중구, 호염구)], 말초혈액단핵구(PBMC), 제대혈 단핵구 세포, 섬유아세포, 전구체 지방세포, 간세포, 피부 각질세포, 간엽성 줄기세포, 지방 줄기세포, 또는 각종 암 세포주를 사용할 수 있고, 바람직하게는 T 세포, 보다 바람직하게는 CD4+, CD8+ T 세포, 및 이팩터 T 세포, 기억 T 세포, 또는 조절 T 세포일 수 있으나, 이에 제한되는 것은 아니다. 적합한 포유동물 숙주 세포를 선택하는 방법 및 세포의 형질전환, 배양, 증폭, 스크리닝 및 정제 방법은 당업계에 공지되어 있다.In the present invention, the host cells include immune cells [T cells, dendritic cells, B cells, hematopoietic stem cells, macrophages, monocytes, NK cells or hematopoietic cells (neutrophils, basophils)], peripheral blood monocytes (PBMC), umbilical cord blood monocytes Cells, fibroblasts, precursor adipocytes, hepatocytes, skin keratinocytes, mesenchymal stem cells, adipocyte stem cells, or various cancer cell lines can be used, preferably T cells, more preferably CD4 +, CD8 + T cells, and It may be an effector T cell, a memory T cell, or a regulatory T cell, but is not limited thereto. Methods for selecting suitable mammalian host cells and methods for transforming, culturing, amplifying, screening and purifying cells are known in the art.

본 발명의 또 다른 구현 예에 따르면, 본 발명에서 제공하는 형질 전환된 숙주 세포로부터 발현되는 폴리펩티드에 관한 것이다. According to another embodiment of the present invention, the present invention relates to a polypeptide expressed from a transformed host cell.

본 발명에서 상기 숙주 세포로부터 발현되는 폴리펩티드는 서열번호 15로 표시될 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the polypeptide expressed from the host cell may be represented by SEQ ID NO: 15, but is not limited thereto.

본 발명의 또 다른 구현 예에 따르면, 본 발명에서 제공하는 형질 전환된 숙주 세포를 유효 성분으로 포함하는 암의 예방 또는 치료용 약학 조성물에 관한 것이다. According to another embodiment of the present invention, the present invention relates to a pharmaceutical composition for preventing or treating cancer comprising the transformed host cell provided as an active ingredient.

본 발명에서 제공하는 형질 전환된 숙주 세포, 바람직하게는 형질 전환된 T 세포 또는 NK 세포를 사용하는 경우 암 세포 중에서도 특히, 췌장암 또는 담관계암 세포를 특이적으로 인식 및 사멸시켜, 췌장암 또는 담관계암을 효과적으로 예방 또는 치료할 수 있다. When using the transformed host cells, preferably transformed T cells or NK cells provided by the present invention, among the cancer cells, pancreatic cancer or bile duct cancer cells are specifically recognized and killed, and thus pancreatic cancer or bile ducts Cancer can be effectively prevented or treated.

본 발명에서 "예방"은 본 발명의 조성물의 투여로 암을 억제하거나 진행을 지연시키는 모든 행위를 의미한다. In the present invention, "prevention" refers to all actions that inhibit cancer or delay progression by administration of the composition of the present invention.

본 발명에서 "치료" 및 "개선"은 본 발명의 조성물의 투여로 암의 증상이 호전 또는 이롭게 변경되는 모든 행위를 의미한다."Treatment" and "improvement" in the present invention means all actions in which symptoms of cancer are improved or beneficially changed by administration of the composition of the present invention.

본 발명의 약학 조성물은 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 약학 조성물로 제제화할 수 있다. 약제학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올, 리포좀 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있으며, 표적 기관에 특이적으로 작용할 수 있도록 표적 기관 특이적 항체 또는 기타 리간드를 상기 담체와 결합시켜 사용할 수 있다. 더 나아가 당해 기술 분야의 적정한 방법으로 또는 레밍턴의 문헌(Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.For administration, the pharmaceutical composition of the present invention may be formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration. Pharmaceutically acceptable carriers can be used by mixing one or more of these components: saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and, if necessary, antioxidants Other buffers, such as buffers and bacteriostatic agents, can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be added in addition to formulated into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, and act specifically on target organs. Target organ-specific antibodies or other ligands can be used in combination with the carrier. Furthermore, it can be preferably formulated according to each disease or ingredient by using a method suitable in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA. have.

본 발명의 약학 조성물은 액제, 현탁제, 분산액, 유제, 겔제, 주사 가능한 액제 및 활성 화합물의 서방출형 제제 등이 될 수 있으며, 바람직하게는 주사제가 될 수 있다.The pharmaceutical composition of the present invention may be a liquid, suspension, dispersion, emulsion, gel, injectable liquid and sustained-release preparation of the active compound, preferably an injection.

본 발명의 약학 조성물을 주사제로 제제화하는 경우, 주사제 처방의 유통에 따른 제품 안정성을 확보하기 위하여 주사제로 사용 가능한 산수용액 또는 인산염 등의 완충용액을 사용하여 pH를 조절함으로써 물리적으로나 화학적으로 매우 안정한 주사제로 제조될 수 있다.When the pharmaceutical composition of the present invention is formulated as an injectable agent, a physically or chemically very stable injectable agent is prepared by adjusting the pH using a buffer solution such as an aqueous acid solution or a phosphate that can be used as an injectable agent to secure product stability according to the distribution of the injectable formulation. Can be manufactured as.

보다 구체적으로, 상기 주사제는 안정화제 또는 용해 보조제와 함께 주사용수에 용해시킨 후, 멸균처리, 특히 고온감압멸균법 또는 무균여과법에 의해 멸균처리하여 제조될 수 있다. 상기 주사용수로는 주사용 증류수 또는 주사용 완충용액, 예를 들어 pH 3.5 내지 7.5 범위의 인산염 완충용액 또는 인산이수소나트륨(NaH2PO4)-구연산 완충용액을 사용할 수 있다. 사용되는 인산염은 나트륨염 또는 칼륨염 형태이거나 무수물 또는 수화물 형태이어도 무방하고, 구연산 또는 무수물 또는 수화물 형태이어도 무방하다.More specifically, the injection may be prepared by dissolving it in water for injection together with a stabilizer or a dissolution aid, followed by sterilization by sterilization, in particular, autoclaving or aseptic filtration. As the water for injection, distilled water for injection or a buffer solution for injection, for example, a phosphate buffer solution having a pH in the range of 3.5 to 7.5 or sodium dihydrogen phosphate (NaH2PO4) -citric acid buffer solution may be used. The phosphate salt used may be in the form of a sodium salt or a potassium salt, in the form of an anhydride or hydrate, or in the form of a citric acid or anhydride or hydrate.

또한, 본 발명에서 사용되는 안정화제는 나트륨 피로설파이트(sodium pyrosulfite), 중아황산나트륨(NaHSO3), 메타중아황산나트륨(Na2S2O3) 또는 에틸렌디아민테트라아세트산(ethylenediaminetetraacetic acid)을 포함하고, 용해 보조제는 수산화나트륨(NaOH), 탄산수소나트륨(NaHCO3), 탄산나트륨(NaCO3) 또는 수산화칼륨(KOH)과 같은 염기, 또는 염산(HCl) 또는 아세트산(CH3COOH)과 같은 산을 포함한다.In addition, the stabilizers used in the present invention include sodium pyrosulfite, sodium bisulfite (NaHSO 3 ), sodium metabisulfite (Na 2 S 2 O 3 ) or ethylenediaminetetraacetic acid, Dissolution aids include bases such as sodium hydroxide (NaOH), sodium hydrogen carbonate (NaHCO 3 ), sodium carbonate (NaCO 3 ) or potassium hydroxide (KOH), or acids such as hydrochloric acid (HCl) or acetic acid (CH 3 COOH). .

본 발명에 따른 주사제는 생체흡수성, 생체 분해성, 생체적합성으로 제형화될 수 있다. 생체흡수성이라 함은 주사제가 체내에서, 분산된 주사제의 분해 또는 분해 없이, 초기 적용에서 사라질 수 있음을 의미하는 것이다. 생체 분해성은 가수분해 또는 효소 분해에 의해 주사제가 체내에서 파쇄 또는 분해될 수 있음을 의미한다. 생체적 합성은 성분 모두가 체내에서 무독성임을 의미한다.The injection according to the present invention may be formulated as bioabsorbable, biodegradable, biocompatible. By bioabsorbability is meant that the injection may disappear in the body, without disintegration or degradation of the dispersed injection, in initial application. Biodegradability means that the injection may be broken or degraded in the body by hydrolysis or enzymatic degradation. Biosynthetic means that all ingredients are non-toxic in the body.

본 발명에 따른 주사제는 통상의 충진제, 중량제, 결합제, 습윤제, 계면활성제 등의 희석제, 또는 부형제 등을 사용하여 제조할 수 있다.The injection according to the present invention can be prepared by using a conventional filler, weight, binder, wetting agent, diluent such as surfactant, or excipient.

본 발명의 조성물 또는 유효성분은 목적에 따라 정맥내, 동맥내, 복강내, 근육내, 흉골내, 경피, 비측내, 피하, 자궁내 경막, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로 등을 통해 통상적인 방식으로 투여할 수 있으며, 바람직하게는 정맥내로 투여될 수 있다. 본 발명의 조성물 또는 유효성분은 주사 또는 카테터로 투여될 수 있다.The composition or active ingredient of the present invention may be intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, subcutaneous, intrauterine dura, inhalation, topical, rectal, oral, intraocular or intradermal route depending on the purpose. It can be administered in a conventional manner through the back, and can preferably be administered intravenously. The composition or active ingredient of the present invention can be administered by injection or catheter.

본 발명의 조성물에 있어서, 유효성분의 투여량은 체중 60 kg 성인기준으로 상기 약학 조성물 내에 포함되는 형질 전환된 숙주 세포가 1 x 101 ~ 1 x 1050개/ kg, 바람직하게는 1 x 101 ~ 1 x 1030개/ kg, 보다 바람직하게는 1 x 105 ~ 1 x 1020개/ kg, 가장 바람직하게는 1 x 107 ~ 1 x 109개/ kg의 범위 내로 투여될 수 있도록 조절할 수 있다. 다만, 투여될 최적의 투여량은 당업자에 의해 쉽게 결정될 수 있으며, 질환의 종류, 질환의 중증도, 조성물에 함유된 유효성분 및 다른 성분의 함량, 제형의 종류, 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다.In the composition of the present invention, the dosage of the active ingredient is 1 x 10 1 to 1 x 10 50 cells / kg, preferably 1 x 10, in which the transformed host cells included in the pharmaceutical composition are based on an adult weight of 60 kg. 1 to 1 x 10 30 pieces / kg, more preferably 1 x 10 5 to 1 x 10 20 pieces / kg, and most preferably 1 x 10 7 to 1 x 10 9 pieces / kg. Can be adjusted. However, the optimal dosage to be administered can be easily determined by those skilled in the art, the type of disease, the severity of the disease, the content of active ingredients and other ingredients in the composition, the type of formulation, and the patient's age, weight, general health It can be adjusted according to various factors, including condition, sex and diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, and drugs used simultaneously.

본 발명의 약학 조성물에 있어서 유효성분은, 조성물 총 중량에 대하여 0.001 내지 50 중량%로 함유될 수 있다. 그러나 함량은 이에 제한되지 않는다.In the pharmaceutical composition of the present invention, the active ingredient may be contained in an amount of 0.001 to 50% by weight based on the total weight of the composition. However, the content is not limited to this.

또한, 본 발명의 약학 조성물은 1종 이상의 항암제를 더 포함할 수 있다.In addition, the pharmaceutical composition of the present invention may further include one or more anti-cancer agents.

본 발명에서 상기 항암제로는 나이트로젠 머스타드, 이마티닙, 옥살리플라틴, 리툭시맙, 엘로티닙, 네라티닙, 라파티닙, 제피티닙, 반데타닙, 니로티닙, 세마사닙, 보수티닙, 악시티닙, 세디라닙, 레스타우르티닙, 트라스투주맙, 게피티니브, 보르테조밉, 수니티닙, 카보플라틴, 베바시주맙, 시스플라틴, 세툭시맙, 비스쿰알붐, 아스파라기나제, 트레티노인, 하이드록시카바마이드, 다사티닙, 에스트라머스틴, 겜투주맵오조가마이신, 이브리투맙튜세탄, 헵타플라틴, 메칠아미노레불린산, 암사크린, 알렘투주맙, 프로카르바진, 알프로스타딜, 질산홀뮴 키토산, 젬시타빈, 독시플루리딘, 페메트렉세드, 테가푸르, 카페시타빈, 기메라신, 오테라실, 아자시티딘, 메토트렉세이트, 우라실, 시타라빈, 플루오로우라실, 플루다가빈, 에노시타빈, 플루타미드, 데시타빈, 머캅토푸린, 티오구아닌, 클라드리빈, 카르퍼, 랄티트렉세드, 도세탁셀, 파클리탁셀, 이리노테칸, 벨로테칸, 토포테칸, 비노렐빈, 에토포시드, 빈크리스틴, 빈블라스틴, 테니포시드, 독소루비신, 이다루비신, 에피루비신, 미톡산트론, 미토마이신, 블레로마이신, 다우노루비신, 닥티노마이신, 피라루비신, 아클라루비신, 페프로마이신, 템시롤리무스, 테졸로마이드, 부설판, 이포스파미드, 사이클로포스파미드, 멜파란, 알트레트민, 다카바진, 치오테파, 니무스틴, 클로람부실, 미토락톨, 레우코보린, 트레토닌, 엑스메스탄, 아미노글루테시미드, 아나그렐리드, 나벨빈, 파드라졸, 타시펜, 토레미펜, 테스토락톤, 아나스트로졸, 레트로졸, 보로졸, 비칼루타미드, 로무스틴 및 카르무스틴으로 이루어진 군에서 선택된 1종 이상을 사할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the anticancer agents include nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nilotinib, semasanib, bosutinib, axitinib, Cediranib, Restautinib, Trastuzumab, Gefitinib, Bortezomib, Sunitinib, Carboplatin, Bevacizumab, Cisplatin, Cetuximab, Biscumalbum, Asparaginase, Tretinoin, Hydroxycarbamide , Dasatinib, Estramustine, gemtuzumab ozogamycin, Ibritumab tucetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, Gemcitabine, doxyfluridine, pemetrexed, tegapur, capecitabine, gimeracin, oteracyl, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludagabine, enocitabine, Flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carper, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine , Teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleomycin, daunorubicin, dactinomycin, pyrarubicin, aclarubicin, pepromycin, temsirolimus , Tezolomide, busulfan, iphosphamide, cyclophosphamide, melphalan, altretmine, dacarbazine, chiotepa, nimustine, chlorambucil, mitoractol, leucovorin, tretonin, exmestan , Consisting of aminoglutesimide, anagrelide, navelvin, padrazol, tasifene, toremifene, testolactone, anastrozole, letrozole, borosol, bicalutamide, lomustine and carmustine One or more selected from the group may be used, but is not limited thereto.

이하, 본 발명을 하기의 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다. Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

실시예Example

[실시예 1] Lenti-K030517S-EGFRt-AT-Free의 제작[Example 1] Preparation of Lenti-K030517S-EGFRt-AT-Free

서열번호 13으로 표시되는 EF-1 알파 프로모터-신호 펩타이드-CA19-9 결합 도메인(scFv)-CD8 힌지 영역-CD28 막통과 도메인-4-1BB-CD3ζ-T2A-EGFRt로 구성되는 1665bp 크기의 키메라 항원 수용체 카세트를 제작한 뒤, 이를 pCAR-EGFRt 렌티바이러스 벡터의 EcoRI/SmaI 위치에 삽입시켰다. A chimeric antigen of 1665 bp in size consisting of the EF-1 alpha promoter-signal peptide-CA19-9 binding domain (scFv) -CD8 hinge region-CD28 transmembrane domain-4-1BB-CD3ζ-T2A-EGFRt shown in SEQ ID NO: 13 After the receptor cassette was constructed, it was inserted into the EcoRI / SmaI site of the pCAR-EGFRt lentiviral vector.

이후 상기 키메라 항원 수용체 카세트가 벡터 내에 제대로 삽입되었는 지 확인하기 위하여, 상기 벡터에 EcoRI/BamHI 제한 효소를 처리한 뒤 겔 전기영동한 후 실시예 1에서 제작 삽입된 키메라 항원 수용체 카세트를 확인한 결과를 도 3에 나타내었다. Subsequently, in order to confirm that the chimeric antigen receptor cassette was properly inserted into the vector, the result of confirming the inserted chimeric antigen receptor cassette prepared in Example 1 after gel electrophoresis after treatment with EcoRI / BamHI restriction enzyme in the vector is shown. It is shown in 3.

도 3에서 보는 바와 같이, pCAR-EGFRt 렌티바이러스 벡터의 EcoRI/SmaI 위치에 키메라 항원 수용체 카세트를 삽입하였으나, 삽입 후 SmaI 사이트(site)가 소멸되어 EcoRI//BamHI으로 확인한 결과를 볼 수 있었다. As shown in FIG. 3, the chimeric antigen receptor cassette was inserted into the EcoRI / SmaI position of the pCAR-EGFRt lentiviral vector, but after insertion, the SmaI site disappeared, and the result confirmed by EcoRI // BamHI was seen.

[실시예 2] 췌장암 세포주에서 상기 키메라 항원 수용체 카세트가 타겟하는 CA 19-9의 발현 분석[Example 2] Expression analysis of CA 19-9 targeted by the chimeric antigen receptor cassette in a pancreatic cancer cell line

인체 유래 췌장암 세포주 BxPC3, Capan2, Cfpac, Hpac1, Miapaca2 및 Panc1에서 상기 실시예 1에서 제작된 키메라 항원 수용체가 타겟하는 CA 19-9 세포막 항원이 발현되는 지 확인하기 위하여 이하의 실험을 수행하였다. 1 X 2 cm 챔버 슬라이드(chamber slide)에 상기 각각의 췌장암 세포주를 1 X 104 씩 분주하여 챔버 바닥에 부착을 유도한 후 면역 형광 염색(immunofluorescence stain)을 수행하였다. 이때, CA 19-9 특이 항체는 Abcam 항-루이스 A 항체(anti-Lewis a antibody)를 이용하여 1:500의 희석배율로 실험하였다. PBS 용액으로 3회 세척 후 FITC-컨쥬게이트 항체(FITC-conjugated antibody) (1:500)를 2차 항체로 하여 1차 항체와 결합을 유도한 후 형광 현미경으로 관찰하여 그 사진을 도 4에 나타내었다. The following experiments were performed to confirm that the chimeric antigen receptors prepared in Example 1 in the human-derived pancreatic cancer cell lines BxPC3, Capan2, Cfpac, Hpac1, Miapaca2, and Panc1 target CA 19-9 cell membrane antigens. Each of the pancreatic cancer cell lines was divided by 1 X 10 4 on a 1 X 2 cm chamber slide to induce adhesion to the bottom of the chamber, and then immunofluorescence staining was performed. At this time, the CA 19-9 specific antibody was tested at a dilution ratio of 1: 500 using Abcam anti-Lewis a antibody. After washing with PBS solution three times, FITC-conjugated antibody (FITC-conjugated antibody) (1: 500) was used as a secondary antibody to induce binding with the primary antibody, and observed by a fluorescence microscope to show the picture in FIG. 4 Did.

그 결과 도 4에서 보는 바와 같이, 상기 키메라 항원 수용체 카세트가 타겟하는 CA 19-9의 세포막 항원은 상기 췌장 담도암 조직 세포주의 대략 70%에서 발현하는 결과를 보였다. As a result, as shown in FIG. 4, the cell membrane antigen of CA 19-9 targeted by the chimeric antigen receptor cassette was expressed in approximately 70% of the pancreatic biliary cancer tissue cell line.

이로부터 본 발명에 따른 상기 키메라 항원 수용체 카세트가 삽입된 T 세포가 이들 암 세포주를 공격할 수 있음을 알 수 있었다. From this, it was found that T cells into which the chimeric antigen receptor cassette according to the present invention was inserted can attack these cancer cell lines.

[실시예 3] CA 19-9 세포막 항원을 타겟하는 키메라 항원 수용체가 발현된 T 세포(Anti CA19-9 CAR-T cell)[Example 3] T cell expressing a chimeric antigen receptor targeting a CA 19-9 cell membrane antigen (Anti CA19-9 CAR-T cell)

형질 도입(transduction)을 위해 사용할 렌티바이러스(lentivirus)의 생성을 위해 가장 수율이 높은 Lenti-X 293 FT 세포 (1X106)를 접종하여 밤새(overnight) 배양하였다. 배양 후 리포펙타민 3000 형질 도입 키트(lipofectamine 3000 transduction kit)를 이용하여 상기 실시예 1에서 제작한 Lenti-K030517S-EGFRt-AT-Free 플라스미드(plasmid)로 형질 전환을 시행하고 48 시간 동안 배양하여 293 FT 세포에서 렌티바이러스가 생성되도록 하였다. For the production of lentivirus to be used for transduction, Lenti-X 293 FT cells (1X10 6 ) having the highest yield were inoculated and cultured overnight. After cultivation, the Lenti-K030517S-EGFRt-AT-Free plasmid prepared in Example 1 was transformed using the lipofectamine 3000 transduction kit, and cultured for 48 hours. Lentivirus was produced in FT cells.

한편, 말초 혈액에서 T 세포를 분리하여 유세포 분석기를 이용해 상기 T 세포의 순도를 확인하였고, 그 결과는 도 5에 나타내었다. 분리된 T 세포를 CD3/CD28 macs 비드(bead)를 이용하여 활성화하여 24 시간을 배양하였고, 활성화 전, 후의 T 세포를 현미경으로 저배율(좌측)과 고배율(우측)로 관찰하여 그 결과는 도 6에 나타내었다. Meanwhile, T cells were separated from peripheral blood to confirm the purity of the T cells using a flow cytometer, and the results are shown in FIG. 5. The isolated T cells were activated using CD3 / CD28 macs beads to incubate for 24 hours, and the T cells before and after activation were observed under a microscope at low magnification (left) and high magnification (right), and the results are shown in FIG. 6. It is shown in.

도 5에서 보는 바와 같이 T 세포의 순도는 97~98% 로 확인되었으며, 도 6의 아랫 배열 현미경 사진에서 나타난 바와 같이, 많은 양의 활성화된 T 세포 (activaed T cells)가 콜로니 (colony) 상태로 보이면서(갈색 부분) 성장하고 있음이 확인되었다. As shown in Figure 5, the purity of the T cells was confirmed to be 97-98%, as shown in the lower array micrograph of Figure 6, a large amount of activated T cells (activaed T cells) in the colony (colony) state It was confirmed that it was growing while being visible (brown part).

상기와 같이 활성화되어 배양된 T 세포에서 얻어진 3 X 105개의 T 세포에 렌티바이러스 입자를 각각 5 또는 10 MOI의 양으로 양성율을 비교하여 형질 도입을 수행하였다. 형질 도입의 수행 후 키메라 항원 수용체 카세트에 탑재된 EGFR 발현에 대한 유세포 분석(flow cytometry)을 수행하여 EGFRt를 발현하는 세포 분획을 측정하여 그 결과를 도 7에 나타내었다. As described above, the lentiviral particles in 3 X 10 5 T cells obtained from activated and cultured T cells were compared with a positive rate of 5 or 10 MOI, respectively, and transfection was performed. After performing the transduction, flow cytometry was performed on the expression of EGFR mounted on the chimeric antigen receptor cassette to measure the cell fraction expressing EGFRt, and the results are shown in FIG. 7.

도 7에서 보는 바와 같이 EGFRt 염색으로 형질 전환된 T 세포의 분석상 5 MOI에서는 13.8%, 10 MOI에서는 31.2%의 발현율을 보임으로써 CA19-9을 타켓하는 CAR-T 세포가 안정적으로 생성된 것을 확인할 수 있었다.As shown in FIG. 7, it was confirmed that CAR-T cells targeting CA19-9 were stably produced by showing the expression rate of 13.8% at 5 MOI and 31.2% at 10 MOI in the analysis of T cells transformed with EGFRt staining. Could.

이를 통하여 본 발명에 따른 키메라 항원 수용체가 높은 순도의 T 세포에서 안정적으로 삽입되어 상기 제작된 키메라 항원 수용체가 발현되고 있음을 알 수 있었다.Through this, it was found that the chimeric antigen receptor according to the present invention was stably inserted in high-purity T cells to express the prepared chimeric antigen receptor.

이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Since the specific parts of the present invention have been described in detail above, it is clear that for those skilled in the art, this specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

[서열목록][Sequence list]

서열번호 1: SEQ ID NO: 1:

MALPVTALLLPLALLLHAARPMALPVTALLLPLALLLHAARP

서열번호 2: SEQ ID NO: 2:

QVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHWVRQPPGKGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNSLRLEDTAFYYCAKDIRRFSTGGAEFEYWGQGTLVTVSSQVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHWVRQPPGKGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNSLRLEDTAFYYCAKDIRRFSTGGAEFEYWGQGTLVTVSS

서열번호 3: SEQ ID NO: 3:

QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSRSGTSASLAISGLRSEDEADYYCAAWDDSLGGHYVFGTGTKVTVLQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSRSGTSASLAISGLRSEDEADYYCAAWDDSLGGHYVFGTGTKVTVL

서열번호 4: SEQ ID NO: 4:

SGGGSGGGGSGGGGSSGGGSGGGGSGGGGS

서열번호 5: SEQ ID NO: 5

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

서열번호 6: SEQ ID NO: 6:

FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

서열번호 7: SEQ ID NO: 7:

QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSRSGTSASLAISGLRSEDEADYYCAAWDDSLGGHYVFGTGTKVTVLQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSRSGTSASLAISGLRSEDEADYYCAAWDDSLGGHYVFGTGTKVTVL

서열번호 8: SEQ ID NO: 8:

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

서열번호 9: SEQ ID NO: 9:

MALPVTALLLPLALLLHAARPQVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHWVRQPPGKGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNSLRLEDTAFYYCAKDIRRFSTGGAEFEYWGQGTLVTVSSSGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSRSGTSASLAISGLRSEDEADYYCAAWDDSLGGHYVFGTGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSRSGTSASLAISGLRSEDEADYYCAAWDDSLGGHYVFGTGTKVTVLRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRMALPVTALLLPLALLLHAARPQVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHWVRQPPGKGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNSLRLEDTAFYYCAKDIRRFSTGGAEFEYWGQGTLVTVSSSGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSRSGTSASLAISGLRSEDEADYYCAAWDDSLGGHYVFGTGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSRSGTSASLAISGLRSEDEADYYCAAWDDSLGGHYVFGTGTKVTVLRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

서열번호 10: SEQ ID NO: 10

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCAGCTGGTTGAATCTGGCGGAGGATCTGTGCAGCCTGGCAGAAGCCTGAGACTGAGCTGTGAAGCCTCCGGCTTCACCTTTGAGGCCTACGCCATGCACTGGGTCCGACAGCCACCTGGCAAAGGCCTTGAATGGGTGTCCAGCATCAACTGGAACAGCGGCAGAATCGCCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAGAAACAGCCTGTACCTGCAGATGAACAGCCTGCGGCTGGAAGATACCGCCTTCTACTACTGCGCCAAGGACATCAGACGGTTCAGCACAGGCGGAGCCGAGTTTGAGTATTGGGGCCAGGGAACCCTGGTCACCGTTTCTAGCGGCGGAGGTGGAAGCGGAGGCGGAGGTAGTGGTGGTGGCGGATCTCAGTCTGTGCTGACACAGCCTCCAAGCGCCTCTGGAACACCTGGCCAGAGAGTGACCATCAGCTGTAGCGGCAGCAGCAGCAACATCGGCAGCAACTTCGTGTACTGGTATCAGCAGCTGCCCGGCACAGCCCCTAAACTGCTGATCTACCGGAACAACCAGCGGCCTAGCGGCGTGCCAGATAGATTTTCTGGCAGCAGAAGCGGCACCTCTGCCAGCCTGGCTATCTCTGGCCTGAGAAGCGAGGACGAGGCCGACTATTATTGCGCCGCCTGGGATGATTCTCTCGGCGGCCACTATGTGTTTGGCACCGGCACCAAAGTGACCGTGCTTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTGAGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTACCGGAGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTGGCCCCATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCAGCTGGTTGAATCTGGCGGAGGATCTGTGCAGCCTGGCAGAAGCCTGAGACTGAGCTGTGAAGCCTCCGGCTTCACCTTTGAGGCCTACGCCATGCACTGGGTCCGACAGCCACCTGGCAAAGGCCTTGAATGGGTGTCCAGCATCAACTGGAACAGCGGCAGAATCGCCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAGAAACAGCCTGTACCTGCAGATGAACAGCCTGCGGCTGGAAGATACCGCCTTCTACTACTGCGCCAAGGACATCAGACGGTTCAGCACAGGCGGAGCCGAGTTTGAGTATTGGGGCCAGGGAACCCTGGTCACCGTTTCTAGCGGCGGAGGTGGAAGCGGAGGCGGAGGTAGTGGTGGTGGCGGATCTCAGTCTGTGCTGACACAGCCTCCAAGCGCCTCTGGAACACCTGGCCAGAGAGTGACCATCAGCTGTAGCGGCAGCAGCAGCAACATCGGCAGCAACTTCGTGTACTGGTATCAGCAGCTGCCCGGCACAGCCCCTAAACTGCTGATCTACCGGAACAACCAGCGGCCTAGCGGCGTGCCAGATAGATTTTCTGGCAGCAGAAGCGGCACCTCTGCCAGCCTGGCTATCTCTGGCCTGAGAAGCGAGGACGAGGCCGACTATTATTGCGCCGCCTGGGATGATTCTCTCGGCGGCCACTATGTGTTTGGCACCGGCACCAAAGTGACCGTGCTTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGA CAGTGGCCTTCATCATCTTTTGGGTGAGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTACCGGAGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTGGCCCCATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCT

서열번호 11: SEQ ID NO: 11:

ATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTCGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTCGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGA TGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATG

서열번호 12: SEQ ID NO: 12

GAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTGGCCCCGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTGGCCCC

서열번호 13: SEQ ID NO: 13

GAGTAATTCATACAAAAGGACTCGCCCCTGCCTTGGGGAATCCCAGGGACCGTCGTTAAACTCCCACTAACGTAGAACCCAGAGATCGCTGCGTTCCCGCCCCCTCACCCGCCCGCTCTCGTCATCACTGAGGTGGAGAAGAGCATGCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCTTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGATTCGAATTCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCAGCTGGTTGAATCTGGCGGAGGATCTGTGCAGCCTGGCAGAAGCCTGAGACTGAGCTGTGAAGCCTCCGGCTTCACCTTTGAGGCCTACGCCATGCACTGGGTCCGACAGCCACCTGGCAAAGGCCTTGAATGGGTGTCCAGCATCAACTGGAACAGCGGCAGAATCGCCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAGAAACAGCCTGTACCTGCAGATGAACAGCCTGCGGCTGGAAGATACCGCCTTCTACTACTGCGCCAAGGACATCAGACGGTTCAGCACAGGCGGAGCCGAGTTTGAGTATTGGGGCCAGGGAACCCTGGTCACCGTTTCTAGCGGCGGAGGTGGAAGCGGAGGCGGAGGTAGTGGTGGTGGCGGATCTCAGTCTGTGCTGACACAGCCTCCAAGCGCCTCTGGAACACCTGGCCAGAGAGTGACCATCAGCTGTAGCGGCAGCAGCAGCAACATCGGCAGCAACTTCGTGTACTGGTATCAGCAGCTGCCCGGCACAGCCCCTAAACTGCTGATCTACCGGAACAACCAGCGGCCTAGCGGCGTGCCAGATAGATTTTCTGGCAGCAGAAGCGGCACCTCTGCCAGCCTGGCTATCTCTGGCCTGAGAAGCGAGGACGAGGCCGACTATTATTGCGCCGCCTGGGATGATTCTCTCGGCGGCCACTATGTGTTTGGCACCGGCACCAAAGTGACCGTGCTTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTGAGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTACCGGAGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTGGCCCCGAGTAATTCATACAAAAGGACTCGCCCCTGCCTTGGGGAATCCCAGGGACCGTCGTTAAACTCCCACTAACGTAGAACCCAGAGATCGCTGCGTTCCCGCCCCCTCACCCGCCCGCTCTCGTCATCACTGAGGTGGAGAAGAGCATGCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCTTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGC GGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGATTCGAATTCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCAGCTGGTTGAATCTGGCGGAGGATCTGTGCAGCCTGGCAGAAGCCTGAGACTGAGCTGTGAAGCCTCCGGCTTCACCTTTGAGGCCTACGCCATGCACTGGGTCCGACAGCCACCTGGCAAAGGCCTTGAATGGGTGTCCAGCATCAACTGGAACAGCGGCAGAATCGCCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAGAAACAGCCTGTACCTGCAGATGAACAGCCTGCGGCTGGAAGATACCGCCTTCTACTACTGCGCCAAGGACATCAGACGGTTCAGCACAGGCGGAGCCGAGTTTGAGTATTGGGGCCAGGGAACCCTGGTCACCGTTTCTAGCGGCGGAGGTGGAAGCGGAGGCGGAGGTAGTGGTGGTGGCGGATCTCAGTCTGTGCTGACACAGCCTCCAAGCGCCTCTGGAACACCTGGCCAGAGAGTGACCATCAGCTGTAGCGGCAGCAGCAGCAACATCGGCAGCAACTTCGTGTACTGGTATCAGCAGCTGCCCGGCACAGCCCCTAAACTGCTGATCTACCGGAACAACCAGCGGCCTAGCGGCGTGCC AGATAGATTTTCTGGCAGCAGAAGCGGCACCTCTGCCAGCCTGGCTATCTCTGGCCTGAGAAGCGAGGACGAGGCCGACTATTATTGCGCCGCCTGGGATGATTCTCTCGGCGGCCACTATGTGTTTGGCACCGGCACCAAAGTGACCGTGCTTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTGAGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTACCGGAGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAAC CCTGGCCCC

서열번호 14:SEQ ID NO: 14

GAGTAATTCATACAAAAGGACTCGCCCCTGCCTTGGGGAATCCCAGGGACCGTCGTTAAACTCCCACTAACGTAGAACCCAGAGATCGCTGCGTTCCCGCCCCCTCACCCGCCCGCTCTCGTCATCACTGAGGTGGAGAAGAGCATGCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCTTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGATTCGAATTCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCAGCTGGTTGAATCTGGCGGAGGATCTGTGCAGCCTGGCAGAAGCCTGAGACTGAGCTGTGAAGCCTCCGGCTTCACCTTTGAGGCCTACGCCATGCACTGGGTCCGACAGCCACCTGGCAAAGGCCTTGAATGGGTGTCCAGCATCAACTGGAACAGCGGCAGAATCGCCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAGAAACAGCCTGTACCTGCAGATGAACAGCCTGCGGCTGGAAGATACCGCCTTCTACTACTGCGCCAAGGACATCAGACGGTTCAGCACAGGCGGAGCCGAGTTTGAGTATTGGGGCCAGGGAACCCTGGTCACCGTTTCTAGCGGCGGAGGTGGAAGCGGAGGCGGAGGTAGTGGTGGTGGCGGATCTCAGTCTGTGCTGACACAGCCTCCAAGCGCCTCTGGAACACCTGGCCAGAGAGTGACCATCAGCTGTAGCGGCAGCAGCAGCAACATCGGCAGCAACTTCGTGTACTGGTATCAGCAGCTGCCCGGCACAGCCCCTAAACTGCTGATCTACCGGAACAACCAGCGGCCTAGCGGCGTGCCAGATAGATTTTCTGGCAGCAGAAGCGGCACCTCTGCCAGCCTGGCTATCTCTGGCCTGAGAAGCGAGGACGAGGCCGACTATTATTGCGCCGCCTGGGATGATTCTCTCGGCGGCCACTATGTGTTTGGCACCGGCACCAAAGTGACCGTGCTTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTGAGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTACCGGAGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTGGCCCCATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTGGCCCCATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTCGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGGAGTAATTCATACAAAAGGACTCGCCCCTGCCTTGGGGAATCCCAGGGACCGTCGTTAAACTCCCACTAACGTAGAACCCAGAGATCGCTGCGTTCCCGCCCCCTCACCCGCCCGCTCTCGTCATCACTGAGGTGGAGAAGAGCATGCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCTTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGC GGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGATTCGAATTCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCAGCTGGTTGAATCTGGCGGAGGATCTGTGCAGCCTGGCAGAAGCCTGAGACTGAGCTGTGAAGCCTCCGGCTTCACCTTTGAGGCCTACGCCATGCACTGGGTCCGACAGCCACCTGGCAAAGGCCTTGAATGGGTGTCCAGCATCAACTGGAACAGCGGCAGAATCGCCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAGAAACAGCCTGTACCTGCAGATGAACAGCCTGCGGCTGGAAGATACCGCCTTCTACTACTGCGCCAAGGACATCAGACGGTTCAGCACAGGCGGAGCCGAGTTTGAGTATTGGGGCCAGGGAACCCTGGTCACCGTTTCTAGCGGCGGAGGTGGAAGCGGAGGCGGAGGTAGTGGTGGTGGCGGATCTCAGTCTGTGCTGACACAGCCTCCAAGCGCCTCTGGAACACCTGGCCAGAGAGTGACCATCAGCTGTAGCGGCAGCAGCAGCAACATCGGCAGCAACTTCGTGTACTGGTATCAGCAGCTGCCCGGCACAGCCCCTAAACTGCTGATCTACCGGAACAACCAGCGGCCTAGCGGCGTGCC AGATAGATTTTCTGGCAGCAGAAGCGGCACCTCTGCCAGCCTGGCTATCTCTGGCCTGAGAAGCGAGGACGAGGCCGACTATTATTGCGCCGCCTGGGATGATTCTCTCGGCGGCCACTATGTGTTTGGCACCGGCACCAAAGTGACCGTGCTTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTGAGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTACCGGAGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAAC CCTGGCCCCATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTGGCCCCATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTCGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAG ACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATG

서열번호 15: SEQ ID NO: 15

MALPVTALLLPLALLLHAARPQVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHWVRQPPGKGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNSLRLEDTAFYYCAKDIRRFSTGGAEFEYWGQGTLVTVSSSGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSRSGTSASLAISGLRSEDEADYYCAAWDDSLGGHYVFGTGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSRSGTSASLAISGLRSEDEADYYCAAWDDSLGGHYVFGTGTKVTVLRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMWLQSLLLLGTVACSISRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMMALPVTALLLPLALLLHAARPQVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHWVRQPPGKGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNSLRLEDTAFYYCAKDIRRFSTGGAEFEYWGQGTLVTVSSSGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSRSGTSASLAISGLRSEDEADYYCAAWDDSLGGHYVFGTGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSRSGTSASLAISGLRSEDEADYYCAAWDDSLGGHYVFGTGTKVTVLRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMWLQSLLLLGTVACSISRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

<110> SMT bio <120> Chimera antigen receptors for treating pancreatic cancer or biliary tract cancer <130> DPB174278k01 <150> KR 10-2018-0010901 <151> 2018-01-29 <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> n-terminal CD8-alpha signal peptide <400> 1 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 2 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> VH domain <400> 2 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Glu Ala Tyr 20 25 30 Ala Met His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asn Trp Asn Ser Gly Arg Ile Ala Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Leu Glu Asp Thr Ala Phe Tyr Tyr Cys 85 90 95 Ala Lys Asp Ile Arg Arg Phe Ser Thr Gly Gly Ala Glu Phe Glu Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 3 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL domain <400> 3 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 Ser Asn 20 25 30 Phe Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Arg 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 Ala Ala Trp Asp Asp Ser Leu 85 90 95 Gly Gly His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 4 Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 5 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> hinge region <400> 5 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 6 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> transmembrane domain <400> 6 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser 20 25 30 Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly 35 40 45 Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala 50 55 60 Ala Tyr Arg Ser 65 <210> 7 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> co-stimulatory domain <400> 7 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 Ser Asn 20 25 30 Phe Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Arg 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 Ala Ala Trp Asp Asp Ser Leu 85 90 95 Gly Gly His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 8 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> signal transduction domain <400> 8 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 9 <211> 606 <212> PRT <213> Artificial Sequence <220> <223> chimeric antigen receptor <400> 9 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser 20 25 30 Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe 35 40 45 Thr Phe Glu Ala Tyr Ala Met His Trp Val Arg Gln Pro Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Ser Ser Ile Asn Trp Asn Ser Gly Arg Ile Ala 65 70 75 80 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Arg Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Leu Glu Asp Thr 100 105 110 Ala Phe Tyr Tyr Cys Ala Lys Asp Ile Arg Arg Phe Ser Thr Gly Gly 115 120 125 Ala Glu Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130 135 140 Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 145 150 155 160 Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg 165 170 175 Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Phe 180 185 190 Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile 195 200 205 Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly 210 215 220 Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser 225 230 235 240 Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Gly 245 250 255 Gly His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Thr Thr 260 265 270 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 275 280 285 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 290 295 300 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val 305 310 315 320 Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala 325 330 335 Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser 340 345 350 Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His 355 360 365 Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln 370 375 380 Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg 385 390 395 400 Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Phe 405 410 415 Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile 420 425 430 Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly 435 440 445 Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser 450 455 460 Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Gly 465 470 475 480 Gly His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Arg Val 485 490 495 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn 500 505 510 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 515 520 525 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 530 535 540 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 545 550 555 560 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 565 570 575 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 580 585 590 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 595 600 605 <210> 10 <211> 1716 <212> DNA <213> Artificial Sequence <220> <223> chimeric antigen receptor <400> 10 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccgcaggtgc agctggttga atctggcgga ggatctgtgc agcctggcag aagcctgaga 120 ctgagctgtg aagcctccgg cttcaccttt gaggcctacg ccatgcactg ggtccgacag 180 ccacctggca aaggccttga atgggtgtcc agcatcaact ggaacagcgg cagaatcgcc 240 tacgccgact ctgtgaaggg cagattcacc atcagccggg acaacgccag aaacagcctg 300 tacctgcaga tgaacagcct gcggctggaa gataccgcct tctactactg cgccaaggac 360 atcagacggt tcagcacagg cggagccgag tttgagtatt ggggccaggg aaccctggtc 420 accgtttcta gcggcggagg tggaagcgga ggcggaggta gtggtggtgg cggatctcag 480 tctgtgctga cacagcctcc aagcgcctct ggaacacctg gccagagagt gaccatcagc 540 tgtagcggca gcagcagcaa catcggcagc aacttcgtgt actggtatca gcagctgccc 600 ggcacagccc ctaaactgct gatctaccgg aacaaccagc ggcctagcgg cgtgccagat 660 agattttctg gcagcagaag cggcacctct gccagcctgg ctatctctgg cctgagaagc 720 gaggacgagg ccgactatta ttgcgccgcc tgggatgatt ctctcggcgg ccactatgtg 780 tttggcaccg gcaccaaagt gaccgtgctt accacgacgc cagcgccgcg accaccaaca 840 ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 900 gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatttctg ggtgctggtc 960 gttgtgggcg gcgtgctggc ctgctacagc ctgctggtga cagtggcctt catcatcttt 1020 tgggtgagga gcaagcggag cagactgctg cacagcgact acatgaacat gaccccccgg 1080 aggcctggcc ccacccggaa gcactaccag ccctacgccc ctcccaggga tttcgccgcc 1140 taccggagca aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 1200 ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 1260 ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtacaagcag 1320 ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1380 gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1440 gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 1500 atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 1560 gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg cgagggcaga 1620 ggcagcctgc tgacatgtgg cgacgtggaa gagaaccctg gccccatgtg gctgcagagc 1680 ctgctgctct tgggcactgt ggcctgcagc atctct 1716 <210> 11 <211> 1056 <212> DNA <213> Artificial Sequence <220> <223> EGFRt <400> 11 atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc tcgcaaagtg 60 tgtaacggaa taggtattgg tgaatttaaa gactcactct ccataaatgc tacgaatatt 120 aaacacttca aaaactgcac ctccatcagt ggcgatctcc acatcctgcc ggtggcattt 180 aggggtgact ccttcacaca tactcctcct ctggatccac aggaactgga tattctgaaa 240 accgtaaagg aaatcacagg gtttttgctg attcaggctt ggcctgaaaa caggacggac 300 ctccatgcct ttgagaacct agaaatcata cgcggcagga ccaagcaaca tggtcagttt 360 tctcttgcag tcgtcagcct gaacataaca tccttgggat tacgctccct caaggagata 420 agtgatggag atgtgataat ttcaggaaac aaaaatttgt gctatgcaaa tacaataaac 480 tggaaaaaac tgtttgggac ctccggtcag aaaaccaaaa ttataagcaa cagaggtgaa 540 aacagctgca aggccacagg ccaggtctgc catgccttgt gctcccccga gggctgctgg 600 ggcccggagc ccagggactg cgtctcttgc cggaatgtca gccgaggcag ggaatgcgtg 660 gacaagtgca accttctgga gggtgagcca agggagtttg tggagaactc tgagtgcata 720 cagtgccacc cagagtgcct gcctcaggcc atgaacatca cctgcacagg acggggacca 780 gacaactgta tccagtgtgc ccactacatt gacggccccc actgcgtcaa gacctgcccg 840 gcaggagtca tgggagaaaa caacaccctg gtctggaagt acgcagacgc cggccatgtg 900 tgccacctgt gccatccaaa ctgcacctac ggatgcactg ggccaggtct tgaaggctgt 960 ccaacgaatg ggcctaagat cccgtccatc gccactggga tggtgggggc cctcctcttg 1020 ctgctggtgg tggccctggg gatcggcctc ttcatg 1056 <210> 12 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> T2A linker <400> 12 gagggcagag gcagcctgct gacatgtggc gacgtggaag agaaccctgg cccc 54 <210> 13 <211> 3009 <212> DNA <213> Artificial Sequence <220> <223> chimeric antigen receptor cassette <400> 13 gagtaattca tacaaaagga ctcgcccctg ccttggggaa tcccagggac cgtcgttaaa 60 ctcccactaa cgtagaaccc agagatcgct gcgttcccgc cccctcaccc gcccgctctc 120 gtcatcactg aggtggagaa gagcatgcgt gaggctccgg tgcccgtcag tgggcagagc 180 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct 240 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc 300 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca 360 acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct 420 ttacgggtta tggcccttgc gtgccttgaa ttacttccac gcccctggct gcagtacgtg 480 attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa 540 ggagcccctt cgcctcgtgc ttgagttgag gcctggcttg ggcgctgggg ccgccgcgtg 600 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa 660 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc 720 caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg 780 tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg 840 gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc 900 ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg 960 cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg 1020 ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac 1080 tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg 1140 tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg 1200 gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt 1260 gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca 1320 tttcaggtgt cgtgattcga attcatggcc ttaccagtga ccgccttgct cctgccgctg 1380 gccttgctgc tccacgccgc caggccgcag gtgcagctgg ttgaatctgg cggaggatct 1440 gtgcagcctg gcagaagcct gagactgagc tgtgaagcct ccggcttcac ctttgaggcc 1500 tacgccatgc actgggtccg acagccacct ggcaaaggcc ttgaatgggt gtccagcatc 1560 aactggaaca gcggcagaat cgcctacgcc gactctgtga agggcagatt caccatcagc 1620 cgggacaacg ccagaaacag cctgtacctg cagatgaaca gcctgcggct ggaagatacc 1680 gccttctact actgcgccaa ggacatcaga cggttcagca caggcggagc cgagtttgag 1740 tattggggcc agggaaccct ggtcaccgtt tctagcggcg gaggtggaag cggaggcgga 1800 ggtagtggtg gtggcggatc tcagtctgtg ctgacacagc ctccaagcgc ctctggaaca 1860 cctggccaga gagtgaccat cagctgtagc ggcagcagca gcaacatcgg cagcaacttc 1920 gtgtactggt atcagcagct gcccggcaca gcccctaaac tgctgatcta ccggaacaac 1980 cagcggccta gcggcgtgcc agatagattt tctggcagca gaagcggcac ctctgccagc 2040 ctggctatct ctggcctgag aagcgaggac gaggccgact attattgcgc cgcctgggat 2100 gattctctcg gcggccacta tgtgtttggc accggcacca aagtgaccgt gcttaccacg 2160 acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 2220 cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 2280 gcctgtgatt tctgggtgct ggtcgttgtg ggcggcgtgc tggcctgcta cagcctgctg 2340 gtgacagtgg ccttcatcat cttttgggtg aggagcaagc ggagcagact gctgcacagc 2400 gactacatga acatgacccc ccggaggcct ggccccaccc ggaagcacta ccagccctac 2460 gcccctccca gggatttcgc cgcctaccgg agcaaacggg gcagaaagaa actcctgtat 2520 atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 2580 tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 2640 gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 2700 cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 2760 aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 2820 gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 2880 ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 2940 gccctgcccc ctcgcgaggg cagaggcagc ctgctgacat gtggcgacgt ggaagagaac 3000 cctggcccc 3009 <210> 14 <211> 4170 <212> DNA <213> Artificial Sequence <220> <223> chimeric antigen receptor cassette <400> 14 gagtaattca tacaaaagga ctcgcccctg ccttggggaa tcccagggac cgtcgttaaa 60 ctcccactaa cgtagaaccc agagatcgct gcgttcccgc cccctcaccc gcccgctctc 120 gtcatcactg aggtggagaa gagcatgcgt gaggctccgg tgcccgtcag tgggcagagc 180 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct 240 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc 300 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca 360 acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct 420 ttacgggtta tggcccttgc gtgccttgaa ttacttccac gcccctggct gcagtacgtg 480 attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa 540 ggagcccctt cgcctcgtgc ttgagttgag gcctggcttg ggcgctgggg ccgccgcgtg 600 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa 660 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc 720 caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg 780 tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg 840 gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc 900 ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg 960 cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg 1020 ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac 1080 tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg 1140 tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg 1200 gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt 1260 gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca 1320 tttcaggtgt cgtgattcga attcatggcc ttaccagtga ccgccttgct cctgccgctg 1380 gccttgctgc tccacgccgc caggccgcag gtgcagctgg ttgaatctgg cggaggatct 1440 gtgcagcctg gcagaagcct gagactgagc tgtgaagcct ccggcttcac ctttgaggcc 1500 tacgccatgc actgggtccg acagccacct ggcaaaggcc ttgaatgggt gtccagcatc 1560 aactggaaca gcggcagaat cgcctacgcc gactctgtga agggcagatt caccatcagc 1620 cgggacaacg ccagaaacag cctgtacctg cagatgaaca gcctgcggct ggaagatacc 1680 gccttctact actgcgccaa ggacatcaga cggttcagca caggcggagc cgagtttgag 1740 tattggggcc agggaaccct ggtcaccgtt tctagcggcg gaggtggaag cggaggcgga 1800 ggtagtggtg gtggcggatc tcagtctgtg ctgacacagc ctccaagcgc ctctggaaca 1860 cctggccaga gagtgaccat cagctgtagc ggcagcagca gcaacatcgg cagcaacttc 1920 gtgtactggt atcagcagct gcccggcaca gcccctaaac tgctgatcta ccggaacaac 1980 cagcggccta gcggcgtgcc agatagattt tctggcagca gaagcggcac ctctgccagc 2040 ctggctatct ctggcctgag aagcgaggac gaggccgact attattgcgc cgcctgggat 2100 gattctctcg gcggccacta tgtgtttggc accggcacca aagtgaccgt gcttaccacg 2160 acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 2220 cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 2280 gcctgtgatt tctgggtgct ggtcgttgtg ggcggcgtgc tggcctgcta cagcctgctg 2340 gtgacagtgg ccttcatcat cttttgggtg aggagcaagc ggagcagact gctgcacagc 2400 gactacatga acatgacccc ccggaggcct ggccccaccc ggaagcacta ccagccctac 2460 gcccctccca gggatttcgc cgcctaccgg agcaaacggg gcagaaagaa actcctgtat 2520 atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 2580 tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 2640 gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 2700 cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 2760 aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 2820 gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 2880 ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 2940 gccctgcccc ctcgcgaggg cagaggcagc ctgctgacat gtggcgacgt ggaagagaac 3000 cctggcccca tgtggctgca gagcctgctg ctcttgggca ctgtggcctg cagcatctct 3060 gagggcagag gcagcctgct gacatgtggc gacgtggaag agaaccctgg ccccatgtgg 3120 ctgcagagcc tgctgctctt gggcactgtg gcctgcagca tctctcgcaa agtgtgtaac 3180 ggaataggta ttggtgaatt taaagactca ctctccataa atgctacgaa tattaaacac 3240 ttcaaaaact gcacctccat cagtggcgat ctccacatcc tgccggtggc atttaggggt 3300 gactccttca cacatactcc tcctctggat ccacaggaac tggatattct gaaaaccgta 3360 aaggaaatca cagggttttt gctgattcag gcttggcctg aaaacaggac ggacctccat 3420 gcctttgaga acctagaaat catacgcggc aggaccaagc aacatggtca gttttctctt 3480 gcagtcgtca gcctgaacat aacatccttg ggattacgct ccctcaagga gataagtgat 3540 ggagatgtga taatttcagg aaacaaaaat ttgtgctatg caaatacaat aaactggaaa 3600 aaactgtttg ggacctccgg tcagaaaacc aaaattataa gcaacagagg tgaaaacagc 3660 tgcaaggcca caggccaggt ctgccatgcc ttgtgctccc ccgagggctg ctggggcccg 3720 gagcccaggg actgcgtctc ttgccggaat gtcagccgag gcagggaatg cgtggacaag 3780 tgcaaccttc tggagggtga gccaagggag tttgtggaga actctgagtg catacagtgc 3840 cacccagagt gcctgcctca ggccatgaac atcacctgca caggacgggg accagacaac 3900 tgtatccagt gtgcccacta cattgacggc ccccactgcg tcaagacctg cccggcagga 3960 gtcatgggag aaaacaacac cctggtctgg aagtacgcag acgccggcca tgtgtgccac 4020 ctgtgccatc caaactgcac ctacggatgc actgggccag gtcttgaagg ctgtccaacg 4080 aatgggccta agatcccgtc catcgccact gggatggtgg gggccctcct cttgctgctg 4140 gtggtggccc tggggatcgg cctcttcatg 4170 <210> 15 <211> 976 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 15 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser 20 25 30 Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe 35 40 45 Thr Phe Glu Ala Tyr Ala Met His Trp Val Arg Gln Pro Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Ser Ser Ile Asn Trp Asn Ser Gly Arg Ile Ala 65 70 75 80 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Arg Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Leu Glu Asp Thr 100 105 110 Ala Phe Tyr Tyr Cys Ala Lys Asp Ile Arg Arg Phe Ser Thr Gly Gly 115 120 125 Ala Glu Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130 135 140 Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 145 150 155 160 Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg 165 170 175 Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Phe 180 185 190 Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile 195 200 205 Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly 210 215 220 Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser 225 230 235 240 Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Gly 245 250 255 Gly His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Thr Thr 260 265 270 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 275 280 285 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 290 295 300 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val 305 310 315 320 Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala 325 330 335 Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser 340 345 350 Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His 355 360 365 Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln 370 375 380 Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg 385 390 395 400 Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Phe 405 410 415 Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile 420 425 430 Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly 435 440 445 Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser 450 455 460 Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Gly 465 470 475 480 Gly His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Arg Val 485 490 495 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn 500 505 510 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 515 520 525 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 530 535 540 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 545 550 555 560 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 565 570 575 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 580 585 590 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Glu Gly 595 600 605 Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro 610 615 620 Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 625 630 635 640 Ser Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser 645 650 655 Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser 660 665 670 Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser 675 680 685 Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys 690 695 700 Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu 705 710 715 720 Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly 725 730 735 Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn 740 745 750 Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp 755 760 765 Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn 770 775 780 Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser 785 790 795 800 Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala 805 810 815 Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val 820 825 830 Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn 835 840 845 Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile 850 855 860 Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr 865 870 875 880 Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly 885 890 895 Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn 900 905 910 Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys 915 920 925 His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys 930 935 940 Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly 945 950 955 960 Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met 965 970 975 <110> SMT bio <120> Chimera antigen receptors for treating pancreatic cancer or          biliary tract cancer <130> DPB174278k01 <150> KR 10-2018-0010901 <151> 2018-01-29 <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> n-terminal CD8-alpha signal peptide <400> 1 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu   1 5 10 15 His Ala Ala Arg Pro              20 <210> 2 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> VH domain <400> 2 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Arg   1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Glu Ala Tyr              20 25 30 Ala Met His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ser Ile Asn Trp Asn Ser Gly Arg Ile Ala Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ser Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Leu Glu Asp Thr Ala Phe Tyr Tyr Cys                  85 90 95 Ala Lys Asp Ile Arg Arg Phe Ser Thr Gly Gly Ala Glu Phe Glu Tyr             100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 3 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL domain <400> 3 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 Ser Asn              20 25 30 Phe Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Arg 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 Ala Ala Trp Asp Asp Ser Leu                  85 90 95 Gly Gly His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu             100 105 110 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 4 Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 15 <210> 5 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> hinge region <400> 5 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala   1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly              20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp          35 40 45 <210> 6 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> transmembrane domain <400> 6 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu   1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser              20 25 30 Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly          35 40 45 Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala      50 55 60 Ala Tyr Arg Ser  65 <210> 7 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> co-stimulatory domain <400> 7 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 Ser Asn              20 25 30 Phe Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Arg 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 Ala Ala Trp Asp Asp Ser Leu                  85 90 95 Gly Gly His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu             100 105 110 <210> 8 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> signal transduction domain <400> 8 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly   1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr              20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys          35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys      50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg  65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala                  85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg             100 105 110 <210> 9 <211> 606 <212> PRT <213> Artificial Sequence <220> <223> chimeric antigen receptor <400> 9 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu   1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser              20 25 30 Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe          35 40 45 Thr Phe Glu Ala Tyr Ala Met His Trp Val Arg Gln Pro Pro Gly Lys      50 55 60 Gly Leu Glu Trp Val Ser Ser Ile Asn Trp Asn Ser Gly Arg Ile Ala  65 70 75 80 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala                  85 90 95 Arg Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Leu Glu Asp Thr             100 105 110 Ala Phe Tyr Tyr Cys Ala Lys Asp Ile Arg Arg Phe Ser Thr Gly Gly         115 120 125 Ala Glu Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser     130 135 140 Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 145 150 155 160 Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg                 165 170 175 Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Phe             180 185 190 Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile         195 200 205 Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly     210 215 220 Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser 225 230 235 240 Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Gly                 245 250 255 Gly His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Thr Thr             260 265 270 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln         275 280 285 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala     290 295 300 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val 305 310 315 320 Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala                 325 330 335 Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser             340 345 350 Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His         355 360 365 Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln     370 375 380 Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg 385 390 395 400 Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Phe                 405 410 415 Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile             420 425 430 Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly         435 440 445 Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser     450 455 460 Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Gly 465 470 475 480 Gly His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Arg Val                 485 490 495 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn             500 505 510 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val         515 520 525 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg     530 535 540 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 545 550 555 560 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg                 565 570 575 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys             580 585 590 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg         595 600 605 <210> 10 <211> 1716 <212> DNA <213> Artificial Sequence <220> <223> chimeric antigen receptor <400> 10 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccgcaggtgc agctggttga atctggcgga ggatctgtgc agcctggcag aagcctgaga 120 ctgagctgtg aagcctccgg cttcaccttt gaggcctacg ccatgcactg ggtccgacag 180 ccacctggca aaggccttga atgggtgtcc agcatcaact ggaacagcgg cagaatcgcc 240 tacgccgact ctgtgaaggg cagattcacc atcagccggg acaacgccag aaacagcctg 300 tacctgcaga tgaacagcct gcggctggaa gataccgcct tctactactg cgccaaggac 360 atcagacggt tcagcacagg cggagccgag tttgagtatt ggggccaggg aaccctggtc 420 accgtttcta gcggcggagg tggaagcgga ggcggaggta gtggtggtgg cggatctcag 480 tctgtgctga cacagcctcc aagcgcctct ggaacacctg gccagagagt gaccatcagc 540 tgtagcggca gcagcagcaa catcggcagc aacttcgtgt actggtatca gcagctgccc 600 ggcacagccc ctaaactgct gatctaccgg aacaaccagc ggcctagcgg cgtgccagat 660 agattttctg gcagcagaag cggcacctct gccagcctgg ctatctctgg cctgagaagc 720 gaggacgagg ccgactatta ttgcgccgcc tgggatgatt ctctcggcgg ccactatgtg 780 tttggcaccg gcaccaaagt gaccgtgctt accacgacgc cagcgccgcg accaccaaca 840 ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 900 gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatttctg ggtgctggtc 960 gttgtgggcg gcgtgctggc ctgctacagc ctgctggtga cagtggcctt catcatcttt 1020 tgggtgagga gcaagcggag cagactgctg cacagcgact acatgaacat gaccccccgg 1080 aggcctggcc ccacccggaa gcactaccag ccctacgccc ctcccaggga tttcgccgcc 1140 taccggagca aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 1200 ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 1260 ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtacaagcag 1320 ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1380 gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1440 gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 1500 atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 1560 gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg cgagggcaga 1620 ggcagcctgc tgacatgtgg cgacgtggaa gagaaccctg gccccatgtg gctgcagagc 1680 ctgctgctct tgggcactgt ggcctgcagc atctct 1716 <210> 11 <211> 1056 <212> DNA <213> Artificial Sequence <220> <223> EGFRt <400> 11 atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc tcgcaaagtg 60 tgtaacggaa taggtattgg tgaatttaaa gactcactct ccataaatgc tacgaatatt 120 aaacacttca aaaactgcac ctccatcagt ggcgatctcc acatcctgcc ggtggcattt 180 aggggtgact ccttcacaca tactcctcct ctggatccac aggaactgga tattctgaaa 240 accgtaaagg aaatcacagg gtttttgctg attcaggctt ggcctgaaaa caggacggac 300 ctccatgcct ttgagaacct agaaatcata cgcggcagga ccaagcaaca tggtcagttt 360 tctcttgcag tcgtcagcct gaacataaca tccttgggat tacgctccct caaggagata 420 agtgatggag atgtgataat ttcaggaaac aaaaatttgt gctatgcaaa tacaataaac 480 tggaaaaaac tgtttgggac ctccggtcag aaaaccaaaa ttataagcaa cagaggtgaa 540 aacagctgca aggccacagg ccaggtctgc catgccttgt gctcccccga gggctgctgg 600 ggcccggagc ccagggactg cgtctcttgc cggaatgtca gccgaggcag ggaatgcgtg 660 gacaagtgca accttctgga gggtgagcca agggagtttg tggagaactc tgagtgcata 720 cagtgccacc cagagtgcct gcctcaggcc atgaacatca cctgcacagg acggggacca 780 gacaactgta tccagtgtgc ccactacatt gacggccccc actgcgtcaa gacctgcccg 840 gcaggagtca tgggagaaaa caacaccctg gtctggaagt acgcagacgc cggccatgtg 900 tgccacctgt gccatccaaa ctgcacctac ggatgcactg ggccaggtct tgaaggctgt 960 ccaacgaatg ggcctaagat cccgtccatc gccactggga tggtgggggc cctcctcttg 1020 ctgctggtgg tggccctggg gatcggcctc ttcatg 1056 <210> 12 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> T2A linker <400> 12 gagggcagag gcagcctgct gacatgtggc gacgtggaag agaaccctgg cccc 54 <210> 13 <211> 3009 <212> DNA <213> Artificial Sequence <220> <223> chimeric antigen receptor cassette <400> 13 gagtaattca tacaaaagga ctcgcccctg ccttggggaa tcccagggac cgtcgttaaa 60 ctcccactaa cgtagaaccc agagatcgct gcgttcccgc cccctcaccc gcccgctctc 120 gtcatcactg aggtggagaa gagcatgcgt gaggctccgg tgcccgtcag tgggcagagc 180 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct 240 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc 300 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca 360 acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct 420 ttacgggtta tggcccttgc gtgccttgaa ttacttccac gcccctggct gcagtacgtg 480 attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa 540 ggagcccctt cgcctcgtgc ttgagttgag gcctggcttg ggcgctgggg ccgccgcgtg 600 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa 660 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc 720 caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg 780 tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg 840 gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc 900 ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg 960 cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg 1020 ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac 1080 tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg 1140 tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg 1200 gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt 1260 gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca 1320 tttcaggtgt cgtgattcga attcatggcc ttaccagtga ccgccttgct cctgccgctg 1380 gccttgctgc tccacgccgc caggccgcag gtgcagctgg ttgaatctgg cggaggatct 1440 gtgcagcctg gcagaagcct gagactgagc tgtgaagcct ccggcttcac ctttgaggcc 1500 tacgccatgc actgggtccg acagccacct ggcaaaggcc ttgaatgggt gtccagcatc 1560 aactggaaca gcggcagaat cgcctacgcc gactctgtga agggcagatt caccatcagc 1620 cgggacaacg ccagaaacag cctgtacctg cagatgaaca gcctgcggct ggaagatacc 1680 gccttctact actgcgccaa ggacatcaga cggttcagca caggcggagc cgagtttgag 1740 tattggggcc agggaaccct ggtcaccgtt tctagcggcg gaggtggaag cggaggcgga 1800 ggtagtggtg gtggcggatc tcagtctgtg ctgacacagc ctccaagcgc ctctggaaca 1860 cctggccaga gagtgaccat cagctgtagc ggcagcagca gcaacatcgg cagcaacttc 1920 gtgtactggt atcagcagct gcccggcaca gcccctaaac tgctgatcta ccggaacaac 1980 cagcggccta gcggcgtgcc agatagattt tctggcagca gaagcggcac ctctgccagc 2040 ctggctatct ctggcctgag aagcgaggac gaggccgact attattgcgc cgcctgggat 2100 gattctctcg gcggccacta tgtgtttggc accggcacca aagtgaccgt gcttaccacg 2160 acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 2220 cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 2280 gcctgtgatt tctgggtgct ggtcgttgtg ggcggcgtgc tggcctgcta cagcctgctg 2340 gtgacagtgg ccttcatcat cttttgggtg aggagcaagc ggagcagact gctgcacagc 2400 gactacatga acatgacccc ccggaggcct ggccccaccc ggaagcacta ccagccctac 2460 gcccctccca gggatttcgc cgcctaccgg agcaaacggg gcagaaagaa actcctgtat 2520 atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 2580 tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 2640 gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 2700 cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 2760 aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 2820 gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 2880 ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 2940 gccctgcccc ctcgcgaggg cagaggcagc ctgctgacat gtggcgacgt ggaagagaac 3000 cctggcccc 3009 <210> 14 <211> 4170 <212> DNA <213> Artificial Sequence <220> <223> chimeric antigen receptor cassette <400> 14 gagtaattca tacaaaagga ctcgcccctg ccttggggaa tcccagggac cgtcgttaaa 60 ctcccactaa cgtagaaccc agagatcgct gcgttcccgc cccctcaccc gcccgctctc 120 gtcatcactg aggtggagaa gagcatgcgt gaggctccgg tgcccgtcag tgggcagagc 180 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct 240 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc 300 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca 360 acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct 420 ttacgggtta tggcccttgc gtgccttgaa ttacttccac gcccctggct gcagtacgtg 480 attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa 540 ggagcccctt cgcctcgtgc ttgagttgag gcctggcttg ggcgctgggg ccgccgcgtg 600 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa 660 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc 720 caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg 780 tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg 840 gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc 900 ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg 960 cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg 1020 ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac 1080 tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg 1140 tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg 1200 gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt 1260 gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca 1320 tttcaggtgt cgtgattcga attcatggcc ttaccagtga ccgccttgct cctgccgctg 1380 gccttgctgc tccacgccgc caggccgcag gtgcagctgg ttgaatctgg cggaggatct 1440 gtgcagcctg gcagaagcct gagactgagc tgtgaagcct ccggcttcac ctttgaggcc 1500 tacgccatgc actgggtccg acagccacct ggcaaaggcc ttgaatgggt gtccagcatc 1560 aactggaaca gcggcagaat cgcctacgcc gactctgtga agggcagatt caccatcagc 1620 cgggacaacg ccagaaacag cctgtacctg cagatgaaca gcctgcggct ggaagatacc 1680 gccttctact actgcgccaa ggacatcaga cggttcagca caggcggagc cgagtttgag 1740 tattggggcc agggaaccct ggtcaccgtt tctagcggcg gaggtggaag cggaggcgga 1800 ggtagtggtg gtggcggatc tcagtctgtg ctgacacagc ctccaagcgc ctctggaaca 1860 cctggccaga gagtgaccat cagctgtagc ggcagcagca gcaacatcgg cagcaacttc 1920 gtgtactggt atcagcagct gcccggcaca gcccctaaac tgctgatcta ccggaacaac 1980 cagcggccta gcggcgtgcc agatagattt tctggcagca gaagcggcac ctctgccagc 2040 ctggctatct ctggcctgag aagcgaggac gaggccgact attattgcgc cgcctgggat 2100 gattctctcg gcggccacta tgtgtttggc accggcacca aagtgaccgt gcttaccacg 2160 acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 2220 cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 2280 gcctgtgatt tctgggtgct ggtcgttgtg ggcggcgtgc tggcctgcta cagcctgctg 2340 gtgacagtgg ccttcatcat cttttgggtg aggagcaagc ggagcagact gctgcacagc 2400 gactacatga acatgacccc ccggaggcct ggccccaccc ggaagcacta ccagccctac 2460 gcccctccca gggatttcgc cgcctaccgg agcaaacggg gcagaaagaa actcctgtat 2520 atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 2580 tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 2640 gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 2700 cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 2760 aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 2820 gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 2880 ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 2940 gccctgcccc ctcgcgaggg cagaggcagc ctgctgacat gtggcgacgt ggaagagaac 3000 cctggcccca tgtggctgca gagcctgctg ctcttgggca ctgtggcctg cagcatctct 3060 gagggcagag gcagcctgct gacatgtggc gacgtggaag agaaccctgg ccccatgtgg 3120 ctgcagagcc tgctgctctt gggcactgtg gcctgcagca tctctcgcaa agtgtgtaac 3180 ggaataggta ttggtgaatt taaagactca ctctccataa atgctacgaa tattaaacac 3240 ttcaaaaact gcacctccat cagtggcgat ctccacatcc tgccggtggc atttaggggt 3300 gactccttca cacatactcc tcctctggat ccacaggaac tggatattct gaaaaccgta 3360 aaggaaatca cagggttttt gctgattcag gcttggcctg aaaacaggac ggacctccat 3420 gcctttgaga acctagaaat catacgcggc aggaccaagc aacatggtca gttttctctt 3480 gcagtcgtca gcctgaacat aacatccttg ggattacgct ccctcaagga gataagtgat 3540 ggagatgtga taatttcagg aaacaaaaat ttgtgctatg caaatacaat aaactggaaa 3600 aaactgtttg ggacctccgg tcagaaaacc aaaattataa gcaacagagg tgaaaacagc 3660 tgcaaggcca caggccaggt ctgccatgcc ttgtgctccc ccgagggctg ctggggcccg 3720 gagcccaggg actgcgtctc ttgccggaat gtcagccgag gcagggaatg cgtggacaag 3780 tgcaaccttc tggagggtga gccaagggag tttgtggaga actctgagtg catacagtgc 3840 cacccagagt gcctgcctca ggccatgaac atcacctgca caggacgggg accagacaac 3900 tgtatccagt gtgcccacta cattgacggc ccccactgcg tcaagacctg cccggcagga 3960 gtcatgggag aaaacaacac cctggtctgg aagtacgcag acgccggcca tgtgtgccac 4020 ctgtgccatc caaactgcac ctacggatgc actgggccag gtcttgaagg ctgtccaacg 4080 aatgggccta agatcccgtc catcgccact gggatggtgg gggccctcct cttgctgctg 4140 gtggtggccc tggggatcgg cctcttcatg 4170 <210> 15 <211> 976 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 15 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu   1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser              20 25 30 Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe          35 40 45 Thr Phe Glu Ala Tyr Ala Met His Trp Val Arg Gln Pro Pro Gly Lys      50 55 60 Gly Leu Glu Trp Val Ser Ser Ile Asn Trp Asn Ser Gly Arg Ile Ala  65 70 75 80 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala                  85 90 95 Arg Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Leu Glu Asp Thr             100 105 110 Ala Phe Tyr Tyr Cys Ala Lys Asp Ile Arg Arg Phe Ser Thr Gly Gly         115 120 125 Ala Glu Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser     130 135 140 Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 145 150 155 160 Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg                 165 170 175 Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Phe             180 185 190 Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile         195 200 205 Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly     210 215 220 Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser 225 230 235 240 Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Gly                 245 250 255 Gly His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Thr Thr             260 265 270 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln         275 280 285 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala     290 295 300 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val 305 310 315 320 Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala                 325 330 335 Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser             340 345 350 Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His         355 360 365 Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln     370 375 380 Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg 385 390 395 400 Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Phe                 405 410 415 Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile             420 425 430 Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly         435 440 445 Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser     450 455 460 Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Gly 465 470 475 480 Gly His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Arg Val                 485 490 495 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn             500 505 510 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val         515 520 525 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg     530 535 540 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 545 550 555 560 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg                 565 570 575 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys             580 585 590 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Glu Gly         595 600 605 Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro     610 615 620 Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 625 630 635 640 Ser Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser                 645 650 655 Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser             660 665 670 Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser         675 680 685 Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys     690 695 700 Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu 705 710 715 720 Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly                 725 730 735 Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn             740 745 750 Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp         755 760 765 Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn     770 775 780 Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser 785 790 795 800 Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala                 805 810 815 Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val             820 825 830 Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn         835 840 845 Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile     850 855 860 Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr 865 870 875 880 Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly                 885 890 895 Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn             900 905 910 Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys         915 920 925 His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys     930 935 940 Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly 945 950 955 960 Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met                 965 970 975

Claims (32)

EF-1α 프로모터; n-터미널 CD8α 신호 펩타이드(n-terminal CD8α signal peptide); CA 19-9 특이적 단쇄 가변 단편(scFv); CD8 힌지; CD28 막통과 도메인; 4-1BB; CD3ζ; T2A 및 EGFRt를 암호화하는 폴리뉴클레오타이드로 구성되며, 서열번호 13으로 표시되는 췌장암 세포 특이적인 키메라 항원 수용체 발현 카세트.EF-1α promoter; n-terminal CD8α signal peptide; CA 19-9 specific short chain variable fragment (scFv); CD8 hinge; CD28 transmembrane domain; 4-1BB; CD3ζ; A pancreatic cancer cell-specific chimeric antigen receptor expression cassette consisting of polynucleotides encoding T2A and EGFRt, represented by SEQ ID NO: 13. 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 제1항의 발현 카세트를 포함하는, 벡터.A vector comprising the expression cassette of claim 1. 제26항에 있어서,
상기 벡터는 플라스미드, 트랜스포손, 코스미드 또는 바이러스 벡터인, 벡터. The method of claim 26,
The vector is a plasmid, transposon, cosmid or viral vector. 제26항에 있어서,
상기 벡터는 레트로바이러스 벡터, 아데노바이러스 벡터, 아데노-관련 바이러스(AAC) 벡터, 렌티바이러스 벡터, 폭스바이러스 벡터, 시미안 바이러스 벡터, 백시니아 바이러스 벡터 또는 센다이 바이러스 벡터, 엡스타인-바르(Epstein-Barr) 바이러스(EBV) 벡터 또는 HSV 벡터인, 벡터. The method of claim 26,
The vectors include retrovirus vectors, adenovirus vectors, adeno-associated virus (AAC) vectors, lentiviral vectors, poxvirus vectors, simian virus vectors, vaccinia virus vectors or Sendai virus vectors, Epstein-Barr A vector, which is a viral (EBV) vector or an HSV vector. 제26항의 벡터가 형질 전환된 숙주 세포. A host cell transformed with the vector of claim 26. 제29항에 있어서,
상기 숙주 세포는 T 세포 또는 NK 세포인, 형질 전환된 숙주 세포. The method of claim 29,
The host cell is a T cell or NK cell, a transformed host cell. 제29항의 숙주 세포를 유효 성분으로 포함하는, 암의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating cancer, comprising the host cell of claim 29 as an active ingredient. 제31항에 있어서,
상기 암은 췌장암 또는 담관계암인, 약학 조성물. The method of claim 31,
The cancer is pancreatic cancer or bile duct cancer, pharmaceutical composition.
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