TW201806970A - Chimeric antigen receptor and use thereof - Google Patents
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Abstract
Description
揭示關於嵌合抗原受體、嵌合抗原受體T細胞及抗體之技術。 Techniques for chimeric antigen receptors, chimeric antigen receptor T cells, and antibodies are disclosed.
伴隨免疫學之發展，對癌之各種免疫療法的開發有所進展。其中尤以將具有識別癌細胞與正常細胞並殺傷癌細胞之能力的T細胞移入患者之過繼免疫療法(adoptive immunotherapy)，作為可對應於癌之轉移/復發的副作用小之治療戰略而受到期待。但是，由癌患者上得到足夠發揮治療效果之質及量的T細胞係有困難，此為阻礙本治療法之臨床應用的原因之一。作為克服該問題之方法，近年來，大量配製可藉由嵌合抗原受體(CAR)基因之導入來攻擊癌細胞的T細胞，且將該表現CAR之T(CAR-T)細胞利用作為細胞醫藥之過繼免疫療法受到注目。 With the development of immunology, the development of various immunotherapy for cancer has made progress. Among them, adoptive immunotherapy for transplanting T cells capable of recognizing cancer cells and normal cells and killing cancer cells into patients is expected as a treatment strategy with a small side effect corresponding to cancer metastasis / recurrence. However, it is difficult to obtain sufficient quality and quantity of T cell lines from cancer patients, which is one of the reasons hindering the clinical application of this treatment method. As a method to overcome this problem, in recent years, a large number of T cells capable of attacking cancer cells by introduction of a chimeric antigen receptor (CAR) gene have been prepared, and CAR (T-CAR) cells expressing CAR have been used as cells. Medical adoptive immunotherapy has attracted attention.
目前研究有進展的CAR-T細胞療法之標的疾病多為造血系癌，特別是對B細胞淋巴瘤以CD19為標的之CAR-T細胞療法，於臨床試驗中被報告顯示出於以往之過繼免疫療法中無法得到之顯著效果。但是，顯示CAR-T細胞療法對固態腫瘤之有效性的臨床研究報告尚少。此可認 為係因為所移入之CAR-T細胞，於造血系腫瘤中容易與癌細胞接觸，相對於此，欲與固態腫瘤之癌細胞直接接觸，必須浸潤於血管外，進而通過間質組織之故。血液癌之罹患率為全部癌患者之未達5%，固態腫瘤佔其他之癌種類的多數。因而，對固態腫瘤之CAR-T細胞療法的開發受到需求。 The currently researched CAR-T cell therapy targets diseases that are mostly hematopoietic cancers, especially CAR-T cell therapy with CD19 as the target for B-cell lymphomas. It has been reported in clinical trials that it is due to past adoptive immunity. Significant results not available in therapy. However, few clinical studies have shown the effectiveness of CAR-T cell therapy in solid tumors. Recognizable This is because the CAR-T cells that are transplanted are easy to contact with cancer cells in hematopoietic tumors. In contrast, in order to directly contact cancer cells with solid tumors, they must infiltrate the blood vessels and then pass through the interstitial tissue. The incidence of blood cancer is less than 5% of all cancer patients, and solid tumors account for the majority of other cancer types. Therefore, the development of CAR-T cell therapy for solid tumors is in demand.
[非專利文獻1]Kanagawa, N. et al., Cancer Gene Ther., 20(1): 57-64 (2013) [Non-Patent Document 1] Kanagawa, N. et al., Cancer Gene Ther., 20 (1): 57-64 (2013)
[非專利文獻2]N Engl J Med. 2014 Oct 16; 371(16): 1507-17. [Non-Patent Document 2] N Engl J Med. 2014 Oct 16; 371 (16): 1507-17.
於如上述之現狀下，提供對癌等疾病之治療有效的新穎CAR及CAR-T細胞等係為課題之1。 Under the current situation as described above, it is the first task to provide novel CAR and CAR-T cells that are effective in the treatment of cancer and other diseases.
為了解決上述課題而重複努力研究的結果，發現了具有特定CDR序列之CAR及CAR-T細胞。於該見解上重複改良與探討，而提供以下述為代表之發明。 As a result of repeated efforts to solve the above-mentioned problems, CAR and CAR-T cells having specific CDR sequences were found. Based on this knowledge, improvements and discussions are repeated to provide inventions represented by the following.
項1. Item 1.
一種嵌合抗原受體，其係選自由下述任一者之嵌合抗原受體A~F所成之群：嵌合抗原受體A，其包含含有：包含具有序列編號1之胺基酸序列的胺基酸序列之輕鏈CDR1、包含具有序列編號2之胺基酸序列的胺基酸序列之輕鏈CDR2、及包含具有序列編號3之胺基酸序列的胺基酸序列之輕鏈CDR3之重鏈可變區域、以及/或含有：包含具有序列編號5之胺基酸序列的胺基酸序列之重鏈CDR1、包含具有序列編號6之胺基酸序列的胺基酸序列之重鏈CDR2、及包含具有序列編號7之胺基酸序列的胺基酸序列之重鏈CDR3之輕鏈可變區域；嵌合抗原受體B，其包含含有：包含具有序列編號21之胺基酸序列的胺基酸序列之輕鏈CDR1、包含具有序列編號22之胺基酸序列的胺基酸序列之輕鏈CDR2、及包含具有序列編號23之胺基酸序列的胺基酸序列之輕鏈CDR3 之重鏈可變區域、以及/或含有：包含具有序列編號25之胺基酸序列的胺基酸序列之重鏈CDR1、包含具有序列編號26之胺基酸序列的胺基酸序列之重鏈CDR2、及包含具有序列編號27之胺基酸序列的胺基酸序列之重鏈CDR3之輕鏈可變區域；嵌合抗原受體C，其包含含有：包含具有序列編號41之胺基酸序列的胺基酸序列之輕鏈CDR1、包含具有序列編號42之胺基酸序列的胺基酸序列之輕鏈CDR2、及包含具有序列編號43之胺基酸序列的胺基酸序列之輕鏈CDR3之重鏈可變區域、以及/或含有：包含具有序列編號45之胺基酸序列的胺基酸序列之重鏈CDR1、包含具有序列編號46之胺基酸序列的胺基酸序列之重鏈CDR2、及包含具有序列編號47之胺基酸序列的胺基酸序列之重鏈CDR3之輕鏈可變區域；嵌合抗原受體D，其包含 含有：包含具有序列編號61之胺基酸序列的胺基酸序列之輕鏈CDR1、包含具有序列編號62之胺基酸序列的胺基酸序列之輕鏈CDR2、及包含具有序列編號63之胺基酸序列的胺基酸序列之輕鏈CDR3之重鏈可變區域、以及/或含有：包含具有序列編號65之胺基酸序列的胺基酸序列之重鏈CDR1、包含具有序列編號66之胺基酸序列的胺基酸序列之重鏈CDR2、及包含具有序列編號67之胺基酸序列的胺基酸序列之重鏈CDR3之輕鏈可變區域；嵌合抗原受體E，其包含含有：包含具有序列編號81之胺基酸序列的胺基酸序列之輕鏈CDR1、包含具有序列編號82之胺基酸序列的胺基酸序列之輕鏈CDR2、及包含具有序列編號83之胺基酸序列的胺基酸序列之輕鏈CDR3之重鏈可變區域、以及/或含有：包含具有序列編號85之胺基酸序列的胺基酸序列之重鏈CDR1、 包含具有序列編號86之胺基酸序列的胺基酸序列之重鏈CDR2、及包含具有序列編號87之胺基酸序列的胺基酸序列之重鏈CDR3之輕鏈可變區域；或嵌合抗原受體F，其包含含有：包含具有序列編號101之胺基酸序列的胺基酸序列之輕鏈CDR1、包含具有序列編號102之胺基酸序列的胺基酸序列之輕鏈CDR2、及包含具有序列編號103之胺基酸序列的胺基酸序列之輕鏈CDR3之重鏈可變區域、以及/或含有：包含具有序列編號105之胺基酸序列的胺基酸序列之重鏈CDR1、包含具有序列編號106之胺基酸序列的胺基酸序列之重鏈CDR2、及包含具有序列編號107之胺基酸序列的胺基酸序列之重鏈CDR3之輕鏈可變區域。 A chimeric antigen receptor is selected from the group consisting of chimeric antigen receptors A to F of any one of the following: chimeric antigen receptor A, comprising: containing an amino acid having the sequence number 1 CDR1 of the light chain of the amino acid sequence of the sequence, CDR2 of the light chain including the amino acid sequence having the amino acid sequence of SEQ ID NO: 2, and the light chain of the amino acid sequence including the amino acid sequence of the serial number 3 The heavy chain variable region of CDR3 and / or contains: heavy chain CDR1 containing an amino acid sequence having the amino acid sequence of SEQ ID NO: 5; Chain CDR2, and a light chain variable region of a heavy chain CDR3 comprising an amino acid sequence having the amino acid sequence of SEQ ID NO: 7; a chimeric antigen receptor B comprising: CDR1 of the light chain of the amino acid sequence of the sequence, CDR2 of the light chain including the amino acid sequence having the amino acid sequence of sequence number 22, and CDR2 of the light chain including the amino acid sequence of the amino acid sequence of sequence number 23 CDR3 Heavy chain variable region, and / or heavy chain CDR1 comprising an amino acid sequence having the amino acid sequence of SEQ ID NO: 25, and heavy chain comprising an amino acid sequence having the amino acid sequence of SEQ ID NO: 26 CDR2, and a light chain variable region of a heavy chain CDR3 comprising an amino acid sequence having the amino acid sequence of SEQ ID NO: 27; a chimeric antigen receptor C, comprising: containing an amino acid sequence having the amino acid sequence of 41 CDR1 of the amino acid sequence of the light chain, CDR2 of the light chain including the amino acid sequence of the amino acid sequence of SEQ ID NO. 42, and CDR3 of the light chain of the amino acid sequence of the amino acid sequence of SEQ ID NO: 43 Heavy chain variable region, and / or heavy chain CDR1 containing an amino acid sequence having the amino acid sequence of sequence number 45, and heavy chain containing an amino acid sequence having the amino acid sequence of sequence number 46 CDR2, and a light chain variable region of a heavy chain CDR3 comprising an amino acid sequence having the amino acid sequence of SEQ ID NO: 47; a chimeric antigen receptor D, comprising Contains: a light chain CDR1 comprising an amino acid sequence having the amino acid sequence of sequence number 61, a light chain CDR2 comprising an amino acid sequence having the amino acid sequence of sequence number 62, and an amine including the amino acid sequence of 63 Heavy chain variable region of the light chain CDR3 of the amino acid sequence of the amino acid sequence, and / or contains: CDR1 of the heavy chain including the amino acid sequence of the amino acid sequence having the amino acid sequence of 65, A heavy chain CDR2 of the amino acid sequence of the amino acid sequence, and a light chain variable region of the heavy chain CDR3 including the amino acid sequence of the amino acid sequence of SEQ ID NO: 67; a chimeric antigen receptor E, comprising Contains: a light chain CDR1 comprising an amino acid sequence having the amino acid sequence of sequence number 81, a light chain CDR2 comprising an amino acid sequence having the amino acid sequence of sequence number 82, and an amine having the amino acid sequence of 83 Heavy chain variable region of the light chain CDR3 of the amino acid sequence of the amino acid sequence, and / or contains: a heavy chain CDR1 comprising an amino acid sequence having the amino acid sequence of SEQ ID NO: 85; A heavy chain CDR2 comprising an amino acid sequence having an amino acid sequence of SEQ ID NO: 86, and a light chain variable region of a heavy chain CDR3 comprising an amino acid sequence having an amino acid sequence of SEQ ID 87; or chimeric An antigen receptor F comprising a light chain CDR1 comprising an amino acid sequence comprising an amino acid sequence having the sequence number 101, a light chain CDR2 comprising an amino acid sequence having an amino acid sequence of the sequence number 1022, and A heavy chain variable region comprising a light chain CDR3 having an amino acid sequence having the amino acid sequence of sequence number 103, and / or a heavy chain CDR1 containing an amino acid sequence having the amino acid sequence of sequence number 105 A heavy chain CDR comprising an amino acid sequence having an amino acid sequence of sequence number 106, and a light chain variable region of a heavy chain CDR3 including an amino acid sequence having an amino acid sequence of sequence number 107.
項2. Item 2.
一種如項1之嵌合抗原受體A。 A chimeric antigen receptor A as described in item 1.
項3. Item 3.
一種嵌合抗原受體T細胞或嵌合抗原受體NK細胞，其 具有如項1或2之嵌合抗原受體。 A chimeric antigen receptor T cell or a chimeric antigen receptor NK cell, which A chimeric antigen receptor according to item 1 or 2.
項4. Item 4.
一種多核苷酸，其編碼如項1或2之嵌合抗原受體。 A polynucleotide encoding a chimeric antigen receptor according to item 1 or 2.
項5. Item 5.
一種醫藥組成物，其含有如項3之嵌合抗原受體T細胞或嵌合抗原受體NK細胞。 A pharmaceutical composition comprising a chimeric antigen receptor T cell or a chimeric antigen receptor NK cell according to item 3.
項6. Item 6.
如項5之醫藥組成物，其係癌之治療或預防用。 The pharmaceutical composition according to item 5, which is used for treating or preventing cancer.
提供有效於癌(較佳為固態癌)之治療的手段。 Provide means effective for the treatment of cancer, preferably solid cancer.
[圖1]表示嵌合抗原受體A所具有的scFV之胺基酸序列。實線之下線表示之區域為輕鏈可變區域。虛線之下線表示之區域為重鏈可變區域。無下線之區域為連結子(linker)。粗體字之大字型表示之區域，如其下所示，為輕鏈及重鏈之CDR1~3。 Fig. 1 shows the amino acid sequence of scFV possessed by the chimeric antigen receptor A. The area indicated by the solid line is the light chain variable region. The region indicated by the line below the dotted line is the heavy chain variable region. The area without offline is a linker. The areas indicated by the large font in bold type are, as shown below, the CDRs 1-3 of the light and heavy chains.
[圖2]表示嵌合抗原受體B所具有的scFV之胺基酸序列。實線之下線表示之區域為輕鏈可變區域。虛線之下線表示之區域為重鏈可變區域。無下線之區域為連結子。粗體字之大字型表示之區域，如其下所示，為輕鏈及重鏈之CDR1~3。 Fig. 2 shows the amino acid sequence of scFV possessed by the chimeric antigen receptor B. The area indicated by the solid line is the light chain variable region. The region indicated by the line below the dotted line is the heavy chain variable region. Areas without offline are linkers. The areas indicated by the large font in bold type are, as shown below, the CDRs 1-3 of the light and heavy chains.
[圖3]表示嵌合抗原受體C所具有的scFV之胺基酸序列。實線之下線表示之區域為輕鏈可變區域。虛線之下線表示之區域為重鏈可變區域。無下線之區域為連結子。粗體字之大字型表示之區域，如其下所示，為輕鏈及重鏈之CDR1~3。 Fig. 3 shows the amino acid sequence of scFV possessed by the chimeric antigen receptor C. The area indicated by the solid line is the light chain variable region. The region indicated by the line below the dotted line is the heavy chain variable region. Areas without offline are linkers. The areas indicated by the large font in bold type are, as shown below, the CDRs 1-3 of the light and heavy chains.
[圖4]表示嵌合抗原受體D所具有的scFV之胺基酸序列。實線之下線表示之區域為輕鏈可變區域。虛線之下線表示之區域為重鏈可變區域。無下線之區域為連結子。粗體字之大字型表示之區域，如其下所示，為輕鏈及重鏈之CDR1~3。 Fig. 4 shows the amino acid sequence of scFV possessed by the chimeric antigen receptor D. The area indicated by the solid line is the light chain variable region. The region indicated by the line below the dotted line is the heavy chain variable region. Areas without offline are linkers. The areas indicated by the large font in bold type are, as shown below, the CDRs 1-3 of the light and heavy chains.
[圖5]表示嵌合抗原受體E所具有的scFV之胺基酸序列。實線之下線表示之區域為輕鏈可變區域。虛線之下線表示之區域為重鏈可變區域。無下線之區域為連結子。粗體字之大字型表示之區域，如其下所示，為輕鏈及重鏈之CDR1~3。 Fig. 5 shows the amino acid sequence of scFV possessed by the chimeric antigen receptor E. The area indicated by the solid line is the light chain variable region. The region indicated by the line below the dotted line is the heavy chain variable region. Areas without offline are linkers. The areas indicated by the large font in bold type are, as shown below, the CDRs 1-3 of the light and heavy chains.
[圖6]表示嵌合抗原受體F所具有的scFV之胺基酸序列。實線之下線表示之區域為輕鏈可變區域。虛線之下線表示之區域為重鏈可變區域。無下線之區域為連結子。粗體字之大字型表示之區域，如其下所示，為輕鏈及重鏈之CDR1~3。 Fig. 6 shows the amino acid sequence of scFV possessed by the chimeric antigen receptor F. The area indicated by the solid line is the light chain variable region. The region indicated by the line below the dotted line is the heavy chain variable region. Areas without offline are linkers. The areas indicated by the large font in bold type are, as shown below, the CDRs 1-3 of the light and heavy chains.
[圖7]表示編碼嵌合抗原受體A所具有的scFV之胺基酸序列的鹼基序列。實線之下線表示之區域為輕鏈可變區域。虛線之下線表示之區域為重鏈可變區域。無下線之區域為連結子。粗體字之大字型表示之區域，如其下所示， 為輕鏈及重鏈之CDR1~3。 FIG. 7 shows a base sequence encoding an amino acid sequence of scFV possessed by the chimeric antigen receptor A. FIG. The area indicated by the solid line is the light chain variable region. The region indicated by the line below the dotted line is the heavy chain variable region. Areas without offline are linkers. The area indicated by the large font in bold type, as shown below, CDR1 ~ 3 for light and heavy chains.
[圖8]表示編碼嵌合抗原受體B所具有的scFV之胺基酸序列的鹼基序列。實線之下線表示之區域為輕鏈可變區域。虛線之下線表示之區域為重鏈可變區域。無下線之區域為連結子。粗體字之大字型表示之區域，如其下所示，為輕鏈及重鏈之CDR1~3。 FIG. 8 shows a base sequence encoding an amino acid sequence of scFV possessed by the chimeric antigen receptor B. FIG. The area indicated by the solid line is the light chain variable region. The region indicated by the line below the dotted line is the heavy chain variable region. Areas without offline are linkers. The areas indicated by the large font in bold type are, as shown below, the CDRs 1-3 of the light and heavy chains.
[圖9]表示編碼嵌合抗原受體C所具有的scFV之胺基酸序列的鹼基序列。實線之下線表示之區域為輕鏈可變區域。虛線之下線表示之區域為重鏈可變區域。無下線之區域為連結子。粗體字之大字型表示之區域，如其下所示，為輕鏈及重鏈之CDR1~3。 FIG. 9 shows a base sequence encoding an amino acid sequence of scFV possessed by the chimeric antigen receptor C. FIG. The area indicated by the solid line is the light chain variable region. The region indicated by the line below the dotted line is the heavy chain variable region. Areas without offline are linkers. The areas indicated by the large font in bold type are, as shown below, the CDRs 1-3 of the light and heavy chains.
[圖10]表示編碼嵌合抗原受體D所具有的scFV之胺基酸序列的鹼基序列。實線之下線表示之區域為輕鏈可變區域。虛線之下線表示之區域為重鏈可變區域。無下線之區域為連結子。粗體字之大字型表示之區域，如其下所示，為輕鏈及重鏈之CDR1~3。 Fig. 10 shows a base sequence encoding an amino acid sequence of scFV possessed by the chimeric antigen receptor D. The area indicated by the solid line is the light chain variable region. The region indicated by the line below the dotted line is the heavy chain variable region. Areas without offline are linkers. The areas indicated by the large font in bold type are, as shown below, the CDRs 1-3 of the light and heavy chains.
[圖11]表示編碼嵌合抗原受體E所具有的scFV之胺基酸序列的鹼基序列。實線之下線表示之區域為輕鏈可變區域。虛線之下線表示之區域為重鏈可變區域。無下線之區域為連結子。粗體字之大字型表示之區域，如其下所示，為輕鏈及重鏈之CDR1~3。 Fig. 11 shows a base sequence encoding an amino acid sequence of scFV possessed by the chimeric antigen receptor E. The area indicated by the solid line is the light chain variable region. The region indicated by the line below the dotted line is the heavy chain variable region. Areas without offline are linkers. The areas indicated by the large font in bold type are, as shown below, the CDRs 1-3 of the light and heavy chains.
[圖12]表示編碼嵌合抗原受體F所具有的scFV之胺基酸序列的鹼基序列。實線之下線表示之區域為輕鏈可變區域。虛線之下線表示之區域為重鏈可變區域。無下線之區 域為連結子。粗體字之大字型表示之區域，如其下所示，為輕鏈及重鏈之CDR1~3。 FIG. 12 shows a base sequence encoding an amino acid sequence of scFV possessed by the chimeric antigen receptor F. FIG. The area indicated by the solid line is the light chain variable region. The region indicated by the line below the dotted line is the heavy chain variable region. No offline area Domains are linkers. The areas indicated by the large font in bold type are, as shown below, the CDRs 1-3 of the light and heavy chains.
[圖13]表示pMXs-IG/CAR[mV-(h28)-h28-h3Z]之構築步驟。 [Figure 13] Construction steps of pMXs-IG / CAR [mV- (h28) -h28-h3Z].
[圖14]表示pMXs-IG/CAR[hV-(h28)-h28-h3Z]之構築步驟。 [Fig. 14] shows the construction steps of pMXs-IG / CAR [hV- (h28) -h28-h3Z].
[圖15]表示pMXs-IG/CAR[mV-(h8α)-h137-h3Z]之構築步驟。 [Fig. 15] Construction steps of pMXs-IG / CAR [mV- (h8α) -h137-h3Z].
[圖16]表示pMXs-IG/CAR[hV-(h8α)-h137-h3Z]之構築步驟。 [Fig. 16] Construction steps of pMXs-IG / CAR [hV- (h8α) -h137-h3Z].
[圖17]表示編碼各CAR之pMXs-IG載體的構造。 Fig. 17 shows the structure of pMXs-IG vectors encoding each CAR.
[圖18]表示測定CAR-T細胞所致之in-vivo抗腫瘤效果的結果。 Fig. 18 shows the results of measuring the anti-tumor effect of in-vivo caused by CAR-T cells.
1.嵌合抗原受體 Chimeric antigen receptor
嵌合抗原受體(CAR)，係指N末端側具有直列結合單株抗體可變區域之輕鏈(VL)與重鏈(VH)而得的單鏈抗體(scFv)、C末端側具有T細胞受體(TCR)ζ鏈之嵌合蛋白。表現CAR之T細胞，係稱為CAR-T細胞。 Chimeric antigen receptor (CAR) refers to a single-chain antibody (scFv) obtained by binding the light chain (VL) and heavy chain (VH) of a variable region of a monoclonal antibody in-line at the N-terminus side, with T at the C-terminus side Chimeric protein of the cell receptor (TCR) zeta chain. T cells that express CAR are called CAR-T cells.
對嵌合抗原受體A~F所具有之scFV區域之胺基酸序列及編碼其之鹼基序列所賦予的序列編號係如下述表1所示。表內之數字意指序列編號。AA意指胺基酸序列。 V意指可變區域。scFV意指scFV區域全體。 The sequence numbers assigned to the amino acid sequences of the scFV regions of the chimeric antigen receptors A to F and the base sequences encoding them are shown in Table 1 below. The numbers in the table mean serial numbers. AA means amino acid sequence. V means a variable region. scFV means the entire scFV region.
嵌合抗原受體A，較佳具有選自由包含具有序列編號1之胺基酸序列的胺基酸序列之輕鏈CDR1、包含具有序列編號2之胺基酸序列的胺基酸序列之輕鏈CDR2、包含具有序列編號3之胺基酸序列的胺基酸序列之輕鏈CDR3、包含具有序列編號5之胺基酸序列的胺基酸序列之重鏈CDR1、包含具有序列編號6之胺基酸序列的胺基酸序列之重鏈CDR2、及包含具有序列編號7之胺基酸序列的胺基酸序列之重鏈CDR3所成之群的至少一種CDR，更佳為具有2種以上、更佳為具有3種以上、更佳為具有4種以上、更佳為具有5種以上、更佳為具有全部的CDR。適宜之一實施形態中，嵌合抗原受體A較佳具有：具有序列編號4之胺基酸序列的輕鏈可變區域及/或具有序列編號8之胺基酸序列的重鏈可變區域。適宜之一實施形態中，嵌合抗原受體A較佳具有：具有序列編號10表示之胺基酸序列的scFV構造。 The chimeric antigen receptor A preferably has a light chain selected from the light chain CDR1 comprising an amino acid sequence having an amino acid sequence having the sequence number 1 and a light chain comprising an amino acid sequence having an amino acid sequence having the sequence number 2 CDR2, a light chain CDR3 containing an amino acid sequence having the amino acid sequence of sequence number 3, a CDR1 containing a heavy chain having an amino acid sequence having the amino acid sequence of sequence number 5, CDR1, including an amine group having the sequence number 6 At least one CDR of the group consisting of the heavy chain CDR2 of the amino acid sequence of the acid sequence and the heavy chain CDR3 including the amino acid sequence of the amino acid sequence of SEQ ID NO: 7, more preferably two or more, It is preferable that it has 3 or more types, it is more preferable that it has 4 or more types, it is more preferable that it has 5 or more types, and it is more preferable that it has all the CDRs. In a suitable embodiment, the chimeric antigen receptor A preferably has a light chain variable region having an amino acid sequence of sequence number 4 and / or a heavy chain variable region having an amino acid sequence of sequence number 8 . In a suitable embodiment, the chimeric antigen receptor A preferably has a scFV structure having an amino acid sequence represented by SEQ ID NO: 10.
一實施形態中，嵌合抗原受體A之輕鏈可變區 域之胺基酸序列，與序列編號4之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體A之重鏈可變區域之胺基酸序列，與序列編號8之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體A，與序列編號10之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體A所具有之連結子之胺基酸序列，只要維持作為嵌合抗原受體之功能，則為任意。 In one embodiment, the light chain variable region of the chimeric antigen receptor A The amino acid sequence of the domain has the identity of 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more with the amino acid sequence of SEQ ID NO. In one embodiment, the amino acid sequence of the variable region of the heavy chain of the chimeric antigen receptor A and the amino acid sequence of sequence number 8 have 90% or more, preferably 95% or more, and preferably 98% or more The identity of 99% or more is preferred. In one embodiment, the chimeric antigen receptor A has an identity of 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more with the amino acid sequence of SEQ ID NO: 10. In one embodiment, the amino acid sequence of the linker possessed by the chimeric antigen receptor A is arbitrary as long as it maintains its function as a chimeric antigen receptor.
嵌合抗原受體B，較佳具有選自由包含具有序列編號21之胺基酸序列的胺基酸序列之輕鏈CDR1、包含具有序列編號22之胺基酸序列的胺基酸序列之輕鏈CDR2、包含具有序列編號23之胺基酸序列的胺基酸序列之輕鏈CDR3、包含具有序列編號25之胺基酸序列的胺基酸序列之重鏈CDR1、包含具有序列編號26之胺基酸序列的胺基酸序列之重鏈CDR2、及包含具有序列編號27之胺基酸序列的胺基酸序列之重鏈CDR3所成之群的至少一種CDR，更佳為具有2種以上、更佳為具有3種以上、更佳為具有4種以上、更佳為具有5種以上、更佳為具有全部的CDR。適宜之一實施形態中，嵌合抗原受體B較佳具有：具有序列編號24之胺基酸序列的輕鏈可變區域及/或具有序列編號28之胺基酸序列的重鏈可變區域。適宜之一實施形態中，嵌合抗原受體B較佳具有：具有序列編號30表示 之胺基酸序列的scFV構造。 The chimeric antigen receptor B preferably has a light chain selected from the light chain CDR1 comprising an amino acid sequence having an amino acid sequence having the sequence number 21, and a light chain comprising an amino acid sequence having an amino acid sequence having the sequence number 22 CDR2, a light chain CDR3 containing an amino acid sequence having the amino acid sequence of sequence number 23, a CDR1 containing a heavy chain having an amino acid sequence having the amino acid sequence of sequence number 25, CDR1, including an amine group having the sequence number 26 At least one CDR of a group formed by a heavy chain CDR2 of an amino acid sequence of an acid sequence and a heavy chain CDR3 including an amino acid sequence having an amino acid sequence of SEQ ID NO: 27, more preferably having two or more types, more It is preferable that it has 3 or more types, it is more preferable that it has 4 or more types, it is more preferable that it has 5 or more types, and it is more preferable that it has all the CDRs. In a suitable embodiment, the chimeric antigen receptor B preferably has a light chain variable region having an amino acid sequence of SEQ ID NO: 24 and / or a heavy chain variable region having an amino acid sequence of SEQ ID NO: 28 . In a suitable embodiment, the chimeric antigen receptor B preferably has: ScFV construction of amino acid sequences.
一實施形態中，嵌合抗原受體B之輕鏈可變區域之胺基酸序列，與序列編號24之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體B之重鏈可變區域之胺基酸序列，與序列編號28之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體B，與序列編號30之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體B所具有之連結子之胺基酸序列，只要維持作為嵌合抗原受體之功能，則為任意。 In one embodiment, the amino acid sequence of the light chain variable region of the chimeric antigen receptor B and the amino acid sequence of SEQ ID NO. 24 have 90% or more, preferably 95% or more, and preferably 98% or more The identity of 99% or more is preferred. In one embodiment, the amino acid sequence of the heavy chain variable region of the chimeric antigen receptor B and the amino acid sequence of SEQ ID NO: 28 have 90% or more, preferably 95% or more, and preferably 98% or more The identity of 99% or more is preferred. In one embodiment, the chimeric antigen receptor B has an identity of 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more with the amino acid sequence of SEQ ID NO: 30. In one embodiment, the amino acid sequence of the linker possessed by the chimeric antigen receptor B is arbitrary as long as the function as the chimeric antigen receptor is maintained.
嵌合抗原受體C，較佳具有選自由包含具有序列編號41之胺基酸序列的胺基酸序列之輕鏈CDR1、包含具有序列編號42之胺基酸序列的胺基酸序列之輕鏈CDR2、包含具有序列編號43之胺基酸序列的胺基酸序列之輕鏈CDR3、包含具有序列編號45之胺基酸序列的胺基酸序列之重鏈CDR1、包含具有序列編號46之胺基酸序列的胺基酸序列之重鏈CDR2、及包含具有序列編號47之胺基酸序列的胺基酸序列之重鏈CDR3所成之群的至少一種CDR，更佳為具有2種以上、更佳為具有3種以上、更佳為具有4種以上、更佳為具有5種以上、更佳為具有全部的CDR。適宜之一實施形態中，嵌合抗原受體C較佳具有：具有序列編號44之胺基酸序列的輕鏈可變區域及/或具有 序列編號48之胺基酸序列的重鏈可變區域。適宜之一實施形態中，嵌合抗原受體C較佳具有：具有序列編號50表示之胺基酸序列的scFV構造。 The chimeric antigen receptor C preferably has a light chain selected from the light chain CDR1 comprising an amino acid sequence having an amino acid sequence having the number 41 and a light chain comprising an amino acid sequence having an amino acid sequence having the number 42 CDR2, a light chain CDR3 containing an amino acid sequence having the amino acid sequence of SEQ ID 43, a CDR1 heavy chain containing an amino acid sequence having the amino acid sequence of 45, CDR1, and an amino group having the sequence number 46 At least one CDR of the group consisting of the heavy chain CDR2 of the amino acid sequence of the acid sequence and the heavy chain CDR3 including the amino acid sequence of the amino acid sequence of SEQ ID NO: 47, more preferably two or more, It is preferable that it has 3 or more types, it is more preferable that it has 4 or more types, it is more preferable that it has 5 or more types, and it is more preferable that it has all the CDRs. In a suitable embodiment, the chimeric antigen receptor C preferably has a light chain variable region having an amino acid sequence of SEQ ID NO: 44 and / or Heavy chain variable region of the amino acid sequence of SEQ ID NO: 48. In a suitable embodiment, the chimeric antigen receptor C preferably has a scFV structure having an amino acid sequence represented by SEQ ID NO: 50.
一實施形態中，嵌合抗原受體C之輕鏈可變區域之胺基酸序列，與序列編號44之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體C之重鏈可變區域之胺基酸序列，與序列編號48之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體C，與序列編號50之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體C所具有之連結子之胺基酸序列，只要維持作為嵌合抗原受體之功能，則為任意。 In one embodiment, the amino acid sequence of the light chain variable region of the chimeric antigen receptor C and the amino acid sequence of sequence number 44 have 90% or more, preferably 95% or more, and preferably 98% or more The identity of 99% or more is preferred. In one embodiment, the amino acid sequence of the heavy chain variable region of the chimeric antigen receptor C and the amino acid sequence of SEQ ID NO: 48 have 90% or more, preferably 95% or more, and preferably 98% or more The identity of 99% or more is preferred. In one embodiment, the chimeric antigen receptor C has an identity of 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more with the amino acid sequence of SEQ ID NO: 50. In one embodiment, the amino acid sequence of the linker possessed by the chimeric antigen receptor C is arbitrary as long as it maintains its function as a chimeric antigen receptor.
嵌合抗原受體D，較佳具有選自由包含具有序列編號61之胺基酸序列的胺基酸序列之輕鏈CDR1、包含具有序列編號62之胺基酸序列的胺基酸序列之輕鏈CDR2、包含具有序列編號63之胺基酸序列的胺基酸序列之輕鏈CDR3、包含具有序列編號65之胺基酸序列的胺基酸序列之重鏈CDR1、包含具有序列編號66之胺基酸序列的胺基酸序列之重鏈CDR2、及包含具有序列編號67之胺基酸序列的胺基酸序列之重鏈CDR3所成之群的至少一種CDR，更佳為具有2種以上、更佳為具有3種以上、更佳為具有4種以上、更佳為具有5種以上、更佳為具有全部的 CDR。適宜之一實施形態中，嵌合抗原受體D較佳具有：具有序列編號64之胺基酸序列的輕鏈可變區域及/或具有序列編號68之胺基酸序列的重鏈可變區域。適宜之一實施形態中，嵌合抗原受體D較佳具有：具有序列編號70表示之胺基酸序列的scFV構造。 The chimeric antigen receptor D preferably has a light chain selected from the light chain CDR1 comprising an amino acid sequence having an amino acid sequence having the sequence number 61, and a light chain comprising an amino acid sequence having an amino acid sequence of the number 62 CDR2, a light chain CDR3 containing an amino acid sequence having the amino acid sequence of sequence number 63, a CDR1 heavy chain containing an amino acid sequence having the amino acid sequence of sequence number 65, and an amine group having the amino acid sequence of 66 At least one CDR of the group consisting of the heavy chain CDR2 of the amino acid sequence of the acid sequence, and the heavy chain CDR3 including the amino acid sequence of the amino acid sequence of SEQ ID NO: 67, more preferably at least one CDR, There are preferably 3 or more types, more preferably 4 or more types, more preferably 5 or more types, and even more preferably all CDR. In a suitable embodiment, the chimeric antigen receptor D preferably has: a light chain variable region having an amino acid sequence of sequence number 64 and / or a heavy chain variable region having an amino acid sequence of sequence number 68 . In a suitable embodiment, the chimeric antigen receptor D preferably has a scFV structure having an amino acid sequence represented by SEQ ID NO: 70.
一實施形態中，嵌合抗原受體D之輕鏈可變區域之胺基酸序列，與序列編號64之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體D之重鏈可變區域之胺基酸序列，與序列編號68之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體D，與序列編號70之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體D所具有之連結子之胺基酸序列，只要維持作為嵌合抗原受體之功能，則為任意。 In one embodiment, the amino acid sequence of the light chain variable region of the chimeric antigen receptor D and the amino acid sequence of sequence number 64 have 90% or more, preferably 95% or more, and preferably 98% or more The identity of 99% or more is preferred. In one embodiment, the amino acid sequence of the variable region of the heavy chain of the chimeric antigen receptor D and the amino acid sequence of sequence number 68 have 90% or more, preferably 95% or more, and preferably 98% or more The identity of 99% or more is preferred. In one embodiment, the chimeric antigen receptor D has 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity with the amino acid sequence of SEQ ID NO: 70. In one embodiment, the amino acid sequence of the linker possessed by the chimeric antigen receptor D is arbitrary as long as it maintains its function as a chimeric antigen receptor.
嵌合抗原受體E，較佳具有選自由包含具有序列編號81之胺基酸序列的胺基酸序列之輕鏈CDR1、包含具有序列編號82之胺基酸序列的胺基酸序列之輕鏈CDR2、包含具有序列編號83之胺基酸序列的胺基酸序列之輕鏈CDR3、包含具有序列編號85之胺基酸序列的胺基酸序列之重鏈CDR1、包含具有序列編號86之胺基酸序列的胺基酸序列之重鏈CDR2、及包含具有序列編號87之胺基酸序列的胺基酸序列之重鏈CDR3所成之群的至少一種 CDR，更佳為具有2種以上、更佳為具有3種以上、更佳為具有4種以上、更佳為具有5種以上、更佳為具有全部的CDR。適宜之一實施形態中，嵌合抗原受體E較佳具有：具有序列編號84之胺基酸序列的輕鏈可變區域及/或具有序列編號88之胺基酸序列的重鏈可變區域。適宜之一實施形態中，嵌合抗原受體E較佳具有：具有序列編號90表示之胺基酸序列的scFV構造。 The chimeric antigen receptor E preferably has a light chain CDR1 selected from a light chain comprising an amino acid sequence having an amino acid sequence having the sequence number 81, and a light chain comprising an amino acid sequence having an amino acid sequence of the number 82 CDR2, a light chain CDR3 containing an amino acid sequence having the amino acid sequence of sequence number 83, a CDR1 heavy chain containing an amino acid sequence having the amino acid sequence of sequence number 85, and an amine group having the amino acid sequence of 86 At least one of the group consisting of a heavy chain CDR2 of an amino acid sequence of an acid sequence, and a heavy chain CDR3 comprising an amino acid sequence having an amino acid sequence of SEQ ID NO: 87 The CDR is more preferably two or more, more preferably three or more, more preferably four or more, more preferably five or more, and most preferably all CDRs. In a suitable embodiment, the chimeric antigen receptor E preferably has a light chain variable region having an amino acid sequence of sequence number 84 and / or a heavy chain variable region having an amino acid sequence of sequence number 88. . In a suitable embodiment, the chimeric antigen receptor E preferably has a scFV structure having an amino acid sequence represented by SEQ ID NO: 90.
一實施形態中，嵌合抗原受體E之輕鏈可變區域之胺基酸序列，與序列編號84之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體E之重鏈可變區域之胺基酸序列，與序列編號88之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體E，與序列編號90之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體E所具有之連結子之胺基酸序列，只要維持作為嵌合抗原受體之功能，則為任意。 In one embodiment, the amino acid sequence of the light chain variable region of the chimeric antigen receptor E has 90% or more, preferably 95% or more, and preferably 98% or more of the amino acid sequence of SEQ ID NO: 84. The identity of 99% or more is preferred. In one embodiment, the amino acid sequence of the heavy chain variable region of the chimeric antigen receptor E and the amino acid sequence of sequence number 88 have 90% or more, preferably 95% or more, and preferably 98% or more The identity of 99% or more is preferred. In one embodiment, the chimeric antigen receptor E has the identity of 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more with the amino acid sequence of SEQ ID NO: 90. In one embodiment, the amino acid sequence of the linker possessed by the chimeric antigen receptor E is arbitrary as long as it maintains its function as a chimeric antigen receptor.
嵌合抗原受體F，較佳具有選自由包含具有序列編號101之胺基酸序列的胺基酸序列之輕鏈CDR1、包含具有序列編號102之胺基酸序列的胺基酸序列之輕鏈CDR2、包含具有序列編號103之胺基酸序列的胺基酸序列之輕鏈CDR3、包含具有序列編號105之胺基酸序列的胺基酸序列之重鏈CDR1、包含具有序列編號106之胺基酸序列 的胺基酸序列之重鏈CDR2、及包含具有序列編號107之胺基酸序列的胺基酸序列之重鏈CDR3所成之群的至少一種CDR，更佳為具有2種以上、更佳為具有3種以上、更佳為具有4種以上、更佳為具有5種以上、更佳為具有全部的CDR。適宜之一實施形態中，嵌合抗原受體F較佳具有：具有序列編號104之胺基酸序列的輕鏈可變區域及/或具有序列編號108之胺基酸序列的重鏈可變區域。適宜之一實施形態中，嵌合抗原受體F較佳具有：具有序列編號110表示之胺基酸序列的scFV構造。 The chimeric antigen receptor F preferably has a light chain CDR1 selected from a light chain comprising an amino acid sequence having an amino acid sequence having the sequence number 101, and a light chain comprising an amino acid sequence having an amino acid sequence of the number 102 CDR2, a light chain CDR3 containing an amino acid sequence having the amino acid sequence of sequence number 103, a CDR1 heavy chain containing an amino acid sequence having the amino acid sequence of sequence number 105, and an amine group having the serial number 106 Acid sequence At least one CDR of the group consisting of the heavy chain CDR2 of the amino acid sequence and the heavy chain CDR3 including the amino acid sequence having the amino acid sequence of SEQ ID NO: 107, more preferably having two or more types, more preferably It has three or more CDRs, more preferably four or more, more preferably five or more, and even more preferably all CDRs. In a suitable embodiment, the chimeric antigen receptor F preferably has a light chain variable region having an amino acid sequence of sequence number 104 and / or a heavy chain variable region having an amino acid sequence of sequence number 108. . In a suitable embodiment, the chimeric antigen receptor F preferably has a scFV structure having an amino acid sequence represented by SEQ ID NO: 110.
一實施形態中，嵌合抗原受體F之輕鏈可變區域之胺基酸序列，與序列編號104之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體F之重鏈可變區域之胺基酸序列，與序列編號108之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體F，與序列編號110之胺基酸序列具有90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性。一實施形態中，嵌合抗原受體F所具有之連結子之胺基酸序列，只要維持作為嵌合抗原受體之功能，則為任意。 In one embodiment, the amino acid sequence of the light chain variable region of the chimeric antigen receptor F and the amino acid sequence of SEQ ID NO. 104 have 90% or more, preferably 95% or more, and preferably 98% or more The identity of 99% or more is preferred. In one embodiment, the amino acid sequence of the heavy chain variable region of the chimeric antigen receptor F and the amino acid sequence of SEQ ID NO: 108 have 90% or more, preferably 95% or more, and preferably 98% or more The identity of 99% or more is preferred. In one embodiment, the chimeric antigen receptor F has 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity with the amino acid sequence of SEQ ID NO: 110. In one embodiment, the amino acid sequence of the linker possessed by the chimeric antigen receptor F is arbitrary as long as it maintains its function as a chimeric antigen receptor.
胺基酸之同一性，可使用市售或可透過網際網路利用的解析工具(例如FASTA、BLAST、PSI-BLAST、SSEARCH等之軟體)來計算。例如，於BLAST檢索一般所使用的主要初期條件，係如以下所示。亦即，於 Advanced BLAST 2.1中，程式使用blastp，Expect值設為10、Filter全部設為OFF，Matrix使用BLOSUM62，將Gap existence cost、Per residue gap cost及Lambda ratio分別設為11、1、0.85(預設值)，其他各種參數亦設定為預設值來進行檢索，藉此可算出胺基酸序列之同一性之值(%)。 The amino acid identity can be calculated using commercially available analysis tools (such as software such as FASTA, BLAST, PSI-BLAST, SSEARCH, etc.) that can be used through the Internet. For example, the main initial conditions commonly used in BLAST searches are as follows. That is, in In Advanced BLAST 2.1, the program uses blastp, Expect is set to 10, and Filter is set to OFF, Matrix uses BLOSUM62, and Gap existence cost, Per residue gap cost, and Lambda ratio are set to 11, 1, 0.85 (default values). , Various other parameters are also set to the preset value for searching, thereby the value (%) of the identity of the amino acid sequence can be calculated.
嵌合抗原受體A~C所具有的scFV(序列編號10、30及50之胺基酸序列)，係各來自於會特異性辨識血管內皮增殖因子受體(VEGFR2)之單株抗體。VEGFR2於腫瘤新生血管有高表現。嵌合抗原受體D~F所具有的scFV(序列編號70、90及110之胺基酸序列)，係各來自會特異性辨識血管內皮細胞特異性受體(Robo4)之單株抗體。Robo4亦作為腫瘤血管特異性標記而為人所知。因而，嵌合抗原受體A~F可特異性辨識癌組織(腫瘤組織)。 The scFVs (amino acid sequences of sequence numbers 10, 30, and 50) possessed by the chimeric antigen receptors A to C are derived from individual antibodies that specifically recognize vascular endothelial growth factor receptor (VEGFR2). VEGFR2 has high performance in tumor neovascularization. The scFVs (amino acid sequences of sequence numbers 70, 90, and 110) possessed by the chimeric antigen receptors D to F are derived from individual antibodies that specifically recognize vascular endothelial cell-specific receptors (Robo4). Robo4 is also known as a tumor vessel-specific marker. Therefore, the chimeric antigen receptors A to F can specifically recognize cancer tissue (tumor tissue).
嵌合抗原受體A~F，較佳為具有自其N末端起依序配置有scFv區域、間隔序列(spacer sequence)、膜貫通結構域、共刺激因子之細胞內結構域，以及TCR之細胞內結構域的構造。scFv區域與膜貫通結構域之間所設置的間隔序列之長度及構成其之胺基酸殘基的種類，只要不阻礙嵌合抗原受體之功能，則無限制。例如，間隔序列可設計為10個~25個左右之胺基酸殘基。 The chimeric antigen receptors A to F are preferably cells having an scFv region, a spacer sequence, a membrane penetrating domain, a co-stimulatory factor intracellular domain, and a TCR arranged sequentially from the N-terminus. Construction of the inner domain. The length of the spacer sequence provided between the scFv region and the membrane penetrating domain and the type of amino acid residues constituting the spacer sequence are not limited as long as the function of the chimeric antigen receptor is not hindered. For example, the spacer sequence can be designed as about 10 to 25 amino acid residues.
膜貫通結構域之種類，只要不阻礙嵌合抗原受體之功能，則無限制。例如，可使用於T細胞等表現之CD28、CD3ζ、CD4、CD8α等。此等之膜貫通結構域，只要不阻礙嵌合抗原受體之功能，則亦可適當導入變異。 The type of the membrane penetrating domain is not limited as long as it does not hinder the function of the chimeric antigen receptor. For example, CD28, CD3ζ, CD4, CD8α, etc. can be used for the expression of T cells. These membrane penetrating domains may be appropriately introduced with a mutation as long as they do not hinder the function of the chimeric antigen receptor.
共刺激因子之細胞內結構域，只要係來自T細胞等所具有的共刺激因子的細胞內結構域即可，並無特殊限定。例如可適當選擇選自由OX40、4-1BB、CD27、CD278及CD28等所成之群的1種以上來使用。此等之共刺激因子之細胞內結構域，只要不阻礙嵌合抗原受體之功能，則亦可適當導入變異。 The intracellular domain of the costimulatory factor is not particularly limited as long as it is derived from the intracellular domain of a costimulatory factor possessed by T cells and the like. For example, one or more members selected from the group consisting of OX40, 4-1BB, CD27, CD278, and CD28 can be appropriately selected and used. The intracellular domains of these costimulatory factors can be appropriately introduced into the mutation as long as they do not hinder the function of the chimeric antigen receptor.
TCR之細胞內結構域，例如可為來自亦稱為TCRζ鏈之CD3等的細胞內結構域。CD3，只要不阻礙嵌合抗原受體之功能，則亦可適當導入變異。對CD3導入變異時，較佳為以包含ITAM(immunoreceptor tyrosine-based activation motif)的方式進行。 The intracellular domain of TCR may be, for example, an intracellular domain derived from CD3, which is also called a TCRζ chain. As long as CD3 does not hinder the function of the chimeric antigen receptor, mutations can also be appropriately introduced. When introducing mutation into CD3, it is preferable to carry out the method including ITAM (immunoreceptor tyrosine-based activation motif).
利用特定scFV之嵌合抗原受體及製造表現其之CAR-T細胞的技術係為公知。例如，可利用非專利文獻1及2所揭示之方法來製造嵌合抗原受體A~F。 Techniques for utilizing chimeric antigen receptors of specific scFVs and producing CAR-T cells expressing them are well known. For example, the methods disclosed in Non-Patent Documents 1 and 2 can be used to produce chimeric antigen receptors A to F.
2.編碼嵌合抗原受體之多核苷酸 2. A polynucleotide encoding a chimeric antigen receptor
編碼嵌合抗原受體A之多核苷酸，只要係編碼上述之嵌合抗原受體A，則無特殊限制。一實施形態中，編碼嵌合抗原受體A之多核苷酸，較佳具有選自由編碼具有序列編號11之鹼基序列的輕鏈CDR1之區域、編碼具有序列編號12之鹼基序列的輕鏈CDR2之區域、編碼具有序列編號13之鹼基序列的輕鏈CDR3之區域、編碼具有序列編號15之鹼基序列的重鏈CDR1之區域、編碼具有序列編號16之鹼基序列的重鏈CDR2之區域、及編碼具有序列編號17之 鹼基序列的重鏈CDR3之區域所成之群的至少一種區域，更佳為具有2種以上、更佳為具有3種以上、更佳為具有4種以上、更佳為具有5種以上、更佳為具有全部之區域。適宜之一實施形態中，編碼嵌合抗原受體A之多核苷酸，較佳具有編碼具有序列編號14之鹼基序列的輕鏈可變區域之區域及/或編碼具有序列編號18之鹼基序列的重鏈可變區域之區域。適宜之一實施形態中，編碼嵌合抗原受體A之多核苷酸，較佳具有序列編號20之鹼基序列。 The polynucleotide encoding the chimeric antigen receptor A is not particularly limited as long as it encodes the aforementioned chimeric antigen receptor A. In one embodiment, the polynucleotide encoding the chimeric antigen receptor A preferably has a light chain selected from the region consisting of a region encoding CDR1 of a light chain having a base sequence having sequence number 11 and the region encoding a base sequence having a sequence number 12 Region of CDR2, region of light chain CDR3 with base sequence of sequence number 13, region of heavy chain CDR1 with base sequence of sequence number 15, region of heavy chain CDR2 with base sequence of sequence number 16 Area and code with sequence number 17 At least one region of the group formed by the regions of the heavy chain CDR3 of the base sequence is more preferably two or more species, more preferably three or more species, more preferably four or more species, more preferably five or more species, It is more preferable to have all areas. In a suitable embodiment, the polynucleotide encoding the chimeric antigen receptor A preferably has a region encoding a light chain variable region having a base sequence of SEQ ID NO: 14 and / or a base encoding a base of SEQ ID NO: 18 The region of the heavy chain variable region of a sequence. In a suitable embodiment, the polynucleotide encoding the chimeric antigen receptor A preferably has a base sequence of SEQ ID NO: 20.
一實施形態中，編碼嵌合抗原受體A之多核苷酸，具有與序列編號11~20之鹼基序列具有80%以上、較佳為85%以上、較佳為90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性的鹼基序列。 In one embodiment, the polynucleotide encoding the chimeric antigen receptor A has a base sequence with sequence numbers 11 to 20 of 80% or more, preferably 85% or more, preferably 90% or more, and more preferably A base sequence having an identity of 95% or more, preferably 98% or more, and preferably 99% or more.
編碼嵌合抗原受體B之多核苷酸，只要係編碼上述之嵌合抗原受體B，則無特殊限制。一實施形態中，編碼嵌合抗原受體B之多核苷酸，較佳具有選自由編碼具有序列編號31之鹼基序列的輕鏈CDR1之區域、編碼具有序列編號32之鹼基序列的輕鏈CDR2之區域、編碼具有序列編號33之鹼基序列的輕鏈CDR3之區域、編碼具有序列編號35之鹼基序列的重鏈CDR1之區域、編碼具有序列編號36之鹼基序列的重鏈CDR2之區域、及編碼具有序列編號37之鹼基序列的重鏈CDR3之區域所成之群的至少一種區域，更佳為具有2種以上、更佳為具有3種以上、更佳為具有4種以上、更佳為具有5種以上、更佳為具有全部之區域。適宜之一實施形態中，編碼嵌合抗原受體B之多核苷 酸，較佳具有編碼具有序列編號34之鹼基序列的輕鏈可變區域之區域及/或編碼具有序列編號38之鹼基序列的重鏈可變區域之區域。適宜之一實施形態中，編碼嵌合抗原受體B之多核苷酸，較佳具有序列編號40之鹼基序列。 The polynucleotide encoding the chimeric antigen receptor B is not particularly limited as long as it encodes the chimeric antigen receptor B described above. In one embodiment, the polynucleotide encoding the chimeric antigen receptor B preferably has a light chain selected from the group consisting of a region encoding a light chain CDR1 having a base sequence having the sequence number 31, and a light chain encoding a base sequence having the sequence number 32. Region of CDR2, region of light chain CDR3 with base sequence of sequence number 33, region of heavy chain CDR1 with base sequence of sequence number 35, region of heavy chain CDR2 with base sequence of sequence number 36 A region and at least one region of a group formed by a region encoding a heavy chain CDR3 having a base sequence of SEQ ID NO: 37, more preferably having 2 or more species, more preferably having 3 or more species, and even more preferably having 4 or more species It is more preferable to have 5 or more types, and it is more preferable to have all the regions. In a suitable embodiment, a polynucleoside encoding a chimeric antigen receptor B The acid preferably has a region encoding a light chain variable region having a base sequence of SEQ ID NO: 34 and / or a region encoding a heavy chain variable region having a base sequence of SEQ ID NO: 38. In a suitable embodiment, the polynucleotide encoding the chimeric antigen receptor B preferably has a base sequence of SEQ ID NO: 40.
一實施形態中，編碼嵌合抗原受體B之多核苷酸，具有與序列編號31~40之鹼基序列具有80%以上、較佳為85%以上、較佳為90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性的鹼基序列。 In one embodiment, the polynucleotide encoding the chimeric antigen receptor B has a base sequence with a sequence number of 31 to 40 having 80% or more, preferably 85% or more, preferably 90% or more, and more preferably A base sequence having an identity of 95% or more, preferably 98% or more, and preferably 99% or more.
編碼嵌合抗原受體C之多核苷酸，只要係編碼上述之嵌合抗原受體C，則無特殊限制。一實施形態中，編碼嵌合抗原受體C之多核苷酸，較佳具有選自由編碼具有序列編號51之鹼基序列的輕鏈CDR1之區域、編碼具有序列編號52之鹼基序列的輕鏈CDR2之區域、編碼具有序列編號53之鹼基序列的輕鏈CDR3之區域、編碼具有序列編號55之鹼基序列的重鏈CDR1之區域、編碼具有序列編號56之鹼基序列的重鏈CDR2之區域、及編碼具有序列編號57之鹼基序列的重鏈CDR3之區域所成之群的至少一種區域，更佳為具有2種以上、更佳為具有3種以上、更佳為具有4種以上、更佳為具有5種以上、更佳為具有全部之區域。適宜之一實施形態中，編碼嵌合抗原受體C之多核苷酸，較佳具有編碼具有序列編號54之鹼基序列的輕鏈可變區域之區域及/或編碼具有序列編號58之鹼基序列的重鏈可變區域之區域。適宜之一實施形態中，編碼嵌合抗原受體C之多核苷酸，較佳具有序列編號60之鹼基序列。 The polynucleotide encoding the chimeric antigen receptor C is not particularly limited as long as it encodes the aforementioned chimeric antigen receptor C. In one embodiment, the polynucleotide encoding the chimeric antigen receptor C preferably has a light chain selected from the region consisting of a region encoding CDR1 of a light chain having a base sequence having the sequence number 51 and the light chain encoding a base sequence having a sequence number 52. Region of CDR2, region of light chain CDR3 with base sequence of sequence number 53, region of heavy chain CDR1 with base sequence of sequence number 55, region of heavy chain CDR2 with base sequence of sequence number 56 A region and at least one region of a group formed by a region encoding a heavy chain CDR3 having a base sequence of SEQ ID NO: 57; more preferably, two or more regions; more preferably, three or more regions; and more preferably, four or more regions. It is more preferable to have 5 or more types, and it is more preferable to have all the regions. In a suitable embodiment, the polynucleotide encoding the chimeric antigen receptor C preferably has a region encoding a light chain variable region having a base sequence of SEQ ID NO: 54 and / or encoding a base having SEQ ID NO: 58 The region of the heavy chain variable region of a sequence. In a suitable embodiment, the polynucleotide encoding the chimeric antigen receptor C preferably has a base sequence of SEQ ID NO: 60.
一實施形態中，編碼嵌合抗原受體C之多核苷酸，具有與序列編號51~60之鹼基序列具有80%以上、較佳為85%以上、較佳為90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性的鹼基序列。 In one embodiment, the polynucleotide encoding the chimeric antigen receptor C has a base sequence with a sequence number of 51 to 60 which is 80% or more, preferably 85% or more, preferably 90% or more, and more preferably A base sequence having an identity of 95% or more, preferably 98% or more, and preferably 99% or more.
編碼嵌合抗原受體D之多核苷酸，只要係編碼上述之嵌合抗原受體D，則無特殊限制。一實施形態中，編碼嵌合抗原受體D之多核苷酸，較佳具有選自由編碼具有序列編號71之鹼基序列的輕鏈CDR1之區域、編碼具有序列編號72之鹼基序列的輕鏈CDR2之區域、編碼具有序列編號73之鹼基序列的輕鏈CDR3之區域、編碼具有序列編號75之鹼基序列的重鏈CDR1之區域、編碼具有序列編號76之鹼基序列的重鏈CDR2之區域、及編碼具有序列編號77之鹼基序列的重鏈CDR3之區域所成之群的至少一種區域，更佳為具有2種以上、更佳為具有3種以上、更佳為具有4種以上、更佳為具有5種以上、更佳為具有全部之區域。適宜之一實施形態中，編碼嵌合抗原受體D之多核苷酸，較佳具有編碼具有序列編號74之鹼基序列的輕鏈可變區域之區域及/或編碼具有序列編號78之鹼基序列的重鏈可變區域之區域。適宜之一實施形態中，編碼嵌合抗原受體D之多核苷酸，較佳具有序列編號80之鹼基序列。 The polynucleotide encoding the chimeric antigen receptor D is not particularly limited as long as it encodes the aforementioned chimeric antigen receptor D. In one embodiment, the polynucleotide encoding the chimeric antigen receptor D preferably has a light chain selected from the region consisting of a region encoding CDR1 of a light chain having a base sequence having sequence number 71 and the light chain encoding a base sequence having a sequence number 72. Region of CDR2, region of light chain CDR3 with base sequence of sequence number 73, region of heavy chain CDR1 with base sequence of sequence number 75, region of heavy chain CDR2 with base sequence of sequence number 76 A region and at least one region of a group formed by a region encoding a heavy chain CDR3 having a base sequence of SEQ ID NO: 77, more preferably having 2 or more species, more preferably having 3 or more species, and more preferably having 4 or more species It is more preferable to have 5 or more types, and it is more preferable to have all the regions. In a suitable embodiment, the polynucleotide encoding the chimeric antigen receptor D preferably has a region encoding a light chain variable region having a base sequence of SEQ ID NO: 74 and / or encoding a base having SEQ ID NO: 78 The region of the heavy chain variable region of a sequence. In a suitable embodiment, the polynucleotide encoding the chimeric antigen receptor D preferably has a base sequence of SEQ ID NO: 80.
一實施形態中，編碼嵌合抗原受體D之多核苷酸，具有與序列編號71~80之鹼基序列具有80%以上、較佳為85%以上、較佳為90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性的鹼基序列。 In one embodiment, the polynucleotide encoding the chimeric antigen receptor D has a base sequence with a sequence number of 71 to 80 having 80% or more, preferably 85% or more, preferably 90% or more, and more preferably A base sequence having an identity of 95% or more, preferably 98% or more, and preferably 99% or more.
編碼嵌合抗原受體E之多核苷酸，只要係編碼上述之嵌合抗原受體E，則無特殊限制。一實施形態中，編碼嵌合抗原受體E之多核苷酸，較佳具有選自由編碼具有序列編號91之鹼基序列的輕鏈CDR1之區域、編碼具有序列編號92之鹼基序列的輕鏈CDR2之區域、編碼具有序列編號93之鹼基序列的輕鏈CDR3之區域、編碼具有序列編號95之鹼基序列的重鏈CDR1之區域、編碼具有序列編號96之鹼基序列的重鏈CDR2之區域、及編碼具有序列編號97之鹼基序列的重鏈CDR3之區域所成之群的至少一種區域，更佳為具有2種以上、更佳為具有3種以上、更佳為具有4種以上、更佳為具有5種以上、更佳為具有全部之區域。適宜之一實施形態中，編碼嵌合抗原受體E之多核苷酸，較佳具有編碼具有序列編號94之鹼基序列的輕鏈可變區域之區域及/或編碼具有序列編號98之鹼基序列的重鏈可變區域之區域。適宜之一實施形態中，編碼嵌合抗原受體E之多核苷酸，較佳具有序列編號100之鹼基序列。 The polynucleotide encoding the chimeric antigen receptor E is not particularly limited as long as it encodes the aforementioned chimeric antigen receptor E. In one embodiment, the polynucleotide encoding the chimeric antigen receptor E preferably has a light chain selected from the region consisting of a region encoding CDR1 of a light chain having a base sequence having the sequence number 91, and a light chain encoding a base sequence having a sequence number 92 Region of CDR2, region of light chain CDR3 with base sequence of sequence number 93, region of heavy chain CDR1 with base sequence of sequence number 95, region of heavy chain CDR2 with base sequence of sequence number 96 A region and at least one region of a group formed by a region encoding a heavy chain CDR3 having a base sequence of SEQ ID NO: 97, more preferably having 2 or more species, more preferably having 3 or more species, and more preferably having 4 or more species It is more preferable to have 5 or more types, and it is more preferable to have all the regions. In a suitable embodiment, the polynucleotide encoding the chimeric antigen receptor E preferably has a region encoding a light chain variable region having a base sequence of SEQ ID NO: 94 and / or a base encoding a sequence of 98 The region of the heavy chain variable region of a sequence. In a suitable embodiment, the polynucleotide encoding the chimeric antigen receptor E preferably has a base sequence of SEQ ID NO: 100.
一實施形態中，編碼嵌合抗原受體E之多核苷酸，具有與序列編號91~100之鹼基序列具有80%以上、較佳為85%以上、較佳為90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性的鹼基序列。 In one embodiment, the polynucleotide encoding the chimeric antigen receptor E has a base sequence with a sequence number of 91 to 100 which is 80% or more, preferably 85% or more, preferably 90% or more, and more preferably A base sequence having an identity of 95% or more, preferably 98% or more, and preferably 99% or more.
編碼嵌合抗原受體F之多核苷酸，只要係編碼上述之嵌合抗原受體F，則無特殊限制。一實施形態中，編碼嵌合抗原受體F之多核苷酸，較佳具有選自由編碼具有序列編號111之鹼基序列的輕鏈CDR1之區域、編碼具有 序列編號112之鹼基序列的輕鏈CDR2之區域、編碼具有序列編號113之鹼基序列的輕鏈CDR3之區域、編碼具有序列編號115之鹼基序列的重鏈CDR1之區域、編碼具有序列編號116之鹼基序列的重鏈CDR2之區域、及編碼具有序列編號117之鹼基序列的重鏈CDR3之區域所成之群的至少一種區域，更佳為具有2種以上、更佳為具有3種以上、更佳為具有4種以上、更佳為具有5種以上、更佳為具有全部之區域。適宜之一實施形態中，編碼嵌合抗原受體F之多核苷酸，較佳具有編碼具有序列編號114之鹼基序列的輕鏈可變區域之區域及/或編碼具有序列編號118之鹼基序列的重鏈可變區域之區域。適宜之一實施形態中，編碼嵌合抗原受體F之多核苷酸，較佳具有序列編號120之鹼基序列。 The polynucleotide encoding the chimeric antigen receptor F is not particularly limited as long as it encodes the aforementioned chimeric antigen receptor F. In one embodiment, the polynucleotide encoding the chimeric antigen receptor F preferably has a region selected from the group consisting of a light chain CDR1 encoding a base sequence having the sequence number 111, Region of light chain CDR2 with base sequence of SEQ ID NO: 112, region of light chain CDR3 with base sequence of SEQ ID NO: 113, region of heavy chain CDR1 with base sequence of SEQ ID NO: 115, and sequence number A region consisting of a region of the heavy chain CDR2 of the base sequence 116 and a region encoding at least one region of the region encoding the heavy chain CDR3 of the base sequence of sequence number 117, more preferably having two or more species, more preferably having 3 More than one species, more preferably four or more species, more preferably five or more species, and even more preferably all regions. In a suitable embodiment, the polynucleotide encoding the chimeric antigen receptor F preferably has a region encoding a light chain variable region having a base sequence of SEQ ID NO: 114 and / or a base encoding a base of SEQ ID NO: 118. The region of the heavy chain variable region of a sequence. In a suitable embodiment, the polynucleotide encoding the chimeric antigen receptor F preferably has a base sequence of SEQ ID NO: 120.
一實施形態中，編碼嵌合抗原受體F之多核苷酸，具有與序列編號111~120之鹼基序列具有80%以上、較佳為85%以上、較佳為90%以上、較佳為95%以上、較佳為98%以上、較佳為99%以上之同一性的鹼基序列。 In one embodiment, the polynucleotide encoding the chimeric antigen receptor F has a base sequence with a sequence number of 111 to 120 with 80% or more, preferably 85% or more, preferably 90% or more, and more preferably A base sequence having an identity of 95% or more, preferably 98% or more, and preferably 99% or more.
鹼基序列之同一性，可使用市售或可透過電信電路(網際網路)利用之解析工具來算出。例如，具體而言，於Advanced BLAST 2.1中，程式使用blastn，且將各種參數設定為預設值來進行檢索，藉此可算出核苷酸序列之相同性的值(%)。多核苷酸，可依表現其之載體或細胞的種類來最佳化密碼子使用頻度。 The identity of the base sequence can be calculated using a commercially available analysis tool or an analysis tool available through a telecommunications circuit (Internet). For example, specifically, in Advanced BLAST 2.1, the program uses blastn, and various parameters are set to preset values for searching, whereby the value (%) of the nucleotide sequence identity can be calculated. Polynucleotides can be optimized for codon usage frequency depending on the type of vector or cell in which they are expressed.
多核苷酸之狀態並無特殊限定，例如可為經單離者、亦可為接入載體內者。載體之種類及用途並無特 殊限定。例如，載體可為質體載體或病毒載體(例如腺病毒或反轉錄病毒)。又，載體例如可為選殖用載體或表現用載體。表現用載體可列舉大腸菌或放線菌等之原核細胞用之載體，或酵母細胞、昆蟲細胞或哺乳類細胞等之真核細胞用之載體。多核苷酸亦可施以任意之修飾，例如，可於5’末端側適當附加編碼訊息胜肽之鹼基序列。 The state of the polynucleotide is not particularly limited, and may be, for example, a single detached person or a person inserted into a vector. The type and use of the carrier are not specific Specially limited. For example, the vector may be a plastid vector or a viral vector (eg, an adenovirus or a retrovirus). The vector may be, for example, a breeding vector or a expression vector. Examples of the expression vector include vectors for prokaryotic cells such as coliform or actinomycete, and vectors for eukaryotic cells such as yeast cells, insect cells, and mammalian cells. The polynucleotide may be subjected to any modification, for example, a base sequence encoding a message peptide may be appropriately added to the 5 'end side.
多核苷酸亦可為被攝入宿主細胞之狀態。宿主細胞含有多核苷酸之態樣並無特殊限定。例如，宿主細胞能夠以載體之形態具有多核苷酸，亦能夠以整合於宿主細胞內之基因體DNA的形態具有多核苷酸。宿主細胞之種類為任意，無特殊限制。例如，宿主細胞可為酵母細胞、昆蟲細胞及哺乳類細胞等之真核細胞，以及大腸菌及放線菌等之原核細胞。一實施形態中，宿主細胞較佳為真核細胞(例如哺乳類、人類)，例如較佳為T細胞或NK細胞等。含有多核苷酸之宿主細胞，例如可於任意之宿主細胞中導入該多核苷酸(例如以載體之形態)而得到。 The polynucleotide may also be in a state of being taken up into a host cell. The form in which the host cell contains the polynucleotide is not particularly limited. For example, a host cell can have a polynucleotide in the form of a vector, and can also have a polynucleotide in the form of a genomic DNA integrated into the host cell. The type of the host cell is arbitrary and is not particularly limited. For example, the host cells may be eukaryotic cells such as yeast cells, insect cells, and mammalian cells, and prokaryotic cells such as coliforms and actinomycetes. In one embodiment, the host cell is preferably a eukaryotic cell (for example, mammals, humans), for example, a T cell or a NK cell. A host cell containing a polynucleotide can be obtained by introducing the polynucleotide (for example, in the form of a vector) into any host cell.
宿主細胞可表現編碼嵌合抗原受體之多核苷酸、亦可為不表現的狀態。於宿主細胞內表現編碼嵌合抗原受體之多核苷酸時，較佳為構成嵌合抗原受體之scFv區域露出於細胞外側，且膜貫通結構域、共刺激因子，及TCR之細胞內結構域係存在於細胞膜或細胞內。 The host cell may express a polynucleotide encoding a chimeric antigen receptor, or may be in a non-expressing state. When a polynucleotide encoding a chimeric antigen receptor is expressed in a host cell, it is preferred that the scFv region constituting the chimeric antigen receptor is exposed outside the cell, and the membrane penetrating domain, costimulatory factor, and intracellular structure of TCR Domain lines exist in the cell membrane or inside the cell.
3.CAR-T細胞 3.CAR-T cells
提供表現上述嵌合抗原受體A~F之任一者的T細胞 (CAR-T細胞)。表現嵌合抗原受體之T細胞等係以scFv區域辨識抗原後，將其辨識訊息通過ζ鏈傳達至T細胞等之內部。scFv區域辨識其抗原決定位時，透過膜貫通結構域及共刺激因子於細胞內使誘發細胞傷害活性之訊息作動，且與其連動地，該細胞係對表現同樣抗原決定位之其他細胞或組織發揮攻擊或細胞傷害活性。 T cells (CAR-T cells) expressing any of the aforementioned chimeric antigen receptors A to F are provided. T cells and the like expressing chimeric antigen receptors recognize antigens in the scFv region, and then transmit their identification information to the inside of the T cells and the like through the ζ chain. When the scFv region recognizes its epitope, the cell through the membrane penetrating domain and the co-stimulatory factor activates the message of inducing cellular nociceptive activity in the cell, and in conjunction with it, the cell line exerts its effect on other cells or tissues showing the same epitope Attack or cellular damage activity.
發揮如此功能之細胞為CTL的情況時，係稱為嵌合抗原受體T細胞(CAR-T細胞)。NK細胞等之具有發揮細胞傷害活性之可能性的細胞亦與嵌合抗原受體T細胞同樣地，藉由scFv區域與其抗原決定位結合，可發揮細胞傷害活性。因此，含有編碼嵌合抗原受體之多核苷酸的宿主細胞(特別是具有細胞傷害活性之宿主細胞)，係有用於作為醫藥組成物之有效成分。含有編碼嵌合抗原受體之多核苷酸的宿主細胞(例如CAR-T細胞)，可參考非專利文獻1及2等所記載之公知方法來製造。 When the cell performing such a function is CTL, it is called a chimeric antigen receptor T cell (CAR-T cell). Similar to chimeric antigen receptor T cells, cells that have the possibility of exhibiting cytotoxic activity, such as NK cells, can exert cytotoxic activity by binding the scFv region to its epitope. Therefore, a host cell containing a polynucleotide encoding a chimeric antigen receptor (especially a host cell having cytotoxic activity) is an active ingredient for use as a pharmaceutical composition. Host cells (for example, CAR-T cells) containing a polynucleotide encoding a chimeric antigen receptor can be produced by referring to known methods described in Non-Patent Documents 1 and 2.
如此之CAR-T細胞等，係特異性地辨識癌組織(腫瘤組織)，因此有用於腫瘤等之治療或預防。腫瘤之種類並無特殊限制，包含固態癌及血液癌。固態癌可列舉例如肺癌、大腸癌、卵巢癌、乳癌、腦腫瘤、胃癌、肝癌、舌癌、甲狀腺癌、腎臟癌、攝護腺癌、子宮癌、骨肉瘤、軟骨肉瘤、及橫紋肌肉瘤。 Since such CAR-T cells specifically recognize cancer tissue (tumor tissue), they are useful for the treatment or prevention of tumors and the like. There are no particular restrictions on the type of tumor, including solid cancer and blood cancer. Examples of solid cancer include lung cancer, colorectal cancer, ovarian cancer, breast cancer, brain tumor, gastric cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, and rhabdomyosarcoma.
4.醫藥組成物 4. Pharmaceutical composition
提供含有上述CAR-T細胞等之醫藥組成物及利用其之 疾病之治療或預防方法。醫藥組成物中之上述CAR-T細胞的含量，可考慮作為對象之疾病(例如固態癌)之種類、作為目的之治療效果、投與方法、治療期間、患者之年齡，及患者之體重等來適當設定。例如，以醫藥組成物全體為100重量份時，醫藥組成物中之抗體的含量，可為0.001重量份~10重量份左右。醫藥組成物中之細胞的含量，例如可為1細胞/mL~104細胞/mL左右。 Provide a pharmaceutical composition containing the above CAR-T cells and a method for treating or preventing a disease using the same. The content of the CAR-T cells in the pharmaceutical composition can be determined by considering the type of the target disease (such as solid cancer), the intended therapeutic effect, the method of administration, the period of treatment, the age of the patient, and the weight of the patient. Set appropriately. For example, when the entire pharmaceutical composition is 100 parts by weight, the content of the antibody in the pharmaceutical composition may be about 0.001 to 10 parts by weight. The content of cells in the pharmaceutical composition may be, for example, about 1 cell / mL to 10 4 cells / mL.
醫藥組成物之投與形態，只要可得到所期望之效果，則無特殊限制，能夠以經口投與及非經口投與(例如靜脈注射、肌肉注射、皮下投與、直腸投與、經皮投與、局部投與)的任一投與路徑對包含人類之哺乳類投與。由於有效成分為細胞，故較佳之投與形態為非經口投與、更佳為靜脈注射。用於經口投與及非經口投與之劑型及其製造方法係為所屬技術領域中具有通常知識者眾所周知，可藉由與藥學上容許之醫藥載體等混合等，遵照一般方法來製造本發明之抗體或細胞。 The administration form of the pharmaceutical composition is not particularly limited as long as the desired effect can be obtained, and can be administered orally and parenterally (e.g., intravenous, intramuscular, subcutaneous, rectal, or Skin administration, local administration) to administration of mammals including humans. Since the active ingredient is a cell, the preferred form of administration is parenteral, and more preferably intravenous injection. Dosage forms for oral administration and parenteral administration, and a method for producing the same are well known to those having ordinary knowledge in the technical field, and can be produced by mixing with a pharmaceutically acceptable pharmaceutical carrier, etc., in accordance with general methods. Antibodies or cells of the invention.
用於非經口投與之劑型，可列舉注射用製劑(例如點滴注射劑、靜脈注射劑、肌肉注射劑、皮下注射劑、皮內注射劑)、外用劑(例如軟膏劑、泥罨劑、洗劑)、栓劑吸入劑、眼劑、眼軟膏劑、點鼻劑、點耳劑、脂質體劑等。例如，注射用製劑，可將抗體或細胞溶解或懸浮於注射用蒸餾水而調製，並依需要添加溶解輔助劑、緩衝劑、pH調整劑、等張化劑、無痛化劑、保存劑及安定化劑等。醫藥組成物亦可作為用時調製用之冷凍乾燥製劑。 Dosage forms for parenteral administration include injection preparations (e.g., drip injections, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), external preparations (e.g., ointments, lozenges, lotions), suppositories Inhalants, eye preparations, eye ointments, nose drops, ear drops, liposomes, etc. For example, injection preparations can be prepared by lysing or suspending antibodies or cells in distilled water for injection, and adding a dissolution aid, buffer, pH adjuster, isotonicity agent, painless agent, preservative, and stabilizer as needed. Agent. The pharmaceutical composition can also be used as a freeze-dried preparation for preparation.
醫藥組成物，亦可進一步含有對疾病之治療或預防有效之其他藥劑。又，醫藥組成物，亦可依需要摻合殺菌劑、消炎劑、細胞賦活劑、維生素類及胺基酸等之成分。 The pharmaceutical composition may further contain other agents effective for the treatment or prevention of diseases. In addition, the pharmaceutical composition may be blended with components such as a bactericide, an anti-inflammatory agent, a cell activating agent, a vitamin, and an amino acid, as needed.
醫藥組成物之製劑化所用的醫藥載體，可使用該技術領域中通常使用之賦形劑、結合劑、崩解劑、潤滑劑、著色劑、調味調臭劑，或依需要之安定化劑、乳化劑、吸收促進劑、界面活性劑、pH調整劑、防腐劑、抗氧化劑、增量劑、濕潤化劑、表面活性化劑、分散劑、緩衝劑、保存劑、溶解輔助劑、無痛化劑等。 The pharmaceutical carrier used for the formulation of the pharmaceutical composition may use excipients, binding agents, disintegrating agents, lubricants, colorants, flavoring and deodorizing agents commonly used in the technical field, or stabilizing agents as needed, Emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, extenders, wetting agents, surfactants, dispersants, buffering agents, preservatives, dissolution aids, painless agents Wait.
使用醫藥組成物來治療或預防的疾病之種類，只要可達成其治療或預防，則無特殊限定。具體的對象疾病，例如可列舉腫瘤。腫瘤之種類並無特殊限制，包含固態癌及血液癌。固態癌可列舉例如肺癌、大腸癌、卵巢癌、乳癌、腦腫瘤、胃癌、肝癌、舌癌、甲狀腺癌、腎臟癌、攝護腺癌、子宮癌、骨肉瘤、軟骨肉瘤及橫紋肌肉瘤。 The type of disease to be treated or prevented using the pharmaceutical composition is not particularly limited as long as the treatment or prevention can be achieved. Specific target diseases include, for example, tumors. There are no particular restrictions on the type of tumor, including solid cancer and blood cancer. Examples of solid cancer include lung cancer, colorectal cancer, ovarian cancer, breast cancer, brain tumor, gastric cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, and rhabdomyosarcoma.
醫藥組成物之投與對象(被試驗體)，例如為罹患了上述疾病之動物或有罹患的可能性之動物。「有罹患的可能性」，例如可藉由後述診斷方法決定。動物係指例如哺乳類動物，較佳為人類。 The subject (subject) to which the pharmaceutical composition is administered is, for example, an animal suffering from the above-mentioned disease or an animal that is likely to be affected. The "possibility of suffering" can be determined, for example, by a diagnostic method described later. Animal means, for example, mammals, preferably humans.
醫藥組成物之投與量，例如可基於投與路徑、疾病之種類、症狀之程度、患者之年齡、性別、體重、疾病之嚴重度、藥物動態及毒物學的特徵等之藥理學 的見解、藥物傳輸系統利用之有無、以及是否作為與其他藥物之組合的一部分投與等各種因子，而由臨床醫師來決定。醫藥組成物之投與量，例如有效成分為抗體時，每一日可為1μg/kg(體重)~10g/kg(體重)左右。又，有效成分為細胞(VI)時，可為104細胞/kg(體重)~109細胞/kg(體重)左右。醫藥組成物之投與時程，亦可考量與其投與量相同之要因來決定。例如，能夠以上述之每1日投與量，於1日~1個月投與1次。 The dosage of a pharmaceutical composition can be based on pharmacological insights such as the route of administration, the type of disease, the degree of symptoms, the age, sex, weight of the patient, the severity of the disease, drug dynamics, and toxicological characteristics. Various factors, such as the availability of a drug delivery system and whether it is administered as part of a combination with other drugs, are at the discretion of the clinician. The dosage of the pharmaceutical composition, for example, when the active ingredient is an antibody, may be about 1 μg / kg (body weight) to 10 g / kg (body weight) per day. When the active ingredient is cells (VI), it may be about 10 4 cells / kg (body weight) to about 10 9 cells / kg (body weight). The administration schedule of a pharmaceutical composition can also be determined by considering the same factors as the administration amount. For example, it is possible to administer the above-mentioned dosing amount every day from once a day to one month.
以下，藉由實施例以更詳細說明本發明，但本發明不限制於此等。 Hereinafter, the present invention will be described in more detail through examples, but the present invention is not limited thereto.
1.pMXs-IG/CAR[hV-(h28)-h28-h3Z]之構築 1. The construction of pMXs-IG / CAR [hV- (h28) -h28-h3Z]
由Open Biosystems公司購入Human CD3zeta chain cDNA及Human CD28 cDNA，hCD3zeta STD及hCD28 HD-hCD28 TMD-hCD28 STD之基因，係使用表2所示之Primer組及KOD Plus(TOYOBO,Inc)，以hCD3zeta STD之5’末端與hCD28 HD-hCD28 TMD-hCD28 STD之3’末端具備相同鹼基序列的方式，製作各編碼該等之基因片段。接著，使用此等之基因片段與KOD Plus進行assembly PCR，製作使上游具有Sac II、下游具有Not I之限制酵素位點的hCD28 HD-hCD28 TMD-hCD28 STD與CD3zeta STD基因連結而得的基因片段(Insert 1)(圖13)。將具備anti-mVEGFR2 scFv且以platelet-derived growth factor receptor(PDGFR)為膜貫通結構域之pMXs-IG/anti-mVEGFR2 scFv，以限制酵素Sac II與Not I切斷，藉由使用了DNA Ligation kit Ver.2.1(TAKARA BIO,Inc)之接合反應接入Insert 1，以構築pMXs-IG/CAR[mV-(h28)-h28-h3Z]。 Human CD3zeta chain cDNA and Human CD28 cDNA, hCD3zeta STD and hCD28 HD-hCD28 TMD-hCD28 STD genes were purchased from Open Biosystems, using the Primer group and KOD Plus (TOYOBO, Inc) shown in Table 2, and hCD3zeta STD The 5 'end and the 3' end of the hCD28 HD-hCD28 TMD-hCD28 STD have the same base sequence, and each gene fragment encoding these is produced. Next, use these gene fragments to perform assembly PCR with KOD Plus to produce a gene fragment obtained by linking hCD28 HD-hCD28 TMD-hCD28 STD with the restriction enzyme site of Sac II upstream and Not I downstream to the CD3zeta STD gene. (Insert 1) (Figure 13). Will have anti-mVEGFR2 scFv and pMXs-IG / anti-mVEGFR2 scFv with platelet-derived growth factor receptor (PDGFR) as the membrane penetrating domain to cut off the enzymes Sac II and Not I, using DNA Ligation kit Ver.2.1 (TAKARA The junction reaction of BIO, Inc) was inserted into Insert 1 to construct pMXs-IG / CAR [mV- (h28) -h28-h3Z].
對於具有anti-hVEGFR2 scFv之基因序列(序列編號20)的pET15b-mouse anti-hVEGFR2-scFv(aV2-95h)，使用表2所示之Primer組及KOD Plus進行PCR，得到上游具備Sfi I及下游具備Sac II之限制酵素位點的基因片段(Insert 2)(圖14)。將pMXs-IG/CAR[mV-(h28)-h28-h3Z]以限制酵素Sfi I與Sac II切斷，藉由使用了DNA Ligation kit Ver.2.1之接合反應接入Insert 2，以構築pMXs-IG/CAR[hV-(h28)-h28-h3Z]。 For pET15b-mouse anti-hVEGFR2-scFv (aV2-95h) with the gene sequence (sequence number 20) of anti-hVEGFR2 scFv, PCR was performed using the Primer group and KOD Plus shown in Table 2 to obtain Sfi I upstream and downstream Gene fragment with Sac II restriction enzyme site (Insert 2) (Figure 14). PMXs-IG / CAR [mV- (h28) -h28-h3Z] was cut off by restriction enzymes Sfi I and Sac II, and inserted into Insert 2 through the conjugation reaction using DNA Ligation kit Ver.2.1 to construct pMXs- IG / CAR [hV- (h28) -h28-h3Z].
2.pMXs-IG/CAR[hV-(h8α)-h137-h3Z]之構築 2.Construction of pMXs-IG / CAR [hV- (h8α) -h137-h3Z]
使用具有使hCD8αHD及TMD之基因與CD137 STD之基因連結而得的序列之質體pCR4-TOPO/hCD8αHD-TMD-hCD137-hCD3zeta與表2所示之Primer組及KOD Plus，進行PCR，製作上游具備Sac II及下游具備Not I之限制酵素位點，且編碼hCD8αHD及TMD與CD137 STD之基因片段(Insert 3)(圖15)。將pMXs-IG/CAR[mV-(h28)-h28-h3Z]以限制酵素Sac II與Not I切斷，藉由使用了DNA Ligation kit Ver.2.1之接合反應接入Insert 3，以構築pMXs-IG/CAR[mV-(h8α)-h137-h3Z]。 PCR was performed using a plastid pCR4-TOPO / hCD8αHD-TMD-hCD137-hCD3zeta with a sequence obtained by linking the genes of hCD8αHD and TMD to the genes of CD137 STD, and the Primer group and KOD Plus shown in Table 2 were used to prepare upstream equipment Sac II and downstream have Not I restriction enzyme sites and gene fragments encoding hCD8αHD and TMD and CD137 STD (Insert 3) (Figure 15). PMXs-IG / CAR [mV- (h28) -h28-h3Z] was cut off with restriction enzymes Sac II and Not I, and inserted into Insert 3 via a conjugation reaction using DNA Ligation kit Ver. 2.1 to construct pMXs- IG / CAR [mV- (h8α) -h137-h3Z].
將pMXs-IG/CAR[hV-(h28)-h28-h3Z]以限制酵素EcoR I與Sac II切斷，得到具有anti-hVEGFR2 scFv之基因序列(序列編號20)的基因片段(Insert 4)(圖16)。將pMXs-IG/CAR[mV-(h8α)-h137-h3Z]以限制酵素EcoR I與Sac II切斷，藉由使用了DNA Ligation kit Ver.2.1之接合 反應接入Insert 4，以構築pMXs-IG/CAR[hV-(h8α)-h137-h3Z]。 PMXs-IG / CAR [hV- (h28) -h28-h3Z] was cut with restriction enzymes EcoR I and Sac II to obtain a gene fragment (Insert 4) with the gene sequence (sequence number 20) of anti-hVEGFR2 scFv ( Figure 16). PMXs-IG / CAR [mV- (h8α) -h137-h3Z] was cut by restriction enzymes EcoR I and Sac II, and ligated using DNA Ligation kit Ver.2.1 The reaction was inserted into Insert 4 to construct pMXs-IG / CAR [hV- (h8α) -h137-h3Z].
由圖17所示之編碼CAR[mV-(m28)-m28-m3Z]、CAR[mV-(h28)-h28-h3Z]、CAR[hV-(h28)-h28-h3Z]、CAR[mV-(h8α)-h137-h3Z]、CAR[hV-(h8α)-h137-h3Z]之基因的質體，使用表2所示之引子及DNA聚合酶KOD plus(Toyobo,Inc)進行PCR，製作包含T7 promoter sequence及CAR基因之DNA模板，使用mMESSAGE mMACHINE(R)T7 ULTRA Transcription Kit(Ambion,Inc)，遵照一般方法純化mRNA，以Nuclease Free Water(Ambion,Inc)懸浮後，於-20℃保管。如此方式地得到CAR-mRNA。 The codes CAR [mV- (m28) -m28-m3Z], CAR [mV- (h28) -h28-h3Z], CAR [hV- (h28) -h28-h3Z], CAR [mV- (h8α) -h137-h3Z] and CAR [hV- (h8α) -h137-h3Z] gene plastids were subjected to PCR using the primers shown in Table 2 and the DNA polymerase KOD plus (Toyobo, Inc). T7 promoter sequence and the DNA template of the CAR gene were purified using the mMESSAGE mMACHINE (R) T7 ULTRA Transcription Kit (Ambion, Inc) in accordance with general methods, suspended in Nuclease Free Water (Ambion, Inc), and stored at -20 ° C. CAR-mRNA was obtained in this way.
3.CAR-mRNA之導入 3. Introduction of CAR-mRNA
將CD8+ T細胞以Opti-MEM(Life Technology,Inc)進行3次懸浮、離心、去除上清的操作，完全去除血清中所含有的蛋白等，使用Opti-MEM懸浮為5.7×106-5.7×107cells/mL。又，將含80pg-4μg/μL之CAR-mRNA的Nuclease Free Water 25μL與細胞懸浮液175μL混合，於正要進行電穿孔之前對4mm比色管(BEX,Inc)以氣泡不會進入的方式填充200μL。對比色管電極用腔室(CU500；Nepagene,Inc)***比色管，使用電穿孔器(CUY21Pro-Vitro；Nepagene,Inc)測定電阻，確認無異常後，進行EP，於3小時後、6小時後、12小時後、24小時後、36小時後、48小時後、72小 時後藉由FCM測定CAR表現強度，以台盼藍色素排除法測定細胞生存率。 CD8 + T cells were suspended, centrifuged, and the supernatant was removed three times with Opti-MEM (Life Technology, Inc) to completely remove proteins contained in the serum. The suspension was 5.7 × 10 6 -5.7 × using Opti-MEM. 10 7 cells / mL. In addition, 25 μL of Nuclease Free Water containing 80 pg-4 μg / μL of CAR-mRNA was mixed with 175 μL of cell suspension, and 4 mm colorimetric tubes (BEX, Inc) were filled so that air bubbles would not enter before electroporation. 200 μL. The cuvette electrode chamber (CU500; Nepagene, Inc) was inserted into the cuvette, and the resistance was measured using an electroporator (CUY21Pro-Vitro; Nepagene, Inc). After confirming that there was no abnormality, EP was performed, and 3 hours and 6 hours later. After 12 hours, 24 hours, 36 hours, 48 hours, and 72 hours, the CAR expression intensity was measured by FCM, and the cell survival rate was determined by trypan blue exclusion method.
4.in vivo抗腫瘤效果 4.in vivo antitumor effect
將黑色素瘤B16BL6細胞3×105個移植於C57BL/6小鼠之腹部皮內，自腫瘤接種起7日後於腫瘤之長徑達到5.0~6.0mm左右之時間點，將表現CAR[mV-(m28)-m28-m3Z]之小鼠CAR-T細胞以5×106 cells/500μL/mouse進行單次投與、或隔2日進行計2次之追加投與，之後經時測定腫瘤之長徑及短徑，遵照以下所示之式算出腫瘤體積。又，製作無處置群與投與未導入CAR-mRNA之CD8+ T細胞之群，作為控制組。小鼠CAR-T細胞係懸浮於RPMI1640進行投與。腫瘤體積(mm3)=(腫瘤長徑；mm)×(腫瘤短徑；mm)2×0.5236 3 × 10 5 melanoma B16BL6 cells were transplanted into the abdominal skin of C57BL / 6 mice. After 7 days from the tumor inoculation, the time point when the length of the tumor reached about 5.0 to 6.0 mm will show CAR [mV- ( m28) -m28-m3Z] mouse CAR-T cells were administered at a single dose of 5 × 10 6 cells / 500 μL / mouse, or additional doses were counted twice every other day, and the tumor length was measured over time. Diameter and short diameter, the tumor volume was calculated according to the formula shown below. In addition, a control group was prepared as a control group, and a group of CD8 + T cells to which CAR-mRNA was not introduced was administered. The mouse CAR-T cell line was suspended in RPMI1640 for administration. Tumor volume (mm 3 ) = (tumor long diameter; mm) × (tumor short diameter; mm) 2 × 0.5236
試驗結果示於圖18。於單次投與CAR-T細胞之荷癌小鼠中，相較於控制組群而言，顯著地抑制腫瘤之增殖，進一步進行2次之追加投與時，可得更強之抗腫瘤效果。因此，我們所製作之CAR-T細胞，暗示了以下可能性：雖CAR之表現為暫時性(transient)，但僅以單次投與即發揮有效性，若進而複數次投與時，可成為充分顯示效果的細胞醫藥。 The test results are shown in FIG. 18. In cancer-bearing mice administered with CAR-T cells in a single administration, compared with the control group, tumor proliferation was significantly inhibited. When two additional administrations were performed, stronger antitumor effects were obtained. . Therefore, the CAR-T cells we have produced suggest the following possibility: Although the manifestation of CAR is transient, it can only be effective with a single administration. Cell medicine with full effect.
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<400> 20 <400> 20
<210> 21 <210> 21
<211> 11 <211> 11
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 21 <400> 21
<210> 22 <210> 22
<211> 7 <211> 7
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 22 <400> 22
<210> 23 <210> 23
<211> 9 <211> 9
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 23 <400> 23
<210> 24 <210> 24
<211> 108 <211> 108
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 24 <400> 24
<210> 25 <210> 25
<211> 10 <211> 10
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 25 <400> 25
<210> 26 <210> 26
<211> 17 <211> 17
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 26 <400> 26
<210> 27 <210> 27
<211> 9 <211> 9
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 27 <400> 27
<210> 28 <210> 28
<211> 118 <211> 118
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 28 <400> 28
<210> 29 <210> 29
<211> 15 <211> 15
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 29 <400> 29
<210> 30 <210> 30
<211> 241 <211> 241
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 30 <400> 30
<210> 31 <210> 31
<211> 33 <211> 33
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 31 <400> 31
<210> 32 <210> 32
<211> 21 <211> 21
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 32 <400> 32
<210> 33 <210> 33
<211> 27 <211> 27
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 33 <400> 33
<210> 34 <210> 34
<211> 324 <211> 324
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 34 <400> 34
<210> 35 <210> 35
<211> 30 <211> 30
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 35 <400> 35
<210> 36 <210> 36
<211> 51 <211> 51
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 36 <400> 36
<210> 37 <210> 37
<211> 27 <211> 27
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 37 <400> 37
<210> 38 <210> 38
<211> 354 <211> 354
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 38 <400> 38
<210> 39 <210> 39
<211> 45 <211> 45
<212> DNA <212> DNA
<213> Artificial <213> Artificial
<220> <220>
<223> Linker <223> Linker
<400> 39 <400> 39
<210> 40 <210> 40
<211> 723 <211> 723
<212> DNA <212> DNA
<213> Artificial <213> Artificial
<220> <220>
<223> ScFV <223> ScFV
<400> 40 <400> 40
<210> 41 <210> 41
<211> 11 <211> 11
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 41 <400> 41
<210> 42 <210> 42
<211> 7 <211> 7
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 42 <400> 42
<210> 43 <210> 43
<211> 9 <211> 9
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 43 <400> 43
<210> 44 <210> 44
<211> 108 <211> 108
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 44 <400> 44
<210> 45 <210> 45
<211> 10 <211> 10
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 45 <400> 45
<210> 46 <210> 46
<211> 17 <211> 17
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 46 <400> 46
<210> 47 <210> 47
<211> 12 <211> 12
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 47 <400> 47
<210> 48 <210> 48
<211> 121 <211> 121
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 48 <400> 48
<210> 49 <210> 49
<211> 15 <211> 15
<212> PRT <212> PRT
<213> Artificial <213> Artificial
<220> <220>
<223> Linker <223> Linker
<400> 49 <400> 49
<210> 50 <210> 50
<211> 244 <211> 244
<212> PRT <212> PRT
<213> Artificial <213> Artificial
<220> <220>
<223> Linker <223> Linker
<400> 50 <400> 50
<210> 51 <210> 51
<211> 33 <211> 33
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 51 <400> 51
<210> 52 <210> 52
<211> 21 <211> 21
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 52 <400> 52
<210> 53 <210> 53
<211> 27 <211> 27
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 53 <400> 53
<210> 54 <210> 54
<211> 324 <211> 324
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 54 <400> 54
<210> 55 <210> 55
<211> 30 <211> 30
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 55 <400> 55
<210> 56 <210> 56
<211> 51 <211> 51
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 56 <400> 56
<210> 57 <210> 57
<211> 36 <211> 36
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 57 <400> 57
<210> 58 <210> 58
<211> 363 <211> 363
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 58 <400> 58
<210> 59 <210> 59
<211> 45 <211> 45
<212> DNA <212> DNA
<213> Artificial <213> Artificial
<220> <220>
<223> Linker <223> Linker
<400> 59 <400> 59
<210> 60 <210> 60
<211> 732 <211> 732
<212> DNA <212> DNA
<213> Artificial <213> Artificial
<220> <220>
<223> scFV <223> scFV
<400> 60 <400> 60
<210> 61 <210> 61
<211> 10 <211> 10
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 61 <400> 61
<210> 62 <210> 62
<211> 7 <211> 7
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 62 <400> 62
<210> 63 <210> 63
<211> 9 <211> 9
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 63 <400> 63
<210> 64 <210> 64
<211> 107 <211> 107
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 64 <400> 64
<210> 65 <210> 65
<211> 10 <211> 10
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 65 <400> 65
<210> 66 <210> 66
<211> 17 <211> 17
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 66 <400> 66
<210> 67 <210> 67
<211> 9 <211> 9
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 67 <400> 67
<210> 68 <210> 68
<211> 118 <211> 118
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 68 <400> 68
<210> 69 <210> 69
<211> 15 <211> 15
<212> PRT <212> PRT
<213> Artificial <213> Artificial
<220> <220>
<223> Linker <223> Linker
<400> 69 <400> 69
<210> 70 <210> 70
<211> 240 <211> 240
<212> PRT <212> PRT
<213> Artificial <213> Artificial
<220> <220>
<223> scFV <223> scFV
<400> 70 <400> 70
<210> 71 <210> 71
<211> 30 <211> 30
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 71 <400> 71
<210> 72 <210> 72
<211> 21 <211> 21
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 72 <400> 72
<210> 73 <210> 73
<211> 27 <211> 27
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 73 <400> 73
<210> 74 <210> 74
<211> 321 <211> 321
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 74 <400> 74
<210> 75 <210> 75
<211> 30 <211> 30
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 75 <400> 75
<210> 76 <210> 76
<211> 51 <211> 51
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 76 <400> 76
<210> 77 <210> 77
<211> 27 <211> 27
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 77 <400> 77
<210> 78 <210> 78
<211> 354 <211> 354
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 78 <400> 78
<210> 79 <210> 79
<211> 45 <211> 45
<212> DNA <212> DNA
<213> Artificial <213> Artificial
<220> <220>
<223> Linker <223> Linker
<400> 79 <400> 79
<210> 80 <210> 80
<211> 720 <211> 720
<212> DNA <212> DNA
<213> Artificial <213> Artificial
<220> <220>
<223> scFV <223> scFV
<400> 80 <400> 80
<210> 81 <210> 81
<211> 10 <211> 10
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 81 <400> 81
<210> 82 <210> 82
<211> 7 <211> 7
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 82 <400> 82
<210> 83 <210> 83
<211> 9 <211> 9
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 83 <400> 83
<210> 84 <210> 84
<211> 107 <211> 107
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 84 <400> 84
<210> 85 <210> 85
<211> 10 <211> 10
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 85 <400> 85
<210> 86 <210> 86
<211> 17 <211> 17
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 86 <400> 86
<210> 87 <210> 87
<211> 13 <211> 13
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 87 <400> 87
<210> 88 <210> 88
<211> 122 <211> 122
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 88 <400> 88
<210> 89 <210> 89
<211> 15 <211> 15
<212> PRT <212> PRT
<213> Artificial <213> Artificial
<220> <220>
<223> Linker <223> Linker
<400> 89 <400> 89
<210> 90 <210> 90
<211> 244 <211> 244
<212> PRT <212> PRT
<213> Artificial <213> Artificial
<220> <220>
<223> scFV <223> scFV
<400> 90 <400> 90
<210> 91 <210> 91
<211> 30 <211> 30
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 91 <400> 91
<210> 92 <210> 92
<211> 21 <211> 21
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 92 <400> 92
<210> 93 <210> 93
<211> 27 <211> 27
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 93 <400> 93
<210> 94 <210> 94
<211> 321 <211> 321
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 94 <400> 94
<210> 95 <210> 95
<211> 30 <211> 30
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 95 <400> 95
<210> 96 <210> 96
<211> 51 <211> 51
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 96 <400> 96
<210> 97 <210> 97
<211> 39 <211> 39
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 97 <400> 97
<210> 98 <210> 98
<211> 366 <211> 366
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 98 <400> 98
<210> 99 <210> 99
<211> 45 <211> 45
<212> DNA <212> DNA
<213> Artificial <213> Artificial
<220> <220>
<223> Linker <223> Linker
<400> 99 <400> 99
<210> 100 <210> 100
<211> 732 <211> 732
<212> DNA <212> DNA
<213> Artificial <213> Artificial
<220> <220>
<223> scFV <223> scFV
<400> 100 <400> 100
<210> 101 <210> 101
<211> 11 <211> 11
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 101 <400> 101
<210> 102 <210> 102
<211> 7 <211> 7
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 102 <400> 102
<210> 103 <210> 103
<211> 9 <211> 9
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 103 <400> 103
<210> 104 <210> 104
<211> 108 <211> 108
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 104 <400> 104
<210> 105 <210> 105
<211> 10 <211> 10
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 105 <400> 105
<210> 106 <210> 106
<211> 17 <211> 17
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 106 <400> 106
<210> 107 <210> 107
<211> 8 <211> 8
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 107 <400> 107
<210> 108 <210> 108
<211> 117 <211> 117
<212> PRT <212> PRT
<213> Mus musculus <213> Mus musculus
<400> 108 <400> 108
<210> 109 <210> 109
<211> 15 <211> 15
<212> PRT <212> PRT
<213> Artificial <213> Artificial
<220> <220>
<223> Linker <223> Linker
<400> 109 <400> 109
<210> 110 <210> 110
<211> 240 <211> 240
<212> PRT <212> PRT
<213> Artificial <213> Artificial
<220> <220>
<223> scFV <223> scFV
<400> 110 <400> 110
<210> 111 <210> 111
<211> 33 <211> 33
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 111 <400> 111
<210> 112 <210> 112
<211> 21 <211> 21
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 112 <400> 112
<210> 113 <210> 113
<211> 27 <211> 27
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 113 <400> 113
<210> 114 <210> 114
<211> 324 <211> 324
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 114 <400> 114
<210> 115 <210> 115
<211> 30 <211> 30
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 115 <400> 115
<210> 116 <210> 116
<211> 51 <211> 51
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 116 <400> 116
<210> 117 <210> 117
<211> 24 <211> 24
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 117 <400> 117
<210> 118 <210> 118
<211> 351 <211> 351
<212> DNA <212> DNA
<213> Mus musculus <213> Mus musculus
<400> 118 <400> 118
<210> 119 <210> 119
<211> 45 <211> 45
<212> DNA <212> DNA
<213> Artificial <213> Artificial
<220> <220>
<223> Linker <223> Linker
<400> 119 <400> 119
<210> 120 <210> 120
<211> 720 <211> 720
<212> DNA <212> DNA
<213> Artificial <213> Artificial
<220> <220>
<223> scFV <223> scFV
<400> 120 <400> 120
<210> 121 <210> 121
<211> 49 <211> 49
<212> DNA <212> DNA
<213> artificial <213> artificial
<220> <220>
<223> primer <223> primer
<400> 121 <400> 121
<210> 122 <210> 122
<211> 38 <211> 38
<212> DNA <212> DNA
<213> Artificial <213> Artificial
<220> <220>
<223> primer <223> primer
<400> 122 <400> 122
<210> 123 <210> 123
<211> 50 <211> 50
<212> DNA <212> DNA
<213> artificial <213> artificial
<220> <220>
<223> primer <223> primer
<400> 123 <400> 123
<210> 124 <210> 124
<211> 19 <211> 19
<212> DNA <212> DNA
<213> artificial <213> artificial
<220> <220>
<223> primer <223> primer
<400> 124 <400> 124
<210> 125 <210> 125
<211> 43 <211> 43
<212> DNA <212> DNA
<213> artificial <213> artificial
<220> <220>
<223> primer <223> primer
<400> 125 <400> 125
<210> 126 <210> 126
<211> 45 <211> 45
<212> DNA <212> DNA
<213> artificial <213> artificial
<220> <220>
<223> primer <223> primer
<400> 126 <400> 126
<210> 127 <210> 127
<211> 30 <211> 30
<212> DNA <212> DNA
<213> artificial <213> artificial
<220> <220>
<223> primer <223> primer
<400> 127 <400> 127
<210> 128 <210> 128
<211> 31 <211> 31
<212> DNA <212> DNA
<213> artificial <213> artificial
<220> <220>
<223> primer <223> primer
<400> 128 <400> 128
Claims (6)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2016095125 | 2016-05-11 | ||
| JP2016-095125 | 2016-05-11 |
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| TW201806970A true TW201806970A (en) | 2018-03-01 |
| TWI756223B TWI756223B (en) | 2022-03-01 |
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| TW106115500A TWI756223B (en) | 2016-05-11 | 2017-05-10 | Chimeric antigen receptors, and their utilization |
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| JP (1) | JP7054143B2 (en) |
| TW (1) | TWI756223B (en) |
| WO (1) | WO2017195749A1 (en) |
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| EP3988650A4 (en) * | 2019-06-21 | 2022-09-21 | Osaka University | Method for preparing artificial recombinant rna virus that stably holds foreign gene |
| EP4229092A1 (en) * | 2020-10-15 | 2023-08-23 | Cornell University | Therapeutic cemip antibodies |
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| JP5934099B2 (en) | 2009-10-01 | 2016-06-15 | アメリカ合衆国 | Anti-vascular endothelial growth factor receptor-2 chimeric antigen receptor and its use for the treatment of cancer |
| US20160237407A1 (en) * | 2015-02-17 | 2016-08-18 | Batu Biologics, Inc. | Universal donor chimeric antigen receptor cells |
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2017
- 2017-05-09 WO PCT/JP2017/017456 patent/WO2017195749A1/en active Application Filing
- 2017-05-09 JP JP2018517017A patent/JP7054143B2/en active Active
- 2017-05-10 TW TW106115500A patent/TWI756223B/en active
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| Publication number | Publication date |
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| WO2017195749A1 (en) | 2017-11-16 |
| JPWO2017195749A1 (en) | 2019-05-09 |
| JP7054143B2 (en) | 2022-04-13 |
| TWI756223B (en) | 2022-03-01 |
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