MSCV Retrovirus Chimeric Antigen Rec件話eptor (CAR) Expressio新人n Vector

Utilizing chimeric拿唱 antigen receptor (CAR) vectors to pr熱門oduce engineered T cel器街ls (also known as CAR T cells)著都 that can recognize tumor-associat姐的ed antigens has 費可emerged as a promising appr下也oach in the treatment of canc謝上er. In CAR T-cell therapy, T c知離ells derived from either patients 花空(autologous) or 去車healthy donors (allogeneic) are他的 modified to express CAR, a chimeri老如c construct which combines antigen bi近裡nding with T cell acti朋做vation for targ畫校eting tumor cells.

Structurally, a CAR consists of年理 four main components: (1) an extrace山資llular antigen recognition do司亮main made up of an ant師車ibody-derived single chain v湖紙ariable fragment (scFv) of kn就但own specificity. T書愛he scFv facilitates 下鄉antigen binding and is compos裡理ed of the variable li河少ght chain and heavy chain regi術少ons of an antigen-specific monoclonal a著要ntibody connected by a flex秒綠ible linker; (2) an extracellul黑亮ar hinge or spacer wh跳制ich connects the scFv with the transmem友動brane domain and prov電師ides flexibility 冷動and stability to the CAR structure拍我; (3) a transmembrane d算放omain which anchors the CA司離R to the plasma mem黃資brane and bridges the extrace林件llular hinge as well as antigen bindi又影ng domain with the intr呢可acellular signaling domai城船n. It plays a criti科你cal role in enhancing receptor e做弟xpression and s拿還tability; (4) and an intrace笑快llular signaling domain which is typi銀相cally derived from the CD3 zeta c笑去hain of the T cell receptor (TCR) and c雨樂ontains immunor人林eceptor tyrosine-ba市光sed activation motifs喝又 (ITAMs). The ITAMs b視秒ecome phosphorylated a謝火nd activate downstream 河算signaling upon antigen binding, 訊相leading to the subseq中市uent activation 爸報of T cells. In addition, the intracel就鐵lular region may co拍讀ntain one or more costi雪相mulatory domains (derived from CD28, 美靜CD137 etc.) in ta光還ndem with the CD3 zeta signalin鐵讀g domain for impr老吃oving T cell proliferation 生女and persistence.

The structure of CAR 吃上has evolved over the past few yea什笑rs based on modifi生歌cations to the composit用飛ion of the intra但見cellular domains. The first-generation 子男CARs consisted of only a s鄉見ingle intracellular CD3 zeta-山朋derived signaling domain. Whil好們e these CARs could activate T cells, th森紙ey exhibited poor anti-tu微對mor activity in v行大ivo due to the low cytotoxicity and pr請兒oliferation of T cells expressin如外g such CARs. This led t錢靜o the advent of the second-generati舞城on CARs which included an看答 intracellular 窗我costimulatory domain in a技嗎ddition to the CD3 zeta signaling doma音劇in leading to a significant imp訊北rovement in the in vivo prolifera船動tion, expansion and persistence of T ce草黑lls expressing second generation C子藍ARs. To further optimize the an湖分ti-tumor efficacy of CAR通兵-T cells, third generation CARs wer公民e developed which included tw綠公o intracellular, c少樹is-acting costimulatory domai街男ns in addition to CD3 ze子討ta. Thereafter, four紙舞th generation CARs wer離拿e derived from second-generation C東聽ARs by modifying the匠來ir intracellular domai市相n for inducible or constit中森utive expression of cytokines醫是. The fifth and th員可e latest generation空綠 of CARs are also derived f山討rom second-generation山做 CARs by the incorpora農家tion of intracellular domains又黃 of cytokine receptors.

Our MSCV retrov村習irus CAR expression vect得信or is a highly efficient tool for r資議etrovirus-based de看遠livery of second-generation CAR ex得廠pression cassett廠開es into T cells. 筆廠MSCV retroviral vecto你白rs are derived from the murine PCC4-cel微藍l passaged myel但美oproliferative sarcoma virus (PCMV) 吃黃based MESV retrov影錢iral vectors and Moloney murine leuke湖很mia virus (MMLV哥門) based LN retroviral ve問技ctors. The inclusi藍影on of an extended hybri身紅d packaging signal derived from the LN愛很 vectors helps to achieve h河拍igher viral titer wit日看h the MSCV retroviral vector 服銀compared to traditional retro玩計viral vectors. Additiona火關lly, the presence of a strat謝物egically designed 5’LTR derived from 雜新the PCMV virus in the MSC道線V retroviral vector co飛見ntributes to transcriptiona家門l activation of target genes in plu城吃ripotent cell lines such as ES or EC ce老少lls.

The MSCV retrovirus CAR express司有ion vector is first constr暗森ucted as a plasmid in E. coli wher白暗e the entire CAR expression casset但視te including the scFv region, the h校男inge, the trans村路membrane domain 都舞and the intracellular CD3 zeta signali樂場ng domain as well銀來 as the costimulatory domain is cl數好oned in between the two M雨少SCV long terminal repeats (L美村TRs). It is then transfected into 舊謝packaging cells又物 along with several he著光lper plasmids. Ins店水ide the packaging cell刀子s, vector DNA located bet們嗎ween the two LTRs is transcribed 化紅into RNA, and vira是弟l proteins expressed by the helper愛姐 plasmids further pac書分kage the RNA into virus. Live virus is 玩件then released into the supern我通atant, which can be used志喝 to infect targ舊嗎et cells directly o不為r after concentration內器.

When the virus is added to target cells冷都, the RNA cargo is shuttled into ce放校lls where it is reverse transcr跳算ibed into DNA and ra有離ndomly integrate化草d into the host gen老匠ome. Any gene(s) that were placed姐河 in-between the 內高two LTRs during vector cloning ar在些e permanently inserted into host DNA al少可ongside the rest of viral ge門現nome.

By design, MSCV retrovi西影ral vectors lack the genes都什 required for viral packaging 爸著and transduction (these genes are i舊請nstead carried by helper plas房西mids used during virus packaging).件可 As a result, virus produced from工暗 retroviral vectors has the important 有著safety feature o和坐f being replication incompetent大黃 (meaning that they can transduc了市e target cells but cannot re師很plicate in them)醫飛.

For further info好做rmation about this vector syste煙她m, please refer to the pape時河rs below.