Tol2 Chimeric Antig資西en Receptor (CAR)女著 Expression Vector

Utilizing chimeric antigen receptor 兵暗(CAR) vectors to produce e黑資ngineered T cells (also件歌 known as CAR T cells) that can rec秒短ognize tumor-associ都亮ated antigens has emerged a影年s a promising approach in t做物he treatment of cancer. In飛會 CAR T-cell therapy, T cells derived fr作外om either patient知計s (autologous) or he制吃althy donors (a子時llogeneic) are modified to expr機拿ess CAR, a chimeric cons兵河truct which combines a林著ntigen binding with T 制書cell activation for target很暗ing tumor cells.

Structurally, a CAR consists 裡討of four main components:要討 (1) an extracellular antig湖還en recognition domai科又n made up of an antibody-deri呢大ved single chain v多校ariable fragment (scFv) of known sp請不ecificity. The sc中拿Fv facilitates anti微靜gen binding and is composed of t北窗he variable light chain and he說火avy chain regions of 內市an antigen-specific monoclonal an靜現tibody connected by a fl鄉公exible linker; (2) an extracel喝影lular hinge or space不離r that connects the scFv大秒 with the transmembra道麗ne domain and p農嗎rovides flexibility and stability t線得o the CAR structure; (3) a tran哥好smembrane domain which anchors the 老體CAR to the plasma membran是做e and bridges the extracellu山笑lar hinge as well as antigen binding do能下main with the intra內跳cellular signaling domai草討n. It plays a cr她長itical role in enhancing 時錢receptor expressio線子n and stability, and (4) an intrace船錢llular signaling domain畫去 that is typica風厭lly derived from the CD3 zeta chain服科 of the T cell receptor (TCR)短民 and contains immunorec鐵去eptor tyrosine-based ac短吧tivation motifs (ITAM區問s). The ITAMs become phosphor老南ylated and activate downstream s熱西ignaling upon antigen binding, leadi通醫ng to the subsequent activation of T ce紅年lls. In addition, the intracellula姐光r region may contain one or more co訊見stimulatory domain輛訊s (derived from CD28, CD137, etc男你.) in tandem with the CD3 ze遠在ta signaling do個生main for improv裡民ing T cell prolifer和影ation and persistence.

The structure of CAR ha商從s evolved over the past few years base們腦d on modifications to the com影知position of the intracellula就街r domains. The first-generat技司ion CARs consiste電遠d of only a single intracellular CD3 又子zeta-derived signa睡厭ling domain. While these CARs could 拿國activate T cells, they exhibited poor 要廠anti-tumor activity in vivo d答近ue to the low cytotoxicity and proli飛通feration of T cells 來靜expressing such CAR但是s. This led to the advent o美票f second-generation CAR舞火s which included an intracellula你靜r costimulatory d短自omain in addition to the CD3 zeta sig現長naling domain leading to a signifi又子cant improvement in the in vivo p就兵roliferation, exp北湖ansion, and per間店sistence of T c道玩ells expressing second-gen話小eration CARs. To further optimi看哥ze the anti-tumor efficacy of CA明為R-T cells, third舞計-generation CARs were 那站developed which included two i兵來ntracellular, cis-ac朋拿ting costimulatory domains in 內玩addition to CD3 zeta. Th熱輛ereafter, fourth-generation CARs 術麗were derived from second-generation技裡 CARs by modifying their靜醫 intracellular domain for inducible物藍 or constitutive expr照老ession of cytokines. The fifth and街山 the latest generation公呢 of CARs are also derived from 身話second-generation CAR房票s by the incorporation of intracel兒西lular domains of cytokine receptors.

Our Tol2 CAR expressio相務n vector is a highly efficient tool for雪火 achieving non-viral輛友, transposon-based delivery of second-懂車generation CAR expression cassettes黑坐 into T cells. This vector system is d上但erived from the Tol2 transposon, whi飛低ch is originally 舊在isolated from th草店e teleost fish, medaka (Oryzi懂議as latipes). Based on sequence費大 homology, the Tol2 transposon was fo車商und to be close章錢ly related to th區對e hAT family of non對開-autonomous elements found throu亮北ghout vertebrate geno地這mes.

The Tol2 system comprises two component人這s: the transposon p理訊lasmid and the transposase. The trans相多poson plasmid contains two inve黃白rted terminal repeats (紙中ITRs) bracketing the region to be tran哥科sposed. The transposase can be delive司內red into target cells through two 山制methods. The helper plasmid can be tra風女nsiently transfected int山得o cells. Alternatively北站, target cells can be injected wi讀那th transposase mRNA generated by in好房 vitro transcription from the helper煙習 plasmid. When the t海醫ransposon and helpe拿離r plasmids are co-transfected i劇謝nto target cells, the transposa報和se produced from the hel新子per plasmid wou玩光ld recognize the two ITRs on the綠分 transposon and insert the flanked reg事很ion including the two ITRs into the h藍小ost genome. At each insertio友志n site, the Tol2 transposase creat鐘煙es an 8 bp duplicatio路靜n, resulting in identical 8 bp direct黑她 repeats flanking each transposon in黃關tegration site in the genome. In見暗sertion occurs without 視匠any significant bias for t風呢he insertion sit學可e sequence. This is unlik體森e transposon systems whic聽但h have specific target c議們onsensus sites. Fo都場r example, piggyBac transposo業劇ns are typically inserte影嗎d at sites containing the 多讀sequence TTAA. T務機hrough both methods of delivering離我 transposase, it is expresse長她d for only a short time. Upon河東 the loss of the helper plas對問mid or degradation of木拍 transposase mRNA, the in拿理tegration of the tran亮放sposon into the 好鄉host genome bec又機omes permanent.

Tol2 is a class II transposon樹關, meaning that it moves 照化in a cut-and-paste manner, hopp畫街ing from place to place without lea長線ving copies behind. (In contr用遠ast, class I tr他什ansposons move in 科月a copy-and-paste樹土 manner.) If Tol2 transposase is r間電eintroduced into the cells, the 兒制transposon could get excise不科d from the genome of some cell一妹s, resulting in either precise or 農笑imprecise excisions with ind對喝els created.

For further information about t明中his vector system, please refer to the 畫場papers below.

Delivery method: Tol2-based CAR expression vec時吃tors are typically delivered into花科 T cells by electrop時雪oration which typi明少cally results in a low好跳 to medium frequency of 作海cells with successful integr醫房ation of the CAR expressio裡個n cassette. Additionally, ele工好ctroporation can be toxic to T cells 暗車which in turn could lead to r吃月educed yield of functional CAR T ce船區lls. Therefore, it may be nece友可ssary to culture the T cells ex vivo 多風for extended periods to attain 人樂the quantities needed務很 for infusion, which over time could al新也ter cell phenotype leadin司工g to reduced long-term memory什歌 function.