MMLV Retrovirus Chimeric一水 Antigen Receptor (CAR)海習 Expression Vector

Utilizing chimeric 愛門antigen receptor (CAR) vect要新ors to produce engineered身我 T cells (also known as CAR道可 T cells) that can recognize tumor-asso亮舊ciated antigens has emerged as a 山畫promising approach in the tr裡火eatment of cancer. In CAR T-cell the吧件rapy, T cells derived from either patie慢車nts (autologous)票頻 or healthy donors (allogene錢高ic) are modified t又上o express CAR, a有暗 chimeric construct whic關不h combines antigen 北哥binding with T cel放放l activation for targeting 樂唱tumor cells.

Structurally, a CAR consists of 樹件four main components: (自東1) an extracellular antigen科舞 recognition domain made城分 up of an antibody-derived single chain農放 variable fragment (scFv) of known 農問specificity. The scFv facilitat鐵化es antigen binding風很 and is composed of the variabl作弟e light chain and heavy chain region花銀s of an antigen-specific mon動那oclonal antibody connected黑農 by a flexible linker; (2) 購什an extracellular hin來身ge or spacer which日資 connects the scFv with 自樹the transmembrane doma做市in and provides flexi村街bility and stability to輛美 the CAR structure; (3) a transmembran姐做e domain which anchors the CAR to the p姐刀lasma membrane an飛月d bridges the extracel腦很lular hinge as well as a水機ntigen binding domain with the intra年計cellular signaling doma坐森in. It plays a critical role in 聽愛enhancing receptor expressio嗎化n and stability; (4) and an intra她吃cellular signaling domain which is 也行typically derived from the CD3 zeta ch林銀ain of the T cell recept現場or (TCR) and contains immu子老noreceptor tyrosine-based activation m在慢otifs (ITAMs). The ITAMs becom場家e phosphorylated and activate d師女ownstream signaling upon antigen bindin做林g, leading to the 務生subsequent acti女用vation of T cells. 市民In addition, the i呢樹ntracellular region may cont器購ain one or more costimulatory明我 domains (derived from CD28, CD13動吧7 etc.) in tandem with the CD3 zeta國相 signaling domain for impr窗了oving T cell pr月吃oliferation and persistence店也.

The structure of CAR has evolved o說好ver the past few years b光對ased on modifications 匠雜to the composition of the intracel藍校lular domains. T們大he first-generati店鄉on CARs consisted of要麗 only a single intracel內雪lular CD3 zeta-derived signa妹妹ling domain. While外裡 these CARs could activate T ce科笑lls, they exhibited poor anti-tumor ac舊拍tivity in vivo due to the low cytot一腦oxicity and proliferation of T cells 亮水expressing such CARs. This led to兵空 the advent of the se低章cond-generation CA報站Rs which included an 開暗intracellular co內讀stimulatory domain in additio多務n to the CD3 zeta sign也上aling domain leading to a s音白ignificant impr影的ovement in the in vivo pro鐵紅liferation, expansion and persist術樂ence of T cells expr老民essing second generation CAR玩姐s. To further optimize the自體 anti-tumor efficacy of CAR山爸-T cells, third gen地訊eration CARs were developed wh務她ich included two intr樂熱acellular, cis-acting costimulatory do下短mains in addition to CD3 zeta.微國 Thereafter, fourth家火 generation CARs were d路鐵erived from second-genera費地tion CARs by modifying 資亮their intracellular domain fo懂議r inducible or co坐黑nstitutive expression of cytokines. The風物 fifth and the latest generation動知 of CARs are also derived呢海 from second-genera體生tion CARs by the incorporation 船坐of intracellular domains of cytokine 微術receptors.

Our MMLV retroviru讀黃s CAR expression vector is derived章通 from the Moloney mur線和ine leukemia virus, which is a 黑聽member of the retrovirus family and 現友is highly suitable for retrovi農妹rus-mediated delivery of s房靜econd-generation CAR expression casset章嗎tes into T cells. Wi們民ldtype MMLV virus has a plus-st員輛rand linear RNA genome.

The MMLV retrovirus CAR expre哥男ssion vector is first constructed as得跳 a plasmid in E訊頻. coli where the entire CAR e短技xpression cassette including the sc子得Fv region, the hinge, the 煙亮transmembrane domain a綠師nd the intracellular CD3 zeta sign秒不aling domain as well購訊 as the costimulatory domain 林購is cloned in between the two M影新MLV long terminal repeats (LTRs). It is也那 then transfected into p市習ackaging cells along wi朋時th several help舞風er plasmids. Inside t我門he packaging cells, vec章事tor DNA located b服自etween the two LTRs is 高通transcribed into RNA, and vi就用ral proteins expre拍空ssed by the helper plasmids fur報湖ther package the RNA into v懂弟irus. Live virus is then月高 released into the supe場雨rnatant, which 還外can be used to inf報妹ect target cells directly or af現南ter concentration.

When the virus is added to 說學target cells, the RNA cargo is是現 shuttled into cells where it is re很用verse transcribed in暗還to DNA and randomly inte微月grated into the host genome. Any 山金gene(s) that were placed in-between th吧輛e two LTRs during vector cloning a都村re permanently inserted志坐 into host DNA alongside姐樂 the rest of viral genome.

By design, MMLV retr銀我oviral vectors lack the ge森數nes required for vir妹信al packaging and tran長習sduction (these genes are instead又我 carried by helper plasm作站ids used during viru議暗s packaging). As a re拿東sult, virus produced from MMLV re內問troviral vectors has t他鄉he important safety featur在門e of being replication incompeten國就t (meaning that th歌計ey can transduce target c信音ells but cannot replicate in them)和男.

For further infor放雜mation about this vector system愛體, please refer to the pap農那ers below.