PiggyBac Antibody Heavy and Ligh弟能t Chain Coexpression Vector

Recombinant antibodi公嗎es have extensive applications in the 看的fields of diagnostics an唱文d therapeutic medicine. Currently, 自爸there are about 180 mon動爸oclonal antibody pr制秒oducts approved worldwide tar著廠geting specific proteins, such 知女as PD-L1, HER2, IL-術影17R, and VEGF. The developm花文ent of genetic engineering h微說as facilitated successful pro動那duction of these therapeutic 空公antibodies, which have i志區deal tumor penetrati訊校on behavior, short re校民tention times, and reduced i什間mmunogenicity. These th弟樂erapeutic recombinant antib綠紅odies are mainly 個聽produced in mammalian cells in vit電西ro (e.g., CHO c文好ells), since the post-transcrip光愛tional modification in this離請 expression system is closest to 西自the in vivo processes in hum票得ans.

All antibodies contain heavy and ligh也空t chains, both of which consist of vari術了able (V) and consta商黑nt (C) regions. 他購The antigen-binding site co兒男nsists of VH+VL dimers where V行校H and VL represent v大拍ariable fragment heavy chains錢民 and light chai服我ns, respectively. Within these靜離 fragments, most sequence variation is 有對located in complementarity determin道一ing regions (CDRs)刀得, which influences antige這事n-binding specificity. In mammals話機, antibodies are divided into fiv站來e isotypes (IgG, IgA,看短 IgM, IgD and IgE) base現報d on the heavy chain C region seque體花nce. Of the five 車媽immunoglobulin (antibod離海y) isotypes, IgG i在相s the most abundant in circulation. Mo家城reover, almost all back女土bones of the approved 費動therapeutic antib街紅odies are IgG. An antibody of a giv她很en isotype either has lambda (λ生子) or kappa (κ) light chains which金得 are encoded by the light chain C re子老gion. While the heavy cha這廠in is typically associa站呢ted with target specificity, t歌劇he light chain is associate哥山d with preventing self-reactivi到學ty and enhancing receptor act鐘日ivation. The ratio花靜 of the two types of light chains varie土靜s from species to species.

VectorBuilder’s piggyBac antibod鄉很y heavy and light chain c請司oexpression vect街書or system is a highly efficient tool f山說or achieving non-viral, transposon-base河什d delivery of antibody heav電雨y and light chain expre民數ssion cassettes into mammalian cells. T內懂his system contains科腦 two vectors, both of which are e她廠ngineered as E. coli plas工司mids. The first vector is re什玩ferred to as the helper PBase plasmi生小d, encoding the tra花河nsposase. The second ve兵購ctor is referred to a章間s the piggyBac transposon plasmid物購 which contains two terminal repeats 車議(TRs) bracketing the antibo很河dy heavy and light chain expression 我費cassettes to be transposed. The cod是視ing regions for heavy an那可d light chains 錢市are separated by 空拿a polyadenylation signal. The transcr雪高iption of the heavy and light姐明 chains is respec窗綠tively driven by separate promoters北站. In the vector des綠短ign process, by cus知金tomizing sequences encoding 做銀the heavy chain and ligh樹生t chain variable regions城內, customers may ge舞靜nerate vectors to sati金看sfy their personalize爸工d need for antigen-bind得站ing specificities. T高說he heavy and light ch子生ain constant regions 相場may either be selected from our po飛身pular constant region database (裡快containing human and mou民兒se IgG1 and mouse IgG2a a線花nd 2b heavy cha從機ins; and containing human and mouse κ木睡 and human and mouse λ2 an答林d mouse λ1 light懂雪 chains) or be pasted by cust她鄉omers using their own sequence. Add音算itionally, signal peptides (e.g., IL-2 有黑sig) can be added into the v少請ector to increase sec藍在retion of the r到腦ecombinant antibody.

When the helper PBase plasmid and the間市 piggyBac transposo弟就n plasmids are co-trans門紙fected into target cells, the trans跳山posase from the helper plasmid w草線ill recognize the 玩森two TRs on the transposon and上秒 insert the flanked antib有白ody expression cassettes in媽為to the host genome. Insertion t但我ypically occurs at host c自章hromosomal sites 場男that contain the TTA用樹A sequence, which i光歌s duplicated on the two flanks of the 通票integrated fragmen見能t. PiggyBac is a女費 class II transposon, meaning 睡雨that it moves in a cut-an腦理d-paste manner, hopping 技水from place to place with花看out leaving copies behind. (In con商土trast, class I transpos快坐ons move in copy-and-paste manner船煙). Because the h森看elper plasmid is only transiently內房 transfected into host cells上弟, it will get lost over time. With藍拍 the loss of the helper p放火lasmid, the integration of genes encodi間你ng the antibody heavy and light chains多錢 becomes perman風知ent in the genome o厭得f host cells.

For further information about this 長年vector system, please r妹街efer to the papers below.

Our piggyBac expression吃什 vector is optimized for high分水-efficiency transfect從工ion and high yield of光拿 monoclonal antibody heavy and light ch農信ains in mammalian cells. By customizing一路 sequences encoding 船愛the variable heavy and light chain司冷s into the vector, custome一從rs may obtain antibodies wit請吃h high-affinity to target a水大ntigens.

Permanent integration of vector DNA你畫: Conventional transfec動我tion results in almost entire他放ly transient delivery of DNA into ho兵雜st cells due to the loss of DN有了A over time. This problem i什舊s especially prominent in ra慢科pidly dividing cells. In contrast,一間 transfection of mammalia喝拿n cells with the piggyBac transposon關頻 plasmid along with the helpe風公r plasmid can deliver ge妹錢nes carried on the transposon permane兵雜ntly into host cells due to the i鄉作ntegration of the transposon into藍學 the host genome.

Technical simplicity: Delivering plasmid vec是暗tors into cells by conventional transf玩來ection is technica笑間lly straight forward, and far報都 easier than viru海黑s-based vectors which requir少文e the packaging of 他微live viruses. Moreover, prese厭來nting coding regions for 輛分both heavy and light ch空中ains on a single plasmid fur明亮ther simplifies the transfection pro信拿cedure.

Reproducibility and scalabi水相lity: The recombinant pro懂東tein is harvested directly from transf影北ected host cells. Therefore, reproducib內能ility of different batch書做es can be easily achieved. M舊和oreover, the amplif山媽ication of host cells allows for高兒 large-scale antibody product舊中ion.