BIOT 5031

Report on

Expression Vectors for Human-Mouse Chimeric Antibodies

Kundnani Deepali Lalchand 1187435 12/2/2013

Expression vectors for Human-Mouse Chimeric Antibodies

As we know the use of antibodies have vastly increased in the past years and scientists are trying to combat various issues in the production of Antibodies and their applications, one of the main being the inherent immunogenicity in patients that hinders long term administration in immunosuppressive therapy. To reduce these reactions human mouse chimeric antibodies containing constant regions of Human antibody and Variable region from organism(murine) with an immune response to the desired antigen are used and a system able to rapidly produce high levels of recombinant antibody is needed. or e!pressions of engineered "g# in $H%($hinese Hamster %varian) cells two systems have been fre&uently utili'ed, one of which is two vector system with each "g# chain on separate plasmid and other being one vector system with both "g# chains on the same plasmid. "n this study two vector system has been adopted because of ease of insertion of se&uences into the vectors and e!pression of variable regions of antibodies in different immunoglobin isotypes.

(ecombinant )*A technology was used in creating e!pression vectors (p+(,#$-V H for heavy chain fragments and p+(,#$-VL for light chain fragments) derived from p.V/,dhfr due to ease of production of antibodies and being able to co amplify the desired gene with )H ((dihydrofolate reductase) in dhfr,$H% cells. p.V/ contains .V01 promotor which can be weakened by deleting its enhancer se&uence by .ph ". ph$2V,2$.,3#H polyA was inserted into the vectors from pc)*A4 which provides a human $ytomegalovirus 5romotor to the gene inserted in the 2$. region. Heavy chain vector contains weakened )H ( gene and light chain e!pression vector contains *eo( which aids the cotransfection of these two vectors in the

mammalian cells. )esired constant and variable region genes were obtained by (T,5$( and were inserted into the 2$. regions of respective vectors by using 6ho" endonucleases.

Transient antibody e!pressions was employed in $%.7 cells and stable e!pression in dhfr ,$H% cells by using 8ipofectA2"*-T2 and grown on )2-2 9 :1; $. under #0:< selection

selection for *eo( in the vectors. The secretion of the antibodies was detected by -8".A and found to be over : =g>m8.

?ith the use of 2T6 (2ethotre!ate) in dhfr, cells, )H ( gene as a fact amplifies (and co, amplifies the gene with it) @11,/111 folds and finally with a several :11 fold amplification in protein production. .o 2T6 was added in increasing &uantities of 4 ! :1 ,< to :1,A 2 which resulted into co,amplification of product gene having h$2V(human cytomegalovirus) promotor along with )H ( gene having weak .V01 promotor and the production of H$Ab was over 41 =g>m8 for 2T6 4 ! :1,< 2 with a highest production of :11 =g>m8for 2T6 at :1,A2. urther screening was done to select a high e!pressing clone from the pools obtained and after 4 rounds of screening the production was found to be :11 mg>8 at :1,A 2 2T6.

The methods used for detection made use of both light and heavy chain recognition. -8".A was employed to assess the productivity of H$Ab using plates coated with goat antihuman kappa light chain antibody and after the sample was let for the reaction between the former antibody and H$Ab produced, the signala was detected using #oat Antihuman "g#( c specific) ,pero!idase Antibody. ?estern blot was further used to analyse the weight of recombinanat "g chain. irst the molecule was isolated using a column and was detected by .). 5A#- under

reducing conditions. The molecular weight found were @1 k)a for heavy chain and /: k)a for light chain. These two tests confirmed the correct structure and correct assembly of the heavy and light chains in the production of the recombinant "g molecule.

The e!pressions vectors used in this study have been optimi'ed to ma!imi'e the production of the recombinant product by using )H ( amplification system and as the amplification is done in the telomeric region, it ensures hereditary stability and weakening of )H ( production aided the increase in recombinant proteins at low 2T6 levels (h$2V being a strong promotor). These vectors were discovered to to be able to e!press different isotypes of "g as they contain restriction sites facilitating the e!change of $ regions. Bse of double selection markers ()H ( and *eo) came to an advantage as they may balance the respective chain e!pressions and selection with both markers will help deleting pseudo,clones. However lack of knowledge of events leading to homologous recombination may lead to disproportionate amplification. To overcome this aspect, weakened )H ( was included in the heavy chain vector so as to produce more of heavy chains to balance the production of both the chains.

This e!pression system is lesstime consuming and more cost effective in mass production of full length antibody molecules in stable pool and the vectors have proved to have following characteristicsC ease of e!pression of H$Ab by e!changing variable regions, transient and stable e!pression, e!pression of variable regions of various isotypes, balanced e!pression of two chains of antibodies and effective amplification and mass production of recombinant antibodies.

References:
6iong H, (an +, 6ing D, -!pression Vectors for Human,mouse $himeric Antibodies (/11@), Journal of Biochemistry and Molecular biology, 38-4, pp. 0:0,0:E