Expression Vectors for Human-Mouse Chimeric Antibodies

8/13/2019 Expression Vectors for Human-Mouse Chimeric Antibodies

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BIOT 5031

Report on

Expression Vectors forHuman-Mouse ChimericAntibodiesKundnani Deepali Lalchand118743512/2/2013


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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+#$-VHfor heavy

chain fragments and p+#$-VLfor light chain fragments) derived from pV/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. pV/ contains V01 promotor which can be

weakened by deleting its enhancer se&uence by ph ". ph$2V2$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


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mammalian cells. esired constant and variable region genes were obtained by T5$ 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 *eoin 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 :12 which

resulted into coamplification 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 :12. 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 :12 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


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reducing conditions. The molecular weight found were @1 ka for heavy chain and /: ka 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 pseudoclones. 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.


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References:

6iong H, an +, 6ing D, -!pression Vectors for Humanmouse $himeric Antibodies (/11@),

Journal of Biochemistry and Molecular biology, 38-4, pp. 0:00:E


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Expression Vectors for Human-Mouse Chimeric Antibodies