US 12,264,193 B2
Method for producing a recombinant allotypespecific rabbit monoclonal antibody
Fernando Jose Rebelo Do Couto, Pleasanton, CA (US)
Assigned to ABCAM LIMITED, Cambridge (GB)
Appl. No. 15/533,983
Filed by ABCAM LIMITED, Cambridge (GB)
PCT Filed Dec. 10, 2015, PCT No. PCT/GB2015/053787
§ 371(c)(1), (2) Date Jun. 7, 2017,
PCT Pub. No. WO2016/092315, PCT Pub. Date Jun. 16, 2016.
Claims priority of application No. 1422075 (GB), filed on Dec. 11, 2014.
Prior Publication US 2018/0009875 A1, Jan. 11, 2018
Int. Cl. C12N 15/11 (2006.01); C07K 16/00 (2006.01); C12N 5/20 (2006.01); C12N 15/10 (2006.01)
CPC C07K 16/00 (2013.01) [C07K 2317/10 (2013.01); C07K 2317/20 (2013.01); C07K 2317/51 (2013.01); C07K 2317/515 (2013.01)] 11 Claims
 
1. A method for producing a recombinant antibody having a b4 allotype, the method comprising the steps of:
a) providing a rabbit antibody-producing B-lymphocyte having a b4 allotype;
b) fusing the B-lymphocyte of step a) with a rabbit fusion partner having a b5 allotype to produce a hybridoma cell capable of producing an antibody;
c) making total cDNA from the hybridoma;
d) amplifying the heavy chain and the b4 light chain from the cDNA of step c) using:
(i) a light chain primer pair that comprises a forward primer having at the 3′ end the nucleotide sequence of SEQ ID NO: 1 and a reverse primer of 15-60 nucleotides in length having at the 3′ end the nucleotide sequence of SEQ ID NO: 12, 21 or 22, wherein the 3′ terminal nucleotide of the reverse primer is the 3′ terminal nucleotide of SEQ ID NO: 12, 21 or 22, and
(ii) a heavy chain primer pair that amplifies the coding sequence for the variable domain of the heavy chain;
e) cloning the amplified heavy chain and the light chain obtained in step d) into one or more plasmids downstream of an expression control sequence;
f) transfecting the plasmids into a mammalian host cell capable of expressing an antibody;
g) culturing the mammalian host cells under conditions suitable for antibody production;
h) isolating the antibodies produced in step g);
i) purifying the antibodies obtained in step h).