Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells.
Isolation of antigen-specific monoclonal antibodies (mAbs) and antibody fragments relies on high-throughput screening of immortalized B cells or recombinant antibody libraries. We bypassed the screening step by using high-throughput DNA sequencing and bioinformatic analysis to mine antibody variable region (V)-gene repertoires from bone marrow plasma cells (BMPC) of immunized mice. BMPCs, which cannot be immortalized, produce the vast majority of circulating antibodies. We found that the V-gene repertoire of BMPCs becomes highly polarized after immunization, with the most abundant sequences represented at frequencies between approximately 1% and >10% of the total repertoire. We paired the most abundant variable heavy (V(H)) and variable light (V(L)) genes based on their relative frequencies, reconstructed them using automated gene synthesis, and expressed recombinant antibodies in bacteria or mammalian cells. Antibodies generated in this manner from six mice, each immunized with one of three antigens were overwhelmingly antigen specific (21/27 or 78%). Those generated from a mouse with high serum titers had nanomolar binding affinities.
Reddy
Sai T
ST
Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA.
Ge
Xin
X
Miklos
Aleksandr E
AE
Hughes
Randall A
RA
Kang
Seung Hyun
SH
Hoi
Kam Hon
KH
Chrysostomou
Constantine
C
Hunicke-Smith
Scott P
SP
Iverson
Brent L
BL
Tucker
Philip W
PW
Ellington
Andrew D
AD
Georgiou
George
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D000941:Antigens / Q000276:immunology; D001483:Base Sequence; D022801:Complementarity Determining Regions / Q000276:immunology; D059014:High-Throughput Nucleotide Sequencing; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007114:Immunization; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010950:Plasma Cells / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000941:Antigens; D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region
Nat Med. 2010 Jan;16(1):123-8
20023635
J Immunol Methods. 2007 Apr 10;321(1-2):41-59
17336321
Nucleic Acids Res. 2008 Jul 1;36(Web Server issue):W503-8
18503082
J Immunol Methods. 1997 Feb 14;201(1):35-55
9032408
Proc Natl Acad Sci U S A. 2009 Dec 1;106(48):20216-21
19875695
J Mol Biol. 2008 May 2;378(3):622-33
18384812
Sci Transl Med. 2009 Dec 23;1(12):12ra23
20161664
Annu Rev Immunol. 2005;23:367-86
15771575
J Mol Biol. 2006 Mar 24;357(2):607-20
16445941
Nature. 1997 Jul 10;388(6638):133-4
9217150
J Exp Med. 2006 Oct 30;203(11):2419-24
17030950
Science. 2009 May 8;324(5928):807-10
19423829
Nature. 1975 Aug 7;256(5517):495-7
1172191
Nat Biotechnol. 2006 Jun;24(6):703-7
16699501
Nature. 2008 May 29;453(7195):667-71
18449194
Nat Biotechnol. 2003 Feb;21(2):163-70
12536217
Nat Biotechnol. 2005 Sep;23(9):1105-16
16151404
Nat Rev Immunol. 2006 Oct;6(10):741-50
16977339
Nature. 1991 Aug 15;352(6336):624-8
1907718
Nat Biotechnol. 2007 May;25(5):563-5
17435747
Nat Biotechnol. 2008 Oct;26(10):1135-45
18846087
Proc Natl Acad Sci U S A. 2004 Jun 22;101(25):9193-8
15197275
J Immunol Methods. 1999 Dec 10;231(1-2):119-35
10648932
Biotechnol Bioeng. 2010 Jun 15;106(3):347-57
20198660
Nat Med. 2004 Aug;10(8):871-5
15247913
Nat Med. 2009 Sep;15(9):1088-92
19684583
Nucleic Acids Res. 2003 Nov 15;31(22):e143
14602936
J Immunol Methods. 2003 May 1;276(1-2):185-96
12738372
J Mol Biol. 2006 May 5;358(3):764-72
16563430
Nat Biotechnol. 2009 Dec;27(12):1091-4
20010582
Nat Rev Immunol. 2005 Mar;5(3):230-42
15738953
Protein Sci. 2007 Mar;16(3):379-90
17242375