Highly enhanced cytotoxicity of a dimeric bispecific diabody, the hEx3 tetrabody.
We previously reported the utility for cancer immunotherapy of a humanized bispecific diabody (hEx3) that targets epidermal growth factor receptor and CD3. Here, we used dynamic and static light scattering measurements to show that the multimer fraction observed in hEx3 in solution is a monodisperse tetramer. The multimerization into tetramers increased the inhibition of cancer cell growth by the hEx3 diabody. Furthermore, 1:2 stoichiometric binding for both antigens was observed in a thermodynamic analysis, indicating that the tetramer has bivalent binding activity for each target, and the structure may be in a circular configuration, as is the case for the single-chain Fv tetrabody. In addition to enhanced cytotoxicity, the functional affinity and stability of the hEx3 tetrabody were superior to those of the hEx3 diabody. The increase in molecular weight is also expected to improve the pharmacokinetics of the bispecific diabody, making the hEx3 tetrabody attractive as a therapeutic antibody fragment for cancer immunotherapy.
Asano
Ryutaro
R
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan.
Ikoma
Keiko
K
Sone
Yukiko
Y
Kawaguchi
Hiroko
H
Taki
Shintaro
S
Hayashi
Hiroki
H
Nakanishi
Takeshi
T
Umetsu
Mitsuo
M
Katayose
Yu
Y
Unno
Michiaki
M
Kudo
Toshio
T
Kumagai
Izumi
I
D018033:Antibodies, Bispecific / Q000235:genetics / Q000627:therapeutic use / Q000633:toxicity*; D000941:Antigens / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D002455:Cell Division / Q000187:drug effects; D002850:Chromatography, Gel; D019281:Dimerization; D066246:ErbB Receptors / Q000276:immunology; D006801:Humans; D015979:Killer Cells, Lymphokine-Activated / Q000187:drug effects / Q000276:immunology; D007700:Kinetics; D008027:Light; D009369:Neoplasms / Q000188:drug therapy / Q000276:immunology / Q000473:pathology; D017510:Protein Folding; D011994:Recombinant Proteins / Q000627:therapeutic use; D012542:Scattering, Radiation; D012984:Software; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
D018033:Antibodies, Bispecific; D000941:Antigens; D017252:CD3 Complex; D011994:Recombinant Proteins; D066246:ErbB Receptors
Cancer Res. 2000 Aug 15;60(16):4336-41
10969772
Protein Eng. 2000 Aug;13(8):565-74
10964986
Protein Eng. 2003 Jan;16(1):47-56
12646692
J Biochem. 2002 Dec;132(6):903-9
12473192
Blood. 1999 Oct 15;94(8):2562-8
10515858
Proc Natl Acad Sci U S A. 1997 Sep 2;94(18):9637-42
9275175
Clin Cancer Res. 2006 Jul 1;12(13):4036-42
16818703
J Biol Chem. 2008 Jan 11;283(2):1156-66
17947238
Proc Natl Acad Sci U S A. 2004 May 18;101(20):7675-80
15136729
J Biol Chem. 2004 Dec 24;279(52):53907-14
15471859
Immunotechnology. 1996 Feb;2(1):21-36
9373325
J Immunol Methods. 1999 Dec 10;231(1-2):177-89
10648937
Anal Biochem. 1989 May 15;179(1):131-7
2757186
J Mol Biol. 2003 Jun 27;330(1):99-111
12818205
Cancer Res. 2005 Apr 1;65(7):2882-9
15805290
J Biol Chem. 2007 Sep 21;282(38):27659-65
17644522
Protein Eng Des Sel. 2004 Apr;17(4):357-66
15126676
J Immunol Methods. 2004 Feb 1;285(1):111-27
14871540
Methods Enzymol. 1986;121:210-28
3724461
FEBS J. 2010 Jan;277(2):477-87
20015073
Adv Drug Deliv Rev. 2003 Feb 10;55(2):171-97
12564976
Trends Biotechnol. 2004 May;22(5):238-44
15109810
J Immunother. 2008 Oct;31(8):752-61
18779744
Protein Sci. 2003 Apr;12(4):734-47
12649432
Biomol Eng. 2001 Oct 15;18(3):95-108
11566601
Protein Eng. 2001 Oct;14(10):815-23
11739902
J Immunol. 2002 Jul 1;169(1):137-44
12077238
Biomol Eng. 2001 Sep;18(2):31-40
11535414
J Immunol Methods. 1999 Dec 10;231(1-2):249-60
10648942
J Mol Biol. 1999 Oct 15;293(1):41-56
10512714
Leukemia. 2004 Mar;18(3):513-20
14981526
Protein Eng Des Sel. 2008 Oct;21(10):597-603
18621782
Cancer Lett. 2002 Mar 8;177(1):29-39
11809528
Cancer Immunol Immunother. 2008 Jan;57(1):43-52
17579857
Cancer Res. 1981 Jun;41(6):2073-8
7237414
J Immunol. 2003 Jul 15;171(2):1070-7
12847281
J Mol Biol. 2004 Oct 29;343(4):985-96
15476815
J Biochem. 2000 Apr;127(4):673-9
10739961
Structure. 1994 Dec 15;2(12):1217-26
7704531