Affinity-matured anti-glycoprotein NMB recombinant immunotoxins targeting malignant gliomas and melanomas.
Glycoprotein NMB (GPNMB), a transmembrane glycoprotein highly expressed in high-grade gliomas (HGGs), is an attractive target in cancer immunotherapy. We isolated a GPNMB-specific scFv clone, G49, from a human synthetic phage-display library. To obtain mutant single-chain variable-fragment antibodies (scFvs) with improved affinity and immunotoxins with increased activity, we subjected G49 to in vitro affinity maturation by a complementarity-determining-region (CDR) random-mutagenesis technique. Using light-chain CDR3 mutagenesis, cell-based panning by phage display, subsequent heavy-chain CDR1 mutagenesis, and flow-cytometric selection by yeast-surface display, we generated the mutant scFv clone 902V, with an overall 11-fold increase in affinity for GPNMB. Clone 902V was further randomized throughout the whole scFv by error-prone PCR, and one mutant, F6V, was selected by yeast-surface display. F6V scFv, differing from 902V by one amino-acid change in the light-chain CDR2, exhibited an affinity for GPNMB of 0.30 nM. The F6V mutant scFv clone was fused with a truncated form of Pseudomonas exotoxin A to form the immunotoxin F6V-PE38. F6V-PE38 demonstrated significant protein-synthesis-inhibition activity on GPNMB-expressing glioma and malignant melanoma cells (IC(50) = 0.5 ng/ml [8 pM]), a 60-fold improvement over G49 activity, but no cytotoxicity on GPNMB-negative cells. Furthermore, F6V-PE38 exhibited significant antitumor activity against subcutaneous malignant glioma xenografts in two nude-mouse models and a melanoma neoplastic meningitis model in athymic rats. These GPNMB-specific scFv antibodies and immunotoxins hold promise as reagents in targeted therapy for HGGs and other GPNMB-expressing malignancies.
Kuan
Chien-Tsun
CT
Preston Robert Tisch Brain Tumor Center at Duke, Duke University Medical Center, Durham, NC 27710, USA. kuan@duke.edu
Wakiya
Kenji
K
Keir
Stephen T
ST
Li
Jianjun
J
Herndon
James E
JE
Pastan
Ira
I
Bigner
Darell D
DD
D000818:Animals; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D005434:Flow Cytometry; D005910:Glioma / Q000473:pathology*; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D008545:Melanoma / Q000473:pathology*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D051379:Mice; D008819:Mice, Nude; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D020349:Surface Plasmon Resonance
C503042:GPNMB protein, human; D000922:Immunotoxins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins
Curr Opin Immunol. 1995 Apr;7(2):248-54
7546385
Nat Biotechnol. 1999 Jun;17(6):568-72
10385321
J Neurooncol. 2010 May;98(1):1-7
19898744
Blood. 1992 Nov 1;80(9):2315-20
1384801
Clin Cancer Res. 2000 Jul;6(7):2835-43
10914732
J Biol Chem. 2005 Jan 7;280(1):607-17
15491997
J Biol Chem. 2003 May 2;278(18):15951-7
12590137
J Neuropathol Exp Neurol. 1981 May;40(3):201-29
6260907
J Mol Biol. 1996 Jul 19;260(3):359-68
8757799
J Immunol. 1995 Aug 15;155(4):1994-2004
7636250
Clin Cancer Res. 2006 Feb 15;12(4):1373-82
16489096
Cancer Res. 1992 Jun 15;52(12):3402-8
1596900
Int J Cancer. 1995 Jan 3;60(1):73-81
7814155
J Mol Biol. 1995 Dec 1;254(3):392-403
7490758
Genome Res. 2000 Sep;10(9):1393-402
10984457
Proc Natl Acad Sci U S A. 1986 Mar;83(5):1320-4
3006045
Cancer Res. 1983 Jul;43(7):3327-34
6303581
Cell. 1992 Jun 26;69(7):1143-57
1535555
Nat Biotechnol. 2003 Feb;21(2):163-70
12536217
Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3576-80
1565653
Science. 1991 Jun 21;252(5013):1657-62
2047874
J Biol Chem. 1992 Jun 25;267(18):12420-3
1618748
Semin Oncol. 2003 Aug;30(4):545-57
12939723
Clin Cancer Res. 1999 Jun;5(6):1539-49
10389943
Science. 1991 Nov 22;254(5035):1173-7
1683495
Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14815-20
8962138
Cancer Chemother Pharmacol. 1994;34(2):171-4
8194169
J Natl Cancer Inst. 1984 Mar;72(3):673-7
6583450
Methods Mol Biol. 2000;145:219-40
10820725
Proc Natl Acad Sci U S A. 1998 May 26;95(11):6037-42
9600913
Int J Radiat Oncol Biol Phys. 1992;22(1):225-30
1309204
Int J Med Microbiol. 2004 Apr;293(7-8):577-82
15149034
J Biol Chem. 1990 Sep 5;265(25):15198-202
2118522
Clin Cancer Res. 2002 Apr;8(4):995-1002
11948105
J Neurosurg. 1998 Jan;88(1):1-10
9420066
Annu Rev Immunol. 1994;12:433-55
8011287
Cancer Res. 1997 Sep 15;57(18):4130-40
9307304
Clin Cancer Res. 1999 Sep;5(9):2646-52
10499644
EMBO J. 1994 Jul 15;13(14):3245-60
8045255
Cancer Metastasis Rev. 1999;18(4):451-64
10855788
Clin Cancer Res. 2006 Apr 1;12(7 Pt 1):1970-82
16609006
J Mol Biol. 1999 Jan 22;285(3):895-901
9887257
J Clin Oncol. 1998 Jun;16(6):2202-12
9626222
Clin Cancer Res. 2000 Feb;6(2):326-34
10690507