An anti-GD2 single chain Fv selected by phage display and fused to Pseudomonas exotoxin A develops specific cytotoxic activity against neuroblastoma derived cell lines

2001 ◽  
Author(s):  
M. Tur ◽  
S. Sasse ◽  
M. Stocker ◽  
K. Djabelkhir ◽  
M. Huhn ◽  
...  
Keyword(s):  
Phage Display ◽  
Cytotoxic Activity ◽  
Cell Lines ◽  
Exotoxin A ◽  
Single Chain ◽  
Pseudomonas Exotoxin ◽  
Pseudomonas Exotoxin A ◽  
Single Chain Fv

scholarly journals Targeting of interleukin-13 receptor on human renal cell carcinoma cells by a recombinant chimeric protein composed of interleukin-13 and a truncated form of Pseudomonas exotoxin A (PE38QQR)

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4333-4339 ◽  
Author(s):  
RK Puri ◽  
P Leland ◽  
NI Obiri ◽  
SR Husain ◽  
RJ Kreitman ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Cell Lines ◽  
Renal Cell ◽  
Chimeric Protein ◽  
Exotoxin A ◽  
Pseudomonas Exotoxin ◽  
Human Renal Cell Carcinoma ◽  
Pseudomonas Exotoxin A ◽  
Interleukin 13

We have previously shown that human renal cell carcinoma (RCC) cells express large numbers of interleukin-13 receptors (IL-13R), a newly described hemopoietic growth factor receptor. To target tumor cells that express IL-13R, we have produced a chimeric protein composed of human IL-13 and a derivative of Pseudomonas exotoxin A, termed PE38QQR. We report here that IL13-PE38QQR is highly cytotoxic to many human RCC cell lines. IL-13R-negative cell lines or cell lines expressing low numbers of IL-13R ( < 300 sites/cell) that include human bone marrow- derived cells were not susceptible to the cytotoxic effect of IL 13- PE38QQR. The sensitivity of RCC cells to IL13-PE38QQR correlated positively with the density of IL-13R. The cytotoxic activity of IL13- PE38QQR was competed by an excess of IL-13 in a protein synthesis inhibition assay and confirmed by a clonogenic assay. Even though IL-13 and IL-4 are homologues and IL-4R and IL-13R have been proposed to share a receptor subunit, IL-4 did not compete for the cytotoxicity mediated by IL13-toxin on RCC. IL13-PE38QQR competes for [125I]-IL-13 binding sites on RCC cells, although at a lower affinity than the wild- type recombinant cytokine. Human T-cell, B-cell, and monocytic cell lines are unresponsive to the cytotoxic action of IL13-PE38QQR. Thus, our results indicate that IL13-PE38QQR is highly cytotoxic to human RCC cells, although it is not cytotoxic to a variety of normal hematopoietic cells. IL13-PE38QQR should be further investigated preclinically for the treatment of human RCCs.

scholarly journals Pseudomonas exotoxin A. Membrane binding, insertion, and traversal.

1986 ◽  
Vol 261 (24) ◽  
pp. 11404-11408 ◽  
Author(s):  
Z T Farahbakhsh ◽  
R L Baldwin ◽  
B J Wisnieski
Keyword(s):  
Membrane Binding ◽  
Exotoxin A ◽  
Pseudomonas Exotoxin ◽  
Pseudomonas Exotoxin A

scholarly journals Characterization of LL25X, a Newly Isolated Anti-SIV Monoclonal Antibody, to Determine Binding Specificity

Elements ◽  
2017 ◽  
Vol 13 (1) ◽  
Author(s):  
Zackary Tajin Park
Keyword(s):  
Phage Display ◽  
Mammalian Cells ◽  
Expression Vector ◽  
Restriction Enzymes ◽  
Phage Display Library ◽  
Single Chain ◽  
Phage Display Technology ◽  
Mammalian Expression ◽  
Mammalian Expression Vector ◽  
Single Chain Fv

A phage display library was previously constructed from an SIV-infected rhesus macaque. Several single chain Fv (scFv), including SU24, SU343 and LL25X, were selected using phage display technology. Sequences corresponding to SU24, SU343 and LL25X were optimized for expression in a mammalian system and commercially synthesized. SU24 and SU343 had previously been cloned into a mammalian expression vector. In this study, we aimed to characterize the specificity of SU24, SU343, and LL25X.. The codon-optimized version of the scFv LL25X gene sequence was cloned into a mammalian expression vector (pCEP4).  LL25X DNA was amplified by PCR, and the PCR product and mammalian expression vector were both digested with KpnI/SapI restriction enzymes. Digested fragments were purified, and the fragments were ligated using T4DNA ligase. E. coli cells were transformed with the ligation reaction. Single colonies were selected on LB agar plates containing the selective antibiotic (ampicillin). Positive colonies were identified after DNA mini-preparation and test-digestion with KpnI and SapI. Sanger sequencing confirmed cloning results and DNA sequence accuracy. Following transfection of mammalian cells (293T), LL25X-Fc cells, and purifying our protein, the binding of LL25X-Fc to the SIV gp140 envelope protein was confirmed via ELISA and Western Blotting.

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