A single chain immunotoxin, targeting the melanoma-associated chondroitin sulfate proteoglycan, is a potent inducer of apoptosis in cultured human melanoma cells

Abstract

A recombinant immunotoxin was constructed by fusing a single-chain Fv (scFv) antibody fragment, specific for the melanoma-associated chondroitin sulfate proteoglycan (MCSP), to a truncated variant of Pseudomonas Exotoxin A (ETA'), carrying a C-terminal KDEL peptide for improved intracellular transport. The resulting immunotoxin, MCSP-ETA', induced antigen-specific, potent apoptosis in the cultured human melanoma-derived cell lines A2058 and A375M, and treatment with a single dose of the agent eliminated up to 80 % of these cells within 72 h. The dose needed for half-maximum killing (EC50) was approximately 1 nM for both cell lines. MCSP-ETA' also displayed cytotoxic activity against cultured primary melanoma cells from patients with advanced disease, with net cell death reaching up to 70 % within 96 h after treatment with a single dose of 14 nM. MCSP-ETA' induced cell death synergistically with Cyclosporin A (CsA), both in established human melanoma cell lines and cultured primary melanoma cells. The distinctive antigen-restricted induction of apoptosis and the synergy with CsA justify further evaluation of this novel agent with regard to its potential applications for the treatment of melanoma and other MCSP-positive malignancies.

Novel EGFR-specific immunotoxins based on panitumumab and cetuximab show in vitro and ex vivo activity against different tumor entities

“…Functional scFv2112-ETAʹ and scFv1711-ETAʹ were successfully isolated from the periplasmic space of E. coli under osmotic stress. The yield following purification by IMAC was 0.2-0.3 mg/l bacterial culture, which was comparable to or higher than yields reported for other recombinant scFv-ETA proteins (Schmidt et al 1997;Schwenkert et al 2008;Stein et al 2010;Wolf et al 2010). Specific binding to the five selected EGFR + cancer cell lines was confirmed.…”

“…ETA-based ITs are renowned for their pro-apoptotic effects (Kreitman 2006;Stein et al 2010). Many groups use ETA-based ITs, including some that target EGFR (Azemar et al 2000;Chandramohan and Bigner 2013;Nachreiner et al 2008;Schwenkert et al 2008;Stein et al 2010;Wolf et al 2006). We have recently shown that the EGFR-specific IT 425(scFv)-ETAʹ is effective in vitro, ex vivo and in vivo, which is why we chose this construct and its corresponding 425(scFv)-SNAP fusion protein as internal references in our experiments (Bruell et al 2003(Bruell et al , 2005Hussain et al 2011;Kampmeier et al 2009Kampmeier et al , 2010Niesen et al 2014;Pardo et al 2012).…”

Novel conjugates of single‐chain Fv antibody fragments specific for stem cell antigen CD123 mediate potent death of acute myeloid leukaemia cells

“…The expression vector pet27b(+)‐STREP‐His‐CD123‐ETA‐KDEL, coding for the CD123‐specific immunotoxin, was generated following procedures established earlier in our laboratory (Schwenkert et al , 2008). In this construct, domain Ia of Pseudomonas exotoxin A and the endoplasmatic reticulum (ER) retention motif REDLK were replaced by the scFv and the eukaryotic ER‐retention motif KDEL, respectively (Fig 1A).…”

“…The sequence coding for the CD123‐specific scFv was excised from the pAK400‐CD123scFv expression construct and cloned as an SfiI‐cassette into the vector pASK6‐20aa‐linker, a vector containing coding sequences for an N‐terminal STREP‐and hexa‐histidine‐tag and a 20 amino acid linker 4(G 4 S). The sequence coding for the ompA‐leader, the tags, the CD123‐specific scFv, and the linker was digested with XbaI and NotI and ligated into the expression vector pet27b(+)‐STREP‐His‐MCSP‐ETA‐KDEL (Schwenkert et al , 2008), creating the vector pet27b(+)‐STREP‐His‐CD123‐ETA‐KDEL. To generate the expression vector for the bsscFv [123 × ds16], the cDNA coding for the CD123‐specific scFv was excised from the vector pAK400‐CD123scFv and cloned as an SfiI cassette into the vector pSecTag2HygroC‐STREP‐His‐dsCD19 × dsCD16 (Bruenke et al , 2005).…”

A novel approach for targeted elimination of CSPG4‐positive triple‐negative breast cancer cells using a MAP tau‐based fusion protein

“…The αCSPG4(scFv)‐ETA′ expression plasmid (Fig. a) was kindly provided by Prof. Dr. Georg Fey (University Nürnberg‐Erlangen, Germany) . The Pseudomonas aeruginosa exotoxin A (ETA′) was replaced by the coding sequence of the MAP tau protein via NotI/BlpI cloning.…”