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

Abstract

SummaryFour new single-chain Fv antibody fragments (scFvs) specific for the human leucocyte surface antigen CD123 (interleukin-3 receptor a) were generated to achieve preferential targeting of leukaemia stem cells (LSCs) in acute myeloid leukaemia (AML). The scFvs were isolated from a phage display library generated with spleen RNA from mice, immunized with a fusion protein consisting of the extracellular domain of CD123 and the Fc domain of a human immunoglobulin G1. The scFvs displayed CD123-specific binding on tumour cells (binding constants (K D ) 4AE5-101 nmol/l). The scFv with the highest affinity was used to design two cell death-inducing molecules. First, an immunotoxin, a fusion protein with truncated Pseudomonas Exotoxin A, induced potent apoptosis of AML-derived MOLM-13 and SKNO-1 cells at nanomolar concentrations. Second, the fusion to another scFv, specific for the low affinity

Targeted therapy aimed at cancer stem cells: Wilms’ tumor as an example

“…This discovery was followed by several efforts aimed at targeting the hematopoietic cancer stem cell markers, such as CD44 and CD123 in AML [64][65][66][67][68]. Further studies have since been performed by targeting CSCs in several solid tumors such as pancreatic, breast, prostate and colon cancers, melanoma, glioma, hepatocellular carcinoma, and others.…”

A recombinant trispecific single‐chain Fv derivative directed against CD123 and CD33 mediates effective elimination of acute myeloid leukaemia cells by dual targeting

“…To generate the vector pSecTag2HygroC-STREP-His-CD123 · dsCD16 · CD123, the sequence coding for the CD123-specific scFv was amplified by polymerase chain reaction (PCR) from the vector pAK400-CD123scFv and ligated into pSecTag2-HygroC-STREP-His-CD123 · dsCD16 · CD33, using XhoI/ EcoRV restriction sites, and replacing the coding sequence for the C-terminal CD33-specific scFv. For construction of the expression vector pSecTag2HygroC-hCD123ex-DsRed, cDNA coding for the extracellular domain of CD123 was amplified by PCR using the existing vector pSecTag2HygroC-hCD123ex-Fc (Stein et al, 2010) and ligated into the vector pSecTag2HygroC-CD33ex-DsRed (Singer et al, 2010), replacing the extracellular domain of CD33. Correct construction of the final constructs was confirmed by DNA sequence analysis on an Applied Biosystems automated DNA sequencer (ABI Prism 310 Genetic Analyser; Perkin-Elmer, Ueberlingen, Germany).…”

“…For the construction of the sctb [123 · ds16 · 33] expression vector the sequence coding for the CD123-specific scFv was excised from the vector pAK400-CD123scFv (Stein et al, 2010) and cloned as a SfiI-cassette into the existing vector pSecTag2HygroC-STREP-His-CD33 · dsCD16 · CD33 (Singer et al, 2010) replacing the coding sequence for the N-terminal CD33-specific scFv. Thereby the vector pSecTag2HygroC-STREP-His-CD123 · dsCD16 · CD33 was produced.…”

“…MOLM-13 cells were cultured in RPMI 1640 Glutamax-I medium containing 20% FCS, 100 units/ml penicillin and 100 lg/ml streptomycin (Invitrogen). Human 293, 293T and 293 cells stably transfected with CD123 (Stein et al, 2010) were maintained in Dulbecco's modified Eagle medium (Invitrogen) Glutamax-I medium containing 10% FCS, 100 units/ml penicillin and 100 lg/ml streptomycin (Invitrogen). The medium for the CD123 expressing cells further contained 400 lg/ml Geneticin (Invitrogen).…”

“…The fact that both antigens, CD33 and CD123, are expressed on AML-LSCs offers novel prospects for the treatment of AML. So far an immunotoxin (Du et al, 2007), a fusion of IL-3 with a truncated version of diphtheria toxin (Feuring-Buske et al, 2002;Frankel et al, 2008), a neutralizing full-length antibody (7G3; Jin et al, 2009), and a bispecific single-chain Fv (bsscFv; Stein et al, 2010) directed against CD123 have been described, and the monoclonal antibody (mAb) 7G3 has been tested in a phase I clinical study (Roberts et al, 2008; http://clinicaltrials.gov/ ct2/show/NCT00401739?term=CSL360&rank=1). Well-studied antibody-derived agents directed against CD33 include, apart from GO an immunotoxin (Schwemmlein et al, 2006), a fusion protein between a single chain Fv antibody fragment (scFv) and the sTRAIL death receptor ligand (ten Cate et al, 2009), and siRNA-loaded liposomes coated with CD33-directed scFvs (Rothdiener et al, 2010).…”