A chemical conjugate between HER2-targeting antibody fragment and Pseudomonas exotoxin A fragment demonstrates cytotoxic effects on HER2-expressing breast cancer cells

Abstract

Conventionally, immunotoxins have been produced as a single polypeptide from fused genes of an antibody fragment and a toxin. In this study, we adopted a unique approach of chemical conjugation of a toxin protein and an antibody fragment. The two genes were separately expressed in Escherichia coli and purified to high levels of purity. The two purified proteins were conjugated using a chemical linker. The advantage of this approach is its ability to overcome the problem of low recombinant immunotoxin production observed in some immunotoxins. Another advantage is that various combinations of immunotoxins can be prepared with fewer efforts, because the chemical conjugation of components is relatively simpler than the processes involved in cloning, expression, and purification of multiple immunotoxins. As a proof of concept, the scFv of trastuzumab and the PE24 fragment of Pseudomonas exotoxin A were separately produced using E. coli and then chemically crosslinked. The new immunotoxin was tested on four breast cancer cell lines variably expressing HER2. The chemically crosslinked immunotoxin exhibited cytotoxicity in proportion to the expression level of HER2. In conclusion, the present study revealed an alternative method of generating an immunotoxin that could effectively reduce the viability of HER2-expressing breast cancer cells. These results suggest the effectiveness of this method of immunotoxin crosslinking as a suitable alternative for producing immunotoxins. [BMB

Soluble Cytoplasmic Expression and Purification of Immunotoxin HER2(scFv)-PE24B as a Maltose Binding Protein Fusion

“…The effects of individual proteins PE24B and HER2(scFv) were also tested for comparison. The purification of HER2(scFv) and PE24B has been described previously [ 24 ]. HER2(scFv)-PE24B showed strong cytotoxicity against all cell lines ( Figure 7 ).…”

“…One approach is to produce the immuno domain and the toxin domain separately, then chemically conjugate them. As a proof of concept, we previously produced HER2(scFv) and PE24B separately and conjugated them chemically [ 24 ]. The question is then whether the cytotoxic activities of the chemically conjugated immunotoxin are comparable to those of recombinant immunotoxin.…”

“…Also, the codon-optimized PE24B with a furin recognition sequence was synthesized and cloned into the pUC57 vector (Genscript). Next, the furin-PE24B gene [ 24 ] was amplified by PCR to include attB5 and attB2 at both ends. The PCR product was combined with the pDONR221-P5P2 vector to generate entry vector 2.…”

“…The correct sequences of all expression vectors were confirmed by DNA sequencing (Macrogen, Daejeon, Korea). Construction of the HER2(scFv) and PE24B expression vectors was described previously [ 24 ].…”

Critical Issues in the Development of Immunotoxins for Anticancer Therapy

“…An alternative approach to overcome the expression host limitations is to prepare separately an antibody and toxin and then ligate them by means of an enzyme, such as sortase A 155 or split inteins, 156 or via a chemical linker. 157 In this case, IgG or antibody fragments can be produced using conventional mammalian cell cultures, and high-quality toxins can be produced in bacteria. Given that the protein ligation method yields a homogenous product, it may offer an alternative route to a new generation of ITs.…”