Redesigned anti-human placental alkaline phosphatase single-chain Fv: soluble expression, characterization and in vivo tumour targeting

Abstract

Although much progress has been made in the production of recombinant antibodies and their fusions, there are still problems with solubility and folding. Useful antibodies produced from cloned hybridomas do not always result in scFvs behaving favourably. We report here further work on an scFv (H17E2) against the oncofetal antigen human placental alkaline phosphatase. The overall expression was greatly improved and the H17E2 scFv was redesigned by manipulation of the interdomain linker, resulting in much higher expression levels of the soluble scFv in its active conformation at 0.2-0.5 mg/l of bacterial culture. We show that the new soluble version of this scFv has similar characteristics to the refolded version in terms of antigen and tumour cell binding, stability and in vivo pharmacokinetics. The final tumour uptake behaviour of these scFvs is superior to that of the parental whole antibody with respect to tumour:organ ratios, but still requires further development before considering it as a suitable molecule for clinical use in ovarian or testicular cancer.

Design, characterization and anti-tumour cytotoxicity of a panel of recombinant, mammalian ribonuclease-based immunotoxins

“…Polyclonal antibodies against the native and denatured form of bovine seminal ribonuclease for detection in ELISAs and western blotting were prepared as described in Deonarain and Epenetos (1995). The H 17E2 scFv plasmid constructs and proteins have been described previously (Deonarain et al, 1997). Pure native seminal ribonuclease was a gift from Professor K Scheit (Max-Plank Institute, Heidelberg, Germany).…”

“…A XhoIlEcoRI cassette containing the gene for the processed form of the enzyme was obtained by polymerase chain reaction (PCR) amplification with the following conditions: 30 cycles of 94°C (1 min), 72°C (1 min), 65°C (1 min) preceded by a 'hot start' and followed by 3 min at 72°C from this plasmid using the primers BSRNXHO 5'-GTGCGGGTCCTCGAGATCAAGCG-CAAGGAATCTGCAGCTGCC-3' and BSRCECO 5'-GAAGC-GGTGGAATTCACTGTTCTGGCCTCAGGT-3'. The gene for the BSRNase was fused to the C-terminus of the kappa light-chain variable region of the insolubly expressed H17E2 scFv gene in plasmid pSPH17E2 (Deonarain et al, 1997) to form the plasmid pSPH17-BSR. An N-terminal linker/spacer (Ala-Pro-Ala-Ala-SerPro-Ala-Asp-Ala), to separate the scFv from the BSRNase, was incorporated by amplifying the BSRNase gene with the N-terminal primer BSRLINK 5'-GTGCGGGTCCTCGAGAT-CAAGGCACCTGCTGCCTCCCCGGCAGACGCTAA GGATC-TGCAGCTGCC-3' and the C-terminal primer BSRCECO.…”

“…These alterations were JrC-I-----) -Bovine seminal monomerr Figure 1 (A) Construction of the various scFv H17-bovine seminal ribonuclease fusion genes. The parental scFv-expressing plasmid is pSPH17 (Deonarain et al, 1997) and all fusion constructs are derived from this. Each BSRNase cassette was obtained by PCR amplification of the gene from the plasmid pBSV5 (Preuss et al, 1990) using the oligonucleotides described in the text.…”

“…All of the constructs were sequenced by dideoxy sequencing to ensure that no spurious alterations had arisen. The construction of these clones is illustrated in Figure IA Expression of recombinant protein E. coli BL21(XDE3) cells (Studier and Moffat, 1986) were transformed by the plasmids for the scFv (Deonarain et al, 1997) and fusion proteins. Single colonies were picked, re-streaked and grown overnight at 37°C in L-Broth supplemented with 100 gg ml' ampicillin.…”

“…The antibody used is the well-studied H17E2 Epenetos et al, 1986). The single-chain Fv has been constructed (Savage et al, 1993) and characterized in detail in vitro and in vivo (Deonarain et al, 1997). We have previously shown that this recombinant antibody localizes to tumours more rapidly than the IgG form in a mouse xenograft model of human cancer (Deonarain et al, 1997); it can be used to target interleukin 2 to tumour cells (Boleti et al, 1986) and diagnostically in immunohistochemical staining for PLAP-positive tumours (Spooner et al, 1994).…”