Mammalian cell expression of dimeric small immune proteins (SIP)

Abstract

We have designed and expressed bivalent small immune proteins (SIP) based on scFv fragments connected through a short linker of four amino acids to the CH3 domain of the human immunoglobulin gamma 1 H-chain. Three different versions have been designed and expressed in mammalian cells. In one construct a cysteine residue was included in the last amino acid of the flexible 15-amino acid long linker connecting the V(L) and V(H) domains, thus creating a disulphide bond stabilized molecule. A version with a shorter (five amino acids) V(L)/V(H) linker was also produced and shown to be efficiently assembled and secreted. All three SIPs form dimers retaining their antigenic specificity in Western blotting and having a comparable functional affinity (avidity) as determined by ELISA.

Controlling complement resistance in cancer by using human monoclonal antibodies that neutralize complement-regulatory proteins CD55 and CD59

“…The coding sequences of the scFv TS-55 and TS-59 were cloned into a modified version of the PUT-SEC vector [19] containing the hinge-CH2-CH3 Fc domains of human IgG1. After subcloning into pcDNA3, the resulting constructs were transfected into HEK293T cell line and stable clones expressing soluble dimeric MB were obtained for both constructs.…”

“…The PUT-SEC [19] plasmid vector was modified as follows: the BspEI site was exchanged with BssHII by inverse PCR using the primers PUT-ApaLI (ATCCGAGTGCACACCTGTGGAGA-GAAAGGCAAAG) and PUT-BssHII (TCCTCAGCGCGCGGCT-CTGGTGGCAGACCGAAGG). The human IgG1 CH2 human gene was amplified by PCR with the primers HuGCH2-s (AGGCGGCGCGCGACAAAACTCACACATGCCCACCGTGCC-CA) and HuGCH3-a (ACGTCGATCGCCTGCTGAATTCTTAAG-TACTATCCAGGCCCAGCAGTGGGTTTGGGATTGGTTTGCCA-CTAGTTTTACCCGGGGACAGGGAGAG), which introduces the SV5 tag sequence for mAb SV5 recognition [34] and SpeI, EcoRI and PvuI sites at the 3 0 end.…”

Anti-idiotypic antibodies induced by genetic immunisation are directed exclusively against combined VL/VH determinants

“…In addition, this scFvhuCH3 design (called SIP) 17 leads to efficient expression and secretion of the encoded protein.…”

“…This is the case of our immunising soluble scFv molecules, which are efficiently secreted in tissue culture supernatants of transfected cells maintaining the antigen-binding capacity. 17 It was then interesting to examine whether the observed reactivity was peculiar to genetic vaccination. To analyse with the same system the reactivity of sera induced by the classical protocol of protein immunisation, we immunised mice with the scFv BCL1 -huCH3 protein purified from SP2/0 cells transfected with pBCL1 ( Figure 1a).…”

Characterisation and radioimmunotherapy of L19-SIP, an anti-angiogenic antibody against the extra domain B of fibronectin, in colorectal tumour models

“…These include a complete human IgG, dimeric scFv ((scFv) 2 ) and a small immunoprotein (SIP). The L19-SIP is an 80 kDa fragment composed of two scFv fused to the human A s2 -CH4 domain to provide a covalent stabilisation of the dimer (Li et al, 1997;Borsi et al, 2002).…”