Bispecific binding constructs with selectively cleavable linkers
A bispecific, construct-based technology for protein engineering applications
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example 1
[0300] Generation and expression of bispecific HHLL-binding constructs with protease cleavage sites
[0301] Different forms of open reading frames ( Figure 1-Figure 3 ) was ordered by gene synthesis and subcloned into a mammalian expression vector containing an IgG-derived signal peptide for secreted expression into cell culture supernatants. Sequence-verified plasmid clones were transiently transfected into 293HEK cells, or stably transfected into CHO cells, and cell culture supernatants were harvested 3 days after transient expression or 6 days after stable transfection. Cell culture supernatants were stored at -80 °C until protein purification.
[0302] Figure 1-Figure 3 The single-chain pro-bispecific binding construct format in the absence of MMP2 / 9 (ie, no protease cleavage) and the fragment produced in the presence of MMP2 / 9 are shown. Form A contains the following domains from N-terminus to C-terminus: CD3 e (a.a.1-6 or a.a.1-27) peptide-L0-anti-CD3 VH-L1-anti-MS...
example 2
[0304] spectrum analysis
[0305]Protein purification by protein A affinity chromatography followed by size exclusion chromatography ( Figure 3-Figure 12 ). Signal (blue) peaks were pooled according to OD280nm and MW analyzed by SDS-PAGE. Protein monomer peaks were formed in 10 mM citrate, 75 mM lysine, 4% trehalose to be aliquoted for storage at -80°C. Figure 4-Figure 13 The results are depicted separately for the following constructs: N4J, N7A, V1E, B1U, Z9P, O7H, W9A, B2P, T7U, L2G, with the indicated expression of the various constructs.
example 3
[0307] Gel / blot size analysis to determine if the cleavage site is functional in vitro
[0308] To determine the in vitro cleavage of the bispecific binding constructs, the purified bispecific binding constructs were incubated with recombinant MMP-9 at a molar ratio of 1:1 at 37° C. for 18 h (or PBS as a control). Then, the samples were denatured at 95°C for 5 min and applied to non-reducing SDS-PAGE ( Figure 13 - Figure 1 4). It is shown that the bispecific binding construct is in the absence of MMP9 (-MMP9), in its pro- (i.e. no protease cleavage) conformation and in the presence of MMP9 (+MMP9), in its active form (i.e. Cleavage by protease) expected MW. Samples incubated with MMP9 were not subsequently purified, indicated by an additional MMP9-specific band (67, 82 kDa). V1E(-MMP9) showed a lower MW than expected, and there was no difference in its activated (+MMP9) conformation. The result is in Figure 14A and Figure 14B depicted in .
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