Mechano-bioconjugation Strategy Empowering Fusion Protein Therapeutics with Aggregation Resistance, Prolonged Circulation, and Enhanced Antitumor Efficacy

Abstract

Bioconjugation is a powerful protein modification strategy to improve protein properties. Herein, we report mechano-bioconjugation as a novel approach to empower fusion protein therapeutics and demonstrate its utility by a protein heterocatenane (cat-IFN-ABD) containing interferon-α2b (IFN) mechanically interlocked with a consensus albumin-binding domain (ABD). The conjugate was selectively synthesized in cellulo following a cascade of post-translational events using a pair of heterodimerizing p53dim variants and two orthogonal split-intein reactions. The catenane topology was proven by combined techniques of LC−MS, SDS-PAGE, SEC, and controlled proteolytic digestion. Not only did cat-IFN-ABD retain activities comparable to those of the wild-type IFN and ABD, the conjugate also exhibited enhanced aggregation resistance and prolonged circulation time over the simple linear and cyclic fusions. Consequently, cat-IFN-ABD potently inhibited tumor growth in the mouse xenograft model. Therefore, mechano-bioconjugation by catenation accomplishes function integration with additional benefits, providing an alternative pathway for developing advanced protein therapeutics.

A Single-domain Protein Catenane of Dihydrofolate Reductase

“…Cellular Synthesis of cat-DHFR. The streamlined biosynthesis using two pairs of mutually orthogonal split-intein pairs 25,26 provides a straightforward way to synthesize cat-DHFR using split DHFR itself as the entangled template. To ensure the reconstitution and cyclization of the two polypeptide chains while keeping the correct spatial relationship, the length of the new loop and the sites of ring closure should be carefully chosen (Figure 1a).…”

“…We thus had to resort to indirect ways of characterization, such as proteolytic digestion experiment similar to previous reports. 15,25,26,28,29,32,48 A tobacco etch virus (TEV) protease recognition site was pre-programmed in the first ring of cat-DHFR. Upon cleavage, cat-DHFR was converted to a linear fragment and a cyclic fragment as expected, which was confirmed by the SDS-PAGE analysis and LC-MS spectra (Figure 2e).…”

“…29 Recently, we reported the use of mutually orthogonal split-intein ligation pairs to facilitate a streamlined synthesis of protein heterocatenanes with minimal residuals from ligation. 25,26 We envision that the need for an exogenous entangling motif as the template for catenane synthesis may be further eliminated by introducing artificial entanglement into the original domain via rewiring the loop region. Using the original fold itself as the entangling template, single-domain protein catenanes may be designed and synthesized, even with identical composition to that of the linear wild-type.…”

“…[2][3][4] In recent years, topology has thus been recognized as a unique dimension for protein engineering. [5][6][7][8][9] Thanks to the "assembly-reaction" synergy, the design and synthesis of artificial topological proteins have advanced tremendously, 4,[10][11][12] which offers access to a variety of topological proteins including star proteins, 13 cyclic proteins, [14][15][16][17][18][19][20][21][22] lasso proteins, 23,24 protein catenanes, [25][26][27][28][29][30][31] and protein pretzelanes. 32 The approach often involves the use of genetically encoded entangling motifs and reaction motifs.…”

A Single-domain Protein Catenane of Dihydrofolate Reductase

“…[2][3][4] In recent years, topology has thus been recognized as a unique dimension for protein engineering. [5][6][7][8][9] Thanks to the "assembly-reaction" synergy, the design and synthesis of artificial topological proteins have advanced tremendously, 4,[10][11][12] which offers access to a variety of topological proteins including star proteins, 13 cyclic proteins, [14][15][16][17][18][19][20][21][22] lasso proteins, 23,24 protein catenanes, [25][26][27][28][29][30][31] and protein pretzelanes. 32 The approach often involves the use of genetically encoded entangling motifs and reaction motifs.…”