Computational stabilization of T cell receptors allows pairing with antibodies to form bispecifics

Abstract

Recombinant T cell receptors (TCRs) can be used to redirect naïve T cells to eliminate virally infected or cancerous cells; however, they are plagued by low stability and uneven expression. Here, we use molecular modeling to identify mutations in the TCR constant domains (Cα/Cβ) that increase the unfolding temperature of Cα/Cβ by 20°C, improve the expression of four separate α/β TCRs by 3-to 10-fold, and improve the assembly and stability of TCRs with poor intrinsic stability. The stabilizing mutations rescue the expression of TCRs destabilized through variable domain mutation. The improved stability and folding of the TCRs reduces glycosylation, perhaps through conformational stabilization that restricts access to N-linked glycosylation enzymes. The Cα/Cβ mutations enables antibody-like expression and assembly of well-behaved bispecific molecules that combine an anti-CD3 antibody with the stabilized TCR. These TCR/CD3 bispecifics can redirect T cells to kill tumor cells with target HLA/peptide on their surfaces in vitro.

Designed, highly expressing, thermostable dengue virus 2 envelope protein dimers elicit quaternary epitope antibodies

“…T280P is at the base of the kl loop, and in the SC.10 structure, the backbone at residue 280 is shifted to create a new backbone side chain hydrogen bond between the P280 backbone carbonyl and the T189 side chain hydroxyl in an adjacent loop. In addition to the structural rigidity that prolines can provide, the new hydrogen bond may be helping to stabilize the sE dimer ( 37 , 38 ). As predicted by the Rosetta model, the two mutations in IntFc2, T262R and A259W, lead to canonical pi-cation interactions across the central dimer interface between R262 on one chain and W259 on the other chain ( Fig.…”

Engineering soluble T‐cell receptors for therapy

“…Although the effects on soluble TCR stability were not investigated, structural modelling and mutational screening were used to demonstrate that cell surface expression levels of poorly expressed TCRs could be rescued through the introduction of three residues present in the dominant TCR pairings: L96α (which resulted in a stabilisation at the Vα‐Cα interface) and R9β/Y10β (which resulted in a stabilisation at the Vβ‐Cβ interface). Additionally, a recent computational analysis of crystal structures and in silico modelling have been successful in improving both the yield and stability of soluble TCR heterodimers, even in the absence of the engineered Boulter disulfide [ 57 ]. The entire C domains were computationally screened to find positions where alternative residues were predicted to stabilise the TCR.…”

Soluble Expression of Fc-Fused T Cell Receptors Allows Yielding Novel Bispecific T Cell Engagers

“…Mammalian cells are the most attractive tool for the production of protein drugs [18], and TCRs can be expressed in mammalian cells in their soluble form [19,20]. Specifically, Froning K, et al [19] produced TCR/aCD3 in mammalian cells that provided a novel strategy for TCR/aCD3 generation, but the biological activity of this mammalian cellproduced TCR/aCD3 and the universality of this technology have not been well studied.…”

“…Mammalian cells are the most attractive tool for the production of protein drugs [18], and TCRs can be expressed in mammalian cells in their soluble form [19,20]. Specifically, Froning K, et al [19] produced TCR/aCD3 in mammalian cells that provided a novel strategy for TCR/aCD3 generation, but the biological activity of this mammalian cellproduced TCR/aCD3 and the universality of this technology have not been well studied. Inspired by this research, we achieved the soluble expression of the two components of TCR/aCD3 (TCRs and CD3ε-specific antibody) in mammalian cells, and click chemical reaction was then used to re-assemble the two components to generate TCR/aCD3.…”