Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle

Abstract

Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anticancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.

A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv)

“…This observation is complementary to previous studies that employed multiple framework-based mutations on the basis of structural analyses and did not attempt to achieve improvements via CDR mutagenesis (11,12). This study shows that the low stability of scFvs built on theoretically stable v-domain scaffolds (14), whether fully human or humanized, can be driven by as little as a single germline side chain clashing with the V H -CDR3. As V H -CDR3 structures are still problematic to model, particularly for human antibodies, which are poorly represented in the PDB database (36), such clashes in CDRs can be rapidly identified and ameliorated by CDR mutagenesis, and subsequently rationalized by structural biology.…”

“…It has been shown that even molecules built on v-domain frameworks of high theoretical stability can suffer from poor development characteristics (14). In the study presented here, we have delineated the factors that led to improved target binding affinity versus scFv stability.…”

Exploration and Modulation of Antibody Fragment Biophysical Properties by Replacing the Framework Region Sequences

“…Thus, creating better complementarity between the AAs of the same domain or at the interface between the variable domains may improve stability of scFv (16). Moreover, the scFv S1D4 exhibited a very high thermal stability [19,41,42] even without additional disulfide bridge [43], which could greatly increase long-term storage stability compared to other scFvs [30]. In terms of chemical stability, clusters II and III substitutions were deleterious.…”

ALTHEA Gold Libraries™: antibody libraries for therapeutic antibody discovery

“…Interestingly, Y was shown to be a destabilizing residue in certain positions of the antigen-binding site. 43 Therefore, a phage display library for therapeutic antibody discovery needs to balance a high Y content, to generate versatile antigen-binding sites capable of producing highly specific and high affinity antibodies, but with not too high Y content, so that the stability and solubility of the potential therapeutic antibodies are not compromised. In our case, the filtration process seemed to have corrected the excess of Y and hydrophobic residues designed based on the available information on the structures, human antibody sequences and the repertoire of human germline genes.…”