Drug-like antibodies with high affinity, diversity and developability directly from next-generation antibody libraries

Abstract

Therapeutic antibodies must have “drug-like” properties. These include high affinity and specificity for the intended target, biological activity, and additional characteristics now known as “developability properties”: long-term stability and resistance to aggregation when in solution, thermodynamic stability to prevent unfolding, high expression yields to facilitate manufacturing, low self-interaction, among others. Sequence-based liabilities may affect one or more of these characteristics. Improving the stability and developability of a lead antibody is typically achieved by modifying its sequence, a time-consuming process that often results in reduced affinity. Here we present a new antibody library format that yields high-affinity binders with drug-like developability properties directly from initial selections, reducing the need for further engineering or affinity maturation. The innovative semi-synthetic design involves grafting natural complementarity-determining regions (CDRs) from human antibodies into scaffolds based on well-behaved clinical antibodies. HCDR3s were amplified directly from B cells, while the remaining CDRs, from which all sequence liabilities had been purged, were replicated from a large next-generation sequencing dataset. By combining two in vitro display techniques, phage and yeast display, we were able to routinely recover a large number of unique, highly developable antibodies against clinically relevant targets with affinities in the subnanomolar to low nanomolar range. We anticipate that the designs and approaches presented here will accelerate the drug development process by reducing the failure rate of leads due to poor antibody affinities and developability. Abbreviations: AC-SINS: affinity-capture self-interaction nanoparticle spectroscopy; CDR: complementarity-determining region; CQA: critical quality attribute; ELISA: enzyme-linked immunoassay; FACS: fluorescence-activated cell sorting; Fv: fragment variable; GM-CSF: granulocyte-macrophage colony-stimulating factor; HCDR3: heavy chain CDR3; IFN2a: interferon α-2; IL6: interleukin-6; MACS: magnetic-activated cell sorting; NGS: next generation sequencing; PCR: polymerase chain reaction; SEC: size-exclusion chromatography; SPR: surface plasmon resonance; TGFβ-R2: transforming growth factor β-R2; VH: variable heavy; VK: variable kappa; VL: variable light; Vl: variable lambda;

Automated optimisation of solubility and conformational stability of antibodies and proteins

“…We noted that the two Nb.b201 variants with worsened CIC retention times, but increased AMS precipitation midpoint, were both mutations to arginine in the CDRs, which are well-known to contribute to poor specificity 60,[69][70][71] . This type of liability is easy to spot computationally, as most software for molecular viewing can highlight patches of positive charges.…”

“…Notably, these two variants with higher CIC RTs than that of the WT are both mutations to arginine. Arginine residues in antibody CDRs have been associated with reduced specificity by several investigations 60,[69][70][71] . Therefore, it is perhaps not surprising that these arginine variants perform worse in cross-interaction assays than they do in relative solubility assays such as the AMS precipitation.…”

Direct selection of functional fluorescent-protein antibody fusions by yeast display

“…These reprise the antigen-binding properties of full-length antibodies in a smaller, single gene construct [2][3][4] and are usually selected from immune or naïve libraries using phage display [5][6][7][8][9][10][11][12]. The properties of selected antibodies can be significantly enhanced by combining phage and yeast display, which provides greater control over selection parameters and improves the affinity and diversity of selected scFvs [13][14][15][16][17].…”

“…Independently of the library used [16,35], we routinely combine phage with yeast display, yielding one to thousands of different binders [14][15][16][17][36][37][38]. scFv affinity and the degree of scFv diversity depends upon the library used.…”

Simultaneous affinity maturation and developability enhancement using natural liability-free CDRs

“…This relies on a new diversification strategy exploiting defined collections of natural CDRs purged of sequence liabilities, which we have previously used to generate highly effective naïve antibody libraries yielding subnanomolar antibodies. 23 , 24 In this approach, diversity is targeted based on the germline gene family of the antibody to be affinity matured, and not to the specific antibody sequence, i.e., two antibodies belonging to the same germline gene family will use the same source of CDR diversity, however different the individual CDR sequences of the parental molecules are.…”