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;

Affinity maturation is often a necessary step for the development of potent therapeutic molecules. Many different diversification strategies have been used for antibody affinity maturation, including error-prone PCR, chain shuffling, and targeted complementary-determining region (CDR) mutation. Although effective, they can negatively impact antibody stability or alter epitope recognition. Moreover, they do not address the presence of sequence liabilities, such as glycosylation, asparagine deamidation, aspartate isomerization, aggregation motifs, and others. Such liabilities, if present or inadvertently introduced, can potentially create the need for new rounds of engineering, or even abolish the value of the antibody as a therapeutic molecule. Here, we demonstrate a sequence agnostic method to improve antibody affinities, while simultaneously eliminating sequence liabilities and retaining the same epitope binding as the parental antibody. This was carried out using a defined collection of natural CDRs as the diversity source, purged of sequence liabilities, and matched to the antibody germline gene family. These CDRs were inserted into the lead molecule in one or two sites at a time (LCDR1-2, LCDR3, HCDR1-2) while retaining the HCDR3 and framework regions unchanged. The final analysis of 92 clones revealed 81 unique variants, with each of 24 tested variants having the same epitope specificity as the parental molecule. Of these, the average affinity improved by over 100 times (to 96 pM), and the best affinity improvement was 231-fold (to 32 pM).

Here, we describe a new VHH library for therapeutic discovery which optimizes humanness, stability, affinity, diversity, developability, and facile purification using protein A in the absence of an Fc domain. Four therapeutic humanized VHHs were used as scaffolds, into which we inserted human HCDR1s, HCDR2s and HCDR3s. The HCDR1 and HCDR2 sequences were derived from human VH3 family next-generation sequencing datasets informatically purged of sequence liabilities, synthesized as array-based oligonucleotides, cloned as single CDR libraries into each of the parental scaffolds and filtered for protein A binding by yeast display to ensure correct folding and display. After filtering, the CDR1 and CDR2 libraries were combined with amplified human HCDR3 from human CD19⁺ IgM⁺ B cells. This library was further improved by eliminating long consecutive stretches of tyrosines in CDR3 and enriching for CDR1-2 diversity with elevated tolerance to high temperatures. A broad diversity of high affinity (100 pM-10 nM), developable binders was directly isolated, with developability evaluated for most assays using the isolated VHHs, rather than fused to Fc, which is customary. This represents the first systematic developability assessment of isolated VHH molecules.

Antibody complementarity determining region diversity has been considered to be the most important metric for the production of a functional antibody library. Generally, the greater the antibody library diversity, the greater the probability of selecting a diverse array of high affinity leads. According to this paradigm, the primary means of elevating library diversity has been by increasing the number of donors. In the present study we explored the possibility of creating an in vitro antibody library from a single healthy individual, showing that the number of lymphocytes, rather than the number of donors, is the key criterion in the production of a diverse and functional antibody library. We describe the construction of a high-quality phage display library comprising 5 × 10⁹human antibodies by applying an efficient B cell extraction protocol from a single donor and a targeted V-gene amplification strategy favoring specific antibody families for their improved developability profiles. Each step of the library generation process was followed and validated by next generation sequencing to monitor the library quality and diversity. The functionality of the library was tested using several therapeutically relevant targets for which a vast number of different antibodies with desired biophysical properties were obtained.

Accurate and efficient affinity measurement techniques are essential for the biophysical characterization of therapeutic monoclonal antibodies, one of the fastest growing drug classes. Surface plasmon resonance (SPR) is widely used for determining antibody affinity, but does not perform well with extremely high affinity (low picomolar to femtomolar range) molecules. In this study, we compare the SPR-based Carterra LSA and the kinetic exclusion assay (KinExA) for measuring the affinities of 48 antibodies generated against the SARS-CoV-2 receptor-binding domain. These data reveal that high-affinity antibodies can be generated straight from selections using high-quality in vitro library platforms with 54% correspondence between affinities measured using LSA and KinExA. Generally, where there was a 2-fold or greater difference between LSA and KinExA, KinExA reported that affinities were tighter. We highlight the differences between LSA and KinExA, identifying the benefits and pitfalls of each in terms of dynamic range and throughput. Furthermore, we demonstrate for the first time that single-point screening with KinExA can significantly improve throughput while maintaining a strong correlation with full binding curve equilibrium measurements, enabling the accurate rank-ordering of clones with exceptionally tight binding properties.