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

ABSTRACT

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).

Abbreviations

CDR: complementarity determining region; FACS: fluorescence-activated cell sorting; k_a: association rate; k_d: dissociation rate; K_D: dissociation constant; scFv: single-chain variable fragment; SPR: surface plasmon resonance

KEYWORDS:

• Affinity maturation
• CDR shuffling
• developability
• liability
• library construction
• off-rate
• scFv

Disclosure statement

AART and CLL received financing from Specifica Inc.

SD, MFE, FF, LPS, LN, EM, TM, ADA, KP, and ARMB are employed by Specifica Inc.

RAB, and HGN are employed by Incyte Research Institute. SS was employed by Incyte Research Institute at the time this work was carried out.

SD, MFE, FF, LN, and ARMB are shareholders at Specifica Inc.

AART, SD, and ARMB are inventors on a patent application describing the technology

The antibodies and methods in this work are part of patent applications US 63/323,632 and US 63/218,919

RAB and HGN are shareholders at Incyte Research Institute

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2022.2115200

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.