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.

Keywords: Antibody; antibody discovery; antibody therapeutics; developability; drug discovery; phage display; yeast display.

Figure 1.

Schematic representation of library design and assembly. LCDR1-3 and HCDR1-2 are from sequences replicated from the naïve repertoire with the liabilities removed. They also undergo a filtering step using yeast display. HCDR3 is recovered from 10 healthy donors. The pieces are assembled to form the VH and VL and subsequently assembled as a scFv. The CDRs are all embedded in a scaffold derived from a developable therapeutic antibody

Figure 2.

(a and b) Proportion of chains showing a liability in at least one of the CDRs in the human naïve repertoire (CDR1-2 for heavy chain and CDR1-3 for light chain), as assessed from a phage display library created from 40 healthy human donors. (c, d, and e) Segmentation of liabilities by V family and CDR

Figure 3.

Analysis of the clinical antibody heavy (a) and light (b) V germline genes in comparison with the human naïve repertoire and the frequency of developable clinical antibodies (defined as having no more than one red flag). Chosen V domains are highlighted in red. (c) V germline genes of the clinical antibodies selected to be used as scaffolds in the new phage display library

Figure 4.

Schematic representation of how each single-CDR library was built and sorted. (a) Design of the six yeast display vectors created for each of four scaffolds: one vector has the original clinical antibody reformatted as a single chain and the other five have one CDR replaced by Type II restriction enzyme sites for scarless insertion of CDR libraries and filtering, represented by a white gap. (b) Workflow for creation and filtering of each single-CDR library: liability-free, replicated natural CDRs are inserted into the open yeast display vector by homologous recombination and filtered for high expression using FACS or MACS. (c) Flow cytometry analysis of the four chosen therapeutic antibodies displayed as scFv on the yeast surface. (d) Flow cytometry analysis of the five single-CDR libraries corresponding to abrilumab (Lib1), comparing the parent therapeutic scaffold with the non-enriched libraries and the FACS/MACS libraries enriched for higher levels of display

Figure 5.

Comparison of CDR distribution and dominance between the natural naïve library, the non-enriched library and enriched single-CDR libraries. Data are shown for the libraries built using the abrilumab scaffold (Lib1, IGHV1-24, IGKV1-12)

Figure 6.

(a) Schematic representation of HCDR3 diversity rescue from 10 human donors. First, peripheral blood is submitted to leukapheresis; then recovered cells are purified by magnetic activated cell sorting (MACS) recognizing the CD19 marker for B-cells. The RNA is extracted, reverse transcribed with an IgM CH1 specific primer and the HCDR3 is amplified by PCR with primers specific to different germline families used in the library. (b) Saturation analysis of the HCDR3 deep sequencing results. (c) HCDR3 amino acid length distribution in library

Figure 7.

(a) Schematic representation of the selection process combining two rounds of phage display and yeast display. (b) Flow cytometry analysis of the final selected populations against each antigen at varying concentrations. Display is detected with anti-SV5 antibody labeled with PE and binding is detected with streptavidin labeled with alexa-633. (c) Levenshtein distance of merged CDRs between clones selected against GM-CSF. (d) Levenshtein distance of HCDR3 between clones selected against GM-CSF. (e) Surface plasmon resonance affinity plot for test clones from GM-CSF, IFN-2⍺, IL6 and TGFβ-R2. The diagonal lines (isoaffinity) represent the affinity (K_D) of the antibodies, x-axis show dissociation constant (k_d) and y-axis shows association constant (k_a)

Figure 8.

Developability profile of selected clones from the library. Measurement(s) of the selected clones (named A to D) are compared to the parental clinical scaffold (named P) and the therapeutic limit. The therapeutic limit is defined as two times the standard deviation of all measurements of the parental mAbs (Lib1-P, Lib2-P, Lib3-P, Lib4-P), represented by the horizontal line extending across each plot in the direction of better (blue) or worse (orange) developability. (a) AC-SINS, AC-SINS at 300 mM salt and polyspecificity results are derived from independent experiments (N = 3 for AC-SINS at 300 mM salt and AC-SINS; N = 2 for polyspecificity). The middle line, box limits and whiskers of the boxplot represent the mean, one standard deviation and two standard deviations of the repeat measurements, respectively. (b) HEKt, Tm, Freeze-Thaw and AS represent the final measured or calculated values from single experiments and depicted by thick horizontal line. Colors indicate whether the test mAb measurement(s) is better (dark blue box; dark blue line), worse (light orange box; light orange line) or within two standard deviations (light blue box; black line) of the therapeutic limit. The parental mAbs (red box; red line) are distinctly colored to provide reference for the test mAbs