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. 2019 Aug 26;8(3):45.
doi: 10.3390/antib8030045.

Back-to-Germline (B2G) Procedure for Antibody Devolution

Anja Schrade  1 , Alexander Bujotzek  1 , Christian Spick  1 , Martina Wagner  1 , Johannes Goerl  1 , Xenia Wezler  1 , Guy Georges  1 , Roland E Kontermann  2 , Ulrich Brinkmann  3
Affiliations

Back-to-Germline (B2G) Procedure for Antibody Devolution

Anja Schrade et al. Antibodies (Basel). .

Abstract

Bispecific antibodies (bsAbs) with avidity-enhanced specificity can be used to address target cells with increased specificity, ideally binding efficiently to cells that express two cognate antigens, yet not to cells that express only one of those. Building blocks required to generate such bsAbs are binders that recognize the two antigens with high specificity yet with various (including very low monovalent) affinities. The herein described 'back-to-germline' (B2G) procedure defines such derivatives. It converts parent antibodies with high specificity to derivatives that retain specificity but modulate affinity. The approach defines mutations to be introduced into antibody complementarity-determining regions (CDRs) regions without requiring structures of antibody-antigen complexes. Instead, it reverses the B-cell maturation process that increases affinities, with preference on CDR residues with high antigen contact probability. Placing germline residues at those positions generates VH and VL domains and Fv-combinations thereof that retain specificities but are 'de-matured' to different degrees. De-maturation influences on-rates and off-rates, and can produce entities with extremely low affinity for which binding can only be detected in bivalent formats. A comparison with alanine replacement in CDRs (so far, the most frequently applied technology) indicates that B2G may be more reliable/predictable without introduction of stickiness or poly-reactivity. The applicability for generating sets of affinity-modulated monospecific variants is exemplarily shown for antibodies that bind CD138, Her2/neu, and EGFR.

Keywords: affinity; antibody; antigen binding; maturation; protein engineering; structure.

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Conflict of interest statement

A.S., A.B., C.S., M.W., J.G., X.W., G.G., and U.B. are employees of Roche. Roche has an interest in targeted therapies.

Figures

Figure 1
Figure 1
Antibody maturation (A) in B cells and (B) de-maturation by B2G.
Figure 2
Figure 2
On-/Off-rate plots showing monovalent binding of B2GL variants to antigens. Surface Plasmon Resonance was applied to measure differences in binding kinetics of (A) <CD138>, (B) <Her2/neu>, and (C) <EGFR> variants. As a reference, note the dashed line indicating KD values of parental IgGs.
Figure 3
Figure 3
Binding of parent and -B2G variants to the hCD138 antigen. Surface Plasmon Resonance with monomeric CD138 protein as analyte assesses (A) monovalent and (B) bivalent binding. SPR profiles of Hg-Lw and Hg-Lg variants show a weak remaining resonance when analyzed in the bivalent assay.
Figure 4
Figure 4
On-/Off-rate plots showing bivalent binding of B2GL variants to antigens. Surface Plasmon Resonance was applied to measure differences in binding kinetics of (A) <CD138>, (B) <Her2/neu>, and (C) <EGFR> variants. As a reference, note the dashed line indicating KD values of parental IgGs.
Figure 5
Figure 5
ELISA-based poly-reactivity assessment of parental IgG and B2GL variants. Poly-reactivity for indicated variants was assessed using non-specific antigens and specific antigen (human Syndecan-1, R&D-Systems, 2780-TS) as a positive control. The B2G variants of do not elicit increased or additional nonspecific signals when compared to the parental IgG Hw-Lw. PTH = Parathyroid hormone.
Figure 6
Figure 6
Comparison of binding kinetics and SPR profiles of B2GL variants vs. alanine replacement variants. Shown are (A) on-/off-rate plots and (B) SPR profiles based on affinity-mediated and avidity-mediated binding kinetics.
See this image and copyright information in PMC

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