Abstract
Human autonomous VH/VL single-domain antibodies (sdAbs) are attractive therapeutic molecules, but often suffer from suboptimal stability, solubility and affinity for cognate antigens. Most commonly, human sdAbs have been isolated from in vitro display libraries constructed via synthetic randomization of rearranged VH/VL domains. Here, we describe the design and characterization of three novel human VH/VL sdAb libraries through a process of: (i) exhaustive biophysical characterization of 20 potential VH/VL sdAb library scaffolds, including assessment of expression yield, aggregation resistance, thermostability and tolerance to complementarity-determining region (CDR) substitutions; (ii) in vitro randomization of the CDRs of three VH/VL sdAb scaffolds, with tailored amino acid representation designed to promote solubility and expressibility; and (iii) systematic benchmarking of the three VH/VL libraries by panning against five model antigens. We isolated ≥1 antigen-specific human sdAb against four of five targets (13 VHs and 7 VLs in total); these were predominantly monomeric, had antigen-binding affinities ranging from 5 nM to 12 µM (average: 2–3 µM), but had highly variable expression yields (range: 0.1–19 mg/L). Despite our efforts to identify the most stable VH/VL scaffolds, selection of antigen-specific binders from these libraries was unpredictable (overall success rate for all library-target screens: ~53%) with a high attrition rate of sdAbs exhibiting false positive binding by ELISA. By analyzing VH/VL sdAb library sequence composition following selection for monomeric antibody expression (binding to protein A/L followed by amplification in bacterial cells), we found that some VH/VL sdAbs had marked growth advantages over others, and that the amino acid composition of the CDRs of this set of sdAbs was dramatically restricted (bias toward Asp and His and away from aromatic and hydrophobic residues). Thus, CDR sequence clearly dramatically impacts the stability of human autonomous VH/VL immunoglobulin domain folds, and sequence-stability tradeoffs must be taken into account during the design of such libraries.
Highlights
The concept of an autonomous single immunoglobulin variable domain as the smallest representation of an antigen-binding-competent antibody was first described by Ward et al in the mouse [1]
To better understand potential constraints on the complementarity-determining region (CDR) sequences of monomeric and stable human VH/VL sdAbs, we examined the effects of stability selection on randomized sequence diversity of two VH/VL sdAb libraries (VL383SS and VHB82SS)
Using an arbitrary frequency cutoff (0.00006% for VL383SS and 0.00004% for VHB82SS; sdAbs at frequencies greater than these cutoffs were not present in any other dataset) to identify unique sdAb sequences enriched by stability selection, we found that different sdAb clones were selected in the two replicate pannings, both sets of sdAbs were heavily biased toward the parental VH/VL sdAb scaffold’s CDR3 length (9 residues for the VL383SS library and 10 residues for the VHB82SS library, which is the shortest CDR3 length possible in the library design and the nearest to the VHB82SS scaffold’s CDR3 length of 6 residues; Figure S4 in Supplementary Material)
Summary
The concept of an autonomous single immunoglobulin variable domain (single-domain antibodies or sdAbs) as the smallest representation of an antigen-binding-competent antibody was first described by Ward et al in the mouse [1]. There are many examples of fully human antibodies (primarily VHs) isolated from such libraries against a variety of targets, including α-amylase [12], β-galactosidase [13, 14], Candida albicans MP65 and SAP-2 [15], carbonic anhydrase [12], CD154 [16], CD28 [17], CD40 [18, 19], CD40L [20], Clostridium difficile toxin B [21], EGFR [22], glypican-2 [23], glypican-3 [24], human serum albumin (HSA) [25,26,27], lysozyme [28,29,30], maltose-binding protein [31], MDM4 [32], mesothelin [33], TNF-α [34], TNFR1 [35], and VEGF [22]. The stochastic process of selecting binders from human VH/VL sdAb libraries is likely a consequence of fundamental tradeoffs between CDR sequence and human VH/VL sdAb stability and aggregation resistance
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