Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries

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.Keywords: single-domain antibody, synthetic antibody, human Vh/Vl, phage display, protein engineering

Antibody Structure and Function: The Basis for Engineering Therapeutics

“…Formulation studies suggest that a Fab T m value can have implications for the long-term physical stability of a corresponding mAb. Thus, the CDR sequence selection can impact the stability of the VH-VL domain, and sequence-stability tradeoffs must be considered during the design of such libraries [290].…”

Evaluation of a noncanonical Cys40‐Cys55 disulfide linkage for stabilization of single‐domain antibodies

“…We first attempted to introduce the Cys40‐Cys55 disulfide linkage into a large panel of sdAbs (9 V H Hs, 12 V H s, and 11 V L s; Table ) to examine the effects of this linkage on sdAb thermostability. Our panel consisted of V H Hs against Clostridium difficile toxin A and human IGF1R, the blood–brain barrier‐transmigrating V H H FC5, and several previously described autonomous human V H and V L domains . Eleven V L s were originally slated for testing, but after introduction of Cys40 and Cys55 residues into their coding sequences, only two showed detectable expression in 1‐L Escherichia coli TG1 cultures.…”

“…The coding sequences of sdAbs were synthesized and cloned into the pSJF2H expression vector by GenScript USA (Piscataway, NJ). C‐terminally c‐Myc‐ and His 6 ‐tagged sdAbs were expressed in the periplasm of E. coli TG1 cells and purified by immobilized metal ion affinity chromatography (IMAC) as previously described . Briefly, competent E. coli TG1 cells were transformed with each expression vector, and 10 mL of 2×YT media containing 100 μg/mL ampicillin were inoculated with a single colony and grown overnight at 37°C with 220 rpm shaking.…”

“…Single‐domain antibodies (sdAbs), the variable domains of camelid and shark heavy chain‐only antibodies, are typically highly thermostable in comparison with other types of antibody fragments. However, additional stability may be of value in some applications (e.g., oral or inhaled administration), especially for synthetic sdAbs, some of which tend to have lower melting temperatures ( T m s) than naturally occurring sdAbs . In the mid‐2000s, two groups independently identified a rare naturally occurring noncanonical disulfide linkage formed between Cys54 and Cys78 of a camelid sdAb, and showed that introducing this linkage improved the thermostability of other sdAbs while sparing antigen binding .…”

Expanding the Boundaries of Biotherapeutics with Bispecific Antibodies

“…By replacing amino acid residues in the framework of the V H domain [ 91 – 93 ], and sometimes also requiring substitutions in the CDRs [ 94 , 95 ], it is possible to obtain monomeric human V H domains. A number of strategies have been developed over time to further increase the stability and solubility of human V H or V L domains [ 96 , 97 ].…”