Chemical Diversification of Simple Synthetic Antibodies

Abstract

Antibodies possess properties that make them valuable as therapeutics, diagnostics, and basic research tools. However, antibody chemical reactivity and covalent antigen binding are constrained, or even prevented, by the narrow range of chemistries encoded in the canonical amino acids. In this work, we investigate strategies for leveraging an expanded range of chemical functionality to augment antibody binding properties. Using yeast displayed antibodies, we explored the presentation of noncanonical amino acids (ncAAs) in or near antibody complementarity determining regions (CDRs) and evaluated the properties of the resulting constructs. To enable systematic characterization of ncAA incorporation sites, we first investigated whether diversification of a single antibody loop would support isolation of binding clones. We constructed a billion-member library containing canonical amino acid diversity and loop length diversity only within the 3rd complementarity determining region of the heavy chain (CDR-H3). Screens against a series of immunoglobulins from three species resulted in the isolation of antibodies exhibiting moderate affinities (double-to triple-digit nanomolar affinities) and, in several cases, single-species specificity. These findings confirmed that antibody specificity can be mediated by a single CDR. With this constrained diversity, we were able to utilize additional CDRs for the installation of chemically reactive and photo-crosslinkable ncAAs. Apparent binding affinities of ncAA-substituted synthetic antibodies on the yeast surface revealed that ncAA incorporation is generally well tolerated. However, changes in binding affinities did occur upon substitution, and varied based on factors including ncAA side chain identity, location of ncAA incorporation, and the ncAA incorporation machinery used. We further investigated chemical modifications facilitated by ncAA installation. Multiple azide-containing ncAAs supported both copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) without abrogation of binding function following the installation of bulky probes. Similarly, several alkyne substitutions facilitated CuAAC without apparent disruption of binding function. Finally, antibodies substituted with a photo-crosslinkable ncAA were evaluated for ultraviolet-mediated crosslinking on the yeast surface. Competition-based assays revealed position-dependent linkages that could not be displaced by excess soluble antigen, strongly suggesting successful crosslinking. Key findings regarding CuAAC reactions and photo-crosslinking on the yeast surface were confirmed using soluble forms of ncAA-substituted clones. These consistent behaviors between the yeast surface and in solution suggest that chemical diversification can be incorporated into yeast display screening approaches. Taken together, our results highlight the power of integrating the use of yeast display and ncAAs in search of proteins with "chemically augmented" binding functions. More specifically, o...

Intracellular Delivery of Antibodies for Selective Cell Signaling Interference

“…Recently, our lab demonstrated that we can secrete scFv-Fcs from yeast that contain noncanonical amino acids (ncAAs) at site-specific locations throughout the construct. [30] Some of these ncAAs contain functional groups that are amenable to "click" chemistry reactions [30,31] that we can leverage to add negatively charged functionalities at selected positions throughout the construct. The constructs in the present work contain additional negative charges only at the C-terminus, but as far as we are aware, no investigations have been made into whether the introduction of negative charge at different locations impact delivery efficiency.…”

Yeast Display Enables Identification of Covalent Single Domain Antibodies Against Botulinum Neurotoxin Light Chain A

“…52 Guided by these sdAb-LC/A complex structures, we identified substitution positions in sdAbs that we deemed likely to result in photocrosslinking to LC/A based on 1) our recent developments in studying AzF-mediated photocrosslinking events on the yeast surface; 33 and 2) access to the AcFRS orthogonal translation system (OTS) to introduce this ncAA in response to the TAG codon in S. cerevisiae (Figure 1b, c). 33,53,54 . In addition, we chose several positions (JPU-A5 Q1; JPU-C10 Q1, Q44 and Y111; ciA-H7 Q1) that lie further away from the sdAb-LC/A interface with future investigations of conjugation-mediated augmentation of sdAb properties in mind, but still evaluated the properties of the resulting clones in this work.…”

“…The mutant plasmids were constructed by introducing TAG codons at the selected positions using typical Gibson assembly procedures (see also Materials and Methods). S. cerevisiae RJY100 57 was transformed with the corresponding pCTCON2 plasmid and pRS315_KanRMod_AcFRS, 33,53,54 which encodes for the AcFRS tRNA aminoacyl synthetase and the corresponding orthogonal tRNACUA for incorporation of ncAAs in response to the TAG codon.…”

“…Upon irradiation with light, photoreactive groups form reactive species that covalently engage with nearby residues and can convert a noncovalent interaction into a covalent one. Photocrosslinking has proven useful for in vitro investigations and protein profiling, [25][26][27][28][29][30][31][32][33] but its dependence on short-wavelength irradiation limits its use in therapeutics and other in vivo applications. Conversely, spontaneous crosslinking is mediated by proximity-enhanced reactivity to initiate covalent bond formation.…”

“…49,50 In this work, we used two ncAAs, 4-azido-L-phenylalanine (AzF) and O-(2-bromoethyl)-Ltyrosine (OBeY) (Figure 1a), to introduce crosslinking functionality into sdAbs via light-mediated and spontaneous crosslinking, respectively. 25,33,35,36,39 Assays in yeast display format revealed numerous ncAA-substituted sdAb variants that retained binding function and led to the identification of photocrosslinkable and spontaneously crosslinkable variants exhibiting timedependent crosslinking behaviors. Corroboration of key observations on the yeast surface with solution-phase experiments indicated that this display platform can be used to discriminate between reversible and irreversible covalent binding agents and to characterize their timedependent behavior.…”