Reduction of Nonspecificity Motifs in Synthetic Antibody Libraries.


Abstract

Successful antibody development requires both functional binding and desirable biophysical characteristics. In the current study, we analyze the causes of one hurdle to clinical development, off-target reactivity, or nonspecificity. We used a high-throughput nonspecificity assay to isolate panels of nonspecific antibodies from two synthetic single-chain variable fragment libraries expressed on the surface of yeast, identifying both individual amino acids and motifs within the complementarity-determining regions which contribute to the phenotype. We find enrichment of glycine, valine, and arginine as both individual amino acids and as a part of motifs, and additionally enrichment of motifs containing tryptophan. Insertion of any of these motifs into the complementarity-determining region H3 of a "clean" antibody increased its nonspecificity, with greatest increases in antibodies containing Trp or Val motifs. We next applied these rules to the creation of a synthetic diversity library based on natural frameworks with significantly decreased incorporation of such motifs and demonstrated its ability to isolate binders to a wide panel of antigens. This work both provides a greater understanding of the drivers of nonspecificity and provides design rules to increase efficiency in the isolation of antibodies with drug-like properties.

Submission Details

ID: vyj9sxAf3

Submitter: Paul Chang

Submission Date: Sept. 19, 2019, 10:56 a.m.

Version: 1

Publication Details
Kelly RL;Le D;Zhao J;Wittrup KD,J Mol Biol (2018) Reduction of Nonspecificity Motifs in Synthetic Antibody Libraries. PMID:29183788
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
5C2B 2015-11-04 1.4 anti-CXCL13 parental scFv - 3B4
5C6W 2015-11-04 1.54 anti-CXCL13 scFv - E10
4OB5 2014-10-08 1.7 Ontogeny of recognition specificity and functionality for the broadly neutralizing anti-HIV antibody 4E10
4M62 2014-10-08 1.8 Ontogeny of recognition specificity and functionality for the anti-HIV neutralizing antibody 4E10
3UPA 2012-06-20 1.8 A general strategy for the generation of human antibody variable domains with increased aggregation resistance
5WKZ 2018-08-01 1.85 VH1-69 germline antibody predicted from CR6261
4LRN 2014-10-08 1.89 Ontogeny of recognition specificity and functionality for the anti-HIV antibody 4E10
3ZHD 2014-01-08 1.96 The crystal structure of single domain antibody 8-4 scaffold.
3ZHK 2014-01-08 1.96 The crystal structure of single domain antibody 2x1 scaffold
1OHQ 2004-03-11 2.0 Crystal structure of HEL4, a soluble human VH antibody domain resistant to aggregation
7FAB 1994-01-31 2.0 CRYSTAL STRUCTURE OF HUMAN IMMUNOGLOBULIN FRAGMENT FAB NEW REFINED AT 2.0 ANGSTROMS RESOLUTION
3BN9 2008-09-09 2.17 Crystal Structure of MT-SP1 in complex with Fab Inhibitor E2
3ZHL 2014-01-08 2.47 The crystal structure of single domain antibody 8-14 scaffold
3UPC 2012-06-20 2.8 A general strategy for the generation of human antibody variable domains with increased aggregation resistance
1F6L 2002-10-23 2.8 VARIABLE LIGHT CHAIN DIMER OF ANTI-FERRITIN ANTIBODY
4KFZ 2014-01-22 2.8 Crystal structure of LMO2 and anti-LMO2 VH complex
4M8Q 2014-10-08 2.89 Ontogeny of recognition specificity and functionality for the anti-HIV neutralizing antibody 4E10
4ODX 2014-10-08 3.1 4E10 germline encoded precursor no.7 in complex with epitope scaffold T117

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.1 Immunoglobulin heavy variable 1-69 A0A0B4J2H0 HV69D_HUMAN
100.0 Immunoglobulin heavy variable 1-69 P01742 HV169_HUMAN
100.0 Immunoglobulin heavy variable 3-15 A0A0B4J1V0 HV315_HUMAN
90.6 Immunoglobulin heavy variable 3-23 P19181 HV05_CARAU
91.5 Immunoglobulin heavy variable 3-23 A0A0C4DH42 HV366_HUMAN
90.6 Immunoglobulin heavy variable 3-23 A0A0J9YX35 HV64D_HUMAN
91.5 Immunoglobulin heavy variable 3-23 A0A075B6Q5 HV364_HUMAN
90.6 Immunoglobulin heavy variable 3-23 P0DP04 HV43D_HUMAN
100.0 Immunoglobulin heavy variable 3-23 P01764 HV323_HUMAN
91.5 Immunoglobulin heavy variable 4-39 A0A0C4DH34 HV428_HUMAN
91.5 Immunoglobulin heavy variable 4-39 A0A087WSY4 HV432_HUMAN
94.9 Immunoglobulin heavy variable 4-39 P01825 HV459_HUMAN
93.2 Immunoglobulin heavy variable 4-39 P0DP07 HV431_HUMAN
95.8 Immunoglobulin heavy variable 4-39 P0DP08 HVD82_HUMAN
94.9 Immunoglobulin heavy variable 4-39 A0A0C4DH41 HV461_HUMAN
94.9 Immunoglobulin heavy variable 4-39 P0DP06 HVD34_HUMAN
100.0 Immunoglobulin heavy variable 4-39 P01824 HV439_HUMAN
93.2 Immunoglobulin heavy variable 5-51 A0A0J9YXX1 HV5X1_HUMAN
100.0 Immunoglobulin heavy variable 5-51 A0A0C4DH38 HV551_HUMAN
90.6 Immunoglobulin kappa variable 1-39 A0A0B4J2D9 KVD13_HUMAN
90.6 Immunoglobulin kappa variable 1-39 P0DP09 KV113_HUMAN
90.6 Immunoglobulin kappa variable 1-39 P01602 KV105_HUMAN
90.6 Immunoglobulin kappa variable 1-39 A0A0C4DH72 KV106_HUMAN
90.6 Immunoglobulin kappa variable 1-39 P01611 KVD12_HUMAN
91.5 Immunoglobulin kappa variable 1-39 A0A0C4DH69 KV109_HUMAN
91.5 Immunoglobulin kappa variable 1-39 A0A0C4DH73 KV112_HUMAN
100.0 Immunoglobulin kappa variable 1-39 P04432 KVD39_HUMAN
100.0 Immunoglobulin kappa variable 1-39 P01597 KV139_HUMAN
93.2 Immunoglobulin kappa variable 3-20 A0A0C4DH55 KVD07_HUMAN
92.8 Immunoglobulin kappa variable 3-20 A0A0A0MRZ8 KVD11_HUMAN
90.5 Immunoglobulin kappa variable 3-20 P04433 KV311_HUMAN
96.6 Immunoglobulin kappa variable 3-20 A0A0C4DH25 KVD20_HUMAN
100.0 Immunoglobulin kappa variable 3-20 P01619 KV320_HUMAN
100.0 Immunoglobulin lambda variable 1-40 P01703 LV140_HUMAN