An Array of 60,000 Antibodies for Proteome-Scale Antibody Generation and Target Discovery

Abstract

4Antibodies are essential for elucidating the roles of genes decoded by genome 5 sequencing. However, affordable technology for proteome-scale antibody 6 generation does not exist. To address this, we developed the Proteome Epitope 7 Tag Antibody Library (PETAL) and its array. PETAL consists of 62,208 mAbs 8 against 15,199 peptides from diverse proteomes. PETAL harbors binders for a 9 great multitude of proteins in nature due to antibody multispecificity, an intrinsic 10 feature of an antibody. Distinctive combinations of 10,000-20,000 mAbs were 11 found to target specific proteomes by array screening. Phenotype-specific mAb-12 target pairs were discovered for maize and zebrafish samples. 13 Immunofluorescence and flow cytometry mAbs for human membrane proteins 14 and ChIP-seq mAbs for transcription factors were identified from respective 15 proteome-binding PETAL mAbs. Differential screening of cell surface proteomes 16 of tumor and normal tissues discovered internalizing tumor antigens for antibody-17 drug conjugates. By discovering high affinity mAbs at a fraction of current time 18 and cost, PETAL enables proteome-scale antibody generation and target 19 discovery.20 21 22 Keywords: 1 antibody; proteome-scale; multispecificity; antibody microarray; target discovery; 2 antibody-drug conjugate (ADC) 3 1Facilitated by the ever-growing capability of current DNA sequencing 2 technologies, more than 1,300 genomes of animals and 496 genomes of plants 3 and many others have already been sequenced, representing millions of genes, 4 and the number will increase faster from projects such as G10K, i5k and so on 1 . 5To understand the roles of these genes, the functions of the gene-coded proteins 6 need to be explored, and antibodies, especially renewable monoclonal antibodies 7 (mAbs) generated at a proteome-scale, are urgently needed. mAbs produced by 8 hybridoma technologies for human proteins have long been recognized as the 9 most direct tools for diagnostic and therapeutic target discovery 2 . Classic 10 therapeutic targets Sialyl Lewis Y, prostate-specific membrane antigen (PSMA) 11 and, more recently, a novel target for multiple myeloma were discovered by 12 mAbs for cell surface proteins 3-6 . 13Despite the power of mAbs and mAb-based discovery, large-scale 14 generation of mAbs remains difficult since traditional hybridoma development is 15 time-consuming (4-6 months starting from antigens), expensive ($3,000-8,000 16 per antigen), and challenging to scale. Further, mAb generation by immunization 17 typically requires a milligram of purified antigens, a significant challenge for many 18 protein targets, especially for membrane proteins of primary research interest. 19Even for human proteins, a majority of the 6,000 membrane proteins have not 20 been directly explored as diagnostic or therapeutic targets due to a lack of high-21 quality antibodies for applications such as flow cytometry (FACS) and 22 immunofluorescence (IF) 7, 8 . 23The Human Protein Atlas (HPA) offers an alternative approach for proteome-1 sca...

Antibody Binding Epitope Mapping (AbMap) of Hundred Antibodies in a Single Run

“…After success with several widely used antibodies, we then set out to apply AbMap for high-throughput analysis. A total of 202 mouse monoclonal antibodies ( supplemental Table S2 ), produced in 2012 via immunization with selected peptide antigens, were included ( 28 ). The anti-6xHis antibody was used as a positive control.…”

“…In addition, our understanding of antibody multispecificity could be enhanced. According to the conventional concept, the capacity of an antibody to bind several “unrelated” targets is regarded as the multispecificity of the antibody ( 13 , 28 , 31 ). When the antibody-binding linear epitope is not available, the relationship among these “unrelated” targets could not be understood beyond their being recognized by the same antibody.…”

The morphological and physiological basis of delayed pollination overcoming pre‐fertilization cross‐incompatibility in Nicotiana

“…The immunoprecipitation (IP) and liquid chromatography (LC)‐mass spectrometry (MS) mass spectrum (MS) assays were performed following the methods of Wang et al (2019). The LC‐MS/MS data were searched against the NCBI Nicotiana tabacum / Hyoscyamus niger cybrid database, containing 1,520,274 entries.…”

Microarrays and NGS for Drug Discovery

“…Analysis of genetic variant(s) is yet another important approach for identifying mutations in rare diseases, as these could then be used for treatment of such target(s) [170,171]. Epigenetic studies, such as methylation analysis or CHIP-seq analysis, known to be altered in different pathologies, could also aid in identifying targets for specialized treatments/therapies [172,173].…”