Antibody biomarker discovery through in vitro directed evolution of consensus recognition epitopes John T. Ballewa,b, Joseph A. Murrayc, Pekka Collind, Markku Mäkie,f, Martin F. Kagnoffg,h, Katri Kaukinend,e,i, and Patrick S. Daughertya,b,1 aDepartment of Chemical Engineering and bCenter for Bioengineering, Biomolecular Science and Engineering Program, University of California, Santa Barbara, CA 93106; cDivision of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN 55905; dDepartment of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, FIN-33520, Tampere, Finland; eSchool of Medicine, University of Tampere, FIN-33520, Tampere, Finland; fTampere Center for Child Health Research, University of Tampere and Tampere University Hospital, FIN-33520, Tampere, Finland; gLaboratory of Mucosal Immunology, Department of Medicine and hDepartment of Pediatrics, University of California, San Diego, La Jolla, CA 92093; and iDepartment of Medicine, Seinäjoki Central Hospital, FIN-60220, Seinäjoki, Finland Edited by K. Christopher Garcia, Stanford University, Stanford, CA, and approved October 17, 2013 (received for review August 5, 2013) To enable discovery of serum antibodies indicative of disease and their clinical development (11–13). Although approved antibody- simultaneously develop reagents suitable for diagnosis, in vitro based diagnostic assays often exhibit sensitivity and/or specificity directed evolution was applied to identify consensus peptides rec- values in excess of 95% (14, 15), library isolated peptides that ognized by patients’ serum antibodies. Bacterial cell-displayed mimic antigens (mimotopes), used alone or in combination, peptide libraries were quantitatively screened for binders to se- rarely meet these stringent requirements. For example, peptides rum antibodies from patients with celiac disease (CD), using cell- from RPLs selected against serum antibodies from patients with sorting instrumentation to identify two distinct consensus epitope Crohn’s disease (16), multiple sclerosis (12, 17, 18), celiac dis- fi E Y families speci c to CD patients (PEQ and /DxFV /FQ). Evolution of ease (11, 13), rheumatoid arthritis (19), or type-1 diabetes (20– E Y fi fi the /DxFV /FQ consensus epitope identi ed a celiac-speci c epi- 22) have exhibited insufficient diagnostic accuracy. Although tope, distinct from the two CD hallmark antigens tissue transglu- these studies have provided support for continued investigation taminase-2 and deamidated gliadin, exhibiting 71% sensitivity of antibodies as candidate biomarkers, they have not yielded clini- and 99% specificity (n = 231). Expansion of the first-generation cally efficacious diagnostic reagents. Consequently, there remains PEQ consensus epitope via in vitro evolution yielded octapeptides a need for discovery processes to produce antibody detection QPEQAFPE and PFPEQxFP that identified ω- and γ-gliadins, and reagents exhibiting accuracies desired for clinical development. their deamidated forms, as immunodominant B-cell epitopes in Although antibody profiling methods using RPLs, including wheat and related cereal proteins. The evolved octapeptides, but phage and bacterial display, lend themselves to various in vitro not first-generation peptides, discriminated one-way blinded CD directed evolution protocols, this capability has not been n = and non-CD sera ( 78) with exceptional accuracy, yielding 100% exploited using blood specimens from patients. Given this, we fi sensitivity and 98% speci city. Because this method, termed anti- applied bacterial display peptide libraries to first screen for dis- body diagnostics via evolution of peptides, does not require prior ease-specific antibody binding peptides and subsequently to knowledge of pathobiology, it may be broadly useful for de novo evolve peptides to achieve diagnostically useful levels of sensi- discovery of antibody biomarkers and reagents for their detection. tivity and specificity. We selected celiac disease (CD) as a model disease because two distinct antibody specificities, transglutaminase he diagnosis of many diseases relies heavily upon the accu- 2 (TG2) and deamidated gliadin, have been characterized ex- Tracy of antibody detection. Assays to detect antibodies using tensively (23) and serve as clinically important antibody bio- known antigens are used extensively to diagnose infectious and markers. Our results demonstrate that in vitro directed evolution autoimmune diseases. And antibodies exhibiting unique antigen- binding patterns have been shown to occur in diverse human Significance diseases, including oncological (1), inflammatory (2), and neu- rological and psychiatric disorders (3). The utility of antibodies The diagnosis of many diseases is dependent upon accurate in diagnostics derives from their intrinsic affinity and specificity, detection of particular antibodies present in blood. However, biochemical stability, and abundance in blood. Nevertheless, the fi fi the development of biochemical reagents that can reliably identi cation of rare antibody speci cities indicative of disease detect these antibodies has proved remarkably challenging. and the development of reagents for their accurate detection fi This study describes a process to create biochemical reagents have proved exceptionally dif cult (4). Intersubject variability of that can accurately and reliably detect disease-associated anti- antibody specificities is a major challenge to the development of fi bodies, without requiring knowledge of the cause or mechanisms accurate tests. Speci cally, individual genetic and stochastic of disease. Simultaneously, this process enabled identification variations that shape the antibody repertoire introduce hetero- of a critical environmental agent involved in celiac disease. geneity in disease antibody subpopulations (polyclonal variation, fi fi Thus, the process presented here may enable the development speci city, af nity, and titer) that hinders uniform antibody de- of effective diagnostic tests for other medical conditions where tection (5, 6). such tests are lacking and the identification of environmental Random peptide libraries (RPLs) have been proposed as factors involved in disease. a potential source of diagnostic reagents capable of mimicking – diverse biological antigens in the environment (7 9). Individual Author contributions: J.T.B., J.A.M., M.F.K., and P.S.D. designed research; J.T.B. performed peptides identified from RPLs using patient sera have been ca- research; J.A.M., P.C., M.M., and K.K. contributed new reagents/analytic tools; J.T.B. and P.S.D. pable of identifying patients with disease with modest accuracy analyzed data; and J.T.B., M.F.K., and P.S.D. wrote the paper. (9, 10). Diagnostic accuracy can be improved in some cases, The authors declare no conflict of interest. using panels of library-isolated peptides coupled with statistical This article is a PNAS Direct Submission. classification algorithms (11), with the drawback of requiring Freely available online through the PNAS open access option. multiple independent measurements. Despite these advances, 1To whom correspondence should be addressed. E-mail: [email protected]. fi peptides identi ed from random libraries have exhibited in- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sufficient diagnostic efficacy (sensitivity and specificity) to foster 1073/pnas.1314792110/-/DCSupplemental. 19330–19335 | PNAS | November 26, 2013 | vol. 110 | no. 48 www.pnas.org/cgi/doi/10.1073/pnas.1314792110 Downloaded by guest on September 28, 2021 can be applied for de novo generation of reagents that exhibit groups (n = 3 subjects per group) were used only once for library requisite levels of diagnostic sensitivity and specificity for clinical enrichment to favor peptides cross-reactive with antibodies from translation. Finally, our results raise the intriguing possibility that many patients with CD. The X6PEQX6 library was enriched for in vitro evolution of such diagnostic reagents may provide a route IgG- and IgA-specific binders, but IgG binders were more rap- to identify previously unknown environmental antigens involved in idly enriched and cross-reactive to multiple CD groups in com- disease and thereby elucidate pathobiology mechanisms. parison with IgA binders; thus, our subsequent analysis focused on IgG isotype reactivity. From the enriched library population, Results three highly represented consensus motifs were observed: Discovery of Celiac Disease-Specific Peptide Epitopes. A Bacterial dis- PEQxFP, PEQPL, and /VFPEQ (Fig. 2A). To assess the di- play random peptide libraries of the form X15,X12CX3,and agnostic sensitivity and specificity of individual peptides, the re- fl X4CX7CX4 were screened using uorescence-activated cell activity of one representative clone from each motif group was sorting (FACS). For screening, individual patient sera were measured using CD case (n = 18) and non-CD control sera (n = 5) pooled into three groups of CD cases and three groups of non- not used for screening. The PEQxFP motif derived peptide CD sera [i.e., healthy and gastrointestinal (GI)-illness control VWDRGVPEQMFPRKG reacted with 18/18 CD sera, whereas subjects], with each group composed of sera pooled from eight VAWTMGPEQPLVRAL reacted with 11/18, and GQGQAF- subjects. Alternating rounds of library enrichment were per- PEQGSVPIN reacted with 14/18. None of the peptides were re- formed with CD sera using FACS and subtraction with non-CD active with control sera. To increase the information content