Proteomic and Metabolomic Approaches to Biomarker Discovery || Autoantibodies and Biomarker Discovery

C H A
P T E R
23

Autoantibodies and Biomarker DiscoveryJi Qiu, Karen S. Anderson

Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ, USA

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O U T L I N E

Introduction 36
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History of the Detection of Autoantibodies
364
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Immunofluorescent Assays
364 Progressive Purification of Antigens 364 Targeted Detection of Autoantibodies 364
Proteomics Methods for the Detectionof Autoantibodies 365

Phage Display of cDNA Libraries
365 Cellular Fractionation and Immunoblotting 369 Protein Microarrays 369 Peptide and Peptoid Arrays 370
d Metabolomic Approaches to Biomarker Discoveryi.org/10.1016/B978-0-12-394446-7.00023-6 363

Detection of Post-Translational Modifications
371
Association of Autoantibodies with DiseaseStates 372

Autoimmune Disorders
372 Type 1 Diabetes 372 Neurologic Disorders 372 Cancer 373
Challenges and Future Development 374

References 374

INTRODUCTION

The immune system plays a fundamental rolein the maintenance of homeostasis. Dysregula-tion of immune responses is observed inmultiple pathogenic states, including autoimmu-nity, diabetes, neurologic disorders, and cancer.The mechanisms by which the immune systemcontributes to the pathogenesis of these diseasesare being uncovered. Because the effectiveness ofthe immune system to fight infections relies on

both adaptability and memory, there are cleargenetic and environmental components to thedepth and specificity of the immune responseto a given antigen. B cells produce antibodieswith target specificities, embedding genetic vari-ability both in the genomic rearrangement of theVDJ gene segments and the acquisition ofsomatic hypermutations in the variable regions,which leads to at least 1011 potential unique anti-body structures. Antibodies that target self-proteins are called autoantibodies to distinguish

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http://dx.doi.org/10.1016/B978-0-12-394446-7.00023-6

23. AUTOANTIBODIES AND BIOMARKER DISCOVERY364

them from antibodies produced by our immunesystem when encountering foreign antigens.Genomic sequencing has identified approxi-mately 20,000 unique open reading frames inthe human genome as source antigenic struc-tures. Tissue-specific splice variation and poly-morphisms add significant variation to thecomplexity of self-antigens. As a result, ourunderstanding of immune responses requiresa systems immunology proteomics approachthat can produce thousands of protein structuresto identify antibody signatures that are associ-ated with disease. In this chapter, we focus onthe application of diverse proteomic technolo-gies for the discovery of autoantigens.

History of the Detectionof Autoantibodies

Immunofluorescent Assays

The development of techniques for the detec-tion of autoantibodies has focused on diseasesassociated with autoimmunity, where a directrelation of autoantibody titer to disease patho-genesis has been established. The presence ofdisease-specific antibodies in the sera of patientswith autoimmune diseases was first describedmore than half a century ago.1e6 Initial studiesof sera from patients with systemic lupus ery-thematosus (SLE) led to the discovery of themajor class of autoantibodies, antinuclear anti-bodies (ANAs).7,8 ANAs were initially detectedwith immunofluorescent assays (IFA) usingcultured cells probed with patient sera,9e13 butidentification of the specific targets of theseANAs has been limited. Molecular characteriza-tion of autoantigens is required to transformthese cell-based semiquantitative assays intoreproducible, quantitative assays to improvediagnosis and disease monitoring. Determiningthe identities of these autoantigens will furtherour understanding of the underlying mecha-nisms of disease pathogenesis in autoimmunityand devise therapeutics for diseasemanagement.

Progressive Purification of Antigens

Identification of autoantigens has paralleledthe technological development in immuno-assays over the past six decades, from crudecellular fractions14,15 to chromatographicpeaks,16 gel bands,17e20 and the identificationof protein structures. One of the first autoanti-bodies identified was rheumatoid factor (RF),an antibody against the Fc domain of humanimmunoglobulins. Targets of ANAs were identi-fied using crude characterizations of antigens incellular/nuclear extracts by immunodiffusion,probing with sera from patients with Sjogren’ssyndrome and SLE. This work led to thediscovery of several well-known antigens suchas Ro/SSA, La/SSB, Sm, RNP, Scl-70, and Jo-1,which are intracellular antigens complexedwith nucleic acids.21 Development of proteinelectrophoresis, immunoblotting, and immuno-precipitation techniques provided tools to refineour knowledge of autoantigens to the level ofmolecular weight. It was not until the adventof molecular biology that the exact autoantigenscould be identified at the molecular level.

Targeted Detection of Autoantibodies

Initially, candidate autoantigens were chosenbased on prior experimental and/or theoreticalknowledgeof theirpotential roles indiseasedevel-opment. Inparticular, oncogenes thatare critical tocancer development and overexpressed in cancerswere selected for discovery of autoantibodies.These oncogenes were purified as recombinantproteins from E. coli and tested in ELISA orWestern blot for antibodies in the sera of cancerpatients. This hypothesis-driven approach ledto the identification of autoantibodies againstseveral oncogenes in cancers. For example, anti-c-myc autoantibodies were identified by Westernblot in 25 out of 44 sera from patients with colo-rectal cancer, but in 8 out 46 normal donors.22

Autoantibodies against other oncogenes such asras or HER-2/neu have also been reported.23,24

However, the discovery of autoantibody

PROTEOMICS METHODS FOR THE DETECTION OF AUTOANTIBODIES 365

biomarkers in diseases using this approach hasbeen limited to selected proteins. Because the rela-tionship between protein structure, proteinexpression, and antigenicity is not well under-stood, there is a need for broad-based discoveryof autoantibodies without selection bias.

PROTEOMICS METHODS FOR THEDETECTION OF AUTOANTIBODIES

Since the beginning of the 21st century, thedevelopment and application of numerous pro-teomics technologies has enabled high-throughput protein expression for autoantigendetection. Based on the source and type ofantigen repertoires, these technologies aredivided into four categories. Antigens havebeen expressed as whole proteins from cDNAlibraries, separated and isolated from cell lysatesand displayed in protein array format, orpeptides can be displayed in microarray format(see Figure 1). Each method displays uniquestructural determinants and contributes to ouroverall understanding of structural immunoge-nicity (Tables 1 and 2).

Phage Display of cDNA Libraries

After the development of immunofluorescentassays, serological identification of recombi-nantly expressed clones (SEREX), initiallydescribed in 1995, represents one of the earliestdevelopmentof this approach that revolutionizedthe discovery of autoantigens.25 In this strategy,cDNA libraries are generated from diseasetissues, cell lines, or testis tissue and the candidateantigens are expressed either directly in E. coli orusing a lambda phage expression system. Colo-nies or phage plaques containing expressedproteins are then replicated onto nitrocellulosemembranes and probed with patient sera(Figure 1, top). Positive clones are sequenced toidentify the autoantigens. This approachwasfirstapplied to cancer but has been widely used for

other diseases.26e30 The autoantigen NY-ESO-1was first identified in esophageal cancer patientsera and was later detected in the sera of patientswith many different cancers. These findings ledto clinical trials targeting NY-ESO-1 for cancerimmunotherapy.31 One key advantage of SEREXis that the cDNA library expression reflects theexpression levels, mutations, and splice varia-tions that are inherent to the specific tissuesfrom which the library is derived. However,SEREX identification of autoantigens has severallimitations. Full-length cDNAs are rarely clonedinto the expression vectors and truncated expres-sion limits coverage. Many of the autoantibodiesthat have been identified target frame shift poly-peptide products, which have yet not beenproven to be expressed at the protein level in therelevant tissue. E. coli expression machinery haspoor expression of high molecular weightproteins or proteins with multiple domains andlacks chaperones to facilitate proper folding.This limits the amount, type, and conformationsof proteins presented for screening. Plaque-based screening is also not amenable to automa-tion and quantitative analysis.

Several recent advances with phage-basedexpression have resulted in the identification ofmultiple new autoantigens and have facilitatedautomation. Lysates of individual E. coli colonieshave been spotted in microarray format for sero-logic screening.32 The use of filamentous T7 orM13 phage for protein expression results insuperior throughput compared with lambdaphage due to compatibility with biopanning.33,34

More recently, solution-based phage display hasbeen developed to further identify autoantigensexpressed in amore native format. Phage displayis compatible with rapid biopanning for negativeand positive selection and has been integratedwith microarray platforms to improve high-throughputmultiplexing and quantitation. Theseadvances have led to the identification of auto-antibody biomarkers in several cancers suchas ovary and prostate.33e35 In summary, theprimary advantages of phage-based expression

Pooled sera from patients

Cell lysate Multidimensional fractionation in solution or gel

Print fractions on multiple slides

Probe with sera

200 40060080010001200m/z

Identify proteins in informative fractions

Probe library display for recognized antigens

Protein arrays (left) or bead arrays (right) with candidate antigens

Probe with sera Determine sensitivity and specificity of antigens

cDNA library screening and phage display:

Cell fractionation:

Protein microarray:

Print peptide arrays

Peptide display:

Blot 2D gels

Display peptides by phage Probe with sera Determine sensitivity and specificity of peptides

Display protein by phage Biopanning for informative antigens

Phage array screening

FIGURE 1 Methods of antigen identification. In traditional library screening (top row), cDNA libraries from target tissueare expressed by phage, blotted on a membrane or panned in solution, and probed with patient sera. Confirmation ofsensitivity and specificity requires recombinant expression of protein for ELISA analysis. Cell fractionation (second row)involved separation of cell lysates by two-dimensional or three-dimensional separations in solution phase or in gel phase,followed by probing with patient sera. Positive fractions are identified by mass spectrometry and confirmed with ELISA.Protein microarrays (third row) can be printed using recombinant proteins or proteins translated in situ, on slides or on beadarrays. Because the arrays are addressable, no further antigen identification is required. Similarly, peptides (bottom) can beprinted on slides or displayed by T7 phage for rapid serum screening. (Modified from reference #109, reprinted with permission.)


TABLE 1 Comparison of Different Technologies for Autoantibody Discovery

Antigen sources Throughput Multiplexity Target identificationSampleconsumption

cDNA expression

Phage plaque Low. Rounds of plaquescreening.

Medium. Many plaquescan be screened on onemembrane lift.

Easy. DNAsequencing.

High

Phage display& microarray

Medium. Screening onspotted phages.

High Easy. DNAsequencing.

High for panning.Low for arrays.

Bacterial expression& microarray

High. Bacterial coloniesare spotted on glassslides.


Low

Cell lysates

2D Western Low. 2D SDS-PAGE andWestern blot.

High. Hundreds ofspots on each gel.

Medium-hard. Massspec of positive spots.

Medium

Natural protein array Medium. Thousands offractions obtained fromliquid phase separationof cell lysates.

High Medium-hard. Massspec of positivefractions.

Low

Recombinant proteins

Purified proteins High High Easy Low

In vitro expressionmixtures

High High Easy Low

On-chip synthesizedproteins

High High Easy Low

Synthetic peptides

Synthetic peptides/peptoids on arrays

High High Easy for spatiallyaddressed arrays.NMR analysis maybe neededfor peptoid arrays.

Low

Synthetic humanpeptidome & phagedisplay

Low. Rounds of panningand NextGen sequencing.

High Hard. Sophisticatedanalysis of NextGensequencing data.

Medium

Microbial surfacedisplay

Low. Rounds ofpanning.


Medium


systems are the ability to rapidly convert a targetlibrary for screening low numbers of serafor autoantigen discovery without specificequipment. However, limitations such as the

overrepresentation of abundant proteins, frameshifts, and lower throughput have limitedthe ultimate translation of these biomarkers intoclinical practice.

TABLE 2 Comparison of Different Technologies for Autoantibody Discovery

Antigen sources Proteome coverage

Neoantigens fromalternative splicing/mutation/translocation

Post-translationalmodifications

cDNA expression

Phage plaque Low. Limited by bacterialexpression and minimalcoverage of low abundanceproteins. Truncations andframe shifts are prevalent.

Yes No

Phage display & microarray Low. Same as above. Yes No

Bacterial expression &microarray

Medium. Limited bybacterial expression andminimal coverage of lowabundance proteins.

Yes No

Cell lysates

2D Western Medium. Only proteinscompatible with 2D gel.

Yes Yes

Natural protein array Medium. Only proteinscompatible withmultidimensional column-based separation.

Yes Yes


Purified proteins High. Limited byORFeome.

No if without priorknowledge and intentionto include a particularneoantigen.

No

In vitro expressionmixtures

High. Limited byORFeome.

No No

On-chip synthesized proteins High No No in the standard format.Possible to study certainPTMs.

Synthetic peptides

Synthetic peptides/peptoidson arrays

N/A Maybe. If a syntheticpeptide happens to havethe same sequence.

No

Synthetic human peptidome &phage display

High No No

Microbial surface display Low to medium Maybe No



Cellular Fractionation andImmunoblotting

A primary concern with the use of phagedisplay, as well as solution-based display, isthat proteins are not expressed in native formand post-translational modifications are not rep-resented. An alternative approach uses relevanttissue lysates as the source of candidate antigens,which are then probed with patient sera.Initially, autoantigens were observed directly incell lysates using immunodiffusion, but thecomplexity of protein content within lysateslimited this approach until the innovation oftechniques in protein separation by columnchromatography and gel electrophoresis per-mitted sufficient separation of protein content.Currently, lysate preparation requires two-dimensional and three-dimensional proteinseparation coupled with target identification bymass spectrometry.

Two-dimensional immunoblotting (2D-Western) is well established for the assessmentof antibody reactivity and has been applied toautoantigen discovery. Two-dimensional gelelectrophoresis separates proteins in complexbiological samples based on isoelectric pointand molecular weight. Lysate preparation andgel composition can be customized to improvethe separation of specific protein classes.After addition of sera, immunogenic spots areexcised and sequenced for protein identificationby mass spectrometry.36,37 Because immunoblot-ting favors the detection of denatured epitopes,solution-based methods of antigen detectionwere developed. Multidimensional separationin liquid phase by reverse phase/ion exchangechromatography and chromatofocusing inconjunction with arraying thousands of proteinfactions prepared from cell/tissue lysates resultsin native protein arrays that contain relevantpost-translational modifications (PTMs).38 Forboth 2D-Western and native protein arrays, theautoantigens can be prepared with relative easewithout prior cloning and purification. The

antigens are displayed in the physiologicallyrelevant form that initially elicited the humoralimmune response. However, proteins in cell/tissue lysates exist in varying abundance thatdiffers by orders of magnitude, and the dynamicrange of analysis is limited. The signals from lowabundance proteins can be buried by high abun-dant housekeeping proteins. The identificationof target antigens within a specific spot or frac-tion requires deconvolution by mass spectrom-etry and often requires further targetedseparation to confirm the identity of the anti-gens. Translation of these targets to clinicalbiomarker assays is also a major challenge;high-throughput serologic validation assaysrequire recombinant protein production thatmay not display the antigenic PTMs.

Protein Microarrays

The development of protein microarrays hasnow enabled the simultaneous analysis of thou-sands of different proteins on the footprint ofa microscopic slide or in bead-array format.With the development of high-throughputprotein production, it is now possible to produceproteins that are limited only by ORFeomecontent. In the near term, protein arrays display-ing the entire human proteome will soon beavailable, although protein arrays that reflectthe genetic variability of tumors and the splicevariation of tissues will take longer to develop.One natural advantage of protein microarraysis the rapid, multiplexed measurement of anti-body levels in sera.

There are three primary methods forproducing protein microarrays. First, proteinsare expressed in model organisms such asE. coli, yeast, or insect cells, purified, and printedon the arrays.39e44 In vitro protein production isa challenging process that can be hindered byprotein purity, batch-to-batch variation, andlimited stability of purified protein products.The second approach is to use printed antibodyarrays to detect antigen/antibody complexes


within sera, which is limited to antigens thatare present in sera and the availability of captureantibodies for array printing. We, and others,have developed in vitro transcription/translationmethods for expression and capture of targettagged antigens onto an array format: nucleicacid programmable protein arrays (NAPPA);see Figure 2. These arrays have the advantage ofreal-time protein production for serologic detec-tion. However, protein arrays in general havelimited PTMs and are limited by variable proteinexpression, folding, and reproducibility.45e48

A variation on spotting purified proteins is tospot mixtures of proteins that are produced inmultiwell plates using PCR amplified genes andin vitro expression system.42,49,50

Peptide and Peptoid Arrays

One way to bypass the challenges of full-length protein expression is to display peptidesrather than proteins. Peptides may be synthe-sized in vitro as random peptides, peptide-likestructures (peptoids) or as peptides representingthe entire potential human ORFeome. Peptidesmay be also be generated in vivo using phage,E. coli, or yeast, cloned in fusion with a surfaceprotein, and displayed for biopanning.51e53

With the development of massively parallelpeptide synthesis, tens of thousands of peptidescan now be synthesized, immobilized, and

DNA

Add cexpr

sys

FIGURE 2 Serologic detection ofantibodies using programmableprotein arrays. Left: full-lengthtagged cDNAs are printed on slides,and detected with picogreen. Middle:tagged antigens are expressed usingmammalian cell lysate, captured in situand protein expression is confirmedusing antitag antibodies. Right: arraysare probed with sera, and boundimmunoglobulin is detected.

screened with sera for antigenicity.54e56 Peptidearrays can also be generated in vitro using on-chip synthesis technology, similar to oligonucle-otide Affymetrix arrays.57 Peptides spanning theproteome are first identified bioinformatically.DNA sequences encoding these peptides aresynthesized on-chip similar to DNA oligonucle-otide arrays and expressed on the arrays usingin vitro T7-based expression systems. In compar-ison to peptide-printed arrays, longer peptidescan be generated by in vitro synthesis. As analternative, the synthesized DNAs encoding thepeptide libraries can be displayed on the surfaceof phage coat proteins for biopanning.

Onekey feature of antigen/peptidedisplay forantibody detection is that they lead to identifica-tion of the naturally occurring cognate antigen.This identification is critical for understandingthe pathogenesis of the disease. As a practicalalternative to developing biomarkers that arespecific for diseases, display of peptide mimo-topes or peptoids can be used to detect specificantibodies in sera of patients with the disease.Peptoids are similar to peptides but presentchemically synthesized diverse structuralepitopes for antibody selection. Peptoid microar-rays displaying approximately 15,000 peptoidshave been successfully used for the identificationof autoantibodies in Alzheimer’s disease.58

Although the initiating natural antigenic stim-ulus cannot be identified from the mimotopes,

- GST ControlCase

Add sera

Detect IgG

α

ell-freeessiontem


the peptoids themselves can be developed as clin-ically useful probes for identifying selective auto-antibody biomarkers that correlate with disease.

Detection of Post-TranslationalModifications

Most proteins undergo PTMs that are criticalfor their biological functions. Aberrant modifica-tions occur in disease states that may elicithumoral immune responses. Due to the hightissue specificity of PTMs, these antibodies may

TABLE 3 Examples of Autoantibodies Against Post-Transl

Post-translationalmodifications Example protein(s)

Carbamylation Filaggrin

Citrullination Filaggrin/fibrin/vimentin/collagen/a-enolase

Acetylation DEK

Phosphorylation SR proteins

La/SSB

Protease cleavage U1-70K

Calreticulin

Pso27

Aspartyl/isoaspartylisomerization

snRNP

Methylation SmD1, SmD3

Sulfilimine bond Collage IV

Deamidation Transglutaminase

Oxidation LDL

b2 glycoprotein 1

Glycosylation MUC1

Annexin I

Mutation p53

Alternative splicing isoforms CML66

Lengsin

be very disease- and tissue- specific. Many anti-bodies against post-translationally modifiedproteins have been identified in different dis-eases36,59e76(Table 3). The majority of these anti-bodies have been detected using cellularfractionation technologies that rely on lysatesfrom disease cell lines or tissues that containnatively modified proteins. Modified proteinsor peptides from small sets of target proteins ofinterest can then be displayed usingmicroarrays.However, current technology cannot systemati-cally mimic PTM structure at the proteome level.

ationally Modified Proteins

Example disease(s) References

Rheumatoid arthritis 68

Rheumatoid arthritis 74

Juvenile idiopathic arthritis 67

Systemic lupus erythematosus 70



Cancer 63

Psoriasis 62



Goodpasteur’s disease 71

Celiac disease 60

Atherosclerosis 64

Antiphospholipid syndrome 61

Cancer 75

Cancer 36

Cancer 72

Cancer 76

Cancer 69


ASSOCIATION OFAUTOANTIBODIES WITH DISEASE

STATES

Autoimmune Disorders

Thefirst autoantibodies identifiedwere associ-ated with autoimmune rheumatic diseases.21,77

Autoantibodies not only indicate the presenceof disease; in some instances, they are pathogeniccauses of inflammation. Even when the autoanti-bodies are not pathogenic, they may bebiomarkers for antigens that stimulate CD4þ

cellular immune responses. Traditional IFAmethods to detect antinuclear antibodies,together with ELISA or radioimmunoassays, areused for the clinical measurement of these anti-bodies. Several of the antibodies that have beenidentified in autoimmune disorders target post-translational modifications (Tables 3 and 4).Anticitrullinated antibodies (ACA) have beenidentified that are very specific to adult RA,and distinct antibodies to citrullinated proteinsmay be involved in the pathogenesis of thedisease. Clinical testing for ACA using cyclic-citrullinated-peptides detects RA sera witha sensitivity of up to 80% to 90% at 98%specificity.

Type 1 Diabetes

Type 1 diabetes (T1D) is an autoimmunedisease that is caused by autoimmune destruc-tion of insulin-secretion islet cells in early child-hood. Like many autoimmune diseases, bothgenetic and environmental factors are thoughtto play an important role in disease etiology.Several autoantibodies, including anti-GAD65and anti-IA2, have been identified in T1D. Usinggenomics, the ZnT8 antigen was recently identi-fied.78 The presence of these antibodies in seraindicates the transition from genetic risk toimmunological risk. Both the titer and thecombined frequency of these autoantibodies arepredictive of T1D development in high-risk

populations. The five year risk for T1Dapproaches 50% in subjects with detectable anti-bodies against all three antigens. Several clinicaltrials targeting these antigens are ongoing. High-throughput proteomics are being used to iden-tify novel T1D antigens. However, as the threeknown autoantibodies in T1D have excellentperformance as clinical biomarkers, the identifi-cation of new targets may have limited utilityfor disease diagnosis or risk prediction. Newlydiscovered antigens may still uncover mecha-nisms of the etiology of T1D for therapeuticdevelopment.

Neurologic Disorders

A number of neurologic disorders haveevidence of immune-mediated pathogenesis.Functional autoantibodies to the acetylcholinereceptor are pathogenic in myasthenia gravis(MG), as are calcium-channel autoantibodies inLambert-Eaton myasthenic syndrome. Paraneo-plastic neuropathies, primarily associated withsmall cell lung cancer and ovarian cancer,are mediated by cross-reactive autoantibodiestargeting neuronal proteins, such as anti-Hu/ANNA-1, anti-Ri/ANNA-2, and anti-Yo/PCA1.79 However, emerging evidencesuggests that other neurologic disorders, suchas multiple sclerosis, Alzheimer’s dementia,and Parkinson’s disease, may be mediated inpart by B-cell mediated autoimmunity. Multiplesclerosis (MS) is an autoimmune disease causedby autoreactive T cells targeting myelin sheaths.Recent studies have identified autoantibodiesthat catalyze site-specific destruction of myelinbasic protein in MS patients80 and cross-reactwith the LMP-1 protein of Epstein-Barr virus.81

Multiple other autoantibody targets, such asPGAM1,82 mtHSP70,37 and contactin-2,83 havebeen identified in the sera of patients with MSusing proteomic-based approaches. Alzheimer’sdementia is a progressive neurologic diseasemarked by accumulation of beta-amyloid andtau protein in the central nervous system.

TABLE 4 Examples of Autoantibodies Identified using Proteomic Technologies

Antigen Sources Example antigens Example diseases References

cDNA expression

Phage plaque/display SSX-2PC326 and othersIFI16 and othersFive-phage peptide

MelanomaMoyamoya diseaseSjogren’s syndromeColorectal carcinoma

29283027

Phage display & microarray A panel of 22 peptidesUbiquilin 1

Prostate cancerLung

3335

Cell lysates

2D Western Annexin I and IImtHSP70

Lung cancerMultiple sclerosis

3637

Natural protein array LAMR1 Lung cancer 38


Purified proteins RALBP1 and othersKelch-like protein 12 and othersRPS20 and othersPanel of antigensA panel of 10 antigens

Ovarian cancerPrimary biliary cirrhosisAutoimmune hepatitisRheumatoid arthritisAlzheimer’s disease

40394341,4442

In vitro expression mixtures Various Infectious disease(Burkholderia pseudomallei)

50

On-chip synthesized proteins A panel of 28 antigensPanel of antigens

Breast cancerankylosing spondylitis

4548

Synthetic peptides

Synthetic peptides/peptoidson arrays

A panel of peptidesSeveral peptoidsGlycopeptides derived frommucins

Alzheimer’s diseaseAlzheimer’s diseaseColorectal cancer

565855

Synthetic human peptidome &phage display

Various Paraneoplastic neurologicaldisorder

57

Microbial surface display HSP60Plasminogen binding protein(PBP)GRP78

AtherosclerosisAutoimmune pancreatitisProstate cancer

535152

ASSOCIATION OF AUTOANTIBODIES WITH DISEASE STATES 373

Although Alzheimer’s is not considered animmune-mediated disorder, autoantibodies topeptoid libraries have been specifically identi-fied in the sera of Alzheimer’s patients, whichmay be biomarkers for disease diagnosis.58

Cancer

Cancer is the second most common cause ofdeath in the United States. In 2012, there will bean estimated 1.6 million new cases of cancer andover 500,000 deaths (http://www.cancer.org),

http://www.cancer.org


with a total cost over $200 billion.84 Although theestimated five-year overall survival after a cancerdiagnosis is 67%, there are few FDA-approvedbiomarkers for early detection and monitoringfor disease recurrence, which could have signifi-cant impact on clinical outcome. There is alsoa need for biomarkers that identify biologicsubtypes of cancer and to predict responses toa rising number of immune-targeted thera-peutics, such as ipilimumab85 and sipuleucel-T.86

There is growing scientific evidence thatimmune dysregulation functions the pathogen-esis of cancer paradoxically, both contributingto carcinogenesis through chronic inflamma-tion87 as well as controlling cancer progressionwith direct immune surveillance of tumor anti-gens.88e90 Production of cytokines such as IL-6and CSF-1 by tumor cells promotes thedevelopment of immunosuppressive tumor-associated macrophages (TAMs).91,92 In modelsystems, TAMs have been shown to directlypromote tumor progression and metastasis.93e96

In contrast, tumor-infiltrating lymphocytes(TILs) are associated with improved survivalfor lymphoma,97 colorectal,98,99 breast,100 esoph-ageal,89 and ovarian cancers.101 In particular, Bcell signatures15,16 and intratumoral B cell folli-cles102 have been identified in multiple can-cers103e105 and are associated with improvedclinical outcome.106 These data suggest thatcancers can induce an active, specific antibodyresponse to tumor-derived antigens.

There have been multiple proteomics-basedapproaches to identify autoantibody biomarkersthat distinguish sera from cancer patients andsera from healthy controls (Table 4). Specificautoantibody signatures have been identifiedin the sera of patients from multiple cancertypes, often corresponding to overexpressedtumor antigens. These autoantibodies are beingdeveloped as potential biomarkers for earlycancer diagnosis, such as Annexin 1, 14-3-3theta, and LAMR1 in lung cancer,38 TA90 inmelanoma,107 and p53 in various cancers.108

Although promising, these biomarkers awaitvalidation in blinded, multicenter phase IIIbiomarker trials.

CHALLENGES AND FUTUREDEVELOPMENT

Over the past decades, proteomic technolo-gies have greatly contributed to the discoveryof novel autoantibody biomarkers. Emergingmethods enable the discovery of autoantigensat the scale of proteomes. To this end, the fieldof proteomics has expanded our understandingof the repertoire of antigens that can elicit auto-antibodies in different diseases. Emerging datafrom genomics and transcriptomics continuesto broaden the scope of potential immunogenicstructures to include mutated antigens andsplice variants that may have significant disease-and tissue specificity. In addition, proteomicmethods that can systematically display PTMsare likely to identify numerous other autoanti-body targets that are associated with disease.The discovery and validation of these antigenswill improve our knowledge of the structuralcontent that elicits antibody immune responses,and to understand the underlying mechanismsof autoimmunity. These advances have potentialclinical implications for both biomarker develop-ment and immune-targeted therapeutics.

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Proteomic and Metabolomic Approaches to Biomarker Discovery || Autoantibodies and Biomarker Discovery