Protein Microarray Platform Research Articles

Abstract PO2-13-06: Analysis of Antibody Response to SV-BR-1-GM Therapeutic Vaccine in Breast Cancer Patients Using Human Protein Microarrays: Potential Correlations with Therapy Response

Abstract Background: Therapeutic cancer vaccines aim to stimulate the immune system by utilizing tumor antigens to trigger an antitumor response. In our clinical trials, we have focused on evaluating the efficacy of the breast cancer cell line secreting GM-CSF, SV-BR-1-GM as a therapeutic vaccine, and we have observed encouraging clinical outcomes. The SV-BR-1-GM regimen has been used alone (“monotherapy”, ClinicalTrials.gov NCT03066947 – study completed) and in combination with checkpoint inhibitors (“combination”, ClinicalTrials.gov NCT03328026 – study ongoing). To further improve the therapeutic efficacy of this vaccine we have initiated a study to characterize patient’s immune response to this treatment. It has been demonstrated that cancer vaccines can generate both humoral and cellular immune responses. However, predicting immune responses to cancer vaccines, especially when whole cells are employed as immunogens, presents significant challenges. We present here a preliminary analysis of the antibody response to SV-BR-1-GM in breast cancer patients using antigen arrays. Methods: Large-scale protein arrays are versatile and sensitive platforms for antibody specificity evaluation. The HuProt™ Human Proteome Microarray (CDI Laboratories, Inc., Baltimore, Maryland, United States) provides the largest number of unique, full-length, individually purified human proteins on a single microscope slide. This allows thousands of interactions to be profiled in a high-throughput manner. We utilized here an unbiased human protein microarray platform encompassing >21,000 proteins and isoforms from ~19,000 unique genes to identify IgG and IgM responses against self-antigens elicited by treatment. Serum samples from SV-BR-GM-treated patients were analyzed, comparing pre- and post-treatment. The human serum samples were probed at 1:1000 dilution. After sample processing and data collection the raw signal intensities on all arrays were quantile normalized using CDI software. Heatmaps were generated using MetaboAnalyst 5.0, which utilizes the R pheatmap package (version 0.7.7). For paired analysis, fold changes (FCs) were calculated by determining the ratio between paired pre- and post-treatment samples, resulting in one FC per pair. The means of these FCs (pair means) were then computed. ANOVA tests were performed using the RStatix R package, which automatically determines the appropriate Type I, II, or III errors for the analysis. Results: By using this approach, we were able to evaluate a broader range of antigens compared to previous investigations. Using a variety of statistical approaches, potential correlations with patient survival on SV-BR-1-GM were evaluated. Antibody responses to galectin antigens demonstrated the most consistent relationships with survival. Further confirmation with additional patients and prospective analysis are needed to fully understand relationships with clinical benefit and survival. Citation Format: Miguel Lopez-Lago, Mingjin Chang, Giuseppe Del Priore, Vikas Bhardwaj, Patience Cournoo, Pedro Ramos, Tyler Hulett, Charles Wiseman, William Williams. Analysis of Antibody Response to SV-BR-1-GM Therapeutic Vaccine in Breast Cancer Patients Using Human Protein Microarrays: Potential Correlations with Therapy Response [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-13-06.

Humoral immunoprofiling identifies novel biomarkers and an immune suppressive autoantibody phenotype at the site of disease in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a heterogeneous cancer, with minimal response to therapeutic intervention and with 85% of cases diagnosed at an advanced stage due to lack of early symptoms, highlighting the importance of understanding PDAC immunology in greater detail. Here, we applied an immunoproteomic approach to investigate autoantibody responses against cancer-testis and tumor-associated antigens in PDAC using a high-throughput multiplexed protein microarray platform, comparing humoral immune responses in serum and at the site of disease in order to shed new light on immune responses in the tumor microenvironment. We simultaneously quantified serum or tissue IgG and IgA antibody isotypes and subclasses in a cohort of PDAC, disease control and healthy patients, observing inter alia that subclass utilization in tumor tissue samples was predominantly immune suppressive IgG4 and inflammatory IgA2, contrasting with predominant IgG3 and IgA1 subclass utilization in matched sera and implying local autoantibody production at the site of disease in an immune-tolerant environment. By comparison, serum autoantibody subclass profiling for the disease controls identified IgG4, IgG1, and IgA1 as the abundant subclasses. Combinatorial analysis of serum autoantibody responses identified panels of candidate biomarkers. The top IgG panel included ACVR2B, GAGE1, LEMD1, MAGEB1 and PAGE1 (sensitivity, specificity and AUC values of 0.933, 0.767 and 0.906). Conversely, the top IgA panel included AURKA, GAGE1, MAGEA10, PLEKHA5 and XAGE3aV1 (sensitivity, specificity, and AUC values of 1.000, 0.800, and 0.954). Assessment of antigen-specific serum autoantibody glycoforms revealed abundant sialylation on IgA in PDAC, consistent with an immune suppressive IgA response to disease.

Protein microarray allergen profiling in bronchoalveolar lavage fluid and serum of horses with asthma

The diagnostic value of allergen-specific immunoglobulin E (IgE) in horses with asthma is uncertain. A recently developed protein microarray detected abnormally high latex-specific IgE concentrations in the serum of horses with severe asthma. The main objective was to characterize the IgE profiles of asthmatic horses in Switzerland using a protein microarray platform in serum and bronchoalveolar lavage fluid (BALF). The secondary objective was to determine whether serological and BALF allergen-specific IgE concentrations correlated. Forty-four asthmatic and 39 control horses ≥5 years of age. This prospective cross-sectional study investigated the sensitization profiles of horses with asthma compared with environmentally matched healthy controls. Both serum and BALF were analyzed using the protein microarray. Partial least square-discriminant analysis (PLS-DA) was used to identify and rank the importance of the allergens for class detection (ie, asthma vs control), with a variable influence on the projection (VIP) >1 considered significant. The allergens that best discriminated (VIP >1) asthmatic horses from controls were proteins derived from fungi (Aspergillus fumigatus), insects (Culicoides spp.), and latex (Hevea brasiliensis). The serological model predictive ability was markedly inferior (area under the curve [AUC] 0.585, 95% confidence interval [CI]: 0.454-0.747) to that of the BALF (AUC 0.751, 95% CI: 0.582-0.866). The two models shared nine allergens, of which eight showed significant weak to moderate correlations. The concentrations of several allergen-specific IgE were higher in asthmatic horses. The protein microarray performed better on BALF than serum for detection of asthma. Serological IgE concentrations do not closely correlate with BALF concentrations and should be interpreted with caution.

Blood-Based Biomarkers of Repetitive, Subconcussive Blast Overpressure Exposure in the Training Environment: A Pilot Study.

Because of their unknown long-term effects, repeated mild traumatic brain injuries (TBIs), including the low, subconcussive ones, represent a specific challenge to healthcare systems. It has been hypothesized that they can have a cumulative effect, and they may cause molecular changes that can lead to chronic degenerative processes. Military personnel are especially vulnerable to consequences of subconcussive TBIs because their training involves repeated exposures to mild explosive blasts. In this pilot study, we collected blood samples at baseline, 6 h, 24 h, 72 h, 2 weeks, and 3 months after heavy weapons training from students and instructors who were exposed to repeated subconcussive blasts. Samples were analyzed using the reverse and forward phase protein microarray platforms. We detected elevated serum levels of glial fibrillary acidic protein, ubiquitin C-terminal hydrolase L1 (UCH-L1), nicotinic alpha 7 subunit (CHRNA7), occludin (OCLN), claudin-5 (CLDN5), matrix metalloprotease 9 (MMP9), and intereukin-6 (IL-6). Importantly, serum levels of most of the tested protein biomarkers were the highest at 3 months after exposures. We also detected elevated autoantibody titers of proteins related to vascular and neuroglia-specific proteins at 3 months after exposures as compared to baseline levels. These findings suggest that repeated exposures to subconcussive blasts can induce molecular changes indicating not only neuron and glia damage, but also vascular changes and inflammation that are detectable for at least 3 months after exposures whereas elevated titers of autoantibodies against vascular and neuroglia-specific proteins can indicate an autoimmune process.

Heterologous Immune Responses of Serum IgG and Secretory IgA Against the Spike Protein of Endemic Coronaviruses During Severe COVID-19.

Defining immune correlates of disease severity is important to better understand the immunopathogenesis in COVID-19. Here we made use of a protein microarray platform to detect IgG- and IgA-reactive antibodies in sera and saliva respectively, and assess cross-reactivity between SARS-CoV-2 and endemic coronaviruses (eCoVs). IgG responses against the full protein of spike, but not the S1 subunit, were significantly higher in convalescent sera of patients with severe disease compared to mild disease and healthy controls. In addition, we detected reactivity of secretory IgA to eCoVs in saliva of patients with severe disease, not present in patients with moderate disease or seropositive healthy controls. These heterologous immune responses are in line with non-protective cross-reactivity, and support a potential role for immune imprinting in the pathogenesis of severe COVID-19.

Novel Autoantibodies in Idiopathic Small Fiber Neuropathy.

ObjectiveSmall fiber neuropathy (SFN) is clinically and etiologically heterogeneous. Although autoimmunity has been postulated to be pathophysiologically important in SFN, few autoantibodies have been described. We aimed to identify autoantibodies associated with idiopathic SFN (iSFN) by a novel high‐throughput protein microarray platform that captures autoantibodies expressed in the native conformational state.MethodsSera from 58 SFN patients and 20 age‐ and gender‐matched healthy controls (HCs) were screened against >1,600 immune‐related antigens. Fluorescent unit readout and postassay imaging were performed, followed by composite data normalization and protein fold change (pFC) analysis. Analysis of an independent validation cohort of 33 SFN patients against the same 20 HCs was conducted to identify reproducible proteins in both cohorts.ResultsNine autoantibodies were screened with statistical significance and pFC criteria in both cohorts, with at least 50% change in serum levels. Three proteins showed consistently high fold changes in main and validation cohorts: MX1 (FC = 2.99 and 3.07, respectively, p = 0.003, q = 0.076), DBNL (FC = 2.11 and 2.16, respectively, p = 0.009, q < 0.003), and KRT8 (FC = 1.65 and 1.70, respectively, p = 0.043, q < 0.003). Further subgroup analysis into iSFN and SFN by secondary causes (secondary SFN) in the main cohort showed that MX1 is higher in iSFN compared to secondary SFN (FC = 1.61 vs 0.106, p = 0.009).InterpretationNovel autoantibodies MX1, DBNL, and KRT8 are found in iSFN. MX1 may allow diagnostic subtyping of iSFN patients. ANN NEUROL 2022;91:66–77

Identification of Tumor Antigens in Ovarian Cancers Using Local and Circulating Tumor-Specific Antibodies.

Ovarian cancers include several disease subtypes and patients often present with advanced metastatic disease and a poor prognosis. New biomarkers for early diagnosis and targeted therapy are, therefore, urgently required. This study uses antibodies produced locally in tumor-draining lymph nodes (ASC probes) of individual ovarian cancer patients to screen two separate protein microarray platforms and identify cognate tumor antigens. The resulting antigen profiles were unique for each individual cancer patient and were used to generate a 50-antigen custom microarray. Serum from a separate cohort of ovarian cancer patients encompassing four disease subtypes was screened on the custom array and we identified 28.8% of all ovarian cancers, with a higher sensitivity for mucinous (50.0%) and serous (40.0%) subtypes. Combining local and circulating antibodies with high-density protein microarrays can identify novel, patient-specific tumor-associated antigens that may have diagnostic, prognostic or therapeutic uses in ovarian cancer.

Optimization of High-Throughput Multiplexed Phenotyping of Extracellular Vesicles Performed in 96-Well Microtiter Plates

Extracellular vesicles (EVs) are promising biomarkers for several diseases, however, no simple and robust methods exist to characterize EVs in a clinical setting. The EV Array analysis is based on a protein microarray platform, where antibodies are printed onto a solid surface that enables the capture of small EVs (sEVs) by their surface or surface-associated proteins. The EV Array analysis was transferred to an easily handled microtiter plate (MTP) format and a range of optimization experiments were performed within this study. The optimization was performed in a comprehensive analytical setup where the focus was on the selection of additives added to spotting-, blocking-, and incubation buffers as well as the storage of printed antibody arrays under different temperatures from one day to 12 weeks. After ending the analysis, the stability of the fluorescent signal was investigated at different storage conditions for up to eight weeks. The various parameters and conditions tested within this study were shown to have a high influence on each other. The reactivity of the spots was found to be preserved for up to 12 weeks when stored at room temperature and using blocking procedure IV in combination with trehalose in the spotting buffer. Similar preservation could be obtained using glycerol or sciSPOT D1 in the spotting buffers, but only if stored at 4 °C after blocking procedure I. Conclusively, it was found that immediate scanning of the MTPs after analysis was not critical if stored dried, in the dark, and at room temperature. The findings in this study highlight the necessity of performing optimization experiments when transferring an established analysis to a new technological platform.

A digital protein microarray for COVID-19 cytokine storm monitoring.

Despite widespread concern regarding cytokine storms leading to severe morbidity in COVID-19, rapid cytokine assays are not routinely available for monitoring critically ill patients. We report the clinical application of a digital protein microarray platform for rapid multiplex quantification of cytokines from critically ill COVID-19 patients admitted to the intensive care unit (ICU) at the University of Michigan Hospital. The platform comprises two low-cost modules: (i) a semi-automated fluidic dispensing/mixing module that can be operated inside a biosafety cabinet to minimize the exposure of the technician to the virus infection and (ii) a 12-12-15 inch compact fluorescence optical scanner for the potential near-bedside readout. The platform enabled daily cytokine analysis in clinical practice with high sensitivity (<0.4 pg mL-1), inter-assay repeatability (∼10% CV), and rapid operation providing feedback on the progress of therapy within 4 hours. This test allowed us to perform serial monitoring of two critically ill patients with respiratory failure and to support immunomodulatory therapy using the selective cytopheretic device (SCD). We also observed clear interleukin-6 (IL-6) elevations after receiving tocilizumab (IL-6 inhibitor) while significant cytokine profile variability exists across all critically ill COVID-19 patients and to discover a weak correlation between IL-6 to clinical biomarkers, such as ferritin and C-reactive protein (CRP). Our data revealed large subject-to-subject variability in patients' response to COVID-19, reaffirming the need for a personalized strategy guided by rapid cytokine assays.

Detection of putative autoantibodies in systemic lupus erythematous using a novel native-conformation protein microarray platform.

Conventional immunoassays detect autoantibodies related to systemic lupus erythematosus (SLE) via recognition of epitopes on autoantigens expressed in their denatured rather than native conformational state, casting difficulty in evaluating the genuine pathogenicity of the autoantibodies. We aimed to use a novel high-throughput protein microarray platform to identify autoantibodies against native autoantigens in SLE sera. Sera from SLE patients and those of gender-, age-, and ethnicity-matched healthy controls (HC) were screened against more than 1,600 immune-related antigens of native conformation. The relative fluorescent unit readout from post-assay imaging were subjected to bioinformatics pre-processing and composite normalization. A penetrance fold change (pFC) analysis between SLE and HC samples shortlisted 50 autoantigens that were subjected to an unsupervised cluster analysis. Correlations between the pFC of putative autoantigens and clinical parameters including SLE disease activity index (SLEDAI-2K) and recent SLE flares were explored. 381 autoantigens were identified when 15 SLE and 15 HC serum samples were compared. The top 20 autoantigens which elicited autoantibody responses in SLE sera filtered based on the highest pFC were further analyzed. Autoantigens which the putative autoantibodies reacted against are those involved in chromatin organization such as DEK, regulation of transcription activity including REOX4 and ELF4, and negative regulation of NFkB activity such as TRIB3. Additionally, the pFC of these autoantibodies significantly and positively correlated with SLEDAI-2K and recent SLE flares. A high-throughput protein microarray platform allows detection and quantification of putative lupus-related autoantibodies which are of potential pathophysiological and prognostic significance in SLE patients.

Development of a comprehensive protein microarray for immunoglobulin E profiling in horses with severe asthma.

BackgroundSevere asthma in horses, known as severe equine asthma (SEA), is a prevalent, performance‐limiting disease associated with increased allergen‐specific immunoglobulin E (IgE) against a range of environmental aeroallergens.ObjectiveTo develop a protein microarray platform to profile IgE against a range of proven and novel environmental proteins in SEA‐affected horses.AnimalsSix SEA‐affected and 6 clinically healthy Warmblood performance horses.MethodsDeveloped a protein microarray (n = 384) using protein extracts and purified proteins from a large number of families including pollen, bacteria, fungi, and arthropods associated with the horses, environment. Conditions were optimized and assessed for printing, incubation, immunolabeling, biological fluid source, concentration techniques, reproducibility, and specificity.ResultsThis method identified a number of novel allergens, while also identifying an association between SEA and pollen sensitization. Immunolabeling methods confirmed the accuracy of a commercially available mouse anti‐horse IgE 3H10 source (R 2 = 0.91). Biological fluid source evaluation indicated that sera and bronchoalveolar lavage fluid (BALF) yielded the same specific IgE profile (average R 2 = 0.75). Amicon centrifugal filters were found to be the most efficient technique for concentrating BALF for IgE analysis at 40‐fold. Overnight incubation maintained the same sensitization profile while increasing sensitivity. Reproducibility was demonstrated (R 2 = 0.97), as was specificity using protein inhibition assays. Arthropods, fungi, and pollens showed the greatest discrimination for SEA.Conclusions and Clinical ImportanceWe have established that protein microarrays can be used for large‐scale IgE mapping of allergens associated with the environment of horses. This technology provides a sound platform for specific diagnosis, management, and treatment of SEA.

Diagnostic Potential of Transferrin Glycoforms-A Lectin-Based Protein Microarray Approach.

Disease or a specific condition may cause alteration of human transferrin (hTf) glycosylation pattern. A specific analytical platform, lectin-based protein microarray, is designed and optimized for the investigation of hTf glycans, attached to the protein core in their native form. hTf molecules isolated from healthy persons of different age, diabetes mellitus type 2 (T2DM) or colorectal carcinoma (CRC) patients are used for method validation. Reliability of the results is ensured by three criteria for the evaluation of hTf-lectin interactions: i) signal-to-noise ratio above 3, ii) signal intensity above 250 arbitrary units, and iii) hTf concentration ensuring high sensitivity of the assay. Six lectins, out of fourteen tested, satisfy the criteria. hTf is spotted at concentration of 50µg mL-L . When physiological samples (isolated hTf) are analyzed, the highest potential to differentiate between population groups expresses Aleuria aurantia (AAL), Triticum vulgaris (WGA) and Phaseolus vulgaris (PHA-E) lectins. The initial amount of hTf which can be analyzed is very low (75 pg). Results confirm that a very sensitive, high-throughput lectin-based protein microarray platform can be formulated to detect changes in hTf glycan structures which can be considered as biomarkers of ageing or a disease.

Elevated blood plasma levels of tissue factor-bearing extracellular vesicles in patients with atrial fibrillation

BackgroundThe risk of thrombus formation in the left atrial appendage (LAA) in patients with atrial fibrillation (AF) may result from blood stasis, local endocardial changes, and/or changed blood composition. Extracellular vesicles (EVs), especially subtypes exposing tissue factor (TF), have procoagulant capacity. We hypothesized that blood concentrations of TF-bearing EVs and other procoagulant biomarkers are elevated in AF patients, particularly in the LAA lumen. MethodsFrom 13 AF patients and 12 controls a venous blood sample was drawn prior to cardiac surgery. Intraoperatively, venous blood and blood directly from the LAA was drawn. Plasma levels of EVs, including TF- and cell type specific antigen-bearing EVs, were measured using a protein microarray platform. Plasma levels of TF, von Willebrand factor (vWF), cell free deoxyribonucleic acid (cf-DNA), procoagulant phospholipids (PPLs), and total submicron particles were also evaluated. ResultsSignificantly higher EV levels, including a several-fold higher median level of TF-bearing EVs were measured in AF patients compared with controls. Median concentrations of TF and vWF were approximately 40% and 30% higher, respectively, in the AF group than in the control group, while no significant differences in levels of cf-DNA, PPLs, or total submicron particles were observed. No significant differences in levels of any of the measured analytes were observed between intraoperative venous and LAA samples. ConclusionsIncreased plasma concentrations of TF in AF patients are accompanied and probably at least partly explained by increased levels of TF-bearing EVs, which may be mechanistically involved in increased thrombogenicity in AF patients.

Ibrutinib inhibition of ERBB4 reduces cell growth in a WNT5A-dependent manner

Alterations in ERBB family members have been associated with many tumor malignancies. EGFR and ERBB2 have been extensively explored in clinical oncology and several drugs currently target them therapeutically. However, the significance of ERBB4 as a potential therapeutic target remains mostly unexplored, even though ERBB4 is overexpressed or mutated in many solid tumors. Using a unique functional protein microarray platform, we found that ibrutinib inhibits ERBB4 activity in the same nM range as its canonical target, BTK. Cell-based assays revealed that ibrutinib treatment inhibited cell growth and decreased phosphorylation of ERBB4 and downstream targets MEK and ERK in cancer cell lines with high levels of endogenous ERBB4. In vivo, ibrutinib-responsive mouse xenograft tumors showed decreased tumor volumes with ibrutinib treatment. Interestingly, global gene expression comparisons between responsive and non-responsive cells identified a signature featuring the WNT pathway that predicts growth responsiveness to ibrutinib. Non-responsive ERBB4-expressing cell lines featured elevated activity of the WNT pathway, through the overexpression of WNT5A. Moreover, inhibition of WNT5A expression led to an ibrutinib response in non-responsive cell lines. Our data show that inhibiting ERBB4 reduces cell growth in cells that have low WNT5A expression and reveal a link between the ERBB4 and WNT pathways.

Plasmonic nanograting enhanced fluorescence for protein microarray analysis of carcinoembryonic antigen (CEA)

The plasmonic nanograting substrate is demonstrated as a superior promising candidate for developing high-throughput protein microarray platforms.

Abstract IA27: A novel cancer-testis antigen protein microarray for autoimmune profiling in cancers: Diagnosis, stratification, and treatment monitoring

Abstract The cancer-testis antigens are a group of unrelated proteins aberrantly expressed in various cancers in adult somatic tissues. This aberrant expression can trigger spontaneous immune responses, a phenomenon exploited for the development of disease markers and therapeutic vaccines. However, expression levels often vary amongst patients presenting the same cancer type, and these antigens are therefore unlikely to be individually viable as diagnostic or prognostic markers. Nevertheless, patterns of antigen expression may provide correlates of specific cancer types and disease progression. This paper will describe the development and application of a novel, readily customizable cancer-testis antigen microarray platform, together with robust bioinformatics tools, which enable quantification of anti-cancer-testis antigen autoantibody profiles in patient sera. By exploiting the high affinity between autoantibodies and tumor antigens, our highly multiplexed protein microarray platform gives linearity of response over 4 orders of magnitude and an autoantibody quantitation limit in the pg/mL range. By using oriented attachment of folded, recombinant antigens and a polyethylene glycol microarray surface coating, our platform gives minimal non-specific antibody binding. Unlike other proteomics methods, which typically use lower affinity interactions between monoclonal antibodies and tumor antigens for detection, the high sensitivity and specificity realized using our autoantibody-based approach may facilitate the development of better cancer biomarkers, as well as potentially enabling pre-symptomatic diagnosis. We will illustrate the usage of our platform by monitoring the time-dependent response of melanoma patient cohorts to an experimental therapeutic NY-ESO-1-based cancer vaccine, to combination BRAF and MEK inhibitor treatment, and to anti-CTLA4/anti-PD1 vaccination. Citation Format: Jonathan Blackburn. A novel cancer-testis antigen protein microarray for autoimmune profiling in cancers: Diagnosis, stratification, and treatment monitoring [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA27.

Abstract A78: Antibody profiling of melanoma patients using a custom protein microarray platform

Abstract There is substantial evidence that the mainly tumor-restricted aberrant expression of the highly immunogenic cancer-testis (CT) antigens across several different cancer types makes them attractive cancer diagnostic and predictive biomarkers. Therefore, we aimed to measure differences in CT antigen-specific antibody repertoires between melanoma patient samples, and assess whether these could identify novel diagnostic or prognostic biomarkers, which could aid in the detection and management of cancer. In order to detect and quantify CT-antigen-specific antibodies circulating in the serum or plasma of cancer patients, we co-developed a novel CT antigen microarray platform. This tool represents a high-throughput means of profiling antibody repertoires against over 100 cancer-restricted antigens in a highly reproducible manner with high sensitivity (detection limit: 10-100pg/ml) and specificity. In addition, it provides a significant advantage over tissue-based approaches, due to the ease of sample access without the need for invasive procedures. We carried out a retrospective serological study of antibody titers across a large cohort of eighty-eight mainly advanced malignant melanoma patients (8% unknown stage, 2% stage I, 10% stage II, 40% stage III and 40% stage IV) undergoing a variety of distinct cancer treatments (surgery, chemotherapy, radiotherapy, immunotherapy or none), using our recently developed and validated novel CT antigen microarray platform. We successfully identified abundant antibody titers towards two leading CT antigens, NY-ESO-1 and CTAG2 - a highly homologous pair - expressed across 50% (n = 44/88) and 45% (n = 40/88) of all patients, respectively, with reported diagnostic and clinical efficacy predictive biomarker potential. Furthermore, the overlap in antibody titers of this pair, most likely due to a shared immunogenic epitope, validated our array platform, showing consistency amongst our obtained results across patients. Additional abundant titers were found across fifteen further antigens, including several members of the melanoma antigen (MAGE) family, such as MAGEB1 (n = 30/88, 34%), MAGEB6 (n = 29/88, 33%), MAGEB5 (n = 26/88, 29%), MAGEA3 (n = 26/88, 29%) and MAGEA11 (n = 24/88, 27%). Abundant antibody titers towards at least one of these leading seventeen cancer antigens were detected across 82% of this cohort (n = 72/88), thus emphasizing the potential uses of these as diagnostic or prognostic biomarkers, warranting further larger studies. Furthermore, healthy individuals showed no detectable cancer-antigen-specific antibody titers, therefore highlighting the specificity of this tool in a cancer setting. In conclusion, we showed that our novel protein microarray platform represents a sensitive, high-throughput and customizable means to detect and quantify the presence of large panels of cancer-specific human antibodies in serum, obtaining consistently robust, high quality and reproducible data, and demonstrating its potential feasibility and inferred biological significance. Citation Format: Jessica Duarte, Janique Peyper, Katherine Woods, Simon Chang-Hao Tsao, Jonathan Cebon, Jonathan Blackburn. Antibody profiling of melanoma patients using a custom protein microarray platform. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A78.

Validation of Biomarker Proteins Using Reverse Capture Protein Microarrays.

Genomics has revolutionized large-scale and high-throughput sequencing and has led to the discovery of thousands of new proteins. Protein chip technology is emerging as a miniaturized and highly parallel platform that is suited to rapid, simultaneous screening of large numbers of proteins and the analysis of various protein-binding activities, enzyme substrate relationships, and posttranslational modifications. Specifically, reverse capture protein microarrays provide the most appropriate platform for identifying low-abundance, disease-specific biomarker proteins in a sea of high-abundance proteins from biological fluids such as blood, serum, plasma, saliva, urine, and cerebrospinal fluid as well as tissues and cells obtained by biopsy. Samples from hundreds of patients can be spotted in serial dilutions on many replicate glass slides. Each slide can then be probed with one specific antibody to the biomarker of interest. That antibody's titer can then be determined quantitatively for each patient, allowing for the statistical assessment and validation of the diagnostic or prognostic utility of that particular antigen. As the technology matures and the availability of validated, platform-compatible antibodies increases, the platform will move further into the desirable realm of discovery science for detecting and quantitating low-abundance signaling proteins. In this chapter, we describe methods for the successful application of the reverse capture protein microarray platform for which we have made substantial contributions to the development and application of this method, particularly in the use of body fluids other than serum/plasma.

Changes in heterosubtypic antibody responses during the first year of the 2009 A(H1N1) influenza pandemic.

Seropositivity to avian influenza (AI) via low-level antibody titers has been reported in the general population and poultry-exposed individuals, raising the question whether these findings reflect true infection with AI or cross-reactivity. Here we investigated serological profiles against human and avian influenza viruses in the general population using a protein microarray platform. We hypothesized that higher antibody diversity across recent H1 and H3 influenza viruses would be associated with heterosubtypic reactivity to older pandemic- and AI viruses. We found significant heterogeneity in antibody profiles. Increased antibody diversity to seasonal influenza viruses was associated with low-level heterosubtypic antibodies to H9 and H7, but not to H5 AI virus. Individuals exposed to the recent 2009 A(H1N1) pandemic showed higher heterosubtypic reactivity. We show that there is a complex interplay between prior exposures to seasonal and recent pandemic influenza viruses and the development of heterosubtypic antibody reactivity to animal influenza viruses.

Exploration of high-density protein microarrays for antibody validation and autoimmunity profiling

High-density protein microarrays of recombinant human protein fragments, representing 12,412 unique Ensembl Gene IDs, have here been produced and explored. These protein microarrays were used to analyse antibody off-target interactions, as well as for profiling the human autoantibody repertoire in plasma against the antigens represented by the protein fragments. Affinity-purified polyclonal antibodies produced within the Human Protein Atlas (HPA) were analysed on microarrays of three different sizes, ranging from 384 antigens to 21,120 antigens, for evaluation of the antibody validation criteria in the HPA. Plasma samples from secondary progressive multiple sclerosis patients were also screened in order to explore the feasibility of these arrays for broad-scale profiling of autoantibody reactivity. Furthermore, analysis on these near proteome-wide microarrays was complemented with analysis on HuProt™ Human Proteome protein microarrays. The HPA recombinant protein microarray with 21,120 antigens and the HuProt™ Human Proteome protein microarray are currently the largest protein microarray platforms available to date. The results on these arrays show that the Human Protein Atlas antibodies have few off-target interactions if the antibody validation criteria are kept stringent and demonstrate that the HPA-produced high-density recombinant protein fragment microarrays allow for a high-throughput analysis of plasma for identification of possible autoantibody targets in the context of various autoimmune conditions.