A proteome-wide immuno-mass spectrometric identification of serum autoantibodies.

BACKGROUND: Autoantibodies are produced when tolerance to self-antigens is broken and they can be mediators of tissue injury and systemic inflammation. They are excellent biomarkers because they are minimally invasive to screen and are highly abundant in serum due to limited proteolysis and slow clearance. Conventionally used methods of identifying autoantibodies in patient sera include indirect immunofluorescence, enzyme-linked immunoabsorbent assays (ELISAs) and protein microarrays. Here we present a novel proteome-wide immuno-mass spectrometric method to identify serum autoantibody targets. METHODS: Serum samples from patients with inflammatory bowel disease (IBD) were analyzed by ELISA for the presence of autoantibodies to CUB and zona pellucida-like domain-containing protein 1 (CUZD1). Protein was extracted from the human pancreas as well as 16 other human tissues to make a complex tissue lysate protein mixture. Antibodies in patient sera were immobilized and purified on protein G magnetic beads and subsequently incubated with pancreatic lysate containing CUZD1 or the aforementioned complex tissue lysate. After extensive washing, antibody-bound protein antigens were trypsin-digested and identified using shotgun mass spectrometry. RESULTS: The protocol was optimized for the immunoaffinity purification of autoantibody targets from tissue lysate, using CUZD1 from pancreatic lysate and anti-CUZD1 autoantibodies present in IBD patient serum as a proof-of-concept. Pancreatic secretory granule membrane major glycoprotein 2, whose autoantibodies are a known biomarker of Crohn's disease, was also immunoprecipitated from IBD patient serum, as an additional internal positive control. CONCLUSIONS: This study demonstrates the effectiveness of a proteomic approach to identify serum autoantibody targets, using immunoaffinity purification followed by tandem mass spectrometry. Our methodology is applicable for proteome-wide analysis of autoantibody targets in a wide variety of clinical settings.

Correction to: A proteome-wide immuno-mass spectrometric identification of serum autoantibodies.

[This corrects the article DOI: 10.1186/s12014-019-9246-0.].

Optimizing cancer immunotherapy: Is it time for personalized predictive biomarkers?

Cancer immunotherapy, a treatment that selectively augments a patient's anti-tumor immune response, is a breakthrough advancement in personalized medicine. A subset of cancer patients undergoing immunotherapy have displayed robust and long-lasting therapeutic responses. Currently, the spotlight is on the use of blocking antibodies against the T-cell checkpoint molecules, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programed cell death-1 (PD-1)/programed death-ligand 1 (PD-L1), which have been effectively used to combat many cancers types. Despite the overall enthusiasm, immune checkpoint blockade inhibitors suffer from significant limitations such as high cost, serious toxicity in a substantial proportion of patients, and a response rate as low as 10%-40% in some clinical trials. Consequently, there is an urgent and unmet medical need for companion biomarkers that could both predict the response of individual patients to these therapies, and provide the means for precise monitoring of their therapeutic outcome. In this era of precision medicine, predictive biomarkers are a hot commodity because they can effectively separate responders from non-responders, and spare non-responders from serious therapy-related toxicity. Emerging predictive biomarkers for immune checkpoint blockade are: PD-L1 expression, increased amounts of tumor-infiltrating lymphocytes, increased mutational load and mismatch repair deficiency. Other well-studied biomarkers include inflammatory infiltrate, absolute lymphocyte count and lactate dehydrogenase levels. We review recent progress on predictive cancer biomarkers in immunotherapy, with a special emphasis on serum autoantibodies that have the potential to be personalized for optimal clinical outcomes.

Perspectives of Older Adults on COVID-19 and Influenza Vaccination in Ontario, Canada.

INTRODUCTION/OBJECTIVES: Addressing vaccine hesitancy has become an increasingly important public health priority in recent years. There is a paucity of studies that have focused on vaccine hesitancy among older adults, who are known to be at greater risk of complications from infections such as COVID-19. We aim to explore the attitudes and beliefs of older adults regarding COVID-19 and influenza vaccines in Toronto, Ontario. METHODS: Older adults enrolled in the Student Senior Isolation Prevention Partnership (SSIPP) program at the University of Toronto were contacted to participate in a phone survey and semi-structured interview. Survey data was analyzed descriptively, and attitude toward vaccination was compared between sociodemographic groups by using Fisher's exact test. Interview audio files were transcribed verbatim and analyzed inductively for themes and sub-themes. RESULTS: All thirty-three (100%) older adults reported that they had received the first and second doses of the COVID-19 vaccine. Twenty-six (78.8%) participants reported intent to get vaccinated against influenza or had already received the influenza vaccine that year. Notably, only 2 out 7 (28.6%) individuals who did not plan to get vaccinated against influenza believed that vaccines offered by health providers are beneficial and only 3 out of 7 (42.9%) agreed that getting vaccines is a good way to protect oneself from disease. No other significant differences in attitudes among participants were found when compared by gender, ethnicity, or education level. The qualitative data analysis of interview transcripts identified 5 themes that impact vaccine decision making: safety, trust, mistrust, healthcare experience, and information dissemination and education. CONCLUSIONS: Our data showed that older adults in the SSIPP program generally had positive views toward vaccination, especially toward the COVID-19 vaccines. However, several concerns regarding the effectiveness of the vaccines were brought up in interviews, such as the speed at which the vaccines were produced and the inconsistency in government messaging.

Predicting response and toxicity to PD-1 inhibition using serum autoantibodies identified from immuno-mass spectrometry.

Background: Validated biomarkers are needed to identify patients at increased risk of immune-related adverse events (irAEs) to immune checkpoint blockade (ICB). Antibodies directed against endogenous antigens can change after exposure to ICB. Methods: Patients with different solid tumors stratified into cohorts received pembrolizumab every 3 weeks in a Phase II trial (INSPIRE study). Blood samples were collected prior to first pembrolizumab exposure (baseline) and approximately 7 weeks (pre-cycle 3) into treatment. In a discovery analysis, autoantibody target immuno-mass spectrometry was performed in baseline and pre-cycle 3 pooled sera of 24 INSPIRE patients based on clinical benefit (CBR) and irAEs. Results: Thyroglobulin (Tg) and thyroid peroxidase (TPO) were identified as the candidate autoantibody targets. In the overall cohort of 78 patients, the frequency of CBR and irAEs from pembrolizumab was 31% and 24%, respectively. Patients with an anti-Tg titer increase ≥1.5x from baseline to pre-cycle 3 were more likely to have irAEs relative to patients without this increase in unadjusted, cohort adjusted, and multivariable models (OR=17.4, 95% CI 1.8-173.8, p=0.015). Similarly, patients with an anti-TPO titer ≥ 1.5x from baseline to pre-cycle 3 were more likely to have irAEs relative to patients without the increase in unadjusted and cohort adjusted (OR=6.1, 95% CI 1.1-32.7, p=0.035) models. Further, the cohort adjusted analysis showed patients with anti-Tg titer greater than median (10.0 IU/mL) at pre-cycle 3 were more likely to have irAEs (OR=4.7, 95% CI 1.2-17.8, p=0.024). Patients with pre-cycle 3 anti-TPO titers greater than median (10.0 IU/mL) had a significant difference in overall survival (23.8 vs 11.5 months; HR=1.8, 95% CI 1.0-3.2, p=0.05). Conclusions: Patient increase ≥1.5x of anti-Tg and anti-TPO titers from baseline to pre-cycle 3 were associated with irAEs from pembrolizumab, and patients with elevated pre-cycle 3 anti-TPO titers had an improvement in overall survival.

Serum PD-1 Is Elevated after Pembrolizumab Treatment but Has No Predictive Value.

Immune-checkpoint blockade (ICB) uses antibody targeting of specific inhibitory receptors and ligands. The major limitations of ICB, such as high cost, limited success rate, and immune-related adverse events (irAE), highlight the need for predictive biomarkers. We analyzed pre-immunotherapy and post-immunotherapy serum samples of 24 patients treated with pembrolizumab for changes in PD-1 and over 1,000 additional protein markers using a multiplex proximity extension assay (PEA) to identify potential predictive biomarkers of response and/or toxicity. Candidates were selected based on the criteria that at least 2 patients within any of 3 patient groups (responders without irAEs, responders with irAEs, or nonresponders with irAEs) had either a ≥4-fold increase or 4-fold decrease in expression post-immunotherapy. Female and male control samples were used as technical duplicates. A patient group with no response and no irAEs was used to exclude candidates. Following treatment with pembrolizumab, there was a relative increase of PD-1 in the serum of all patients, compared with controls (average 4.4-fold). We identified 7 additional serum proteins that met our candidate selection criteria. These candidate markers did not have any significant association with response or toxicity to pembrolizumab. Overall, we show that serum PD-1 increases post-therapy with pembrolizumab treatment but has no predictive value for response or toxicity in this small set of patients.