**3. Diagnostic utility of autoantibody biomarkers**

### **3.1. Early detection of disease**

affected in inflammatory bowel disease, while myelin - a fatty substance that protects nerve fibres in the brain and spinal cord - is destroyed in Multiple Sclerosis. In RA, connective tissues are affected and in SLE, auto-reactivity usually occurs in skin, heart and lung tissues. Sjogren's syndrome occurs when autoantibodies target secretory glands that produce tears

**Figure 2.** Gender-dependent susceptibility to autoimmune diseases, infectious diseases, inflammatory diseases and

In other diseases, however, the functional role of autoantibodies is less clear. For example, in neurodegenerative diseases such as Parkinson's and Alzheimer's Diseases, increased cellular toxicity is caused by the accumulation and aggregation of misfolded proteins, which might also result in the generation of protective autoantibodies in some patients. For example, in Parkinson's Disease (PD), the protein alpha-synuclein misfolds and aggregates to form Lewy bodies; these bodies form in the brain tissues of PD patients and infiltrate the neurons, disrupting signalling process in the brain. A recent study reported that a defined set of epitopes derived from alpha-synuclein drive cytotoxic T-cell responses in people with PD [23], whilst another recent study reported a decline in anti-alpha-synuclein autoantibodies in PD patients compared to controls, suggesting that in some patients anti-alpha-synuclein autoantibodies

Similarly, in Alzheimer's Disease (AD), the microtubule-associated protein Tau accumulates and aggregates in neurons causing neuronal degeneration. Tau also accumulates and

and saliva, causing extreme dryness and other complications [22].

might play a protective role [24].

cancers.

164 Autoantibodies and Cytokines

Autoantibody production is a key indicator of many diseases and has emerged as an important tool in predicting onset of a number of diseases. Autoantibodies are in principle detectable many years before manifestation of disease or symptoms and have been observed in an ever-widening range of disease areas, which makes novel autoantibodies attractive plausible biomarkers for early diagnosis of a broader spectrum of diseases now. Known autoantibody biomarkers have been reported to predate symptoms in Sjogren's syndrome, rheumatoid arthritis, Alzheimer's disease and cancers, as discussed below:

Sjogren's syndrome is an autoimmune disease that affects parts of the body which produce secretions such as tears and saliva. The symptoms overlap with other autoimmune conditions and can range from mild to severe, causing nausea, fatigue, joint pain as well as excessive dryness of the eyes and mouth. Autoantibodies attack cells in mucous membranes and moisture-secreting glands of the eyes and mouth, causing dryness, irritation and pain. A study published in 2015 concluded that autoantibodies are present up to 18–20 years before the diagnosis of primary Sjogren's syndrome [27]. A total of five autoantibodies were analysed, namely antinuclear antibodies, rheumatoid factor and autoantibodies against Ro 60/SSA, Ro 52/SSA, and La/SSB, with 81% of the patients who became seropositive after diagnosis having autoantibodies in pre-diagnostic serum samples. More importantly, these autoantibodies were present in the earliest available serum sample of 95% of the patients who expressed autoantibodies before diagnosis and before the onset of first symptoms [27].

detection of six autoantibodies directed against prostate cancer with PSA levels, increasing the accuracy of detection from 65% using PSA alone to 81% with both methods [37]. A similar outcome was achieved in breast cancer diagnosis using a panel of six autoantigens to detect ductal carcinoma *in situ* and lung cancer with specificity of 85 and 92%, respectively [38]. It is thus evident that having an increased level of specific circulating autoantibodies may reflect the overall state of the immune response of an individual, whilst the presence of such autoantibodies in otherwise healthy individuals might be an indicator of future autoimmune or

Autoantibody-Based Diagnostic Biomarkers: Technological Approaches to Discovery and Validation

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167

An accurate pathology diagnosis is of central importance in precision medicine, since it should guide choice of the most effective treatment and management regimens. Reliable biomarkers for monitoring and prediction of disease course, stage and progression will be therefore invaluable, particularly in therapeutic decision-making to treat disease at an early stage. Current research has not only established the presence of autoantibodies in several diseases but has also shown that they have the potential to be used as biomarkers capable of diagnosis and staging various degrees of pathology. One such example is Type 1 insulindependent diabetes - a chronic autoimmune disease that impairs the insulin-producing beta cells in the pancreas, preventing the body from producing enough insulin to regulate blood glucose levels. This disease can be characterised into well-defined stages, and the rate of progression to symptomatic disease can be predicted with appreciable accuracy [39]. Stage 1 is defined by the presence of two or more islet autoantibodies and progressing at a variable rate to a second stage of glucose intolerance or dysglycaemia, before becoming clinically

In another example, Cai et al. reported that anti-p53 antibodies develop several years before the clinical diagnosis of certain cancers and suggested that monitoring the change of serum p53 antibodies before and after treatment of patients diagnosed with oesophageal carcinoma with radiotherapy would be useful for evaluating the prognosis and response to the treatment. This study showed that the positive rate of p53-antibodies in patients with oesophageal carcinoma was related to histological grade, stage of the disease and lymph node metastasis but not to age, gender, or site of tumour formation. The study also reported a significant difference in the level of serum p53 antibodies before and after radiotherapy treatment, with the positive rate of p53 antibodies in patients who responded to radiotherapy being much lower than the patients who did not respond to radiotherapy [40]. A separate study by Shimada et al. showed that seropositive oesophageal squamous cell carcinoma patients, whose serum anti-p53 titre did not decrease after surgery, exhibited worse prognosis than patients who showed seroconversion. Thus, a correlative study between the level of tumour autoantibodies and the overall survival outcome of cancer patients (reflected in the change in tumour status or tumour burden related to the therapy) could be extremely informative for evaluating therapeutic interventions [41]. Stage-specific autoantibody biomarkers screening is thus in principle useful in predicting onset of disease, thereby providing an opportunity to intervene

other disorders.

symptomatic (stage 3).

**3.2. Disease staging and treatment monitoring**

and delay or prevent the onset of clinical symptoms.

RA is a chronic autoimmune disease characterised by inflammation of synovial joints, leading to joint erosion and deterioration. Rheumatoid factor and anti-cyclic citrullinated peptide (anti-CCP) are detected in the blood of 80% and 60–70% of RA-affected individuals, respectively. Anti-CCP autoantibodies were detected in some patient sera samples 12–14 years prior to the development of RA and 34–40% of the RA patients were anti-CCP positive prior to disease onset [28].

The presence of autoantibodies has also been implicated in AD. Aβ-autoantibodies were reported to show promise as an effective blood biomarker for AD and a positive association between Aβ-autoantibody titres and cognitive status have been reported [29]. Glial autoantibody markers to glutamate were detected in the plasma of AD patients and, interestingly, the level of that autoantibody in patients with moderate and severe dementia was 2-fold higher than that in patients with mild dementia [30]. In addition, autoantibodies to ATP synthase were reported to be found frequently in the sera of AD patients but not in age-matched healthy subjects or in patients with Parkinson's disease or atherosclerosis, suggesting anti-ATP synthase autoantibodies could be a specific biomarker for AD [29].

In addition to autoimmune diseases, multiple studies have described autoantibody production prior to cancer diagnosis, including in lung cancer [31], prostate cancer [32] and ovarian cancer [33]. Autoantibody production in cancer is thought to be a product of immunosurveillance - a process in which the body's own systems recognise and eliminate abnormal cells during early tumorigenesis [34] - suggesting that detection of disease-associated autoantibodies may be feasible in the asymptomatic stages of cancer and may predate the clinical signs of tumour progression by months or years, thus enabling their use in early diagnosis [35].

By way of example, in healthy mammalian cells, cAMP-dependent protein kinase A (PKA) is an intracellular enzyme, while in most cancers it is secreted into the circulatory system as ECPKA. The level of ECPKA was found to be elevated in various stages of a wide range of cancers including bladder, breast, cervical, colon, esophageal, gastric, liver, lung, ovarian, prostate, pancreatic, renal, small bowel, rectal, adenocystic carcinomas, melanoma, sarcoma, thymoma, liposarcoma, and leiomyosarcoma compared with healthy controls [35]. Extracellular protein kinase A (ECPKA) autoantibody is thus a potential serologic autoantibody for early-stage cancers diagnosis since it presents at high levels before surgical removal of solid tumours and diminishes after tumour removal [36]. An ELISA-based test for anti-ECPKA IgG was developed and the sensitivity and specificity of this biomarker for detecting 20 different cancers were reported to be 90 and 87% respectively, with the anti-ECPKA autoantibody being detected in 90% of the patient samples but in only 13% of the control samples [35].

Autoantibody-based screening for a variety of other cancers has also been carried out in laboratory environments. For example, Xie et al. developed a test platform by combining the detection of six autoantibodies directed against prostate cancer with PSA levels, increasing the accuracy of detection from 65% using PSA alone to 81% with both methods [37]. A similar outcome was achieved in breast cancer diagnosis using a panel of six autoantigens to detect ductal carcinoma *in situ* and lung cancer with specificity of 85 and 92%, respectively [38]. It is thus evident that having an increased level of specific circulating autoantibodies may reflect the overall state of the immune response of an individual, whilst the presence of such autoantibodies in otherwise healthy individuals might be an indicator of future autoimmune or other disorders.

#### **3.2. Disease staging and treatment monitoring**

52/SSA, and La/SSB, with 81% of the patients who became seropositive after diagnosis having autoantibodies in pre-diagnostic serum samples. More importantly, these autoantibodies were present in the earliest available serum sample of 95% of the patients who expressed

RA is a chronic autoimmune disease characterised by inflammation of synovial joints, leading to joint erosion and deterioration. Rheumatoid factor and anti-cyclic citrullinated peptide (anti-CCP) are detected in the blood of 80% and 60–70% of RA-affected individuals, respectively. Anti-CCP autoantibodies were detected in some patient sera samples 12–14 years prior to the development of RA and 34–40% of the RA patients were anti-CCP positive prior to

The presence of autoantibodies has also been implicated in AD. Aβ-autoantibodies were reported to show promise as an effective blood biomarker for AD and a positive association between Aβ-autoantibody titres and cognitive status have been reported [29]. Glial autoantibody markers to glutamate were detected in the plasma of AD patients and, interestingly, the level of that autoantibody in patients with moderate and severe dementia was 2-fold higher than that in patients with mild dementia [30]. In addition, autoantibodies to ATP synthase were reported to be found frequently in the sera of AD patients but not in age-matched healthy subjects or in patients with Parkinson's disease or atherosclerosis, suggesting anti-

In addition to autoimmune diseases, multiple studies have described autoantibody production prior to cancer diagnosis, including in lung cancer [31], prostate cancer [32] and ovarian cancer [33]. Autoantibody production in cancer is thought to be a product of immunosurveillance - a process in which the body's own systems recognise and eliminate abnormal cells during early tumorigenesis [34] - suggesting that detection of disease-associated autoantibodies may be feasible in the asymptomatic stages of cancer and may predate the clinical signs of tumour progression by months or years, thus enabling their use in early diagnosis [35].

By way of example, in healthy mammalian cells, cAMP-dependent protein kinase A (PKA) is an intracellular enzyme, while in most cancers it is secreted into the circulatory system as ECPKA. The level of ECPKA was found to be elevated in various stages of a wide range of cancers including bladder, breast, cervical, colon, esophageal, gastric, liver, lung, ovarian, prostate, pancreatic, renal, small bowel, rectal, adenocystic carcinomas, melanoma, sarcoma, thymoma, liposarcoma, and leiomyosarcoma compared with healthy controls [35]. Extracellular protein kinase A (ECPKA) autoantibody is thus a potential serologic autoantibody for early-stage cancers diagnosis since it presents at high levels before surgical removal of solid tumours and diminishes after tumour removal [36]. An ELISA-based test for anti-ECPKA IgG was developed and the sensitivity and specificity of this biomarker for detecting 20 different cancers were reported to be 90 and 87% respectively, with the anti-ECPKA autoantibody being detected in 90% of the patient samples but in only 13% of the control

Autoantibody-based screening for a variety of other cancers has also been carried out in laboratory environments. For example, Xie et al. developed a test platform by combining the

autoantibodies before diagnosis and before the onset of first symptoms [27].

ATP synthase autoantibodies could be a specific biomarker for AD [29].

disease onset [28].

166 Autoantibodies and Cytokines

samples [35].

An accurate pathology diagnosis is of central importance in precision medicine, since it should guide choice of the most effective treatment and management regimens. Reliable biomarkers for monitoring and prediction of disease course, stage and progression will be therefore invaluable, particularly in therapeutic decision-making to treat disease at an early stage. Current research has not only established the presence of autoantibodies in several diseases but has also shown that they have the potential to be used as biomarkers capable of diagnosis and staging various degrees of pathology. One such example is Type 1 insulindependent diabetes - a chronic autoimmune disease that impairs the insulin-producing beta cells in the pancreas, preventing the body from producing enough insulin to regulate blood glucose levels. This disease can be characterised into well-defined stages, and the rate of progression to symptomatic disease can be predicted with appreciable accuracy [39]. Stage 1 is defined by the presence of two or more islet autoantibodies and progressing at a variable rate to a second stage of glucose intolerance or dysglycaemia, before becoming clinically symptomatic (stage 3).

In another example, Cai et al. reported that anti-p53 antibodies develop several years before the clinical diagnosis of certain cancers and suggested that monitoring the change of serum p53 antibodies before and after treatment of patients diagnosed with oesophageal carcinoma with radiotherapy would be useful for evaluating the prognosis and response to the treatment. This study showed that the positive rate of p53-antibodies in patients with oesophageal carcinoma was related to histological grade, stage of the disease and lymph node metastasis but not to age, gender, or site of tumour formation. The study also reported a significant difference in the level of serum p53 antibodies before and after radiotherapy treatment, with the positive rate of p53 antibodies in patients who responded to radiotherapy being much lower than the patients who did not respond to radiotherapy [40]. A separate study by Shimada et al. showed that seropositive oesophageal squamous cell carcinoma patients, whose serum anti-p53 titre did not decrease after surgery, exhibited worse prognosis than patients who showed seroconversion. Thus, a correlative study between the level of tumour autoantibodies and the overall survival outcome of cancer patients (reflected in the change in tumour status or tumour burden related to the therapy) could be extremely informative for evaluating therapeutic interventions [41]. Stage-specific autoantibody biomarkers screening is thus in principle useful in predicting onset of disease, thereby providing an opportunity to intervene and delay or prevent the onset of clinical symptoms.

#### **3.3. Immune-related adverse events**

Immunotherapies have been changing the outlook for many cancer patients in recent years and immune checkpoint inhibitors represent one of several strategies now targeting the immune system for therapeutic benefits. The immune checkpoint proteins cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and programmed cell death-1 (PD-1) play essential roles in central immune tolerance and are prominent targets for cancer vaccines now since inhibition of CTLA-4 and PD-1 can (re)activate the immune system to target cancer cells. Alone or in combination, clinical trials of anti-CTLA-4 and anti-PD-1 antibodies, such as Ipilimumab, Nivolumab and Pembrolizumab, have shown promising results for the treatment of melanoma, non-small cell lung-, kidney-, prostate- and head and neck cancers, as well as renal cell carcinomas, with reported therapeutic response rates approaching 70% in some cases, albeit positive immunotherapy outcomes remain cancer- and patientspecific [42].

Ipilimumab was the first anti-CTLA-4 antibody to prolong survival in patients with advanced melanoma [43, 44], with long term analysis indicating a 3-year survival of 22% across all patients with sufficient follow-up [45]. Similarly, PD-1 blockade with Nivolumab or Pembrolizumab has improved survival for metastatic melanoma, non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC) patients [46–50]. In one trial, advanced melanoma patients treated with pembrolizumab showed a response rate of 34% and a survival rate of 74% [51]. Nivolumab has been reported to result in increased response rates, survival and progression-free survival when compared to intravenous docetaxel in NSCLC [52], whilst stage III/IV melanoma patients achieved a partial tumour response, with a median progression free survival of 172 days, with only 18% experiencing grade 2 or 4 adverse events [53].

Combination check-point inhibitor treatments, targeting both CTLA-4 and PD-1, have also shown strong promise, with clinical trial data in untreated melanoma patients reporting objective response rates up to 72% (amongst patients with PD-L1-positive tumours) and with median progression-free survival of 11.5 months for ipilimumab plus nivolumab, compared to 2.9 months with ipilimumab alone and 6.9 months with nivolumab alone [54]. However, high grade immune-related adverse events (irAEs) occurred in 55% of those in the combination treatment group [54] and similarly high rates of irAEs have been reported elsewhere for anti-CTLA-4 and anti-PD-1 treatments [55].

Indeed, clinical findings on monoclonal antibody-induced adverse effects in general show that this is a wider phenomenon across different disease areas [56], which potentially compromises the effectiveness of such immunotherapies. Efforts are being channelled therefore towards predicting and monitoring undesirable immunotoxic effects and a panel of potential antibodies associated with irAE has been proposed (**Table 1**). Further exploratory studies involving autoantibody-based immunotoxicity profiling in immunotherapy patients are underway to better characterise the role and diagnostic potential of these circulating autoantibodies in irAE.

**4. Classical technological approaches to autoantibody-based** 

**Table 1.** List of proposed potential seromic autoantibodies that can be used for identifying irAEs [55].

Since its introduction in 1979, immunoblotting, or 'western blotting', has become a ubiquitous protein analysis technique in which proteins are separated by electrophoresis according to

CCP, cyclic citrullinated peptide; GAD, glutamate decarboxylase; RNP, ribonucleoprotein; Sm, small nuclear ribonucleoprotein; SSA, Sjogren's syndrome-related antigen A; Scl, Sclerosis systemic; SSB, Sjogren's syndrome-related

**biomarker discovery and validation**

**4.1. Western blots**

antigen B.

**Immune-related organ** 

Gastro-intestinal None

Skin None

**Antibodies**

Anti-smooth muscle, anti-liver kidney microsomal antibody type 1, anti-liver cytosol

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169

Autoantibody-Based Diagnostic Biomarkers: Technological Approaches to Discovery and Validation

Extractable nuclear antigens (ENA): anti-Sm, anti-RNP: anti-Ro (SSA)

Liver Antinuclear antibodies (ANAs)

Lung Antinuclear antibodies (ANAs)

Polyarthritis Antinuclear antibodies (ANAs)

Renal Antinuclear antibodies (ANAs)

Haematologic syndromes Antinuclear antibodies (ANAs)

type 1

Rheumatoid factor Anti-centromere

Endocrine Anti-GAD, anti-insulin, anti-carbonic anhydrase Anti-21 hydroxylase Anti-pituitary

Anti-La (SSB): anti-Scl70, anti-Jo

Anti-ENA: anti-SSA, SSB, Sm

Coombs' erythrocyte test

Complement fractions C3 CA CH50 Anti-neutrophil cytoplasmic (ANCA)

Anti-CCP, complement fractions C3 C4 CH50

**involved**


CCP, cyclic citrullinated peptide; GAD, glutamate decarboxylase; RNP, ribonucleoprotein; Sm, small nuclear ribonucleoprotein; SSA, Sjogren's syndrome-related antigen A; Scl, Sclerosis systemic; SSB, Sjogren's syndrome-related antigen B.

**Table 1.** List of proposed potential seromic autoantibodies that can be used for identifying irAEs [55].
