WO2023277772A1

WO2023277772A1 - A method for the diagnosis of paraneoplastic pemphigus

Info

Publication number: WO2023277772A1
Application number: PCT/SE2022/050641
Authority: WO
Inventors: Nils LANDEGREN; Takashi Hashimoto; Olof KÄMPE
Original assignee: Biocistronic Ab
Priority date: 2021-06-30
Filing date: 2022-06-28
Publication date: 2023-01-05
Also published as: EP4373954A1

Abstract

The present invention relates to transglutaminase 1 (TGM1) as a new epidermal specific autoantigen, and methods for diagnosis of the severe blistering disease paraneoplastic pemphigus.

Description

A METHOD FOR THE DIAGNOSIS OF PARANEOPLASTIC PEMPHIGUS

FIELD OF THE INVENTION

BACKGROUND TO THE INVENTION

The identification of immune targets is crucial to the understanding of autoimmune disease mechanisms and can spur developments in clinical diagnostics and treatment. Autoantibodies in tissue-specific autoimmune disorders typically targets proteins that are specifically expressed in the affected tissue and oftentimes also possess key functions. Autoimmune diseases may therefore present as phenocopies of monogenic disorders affecting the same proteins, as exemplified by the familial and acquired forms of epidermolysis bullosa caused either by mutations in the collagen VII type 1 alpha chain gene (COL7A1) or by autoantibodies targeting the corresponding protein (1, 2). Thus far targets of autoimmune attack have typically been identified by screening for immunoreactivity in individual disorders. Given the abundant and growing information about gene expression patterns across tissues it was reasoned that a reverse approach, starting with a candidate autoantigen, might serve to reveal a corresponding disease. To explore this possibility the nine-member transglutaminase protein family was chosen, among which five clinically significant autoantigens have been identified. Tissue transglutaminase (TGM2) is the major autoantigen in celiac disease (3), and is widely used for diagnostics. Gluten-sensitivity frequently involves skin manifestations, and in rare cases also symptoms from the nervous system. Dermatitis herpetiformis is associated with autoantibodies targeting epidermal transglutaminase (TGM3) (4), while gluten-related cerebellar ataxia and peripheral neuropathy is linked with autoantibodies against transglutaminase 6 (TGM6) (5). Extending beyond gluten-sensitivity, acquired factor XIII- deficient hemophilia is caused by autoantibodies targeting the catalytic alpha-subunit of coagulation factor FXIII (6), and the prostate-specific TGM4 is a major autoantigen in male patients with autoimmune polyendocrine syndrome type 1 (APS1) (7). Given this range of autoantigens in the transglutaminase gene family, it was of interest to learn whether some of the remaining members also might represent targets in as yet unexplained autoimmune disorders. Attention was focused on TGM1, which is predominantly expressed in squamous epithelia, having an integral function in the assembly of the cornified cell envelope (8,9). Individuals with autosomal recessive congenital ichthyosis due to TGM1 gene mutations (Mendelian Inheritance in Man number 242300) suffer from lifelong severe scaling of the skin (10), suggesting a crucial role for skin barrier integrity.

SUMMARY OF THE INVENTION

The present inventors have identified transglutaminase 1 (TGM1), also known as Protein- glutamine gamma-glutamyltransferase K, as an epidermal autoantigen and demonstrate that TGM1 is a major autoantigen in the severe blistering disease paraneoplastic pemphigus.

TGM1 autoantibodies were specifically detected in patients suffering from paraneoplastic pemphigus.

Accordingly, the present invention provides methods for the diagnosis of paraneoplastic pemphigus. The methods comprise detection of autoantibodies specific for TGM1 in a sample obtained from a subject. The subject can be a human. The sample can be a blood sample, such as a serum sample or plasma sample, or a tissue sample

Antibodies specific for TGM1 can be detected using an immunoassay, such as ELISA, RIA, or radioimmunoprecipitation assays, by surface plasmon resonance, or by electrochemiluminescence.

LEGENDS TO THE FIGURES

Figure 1. Discovery and validation of transglutaminase 1 autoantibodies in paraneoplastic pemphigus. A, Explorative screen for TGMI autoantibodies across multiple autoimmune and idiopathic skin disorders and controls, identifying TGM1 autoantibodies in sera from five patients with paraneoplastic pemphigus. B, Validation in a replication cohort of 88 patients with paraneoplastic pemphigus and controls. TGM1 autoantibodies were detected in sera from 53 out of 97 (55%) patients with paraneoplastic pemphigus in the discovery and replication cohorts combined. Cutoff=index value 10.

C, The target specificity of serum autoantibodies was studied against a panel of transglutaminases in five subjects with paraneoplastic pemphigus as compared to controls, revealing specific reactivity towards TGM1 in paraneoplastic pemphigus. DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides methods for the diagnosis of paraneoplastic pemphigus. The methods comprise detection of autoantibodies specifically binding to TGM1 in a sample obtained from a subject. The subject can be a human. The sample can be a blood sample, such as serum sample or a plasma sample, or a tissue-sample.

Antibodies specifically binding to TGM1 can be detected using an immunoassay, such as ELISA, or RIA, by surface plasmon resonance, electrochemiluminescence, or assays using fluorogenic reporters such as protein microarrays or suspension bead arrays.

The immunoassay used to detect autoantibodies specifically binding to TGM1 according to the invention may be based on standard techniques known in the art. In a preferred embodiment the immunoassay may be an ELISA.

ELISAs are generally well known in the art. In a typical ELISA the TGM1 antigen is immobilised on a solid surface (e.g. the wells of a standard microtiter assay plate, or the surface of a microbead or a microarray) and a sample comprising the sample to be tested for the presence of autoantibodies specifically binding to TGM1 is brought into contact with the immobilised antigen. Any autoantibodies of the desired specificity present in the sample will bind to the immobilised antigen. The bound antibody/antigen complexes may then be detected using any suitable method. In one preferred embodiment a labelled secondary anti-human immunoglobulin antibody, which specifically recognises an epitope common to one or more classes of human immunoglobulins, is used to detect the antibody/antigen complexes. Typically, the secondary antibody will be anti-lgG or anti- IgM. The secondary antibody is usually labelled with a detectable marker, typically an enzyme marker such as, for example, peroxidase or alkaline phosphatase, allowing quantitative detection by the addition of a substrate for the enzyme which generates a detectable product, for example a coloured, chemiluminescent or fluorescent product. Other types of detectable labels known in the art may be used.

In a radioimmunoprecipitation assay the antigen is typically produced in vitro by in vitro transcription of a specific plasmid containing the cDNA sequence of TGM1 or a fragment thereof with suitable promotors, e.g. T7, T3 och SP-6, followed by in vitro translation in the presence of a components from a reticulocyte lysate and a radioactive amino acid, e.g. S35-methionine. The resulting radioactive proteins are then incubated with patient sera, the antibodies collected by Protein A or G bound to a matrix, antibodies against IgG and or IgM and or IgA, collected, washed and the bound radioactivity analysed. Full-length TGM1 or one or more peptides derived from the amino acid sequence of TGM1 can be used as antigen in the detection of autoantibodies specifically binding to TGM1. The amino acid sequence of TGM1 can be found in GenBank Accession No. NP_000350 or SwissProt/UniProt Accesion No. P22735 (TGM1_HUMAN).

Paraneoplastic pemphigus is a severe autoimmune bullous disease characterized by polymorphic mucocutaneus lesions that appear in association with a neoplastic disease (14). The clinical presentation is variable and may resemble pemphigus vulgaris, erythema multiforme, or lichen planus (14,15). Malignant lymphoma is the most commonly associated neoplastic disease, as seen in almost half of the patients, but many other malignant and benign neoplasms have been reported (15). In many cases the underlying neoplastic disease remains undiagnosed at the time of presentation of the mucocutaneous manifestations. It is therefore urgent to establish the correct diagnosis, so that investigations for occult malignancies can be initiated without delay. Autoantibody analysis constitutes a central part of the diagnostic assessment. The present inventors found TGM1 autoantibodies to be 55% sensitive and 100% specific for paraneoplastic pemphigus, as investigated in a broad group of patients with clinically relevant differential diagnoses. Given the high specificity of TGM1 autoantibodies, TGM1 autoantibody analysis will improve the diagnostic precision for paraneoplastic pemphigus alongside the established serological assays.

EXAMPLES

Materials and Methods

Study subjects

Serum samples from patients with autoimmune bullous skin diseases were obtained from a previously described cohort at Kurume University in Japan, which included patients who had visited Kurume University Hospital or been referred for serological tests from other hospitals in Japan, Korea, the USA and European countries over a period of more than 20 years (15, 16). All patients had undergone rigorous serological testing to secure the respective diagnoses. Sera were also included from patients with dermatitis herpetiformis from Hungary (17), patients with atopic dermatitis from Sweden (18), patients with pustulosis palmoplantaris from Sweden (19), patients with celiac disease from Sweden (20), patients with APS1 from Finland, and healthy blood donors from Sweden. The studies were approved by ethical boards in Kurume, Budapest and Stockholm, and were conducted in accordance with the Helsinki declaration.

TGM1 immunohistochemistry

The expression pattern of TGM1 protein was characterized across a broad panel of human tissues. Tissue microarrays (TMAs) containing multiple formalin-fixed paraffin- embedded human tissue samples were constructed as previously described (21), and used for immunohistochemistry using a TGM1 -specific rabbit antibody (Atlas antibodies, HPA040171). The TMAs were first incubated with Ultra V block (TA-125-UB, Thermo Fisher Scientific, Inc.) for 5 minutes, followed by anti-TGM1 at 1:1200 dilution for 30 minutes, and thereafter with labeled horseradish peroxidase-polymer for 30 minutes. The TMAs were next incubated with 3,3'-diaminobenzidine (DAB) solution for 2x5 minutes, counterstained in Mayer’s hematoxylin (01820, Histolab) for 5 minutes using the Autostainer XL (Leica), rinsed in lithium carbonate water (diluted 1:5 from saturated solution) for 1 minute, dehydrated in graded ethanol, and lastly coverslipped (PERTEX, Histolab) using an automated glass coverslipper (CV5030, Leica). The TMAs were scanned using the automated scanning system Aperio XT (Aperio Technologies). Indirect immunofluorescence

Fresh frozen normal human skin samples were stained with sera from patients with different autoimmune bullous skin disorders or healthy controls, according to previously described methodology (11,22).

Transglutaminase radio-ligand binding assays

TGM1 autoantibodies were measured using a radio-ligand binding assay. Human TGM1 cDNA (Origene, SC122560) was cloned into a pTNT expression vector (L5610, Promega), which was used for in vitro transcription and translation in the presence of ³⁵S methionine (Promega TNT Systems) following the manufacturer’s protocol (Promega TNT Systems). Radiolabeled TGM1 protein (30,000 counts per minute (CPM)) was immunoprecipitated with patient or control sera (2.5mI) in 96 well filtration plates (Millipore) using protein A Sepharose (nProtein A Sepharose™ 4 Fast Flow, GE Health Care). All sera were analyzed in duplicate. Positive standards were included in each plate: either a polyclonal TGM1 antibody (Atlas antibodies, HPA040171) in the discovery analysis, or serum from a patient with paraneoplastic pemphigus and confirmed immunoreactivity against TGM1 in the validation analysis. Negative standard, represented by 4% bovine serum albumin (BSA), was also included in each plate. Radioactivity was measured using a liquid scintillation counter (Wallac 1450 MicroBeta PerkinElmer). Autoantibody index values were calculated according to the following: (sample value-negative standard)/(positive standard-negative standard) x 100. The upper limit of the normal range was defined as an index value of 10.

The target specificity of TGM1 autoantibodies was investigated using radio-ligand binding assays for TGM1 (Origene, SC122560), TGM2 (kindly provided by Ake Lernmark at Lund University), TGM4 (Origene, SC303287), TGM5 (Origene, SC308078), and TGM7 (Origene, SC305704). Patient and control sera were added in a volume of 0.625mI per well, and otherwise followed the same procedure as described above. Polyclonal antibodies against TGM1 (Zedira, A018), TGM2 (Zedira, A014), TGM4 (Zedira, A022), TGM5 (Novus Biologicals, NPB1-54329), TGM7 (Zedira, A040) were included as positive standards, each in a volume of 0.625 pi per well. Z-scores were calculated based on the mean (m) and standard deviation (s) of the healthy controls, according the following: Z=(c-m)/s. Index values were thereafter calculated as described above.

Results

The tissue-localization of TGM1 was compared with the binding pattern of serum autoantibodies from patients with different forms of autoimmune bullous disorders. First affinity-purified rabbit TGM1 -antibodies were used to perform immunohistochemistry of a human tissue panel. A distinctive intercellular staining pattern was observed in squamous epithelia, including the epidermis, esophagus, vagina, and cervix uteri, while remaining tissues showed only very faint or no staining. The results were consistent with the available consensus dataset for TGM1 at www.proteinatlas.org, which combines data from several independent investigations of mRNA expression in human tissues (13). Next the tissue localization of TGM1 compared to the pattern of autoantibody reactivity in different bullous disorders was investigated, as detected using indirect immunofluorescence technique on normal human skin tissue sections. Sera from patients with intraepithelial blistering disorders: pemphigus vulgaris, pemphigus vegetans, pemphigus foliaceus and paraneoplastic pemphigus, all displayed an intercellular pattern that was compatible with the TGM1 staining. Conversely, the autoantibody reactivity of sera from patients with subepithelial bullous disorders: bullous pemphigoid, epidermolysis bullosa acquisita, and anti-laminin gamma-1 (p200) pemphigoid, all displayed a linear pattern along the basal lamina that was clearly distinct from the TGM1 staining. The tissue distribution of TGM1 was thus consistent with the autoantibody reactivity of the pemphigus group of disorders but not with that of pemphigoid.

Next a radio-ligand binding assay was used to perform an explorative screen for TGM1 autoantibodies in sera from patients with various acquired skin disorders. A cohort was assembled with the aim to include all major types of pemphigus and to also have broad representation of other forms of autoimmune and idiopathic skin disorders. The discovery cohort comprised patients with pemphigus vulgaris (n=5), pemphigus foliaceus (n=5), intercellular IgA dermatosis (n=6), paraneoplastic pemphigus (n=9), epidermolysis bullosa acquisita (n=5), bullous pemphigoid (n=5), anti-p200 pemphigoid (n=10), pustulosis palmoplantaris (n=21), atopic dermatitis (n=20), dermatitis herpetiformis (n=21), as well as control subjects with celiac disease (n=290) and blood donors (n=111). Among the 507 investigated sera, five demonstrated immunoreactivity against TGM1 - all of which were from patients diagnosed with the severe mucocutaneous disease paraneoplastic pemphigus (FigurelA).

To verify the role of TGM1 autoantibodies in this disease, a replication cohort of 88 patients with paraneoplastic pemphigus together with additional control groups: pemphigus vulgaris (n=20), pemphigus foliaceus (n=20), bullous pemphigoid (n=20), anti laminin gamma-1 (p200) pemphigoid (n=20), epidermolysis bullosa acquisita (n=20), and healthy controls (n=78) was investigated. Autoantibodies against TGM1 were detected in sera from 48 out of 88 patients with paraneoplastic pemphigus in the replication cohort, while all other sera were negative (Figure 1B). Altogether TGM1 autoantibodies were present in sera from 53 out of 97 (55%) patients with paraneoplastic pemphigus while absent in all other disease categories and healthy controls (n=652) in the discovery and replication cohorts combined, suggesting TGM1 autoantibodies was a highly specific biomarker for this disease.

Next it was sought to determine whether TGM1 autoantibody positive patients with paraneoplastic pemphigus differed from the TGM1 autoantibody-negative group in their clinical phenotypes or any other aspect. To this end, associations between TGM1 autoantibody status and available data on age, sex, mucocutaneous manifestations, respiratory complications, associated neoplasms, and previously obtained autoantibody data was assessed using Fishers exact test (Table 1). Table 1. Clinical features and TGM1 autoantibodies in 97 patients with paraneoplastic pemphigus. Associations between TGM1 autoantibody results and clinical features were assessed using Fisher’s exact test.

Number of TGM1 TGM1 p- individuals autoantibody autoantibody value

_ (n=97) positive (n=53) negative (n=44 _

Sex

Male 39 21 18 0.31 Female 48 26 22 0.31 Unknown 10 6 4

Age (years) 10-20 4 1 3 0.32 21-30 0 31-40 4 1 3 0.32 41-50 9 6 3 0.5 51-60 19 12 7 0.44 61-70 26 14 12 1 71-80 16 11 5 0.27 80+ 4 0 4 0.04

Unknown 15 8 7

Respiratory disease

Bronchiolitis obliterans 19 9 10 0.75

Pulmonary embolism 1 1 0 1

Respiratory failure 1 1 0 1

Pulmonary emphysema 1 1 0 1

Pneumonia 1 1 0 1

Asthma 1 1 0 1

None 13 5 8 0.31

Unknown 60 34 26

Mucocutaneous lesions

Erythema 14 5 9 0.07

Blister 9 7 2 0.3

Erosion 4 1 3 0.3

Erythema + Blister 11 7 4 1

Erythema + Erosion 5 3 2 1

Erythema + Blister + Erosion 14 10 4 0.38

Erythema + Erosion + Nail lesion 1 1 0 1

Erythema + Blister + Erosion + Nail lesion 1 1 0 1

Blister-·- Erosion 1 0 1 0.4

None 10 8 4 0.75

Unknown 25 10 15

Site of cutaneous lesions

Trunk 12 6 6 0.52

Extremity 4 3 1 0.65

Head + Trunk 3 1 2 0.56

Trunk + Extremity 22 12 10 0.6

Head + Extremity 2 1 1 1

Head + Trunk + Extremity 15 11 4 0.37

None 12 8 4 0.75

Unknown 27 11 16

Site of mucosal lesion

Oral 31 17 14 0.35

Oral + Ocular 21 14 7 0.61

Oral + Ocular + Nasal 1 1 0 1

Oral + Ocular + Nasal + Genital 3 3 0 0.28

Oral + Genital 4 1 3 0.3 Number of TGM1 TGM1 p- individuals autoantibody autoantibody value

_ (n=97) positive (n=53) negative (n=44 _

Oral + Ocular + Genital 11 9 2 0.19 Oral + Nasal 1 1 0 1 None 3 0 3 0.05 Unknown 22 7 15

Neoplasms

Hodgkin’s lymphoma 1 0 1 0.36

Non-Hodgkin lymphoma 2 0 2 0.12

Follicular lymphoma 15 12 3 0.27

B-cell lymphoma 7 3 4 0.24

Diffuse B-cell lymphoma 4 2 2 0.61

Malignant lymphoma (unknown type) 11 10 1 0.08

Chronic lymphatic leukaemia 3 3 0 0.55

Primary macroglobulinemia 1 1 0 1

Acute lymphatic leukaemia 1 1 0 1

Plasmacytoma 1 1 0 1

Castleman’s disease 6 3 3 0.66

Follicular dendritic cell sarcoma 2 2 0 0.53

Thymoma 4 2 2 0.61

Rectal cancer 1 1 0 1

Gastrointestinal stromal cell tumour 1 1 0 1

Hepatocarcinoma 1 1 0 1

Myofibroblastoma 1 0 1 0.36

Gastric cancer 3 0 3 0.042

Thyroid cancer 1 1 0 1

Breast cancer 1 0 1 0.36

Lung cancer 1 0 1 0.36

Mediastinal tumour 1 1 0 1

Intrapelvic tumour 1 0 1 0.36

Unknown 27 8 19

For many of the collected phenotypes, the number of individuals were too small for meaningful statistical association testing. It was possible to conclude though that TGMI autoantibodies appeared at even rates in women and men, and that they were present at all age categories. There were no significant associations with the characteristics of mucocutaneous manifestations (erythema, blister, or erosion), nor the sites of these lesions (trunk, head, extremity, oral, ocular, and genital). No associations were found with bronchiolitis obliterans or other respiratory complications seen among the patients. Furthermore, TGM1 autoantibodies appeared unrelated to the type of concomitant neoplasm. In all, there was no evidence that TGM1 autoantibody-positive patients differed from the remaining patients with paraneoplastic pemphigus in regards to underlying neoplasms or the clinical presentation. Patients with dermatitis herpetiformis display autoantibody reactivity against both TGM3 and TGM2 (4) whereas patients with APS1 and TGM4 autoantibodies do not show cross reactivity against TGM2 (7). To verify that TGM1 was the primary target of transglutaminase autoantibodies in paraneoplastic pemphigus autoantibody reactivity against a panel of full-length transglutaminase proteins was studied. Five patients with paraneoplastic pemphigus who had all been previously found positive for TGM1 autoantibodies were investigated, and TGM2 autoantibody-positive patients with celiac disease (n=5), TGM4 autoantibody-positive patients with APS1 (n=5), and healthy blood donors (n=5) were included as controls. Paraneoplastic pemphigus sera specifically reacted against TGM1 , while celiac disease and APS1 patient sera only showed binding to TGM2 and TGM4 respectively (Figure 1C). Transglutaminase autoantibodies in paraneoplastic pemphigus thus appeared to be highly specific for TGM1.

References

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2. Hovnanian A, et al. (1992) Genetic linkage of recessive dystrophic epidermolysis bullosa to the type VII collagen gene. J Clin Invest 90(3): 1032-1036.

3. Dieterich W, et al. (1997) Identification of tissue transglutaminase as the autoantigen of celiac disease. Nature medicine 3(7):797-801.

4. Sardy M, Karpati S, Merkl B, Paulsson M, & Smyth N (2002) Epidermal transglutaminase (TGase 3) is the autoantigen of dermatitis herpetiformis. J Exp Med 195(6):747-757.

5. Hadjivassiliou M, et al. (2008) Autoantibodies in gluten ataxia recognize a novel neuronal transglutaminase. Annals of neurology 64(3):332-343.

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Claims

1. A method for the diagnosis of paraneoplastic pemphigus, comprising detection of autoantibodies specific for transglutaminase 1 (TGM1) in a sample obtained from a subject.

2. The method according to claim 1 , wherein the sample is a blood sample, such as a serum sample or a plasma sample, or a tissue-sample.

3. The method according to claim 1, wherein the antibodies specific for transglutaminase 1 (TGM1) is detected using an immunoassay, such as ELISA, RIA, radioimmunoprecipitation assays, or protein arrays.

4. The method according to claim 1 , wherein the antibodies specific for transglutaminase 1 (TGM1) is detected using surface plasmon resonance, or electrochemiluminescence.