Cluster binding studies with two anti-Thomsen-Friedenreich (anti-core-1, CD176, TF) antibodies: Evidence for a multiple TF epitope.

Antibodies to carbohydrate epitopes are often of the IgM isotype and require multiple binding for sufficient avidity. Therefore clusters of epitopes are preferred antigenic sites in these cases. We have examined the type of clusters recognized by two anti-Thomsen-Friedenreich (TF, core-1, CD176) IgM antibodies, NM-TF1 and NM-TF2, using several different sets of TF-carrying synthetic glycoconjugates in ELISA experiments. To our surprise, the single most important factor determining binding strength was a close vicinity of several TF glycans at distances of ≤1 nm. Considering the known dimensions of IgM antibodies, our data strongly suggest that a cluster of up to four TF moieties, presenting as a "multiple epitope", is required to attach to a single combining site in order to result in adequate binding strength. This effect can also be achieved by "surrogate-multiple epitopes" consisting of separate TF-carrying molecules in close vicinity. In addition, it was found that serine-linked TFs are stronger bound than threonine-linked TFs by both antibodies. This peculiar type of cluster recognition may contribute to improved avidity and explicit tumor specificity.

[Anti-p200 pemphigoid]. / Anti-p200-Pemphigoid.

Anti-p200 pemphigoid is a rare autoimmune blistering disease. It belongs to the group of pemphigoid diseases and was first described in 1996. The diagnostic gold standard is the combination of (1) linear deposits of immunoreactants at the dermal epidermal junction by direct immunofluorescence microscopy of a perilesional skin biopsy, (2) detection of circulating autoantibodies binding to the dermal side (blister floor) of human salt split skin by indirect immunofluorescence microscopy, and reactivity with a 200 kDa protein (p200) in extract of human dermis by immunoblotting. In 2009, laminin γ1 was described as an additional target antigen in 90% of anti-p200 pemphigoid patients. Since ex vivo and in vivo studies have shown no direct pathogenic relevance for laminin γ1 antibodies and the preadsorption of patient sera against laminin γ1 does not reduce their reactivity with p200, the molecular identity of p200 still remains to be elucidated. The clinical phenotype of the disease is heterogeneous; in most cases, however, it resembles bullous pemphigoid. Anti-p200 patients are younger and skin lesions more often appear on palms of the hands and soles of the feet than in bullous pemphigoid. Therapy consists of topical and systemic corticosteroids. In addition, the use of daspone and immunosuppressants has been reported.

A sensitive and specific assay for the serological diagnosis of antilaminin 332 mucous membrane pemphigoid.

BACKGROUND: Antilaminin 332 mucous membrane pemphigoid (MMP) is an autoimmune subepidermal blistering disease with predominant mucosal involvement and autoantibodies against laminin 332. Malignancies have been associated with this disease; however, no standardized detection system for antilaminin 332 serum antibodies is widely available. OBJECTIVES: Development of a sensitive and specific assay for the detection of antilaminin 332 antibodies. METHODS: An indirect immunofluorescence (IF) assay using recombinant laminin 332 was developed and probed with a large number of antilaminin 332 MMP patient sera (n = 93), as well as sera from patients with antilaminin 332-negative MMP (n = 153), bullous pemphigoid (n = 20), pemphigus vulgaris (n = 20) and noninflammatory dermatoses (n = 22), and healthy blood donors (n = 100). RESULTS: In the novel IF assay, sensitivities with the laminin 332 heterotrimer and the individual α3, ß3 and γ2 chains were 77%, 43%, 41% and 13%, respectively, with specificities of 100% for each substrate. The sensitivity for the heterotrimer increased when an anti-IgG4 enriched antitotal IgG conjugate was applied. Antilaminin 332 reactivity paralleled disease activity and was associated with malignancies in 25% of patients with antilaminin 332 MMP. CONCLUSIONS: The novel IF-based assay will facilitate the serological diagnosis of antilaminin 332 MMP and may help to identify patients at risk of a malignancy.

Prospective study in bullous pemphigoid: association of high serum anti-BP180 IgG levels with increased mortality and reduced Karnofsky score.

BACKGROUND: Bullous pemphigoid (BP) is a subepidermal blistering disease characterized by autoantibodies against the two hemidesmosomal proteins, BP180 (type XVII collagen) and BP230. The multicentre prospective BLISTER (Bullous Pemphigoid Steroids and Tetracyclines) trial randomized 253 patients with BP to compare the benefits and harms between initial treatment with doxycycline or prednisolone. OBJECTIVES: To analyse distinct autoantibody profiles for the prediction of the disease course in a well-characterized cohort of BP sera. METHODS: One hundred and forty-three patients of the BLISTER trial consented to participate in this serological study. Sera taken at baseline were analysed by (i) indirect immunofluorescence, (ii) anti-BP180 NC16A (16th noncollagenous domain) and anti-BP230 enzyme-linked immunosorbent assay and (iii) immunoblotting with various substrates. Results were then linked with clinical parameters including age, Karnofsky score, number of blisters, related adverse events and mortality. RESULTS: Disease activity correlated with immunoglobulin (Ig)G anti-BP180 levels but not with levels of anti-BP230 IgG and anti-BP180 IgE. High levels of both anti-BP180 IgG and anti-BP230 IgG were associated with a low Karnofsky score. The presence of anti-BP230 IgG was more frequent in older patients. Those with higher total IgE serum levels suffered from fewer adverse events. Higher IgG anti-BP180 levels were associated with an increased 1-year mortality rate. CONCLUSIONS: Analysis of the autoantibody profile is not only of diagnostic relevance but may also be helpful in predicting the course of the disease.

Whole-Genome Expression Profiling in Skin Reveals SYK As a Key Regulator of Inflammation in Experimental Epidermolysis Bullosa Acquisita.

Because of the morbidity and limited therapeutic options of autoimmune diseases, there is a high, and thus far, unmet medical need for development of novel treatments. Pemphigoid diseases, such as epidermolysis bullosa acquisita (EBA), are prototypical autoimmune diseases that are caused by autoantibodies targeting structural proteins of the skin, leading to inflammation, mediated by myeloid cells. To identify novel treatment targets, we performed cutaneous genome-wide mRNA expression profiling in 190 outbred mice after EBA induction. Comparison of genome-wide mRNA expression profiles in diseased and healthy mice, and construction of a co-expression network identified Sykb (spleen tyrosine kinase, SYK) as a major hub gene. Aligned, pharmacological SYK inhibition protected mice from experimental EBA. Using lineage-specific SYK-deficient mice, we identified SYK expression on myeloid cells to be required to induce EBA. Within the predicted co-expression network, interactions of Sykb with several partners (e.g., Tlr13, Jdp2, and Nfkbid) were validated by curated databases. Additionally, novel gene interaction partners of SYK were experimentally validated. Collectively, our results identify SYK expression in myeloid cells as a requirement to promote inflammation in autoantibody-driven pathologies. This should encourage exploitation of SYK and SYK-regulated genes as potential therapeutic targets for EBA and potentially other autoantibody-mediated diseases.

A phase I study of PankoMab-GEX, a humanised glyco-optimised monoclonal antibody to a novel tumour-specific MUC1 glycopeptide epitope in patients with advanced carcinomas.

BACKGROUND: A phase I open-label dose-escalation study was conducted to define the safety, tolerability, and pharmacokinetics (PK) of PankoMab-GEX, a glyco-optimised humanised IgG1, with high affinity to a novel tumour-specific glycopeptide epitope of MUC1 (TA-MUC1) with excellent preclinical anti-tumour activity. PATIENTS AND METHODS: Seventy-four patients with advanced TA-MUC1-positive carcinomas received PankoMab-GEX intravenously every 3 (Q3W), 2 (Q2W), or 1 (QW) week in doses of 1-2200 mg in a three-plus-three dose-escalation design until disease progression (NCT01222624). RESULTS: No maximum tolerated dose was reached. Adverse events were mainly mild-to-moderate infusion-related reactions (IRRs) by the first infusion in 45% of patients. Only one dose-limiting toxicity, a grade III IRR, was observed. PankoMab-GEX exhibited linear PK over all doses. Mean terminal half-life was 189 ± 66 h (Q3W), without dose dependency. A target trough level ≥50 µg/mL was reached after one infusion with doses ≥1700 mg Q3W in 80% of patients. Clinical benefit in 60 evaluable patients included one complete response in a patient with ovarian cancer treated 483 d and confirmed disease stabilisation in 19 patients lasting a median (range) of 23 (10-109) weeks. All but two of the patients with clinical benefit had received a compounded total dose ≥700 mg over a 3-week period, including 8 of 12 (67%) patients with ovarian cancer. CONCLUSION: PankoMab-GEX is safe, well tolerated, and showed promising anti-tumour activity in advanced disease. A phase IIb study is ongoing evaluating the efficacy of PankoMab-GEX as a maintenance therapy in advanced ovarian cancer.

Impact of oral consumption of heat-treated Bacteroides xylanisolvens DSM 23964 on the level of natural TFα-specific antibodies in human adults.

It is now generally accepted that the human body exists in close synergy with the gut microbiome and that this cross-talk plays an essential role in human health and disease. One facet from the many interactions between the microbiome and the immune system is the induction of natural antibodies to commensal bacterial glycans, such as blood group antigens, the alpha-Gal epitope or the Thomsen-Friedenreich (TFα) antigen. Since we have observed that certain species of the commensal genus Bacteroides express the TFα antigen, we examined whether the oral dietary supplementation of a pasteurised Bacteroides xylanisolvens strain might be able to enhance the level of natural anti-TFα antibodies in healthy adults. The data obtained from a double-blind, placebo-controlled study involving 140 healthy volunteers and lasting 8 weeks revealed that the oral uptake of this strain was indeed able to increase the level of TFα-specific immunoglobulin M serum antibodies. The effect was dose-dependent but remained - at any doses - within the physiological range determined before intervention. Furthermore, the effect reverted after stopping the intake. The results support the idea of the microbiome inducing the generation of systemic antigen-specific antibodies against sugar epitopes. They also demonstrate the possibility to modulate essential regulatory or defence processes through dietary supplementation of selected commensal bacteria with the aim to assist human health.

Blood group antigen A type 3 expression is a favorable prognostic factor in advanced NSCLC.

OBJECTIVES: Several blood group-related carbohydrate antigens are prognosis-relevant markers of tumor tissues. A type 3 (repetitive A) is a blood group antigen specific for A1 erythrocytes. Its potential expression in tumor tissues has so far not been examined. MATERIAL AND METHODS: We have evaluated its expression in normal lung and in lung cancer using a novel antibody (A69-A/E8). For comparison an anti-A antibody specific to A types 1 and 2 was used, because its expression on lung cancer tissue has been previously reported to be of prognostic relevance. Resected tissue samples of 398 NSCLC patients were analyzed in immunohistochemistry using tissue microarrays. RESULTS AND CONCLUSIONS: Expression of A type 3 was not observed in non-malignant lung tissues. A type 3 was expressed on tumor cells of around half of NSCLC patients of blood group A1 (p<0.001). Whereas no prognostic effect for A type 1/2 antigen was observed (p=0.562), the expression of A type 3 by tumor cells indicated a highly significant favorable prognosis among advanced NSCLC patients (p=0.011) and in NSCLC patients with lymphatic spread (p=0.014). Univariate prognostic results were confirmed in a Cox proportional hazards model. In this study we present for the first time prognostic data for A type 3 antigen expression in lung cancer patients. Prospective studies should be performed to confirm the prognostic value of A type 3 expression for an improved risk stratification in NSCLC patients.

Clinical outcome and global gene expression data support the existence of the estrogen receptor-negative/progesterone receptor-positive invasive breast cancer phenotype.

The presence or absence of estrogen and progesterone steroid hormone receptor expression (ER, PR) is an essential feature of invasive breast cancer and determines prognosis and endocrine treatment decisions. Among the four ER/PR receptor phenotypes, the ER-/PR+ is infrequent, and its clinical relevance has been controversially discussed. Thus, we investigated its clinical significance and gene expression pattern in large datasets. In a retrospective clinical study of 15,747 breast cancer patients, we determined the ER/PR subtype survival probabilities using Kaplan-Meier and Cox regression analyses. From The Cancer Genome Atlas (TCGA) breast cancer dataset, PAM50 expression signature and pathway analyses were performed to test for distinct molecular features. In our cohort, the ER-/PR+ phenotype has been observed at a frequency of 4.1 % and was associated with an improved 10-year survival for stage I cancers compared to the ER+/PR+ reference subtype (median; 95 % CI 88.1 %; 83-93 vs. 84.3 %; 82-86 %, P = 0.024) as was confirmed by multivariate analysis over the entire follow-up (HR 0.59, 95 % CI 0.38-0.92, P = 0.021). This association lacked significance when including all stages. ER-/PR+ patients treated with antihormonal agents (34.5 %) had shorter survival compared to their non-treated counterparts (Log-rank P = 0.0001). PAM50 signatures suggest a distinct configuration for the ER-/PR+ phenotype. This specific phenotype has been further separated by a set of 59 uniquely expressed genes. Our study supports the notion of the existence of an ER-/PR+ phenotype with clinical and molecular features distinct from the large group of ER+/PR+ patients.

What controls the expression of the core-1 (Thomsen-Friedenreich) glycotope on tumor cells?

Malignant transformation is tightly connected with changes in the glycosylation of proteins and lipids, which in turn are contributing to the invasive and metastatic behavior of tumor cells. One example of such changes is demasking of the otherwise hidden core-1 structure, also known as Thomsen-Friedenreich antigen, which is a highly tumor-specific glycotope and potentially a cancer stem cell marker. This review summarizes what is known about the mechanism(s) of its expression on tumor cells. New data reveal a close connection between tumor metabolism and Golgi function. Based on these data, we suggest that the expression of this antigen is also a marker of aerobic glycolysis.

Safety and tolerance of Bacteroides xylanisolvens DSM 23964 in healthy adults.

We recently presented the strain Bacteroides xylanisolvens DSM 23964 to be safe for use in food. In order to confirm the tolerance of healthy humans to a regular oral intake of the strain B. xylanisolvens DSM 23964, we here report on the safety data of two successive human studies: a randomised and double-blind parallel group-controlled pilot study with 41 volunteers receiving a daily dose of a pasteurised fermented milk product containing up to 8.5×1011B. xylanisolvens DSM 23964 cells for 3 weeks, and a randomised and placebo-controlled double-blind major study with 140 volunteers receiving the same product but spray-dried and containing up to 1012 cells for 6 weeks. In both studies no persistent side effects of any kind were reported. The measured haematological parameters, and the serum concentrations of immunoglobulin and of inflammatory markers (IL-6, CRP, IFN-γ) were unaffected by the supplementation in both studies. A small decrease in the phagocytic activity of granulocytes and a small increase of TNF-α detected in the pilot study were both invalidated by the major study. This study further revealed that the supplementation induced no modification in natural killer cell activity and in liver enzyme values (gamma-glutamyl-transferase, glutamate-oxalacetate transaminase, glutamate-pyruvate transaminase). Our results definitively demonstrate that the pasteurised B. xylanisolvens DSM 23964 strain is safe and well tolerated by healthy human individuals.

Clinical impacts of histological subtyping primary breast cancer.

BACKGROUND: Treatment decisions in breast cancer depend on TNM classification and the assessment of additional variables with have an impact on survival. We examined whether histological subtyping breast cancer as either ductal or lobular is related to disease outcome. PATIENTS AND METHODS: We examined a large data base of 14198 breast cancer patients. RESULTS: Histological sub-classification of invasive breast cancer as either ductal or lobular is not correlated with disease outcome. However, the data further showed that invasive lobular carcinomas have a higher probability of being oestrogen receptor (ER)- and progesterone receptor (PR)-positive and a lower probability of being c-erbB2-positive. They also showed a higher average age at the time of diagnosis in comparison with invasive ductal carcinoma. Local recurrence rates were lower in invasive lobular carcinoma in comparison with invasive ductal carcinoma (3.5% vs. 6.2%; p = 0.031). The multivariable Cox regression analysis showed that ER, PR, nodal status, grade and tumour size predicted disease outcome with statistical significance, while the histological subtype (invasive ductal or lobular) was not a significant predictor of disease outcome. CONCLUSION: Histological sub-classification of invasive breast cancer as either ductal or lobular is not correlated with disease outcome. On the other hand our data gives some indication that lobular and ductal breast cancer appear to be different biological entities.

A novel series of anti-human glycophorin A (CD235a) antibodies defining five extra- and intracellular epitopes.

Glycophorin A (GPA, CD235a) is a major membrane glycoprotein and marker of cells of the erythroid lineage. It is also the target of Plasmodium falciparum and of influenza virus. We describe a novel series of 10 antibodies towards GPA, recognizing four extra- and intracellular peptide epitopes of this molecule (defined by epitope mapping) and one mixed peptide/carbohydrate epitope. All antibodies bind better to the desialylated than to the fully sialylated molecule, including those specific for the intracellular epitope. For some of the antibodies (representing all five epitopes) functional binding constants were determined by Surface Plasmon Resonance. The new panel complements the already known anti-glycophorin antibodies and offers several potential applications for, e.g., differential diagnosis of erythroleukemias, lineage analyses of erythroid cells, isolation of senescent erythrocytes, or a highly sensitive neuraminidase assay.

Modifying antibody specificity by chain shuffling of V / V between antibodies with related specificities.

Histo-blood group antigens are important markers of developmental stages and as such also often of tumours. Generation of antibodies towards these carbohydrate structures is still a challenging task as they may lack specificity, affinity or are only of the IgM class. We have examined four own antibodies to Lewis Y/H type 2 for their fine specificities using a large panel of mono- and oligosaccharides. Sequence alignment to other antibodies with similar specificity revealed an overall limited variation, and that our antibodies constitute a novel set. Based on produced and analysed chimeric mouse-human antibodies, extensive chain shuffling experiments were performed in order to analyse influences of the respective H and L chains on the specificity of the antibodies, and to generate modified antibodies with improved properties. One chIgG1 out of the shuffled antibodies revealed improved specificity and markedly enhanced functional affinity to Lewis Y compared to the parental chIgG1 antibodies. Therefore, the combinatorial approach of chain shuffling provides a platform to improve specificity and/or affinity of anti-carbohydrate antibodies.

A monoclonal antibody to Lewis Y/Lewis b revealing mimicry of the histone H1 to carbohydrate structures.

Antibodies to either peptide or carbohydrate tumour antigens are established tools for diagnostics and therapy. We here describe an antibody (A70-A/A9) recognizing a carbohydrate epitope common to the tumour-associated Lewis Y and Lewis b antigens (Fucalpha1-2Galbeta1-4/3[Fucalpha1-3/4]GlcNAcbeta-). Its specificity was established without doubt with a panel of 86 synthetic mono- and oligosaccharidic structures. This antibody was found to cross-react with the nuclear protein histone H1. Binding to H1 was specific, periodate-insensitive (non-carbohydrate) and saturable. Histone H1 was able to inhibit Lewis Y binding very effectively in a concentration-dependent manner. We conclude that it represents an example of natural peptide mimicry of a carbohydrate epitope. It may explain the observed occurrence of 'anti-histone autoantibodies' in cancer patients.

A multivalent HIV-vaccine: development of a plasmid DNA for the expression of HIV envelope glycoproteins with hypervariable V3-loop domains.

The third hypervariable (V3) loop of the HIV-1 envelope glycoprotein gp120 plays an essential role in the process of viral entry. It contributes to the tropism, coreceptor usage and immune-escape of the virus. We generated a monovalent plasmid DNA and demonstrated the expression of HIV-1 clade B subtype NL4-3 gp120 and gp160 in comparison to a multivalent plasmid DNA encoding for a variety of V3-variants. In contrast to the membrane-anchored gp160, preliminary data demonstrate the monovariant gp120 is expressed in and presented on a human dendritic cell (DC) line, due to a HIVenv-specific re-stimulation of naïve T-cells detected by IFNgamma-ELISPOT assay.

Expression of the Thomsen-Friedenreich (TF) antigen in the human placenta.

The Thomsen-Friedenreich (TF) antigen (or, more precisely, epitope; Gal beta 1-3GalNAc-) has been known for a long time as a pancarcinoma antigen. Here we report the immunohistochemical identification of this carbohydrate antigen in the human placenta. Paraffin-embedded placental and decidual tissues of the first, second and third trimester were incubated with different monoclonal antibodies (A78-G/A7 and HH8) directed against the TF-epitope and stained with an immunohistochemistry system. We found a strong expression of the TF-epitope in the first trimester of pregnancy. In addition, we identified an expression of the TF-antigen in the second trimester of pregnancy but only in a few cases a positive staining in the third trimester of pregnancy.

Enhanced binding of antibodies to the DTR motif of MUC1 tandem repeat peptide is mediated by site-specific glycosylation.

The epithelial mucin MUC1 is an important tumor marker of breast cancer and other carcinomas. Its immunodominant DTR motif, which is the principal target for immunotherapeutic approaches, has been assumed until recently not to be glycosylated in both normal and tumor MUC1 and to acquire its immunogenic conformation by virtue of a certain number of tandem repeats. We present evidence that the antigenicity of the single repeat toward a considerable number of antibodies to the DTR motif is greatly enhanced if it is glycosylated within this motif, and only in this position. Twenty-eight monoclonal anti-MUC1 antibodies with DTR specificity were tested for binding to synthetic 21-mer (AHG21) or 20-mer (HGV20) tandem repeat peptides O-glycosylated with galactose beta1-3N-acetylgalactosamine alpha or N-acetylgalactosamine alpha at defined Thr or Ser positions. Binding was measured in ELISA experiments using the glycopeptides as plate-immobilized antigens or as inhibitors in solution. At least 12 antibodies revealed significantly enhanced binding to the peptides glycosylated at the DTR motif (Thr-10) as compared to positional isomers glycosylated at Thr-5, Ser-6, Ser-16, or Thr-17 and to the nonglycosylated peptides. Six antibodies (VU-3-C6, A76-A/C7, Ma552, VU-11-D1, VU-12-E1, and VU-11-E2) that were unreactive with the monomeric repeat peptide did bind to the DTR-glycosylated peptide. Several lines of evidence suggest that glycosylation with N-acetylgalactosamine is sufficient for the observed enhancement effect. Our results are of special interest in conjunction with the recent observation that the DTR motif of lactation-associated MUC1 is O-glycosylated in vivo (Müller et al., J. Biol. Chem., 272: 24780-24793, 1997). They may have consequences for the design of efficient tumor vaccines.