Monoklonale Antikörper und rekombinante Antikörperfusionsproteine für die Diagnose des Mammakarzinoms und die Therapie des Rhabdomyosarkoms

Monoclonal antibodies (mab) and antibody fusion proteins gain particular significance in pharmaceutical industry with diagnosis and therapy of cancer being the most interesting fields of research and development. Their high specificity make them ideal tools for target directed cancer therapeutics as well as for identification of novel specific tumour markers. The identification and characterisation of new reliable tumour-associated markers remain the most challenging tasks. The aim of this work was the generation and characterisation of mabs against a potential tumour antigen, namely RAI3. RAI3 belongs to the GPCR family the most widespread and abundant receptor family known. Recent studies underline an exceptional role of RAI3 in the growth of mammary carcinoma making it a potential candidate for an antibody-based therapy as well as a novel diagnostic marker. The exact molecular mechanism of RAI3 remains unclear and is a matter of some controversy. In this work we generated five mabs against RAI3 using classical hybridoma technology including immunisation of BALB/c mice with recombinant full-length protein integrated into liposomes. Their specific binding properties on recombinant protein as well as cell lysates and whole cells were characterised using ELISA, Western blot and immunofluorescence techniques. All mabs showed high specificity and affinities against RAI3 in the nanomolar range. However, cell surface binding on RAI3 positive cells in flow cytometry could not be observed. Therefore, further investigations focussed on the application of the antibodies in a diagnostic approach on tumour tissues. A method for staining of paraffin-embedded tissue sections was successfully established. Mab 24 2.3 was subsequently used for the immunohistochemical analysis of 147 breast carcinoma and 44 normal breast tissues using a tissue microarray. We found RAI3 was abundantly expressed in breast carcinoma although median staining score did not differ in normal and tumour tissue. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. Based on these findings, the role of RAI3 as a tumour marker remains unclear. The generated mabs could serve as important tools for further studies on a variety of tissues to reveal the functional role of RAI3 expression in human cancer. In a second part of this work the production and characterisation of a novel fAChR-specific immunotoxin, scFv35-ETA’, is described. The fAChR is the fetal precursor of the nicotinic AChR and has already been identified as a specific maker in rhabdomyosarcoma (RMS). RMS is the most common malignant soft tissue tumour of childhood. The investigated immunotoxin is composed of the human autoantibody-based scFv35 and a deletion mutant of Pseudomonas Exotoxin A (ETA’). The immunotoxin was successfully expressed in E.coli and purified from cell lysates following a functional characterisation towards specificity, affinity and cytotoxicity. The analysis confirmed specific binding of the immunotoxin with high functional affinities (Kd= 4.3 nM) on RMS cell lines of the embryonal as well as the alveolar subtype. In an XTT proliferation assay a specific inhibition of cell growth was demonstrated that could be prevented by co-incubation of scFv35 without a toxic domain. A collection of different cell lines were tested that showed varying sensitivities towards the immunotoxins with IC50 values ranging from 0.13 nM (FL-OH-1) to 113 nM (RH30). A further investigation on the cytotoxic potential of the immunotoxins revealed the induction of apoptosis. These promising results show the great potential of this novel immunotoxin making it an interesting candidate for further preclinical and clinical development. The human origin of scFv35 allows the development of a fully human immunotoxin by fusion to human toxins or effector domains. Recent observations of rhabdoid transdifferentiation of metastatic malignant melanoma could reveal a broadened spectrum of application for fAChR-specific therapeutics

Monoklonale Antikörper und rekombinante Antikörperfusionsproteine für die Diagnose des Mammakarzinoms und die Therapie des Rhabdomyosarkoms

Monoclonal antibodies (mab) and antibody fusion proteins gain particular significance in pharmaceutical industry with diagnosis and therapy of cancer being the most interesting fields of research and development. Their high specificity make them ideal tools for target directed cancer therapeutics as well as for identification of novel specific tumour markers. The identification and characterisation of new reliable tumour-associated markers remain the most challenging tasks. The aim of this work was the generation and characterisation of mabs against a potential tumour antigen, namely RAI3. RAI3 belongs to the GPCR family the most widespread and abundant receptor family known. Recent studies underline an exceptional role of RAI3 in the growth of mammary carcinoma making it a potential candidate for an antibody-based therapy as well as a novel diagnostic marker. The exact molecular mechanism of RAI3 remains unclear and is a matter of some controversy. In this work we generated five mabs against RAI3 using classical hybridoma technology including immunisation of BALB/c mice with recombinant full-length protein integrated into liposomes. Their specific binding properties on recombinant protein as well as cell lysates and whole cells were characterised using ELISA, Western blot and immunofluorescence techniques. All mabs showed high specificity and affinities against RAI3 in the nanomolar range. However, cell surface binding on RAI3 positive cells in flow cytometry could not be observed. Therefore, further investigations focussed on the application of the antibodies in a diagnostic approach on tumour tissues. A method for staining of paraffin-embedded tissue sections was successfully established. Mab 24 2.3 was subsequently used for the immunohistochemical analysis of 147 breast carcinoma and 44 normal breast tissues using a tissue microarray. We found RAI3 was abundantly expressed in breast carcinoma although median staining score did not differ in normal and tumour tissue. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. Based on these findings, the role of RAI3 as a tumour marker remains unclear. The generated mabs could serve as important tools for further studies on a variety of tissues to reveal the functional role of RAI3 expression in human cancer. In a second part of this work the production and characterisation of a novel fAChR-specific immunotoxin, scFv35-ETA’, is described. The fAChR is the fetal precursor of the nicotinic AChR and has already been identified as a specific maker in rhabdomyosarcoma (RMS). RMS is the most common malignant soft tissue tumour of childhood. The investigated immunotoxin is composed of the human autoantibody-based scFv35 and a deletion mutant of Pseudomonas Exotoxin A (ETA’). The immunotoxin was successfully expressed in E.coli and purified from cell lysates following a functional characterisation towards specificity, affinity and cytotoxicity. The analysis confirmed specific binding of the immunotoxin with high functional affinities (Kd= 4.3 nM) on RMS cell lines of the embryonal as well as the alveolar subtype. In an XTT proliferation assay a specific inhibition of cell growth was demonstrated that could be prevented by co-incubation of scFv35 without a toxic domain. A collection of different cell lines were tested that showed varying sensitivities towards the immunotoxins with IC50 values ranging from 0.13 nM (FL-OH-1) to 113 nM (RH30). A further investigation on the cytotoxic potential of the immunotoxins revealed the induction of apoptosis. These promising results show the great potential of this novel immunotoxin making it an interesting candidate for further preclinical and clinical development. The human origin of scFv35 allows the development of a fully human immunotoxin by fusion to human toxins or effector domains. Recent observations of rhabdoid transdifferentiation of metastatic malignant melanoma could reveal a broadened spectrum of application for fAChR-specific therapeutics

Production and characterisation of monoclonal antibodies against RAI3 and its expression in human breast cancer

Abstract Background RAI3 is an orphan G-protein coupled receptor (GPCR) that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens. Methods We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA), western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147) and normal breast tissues (n = 44) using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50) as well as lymph node metastases (n = 3) for RAI3 mRNA expression. Results The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. Conclusion We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic analysis of RAI3 expression in normal and cancerous human breast tissue at both the mRNA and protein levels.</p