Focà, Giuseppina (2015) Anti-Cripto monoclonal antibodies and their fragments: structural and functional studies. [Tesi di dottorato]
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Focà Giuseppina 2015.pdf Download (7MB) | Anteprima |
| Tipologia del documento: | Tesi di dottorato | ||||||
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| Lingua: | English | ||||||
| Titolo: | Anti-Cripto monoclonal antibodies and their fragments: structural and functional studies | ||||||
| Autori: |
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| Data: | 30 Marzo 2015 | ||||||
| Numero di pagine: | 189 | ||||||
| Istituzione: | Università degli Studi di Napoli Federico II | ||||||
| Dipartimento: | Farmacia | ||||||
| Scuola di dottorato: | Scienze farmaceutiche | ||||||
| Dottorato: | Scienza del farmaco | ||||||
| Ciclo di dottorato: | 27 | ||||||
| Coordinatore del Corso di dottorato: |
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| Tutor: |
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| Data: | 30 Marzo 2015 | ||||||
| Numero di pagine: | 189 | ||||||
| Parole chiave: | human Cripto-1, mAb, Fab, ADC | ||||||
| Settori scientifico-disciplinari del MIUR: | Area 05 - Scienze biologiche > BIO/10 - Biochimica Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare Area 03 - Scienze chimiche > CHIM/08 - Chimica farmaceutica |
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| Depositato il: | 10 Apr 2015 11:35 | ||||||
| Ultima modifica: | 28 Apr 2018 01:00 | ||||||
| URI: | http://www.fedoa.unina.it/id/eprint/10263 | ||||||
| DOI: | 10.6093/UNINA/FEDOA/10263 |
Abstract
Human Cripto-1 (CR-1) is the founding member of the epidermal growth factors (EGF-CFC) superfamily, composed of structurally related protein members. CR-1 is involved in many biological functions. Among the others, it is a key regulator of the embryogenesis, promoting the differentiation of embryonic stem cells and the organogenesis of brain and heart. Interestingly, CR-1 also promotes tumorigenesis, regulating cell proliferation, migration, epithelial-mesenchimal transition, survival and angiogenesis. The functional part of CR-1 protein consists of two central Cysteine Rich Domains, EGF-like and CFC, that independently interact with several partners. The increasing interest on CR-1 as a novel diagnostic and therapeutic target is derived from the evidence that it is highly expressed in many human solid tumors, while it is barely present or totally absent in normal adult tissues. CR-1 can promote tumorigenesis by activation of several pathways; we have focused our attention on the CR-1/ALK4 interaction which triggers the Smads cascade, leading to the activation of gene transcription encoding tumor-related factors (Nodal, Lefty). Thus, the aim of this study was the generation and development of smart and highly selective biomolecules such as mAbs and their fragments that target the CFC domain of CR-1, directly involved in the CR-1/ALK4 as well as in the CR-1/ GRP78 interactions. For this purpose, as starting point, a set of monoclonal antibodies was generated by the hybridoma technique, using as antigen the synthetic and correctly folded human CFC [112-150] domain. A screening was performed to identify the mAbs with the highest affinity toward the full-length recombinant human CR-1 protein (rhCR-1) and with the highest specificity toward the two "hot spots" residues (H120 and W123) of the CFC domain. We thus identified two mAbs, named 10D1 and 1B4. 10D1 and 1B4 interacted very strongly with CR-1, as they were characterized by KD of 70 pM and 200 pM, respectively. A subsequent comparative epitope study, combining a computational and a biochemical approach, confirmed that both mAbs recognized the region of the CFC domain containing the H120 and W123, suggesting that these molecules may act as potential blocking antibodies and/or at least be the starting point to develop potential therapeutic tools to be employed on cancer cells over-expressing CR-1. In addition, preliminary in vitro assays suggested that the 1B4 mAb had such properties, as it promoted cell death on GEO cancer cell lines. The second phase of this study was focused on the generation of functional antibody fragments of the 1B4 mAb. A first proteolytic approach was unsuccessful, leading to a partially inactive Fab fragment. We thus next moved to generate an engineered recombinant chimeric Fab fragment of the same mAb (1B4 rFab) that only retained a partial binding activity (KD 6 nM) toward the target protein. Using this recombinant fragment as template, a new innovative dimeric format (1B4 rFab2) was created exploiting an elegant conjugation strategy based on the use of the M-TGase enzyme. This dimeric 1B4 rFab2 protein displayed to rhCR1 a binding affinity comparable to that one of the whole 1B4 antibody. Finally, with the aim of increasing the cell killing potential of the 1B4 rFab and to exploit its CR-1 specificity in tumor homing approaches, we designed and realized a strategy to generate an ADC (Antibody Drug Conjugate), arming the 1B4 rFab with a payload consisting of the cytotoxic agent Doxorubicin.
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