Immunotherapy has had a revolutionary impact on cancer treatment, providing valuable tools to be used in combination or as an alternative to chemotherapy (Weiner G.J., 2015). Such wide success has been demonstrated by the fact that, since 1997, twelve monoclonal antibodies have been approved by the FDA for the treatment of a variety of solid tumors and haematological malignancies, along with an increasing number of new antibodies that are now being tested at different stages of clinical trials (ClinicalTrials.gov). Nevertheless, there are limitations to the use of monoclonal antibodies mainly due to their big size, which is detrimental especially for their applications in clinics and diagnostics. For these reasons, the arise of recombinant antibodies, which conjugate small size with antigen specificity has represented a big achievement in the oncological setting. Another crucial point is the identification of cell antigens, which can be exploited to target cancer cells. To this purpose, we have focused on inotropic Glutamate Receptor 4 (iGluR4), which is involved in many CNS pathological conditions and, moreover, has recently emerged to be implicated in many aspects of cancer progression (Stepulak A. et al., 2011; Stepulak A. et al., 2014; Ribeiro MP et al., 2016). We have also focused on hERG1 voltage-gated ion channel, that it is known to be a role player in cancer progression (Bianchi L. et al., 1998; Lastraioli E. et al., 2015). More recently, it has emerged as a novel target for cancer therapy and as a marker for patients’ stratification (Arcangeli A. et al., 2009; Pointer KB et al., 2016). Moreover, it has been demonstrated that hERG1 forms complexes in particular with β1 subunit of integrin receptors, thus mediating its effects in cancer cell behavior (Crociani et al. 2013). The present work has focused on developing new antibodies, that have demonstrated their potential from a diagnostic or a therapeutic point of view. We have produced two clones B5 and C4 of a monoclonal antibody directed against ionotropic Glutamate Receptor 4 (iGluR4), providing evidences that this molecule is able to recognize the antigen, also in a pathological scenario such as that of tuberous sclerosis complex (TSC) disease. Moreover, C4 anti-iGluR4 clone has emerged as a Abstract possible positive allosteric modulator of the receptor; such behavior will be better characterized in the future, to exploit its potential application as a channel regulator (Stepulak A. et al., 2014). We are also assembling a construct for the production of a scFv-iGluR4 directed against the same antigen as the monoclonal antibody, to overcome the problems related to the passage of the blood brain barrier (BBB). In fact, it is known that recombinant protein therapeutics are too large to cross the BBB, however, recombinant proteins can be reengineered as BBB-penetrating IgG fusion proteins, where the IgG part is a genetically engineered monoclonal antibody (MAb) (Pardridge WM and Boado RJ, 2012). The recombinant antibody we have produced will be conjugated with NPs (already capable to cross an in vitro model of BBB), to set up a drug-delivery system. In parallel, we have developed a single-chain variable fragment antibody, scFv-hERG1- G3 (scFv-hERG1 construct), that has already been validated, after labelling with Alexa 488 fluorophore, as a tool for optical in vivo imaging (Lastraioli E. et al., 2016). Moreover, we have produced a new antibody scFv-hERG1-D8-Cys (scFv-hERG1-Cys construct) (via mutagenesis of the scFv-hERG1 construct), in which we have reintroduced one of the cysteine that is in a fundamental position for the formation of the disulfide bonds, within the VH chain of the scFv antibody. In fact, scFv-hERG1-G3 antibody, in this position, showed a phenylalanine amino acid. We have given evidence that the restoration of the Cys deeply affects the protein yield, without affecting the antibody binding capacities. scFv-hERG1-D8-Cys has also demonstrated to have effects on cell growth and invasiveness, as demonstrated by the viability experiments performed on 2D tumour cell lines and on 3D spheroid tumour cell cultures (patent in preparation). In the third part of this work, we have focused on the expression and characterization of a scDb bispecific antibody directed against hERG1-β1 oncogenic unit. The antibody has been also characterized with IF experiments performed on different substrates, showing the capacity to recognize hERG1-β1complex. These evidences allow us to conclude that both scFv-hERG1-D8-Cys and anti-scDbhERG1- β1, after a proper validation, will represent valuable tools for cancer therapy.

Novel molecular tools in cancer therapy: diagnostic and therapeutic applications of antibodies targeting ion channels and receptors / Claudia Duranti. - (2017).

Novel molecular tools in cancer therapy: diagnostic and therapeutic applications of antibodies targeting ion channels and receptors.

DURANTI, CLAUDIA
2017

Abstract

Immunotherapy has had a revolutionary impact on cancer treatment, providing valuable tools to be used in combination or as an alternative to chemotherapy (Weiner G.J., 2015). Such wide success has been demonstrated by the fact that, since 1997, twelve monoclonal antibodies have been approved by the FDA for the treatment of a variety of solid tumors and haematological malignancies, along with an increasing number of new antibodies that are now being tested at different stages of clinical trials (ClinicalTrials.gov). Nevertheless, there are limitations to the use of monoclonal antibodies mainly due to their big size, which is detrimental especially for their applications in clinics and diagnostics. For these reasons, the arise of recombinant antibodies, which conjugate small size with antigen specificity has represented a big achievement in the oncological setting. Another crucial point is the identification of cell antigens, which can be exploited to target cancer cells. To this purpose, we have focused on inotropic Glutamate Receptor 4 (iGluR4), which is involved in many CNS pathological conditions and, moreover, has recently emerged to be implicated in many aspects of cancer progression (Stepulak A. et al., 2011; Stepulak A. et al., 2014; Ribeiro MP et al., 2016). We have also focused on hERG1 voltage-gated ion channel, that it is known to be a role player in cancer progression (Bianchi L. et al., 1998; Lastraioli E. et al., 2015). More recently, it has emerged as a novel target for cancer therapy and as a marker for patients’ stratification (Arcangeli A. et al., 2009; Pointer KB et al., 2016). Moreover, it has been demonstrated that hERG1 forms complexes in particular with β1 subunit of integrin receptors, thus mediating its effects in cancer cell behavior (Crociani et al. 2013). The present work has focused on developing new antibodies, that have demonstrated their potential from a diagnostic or a therapeutic point of view. We have produced two clones B5 and C4 of a monoclonal antibody directed against ionotropic Glutamate Receptor 4 (iGluR4), providing evidences that this molecule is able to recognize the antigen, also in a pathological scenario such as that of tuberous sclerosis complex (TSC) disease. Moreover, C4 anti-iGluR4 clone has emerged as a Abstract possible positive allosteric modulator of the receptor; such behavior will be better characterized in the future, to exploit its potential application as a channel regulator (Stepulak A. et al., 2014). We are also assembling a construct for the production of a scFv-iGluR4 directed against the same antigen as the monoclonal antibody, to overcome the problems related to the passage of the blood brain barrier (BBB). In fact, it is known that recombinant protein therapeutics are too large to cross the BBB, however, recombinant proteins can be reengineered as BBB-penetrating IgG fusion proteins, where the IgG part is a genetically engineered monoclonal antibody (MAb) (Pardridge WM and Boado RJ, 2012). The recombinant antibody we have produced will be conjugated with NPs (already capable to cross an in vitro model of BBB), to set up a drug-delivery system. In parallel, we have developed a single-chain variable fragment antibody, scFv-hERG1- G3 (scFv-hERG1 construct), that has already been validated, after labelling with Alexa 488 fluorophore, as a tool for optical in vivo imaging (Lastraioli E. et al., 2016). Moreover, we have produced a new antibody scFv-hERG1-D8-Cys (scFv-hERG1-Cys construct) (via mutagenesis of the scFv-hERG1 construct), in which we have reintroduced one of the cysteine that is in a fundamental position for the formation of the disulfide bonds, within the VH chain of the scFv antibody. In fact, scFv-hERG1-G3 antibody, in this position, showed a phenylalanine amino acid. We have given evidence that the restoration of the Cys deeply affects the protein yield, without affecting the antibody binding capacities. scFv-hERG1-D8-Cys has also demonstrated to have effects on cell growth and invasiveness, as demonstrated by the viability experiments performed on 2D tumour cell lines and on 3D spheroid tumour cell cultures (patent in preparation). In the third part of this work, we have focused on the expression and characterization of a scDb bispecific antibody directed against hERG1-β1 oncogenic unit. The antibody has been also characterized with IF experiments performed on different substrates, showing the capacity to recognize hERG1-β1complex. These evidences allow us to conclude that both scFv-hERG1-D8-Cys and anti-scDbhERG1- β1, after a proper validation, will represent valuable tools for cancer therapy.
2017
Annarosa Arcangeli
Claudia Duranti
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/1077157
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