Development of polypeptide-based therapeutics for the treatment of castration resistant prostate cancer
- Autores: Sonia Vicente Ruiz Salvador
- Directores de la Tesis: María J. Vicent Docon (dir. tes.), Ana Armiñán de Benito (codir. tes.)
- Lectura: En la Universitat de València ( España ) en 2020
- Idioma: español
- Tribunal Calificador de la Tesis: Fabian Kiessling (presid.), Silvia Muro Galindo (secret.), Antonio José Conde Moreno (voc.)
- Programa de doctorado: Programa de Doctorado en Biomedicina y Biotecnología por la Universitat de València (Estudi General)
- Materias:
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- Resumen
Prostate Cancer (PCa) is currently the most prevalent non-cutaneous malignancy and the second most prevalent cancer in men. Advances in early diagnosis, identification of biomarkers for specific subtypes, and the application of effective treatments represent critical parameters in the clinical management of PCa patients. Effective treatment options for cancer in general now include combination-based therapies, given the general complexity of cancers and the rise of resistance to some single-drug approaches. Furthermore, the modification of existing drugs, including polymer conjugation, can allow for greater stability in plasma, enhanced tumor targeting, altered cell trafficking, and improved pharmacokinetics to foster improvements in patient outcomes.
Studies have established the TMPRSS2 (androgen-dependent serine protease) and ERG (ETS family transcription factor) fusion gene (T2E) as a potential biomarker of metastatic castration-resistant PCa, with overexpression observed in 50-70% of PCa cases. In these cases, the androgen receptor (AR) and insulin-like growth factor 1 receptor (IGF-1R) reciprocal signaling pathways potently drive tumor progression; therefore, the dual inhibition of these pathways may represent a promising means to inhibit PCa progression.
The inhibition of IGF-1R signaling with an anti-IGF-1R-specific monoclonal antibody (mAb - AVE1642) has shown potential in the treatment of T2E-positive PCa patients; however, patients treated with anti-IGF-1R inhibitors in several clinical trials only experienced a partial response to therapy and suffered from significant side toxicities. The application of nanomedicine to PCa has the potential to overcome many of the limitations of current therapies in both localized and metastatic disease, and we hypothesized that the polymer-modification of AVE1642 might improve therapeutic outcomes in the T2E-positive PCa subtype.
To this end, we conjugated AVE1642 with a biodegradable and biocompatible polypeptide (poly-L-glutamic acid (PGA)) to create PGA-AVE1642, which we then evaluated as an optimized treatment approach. While we aimed to generate an advanced therapeutic approach, we also sought to fully understand the effect of PGA conjugation to a mAb regarding pharmacological activity in vitro and in vivo.
Our initial in vitro analyses showed similar cell viability in the T2E-positive VCaP PCa cell line treated with both AVE1642 and PGA-AVE1642, confirming that PGA-conjugation does not impede AVE1642 function. PGA-AVE1642 maintained a general specificity for T2E-positive cells, but instead of binding to IGF-1R and becoming internalized rapidly as occurs for unmodified AVE1642, PGA-AVE1642 displayed enhanced stability in plasma and stronger binding to IGF-1R, which maintained PGA-AVE1642 at the cell membrane and prevented IGF-1R internalization.
We also discovered that PGA-conjugation of AVE1642 altered cellular trafficking; while confocal/STORM microscopy in VCaP cells established that AVE1642 mainly colocalized with endosomes and clathrin, PGA-AVE1642 mostly colocalized with caveolin-1 but not with endosomes, demonstrating PGA-conjugation modifies IGF-1R mediated endocytosis probably due to a modified interaction with the receptor. Furthermore, altered trafficking coincided with a differential signaling pathway inhibition pattern upon PGA conjugation. While unmodified AVE1642 inhibited the PI3K pathway downstream of IGF-1R inhibition, PGA-AVE1642 inhibited both the PI3K and MAPK pathways, thereby displaying increased anti-tumorigenic potential.
Subsequent in vivo analyses in a newly developed orthotopic PCa mouse model employing luciferase-expressing VCaP cells revealed higher anti-tumoral activity for PGA-AVE1642 compared with AVE1642 using an equivalent dose. Again, we found that PGA-AVE1642 treatment inhibited both the PI3K and MAPK pathways; however, subsequent tumor microenvironmental studies revealed that PGA-AVE1642 treatment also potently inhibited tumor blood vessel functionality and maturity, cell proliferation, and angiogenesis when compared to treatment with unmodified AVE1642.
Overall, polymer conjugation improves AVE1642 antitumoral activity both in vitro and in vivo by stronger IGF-1R inhibition, which avoids the activation of MAPK and PI3K downstream signaling pathways preventing cancer cell progression. Furthermore, our results suggest that polymer conjugation of AVE1642 promotes alterations to the tumor microenvironment in PCa tumor models, thereby enhancing therapeutic efficacy.
As a means to further improve PCa treatment with PGA-AVE1642, we looked to evaluate a combination therapy-based approach through the additional inhibition of the AR signaling pathway via treatment with the anti-androgen drug abiraterone. Polymer-based combination therapies employing synergistic drugs aimed at different pharmacological targets represent an exciting means to target tumor cells effectively. As a first step towards the generation of a PGA-antibody conjugate, we evaluated the combination of AVE1642 or PGA-AVE1642 with free abiraterone in vitro and in vivo.
Our in vitro analyses provided evidence for a synergistic effect for both combination therapies in T2E-positive PCa cells; however, we only found synergy with regards to primary tumor growth for free AVE1642 in combination with abiraterone, and not for PGA-AVE1642. This is most probably due to the differential molecular mechanism achieved upon conjugation that it is already providing the role that abiraterone plays in T2E-positive PCa tumors. Both combination therapies (AVE1642 or PGA-AVE1642 with abiraterone) provided for dual PI3K and MAPK pathway inhibition in vivo. Exploration on the effect on tumor microenvironment is still ongoing as even if not synergistic, significant differences have been encountered when abiraterone is present not only for free but also for conjugated AVE1642.
Overall, our studies have demonstrated that polymer-conjugation of a monoclonal antibody alters the function and improves anti-tumorigenic capacity. Furthermore, we have also provided evidence for the anti-tumor efficacy of a new combination therapy for advanced PCa based on the inhibition of both IGF-1R and AR pathways.
