Periodic Reporting for period 1 - NANOTAM (Development and Evaluation of Nanomedicines for Cancer Treatment through Immunomodulation: Targeting Tumor-Associated Macrophages)
Reporting period: 2015-05-20 to 2017-05-19
Recent clinical and experimental research establishes chronic inflammation and immune suppression as dominant effects in the tumor microenvironment. The infiltration of Tumor-Associated Macrophages (TAM) in tumor tissues has been shown to support tumor growth, invasion and metastasis. These discoveries have established the therapeutic targeting of TAM, either by depletion, inhibition of their recruitment or by re-stimulating their cytotoxic function, as one of the most promising approaches to combat cancer. However, up-to-now, these ambitious objectives have not been efficiently achieved, due to a deficient “targeting” and/or “perdurable” dose and “long-lasting” effect of the drugs used at the right location (TAM in the tumor). To address this challenge, this NANOTAM project aims to combine the last advances in the fields of Nanotechnology and Cancer Immunotherapy with the ultimate goal of providing new solutions to the patients with cancer.
This NANOTAM project was designed to develop novel Therapeutic Nanostructures (TNs) to improve the specificity of the targeting towards TAM and to increase the effectiveness current antitumoral drugs. Considering the specific molecular characteristics of TAM we have designed on a molecular basis a series of original targeting and therapeutic strategies to reach and re-educate or kill TAM in vivo. The use of nanotechnology in NANOTAM has allowed to address the main challenge of combining the selected molecular strategies in an unique TN. The final goal of this TNs is to re-educate or kill TAM, and consequently, induce the effective switching of the tumor-promoting immune suppressive microenvironment, characteristic of tumors rich in macrophages, to one that kills tumor cells, is anti-angiogenic and promotes adaptive immune responses.
This NANOTAM project was designed to develop novel Therapeutic Nanostructures (TNs) to improve the specificity of the targeting towards TAM and to increase the effectiveness current antitumoral drugs. Considering the specific molecular characteristics of TAM we have designed on a molecular basis a series of original targeting and therapeutic strategies to reach and re-educate or kill TAM in vivo. The use of nanotechnology in NANOTAM has allowed to address the main challenge of combining the selected molecular strategies in an unique TN. The final goal of this TNs is to re-educate or kill TAM, and consequently, induce the effective switching of the tumor-promoting immune suppressive microenvironment, characteristic of tumors rich in macrophages, to one that kills tumor cells, is anti-angiogenic and promotes adaptive immune responses.
In this NANOTAM project, we have designed and developed 10 new empty polymeric Therapeutic Nanostructures (TNs) which can be decorated with different targeting ligands and at the same time loaded with immunomodulatory or chemotherapeutic drugs. Hyaluronic Acid Therapeutic Nanostructures (HA-TNs) decorated with Mannose or with the t-LyP-1 peptide have been generated and evaluated for the in vitro and in vivo targeting of Tumor Associated Macrophages (TAM). These HA-TNs have been successfully engineered to encapsulate 2 chemotherapeutic drugs (Docetaxel, DCX and modified Gemcitabine, GEM-C14) and 1 immunostimulatory drug (Imiquimod, IMQ).
In vitro methodologies and protocols have been adapted and implemented for the toxicological and immunomodulatory evaluation of polymeric-TNs. Non-toxic TNs have been identified and evaluated for their ability to re-educate protumoral M2-like macrophages into antitumoral M1-like macrophages. The toxicity and induction of M1-polarization (ability to re-educate TAM) by HA-TNs loaded with DCX, GEM-C14 or IMQ were observed at similar concentrations in vitro.
Flow cytometry and confocal techniques have been used to evaluate the ability of HA-TNs decorated with different targeting ligands (Mannose or t-LyP-1) to be uptaken by M0, M1 or M2 macrophages in vitro, demonstrating a better ability for the Mannose-HA-TNs than the t-LyP-1-HA-TNs to be internalized by M2 macrophages.
In vivo biocompatibility/toxicological analysis of TNs, based on anatomical or behavioural observations, and biochemical analysis for IMQ, demonstrated no signs of toxicity for the intravenous administration (i.v.) of any TN.
In vivo biodistribution experiments have been performed by i.v. injection of DiR-loaded TNs into MN/MCA fibrosarcoma bearing mice. The IVIS evaluation demonstrated a higher accumulation of Mannose-HA-TNs and t-LyP-1-HA-TNs in the tumor, but also undesirable accumulation in liver and spleen. Of note, higher accumulation for the Mannose-HA-TNs was also observed in the lung.
Evaluation of TNs loaded with DCX, GEM-C14 or IMQ, decorated or not with t-LyP-1 peptide, using immunocompetent murine in vivo models of fibrosarcoma demonstrated a significant antitumoral effect only for the i.v. administration of GEM-C14-loaded-TNs. On-going experiments with Mannose-decorated-HA-TNs are expected to improve the amount of drugs reaching TAM in tumors and to achieve a higher antitumoral efficacy. The decoration of TNs with t-LyP-1 did not show improved antitumoral efficacy in our in vivo murine model (correlated with the poor ability observed in vitro for this targeting ligand to improve the recognition and uptake of TNs by TAM). Additional experiments with Mannose-HA-DCX-TNs must be performed modifying doses and times of injections. The use of IMQ free or loaded into TNs, administered intravenously or intratumorally, did not show significant antitumoral effect in fibrosarcoma bearing mice, however a significant immunostimulatory response was observed in some mice. Thus, we do not consider these HA-IMQ-TNs effective as monotherapy, however their immunostimulatory effect could be exploited for combination therapies (i.e. with immunogenic cell death activators).
Overall, the observations and ideas arising from our experimental research investigation have been published in two review manuscripts (1 under revision). Additional work is required for the preparation of our results for their publication in an original research manuscript and/or to explore the possibilities for patenting, exploitation and/or commercialization.
In vitro methodologies and protocols have been adapted and implemented for the toxicological and immunomodulatory evaluation of polymeric-TNs. Non-toxic TNs have been identified and evaluated for their ability to re-educate protumoral M2-like macrophages into antitumoral M1-like macrophages. The toxicity and induction of M1-polarization (ability to re-educate TAM) by HA-TNs loaded with DCX, GEM-C14 or IMQ were observed at similar concentrations in vitro.
Flow cytometry and confocal techniques have been used to evaluate the ability of HA-TNs decorated with different targeting ligands (Mannose or t-LyP-1) to be uptaken by M0, M1 or M2 macrophages in vitro, demonstrating a better ability for the Mannose-HA-TNs than the t-LyP-1-HA-TNs to be internalized by M2 macrophages.
In vivo biocompatibility/toxicological analysis of TNs, based on anatomical or behavioural observations, and biochemical analysis for IMQ, demonstrated no signs of toxicity for the intravenous administration (i.v.) of any TN.
In vivo biodistribution experiments have been performed by i.v. injection of DiR-loaded TNs into MN/MCA fibrosarcoma bearing mice. The IVIS evaluation demonstrated a higher accumulation of Mannose-HA-TNs and t-LyP-1-HA-TNs in the tumor, but also undesirable accumulation in liver and spleen. Of note, higher accumulation for the Mannose-HA-TNs was also observed in the lung.
Evaluation of TNs loaded with DCX, GEM-C14 or IMQ, decorated or not with t-LyP-1 peptide, using immunocompetent murine in vivo models of fibrosarcoma demonstrated a significant antitumoral effect only for the i.v. administration of GEM-C14-loaded-TNs. On-going experiments with Mannose-decorated-HA-TNs are expected to improve the amount of drugs reaching TAM in tumors and to achieve a higher antitumoral efficacy. The decoration of TNs with t-LyP-1 did not show improved antitumoral efficacy in our in vivo murine model (correlated with the poor ability observed in vitro for this targeting ligand to improve the recognition and uptake of TNs by TAM). Additional experiments with Mannose-HA-DCX-TNs must be performed modifying doses and times of injections. The use of IMQ free or loaded into TNs, administered intravenously or intratumorally, did not show significant antitumoral effect in fibrosarcoma bearing mice, however a significant immunostimulatory response was observed in some mice. Thus, we do not consider these HA-IMQ-TNs effective as monotherapy, however their immunostimulatory effect could be exploited for combination therapies (i.e. with immunogenic cell death activators).
Overall, the observations and ideas arising from our experimental research investigation have been published in two review manuscripts (1 under revision). Additional work is required for the preparation of our results for their publication in an original research manuscript and/or to explore the possibilities for patenting, exploitation and/or commercialization.
NANOTAM, has aimed to create novel nanomedicines, to develop human and capital infrastructure needed for technology development, and to find new solutions for the cure of cancer through the manipulation of the patient’s immune system. With this purpose, in the frame of NANOTAM, Dr. Andón has gathered the support of leading researchers in the fields of nanomedicine and cancer immunotherapy in two european countries (Italy and Spain). His work has provided a bi-directional transfer of knowledge between the host and partner institutions, which is beneficial for the applicant but also for the institutions and for the EU, enhancing the collaboration of excellent researchers from different EU countries, who are now working together on the elaboration of future projects.
Long-term objectives of NANOTAM are to provide new perspectives for research in the fields of nanomedicine and cancer immunotherapy in the EU, with a special focus in linking both disciplines. This project is in line with the Horizon2020 priorities, promoting: Excellence in Science (training of excellent researchers), Industrial Leadership (nanotechnologies) and Societal Challenges (health). Although, no patentable results have still been achieved, the novel approaches for the design of Therapeutic Nanostructures (TNs) evaluation methodologies and research experience arising along NANOTAM, is expected to provide a reference for the use of nanomedicines with immunomodulatory properties for treating cancer, and possibly other immune-related diseases. The ultimate goal, “benefit” of NANOTAM, the institutions and the work of all participants implicated in this proposal has been: to provide solutions to the patients with cancer and improve their lives.
Long-term objectives of NANOTAM are to provide new perspectives for research in the fields of nanomedicine and cancer immunotherapy in the EU, with a special focus in linking both disciplines. This project is in line with the Horizon2020 priorities, promoting: Excellence in Science (training of excellent researchers), Industrial Leadership (nanotechnologies) and Societal Challenges (health). Although, no patentable results have still been achieved, the novel approaches for the design of Therapeutic Nanostructures (TNs) evaluation methodologies and research experience arising along NANOTAM, is expected to provide a reference for the use of nanomedicines with immunomodulatory properties for treating cancer, and possibly other immune-related diseases. The ultimate goal, “benefit” of NANOTAM, the institutions and the work of all participants implicated in this proposal has been: to provide solutions to the patients with cancer and improve their lives.