Periodic Reporting for period 2 - CellularNanoMachines (Development of Stimuli-Responsive Nanoparticle-carrying T lymphocytes in the Fight against Cancer)
Período documentado: 2021-01-04 hasta 2022-01-03
Cancer immunotherapy has been recently erected as the fourth pillar of treatment modalities, along with radiotherapy, surgery, and chemotherapy. Since 2010, numerous clinical trials infusing ex vivo expanded tumor-specific T lymphocytes have demonstrated tumor regression in patients with blood-related tumors. Unfortunately, treatment of solid tumors with similar approaches has yielded less favorable results. Poor trafficking of T cells to the tumor tissue as well as their low ability to survive and/or expand in the hostile tumor microenvironment are two of the main limitations raised in initial clinical trials. Therefore, new strategies able to control and modulate the lifespan and function of antitumor immune cells in solid tumors are urgently needed.
Here, we aimed to find innovative ways to make smart cell-based machines, that is, hybrid systems capable of producing synergistic effects between nanoparticles (NPs) and immune cells towards aforementioned clinical needs. The design would consist of human T cells containing magnetic NPs (coined “magnetic cells”). The tumor-homing “magnetic cells” (guided by an external magnetic field) would facilitate T cell infiltration into tumors to improve T cell infiltration in tumors and potentially enhance the performance of cell therapies.
More specifically, the research objectives for this project can be listed as follows:
(1) to design, synthesize and characterize independent NPs based on iron oxide materials;
(2) to synthesize and characterize polymers containing “clickable” groups for cell surface-grafting and NP-anchoring;
(3) to internalize the different NPs into T cells using innovative bioconjugate chemistry, and;
(4) to evaluate and validate—iterative loop—the accumulation of these NP-cell hybrid systems into tumors using PET, and/or optical imaging techniques.
After the completion of this project, a family of water-soluble zinc-doped iron oxide NPs have been prepared. These NPs have been fully-characterized (TEM, DLS, z-potential, magnetism) and demonstrated optimal biocompatible properties as well as excellent magnetic performance. In vitro studies (using confocal microscopy and flow cytometry) also demonstrated that these NPs can interact with T cells of different origin, and that, the resulting hybrid “NP-cell” adducts can be efficiently magnetized and moved towards a NdFeB magnet. Using optical and nuclear in vivo imaging techniques, we demonstrated that these “magnetic cells” could circulate to tumors more efficiently than control T cells (without NPs). This improved targeting behavior could be attributed to the influence that an external magnetic field exerts on the magnetic NPs, attracting the hybrid systems (NP-T cells) to the targeted area.
Here, we aimed to find innovative ways to make smart cell-based machines, that is, hybrid systems capable of producing synergistic effects between nanoparticles (NPs) and immune cells towards aforementioned clinical needs. The design would consist of human T cells containing magnetic NPs (coined “magnetic cells”). The tumor-homing “magnetic cells” (guided by an external magnetic field) would facilitate T cell infiltration into tumors to improve T cell infiltration in tumors and potentially enhance the performance of cell therapies.
More specifically, the research objectives for this project can be listed as follows:
(1) to design, synthesize and characterize independent NPs based on iron oxide materials;
(2) to synthesize and characterize polymers containing “clickable” groups for cell surface-grafting and NP-anchoring;
(3) to internalize the different NPs into T cells using innovative bioconjugate chemistry, and;
(4) to evaluate and validate—iterative loop—the accumulation of these NP-cell hybrid systems into tumors using PET, and/or optical imaging techniques.
After the completion of this project, a family of water-soluble zinc-doped iron oxide NPs have been prepared. These NPs have been fully-characterized (TEM, DLS, z-potential, magnetism) and demonstrated optimal biocompatible properties as well as excellent magnetic performance. In vitro studies (using confocal microscopy and flow cytometry) also demonstrated that these NPs can interact with T cells of different origin, and that, the resulting hybrid “NP-cell” adducts can be efficiently magnetized and moved towards a NdFeB magnet. Using optical and nuclear in vivo imaging techniques, we demonstrated that these “magnetic cells” could circulate to tumors more efficiently than control T cells (without NPs). This improved targeting behavior could be attributed to the influence that an external magnetic field exerts on the magnetic NPs, attracting the hybrid systems (NP-T cells) to the targeted area.
Different academic results from the different WPs of the proposal have been obtained. Overall, the results can be described as follows:
WP 1: Several zinc-doped iron oxide NPs have been synthesized using thermal decomposition methods. The NPs have been characterized by TEM, DLS and UV-vis. The size of the different nanoparticles varies between 5 and 50 nm. Their magnetic characterization has also been performed, allowing the possibility to select the NPs with the best magnetic performance for following biological applications.
In addition, several strategies have been sought to develop water soluble NPs. The first one was to encapsulate the NPs with a polymer shell of poly(maleic anhydride-alt-1-octadecene). Unfortunately, the resulting NPs tend to precipitate after activation with NHS/EDC (for subsequent bioconjugation). We changed the solubilization method and encapsulate the hydrophobic NPs in the inner core of phospholipid-base micelles. This method has proven really useful in giving exceptional water soluble NPs. Alternatively, the use of a positively-charged polymer, PEI, was also assessed. Unfortunately, this method was not able to produce optimal amounts of water-soluble NPs for in vivo studies and had to be discarded.
For the bifunctional stimuli-responsive likers, a similar ligand with a disulfide bridge (reduction responsive mechanism) was prepared. The release mechanism of this ligand has also been tested both in vitro and in vivo and, the ligand seems to work properly.
WP 2: The ability to prepare polymers on Jurkat T cells (used as a model of human T cells) was assessed, but the viability of these cells was seriously compromised. In different experimental conditions tested, viabilities were always lower than 50%, which was not optimal for later in vivo studies. Additionally, it was thought that there is no need to incorporate polymers with multiple clickable groups because one reactive group per anchor site would be enough for the NPs to incorporate on T cells. This strategy has been validated and a method optimized to click NPs and also small molecules of different origin on the plasma membrane of T cells (Jurkat, human primary and CAR) T cells. Also, cancer cells have also shown ability to react with tetrazine-containing small molecules.
I also acquired experience working with immune cells and isolate T cells from PBMCs. Also, I am now able to work with cells for in vitro and in vivo studies.
WP 3: Different in vitro studies using confocal microscopy and flow cytometry were performed. These studies demonstrated that the prepared NPs can efficiently interact with T cells from different origin: Jurkat cells, human primary T cells, and CAR T cells.
WP 4: Extensive in vivo studies were performed to study the possibility of improving T cell accumulation in tumors using magnetic NPs and an external magnetic field.
Generally, NSG mice bearing two PC3-PSMA+ tumors (one on each flank) were used. Then, different formulations were injected intravenously and all the groups had a NdFeB magnet placed only on the left tumor. After different time points, animals were followed accordingly to the imaging technique being used.
Moreover, different radiolabeling strategies had to be optimized and implemented for NP, cell and NP-cell labelings.
During these 3 years, I have supervised 3 students in the two different groups I have been in. I also gave one Department talks and participated in two conferences, the last one the World Molecular Imaging Conference 2021 (WMIC 21) with a poster. Moreover, I participated in different publications and a recent review. I was also included in the 30 early career professionals selected as “Ones to Watch” for 2021 by the SNMMI.
WP 1: Several zinc-doped iron oxide NPs have been synthesized using thermal decomposition methods. The NPs have been characterized by TEM, DLS and UV-vis. The size of the different nanoparticles varies between 5 and 50 nm. Their magnetic characterization has also been performed, allowing the possibility to select the NPs with the best magnetic performance for following biological applications.
In addition, several strategies have been sought to develop water soluble NPs. The first one was to encapsulate the NPs with a polymer shell of poly(maleic anhydride-alt-1-octadecene). Unfortunately, the resulting NPs tend to precipitate after activation with NHS/EDC (for subsequent bioconjugation). We changed the solubilization method and encapsulate the hydrophobic NPs in the inner core of phospholipid-base micelles. This method has proven really useful in giving exceptional water soluble NPs. Alternatively, the use of a positively-charged polymer, PEI, was also assessed. Unfortunately, this method was not able to produce optimal amounts of water-soluble NPs for in vivo studies and had to be discarded.
For the bifunctional stimuli-responsive likers, a similar ligand with a disulfide bridge (reduction responsive mechanism) was prepared. The release mechanism of this ligand has also been tested both in vitro and in vivo and, the ligand seems to work properly.
WP 2: The ability to prepare polymers on Jurkat T cells (used as a model of human T cells) was assessed, but the viability of these cells was seriously compromised. In different experimental conditions tested, viabilities were always lower than 50%, which was not optimal for later in vivo studies. Additionally, it was thought that there is no need to incorporate polymers with multiple clickable groups because one reactive group per anchor site would be enough for the NPs to incorporate on T cells. This strategy has been validated and a method optimized to click NPs and also small molecules of different origin on the plasma membrane of T cells (Jurkat, human primary and CAR) T cells. Also, cancer cells have also shown ability to react with tetrazine-containing small molecules.
I also acquired experience working with immune cells and isolate T cells from PBMCs. Also, I am now able to work with cells for in vitro and in vivo studies.
WP 3: Different in vitro studies using confocal microscopy and flow cytometry were performed. These studies demonstrated that the prepared NPs can efficiently interact with T cells from different origin: Jurkat cells, human primary T cells, and CAR T cells.
WP 4: Extensive in vivo studies were performed to study the possibility of improving T cell accumulation in tumors using magnetic NPs and an external magnetic field.
Generally, NSG mice bearing two PC3-PSMA+ tumors (one on each flank) were used. Then, different formulations were injected intravenously and all the groups had a NdFeB magnet placed only on the left tumor. After different time points, animals were followed accordingly to the imaging technique being used.
Moreover, different radiolabeling strategies had to be optimized and implemented for NP, cell and NP-cell labelings.
During these 3 years, I have supervised 3 students in the two different groups I have been in. I also gave one Department talks and participated in two conferences, the last one the World Molecular Imaging Conference 2021 (WMIC 21) with a poster. Moreover, I participated in different publications and a recent review. I was also included in the 30 early career professionals selected as “Ones to Watch” for 2021 by the SNMMI.
Overall, we believe that these hybrid “nanoparticle-immune cell” systems are an accessible and simple manner to improve T cell trafficking to solid tumors. These results provide a step forward in magnetic control of the biodistribution of human T cells, facilitating the design of more effective cell therapies. Cancer immunotherapy is a rapidly growing field in Oncology. In the last 10 years, this field has witnessed the clinical approval of different monoclonal antibodies and cell-based therapies for multiple tumor types. However, such treatments are often very expensive. For example, current CAR-T cell therapies can cost between $350,000 and $2 million. Moreover, at this moment, these therapies are limited to specific antigens (e.g. PSMA, CD33). So, these results would potentially provide an alternative for cell transfer therapies to hospitals with no experience or infrastructure in cell engineering as well as to lower the total costs of these therapies.