Matthew T. Silvestrini

Devices for cell transplantation into the central nervous system: Design considerations and emerging technologies

Successful use of cell-based therapies for the treatment of neurological diseases is dependent upon effective delivery to the central nervous system (CNS). The CNS poses several challenges to the delivery of cell-based therapeutics, including the blood–brain barrier, anatomic complexity, and regional specificity. Targeted delivery methods are therefore required for the selective treatment of specific CNS regions. In addition, CNS tissues are mechanically and physiologically delicate and even minor injury to normal brain or spinal cord can cause devastating neurological deficits. Targeted delivery methods must therefore minimize tissue trauma. At present, direct injection into brain or spinal cord parenchyma promises to be the most versatile and accurate method of targeted CNS therapeutic delivery. While direct injection methods have already been employed in clinical trials of cell transplantation for a wide variety of neurological diseases, there are many shortcomings with the devices and surgical approaches currently used. Some of these technical limitations may hinder the clinical development of cell transplantation therapies despite validity of the underlying biological mechanisms. In this review, we discuss some of the important technical considerations of CNS injection devices such as targeting accuracy, distribution of infused therapeutic, and overall safety to the patient. We also introduce and discuss an emerging technology – radially branched deployment – that may improve our ability to safely distribute cell-based therapies and other therapeutic agents to the CNS. Finally, we speculate on future technological developments that may further enhance the efficacy of CNS therapeutic delivery.

Nanoengineered Surfaces Enhance Drug Loading and Adhesion

To circumvent the barriers encountered by macromolecules at the gastrointestinal mucosa, sufficient therapeutic macromolecules must be delivered in close proximity to cells.(1) Previously, we have shown that silicon nanowires penetrate the mucous layer and adhere directly to cells under high shear.(2) In this work, we characterize potential reservoirs and load macromolecules into interstitial space between nanowires. We show significant increases in loading capacity due to nanowires while retaining adhesion of loaded particles under high shear.

Radially Branched Deployment for More Efficient Cell Transplantation at the Scale of the Human Brain

Background: In preclinical studies, cell transplantation into the brain has shown great promise for the treatment of a wide range of neurological diseases. However, the use of a straight cannula and syringe for cell delivery to the human brain does not approximate cell distribution achieved in animal studies. This technical deficiency may limit the successful clinical translation of cell transplantation. Objective: To develop a stereotactic device that effectively distributes viable cells to the human brain. Our primary aims were to (1) minimize the number of transcortical penetrations required for transplantation, (2) reduce variability in cell dosing and (3) increase cell survival. Methods: We developed a modular cannula system capable of radially branched deployment (RBD) of a cell delivery catheter at variable angles from the longitudinal device axis. We also developed an integrated catheter-plunger system, eliminating the need for a separate syringe delivery mechanism. The RBD prototype was evaluated in vitro and in vivo with subcortical injections into the swine brain. Performance was compared to a 20G straight cannula with dual side ports, a device used in current clinical trials. Results: RBD enabled therapeutic delivery in a precise ‘tree-like’ pattern branched from a single initial trajectory, thereby facilitating delivery to a volumetrically large target region. RBD could transplant materials in a radial pattern up to 2.0 cm from the initial penetration tract. The novel integrated catheter-plunger system facilitated manual delivery of small and precise volumes of injection (1.36 ± 0.13 µl per cm of plunger travel). Both dilute and highly concentrated neural precursor cell populations tolerated transit through the device with high viability and unaffected developmental potential. While reflux of infusate along the penetration tract was problematic with the use of the 20G cannula, RBD was resistant to this source of cell dose variability in agarose. RBD enabled radial injections to the swine brain when used with a modern clinical stereotactic system. Conclusions: By increasing the total delivery volume through a single transcortical penetration in agarose models, RBD strategy may provide a new approach for cell transplantation to the human brain. Incorporation of RBD or selected aspects of its design into future clinical trials may increase the likelihood of successful translation of cell-based therapy to the human patient.

Priming is key to effective incorporation of image-guided thermal ablation into immunotherapy protocols

Focal therapies play an important role in the treatment of cancers where palliation is desired, local control is needed, or surgical resection is not feasible. Pairing immunotherapy with such focal treatments is particularly attractive; however, there is emerging evidence that focal therapy can have a positive or negative impact on the efficacy of immunotherapy. Thermal ablation is an appealing modality to pair with such protocols, as tumors can be rapidly debulked (cell death occurring within minutes to hours), tumor antigens can be released locally, and treatment can be conducted and repeated without the concerns of radiation-based therapies. In a syngeneic model of epithelial cancer, we found that 7 days of immunotherapy (TLR9 agonist and checkpoint blockade), prior to thermal ablation, reduced macrophages and myeloid-derived suppressor cells and enhanced IFN-γ-producing CD8+ T cells, the M1 macrophage fraction, and PD-L1 expression on CD45+ cells. Continued treatment with immunotherapy alone or with immunotherapy combined with ablation (primed ablation) then resulted in a complete response in 80% of treated mice at day 90, and primed ablation expanded CD8+ T cells as compared with all control groups. When the tumor burden was increased by implantation of 3 orthotopic tumors, successive primed ablation of 2 discrete lesions resulted in survival of 60% of treated mice as compared with 25% of mice treated with immunotherapy alone. Alternatively, when immunotherapy was begun immediately after thermal ablation, the abscopal effect was diminished and none of the mice within the cohort exhibited a complete response. In summary, we found that immunotherapy begun before ablation can be curative and can enhance efficacy in the presence of a high tumor burden. Two mechanisms have potential to impact the efficacy of immunotherapy when begun immediately after thermal ablation: mechanical changes in the tumor microenvironment and inflammatory-mediated changes in immune phenotype.

CpG expedites regression of local and systemic tumors when combined with activatable nanodelivery.

Ultrasonic activation of nanoparticles provides the opportunity to deliver a large fraction of the injected dose to insonified tumors and produce a complete local response. Here, we evaluate whether the local and systemic response to chemotherapy can be enhanced by combining such a therapy with locally-administered CpG as an immune adjuvant. In order to create stable, activatable particles, a complex between copper and doxorubicin (CuDox) was created within temperature-sensitive liposomes. Whereas insonation of the CuDox liposomes alone has been shown to produce a complete response in murine breast cancer after 8 treatments of 6 mg/kg delivered over 4 weeks, combining this treatment with CpG resolved local cancers within 3 treatments delivered over 7 days. Further, contralateral tumors regressed as a result of the combined treatment, and survival was extended in systemic disease. In both the treated and contralateral tumor site, the combined treatment increased leukocytes and CD4+ and CD8+ T-effector cells and reduced myeloid-derived suppressor cells (MDSCs). Taken together, the results suggest that this combinatorial treatment significantly enhances the systemic efficacy of locally-activated nanotherapy.

Toward Personalized Peptide-Based Cancer Nanovaccines: A Facile and Versatile Synthetic Approach

Personalized cancer vaccines (PCVs) are receiving attention as an avenue for cancer immunotherapy. PCVs employ immunogenic peptide epitopes capable of stimulating the immune system to destroy cancer cells with great specificity. Challenges associated with effective delivery of these peptides include poor solubility of hydrophobic sequences, rapid clearance, and poor immunogenicity, among others. The incorporation of peptides into nanoparticles has the potential to overcome these challenges, but the broad range of functionalities found in amino acids presents a challenge to conjugation due to possible interferences and lack of reaction specificity. Herein, a facile and versatile approach to generating nanosized PCVs under mild nonstringent conditions is reported. Following a simple two-step semibatch synthetic approach, amphiphilic hyperbranched polymer-peptide conjugates were prepared by the conjugation of melanoma antigen peptides, either TRP2 (hydrophobic) or MUT30 (hydrophilic), to an alkyne functionalized core via strain-promoted azide-alkyne click chemistry. Self-assembly of the amphiphiles gave spherical nanovaccines (by transmission electron microscopy) with sizes in the range of 10-30 nm (by dynamic light scattering). Fluorescently labeled nanovaccines were prepared to investigate the cellular uptake by antigen presenting cells (dendritic cells), and uptake was confirmed by flow cytometry and microscopy. The TRP2 nanovaccine was taken up the most followed by MUT30 nanoparticles and, finally, nanoparticles without peptide. The nanovaccines showed good biocompatibility against B16-F10 cells, yet the TRP2 peptide showed signs of toxicity, possibly due to its hydrophobicity. A test for immunogenicity revealed that the nanovaccines were poorly immunogenic, implying the need for an adjuvant when administered in vivo. Treatment of mice with melanoma tumors showed that in combination with adjuvant, CpG, groups with the peptide nanovaccines slowed tumor growth and improved survival (up to 24 days, TRP2) compared to the untreated group (14 days).

Abstract LB-052: Activatable nanodelivery combined with CpG-ODN and anti-PD-1 achieves a complete response in directly-treated and contralateral tumors in a murine breast cancer model

We demonstrate for the first time that blocking of the programmed death-1 (PD-1) pathway in conjunction with immunogenic cell death induced by CpG-ODN and activatable nanodelivery of doxorubicin can generate curative responses in both primary and contralateral tumors. Activatable nanotherapeutics are attractive since the toxicity of chemotherapeutics can be constrained to a small region; combining such a strategy with immunotherapy is the goal of this study. We have previously shown that administration of CpG-ODN as an adjuvant, together with local release of doxorubicin from temperature sensitive liposomes (TSL) resulted in regression of directly-treated tumors, suppressed growth of contralateral tumors and reduced chemotherapeutic-mediated toxicity in a murine breast cancer model.1 Increases in cytotoxic CD8+ T lymphocytes and a reduction in regulatory T cells and myeloid-derived suppressor cells were observed in both directly treated and contralateral tumors. This combinatorial approach was curative for directly-treated tumors and overall survival was significantly extended, however, the contralateral tumor returned in all treated mice. The following is the protocol explored for the addition of anti-PD1: immune intact FVB/n mice with bilateral invasive neu deletion syngeneic transplanted tumors were treated with a combination of anti-PD1 (aPD-1, 200 μg, i.p.) and intratumoral administration of CpG-ODN (100 μg, i.t.) on days 0, 7, 14 and 0, 3, 7, 10, 17 and 24, respectively. Doxorubicin TSL were prepared from DPPC:MPPC:DSPE-PEG2k, 86:10:4 in the presence of copper (II) gluconate and triethanolamine at 0.2 mg-drug/mg-lipid and administrated i.v. at 6 mg doxorubicin/kg body weight on days 10, 17, 24. The formation of a complex between doxorubicin and copper was created to enhance the circulation and stability of TSL and to reduce systemic toxicity. To trigger drug release, hyperthermia was induced in the primary tumor with ultrasound (peak ultrasound pressure of 1.1 MPa at a frequency of 1.5 MHz) at 42°C for 5 min prior to and 20 min post drug injection with a variable duty cycle. Immediately afterwards, 100 μg of CpG-ODN 1826 was administered intratumorally to the insonified tumor. Upon treatment with this combination of locally-released doxorubicin, local administration of CpG-ODN and systemic aPD-1, 100% of treated and contralateral tumors regressed by at least 80%; further, all of the directly-treated tumors and 50% of the contralateral tumors were eliminated without recurrence. Thus, a 50% complete response rate was achieved, with tumor regression observed immediately after the incorporation of the doxorubicin treatment. By contrast, administration of CpG-ODN and systemic aPD-1 alone resulted in regression of 66% of treated and contralateral tumors. *MS and AK contributed equally to this work. 1. J Control Release (2015); 220: 253-264. Citation Format: Matthew T. Silvestrini, Azadeh Kheirolomoom, Elizabeth S. Ingham, Lisa M. Mahakian, Sarah M. Tam, Josquin Foiret, Samantha Tucci, Neil E. Hubbard, Alexander D. Borowsky, Katherine W. Ferrara. Activatable nanodelivery combined with CpG-ODN and anti-PD-1 achieves a complete response in directly-treated and contralateral tumors in a murine breast cancer model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-052.

CD8+ T-Cell Density Imaging with 64Cu-Labeled Cys-Diabody Informs Immunotherapy Protocols

Purpose: Noninvasive and quantitative tracking of CD8+ T cells by PET has emerged as a potential technique to gauge response to immunotherapy. We apply an anti-CD8 cys-diabody, labeled with 64Cu, to assess the sensitivity of PET imaging of normal and diseased tissue. Experimental Design: Radiolabeling of an anti-CD8 cys-diabody (169cDb) with 64Cu was developed. The accumulation of 64Cu-169cDb was evaluated with PET/CT imaging (0, 5, and 24 hours) and biodistribution (24 hours) in wild-type mouse strains (n = 8/group studied with imaging and IHC or flow cytometry) after intravenous administration. Tumor-infiltrating CD8+ T cells in tumor-bearing mice treated with CpG and αPD-1 were quantified and mapped (n = 6–8/group studied with imaging and IHC or flow cytometry). Results: We demonstrate the ability of immunoPET to detect small differences in CD8+ T-cell distribution between mouse strains and across lymphoid tissues, including the intestinal tract of normal mice. In FVB mice bearing a syngeneic HER2-driven model of mammary adenocarcinoma (NDL), 64Cu-169cDb PET imaging accurately visualized and quantified changes in tumor-infiltrating CD8+ T cells in response to immunotherapy. A reduction in the circulation time of the imaging probe followed the development of treatment-related liver and splenic hypertrophy and provided an indication of off-target effects associated with immunotherapy protocols. Conclusions: 64Cu-169cDb imaging can spatially map the distribution of CD8+ T cells in normal organs and tumors. ImmunoPET imaging of tumor-infiltrating cytotoxic CD8+ T cells detected changes in T-cell density resulting from adjuvant and checkpoint immunotherapy protocols in our preclinical evaluation. Clin Cancer Res; 24(20); 4976–87. ©2018 AACR.

Combining ultrasound ablation with immunotherapy: Opportunities and challenges

Thermal ablation provides local control of cancer; however, pairing ablation with immunotherapy is attractive as there is emerging evidence that focal therapy enhances the efficacy of immunotherapy. The goals of local treatment are to rapidly debulk the tumor and release tumor antigen to stimulate immune recognition. We seek to evaluate combination protocols and their tumor suppressive effects.

Abstract 576: Neoadjuvant immunotherapy improves efficacy of image-guided thermal ablation to generate curative responses in a murine breast cancer model

Magnetic resonance-guided focused ultrasound (MRgFUS) facilitates local tumor control via thermal ablation, however, the anti-tumor immune effects induced are weak and unable to consistently generate robust objective responses in distant lesions. Here, we set out to optimize a therapeutic approach for employing immunotherapy with thermal ablation for systemic cancer treatment. We assessed the efficacy of implementing MRgFUS ablation with blockade of the PD-1/PD-L1 axis (anti-PD-1) and activation of TLR9 (CpG oligonucleotide) under various protocols and in multiple models of murine cancer. Anti-PD-1 (200 µg, i.p., days 21 & 28) and CpG (100 µg, i.t., days 21, 24 and 28) were administered coincidentally with MRgFUS ablation (3 MHz central frequency, circular pattern with R=2 mm, 1 revolution per second, 65oC for 1 min, days 21 and 28) over the course of a week in bilateral syngeneic neu deletion line (NDL), 4T-1 and B16 tumor bearing mice. Additionally, we evaluated the administration of immunotherapy prior to a course of thermal ablation (i.e., “primed ablation”), where anti-PD-1 (as above on days 21, 28 & 35), CpG (as above on days 21, 24, 28, 31, 38 and 45) and MRgFUS ablation (as above on days 31, 38 and 45) were administered in bilateral NDL tumor-bearing mice. Primed ablation generated a robust anti-tumor immune response in distant lesions two weeks after the start of treatment, where a threefold increase in tumor infiltrating leukocytes (reaching 40% CD8+ and 20% CD4+ T-cells) was observed. This led to a complete response in 80% of treated mice within 70 days after treatment commenced. This effect was also observed in animals with high tumor burden and when thermal ablation was performed sequentially at multiple independent sites; 80% of untreated lesions were eradicated at 50 days after the start of treatment. However, therapeutic efficacy was limited when thermal ablation was performed coincident with the first dose of immunotherapy; this protocol was not curative in any murine model. To elucidate the mechanism for this effect, we employed tumor histology and positron emission tomography immediately after MRgFUS ablation. We found that thermal ablation induced stromal inflammation, and the loss of cell-cell adhesion and local vascular integrity, which impacted the intratumoral transport of small molecules and proteins for 48 hours post treatment. These data suggest that tumor debulking using image-guided thermal therapy can be successfully incorporated within a curative protocol in which immunotherapy is initiated before ablation. Citation Format: Matthew T. Silvestrini, Elizabeth S. Ingham, Lisa M. Mahakian, Azadeh Kheirolomoom, Yu Liu, Brett Z. Fite, Sarah M. Tam, Samantha Tucci, Katherine D. Watson, Andrew W. Wong, Arta M. Monjazeb, Neil E. Hubbard, William J. Murphy, Alexander D. Borowsky, Katherine W. Ferrara. Neoadjuvant immunotherapy improves efficacy of image-guided thermal ablation to generate curative responses in a murine breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 576. doi:10.1158/1538-7445.AM2017-576