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Background: Although combination immunotherapies incorporating local and systemic components have shown promising results in treating solid tumors, varied tumor microenvironments (TMEs) can impact immunotherapeutic efficacy. Method: We designed and evaluated treatment strategies for breast and pancreatic cancer combining magnetic resonance-guided focused ultrasound (MRgFUS) ablation and antibody therapies. With a combination of single-cell sequencing, spectral flow cytometry, and histological analyses, we profiled an immune-suppressed KPC (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre) pancreatic adenocarcinoma (MT4) model and a dense epithelial neu deletion (NDL) HER2+ mammary adenocarcinoma model with a greater fraction of lymphocytes, natural killer cells and activated dendritic cells. We then performed gene ontology analysis, spectral and digital cytometry to assess the immune response to combination immunotherapies and correlation with survival studies. Result: Based on gene ontology analysis, adding ablation to immunotherapy enriched immune cell migration pathways in the pancreatic cancer model and extensively enriched wound healing pathways in the breast cancer model. With CIBERSORTx digital cytometry, aCD40 + aPD-1 immunotherapy combinations enhanced dendritic cell activation in both models. In the MT4 TME, adding the combination of aCD40 antibody and checkpoint inhibitors (aPD-1 and aCTLA-4) with ablation was synergistic, increasing activated natural killer cells and T cells in distant tumors. Furthermore, ablation with immunotherapy upregulated critical Ly6c myeloid remodeling phenotypes that enhance T-cell effector function and increased granzyme and protease encoding genes by as much as 100-fold. Ablation combined with immunotherapy then extended survival in the MT4 model to a greater extent than immunotherapy alone. Conclusion: In summary, TME profiling informed a successful multicomponent treatment protocol incorporating ablation and facilitated differentiation of TMEs in which ablation is most effective.

Raman spectroscopy provides excellent specificity forin vivopreclinical imaging through a readout of fingerprint-like spectra. To achieve sufficient sensitivity forin vivoRaman imaging, metallic gold nanoparticles larger than 10 nm were employed to amplify Raman signals via surface-enhanced Raman scattering (SERS). However, the inability to excrete such large gold nanoparticles has restricted the translation of Raman imaging. Here we present Raman-active metallic gold supraclusters that are biodegradable and excretable as nanoclusters. Although the small size of the gold nanocluster building blocks compromises the electromagnetic field enhancement effect, the supraclusters exhibit bright and prominent Raman scattering comparable to that of large gold nanoparticle-based SERS nanotags due to high loading of NIR-resonant Raman dyes and much suppressed fluorescence background by metallic supraclusters. The bright Raman scattering of the supraclusters was pH-responsive, and we successfully performedin vivoRaman imaging of acidic tumors in mice. Furthermore, in contrast to large gold nanoparticles that remain in the liver and spleen, the supraclusters dissociated into small nanoclusters, and 73% of the administered dose to mice was excreted over 4 months. The highly excretable Raman supraclusters demonstrated here offer great potential for clinical applications ofin vivoRaman imaging by replacing non-excretable large gold nanoparticles.

The development of gene delivery vehicles with high organ specificity when administered systemically is a critical goal for gene therapy. We combine optical and positron emission tomography (PET) imaging of 1) reporter genes and 2) capsid tags to assess the temporal and spatial distribution and transduction of adeno-associated viruses (AAVs). AAV9 and two engineered AAV vectors (PHP.eB and CAP-B10) that are noteworthy for maximizing blood-brain barrier transport were compared. CAP-B10 shares a modification in the 588 loop with PHP.eB, but also has a modification in the 455 loop, added with the goal of reducing off-target transduction. PET and optical imaging revealed that the additional modifications retained brain receptor affinity. In the liver, the accumulation of AAV9 and the engineered AAV capsids was similar (∼15% of the injected dose per cc and not significantly different between capsids at 21 h). However, the engineered capsids were primarily internalized by Kupffer cells rather than hepatocytes, and liver transduction was greatly reduced. PET reporter gene imaging after engineered AAV systemic injection provided a non-invasive method to monitor AAV-mediated protein expression over time. Through comparison with capsid tagging, differences between brain localization and transduction were revealed. In summary, AAV capsids bearing imaging tags and reporter gene payloads create a unique and powerful platform to assay the pharmacokinetics, cellular specificity and protein expression kinetics of AAV vectors in vivo, a key enabler for the field of gene therapy.

Resiquimod (R848) is a toll-like receptor 7 and 8 (TLR7/8) agonist with potent antitumor and immunostimulatory activity. However, systemic delivery of R848 is poorly tolerated because of its poor solubility in water and systemic immune activation. In order to address these limitations, we developed an intravenously-injectable formulation with R848 using thermosensitive liposomes (TSLs) as a delivery vehicle. R848 was remotely loaded into TSLs composed of DPPC: DSPC: DSPE-PEG2K (85:10:5, mol%) with 100mM FeSO4 as the trapping agent inside. The final R848 to lipid ratio of the optimized R848-loaded TSLs (R848-TSLs) was 0.09 (w/w), 10-fold higher than the previously-reported values. R848-TSLs released 80% of R848 within 5min at 42°C. These TSLs were then combined with αPD-1, an immune checkpoint inhibitor, and ultrasound-mediated hyperthermia in a neu deletion (NDL) mouse mammary carcinoma model (Her2+, ER/PR negative). Combined with αPD-1, local injection of R848-TSLs showed superior efficacy with complete NDL tumor regression in both treated and abscopal sites achieved in 8 of 11 tumor bearing mice over 100days. Immunohistochemistry confirmed enhanced CD8+ T cell infiltration and accumulation by R848-TSLs. Systemic delivery of R848-TSLs, combined with local hyperthermia and αPD-1, inhibited tumor growth and extended median survival from 28days (non-treatment control) to 94days. Upon re-challenge with reinjection of tumor cells, none of the previously cured mice developed tumors, as compared with 100% of age-matched control mice. The dose of R848 (10μg for intra-tumoral injection or 6mg/kg for intravenous injection delivered up to 4 times) was well-tolerated without weight loss or organ hypertrophy. In summary, we developed R848-TSLs that can be administered locally or systematically, resulting in tumor regression and enhanced survival when combined with αPD-1 in mouse models of breast cancer.

Adeno-associated viruses (AAVs) are typically single-stranded deoxyribonucleic acid (ssDNA) encapsulated within 25-nm protein capsids. Recently, tissue-specific AAV capsids (e.g. PHP.eB) have been shown to enhance brain delivery in rodents via the LY6A receptor on brain endothelial cells. Here, we create a non-invasive positron emission tomography (PET) methodology to track viruses. To provide the sensitivity required to track AAVs injected at picomolar levels, a unique multichelator construct labeled with a positron emitter (Cu-64, t1/2 = 12.7 h) is coupled to the viral capsid. We find that brain accumulation of the PHP.eB capsid 1) exceeds that reported in any previous PET study of brain uptake of targeted therapies and 2) is correlated with optical reporter gene transduction of the brain. The PHP.eB capsid brain endothelial receptor affinity is nearly 20-fold greater than that of AAV9. The results suggest that novel PET imaging techniques can be applied to inform and optimize capsid design., Adeno-associated viruses (AAVs) can be targeted in a tissue-specific manner, but their tissue accumulation cannot be assessed in a non-invasive manner. Here the authors conjugate a multivalent chelator labelled with Cu-64 to the surface of AAVs and image the brain accumulation of the PHB.eB capsid by PET.

Immunotherapy is an important cancer treatment strategy; nevertheless, the lack of robust immune cell infiltration in the tumor microenvironment remains a factor in limiting patient response rates. In vivo gene delivery protocols can amplify immune responses and sensitize tumors to immunotherapies, yet non-viral transfection methods often sacrifice transduction efficiency for improved safety tolerance. To improve transduction efficiency, we optimized a strategy employing low ultrasound transmission frequency-induced bubble oscillation to introduce plasmids into tumor cells. Differential centrifugation isolated size-specific microbubbles. The diameter of the small microbubble population was 1.27 ± 0.89 μm and that of larger population was 4.23 ± 2.27 μm. Upon in vitro insonation with the larger microbubble population, 29.7% of cancer cells were transfected with DNA plasmids, higher than that with smaller microbubbles (18.9%, P

Introduction: Adoptive cell transfer (ACT) of T cells has emerged in recent years as a powerful immunotherapy against cancer. Currently, T cells are harvested from the patient or donor and are genetically modified ex vivo to enhance their cancer-fighting capabilities. However, this process is costly and more complex than administering off-the-shelf therapies such as small molecule drugs or monoclonal antibodies. The development of methods to transfect T cells in vivo remains an important endeavor for immunotherapies. Ionizable lipid nanoparticles (LNPs) have recently played a key role as carrier vehicles for mRNA in the Pfizer-BioNTech and Moderna mRNA vaccines. While ionizable LNPs delivering mRNA have now been FDA-approved for non-targeted immune cell delivery, their potential for targeting specific immune cell subtypes has yet to be fully realized. Delivering therapeutic genes that could reprogram T cells in vivo to recognize disease-relevant antigens would be of great clinical significance in reducing the lead time and cost of current ACT therapies. Methods: Ionizable LNPs were synthesized to encapsulate mCherry or Firefly Luciferase (FLuc) reporter gene mRNA with a fluorescent Cy7-labeled lipid on the LNP surface. CD3 monoclonal antibody, F(ab’)2 CD3e fragment, or CD8 diabody was conjugated to the LNP surface. In vitro, the aCD3- or aCD8-LNPs were incubated with Jurkat or TK-1 cell lines, respectively. LNP uptake was imaged using fluorescent confocal microscopy, and mCherry expression was quantified via flow cytometry. For in vivo delivery, the ionizable LNPs were injected i.v., and reporter gene expression and cell activation were analyzed via flow cytometry and bioluminescence imaging. Results: Confocal microscopy of ionizable LNP uptake in vitro highlighted differences in the internalization of the aCD3- and aCD8-LNPs in their respective cell lines, and transfection was observed with each targeting ligand. In murine models, aCD3-LNPs led to FLuc expression in the spleen and liver with increased accumulation in lymph nodes. aCD3-LNPs also transfected mCherry mRNA and activated splenic and circulating T cells. Cytokine concentrations were elevated in blood 5 and 24 h after aCD3-LNP injection. The impact of LNP lipid composition on transfection will be detailed in the presentation. Conclusions: We successfully packaged mRNAs of mCherry and FLuc reporter genes within ionizable LNPs and targeted them to T cells via the CD3 or CD8 receptor. The ionizable LNPs were capable of transfecting T cells in vitro and in vivo while causing transient activation, depletion, migration, cytokine release, and phenotypic shifts. Working towards the goal of receptor-mediated T cell targeting in vivo, we elucidate the circulation time, transfection efficiency, and activating potential of ionizable LNPs targeted to T cells in the spleen and blood via two potential targets, the CD3 and CD8 receptors. Citation Format: Elise R. Robinson, Aris J. Kare, Azadeh Kheirolomoom, Mohammed Inayathullah, Spencer K. Tumbale, Bo Wu, Marina N. Raie, Jai W. Seo, Felix B. Salazar, Ramasamy Paulmurugan, Anna M. Wu, Katherine W. Ferrara. CD3 and CD8 targeting of ionizable lipid nanoparticles for in vivo mRNA delivery to T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5372.

Early cancer detection can dramatically increase treatment options and survival rates for patients, yet detection of early-stage tumors remains difficult. Here, we demonstrate a two-step strategy to detect and locate cancerous lesions by delivering tumor-activatable minicircle (MC) plasmids encoding a combination of blood-based and imaging reporter genes to tumor cells. We genetically engineered the MCs, under the control of the pan-tumor-specific Survivin promoter, to encode: 1) Gaussia Luciferase (GLuc), a secreted biomarker that can be easily assayed in blood samples; and 2) Herpes Simplex Virus Type 1 Thymidine Kinase mutant (HSV-1 sr39TK), a PET reporter gene that can be used for highly sensitive and quantitative imaging of the tumor location. We evaluated two methods of MC delivery, complexing the MCs with the chemical transfection reagent jetPEI or encapsulating the MCs in extracellular vesicles (EVs) derived from a human cervical cancer HeLa cell line. MCs delivered by EVs or jetPEI yielded significant expression of the reporter genes in cell culture versus MCs delivered without a transfection reagent. Secreted GLuc correlated with HSV-1 sr39TK expression with R2 = 0.9676. MC complexation with jetPEI delivered a larger mass of MC for enhanced transfection, which was crucial for in vivo animal studies, where delivery of MCs via jetPEI resulted in GLuc and HSV-1 sr39TK expression at significantly higher levels than controls. To the best of our knowledge, this is the first report of the PET reporter gene HSV-1 sr39TK delivered via a tumor-activatable MC to tumor cells for an early cancer detection strategy. This work explores solutions to endogenous blood-based biomarker and molecular imaging limitations of early cancer detection strategies and elucidates the delivery capabilities and limitations of EVs.

Resiquimod is an immunopotent toll-like receptor 7/8 agonist with antitumor activity. Despite being potent against skin cancers, it is poorly tolerated systemically due to toxicity. Integrating resiquimod into nanoparticles presents an avenue to circumvent the toxicity problem. Herein, the preparation of degradable nanoparticles with covalently bound resiquimod and their systemic application in cancer immunotherapy is reported. Dispersion in water of amphiphilic constructs integrating resiquimod covalently bound via degradable amide or ester linkages yields immune-activating nanoparticles. The degradable agonist-nanoparticle bonds allow the release of resiquimod from the carrier nanoparticles. In vitro assays with antigen presenting cells demonstrate that the nanoparticles retain the immunostimulatory activity of resiquimod. Systemic administration of the nanoparticles and checkpoint blockade (aPD-1) to a breast cancer mouse model with multiple established tumors triggers antitumor activity evidenced by suppressed tumor growth and enhanced CD8+ T-cell infiltration. Nanoparticles with ester links, which hydrolyze more readily, yield a stronger immune response with 75% of tumors eliminated when combined with aPD-1. The reduced tumor growth and the presence of activated CD8+ T-cells across multiple tumors suggest the potential for treating metastatic cancer.

BACKGROUND: A novel [(64)Cu]Cu-NOTA-aCD40 immunoPET tracer was developed to image a CD40(+) pancreatic tumor model in C57BL/6 mice and to study the biodistribution profile of the agonist CD40 (aCD40) monoclonal antibody (mAb) alone or combined with other mAbs. PROCEDURES: Copper-64 ([(64)Cu]Cu) labeled NOTA-aCD40 and NOTA-IgG (10 μg; 7 MBq) were injected intravenously into C57BL/6 mice with subcutaneous mT4 tumors to assess specificity 48 h post injection (p.i.) through positron emission tomography/computed tomography (PET/CT) imaging and biodistribution studies (n = 5). [(64)Cu]Cu-NOTA-aCD40 was injected alone or simultaneously in combination with a therapeutic mass of cold aCD40 (100 μg), aPD-1 (200 μg) and aCTLA-4 (200 μg) mAbs. A group of mice with or without tumor received the second round of injections 1 or 3 weeks apart, respectively. PET/CT imaging and biodistribution studies were performed at 48 h p.i. The organ dose for [(64)Cu]Cu was estimated based on biodistribution studies with 2 μg [(64)Cu]Cu-NOTA-aCD40 (corresponds to 5 mg patient dose) in non-tumor bearing mice. RESULTS: [(64)Cu]Cu-NOTA-aCD40 accumulation was 2.3- and 7.8-fold higher than [(64)Cu]Cu-NOTA-IgG in tumors and spleen, respectively, indicating the specificity of aCD40 mAb in a mouse pancreatic tumor model. Tumor accumulation of [(64)Cu]Cu-NOTA-aCD40 was 21.2 ± 7.3 %ID/g at 48 h after injection. Co-injection of [(64)Cu]Cu-NOTA-aCD40 with cold aCD40 mAb alone or with PD-1 and CTLA-4 mAbs reduced both spleen and tumor uptake, whereas liver uptake was increased. With the second round of injections, the liver was the only organ with substantial uptake. With a 2 μg administered dose of [(64)Cu]Cu-NOTA-aCD40 in a dosimetry study, the liver to spleen ratio was greater compared to the 10 μg dose (2.8 vs 0.37; respectively). The human equivalent for the highest dose organ (liver) was 198 ± 28.7 μSv/MBq. CONCLUSIONS: A CD40-immunoreactive [(64)Cu]Cu-NOTA-aCD40 probe was developed. The ratio of spleen to liver accumulation exceeded that of the IgG isotype and was greatest with a single small, injected mass. The safety of human patient imaging with [(64)Cu]Cu was established based on extrapolation of the organ specificity to human imaging.

Robust cytotoxic T cell infiltration has proven to be difficult to achieve in solid tumors. We set out to develop a flexible protocol to efficiently transfect tumor and stromal cells to produce immune-activating cytokines, and thus enhance T cell infiltration while debulking tumor mass. By combining ultrasound with tumor-targeted microbubbles, membrane pores are created and facilitate a controllable and local transfection. Here, we applied a substantially lower transmission frequency (250 kHz) than applied previously. The resulting microbubble oscillation was significantly enhanced, reaching an effective expansion ratio of 35 for a peak negative pressure of 500 kPa in vitro. Combining low-frequency ultrasound with tumor-targeted microbubbles and a DNA plasmid construct, 20% of tumor cells remained viable, and ∼20% of these remaining cells were transfected with a reporter gene both in vitro and in vivo. The majority of cells transfected in vivo were mucin 1 + /CD45 − tumor cells. Tumor and stromal cells were then transfected with plasmid DNA encoding IFN-β, producing 150 pg/10 6 cells in vitro, a 150-fold increase compared to no-ultrasound or no-plasmid controls and a 50-fold increase compared to treatment with targeted microbubbles and ultrasound (without IFN-β). This enhancement in secretion exceeds previously reported fourfold to fivefold increases with other in vitro treatments. Combined with intraperitoneal administration of checkpoint inhibition, a single application of IFN-β plasmid transfection reduced tumor growth in vivo and recruited efficacious immune cells at both the local and distant tumor sites.

Ex vivo programming of T cells can be efficacious but is complex and expensive; therefore, the development of methods to transfect T cells in situ is important. We developed and optimized anti-CD3-targeted lipid nanoparticles (aCD3-LNPs) to deliver tightly packed, reporter gene mRNA specifically to T cells. In vitro, targeted LNPs efficiently delivered mCherry mRNA to Jurkat T cells, and T-cell activation and depletion were associated with aCD3 antibody coating on the surface of LNPs. aCD3-LNPs, but not non-targeted LNPs, accumulated within the spleen following systemic injection, with mCherry and Fluc signals visible within 30 minutes after injection. At 24 h after aCD3-LNP injection, 2-4% of all splenic T cells and 2-7% of all circulating T cells expressed mCherry, and this was dependent on aCD3 coating density. Targeting and transfection were accompanied by systemic CD25(+), OX40(+), and CD69(+) T-cell activation with temporary CD3e ligand loss and depletion of splenic and circulating subsets. Migration of splenic CD8a(+) T cells from the white-pulp to red-pulp, and differentiation from naïve to memory and effector phenotypes, followed upon aCD3-LNP delivery. Additionally, aCD3-LNP injection stimulated the secretion of myeloid-derived chemokines and T-helper cytokines into plasma. Lastly, we administered aCD3-LNPs to tumor bearing mice and found that transfected T cells localized within tumors and tumor-draining lymph nodes following immunotherapy treatment. In summary, we show that CD3-targeted transfection is feasible, yet associated with complex immunological consequences that must be further studied for potential therapeutic applications.

The treatment of pancreatic cancer with gemcitabine is hampered by its rapid metabolism in vivo, the dense stroma around the tumor site which prevents the drug from reaching the cancerous cells and drug resistance. To address these challenges, this study describes the preparation of a retinoid prodrug of gemcitabine, GemRA (gemcitabine conjugated to retinoic acid), and its formulation into a nanoparticulate system applicable for pancreatic cancer treatment. Retinoic acid targets stellate cells which are part of the stroma and can thus augment the delivery of gemcitabine. GemRA dissolved in dimethylsulfoxide presented efficacy towards PANC-1 (human) and mT4 (mouse) pancreatic cancer cell lines but its poor solubility in aqueous solution affects its applicability. Thus, the preparation of the nanoparticles was initially attempted through self-assembly of GemRA, which resulted in the formation of unstable aggregates that precipitated during preparation. As a result, encapsulation of the drug into micelles of polyethylene glycol-retinoic acid (PGRA) amphiphilic conjugates was accomplished and resulted in successful incorporation of GemRA into nanoparticles of ca. 33 nm by dynamic light scattering and 25 nm by transmission electron microscopy. The nanoparticles had good stability in aqueous media and protected gemcitabine from the enzymatic action of cytidine deaminase, which converts gemcitabine to its inactive metabolite upon circulation. Cellular uptake of the nanoparticles by PANC-1 cells was confirmed by fluorescence spectroscopy and flow cytometry. Treatment of PANC-1 cells in vitro with the prodrug-loaded nanoparticles resulted in a significant reduction in cell viability (IC50 ca. 5 μM) compared to treatment with gemcitabine (IC50 > 1000 μM). The ability of the GemRA-loaded nanoparticles to induce cellular apoptosis of treated PANC-1 cells was ascertained via a TUNEL assay suggesting these nanoparticles are effective in pancreatic cancer treatment.

A successful chemotherapy-immunotherapy solid-tumor protocol should accomplish the following goals: debulk large tumors, release tumor antigen for cross-presentation and cross-priming, release cancer-suppressive cytokines and enhance anti-tumor immune cell populations. Thermally-activated drug delivery particles have the potential to synergize with immunotherapeutics to accomplish these goals; activation can release chemotherapy within bulky solid tumors and can enhance response when combined with immunotherapy. We set out to determine whether a single protocol, combining locally-activated chemotherapy and agonist immunotherapy, could accomplish these goals and yield a potentially translational therapy. For effective delivery of free doxorubicin to tumors with minimal toxicity, we stabilized doxorubicin with copper in temperature-sensitive liposomes that rapidly release free drug in the vasculature of cancer lesions upon exposure to ultrasound-mediated hyperthermia. We found that in vitro exposure of tumor cells to hyperthermia and doxorubicin resulted in immunogenic cell death and the local release of type I interferons across murine cancer cell lines. Following intravenous injection, local activation of the liposomes within a single tumor released doxorubicin and enhanced cross-presentation of a model antigen at distant tumor sites. While a variety of protocols achieved a complete response in more than 50% of treated mice, the complete response rate was greatest (90%) when 1 week of immunotherapy priming preceded a single activatable chemotherapeutic administration. While repeated chemotherapeutic delivery reduced local viable tumor, the complete response rate and a subset of tumor immune cells were also reduced. Taken together, the results suggest that activatable chemotherapy can enhance adjuvant immunotherapy; however, in a murine model the systemic adaptive immune response was greatest with a single administration of chemotherapy.

Squalenoylation of gemcitabine, a front-line therapy for pancreatic cancer, allows for improved cellular-level and system-wide drug delivery. The established methods to conjugate squalene to gemcitabine and to form nanoparticles (NPs) with the squalenoylated gemcitabine (SqGem) conjugate are cumbersome, time-consuming and can be difficult to reliably replicate. Further, the creation of multi-functional SqGem-based NP theranostics would facilitate characterization of in vivo pharmacokinetics and efficacy. Methods: Squalenoylation conjugation chemistry was enhanced to improve reliability and scalability using tert-butyldimethylsilyl (TBDMS) protecting groups. We then optimized a scalable microfluidic mixing platform to produce SqGem-based NPs and evaluated the stability and morphology of select NP formulations using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Cytotoxicity was evaluated in both PANC-1 and KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx-Cre) pancreatic cancer cell lines. A 64Cu chelator (2-S-(4-aminobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) was squalenoylated and used with positron emission tomography (PET) imaging to monitor the in vivo fate of SqGem-based NPs. Results: Squalenoylation yields of gemcitabine increased from 15% to 63%. Cholesterol-PEG-2k inclusion was required to form SqGem-based NPs using our technique, and additional cholesterol inclusion increased particle stability at room temperature; after 1 week the PDI of SqGem NPs with cholesterol was ~ 0.2 while the PDI of SqGem NPs lacking cholesterol was ~ 0.5. Similar or superior cytotoxicity was achieved for SqGem-based NPs compared to gemcitabine or Abraxane® when evaluated at a concentration of 10 µM. Squalenoylation of NOTA enabled in vivo monitoring of SqGem-based NP pharmacokinetics and biodistribution. Conclusion: We present a scalable technique for fabricating efficacious squalenoylated-gemcitabine nanoparticles and confirm their pharmacokinetic profile using a novel multifunctional 64Cu-SqNOTA-SqGem NP.

Optimal pairing of chemotherapy with immunotherapy can harness potential synergies and augment tumor immunity in solid tumors with low mutation burden by i) reducing tumor bulk, ii) releasing tumor antigen for cross-presentation and cross-priming, iii) releasing cancer-suppressive cytokines/chemokines, and iv) retaining circulating immune cell populations. Temperature-sensitive liposomes (TSL) have the potential to accomplish these goals as ultrasound (US) insonation or other treatment modalities can release the chemotherapy within bulky solid tumors in response to hyperthermia to maximize the local response and minimize off-target effects. Here we used Doxorubicin (Dox), stabilized within the core of TSL by forming a drug complex with copper (CuDox), to mediate immunogenic cell death (ICD) and release of type I interferons (IFNs). This activatable chemotherapy (CuDox-TSL+US) was combined with immunotherapy using CpG-ODN, a toll-like receptor 9 agonist, and a PD-1 antibody (αPD-1) for checkpoint blockade. We assessed the schedule and sequence of the chemo-immunotherapy protocol to maximize therapeutic outcome. CuDox-TSL were made of DPPC:MPPC:DSPE-PEG2k, 86:10:4 in the presence of copper (II) gluconate and triethanolamine at 0.2 mg-Dox/mg-lipid. Mice with bilateral invasive neu deletion (NDL) tumors (4 mm) were treated with an i.v. administration of CuDox-TSL at 6 mg Dox/kg body weight. One tumor was insonified with a 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. Upon completion of US-hyperthermia, 100 µg of CpG-ODN 1826 was injected intratumorally to the insonified tumor. Three days later αPD-1 (200 µg, i.p.) was administrated. We found that Dox in combination with hyperthermia induced release of ICD markers and type I IFNs in cell culture of various murine cancer cell lines. Following intravenous administration of CuDox-TSL, ultrasonic activation of liposomes in the treated tumors promoted release and cross-presentation of a model tumor antigen by antigen-presenting cells in distant tumors. While a variety of chemo-immunotherapy protocols achieved a complete response in more than 50% of treated mice, the complete response rate was greatest (90%) when one week of immunotherapy priming preceded activatable chemotherapeutic administration. Repeated chemotherapeutic delivery, while effectively reducing viable tumor, also reduced efficacy and the fraction of viable CD8+ T-cells as indicated by flow cytometric analysis. Taken together, the results suggest that a single-dose administration of activatable chemotherapy after immune priming enhances survival in a murine model of breast cancer. In clinical practice, however, assessment of macrophage and T-cell populations over the course of treatment could help inform dose timing and number of treatment repetitions. Citation Format: Azadeh Kheirolomoom, Matthew T. Silvestrini, Elizabeth S. Ingham, Lisa M. Mahakian, Sarah M. Tam, Spencer K. Tumbale, Josquin Foiret, Neil E. Hubbard, Alexander D. Borowsky, William J. Murphy, Katherine W. Ferrara. Localized nanodelivery combined with immunotherapy promotes curative anti-tumor responses in a murine breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3952.

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.

Ultrasound molecular imaging has great potential to impact early disease diagnosis, evaluation of disease progression and the development of target-specific therapy. In this paper, two neuropilin-1 (NRP) targeted peptides, CRPPR and ATWLPPR, were conjugated onto the surface of lipid microbubbles (MBs) to evaluate molecular imaging of tumor angiogenesis in a breast cancer model. Development of a molecular imaging agent using CRPPR has particular importance due to the previously demonstrated internalizing capability of this and similar ligands. Invitro, CRPPR MBs bound to an NRP-expressing cell line 2.6 and 15.6 times more than ATWLPPR MBs and non-targeted (NT) MBs, respectively, and the binding was inhibited by pretreating the cells with an NRP antibody. Invivo, the backscattered intensity within the tumor, relative to nearby vasculature, increased over time during the ∼6min circulation of the CRPPR-targeted contrast agents providing high contrast images of angiogenic tumors. Approximately 67% of the initial signal from CRPPR MBs remained bound after the majority of circulating MBs had cleared (8min), 8 and 4.5 times greater than ATWLPPR and NT MBs, respectively. Finally, at 7-21 days after the first injection, we found that CRPPR MBs cleared faster from circulation and tumor accumulation was reduced likely due to a complement-mediated recognition of the targeted microbubble and a decrease in angiogenic vasculature, respectively. In summary, we find that CRPPR MBs specifically bind to NRP-expressing cells and provide an effective new agent for molecular imaging of angiogenesis.

Activatable nanotherapeutics provide the opportunity to deliver anthracyclines to a well-controlled region of interest and therefore to locally stimulate immunogenic cell death. Such a strategy can be combined with immunotherapy for both local tumor control and creation of an abscopal effect. Our group previously reported that combining local administration of a CpG oligonucleotide with local release of doxorubicin from a temperature-sensitive liposomal nanoparticle (TSL) led to the elimination of directly treated lesions in a syngeneic murine model of mammary adenocarcinoma.1 While this therapeutic approach increased the numbers of tumor infiltrating CD8+ T lymphocytes in distant lesions and extended survival, distant tumors returned with a growth delay of approximately 14 days and T-regulatory cells were not reduced by treatment. Therefore, checkpoint blockade of the programmed death-1 (PD-1) pathway (anti-PD-1) was incorporated in the protocol. CuDox-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. A complex between doxorubicin and copper was created to enhance the circulation and stability of TSL and to reduce systemic toxicity. On day 21, mice with bilateral invasive neu deletion (NDL) tumors (~4 mm) were treated with i.v. administration of CuDox-TSL at 6 mg doxorubicin/kg body weight. Ultrasound (US) hyperthermia (1.1 MPa, 1.5 MHz, achieving 42°C for 5 min before and 20 min after drug injection) triggered drug release. Immediately after US, 100 µg of CpG-ODN 1826 was administered intratumorally to the insonified tumor; anti-PD-1 (200 µg, i.p.) was administrated three days later. Flow cytometry and immunohistochemistry were performed on day 28 after one treatment with each component and survival was assessed after treatment was repeated for three weeks. While control mice survived 35 days, 50% of treated mice were tumor-free after 100 days. All primary tumors and 50% of the contralateral tumors regressed and were eliminated by day 63. With the incorporation of immunotherapy, three doses of chemotherapy were sufficient to eliminate all directly treated tumors, as compared with 8 doses without immunotherapy. After a single treatment with each component, a substantial change was observed in systemic immune cells, including a significant increase in natural killer and dendritic cells in distant lymph nodes, in CD8+ T cells in tumors and associated lymph nodes and in IFNγ-secreting CD4+ T cells in the treated tumor and distant lymph nodes. The results demonstrate that activatable chemotherapy can be paired with an immune adjuvant and PD-1 blockade to generate a curative response in primary and distant tumors. Further, local chemotherapy, as potentiated by activatable liposomes does not impede a systemic CD8+ T cell response. [1] Journal of Controlled Release (2015); 220: 253-264. Citation Format: Azadeh Kheirolomoom, Matthew Silvestrini, Lisa Mahakian, Elizabeth Ingham, Josquin Foiret, Spencer Tumbale, Sarah Tam, Neil Hubbard, Alexander Borowsky, Katherine Ferrara. Protocol optimization combining activatable nanodelivery with immunotherapy 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 4603. doi:10.1158/1538-7445.AM2017-4603