Off-target Tissues Research Articles

Radiolabeled GRPR Antagonists for Imaging of Disseminated Prostate Cancer - Influence of Labeling Chemistry on Targeting Properties.

Radionuclide molecular imaging of Gastrin-Releasing Peptide Receptor (GRPR) expression promises unparalleled opportunities for visualizing subtle prostate tumors, which due to small size, adjacent benign tissue, or a challenging location would otherwise remain undetected by conventional imaging. Achieving high imaging contrast is essential for this purpose and the molecular design of any probe for molecular imaging of prostate cancer should be aimed at obtaining as high tumor-to-organ ratios as possible. This short review summarizes the key imaging modalities currently used in prostate cancer, with a special focus on radionuclide molecular imaging. Emphasis is laid mainly on the issue of radiometals labeling chemistry and its influence on the targeting properties and biodistribution of radiolabeled GRPR antagonists for imaging of disseminated prostate cancer. A comprehensive literature search of the PubMed/MEDLINE, and Scopus library databases was conducted to find relevant articles. The combination of radionuclide, chelator and required labeling chemistry was shown to have a significant influence on the stability, binding affinity and internalization rate, off-target interaction with normal tissues and blood proteins, interaction with enzymes, activity uptake and retention in excretory organs and activity uptake in tumors of radiolabeled bombesin antagonistic analogues. Labeling chemistry has a very strong impact on the biodistribution profile of GRPRtargeting peptide based imaging probes and needs to be considered when designing a targeting probe for high contrast molecular imaging. Taking into account the complexity of in vivo interactions, it is not currently possible to accurately predict the optimal labeling approach. Therefore, a detailed in vivo characterization and optimization is essential for the rational design of imaging agents.

Design and preclinical evaluation of nanostars for the passive pretargeting of tumor tissue

IntroductionPretargeting strategies that do not rely on the expression of molecular targets have expanded imaging and therapy options for cancer patients. Nanostars with designed multivalency and which highly accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect may therefore be the ideal vectors for the development of a passive pretargeting approach. MethodsNanostars were synthesized, consisting of 7–8 center-cross-linked arms that were modified with trans-cyclooctene (TCO) using poly(ethylene glycol) (PEG) linkers of 12 or 106 monomer units or without linker. The bioorthogonal click reaction with radiofluorinated 2,2′-(7-(2-(tetrazine-poly(ethyleneglycol)11-amino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid ([18F]F-Tz-PEG11-NODA) or 2,2′-(7-(2-(tetrazine-amino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid ([18F]F-Tz-NODA) was measured by ex vivo biodistribution studies and positron emission tomography (PET) in mice bearing tumors with high EPR characteristics. Bioorthogonal masking was performed using a tetrazine-functionalized dextran polymer (Tz-DP). ResultsHighest tumor accumulation of [18F]F-Tz-PEG11-NODA was observed for nanostars functionalized with TCO without linker, with a tumor uptake of 3.2 ± 0.4%ID/g and a tumor-to-muscle ratio of 12.8 ± 4.2, tumor-to-large intestine ratio of 0.5 ± 0.3 and tumor-to-kidney ratio of 2.0 ± 0.3, being significantly higher than for nanostars functionalized with TCO-PEG12 (P < 0.05) or TCO-PEG106 (P < 0.05). Tumor uptake and tumor-to-tissue ratios did not improve upon bioorthogonal masking with Tz-DP or when using a smaller, more lipophilic tetrazine([18F]F-Tz-NODA). ConclusionsA pretargeting strategy was developed based on the passive delivery of TCO-functionalized nanostars. Such a strategy would allow for the imaging and treatment of tumors with apparent EPR characteristics, with high radioactive tumor doses and minimal doses to off-target tissues.

Effects of Histotripsy on Local Tumor Progression in an in vivo Orthotopic Rodent Liver Tumor Model.

Objective and Impact Statement.This is the first longitudinal study investigating the effects of histotripsy on local tumor progression in an in vivo orthotopic, immunocompetent rat hepatocellular carcinoma (HCC) model.Introduction.Histotripsy is the first noninvasive, nonionizing, nonthermal, mechanical ablation technique using ultrasound to generate acoustic cavitation to liquefy the target tissue into acellular debris with millimeter accuracy. Previously, histotripsy has demonstrated in vivo ablation of noncancerous liver tissue.Methods.N1-S1 HCC tumors were generated in the livers of immunocompetent rats (n = 6, control; n = 15, treatment). Real-time ultrasound-guided histotripsy was applied to ablate either 100% tumor volume + up to 2mm margin (n = 9, complete treatment) or 50-75% tumor volume (n = 6, partial treatment) by delivering 1-2 cycle histotripsy pulses at 100 Hz PRF (pulse repetition frequency) with p – ≥30MPa using a custom 1MHz transducer. Rats were monitored weekly using MRI (magnetic resonance imaging) for 3 months or until tumors reached ~25mm.Results.MRI revealed effective post-histotripsy reduction of tumor burden with near-complete resorption of the ablated tumor in 14/15 (93.3%) treated rats. Histopathology showed <5mm shrunken, non-tumoral, fibrous tissue at the treatment site at 3 months. Rats with increased tumor burden (3/6 control and 1 partial treatment) were euthanized early by 2-4 weeks. In 3 other controls, histology revealed fibrous tissue at original tumor site at 3 months. There was no evidence of histotripsy-induced off-target tissue injury.Conclusion.Complete and partial histotripsy ablation resulted in effective tumor removal for 14/15 rats, with no evidence of local tumor progression or recurrence.

Controlled Tyrosine Kinase Inhibitor Delivery to Liver Cancer Cells by Gate-Capped Mesoporous Silica Nanoparticles.

Hepatocellular carcinoma is the most common type of primary malignancy in the liver and one of the most common types of cancer worldwide. Its readily increasing mortality rate highlights the urgent need for the development of efficient therapeutic strategies. Tyrosine kinase inhibitors (TKIs) such as sorafenib and sunitinib are used as efficient angiogenesis inhibitors for this purpose. However, despite their pharmacological effects, their transfer into clinical practice is characterized by their poor aqueous solubility and accumulation in off-target tissues, resulting in unfavorable side effects. Here, we report a nanocomposite made of amine-functionalized mesoporous silica nanocomposites (MSNs) that are surface-coated with cerium oxide nanoparticles (CNPs) for the controlled delivery and release of TKIs. Amine-functionalized MSNs were prepared using a sol-gel method and loaded with TKIs. To trap drug molecules into the mesoporous structure, CNPs were covalently conjugated to the surface of MSNs. The synthesis and functionalization steps were controlled using different characterization methods, confirming the desired morphology and structure, the identity of functional groups on the surface, successful coating, and appropriate loading efficiency. Under physiological conditions, CNP-capped MSNs demonstrated a sustained drug release over time as a result of CNPs' gatekeeping effect on the payloads. Strong cellular interactions with different liver cancer cells and enhanced cellular uptake were also observed in vitro for the gate-capped MSNs. Internalization of nanocomposites induced cell death via the production of reactive oxygen species, and subsequent activation of apoptosis pathways. This study demonstrates that gate-capped MSNs are promising chemotherapeutic vehicles characterized by a sustained drug release profile and high cellular internalization.

Surface-PASylation of ferritin to form stealth nanovehicles enhances in vivo therapeutic performance of encapsulated ellipticine

Surface functionalisations substantially influence the performance of drug delivery vehicles by improving their biocompatibility, selectivity and circulation in bloodstream. Herein, we present the study of in vitro and in vivo behaviour of a highly potent cytostatic alkaloid ellipticine (Elli) encapsulated in internal cavity of ferritin (FRT)-based nanocarrier (hereinafter referred to as FRTElli). In addition, FRTElli surface was functionalised with three different molecular coatings: two types of protective PAS peptides (10- or 20-residues lengths) with sequences comprising amino acids proline (P), alanine (A) and serine (S) (to form PAS-10-FRTElli or PAS-20-FRTElli, respectively), or polyethylene glycol (PEG-FRTElli). All three surface modifications of FRT disposed sufficient encapsulation efficiency of Elli with no premature cumulative release of cargo. Noteworthy, all tested surface modifications displayed beneficial effects on the in vitro biocompatibility. PAS-10-FRTElli exhibited markedly reduced uptake by macrophages compared to PAS-20-FRTElli, PEG-FRTElli or unmodified FRTElli. The exceptional properties of PAS-10-FRTElli were validated by an array of in vitro analyses including formation of protein corona, uptake efficiency or screenings of selectivity of cytotoxicity. In murine preclinical model bearing triple-negative breast cancer (MDA-MB-231) xenograft, compared to free Elli or FRTElli, PAS-10-FRTElli displayed enhanced accumulation of Elli within tumour tissue, while hampering the uptake of Elli into off-target tissues. Noteworthy, PAS-10-FRTElli led to decreased in vivo complement (C3) activation and protein corona formation. Taken together, presented in vivo results indicate that PAS-10-FRTElli represents a promising stealth platform for translation into clinical settings.

Comparison of Gene Delivery to the Kidney by Adenovirus, Adeno-Associated Virus, and Lentiviral Vectors After Intravenous and Direct Kidney Injections.

There are many kidney diseases that might be addressed by gene therapy. However, gene delivery to kidney cells is inefficient. This is due, in part, to the fact that the kidney excludes molecules above 50 kDa and that most gene delivery vectors are megaDaltons in mass. We compared the ability of adeno-associated virus (AAV), adenovirus (Ad), and lentiviral (LV) vectors to deliver genes to renal cells. When vectors were delivered by the intravenous (IV) route in mice, weak luciferase activity was observed in the kidney with substantially more in the liver. When gene delivery was observed in the kidney, expression was primarily in the glomerulus. To avoid these limitations, vectors were injected directly into the kidney by retrograde ureteral (RU) and subcapsular (SC) injections in mice. Small AAV vectors transduced the kidney, but also leaked from the organ and mediated higher levels of transduction in off-target tissues. Comparison of AAV2, 6.2, 8, and rh10 vectors by direct kidney injection demonstrated highest delivery by AAV6.2 and 8. Larger Ad and LV vectors transduced kidney cells and mediated less off-target tissue transduction. These data demonstrate the utility of direct kidney injections to circumvent the kidney size exclusion barrier. They also identify the effects of vector size on on-target and off-target transduction. This lays the foundation for the use of different vector platforms for gene therapy of diverse kidney diseases.

Importance of thorough tissue and cellular level characterization of targeted drugs in the evaluation of pharmacodynamic effects.

Targeted nanoparticle delivery is a promising strategy for increasing efficacy and limiting side effects of therapeutics. When designing a targeted liposomal formulation, the in vivo biodistribution of the particles must be characterized to determine the value of the targeting approach. Peroxisome proliferator-activated receptor (PPAR) agonists effectively treat metabolic syndrome by decreasing dyslipidemia and insulin resistance but side effects have limited their use, making them a class of compounds that could benefit from targeted liposomal delivery. The adipose targeting sequence peptide (ATS) could fit this role, as it has been shown to bind to adipose tissue endothelium and induce weight loss when delivered conjugated to a pro-apoptotic peptide. To date, however, a full assessment of ATS in vivo biodistribution has not been reported, leaving important unanswered questions regarding the exact mechanisms whereby ATS targeting enhances therapeutic efficacy. We designed this study to evaluate the biodistribution of ATS-conjugated liposomes loaded with the PPARα/γ dual agonist tesaglitazar in leptin-deficient ob/ob mice. The ATS-liposome biodistribution in adipose tissue and other organs was examined at the cellular and tissue level using microscopy, flow cytometry, and fluorescent molecular tomography. Changes in metabolic parameters and gene expression were measured by target and off-target tissue responses to the treatment. Unexpectedly, ATS targeting did not increase liposomal uptake in adipose relative to other tissues, but did increase uptake in the kidneys. Targeting also did not significantly alter metabolic parameters. Analysis of the liposome cellular distribution in the stromal vascular fraction with flow cytometry revealed high uptake by multiple cell types. Our findings highlight the need for thorough study of in vivo biodistribution when evaluating a targeted therapy.

An Injectable Cytokine Trap for Local Treatment of Autoimmune Disease

Systemic cytokine therapy is limited by toxicity due to activation of unwanted immune cells in off-target tissues. Injectable nanomaterials that interact with the immune system have potential to offer improved pharmacokinetics and cell specificity compared to systemic cytokine therapy by instead capturing and potentiating endogenous cytokine. Here we demonstrate the use of high aspect ratio polycaprolactone nanowires conjugated to cytokine-binding antibodies that assemble into porous matrices when injected into the subcutaneous space. Nanowires are well tolerated in vivo over several weeks, incite minimal foreign body response and resist clearance. Nanowires conjugated with JES6-1, an anti-interleukin-2 (IL-2) antibody, were designed to capture endogenous IL-2 and selectively activate tissue resident regulatory T cells (Tregs). Together these nanowire-antibody matrices were capable of sequestering endogenous IL-2 in the skin and were successful in rebalancing local immune compartments to a more suppressive, Treg-mediated phenotype in both wild type and transgenic murine autoimmune disease models.

Utilizing a Kidney-Targeting Peptide to Improve Renal Deposition of a Pro-Angiogenic Protein Biopolymer.

Elastin-like polypeptides (ELP) are versatile protein biopolymers used in drug delivery due to their modular nature, allowing fusion of therapeutics and targeting agents. We previously developed an ELP fusion with vascular endothelial growth factor (VEGF) and demonstrated its therapeutic efficacy in translational swine models of renovascular disease and chronic kidney disease. The goal of the current work was to refine renal targeting and reduce off-target tissue deposition of ELP–VEGF. The ELP–VEGF fusion protein was modified by adding a kidney-targeting peptide (KTP) to the N-terminus. All control proteins (ELP, KTP–ELP, ELP–VEGF, and KTP–ELP–VEGF) were also produced to thoroughly assess the effects of each domain on in vitro cell binding and activity and in vivo pharmacokinetics and biodistribution. KTP–ELP–VEGF was equipotent to ELP–VEGF and free VEGF in vitro in the stimulation of primary glomerular microvascular endothelial cell proliferation, tube formation, and extracellular matrix invasion. The contribution of each region of the KTP–ELP–VEGF protein to the cell binding specificity was assayed in primary human renal endothelial cells, tubular epithelial cells, and podocytes, demonstrating that the VEGF domain induced binding to endothelial cells and the KTP domain increased binding to all renal cell types. The pharmacokinetics and biodistribution of KTP–ELP–VEGF and all control proteins were determined in SKH-1 Elite hairless mice. The addition of KTP to ELP slowed its in vivo clearance and increased its renal deposition. Furthermore, addition of KTP redirected ELP–VEGF, which was found at high levels in the liver, to the kidney. Intrarenal histology showed similar distribution of all proteins, with high levels in blood vessels and tubules. The VEGF-containing proteins also accumulated in punctate foci in the glomeruli. These studies provide a thorough characterization of the effects of a kidney-targeting peptide and an active cytokine on the biodistribution of these novel biologics. Furthermore, they demonstrate that renal specificity of a proven therapeutic can be improved using a targeting peptide.

A side-by-side evaluation of [18F]FDOPA enantiomers for non-invasive detection of neuroendocrine tumors by positron emission tomography

Neuroendocrine tumors (NETs) are an extremely heterogenous group of malignancies with variable clinical behavior. Molecular imaging of patients with NETs allows for effective patient stratification and treatment guidance and is crucial in selection of targeted therapies. Positron emission tomography (PET) with the radiotracer L-[18F]FDOPA is progressively being utilized for non-invasive in vivo visualization of NETs and pancreatic β-cell hyperplasia. While L-[18F]FDOPA-PET is a valuable tool for disease detection and management, it also exhibits significant diagnostic limitations owing to its inherent physiological uptake in off-target tissues. We hypothesized that the D-amino acid structural isomer of that clinical tracer, D-[18F]FDOPA, may exhibit superior clearance capabilities owing to a reduced in vivo enzymatic recognition and enzyme-mediated metabolism. Here, we report a side-by-side evaluation of D-[18F]FDOPA with its counterpart clinical tracer, L-[18F]FDOPA, for the non-invasive in vivo detection of NETs. In vitro evaluation in five NET cell lines, including invasive small intestinal neuroendocrine carcinomas (STC-1), insulinomas (TGP52 and TGP61), colorectal adenocarcinomas (COLO-320) and pheochromocytomas (PC12), generally indicated higher overall uptake levels of L-[18F]FDOPA, compared to D-[18F]FDOPA. While in vivo PET imaging and ex vivo biodistribution studies in PC12, STC-1 and COLO-320 mouse xenografts further supported our in vitro data, they also illustrated lower off-target retention and enhanced clearance of D-[18F]FDOPA from healthy tissues. Cumulatively our results indicate the potential diagnostic applications of D-[18F]FDOPA for malignancies where the utility of L-[18F]FDOPA-PET is limited by the physiological uptake of L-[18F]FDOPA, and suggest D-[18F]FDOPA as a viable PET imaging tracer for NETs.

99mTc-Labeled LyP-1 for SPECT Imaging of Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC), the most aggressive breast cancer type, is associated with high mortality and recurrence rates. An active-targeted strategy based on homing peptides is an effective approach to diagnose and treat cancer as it can deliver imaging agents or therapeutic drugs into desired tissues and accumulate less into off-target tissues. As a homing peptide, LyP-1 has shown properties of targeting, internalization, and proapoptosis to TNBC. In the study, we designed a Technetium-99m- (99mTc-) labeled LyP-1 and investigated its feasibility for targeted single-positron emission computed tomography (SPECT) imaging of TNBC. The results showed that the LyP-1 peptide had acceptable biocompatibility in the studied concentration range and could specifically bind to TNBC cells in vitro. 99mTc-labeled LyP-1 showed high radiochemical purity and stability and could be used as a probe for targeted SPECT imaging of TNBC cells in vitro and in a TNBC tumor-bearing mouse model. Our findings indicate that this active-targeted strategy has great potential to be developed into a new imaging tool for TNBC diagnosis.

Exercise stimulates beneficial adaptations to diminish doxorubicin-induced cellular toxicity.

Doxorubicin (DOX) is a highly effective antitumor agent used for the treatment of a wide range of cancers. Unfortunately, DOX treatment results in cytotoxic side effects due to its accumulation within off-target tissues. DOX-induced cellular toxicity occurs as a result of increased oxidative damage, resulting in apoptosis and cell death. While there is no standard-of-care practice to prevent DOX-induced toxicity to healthy organs, exercise has been shown to prevent cellular dysfunction when combined with DOX chemotherapy. Endurance exercise stimulates numerous biochemical adaptations that promote a healthy phenotype in several vulnerable tissues without affecting the antineoplastic properties of DOX. Therefore, for the development of an effective strategy to combat the pathological effects of DOX, it is important to determine the appropriate exercise regimen to prescribe to cancer patients receiving DOX therapy and to understand the mechanisms responsible for exercise-induced protection against DOX toxicity to noncancer cells. This review summarizes the cytotoxic effects of DOX on the heart, skeletal muscle, liver, and kidneys and discusses the current understanding of the clinical benefits of regular physical activity and the potential mechanisms mediating the positive effects of exercise on each organ system.

Abstract LB-002: SQ3370 enhances the safety of chemotherapeutics via local activation therapy

Abstract With systemic chemotherapy, only 1-2% of the administered dose actually reaches a localized tumor, while the remaining leads to adverse off-target toxicities, including immunosuppression. Hence, there is a critical need to locally deliver cytotoxics directly to the tumor. Our patented approach (SQ3370) consists of: (1) SQL70 - a drug-activating biomaterial carrying no payload (2) SQP33 - a chemically-modified prodrug of doxorubicin (Dox) with attenuated activity. SQL70 is injected at the tumor site followed by SQP33 administered systemically. The prodrug first concentrates to the biomaterial at the tumor site due to their complementary chemical reactivities. The active drug is then spontaneously released over multiple days, providing sustained local delivery directly to the tumor region while reducing systemic side effects. Previously, we have shown that the greater safety of SQP33 allows it to be given at over 38 times the dose of standard Dox in SQL70-injected mice. Pharmacokinetic and biodistribution studies in SQ3370-treated rodents and dogs show that SQP33 disappears from plasma within the first hour of administration, likely due to the rapid concentration to SQL70. Without SQL70, SQP33 shows minimal spontaneous conversion to activated Dox. These studies also indicate that a single injection of SQL70 can activate multiple doses of SQP33, maximizing the local therapeutic index. Recent studies demonstrate that SQ3370 treatment enhances therapeutic response and survival in tumor-bearing mice: In a syngeneic MC38 colorectal cancer model, SQ3370 slowed tumor progression in 8/10 mice, showing improved efficacy compared to standard Dox or anti-PD-1 therapy. In mice bearing dual tumors, with one tumor site injected locally with SQL70, we observed that SQP33 can induce a response in both primary and secondary tumors, suggestive of an anti-tumor immune activation effect. Furthermore, we found that SQ3370 synergizes with anti-PD-1, expanding potential treatment options in the clinic and highlighting the advantages of immune-sparing cytotoxic therapy. Collectively, our results demonstrate that SQ3370 enables delivery of cytotoxic drugs to a target site while limiting exposure in off-target tissues in small and large animals, leading to improvements in both safety and efficacy. Citation Format: Nathan A. Yee, Sangeetha Srinivasan, Maksim Royzen, Jose M. Mejia Oneto. SQ3370 enhances the safety of chemotherapeutics via local activation therapy [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 LB-002.

Abstract 933: Surface-tethered IL-12 improves tumor-specific T-cell therapy and enhances inflammatory activity in tumors without inducing systemic toxicities

Abstract Introduction Interleukin-12 (IL-12) is a potent cytokine that augments anti-tumor immune responses by promoting CD4 T cell Th1 differentiation, increasing CD8 T cell and NK cell cytotoxicity, inducing MHC expression on antigen presenting cells, and reprogramming myeloid-derived suppressor cells. However, the clinical utility of IL-12 administered systemically or produced by genetically engineered tumor-specific T cells has been limited by toxicities. DeepTM IL-12 is a fusion protein between IL-12 and a Fab antibody against CD45, an abundant surface receptor on T cells. This tethers IL-12 onto tumor-specific T cells prior to adoptive cell transfer (ACT) with the aim of reducing systemic exposure and focusing IL-12 function on the tumor microenvironment. Here, we describe the superior safety and efficacy profiles of Deep IL-12 tethered T cells in an immune competent adoptive cell therapy model for solid tumors. Methods The safety and efficacy of Deep IL-12 Primed T cells were evaluated in the PMEL/B16-F10 cancer model. CD8 PMEL T cells contain a TCR that recognizes the gp100 antigen expressed in B16-F10 melanoma cells. Deep IL-12 was loaded onto PMEL CD8 T cells to generate Deep IL-12 Primed PMEL T cells which were then adoptively transferred into mice bearing B16-F10 tumors. We compared tumor size, body weight change, cytokine release, circulating toxicity biomarkers, immune cell activity and histopathology in mice treated with Deep IL-12 Primed T cells and PMEL T cells alone or co-administered with recombinant IL-12. Results Deep IL-12 Primed PMEL T cells significantly improved tumor growth inhibition and overall survival in mice bearing established B16-F10 tumors as compared with PMEL T cell therapy alone or combined with systemic co-administration of IL-12. Deep IL-12 Primed PMEL T cells were well-tolerated, as administration of at least 40 million Deep IL-12 Primed PMEL T cells did not cause overt toxicities in non-tumor bearing mice. Deep IL-12 Primed PMEL T cells, but not PMEL T cells combined with systemic IL-12, resulted in elevated and sustained induction of IFNγ in the tumor microenvironment. In contrast, IFNγ induction in serum was transient and returned to baseline levels within four days of ACT. Interestingly, Deep IL-12 Priming increased both accumulation and activity of the adoptively transferred PMEL T cells in the tumor microenvironment but not in off-target healthy tissues such as the spleen. Further toxicological analysis of Deep IL-12 Primed tumor-reactive T cells will be discussed. Conclusions Deep IL-12 Priming enables tethering of IL-12 to the surface of tumor-specific T cells prior to ACT. This improves anti-tumor efficacy by promoting T cell function selectively in the tumor microenvironment and avoids toxicity by limiting systemic IL-12 exposure, resulting in a highly favorable benefit-risk profile for Deep IL-12 Primed T cells. Citation Format: De-Kuan Chang, Gulzar Ahmad, Jonathan Nardozzi, Katharine Sackton, Jesse Lyons, Karsten Sauer, Thomas Andresen, Douglas Jones. Surface-tethered IL-12 improves tumor-specific T-cell therapy and enhances inflammatory activity in tumors without inducing systemic toxicities [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 933.

Abstract 5101: CAB-CAR-T: The prioritization of cell surface protein targets for conditionally active biologics to treat all solid tumors

Abstract Introduction Proteins on the surface of cancer cells represent viable targets for conditionally active biologic (CAB) CAR-T therapy that only work in the tumor microenvironment. By modeling properties of cell surface proteins from TCGA datasets we seek to identify optimal targets across all TCGA cancer cohorts that when used in CAB-CAR-T therapies will provide the greatest number of treatment options for patients across all cancer malignancies. Method Cell surface proteins(n=1086) were identified and meta data specific to each gene was organized from a variety of public databases for data modeling. Various approaches based on ideal CAB-CAR-T properties are used to rank the cell surface proteins as therapeutic targets. The 31 TCGA cohorts representing the most comprehensive collection of genomic profiled tumor samples and outcomes of all cancers is used to rank cell surface proteins for the percentage of patients in each cohort who would be eligible for treatment based on predetermined mRNA cutoffs. Various approaches were used to filter the ranked list based on ideal CAB-CAR-T properties: known antibodies that can be used for initial CAB-CAR-T development, non-receptor as a static protein structure, highly expressed in CCLE indicating mRNA expression is a feature of cancer cell lines and low expression in critical tissues like Heart, Lung, Liver, Muscle etc. Different ranked lists of cell surface proteins were used to determine the number of CAB-CAR-T products required to treat 90% of patients in TCGA cohorts. A patient with the highest mRNA expression above the mean plus one standard deviation as determine across all TCGA samples is assigned to that specific protein biomarker as eligible for treatment and removed from the list of patients still to be treated. A bootstrap p-value for the ranked lists was determined by calculating the minimum number of randomly selected cell surface proteins that would give 90% coverage of the TCGA cohort. Results It was shown that it is reasonable to find ranked list of genes with high mRNA expression in TCGA and minimum expression in off-target critical tissue that 5-7 CAB-CAR-T products could be used to treat 90% of TCGA patients. To achieve 100% treatment coverage each additional CAB-CAR-T product added to the list had minimum inclusion of additional patients for treatment. Conclusions By modeling various properties of cell surface proteins to establish future development of CAB-CAR-T products it is reasonable to expect 90% patient coverage with 10 distinct therapies. Further modeling will be performed to exam combination therapies where tumor heterogeneity is an important criteria for the ranked list to have efficacy, with a goal of maximizing complete responses (CRs) and minimize the chance of relapse in the future. Citation Format: Hiba A. Shaban, Laurence Jadin, James Onuffer, Farzad Haerizadeh, Alissa Kerner, Gregory Schreiber, Gregory Frost, Scooter Willis. CAB-CAR-T: The prioritization of cell surface protein targets for conditionally active biologics to treat all solid tumors [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 5101.

Use of magnetic fields and nanoparticles to trigger drug release and improve tumor targeting.

Drug delivery strategies aim to maximize a drug's therapeutic index by increasing the concentration of drug at target sites while minimizing delivery to off-target tissues. Because biological tissues are minimally responsive to magnetic fields, there has been a great deal of interest in using magnetic nanoparticles in combination with applied magnetic fields to selectively control the accumulation and release of drug in target tissues while minimizing the impact on surrounding tissue. In particular, spatially variant magnetic fields have been used to encourage accumulation of drug-loaded magnetic nanoparticles at target sites, while time-variant magnetic fields have been used to induce drug release from thermally sensitive nanocarriers. In this review, we discuss nanoparticle formulations and approaches that have been developed for magnetic targeting and/or magnetically induced drug release, as well as ongoing challenges in using magnetism for therapeutic applications. This article is categorized under: Diagnostic Tools > in vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

&lt;p&gt;CPX-351: a nanoscale liposomal co-formulation of daunorubicin and cytarabine with unique biodistribution and tumor cell uptake properties&lt;/p&gt;

Combination regimens are a standard of care for many cancers. However, components of such regimens are typically first developed individually and subsequently combined using strategies to minimize toxicity. Little or no consideration is given to strategies that potentially maximize efficacy. In contrast, CPX-351 (Vyxeos®) is a dual-drug liposomal encapsulation of cytarabine and daunorubicin that was rationally designed to improve efficacy over the traditional 7+3 cytarabine/daunorubicin chemotherapy regimen for patients with acute myeloid leukemia (AML). The notable clinical efficacy of CPX-351 is achieved through maintenance of a synergistic 5:1 molar ratio of cytarabine and daunorubicin within the liposome after intravenous injection. The CPX-351 liposome, which is formulated to contain bilayers of distearoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol at a 7:2:1 molar ratio and remains in a gel phase at body temperature, provides stability without polyethylene glycol, controlled release of cytarabine and daunorubicin, limited systemic drug distribution, and preferential internalization within malignant myeloblasts in the bone marrow via active uptake of liposomes into cytoplasmic vacuoles. Thus, the CPX-351 liposome protects cytarabine and daunorubicin from metabolism and elimination, while overcoming pharmacokinetic differences between the two agents. In clinical studies, these liposome properties markedly increased the elimination half-life of CPX-351 versus free cytarabine and daunorubicin and maintained a synergistic drug ratio for over 24 hrs after administration. Preferential uptake of liposomes by leukemia cells suggests that relatively large amounts of cytarabine and daunorubicin enter malignant cells via liposomes, potentially bypassing P-glycoprotein-based efflux pumps, which are important mediators of chemotherapy resistance, and contribute to the rapid clearance of leukemia cells from the circulation and bone marrow. These pharmacologic advantages, a direct consequence of properties of the encapsulating liposome, may explain the efficacy of CPX-351 in patients with newly diagnosed high-risk/secondary AML and the reduced drug exposure in off-target tissues that contribute to a manageable safety profile.

Intracameral Sustained-Release Bimatoprost Implant Delivers Bimatoprost to Target Tissues with Reduced Drug Exposure to Off-Target Tissues.

Purpose: To explore the ocular distribution of bimatoprost after intracameral administration of a biodegradable sustained-release bimatoprost implant (Bimatoprost SR) versus repeated topical administration of bimatoprost 0.03% ophthalmic solution in dogs. Bimatoprost SR and topical bimatoprost 0.03% previously were shown to have similar intraocular pressure-lowering effects in humans in a phase 1/2 clinical trial.Methods: Twenty-four beagle dogs received either once-daily topical bimatoprost 0.03% for 7 days or a bilateral intracameral administration of Bimatoprost SR (15 μg). At predetermined time points, ocular tissues were collected and concentrations of bimatoprost and bimatoprost acid were quantified using liquid chromatography–tandem mass spectrometry.Results: Bimatoprost SR administration enhanced delivery of study drug to a site of action [iris–ciliary body (ICB)] compared with topical bimatoprost (Cmax [bimatoprost+bimatoprost acid] = 18,200 and 4.13 ng/g, respectively). However, distribution of drug to tissues associated with prostaglandin analog (PGA)-related side effects (i.e., bulbar conjunctiva, eyelid margins, and periorbital fat) was limited following Bimatoprost SR administration (Cmax [bimatoprost+bimatoprost acid] = BLQ [beneath the limit of quantitation] to 0.354 ng/g) compared with topical dosing (Cmax [bimatoprost+bimatoprost acid] = 36.6–2,110 ng/g).Conclusions: Bimatoprost SR administration in dogs selectively delivered drug to the ICB with low or undetectable drug levels in ocular surface and extraocular tissues. Use of Bimatoprost SR for glaucoma treatment may reduce the incidence of adverse events typically associated with topical PGAs by targeting bimatoprost delivery to the key site of action of the PGA class and reducing exposure to off-target tissues.

Rational design of a super-contrast NIR-II fluorophore affords high-performance NIR-II molecular imaging guided microsurgery.

In vivo molecular imaging in the "transparent" near-infrared II (NIR-II) window has demonstrated impressive benefits in reaching millimeter penetration depths with high specificity and imaging quality. Previous NIR-II molecular imaging generally relied on high hepatic uptake fluorophores with an unclear mechanism and antibody-derived conjugates, suffering from inevitable nonspecific retention in the main organs/skin with a relatively low signal-to-background ratio. It is still challenging to synthesize a NIR-II fluorophore with both high quantum yield and minimal liver-retention feature. Herein, we identified the structural design and excretion mechanism of novel NIR-II fluorophores for NIR-II molecular imaging with an extremely clean background. With the optimized renally excreted fluorophore-peptide conjugates, superior NIR-II targeting imaging was accompanied by the improved signal-to-background ratio during tumor detection with reducing off-target tissue exposure. An unprecedented NIR-II imaging-guided microsurgery was achieved using such an imaging platform, which provides us with a great preclinical example to accelerate the potential clinical translation of NIR-II imaging.

ROS-sensitive thioketal-linked polyphosphoester-doxorubicin conjugate for precise phototriggered locoregional chemotherapy

Minimizing drug leakage at off-target sites and triggering sufficient drug release in tumor tissue are major objectives for effective nanoparticle (NP)-based cancer therapy. The current covalent and cleavable drug-NP conjugation strategy is promising but lacks high controllability to realize tumor-specific release. As a proof-of-concept, the reactive oxygen species (ROS)-activatable thioketal (TK) bond was explored as the linkage between doxorubicin (DOX) and polyphosphoester (PPE-TK-DOX). The Ce6@PPE-TK-DOX NPs constructed by co-self-coassembly of PPE-TK-DOX and the photosensitizer Ce6 efficiently prevented premature drug leakage in the off-target tissue and cells because of the high stability of the TK bond under physiological conditions. Once circulating into the tumor site, the 660-nm red light was precisely employed to irradiate the tumor area under the guidance of fluorescence/magnetic resonance (MR) dual-model imaging, which can induce localized ROS generation, resulting in rapid cleavage of the TK bond. Consequently, the DOX prodrug was locoregionally released and activated, achieving tumor-specific drug delivery with high controllability by light. Such phototriggered prodrug release and activation at the desired site significantly enhanced the therapeutic efficacy and minimized the side effect, providing new avenues to develop drug delivery systems for remote on-demand drug delivery in vivo.