Widespread in vivo efficacy of The-0504: A conditionally-activatable nanoferritin for tumor-agnostic targeting of CD71-expressing cancers.

Background: Cancer is still among the leading causes of death all over the world. Improving chemotherapy and minimizing associated toxicities are major unmet medical needs. Recently, we provided a preliminary preclinical evaluation of a human ferritin (HFt)-based drug carrier (The-0504) that selectively delivers the wide-spectrum topoisomerase I inhibitor Genz-644282 to CD71-expressing tumors. The-0504 has so far been evaluated on four different human tumor xenotransplant models (breast, colorectal, pancreatic and liver cancers). Methods: Herein, we extend our studies, by: (a) testing DNA damage in vitro, (b) treating eight additional tumor xenograft models in vivo with The-0504; (c) performing pharmacokinetic (PK) studies in rats; and (d) evaluating The-0504 anti-tumor xenotransplant efficacy by optimizing its administration schedule based on PK considerations. Results: Immunofluorescence demonstrated that The-0504 induces foci expressing the DNA double-strand break marker γH2AX. Expression increases up to 4-fold and is more persistent as compared to free Genz-644282. In vivo studies confirmed a remarkable anti-tumor activity of The-0504, resulting in tumor eradication in most murine xenograft models, regardless of embryological origin (e.g. epithelial, mesenchymal or neuroendocrine), and molecular subtypes. PK studies demonstrated a long persistence of The-0504 in rat serum (half-life of about 40 h as compared to 15 h of the free drug), with a 400-fold increase in peak concentrations as compared to the free drug. On this basis, we reduced The-0504 administration frequency from twice to once per week, with no appreciable loss in therapeutic efficacy in mice. Conclusion: The results presented here confirm that The-0504 is highly active against several human tumor xenotransplants, even when administered less frequently than previously reported. The-0504 may be a good candidate for further clinical development in a tumor histotype-agnostic setting.

G Protein-Coupled Receptors and the Rise of Type 2 Diabetes in Children.

The human genome counts hundreds of GPCRs specialized to sense thousands of different extracellular cues, including light, odorants and nutrients in addition to hormones. Primordial GPCRs were likely glucose transporters that became sensors to monitor the abundance of nutrients and direct the cell to switch from aerobic metabolism to fermentation. Human ß cells express multiple GPCRs that contribute to regulate glucose homeostasis, cooperating with many others expressed by a variety of cell types and tissues. These GPCRs are intensely studied as pharmacological targets to treat type 2 diabetes in adults. The dramatic rise of type 2 diabetes incidence in pediatric age is likely correlated to the rapidly evolving lifestyle of children and adolescents of the new century. Current pharmacological treatments are based on therapies designed for adults, while youth and puberty are characterized by a different hormonal balance related to glucose metabolism. This review focuses on GPCRs functional traits that are relevant for ß cells function, with an emphasis on aspects that could help to differentiate new treatments specifically addressed to young type 2 diabetes patients.

Neutralization of NET-associated human ARG1 enhances cancer immunotherapy.

Myeloid cells can restrain antitumor immunity by metabolic pathways, such as the degradation of l-arginine, whose concentrations are regulated by the arginase 1 (ARG1) enzyme. Results from preclinical studies indicate the important role of arginine metabolism in pancreatic ductal adenocarcinoma (PDAC) progression, suggesting a potential for clinical application; however, divergent evolution in ARG1 expression and function in rodents and humans has restricted clinical translation. To overcome this dichotomy, here, we show that neutrophil extracellular traps (NETs), released by spontaneously activated neutrophils isolated from patients with PDAC, create a microdomain where cathepsin S (CTSS) cleaves human (h)ARG1 into different molecular forms endowed with enhanced enzymatic activity at physiological pH. NET-associated hARG1 suppresses T lymphocytes whose proliferation is restored by either adding a hARG1-specific monoclonal antibody (mAb) or preventing CTSS-mediated cleavage, whereas small-molecule inhibitors are not effective. We show that ARG1 blockade, combined with immune checkpoint inhibitors, can restore CD8+ T cell function in ex vivo PDAC tumors. Furthermore, anti-hARG1 mAbs increase the frequency of adoptively transferred tumor-specific CD8+ T cells in tumor and enhance the effectiveness of immune checkpoint therapy in humanized mice. Thus, this study shows that extracellular ARG1, released by activated myeloid cells, localizes in NETs, where it interacts with CTSS that in turn cleaves ARG1, producing major molecular forms endowed with different enzymatic activity at physiological pH. Once exocytosed, ARG1 activity can be targeted by mAbs, which bear potential for clinical application for the treatment of PDAC and require further exploration.

D2B-Functionalized Gold Nanoparticles: Promising Vehicles for Targeted Drug Delivery to Prostate Cancer.

Despite the multitude of therapeutic agents available to treat prostate cancer (PC), there are still no effective and safe measures to treat the tumor. It remains a challenge to develop a simple approach to target PC with specific antibodies. In our study, D2B monoclonal antibodies against a prostate-specific membrane antigen (PSMA) were used. We investigated the functionalization of gold nanoparticles (AuNPs) with D2B to generate favorable physicochemical and biological properties that mediate specific binding to PC. For this purpose, AuNPs with a size of about 25 nm were synthesized in water using sodium citrate as a reducing and stabilizing agent and then coated with D2B. Major physicochemical properties of naked and D2B-coated AuNPs were investigated by ultraviolet-visible (UV-vis) spectroscopy, dynamic light scattering (DLS), and zeta potential measurements. The successful binding of D2B to AuNPs-citrate caused a 15 nm red shift in the UV-vis. This was assessed by DLS as an increase in zeta potential from ∼-45 to ∼-23 mV and in the size of AuNPs from ∼25 to ∼63 nm. Scanning electron microscopy confirmed the size shift of AuNPs, which was detected as an exterior organic layer of D2Bs surrounding each AuNP. Even at high exposure levels of the bioconjugates, PSMA-PC-3 cells exhibited minimal cytotoxicity. The specific and dose-dependent binding of AuNPs-D2B to PC-3-PSMA cells was validated by flow cytometry analysis. Our data provide effective drug delivery systems in PC theranostics.

Development and Characterization of 99mTc-scFvD2B as a Potential Radiopharmaceutical for SPECT Imaging of Prostate Cancer.

Previously, we demonstrated that the 177Lu-labeled single-chain variable fragment of an anti-prostate-specific membrane antigen (PSMA) IgG D2B antibody (scFvD2B) showed higher prostate cancer (PCa) cell uptake and tumor radiation doses compared to 177Lu-labeled Glu-ureide-based PSMA inhibitory peptides. To obtain a 99mTc-/177Lu-scFvD2B theranostic pair, this research aimed to synthesize and biochemically characterize a novel 99mTc-scFvD2B radiotracer. The scFvD2B-Tag and scFvD2B antibody fragments were produced and purified. Then, two HYNIC derivatives, HYNIC-Gly-Gly-Cys-NH2 (HYNIC-GGC) and succinimidyl-HYNIC (S-HYNIC), were used to conjugate the scFvD2B-Tag and scFvD2B isoforms, respectively. Subsequently, chemical characterization, immunoreactivity tests (affinity and specificity), radiochemical purity tests, stability tests in human serum, cellular uptake and internalization in LNCaP(+), PC3-PIP(++) or PC3(-) PCa cells of the resulting unlabeled HYNIC-scFvD2B conjugates (HscFv) and 99mTc-HscFv agents were performed. The results showed that incorporating HYNIC as a chelator did not affect the affinity, specificity or stability of scFvD2B. After purification, the radiochemical purity of 99mTc-HscFv radiotracers was greater than 95%. A two-sample t-test of 99mTc-HscFv1 and 99mTc-HscFv1 uptake in PC3-PIP vs. PC3 showed a p-value < 0.001, indicating that the PSMA receptor interaction of 99mTc-HscFv agents was statistically significantly higher in PSMA-positive cells than in the negative controls. In conclusion, the results of this research warrant further preclinical studies to determine whether the in vivo pharmacokinetics and tumor uptake of 99mTc-HscFv still offer sufficient advantages over HYNIC-conjugated peptides to be considered for SPECT/PSMA imaging.

Human blood biocompatibility and immunogenicity of scFvD2B PEGylated gold nanoparticles.

Single chain variable D2B antibody fragments (scFvD2Bs) exhibit high affinity binding to prostate specific membrane antigens overexpressed in metastatic prostate cancer (PC). Conjugation of scFvD2B to gold nanoparticles (AuNPs) would enhance its stability and plasma half-life circulation to shuttle theranostic agents in PC. In this study, we synthesized PEGylated scFvD2B-AuNPs (AuNPs-scFvD2B-PEG) and tested their integrity, biocompatibility, and immunogenicity in freshly withdrawn human blood. Prior to blood incubation, Zeta potential measurements, UV-Vis spectroscopy, and dynamic light scattering (DLS) were used to assess the physicochemical properties of our nano-complexes in the presence or absence of PEGylation. A surface plasmon resonance band shift of 2 and 4 nm confirmed the successful coating for AuNPs-scFvD2B and AuNPs-scFvD2B-PEG, respectively. Likewise, DLS revealed a size increase of ∼3 nm for AuNPs-scFvD2B and ∼19 nm for AuNPs-scFvD2B-PEG. Zeta potential increased from -34 to -19 mV for AuNPs-scFvD2B and reached -3 mV upon PEGylation. Similar assessment measures were applied post-incubation in human blood with additional immunogenicity tests, such as hemolysis assay, neutrophil function test, and pyridine formazan extraction. Interestingly, grafting PEG chains on AuNPs-scFvD2B precluded the binding of blood plasma proteins and reduced neutrophil activation level compared with naked AuNPs-citrate counterparts. Most likely, a hydrated negative PEG cloud shielded the NPs rendering blood compatiblility with less than 10% hemolysis. In conclusion, the biocompatible AuNPs-scFvD2B-PEG presents promising characteristics for PC targeted therapy, with minimal protein adsorption affinity, low immunorecognition, and reduced hemolytic activity.

Mitoxantrone-Loaded Nanoferritin Slows Tumor Growth and Improves the Overall Survival Rate in a Subcutaneous Pancreatic Cancer Mouse Model.

Pancreatic cancer (PC) represents an intriguing topic for researchers. To date, the prognosis of metastasized PC is poor with just 7% of patients exceeding a five-year survival period. Thus, molecular modifications of existing drugs should be developed to change the course of the disease. Our previously generated nanocages of Mitoxantrone (MIT) encapsulated in human H-chain Ferritin (HFt), designated as HFt-MP-PASE-MIT, has shown excellent tumor distribution and extended serum half-life meriting further investigation for PC treatment. Thus, in this study, we used the same nano-formulation to test its cytotoxicity using both in vitro and in vivo assays. Interestingly, both encapsulated and free-MIT drugs demonstrated similar killing capabilities on PaCa44 cell line. Conversely, in vivo assessment in a subcutaneous PaCa44 tumor model of PC demonstrated a remarkable capability for encapsulated MIT to control tumor growth and improve mouse survival with a median survival rate of 65 vs. 33 days for loaded and free-MIT, respectively. Interestingly, throughout the course of mice treatment, MIT encapsulation did not present any adverse side effects as confirmed by histological analysis of various murine tissue organs and body mass weights. Our results are promising and pave the way to effective PC targeted chemotherapy using our HFt nanodelivery platforms.

PSMA-Specific CAR-Engineered T Cells for Prostate Cancer: CD28 Outperforms Combined CD28-4-1BB "Super-Stimulation".

Prostate cancer (PCa) is the second leading cause of malignancy-related mortality in males in the Western world. Although treatment like prostatectomy and radiotherapy for localized cancer have good results, similar positive outcomes are not achieved in metastatic PCa. Consequently, these aggressive and metastatic forms of PCa urgently need new methods of treatment. We already described an efficient and specific second-generation (2G) Chimeric Antigen Receptor (CAR) against Prostate Specific Membrane Antigen (PSMA), a glycoprotein overexpressed in prostate cancer and also present on neovasculature of several tumor entities. In an attempt to improve efficacy and in vivo survival of anti-PSMA 2G CAR-T cells, we developed a third generation (3G) CAR containing two costimulatory elements, namely CD28 and 4-1BB co-signaling domains, in addition to CD3ζ. Differently from what described for other 3G receptors, our third generation CAR disclosed an antitumor activity in vitro similar to the related 2G CAR that comprises the CD28 co-signaling domain only. Moreover, the additional costimulatory domain produced detrimental effects, which could be attributed to an increased activation-induced cell death (AICD). Indeed, such "superstimulation" resulted in an exhausted phenotype of CAR-T cells, after prolonged in vitro restimulation, a higher frequency of cell death, and an impairment in yielding sufficient numbers of transgenic T lymphocytes. Thus, the optimal combination of costimulatory domains for CAR development should be assessed cautiously and evaluated case-by-case.

Design and Evaluation of 223Ra-Labeled and Anti-PSMA Targeted NaA Nanozeolites for Prostate Cancer Therapy-Part II. Toxicity, Pharmacokinetics and Biodistribution.

Metastatic castration-resistant prostate cancer (mCRPC) is a progressive and incurable disease with poor prognosis for patients. Despite introduction of novel therapies, the mortality rate remains high. An attractive alternative for extension of the life of mCRPC patients is PSMA-based targeted radioimmunotherapy. In this paper, we extended our in vitro study of 223Ra-labeled and PSMA-targeted NaA nanozeolites [223RaA-silane-PEG-D2B] by undertaking comprehensive preclinical in vitro and in vivo research. The toxicity of the new compound was evaluated in LNCaP C4-2, DU-145, RWPE-1 and HPrEC prostate cells and in BALB/c mice. The tissue distribution of 133Ba- and 223Ra-labeled conjugates was studied at different time points after injection in BALB/c and LNCaP C4-2 tumor-bearing BALB/c Nude mice. No obvious symptoms of antibody-free and antibody-functionalized nanocarriers cytotoxicity and immunotoxicity was found, while exposure to 223Ra-labeled conjugates resulted in bone marrow fibrosis, decreased the number of WBC and platelets and elevated serum concentrations of ALT and AST enzymes. Biodistribution studies revealed high accumulation of 223Ra-labeled conjugates in the liver, lungs, spleen and bone tissue. Nontargeted and PSMA-targeted radioconjugates exhibited a similar, marginal uptake in tumour lesions. In conclusion, despite the fact that NaA nanozeolites are safe carriers, the intravenous administration of NaA nanozeolite-based radioconjugates is dubious due to its high accumulation in the lungs, liver, spleen and bones.

Preclinical dosimetric studies of 177 Lu-scFvD2B and comparison with 177 Lu-PSMA-617 and 177 Lu-iPSMA endoradiotherapeutic agents.

PURPOSE: Internal dosimetry has become a very important tool to evaluate the risks and benefits of new endoradiotherapeutic agents. Nowadays, some of the most successful targeted radionuclide therapy (TRT) agents are 177 Lu-DOTA conjugates based on low molecular weight (LMW) Glu-ureido PSMA inhibitors. It has, however, been demonstrated that the DOTA chelating moiety reduces the internalization of the LMW-PSMA agent and its radiation dose to the tumor. Previously, we reported that 177 Lu-scFvD2B, an antibody-based construct, demonstrated statistically significant higher cell uptake and internalization in LNCaP prostate cancer (PCa) cells (PSMA-positive) when compared to the LMW-PSMA agents, 177 Lu-PSMA-617 and 177 Lu-iPSMA, two of the endoradiotherapeutic agents which currently are the most used in PCa therapy. The aim of this study is to estimate the preclinical 177 Lu-scFvD2B organ and tumor-absorbed doses, and to compare the values with those of 177 Lu-PSMA-617 and 177 Lu-iPSMA. METHODS: 177 Lu-scFvD2B, 177 Lu-PSMA-617, and 177 Lu-iPSMA were prepared and their radiochemical purity determined. Biodistribution studies of each radiopharmaceutical were then carried out in healthy mice to define the main source organs (SO) and to calculate the number of disintegrations in each source organs per unit of administered activity (NSO ). Absorbed dose in the main organs were then calculated for each 177 Lu-conjugate by means of OLINDA/EXM 2.1.1 software, using the calculated NSO for both the adult male and the mouse phantoms as program inputs. Images of mice bearing micropulmonary tumors injected with 177 Lu-conjugates were also obtained. Tumor standardized uptake values (SUV) for the different conjugates, obtained from the 3D SPECT image reconstruction of these mice, were used as the number of disintegrations in a tumor site per unit of administered activity (NT ). The tumor-absorbed dose was calculated using the published electron dose S-values for sphere models with diameters ranging from 10 µm to 10 mm and considering a uniform activity distribution and tumor density equivalent to water density. RESULTS: All 177 Lu-labeled agents were obtained in high yield (98%). Dosimetric studies carried out using mouse phantoms demonstrated that organ absorbed doses of 177 Lu-scFvD2B were from 1.4 to 2.3 times higher than those for 177 Lu-iPSMA and from 1.5 to 2.6 times higher than those for 177 Lu-PSMA-617. However, the 177 Lu-scFvD2B values of tumor-absorbed doses for all investigated tumor sizes were from 2.8 to 3.0 times greater than those calculated for 177 Lu-iPSMA and 177 Lu-PSMA-617, respectively. Moreover, 177 Lu-scFvD2B showed the highest tumor/kidney ratio when compared to those reported for 177 Lu-albumin conjugates. CONCLUSIONS: In this preclinical study, we demonstrated the potential of 177 Lu-scFvD2B as a therapeutic agent for PSMA-expressing tumors, due to its higher tumor-absorbed dose when compared with 177 Lu-LMW agents.

Polymer-coated silver-iron nanoparticles as efficient and biodegradable MRI contrast agents.

Bimetallic nanoparticles allow new and synergistic properties compared to the monometallic equivalents, often leading to unexpected results. Here we present on silver-iron nanoparticles coated with polyethylene glycol, which exhibit a high transverse relaxivity (316 ± 13 mM-1s-1, > 3 times that of the most common clinical benchmark based on iron oxide), excellent colloidal stability and biocompatibility in vivo. Ag-Fe nanoparticles are obtained through a one-step, low-cost laser-assisted synthesis, which makes surface functionalization with the desired biomolecules very easy. Besides, Ag-Fe nanoparticles show biodegradation over a few months, as indicated by incubation in the physiological environment. This is crucial for nanomaterials removal from the living organism and, in fact, in vivo biodistribution studies evidenced that Ag-Fe nanoparticles tend to be cleared from liver over a period in which the benchmark iron oxide contrast agent persisted. Therefore, the Ag-Fe NPs offer positive prospects for solving the problems of biopersistence, contrast efficiency, difficulties of synthesis and surface functionalization usually encountered in nanoparticulate contrast agents.

High activity and low toxicity of a novel CD71-targeting nanotherapeutic named The-0504 on preclinical models of several human aggressive tumors.

BACKGROUND: Ferritin receptor (CD71) is an example of a very attractive cancer target, since it is highly expressed in virtually all tumor types, including metastatic loci. However, this target can be considered to be inaccessible to conventional target therapies, due to its presence in many healthy tissues. Here, we describe the preclinical evaluation of a tumor proteases-activatable human ferritin (HFt)-based drug carrier (The-0504) that is able to selectively deliver the wide-spectrum topoisomerase I inhibitor Genz-644282 to CD71-expressing tumors, preventing the limiting toxic effects associated with CD71-targeting therapies. METHODS: CD71 expression was evaluated using flow cytometry and immunohistochemistry techniques. The-0504 antiproliferative activity towards several cancer cell lines was assessed in vitro. The-0504 antitumor efficacy and survival benefit were evaluated in different human tumors, which had been grown either as xenografts or patient-derived xenografts in mice. The-0504 toxicology profile was investigated in multiple-cycle repeat-dose study in rodents. RESULTS: In vitro studies indicate that The-0504 is highly specific for CD71 expressing cells, and that there is a relationship between CD71 levels and The-0504 anticancer activity. In vivo treatments with The-0504 showed a remarkable efficacy, eradicating several human tumors of very diverse and aggressive histotypes, such as pancreas, liver and colorectal carcinomas, and triple-negative breast cancer. CONCLUSIONS: Durable disease-free survival, persistent antitumor responses after discontinuation of treatment and favorable toxicology profile make The-0504 an ideal candidate for clinical development as a novel, CD71-targeted, low-toxicity alternative to chemotherapy.

Engineered Human Nanoferritin Bearing the Drug Genz-644282 for Cancer Therapy.

Gastrointestinal tumors, including pancreatic and colorectal cancers, represent one of the greatest public health issues worldwide, leading to a million global deaths. Recent research demonstrated that the human heavy chain ferritin (HFt) can encapsulate different types of drugs in its cavity and can bind to its receptor, CD71, in several solid and hematological tumors, thus highlighting the potential use of ferritin for tumor-targeting therapies. Here, we describe the development and characterization of a novel nanomedicine based on the HFt that is named The-0504. In particular, this novel system is a nano-assembly comprising an engineered version of HFt that entraps about 80 molecules of a potent, wide-spectrum, non-camptothecin topoisomerase I inhibitor (Genz-644282). The-0504 can be produced by a standardized pre-industrial process as a pure and homogeneously formulated product with favourable lyophilization properties. The preliminary anticancer activity was evaluated in cultured cancer cells and in a mouse model of pancreatic cancer. Overall results reported here make The-0504 a candidate for further preclinical development against CD-71 expressing deadly tumors.

SERRS multiplexing with multivalent nanostructures for the identification and enumeration of epithelial and mesenchymal cells.

Liquid biopsy represents a new frontier of cancer diagnosis and prognosis, which allows the isolation of tumor cells released in the blood stream. The extremely low abundance of these cells needs appropriate methodologies for their identification and enumeration. Herein we present a new protocol based on surface enhanced resonance Raman scattering (SERRS) gold multivalent nanostructures to identify and enumerate tumor cells with epithelial and mesenchimal markers. The validation of the protocol is obtained with spiked samples of peripheral blood mononuclear cells (PBMC). Gold nanostructures are functionalized with SERRS labels and with antibodies to link the tumor cells. Three types of such nanosystems were simultaneously used and the protocol allows obtaining the identification of all individual tumor cells with the help of a Random Forest ensemble learning method.

Design and Evaluation of 223Ra-Labeled and Anti-PSMA Targeted NaA Nanozeolites for Prostate Cancer Therapy-Part I.

Prostate cancer is the second most frequent malignancy in men worldwide. Unfortunately, current therapies often lead to the onset of metastatic castration-resistant prostate cancer (mCRPC), causing significant mortality. Therefore, there is an urgent need for new and targeted therapies that are advantageous over the current ones. Recently, the PSMA-targeted radioligand therapy of mCRPC has shown very promising results. In line with this, we described the synthesis of a new radioimmunoconjugate, 223RaA-silane-PEG-D2B, for targeted mCRPC therapy. The new compound consists of a NaA zeolite nanocarrier loaded with the α-particle emitting Ra-223 radionuclide, functionalized with the anti-PSMA D2B antibody. Physicochemical properties of the synthesized compound were characterized by standard methods (HR-SEM, TEM, XRD, FTIR, EDS, NTA, DLS, BET, TGA). The targeting selectivity, the extent of internalization, and cytotoxicity were determined in LNCaP C4-2 (PSMA+) and DU-145 (PSMA-) cells. Our results supported the 223RaA-silane-PEG-D2B synthesis and revealed that the final product had a diameter ca. 120 nm and specific activity 0.65 MBq/1mg. The product was characterized by a high yield of stability (>95% up to 12 days). The conjugation reaction resulted in approximately 50 antibodies/nanoparticle. The obtained radioimmunoconjugate bound specifically and internalized into PSMA-expressing LNCaP C4-2 cells, but not into PSMA-negative DU-145 cells. 223RaA-silane-PEG-D2B demonstrated also potent cytotoxicity in LNCaP C4-2 cells. These promising results require further in vivo evaluation of 223RaA-silane-PEG-D2B with regard to its toxicity and therapeutic efficacy.

Anti-PSMA CAR-engineered NK-92 Cells: An Off-the-shelf Cell Therapy for Prostate Cancer.

Prostate cancer (PCa) has become the most common cancer among males in Europe and the USA. Adoptive immunotherapy appears a promising strategy to control the advanced stages of the disease by specifically targeting the tumor, in particular through chimeric antigen receptor T (CAR-T) cell therapy. Despite the advancements of CAR-T technology in the treatment of hematological malignancies, solid tumors still represent a challenge. To overcome current limits, other cellular effectors than T lymphocytes are under study as possible candidates for CAR-engineered cancer immunotherapy. A novel approach involves the NK-92 cell line, which mediates strong cytotoxic responses against a variety of tumor cells but has no effect on non-malignant healthy counterparts. Here, we report a novel therapeutic approach against PCa based on engineering of NK-92 cells with a CAR recognizing the human prostate-specific membrane antigen (PSMA), which is overexpressed in prostatic neoplastic cells. More importantly, the potential utility of NK-92/CAR cells to treat PCa has not yet been explored. Upon CAR transduction, NK-92/CAR cells acquired high and specific lytic activity against PSMA-expressing prostate cancer cells in vitro, and also underwent degranulation and produced high levels of IFN-γ in response to antigen recognition. Lethal irradiation of the effectors, a safety measure requested for the clinical application of retargeted NK-92 cells, fully abrogated replication but did not impact on phenotype and short-term functionality. PSMA-specific recognition and antitumor activity were retained in vivo, as adoptive transfer of irradiated NK-92/CAR cells in prostate cancer-bearing mice restrained tumor growth and improved survival. Anti-PSMA CAR-modified NK-92 cells represent a universal, off-the-shelf, renewable, and cost-effective product endowed with relevant potentialities as a therapeutic approach for PCa immunotherapy.

Development of 177Lu-scFvD2B as a Potential Immunotheranostic Agent for Tumors Overexpressing the Prostate Specific Membrane Antigen.

The clinical translation of theranostic 177Lu-radiopharmaceuticals based on inhibitors of the prostate-specific membrane antigen (PSMA) has demonstrated positive clinical responses in patients with advanced prostate cancer (PCa). However, challenges still remain, particularly regarding their pharmacokinetic and dosimetric properties. We developed a potential PSMA-immunotheranostic agent by conjugation of a single-chain variable fragment of the IgGD2B antibody (scFvD2B) to DOTA, to obtain a 177Lu-labelled agent with a better pharmacokinetic profile than those previously reported. The labelled conjugated 177Lu-scFvD2B was obtained in high yield and stability. In vitro, 177Lu-scFvD2B disclosed a higher binding and internalization in LNCaP (PSMA-positive) compared to PC3 (negative control) human PCa cells. In vivo studies in healthy nude mice revealed that 177Lu-scFvD2B present a favorable biokinetic profile, characterized by a rapid clearance from non-target tissues and minimal liver accumulation, but a slow wash-out from kidneys. Micro-SPECT/CT imaging of mice bearing pulmonary microtumors evidenced a slow uptake by LNCaP tumors, which steadily rose up to a maximum value of 3.6 SUV at 192 h. This high and prolonged tumor uptake suggests that 177Lu-scFvD2B has great potential in delivering ablative radiation doses to PSMA-expressing tumors, and warrants further studies to evaluate its preclinical therapeutic efficacy.

Simple and Rapid Non-Enzymatic Procedure Allows the Isolation of Structurally Preserved Connective Tissue Micro-Fragments Enriched with SVF.

The stromal vascular fraction (SVF) consists of a heterogeneous population of stem and stromal cells, generally obtained from adipose tissue by enzymatic digestion. For human cell-based therapies, mechanical process methods to obtain SVF represent an advantageous approach because they have fewer regulatory restrictions for their clinical use. The aim of this study was to characterize a novel commercial system for obtaining SVF from adipose tissue by a mechanical approach without substantial manipulations. Lipoaspirate samples collected from 27 informed patients were processed by a simple and fast mechanical system (by means of Hy-Tissue SVF). The Hy-Tissue SVF product contained a free cell fraction and micro-fragments of stromal connective tissue. The enzymatic digestion of the micro-fragments increased the yield of free cells (3.2 times) and CFU-F (2.4 times). Additionally, 10% of free cells from SVF were positive for CD34+, suggesting the presence of endothelial cells, pericytes, and potential adipose-derived stem cells (ADSC). Moreover, the SVF cells were able to proliferate and differentiate in vitro toward adipocytes, osteocytes, and chondrocytes. The immunophenotypic analysis of expanded cells showed positivity for typical mesenchymal stem cell markers. The Hy-Tissue SVF system allows the isolation of stromal vascular fraction, making this product of potential interest in regenerative medicine.

Development and characterization of a theranostic multimodal anti-PSMA targeting agent for imaging, surgical guidance, and targeted photodynamic therapy of PSMA-expressing tumors.

Rationale: Prostate cancer (PCa) recurrences after surgery frequently occur. To improve the outcome after surgical resection of the tumor, the theranostic multimodal anti-PSMA targeting agent 111In-DTPA-D2B-IRDye700DX was developed and characterized for both pre- and intra-operative tumor localization and eradication of (residual) tumor tissue by PSMA-targeted photodynamic therapy (tPDT), which is a highly selective cancer treatment based on targeting molecules conjugated to photosensitizers that can induce cell destruction upon exposure to near-infrared (NIR) light. Methods: The anti-PSMA monoclonal antibody D2B was conjugated with IRDye700DX and DTPA and subsequently radiolabeled with 111In. To determine the optimal dose and time point for tPDT, BALB/c nude mice with PSMA-expressing (PSMA+) s.c. LS174T-PSMA xenografts received the conjugate (24-240 µg/mouse) intravenously (8 MBq/mouse) followed by µSPECT/CT, near-infrared fluorescence imaging, and ex vivo biodistribution at 24, 48, 72 and 168 h p.i. Tumor growth of LS174T-PSMA xenografts and overall survival of mice treated with 1-3 times of NIR light irradiation (50, 100, 150 J/cm2) 24 h after injection of 80 µg of DTPA-D2B-IRDye700DX was compared to control conditions. Results: Highest specific tumor uptake was observed at conjugate doses of 80 µg/mouse. Biodistribution revealed no significant difference in tumor uptake in mice at 24, 48, 72 and 168 h p.i. PSMA+ tumors were clearly visualized with both µSPECT/CT and NIR fluorescence imaging. Overall survival in mice treated with 80 µg of DTPA-D2B-IRDye700DX and 1x 150 J/cm2 of NIR light at 24 h p.i. was significantly improved compared to the control group receiving neither conjugate nor NIR light (73 days vs. 16 days, respectively, p=0.0453). Treatment with 3x 150 J/cm2 resulted in significantly prolonged survival compared to treatment with 3x 100 J/cm2 (p = 0.0067) and 3x 50 J/cm2 (p = 0.0338). Principal conclusions:111In-DTPA-D2B-IRDye700DX can be used for pre- and intra-operative detection of PSMA+ tumors with radionuclide and NIR fluorescence imaging and PSMA-targeted PDT. PSMA-tPDT using this multimodal agent resulted in significant prolongation of survival and shows great potential for treatment of (metastasized) prostate cancer.

Induction of immunosuppressive functions and NF-κB by FLIP in monocytes.

Immunosuppression is a hallmark of tumor progression, and treatments that inhibit or deplete monocytic myeloid-derived suppressive cells could promote anti-tumor immunity. c-FLIP is a central regulator of caspase-8-mediated apoptosis and necroptosis. Here we show that low-dose cytotoxic chemotherapy agents cause apoptosis linked to c-FLIP down-regulation selectively in monocytes. Enforced expression of c-FLIP or viral FLIP rescues monocytes from cytotoxicity and concurrently induces potent immunosuppressive activity, in T cell cultures and in vivo models of tumor progression and immunotherapy. FLIP-transduced human blood monocytes can suppress graft versus host disease. Neither expression of FLIP in granulocytes nor expression of other anti-apoptotic genes in monocytes conferred immunosuppression, suggesting that FLIP effects on immunosuppression are specific to monocytic lineage and distinct from death inhibition. Mechanistically, FLIP controls a broad transcriptional program, partially by NF-κB activation. Therefore, modulation of FLIP in monocytes offers a means to elicit or block immunosuppressive myeloid cells.