Engineered soluble, trimerized 4-1BBL variants as potent immunomodulatory agents.

Targeting co-stimulatory receptors promotes the activation and effector functions of anti-tumor lymphocytes. 4-1BB (CD137/TNFSF9), a member of the tumor necrosis factor receptor superfamily (TNFR-SF), is a potent co-stimulatory receptor that plays a prominent role in augmenting effector functions of CD8+ T cells, but also CD4+ T cells and NK cells. Agonistic antibodies against 4-1BB have entered clinical trials and shown signs of therapeutic efficacy. Here, we have used a T cell reporter system to evaluate various formats of 4-1BBL regarding their capacity to functionally engage its receptor. We found that a secreted 4-1BBL ectodomain harboring a trimerization domain derived from human collagen (s4-1BBL-TriXVIII) is a strong inducer of 4-1BB co-stimulation. Similar to the 4-1BB agonistic antibody urelumab, s4-1BBL-TriXVIII is very potent in inducing CD8+ and CD4+ T cell proliferation. We provide first evidence that s4-1BBL-TriXVIII can be used as an effective immunomodulatory payload in therapeutic viral vectors. Oncolytic measles viruses encoding s4-1BBL-TriXVIII significantly reduced tumor burden in a CD34+ humanized mouse model, whereas measles viruses lacking s4-1BBL-TriXVIII were not effective. Natural soluble 4-1BB ligand harboring a trimerization domain might have utility in tumor therapy especially when delivered to tumor tissue as systemic administration might induce liver toxicity.

Vaccinia virus-mediated melanin production allows MR and optoacoustic deep tissue imaging and laser-induced thermotherapy of cancer.

We reported earlier the delivery of antiangiogenic single chain antibodies by using oncolytic vaccinia virus strains to enhance their therapeutic efficacy. Here, we provide evidence that gene-evoked production of melanin can be used as a therapeutic and diagnostic mediator, as exemplified by insertion of only one or two genes into the genome of an oncolytic vaccinia virus strain. We found that produced melanin is an excellent reporter for optical imaging without addition of substrate. Melanin production also facilitated deep tissue optoacoustic imaging as well as MRI. In addition, melanin was shown to be a suitable target for laser-induced thermotherapy and enhanced oncolytic viral therapy. In conclusion, melanin as a mediator for thermotherapy and reporter for different imaging modalities may soon become a versatile alternative to replace fluorescent proteins also in other biological systems. After ongoing extensive preclinical studies, melanin overproducing oncolytic virus strains might be used in clinical trials in patients with cancer.

Inducible gene expression in tumors colonized by modified oncolytic vaccinia virus strains.

UNLABELLED: Exogenous gene induction of therapeutic, diagnostic, and safety mechanisms could be a considerable improvement in oncolytic virotherapy. Here, we introduced a doxycycline-inducible promoter system (comprised of a tetracycline repressor, several promoter constructs, and a tet operator sequence) into oncolytic recombinant vaccinia viruses (rVACV), which were further characterized in detail. Experiments in cell cultures as well as in tumor-bearing mice were analyzed to determine the role of the inducible-system components. To accomplish this, we took advantage of the optical reporter construct, which resulted in the production of click-beetle luciferase as well as a red fluorescent protein. The results indicated that each of the system components could be used to optimize the induction rates and had an influence on the background expression levels. Depending on the given gene to be induced in rVACV-colonized tumors of patients, we discuss the doxycycline-inducible promoter system adjustment and further optimization. IMPORTANCE: Oncolytic virotherapy of cancer can greatly benefit from the expression of heterologous genes. It is reasonable that some of those heterologous gene products could have detrimental effects either on the cancer patient or on the oncolytic virus itself if they are expressed at the wrong time or if the expression levels are too high. Therefore, exogenous control of gene expression levels by administration of a nontoxic inducer will have positive effects on the safety as well as the therapeutic outcome of oncolytic virotherapy. In addition, it paves the way for the introduction of new therapeutic genes into the genome of oncolytic viruses that could not have been tested otherwise.

Combination of fractionated irradiation with anti-VEGF expressing vaccinia virus therapy enhances tumor control by simultaneous radiosensitization of tumor associated endothelium.

Oncolytic viruses are currently in clinical trials for a variety of tumors, including high grade gliomas. A characteristic feature of high grade gliomas is their high vascularity and treatment approaches targeting tumor endothelium are under investigation, including bevacizumab. The aim of this study was to improve oncolytic viral therapy by combining it with ionizing radiation and to radiosensitize tumor vasculature through a viral encoded anti-angiogenic payload. Here, we show how vaccinia virus-mediated expression of a single-chain antibody targeting VEGF resulted in radiosensitization of the tumor-associated vasculature. Cell culture experiments demonstrated that purified vaccinia virus encoded antibody targeting VEGF reversed VEGF-induced radioresistance specifically in endothelial cells but not tumor cells. In a subcutaneous model of U-87 glioma, systemically administered oncolytic vaccinia virus expressing anti-VEGF antibody (GLV-1h164) in combination with fractionated irradiation resulted in enhanced tumor growth inhibition when compared to nonanti-VEGF expressing oncolytic virus (GLV-1h68) and irradiation. Irradiation of tumor xenografts resulted in an increase in VACV replication of both GLV-1h68 and GLV-1h164. However, GLV-1h164 in combination with irradiation resulted in a drastic decrease in intratumoral VEGF levels and tumor vessel numbers in comparison to GLV-1h68 and irradiation. These findings demonstrate the incorporation of an oncolytic virus expressing an anti-VEGF antibody (GLV-1h164) into a fractionated radiation scheme to target tumor cells by enhanced VACV replication in irradiated tumors as well as to radiosensitize tumor endothelium which results in enhanced efficacy of combination therapy of human glioma xenografts.

Growth inhibition of different human colorectal cancer xenografts after a single intravenous injection of oncolytic vaccinia virus GLV-1h68.

BACKGROUND: Despite availability of efficient treatment regimens for early stage colorectal cancer, treatment regimens for late stage colorectal cancer are generally not effective and thus need improvement. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) strains is a promising new strategy for therapy of a variety of human cancers. METHODS: Oncolytic efficacy of replication-competent vaccinia virus GLV-1h68 was analyzed in both, cell cultures and subcutaneous xenograft tumor models. RESULTS: In this study we demonstrated for the first time that the replication-competent recombinant VACV GLV-1h68 efficiently infected, replicated in, and subsequently lysed various human colorectal cancer lines (Colo 205, HCT-15, HCT-116, HT-29, and SW-620) derived from patients at all four stages of disease. Additionally, in tumor xenograft models in athymic nude mice, a single injection of intravenously administered GLV-1h68 significantly inhibited tumor growth of two different human colorectal cell line tumors (Duke's type A-stage HCT-116 and Duke's type C-stage SW-620), significantly improving survival compared to untreated mice. Expression of the viral marker gene ruc-gfp allowed for real-time analysis of the virus infection in cell cultures and in mice. GLV-1h68 treatment was well-tolerated in all animals and viral replication was confined to the tumor. GLV-1h68 treatment elicited a significant up-regulation of murine immune-related antigens like IFN-γ, IP-10, MCP-1, MCP-3, MCP-5, RANTES and TNF-γ and a greater infiltration of macrophages and NK cells in tumors as compared to untreated controls. CONCLUSION: The anti-tumor activity observed against colorectal cancer cells in these studies was a result of direct viral oncolysis by GLV-1h68 and inflammation-mediated innate immune responses. The therapeutic effects occurred in tumors regardless of the stage of disease from which the cells were derived. Thus, the recombinant vaccinia virus GLV-1h68 has the potential to treat colorectal cancers independently of the stage of progression.

Functional hyper-IL-6 from vaccinia virus-colonized tumors triggers platelet formation and helps to alleviate toxicity of mitomycin C enhanced virus therapy.

BACKGROUND: Combination of oncolytic vaccinia virus therapy with conventional chemotherapy has shown promise for tumor therapy. However, side effects of chemotherapy including thrombocytopenia, still remain problematic. METHODS: Here, we describe a novel approach to optimize combination therapy of oncolytic virus and chemotherapy utilizing virus-encoding hyper-IL-6, GLV-1h90, to reduce chemotherapy-associated side effects. RESULTS: We showed that the hyper-IL-6 cytokine was successfully produced by GLV-1h90 and was functional both in cell culture as well as in tumor-bearing animals, in which the cytokine-producing vaccinia virus strain was well tolerated. When combined with the chemotherapeutic mitomycin C, the anti-tumor effect of the oncolytic virotherapy was significantly enhanced. Moreover, hyper-IL-6 expression greatly reduced the time interval during which the mice suffered from chemotherapy-induced thrombocytopenia. CONCLUSION: Therefore, future clinical application would benefit from careful investigation of additional cytokine treatment to reduce chemotherapy-induced side effects.

Bacterial glucuronidase as general marker for oncolytic virotherapy or other biological therapies.

BACKGROUND: Oncolytic viral tumor therapy is an emerging field in the fight against cancer with rising numbers of clinical trials and the first clinically approved product (Adenovirus for the treatment of Head and Neck Cancer in China) in this field. Yet, until recently no general (bio)marker or reporter gene was described that could be used to evaluate successful tumor colonization and/or transgene expression in other biological therapies. METHODS: Here, a bacterial glucuronidase (GusA) encoded by biological therapeutics (e.g. oncolytic viruses) was used as reporter system. RESULTS: Using fluorogenic probes that were specifically activated by glucuronidase we could show 1) preferential activation in tumors, 2) renal excretion of the activated fluorescent compounds and 3) reproducible detection of GusA in the serum of oncolytic vaccinia virus treated, tumor bearing mice in several tumor models. Time course studies revealed that reliable differentiation between tumor bearing and healthy mice can be done as early as 9 days post injection of the virus. Regarding the sensitivity of the newly developed assay system, we could show that a single infected tumor cell could be reliably detected in this assay. CONCLUSION: GusA therefore has the potential to be used as a general marker in the preclinical and clinical evaluation of (novel) biological therapies as well as being useful for the detection of rare cells such as circulating tumor cells.

Specific antibody-receptor interactions trigger InlAB-independent uptake of Listeria monocytogenes into tumor cell lines.

BACKGROUND: Specific cell targeting is an important, yet unsolved problem in bacteria-based therapeutic applications, like tumor or gene therapy. Here, we describe the construction of a novel, internalin A and B (InlAB)-deficient Listeria monocytogenes strain (Lm-spa+), which expresses protein A of Staphylococcus aureus (SPA) and anchors SPA in the correct orientation on the bacterial cell surface. RESULTS: This listerial strain efficiently binds antibodies allowing specific interaction of the bacterium with the target recognized by the antibody. Binding of Trastuzumab (Herceptin®) or Cetuximab (Erbitux®) to Lm-spa+, two clinically approved monoclonal antibodies directed against HER2/neu and EGFR/HER1, respectively, triggers InlAB-independent internalization into non-phagocytic cancer cell lines overexpressing the respective receptors. Internalization, subsequent escape into the host cell cytosol and intracellular replication of these bacteria are as efficient as of the corresponding InlAB-positive, SPA-negative parental strain. This specific antibody/receptor-mediated internalization of Lm-spa+ is shown in the murine 4T1 tumor cell line, the isogenic 4T1-HER2 cell line as well as the human cancer cell lines SK-BR-3 and SK-OV-3. Importantly, this targeting approach is applicable in a xenograft mouse tumor model after crosslinking the antibody to SPA on the listerial cell surface. CONCLUSIONS: Binding of receptor-specific antibodies to SPA-expressing L. monocytogenes may represent a promising approach to target L. monocytogenes to host cells expressing specific receptors triggering internalization.

Enterobacterial tumor colonization in mice depends on bacterial metabolism and macrophages but is independent of chemotaxis and motility.

Despite promising results and increasing attention in bacterial cancer therapy, surprisingly little is known about initial tumor colonization and the interaction between bacteria and surrounding tumor tissue. Here, we analyzed the role of chemotaxis, motility, and metabolism both in Escherichia coli and Salmonella enterica serovar Typhimurium strains upon intravenous injection into tumor-bearing mice. In contrast to previous models, we found that chemotaxis and motility do not play a significant role in tumor colonization and bacterial distribution within the tumor. Rather, the whole colonization and intratumoral migration process seems to be a passive mechanism that is influenced by the reticuloendothelial system of the host, by the tumor microenvironment and by the bacterial metabolism. These conclusions were supported by experimental data demonstrating that disruption of the basic branch of the aromatic amino acid biosynthetic pathway and depletion of macrophages, in contrast to flagellar mutations, led to significant changes in bacterial accumulation in tumors of live mice.

Regression of human prostate tumors and metastases in nude mice following treatment with the recombinant oncolytic vaccinia virus GLV-1h68.

Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In the current study, we analyzed the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 against two human prostate cancer cell lines DU-145 and PC-3 in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 was able to infect, replicate in, and lyse these prostate cancer cells in culture. In DU-145 and PC-3 tumor xenograft models, a single intravenous injection with GLV-1h68 resulted in a significant reduction of primary tumor size. In addition, the GLV-1h68-infection led to strong inflammatory and oncolytic effects resulting in drastic reduction of regional lymph nodes with PC-3 metastases. Our data documented that the GLV-1h68 virus has a great potential for treatment of human prostate carcinoma.

A novel recombinant vaccinia virus expressing the human norepinephrine transporter retains oncolytic potential and facilitates deep-tissue imaging.

Noninvasive and repetitive monitoring of a virus in target tissues and/or specific organs of the body is highly desirable for the development of safe and efficient cancer virotherapeutics. We have previously shown that the oncolytic vaccinia virus GLV-1h68 can target and eradicate human tumors in mice and that its therapeutic effects can be monitored by using optical imaging. Here, we report on the development of a derivative of GLV-1h68, a novel recombinant vaccinia virus (VACV) GLV-1h99, which was constructed to carry the human norepinephrine transporter gene (hNET) under the VACV synthetic early promoter placed at the F14.5L locus for deep-tissue imaging. The hNET protein was expressed at high levels on the membranes of cells infected with this virus. Expression of the hNET protein did not negatively affect virus replication, cytolytic activity in cell culture, or in vivo virotherpeutic efficacy. GLV-1h99-mediated expression of the hNET protein in infected cells resulted in specific uptake of the radiotracer [131I]-meta-iodobenzylguanidine (MIBG). In mice, GLV-1h99-infected tumors were readily imaged by [124I]-MIBG positron emission tomography. To our knowledge, GLV-1h99 is the first oncolytic virus expressing the hNET protein that can efficiently eliminate tumors and simultaneously allow deep-tissue imaging of infected tumors.

Colonization of experimental murine breast tumours by Escherichia coli K-12 significantly alters the tumour microenvironment.

The successful application of live bacteria in cancer therapy requires a more detailed understanding of bacterial interaction with the tumour microenvironment. Here, we analysed the effect of Escherichia coli K-12 colonization on the tumour microenvironment by immunohistochemistry and fluorescence microscopy in the murine 4T1 breast carcinoma model. We described the colonization of tumour-bearing mice, as well as the spatiotemporal distribution of E. coli K-12 in the 4T1 tumour tissue over a period of 14 days. The colonization resulted within 3 days in large avascular necrotic tissue, redistribution of hypoxic areas and an enhanced collagen IV deposition within the colonized tumour tissue, which changed the tumoral perfusion of systemically injected immunoglobulins. In addition, E. coli K-12 colonization led to the redistribution of tumour-associated macrophages, forming a granulation tissue around bacterial colonies, and also to an increase in TNFalpha and matrix metalloproteinase 9 expression. Colonization of 4T1 tumours by E. coli K-12 resulted in strong reduction of pulmonary metastatic events. These new insights will contribute to the general understanding of the tumour-microbe cross-talk and to the design of bacterial strains with enhanced anticancer efficiency.

Escherichia coli Nissle 1917 facilitates tumor detection by positron emission tomography and optical imaging.

PURPOSE: Bacteria-based tumor-targeted therapy is a modality of growing interest in anticancer strategies. Imaging bacteria specifically targeting and replicating within tumors using radiotracer techniques and optical imaging can provide confirmation of successful colonization of malignant tissue. EXPERIMENTAL DESIGN: The uptake of radiolabeled pyrimidine nucleoside analogues and [18F]FDG by Escherichia coli Nissle 1917 (EcN) was assessed both in vitro and in vivo. The targeting of EcN to 4T1 breast tumors was monitored by positron emission tomography (PET) and optical imaging. The accumulation of radiotracer in the tumors was correlated with the number of bacteria. Optical imaging based on bioluminescence was done using EcN bacteria that encode luciferase genes under the control of an l-arabinose-inducible P(BAD) promoter system. RESULTS: We showed that EcN can be detected using radiolabeled pyrimidine nucleoside analogues, [18F]FDG and PET. Importantly, this imaging paradigm does not require transformation of the bacterium with a reporter gene. Imaging with [18F]FDG provided lower contrast than [18F]FEAU due to high FDG accumulation in control (nontreated) tumors and surrounding tissues. A linear correlation was shown between the number of viable bacteria in tumors and the accumulation of [18F]FEAU, but not [18F]FDG. The presence of EcN was also confirmed by bioluminescence imaging. CONCLUSION: EcN can be imaged by PET, based on the expression of endogenous E. coli thymidine kinase, and this imaging paradigm could be translated to patient studies for the detection of solid tumors. Bioluminescence imaging provides a low-cost alternative to PET imaging in small animals.

Listeria monocytogenes as novel carrier system for the development of live vaccines.

Listeria monocytogenes is a facultative intracellular bacterium that enters a variety of non-professional mammalian cells by triggered phagocytosis ("zipper mechanism") and replicates in the cytosol of the infected host cells. Therefore, it is a promising vaccine vector for the presentation of passenger antigens to the MHC class II and especially class I pathways. Here, we review recent progress made in our laboratory on the development of novel attenuated L. monocytogenes carrier strains for the delivery of heterologous antigens or antigen-encoding DNA and RNA to eukaryotic host cells. Based on the deletion of the chromosomal copy of the tryptophanyl-tRNA synthetase gene (trpS) and plasmid-based in trans complementation of the same, we were able to establish a balanced-lethal plasmid system in L. monocytogenes. Safety concerns in the antigen delivery in vivo were addressed by chromosomal deletion of genes in the basic branch of the aromatic amino acid pathway, resulting in safe, attenuated L. monocytogenes carrier strains. Furthermore, plasmid-based expression of a cytosolically expressed phage lysin resulted in a self-destructing carrier strain that has been successfully used for the delivery of antigens as well as antigen-encoding plasmid DNA and particularly mRNA, therefore overcoming bottlenecks that have been shown to exist for bacteria-mediated DNA delivery.

Prodrug converting enzyme gene delivery by L. monocytogenes.

BACKGROUND: Listeria monocytogenes is a highly versatile bacterial carrier system for introducing protein, DNA and RNA into mammalian cells. The delivery of tumor antigens with the help of this carrier into tumor-bearing animals has been successfully carried out previously and it was recently reported that L. monocytogenes is able to colonize and replicate within solid tumors after local or even systemic injection. METHODS: Here we report on the delivery of two prodrug converting enzymes, purine-deoxynucleoside phosphorylase (PNP) and a fusion protein consisting of yeast cytosine deaminase and uracil phosphoribosyl transferase (FCU1) into cancer cells in culture by L. monocytogenes. Transfer of the prodrug converting enzymes was achieved by bacterium mediated transfer of eukaryotic expression plasmids or by secretion of the proteins directly into the host cell cytosol by the infecting bacteria. RESULTS: The results indicate that conversion of appropriate prodrugs to toxic drugs in the cancer cells occured after both procedures although L. monocytogenes-mediated bactofection proved to be more efficient than enzyme secretion 4T1, B16 and COS-1 tumor cells. Exchanging the constitutively PCMV-promoter with the melanoma specific P4xTETP-promoter resulted in melanoma cell-specific expression of the prodrug converting enzymes but reduced the efficiencies. CONCLUSION: These experiments open the way for bacterium mediated tumor specific activation of prodrugs in live animals with tumors.

Use of GLV-1h68 for Vaccinia Virotherapy and Monitoring.

Herein we describe the use of the vaccinia virus strain GLV-1h68 as a theragnostic agent in cancer models. To date, GLV-1h68 has been used successfully in more than 50 xenograft tumor models. The recombinant vaccinia virus strain has been equipped with heterologous expression cassettes for a luciferase-fluorescent protein fusion gene, bacterial beta-galactosidase, as well as a bacterial glucuronidase. The methods to investigate and monitor GLV-1h68 mediated virotherapy, including optical imaging and biomarker analysis, will be presented in detail.

Doxycycline Inducible Melanogenic Vaccinia Virus as Theranostic Anti-Cancer Agent.

We reported earlier the diagnostic potential of a melanogenic vaccinia virus based system in magnetic resonance (MRI) and optoacoustic deep tissue imaging (MSOT). Since melanin overproduction lead to attenuated virus replication, we constructed a novel recombinant vaccinia virus strain (rVACV), GLV-1h462, which expressed the key enzyme of melanogenesis (tyrosinase) under the control of an inducible promoter-system. In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models. Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography. At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system.

Drug-Encoded Biomarkers for Monitoring Biological Therapies.

Blood tests are necessary, easy-to-perform and low-cost alternatives for monitoring of oncolytic virotherapy and other biological therapies in translational research. Here we assessed three candidate proteins with the potential to be used as biomarkers in biological fluids: two glucuronidases from E. coli (GusA) and Staphylococcus sp. RLH1 (GusPlus), and the luciferase from Gaussia princeps (GLuc). The three genes encoding these proteins were inserted individually into vaccinia virus GLV-1h68 genome under the control of an identical promoter. The three resulting recombinant viruses were used to infect tumor cells in cultures and human tumor xenografts in nude mice. In contrast to the actively secreted GLuc, the cytoplasmic glucuronidases GusA and GusPlus were released into the supernatants only as a result of virus-mediated oncolysis. GusPlus resulted in the most sensitive detection of enzyme activity under controlled assay conditions in samples containing as little as 1 pg/ml of GusPlus, followed by GusA (25 pg/ml) and GLuc (≥375 pg/ml). Unexpectedly, even though GusA had a lower specific activity compared to GusPlus, the substrate conversion in the serum of tumor-bearing mice injected with the GusA-encoding virus strains was substantially higher than that of GusPlus. This was attributed to a 3.2 fold and 16.2 fold longer half-life of GusA in the blood stream compared to GusPlus and GLuc respectively, thus a more sensitive monitor of virus replication than the other two enzymes. Due to the good correlation between enzymatic activity of expressed marker gene and virus titer, we conclude that the amount of the biomarker protein in the body fluid semiquantitatively represents the amount of virus in the infected tumors which was confirmed by low light imaging. We found GusA to be the most reliable biomarker for monitoring oncolytic virotherapy among the three tested markers.

PET imaging with [68Ga]NOTA-RGD for prostate cancer: a comparative study with [¹8F]fluorodeoxyglucose and [¹8F]fluoroethylcholine.

The α(v)ß3 integrin is highly expressed in prostate cancer (PCa), in which it is a key player in tumour invasion, angiogenesis and metastasis formation. Therefore, α(v)ß3 integrin is considered a very promising target for molecular imaging of PCa. This study tested the potential of the novel α(v)ß3 integrin affine agent [68Ga]NOTA-RGD in comparison with the established [¹8F]fluoroethylcholine (FEC) and [¹8F]fluorodeoxyglucose (FDG) for assessing PCa using positron emission tomography (PET). [68Ga]NOTA-RGD showed a lower uptake in PC-3 and DU-145 cells compared with FEC and FDG. µPET imaging studies showed a good delineation of the PCa xenografts in mice. The means tumor-to-muscle and tumor-to-bone-ratio amounted 5.1 ± 1.4 and 5.2 ± 1.2 for [68Ga]NOTA-RGD compared with 2.6 ± 0.9 and 2.9 ± 1.6 for FDG, and 2.4 ± 0.7 and 0.8 ± 0.2 for FEC, respectively. The uptake of [68Ga]NOTA-RGD into tumor was fully inhibited by c(RGDfV), known to bind specifically to α(v)ß3 integrin, confirming the specificity of the tumor uptake in vivo. These results suggest that [68Ga]NOTA-RGD is a promising candidate for PET imaging of α(v)ß3 integrin expression in PCa and warrant further in vivo validations to ascertain its potential as an imaging agent for clinical use. The simple and fast preparation of [68Ga]NOTA-RGD may greatly facilitate its translation to a clinical setting.