Fine‐tuning of the automated [18F]PSMA‐1007 radiosynthesis

Radiolabeled prostate‐specific membrane antigen (PSMA) targeting PET‐tracers have become desirable radiopharmaceuticals for the imaging of prostate cancer (PC). Recently, the PET radiotracer [¹⁸F]PSMA‐1007 was introduced as an alternative to [⁶⁸Ga]Ga‐PSMA‐11, for staging and diagnosing biochemically recurrent PC. We incorporated a one‐step procedure for [¹⁸F]PSMA‐1007 radiosynthesis, using both Synthra RNplus and GE TRACERlab FxFN automated modules, in accordance with the recently described radiolabeling procedure. Although the adapted [¹⁸F]PSMA‐1007 synthesis resulted in repeatable radiochemical yields (55 ± 5%, NDC), suboptimal radiochemical purities of 87 ± 8% were obtained using both modules. As described here, modifications made to the radiolabeling and the solid‐phase extraction purification steps reduced synthesis time to 32 minutes and improved radiochemical purity to 96.10%, using both modules, without shearing the radiochemical yield.     

Preclinical assessment of 68Ga‐PSMA‐617 entrapped in a microemulsion delivery system for applications in prostate cancer PET/CT imaging

It has in recent years been reported that microemulsion (ME) delivery systems provide an opportunity to improve the efficacy of a therapeutic agent whilst minimising side effects and also offer the advantage of favourable treatment regimens. The prostate‐specific membrane antigen (PSMA) targeting agents PSMA‐11 and PSMA‐617, which accumulate in prostate tumours, allow for [⁶⁸Ga]Ga³⁺‐radiolabelling and positron emission tomography/computed tomography (PET) imaging of PSMA expression in vivo. We herein report the formulation of [⁶⁸Ga]Ga‐PSMA‐617 into a ME ≤40 nm including its evaluation for improved cellular toxicity and in vivo biodistribution. The [⁶⁸Ga]Ga‐PSMA‐617‐ME was tested in vitro for its cytotoxicity to HEK293 and PC3 cells. [⁶⁸Ga]Ga‐PSMA‐617‐ME was administered intravenously in BALB/c mice followed by microPET/computed tomography (CT) imaging and ex vivo biodistribution determination. [⁶⁸Ga]Ga‐PSMA‐617‐ME indicated negligible cellular toxicity at different concentrations. A statistically higher tolerance towards the [⁶⁸Ga]Ga‐PSMA‐617‐ME occurred at 0.125 mg/mL by HEK293 cells compared with PC3 cells. The biodistribution in wild‐type BALB/C mice showed the highest amounts of radioactivity (%ID/g) presented in the kidneys (31%) followed by the small intestine (10%) and stomach (9%); the lowest uptake was seen in the brain (0.5%). The incorporation of [⁶⁸Ga]Ga‐PSMA‐617 into ME was successfully demonstrated and resulted in a stable nontoxic formulation as evaluated by in vitro and in vivo means.     

Preparation of the alpha-emitting prostate-specific membrane antigen targeted radioligand [212Pb]Pb-NG001 for prostate cancer

Prostate-specific membrane antigen (PSMA) is the most promising target for radioligand therapy of prostate cancer. The aim of this study was to prepare a small molecular ligand p-SCN-Bn-TCMC-PSMA (NG001) and compare it with the commonly used DOTA-based PSMA-617. The PSMA-targeting ability of the ²¹²Pb-labelled ligands was evaluated using PSMA-positive C4-2 human prostate cancer cells. Lead-212 is an in vivo generator of alpha particles by its daughter nuclides ²¹²Bi and ²¹²Po. NG001 was synthesized by conjugating the isothiocyanato group of p-SCN-Bn-TCMC to the amino group of a glutamate-urea-based PSMA-binding entity. Molecular size, chelator unit and chelator linking method are different in NG001 and PSMA-617. Both ligands were efficiently labelled with ²¹²Pb using a ²²⁴Ra/²¹²Pb-solution generator in transient equilibrium with progeny. Lead-212-labelled NG001 was purified with a yield of 85.9±4.7% and with 0.7±0.2% of ²²⁴Ra. Compared with [²¹²Pb]Pb-PSMA-617, [²¹²Pb]Pb-NG001 displayed a similar binding and internalization in C4-2 cells, with comparable tumour uptake in mice bearing C4-2 tumours, but almost a 2.5-fold lower kidney uptake. Due to the rapid normal tissue clearance and tumour cell internalization, any significant translocalization of ²¹²Bi was not detected in mice. In conclusion, the obtained results warrant further preclinical studies to evaluate the therapeutic efficacy of [²¹²Pb]Pb-NG001.     

Prospects of medium specific activity 177Lu in targeted therapy of prostate cancer using 177Lu‐labeled PSMA inhibitor

Targeted radionuclide therapy using ¹⁷⁷Lu‐labeled peptidomimetic inhibitor of prostate specific membrane antigen (PSMA) viz. PSMA‐617 is emerging as one the most effective strategies for management of metastatic prostate cancer, which is one of the leading causes of cancer related death. The aim of the present study is to develop a robust and easily adaptable protocol for formulation of therapeutic dose of ¹⁷⁷Lu‐PSMA‐617 at hospital radiopharmacy using moderate specific activity ¹⁷⁷Lu available at an affordable cost. Extensive radiochemical studies were performed to optimize the required [PSMA‐617] / [Lu] ratio and other parameters to formulate 7.4 GBq dose of ¹⁷⁷Lu‐PSMA‐617. Based on these, 7.4 GBq therapeutic dose of ¹⁷⁷Lu‐PSMA‐617 was formulated by incubating 160 µg of PSMA‐617 with indigenously produced ¹⁷⁷LuCl₃ (555 GBq/µg specific activity of ¹⁷⁷Lu) at 90 °C for 30 min. The radiochemical purity of the formulation was 98.3 ± 0.6% (n = 7) which was retained to the extent of >95% after 7 d in normal saline at room temperature and >96% after 2 d in human serum at 37 °C. Preliminary clinical studies showed specific targeting of the agent in the lesion sites and similar physiological distribution as in diagnostic ⁶⁸Ga‐PSMA‐11 PET scans performed earlier. The developed optimized protocol for formulating therapeutic dose of ¹⁷⁷Lu‐PSMA‐617 could be useful for large number of nuclear medicine therapy clinics across the world having access to moderate specific activity ¹⁷⁷Lu at an affordable cost.     

Dual‐time‐point 64Cu‐PSMA‐617‐PET/CT in patients suffering from prostate cancer

Regardless of its high positron energy, ⁶⁸Ga‐labeled PSMA ligands have become standard of care in metabolic prostate cancer imaging. ⁶⁴Cu, a radionuclide with a much longer half‐life (12.7 h), is available for PSMA labeling allowing imaging much later than ⁶⁸Ga. In this study, the diagnostic performance of ⁶⁴Cu‐labeled PSMA was compared between early and late scans. Sixteen men (median age: 70 y) with prostate cancer in different stages underwent ⁶⁴Cu‐PSMA‐617‐PET/CT 2 and 22 hours post tracer injection. Pathologic and physiologic uptakes were analyzed for both points of time. Pathologic tracer accumulations occurred in 12 patients. Five patients presented with pathologic uptake in 17 different lymph nodes, two patients showed pathologic bone uptake in nine lesions, and seven patients had pathologic PSMA uptake in eight prostatic lesions. Physiologic uptake of the renal parenchyma, urine bladder, and salivary glands decreased over time, while the physiologic uptake of liver and bowel increased. In the present study, ⁶⁴Cu‐PSMA‐617‐PET demonstrated to be feasible for imaging prostate cancer for both the primary tumor site and metastases. Later imaging showed no additional, clinically relevant benefit compared with the early scans. At least the investigated time points we chose did not vindicate the additional expenditure.     

Development of polymeric microbubbles targeted to prostate-specific membrane antigen as prototype of novel ultrasound contrast agents

Ultrasound-targeted microbubbles (MBs) offer new opportunities to enhance the capabilities of diagnostic ultrasound (US) imaging to specific pathological tissue. Herein, we report on the design and development of a novel prototype of US contrast agent based on polymeric MBs targeted to prostate-specific membrane antigen (PSMA) for use in the diagnosis of prostate cancer (PCa). First, a set of air-filled MBs by a variety of biocompatible polymers were prepared and characterized in terms of morphology and echogenic properties after exposure to US. MBs derived from poly(D,L-lactic-co-glycolic acid) (PLGA)-poly(ethylene glycol) (PEG) copolymer resulted as the most effective in terms of reflectivity. Such polymer was therefore preconjugated with a urea-based PSMA inhibitor molecular probe (DCL), and the obtained MBs were investigated in vitro for their targeting efficacy toward PSMA positive PCa (LNCaP) cells. Fluorescence microscopy proved a specific and efficient adhesion of targeted MBs to LNCaP cells. To our knowledge, this work reports the first model of polymeric MBs appropriately engineered to target PSMA, which might be further optimized and used for PCa diagnosis and potential carriers for selective drug delivery.     

Bispecific radioligands targeting prostate‐specific membrane antigen and gastrin‐releasing peptide receptors on the surface of prostate cancer cells

Metastases of prostate cancer usually show highly heterogeneous or partly lost prostate‐specific membrane antigen (PSMA) expression. In order to image and treat both PSMA positive and negative tissues, the extension of PSMA tracer specificity to other receptors, also highly expressed on the surface of prostate cancer cells, has been suggested. Prostate cancer cells usually express both PSMA and gastrin‐releasing peptide (GRP) receptors; thus, bispecific heterodimeric molecules, addressing both targets at the same time, may significantly improve prostate cancer imaging and therapy. This article summarizes preclinical data regarding low‐molecular weight molecules targeting PSMA and GRP receptors.     

Targeting novel antigens for prostate cancer treatment: focus on prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a relatively omnipresent, but unique Type II dimeric transmembrane protein with a multiplicity of functions and has been shown to be a reasonable target for immunological approaches such as vaccines or more directed therapy with radioactively labelled monoclonal antibodies against PSMA. Given the abundance of various glycoprotein and carbohydrate antigens expressed on the surface of prostate cancer cells and cell lines, PSMA stands out as another 'self' antigen which is not only expressed on cancer cells, but on neovasculature. Although vaccines are varied in their design and target goal, recent technology has afforded researchers the opportunity to induce recruitment of multiple effector cell populations, cytokines and factors which can elicit both cellular and humoral responses. This review serves to present unique approaches in vaccine development which can induce immunological responsiveness with potential impact on disease progression and to introduce PSMA as a potential target for multimodality therapies.     

Targeting novel antigens for prostate cancer treatment: focus on prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a relatively omnipresent, but unique Type II dimeric transmembrane protein with a multiplicity of functions and has been shown to be a reasonable target for immunological approaches such as vaccines or more directed therapy with radioactively labelled monoclonal antibodies against PSMA. Given the abundance of various glycoprotein and carbohydrate antigens expressed on the surface of prostate cancer cells and cell lines, PSMA stands out as another ‘self’ antigen which is not only expressed on cancer cells, but on neovasculature. Although vaccines are varied in their design and target goal, recent technology has afforded researchers the opportunity to induce recruitment of multiple effector cell populations, cytokines and factors which can elicit both cellular and humoral responses. This review serves to present unique approaches in vaccine development which can induce immunological responsiveness with potential impact on disease progression and to introduce PSMA as a poten-tial target for multimodality therapies.     

The utility of radiolabeled PSMA ligands for tumor imaging

Prostate-specific membrane antigen (PSMA) is a glycosylated type-II transmembrane protein expressed in prostatic tissue and significantly overexpressed in several prostate cancer cells. Despite its name, PSMA has also been reported to be overexpressed in endothelial cells of benign and malignant non-prostate disease. So its clinical use was extended to detection, staging, and therapy of various tumor types. Recently small molecules targeting PSMA have been developed as imaging probes for diagnosis of several malignancies. Preliminary studies are emerging improved diagnostic sensitivity and specificity of PSMA imaging, leading to a change in patient management. In this review, we evaluated the first preclinical and clinical studies on PSMA ligands resulting future perspectives radiolabeled PSMA in staging and molecular characterization, based on histopathologic examinations of PSMA expression.     

GCPII imaging and cancer

Glutamate carboxypeptidase II (GCPII) in the central nervous system is referred to as the prostate-specific membrane antigen (PSMA) in the periphery. PSMA serves as a target for imaging and treatment of prostate cancer and because of its expression in solid tumor neovasculature has the potential to be used in this regard for other malignancies as well. An overview of GCPII/PSMA in cancer, as well as a discussion of imaging and therapy of prostate cancer using a wide variety of PSMA-targeting agents is provided.     

Targeted biocompatible nanoparticles for the delivery of (-)-epigallocatechin 3-gallate to prostate cancer cells

Molecular targeted cancer therapy mediated by nanoparticles (NPs) is a promising strategy to overcome the lack of specificity of conventional chemotherapeutic agents. In this context, the prostate-specific membrane antigen (PSMA) has demonstrated a powerful potential for the management of prostate cancer (PCa). Cancer chemoprevention by phytochemicals is emerging as a suitable approach for the treatment of early carcinogenic processes. Since (-)-epigallocatechin 3-gallate (EGCG) has shown potent chemopreventive efficacy for PCa, we designed and developed novel targeted NPs in order to selectively deliver EGCG to cancer cells. Herein, to explore the recent concept of "nanochemoprevention", we present a study on EGCG-loaded NPs consisting of biocompatible polymers, functionalized with small molecules targeting PSMA, that exhibited a selective in vitro efficacy against PSMA-expressing PCa cells. This approach could be beneficial for high risk patients and would fulfill a significant therapeutic need, thus opening new perspectives for novel and effective treatment for PCa.     

Establishment and characterization of a new human prostatic cancer cell line: DuCaP

BACKGROUND: The lack of appropriate, clinically relevant, cell-based model systems has limited prostate cancer research and the development of new therapeutic modalities. Here we report the isolation and characterization of a new adherent prostate cancer cell line, derived from the dura mater of a cancer patient. METHODS: Prostate cancer tissue was harvested at autopsy from a metastatic lesion to the dura mater of a patient with hormone refractory prostate cancer. This tissue was xenografted into SCID mice and later harvested and plated on tissue culture dishes. For characterization, soft agar clonegenic assay, in vivo xenograft growth, in vitro doubling time, karyotype analysis, immunocytochemistry for cytokeratin-18, androgen receptor, and PAP (prostatic acid phosphatase) expression, RT PCR for PAP, PSMA (prostate specific membrane antigen), expression and northern and western blot analysis to determine expression of Rb and p53, were performed. RESULTS: DuCap grows in vitro (passage 55), forms colonies in soft agar, produces tumors in SCID mice (xenograft passage 12), and is androgen sensitive. DNA content was hypertriploid. PSA was detected in mouse serum and media. Cells were AR, PAP and cytokeratin-18 positive by immunocytochemistry. PSMA and PAP were detected by RT-PCR. AR, P53, and Rb were expressed in Northern blot analysis. P53 protein was detected in Western blot analysis but Rb protein was not. CONCLUSIONS: This cell line exhibits many phenotypic characteristics of clinical prostate carcinoma, including expression of PSA, PSMA, PAP and AR.     

嵌合抗原受体修饰T细胞治疗前列腺癌的研究进展

摘要：嵌合抗原受体修饰T细胞（CAR-T）是近年来免疫治疗的热点,在血液系统疾病的治疗中取得了显著的临床疗效。目前,CAR-T在前列腺癌（PCa）治疗的研究中也取得了较大进展,其中以前列腺特异性膜抗原（PSMA）和前列腺干细胞抗原（PSCA）为靶点的CAR-T治疗进展较快,其可显著抑制肿瘤细胞生长,一定程度地延缓疾病进展。本文综述了前列腺肿瘤相关抗原（TAA）在前列腺癌CAR-T治疗中的最新研究进展,并对CAR-T在前列腺癌治疗中的挑战予以简要探讨,以期为临床提供参考。     

Targeting of biotinylated oligonucleotides to prostate tumors with antibody-based delivery vehicles

Specific delivery of therapeutic agents to tumor sites remains a problem and requires a nontoxic carrier able to bind a specific tumor cell marker. Although antibodies have been utilized as protein carriers for different types of drugs, they have not been employed for delivery of antisense oligonucleotides (ODNs). In this study, we modified monoclonal antibodies to target biotinylated ODNs to prostatic tumors which express prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA). Modified antibodies retained immunoreactivity and demonstrated an ability to form complexes with ODNs. Biodistribution of (35)S-oligonucleotide-antibody complexes was also examined in nude mice bearing human derived LNCaP prostatic tumors. Delivering ODNs with modified prostate specific antibodies produced greater tissue uptake and higher blood levels with both PSA and PSMA directed antibodies. While tumor uptake was not significantly improved after 24 h, the overall results of this study provide some additional insights into the complexity of the antibody-mediated delivery of ODNs in vivo. The strategies employed here for the selective delivery of ODNs warrant further investigation.     

Targeting of biotinylated oligonucleotides to prostate tumors with antibody-based delivery vehicles

Specific delivery of therapeutic agents to tumor sites remains a problem and requires a nontoxic carrier able to bind a specific tumor cell marker. Although antibodies have been utilized as protein carriers for different types of drugs, they have not been employed for delivery of antisense oligonucleotides (ODNs). In this study, we modified monoclonal antibodies to target biotinylated ODNs to prostatic tumors which express prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA). Modified antibodies retained immunoreactivity and demonstrated an ability to form complexes with ODNs. Biodistribution of ³⁵S-oligonucleotide–antibody complexes was also examined in nude mice bearing human derived LNCaP prostatic tumors.

Delivering ODNs with modified prostate specific antibodies produced greater tissue uptake and higher blood levels with both PSA and PSMA directed antibodies. While tumor uptake was not significantly improved after 24 h, the overall results of this study provide some additional insights into the complexity of the antibody-mediated delivery of ODNs in vivo. The strategies employed here for the selective delivery of ODNs warrant further investigation.     

Development of a high-performance liquid chromatography method for rapid radiochemical purity measurement of [18F]PSMA-1007, a PET radiopharmaceutical for detection of prostate cancer

Since first becoming commercially available in 2018, the PET radiopharmaceutical [¹⁸F]PSMA-1007 has been used widely for the diagnosis and staging of prostate cancer. A pharmacopoeia monograph first became available in 2021, prescribing a radiochemical purity specification of >91%, based on analytical results from both TLC (for [¹⁸F]fluoride impurity alone) and HPLC (for all other ¹⁸F-impurities). Though this monograph has provided clarity for the quality control testing of [¹⁸F]PSMA-1007, it prescribes a HPLC method using phosphate buffer mobile phase that may present a risk of precipitation of phosphate salts in the HPLC system. The method also requires specialised hardware not immediately available to all laboratories. This work describes the development of a simple, rapid reversed-phase HPLC method utilising 0.1 M ammonium formate mobile phase for the accurate assessment of both [¹⁸F]fluoride impurity and overall radiochemical purity in a single test. This method is especially useful for assessment of product stability over time. A more accurate TLC method for [¹⁸F]fluoride impurity is also described.     

前列腺特异膜抗原基因的表达

目的克隆前列腺特异膜抗原（PSMA）基因的编码区序列,构建PSMA真核表达载体。方法用RT．PCR方法扩增前列腺癌组织标本中的PSMAeDNA序列,并将其克隆至真核表达载体pcDNA3．0。用脂质体转染法,把pcDNA3．0-PSMA转染哺乳动物细胞,鉴定PSMA蛋白的表达。结果两条引物之间的片断长度为2279bp,与预期长度一致。将克隆的编码区序列与Genebank的PSMA序列同源性为99．7％;表达的蛋白质为膜蛋白,免疫印迹表明表达蛋白质的相对分子质量为100kD。结论扩增PSMA编码区序列,可构建PSMA真核表达载体,建立PSMA稳定表达的细胞株。PSMA具有良好的抗原性。     

Mass spectral analyses of labile DOTA-NHS and heterogeneity determination of DOTA or DM1 conjugated anti-PSMA antibody for prostate cancer therapy

Prostate specific membrane antigen (PSMA) is a well-characterized glycoprotein overexpressed on the surface of prostate cancer cells. The novel radiopharmaceutical 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid (DOTA) radiolabeled with Yttrium (90Y) or Indium (111In) conjugated with anti-PSMA genetically engineered humanized monoclonal antibody (huJ591) has been investigated to target prostate cancer cells. The immunoconjugate of huJ591 with the analog of the cytotoxic drug maytansine, DM1 (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine) has also been developed at Millennium Pharmaceuticals. Activation of the DOTA molecule, resulting in 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid mono-(N-hydroxysuccinimidyl) ester (DOTA-NHS), allows conjugation with the anti-PSMA antibody through lysine residues in the antibody. The objectives of the study were to characterize the unstable chemical properties of DOTA-NHS before bioconjugation with huJ591, evaluate the binding profiles of DOTA to huJ591, and calculate trace metal elements (which may disturb 90Y or 111In labeling efficacy to the DOTA-huJ591 conjugate). A novel LC/MS/MS (Liquid Chromatography/Mass Spectrometry/Mass Spectrometry) quantitation method was developed to monitor the stability of DOTA-NHS in solid form and its bioconjugation chemistry reactions. Meanwhile, metal analysis was quantified by Inductively Coupled Plasma Mass Spectrometry (ICP/MS) to estimate the amounts of trace metals in DOTA-NHS and ensure radiolabeling efficiency of the conjugate at the radiopharmacy. MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry) was used to identify levels of DOTA or DM1conjugation in DOTA-huJ591 and DM1-huJ591 conjugates, respectively.