Prostate-specific Membrane Antigen Protein Research Articles

Phase 1 clinical trial of AMG 340, a prostate-specific membrane antigen (PSMA)-targeted T-cell engager with a novel low-affinity CD3 binding domain designed to mitigate toxicity for the treatment of metastatic castration-resistant prostate cancer (mCRPC).

e14587 Background: PSMA, a transmembrane protein highly expressed on the surface of prostate cancer cells, is a validated target in prostate cancer; however, achieving a favorable therapeutic index has been challenging for PSMA-directed T-cell engagers (TCE’s). AMG 340, a PSMA-targeted TCE with a novel low-affinity CD3 binding domain specifically engineered to mitigate cytokine release syndrome (CRS) toxicity and maintain efficacy was investigated in a phase 1, open-label, dose-escalation study (NCT04740034). Methods: Patients (pts) with mCRPC who had received ≥2 prior lines of therapy were treated with AMG 340 monotherapy (1.5–800 mg intravenously every 3 weeks [Q3W]) during dose escalation. The primary outcome measures were dose-limiting toxicities, treatment-emergent adverse events (TEAEs), and pharmacokinetic profile. Secondary outcomes included prostate antigen specific (PSA) response and objective response per response evaluation criteria in solid tumors (RECIST). Results: As of September 6, 2023, a total of 42 pts (mean ± SD age, 68.5±7.8 years) received AMG 340 step up dosing across 11 cohorts. At doses up to 800 mg Q3W, AMG 340 had a manageable side effect profile. Any grade TEAEs reported in ≥30% of pts included CRS (52%), nausea (52%), vomiting (52%), fatigue (48%), diarrhea (38%), increased aspartate aminotransferase (36%), increased alanine aminotransferase (33%), and hypocalcemia (31%). Treatment-related toxicities included CRS (52%; Grade 1, 26%; Grade 2, 24%; Grade ≥3, 2%) and thrombocytopenia (24%; Grade 2, 10%; Grade ≥3, 14%). Step dosing was implemented at doses >40 mg and successfully mitigated CRS incidence and severity after the first dose. The maximal tolerated dose was not reached, as increasing the dose beyond 800 mg was predicted to minimally increase the potential AMG 340 activity. Exposure increased in an approximately dose-proportional manner with no evidence of clinically significant anti-drug antibodies. AMG 340 demonstrated evidence for pharmacodynamic engagement as shown by cytokine induction. Of the 41 PSA evaluable pts, PSA responses were seen across dose levels (PSA50, n=4), but were not dose dependent. Eight of 42 pts remained on treatment after 6 months. Among 27 RECIST evaluable pts, no RECIST responses were observed (n=14 stable disease, n=0 partial or complete response). Conclusions: Despite reaching high doses, no dose response was observed, and AMG 340 did not generate sufficient clinical activity to warrant further exploration. However, AMG 340 demonstrated manageable CRS toxicity showing that decreased CD3-binding strength mitigates CRS, the main toxicity of TCEs. Clinical studies are ongoing evaluating the PSMA protein as a molecular target for prostate cancer. Clinical trial information: NCT04740034 .

Frequent Amplification and Overexpression of PSMA in Basallike Breast Cancer from Analysis of The Cancer Genome Atlas.

Prostate-specific membrane antigen (PSMA) is frequently overexpressed in nonprostate malignancies. This preclinical study investigated the molecular basis of the application of PMSA-targeting radiopharmaceuticals in breast cancer subtypes. Methods: The somatic copy number status and the transcriptomic and protein expressions of FOLH1 (gene name of PSMA) were analyzed across breast cancer subtypes in 998 patients from The Cancer Genome Atlas dataset. Results: FOLH1 was frequently amplified in basallike breast cancer (BLBC) (32%) compared with luminal and human epidermal growth factor receptor 2-positive subtypes (16% and 17%, respectively; P < 0.01). FOLH1 expression was higher in BLBC (P < 0.001) and was negatively correlated with estrogen-receptor and progesterone-receptor expressions. Consistently, the PSMA protein level was higher in BLBC (P < 0.05). Interestingly, FOLH1 expression was associated with relapse-free and distant metastasis-free survival in patients with BLBC. Conclusion: The BLBC subtype exhibited frequent amplification and overexpression of PSMA, supporting the exploration of PSMA-targeting radiopharmaceuticals in this aggressive breast cancer subtype.

Enhancing the anticancer effect of androgen deprivation therapy by monocarboxylate transporter 1 inhibitor in prostate cancer cells.

Tumor initiation and progression necessitate a metabolic shift in cancer cells. Consequently, the progression of prostate cancer (PCa), a leading cause of cancer-related deaths in males globally, involves a shift from lipogenic to glycolytic metabolism. Androgen deprivation therapy (ADT) serves as the standard treatment for advanced-stage PCa. However, despite initial patient responses, castrate resistance emerges ultimately, necessitating novel therapeutic approaches. Therefore, in this study, we aimed to investigate the role of monocarboxylate transporters (MCTs) in PCa post-ADT and evaluate their potential as therapeutic targets. PCa cells (LNCaP and C4-2 cell line), which has high prostate-specific membrane antigen (PSMA) and androgen receptor (AR) expression among PCa cell lines, was used in this study. We assessed the expression of MCT1 in PCa cells subjected to ADT using charcoal-stripped bovine serum (CSS)-containing medium or enzalutamide (ENZ). Furthermore, we evaluated the synergistic anticancer effects of combined treatment with ENZ and SR13800, an MCT1 inhibitor. Short-term ADT led to a significant upregulation in folate hydrolase 1 (FOLH1) and solute carrier family 16 member 1 (SLC16A1) gene levels, with elevated PSMA and MCT1 protein levels. Long-term ADT induced notable changes in cell morphology with further upregulation of FOLH1/PSMA and SLC16A1/MCT1 levels. Treatment with ENZ, a nonsteroidal anti-androgen, also increased PSMA and MCT1 expression. However, combined therapy with ENZ and SR13800 led to reduced PSMA level, decreased cell viability, and suppressed expression of cancer stem cell markers and migration indicators. Additionally, analysis of human PCa tissues revealed a positive correlation between PSMA and MCT1 expression in tumor regions. Our results demonstrate that ADT led to a significant upregulation in MCT1 levels. However, the combination of ENZ and SR13800 demonstrated a promising synergistic anticancer effect, highlighting a potential therapeutic significance for patients with PCa undergoing ADT.

Abstract 3696: Liquid biopsy biomarker analysis during treatment with 177Lu-PSMA-617 in castrate resistant prostate cancer

Abstract Background: Prostate-Specific Membrane Antigen (PSMA) is a highly expressed prostate cancer (PCa) cell surface protein regulated by androgen receptor signaling and the target of the recently approved radioligand therapy 177Lu-PSMA-617. While some patients have dramatic responses to this therapy, rates of upfront or acquired resistance are high even in patients with PSMA-PET-avid disease, and better biomarkers of 177Lu PSMA-617 response and resistance are needed to select patients more effectively for this therapy. To address this need, we evaluated the relationship between longitudinal circulating tumor cell (CTC) androgen signaling and PSMA expression to 177Lu-PSMA-617 response or resistance in patients with metastatic prostate cancer. We leveraged a CTC androgen signaling signature that we have previously shown is prognostic for patients with metastatic prostate cancer treated with AR signaling inhibitors. Methods: Longitudinal blood samples were collected from 20 patients for circulating tumor cell (CTC) analysis at C1D1, C2D1, C4D1, and C6D1. CTCs were isolated with antibodies to EpCAM followed by parallel enumeration, analysis of cell surface expression of PSMA and RNA extraction for AR or neuroendocrine marker transcriptional profiling. Results: CTC samples were assessed for PSMA cell surface protein expression prior to treatment and on treatment. FOLH1 (PSMA) gene expression was detected from CTCs at baseline that persisted through treatment. Gene expression representative of AR pathway activation was present in 70% of the cohort at baseline and 50% of patients at C2D1. Concordant with these observations, no significant decrease in CTC number or PSMA protein expression was seen from baseline to C2D1. However, increased CTC AR pathway activation at C2D1 was associated with increased mean serum PSA (155 vs 21.93 ng/ml) and was associated with decreased duration of response. Conclusions: Improved tools to predict who will respond to 177Lu-PSMA-617 and detect early treatment resistance and disease progression are needed. Here, we show that persistent CTC burden, CTC AR signaling, and CTC PSMA expression are common early in treatment with 177Lu-PSMA-617 and may be associated with lower likelihood of clinical response. Further ongoing studies are needed to elucidate the relationship between these liquid biopsy results and clinical outcomes. Citation Format: Jamie M. Sperger, Marina N. Sharifi, Luke Nunamaker, Viridiana Carreno, Alex H. Chang, Shannon R. Reese, Charlotte Linebarger, Amy K. Taylor, Grace C. Bliitzer, John Floberg, Shuang G. Zhao, Joshua M. Lang. Liquid biopsy biomarker analysis during treatment with 177Lu-PSMA-617 in castrate resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3696.

Exploring the role of combined external beam radiotherapy and targeted radioligand therapy with [177Lu]Lu-PSMA-617 for prostate cancer - from bench to bedside.

Management of prostate cancer (PC) might be improved by combining external beam radiotherapy (EBRT) and prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT) with lutetium-177 (177Lu)-labeled PSMA inhibitors. We hypothesized a higher efficacy of the combination due to augmentation of the radiation dose to the tumor and interactions of EBRT with PSMA expression potentially increasing radiopharmaceutical uptake. Therefore, this study analyzed the influence of radiation on PSMA expression levels in vitro. The results were translated to evaluate the efficacy of the combination of photon EBRT and [177Lu]Lu-PSMA-617 in a murine PC xenograft model. Finally, a clinical case report on a combined elective field EBRT with RLT dose escalation illustrates a proof-of-concept. Methods: PSMA gene and protein expression were assessed in human PSMA-overexpressing LNCaP cells after irradiation using reverse transcription quantitative polymerase chain reaction (RT-qPCR), flow cytometry and On-Cell Western assays. In the in vivo therapy study, LNCaP tumor-bearing BALB/c nu/nu mice were irradiated once with 2 Gy X-ray EBRT and injected with 40 MBq [177Lu]Lu-PSMA-617 after 4 h or received single or no treatment (n = 10 each). Tumor-absorbed doses by [177Lu]Lu-PSMA-617 were calculated according to the Medical Internal Radiation Dosimetry (MIRD) formalism after deriving time-activity curves using a gamma probe. An exemplified patient case is demonstrated where fractionated EBRT (54 Gy to prostate; 45 Gy to pelvic lymphatics) and three cycles of [177Lu]Lu-PSMA-617 (3.4-6.0 GBq per cycle) were sequentially combined under concurrent androgen deprivation for treating locally advanced PC. Results: At 4 h following irradiation with 2-8 Gy, LNCaP cells displayed a PSMA protein upregulation by around 18% relative to non-irradiated cells, and a stronger upregulation on mRNA level (up to 2.6-fold). This effect was reversed by 24 h when PSMA protein levels were downregulated by up to 22%. Mice treated with the combination therapy showed significantly improved outcomes regarding tumor control and median survival (p < 0.0001) as compared to single or no treatment. Relative to monotherapy with PSMA-RLT or EBRT, the tumor doubling time was prolonged 1.7- or 2.7-fold and the median survival was extended by 24% or 60% with the combination, respectively. Additionally, tumors treated with EBRT exhibited a 14% higher uptake of the radiopharmaceutical as evident from the calculated tumor-absorbed dose, albeit with high variability in the data. Concerning the patient case, the tri-modality treatment was well tolerated and the patient responded with a long-lasting complete biochemical remission for five years following end of PSMA-RLT. The patient then developed a biochemical relapse with oligo-recurrent disease on follow-up imaging. Conclusion: The present preclinical and clinical data demonstrate that the combination of EBRT with dose escalation by PSMA-RLT improves tumor control and potentially prolongs survival. This may pave the way for further clinical investigations of this approach to explore the curative potential of the combination therapy.

Tumoral Ki67 and PSMA Expression in Fresh Pre-PSMA-RLT Biopsies and Its Relation With PSMA-PET Imaging and Outcomes of PSMA-RLT in Patients With mCRPC.

Prostate specific membrane antigen (PSMA) directed radioligand therapy (RLT) is a novel therapy for metastatic castration-resistant prostate cancer (mCRPC) patients. However, it is still poorly understood why approximately 40% of the patients does not respond to PSMA-RLT. The aims of this study were to evaluate the pretreatment PSMA expression on immunohistochemistry (IHC) and PSMA uptake on PET/CT imaging in mCRPC patients who underwent PSMA-RLT. We correlated these parameters and a cell proliferation marker (Ki67) to the therapeutic efficacy of PSMA-RLT. In this retrospective study, mCRPC patients who underwent PSMA-RLT were analyzed. Patients biopsies were scored for immunohistochemical Ki67 expression, PSMA staining intensity and percentage of cells with PSMA expression. Moreover, the PSMA tracer uptake of the tumor lesion(s) and healthy organs on PET/CT imaging was assessed. The primary outcome was to evaluate the association between histological PSMA protein expression of tumor in pre-PSMA-RLT biopsies and the PSMA uptake on PSMA PET/CT imaging of the biopsied lesion. Secondary outcomes were to assess the relationship between PSMA expression and Ki67 on IHC and the progression free survival (PFS) and overall survival (OS) following PSMA-RLT. In total, 22 mCRPC patients were included in this study. Nineteen (86%) patients showed a high and homogenous PSMA expression of >80% on IHC. Three (14%) patients had low PSMA expression on IHC. Although there was limited PSMA uptake on PET/CT imaging, these 3 patients had lower PSMA uptake on PET/CT imaging compared to the patients with high PSMA expression on IHC. Yet, no correlation was found between PSMA uptake on PET/CT imaging and PSMA expression on IHC (SUVmax: R2=0.046 and SUVavg: R2=0.036). The 3 patients had a shorter PFS compared to the patients with high PSMA expression on IHC (HR: 4.76, 95% CI: 1.14-19.99; P=.033). Patients with low Ki67 expression had a longer PFS and OS compared to patients with a high Ki67 expression (HR: 0.40, 95% CI: 0.15-1.06; P=.013) CONCLUSION: The PSMA uptake on PSMA-PET/CT generally followed the PSMA expression on IHC. However, heterogeneity may be missed on PSMA-PET/CT. Immunohistochemical PSMA and Ki67 expression in fresh tumor biopsies, may contribute to predict treatment efficacy of PSMA-RLT in mCRPC patients. This needs to be further explored in prospective cohorts.

Improved Prostate-Specific Membrane Antigen (PSMA) Stimulation Using a Super Additive Effect of Dutasteride and Lovastatin In Vitro.

Prostate-specific membrane antigen (PSMA)-based imaging improved the detection of primary, recurrent and metastatic prostate cancer. However, in certain patients, a low PSMA surface expression can be a limitation for this promising diagnostic tool. Pharmacological induction of PSMA might be useful to further improve the detection rate of PSMA-based imaging. To achieve this, we tested dutasteride (Duta)-generally used for treatment of benign prostatic enlargement-and lovastatin (Lova)-a compound used to reduce blood lipid concentrations. We aimed to compare the individual effects of Duta and Lova on cell proliferation as well as PSMA expression. In addition, we tested if a combination treatment using lower concentrations of Duta and Lova can further induce PSMA expression. Our results show that a treatment with ≤1 μM Duta and ≥1 μM Lova lead to a significant upregulation of whole and cell surface PSMA expression in LNCaP, C4-2 and VCaP cells. Lower concentrations of Duta and Lova in combination (0.5 μM Duta + 0.5 μM Lova or 0.5 μM Duta + 1 μM Lova) were further capable of enhancing PSMA protein expression compared to a single compound treatment using higher concentrations in all tested cell lines (LNCaP, C4-2 and VCaP).

18F-DCFPyL (PSMA) PET as a radiotherapy response assessment tool in metastatic prostate cancer.

Prostate Specific Membrane Antigen (PSMA) - positron emission tomography (PET) guides metastasis-directed radiotherapy (MDRT) in prostate cancer (PrCa). However, its value as a treatment response assessment tool after MDRT remains unclear. Importantly, there is limited understanding of the potential of radiotherapy (RT) to alter PSMA gene (folate hydrolase 1; FOLH1) expression. We reviewed a series of 11 men with oligo-metastatic PrCa (25 metastasis sites) treated with MDRT before re-staging with 18F-DCFPyL (PSMA) PET upon secondary recurrence. Acute effects of RT on PSMA protein and mRNA levels were examined with qPCR and immunoblotting in human wild-type androgen-sensitive (LNCap), castrate-resistant (22RV1) and castrate-resistant neuroendocrine (PC3 and DU145) PrCa cell lines. Xenograft tumors were analyzed with immunohistochemistry. Further, we examined PSMA expression in untreated and irradiated radio-resistant (RR) 22RV1 (22RV1-RR) and DU145 (DU145-RR) cells and xenografts selected for survival after high-dose RT. The majority of MDRT-treated lesions showed lack of PSMA-PET/CT avidity, suggesting treatment response even after low biological effective dose (BED) MDRT. We observed similar high degree of heterogeneity of PSMA expression in both human specimens and in xenograft tumors. PSMA was highly expressed in LNCap and 22RV1 cells and tumors but not in the neuroendocrine PC3 and DU145 models. Single fraction RT caused detectable reduction in PSMA protein but not in mRNA levels in LNCap cells and did not significantly alter PSMA protein or mRNA levels in tissue culture or xenografts of the other cell lines. However, radio-resistant 22RV1-RR cells and tumors demonstrated marked decrease of PSMA transcript and protein expression over their parental counterparts. PSMA-PET may be a promising tool to assess RT response in oligo-metastatic PrCa. However, future systematic investigation of this concept should recognize the high degree of heterogeneity of PSMA expression within prostate tumors and the risk for loss of PSMA expression in tumor surviving curative courses of RT.

Consecutive Prostate-Specific Membrane Antigen (PSMA) and Antigen Receptor (AR) PET Imaging Shows Positive Correlation with AR and PSMA Protein Expression in Primary Hormone-Naïve Prostate Cancer.

The present study was carried out to investigate whether PET imaging can be used as a potential substitute for immunohistochemical analysis of tumor samples in prostate cancer (PC) patients. Correlation between imaging signals of 2 PET tracers and the corresponding target structures was assessed. The first tracer was [68Ga]Ga-PSMA (prostate-specific membrane antigen)-HBED-CC (N,N'-bis [2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid) [68Ga]Ga-PSMAHBED-CC ([68Ga]PSMA), which is already implemented in clinical routines. The second tracer was 16β-[18F]fluoro-5α-dihydrotestosterone (16β-[18F]FDHT), which binds to the androgen receptor (AR). The AR is particularly interesting in PC, because AR expression status and its shift during therapy might directly influence patient care. Methods: This prospective, explorative clinical study included 10 newly diagnosed PC patients. Each patient underwent [68Ga]PSMA PET/MRI and [18F]FDHT PET/MRI scans before prostatectomy. Cancer SUVs were determined and related to background SUVs. After prostatectomy, tumor tissue was sampled, and AR and prostate-specific membrane antigen (PSMA) expression was determined. AR and PSMA expression was evaluated quantitatively with the open-source bioimage analysis software QuPath and with a 4-tier rating system. Correlation between imaging signals and marker expression was statistically assessed. Results: For [18F]FDHT, the SUVmax/SUVbackground ratio showed a significant, strong correlation (r = 0.72; P = 0.019) with the AR optical density of the correlating tissue sample. The correlation between PSMA optical density and the [68Ga]PSMA SUVmax/SUVbackground ratio was not significant (P = 0.061), yet a positive correlation trend could be observed (r = 0.61). SUVmax/SUVbackground ratios were higher for [68Ga]PSMA (mean ± SD, 34.9 ± 24.8) than for [18F]FDHT (4.8 ± 1.2). In line with these findings, the tumor detection rates were 90% for the [68Ga]PSMA PET scan but only 40% for the [18F]FDHT PET scan. The 4-tier rating of PSMA staining intensity yielded very homogeneous results, with values of 3+ for most subjects (90%). AR staining was rated as 1+ in 2 patients (20%), 2+ in 4 patients (40%), and 3+ in 4 patients (40%). Conclusion: [18F]FDHT PET may be useful for monitoring AR expression and alterations in AR expression during treatment of PC patients. This approach may facilitate early detection of treatment resistance and allows for adaptation of therapy to prevent cancer progression. [18F]FDHT PET is inferior to [68Ga]PSMA PET for primary PC diagnosis, but the correlation between [68Ga]PSMA SUVs and PSMA expression is weaker than that between [18F]FDHT and the AR.

The current status of prostate cancer treatment and PSMA theranostics

In the treatment of cancer, understanding the disease status, or accurate staging, is extremely important, and various imaging techniques are used. Computed tomography (CT), magnetic resonance imaging, and scintigrams are commonly used for solid tumors, and advances in these technologies have improved the accuracy of diagnosis. In the clinical practice of prostate cancer, CT and bone scans have been considered especially important for detecting metastases. Nowadays, CT and bone scans are called conventional methods because positron emission tomography (PET), especially prostate-specific membrane antigen (PSMA)/PET, is extremely sensitive in detecting metastases. Advances in functional imaging, such as PET, are advancing the diagnosis of cancer by allowing information to be added to the morphological diagnosis. Furthermore, PSMA is known to be upregulated depending on the malignancy of the prostate cancer grade and resistance to therapy. Therefore, it is often highly expressed in castration-resistant prostate cancer (CRPC) with poor prognosis, and its therapeutic application has been attempted for around two decades. PSMA theranostics refers to a type of cancer treatment that combines both diagnosis and therapy using a PSMA. The theranostic approach uses a radioactive substance attached to a molecule that targets PSMA protein on cancer cells. This molecule is injected into the patient’s bloodstream and can be used for both imaging the cancer cells with a PET scan (PSMA PET imaging) and delivering radiation directly to the cancer cells (PSMA-targeted radioligand therapy), with the aim of minimizing damage to healthy tissue. Recently, in an international phase III trial, the impact of 177Lu-PSMA-617 therapy was studied in patients with advanced PSMA-positive metastatic CRPC who had previously been treated with specific inhibitors and regimens. The trial revealed that 177Lu-PSMA-617 significantly extended both progression-free survival and overall survival compared to standard care alone. Although there was a higher incidence of grade 3 or above adverse events with 177Lu-PSMA-617, it did not negatively impact the patients’ quality of life. PSMA theranostics is currently being studied and used primarily for the treatment of prostate cancer, but it has the potential to be applied to other types of cancers as well.

Investigating PSMA differential expression in canine uroepithelial carcinomas to aid disease-based stratification and guide therapeutic selection

BackgroundIn male dogs, uroepithelial cancers include invasive urothelial carcinoma (iUC) and prostate carcinoma (PCA). The inability to distinguish iUC involving the prostate from PCA results in indiscriminate clinical management strategies that could be suboptimal as first-line chemotherapy for iUC (cisplatin) and PCA (docetaxel) differ in people. Prostate specific membrane antigen (PSMA) is a transmembrane protein, and its overexpression has been identified in human prostate carcinoma and neovasculature associated with solid tumor growth. This study investigates whether differential PSMA expression exists between presumptive canine iUC and PCA among cell lines and archived patient samples, which might allow for improved accuracy in disease-based stratification and optimal chemotherapy selection. Additionally, in vitro sensitivities of reported canine iUC and PCA cell lines to uroepithelial directed chemotherapeutic agents were characterized.ResultsNormalized PSMA gene and protein expressions were not significantly different between 5 iUC and 4 PCA cell lines. PSMA protein expression was uniformly observed in uroepithelial cancers regardless of anatomic origin from archived patient samples, further confirming that PSMA cannot differentiate iUC from PCA. In vitro sensitivity of cell lines to uroepithelial directed chemotherapeutics revealed that vinblastine exerted the broadest cytotoxic activity.ConclusionsDifferential expression of PSMA was not identified between canine iUC and PCA cell lines or archived patient samples, and PSMA alone cannot be used for disease stratification. Nonetheless given its conserved overexpression, PSMA may be a targetable surface marker for both canine iUC and PCA. Lastly, in uroepithelial carcinomas, vinblastine might exert the broadest anticancer activity regardless of cellular origin.

Pharmacological Optimization of PSMA-Based Radioligand Therapy.

Prostate cancer (PCa) is the most common malignancy in men of middle and older age. The standard treatment strategy for PCa ranges from active surveillance in low-grade, localized PCa to radical prostatectomy, external beam radiation therapy, hormonal treatment and chemotherapy. Recently, the use of prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT) for metastatic castration-resistant PCa has been approved. PSMA is predominantly, but not exclusively, expressed on PCa cells. Because of its high expression in PCa, PSMA is a promising target for diagnostics and therapy. To understand the currently used RLT, knowledge about pharmacokinetics (PK) and pharmacodynamics (PD) of the PSMA ligand and the PSMA protein itself is crucial. PK and PD properties of the ligand and its target determine the duration and extent of the effect. Knowledge on the concentration-time profile, the target affinity and target abundance may help to predict the effect of RLT. Increased specific binding of radioligands to PSMA on PCa cells may be associated with better treatment response, where nonspecific binding may increase the risk of toxicity in healthy organs. Optimization of the radioligand, as well as synergistic effects of concomitant agents and an improved dosing strategy, may lead to more individualized treatment and better overall survival.

The oncological characteristics of non-prostate-specific membrane antigen (PSMA)-expressing primary prostate cancer on preoperative PSMA positron emission tomography/computed tomography.

The oncological characteristics of non-prostate-specific membrane antigen (PSMA)-expressing primary prostate cancer on preoperative PSMA positron emission tomography/computed tomography.

Abstract 301: Repurposing engineered thymoquinone nanoparticles to inhibit drug-resistant prostate cancer

Abstract Despite advanced treatment strategies and therapeutics for prostate cancer (PCa), the metastatic castration-resistant prostate cancer (mCRPC) mechanism remains obscure. Although docetaxel (DTX) and other anti-mitotic chemotherapeutic drug are considered the first line drugs for PCa patients; the resulting side effects and drug resistance increase the demand for an alternative such as natural compounds for effective treatment. However, the natural compounds that achieved remarkable height to overcome drug resistance, their bioavailability and targeted delivery during treatment has become a new challenge. As prostate-specific membrane antigen (PSMA) receptors are expressed on the surface of most PCa cells, we anticipated that these receptors may provide the potential target for effective therapy. Considering these, we employed the natural compound, thymoquinone (TQ)-encapsulated planetary ball-milled nanoparticles (PBM-NPs) formulated with starch (FDA approved, natural polysaccharides) inner core and biocompatible and biodegradable co-polymers poly (ε-caprolactone)/poly (ethylene glycol), functionalized with a PSMA aptamer (A10), which can selectively recognize and bind to PSMA membrane. The functionalities of this strategy were analyzed by targeting A10-TQ-PBM-NP in DTX-resistant cells (C4-2B-R and LNCaP-R), which overexpresses PSMA protein and multidrug resistance (MDR) gene ABCB1, and validated through flow cytometry, immunofluorescence, qRT-PCR, and western blot techniques. The results showed that the aptamer-based nano delivery of a TQ can modulate the cellular and molecular pathways responsible for DTX resistance in PCa cells through the EGFR and ZEB1 dependent mechanism. The A10-TQ-PBM-NPs counteract ATP-binding cassette (ABC)-transporter-mediated chemoresistance in PCa cells. In both cell lines, these NPs rapidly internalized and induced significant cell death at five times lower concentrations than non-conjugated PBM-TQ-NP. Further, A10-TQ-PBM-NPs have high selectivity to the PSMA receptor and downregulated the EGFR dependent pathway, Akt-1, ERK1/2, NF-kB, STAT3, and epithelial-mesenchymal transition (EMT) markers in both C4-2B-R and LNCaP-R cells, thus constraining cell survival, proliferation, and migration. The A10-TQ-PBM-NP bioconjugates also reveal remarkable efficacy and reduced toxicity as measured by body weight loss in the LNCaP-R cells xenograft mice. Altogether, our findings demonstrate that targeting PCa cells with PBM-NPs may reverse the MDR and could be used as potential therapeutic options for patients. Citation Format: Santosh Kumar Singh, Manoj Kumar Mishra, Rajesh Singh. Repurposing engineered thymoquinone nanoparticles to inhibit drug-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 301.

Abstract 5577: PSMA targeting CAR T cell immunotherapeutic strategy for prostate cancer

Abstract Introduction: Prostate cancer is the third most frequent cause of cancer-related death in men worldwide. Since prostate-specific membrane antigen (PSMA) shows a significant overexpression on prostatic cancer cells, PSMA can be considered a promising target for prostate cancer imaging and treatment. Although chimeric antigen receptor (CAR) T cells have shown great clinical outcomes in hematological malignancies, there is still limited efficacy in solid tumors due to the lack of tumor-specific antigens, low T cell infiltration in the tumors, immune suppressive tumor microenvironment, and on-target off-tumor toxicity in healthy tissues. The antigen recognition module of CAR T cells is usually a single-chain variable fragment (scFv). In contrast, the variable domains of heavy-chain antibodies (VHH) are small, stable, single-domain antibody fragments with high affinity and do not require the supramolecular assembly. Severe fatalities recently halted clinical trials of PSMA CAR T cells, so affinity tuning may be required to avoid CAR T targeting normal tissues. Here, we developed VHH-based CAR T cells targeting PSMA, able to co-express human somatostatin receptor 2 (SSTR2) actioning as a PET reporter for tracking CAR T cell distribution, as an immunotherapeutic strategy for prostate cancer. Methods: Primary human T cells were isolated and transduced with PSMA CAR lentivirus at 24 and 48 hours after activation with anti-CD3/CD28 Dynabeads. Mouse models of 8505C cell line transduced to overexpress PSMA were utilized to test the efficacy of CAR T cells. Tumor growth was monitored regularly by bioluminescence imaging. PET/CT imaging was performed to monitor T cell localization and biodistribution using ¹⁸F-NOTA-octreotide radiotracer targeting SSTR2 introduced in CAR T cells. Results: By alanine substitution of CDR3 in the VHH, we identified several low affinity candidates. The affinity of the VHH-based PSMA CAR was determined to be 22 nM by a saturation binding assay using monomeric human PSMA protein. PSMA CAR expression in primary human T cells was higher than 90%. Compared to non-treated mice, PSMA targeting CAR T cells led to tumor reduction and improved survival in animal models. Moreover, high levels of T cell infiltration and localization in tumor tissues were found, based on PET/CT imaging. Considering possible treatment-associated toxicities, which led to 2 patient deaths and the halt of one phase 1 trial of PSMA CAR T cells, we will affinity tune VHH-based CAR to improve the safety profile. Thus, additional studies will be performed to test both activity and safety of affinity variants of PSMA CAR T cells. Conclusions: We developed PSMA targeting CAR T cells able to induce potent and specific killing of prostate cancer cells. This strategy demonstrated significant therapeutic efficacy in vivo against animal models. Therefore, CAR T cells targeting PSMA may be a promising treatment strategy for patients with prostate cancer. Citation Format: Juan Gonzalez-Valdivieso, Yanping Yang, Yogindra Vedvyas, Yago Alcaina, Moonsoo M. Jin. PSMA targeting CAR T cell immunotherapeutic strategy for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5577.

Prostate-Specific Membrane Antigen Expression and Response to DNA Damaging Agents in Prostate Cancer.

Prostate-specific membrane antigen (PSMA) targeting therapies such as Lutetium-177 (177Lu)-PSMA-617 are affecting outcomes from metastatic castration-resistant prostate cancer (mCRPC). However, a significant subset of patients have prostate cancer cells lacking PSMA expression, raising concerns about treatment resistance attributable at least in part to heterogeneous PSMA expression. We have previously demonstrated an association between high PSMA expression and DNA damage repair defects in mCRPC biopsies and therefore hypothesized that DNA damage upregulates PSMA expression. To test this relationship between PSMA and DNA damage we conducted a screen of 147 anticancer agents (NCI/NIH FDA-approved anticancer "Oncology Set") and treated tumor cells with repeated ionizing irradiation. The topoisomerase-2 inhibitors, daunorubicin and mitoxantrone, were identified from the screen to upregulate PSMA protein expression in castration-resistant LNCaP95 cells; this result was validated in vitro in LNCaP, LNCaP95, and 22Rv1 cell lines and in vivo using an mCRPC patient-derived xenograft model CP286 identified to have heterogeneous PSMA expression. As double-strand DNA break induction by topoisomerase-2 inhibitors upregulated PSMA, we next studied the impact of ionizing radiation on PSMA expression; this also upregulated PSMA protein expression in a dose-dependent fashion. The results presented herein are the first, to our knowledge, to demonstrate that PSMA is upregulated in response to double-strand DNA damage by anticancer treatment. These data support the study of rational combinations that maximize the antitumor activity of PSMA-targeted therapeutic strategies by upregulating PSMA.

Impact of Androgen Receptor Activity on Prostate-Specific Membrane Antigen Expression in Prostate Cancer Cells.

Prostate-specific membrane antigen (PSMA) is an essential molecular regulator of prostate cancer (PCa) progression coded by the FOLH1 gene. The PSMA protein has become an important factor in metastatic PCa diagnosis and radioligand therapy. However, low PSMA expression is suggested to be a resistance mechanism to PSMA-based imaging and therapy. Clinical studies revealed that androgen receptor (AR) inhibition increases PSMA expression. The mechanism has not yet been elucidated. Therefore, this study investigated the effect of activation and inhibition of androgen signaling on PSMA expression levels in vitro and compared these findings with PSMA levels in PCa patients receiving systemic therapy. To this end, LAPC4, LNCaP, and C4-2 PCa cells were treated with various concentrations of the synthetic androgen R1881 and antiandrogens. Changes in FOLH1 mRNA were determined using qPCR. Open access databases were used for ChIP-Seq and tissue expression analysis. Changes in PSMA protein were determined using western blot. For PSMA staining in patients’ specimens, immunohistochemistry (IHC) was performed. Results revealed that treatment with the synthetic androgen R1881 led to decreased FOLH1 mRNA and PSMA protein. This effect was partially reversed by antiandrogen treatment. However, AR ChIP-Seq analysis revealed no canonical AR binding sites in the regulatory elements of the FOLH1 gene. IHC analysis indicated that androgen deprivation only resulted in increased PSMA expression in patients with low PSMA levels. The data demonstrate that AR activation and inhibition affects PSMA protein levels via a possible non-canonical mechanism. Moreover, analysis of PCa tissue reveals that low PSMA expression rates may be mandatory to increase PSMA by androgen deprivation.

Spherical Nucleic Acid Vaccine Structure Markedly Influences Adaptive Immune Responses of Clinically Utilized Prostate Cancer Targets.

Cancer vaccines, which activate the immune system against a target antigen, are attractive for prostate cancer, where multiple upregulated protein targets are identified. However, many clinical trials implementing peptides targeting these proteins have yielded suboptimal results. Using spherical nucleic acids (SNAs), we explore how precise architectural control of vaccine components can activate a robust antigen-specific immune response in comparison to clinical formulations of the same targets. The SNA vaccines incorporate peptides for human prostate-specific membrane antigen (PSMA) or T-cell receptor γ alternate reading frame protein (TARP) into an optimized architecture, resulting in high rates of immune activation and cytolytic ability in humanized mice and human peripheral blood mononuclear cells (hPBMCs). Specifically, administered SNAs elevate the production and secretion of cytokines and increase polyfunctional cytotoxic T cells and effector memory. Importantly, T cells raised from immunized mice potently kill targets, including clinically relevant cells expressing the whole PSMA protein. Treatment of hPBMCs increases costimulatory markers and cytolytically active T cells. This work demonstrates the importance of vaccine structure and its ability to reformulate and elevate clinical targets. Moreover, it encourages the field to reinvestigate ineffective peptide targets and repackage them into optimally structured vaccines to harness antigen potency and enhance clinical outcomes.

Imaging of prostate cancer: optimizing affinity to prostate specific membrane antigen by spacer modifications in a tumor spheroid model

Early diagnosis of prostate cancer (PCa) is crucial for staging, treatment and management of patients. Prostate specific membrane antigen (PSMA), highly over-expressed on PCa cells, is an excellent target for selective imaging of PCa. In recent years, various scaffolds have been explored as potential carriers to target diagnostic and therapeutic agents to PSMA+ tumour cells. Numerous fluorescent or radioisotope probes linked via a peptide linker have been developed that selectively binds to PCa cells. However, there are very few reports that examine the effects of chemical modifications in the peptide linker of an imaging probe on its affinity to PSMA protein. This report systematically investigates the impact of hydrophobic aromatic moieties in the peptide linker on PSMA affinity and in vitro performance. For this, a series of fluorescent bioconjugates 12–17 with different aromatic spacers were designed, synthesized, and their interactions within the PSMA pocket were first analysed in silico. Cell uptake studies were then performed for 12–17 in PSMA+ cell lines and 3D tumour models in vitro. Binding affinity values of 12–17 were found to be in the range of 36 to 157.9 nM, and 12 with three aromatic groups in the spacer exhibit highest affinity (KD = 36 nM) compared to 17 which is devoid of aromatic groups. These studies suggest that aromatic groups in the spacer region can significantly affect deep tissue imaging of fluorescent bioconjugates. Bioconjugate 12 can be a promising diagnostic tool, and conjugation to near-infrared agents would further its applications in deep-tissue imaging and surgery. Communicated by Ramaswamy H. Sarma

Impact of short-term Dutasteride treatment on prostate-specific membrane antigen expression in a mouse xenograft model.

BackgroundDutasteride has been shown to increase expression of the prostate‐specific membrane antigen (PSMA) in prostate cancer cells in previous in vitro studies. This 5‐alpha‐reductase inhibitor is commonly used for the treatment of symptomatic benign prostatic enlargement. The modulation of PSMA expression might affect PSMA‐based prostate cancer imaging and therapy.AimThe purpose of this work was to further analyze concentration‐dependent effects of Dutasteride on PSMA expression in a mouse xenograft model.Methods and resultsFour groups of mice bearing LNCaP xenografts were treated for 14 days with daily intraperitoneal injections of either vehicle control or different concentrations of Dutasteride (0.1, 1, 10 mg/kg). Total expression of PSMA, androgen receptor (AR), and caspase‐3 protein was analyzed using immunoblotting (WES). In addition, PSMA, cleaved caspase‐3 and Ki‐67 expression was assessed and quantified by immunohistochemistry. Tumor size was measured by caliper on day 7 and 14, tumor weight was assessed following tissue harvesting.The mean PSMA protein expression in mice increased significantly after treatment with 1 mg/kg (10‐fold) or 10 mg/kg (sixfold) of Dutasteride compared to vehicle control. The mean fluorescence intensity significantly increased by daily injections of 0.1 mg/kg Dutasteride (1.6‐fold) as well as 1 and 10 mg/kg Dutasteride (twofold). While the reduction in tumor volume following treatment with high concentrations of 10 mg/kg Dutasteride was nonsignificant, no changes in AR, caspase‐3, cleaved caspase‐3, and Ki‐67 expression were observed.ConclusionShort‐term Dutasteride treatments with concentrations of 1 and 10 mg/kg significantly increase the total PSMA protein expression in a mouse LNCaP xenograft model. PSMA fluorescence intensity increases significantly even using lower daily concentrations of 0.1 mg/kg Dutasteride. Further investigations are needed to elucidate the impact of Dutasteride treatment on PSMA expression in patients.