Prostate-specific Membrane Antigen Binding Research Articles

68Ga/177Lu-Labeled Bivalent Agents for Targeting Hypoxia and PSMA-Binding in Prostate Cancer.

Prostate-specific membrane antigen (PSMA) is an excellent target for cancer detection and therapy. Hypoxia is prevalent in solid tumors, and various nitroimidazole (NI) radioligands can be trapped inside hypoxic cells for diagnosis and therapy. To enhance tumor uptake and retention, we designed bivalent agents (compounds 1-8) incorporating a hypoxia-sensitive NI-moiety and a PSMA-targeting group. Ligands 1-8 were successfully prepared and labeled with 68Ga or 177Lu. Among them, [68Ga]Ga-8 ([68Ga]Ga-AAZTA-NI-PSMA-093) demonstrated significantly higher cellular accumulation under hypoxic conditions than under normoxic conditions, suggesting hypoxia-selective trapping by the introduction of NI group. PET/CT imaging at 60 min postinjection of [68Ga]Ga-8 revealed high tumor uptake (SUVmax: 10.68%ID/mL) in the tumor-bearing mice model. SPECT/CT imaging of [177Lu]Lu-8 at 24 and 48 h postinjection demonstrated excellent accumulation and retention. Preliminary studies indicate that [68Ga]Ga/[177Lu]Lu-8 may be promising bivalent agents targeting hypoxia and PSMA binding for diagnosis and radiotherapy.

Prostate-Specific Membrane Antigen Radioligand Therapy in Non-Prostate Cancers: Where Do We Stand?

The term theragnostic refers to the combination of a predictive imaging biomarker with a therapeutic agent. The promising application of prostate-specific membrane antigen (PSMA)-based radiopharmaceuticals in the imaging and treatment of prostate cancer (PCa) patients opens the way to investigate a possible role of PSMA-based radiopharmaceuticals in cancers beyond the prostate. Therefore, the aim of this review was to evaluate the role of 177Lu-PSMA radioligand therapy (RLT) in malignancies other than prostate cancer by evaluating preclinical, clinical studies, and ongoing clinical trials. An extensive literature search was performed in three different databases using different combinations of the following terms: "Lu-PSMA", "177Lu-PSMA", "preclinical", "mouse", "salivary gland cancer", "breast cancer", "glioblastoma", "solid tumour", "renal cell carcinoma", "HCC", "thyroid", "salivary", "radioligand therapy", and "lutetium-177". The search had no beginning date limit and was updated to April 2024. Only articles written in English were included in this review. A total of four preclinical studies were selected (breast cancer model n = 3/4). PSMA-RLT significantly reduced cell viability and had anti-angiogenic effects, especially under hypoxic conditions, which increase PSMA binding and uptake. Considering the clinical studies (n = 8), the complexity of evaluating PSMA-RLT in cancers other than prostate cancer was clearly revealed, since in most of the presented cases a sufficient tumour radiation dose was not achieved. However, encouraging results can be found in some types of diseases, such as thyroid cancer. Some clinical trials are still ongoing, and results from prospective larger cohorts of patients are awaited. The need for larger patient cohorts and more RLT cycles administered underscores the need for further comprehensive studies. Given the very preliminary results of both preclinical and clinical studies, ongoing clinical trials in the near future may provide stronger evidence of both the safety and therapeutic efficacy of PSMA-RLT in malignancies other than prostate cancer.

Prostate-Specific Membrane Antigen Targeted StarPEG Nanocarrier for Imaging and Therapy of Prostate Cancer.

The tumor uptake of large non-targeted nanocarriers primarily occurs through passive extravasation, known as the enhanced permeability and retention (EPR) effect. Prior studies demonstrated improved tumor uptake and retention of 4-arm 40 kDa star polyethylene glycol (StarPEG) polymers for cancer imaging by adding prostate-specific membrane antigen (PSMA) targeting small molecule ligands. To test PSMA-targeted delivery and therapeutic efficacy, StarPEG nanodrugs with/without three copies of PSMA-targeting ligands, ACUPA, are designed and synthesized. For single-photon emission computed tomography (SPECT) imaging and therapy, each nanocarrier is labeled with 177Lu using DOTA radiometal chelator. The radiolabeled nanodrugs, [177Lu]PEG-(DOTA)1 and [177Lu]PEG-(DOTA)1(ACUPA)3, are evaluated in vitro and in vivo using PSMA+ PC3-Pip and/or PSMA- PC3-Flu cell lines, subcutaneous xenografts and disseminated metastatic models. The nanocarriers are efficiently radiolabeled with 177Lu with molar activities 10.8-15.8 MBq/nmol. Besides excellent in vitro PSMA binding affinity (kD = 51.7nM), the targeted nanocarrier, [177Lu]PEG-(DOTA)1(ACUPA)3, demonstrated excellent in vivo SPECT imaging contrast with 21.3% ID/g PC3-Pip tumors uptake at 192h. Single doses of 18.5MBq [177Lu]PEG-(DOTA)1(ACUPA)3 showed complete resolution of the PC3-Pip xenografts observed up to 138days. Along with PSMA-targeted excellent imaging contrast, these results demonstrated high treatment efficacy of [177Lu]PEG-(DOTA)1(ACUPA)3 for prostate cancer, with potential for clinical translation.

Abstract 6725: NTX-470, a novel multispecific T cell engager expressed from mRNA targets PSMA and STEAP1 prostate cancer antigens to generate enhanced functional activity

Abstract Prostate cancer is the most common cancer in American men and the second leading cause of cancer death in men in western countries. Most metastatic patients develop castration resistant prostate cancer, which despite advances in immunotherapy, is limited in treatment options and represents a critical unmet need. Bi-specific T cell engagers (TCEs) targeting prostate cancer antigens represent a modality that has demonstrated preliminary clinical activity. Clinically validated antigens that are highly expressed in prostate cancers include Prostate-Specific Membrane Antigen (PSMA) and Six Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1). TCEs targeting either PSMA or STEAP1 individually have shown promising clinical activity, but challenges with durability and toxicity have been reported, potentially attributable to antigen escape, target-independent T cell activation (bystander activation) and specificity issues. Contrary to its name, PSMA expression is not exclusive to prostate cells and can be found in several other tissues and/or conditions, including normal nonprostatic epithelial cells, inflammation/infection, nonprostatic neoplastic cells and nonprostatic tumor-associated neovasculature. The widespread expression of PSMA may limit the therapeutic window of PSMA-specific TCEs and drive immunotoxicity by increased on-target, off-tumor binding. To address these issues, Nutcracker Therapeutics (NTX) has developed novel and highly complex multispecific TCE molecules that target both PSMA and STEAP1, which can be expressed using NTX’s proprietary mRNA therapeutics platform. A wide library of molecules was designed with attenuated PSMA and CD3 binding to mitigate on-target, off-tumor binding and target-independent T cell activation. Molecules were screened and selected based on their propensity to cause bystander T cell activation and the strength of PSMA binding. We identified a small number of molecules that are capable of engaging CD3 T cells but display minimal activity in the absence of STEAP1 or PSMA expressing target cells, including molecules that retain low bystander activity and attenuated PSMA binding yet maintain robust target cell killing. These molecules include NTX-470, which targets both STEAP1 and PSMA on tumor cells with minimal bystander activity. Citation Format: Chris Rae, Sudha Adusumilli, Srinivasa Bandi, Shruti Lal, Pragyesh Dhungel, Kimmy Ferry, Shima Gholizadeh, Anushtha Sharma, Adrienne Sallets, Weiqun Liu, Evan McCartney-Melstad, Pei-Ken Hsu, Ray Low, Colin McKinlay, Meredith Leong, David Y. Oh, Lawrence Fong, Guna Kannan, Samuel Deutsch, Ole Haabeth. NTX-470, a novel multispecific T cell engager expressed from mRNA targets PSMA and STEAP1 prostate cancer antigens to generate enhanced functional activity [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 6725.

A novel PSMA-targeting tracer with highly negatively charged linker demonstrates decreased salivary gland uptake in mice compared to [68Ga]Ga-PSMA-11

BackgroundThe current generation of radiolabeled PSMA-targeting therapeutic agents is limited by prominent salivary gland binding, which results in dose-limiting xerostomia from radiation exposure. JB-1498 is a urea-based small molecule with a highly negatively charged linker targeting prostate specific membrane antigen (PSMA). Prior work on a similar tracer with the same negatively charged linker demonstrated low normal organ/soft tissue background uptake compared to [68Ga]Ga-PSMA-11. The purpose of this study was to investigate if [68Ga]Ga-JB-1498 had reduced salivary gland uptake in mice compared to [68Ga]Ga-PSMA-11.ResultsJB-1498 demonstrated high affinity for PSMA binding and tumor uptake in a murine tumor model. In an initial biodistribution study with low molar activity, [68Ga]Ga-JB-1498 demonstrated salivary gland uptake of 0.13 ± 0.01%ID/g. In a second biodistribution study in non-tumor-bearing mice with high molar activity, [68Ga]Ga-JB1498 demonstrated salivary gland uptake of 0.39 ± 0.24% ID/g and kidney activity of 10.12 ± 1.73% ID/g at one hour post IV injection. This salivary gland uptake is significantly less than the published uptake of [68Ga]Ga-PSMA-11. Micro-PET visually confirmed the findings of the biodistribution studies. Dynamic micro-PET imaging demonstrated gradually decreasing [68Ga]Ga-JB1498 activity in salivary glands and kidneys, compared to gradually increasing [68Ga]Ga-PSMA-11 activity in these two organs during the first hour.ConclusionBiodistribution and micro-PET imaging of [68Ga]Ga-JB-1498 demonstrate significantly decreased salivary gland uptake and different pharmacokinetic behavior in kidneys and salivary glands in mice compared to [68Ga]Ga-PSMA-11. Our findings suggest that constructing a PSMA-targeting molecule with a highly negatively charged linker is a promising strategy to reduce salivary gland uptake of GCP-II/PSMA ligands in theranostic applications.

Synthesis and Evaluation of 99mTc-Labeled PSMA-Targeted Tracers Based on the Lys-Urea-Aad Pharmacophore for Detecting Prostate Cancer with Single Photon Emission Computed Tomography.

Prostate-specific membrane antigen (PSMA) is a well-validated prostate cancer marker but reported PSMA-targeted tracers derived from the Lys-urea-Glu pharmacophore including the clinically validated [99mTc]Tc-EDDA/HYNIC-iPSMA have high off-target uptake in kidneys, spleen, and salivary glands. In this study, we synthesized and evaluated three novel 99mTc-labeled PSMA-targeted tracers and investigated if the tracers derived from the Lys-urea-Aad pharmacophore could have minimized uptake in off-target organs/tissues. In vitro competition binding assays showed that compared with HYNIC-iPSMA, the three novel ligands had slightly weaker PSMA binding affinity (average Ki = 3.11 vs. 8.96-11.6 nM). Imaging and ex vivo biodistribution studies in LNCaP tumor-bearing mice showed that [99mTc]Tc-EDDA/HYNIC-iPSMA and the three novel tracers successfully visualized LNCaP tumor xenografts in SPECT images and were excreted mainly via the renal pathway. The average tumor uptake at 1 h post-injection varied from 5.40 to 18.8%ID/g, and the tracers derived from the Lys-urea-Aad pharmacophore had much lower uptake in the spleen and salivary glands. Compared with the clinical tracer [99mTc]Tc-EDDA/HYNIC-iPSMA, the Lys-urea-Aad-derived [99mTc]Tc-EDDA-KL01127 had lower background uptake and superior tumor-to-background contrast ratios and is therefore promising for clinical translation to detect prostate cancer lesions with SPECT.

NIR-II-triggered photothermal therapy with Au@PDA/PEG-PI for targeted downregulation of PSMA in prostate cancer.

Although positron emission tomography (PET) imaging products targeting prostate-specific membrane antigen (PSMA) have been approved for marketing, clinical challenges remain in the study of its use as a therapeutic target, such as the complex synthesis process and side effects after treatment. Here, we developed a strategy for targeted photothermal therapy (PTT) using PSMA as the target. The results of molecular docking demonstrated that the synthesized PEG modified urea-based PSMA inhibitor (small molecular PSMA inhibitor, PI) PI-PEG has a high affinity energy (binding energy=- 8.3 kcal mol-1) for the PSMA target. Therefore, modification of PI-PEG onto the surface of gold@polydopamine (Au@PDA) with NIR-II absorption could enable targeted PTT against PSMA. This work revealed that the prepared Au@PDA/PEG-PI were not only highly selective for PSMA, but also could efficiently ablate PSMA expression by targeted PTT at the maximum permissible exposure (MPE) of the NIR-II laser. Moreover, Au@PDA/PEG-PI also have potential for photoacoustic (PA) imaging and computed tomography (CT) imaging. As the first strategy to downregulate the expression of PSMA and successfully inhibit prostate cancer by targeted PTT, this study case provides a new idea for the clinical translation of PSMA as an integrated target for tumor diagnosis and anti-tumor treatment. STATEMENT OF SIGNIFICANCE: (1) Au@PDA/PEG-PI NPs were the novel PTT agent to target PSMA and successfully down-regulate PSMA expression. (2) Molecular docking results demonstrated that PI-PEG inhibitors have a high affinity energy for PSMA (binding energy=- 8.3 kcal mol-1). (3) Au@PDA/PEG-PI NPs can be targeted for efficient PTT at the MPE of the NIR-II laser. (4) Au@PDA/PEG-PI NPs also have the potential for PA and CT imaging.

Prostate specific membrane antigen binding radiopharmaceuticals: Current data and new concepts.

Prostate specific membrane antigen (PSMA) represents a validated target for prostate cancer therapeutics. The phase III VISION study with 177lutetium (177Lu)-PSMA-617 represented a pivotal step forward and the FDA has now approved this agent in advanced metastatic castrate-resistant prostate cancer (mCRPC). A number of other PSMA targeted radiopharmaceuticals are now under development. Some of these agents are targeted to PSMA via monoclonal antibodies such as J591 and TLX591. Others are targeted to PSMA via small molecules such as PSMA-617, PSMA I&T, MIP-1095, etc. In addition to the use of various ligands, multiple isotopes are now in clinical trials. Beta emitters in development include 177Lu, 131iodide (131I), and 67copper (67Cu). Targeted alpha emitters potentially include 225actinium (225Ac), 227thorium (227Th), and 212lead (212Pb). Phase III trials are underway with both 177Lu-PSMA-617 and 177Lu-PSMA I&T in mCRPC. Single dose phase I trials are complete with 225Ac-J591 but additional data are need to launch a phase III. Data are promising with 225Ac-PSMA-617 but concerns remain over salivary and renal toxicity. Tandem therapies are also considered combining both beta and alpha-targeted therapy. Taken together the field of PSMA targeted radiopharmaceuticals is rapidly developing. The targeted alpha therapies are particularly promising and several developmental paths forward are being considered in the near future.

Prostate-Specific Membrane Antigen Targeted Deep Tumor Penetration of Polymer Nanocarriers.

Tumoral uptake of large-size nanoparticles is mediated by the enhanced permeability and retention (EPR) effect, with variable accumulation and heterogenous tumor tissue penetration depending on the tumor phenotype. The performance of nanocarriers via specific targeting has the potential to improve imaging contrast and therapeutic efficacy in vivo with increased deep tissue penetration. To address this hypothesis, we designed and synthesized prostate cancer-targeting starPEG nanocarriers (40 kDa, 15 nm), [89Zr]PEG-(DFB)3(ACUPA)1 and [89Zr]PEG-(DFB)1(ACUPA)3, with one or three prostate-specific membrane antigen (PSMA)-targeting ACUPA ligands. The in vitro PSMA binding affinity and in vivo pharmacokinetics of the targeted nanocarriers were compared with a nontargeted starPEG, [89Zr]PEG-(DFB)4, in PSMA+ PC3-Pip and PSMA- PC3-Flu cells, and xenografts. Increasing the number of ACUPA ligands improved the in vitro binding affinity of PEG-derived polymers to PC3-Pip cells. While both PSMA-targeted nanocarriers significantly improved tissue penetration in PC3-Pip tumors, the multivalent [89Zr]PEG-(DFB)1(ACUPA)3 showed a remarkably higher PC3-Pip/blood ratio and background clearance. In contrast, the nontargeted [89Zr]PEG-(DFB)4 showed low EPR-mediated accumulation with poor tumor tissue penetration. Overall, ACUPA conjugated targeted starPEGs significantly improve tumor retention with deep tumor tissue penetration in low EPR PC3-Pip xenografts. These data suggest that PSMA targeting with multivalent ACUPA ligands may be a generally applicable strategy to increase nanocarrier delivery to prostate cancer. These targeted multivalent nanocarriers with high tumor binding and low healthy tissue retention could be employed in imaging and therapeutic applications.

New PSMA-Targeting Ligands: Transformation from Diagnosis (Ga-68) to Radionuclide Therapy (Lu-177).

Prostate-specific membrane antigen (PSMA) is a promising target for the diagnosis and radionuclide therapy of prostate cancer. This study reports conversion of a previously reported 68Ga-imaging agent, [68Ga]Ga-P16-093, to a Lu-177 radionuclide therapeutic agent. Substitution of the HBED-CC metal chelating group with DOTA(GA)2 led to P17-087 (4) and P17-088 (7). Both agents showed excellent PSMA binding affinity (IC50 = 10-30 nM) comparable to that of recently FDA-approved [177Lu]Lu-PSMA-617 (Pluvicto). Biodistribution studies in PSMA expressing tumor bearing mice showed that [177Lu]Lu-4 exhibited very high tumor uptake and a fast blood clearance similar to those of [177Lu]Lu-PSMA-617. Conversely, [177Lu]Lu-7, containing an albumin binder, extended its blood half-life and exhibited significantly higher uptake and longer tumor residence time than [177Lu]Lu-4 and [177Lu]Lu-PSMA-617. The switch from chelator HBED-CC to DOTA(GA)2 and the switch from the imaging isotope gallium-68 to the therapeutic isotope lutetium-177 have successfully transformed a PSMA-targeting agent from diagnosis to promising radionuclide therapeutic agents.

Refined Chelator Spacer Moieties Ameliorate the Pharmacokinetics of PSMA-617.

Prostate-specific membrane antigen (PSMA) binding tracers are promising agents for the targeting of prostate tumors. To further optimize the clinically established radiopharmaceutical PSMA-617, novel PSMA ligands for prostate cancer endoradiotherapy were developed. A series of PSMA binding tracers that comprise a benzyl group at the chelator moiety were obtained by solid-phase synthesis. The compounds were labeled with 68Ga or 177Lu. Competitive cell-binding assays and internalization assays were performed using the cell line C4-2, a subline of the PSMA positive cell line LNCaP (human lymph node carcinoma of the prostate). Positron emission tomography (PET) imaging and biodistribution studies were conducted in a C4-2 tumor bearing BALB/c nu/nu mouse model. All 68Ga-labeled ligands were stable in human serum over 2 h; 177Lu-CA030 was stable over 72 h. The PSMA ligands revealed inhibition potencies [Ki] (equilibrium inhibition constants) between 4.8 and 33.8 nM. The percentage of internalization of the injected activity/106 cells of 68Ga-CA028, 68Ga-CA029, and 68Ga-CA030 was 41.2 ± 2.7, 44.3 ± 3.9, and 53.8 ± 5.4, respectively; for the comparator 68Ga-PSMA-617, 15.5 ± 3.1 was determined. Small animal PET imaging of the compounds showed a high tumor-to-background contrast. Organ distribution studies revealed high specific uptake in the tumor, that is, approximately 34.4 ± 9.8% of injected dose per gram (%ID/g) at 1 h post injection for 68Ga-CA028. At 1 h p.i., 68Ga-CA028 and 68Ga-CA030 demonstrated lower kidney uptake than 68Ga-PSMA-617, but at later time points, kidney time–activity curves converge. In line with the preclinical data, first diagnostic PET imaging using 68Ga-CA028 and 68Ga-CA030 revealed high-contrast detection of bone and lymph node lesions in patients with metastatic prostate cancer. The novel PSMA ligands, in particular CA028 and CA030, are promising agents for targeting PSMA-positive tumor lesions as shown in the preclinical evaluation and in a first patient, respectively. Thus, clinical translation of 68Ga-CA028 and 68Ga/177Lu-CA030 for diagnostics and endoradiotherapy of prostate cancer in larger cohorts of patients is warranted.

DOTA Conjugate of Bisphosphonate and PSMA-Inhibitor: A Promising Combination for Therapy of Prostate Cancer Related Bone Metastases.

Prostate cancer (PCa) is one of the most common cancer types worldwide. 90% of men with late stage PCa will develop bone metastases. Since the expression level of PSMA (prostate-specific membrane antigen) in bone metastases can vary significantly, a compound is being searched for which accumulates in bone metastases independently of PSMA level. With DOTA-L-Lys(SA.Pam)-PSMA-617, we present a compound that, in addition to a PSMA inhibitor as a target vector, also contains a bisphosphonate that is established as a bone tracer and thus combines the advantages of PSMA targeting and bone targeting. This is a class of small molecules combining targeting of two different targets with the potential advantages for treatment of biologically heterogeneous bone metastasis from prostate cancer. The molecule can be labeled with lutetium-177 and used for the therapy of PCa-related bone metastases. DOTA-L-Lys(SA.Pam)-PSMA-617 was synthesized and radiolabelled in 1 M ammonium acetate buffer pH 5.5 at 95°C. Different amounts of precursor were evaluated. Complex stability was evaluated in three different media. LogD7.4 value was evaluated via the determination of the equilibrium distribution in a PBS/n-octanol mixture. A hydroxyapatite binding assay was used to evaluate the potential binding to bone metastases. In vitro affinity was determined and Ki value was evaluated. To evaluate the binding potential in mice, ex vivo biodistribution studies were carried out in LNCaP tumor-bearing Balb/c mice. [177Lu]Lu-labeling of DOTA-L-Lys(SA.Pam)-PSMA-617 showed quantitative RCY within 10 min and high complex stability over 14 days. The lipophilicity of the labeled compound was similar to the lipophilicity of the reference compound [177Lu]Lu-PSMA-617 and showed an excellent and selective HAP binding of 98.2 ± 0.11%. With a Ki of 42.3 ± 7.7 nM PSMA binding affinity is lower in comparison to [177Lu]Lu-PSMA-617. First ex vivo biodistribution studies with LNCaP tumor-bearing Balb/c mice showed a PSMA dependent tumor accumulation of 4.2 ± 0.7%ID/g and a femur accumulation of 3.4 ± 0.4%ID/g. [177Lu]Lu-DOTA-L-Lys(SA.Pam)-PSMA-617 is a promising compound for therapy of PCa related bone and tissue metastases. Accumulation on the bone metastases via two mechanisms also enables the treatment of bone metastases that show little or no PSMA expression.

Theranostic Small-Molecule Prodrug Conjugates for Targeted Delivery and Controlled Release of Toll-like Receptor 7 Agonists.

We previously reported the design and synthesis of a small-molecule drug conjugate (SMDC) platform that demonstrated several advantages over antibody–drug conjugates (ADCs) in terms of in vivo pharmacokinetics, solid tumor penetration, definitive chemical structure, and adaptability for modular synthesis. Constructed on a tri-modal SMDC platform derived from 1,3,5-triazine (TZ) that consists of a targeting moiety (Lys-Urea-Glu) for prostate-specific membrane antigen (PSMA), here we report a novel class of chemically identical theranostic small-molecule prodrug conjugates (T-SMPDCs), [18/19F]F-TZ(PSMA)-LEGU-TLR7, for PSMA-targeted delivery and controlled release of toll-like receptor 7 (TLR7) agonists to elicit de novo immune response for cancer immunotherapy. In vitro competitive binding assay of [19F]F-TZ(PSMA)-LEGU-TLR7 showed that the chemical modification of Lys-Urea-Glu did not compromise its binding affinity to PSMA. Receptor-mediated cell internalization upon the PSMA binding of [18F]F-TZ(PSMA)-LEGU-TLR7 showed a time-dependent increase, indicative of targeted intracellular delivery of the theranostic prodrug conjugate. The designed controlled release of gardiquimod, a TLR7 agonist, was realized by a legumain cleavable linker. We further performed an in vivo PET/CT imaging study that showed significantly higher uptake of [18F]F-TZ(PSMA)-LEGU-TLR7 in PSMA+ PC3-PIP tumors (1.9 ± 0.4% ID/g) than in PSMA− PC3-Flu tumors (0.8 ± 0.3% ID/g) at 1 h post-injection. In addition, the conjugate showed a one-compartment kinetic profile and in vivo stability. Taken together, our proof-of-concept biological evaluation demonstrated the potential of our T-SMPDCs for cancer immunomodulatory therapies.

Abstract CT143: Results of combined targeting of prostate-specific membrane antigen (PSMA) with alpha-radiolabeled antibody 225Ac-J591 and beta-radiolabeled ligand 177Lu-PSMA I&T: preclinical and initial phase 1 clinical data in patients with metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background: PSMA is overexpressed by the majority of PC and may be targeted by both antibodies (mAb) and small molecule ligands (SML), each with differing PSMA binding sites, kinetics, and biodistributions. mAbs have long circulating times (exposing organs such a bone marrow) but are too large to target normal tissue luminal PSMA expression in salivary glands, small bowel, and kidney; SMLs rapidly diffuse to all PSMA+ sites and are excreted by the kidneys within hours. Alpha emitters have high potency over a short range whereas beta emitters have lower energy over a significantly longer range. We hypothesized that combining mAb and SML targeting plus combining alpha and beta emitters offered complementary benefits and would be safe and effective. Methods: A series of cell line and xenograft studies were performed to test mAb vs SML vs combo binding, uptake, and efficacy. A phase I/II clinical trial was initiated, enrolling patients with progressive, PSMA+ (by PSMA PET) mCRPC following potent AR pathway inhibition and chemotherapy (or chemo ineligible/refused)[NCT04886986]. The primary endpoint of phase I is assessment of dose-limiting toxicity (DLT) with secondary endpoints of response, progression-free and overall survival, genomic and imaging correlatives, and patient-reported outcomes. Results: Binding studies demonstrated that mAb plus SML binding was additive rather than competitive in LNCaP and CWR22Rv1 cell lines. Uptake of 177Lu-mAb plus -SML in LNCaP, CWR22rv1, and PC3/PSMA xenograft models demonstrated synergism, with 44-65% greater tumor radioactivity in combination than the sum of the individual agents. Intracellular tracking studies indicate that the mAb re-directs SML to lysosomes from recycling endosomes, maintains retention of SML in tumor cells, and, therefore, prolongs intra-tumoral radioactivity exposure. Survival was prolonged in animals receiving 225Ac-mAb plus 177Lu-SML vs either drug alone (or control). Nine men with median age 68 (range 55-87), PSA 140 (2.4-9614) have been enrolled in both of 2 planned dose-escalation cohorts. 89% with bone, 44% lymph node, 22% liver, 22% lung metastases; 88% with detectable CTC count (75% unfavorable). All had at least 1 metastatic lesion with PSMA PET SUVmax > liver SUVmean, with SUVmax of the single hottest lesion ranging from 11.6-69.9. For the primary endpoint, 0 of 3 with DLT in cohort 1, and with follow up ongoing, no DLT has occurred to date in 6 patients in cohort 2. Of evaluable patients, 5 of 6 with PSA decline (8-97% decline). Conclusions: Targeting PSMA with the combination of mAb and SML leads to synergistic radioactivity in preclinical studies. The combination on mAb and SML radiolabeled with alpha and beta emitters appears safe with short-term follow up with phase 2 enrollment planned. Citation Format: Scott T. Tagawa, Edward Fung, Muhammad O. Niaz, Mahelia Bissassar, Sharon Singh, Amie Patel, Angela Tan, Juana Martinez Zuloaga, Sandra Huicochea Castellanos, Jones T. Nauseef, Ana Molina, Cora Sternberg, David M. Nanus, Joseph Osborne, Neil H. Bander. Results of combined targeting of prostate-specific membrane antigen (PSMA) with alpha-radiolabeled antibody 225Ac-J591 and beta-radiolabeled ligand 177Lu-PSMA I&T: preclinical and initial phase 1 clinical data in patients with metastatic castration-resistant prostate cancer (mCRPC) [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 CT143.

Estimation of The Main Effect and Total Effect of a PBPK Model Based on The Uncertainty of Individual Parameter for Treatment Planning in PSMA Therapy

The purpose of this study was to identify the most important physiologically-based pharmacokinetic (PBPK) model parameters determining the absorbed dose (AD) in prostate-specific membrane antigen (PSMA) therapy. The extended-Sobol’ global sensitivity analysis method was used to analyze the sensitivity of the PBPK model parameters obtained from 3 patients. The investigated PBPK model parameters were the blood flow to the organs, PSMA binding rate, biological release rates, and density of organs receptor. The outputs of extended Sobol method were the main effect Si and the total effect STi of the parameter of interests for each ADs. The sampling strategy of extended Sobol has been implemented based on the mean and covariance matrix of the parameters. From the simulations, the most important parameters which determine the ADs to the kidney was the kidney receptor density (Si=0,4, STi=0,8). For tumors, it was shown that tumor receptor density was the most essential parameter (Si=0,7, STi=0,8). In conclusion, measurement of the blood flow and organ receptor densities might be of interest to improve individualized treatment of PSMA therapy.

Hybrid Chelator-Based PSMA Radiopharmaceuticals: Translational Approach.

(1) Background: Prostate-specific membrane antigen (PSMA) has been extensively studied in the last decade. It became a promising biological target in the diagnosis and therapy of PSMA-expressing cancer diseases. Although there are several radiolabeled PSMA inhibitors available, the search for new compounds with improved pharmacokinetic properties and simplified synthesis is still ongoing. In this study, we developed PSMA ligands with two different hybrid chelators and a modified linker. Both compounds have displayed a promising pharmacokinetic profile. (2) Methods: DATA5m.SA.KuE and AAZTA5.SA.KuE were synthesized. DATA5m.SA.KuE was labeled with gallium-68 and radiochemical yields of various amounts of precursor at different temperatures were determined. Complex stability in phosphate-buffered saline (PBS) and human serum (HS) was examined at 37 °C. Binding affinity and internalization ratio were determined in in vitro assays using PSMA-positive LNCaP cells. Tumor accumulation and biodistribution were evaluated in vivo and ex vivo using an LNCaP Balb/c nude mouse model. All experiments were conducted with PSMA-11 as reference. (3) Results: DATA5m.SA.KuE was synthesized successfully. AAZTA5.SA.KuE was synthesized and labeled according to the literature. Radiolabeling of DATA5m.SA.KuE with gallium-68 was performed in ammonium acetate buffer (1 M, pH 5.5). High radiochemical yields (>98%) were obtained with 5 nmol at 70 °C, 15 nmol at 50 °C, and 60 nmol (50 µg) at room temperature. [68Ga]Ga-DATA5m.SA.KuE was stable in human serum as well as in PBS after 120 min. PSMA binding affinities of AAZTA5.SA.KuE and DATA5m.SA.KuE were in the nanomolar range. PSMA-specific internalization ratio was comparable to PSMA-11. In vivo and ex vivo studies of [177Lu]Lu-AAZTA5.SA.KuE, [44Sc]Sc-AAZTA5.SA.KuE and [68Ga]Ga-DATA5m.SA.KuE displayed specific accumulation in the tumor along with fast clearance and reduced off-target uptake. (4) Conclusions: Both KuE-conjugates showed promising properties especially in vivo allowing for translational theranostic use.

Targeted delivery of cytotoxic proteins to prostate cancer via conjugation to small molecule urea-based PSMA inhibitors

Prostate cancer cells are characterized by a remarkably low proliferative rate and the production of high levels of prostate-specific proteases. Protein-based toxins are attractive candidates for prostate cancer therapy because they kill cells via proliferation-independent mechanisms. However, the non-specific cytotoxicity of these potent cytotoxins must be redirected to avoid toxicity to normal tissues. Prostate-Specific Membrane Antigen (PSMA) is membrane-bound carboxypeptidase that is highly expressed by prostate cancer cells. Potent dipeptide PSMA inhibitors have been developed that can selectively deliver and concentrate imaging agents within prostate cancer cells based on continuous PSMA internalization and endosomal cycling. On this basis, we conjugated a PSMA inhibitor to the apoptosis-inducing human protease Granzyme B and the potent Pseudomonas exotoxin protein toxin fragment, PE35. We assessed selective PSMA binding and entrance into tumor cell to induce cell death. We demonstrated these agents selectively bound to PSMA and became internalized. PSMA-targeted PE35 toxin was selectively toxic to PSMA producing cells in vitro. Intratumoral and intravenous administration of this toxin produced marked tumor killing of PSMA-producing xenografts with minimal host toxicity. These studies demonstrate that urea-based PSMA inhibitors represent a simpler, less expensive alternative to antibodies as a means to deliver cytotoxic proteins to prostate cancer cells.

Synthesis and preliminary evaluation of 211At-labeled inhibitors of prostate-specific membrane antigen for targeted alpha particle therapy of prostate cancer

IntroductionThe high potency and short tissue range of α-particles are attractive features for targeted radionuclide therapy, particularly for cancers with micro-metastases. In the current study, we describe the synthesis of a series of 211At-labeled prostate-specific membrane antigen (PSMA) inhibitors and their preliminary evaluation as potential agents for metastatic prostate cancer treatment. MethodsFour novel Glu-urea based PSMA ligands containing a trialkyl stannyl group were synthesized and labeled with 211At, and for comparative purposes, 131I, via halodestannylation reactions with N-chlorosuccinimide as the oxidant. A PSMA inhibitory assay was performed to evaluate PSMA binding of the unlabeled, iodinated compounds. A series of paired-label biodistribution experiments were performed to compare each 211At-labeled PSMA ligand to its 131I-labeled counterpart in mice bearing subcutaneous PC3 PSMA+ PIP xenografts. ResultsRadiochemical yields ranged from 32% to 65% for the 211At-labeled PSMA inhibitors and were consistently lower than those obtained with the corresponding 131I-labeled analogue. Good localization in PC3 PSMA+ PIP but not control xenografts was observed for all labeled molecules studied, which exhibited a variable degree of in vivo dehalogenation as reflected by thyroid and stomach activity levels. Normal tissue uptake and in vivo stability for several of the compounds was markedly improved compared with the previously evaluated compounds, [211At]DCABzL and [*I]DCIBzL. Conclusions and implications for patient careCompared with the first generation compound [211At]DCABzL, several of the novel 211At-labeled PSMA ligands exhibited markedly improved stability in vivo and higher tumor-to-normal tissue ratios. [211At]GV-620 has the most promising characteristics and warrants further evaluation as a targeted radiotherapeutic for prostate cancer.

Extensive preclinical evaluation of lutetium-177-labeled PSMA-specific tracers for prostate cancer radionuclide therapy

PurposeVarious radiolabeled prostate-specific membrane antigen (PSMA)–targeting tracers are clinically applied for prostate cancer (PCa) imaging and targeted radionuclide therapy. The PSMA binding affinities, biodistribution, and DNA-damaging capacities of these radiotracers have not yet been compared in detail. A major concern of PSMA-targeting radiotracers is the toxicity in other PSMA-expressing organs, such as the salivary glands, thus demanding careful evaluation of the most optimal and safest radiotracer. In this extensive preclinical study, we evaluated the clinically applied PSMA-targeting small molecule inhibitors DOTA-PSMA-617 (PSMA-617) and DOTAGA-PSMA-I&T (PSMA-I&T) and the PSMA nanobody DOTA-JVZ-007 (JVZ-007) using PSMA-expressing cell lines, a unique set of PCa patient-derived xenografts (PDX) and healthy human tissues.Methods and resultsIn vitro displacement studies on PSMA-expressing cells and cryosections of a PSMA-positive PDX revealed high and specific binding affinity for all three tracers labeled with lutetium-177 with IC50 values in the nanomolar range. Interestingly, [177Lu]Lu-JVZ-007 could not be displaced by PSMA-617 or PSMA-I&T, suggesting that this tracer targets an alternative binding site. Autoradiography assays on cryosections of human salivary and renal tissues revealed [177Lu]Lu-PSMA-617 to have the lowest binding to these healthy organs compared with [177Lu]Lu-PSMA-I&T. In vivo biodistribution assays confirmed the in vitro results with comparable tumor uptake of [177Lu]Lu-PSMA-617 and [177Lu]Lu-PSMA-I&T at all timepoints, resulting in induction of similar levels of DNA double-strand breaks in the tumors. However, [177Lu]Lu-PSMA-I&T demonstrated approximately 40× higher renal uptake at 4 and 8 h post injection resulting in an unfavorable tumor-to-kidney ratio.Conclusion[177Lu]Lu-PSMA-617 has the most favorable biodistribution in mice as well as more favorable binding characteristics in vitro in PSMA-positive cells and human kidney and salivary gland specimens compared with [177Lu]Lu-PSMA-I&T and [177Lu]Lu-JVZ-007. Based on our preclinical evaluation, [177Lu]Lu-PSMA-617 is the best performing tracer to be taken further into clinical evaluation for PSMA-targeted radiotherapeutic development although with careful evaluation of the tracer binding to PSMA-expressing organs.

Abstract 2779: Non-invasive quantification of pharmacodynamic effects of an antibody targeting PSMA by PET imaging

Abstract Introduction: Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancers (PCa) and a target for the development of a variety of therapeutics. Knowledge of how those therapeutics engage tumor PSMA could improve dose finding and optimization. Here we studied the pharmacodynamic effects of a PSMA monoclonal antibody (5D3, mAb) on target engagement and accessible levels of PSMA in the tumors in PCa and melanoma xenograft models known to express PSMA. We also sought to evaluate effect of tumor burden on the pharmacodynamic effects of the mAb and accessible tumor PSMA levels. To quantify accessible PSMA levels, we used [18F]DCFPyL, an established PSMA binding positron emission tomography (PET) imaging agent. Methods: Prostate (22RV1) and skin (SKMEL3) cancer cells were tested for PSMA expression by flow cytometry. Corresponding tumors were generated in the upper flanks of male NSG mice and used for PET imaging and biodistribution studies. [18F]DCFPyL was synthesized using established procedures. To assess the effect of 5D3 dose on accessible PSMA levels, mice with 22RV1 tumors were treated with a single dose of 0.3, 1, 3, 10 and 30 mg/kg of mAb or saline as a control. Mice with one and three 22RV1 tumors were used to establish low and high tumor burden models, respectively and treated with a single 1 and 10 mg/kg dose of 5D3. All the treatments were performed for 24h. After 5D3 treatment, [18F]DCFPyL was injected and imaging and biodistribution studies were performed at 1 hour after radiotracer injection. All the data were normalized and presented as % of injected dose per gram (%ID/g). Results: We observed a negative correlation between [18F]DCFPyL uptake in tumors and antibody dose, indicating a decrease in accessible tumor PSMA levels with increased mAb dose. Ex vivo biodistribution studies showed high [18F]DCFPyL uptake (13.2±1.4 %ID/g) in saline treated controls and a 25, 46 and 76% reduction in mice treated with 0.3, 1 and 10 mg/kg dose of mAb, respectively. Those findings were validated in SKMEL3 tumors with 4-fold lower PSMA expression. Collectively, these data establish that accessible PSMA levels in the tumors are reduced for [18F]DCFPyL binding with increased 5D3 dose. Next, we studied the effect of tumor burden on antibody dose and accessible PSMA levels at the tumor. In mice bearing one 22RV1 tumor (low tumor burden), [18F]DCFPyL uptake in tumors was reduced by 52% and 77% in mice treated with 1 and 10 mg/kg dose of 5D3, respectively. In mice with three tumors (high tumor burden), [18F]DCFPyL uptake in tumors was reduced by 29% and 56% at 1 and 10 mg/kg dose of 5D3, respectively. No differences were observed in saline treated controls between mice with one or three tumors. These data suggest higher accessible PSMA levels in mice with three tumors compared to mice with one tumor when treated with the same dose of an mAb. These observations indicate that high tumor burden leads to increased antibody processing and accessible PSMA levels in tumors, which could have implication for dosing of PSMA targeted therapeutics. Conclusion: Quantification of accessible PSMA levels, measured using [18F]DCFPyL uptake, can be used as a tool for dose-finding and optimization of PSMA targeted therapeutics. Citation Format: Akhilesh Mishra, Zora Nováková, Ala Lisok, Dhiraj Kumar, Ronnie C. Mease, Cyril Bařinka, Sridhar Nimmagadda. Non-invasive quantification of pharmacodynamic effects of an antibody targeting PSMA by PET imaging [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2779.