PSMA-expressing Prostate Cancer Cells Research Articles

Receptor-Targeted Peptide Conjugates Based on Diphosphines Enable Preparation of 99mTc and 188Re Theranostic Agents for Prostate Cancer.

Benchtop 99Mo/99mTc and 188W/188Re generators enable economical production of molecular theranostic 99mTc and 188Re radiopharmaceuticals, provided that simple, kit-based chemistry exists to radiolabel targeting vectors with these radionuclides. We have previously described a diphosphine platform that efficiently incorporates 99mTc into receptor-targeted peptides. Here, we report its application to label a prostate-specific membrane antigen (PSMA)-targeted peptide with 99mTc and 188Re for diagnostic imaging and systemic radiotherapy of prostate cancer. Methods: Two diphosphine-dipeptide bioconjugates, DP1-PSMAt and DP2-PSMAt, were formulated into kits for radiolabeling with 99mTc and 188Re. The resulting radiotracers were studied invitro, in prostate cancer cells, and invivo in mouse xenograft models, to assess similarity of uptake and biodistribution for each 99mTc/188Re pair of agents. Results: Both DP1-PSMAt and DP2-PSMAt could be efficiently radiolabeled with 99mTc and 188Re using kit-based methods to furnish the isostructural compounds M-DP1-PSMAt and M-DP2-PSMAt (M = [99mTc]Tc, [188Re]Re). All 99mTc/188Re radiotracers demonstrated specific uptake in PSMA-expressing prostate cancer cells, with negligible uptake in prostate cancer cells that did not express PSMA or in which PSMA uptake was blocked. M-DP1-PSMAt and M-DP2-PSMAt also exhibited high tumor uptake (18-30 percentage injected dose per gram at 2 h after injection), low retention in nontarget organs, fast blood clearance, and excretion predominantly via a renal pathway. Importantly, each pair of 99mTc/188Re radiotracers showed near-identical biologic behavior in these experiments. Conclusion: We have prepared and developed novel pairs of isostructural PSMA-targeting 99mTc/188Re theranostic agents. These generator-based theranostic agents have potential to provide access to the benefits of PSMA-targeted diagnostic imaging and systemic radiotherapy in health care settings that do not routinely have access to either reactor-produced 177Lu radiopharmaceuticals or PET/CT infrastructure.

Phase I/II trial of LAVA-1207, a novel bispecific gamma-delta T-cell engager alone, or with low dose IL-2 or pembrolizumab, in metastatic castration resistant prostate cancer (mCRPC).

TPS2672 Background: LAVA-1207 is a humanized bispecific antibody that binds with high affinity to the Vδ2 chain of Vγ9Vδ2-T cells and to prostate-specific membrane antigen (PSMA). It comprises a heterodimer of two fusion proteins, each consisting of a VHH linked to a human IgG1 Fc domain. Preclinical evidence demonstrates that, upon binding both targets, LAVA-1207 leads to potent Vγ9Vδ2-T cell degranulation and cytolytic activity against PSMA-expressing prostate cancer cells. IL-2 is an immune modulator which has been shown to support expansion of activated Vγ9Vδ2-T cells. PD-1 is an inhibitory immune checkpoint receptor that can dampen (Vγ9Vδ2-) T cell reactivity, suggesting that pembrolizumab could potentiate the effect of LAVA-1207. Methods: This trial is a phase 1/2a open label study with a 3+3 design in patients with refractory mCRPC to assess the safety of LAVA-1207 alone or with low dose IL-2 or with pembrolizumab, and to determine the recommended Phase 2a dose (RP2D). Secondary objectives include evaluation of pharmacokinetics, pharmacodynamics, immunogenicity, and preliminary antitumor activity. Exploratory endpoints include evaluation of the effect of study treatment on circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). LAVA-1207 is administered IV every two weeks. Two parallel cohorts assessing LAVA-1207 with low dose subcutaneous IL-2 have been implemented: (1) single IL-2 administration and (2) three IL-2 administrations on consecutive days per cycle for up to four cycles. An additional dose escalation arm evaluates LAVA-1207 in combination with pembrolizumab, 400mg Q6W, IV. Patients with mCRPC that have failed at least one prior AR therapy and taxane-based chemotherapy (unless deemed medically unsuitable to receive a taxane) will be enrolled on the study. Patients should have progressive disease either by PSA or by RECIST 1.1 or appearance of 2 or more bone metastases. Patients must have ECOG performance status of 0-1. PSMA-PET is performed at baseline. Paired biopsies are requested to further assess LAVA-1207 activity. Dose escalation is ongoing. Expansion arm(s) will be included based on available data from part 1 of the study and may include one or more of LAVA-1207, LAVA-1207 with low dose IL-2, or LAVA-1207 in combination with pembrolizumab. Clinical trial information: NCT05369000 .

Photoimmunotherapy of Prostate Cancer With Antibody and Fab Fragments Targeting the Prostate Specific Membrane Antigen.

The standard treatment for localized prostate cancer involves surgical removal of the prostate with curative intent. However, when tumor cells persist in the operation site, there is high risk of local recurrence and tumor spread, leading to stressful follow-up treatments, impaired quality of life, and reduced overall survival. This study examined photoimmunotherapy (PIT) as a new treatment option for prostate cancer cells. We generated conjugates consisting of either a humanized antibody or Fab fragments thereof targeting the prostate specific membrane antigen (PSMA), along with our silicon phthalocyanine photosensitizer dye WB692-CB1. PSMA-expressing prostate cancer cells were incubated with the antibody dye or Fab dye conjugates and cell binding was measured using flow cytometry. Cells were irradiated with varying doses of red light for dye activation, and cytotoxicity was determined by erythrosin B staining and subsequent analysis using a Neubauer counting chamber. Specific cytotoxicity was induced with the antibody dye conjugate in the prostate cancer cells in a light dose-dependent manner. Treatment of the cells with the Fab dye conjugate resulted in lower cytotoxicity, which could be attributed to a reduced binding affinity and a reduced dye uptake of the Fab fragment. Our new antibody dye and Fab dye conjugates offer potential for future intraoperative PIT in patients with localized prostate cancer, with the aim to ensure complete removal of tumor cells from the surgical area, to avoid local recurrence, and to improve clinical outcome.

Abstract 5750: Development of prostate specific membrane antigen-targeted liposomal zinc for the treatment of prostate cancer

Abstract Introduction: Treatment choices for early-stage prostate cancer include mostly watchful waiting, radical prostatectomy, or radiation therapy, whereas for advanced cases hormone therapy followed by chemo/radiotherapy are the preferred options, despite their notable side effects. There is a need for more effective therapy with lower side effects, especially for advanced scenarios. Unlike normal epithelial prostate cells, cancerous ones show reduced zinc (Zn) levels due to ZIP1 zinc transporter downregulation. Zn deficiency allows m-aconitase to remain active, making prostate cancer cells energy efficient and grow. High levels of Zn are needed to inhibit m-aconitase, the enzyme responsible for the first reaction in the Krebs cycle. This study aims to induce energy starvation and cell death of prostate cancer cells by inhibiting m-aconitase using the prostate-specific membrane antigen (PSMA)-targeting liposomal zinc. Methods: Liposomes were prepared by standard reverse phase evaporation method with DSPC, Cholesterol and DSPE-mPEG(2000) lipids. Zinc chloride was used to load Zn+2 into liposome following passive loading method. PSMA-expressing (PC3-PIP) and PSMA-null (PC3-LUC) human prostate cancer cells were used for in vitro studies. In the cell uptake study, cells were incubated with Dil-dye labeled liposomes for 2 hours before analyzing them by microscopy and flow cytometry. For the uptake inhibition study, cells were pre-incubated with PSMA-specific peptide for 2 hours before incubation with liposomes. Cytotoxicity studies were performed with PSMA-targeting Zn-loaded liposomes in PC3-PIP cells. Results: We conjugated the PSMA-specific peptide and control peptide (ASP3) with DSPE-PEG(3400)-NHS, and used them at various amounts (1 to 4 mol% of total lipid) to prepare Zn-loaded PSMA-targeting liposomes (TL) and control liposomes (CL), respectively. The average hydrodynamic diameter of all the liposomes was 120 to 130 nm, and zeta potential ranged from -16 to -12 mV. The encapsulation efficiency and loading capacity of Zn+2 were 0.6% and 8.5%, respectively. Cellular uptake studies showed a higher accumulation of PSMA-targeting liposome (TL) compared to non-targeting liposome (CL) in PSMA-expressing PC3 cells, whereas both liposomes showed low uptake in PSMA non-expressing cell. Furthermore, pre-treatment of PSMA-expressing cells with PSMA-specific peptide reduced the uptake of TL, indicating that the uptake of TL liposome is PSMA dependent. Finally, cytotoxicity studies showed that Zn-loaded TL, induces cell death in the PSMA-expressing cells. Overall, we developed PSMA-targeting Zn-containing liposomes that selectively target and kill PSMA-expressing prostate cancer cells. Future work includes testing the effect of Zn-loaded TL on m-aconitase activity and testing of in vivo efficacy of the formulation in prostate cancer models. Citation Format: Sujan K. Mondal, Alexander L. Klibanov, Anna Moore. Development of prostate specific membrane antigen-targeted liposomal zinc for the treatment of 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 5750.

Ligand-Directed H2 S Probe and Scavenger for Specific Tumor Imaging.

An expanding body of evidence suggests that specifically targeting hydrogen sulfide (H2 S) might potentially benefit both tumor diagnosis and treatment, but there is still a lack of cancer-targeted molecular tools for in vivo applications. Here, we report the first ligand-directed H2 S-specific near-infrared fluorescent sensor PSMA-Cy7-NBD and scavenger PSMA-Py-NBD that target the prostate-specific membrane antigen (PSMA). PSMA-Cy7-NBD displays a 53-fold off-on fluorescence response to H2 S at 803 nm with high specificity. PSMA-Py-NBD can scavenge H2 S fast (k2 =30.8 M-1 s-1 at 25 °C) without interference from biothiols. Both tools are highly water-soluble and can be transported selectively into PSMA-expressing prostate cancer cells. Endogenous H2 S levels in murine 22Rv1 tumor models can be imaged and downregulated by intravenous injection of PSMA-Cy7-NBD and PSMA-Py-NBD, respectively. These tools could potentially help to investigate H2 S cancer biology and with related therapies.

Enhanced cytotoxicity of a Pseudomonas Exotoxin A based immunotoxin against prostate cancer by addition of the endosomal escape enhancer SO1861.

Immunotoxins consist of an antibody or antibody fragment that binds to a specific cell surface structure and a cytotoxic domain that kills the cell after cytosolic uptake. Pseudomonas Exotoxin A (PE) based immunotoxins directed against a variety of tumor entities have successfully entered the clinic. PE possesses a KDEL-like motif (REDLK) that enables the toxin to travel from sorting endosomes via the KDEL-receptor pathway to the endoplasmic reticulum (ER), from where it is transported into the cytosol. There, it ADP-ribosylates the eukaryotic elongation factor 2, resulting in ribosome inhibition and finally apoptosis. One major problem of immunotoxins is their lysosomal degradation causing the need for much more immunotoxin molecules than finally required for induction of cell death. The resulting dose limitations and substantially increased side effects require new strategies to achieve improved cytosolic uptake. Here we generated an immunotoxin consisting of a humanized single chain variable fragment (scFv) targeting the prostate specific membrane antigen (PSMA) and the de-immunized PE variant PE24mut. This immunotoxin, hD7-1(VL-VH)-PE24mut, showed high and specific cytotoxicity in PSMA-expressing prostate cancer cells. We deleted the REDLK sequence to prevent transport to the ER and achieve endosomal entrapment. The cytotoxicity of this immunotoxin, hD7-1(VL-VH)-PE24mutΔREDLK, was greatly reduced. To restore activity, we added the endosomal escape enhancer SO1861 and observed an up to 190,000-fold enhanced cytotoxicity corresponding to a 57-fold enhancement compared to the initial immunotoxin with the REDLK sequence. A biodistribution study with different routes of administration clearly showed that the subcutaneous injection of hD7-1(VL-VH)-PE24mutΔREDLK in mice resulted in the highest tumor uptake. Treatment of mice bearing prostate tumors with a combination of hD7-1(VL-VH)-PE24mutΔREDLK plus SO1861 resulted in inhibition of tumor growth and enhanced overall survival compared to the monotherapies. The endosomal entrapment of non-toxic anti-PSMA immunotoxins followed by enhanced endosomal escape by SO1861 provides new therapeutic options in the future management of prostate cancer.

Modular Smart Molecules for PSMA-Targeted Chemotherapy.

New targeted chemotherapeutics are urgently needed to minimize off-target toxicity and reduce the high-mortality rate associated with metastatic prostate cancer. Herein, we report on the modular synthesis, pharmacokinetics, and efficacy of two small-molecule-drug conjugates (SMDC) targeted to prostate-specific membrane antigen (PSMA) incorporating either: (i) a cathepsin-B-cleavable valine-citrulline (Val-Cit), or (ii) an acid-cleavable phosphoramidate linker. Crucial components used in the design of the conjugates include: (i) CTT1298, a nanomolar affinity ligand that binds irreversibly to PSMA and has proven in past studies to rapidly internalize and shuttle payloads into PSMA-expressing prostate cancer cells, (ii) MMAE, a known potent cytotoxic payload, and (iii) an albumin-binder, proven to improve residence time of drug conjugates. At dose of 0.8 mg/kg (∼250 nmol/kg), the two SMDCs showed significant efficacy in a PSMA(+) PC3-PIP mouse model of human prostate cancer compared with controls, without inducing systemic toxicity. Though localization of the SMDCs was observed in tissues apart from the tumor, release of MMAE was observed predominantly in tumor tissue, at levels that were 2-3 orders of magnitude higher than non-target tissues. Furthermore, SMDC2, which incorporated a novel pH-responsive phosporamidate linker, demonstrated significantly improved efficacy over SMDC1 that has a Val-Cit linker, with a 100% survival over 90 days and 4 out of 8 mice showing complete tumor growth inhibition after 6 weekly doses of 0.8 mg/kg (244 nmol/kg). Our findings demonstrate the potential of irreversible PSMA inhibitors combined with pH-responsive linkers as a way to specifically deliver chemotherapeutic drugs to prostate cancer tumors with minimal toxicity.

PSMA-specific degradable dextran for multiplexed immunotargeted siRNA therapeutics against prostate cancer.

Small interfering RNA (siRNA) is ideal for gene silencing through a sequence-specific RNA interference process. The redundancy and complexity of molecular pathways in cancer create a need for multiplexed targeting that can be achieved with multiplexed siRNA delivery. Here, we delivered multiplexed siRNA with a PSMA-targeted biocompatible dextran nanocarrier to downregulate CD46 and PD-L1 in PSMA expressing prostate cancer cells. The selected gene targets, PD-L1 and CD46, play important roles in the escape of cancer cells from immune surveillance. PSMA, abundantly expressed by prostate cancer cells, allowed the prostate cancer-specific delivery of the nanocarrier. The nanocarrier was modified with acid cleavable acetal bonds for a rapid release of siRNA. Cell imaging and flow cytometry studies confirmed the PSMA-specific delivery of CD46 and PD-L1 siRNA to high PSMA expressing PC-3 PIP cells. Immunoblot, qRT-PCR and flow cytometry methods confirmed the downregulation of CD46 and PD-L1 following treatment with multiplexed siRNA.

Prostate-specific membrane antigen (PSMA) targeted singlet oxygen delivery via endoperoxide tethered ligands.

Singlet oxygen is the primary agent responsible for the therapeutic effects of photodynamic therapy (PDT). In this work, we demonstrate that singlet oxygen release due to thermal endoperoxide cycloreversion can be targeted towards specific features of selected cancer cells, and this targeted singlet oxygen delivery can serve as an effective therapeutic tool. Thus, cytotoxic singlet oxygen can be delivered regioselectively into prostate specific membrane antigen (PSMA) overexpressing lymph node carcinoma (LNCaP) cells. However, unlike typical photodynamic processes, there is no need for light or oxygen. The potential of the approach is exciting, considering the limitations on the availability of light and oxygen in deep-seated tumors.

Prostate-Specific Membrane Antigen-Targeted Turn-on Probe for Imaging Cargo Release in Prostate Cancer Cells.

The tunable nature of phosphoramidate linkers enables broad applicability as pH-triggered controlled-release platforms, particularly in the context of antibody- and small-molecule-drug conjugates (ADCs and SMDCs), where there remains a need for new linker technology. Herein, we explored in-depth the release of turn-on fluorogenic payloads from a homoserinyl-based phosphoramidate acid-cleavable linker. Kinetics of payload release from the scaffold was observed in buffers representing the pH conditions of systemic circulation, early and late endosomes, and lysosomes. It was found that payload release takes place in two key consecutive steps: (1) P-N bond hydrolysis and (2) spacer immolation. These two steps were found to follow pseudo-first-order kinetics and had opposite dependencies on pH. P-N bond hydrolysis increased with decreasing pH, while spacer immolation was most rapid at physiological pH. Despite the contrasting release kinetics of these two steps, maximal payload release was observed at the mildly acidic pH (5.0-5.5), while minimal payload release occurred at physiological pH. We integrated this phosphoramidate-payload linker system into a PSMA-targeted fluorescent turn-on probe to study the intracellular trafficking and release of a fluorescent payload in PSMA-expressing prostate cancer cells. Results showed excellent turn-on and accumulation of the coumarin payload in the late endosomal and lysosomal compartments of these cells. The release properties of this linker mark it as an attractive alternative in the modular design of ADCs and SMDCs, which demand selective intracellular payload release triggered by the pH changes that accompany intracellular trafficking.

Results of an ongoing phase 1/2a dose escalation study of HPN424, a tri-specific half-life extended PSMA-targeting T-cell engager, in patients with metastatic castration-resistant prostate cancer (mCRPC).

5013 Background: HPN424 is a prostate-specific membrane antigen (PSMA)-targeting T cell engager designed to redirect T cells to kill PSMA-expressing prostate cancer cells; engineered with three binding domains: anti-PSMA for tumor cell engagement, anti-albumin for half-life extension and anti-CD3 for T cell engagement. HPN424 is optimized for small size and increased stability compared to other bispecific platforms. Methods: This Ph1/2a study is evaluating HPN424 in mCRPC patients (pts) who have received > 2 prior systemic therapies. Primary endpoints are safety, tolerability and determination of MTD/RP2D. Secondary objectives are pharmacokinetics (PK), pharmacodynamics, immunogenicity and preliminary anti-tumor activity. HPN424 is administered IV once weekly. Tumor assessments include PSA, CT and bone scans every 9-weeks. Results: As of 2/8/21, 80 pts were dosed in 15 cohorts with target doses ranging from 1.3 to 160ng/kg fixed dose, and up to 300ng/kg with step dosing to the target dose after initial priming dose. Pts had received a median of 6 prior systemic regimens, 75% received prior chemotherapy for mCRPC. Median age was 70 (43 – 91). Most common grade > 3 TEAEs were AST increase (18%), anemia (11%) and ALT increase (11%). DLTs include CRS G3 (n = 3), elevated lipase G3 (n = 1) and seizure G3 (n = 1). These events did not limit escalation, MTD has not been reached and escalation continues. All grade CRS occurred in 63% of pts, 4% grade 3 per ASTCT and no Grade 4/5 CRS. CRS G3 events occurred after first administration of target dose (n = 2 fixed dose, n = 1 step dose). Transaminase elevation occurred predominantly during Cycle 1, was transient and had no clinical sequelae. Disease progression was the primary reason for drug discontinuation; 2 pts (3%) discontinued due to TRAE. Reduction in circulating tumor cells (CTC) was seen in 32 of 56 pts (57%) with measurable CTC at baseline. Fifteen of 62 pts (24%) with > 24 weeks follow-up remained on treatment ≥ 24 weeks. Thirteen of 63 pts (21%) with post-baseline levels had PSA declines from baseline, including 3 PSA50, 2 PSA30 responses. In chemo-naïve pts, 5 of 15 (33%) showed PSA declines post-baseline. In the highest fixed dose cohort (160ng/kg) tested to-date, 3 of 7 evaluable pts had PSA declines from baseline and 1 had a confirmed partial response per RECIST. Conclusions: HPN424, a novel half-life extended PSMA-targeting T cell engager, was well tolerated when administered once weekly. AEs were transient, manageable and consistent with class of agent. Grade 3 CRS was observed in 4% of patients, occurring with first administration of target dose. Evidence of antitumor activity included PSA and CTC reductions and treatment duration > 24 weeks in 15/62 pts. Encouraging signals were seen at the highest fixed dose cohort including a confirmed RECIST partial response. NCT03577028 Clinical trial information: NCT03577028.

A prostate-specific membrane antigen (PSMA)-targeted prodrug with a favorable in vivo toxicity profile

Prostate-specific membrane antigen (PSMA) is a promising target for the treatment of advanced prostate cancer (PC) and various solid tumors. Although PSMA-targeted radiopharmaceutical therapy (RPT) has enabled significant imaging and prostate-specific antigen (PSA) responses, accumulating clinical data are beginning to reveal certain limitations, including a subgroup of non-responders, relapse, radiation-induced toxicity, and the need for specialized facilities for its administration. To date non-radioactive attempts to leverage PSMA to treat PC with antibodies, nanomedicines or cell-based therapies have met with modest success. We developed a non-radioactive prodrug, SBPD-1, composed of a small-molecule PSMA-targeting moiety, a cancer-selective cleavable linker, and the microtubule inhibitor monomethyl auristatin E (MMAE). SBPD-1 demonstrated high binding affinity to PSMA (Ki = 8.84 nM) and selective cytotoxicity to PSMA-expressing PC cell lines (IC50 = 3.90 nM). SBPD-1 demonstrated a significant survival benefit in two murine models of human PC relative to controls. The highest dose tested did not induce toxicity in immunocompetent mice. The high specific targeting ability of SBPD-1 to PSMA-expressing tumors and its favorable toxicity profile warrant its further development.

PSMA conjugated combinatorial liposomal formulation encapsulating genistein and plumbagin to induce apoptosis in prostate cancer cells

Although the biomedical sciences have achieved tremendous success in developing novel approaches to managing prostate cancer, this disease remains one of the major health concerns among men worldwide. Liposomal formulations of single drugs have shown promising results in cancer treatment; however, the use of multi drugs has shown a better therapeutic index than individual drugs. The identification of cancer-specific receptors has added value to design targeted drug delivering nanocarriers. We have developed genistein and plumbagin co-encapsulating liposomes (∼120 nm) with PSMA specific antibodies to target prostate cancer cells selectively in this work. These liposomes showed >90 % decrease in PSMA expressing prostate cancer cell proliferation without any appreciable toxicity to healthy cells and human red blood cells. Release of plumbagin and genistein was found to decrease the expression of PI3/AKT3 signaling proteins and Glut-1 receptors (inhibited glucose uptake and metabolism), respectively. The decrease in migration potential of cells and induced apoptosis established the observed anti-proliferative effect in prostate cancer cell lines. The discussed strategy of developing novel, non-toxic, and PSMA specific antibody conjugated liposomes carrying genistein and plumbagin drugs may also be used for encapsulating other drugs and inhibit the growth of different types of cancers.

The PSMA-targeting Half-life Extended BiTE Therapy AMG 160 has Potent Antitumor Activity in Preclinical Models of Metastatic Castration-resistant Prostate Cancer.

Metastatic castration-resistant prostate cancer (mCRPC) remains a disease with high unmet medical need, as most patients do not achieve durable response with available treatments. Prostate-specific membrane antigen (PSMA) is a compelling target for mCRPC. It is highly expressed by primary and metastatic prostate cancer cells, with increased expression after progression on androgen deprivation therapy. We developed AMG 160, a half-life extended, bispecific T-cell engager immuno-oncology therapy that binds PSMA on prostate cancer cells and cluster of differentiation 3 on T cells for treatment of mCRPC. AMG 160 was evaluated in vitro and in mCRPC xenograft models. AMG 160 tolerability was assessed in nonhuman primates (NHP). AMG 160 activity as monotherapy and in combination with a PSMA-imaging agent, novel hormonal therapy, and immune checkpoint blockade was evaluated. AMG 160 induces potent, specific killing of PSMA-expressing prostate cancer cell lines in vitro, with half-maximal lysis of 6-42 pmol/L. In vivo, AMG 160 administered weekly at 0.2 mg/kg engages T cells administered systemically and promotes regression of established 22Rv-1 mCRPC xenograft tumors. AMG 160 is compatible with the imaging agent gallium 68-labeled PSMA-11, and shows enhanced cytotoxic activity when combined with enzalutamide or an anti-programmed death-1 antibody. AMG 160 exhibits an extended half-life and has an acceptable safety profile in NHPs. The preclinical characterization of AMG 160 highlights its potent antitumor activity in vitro and in vivo, and its potential for use with known diagnostic or therapeutic agents in mCRPC. These data support the ongoing clinical evaluation of AMG 160 in patients with mCRPC.See related commentary by Kamat et al., p. 2675.

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

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

Intracellular Delivery of Glucose Oxidase for Enhanced Cytotoxicity toward PSMA-Expressing Prostate Cancer Cells.

Reactive oxygen species (ROS) forming enzymes are of significant interest as anticancer agents due to their potent cytotoxicity. A key challenge in their clinical translation is attaining site-specific delivery and minimizing biodistribution to healthy tissues. Here, complexes composed of the ROS enzyme glucose oxidase (GOX), poly-l-lysine-grafted-polyethylene glycol (PLL-g-PEG), and anti-prostate specific membrane antigen (anti-PSMA) monoclonal antibody are synthesized for localized delivery and uptake in prostate cancer cells. Formation of anti-PSMA-PLL-g-PEG/GOX results in nanoscale complexes ≈30 nm in diameter with a ζ-potential of 6 mV. The anti-PSMA-PLL-g-PEG/GOX complexes show significant cytotoxicity (≈60% reduction in cell viability) against PSMA-expressing LNCaP cells compared to unmodified GOX. Importantly, cytotoxicity in LNCaP cells occurrs concurrently with anti-PSMA-PLL-g-PEG/GOX uptake and increases in intracellular generation of ROS. These results demonstrate that cytotoxicity of ROS inducing enzymes can be enhanced by intracellular delivery compared to equivalent concentrations of free enzyme, providing a novel means for cancer therapy.

Localised delivery of doxorubicin to prostate cancer cells through a PSMA-targeted hyperbranched polymer theranostic.

The therapeutic potential of hyperbranched polymers targeted to prostate cancer and loaded with doxorubicin was investigated. Polyethylene glycol hyperbranched polymers were synthesised via RAFT polymerisation to feature glutamate urea targeting ligands for PSMA on the periphery. The chemotherapeutic, doxorubicin, was attached to the hyperbranched polymers through hydrazone formation, which allowed controlled release of the drug from the polymers invitro endosomal conditions, with 90% release of the drug over 36h. The polymers were able to target to PSMA-expressing prostate cancer cells invitro, and demonstrated comparable cytotoxicity to free doxorubicin. The ability of the hyperbranched polymers to specifically facilitate transport of loaded doxorubicin into the cells was confirmed using live cell confocal imaging, which demonstrated that the drug was able to travel with the polymer into cells by receptor mediated internalisation, and subsequently be released into the nucleus following hydrazone degradation. Finally, the ability of the complex to induce a therapeutic effect on prostate cancer cells was investigated through a long term tumour regression study, which confirmed that the DOX-loaded polymers were able to significantly reduce the volume of subcutaneous prostate tumours invivo in comparison to free doxorubicin and a polymer control, with no adverse toxicity to the animals. This work therefore demonstrates the potential of a hyperbranched polymer system to be utilised for prostate cancer theranostics.

Abstract 3111A: ADCT-401/MEDI3726, a novel pyrrolobenzodiazepine (PBD)-based antibody-drug conjugate (ADC) targeting PSMA-expressing prostate cancers

Abstract Prostate-specific membrane antigen (PSMA) is an attractive target for an ADC approach as it is over-expressed by virtually all prostate cancers and its expression is highest in poorly-differentiated, metastatic and castration-resistant cases. PSMA has limited expression in non-prostatic tissues, it is not secreted or cleaved by PSMA-expressing cells, and it is constitutively internalized, a process that may be accelerated by specific antibody binding. ADCT-401/MEDI3726 is an ADC composed of a monoclonal antibody (J591), directed against human PSMA, site-specifically conjugated (drug-to-antibody ratio of 1.8) to a highly cytotoxic DNA cross-linking PBD dimer with a valine-alanine dipeptide linker. In vitro, ADCT-401/MEDI3726 demonstrated potent and specific cytotoxicity in a panel of PSMA-expressing prostate cancer cell lines, whereas its activity was greatly reduced in PSMA-negative cell lines. In vivo, ADCT-401/MEDI3726 showed strong antitumor activity against CWR22Rv1 and LNCaP human-derived prostate cancer xenograft models. In the CWR22Rv1 model, a tumor with low, heterogeneous PSMA expression, ADCT-401/MEDI3726 achieved dose-dependent antitumor activity when administered as single dose at either 0.33 or 1 mg/kg, which resulted in significant increase in survival compared to the vehicle-treated animals. Moreover, a single dose of ADCT-401/MEDI3726 showed remarkable superior antitumor activity compared to multiple doses of another PSMA-targeted ADC stochastically conjugated to the auristatin payload vcMMAE with a DAR of ~4. In the LNCaP model, ADCT-401/MEDI3726 resulted in dose-dependent antitumor activity when dosed once at 0.11, 0.33 or 1 mg/kg. In the PSMA-negative prostate cancer-derived PC3 xenograft model, ADCT-401/MEDI3726 and an isotope-control ADC showed limited tumor growth inhibition highlighting target-mediated antitumor activity. ADCT-401/MEDI3726 demonstrated potent and specific in vitro and in vivo antitumor activity in prostate cancer-derived models of differing levels of PSMA and this warrants further development of this ADC into the clinic. Citation Format: Francesca Zammarchi, Simon Chivers, Karin Havenith, David G. Williams, Lauren Adams, Maria Mellinas-Gomez, Simon Corbett, Peter Tyrer, Francois D'Hooge, Song Cho, Nazzareno Dimasi, Mary Jane Hinrichs, Phil W. Howard, John A. Hartley, Patrick H. van Berkel. ADCT-401/MEDI3726, a novel pyrrolobenzodiazepine (PBD)-based antibody-drug conjugate (ADC) targeting PSMA-expressing prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3111A. doi:10.1158/1538-7445.AM2017-3111A

PSMA-Targeted Theranostic Nanocarrier for Prostate Cancer.

Herein, we report the use of a theranostic nanocarrier (Folate-HBPE(CT20p)) to deliver a therapeutic peptide to prostate cancer tumors that express PSMA (folate hydrolase 1). The therapeutic peptide (CT20p) targets and inhibits the chaperonin-containing TCP-1 (CCT) protein-folding complex, is selectively cytotoxic to cancer cells, and is non-toxic to normal tissue. With the delivery of CT20p to prostate cancer cells via PSMA, a dual level of cancer specificity is achieved: (1) selective targeting to PSMA-expressing prostate tumors, and (2) specific cytotoxicity to cancer cells with minimal toxicity to normal cells. The PSMA-targeting theranostic nanocarrier can image PSMA-expressing cells and tumors when a near infrared dye is used as cargo. Meanwhile, it can be used to treat PSMA-expressing tumors when a therapeutic, such as the CT20p peptide, is encapsulated within the nanocarrier. Even when these PSMA-targeting nanocarriers are taken up by macrophages, minimal cell death is observed in these cells, in contrast with doxorubicin-based therapeutics that result in significant macrophage death. Incubation of PSMA-expressing prostate cancer cells with the Folate-HBPE(CT20p) nanocarriers induces considerable changes in cell morphology, reduction in the levels of integrin β1, and lower cell adhesion, eventually resulting in cell death. These results are relevant as integrin β1 plays a key role in prostate cancer invasion and metastatic potential. In addition, the use of the developed PSMA-targeting nanocarrier facilitates the selective in vivo delivery of CT20p to PSMA-positive tumor, inducing significant reduction in tumor size.

Design, production, and characterization of a single-chain variable fragment (ScFv) derived from the prostate specific membrane antigen (PSMA) monoclonal antibody J591

A single chain variable fragment (J591 ScFv) that recognizes the extracellular glyco-protein prostate specific membrane antigen (PSMA) was designed, constructed, and expressed in Pichia pastoris. Construction of the J591 ScFv was based on the reported complementarity-determining region (CDR) of the PSMA specific J591 monoclonal antibody (mAb). The nucleotide sequence encoding the J591-derived ScFv was codon-optimized for expression in P. pastoris and a 6× his-tag was added to facilitate affinity purification. A down-scale 2L methanol-induced P. pastoris fermentation yielded 330mg of total protein following a 96h induction. Following Immobolized Metal Affinity Chromatography, functionality of the J591 ScFv was confirmed via Western blot, immunoblot, binding studies, and flow cytometry analysis. The J591 ScFv showed binding affinity and specificity to cell extracts containing PSMA and PSMA-expressing prostate cancer cells. Our results demonstrate that functional J591 ScFv can be produced in P. pastoris for use in diagnostic and targeted therapeutic applications.