Xinning Wang

In Vitro and In Vivo Responses of Advanced Prostate Tumors to PSMA ADC, an Auristatin-Conjugated Antibody to Prostate-Specific Membrane Antigen

Prostate-specific membrane antigen (PSMA) is a membrane protein that is overexpressed manifold in prostate cancer and provides an attractive target for therapy. PSMA ADC is an antibody-drug conjugate (ADC) that consists of a fully human anti-PSMA monoclonal antibody conjugated to monomethylauristatin E through a valine-citrulline linker. In this study, the antitumor activity of PSMA ADC was evaluated against a panel of prostate cancer cell lines in vitro and in a novel in vivo model of taxane-refractory human prostate cancer. In vitro cell killing was efficient for cells with abundant PSMA expression (>105 molecules/cell; IC50 ≤ 0.022 nmol/L) and 1,000-fold less efficient for cells with undetectable PSMA (IC50 > 30 nmol/L). Intermediate potency (IC50 = 0.80 nmol/L) was observed for cells with approximately 104 molecules of PSMA per cell, indicating a threshold PSMA level for selective cell killing. Similar in vitro activity was observed against androgen-dependent and -independent cells that had abundant PSMA expression. In vitro activity of PSMA ADC was also dependent on internalization and proper N-glycosylation/folding of PSMA. In contrast, less potent and nonselective cytotoxic activity was observed for a control ADC, free monomethylauristatin E, and other microtubule inhibitors. PSMA ADC showed high in vivo activity in treating xenograft tumors that had progressed following an initial course of docetaxel therapy, including tumors that were large (>700 mm3) before treatment with PSMA ADC. This study defines determinants of antitumor activity of a novel ADC. The findings here support the clinical evaluation of this agent in advanced prostate cancer. Mol Cancer Ther; 10(9); 1728–39. ©2011 AACR.

A Novel Alphavirus Vaccine Encoding Prostate-Specific Membrane Antigen Elicits Potent Cellular and Humoral Immune Responses

Purpose: Prostate-specific membrane antigen (PSMA) is an attractive target for active immunotherapy. Alphavirus vaccines have shown promise in eliciting immunity to tumor antigens. This study investigated the immunogenicity of alphavirus vaccine replicon particles (VRP) that encode PSMA (PSMA-VRP). Experimental Design: Cells were infected with PSMA-VRP and evaluated for PSMA expression and folate hydrolase activity. Mice were immunized s.c. with PSMA-VRP or purified PSMA protein. Sera, splenocytes, and purified T cells were evaluated for the magnitude, durability, and epitope specificity of the anti-PSMA response. Antibodies were measured by flow cytometry, and cellular responses were measured by IFN-γ enzyme-linked immunospot and chromium release assays. Cellular responses in BALB/c and C57BL/6 mice were mapped using overlapping 15-mer PSMA peptides. A Good Laboratory Practice–compliant toxicology study was conducted in rabbits. Results: PSMA-VRP directed high-level expression of active PSMA. Robust T-cell and B-cell responses were elicited by a single injection of 2 × 105 infectious units, and responses were boosted following repeat immunizations. Anti-PSMA responses were detected following three immunizations with 102 infectious units and increased with increasing dose. PSMA-VRP was more immunogenic than adjuvanted PSMA protein. Responses to PSMA-VRP were characterized by Th-1 cytokines, potent CTL activity, and IgG2a/IgG2b antibodies. T-cell responses in BALB/c and C57BL/6 mice were directed toward different PSMA peptides. Immunogenic doses of PSMA-VRP were well tolerated in mice and rabbits. Conclusions: PSMA-VRP elicited potent cellular and humoral immunity in mice, and specific anti-PSMA responses were boosted on repeat dosing. PSMA-VRP represents a promising approach for immunotherapy of prostate cancer.

Targeted treatment of prostate cancer

Over a half century ago, Charles Huggins demonstrated the response of prostate cancer to androgen deprivation therapy. Subsequently, many discoveries and evolving findings continued to support a research rationale focused on the androgen receptor (AR) as a key target for prostate cancer. More recently, preliminary trials have suggested that other targets could also be useful in the treatment of prostate cancer, and the proposed strategies for treatment have ranged from targeted toxins to immunotherapeutic agents. We provide an overview of some of these approaches, with an emphasis on those that employ prostate specific membrane antigen (PSMA) as a target. J. Cell. Biochem. 102: 571–579, 2007.

Design and Synthesis of a PSMA Inhibitor–Doxorubicin Conjugate for Targeted Prostate Cancer Therapy

Prostate cancer is the most commonly diagnosed cancer and the second leading cause of death in men in the United States after lung cancer. It is estimated that prostate cancer affects approximately 180000 and kills 40000 men in the United States each year. As long as the cancer is confined to the prostate, it can be successfully controlled by surgery or radiation, but there is little effective treatment available for the metastatic disease, particularly if androgen-deprivation therapy fails. 3] Advances in our understanding of tumor immunology have, however, led to new approaches for treatment of prostate and other cancers, including tumor antigen-specific immunotherapy. Several prostate antigens have now been identified that are attractive candidates for both prostate-tumor imaging and therapy. One of the most interesting among these is the prostate-specific membrane antigen (PSMA). PSMA is considerably upregulated in prostate cancers, metastatic disease, and in hormone-refractory prostate cancers. 7] Capromab pendetide (ProstaScint) is a commercially available monoclonal antibody in clinical use for detecting prostate cancer, but it has been cited for complexities associated with its administration and the interpretation of the images obtained. PSMA is a 100 kDa, type II membrane glycoprotein highly expressed by all prostate cancers as well as by nonprostatic tumor neovasculature and the vascular endothelium of virtually all solid sarcoma and carcinoma tumors. PSMA is highly homologous to the neuropeptidase NAALADase (GCP II) that releases the neurotransmitter glutamate from the neuronal peptide NAAG (N-acetyl-l-aspartyl-l-glutamate). PSMA also exhibits folate hydrolase activity whereby it cleaves terminal glutamates from g-linked polyglutamates. A 3.5 < resolution crystal structure of the PSMA ectodomain has been disclosed recently. The homodimer contains a binuclear zinc site, catalytic residues, and a proposed substrate-binding arginine patch, which is similar to our previously modeled three-dimensional structure of the GCP II extracellular domain obtained through homology-based modeling methods. Based upon mechanistic considerations together with the modeling studies, we have developed an array of potent, urea-based inhibitors of NAAG peptidase/PSMA. Since PSMA is significantly upregulated in prostate cancer and metastasis, and is strongly expressed in the neovasculature of solid tumors, we have explored the use of our designed small-molecule inhibitors as PET imaging agents. As these imaging applications proved successful, we turned our attention to the possibility of using these same PSMA inhibitors for the targeting of anticancer drugs to prostate cancer cells. Anticancer drugs, including doxorubicin and others, have limited utility in prostate cancer therapy due to their poor target selectivity and systemic toxicities, such as cardiotoxicity and immunosuppression. Targeted therapy for cancer offers potential improvements over existing chemotherapy regimens since the drug is delivered preferentially to the cancer tissue and side effects can be minimized. Research in this area has produced several cytotoxic conjugates with improved selectivity and drug effects. We sought to synthesize a conjugate between one of our PSMA inhibitors and the anticancer drug doxorubicin in order to test whether such a hybrid molecule would lead to a drug with an improved therapeutic index. We report here the design, synthesis, and biological activity of PSMA–doxorubicin conjugate 1 targeted for prostate cancer therapy. In the design of the PSMA–doxorubicin conjugate, it was important to define the optimal sites on both the doxorubicin and the PSMA inhibitor for attachment of a linker. It is known that acylation of the amino group present in the pyranose ring of doxorubicin results in a decrease in the cytotoxicity of this drug. Our previous SAR work on the NAAG peptidase inhibitors had revealed that the urea formed between glutamate and a variety of phenylalanine derivatives containing diverse substituents on the para-position of the aromatic ring serve as potent (nm) peptidase inhibitors. We elected to join a ureabased PSMA inhibitor made up of p-aminophenylalanine through a glutaric acid linker to doxorubicin (Scheme 1). The amino groups present in these two moieties thus became the key connection points. In pursuing this approach, we imagined that the PSMA inhibitor would serve to target the doxorubicin to the prostate cancer cells. Once it was localized there and internalized, other amidase/peptidase activities (e.g. , PSA, but not the extracellular PSMA, as this would be inhibited by the bioconjugate) would free the doxorubicin. In support of the possibility for internalization, we refer to the recent article by Langer et al. that demonstrates that a nanoparticle–aptamer bioconjugate comprising RNA aptamers that target PSMA can be internalized into LNCaP cells after a 2 h incubation period. Moreover, a previous report has revealed an antibody-mediated enhancement in the rate of PSMA endocytosis in LNCaP cells. With the above considerations in mind, we began the synthesis of our designed, small-molecule–PSMA inhibitor-based bioconjugate with the preparation of the urea derivative 6, as shown in Scheme 2. [a] Dr. S. Jayaprakash, Prof. A. P. Kozikowski Drug Discovery Program Department of Medicinal Chemistry and Pharmacognosy 833 South Wood Street, Chicago, IL 60612 (USA) Fax: (1) 312-996-7107 E-mail : kozikowa@uic.edu [b] Dr. X. Wang, Dr. W. D. Heston The Cleveland Clinic Foundation Department of Cancer Biology 9500 Euclid Avenue, Cleveland, OH 44195 (USA) Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author : experimental procedures and characterization data for compounds 1, 3, 4, 6–10.

The Role of 18F-FDG PET/CT for Initial Staging of Nasal Type Natural Killer/T-Cell Lymphoma: A Comparison with Conventional Staging Methods

The utility of 18F-FDG PET/CT in patients with nasal-type natural killer (NK)/T-cell lymphoma has not been established. Therefore, we evaluated the role of 18F-FDG PET/CT for determining cancer staging by comparing its results to those of conventional staging methods (CSMs) (physical examination, CT with intravenous contrast, biopsies from primary sites, and bone marrow examinations) in patients with nasal-type NK/T-cell lymphoma. Methods: In this study, 52 consecutive patients (34 men, 18 women; mean age, 49.4 y) with newly diagnosed nasal-type NK/T-cell lymphoma were studied. Anatomic regions (n = 1,300; 16 nodal and 9 extranodal regions per patient) were assessed with an 18F-FDG PET/CT scan and with CSMs, and each anatomic region was classified as positive or negative for malignancy. Biopsy and clinical follow-up, including additional imaging studies, were used as the gold standard for diagnosis. Results: Of the 59 nodal and 71 extranodal anatomic regions that were truly positive for malignancy, 18F-FDG PET/CT detected 58 nodal and 69 extranodal. CSMs, however, detected only 44 of the nodal and 61 of the extranodal anatomic regions that were positive for malignancy (nodal comparison of PET/CT vs. CSMs, P < 0.001; extranodal comparison of PET/CT vs. CSMs, P = 0.008). PET/CT scans exhibited a significantly better sensitivity (97.7% vs. 80.7%, P < 0.001) than CSMs for the detection of malignant lesions. PET/CT findings altered the original staging category for 12 patients (21.2%) and affected treatment planning in 23 cases (44.2%). Conclusion: Our study demonstrated that 18F-FDG PET/CT scanning is a valuable modality for staging and treatment planning in patients with nasal-type NK/T-cell lymphoma.

Improving the biodistribution of PSMA-targeting tracers with a highly negatively charged linker

Prostate specific membrane antigen (PSMA) is overexpressed in prostate cancer and in tumor vasculature. Small molecule based inhibitors of PSMA have promised to provide sensitive detection of primary and metastatic prostate tumors. Although significant progress has been made, many of the radiolabeled imaging agents exhibit non‐specific background binding. Prevailing tracer designs focus on high affinity urea‐based inhibitors with strategically placed hydrophobic patches that interact favorably with the substrate tunnel of PSMA. We hypothesized that a novel PSMA inhibitor design incorporating highly negatively charged linkers may minimize non‐specific binding and decrease overall background.

Robotic Real-time Near Infrared Targeted Fluorescence Imaging in a Murine Model of Prostate Cancer: A Feasibility Study

OBJECTIVE To evaluate the detection of near-infrared fluorescence from prostate tumors stained with a prostate-specific membrane antigen (PSMA)-targeted tracer developed in our institution with a novel robotic imaging system. METHODS Prostate cancer cell lines PC3-pip (PSMA positive) and PC3-flu (PSMA negative) were implanted subcutaneously into 6 immunodeficient mice. When tumors reached 5 mm, a PSMA-targeted fluorescent conjugate was injected intravenously. The first 3 mice underwent near-infrared imaging immediately and hourly up to 4 hours after injection to determine the time necessary to obtain peak fluorescence and were killed. The last 3 mice were imaged once preoperatively and were euthanized 120 minutes later. Excision of the tumors was performed by using a novel robotic imaging system to detect near-infrared fluorescence in real time. Specimens were submitted for pathology. RESULTS In the first 3 mice, we found 120 minutes as the time needed to observe peak fluorescence from the PSMA-positive tumors. We identified discrete near-infrared fluorescence from 2 of 3 PSMA-positive tumors with the robotic imaging system. Surgical margins were negative for all excised specimens except for one PSMA-negative tumor. CONCLUSIONS Real-time near-infrared fluorescence imaging of prostate cancer is feasible with a novel robotic imaging system. Further research is needed to optimize the signal intensity detectable from prostate cancer with our tracer. Toxicologic studies are needed before its clinical use.

2-5A ligands - A new concept for the treatment of prostate cancer

Several potent prostate specific membrane antigen (PSMA) inhibitors have been described recently. We generated a PSMA-specific 2-5A ligand called RBI 1033 by linking 2-5A to the N-acetylaspartylglutamate (NAAG)-based inhibitor ZJ-24. We measured the inhibitory activity of RBI 1033 to the folate hydrolase activity of PSMA. Amazingly, we found that compared to ZJ-24 (IC50 = 53.9 nM), RBI 1033 was more than 10 times more potent (IC50 = 4.78 nM) as a folate hydrolase inhibitor, while SMCC 2-5A lacking the ZJ-24 part, did not show much activity (IC50 = 1974 nM). Also, RBI 1033s affinity to PSMA was found to be 10 times higher than ZJ-24 itself.

Structure-Activity Relationships of 2′,5′-Oligoadenylate Analogue Modifications of Prostate-Specific Membrane Antigen (PSMA) Antagonists

Prostate-specific membrane antigen (PSMA) is an ideal biomarker for prostate cancer. A previously reported 2-5A conjugate RBI1033 (3) showed binding affinity more than 10 times higher than the parent urea-based compound (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic acid (1). The purpose of this work is to further optimize the structure of 3 to identify highly selective ligands of PSMA. It was found that conjugates having 2-5A in their structure showed extraordinary improved binding affinity to PSMA compared with compound 1. Removal of 2-5A significantly reduced its biological activity. The results will provide a path to agents for targeted imaging and treatment of prostate cancer.

Selenoprotein P neutralizes lipopolysaccharide and participates in hepatic cell endoplasmic reticulum stress response.

Low serum selenium or selenoprotein P (SePP) levels have been repetitively observed in severe sepsis. The role of SePP in sepsis is incompletely characterized. To test the hypothesis that lipopolysaccharide (LPS) interacts with SePP, we investigated the interaction between LPS and the histidine‐rich (His‐rich) regions of SePP. We demonstrate that both purified SePP and synthetic peptides corresponding to the His‐rich motifs neutralized LPS. In addition, we used a hepatocyte model to study the fate of SePP in response to LPS or endoplasmic reticulum (ER) stress. Our findings indicate that ER stress increases the cellular level of SePP and promotes its nuclear localization.