Sae Kim

Noninvasive measurement of androgen receptor signaling with a positron-emitting radiopharmaceutical that targets prostate-specific membrane antigen

Despite encouraging clinical results with next generation drugs (MDV3100 and abiraterone) that inhibit androgen receptor (AR) signaling in patients with castration-resistant prostate cancer (CRPC), responses are variable and short-lived. There is an urgent need to understand the basis of resistance to optimize their future use. We reasoned that a radiopharmaceutical that measures intratumoral changes in AR signaling could substantially improve our understanding of AR pathway directed therapies. Expanding on previous observations, we first show that prostate-specific membrane antigen (PSMA) is repressed by androgen treatment in multiple models of AR-positive prostate cancer in an AR-dependent manner. Conversely, antiandrogens up-regulate PSMA expression. These expression changes, including increased PSMA expression in response to treatment with the antiandrogen MDV3100, can be quantitatively measured in vivo in human prostate cancer xenograft models through PET imaging with a fully humanized, radiolabeled antibody to PSMA, 64Cu-J591. Collectively, these results establish that relative changes in PSMA expression levels can be quantitatively measured using a human-ready imaging reagent and could serve as a biomarker of AR signaling to noninvasively evaluate AR activity in patients with CRPC.

Docetaxel down‐regulates the expression of androgen receptor and prostate‐specific antigen but not prostate‐specific membrane antigen in prostate cancer cell lines: Implications for PSA surrogacy

Docetaxel (DOC) has potent anti‐tumor efficacy as a result of promoting microtubule assembly and microtubule bundling thereby impairing mitosis. Knowing that some anti‐microtubule agents affect the polarity of prostate‐specific membrane antigen (PSMA) expression and that androgen ablation can up‐regulate PSMA expression, we sought to determine any effect of DOC on PSMA expression in prostate cancer (PC) cell lines as a prelude to a clinical effort. As controls, we also looked at the expression of androgen receptor (AR) and prostate‐specific antigen (PSA).

Saporin toxin-conjugated monoclonal antibody targeting prostate-specific membrane antigen has potent anticancer activity.

Prostate‐specific membrane antigen (PSMA) provides an attractive target for monoclonal antibody targeted therapies in the treatment of prostate cancer (PC). In this study, we generated an immunotoxin by linking a humanized anti‐PSMA monoclonal antibody (hJ591) to the ribosome‐inactivating protein toxin saporin. The hJ591–saporin immunoconjugate was evaluated for antitumor activity against PC cells.

Prostate-specific Membrane Antigen (psma) Expression in the Neovasculature of Gynecologic Malignancies: Implications for Psma-targeted Therapy.

The goal of the study was to examine expression of prostate-specific membrane antigen (PSMA) in neovasculature of gynecologic cancers, as PSMA-targeted therapy has showed a promise in treatment of advanced carcinomas. The study included cervical carcinoma (n=28), vulvar carcinoma (n=20), endometrial carcinoma (n=23), primary ovarian carcinoma (n=21), metastatic ovarian carcinoma (n=25), and normal cervix (n=12) as negative control. All cases were immunostained using anti-CD31 antibody to delineate capillary endothelial cells. In parallel, all cases were immunostained using anti-PSMA antibody. The PSMA staining was assessed in tumor capillaries and in normal tissues and scored as a percentage of CD31 staining. PSMA expression was found in the tumor neovasculature, and no significant expression was identified in vasculature of normal tissues. The extent of PSMA staining in tumor capillaries varied from high expression in ovarian and endometrial cancers, to medium expression in cervical squamous cell carcinomas, and low expression in cervical adenocarcinomas and vulvar cancers. All (100%) cases of primary ovarian carcinoma, ovarian carcinoma metastases, and primary endometrial carcinoma showed PSMA expression in tumor vasculature, which was diffuse in majority of cases. The expression of PSMA in ovarian cancer metastases was similar among different metastatic foci of the same tumor. Fifteen percent of cervical squamous cell carcinoma, 50% of cervical adenocarcinoma, and 75% of vulvar carcinomas showed no capillary expression of PSMA. In conclusion, PSMA is highly and specifically expressed in the neovasculature of ovarian, endometrial, and cervical squamous carcinoma, rendering it a potential therapeutic vascular target.

Abstract 5201: PSMA-targeted alpha radioimmunotherapy for prostate cancer with225Ac-J591

Background: Prostate cancer (PC) is a radiosensitive disease. In addition to external beam and brachytherapy, systemic bone-seeking radioisotopes have been utilized clinically for many years, with radium-223 leading to overall survival benefits. However, the available beta and alpha emitting particles in the clinic target tumors / stroma in bone only. Biologically targeted radiopharmaceutical treatment has shown promising results in our earlier multiple clinical trials using beta-emitting anti-PSMA therapy with Leutetium-177 radiolabeled anti-PSMA monoclonal antibody (mAb) J591, with accurate targeting and tumor response, but treatment is limited by myelosuppression. Both beta and alpha-small molecule PSMA ligand directed therapy has been utilized anecdotally outside of the US, but has not been systemically studied. Based upon their biodistribution, targeting of salivary glands and potentially proximal renal tubules may pose longer term toxicity issues. mAb-based targeted alpha-particle delivery may be advantageous with less expected myelosuppression than seen with β-emitters. Experimental procedures: To optimize labeling of humanized DOTA-J591 mAb, actinium-225 (225Ac) nitrate was obtained from Oak Ridge National Laboratories and was dissolved in HCl solution and then mixed with trimethyl ammonium acetate and L-ascorbic acid. This preparation was incubated with DOTA-J591. The labeling efficiency (LE) was determined by ITLC-SG. If the LE is >60%, 225Ac-DOTA-J591 mAb was purified by gel filtration and sterilized by membrane filtration. In a pilot pre-clinical study, we studied the toxicity in non-tumor bearing BALB/c mice. Results: The radiochemical purity is >95% and the immunoreactivity was >80%. Twenty-five BALB/c mice received from 2.1 to 6.3 KBq of 225Ac-J591. Treatment was well tolerated, with all mice alive and healthy at Day 26. Conclusions: A stable alpha emitting-antibody complex has been produced with 225Ac-J591. Initial mouse safety experiments have been successful. A RIT dose escalation study of PSMA-targeted alpha emitter in a LNCaP xenograft model is underway. Citation Format: Jaspreet S. Batra, He Liu, Sae Kim, Vicente N. Navarro, Shankar Vallabhajosula, Scott T. Tagawa, Neil H. Bander. PSMA-targeted alpha radioimmunotherapy for prostate cancer with 225Ac-J591 [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 5201. doi:10.1158/1538-7445.AM2017-5201

Abstract 1905: Possible cancer stem cells: Folate hydrolase-1 is expressed in a subset of Oct4-positive melanoma cells

Background: Folate hydrolase-1 (FOLH1; NAALADase; PSMA) is a type II transmembrane protein that binds substrates with terminal glutamates. The MXXXL motif on the cytoplasmic N-terminal domain of FOLH1 interacts with clathrin and caveolin-1 to facilitate constitutive internalization upon substrate binding. J591, an established monoclonal antibody (AB) to FOLH1, is highly specific to and is effectively endocytosed after binding to the extracellular domain of FOLH1; J591 is presently being developed in the clinical trial setting, as a vehicle for AB-based brachytherapy in cancers that express FOLH1. Physiological FOLH1 is expressed in cellular regions that are protected by tight-junctions or the blood-brain-barrier, and are therefore not targeted by circulating J591. Functionally, FOLH1 is responsible for cerebral glutamate production. In the oncological setting, FOLH1 is upregulated throughout prostate cancer cellular membranes, and is luminally expressed by cancer neovessels. We have characterized neovascular FOLH1 expression in malignant melanoma (MM), a solid tumor of neural crest (NC) origin. Comparative RTqPCR analysis of normal skin, primary (p), and metastatic (m) MM revealed respective 10.64 and 18.21 -fold increases in FOLH1 gene-expression in pMM (p=0.0041) and mMM (p=0.042) samples as compared to normal skin. Immunohistochemistry of paraffin-embedded MM tumors revealed FOLH1 protein expression in the neovasculature of 4/11 (36%) of pMM and 9/14 (64%) of mMM cases; we noted a subset of keratinocytic and melanocytic FOLH1 positive cells. These results, and the known functional role of glutamate production in a subset of NC-derived tissue, suggest that MM cells may also express cellular FOLH1. Methods: Six (3 BRAF+; 3 BRAF-) MM cell lines were evaluated for cellular FOLH1 and Oct4 expression with J591-based and anti-Oct4 AB immunofluorescence (IF). Oct4 is a homeodomain binding protein encoded by POU5f1, widely accepted as a stem cell marker. FOLH1+ cell lines with permeablized and non-permeablized membranes were incubated with J591. The effect of γ-irradiation (0, 4.5, or 9 Gy) was also tested in FOLH1+ cell lines with harvesting 6 or 24 hours post-irradiation. Results: Ten to 40% of MM cells demonstrated intracellular FOLH1 expression in 2 BRAF-/6 MM cell lines, with a predominant perinuclear and terminal dendritic distribution (e.g., axonal morphology). IF co-labeling with J591 (cytoplasmic) and antibodies to Oct4 (nuclear) identified possible stemness of FOLH1+ / Oct4+ MM cells. RT-PCR analysis of the irradiated cell lines demonstrated ~2-fold increased FOLH1 mRNA levels in 1/2 MM cell lines. Conclusions: Herein is the first demonstration that MM cells express FOLH1. Given that FOLH1 glutamate production is closely linked to NC-originating glial cells, it can be reasonably postulated that this newly identified subset of FOLH1+/Oct4+ MM cells could be NC precursors, or cancer stem cells, for MM. Further, radiation-induced FOLH1 upregulation may increase the therapeutic ratio of AB-based brachytherapy. Citation Format: Marigdalia K. Ramirez-Fort, He Liu, Vicente Navarro, Barbara Meier, Mitch Levesque, Jonathan Moy, Jessica Vissicchio, Emmanuel Contassot, Sae Kim, Talal Syed, Michael Zhang, Vincent Tem, Paul J. Christos, Scott T. Tagawa, Neil H. Bander, Christopher S. Lange, Lars E. French. Possible cancer stem cells: Folate hydrolase-1 is expressed in a subset of Oct4-positive melanoma cells [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 1905. doi:10.1158/1538-7445.AM2017-1905