Antibody J591 Research Articles

The Bioconjugation and Radiosynthesis of <sup>89</sup>Zr-DFO-labeled Antibodies

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET radiopharmaceuticals. Due to their multi-day biological half-life, antibodies must be labeled with positron-emitting radionuclides with relatively long physical decay half-lives. Traditionally, the positron-emitting isotopes 124I (t1/2 = 4.18 d), 86Y (t1/2 = 14.7 hr), and 64Cu (t1/2 = 12.7 hr) have been used to label antibodies for PET imaging. More recently, however, the field has witnessed a dramatic increase in the use of the positron-emitting radiometal 89Zr in antibody-based PET imaging agents. 89Zr is a nearly ideal radioisotope for PET imaging with immunoconjugates, as it possesses a physical half-life (t1/2 = 78.4 hr) that is compatible with the in vivo pharmacokinetics of antibodies and emits a relatively low energy positron that produces high resolution images. Furthermore, antibodies can be straightforwardly labeled with 89Zr using the siderophore-derived chelator desferrioxamine (DFO). In this protocol, the prostate-specific membrane antigen targeting antibody J591 will be used as a model system to illustrate (1) the bioconjugation of the bifunctional chelator DFO-isothiocyanate to an antibody, (2) the radiosynthesis and purification of a 89Zr-DFO-mAb radioimmunoconjugate, and (3) in vivo PET imaging with an 89Zr-DFO-mAb radioimmunoconjugate in a murine model of cancer.

The bioconjugation and radiosynthesis of 89Zr-DFO-labeled antibodies.

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET radiopharmaceuticals. Due to their multi-day biological half-life, antibodies must be labeled with positron-emitting radionuclides with relatively long physical decay half-lives. Traditionally, the positron-emitting isotopes 124I (t1/2=4.18 d), 86Y (t1/2=14.7 hr), and 64Cu (t1/2=12.7 hr) have been used to label antibodies for PET imaging. More recently, however, the field has witnessed a dramatic increase in the use of the positron-emitting radiometal 89Zr in antibody-based PET imaging agents. 89Zr is a nearly ideal radioisotope for PET imaging with immunoconjugates, as it possesses a physical half-life (t1/2=78.4 hr) that is compatible with the in vivo pharmacokinetics of antibodies and emits a relatively low energy positron that produces high resolution images. Furthermore, antibodies can be straightforwardly labeled with 89Zr using the siderophore-derived chelator desferrioxamine (DFO). In this protocol, the prostate-specific membrane antigen targeting antibody J591 will be used as a model system to illustrate (1) the bioconjugation of the bifunctional chelator DFO-isothiocyanate to an antibody, (2) the radiosynthesis and purification of a 89Zr-DFO-mAb radioimmunoconjugate, and (3) in vivo PET imaging with an 89Zr-DFO-mAb radioimmunoconjugate in a murine model of cancer.

Abstract CT304: Anti-prostate-specific membrane antigen (PSMA) monoclonal antibody J591 plus low-dose interleukin-2 (IL-2) in patients with recurrent prostate cancer (PC)

Abstract Purpose J591 is a deimmunized monoclonal antibody that recognizes the extracellular domain of prostate specific membrane antigen and was engineered to induce antibody-dependent cellular cytotoxicity (ADCC). Low dose IL-2 may enhance ADCC by the clonal expansion of natural killer (NK) cells. Immunotherapy may result in survival benefits independent of immediate response. This study was designed to evaluate the efficacy of J591 plus low-dose IL-2 in men with recurrent PC. Experimental Design Men with non-castrate PSA relapse or progressive metastatic castration-resistant PC (mCRPC) received low-dose subcutaneous IL-2 (1.2 x 106 IU/m2/day) daily for 8 weeks with weekly intravenous infusions of J591 (25 mg/m2) on weeks 4-6 (considered 1 cycle). A maximum of 3 cycles of therapy was delivered. Flow cytometry was used to assess NK cell fraction and PSA changes and survival were assessed. Results Seventeen men were treated between 2001-03 with at least 1 cycle (11 PSA relapse, 6 mCRPC) with 1 mCRPC patient invaluable for PSA response due to a protocol violation (9 received 2 cycles, 2 received 3 cycles). Toxicity was mild, mainly limited to fatigue and injection site reactions. PSA declined following therapy in 7 of 16 evaluable men (2 of 5 mCRPC). Flow cytometry revealed an average increase in absolute NK cell count of 107% at week 4 and 117% at the end of cycle 1. NK cell percent change had an inverse relationship with PSA (2-tailed Pearson correlation coefficient r = -0.46; P= 0.13). The actual vs. predicted survival for 5 of the 6 men with mCRPC disease was greater than predicted by the Halabi nomogram (Spearman correlation analysis r=0.29; P=0.57, i.e. nomogram did not predict survival). Conclusions The combination of J591 with low-dose IL-2 was well tolerated in patients with biochemical relapse and mCRPC. In addition, combination therapy expanded NK cell fraction which correlated with PSA decline. Interestingly, long term follow up reveals that 83% of the mCRPC group lived significantly longer than predicted, mostly in an era prior to the approval of life-prolonging therapy. These data support further testing of the hypothesis that PSMA-directed immunotherapy influences outcome in men with advanced PC. Citation Format: Yulian Khagi, Gurveen Kaur, Paul Christos, Naveed H. Akhtar, David M. Nanus, Neil H. Bander, Scott T. Tagawa. Anti-prostate-specific membrane antigen (PSMA) monoclonal antibody J591 plus low-dose interleukin-2 (IL-2) in patients with recurrent prostate cancer (PC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT304. doi:10.1158/1538-7445.AM2014-CT304

Abstract CT413: Lutetium-177-labeled anti-prostate-specific membrane antigen (PSMA) monoclonal antibody J591 (177Lu-J591) for metastatic non-prostate solid tumors

Abstract Introduction: PSMA is a non-secreted cell-surface protein that is expressed by the neovascular endothelium of most solid tumors (but not by vascular endothelium of normal tissue), making it a candidate for vascular-targeted therapy. The purpose of this study was to define the change in tumor perfusion and cellularity after 177Lu-J591 therapy in patients (pts) with advanced non-prostate solid tumors and PSMA expression as determined by radionuclide imaging (with 111In-J591) and/or by immunohistochemistry (IHC). Of note, radiolabeled J591 is hepatically cleared (negating accurate assessment of liver metastases by J591 imaging). Methods: In this pilot study, following confirmation of PSMA expression by 111In-J591 imaging and/or IHC of tumor neovasculature), 14 pts in 2 cohorts received 177Lu-J591 therapy (7 received a single dose at 70 mCi/m2; 7 received 2 fractionated doses at 35 mCi/m2 each). Pts underwent dynamic contrast enhanced (DCE)-MRI and diffusion weighted imaging (DWI) at baseline, then repeated at 1, 4, 8, and 12 wks post treatment to evaluate tumor perfusion (DCE-MRI) and cellularity (Apparent Diffusion Coefficient (ADC) values from DWI). Values were compared with paired t-testing using Wilcoxon signed-rank test for significance. Results: Among the 14 treated pts, tumor types included: lung (5), bladder (2), pancreas (2), colon, GE junction, renal, ovarian carcinomas and melanoma (1 each). Median age was 61.5 with a median of 3 prior therapies including 5 with prior anti-angiogenic agents; 8 pts received prior radiation. Metastatic sites included: bone, lymph nodes, liver, lung, brain, other. 11 of 14 pts had PSMA expression confirmed by imaging, 3 additional by IHC of archival tumor tissue (with liver metastases). DCE-MRI demonstrated that treatment significantly affected tumor perfusion, with decreased perfusion at the 1-wk (n=11, p=0.03) and 4-wk (n=6, p=0.01) post 177Lu-J591 time points. Compared to baseline, DCE slope was lower than baseline at 1 wk (p=0.045) and 4-wk (p=0.02). Week 8 (n=4) was non-significant for both % change and DCE slope and only 2 pts completed the wk 12 time point. In evaluation of cellularity, 5 of 9 pts had an increase in ADC at either the 1- or 4-wk time point (1 stable, 3 decreased). However, paired comparisons at individual time points were non-significant. Conclusion: Anti-PSMA radioimmunotherapy targeting tumor neovasculature is feasible using 177Lu-J591 and can alter tumor perfusion and cellularity, including in those with prior exposure to anti-angiogenic therapy and/or radiation. Refinement in patient selection with improved imaging (anti-PSMA immuno-PET) is ongoing. Citation Format: Orrin Pail, Gurveen Kaur, Jonathan Dyke, Yuliya Jhanwar, Paul Christos, Allyson Ocean, Manish Shah, Tsiporah Shore, Bryan Schneider, Ronald Scheff, Himisha Beltran, Douglas Ballon, Brian Robinson, David M. Nanus, Neil H. Bander, Scott T. Tagawa. Lutetium-177-labeled anti-prostate-specific membrane antigen (PSMA) monoclonal antibody J591 (177Lu-J591) for metastatic non-prostate solid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT413. doi:10.1158/1538-7445.AM2014-CT413

TU-F-12A-01: Quantitative Non-Linear Compartment Modeling of 89Zr- and 124I- Labeled J591 Monoclonal Antibody Kinetics Using Serial Non-Invasive Positron Emission Tomography Imaging in a Pre-Clinical Human Prostate Cancer Mouse Model

Purpose: To examine the binding kinetics of human IgG monoclonal antibody J591 which targets prostate-specific membrane antigen (PSMA) in a pre-clinical mouse cancer model using quantitative PET compartmental analysis of two radiolabeled variants. Methods: PSMA is expressed in normal human prostate, and becomes highly upregulated in prostate cancer, making it a promising therapeutic target. Two forms of J591, radiolabeled with either 8 9Zr or 12 4I, were prepared. 8 9Zr is a radiometal that becomes trapped in the cell upon internalization by the antigen-antibody complex, while radioiodine leaves the cell. Mice with prostate cancer xenografts underwent non-invasive serial imaging on a Focus 120 microPET up to 144 hours post-injection of J591. A non-linear compartmental model describing the binding and internalization of antibody in tumor xenograft was developed and applied to the PET-derived time-activity curves. The antibody-antigen association rate constant (ka), total amount of antigen per gram tumor (Ag_total), internalization rate of antibody-antigen complex, and efflux rate of radioisotope from tumor were fitted using the model. The surface-bound and the internalized activity were also estimated. Results: Values for ka, Ag_total, and internalization rate were found to be similar regardless of radiolabel payload used. The efflux rate, however, was ∼ 9-fold higher for 12 4I-J591 than for 8 9Zr-J591. Time-dependent surface-bound and internalized radiotracer activity were similar for both radiolabels at early times post-injection, but clearly differed beyond 24 hours. Conclusion: Binding and internalization of J591 to PSMA-expressing tumor xenografts were similar when radiolabeled with either 8 9Zr or 12 4I payload. The difference in efflux of radioactivity from tumor may be attributable to differential biological fate intracellularly of the radioisotopes. This has great significance for radioimmunotherapy and antibody-drug conjugates. Further exploration using the model will examine binding and radioisotope residence as antibody dose is increased to antigen saturation. The Center for Targeted Radioimmunotherapy and Theranostics, Ludwig Center for Cancer Immunotherapy, Memorial Sloan Kettering Cancer Center (MSK), NIH (R25-CA096945). Technical services provided by the MSK Small-Animal Imaging Core Facility were supported by the NIH (R24-CA83084, P30-CA08748, and P50-CA92629; Zanzonico). NCI, Center to Reduce Cancer Health Disparity (R21 CA153177-03; Osborne).

Design and preclinical evaluation of a 99mTc-labelled diabody of mAb J591 for SPECT imaging of prostate-specific membrane antigen (PSMA)

BackgroundSensitive and specific detection of nodal status, sites of metastases and low-volume recurrent disease could greatly improve management of patients with advanced prostate cancer. Prostate-specific membrane antigen (PSMA) is a well-established marker for prostate carcinoma with increased levels of expression in high-grade, hormone-refractory and metastatic disease. The monoclonal antibody (mAb) J591 is directed against an extracellular epitope of PSMA and has been shown to efficiently target disseminated disease including metastases in lymph nodes and bone. Its use as a diagnostic imaging agent however is limited due to its slow pharmacokinetics. In this study a diabody derived from mAb J591 was developed as a single photon emission computed tomography (SPECT) tracer with improved pharmacokinetics for the detection of PSMA expression in prostate cancer.MethodsA diabody in VH-VL orientation and with a C-terminal cysteine was expressed in HEK293T cells and purified by a combination of metal ion affinity and size exclusion chromatography. Specificity and affinity were determined in cell binding studies. For SPECT imaging, the diabody was site-specifically labelled with [99mTc(CO)3]+ via the C-terminal His tag and evaluated in a subcutaneous DU145/DU145-PSMA prostate carcinoma xenograft model.ResultsJ591C diabody binds to PSMA-expressing cells with low nanomolar affinity (3.3 ± 0.2 nM). SPECT studies allowed imaging of tumour xenografts with high contrast from 4 h post injection (p.i.). Ex vivo biodistribution studies showed peak tumour uptake of the tracer of 12.1% ± 1.7% injected dose (ID)/g at 8 h p.i. with a tumour to blood ratio of 8.0. Uptake in PSMA-negative tumours was significantly lower with 6.3% ± 0.5% at 8 h p.i. (p < 0.001).ConclusionThe presented diabody has favourable properties required to warrant its further development for antibody-based imaging of PSMA expression in prostate cancer, including PSMA-specific uptake, favourable pharmacokinetics compared to the parental antibody and efficient site-specific radiolabelling with 99mTc.

Anti–Prostate-Specific Membrane Antigen Liposomes Loaded with225Ac for Potential Targeted Antivascular α-Particle Therapy of Cancer

This study evaluates targeted liposomes loaded with the α-particle generator (225)Ac to selectively kill prostate-specific membrane antigen (PSMA)-expressing cells with the aim to assess their potential for targeted antivascular radiotherapy. In this study, PEGylated liposomes were loaded with (225)Ac and labeled with the mouse antihuman PSMA J591 antibody or with the A10 PSMA aptamer. The targeting selectivity, extent of internalization, and killing efficacy of liposomes were evaluated on monolayers of prostate cancer cells intrinsically expressing PSMA (human LNCaP and rat Mat-Lu cells) and on monolayers of HUVEC induced to express PSMA (induced HUVEC). The loading efficiency of (225)Ac into preformed liposomes ranged from 58.0% ± 4.6% to 85.6% ± 11.7% of introduced radioactivity. The conjugation reactions resulted in approximately 17 ± 2 J591 antibodies and 9 ± 2 A10 aptamers per liposome. The average size of liposomes, 107 ± 2 nm in diameter, was not affected by conjugation or loading. LNCaP cells exhibit 2:1:0.5 relative PSMA expression, compared with MatLu and induced HUVEC, respectively, based on flow cytometry detecting association of the J591 antibody. J591-labeled liposomes display higher levels of total specific binding to all cell lines than A10 aptamer-labeled liposomes. Specific cell association of targeted liposomes increases with incubation time. Cytotoxicity studies demonstrate that radiolabeled J591-labeled liposomes are most cytotoxic, with median lethal dose values, after 24 h of incubation, equal to 1.96 (5.3 × 10(-5)), 2.92 × 10(2) (7.9 × 10(-3)), and 2.33 × 10(1) Bq/mL (6.3 × 10(-4) μCi/mL) for LNCaP, Mat-Lu, and induced HUVEC, respectively, which are comparable to the values for the radiolabeled J591 antibody. For A10 aptamer-labeled liposomes, the corresponding values are 3.70 × 10(1) (1.0 × 10(-3)), 1.85 × 10(3) (5.0 × 10(-2)), and 4.07 × 10(3) Bq/mL (1.1 × 10(-1) μCi/mL), respectively. Our studies demonstrate that anti-PSMA-targeted liposomes loaded with (225)Ac selectively bind, become internalized, and kill PSMA-expressing cells including endothelial cells induced to express PSMA. These findings-combined with the unique ability of liposomes to be easily tuned, in terms of size and surface modification, for optimizing biodistributions-suggest the potential of PSMA-targeting liposomes encapsulating α-particle emitters for selective antivascular α radiotherapy.

Combination of the PI3K Inhibitor ZSTK474 with a PSMA-Targeted Immunotoxin Accelerates Apoptosis and Regression of Prostate Cancer

The phosphoinositide 3-kinase (PI3K) pathway is activated in most advanced prostate cancers, yet so far treatments with PI3K inhibitors have been at best tumorostatic in preclinical cancer models and do not show significant antitumor efficacy in clinical trials. Results from tissue culture experiments in prostate cancer cells suggest that PI3K inhibitors should be combined with other cytotoxic agents; however, the general toxicity of such combinations prevents translating these experimental data into preclinical and clinical models. We investigated the emerging concept of tumor-targeted synthetic lethality in prostate cancer cells by using the pan-PI3K inhibitor ZSTK474 and the immunotoxin J591PE, a protein chimera between the single-chain variable fragment of the monoclonal antibody J591 against the prostate-specific membrane antigen (PSMA) and the truncated form of the Pseudomonas aeruginosa exotoxin A (PE38QQR). The combination of ZSTK474 and J591PE increased apoptosis within 6 hours and cell death (monitored at 24-48 hours) in the PSMA-expressing cells LNCaP, C4-2, and C4-2Luc but not in control cells that do not express PSMA (PC3 and BT549 cells). Mechanistic analysis suggested that induction of apoptosis requires Bcl-2-associated death promoter (BAD) dephosphorylation and decreased expression of myeloid leukemia cell differentiation protein 1 (MCL-1). A single injection of ZSTK474 and J591PE into engrafted prostate cancer C4-2Luc cells led to consistent and stable reduction of luminescence within 6 days. These results suggest that the combination of a PI3K inhibitor and a PSMA-targeted protein synthesis inhibitor toxin represents a promising novel strategy for advanced prostate cancer therapy that should be further investigated.

Phase II Study of Lutetium-177–Labeled Anti-Prostate-Specific Membrane Antigen Monoclonal Antibody J591 for Metastatic Castration-Resistant Prostate Cancer

To assess the efficacy of a single infusion of radiolabeled anti-prostate-specific membrane antigen (PSMA) monoclonal antibody J591 (lutetium-177; (177)Lu) by prostate-specific antigen (PSA) decline, measurable disease response, and survival. In this dual-center phase II study, two cohorts with progressive metastatic castration-resistant prostate cancer received one dose of (177)Lu-J591 (15 patients at 65 mCi/m(2), 17 at 70 mCi/m(2)) with radionuclide imaging. Expansion cohort (n = 15) received 70 mCi/m(2) to verify response rate and examine biomarkers. Forty-seven patients who progressed after hormonal therapies (55.3% also received prior chemotherapy) received (177)Lu-J591. A total of 10.6% experienced ≥50% decline in PSA, 36.2% experienced ≥30% decline, and 59.6% experienced any PSA decline following their single treatment. One of 12 with measurable disease experienced a partial radiographic response (8 with stable disease). Sites of prostate cancer metastases were targeted in 44 of 47 (93.6%) as determined by planar imaging. All experienced reversible hematologic toxicity, with grade 4 thrombocytopenia occurring in 46.8% (29.8% received platelet transfusions) without significant hemorrhage. A total of 25.5% experienced grade 4 neutropenia, with one episode of febrile neutropenia. The phase I maximum tolerated dose (70 mCi/m(2)) resulted in more 30% PSA declines (46.9% vs. 13.3%, P = 0.048) and longer survival (21.8 vs. 11.9 months, P = 0.03), but also more grade 4 hematologic toxicity and platelet transfusions. No serious nonhematologic toxicity occurred. Those with poor PSMA imaging were less likely to respond. A single dose of (177)Lu-J591 was well tolerated with reversible myelosuppression. Accurate tumor targeting and PSA responses were seen with evidence of dose response. Imaging biomarkers seem promising.

What's in a Label? Radioimmunotherapy for Metastatic Prostate Cancer

Prostate cancer has a highly tumor-restricted prostate-specific membrane antigen (PSMA) and may be the ideal solid-organ malignancy for treatment with radioimmunotherapy. Encouraging results using lutetium-177-labeled anti-PSMA monoclonal antibody J591 from a phase II study by Tawaga and colleagues support the continued clinical and preclinical development of radioimmunotherapy for solid tumors.

Enzyme-Mediated Methodology for the Site-Specific Radiolabeling of Antibodies Based on Catalyst-Free Click Chemistry

An enzyme- and click chemistry-mediated methodology for the site-selective radiolabeling of antibodies on the heavy chain glycans has been developed and validated. To this end, a model system based on the prostate specific membrane antigen-targeting antibody J591, the positron-emitting radiometal (89)Zr, and the chelator desferrioxamine has been employed. The methodology consists of four steps: (1) the removal of sugars on the heavy chain region of the antibody to expose terminal N-acetylglucosamine residues; (2) the incorporation of azide-modified N-acetylgalactosamine monosaccharides into the glycans of the antibody; (3) the catalyst-free click conjugation of desferrioxamine-modified dibenzocyclooctynes to the azide-bearing sugars; and (4) the radiolabeling of the chelator-modified antibody with (89)Zr. The site-selective labeling methodology has proven facile, reproducible, and robust, producing (89)Zr-labeled radioimmunoconjguates that display high stability and immunoreactivity in vitro (>95%) in addition to highly selective tumor uptake (67.5 ± 5.0%ID/g) and tumor-to-background contrast in athymic nude mice bearing PSMA-expressing subcutaneous LNCaP xenografts. Ultimately, this strategy could play a critical role in the development of novel well-defined and highly immunoreactive radioimmunoconjugates for both the laboratory and clinic.

Abstract 5130: Prostate circulating tumor cells metastasize to bone via E-selectin expressed on endothelial cells.

Abstract Abstract Prostate cancer cells preferentially metastasize to bone. Studies show that the preferential adhesion is due to interactions between prostate tumor cells and E-selectin present on human bone marrow endothelial cells. In our study, we used a parallel flow chamber to identify if prostate circulating tumor cells (CTCs) metastasize using similar interactions. CTCs were investigated based on the fact that majority of prostate tumor cells exhibit prostate specific membrane antigen (PSMA) on their cell surface. Prostate cancer cells, MDA PCa 2b (MDA) were first labeled with anti-PSMA monoclonal antibody J591 conjugated with alexa fluor 488 (J591-488) that recognizes PSMA and is internalized following binding to PSMA. Anti-PSMA labeled MDA cells were then perfused over IL-β stimulated human umbilical vein endothelial cells (HUVECs) using parallel flow chamber under physiological blood flow. Both unlabeled and J591-488 labeled MDA cells showed similar rolling behavior on stimulated HUVECs. This suggests that prostate CTCs can be identified and their interactions with endothelial cells can be studied. Our study will potentially elucidate the mechanism involved in prostate cancer metastasis to bone. Citation Format: Gunjan Gakhar, Guang YuLee, Marco Seandel, Neil Bander, David Nanus. Prostate circulating tumor cells metastasize to bone via E-selectin expressed on endothelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5130. doi:10.1158/1538-7445.AM2013-5130

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.

Phase I trial of zirconium 89 (Zr89) radiolabeled J591 in metastatic castration-resistant prostate cancer (mCRPC).

31 Background: Presently, there are no means of accurately and reproducibly imaging bone metastases for patients with mCRPC. We are investigating a prostate-specific imaging method that can directly visualize metastases using PSMA as a target and Zr89 radiolabeled anti-PSMA antibody J591 as a tracer. Following the FDA roadmap for biomarker development, we report the first human data on the tracer and its preliminary analytic validation. Methods: 5 mCi of Zr89-J591 was administered intravenously and 4 serial PET/CT scans were obtained within the following time intervals post-injection: 2-4 hours, 18-30 hours, 2-5 days, and 6-7 days. Pharmacokinetic (PK) sampling occurred at 5, 30, and 60 minutes post-injection, and at each PET scan. Patients underwent standard cross-sectional imaging, bone scintigraphy, and FDG PET scanning. Metastatic sites were biopsied in order of preference: Zr89-J591 and FDG positivity, then Zr89-J591 and FDG mismatch, and lastly standard imaging and any PET mismatch. Results: Ten patients were scanned, 132 Zr89-J591 positive sites were detected, and 12 lesions (5 bone, 6 node, 1 lung) in 8 patients were biopsied. Tissue correlation: 12/12 biopsies were positive for cancer. 11/12 biopsied lesions (5 bone, 5 node, 1 lung) were positive on Zr89-J591 imaging. 10/12 were positive by standard scans or FDG. 1 lesion was negative by Zr89-J591 PET, but positive by other modalities. Immunokinetics: Blood clearance T1/2α: 7 +/- 4.5 h (1.1-14 h); T1/2β: 62 +/- 13 h (51-89 h); Whole body clearance T1/2: 219 +/- 48 h (153-317 h). Dosimetry: Maximum retention in liver 8 +/- 1.5 SUV; Kidney 4.1 +/- 0.8 SUV; Tumor 8.2 +/- 6.5 SUV (mean SUV in bone 11.13 +/- 7.57 vs. soft tissue 4.31 +/- 2.59). Optimal time for patient imaging after injection, in terms of tumor to background ratios was 7 +/- 1 days. Conclusions: Zr89-J591 imaging demonstrates excellent tumor localization, pathology correlation, and can demonstrate the presence of tumor even when lesions are negative by standard imaging or FDG PET imaging. PK properties of the tracer have been defined. Use of Zr89-J591 to direct biopsies of metastases provides excellent yield. Further studies to examine reproducibility and post-treatment effects are planned. Clinical trial information: NCT01543659.

Phase II trial of 177lutetium radiolabeled anti-PSMA antibody J591 (177Lu-J591) for metastatic castrate-resistant prostate cancer (metCRPC): Survival update and expansion cohort with biomarkers.

121 Background: A phase II trial in men with progressive metCRPC receiving a single dose of 177Lu-J591 at 65 mCi/m2 (15 pts) or 70 mCi/m2 (phase I MTD, 17 pts) was performed without selection for PSMA expression, suggesting a larger than expected dose-response (13 vs 47% &gt;30% PSA decline respectively), leading to an expansion cohort to validate the response rate at 70 mCi/m2. Methods: Endpoints: to validate the PSA and/or measurable disease response at 70 mCi/m2, evaluate circulating tumor cell counts (CellSearch) and pre-treatment PSMA imaging with 111In-J591 in the expansion cohort, and examine overall survival (OS) for all pts. Results: 15 additional pts were treated. Expansion cohort demographics were similar to the initial cohorts, and PSA responses and toxicity were similar to the initial cohort treated at 70 mCi/m2 (see Table), with myelotoxicity improving in all following nadir at 1 month. More PSA declines and longer OS were seen at 70 mCi/m2. 12 of 15 pts had baseline and follow up CTC counts at 4-6 weeks: 66.7% had &gt;50% decline and 25% were unchanged at 0 or 1 (one declined 27%). Although 93.3% had accurate targeting (imaging) of known sites of disease, as seen in initial analysis, a trend for fewer &gt; 30% PSA declines was seen with less intense PSMA imaging. Conclusions: Single dose 177Lu-J591 at70 mCi/m2 was generally well tolerated, with predictable, reversible myelosuppression, and demonstrates anti-tumor activity in pts with progressive metCRPC. A dose-response relationship was confirmed for both toxicity and activity, with improved response and OS at 70 mCi/m2. CTC declines are demonstrated. Selection of pts based upon non-invasive testing (PSMA imaging) may improve the therapeutic profile. Clinical trial information: NCT00195039. [Table: see text]

Bone Marrow Recovery and Subsequent Chemotherapy Following Radiolabeled Anti-Prostate-Specific Membrane Antigen Monoclonal Antibody J591 in Men with Metastatic Castration-Resistant Prostate Cancer

Radioimmunotherapy (RIT) has demonstrated efficacy with acceptable toxicity leading to approval in non-Hodgkin’s lymphoma, but has been slower to develop for the treatment of advanced solid tumors. Prostate cancer (PC) represents a good candidate for RIT based upon high exposure to circulating antibodies at common disease sites with a specific, highly expressed cell-surface antigen of prostate-specific membrane antigen. Four phase I and II trials utilizing 177Lu- or 90Y-J591 have been reported. Long-term toxicity and chemotherapy administration was analyzed. As expected, the only serious toxicity observed was myelosuppression. Grade 4 thrombocytopenia occurred in 33.3% without significant hemorrhage and grade 4 neutropenia occurred in 17.3% with 0.07% febrile neutropenia. Nearly all subjects (97.3%) recovered to grade 0 or 1 platelets and all had complete neutrophil recovery. The majority (81.3%) received chemotherapy at any time, with 61.3% receiving chemotherapy following RIT. Ten subjects underwent bone marrow biopsies at some point in their disease course following RIT for low counts; all had diffuse PC infiltration without evidence of myelodysplasia or leukemia. As expected, myelosuppression occurs following therapeutic doses of RIT for men with metastatic castration-resistant PC. However, toxicity is predictable and self-limited, with the majority of patients who do not refuse able to receive cytotoxic chemotherapy following RIT.

Abstract 2748: Prostate-specific inhibition of PI3 Kinase and protein synthesis as a therapy for advanced hormone-refractory prostate cancer

Abstract Prostate cancer remains the second leading cause of cancer-related death in men in the United States, as at present there are no effective treatments for advanced stage-affected patients. Recurrence usually involves hormonal androgen suppression, which typically translates into androgen-ablation insensitivity with accompanied limited or transient response to systemic chemotherapy. Our preliminary experiments have shown that it is possible to induce massive and rapid apoptosis in the hormone-refractory prostate cancer cells in vitro and in vivo, by using the PI3K inhibitor ZSTK474 in combination with a prostate cancer-selective cytotoxin J591PE38, a fusion protein between two fragments: one from the J591 antibody (that recognizes the Prostate-Specific Membrane Antigen, expressed in nearly all prostate cancers with the highest rate in poorly differentiated, metastatic and hormone-refractory cases), and the other from the Pseudomonas aeruginosa exotoxin A (PE38, that ADP-ribosylates and inhibits the Eukaryotic Elongation Factor 2). The PI3K inhibitor ZSTK474 in combination with the cytotoxin J591PE38 induces massive cell death (around 100%) in prostate cancer C4-2tetLuc and C4-2 cells monitored by Time-Lapse Video Microscopy during 24 hours, when compared to controls. Combined agents at 6 hours synergistically increases Caspase 3 activity, as confirmed by DEVD-Afc enzymatic assay (about 25 and 30 folds in C4-2tetLuc and C4-2 cells respectively, compared to control) and Western blotting (3.28 and 2.45 folds in C4-2tetLuc and C4-2 cells respectively, compared to control). Apoptosis activation was confirmed by detecting the cleaved fragments of Caspase 7 and PARP (compared to control respectively: 4.12 and 3.58 folds for C4-2tetLuc; 5.38 and 13.38 folds for C4-2 cells). No any major effects were found in the PSMA-negative prostate cancer PC-3 and breast cancer BT-549 cells treated with both agents when compared to controls, confirming the specificity of the cytotoxin J591PE38. PI3 Kinase inhibition by ZSTK474 was confirmed in all cell lines used by monitoring the phosphorylation levels of Akt (Thr308) by Western blotting. Prostate cancer C4-2tetLuc xenografts in nude mice showed a potent reduction of luminescence (around 95% at day 7) in tumors treated with a single local injection of both agents, when compared to controls. Apoptosis activation and PI3 Kinase inhibition at 6 hours were confirmed by monitoring the protein levels of cleaved-PARP and the phosphorylation levels of Akt by Western blotting. In summary, in this study we demonstrated the efficacy of a potential combinatorial approach using a specific PI3 Kinase and de novo protein synthesis inhibitor to treat hormone-refractory prostate cancer with constitutive active PI3K/Akt pathway, by generating massive and rapid apoptosis in vitro and in vivo (This work is supported by NIH/NCI grant 3R01 CA 118329 02S2). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2748. doi:1538-7445.AM2012-2748

Targeted, Activatable, In Vivo Fluorescence Imaging of Prostate-Specific Membrane Antigen (PSMA) Positive Tumors Using the Quenched Humanized J591 Antibody–Indocyanine Green (ICG) Conjugate

In patients with prostate cancer, a positive surgical margin is associated with an increased risk of cancer recurrence and poorer outcome, yet margin status cannot be determined during the surgery. An in vivo optical imaging probe that could identify the tumor margins during surgery could result in improved outcomes. The design of such a probe focuses on a highly specific targeting moiety and a near-infrared (NIR) fluorophore that is activated only when bound to the tumor. In this study, we successfully synthesized an activatable monoclonal antibody-fluorophore conjugate consisting of a humanized anti-Prostate-Specific Membrane Antigen (PSMA) antibody (J591) linked to an indocyanine green (ICG) derivative. Prior to binding to PSMA and cellular internalization, the conjugate yielded little light; however, after binding an 18-fold activation was observed permitting the specific detection of PSMA+ tumors up to 10 days after injection of a low dose (0.25 mg/kg) of the reagent. This agent demonstrates promise as a method to image the extent of prostate cancer in vivo and could assist with real-time resection of extracapsular extension of tumor and positive lymph nodes.

Phase I dose-escalation study of docetaxel/predisone and fractionated 177Lu-J591 for metastatic castrate-resistant prostate cancer (metCRPC).

TPS247 Background: Nearly all prostate cancers express prostate specific membrane antigen (PSMA), with limited expression in other organs, making it an ideal therapeutic target. Monoclonal antibody J591 binds the extracellular domain of cells expressing PSMA with optimal affinity and is effectively internalized, with excellent targeting of metastatic sites when radiolabeled (Liu Clin Cancer Res 1997 & 1998, Milowsky JCO 2004, Bander JCO 2005). 177Lu is a radionuclide with gamma and short-range beta emission. A phase II trial of single-dose 177Lu-J591 radioimmunotherapy (RIT) in pts with metCRPC demonstrated efficacy with acceptable toxicity and an apparent dose-response relationship (Tagawa et al, ASCO 2008). It appears that dose-fractionation of 177Lu-J591 allows higher cumulative doses with less myelosuppression (Tagawa et al, submitted ASCO 2010). Docetaxel, which is standard chemotherapy for metCRPC, is a known radiosensitizer. We hypothesize that this combination will have superior clinical efficacy compared to either agent alone. The objective is to determine toxicity profile and maximal tolerated dose (MTD) of 177Lu-J591 plus docetaxel prior to the initiation of a phase II clinical trial. Methods: This is a multi-institutional phase I trial for men with progressive metCRPC that are docetaxel naive or have demonstrated prior docetaxel sensitivity. Docetaxel 75 mg/m2 is given every 3 weeks with prednisone 10 mg daily. Cohorts of 3-6 men receive escalated doses of fractionated 177Lu-J591 (20 mCi/m2 × 2, escalated in 5 mCi/m2 increments, to maximum of 45 mCi/m2 × 2). The initial dose of 177Lu-J591 is administered 2-3 days prior to cycle 3 of docetaxel to take advantage of initial chemotherapy debulking and to optimize radiosensitization (DeNardo Cancer 1997). Cycle 4 of docetaxel is administered upon adequate hematologic recovery. The primary endpoints are determination of MTD and toxicity profile. Secondary endpoints include response rate and survival. Exploratory objectives include the analysis of PSMA expression in circulating tumor cells and PSMA-based nuclear scans as biomarkers of response and/or toxicity with anti-PSMA-based RIT. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration sanofi-aventis

A randomized phase II trial of 177lu radiolabeled monoclonal antibody J591 (177Lu-J591) and ketoconazole in patients (pts) with high-risk castrate biochemically relapsed prostate cancer (PC) after local therapy.

TPS248 Background: Biochemical recurrence without evidence of PC on standard CT/MRI and bone scans after local therapy is common. Salvage radiotherapy affords a cure to a proportion of pts with biochemical relapse, but most suffer disease progression because of micrometastatic PC outside of the radiation field. J591 is a monoclonal antibody that targets the extracellular domain of prostate specific membrane antigen (PSMA). A phase II trial of single-dose 177Lu-J591 radioimmunotherapy (RIT) in pts with progressive, metastatic castration-resistant prostate cancer (CRPC) demonstrated excellent targeting of metastatic sites, efficacy, and acceptable toxicity (Tagawa et al, ASCO 2008). In general, RIT appears to have its greatest impact in the setting of minimal disease (Kaminski NEJM 2005; Leonard JCO2005; Press JCO 2006) and the beta emission of 177Lu is best suited for lesions 1-3 mm in diameter (O'Donoghue J Nuc Med 1995) (i.e. micrometastatic disease). Methods: Men with high-risk CRPC (PSA doubling time < 8 months and/or PSA > 20 [Smith JCO 2005]) and no evidence of disease on CT/MRI and bone scans are randomized in a 2:1 fashion to receive double-blinded 177Lu-J951 vs 111In-J591 (control) and will undergo planar gamma camera imaging with SPECT following infusion. All pts receive ketoconazole plus hydrocortisone. The primary endpoint of the study is 18-month metastasis-free survival. 140 pts will be treated to allow 80% power with a 2-sided alpha of 5% to detect a 25% absolute difference (50% vs. 75% metastasis free) in radiographically apparent metastasis at 18 months (with interim analysis after 50% of pts have at least 12 months follow- up). Secondary/exploratory endpoints include evaluation of radiolabeled J591 imaging to detect sites of metastases not apparent on standard CT/MRI and bone scan, validation of adrenal androgen levels as biomarkers for ketoconazole (Ryan Clin Cancer Res 2007), analysis of circulating tumor cells for PSMA expression and to predict the appearance of radiographic metastases, and exploration of plasma markers of hemostasis, fibrinolysis, and angiogenesis as biomarkers. No significant financial relationships to disclose.