The purpose of this research proposal was to evaluate the potential diagnostic and therapeutic value of radiolabeled monoclonal antibodies (J591, J533, J415) specific to the extracellular domain of PSMA (PSMA(sub ext)). We previously reported all the in vitro studies and some preliminary biodistribution and radioimmunotherapy studies in nude mice. This final report describes all the in vivo studies including biodistribution and radioimmunotherapy evaluations. The pharmacokinetics, biodistribution, and tumor uptake of (131)I and (111)In- labeled MAbs were performed in nude mice bearing LNCaP tumors. There were significant differences in the absolute tumor uptake (%ID/g) among the (131)I-MAbs. By contrast, the tumor uptake of (111)In and (177)Lu labeled MAbs was similar. However, the T/B and tumor/muscle (TIM) ratios were significantly higher with (111)In and (177)Lu compared to (131)I-MAbs. In addition, the ratios were significantly higher with J591 and J415 compared to that of 7E11 (antibody specific to intracellular domain of PSMA). Radioimmunotherapy studies with (131)I-J59l, (90)Y-DOTA-J591, and (177)Lu-DOTA-J591 produced an unambiguous dose-response resulting in tumor shrinkage or slowing the growth over a period of 4-6 weeks. The most important finding is that this therapeutic response was specific since (90)Y-non-specific MAb had no response. These results clearly demonstrate that radiolabeled MAbs specific PSMA(sub ext) are useful for RID and RIT of prostate cancer.

Despite recent advances, advanced prostate cancer is suboptimally responsive to current chemotherapeutic agents. Radiolabeled monoclonal antibody therapy that targets prostate specific membrane antigen (PSMA) shows promise and is an area of active investigation. J591 is a deimmunized IgG monoclonal antibody developed to target the extracellular domain of PSMA. Preclinical and early phase clinical studies utilizing radiolabeled J591 have demonstrated efficacy in targeting tumor cells and decreasing levels of prostate-specific antigen. Radiolabeled J591 is well-tolerated, non-immunogenic, and can be administered in multiple doses. The dose limiting toxicity is reversible myelosuppression with little non-hematologic toxicity. Future studies will include approaches to optimize patient selection and incorporate novel strategies to improve the success of anti-PSMA radioimmunotherapy.

In the fall of 2007, we started the Phase I dose escalation studies with 177Lu-DOTA-huJ591 monoclonal antibodies (mAb) using dose fractionation regimen. In patients with PCa and who have recurrent and/or metastatic disease, 177Lu dose (20-40 mCi/m2) was escalated in 5 different dose levels (3-6 patients at each dose level). So far we have recruited 17 patients in 5 groups. At each dose level, the patients received two doses of 177Lu-J591 mab, 2 weeks apart. The dose of huJ591 MAb remained fixed at 20 mg/dose. We hope to complete this first trial by June 2009 and start the combination therapy protocol almost immediately. The combination therapy protocol was approved by our IRB and was submitted to DOD HSRRB for review and approval. We started recruitment of subjects into the combination therapy protocol. Our goal is to complete the study in by September 2010. The revised SOW is attached.

These studies were designed to improve the outcome of radioimmunotherapy (RIT) in prostate adenocarcinoma by the inclusion of vasoactive peptides in the RIT protocol. To date three peptides able to modify vascular permeability (VP) were tested. Cytotoxicity studies indicated dose-dependent changes in cell metabolic activities after treatment with two agonists interacting with CD88 (C5aAP peptides); whereas one peptide interactive with a formyl peptide receptor-like I did not seem to have any effect on the growth of these cells in vitro. In vivo results confirmed that at least two of these peptides significantly augmented RIT with 131ICC49. The principal reason for improvements was identified as the increased tumor uptake of the radiotracer. However, there is also an indication that the generation of reactive oxygen species play a significant role. The effect of the C5aAP peptide N2's effect on VP and tumor uptake is virtually identical to the effects of the N1 peptide. The N2, however, considerably reduces the radiosensitivity of LNCaP, PC3 and DU145 cells in vitro. The in vivo studies of N2 with and without thiol group modification are in progress.

Prostate cancer diagnosis and treatment options need to be improved as over- and under-treatment, as well as disease recurrence and resistance to current therapies, continue to be challenges. Prostate stem cell antigen (PSCA) is overexpressed in the majority of prostate cancers and correlates with grade, stage, and metastatic potential. Antibodies, which are highly specific for their target, can be labeled with radionuclides and fluorophores for molecular imaging and therapy. This dissertation describes the use of humanized anti-PSCA antibody fragments for dual-modality immuno-positron emission tomography (immunoPET)/fluorescence imaging and for radioimmunotherapy of prostate cancer in preclinical models. Prostate cancer visualization could be improved by using immunoPET for preoperative non-invasive disease detection, and fluorescence imaging for intraoperative guidance of tumor resection; the power of both imaging modalities can be combined on a single agent. In this work, the dual-labeled humanized anti-PSCA A11 cys-minibody (A11 cMb) was evaluated for successive immunoPET/fluorescence imaging in subcutaneous and orthotopic prostate cancer models. A11 cMb was site-specifically conjugated with the near-infrared fluorophore Cy5.5 and radiolabeled with 124I or 89Zr. 124I- and 89Zr-A11 cMb-Cy5.5 were used to detect subcutaneous and intraprostatic PSCA-positive tumors. High contrast immunoPET/fluorescence imaging with 124I-A11 cMb-Cy5.5 identified both high PSCA-expression and moderate PSCA-expression subcutaneous tumors. 89Zr-A11 cMb-Cy5.5 immunoPET showed uptake in the prostate without interfering signal in the bladder, and ex vivo fluorescence imaging clearly showed signal in the tumor and not the surrounding seminal vesicles. These studies support the potential for dual-modality A11 cMb to be translated for preoperative whole-body disease detection and real-time surgical guidance in prostate cancer patients. Prostate cancer that metastasizes often becomes resistant to current therapies and eventually progresses, and alternative therapy options include radioimmunotherapy which delivers a high radiation dose to the tumor with the aim of minimizing dose to normal organs. Anti-PSCA antibody fragments radiolabeled with a cytotoxic radionuclide, such as the beta-emitter 131I or 177Lu, may be effective for radioimmunotherapy with lower toxicity compared to intact antibodies. 124I- and 89Zr-A11 minibody (A11 Mb) immunoPET was used to guide dosimetry studies, and 131I- and 177Lu-A11 Mb was administered to 22Rv1-PSCA s.c. tumor-bearing nude mice to complete dosimetry estimation. 131I-A11 Mb had a higher projected therapeutic index, or tumor-to-radiosensitive tissue dose, and was used in subsequent therapy studies. 131I-A11 Mb inhibited PSCA-positive tumor growth in a dose-dependent manner and extended survival with minimal off-target toxicity. Additionally, preliminary biodistribution studies in knock-in transgenic mice that express human-PSCA were similar to that of nude mice. Therefore, radioimmunotherapy will likely not be toxic to organs with normal PSCA expression (stomach, bladder), and A11 Mb may be suitable for human translation. The results of this work demonstrate the potential of anti-PSCA minibodies as theranostic agents for disease diagnosis, surgical guidance, and treatment. Additionally, the success of anti-PSCA dual-modality imaging and radioimmunotherapy in preclinical models support further evaluation in the clinic.