Purpose: Metastatic castration-resistant prostate cancer (mCRPC) is a lethal, heterogeneous disease with few therapeutic strategies that significantly prolong survival. Innovative therapies for mCRPC are needed; however, the development of new therapies relies on accurate imaging to assess metastasis and monitor response. Standard imaging modalities for prostate cancer require improvement and there remains a need for selective and sensitive imaging probes that can be widely used in patients with mCRPC. Experimental Design: We evaluated the transmembrane protease fibroblast activation protein alpha (FAP) as a targetable cell surface antigen for mCRPC. Genomic and IHC analyses were performed to investigate FAP expression in prostate cancer. Our FAP-targeted antibody imaging probe, [ 89 Zr]Zr-B12 IgG, was evaluated by PET/CT imaging in preclinical prostate cancer models. Results: Analysis of patient data documented FAP overexpression in metastatic disease across tumor subtypes. PET imaging with [ 89 Zr]Zr-B12 IgG demonstrated high tumor uptake and long-term retention of the probe in the preclinical models examined. FAPpositive stroma tumor uptake of [ 89 Zr]Zr-B12 IgG was 5-fold higher than the isotype control with mean %ID/cc of 34.13 AE 1.99 versus 6.12 AE 2.03 (n ¼ 3/group; P ¼ 0.0006) at 72 hours. Ex vivo biodistribution corroborated these results documenting rapid blood clearance by 24 hours and high tumor uptake of [ 89 Zr]Zr-B12 IgG by 72 hours. Conclusions: Our study reveals FAP as a target for imaging the tumor microenvironment of prostate cancer. Validation of [ 89 Zr]Zr-B12 IgG as a selective imaging probe for FAPexpressing tumors presents a new approach for noninvasive PET/CT imaging of mCRPC.

The Ca mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) regulates intracellular trafficking events, including translocation of certain enveloped viruses through the endolysosomal system. Targeting NAADP-evoked Ca signaling may therefore be an effective strategy for discovering novel antivirals as well as therapeutics for other disorders. To aid discovery of novel scaffolds that modulate NAADP-evoked Ca signaling in human cells, we have investigated the potential of using the sea urchin egg homogenate system for a screening campaign. Known pharmacological inhibitors of NAADP-evoked Ca release (but not cADPR- or IP-evoked Ca release) in this invertebrate system strongly correlated with inhibition of MERS-pseudovirus infectivity in a human cell line. A primary screen of 1534 compounds yielded eighteen 'hits' exhibiting >80% inhibition of NAADP-evoked Ca release. A validation pipeline for these candidates yielded seven drugs that inhibited NAADP-evoked Ca release without depleting acidic Ca stores in a human cell line. These candidates displayed a similar penetrance of inhibition in both the sea urchin system and the human cell line, and the extent of inhibition of NAADP-evoked Ca signals correlated well with observed inhibition of infectivity of a Middle East Respiratory syndrome coronavirus (MERS-CoV) pseudovirus. These experiments support the potential of this simple, homogenate system for screening campaigns to discover modulators of NAADP, cADPR and IP-dependent Ca signaling with potential therapeutic value.

Metastatic castration-resistant prostate cancer (mCRPC) has been largely resistant to immunotherapy. Natural killer (NK) cells are cytotoxic lymphocytes that detect and kill transformed cells without prior sensitization, and their infiltration into prostate tumors corresponds with an increased overall survival among patients with mCRPC. We sought to harness this knowledge to develop an approach to NK-cell based immunotherapy for mCRPC. We engineered an NK cell line (NK-92MI) to express CD64, the sole human high-affinity IgG Fcγ receptor (FcγR1), and bound these cells with antibodies to provide interchangeable tumor-targeting elements. NK-92MICD64 cells were evaluated for cell-activation mechanisms and antibody-dependent cell-mediated cytotoxicity (ADCC). A combination of mAbs was used to target the prostate tumor antigen tumor-associated calcium signal transducer 2 (TROP2) and the cancer-associated fibroblast marker fibroblast activation protein alpha (FAP). We found that CD64, which is normally expressed by myeloid cells and associates with the adaptor molecule FcRγ, can be expressed by NK-92MI cells and mediate ADCC through an association with CD3ζ. Cytotoxicity from the combination approach was two-fold higher compared to treatment with NK-92MICD64 cells and either mAb alone, and seven-fold higher than NK-92MICD64 cells alone at an effector–target cell ratio of 20:1. The cytotoxic effect was lost when using isotype control antibodies, indicating a selective targeting mechanism. The combination approach demonstrated efficacy in vivo as well and significantly reduced tumor growth compared with the saline control. This combination therapy presents a potential approach for treating mCRPC and could improve immunotherapy response.

Here, we document the discovery of a monoclonal antibody that selectively binds to both human and murine fibroblast activation protein alpha (FAP), a serine protease that is overexpressed on cancer-associated fibroblasts (CAFs), making it an attractive therapeutic target for the aiding and abetting tumor microenvironment. The lead antibody, B12, was identified from a nai ̈ve murine single-chain variable fragment antibody phage display library screened against recombinant human FAP on magnetic beads. The heavy and light chains of B12 were cloned into full-length human immunoglobulin 1 (IgG) vectors and expressed as a chimeric monoclonal antibody (B12 IgG). We engineered a drugresistant prostate cancer cell line, CWR-R1-EnzR, to express human FAP for antibody characterization and validation (R1-EnzR FAP ). B12 IgG selectively bound to the R1-EnzR FAP cells by flow cytometry and was internalized in vitro by confocal microscopy. B12 IgG was further evaluated as a near-infrared (NIR) optical imaging probe in R1-EnzR FAP and parental xenograft models. High tumor uptake and retention of the NIR probe was observed in the R1-EnzR FAP xenografts, and endogenous expression of murine stromal origin FAP was detected in the parental xenografts. Ex vivo evaluation of these models by immunohistochemistry documented B12 IgG localization to both human and murine FAP-expressing cells.

Natural killer (NK) cells are cytotoxic lymphocytes that detect and kill virally infected or malignant cells. Targeting NK cells to solid tumors can be improved by engineering NK cells to express CD64 (FcγR1), a high affinity receptor for human IgG Fc, in combination with therapeutic monoclonal antibodies (mAbs). CD64 can capture soluble mAbs with two to three orders of magnitude higher affinity than CD16A (FcγIIIA) and mediates tumor cell killing when anti-tumor mAb is bound. This docking platform allows for switchable targeting elements to effectively direct the NK cells to multiple tumor antigens. We are targeting several relevant prostate cancer (PCa) antigens including TROP2, a transmembrane glycoprotein that is overexpressed in metastatic PCa, and FAP, a membrane bound serine protease expressed by cancer-associated fibroblasts in the tumor microenvironment. For all cytotoxicity experiments, we used an immortalized human PCa stromal cell line that highly expresses FAP (hPrCSC-44) and a human PCa cell line that highly expresses TROP2 (DU145). Cell killing was measured using the Delfia EuTDA cytotoxicity assay. First, we tested for mediation of NK92MICD64 cell antibody dependent cell cytotoxicity (ADCC) using a titration of anti-FAP or anti-TROP2 mAb (5 to 0 µg/mL) and saw a concentration dependent effect on cell killing. Next, CD64 capture of soluble anti-FAP or anti-TROP2 mAb was tested by flow cytometry. Over 98% of the NK92MICD64 cells incubated with the therapeutic mAbs were positively stained, indicating that the mAbs dock to CD64 and could direct tumor cell killing. Therapeutic efficacy of anti-FAP or anti-TROP2 mAb bound to NK92MICD64 cells (αFAP-NK92MICD64 and αTROP2-NK92MICD64) was tested by incubating the effector cells with hPrCSC-44 or DU145 cells at several effector:target (E:T) ratios. Potent cytotoxicity was observed and the % specific release was significantly higher than the NK92MICD64 cells alone. Cytotoxicity dropped to baseline levels when the anti-tumor mAb was switched to an isotype control, indicating that the mAb-directed cell killing occurs by an antigen-selective mechanism. After seeing the success of the monotherapies, we wanted to evaluate αFAP-NK92MICD64 and αTROP2-NK92MICD64 cells as a combination therapy. Previous studies have reported a synergistic effect of combination therapies that target both the tumor stroma and malignant cells. To test our approach, hPrCSC-44 and DU145 cells were co-incubated with αFAP-NK92MICD64 and αTROP2-NK92MICD64 cells at several E:T ratios. We observed potent cytotoxicity and % specific release was significantly higher than the NK92MICD64 cells alone. In conclusion, CD64 on NK92MI cells facilitated ADCC and cytokine production demonstrating functional activity in our prostate cancer models. The use of CD64 as a docking platform for therapeutic mAbs was shown to effectively mediate tumor cell killing. Together, these data suggest the potential for our combination cell therapy to be developed as a new therapeutic approach for PCa. Citation Format: Hallie M. Hintz, Aaron M. LeBeau. Engineered NK92MICD64 cell docking platform as a combination therapy approach for prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-382.