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Introduction: Cancer cells can escape tumor-specific T cell responses via engagement of inhibitory immune checkpoints. PD-L1/PD-1 interaction is one of the major checkpoints that limit effector T cell function against cancer cells, and monoclonal antibodies that block this interaction have been approved as therapies in multiple tumor indications. As a next generation therapy, small molecule inhibitors of PD-L1 potentially have advantageous properties compared to approved monoclonal antibodies, such as better penetration into solid tumors, reduced immunogenicity, lack of Fc-mediated side effects, convenience of oral administration, and lower cost of goods. We therefore embarked on an effort to identify and develop an orally available small molecule capable of targeting PD-L1/PD-1 interactions. Methods: Inhibition of the PD-L1/PD-1 interaction was measured using a binding assay, followed by a cell culture system assessing PD-1 inhibition of T cell receptor (TCR) activation. Human T cell responses were assessed in vitro using the mixed lymphocyte reaction (MLR) assay, and a human peripheral blood mononuclear cell (PBMC)-mediated tumor cell killing assay. For in vivo studies CCX559 was dosed orally in a syngeneic tumor model and in a human tumor cell/PBMC co-implantation model in immune deficient mice. Results: Using structural information and focused medicinal chemistry, we identified CCX559 as a potent inhibitor of PD-L1 interaction with PD-1. CCX559 prevented PD-L1/PD-1 inhibition of TCR signaling in a cell-based reporter assay, increased IFNγ secretion in allogeneic MLR assays, and increased tumor cell killing by human PBMCs. We demonstrated that CCX559 potentially employs multiple mechanisms to inhibit PD-L1, which are distinct from those published for human anti-PD-L1 antibodies. In murine tumor models, orally administered CCX559 reduced tumor growth similarly to a clinically-approved anti-human PD-L1 antibody. Summary: CCX559 is a highly potent, small molecule PD-L1 inhibitor that can be orally administered. CCX559 enhanced primary human T cell activity in vitro and demonstrated anti-tumor efficacy in two murine tumor models. Based on its unique mechanism of PD-L1 inhibition, strong anti-tumor activity, desirable drug properties, and good safety profile, we plan to advance CCX559 into clinical development in the first half of 2021. Citation Format: Chris Li, Marta Vilalta, Linda S. Ertl, Yu Wang, Yibin Zeng, Pingchen Fan, Christopher Lange, Darren McMurtrie, Ju Yang, Rebecca Lui, Ryan Scamp, Vicky Chhina, Alice Kumamoto, Ryan Ong, Ton Dang, Ashton Easterday, Niky Zhao, Shirley Liu, Rajinder Singh, Israel Charo, Kathleen Sullivan, Thomas J. Schall, Penglie Zhang. CCX559 is a potent orally-administered small molecule PD-L1 inhibitor that induces anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1274.

Introduction: Antibody-based therapies targeting the Programmed cell Death-1/ Programmed Death-Ligand 1 (PD-1/PD-L1) immune checkpoint axis have achieved great success in cancer immunotherapy in recent years. As a next generation therapy, small molecule inhibitors of PD-1/PD-L1 offer the potential for increased tumor penetration, shorter half-life (to better manage immune related adverse events), and lower cost. We therefore embarked on an effort to identify and develop orally available small molecules capable of targeting PD-L1. Methods: We designed and optimized a number of small molecule PD-L1 inhibitors which potently disrupted the interaction of PD-1 with PD-L1. Active compounds were first profiled by an ELISA assay measuring inhibition of the PD-1/PD-L1 interaction, followed by a functional cell-based reporter assay, mixed lymphocyte reaction (MLR) assay, and human peripheral blood mononuclear cell (PBMC)-mediated tumor cell killing assay. Targeted medicinal chemistry efforts were employed to improve potency and oral bioavailability, and candidate compounds were then evaluated in murine tumor models. Due to the specific reactivity of these compounds to human PD-L1 (but not murine PD-L1), a syngeneic tumor model with murine MC-38 colon tumor cells expressing human PD-L1 (MC38-hPD-L1 tumor model) was used. Results: The optimized PD-L1 inhibitors were highly potent in cell-based reporter assay, the MLR assay and PBMC-mediated tumor cell killing assays. These compounds also possess high oral bioavailability and desirable safety profiles. In the MC38-hPD-L1 tumor model, Lead compounds potently reduced tumor growth similarly to an anti-human PD-L1 antibody, which was used as a positive control for the experiments. The tumor microenvironment analysis by flow cytometry demonstrated that these compounds almost completely occupied human PD-L1 on the tumor cells in vivo, and thus could potently block the interaction of PD-1/PD-L1 and enhance the immune responses against tumor. Summary: We have identified and advanced unique small molecule inhibitors of human PD-L1 by rational design and optimization. Molecules resulting from these efforts exhibited marked inhibition of the PD-1/PD-L1 interaction and signaling in vitro, and potent anti-tumor effects in an animal model. Citation Format: Shijie "Chris" Li, Marta Vilalta, Linda S. Ertl, Yu Wang, Yibin Zeng, Pingchen Fan, Christopher Lange, Darren McMurtrie, Ju Yang, Rebecca Lui, Ryan Ong, Vicky Chhina, Alice Kumamoto, Simon Yau, Ton Dang, Ashton Easterday, Shirley Liu, Rajinder Singh, Israel Charo, Thomas J. Schall, Penglie Zhang. Anti-tumor effect of orally available small molecule PD-L1 inhibitors in a murine model of colon adenocarcinoma [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 5693.

Purpose/Objective(s): TTFields are FDA-approved for the treatment of recurrent and newly diagnosed Glioblastoma (GBM) in the supratentorial brain. The EF-14 trial showed that combining TTFields with adjuvant chemo-radiation leads to a significant increase in overall survival compared to adjuvant chemo-radiation alone. High-grade gliomas also occur in the infratentorium and brainstem of pediatric and adult patients. Brainstem gliomas have a median survival of 9 to 11 months, necessitating the exploration of novel treatment combinations, such as delivering TTFields to the infratentorial brain. We recently showed in a simulation-based study that TTFields can be successfully delivered to the infratentorium. Effective delivery was achieved by placement of the arrays on the vertex, bilateral posterolateral occiput, and superior-posterior neck; the array placement results in TTFields at therapeutic intensities throughout the brainstem and cerebellum of realistic computational head models. The previous simulation-based study was performed using realistic computational models of healthy individuals; it did not provide detailed insight into field distributions within tumor tissue located in the infratentorium. Here, we aim to expand the results of this previous study, by simulating the delivery of TTFields to the infratentorium of adult and pediatric patients with high-grade brainstem gliomas. Materials/Methods: A realistic computational model was created using MRI data of adult and pediatric patients with high-grade brainstem gliomas. Transducer arrays were placed on the model and the delivery of TTFields to the infratentorial brain was then simulated using a commercial numerical solver. The electric field distribution in the supratentorium, infratentorium, and within the tumor were derived from the simulations, and the dose in the tumor analyzed. Results: The distribution of calculated isofield lines demonstrates effective delivery of TTFields to infratentorial brain tumors. Conclusion: Our results provides rationale for clinically investigating the utility of TTFields in treating infratentorial high-grade gliomas. Citation Format: Marigdalia K. Ramirez-Fort, Brittany Cross, Ariel Naveh, Shearwood McClelland III, Melissa Mendez, Roberto Santiago, Sean S. Mahase, Jaime Matta, Ze'ev Bomzon, Christopher S. Lange. Simulating delivery of TTFields to the infratentorium in patients with brainstem gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4430.

Purpose/Objective(s) Prostate-specific membrane antigen (PSMA), also known as folate hydrolase 1 (FOLH1), is a highly specific biomarker and therapeutic target for prostate cancer (PC). J591, a monoclonal antibody specific to PSMA, is the primary method used to detect PSMA expression. It is currently under investigation as a vehicle to deliver targeted therapies to PC cells. Androgen deprivation therapy (ADT) upregulates PSMA. Preclinical models of androgen resistant (AR) xenografts demonstrate PSMA upregulation by ADT increases the therapeutic index of J591-drug conjugates, suggestive of enhanced drug uptake. We sought to evaluate the effect of external beam radiation (EBRT) on PSMA expression in PC cells ± ADT. Methods Androgen sensitive LNCaP and AR CWR22Rv1 PC cell lines were cultured in standard and charcoal-stripped media for two weeks. Both cell lines were grown in both media conditions, and received either no EBRT, EBRT in 2 Gray (Gy) daily fractions to a maximum of 10 Gy, or 7.5 Gy in 1 fraction. Cell surface PSMA expression was measured by flow cytometry as mean fluorescence intensity 24 hours after each 2 Gy fraction, or on days 1, 3, 5, 7, and 9 after 7.5 Gy. Results LNCaP in standard media upregulated PSMA by 2.97-fold (SEM ± 0.72) with fractionated EBRT to a cumulative dose of 4 Gy, peaking on days 2 and 3 after 4 Gy and 6 Gy cumulative doses, respectively. CWR22Rv1 in standard medium, upregulated PSMA expression by 3.21-fold (SEM ± 0.44) after a cumulative dose of 8 Gy, peaking on day 4. Under ADT (charcoal-stripped media), EBRT did not further increase LNCaP cell PSMA expression (maximal fold-change of 1.01 (SEM ± 0.02)) relative to ADT alone. Compared to CWR22Rv1 with ADT alone, ADT plus EBRT upregulated PSMA by 3.73-fold (SEM ± 0.44) after a cumulative fractionated dose of 8 Gy, and peaking on days 4 and 5. However, 7.5 Gy in one fraction did not significantly increase PSMA expression of LNCaP or CWR22Rv1 in standard media. LNCaP showed a maximal fold-change of 1.14 (SEM ± 0.29) on day 9. CWR22Rv1 showed a maximal fold-change of 1.27 (SEM ± 0.02) on day 1. Conclusions We show PSMA expression is independently enhanced by ADT and by low-dose fractionated EBRT, but not with single-fraction ablative EBRT. These observations warrant additional studies using in vitro and in vivo PC models to validate our findings. PSMA upregulation may aid in optimizing J591 targeting for treatment of local and disseminated or micrometastatic disease. Validation of PSMA upregulation by ADT and EBRT supports use of J591-drug conjugates as an adjunct treatment to these standard therapies for PC. Furthermore, if fractionated EBRT upregulates PSMA, then targeted brachytherapy using appropriately selected J591-radionucleotide conjugates may increase its own therapeutic index in a time-dependent manner as a result of regional low-dose rate beta or alpha emission. Citation Format: Marigdalia K. Ramirez-Fort, Sean S. Mahase, M. Junaid Niaz, He Liu, Vincent Navarro, Goran Rac, Harry S. Clarke, Scott T. Tagawa, Joseph M. Jenrette, Neil H. Bander, Christopher S. Lange. External beam radiation therapy and androgen deprivation therapy increase PSMA expression in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1774.

Folate hydrolase-1 (FOLH1) is a type II transmembrane protein. Oncologically, FOLH1 is upregulated throughout prostate cancer cells; it is also luminally expressed by the neovasculature of most solid tumors but not by normal vessels. J591, a monoclonal antibody (AB), is specific to, and is effectively endocytosed after extracellular binding to, FOLH1. J591 is presently being developed in clinical trials as a vehicle for AB-based brachytherapy in FOLH1+ cancers. Merkel cell carcinoma (MCC) is a rare, neuroendocrine tumor; metastatic (m) MCC is associated with poor survival. We characterized FOLH1 expression in MCC to determine its target potential for J591-brachytherapy. Paraffin sections from primary (p) and mMCC were deparaffinized and rehydrated. Samples were stained with 3E6 (DAKO), a mouse IgG1 monoclonal anti-human FOLH1. Mouse IgG1 (10 ug/mL in 1% bovine serum albumin) was used as an isotype-matched negative control. Anti-CD31 (IgG1) was used as a positive control. Kaplan-Meier survival curves were calculated based on patient outcome data and FOLH1 expression. 81 MCC tumors were evaluated. 67% (54/81) of all cases with 77% (24/31) of pMCC and 60% (30/50) of mMCC tumors demonstrated FOLH1+ neovessels. No cellular staining of tumor cells was identified. 34 patients with FOLH1 +/- MCC were demographically homogeneous in terms of sex, age, immunosuppression status, prior therapies, stage at diagnosis, and local or distant recurrences. No significant differences were detected based on FOLH1 status, in regards to MCC specific survival (P=0.905), or overall survival (P=0.687), as measured from time of diagnosis. FOLH1 is expressed in the majority of pMCC and mMCC cases and is nonprognostic. Our findings support further investigation of targeted therapy with J591-brachytherapy for the management of MCC. Citation Format: Barbara Meier, Marigdalia K. Ramirez-Fort, Kristina Lachance, Candice D. Church, Sean S. Mahase, Joseph M. Jenrette, Christopher S. Lange, Lars E. French, Paul Nghiem, Neil H. Bander. Folate hydrolase-1 is a novel target for J591-brachytherapy in Merkel cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2518.

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

We tested the Cancer Stem Cell (CSC) hypothesis using the patented Hybrid Spheroid (HS) Assay (HSA) and by applying Koch's postulates to test its validity. The HSA is an in vitro assay that enables one to take a viable sample of an individual patient's tumor, make a single cell suspension, mix it in known proportions with human fibroblasts (AG1522) and dispense a known number of cells of the mixture into each well of Ultra Low Attachment (ULA) 96-well plates to agglomerate into 1 HS/well, each containing an average of Applying Koch's postulates: (1) Does the patient's tumor contain cells with CSC properties? Yes: (a) The HSs grow to a size consistent with containing a CSC, (b) those that grew contain cells expressing ≥ 1 OKSM factor(s), and (c) such HSs contain differentiated progeny of the initial CSC. (2) Can we isolate and propagate these cells? In progress: testing HS serial growth, dissociation and reculture. (3) Can these cells induce the tumor in vivo? In progress: testing by xenografting individual CSC-containing HSs in NSG mice. (4) Do these cells contain and express specific gene products that give them CSC properties? Yes: Demonstrated for Oct4 and Nanog. (5) If we disrupt these genes, do the cells lose their CSC properties? In progress: (shRNA). (6) If we eliminate the CSCs do we eliminate the cancer? Yes: tested by (a) mathematical modeling of single CSC growth, differentiation of some progeny into ATCs capable of a limited number of divisions, that then become terminal non-proliferative tumor cells in the HSA; if a single CSC survives/remains, the HS will grow, i.e., the cancer will recur (63% for an average of 1 CSC in a HS, with a Poisson distribution, assuming that niche sites remain available), and (b) by measuring surviving fractions after exposure to radiation and/or chemotherapy agents (where sterilization of the CSC prevents the HS from growing ≥ 10 divisions. The HSA was applied to tumor samples taken from individual endometrial adenocarcinoma patients and correctly predicted, based on CSC radioresistance, which patients would fail their standard-of-care treatments. Conclusion: The CSC hypothesis is validated in the HSA. Citation Format: Christopher S. Lange, Talal Syed, Mike Zhang, Christian Sabalvaro, Hana I. Lim, Ghadir Salame, Bhargava Chitti, Ifeanyi Ilonzo, Arsalaan Ansari, Michelle Chan, Catherine Li, Jing Xu, Xiaotong Chen. Testing the cancer stem cell hypothesis using the hybrid spheroid assay and Koch's postulates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3346.