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Theory with ConsequentsbyJustin Lucas SolaDoctor of Philosophy in Criminology, Law & SocietyUniversity of California, Irvine, 2024Associate Professor Bryan L. Sykes, ChairThis article-based dissertation highlights problems with the operationalization of causal theories of neoliberalism. When our theory is incoherent or leads to incorrect predictions, that marks an opportunity to refine our understanding and be able to suggest better policy. The contribution of this approach is to invite productive challenges to our theorization of social problems. Rather than definitively answering a single large research question, I pursue these questions via four articles that test theories of neoliberalism and further our understanding of why Americans want guns.In the introduction I define what I mean by neoliberalism, discuss the motivations of my dissertations, and lastly discuss the four articles in greater depth. In article one I review how theories of neoliberalism have been operationalized in studies of criminology, law & society, and gun ownership, before critiquing some operationalizations’ adequacy to support causal claims. This motivates my dissertation. The second article is Transmitting Desire (Sola 2021), published in Sociological Perspectives. This article introduces a measure of gun desirability by validating it against the known increase in gun purchases during the COVID-19 pandemic and racial justice protests. I argue that this measure is ideal to test theorized causes of gun interest, like neoliberalism.The third article is Firearms, Families, and Financial Distress (Sola and Warner), a manuscript-in-progress. We use a large dataset (from Sola 2021) to test a prominent causal claim from literatures linking gun ownership to neoliberal responsibilization: that men turn to guns in the face of socioeconomic threat. The fourth article is Widespread, bipartisan aversion exists to neighbors owning AR-15s or storing guns insecurely (Sola and Pickett 2024), published in the Proceedings of the National Academy of Sciences. In this article we test whether Americans are comfortable with their neighbors’ gun ownership and gun storage practices. I extend this article with an appendix that utilizes Honest Causal Forests, a machine learning approach to identify causal heterogeneity (Wager and Athey 2018; Brand et al. 2021). I use this method to explore who is comfortable with their neighbor owning an AR-15 rifle. I find a small group of respondents are comfortable with their neighbor’s AR-15, and then test whether such respondents are similar to citizen-protectors predicted by neoliberal responsibilization literature (Carlson 2015; Stroud 2016). The contribution of this approach is to invite productive challenges to our theorization of social problems.

The COVID-19 pandemic, and protests, have marked an unprecedented increase in US gun sales. But America has long been an outlier; the stockpile of private guns climbed to almost 300 million in 2017. Scholars use multiple theories to explain why gun sales have tripled since the early 2000s, and why disruptions like the pandemic might cause gun sales. However, scholars have difficulty evaluating these theories with existing retrospective estimates of gun sales and other measures, limiting their ability to test theory or suggest policy changes.This study uses the known increase in gun sales during the COVID-19 pandemic to introduce and experimentally validate a novel measure of gun desirability. With a sample of 4,240 US residents, this project demonstrates that gun desirability is a valid measure of inclination towards gun ownership, and that a pandemic video vignette significantly increases overall gun desirability relative to a control video vignette. These results serve as a foundation for future scholarship to (1) discern gun desirability trends, (2) evaluate theorized causes of gun desirability, and (3) consider interventions on those conditions that arouse desire for gun ownership.

Supplementary Figures 1-4. Supplementary Figure 1. Analysis of EFNA4 copy number in breast and hepatocellular carcinoma. Supplementary Figure 2. Characterization of PF-06647263 conjugates. Supplementary Figure 3. Biomarkers of PF-06647263 activity in breast PDX tumors. Supplementary Figure 4. Characterization of EFNA4 affinity for EphA and EphB receptors.

Purpose:Gastrointestinal cancers remain areas of high unmet need despite advances in targeted and immunotherapies. Here, we demonstrate potent, tumor-selective efficacy with PF-07062119, a T-cell engaging CD3 bispecific targeting tumors expressing Guanylyl Cyclase C (GUCY2C), which is expressed widely across colorectal cancer and other gastrointestinal malignancies. In addition, to address immune evasion mechanisms, we explore combinations with immune checkpoint blockade agents and with antiangiogenesis therapy.Experimental Design:PF-07062119 activity was evaluated in vitro in multiple tumor cell lines, and in vivo in established subcutaneous and orthotopic human colorectal cancer xenograft tumors with adoptive transfer of human T cells. Efficacy was also evaluated in mouse syngeneic tumors using human CD3ϵ transgenic mice. IHC and mass cytometry were performed to demonstrate drug biodistribution, recruitment of activated T cells, and to identify markers of immune evasion. Combination studies were performed with anti–PD-1/PD-L1 and anti-VEGF antibodies. Toxicity and pharmacokinetic studies were done in cynomolgus macaque.Results:We demonstrate that GUCY2C-positive tumors can be targeted with an anti-GUCY2C/anti-CD3ϵ bispecific, with selective drug biodistribution to tumors. PF-07062119 showed potent T-cell–mediated in vitro activity and in vivo efficacy in multiple colorectal cancer human xenograft tumor models, including KRAS- and BRAF-mutant tumors, as well as in the immunocompetent mouse syngeneic tumor model. PF-07062119 activity was further enhanced when combined with anti–PD-1/PD-L1 treatment or in combination with antiangiogenic therapy. Toxicity studies in cynomolgus indicated a monitorable and manageable toxicity profile.Conclusions:These data highlight the potential for PF-07062119 to demonstrate efficacy and improve patient outcomes in colorectal cancer and other gastrointestinal malignancies.

Mouse body weights of LS1034 tumor bearing mice treated with GUCY2C(M)-CD3 using adoptive T cell transfer

Purpose: Triple-negative breast cancer (TNBC) and ovarian cancer each comprise heterogeneous tumors, for which current therapies have little clinical benefit. Novel therapies that target and eradicate tumor-initiating cells (TIC) are needed to significantly improve survival.Experimental Design: A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined. The TICs were queried for overexpressed antigens, one of which was selected to be the target of an antibody–drug conjugate (ADC). The efficacy of the ADC was evaluated in 15 PDX models to generate hypotheses for patient stratification.Results: We herein identified E-cadherin (CD324) as a surface antigen able to reproducibly enrich for TIC in well-annotated, low-passage TNBC and ovarian cancer PDXs. Gene expression analysis of TIC led to the identification of Ephrin-A4 (EFNA4) as a prospective therapeutic target. An ADC comprising a humanized anti-EFNA4 monoclonal antibody conjugated to the DNA-damaging agent calicheamicin achieved sustained tumor regressions in both TNBC and ovarian cancer PDX in vivo. Non-claudin low TNBC tumors exhibited higher expression and more robust responses than other breast cancer subtypes, suggesting a specific translational application for tumor subclassification.Conclusions: These findings demonstrate the potential of PF-06647263 (anti–EFNA4-ADC) as a first-in-class compound designed to eradicate TIC. The use of well-annotated PDX for drug discovery enabled the identification of a novel TIC target, pharmacologic evaluation of the compound, and translational studies to inform clinical development. Clin Cancer Res; 21(18); 4165–73. ©2015 AACR.

Table S2. Surface Plasmon Resonance binding affinities of anti-GUCY2C/anti-CD3ε bispecifics to human, mouse and cynomolgus GUCY2C

Supplementary Methods

The COVID-19 pandemic and protests have marked an unprecedented increase in U.S. gun sales. But America has long been an outlier; the stockpile of private guns climbed to almost 300 million in 2017. Scholars use multiple theories to explain why gun sales have tripled since the early 2000s, and why disruptions like the pandemic might cause gun sales. However, scholars have difficulty evaluating these theories with existing retrospective estimates of gun sales and other measures, limiting their ability to test theory or suggest policy changes. This study uses the known increase in gun sales during the COVID-19 pandemic to introduce and experimentally validate a novel measure of gun desirability. With a sample of 4,240 U.S. residents, this project demonstrates that gun desirability is a valid measure of inclination toward gun ownership, and that a pandemic video vignette significantly increases overall gun desirability relative to a control video vignette. These results serve as a foundation for future scholarship to (1) discern gun desirability trends, (2) evaluate theorized causes of gun desirability, and (3) consider interventions on those conditions that arouse desire for gun ownership.

INTRODUCTION AND OBJECTIVE:The final analysis of TITAN confirmed improvement in long-term outcomes in metastatic castration-sensitive prostate cancer (mCSPC) with apalutamide (APA) + androgen depri...

Objective: AI has demonstrated great promise in learning sophisticated features and relations in data that would otherwise remain hidden to the human eye. Here, we developed a proprietary AI-based multimodal approach to integrate clinical, digitized hematoxylin-eosin (H&E), and radiology bone scan (rBS) data for outcome prediction in ADT-treated nmCRPC patients. Identifying prostate cancer patients who may not benefit from ADT could improve the medical management of this disease beyond current definitive therapy. Methods: Patients in the ADT+placebo arm from SPARTAN clinical trial on nmCRPC with available clinical, H&E, and rBS were used (n=154). These patients were randomly divided into 70% (n=107) discovery and 30% (n=47) hold-out test datasets. Using the discovery set, we developed and trained a multimodal approach that combines survival convolutional neural networks (SCNNs1) and Cox proportional-hazards model (CPH) to learn ADT outcomes for overall survival (OS) and time to PSA progression (TTP) from the integration of imaging data and 11 traditional clinical features (e.g., tumor stage, Gleason score, PSA). The ability of the trained framework in predicting outcomes and risk stratification was evaluated on the hold-out set. Bootstrap analysis with Wilcoxon signed rank test was used to determine the significance of the multimodal framework’s performance improvement compared to clinical CPH. Results: The multimodal framework was predictive of ADT outcomes for OS and TTP in nmCRPC patients. In SPARTAN’s hold-out set, the multimodal framework significantly improved the predictive power of clinical CPH by 14%—16% across both outcomes (Wilcoxon signed rank P Conclusion: AI-based framework that learns from the integration of different data types improves outcome prediction in ADT-treated nmCRPC. The multimodal approach demonstrates promise in treatment decision support for the early use of androgen receptor-directed therapy and patient selection for clinical trials with novel treatment combinations. Reference: 1. Mobadersany, Pooya, et al. "Predicting cancer outcomes from histology and genomics using convolutional networks." Proceedings of the National Academy of Sciences 115.13 (2018): E2970-E2979. Conflict of interest statement P.M., J.L., D.G., C.A., S.M., S.B., M.K.Y., K.T., N.H., J.Z., J.G., N.K., and S.S.F.Y., are employees of Janssen Pharmaceutical, LLC. Citation Format: Pooya Mobadersany, Justin Lucas, Darshana Govind, Clemente Aguilar-Bonavides, Sharon McCarthy, Sabine Brookman-May, Margaret K. Yu, Ken Tian, Natalie Hutnick, Jose Zamalloa, Joel Greshock, Najat Khan, Stephen S.F. Yip. Artificial intelligence (AI)-based multimodal framework predicts androgen-deprivation therapy (ADT) outcomes in non-metastatic castration resistant prostate cancer (nmCRPC) from SPARTAN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5053.

5066 Background: TITAN, a phase 3 PBO-controlled study in patients (pts) with mCSPC, showed APA + ADT improved radiographic progression-free survival and OS vs PBO + ADT. In this exploratory analysis, we report the relationships between biomarkers and OS in TITAN. Methods: Circulating tumor (ct)DNA and genomic aberrations in 17 PC-related genes, including androgen receptor (AR), were assessed at baseline (BL; 114 pts) and end of study treatment (EOST; 129 pts) using next-generation sequencing. ctDNA was assessed qualitatively; genomic aberrations were assessed within ctDNA-positive samples as inactivation (heterozygous/homozygous deletion or single nucleotide variant [SNV]) or activation (amplification or SNV). Associations of detected ctDNA/aberrations at BL or EOST with OS, and biomarkers at EOST with OS on subsequent therapies, were evaluated using univariate or multivariate analyses and Cox proportional hazards model; results were stratified by treatment arm. Results: Among pts from both treatment groups, 36% had detectable ctDNA, of which 27% had any genomic AR aberration and 24% had AR gene amplification at BL; prevalence of these biomarkers increased significantly from BL to EOST (Table). The most prevalent non-AR aberrations at BL ( TP53, homologous recombination repair [HRR] pathway, PTEN, RB1, and PIK3CA genes) increased at EOST but not significantly (Table). Among assessed aberrations, presence of ctDNA or any AR genomic aberrations at BL (HR, 1.9 or 6.7; all p < 0.05) and any AR genomic aberrations or PI3K pathway activation at EOST (1.7, p < 0.05 or 2.2, p < 0.001) was significantly associated with poor OS in multivariate analyses from both treatment groups. In univariate analyses of pts who received subsequent therapy (chemotherapy: 106; hormonal therapy: 161), worse OS was associated only with PIK3CA activation, PI3K pathway activation, or TP53 inactivation at EOST (3.7, p < 0.05; 2.4, p < 0.05; 3.0, p < 0.01, respectively) in chemo-treated pts. A small sample size in some biomarker subgroups limits interpretation. Conclusions: These hypothesis-generating data from TITAN show that presence of ctDNA or select AR and non-AR biomarkers at BL or EOST were associated with poor OS. The predictive value of these biomarkers for survival in mCSPC needs further confirmation. Clinical trial information: NCT02489318. [Table: see text]

142 Background: In ACIS, APA + AAP improved radiographic progression-free survival in mCRPC vs AAP; difference in overall survival (OS) was not statistically significant. We report a prespecified analysis of androgen receptor (AR) or non-AR genomic/transcriptional aberrations, known to be associated with poor prognosis, and their associations with OS in patients (pts) with mCRPC. Methods: Circulating tumor (ct)DNA and aberrations in 17 PC-relevant genes were assessed using next-generation sequencing at baseline (BL; n = 197) and at end of study treatment (EOST; n = 140) (biomarker population). ctDNA was summarized qualitatively; genomic aberrations were normalized by ctDNA detection. AR splice variant version 7 (ARv7) was detected by quantitative RT-PCR. Cox proportional hazards model assessed association of OS with biomarkers in univariate/multivariate analyses overall and in pts receiving subsequent treatment (tx). Results: BL characteristics of biomarker and total study populations were similar. From BL to EOST: ctDNA detection did not differ (123/196 [63%] to 92/140 [66%], p=0.6); significant increases occurred in ARv7, AR mutations, and any AR aberration (Table). Prevalence of aberrations in PI3K and homologous recombination repair (HRR) pathways did not change from BL to EOST (40/123 [33%] to 34/92 [37%], p=0.5 and 18/123 [15%] to 14/92 [15%], p>0.9, respectively). Worse OS was associated with ctDNA detection or HRR pathway inactivation at BL (HR, 2.5 or 2.2; all p < 0.001) or presence of ctDNA, ARv7, AR amplification or inactivation of TP53, RB1, or RB1/TP35 pathway at EOST (2.2, p < 0.001; 2.1, p < 0.001; 2.6, p < 0.001; 1.5, p < 0.05; 1.7, p < 0.05; 2.1, p < 0.001, respectively) in univariate analyses. EOST ARv7, AR amplification, and inactivation of RB1, TP53, or RB1/TP53 pathway was associated with worse OS in pts receiving subsequent tx. Detection of ctDNA and RB1 inactivation at BL and RB1/TP53 pathway inactivation at EOST was strongly associated with worse OS (2.1, 3.3, 2.9, respectively, all p < 0.01) in multivariate analyses. Conclusions: In ACIS, AAP or APA + AAP tx was associated with increased AR mutations and ARv7 expression. Detection of ctDNA and select AR/non-AR aberrations may predict poor survival in mCRPC. These markers may be used to improve tx selection following confirmation of their predictive value. Clinical trial information: NCT02257736. [Table: see text]

Background: TITAN, a phase 3 placebo (PBO)-controlled study in a broad population of pts with mCSPC (NCT02489318), demonstrated that the addition of APA to androgen deprivation therapy (ADT) significantly improved radiographic progression-free survival (rPFS) (median rPFS in the APA and PBO groups was not reached and was 22.1 months, respectively) and overall survival (OS) compared with PBO + ADT (Chi N Engl J Med 2019). This exploratory analysis investigated molecular signatures of pts with mCSPC who had long-term responses to APA. Methods: Pts (n = 222) in the biomarker cohort of TITAN were characterized as long-term responders (LTR) or early progressors (EP) by separating rPFS into quartiles. The last quartile with longest rPFS (APA, n = 28; PBO, n = 28) represented LTR. The first quartile, with shortest rPFS (APA, n = 17; PBO, n = 21) represented EP. Gene expression profiles of TITAN pts were generated from archival primary prostate tumors; data were summarized based on 110 predefined gene signatures. Mean signature scores between LTR and EP groups were compared within treatment groups using 2 sample t tests to evaluate whether effects were prognostic or specific to APA treatment. Signatures were also evaluated for association with outcome using Cox proportional hazard models in each treatment group. Results: At primary analysis, with median 22.7 months follow-up, median rPFS was, respectively, not reached and 32.9 months in APA and PBO pts in the LTR group, and 10.9 and 3.7 months in APA and PBO pts in the EP group. A higher level of expression (above median) of either polycomb repressor complex 2 activation or the Decipher signature was more frequently associated with EP in both groups. LTR in the APA treatment group, but not those in the PBO group, had lower expression of transcriptional signatures describing myeloid-derived suppressor cells (MDSCs) and cell cycle progression. These results suggest that pts with mCSPC who have relatively lower levels of MDSCs in the tumor, indicative of a less immune-suppressed tumor microenvironment, may have long-term benefit from treatment with APA. Conclusions: Consistent clinical benefit was observed with the addition of APA to ADT in both EP and LTR. Although the data require confirmation in larger studies, these molecular signatures may have utility in selecting pts with mCSPC who may derive the most benefit from APA and other androgen signaling inhibitors. Citation Format: Justin Lucas, Neeraj Agarwal, Felix Feng, Clemente Aguilar-Bonavides, Shibu Thomas, Michael Gormley, Suneel Mundle, Sabine Brookman-May, Sharon A. McCarthy, Anders Bjartell, Hirotsugu Uemura, Simon Chowdhury, Kim N. Chi. Molecular signatures associated with long-term response to apalutamide (APA) in patients (pts) with metastatic castration-sensitive prostate cancer (mCSPC) in TITAN [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 359.

Objectives: There is a paucity of data describing the immunologic subtypes of prostate cancer, and the features that contribute to them, in either the localized or metastatic disease setting. The objective of our effort was to utilize publicly available datasets in both disease settings to better understand the prevalence of immunologic subtypes within each disease setting and to explore biological mechanisms that may regulate them. Methods: Publicly available datasets utilized were The Cancer Genome Atlas (TCGA), for localized disease, and Stand Up To Cancer (SU2C), for metastatic disease. Evaluating a pan-cancer unsupervised clustering analysis of over 5000 tumors in TCGA with 20 biologically distinct immune related transcriptional signatures we identified sample classes with distinct profiles. The expression level of signatures within each sample class was taken as a composite score to identify the immunological characteristics of individual tumors. This characterization was then applied to the SU2C dataset to interrogate 444 mCRPC tumors. Results: The results of the pan-cancer analysis identified 4 individual sample classes; hot, cold, and 2 altered phenotypes. Of the two altered phenotypes identified, one represented only prostate cancer samples, and was the focus of our investigation in this study. The TCGA dataset showed, 60% cold, 12% hot, and 28% altered. A transcriptional signature representing Macrophage Colony Stimulating Factor 1 (CSF-1) response showed positive correlation with effector cells(CD4 & CD8), while it showed a negative correlation with suppressive cell(Tregs & MDSCs) highlighting the potential for positive immune modulation through this pathway. The SU2C dataset showed, 83% cold, 6% hot, and 11% altered. Cancer testis antigens (CTAs) were expressed at a higher level in SU2C compared to TCGA. We also observed positive correlation between the expression of CTAs and immune suppressive mechanisms and a negative correlation with inflammation. These observations were unique to the metastatic disease setting suggesting a role of CTA expression and increased immune suppressive environment in mCRPC. Conclusions: Through characterization of publicly available datasets, TCGA & SU2C, we were able to define frequencies and characteristics of hot, cold, and altered immune subtypes within different disease settings in prostate cancer. Further validation of unique subtypes and mechanisms identified in this study in larger cohorts would be required to understand the true biological impact of these observations. Citation Format: Justin Lucas, Clemente Aguilar, Michael Gormley, Shibu Thomas, Natalie Hutnick, John Loffredo. Transcriptional characterization of immunological features observed in localized and metastatic prostate cancer [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 3197.

Strong evidence exists supporting the important role T cells play in the immune response against tumors. Still, the ability to initiate tumor-specific immune responses remains a challenge. Recent clinical trials suggest that bispecific antibody-mediated retargeted T cells are a promising therapeutic approach to eliminate hematopoietic tumors. However, this approach has not been validated in solid tumors. PF-06671008 is a dual-affinity retargeting (DART®)-bispecific protein engineered with enhanced pharmacokinetic properties to extend in vivo half-life, and designed to engage and activate endogenous polyclonal T cell populations via the CD3 complex in the presence of solid tumors expressing P-cadherin. This bispecific molecule elicited potent P-cadherin expression-dependent cytotoxic T cell activity across a range of tumor indications in vitro, and in vivo in tumor-bearing mice. Regression of established tumors in vivo was observed in both cell line and patient-derived xenograft models engrafted with circulating human T lymphocytes. Measurement of in vivo pharmacodynamic markers demonstrates PF-06671008-mediated T cell activation, infiltration and killing as the mechanism of tumor inhibition.

The clinical success of immune checkpoint inhibitors that target cytotoxic T-lymphocyte associated protein 4 (CTLA4) and programmed cell death protein 1 (PD-1) or programmed death ligand-1 (PD-L1) demonstrates that reactivation of the human immune system delivers durable responses for some patients and represents an exciting approach for cancer treatment. The combination of multiple immunotherapies as well as the combination of immunotherapy with targeted therapy is being pursued vigorously to increase the rate and extend the duration of response. Preclinical in vivo models for immuno-oncology (IO) typically require immunocompetent mice bearing murine syngeneic tumors. To facilitate translation of preclinical studies into human, we characterized the genomic, transcriptomic, and protein expression of a panel of mouse tumor cell lines grown in vitro culture as well as in vivo tumor samples. Our studies identified many genetic and cellular phenotypic differences that distinguish murine syngeneic models from human cancers. For example, only a small fraction of the somatic single nucleotide variants (SNVs) in mouse cell lines directly match SNVs from human actionable cancer genes. At the cellular level, some epithelial tumor models have a more mesenchymal phenotype with relatively low T-lymphocyte infiltration compared to the corresponding human cancers. Furthermore, in contrast to what has been reported for human tumors, we did not observe a correlation between neoantigen load and cytolytic activity in syngeneic models. Finally, the relative immunogenicity of syngeneic tumors does not typically resemble that of human tumors of the same tissue origin. CT26, a colon tumor model, had the highest immunogenicity and was the most responsive model to CTLA4 inhibitor treatment, by contrast to the relatively low immunogenicity and response rate to checkpoint inhibitor therapies in human colon cancers. These differences highlight limitations of syngeneic models for evaluating novel immune therapies and rationalize some of the challenges associated with translating preclinical findings to clinical studies.

8 Background: SPARTAN, a phase 3 placebo (PBO)-controlled study in patients (pts) with nmCRPC, showed that APA plus androgen deprivation therapy (ADT) significantly improves metastasis-free survival compared with PBO + ADT. This exploratory analysis investigated potential biological signatures of pts with long-term responses to APA and PBO. Methods: The biomarker cohort of SPARTAN was characterized as long-term responders (LTR) or early progressors (EP) based on time to metastasis, and separated into quartiles for APA and PBO groups. Pts progressing in the first quartile (APA, 21; PBO, 17), with shortest time to metastatic event, were classified as EP, those progressing in the last quartile (APA, 39; PBO, 20) as LTR. Gene expression profiles were generated from 233 archival primary prostate tumors. Predefined gene signatures indicative of cancer biology were compared between LTR and EP groups within the APA and PBO arms using 2 sample t tests. Signatures associated with LTR and EP were identified using p values of less than 0.05. Results: Median time to metastatic progression was 40.5 months in APA pts and 22 months in PBO pts in the LTR group and 7.3 and 3.6 months in APA and PBO pts, respectively, in the EP group. Signatures categorized into 3 general mechanistic classes (immune regulation, proliferation, and hormone dependence) associated with LTR on APA included increased T cell activity reflected by T cell activation ( p = 0.0045), stimulation ( p = 0.0642), cytokine response ( p = 0.0489), and interferon production (gamma response p = 0.0227 ), and decreased T cell exclusion ( p = 0.0652), low proliferative capacity ( p = 0.0435), and increased hormonal dependence ( p = 0.0485). High risk (DECIPHER p = 0.0406, metastatic potential p = 0.0077), hormone nonresponsive (basal p = 0.0115; androgen receptor activity-low, p = 0.0437), and neuroendocrine-like tumors ( p = 0.0125) were associated with early progression on treatment with PBO. Conclusions: Although the data require confirmation in larger studies, these molecular determinants may have utility in selecting pts with nmCRPC who may derive the most benefit from APA and other androgen signaling inhibitors. Clinical trial information: NCT01946204.

Purpose: Triple-negative breast cancer (TNBC) and ovarian cancer each comprise heterogeneous tumors, for which current therapies have little clinical benefit. Novel therapies that target and eradicate tumor-initiating cells (TIC) are needed to significantly improve survival. Experimental Design: A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined. The TICs were queried for overexpressed antigens, one of which was selected to be the target of an antibody–drug conjugate (ADC). The efficacy of the ADC was evaluated in 15 PDX models to generate hypotheses for patient stratification. Results: We herein identified E-cadherin (CD324) as a surface antigen able to reproducibly enrich for TIC in well-annotated, low-passage TNBC and ovarian cancer PDXs. Gene expression analysis of TIC led to the identification of Ephrin-A4 (EFNA4) as a prospective therapeutic target. An ADC comprising a humanized anti-EFNA4 monoclonal antibody conjugated to the DNA-damaging agent calicheamicin achieved sustained tumor regressions in both TNBC and ovarian cancer PDX in vivo. Non-claudin low TNBC tumors exhibited higher expression and more robust responses than other breast cancer subtypes, suggesting a specific translational application for tumor subclassification. Conclusions: These findings demonstrate the potential of PF-06647263 (anti–EFNA4-ADC) as a first-in-class compound designed to eradicate TIC. The use of well-annotated PDX for drug discovery enabled the identification of a novel TIC target, pharmacologic evaluation of the compound, and translational studies to inform clinical development. Clin Cancer Res; 21(18); 4165–73. ©2015 AACR.

Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer (OVCA), and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor recurrence and metastasis. We demonstrate that protein tyrosine kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase in the Wnt signaling pathway, is enriched on TICs in low-passage TNBC, OVCA, and NSCLC patient-derived xenografts (PDXs). To deliver a potent anticancer drug to PTK7-expressing TICs, we generated a targeted antibody-drug conjugate (ADC) composed of a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker, and Aur0101, an auristatin microtubule inhibitor. The PTK7-targeted ADC induced sustained tumor regressions and outperformed standard-of-care chemotherapy. Moreover, the ADC specifically reduced the frequency of TICs, as determined by serial transplantation experiments. In addition to reducing the TIC frequency, the PTK7-targeted ADC may have additional antitumor mechanisms of action, including the inhibition of angiogenesis and the stimulation of immune cells. Together, these preclinical data demonstrate the potential for the PTK7-targeted ADC to improve the long-term survival of cancer patients.

The authors briefly summarize advances in the broad field of biochirality connecting two poles of the phenomena: the atomic orbitals and the brain’s cognitive function. Analysis of current results allows introducing the new generation of entangled biomarkers ranging from the molecular chirality to laterality of cognitive and executive functions.

Background: Emerging data suggests that predictive biomarkers based on the spatial arrangement of multiple cell types in FFPE tissue sections will be an important component of precision medicine in immune-oncology. Multiplexed immunofluorescence (mIF) facilitates such assessments. If mIF is to play a translational role in research and ultimately clinical practice, it is vital to refine, standardize, and validate an end-to-end workflow that supports large scale multi-site trials and clinical laboratory processes. To this end, six institutions collaborated to develop an automated 6-plex assay focused on the PD-1/PD-L1 axis and assessed its inter- and intra-site reproducibility. Specific attention was paid to assessment of %PD-L1 expression by immune cells (ICs), as pathologists have poor concordance for this parameter as noted in the Blueprint 2 and multi-institutional NCCN studies on PD-L1 IHC. Methods: A 7-color mIF panel (PD-L1, PD-1, CD8, CD68, FoxP3, Cytokeratin, and DAPI) was optimized on a Leica Bond Rx autostainer. Serial sections of tonsil and a lung cancer tissue-microarray (TMA), antibodies and TSA-Opal detection reagents (Akoya Biosciences) were distributed. Cell pellet arrays were also distributed and used to normalize batch variation in intensity measurements. Tonsil and TMA sections were stained at each site and imaged at 20x using a Vectra Polaris. Cells were segmented and phenotyped using image analysis algorithms. In tonsil sections, the average intensity of the top quartile of cells positive for each marker was assessed to identify potential variation in staining intensity. In lung TMAs, cell densities and %PD-L1 expression in immune cells (CD68+ and CD8+ cells) was determined. Results: The average staining intensity coefficients of variation (CV) for all markers within sites was 10% in tonsil samples. Inter-site concordance for tumor cell and immune cell subset densities in TMAs, had an average R2 value of 0.86 and slope of 0.96. Inter-site concordance for %PD-L1+ ICs had an average R2 value of 0.81, and slope of 0.82, in contrast to ICC values of Conclusions: We demonstrate a reproducible end-to-end process for mIF characterization of the PD-1/PD-L1 axis including automated staining, multispectral imaging, and machine-learning-trained image analysis algorithms. This approach improved reproducibility of %PD-L1 IC assessment and brought it in line with %PD-L1 tumor cell assessment by pathologists. Sources of variation were identified and will be discussed. The described approach may serve as a template for assessing reproducibility of emerging mIF panels for other investigative teams, with an eye toward translating such approaches into clinical trials and ultimately into the clinic. Citation Format: Clifford Hoyt, Kristin Roman, Liz Engle, Chichung Wang, Carmen Ballesteros-Merino, Shawn M. Jensen, John McGuire, Yi Zheng, Carla Coltharp, Mei Jiang, Justin Lucas, Edwin Parra, Ignacio Wistuba, Darren Locke, Bernard A. Fox, David L. Rimm, Janis Marie Taube. Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE study): Reproducibility assessment of an automated multiplexed immunofluorescence slide staining, imaging, and analysis workflow [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 LB-318.

For African Americans facing advanced cancer, churches are trusted sources of support and ideal settings to improve access to supportive care. The Support Team model enhances community support for practical, emotional, and spiritual caregiving. We report on focus groups with pastors of 23 Black Churches and explore their perspective on the Support Team model for church members with cancer. Pastors describe the needs of church members facing cancer from a holistic perspective and recognize opportunities for synergistic faith-health collaboration. The results of this study indicate potential benefits of the Support Team model in Black Churches to reduce silent suffering among individuals facing cancer.

Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor regrowth and metastasis. Here we identify Protein Tyrosine Kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase, as an antigen that is enriched on TICs in low-passage patient-derived xenografts (PDX) of TNBC, NSCLC and other tumor types. An anti-PTK7 antibody-drug conjugate (ADC) was generated from a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker and the Aur0101 auristatin microtubule inhibitor. The anti-PTK7 ADC induced sustained regressions of TNBC, NSCLC and ovarian cancer PDX, with improved activity over standard-of-care chemotherapy, and reduced the frequency of TICs as determined by serial transplantation experiments. Moreover, the ADC may have additional mechanisms of action, including an anti-angiogenic effect, that promote anti-tumor immune responses. Together these preclinical results indicate the potential of the anti-PTK7 ADC to improve the long-term survival of cancer patients. The ADC is currently being tested in a Phase 1 clinical trial, from which interim results will be presented. Citation Format: Marc Isaac Damelin, Alex Bankovich, Jeff Bernstein, Justin Lucas, Liang Chen, Sam Williams, Albert Park, Jorge Aguilar, Elana Ernstoff, Manoj Charati, Russell Dushin, Amy Jackson-Fisher, Monette Aujay, Christina Lee, Hanna Ramoth, Milly Milton, Johannes Hampl, Sasha Lazetic, Virginia Pulito, Douglas Armellino, Edward Rosfjord, Magali Guffroy, Hadi Falahatpisheh, Lindsay King, Frank Barletta, Robert Stull, Marybeth Pysz, Paul Escarpe, David Liu, Orit Foord, Brenda Gibson, Eric Powell, Christopher O’Donnell, Xiaohua Xin, Hans Peter Gerber, Puja Sapra, Scott Dylla. A novel PTK7-targeted antibody-drug conjugate eliminates tumor-initiating cells and induces sustained tumor regressions. [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 1220.

Utilizing pharmacodynamic immunohistochemistry (PD-IHC) for in situ & quantitative measures, we explored the mechanism of action of a Dual-Affinity Re-Targeting (DART®) bispecific recombinant antibody engineered with enhanced pharmacokinetic properties to extend in vivo half-life. This bispecific, designated P-cadherin LP-DART, is designed to engage and activate polyclonal T cell populations via the CD3 complex in the presence of P-cadherin expressing tumors. Following administration in mice bearing established human tumors and implanted with human T-cells we examined the localization of P-cadherin LP-DART within the tumor xenografts, P-cadherin expression, quantitation and immunophenotyping of tumor infiltrating lymphocytes (TILs), downstream biomarkers of T-cell effector function and immunoregulatory mechanisms. Cell surface P-cadherin expression was maintained on the established tumor xenografts after the administration of single and multiple doses of the bispecific molecule. Furthermore, we detected P-cadherin LP-DART in the tumors more than one week after administration. Pan lymphocyte IHC and digital image analysis demonstrated P-cadherin LP-DART mediated CD3+ T-cell infiltration, resulting in nearly half of the viable cells in the tumor being TILs. Conversely, we did not detect infiltrating CD3+ human T-cells in normal organs, confirming a specific target mediated T-cell response at the tumor site. Elevated proximal and downstream mediators of drug action (granzyme B and cleaved caspase 3) further support that P-cadherin LP-DART localized within the tumor induces T-cell mediated growth inhibition and sustained regression. Additionally, to study the the tumor response to redirected T-cell mediated killing we measured the up-regulation of critical immune check point pathways after treatment P-cadherin LP-DART. In the in vivo tumor models examined, tumor cells acutely and robustly induced expression of immunoregulatory pathways in response to effector T-cell activity. Taken together, we demonstrate the utility of in situ kinetic PD-IHC methodologies to demonstrate target expression, drug localization, downstream biomarkers of drug action, and provide insights into potential immunoregulatory mechanisms in response to T-cell mediated bispecific immunotherapy. Citation Format: Justin Lucas, Andrea T. Hooper, Jonathon Golas, Bryan Peano, Alan Opsahl, Leslie Obert, Maria Gavriil, Timothy Fisher, Anton Xavier, Michael Cinque, Roger Conant, Judy Lucas, Adam Root, Lioudmila Tchistiakova, Hans Peter Gerber, Chad May. Pharmacodynamics and mechanisms of drug action for bispecific redirected T cell immunotherapy against P-cadherin. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B010.

The purpose of this study was to determine the effects of temperature or 0.25% bupivacaine treatment in combination with supraphysiologic temperatures on chondrocyte viability.Bovine articular chondrocytes in suspension culture were treated with phosphate-buffered saline solution at 20°C, 37°C, 40°C, 42°C, 45°C, 47°C, and 50°C for 15, 30, and 60 minutes or with phosphate-buffered saline solution at 37°C, 45°C, and 50°C for 30 and 60 minutes followed by 0.25% bupivacaine at 20°C for 60 minutes. Chondrocyte viability was analyzed by flow cytometry with the LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR). Annexin V and ethidium double staining determined whether apoptosis or necrosis occurred.Temperatures from 20°C to 42°C did not cause chondrocyte death. Temperatures at or above 45°C caused significant chondrocyte death, particularly at 50°C for 60 minutes, compared with 37°C at 60 minutes (P.01). When the chondrocytes were incubated at 50°C, subsequent exposure to bupivacaine significantly increased chondrocyte death compared with the saline solution-treated control group (P.001). There were additive cytotoxic effects when bupivacaine was combined with supraphysiologic temperatures. It was also found that bupivacaine at supraphysiologic temperatures caused necrosis of articular chondrocytes.Temperatures at or above 45°C caused significant chondrocyte death. Bupivacaine treatment in the presence of 45°C and 50°C temperatures significantly increased necrosis of bovine articular chondrocytes in this in vitro study.Immediate intra-articular injection of bupivacaine after heat-generating procedures may cause damage to the cartilage because of the additive cytotoxic effects of bupivacaine and elevated temperature.

Thermoplastic olefins (TPOs) are polymeric materials utilized for interior and exterior automotive parts. These materials are often painted to protect and enhance their appearance. TPO materials possess a low surface energy because of the aliphatic backbone, and adhesion promoters (APs), such as chlorinated polyolefins, are used to increase paint adhesion and improve paint performance. The impact of the AP structure and processing conditions on the mechanical properties and degree of interpenetration was investigated on cross sectioned coatings. The AP structure was varied by the chlorine content and molecular weight for the same TPO and polyester acrylic, melamine-cured paint system. Two different processing conditions were investigated. Mechanical properties were measured using depth-sensing indentation. Long-range diffusion of coating components across each interface was verified with confocal Raman microscopy. The AP interfaces, AP/base coat and AP/TPO, were chemically and mechanically sharp at the 1 mum lateral resolution of both techniques. Depth-sensing indentation measured a modulus gradient across the top coating cross section. The free air surface of the top coat had a higher modulus than the interior. Processing conditions and polishing to prepare the cross section for measurements were found to affect only the control AP.

Thermoplastic olefins (TPO) have made significant inroads as polymeric materials for interior and exterior automotive parts. Spray applied chlorinated polyolefins (CPO) are often used to improve paint adhesion to the low surface energy TPO substrates. The penetration of the CPO into the substrate is difficult to quantify, but is critical to achieving a good paint/TPO bond. The interphase between each layer in a coated TPO coupon was investigated using a combination of instrumented indentation and confocal Raman microscopy. The degree of CPO interpenetration was Thermoplastic olefins (TPO) have made significant inroads as polymeric materials for interior and exterior automotive parts. Spray applied chlorinated polyolefins (CPO) are often used to improve paint adhesion to the low surface energy TPO substrates. The penetration of the CPO into the substrate is difficult to quantify, but is critical to achieving a good paint/TPO bond. The interphase between each layer in a coated TPO coupon was investigated using a combination of instrumented indentation and confocal Raman microscopy. The degree of CPO interpenetration was altered by using three different CPOs exposed to two different manufacturing methods for the same TPO and base/clear coat system. It was found that the interfaces, CPO/base coat or CPO/TPO, were chemically and mechanically sharp at the 1 μm lateral resolution of both techniques. A gradient in the modulus through the thickness of the clear coat was observed using instrumented indentation.

Introduction: Strong evidence exists supporting the important role T-cells play in the immune response against tumors. Still, the ability to initiate tumor specific immune responses remains a challenge. We have developed a Dual-Affinity Re-Targeting (DART®) protein engineered with enhanced pharmacokinetic properties to extend in vivo half-life, and designed to engage and activate endogenous polyclonal T cell populations via the CD3 complex in the presence of P-cadherin expressing tumors. We designate this bispecific redirecting T-cell molecule as P-cadherin LP-DART. P-cadherin up-regulation has been reported in various tumors, including breast, gastric, endometrial, colorectal and pancreatic cancers, and is correlated with poor survival of patients. Methods: The P-cadherin LP-DART was stably expressed by CHO cells and purified to homogeneity via standard antibody-purification methods. Functional in vitro studies were performed with a panel of human cancer cell lines and human T cells isolated from healthy donors. In vivo tumor growth inhibition studies were performed in immunodeficient athymic nude or NOD-scid IL2Rgammanull (NSG) mice bearing human tumor cell line- or patient derived-xenografts and human T-cells, and treated with P-cadherin LP-DART. Results: P-cadherin LP-DART exhibited binding to a broad panel of cancer cell lines expressing various levels of endogenous cell surface P-cadherin, and comparable binding to a number of human donor derived T-cells expressing CD3. In the presence of T-cells, this bispecific molecule elicited P-cadherin expression level dependent cytotoxic T-lymphocyte (CTL) responses against the different tumor cell lines, and induced antigen dependent T-cell activation and cytokine release. P-cadherin LP-DART also demonstrated potent in vivo anti-tumor activity against implanted tumor xenografts. Significant tumor growth inhibition was observed across a range of potential indications that express P-cadherin. Measurement of in vivo pharmacodynamic markers support P-cadherin LP-DART mediated CTL infiltration and killing as the mechanism of tumor inhibition. Conclusions: P-cadherin LP-DART displays potent in vitro and in vivo redirected T-cell activity against a broad panel of cancer cell lines expressing a range of cell surface P-cadherin levels. These data support further investigation of P-cadherin LP-DART as a potential novel therapeutic treatment for cancers expressing P-cadherin. Citation Format: Timothy S. Fisher, Adam Root, Bryan Peano, Allison Rohner, Justin Lucas, Mark Elliott, Konstantinos Tsaparikos, Hui Wang, Jonathan Golas, Maria Gavriil, Susan Benard, Tao He, Tracey Clark, Nahor Haddish-Berhane, Ralph Alderson, Yinhua Yang, Syd Johnson, Paul Moore, Lioudmila Tchistiakova, Hans-Peter Gerber, Chad May. Bispecific redirected T-cell immunotherapy targeting P-cadherin expressing tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2476. doi:10.1158/1538-7445.AM2015-2476

Clinical development of cancer therapies is associated with attrition rates as high as 80-95%. This high attrition suggests that standard preclinical pharmacology models do not accurately reflect clinical responses. The development of more predictive preclinical models requires several considerations; the relevance of the in vivo model, the administration of test agent, and the interpretation of efficacy data. PDX are cancer models developed from the direct transfer of patient tumor tissue into immunocompromised mice. A collection of PDX models, by retaining the genetic and histologic characteristics of the patients from which they were derived, represents the complexity and heterogeneity of human cancer. To minimize the clinical attrition rates of oncology compounds, we are developing hundreds of PDX models in seven major cancer indications. The collection is being molecularly profiled by RNAseq, WES, and proteomics. Profiling has identified models with robust expression of target proteins or mutant oncogenes that are likely to respond in preclinical efficacy tests. Conversely, the PDX models may provide an understanding of resistance, for example evaluating models with good target expression that fail to respond to therapy. Patient and tumor information, if known, has been collected for each PDX model including age, sex, cancer stage and grade, diagnosis, primary or metastatic site, and prior treatments. In addition to the improvements provided by the PDX models, a preclinical paradigm shift away from treatment with maximally tolerated dose towards clinically relevant dose (CRD), taking into consideration such aspects as exposure, formulation, route and schedule, is critical when attempting to predict clinical outcome from preclinical data. Also essential is the incorporation of clinically meaningful endpoints (regression) when assessing preclinical activity. We have initiated studies on cohorts of non small cell lung and breast PDX models to predict the likely clinical efficacy of candidate compounds for clinical development and to determine the CRD for standard of care (SOC) regimens required to define the most promising Phase II/III combination therapies. Anti-tumor activities were characterized using RECIST criteria of progressive disease (PD), stable disease (SD), partial response (PR), and complete response (CR). Target expression was evaluated by RNA, proteomics and immunohistochemistry. Preliminary results demonstrate a spectrum of responses against experimental therapeutics, including Phase I ADCs and are defining the CRD required for combination treatments with SOC. Identification of the most critical parameters of PDX models predicting clinical outcome will help in validating the utility of ‘n of 1′ studies with the PDX collection, inform patient enrollment strategies, guide combination therapies, and provide insight for identifying new tumor indications. Citation Format: Edward Rosfjord, Xin Han, Danielle Leahy, Erik Upeslacis, Justin Lucas, Jonathon Golas, Andrea Hooper, Fred Immermann, Bingwen Lu, Jeremy Myers, Zhengyan Kan, James Hardwick, Eric Powell, Puja Sapra, Paul Rejto, Hans-Peter Gerber, Judy Lucas. Patient derived xenograft (PDX) models: improving predictability of experimental cancer therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1469. doi:10.1158/1538-7445.AM2015-1469

Triple-negative breast cancer (TNBC) and ovarian cancer comprise heterogeneous tumors, and neither targeted therapies nor traditional chemotherapies have provided consistent clinical benefit. Novel therapies that target and actively eradicate the subpopulation of tumor cells that mediate drug resistance and tumor relapse could significantly improve patient survival. Tumor-initiating cells (TIC) are functionally defined as the subpopulation of cells that drive long-term tumor growth, resistance to therapy and disease relapse. We herein identified CD324 as a surface antigen able to reproducibly enrich for TIC in well annotated, low passage TNBC and ovarian cancer patient-derived xenografts (PDXs). Gene expression analysis of TIC led to the identification of Ephrin-A4 as a prospective therapeutic TIC target. Humanized Ephrin-A4-specific monoclonal antibodies (mAbs) were generated and demonstrated to internalize to mediate the delivery of potent cytotoxins. An antibody-drug conjugate (ADC) comprising a humanized anti-Ephrin-A4 mAb conjugated to the DNA damaging agent calicheamicin achieved sustained tumor regressions in vivo in both TNBC and ovarian cancer PDX. Anti-Ephrin-A4-ADC (PF-06647263) actively reduced TIC frequency as evidenced by limiting dilution analysis in serial transplantation assays. Unexpectedly, TNBC tumors of the non-Claudin low molecular subtype exhibited higher Ephrin-A4 expression and more robust responses to the ADC than other breast cancer subtypes, which suggests a specific translational application for breast tumor subtype classification. Together these findings demonstrate the potential of the Ephrin-A4-targeted calicheamicin conjugate as a first-in-class compound designed to eradicate TIC and improve long-term survival of cancer patients. PF-06647263 is currently being evaluated in a Phase I clinical trial. Citation Format: Marc Damelin, Alex Bankovich, Albert Park, Jorge Aguilar, Wade Anderson, Marianne Santaguida, Sarah Fong, Monette Aujay, Kiran Khandke, Virginia Pulito, Elana Ernstoff, Paul Escarpe, Jeff Bernstein, Marybeth A. Pysz, Wenyan Zhong, Erik Upeslacis, Judy Lucas, Justin Lucas, Timothy Nichols, Kathryn Loving, Orit Foord, Johannes Hampl, Robert Stull, Frank Barletta, Hadi Falahatpisheh, Puja Sapra, Hans Peter Gerber, Scott J. Dylla. An anti-Ephrin-A4 calicheamicin conjugate effectively targets triple-negative breast and ovarian tumor-initiating cells to result in sustained tumor regression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5425. doi:10.1158/1538-7445.AM2015-5425

Antibody drug conjugates (ADCs) are clinically validated as a modality for targeted therapy of solid and hematological cancer due to advancements in target selection, conjugation chemistry and linker technology. However, much about mechanism of action (MoA) is yet to be fully understood. Our goal was to interrogate ADC pharmacokinetics and pharmacodynamics establishing proof of mechanism (PoM) of drug action with a diverse panel of ADCs. Herein, we describe the development of novel immunohistochemical (IHC) methods for in situ visualization of ADCs binding to target expressing cells and their cognate downstream biomarkers of response in formalin fixed paraffin embedded cells/tissues. We demonstrate specific binding of 4 different ADCs spanning 2 solid tumor targets and an endothelial cell target using IHC with anti-human IgG in human tumor xenograft models expressing the respective targets. ADC binding to target is observed as early as 20 minutes after a single dose of ADC at 3 mg/kg. Utilizing an anti-microtubule inhibitor (MTI) payload-specific antibody we additionally detect ADC binding to tumor cells by monitoring the cytotoxic payload. The cell type where the antibodies and payload localized was identified by double and triple IHC. Pharmacodynamic biomarkers of response for two payload classes (DNA damaging agents and MTIs) were detected with antibodies against phospho-Histone H2AX and phospho-Histone H3, respectively - confirming the expected ADC MoAs. Downstream apoptosis of target cells was detected with cleaved caspase 3 IHC. The kinetics of biomarker response and downstream cellular impact was quantified via image analysis with biomarkers evident as early as 24 hours after a single dose for both tumor cell and vascular targets. Furthermore, we observed a correlation between biomarkers of response and efficacy of the ADCs as measured by statistically significant tumor growth inhibition for the 4 ADCs we studied. These data suggest that IHC interrogations of drug action should be used to further the clinical development of ADCs via demonstration of pharmacodynamic activities at the cellular level, establishing PoM data, and enabling predictive preclinical oncology models in order to reduce clinical attrition of ADCs. Citation Format: Jonathon Golas, Andrea T. Hooper, Justin Lucas, Heather Jones, Timothy Nichols, Kiran Khandke, Manoj Charati, Roger Conant, Michael Cinque, Judy Lucas, Marc Damelin, Ken Geles, Caiazzo Teresa, Frank Loganzo, Puja Sapra, Hans-Peter Gerber, Chad May. In situ imaging of antibody drug conjugate (ADC) binding and pharmacodynamic biomarkers of response in models of human cancer. [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 2059. doi:10.1158/1538-7445.AM2014-2059

Tumor-Initiating Cells (TICs), or Cancer Stem Cells (CSCs), are considered a subpopulation of cells within a tumor that are particularly aggressive because when isolated, these cells can form secondary tumors. Along these lines, experiments in which fragments of tumors, derived from primary patient samples, are re-implanted into nude mice, the majority of cells within the fragment die. However, day 3 after re-implantation, a small subpopulation emerges that is able to seed a new tumor. We hypothesized that these cells are unique and rare within the original tumor and consequently present an opportunity to identify novel markers that can identify these aggressive tumor cells. After sorting live human cells from tumor fragments three days after passaging, we used single cell PCR to determine the extent of heterogeneity within the fragment population. We, in fact, found that there is a subpopulation that highly expresses markers of proliferation and stemness. This is in contrast to the major population within the fragment that is enriched for genes associated with hypoxia and stress response. Interestingly, we found that the hypoxia responsive gene, CA9, does not correlate with other markers of hypoxia, such as VEGF, when analyzing cells derived from the fragment. Instead, CA9 was highly expressed among cells that were also enriched for markers such as Ki67, survivin, and Lgr5. To confirm if CA9 does identify a more proliferative, stem-like population, we have sorted CA9+ and CA9- cells from three day PDX fragments and analyzed using DNA microarrays and real-time PCR. We have consistently found, among several different PDX lines, that passaging of tumors followed by sorting CA9+ cells enriches for cells with increased levels of Lgr5, ASCL2, CD133, and Ki67. This is also validated through IHC analysis in which the majority of the cells that are positive for Ki67 are also positive for CA9. We are continuing to investigate the tumorigenicity of these cells by reinjecting CA9+ and CA9- cells from fragments back into immune-compromised mice. Furthermore, we are building a gene list derived from the microarrays to inspect by single cell PCR if any of these genes associate with Lgr5 in a grown tumor. Thus, we believe this is a functionally relevant and novel method for identify novel TIC markers. Citation Format: Julia Friedman, Wenyan Zhong, Christine Loreth, Veronica Diesl, Xin Han, Justin Lucas, Andrea Hooper, Vlad Buklan, Edward Rosfjord, Danielle Leahy, Judy Lucas, Maximillian Follettie, Kim Arndt. CA9 expression highly correlates with cancer stem cell markers during passaging of PDX lines. [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 3487. doi:10.1158/1538-7445.AM2014-3487

Clinical use of vascular targeting antibody drug conjugates (VT-ADCs) has not yet been explored. Herein, we preclinically demonstrate utility, mechanisms and advantages of VT-ADCs as targeted cancer therapeutics. A monoclonal human IgG1 antibody selective for murine VEGFR2 (B3) is non-VEGF-A neutralizing and traffics to the lysosome. B3 was conjugated to a proprietary auristatin microtubule inhibitor with a non-cleavable maleimidocaproyl linker, resulting in the proof-of-concept VT-ADC, B3-mcMTI. In vitro, free-payload and anti-VEGFR2-mcMTI selectively inhibit proliferating endothelial cells (ECs) with pM and low nM IC50s - suggesting that even with a broadly expressed, low-level target, a VT-ADC should be potent and tumor specific. To test this in vivo, we treated three cell line xenografts (CLX, HT29, Ls174T, A498) and a colorectal carcinoma patient derived xenograft (CRC PDX) with B3-mcMTI at 3 mg/kg q4d, resulting in tumor stasis in four models. In the HT29 colon carcinoma CLX and the CRC PDX model, B3-mcMTI was superior to VEGF-neutralizing antibody G6-31 (anti-VEGF mAb). In standard-of-care (SOC) chemotherapy (irinotecan and 5-FU) combination studies in HT29, B3-mcMTI had improved anti-tumor activity over SOC alone, and, moreover, was as effective in inhibiting tumor growth as a single agent ADC as anti-VEGF mAb combined with SOC. In the 4T1 orthotopic anti-VEGF resistant breast carcinoma setting, B3-mcMTI outperformed anti-VEGF mAb, and when combined with SOC (paclitaxel) demonstrated improved activity over single agent without an increase in metastases. B3-mcMTI internalization in endothelial cells is VEGFR2-mediated, with antibody binding and active payload releasing in normal and tumor tissues at concentrations above the in vitro IC50s; however, payload activity as measured by quantitative image analysis of pharmacodynamic biomarkers such as phospho-Histone H3 and cleaved caspase 3 is overwhelmingly localized to tumor ECs. Targeted vessels are smooth muscle invested, suggesting that VT-ADCs may target vasculature that contributes to anti-VEGF resistance. In conclusion, VT-ADCs are effective both as single agents and when combined with SOC, and VT-ADCs may overcome resistance mechanisms to standard anti-angiogenics. Taken together, these data suggest that payloads selective for proliferating ECs enable ADC-mediated targeting of widely expressed EC surface proteins, supporting the clinical pursuit of VT-ADCs. Citation Format: Andrea T. Hooper, Chao-Pei Betty Chang, Kimberly Marquette, Jonathon Golas, Justin Lucas, Timothy Nichols, Judy Lucas, Gavriil Maria, Edward Rosfjord, Anton Xavier, Nathan Scott, Sadhana Jain, Wei Cao, Mauricio Leal, Andreas Maderna, Magali Guffroy, Xiang Zheng, Lioudmila Tchistiakova, Frank Loganzo, Hans-Peter Gerber, Chad May. Targeting the tumor vasculature with antibody drug conjugates. [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 2669. doi:10.1158/1538-7445.AM2014-2669

The effects of surface treatment, particle size, and concentration of titanium dioxide (TiO2) and its dispersion on the photodegradation of two resin systems, epoxy and acrylic urethane, were examined. A suite of techniques, including laser scanning confocal microscopy (LSCM) and attenuated total reflectance -Fourier transform infrared spectroscopy (ATR-FTIR), was used to follow the degradation process of TiO2/polymer films as a function UV exposure. LSCM was used to show that both pigment dispersion and durability of the polymer matrix influenced the generation of pits/holes in pigmented polymer film. The type of pigment had a greater influence on the more durable polymer matrix, the acrylic urethane system. The epoxy system showed the greatest extent of degradation regardless of the TiO2 choice. The LCSM results were supported by the ATR-FTIR data.